Intravenously delivered Multilineage-differentiating stress enduring cells dampen in experimental bronchopulmonary dysplasia rat model

Intravenously delivered Multilineage-differentiating stress enduring cells dampen in experimental bronchopulmonary dysplasia rat model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intravenously delivered Multilineage-differentiating stress enduring cells dampen in experimental bronchopulmonary dysplasia rat model Ryosuke Miura, Atsuto Onoda, Toshihiko Suzuki, Azusa Okamoto, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7875937/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background Neonatal bronchopulmonary dysplasia (BPD) is a lung injury caused by various factors, including intrauterine inflammation, mechanical ventilation, and oxidative stress. BPD results in serious respiratory and neurological dysfunctions and mortality. Recently, some clinical trials have commenced using intravenous delivery of donor-derived multilineage-differentiating stress enduring (Muse) cells. In the present study, we aimed to investigate the therapeutic effects of human Muse cells in a BPD rat model. Methods Rats were put into the incubator within 24 h from birth to expose to hyperoxia (83%) until postnatal day 15. Muse and non-Muse cells, obtained from the bone marrow-mesenchymal stromal cells (MSCs) as stage-specific embryonic antigen-3 (SSEA-3) + and -, respectively, were administered slowly via the right external jugular vein or trachea (Muse cells only) on postnatal day 5. For the vehicle groups, only the acetic acid Ringer's solution was administered. Results Regarding the administration route, intravenous administration was superior to intratracheal administration in terms of improving survival, weight gain, and respiratory function. When administered intravenously, the Muse cells showed superior outcomes in ameliorating respiratory function impairment, lung inflammation, pulmonary hypertension, and anti-inflammatory effects, compared to the non-Muse cells. We also confirmed the engraftment of Muse cells in the lung tissues. A proteomic analysis also showed that Muse cells might have additional effects on the abnormalities in cell adhesion and the blood coagulation/fibrinolytic system caused by BPD. Conclusions Our findings suggested that intravenously transplanted Muse cells provided functional benefits in our experimental BPD rat model. Muse cells BPD Mesenchymal stromal cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Neonatal bronchopulmonary dysplasia (BPD) is a lung injury caused by various factors, including intrauterine inflammation, mechanical ventilation, and oxidative stress.( 1 ) With the advancement in neonatal care, the survival rate of infants born with a birth weight of 90%.( 2 ) However, neonatal BPD remains a serious complication affecting in approximately 40% of surviving infants born at ≤ 28 weeks’ gestation, and its incidence has been relatively stable over the last few decades.( 3 ) Many infants with severe BPD die due to respiratory distress, and even those who are discharged alive require home ventilators and/or home oxygen therapy. Even after hospital discharge, BPD infants have a high likelihood of hospitalization for respiratory infections, and their medical costs are high.( 4 ) Additionally, BPD can lead to impaired respiratory function persisting into adolescence( 3 ) and a low intelligence quotient score in school-aged children.( 5 ) Currently, no definitive treatment exists beyond ventilator management. Systemic administration of steroids may have some short-term treatment effects, yet there are concerns about their long-term neurological outcome,( 6 , 7 ) even with low-dose administration.( 8 ) Therefore, the development of new therapies for BPD is an urgent issue. Recently, stem cell therapy has brought “hope” to patients with various diseases for which no adequate treatment has been available. Even in perinatal and neonatal medicine, numerous animal studies across various diseases have been published.( 9 – 14 ) As for BPD, mesenchymal stromal cells (MSCs) are the primary cell source for stem cell therapy( 15 ). A previous meta-analysis of preclinical studies have revealed that MSCs considerably improved alveolarization and ameliorated pulmonary hypertension, lung tissue inflammation, fibrosis, angiogenesis, and apoptosis.( 16 ) Additionally, more than 10 clinical trials have been started worldwide,( 17 ) and the results for some of these have been already reported.( 18 – 21 ) However, the effect of MSC therapy in these clinical trials did not demonstrate sufficient efficacy in terms of reducing the risk for mortality or moderate/severe BPD.( 21 ) Therefore, to enhance the treatment effects, therapy modifications and/or other strategies should be considered. Multilineage-differentiating stress enduring (Muse) cells, which make up a small fraction of the total MSCs,( 22 ) display endogenous pluripotent-like property and express pluripotent surface marker stage-specific embryonic antigen-3 (SSEA-3).( 23 ) Muse cells express sphingosine-1-phosphate (S1P) receptor 2 and selectively home into the damaged sites by sensing S1P, induced by the injured cells.( 24 , 25 ) Importantly, Muse cells also possess phagocytic activity that allows them to recycle transcription factors from the damaged cells and rapidly differentiate into the same cell type at the homed tissues, thereby replenishing injured cells and repairing the tissues.( 9 , 24 – 29 ) Therefore, Muse cells do not require artificial induction to a pluripotent state or artificial differentiation induction into the target cell type. Besides replacing the damaged cells, the therapy using Muse cells has paracrine effects, as it produces various trophic factors.( 9 ) Moreover, since Muse cells have an immunosuppressive property similar to that of the placenta, specifically the expressions of human leukocyte antigen (HLA)-G and express interferon gamma-induced indoleamine-2,3 dioxygenase, a mediator of immunosuppression,( 26 ) they can survive for a long term in the homed tissue. In fact, intravenously administered allogenic Muse cells could survive for over 6 months in the host tissue without administering any immunosuppressants in preclinical studies.( 9 , 24 ) Based on these attractive properties, clinical trials for several diseases, including neonatal hypoxic–ischemic encephalopathy( 30 ) ( 31 ), adult subacute stroke,( 32 ) acute myocardial infarction,( 33 ) adult epidermolysis bullosa,( 34 ) amyotrophic lateral sclerosis,( 35 ) and cervical spinal cord injury ( 36 ), that utilized the intravenous administration of donor-derived allogeneic Muse cells without HLA matching and immunosuppressant treatment have been initiated in Japan. The effects of stem cell therapy for BPD to date were not derived from the replacement of the injured cells with exogenous MSCs, but from the paracrine effect by the trophic factors secreted from the stem cells.( 37 ) If we can combine the replacement effect using Muse cells with the paracrine effect, we may achieve enhanced therapeutic outcomes. Even in terms of the paracrine effect, the ability of Muse cells to home into the injured sites offers a considerable advantage. In the present study, we evaluated the therapeutic effects of the intravenous administration of Muse cells in hyperoxia-induced BPD rat model and compared these outcomes with those of treatments using non-Muse MSCs (non-Muse cells). In a previous study( 38 ) on BPD using MSCs, the dosage, timing, and route of administration were considered important factors. Moreover, it has been reported that the intratracheal administration of MSCs is more effective than their intravenous administration for the treatment of BPD.( 39 ) Therefore, in the present study, we aimed to compare the therapeutic effects between the intravenous and intratracheal administrations of Muse cells using a BPD rat model. Methods All animal experiments were approved by the Nagoya University Animal Experiment Committee (Protocol Nos. 26126, 27190, 28215, 29012, 30197, 31106, 20032, and M210107-003) and conducted in accordance with the Regulations on Animal Experiments in Nagoya University. The present study complies with the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments). Isolation of human Muse and non-Muse cells Human Muse and non-Muse cells were isolated as described previously.( 22 , 40 ) Briefly, human BM-MSCs (Lonza, PT-2501) were cultured and labelled with green fluorescent protein (GFP), then the human Muse and non-Muse cells were isolated using a fluorescence-activated cell sorter (FACS) as GFP + /SSEA-3 + and GFP + /SSEA-3 − cell fractions, respectively. Animal models and experimental protocol Pregnant Sprague–Dawley rats were obtained from Japan SLC Inc. (Shizuoka, Japan). They were healthy, non-genetically modified, and had not previously undergone any procedures. Pregnant rats at 16 days’ gestation were transported to the animal facility of Nagoya University and acclimatized until delivery on gestational day 21.The rats were housed under 12-h light/12-h dark cycles (9.00 A.M. to 9.00 P.M.) with the temperature controlled at 23°C. The pups were placed in an incubator within 24 h of birth and exposed to hyperoxia (83%), using a high/low oxygen control system for small animal experiments (BioSpherix, Parish, NY) until postnatal day 15. Muse (1 × 10 4 cells; Muse group) or non-Muse cells (1 × 10 4 cells; non-Muse group) in 0.1-mL acetic acid Ringer's solution or vehicle (only 0.1-mL acetic acid Ringer's solution: vehicle group) were administered slowly via the right external jugular vein or trachea on postnatal day 5. The rats in the sham group were housed under room-air conditions. The dams were rotated once every 2 days between the litters in the normoxia and hyperoxia groups to avoid excessive oxygen toxicity. Rats were sacrificed on day 15 or 29 to evaluate the treatment effect. On day 5, pups exposed to hyperoxia were allocated to the vehicle and cell-treated groups, so that the body weights were evenly balanced. The group allocations and experimental procedures were not blinded. However, the evaluations of tissues and bronchoalveolar lavage fluid (BALF) were conducted in a manner blinded to group assignment. The minimum sample for comparing the outcomes between the intravenous and intratracheal injections was calculated based on the preliminary experiments to achieve an 80% power of testing with an error rate of 1.67%, assuming a difference of 0.005 and standard deviation of 0.0065 in the tidal volume (TV) as a primary endpoint. The total sample size was calculated as n = 36. Additionally, in our previous experiments, approximately 20% of the intratracheal rats would occasionally die, particularly during the experimental process; therefore, the number of rats for evaluations was set to 44 per group. The minimum sample for comparing the outcomes between therapies using non-Muse and Muse cells was calculated based on the preliminary experiments to achieve an 80% power of testing with an error rate of 1.25%, assuming a difference of 0.025 and a standard deviation of 0.0020 in the TV as a primary endpoint. In this case, the total sample size was calculated as n = 15. Given that rats receiving intravenous injections rarely dies, the number of rats for evaluations was set to 15 per group. Rats with TV values of > + 4 SD were considered outliers and excluded from the analysis. Two rats were excluded. The humane endpoints in this study were as follows: if severe lethargy (loss of spontaneous activity or inability to feed or drink) persisted or if the animals exhibited signs of severe distress, such as respiratory difficulty, abnormal posture, or convulsions, the experiment was terminated. Animals meeting these criteria were humanely euthanized by CO₂ inhalation. If any animals showed signs of unexpected suffering, they were immediately evaluated and euthanized according to these criteria. Body weight and survival rate The rats’ body weights were evaluated from day 5 to day 15. The cumulative survival rate in each group was also evaluated every other day from birth to day 15. Respiratory function test: whole body plethysmography (WBP) A respiratory function test was performed on day 15. TV was measured using WBP, which is an unrestrained respiratory function analyzer (IOX, emka TECHNOLOGIES Co., Ltd., Paris, France.). Each rat was housed in each animal chamber. After confirming that the respiratory parameters were stable, recording was started and continued for > 10 min. From the total respiratory data, a 2-min segment when the respiratory rate was stable was selected for analysis. Tissue preparation The rats were deeply anesthetized with an intraperitoneal injection of overdosed pentobarbital or an anesthetic mixture comprising medetomidine, midazolam, and butorphanol( 41 ) on day 29. Then, these rats were transcardially perfused with saline. The lungs were fixed using 4% paraformaldehyde via a tracheal catheter and kept at 20-cmH 2 O pressure for 20 min to be inflated, followed by immersion–fixation in 4% paraformaldehyde overnight at 4°C. Subsequently, the right lungs were immersed in 20% and 30% sucrose solutions for 24 h each and embedded in a Tissue-Tek OCT solution (Sakura Finetek, Torrance, CA, USA) for the preparation of 30-µm frozen sections. Contrarily, the left lungs were dehydrated with a graded series of ethanol and xylene and embedded in paraffin for the preparation of 5-µm sections. Tissue morphometry After deparaffinization and rehydration with xylene and graded alcohols to water, the paraffin sections were stained with hematoxylin and eosin (H&E). To assess the alveolar maldevelopment, the tissue volume densities (TVDs) in the left lungs were evaluated. The TVD was evaluated as described previously( 11 ). With 100 evenly spaced points (a 10 × 10 grid), 300 µm apart, in each of the three random fields with six sections (a total of 18 fields), the proportion of lung tissue (alveolar ducts and sacs) in the lungs was evaluated using Stereo Investigator version 2020 stereology software (MicroBrightField, Williston, VT). Differential cell counts of bronchoalveolar lavage fluid On day15, BALF was evaluated. The BALF was collected by instilling 0.6 mL (0.3 × 2) saline via a tracheal tube. After staining with Türk solution, the total cell number of BALF was counted using Burker chambers. Then, a 100-µL aliquot was centrifuged and plated onto the glass slides. Differential cell counts were made, with at least 200 cells per animal, by staining with May–Giemsa ( 42 , 43 ). Quantitative polymerase chain reaction with the Alu sequence specific primer To evaluate the distribution of Muse and non-Muse cells, quantitative polymerase chain reaction (qPCR) assays were performed with Alu elements, which are primate-specific repeats and comprise 11% of the human genome,( 44 ) as previously described.( 9 ) Briefly, genomic DNA from the brain, lung, liver and spleen was collected after the intravenous administration of Muse or non-Muse cells on day 15. PCRs were performed with a volume of 20 µL, containing 10-µL TaqMan Universal Master Mix II (Applied Biosystems, Waltham, MA), 900-nM forward and reverse primers, 250-nM TaqMan probe, and 100-ng target template. Amplification was initiated at 50°C for 2 min and 95°C for 10 min, followed by 45 cycles of an incubation step at 95°C for 15 s and 60°C for 1 min. Immunofluorescence staining Altogether, 30 µm-thick lung tissue sections placed into 12-well plates were used for a histological analysis. To remove the storage solution, the sections were washed three times in PBS for 10 min each. Antigen retrieval was performed by incubating the sections in a HistoVT One solution (Nacalai Tesque, Kyoto, Japan) at 70°C for 30 min, followed by cooling to room temperature. The sections were then blocked using a 10% normal donkey serum (IHR-8135, Immunobioscience, Mukilteo, WA) in PBS, containing 0.01% Triton X for 60 min at room temperature on a shaker. After washing three times in PBS containing 0.01% Triton X, the sections were then incubated with primary Goat anti-GFP antibody (1:50, ab6673, abcam, Cambridge, UK) diluted in the Canget Signal Solution B (TOYOBO CO., LTD., Osaka, Japan) for 24 h at 4°C on a shaker. The sections were washed three times with PBS containing 0.01% Triton X (MP Biomedicals, Inc., Irvine, CA) for 10 min each. The sections were then incubated with secondary Donkey anti-Goat IgG (H&L) Antibody conjugated DyLight™ 488 (1:500, 605-741-002, ROCKLAND, Limerick, PA) diluted in Canget Signal Solution B for 10 h at room temperature on a shaker. To protect the fluorescent signal, all incubations were performed while shielded from light. After the first round of staining, the tissue sections were washed again three times in PBS containing 0.01% Triton X for 10 min each. A second set of primary antibodies, including Mouse anti-STEM121 (1:50, Y40410, Takara Bio Inc., Shiga, Japan), Mouse anti-hPE10 (1:50, 10375, IBL Co., Ltd., Gunma, Japan), Rat anti-hPDPN (1:50, 018-24101, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), Rabbit anti-Ctnnb1 (1:100, 51067-2-AP, Proteintech, Inc., IL), or Rabbit anti-Anxa1 (1:100, 21990-1-AP, Proteintech) were diluted in Canget Signal Solution B and applied to the sections. The sections were incubated for 24 h at 4°C on shaker, while again protected from light. The sections were washed three times in PBS containing 0.01% Triton X for 10 min each. Secondary antibodies including Donkey anti-mouse IgG (H&L) Antibody conjugated DyLight™ 549 (1:500, 610-742-124, ROCKLAND), Rat anti-Rat IgG (H&L) Antibody conjugated DyLight™ 549 (1:500, 612-442-026, ROCKLAND), or Rabbit anti-Rat IgG (H&L) Antibody conjugated DyLight™ 549 (1:500, 611-142-002, ROCKLAND) were diluted in Canget Signal Solution B and applied to the sections. The sections were incubated for 10 h at room temperature on a shaker while again protected from light. Hoechst 33342 (10 µg/mL, Abcam, Cambridge, UK) was used for nuclear counterstaining under light-protected conditions. Finally, the tissue sections were transferred onto glass slides using a glass rod in 0.01-M phosphate buffer with 0.001% Triton X. Excess buffer was removed, and the sections were air-dried on the glass slides. The slides were mounted with a ProLong Glass mounting medium, ensuring no air bubbles or debris were trapped under the coverslip. The mounted slides were allowed to dry in a dark environment for at least 24 h to preserve the fluorescent signals. Assessment of pulmonary hypertension To assess for pulmonary hypertension, the dry weight ratio of the right ventricle (RV) to the left ventricle (LV) plus interventricular septum (IVS) (RV/LV + IVS) and right ventricular systolic pressure (RVSP) were used. The hearts obtained on day 29 were dissected, and the RV was separated from the LV and IVS. These tissues were dried up in a drying oven at 60°C for 48 h. The weight of each tissue was measured, and the RV/LV + S ratio was calculated.( 45 ) To measure the RVSP, the external jugular vein was exposed under isoflurane anesthesia, and a catheter (Natsume Seisakusho Co., Ltd. Tokyo, Japan) was inserted into the RV. The RVSP was measured by a pressure transducer and an amplifier system attached to the catheter (LEG-1000; Nihon Kohden Co, Tokyo, Japan) on day 29. Proteomic analysis Protein preparation Protein lysates of lung tissues were prepared according to a previously reported method.( 46 ) The lung tissue samples were collected from day-15 rats of each group (n = 8/group). The lung tissues were quickly frozen by liquid nitrogen, smashed to a powdered form using the Multi-beads shocker (Yasui Kikai Co., Ltd., Osaka, Japan), and then homogenized in a T-PER protein extraction reagent (20 mL/g of tissue) (Takara Bio. Inc., Shiga, Japan) with a protease inhibitor cocktail (Complete Tablet, EDTA-free, Roche Diagnostics, Basel, Switzerland) on ice. Homogenates were centrifuged at 11,000 × g for 15 min at 4°C to separate the insoluble debris. Then, the supernatants of protein lysates were collected. A pierce detergent removal spin column (Thermo Fisher Scientific, MA) was used to remove the surfactant from the supernatants. The total protein concentrations in the supernatants were determined by the bicinchoninic acid method using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). The protein supernatants of each pup were adjusted to 100 µg /200 µL and labeled with the Tandem Mass Tag™ system (Sixplex TMT, Thermo Fisher Scientific) for liquid chromatography/tandem mass spectrometry (LC/MS/MS). Proteomic analysis Proteomics was conducted based on our previous study.( 46 ) The amounts of all protein types in the lung were detected and quantified by LC/MS/MS. During the LC/MS/MS, the Orbitrap Fusion mass spectrometry system (Thermo Fisher Scientific) combined with the UltiMate 3000 RSLCnano LC system (Dionex Co., Amsterdam, The Netherlands) and a nanocapillary column (150 mm × 75 µm i.d., Nikkyo Technos Co., Tokyo, Japan) via a nanoelectrospray ion source was used. In reversed-phase chromatography, the flow rate of the linear gradient (0 min, 5% B; 100 min, 40% B) of 2% acetonitrile with 0.1% formic acid solvent and 95% acetonitrile with 0.1% formic acid solvent was set at 300 nL/min. Prior to tandem MS analysis, a precursor ion scan was performed at a 400–1600 mass-to-charge ratio (m/z). Tandem MS was performed by quadrupole separation at 0.8 Th, HCD fragmentation at 30% normalized collision energy, and fast scan MS analysis in an ion trap. Only the precursors with charge states 2–6 were sampled for tandem MS. The dynamic exclusion time was set to 15 s with a tolerance of 10 ppm. The instrument was operated at a maximum speed in 3-second cycles. After quantification of protein concentrations, the proteome software Scaffold (version Scaffold_4.4.8, Proteome Software Inc., Portland, OR) was used to validate the tandem MS-based peptide and protein identifications. The proteomic data were analyzed with Proteome Discoverer 1.4 (Thermo Fisher Scientific) and the MASCOT search engine (version 2.6.0, Matrix Science Inc., Boston, MA) to identify the proteins and peptides. The UniProt protein database (release 2021_01) was referenced for the identification, with an acceptable precursor mass range of 10 ppm and an acceptable fragment ion mass range of 0.8 Da. Raw proteomic data were submitted to the Japan Proteome Standard Repository/Database( https://repository.jpostdb.org/ ) ( 47 ). The accession number of jPOST for the proteomic data used in the present study is JPST003674 (PXD061493) . Analytical process and extraction criteria of the proteomic data The expression levels of the detected proteins were statistically compared among the four groups (sham, BPD-vehicle, BPD-Muse, and BPD-non Muse) using the Steel–Dwass test (nonparametric multiple test). The false discovery rate (FDR) was calculated using Storey’s method based on the p-value. To extract the significant effects, the threshold levels of P < 0.05 and FDR < 0.10 were set. The sham-vehicle and BPD-vehicle groups were compared to extract proteins affected or not by BPD. The extracted proteins were divided into those showing significant effects from the Muse cell injection and those that did not, by comparing the BPD-vehicle and BPD-Muse groups. Then, by comparing the BPD-vehicle and BPD-non Muse groups, these proteins were further separated into those exhibting significant effects from the non-Muse cell injections and those that did not, by comparing the BPD-vehicle and BPD-non-Muse groups. The protein groups extracted by this stratified analysis were as follows (Fig. 1 a): proteins affected by BPD (I); proteins not affected by BPD (II); among the proteins affected by BPD, those showing significant effects from the Muse cell injections (III); proteins that did not receive significant effects from the Muse cell injections (IV); proteins showing significant effects from therapy with MSCs, including Muse cells (V); proteins showing significant effects from treatment with only Muse cells (VI); proteins demonstrating significant effects from treatment with MSCs, not including Muse cells (VII); and proteins not showing significant effects from treatment with all MSCs, including Muse cells (VIII); among the proteins not affected by BPD, the proteins showing significant effects from the treatment with Muse cells (IX); proteins not showing significant effects from the treatment with Muse cells (X); proteins not showing significant effects from the treatment with MSCs, including Muse cells (XI); proteins exhibiting significant effects from the treatment with only the Muse cells (XII); proteins showing significant effects from the treatment with MSCs, not including Muse cells (XIII); and Others. The enrichment ratio for the number of proteins affected by the Muse or non-Muse celll injections was defined as the number of proteins affected by BPD /the number of proteins not affected by BPD. Functional analysis In the protein profiles extracted by these 13 conditions, groups I (proteins affected by BPD), III (among the proteins affected by BPD, those showing significant effects from the treatment with only Muse cell injections), and V + VII (among the proteins affected by BPD, those showing significant effects from the treatment with non-Muse cell injections) were used for the functional annotation analysis on the Database for Annotation, Visualization and Integrated Discovery 6.8 (DAVID 6.8, https://david.ncifcrf.gov/ ) (Huang et al., 2009). The annotations database was used on Feb. 19, 2021. Functional analysis using DAVID 6.8 was performed to enrich the proteins of these 12 protein profiles into gene ontology (GO term). The flagged GO terms were then clustered to clarify the relationships among similar annotation and co-association proteins. Among the clustered GO terms, we extracted the groups with the top 5 cluster enrichment scores. Additionally, molecular networks among the proteins extracted in the clustered ontologies were visualized using the Search Tool for the Retrieval of Interacting Genes/Proteins 11.0 (STRING 11.0, https://string-db.org/ ). The results analyzed with STRING were clustered using the Markov cluster algorithm (MCL clustering; inflation parameter is 3). The minimum required interaction score was set to 0.400. The interactions were indicated by eight criteria for linkage, including neighborhood, gene fusion, co-occurrence, co-expression, experiments, databases, text mining, and homology. Information on the distribution and main function of the extracted proteins in the lung was also investigated using the The Human Protein Atlas ( https://www.proteinatlas.org/ ) and Uniprot ( https://www.uniprot.org/ ) databases. Statistical analyses Statistical analyses were performed using the Prism 10 software (GraphPad Software, Boston, MA). The survival rate was evaluated by using the Kaplan–Meier method. Proteomic analysis was evaluated by using the Steel–Dwass test. The other analyses were performed using one-way analysis of variance, followed by the Holm Sidak method. All values were presented as the mean with standard error. Statistical significance was defined as p < 0.05. Results Comparison between the intravenous and intratracheal administrations Body weight and survival rate In the intravenous (iv) group, the body weight on day 15 and survival rate were not significantly different among the sham, vehicle, and Muse groups.(Fig. 2 a, c) However, in the intratracheal (it) groups, regardless of whether the cells were administered or not, a slowing in weight gain (p < 0.05; sham vs vehicle-it and Muse-it) and worsening of survival rate (p < 0.01; sham vs vehicle-it and Muse-it) were observed in the it groups than in the sham group (Fig. 2 b, d). Respiratory function test: WBP For intravenous administration, the TV was significantly lower in the vehicle iv group than the sham group (P < 0.0001) and significantly higher in the Muse-iv group than in the vehicle-iv group (P < 0.05). For the intratracheal administration, although the TV was lower in the vehicle-it group than in the sham group (P < 0.0001), the difference was not significant between the Muse-it and vehicle-it groups (Fig. 2 e). Impact of Muse cells on the lung tissues The alveolar spaces were more enlarged in the hyperoxia-loaded rats (vehicles) than in the sham group, and this enlargement was ameliorated in the Muse groups. TVD was significantly lower in the vehicle group than in the sham group. Comparing the intravenous and intratracheal injections, both injections ameliorated the lung tissue volume density in the Muse group, but not in the vehicle group (iv; p < 0.0001, it; p < 0.0001, Fig. 2 f). Differential cell counts BALF The white blood cell (WBC) count was significantly lower in the Muse group than in the vehicle group for both iv and it injections (iv; p < 0.0001, it; p < 0.01, Fig. 2 g). These results show that, regardless of whether the route of administration was intravenous or intratracheal, Muse cells ameliorated the alveolar-impaired development, and also produced anti-inflammatory effects, as evidenced by lower WBC counts in the BALF of the Muse group. However, the intratracheal injection did not lead to the amelioration in respiratory function in the TV, whereas intravenous injection did. Moreover, body weight gain and survival rate were lower in the Muse-it and vehicle-it groups than in the Muse-iv and vehicle-iv groups. Based on these findings, we conducted subsequent studies on the intravenous administration. Comparison of the intravenous Muse and non-Muse cell administrations with vehicle Respiratory function test: WBP The TV on day 15 was significantly lower in the vehicle group than in the sham (P < 0.01). However, there is no significant difference between the vehicle, non-Muse, and Muse groups (Fig. 3 a). The TV in the Muse group tended to be higher than that in the non-Muse group, although the difference is not significant (p = 0.053). Impact of Muse cells on lung tissue Representative photographs on day 29 are shown in Fig. 3 b. The alveolar spaces of the hyperoxia-loaded rats (vehicles) were more enlarged than those of the sham group, and this enlargement was ameliorated in the Muse group, whereas, in the non-Muse group, the improvement was poor. TVD was significantly lower in the vehicle group than in the sham group, and was significantly higher in the Muse group than in the vehicle and non-Muse groups on day 29 (Fig. 3 c). Differential cell counts BALF We evaluated the differential cell counts of BALF on day 15. The total number of cells,in BALF was increased in the vehicle group and was significantly lower in the Muse group, not in the non-Muse group, than in the vehicle group. The administration of Muse cells, not the non-Muse cells, suppressed the inflammation caused by BPD (Fig. 3 d). Distribution of Muse and non-Muse cells after intravenous administration in BPD rats To evaluate the distribution of Muse and non-Muse cells, qPCR assay with human Alu elements (Alu PCR) was performed at 10 days afte the intravenous administration (day 15). Alu PCR detected human genomic DNA in all of the organs (left lung, brain, liver, and spleen), with the highest amount being found in the lung of the Muse-treated rats. Human DNA was also detected in all of the organs of the non-Muse group. However, the amount of DNA in most organs was significantly lesser than that of the Muse group (Fig. 3 e). Engraftment of Muse and non-Muse cells in the lung. To confirm the engraftment of the Muse and non-Muse cells in the lung, immunofluorescence staining for GFP or STEM121 (human cell marker) was performed on day15. Figure 4 a shows the representative pictures. The GFP or STEM121-positive cells indicated the presence of engrafted Muse or non-Muse cells. The Muse group showed more flattened cells, whereas the non-Muse group showed more spherical cells. Cells were detected in both the non-Muse group and Muse groups, but the number of cells was significantly higher in the Muse group in both markers.(Fig. 4 b, c) Differentiation of Muse cells into type 1 and 2 alveolar epithelial cells Expression of human podoplanin, a marker of human type I alveolar epithelial cells, was confirmed in the GFP-positive squamous cells in the GFP-labeled Muse cells. The human podoplanin-positive cells were observed to localize in a ring-like pattern, suggesting that they may contribute to alveolar repair and formation (Fig. 4 d). The expression of human hPE10, a marker of human type2 alveolar epithelial cells, was also confirmed in the GFP-positive cells in the GFP-labeled Muse cells (Fig. 4 e). Assessment of pulmonary hypertension In BPD, RV hypertrophy develops, evidenced by the elevated dry weight ratio of the RV to the LV plus IVS (RV/LV + IVS). Compared to the vehicle and non-Muse groups, the Muse group showed a significant decrease in the RV/LV + S on day 29 (p < 0.001), suggesting that Muse cell treatment ameliorates RV hypertrophy (Fig. 5 a). In BPD, RVSP also develops. As compared to the sham group, the RVSP in the vehicle group was significantly higher at P29 (p < 0.0001). As compared to the vehicle group, the RVSP in the Muse group was significantly lower on day 29 (p < 0.01), suggesting that Muse cells ameliorate the elevated RVSP caused by BPD (Fig. 5 b). Proteomics The protein profiles of the lungs obtained from the four experimental groups (sham, vehicle, Muse, and non-Muse groups) were analyzed to investigate the target molecules associated with the mechanisms of abnormal respiratory function and thw therapeutic effects of the cells (Fig. 1 a,b). Using LC/MS/MS, 2694 proteins with high-quality signals were detected and quantified; of these, 844 proteins exhibited significant differences between the Sham and Vehicle groups. Of the 844 proteins, 141 proteins were extracted as having been affected by the Muse cell administration and 703 proteins were not significantly affected by its administration. Furthermore, among the 141 proteins extracted, 56 showed significant effects not only from the treatment with Muse cells but also from the treatment with non-Muse cells. In other words, 85 proteins showed significant effects from the treatment with only the Muse cells. Contrarily, 167 proteins were significantly affected by the treatment with only the non-Muse cells. Of the 1850 proteins, not affected by BPD, 115 and 227 proteins were significantly affected from the treatments with Muse and non-Muse cells, respectively; of these, 39 proteins were common between the Muse and non-Muse groups. According to the number of extracted proteins, the enrichment ratio of the proteins affected by Muse cell administration was 2.69, whereas that of the proteins affected by non-Muse cell administration was 2.15 (Fig. 1 c). Functional analysis To understand the biological significance of each extracted protein profile, we performed a functional analysis using the GO term database and its clustering analysis. First, the proteins categorized as Group I, which includes 844 proteins affected by BPD, were evaluated. As top five clusters, cell adhesion-related, ATP synthase-related, cAMP-dependent protein kinase-related, blood coagulation/fibrinolytic system-related, and lipoprotein-related ontologies were extracted through the analysis (Table 1 ). Table 1 Top five clusters of significantly enriched GO terms in proteins affected by BPD. Expression levels of the 844 proteins altered by BPD. Functional features of these 844 proteins were extracted using gene annotation by gene ontology and those clustering analysis. Cluster Enrichment Score Term name p-value Fold Enrichment Cluster 1 14.46 cadherin binding involved in cell-cell adhesion < 0.001 4.28 cell-cell adherens junction < 0.001 4.07 cell-cell adhesion < 0.001 3.97 Cluster 2 2.37 proton-transporting ATP synthase complex, catalytic core F( 1 ) 0.0016 14.97 mitochondrial proton-transporting ATP synthase complex, catalytic core F( 1 ) 0.0016 14.97 mitochondrial proton-transporting ATP synthase complex 0.0024 6.12 proton-transporting ATP synthase activity, rotational mechanism 0.0099 5.69 ATP synthesis coupled proton transport 0.024 4.47 Cluster 3 2.13 cAMP-dependent protein kinase regulator activity 0.0021 13.65 cAMP-dependent protein kinase complex 0.0027 12.83 negative regulation of cAMP-dependent protein kinase activity 0.0047 10.73 cAMP-dependent protein kinase inhibitor activity 0.0054 10.24 protein kinase A catalytic subunit binding 0.034 5.46 cAMP binding 0.036 3.94 Cluster 4 1.64 plasminogen activation 0.0094 8.59 positive regulation of heterotypic cell-cell adhesion 0.0094 8.59 protein polymerization 0.013 7.8 blood coagulation, fibrin clot formation 0.02 12.88 platelet alpha granule 0.027 5.99 fibrinogen complex 0.036 9.62 cellular protein complex assembly 0.056 4.52 positive regulation of peptide hormone secretion 0.063 7.15 Cluster 5 1.62 high-density lipoprotein particle assembly 0.0047 10.73 discoidal high-density lipoprotein particle 0.0058 22.45 reverse cholesterol transport 0.016 7.15 high-density lipoprotein particle remodeling 0.1 5.37 lipid transporter activity 0.16 4.1 (Table 1 is here.) The proteins affected by BPD were classified as Group III, which includes 141 proteins affected by Muse cell administration, and as Group V + VII, which includes 223 proteins affected by non-Muse cell administration. The analysis identified endopeptidase and inflammation-related, filament and cytoskeleton-related, blood coagulation/fibrinolytic system-related, and cell adhesion-related ontologies as the top five clusters of Group III (Table 2 ), whereas, for Group V + VII, these were GTPase-related, ribosome-related, filament-related, cytoskeleton-related, and hydrogen peroxide-related ontologies (Table 3 ). Table 2 Top five enriched GO clusters in BPD-related proteins affected by Muse cells. Cluster Enrichment Score Term name p-value Fold Enrichment Cluster 1 3.04 negative regulation of endopeptidase activity < 0.001 6.82 endopeptidase inhibitor activity 0.0018 16.2 inflammatory response 0.0024 3.82 Cluster 2 2.49 brush border < 0.001 14.4 stress fiber 0.0018 9.65 calmodulin binding 0.04 3.87 myosin complex 0.055 7.84 motor activity 0.072 6.75 Cluster 3 2.2 negative regulation of endopeptidase activity < 0.001 6.82 vasodilation 0.001 20.18 negative regulation of blood coagulation 0.0039 31.54 cysteine-type endopeptidase inhibitor activity 0.048 8.48 positive regulation of cytosolic calcium ion concentration 0.35 2.43 Cluster 4 2.09 stress fiber 0.0018 9.65 actin filament binding 0.0057 5.25 actin cytoskeleton 0.054 3.51 Cluster 5 2.07 cadherin binding involved in cell-cell adhesion 0.0022 4.42 cell-cell adherens junction 0.0033 4.12 cell-cell adhesion 0.085 2.99 Table 3 Top five enriched GO clusters in BPD-related proteins affected by non-Muse cells. Cluster Enrichment Score Term name p-value Fold Enrichment Cluster 1 7.18 small GTPase mediated signal transduction < 0.001 6.19 GTP binding < 0.001 4.55 GTPase activity < 0.001 5.95 GDP binding < 0.001 11.78 Cluster 2 3.25 cytosolic small ribosomal subunit < 0.001 9.53 translation 0.0027 2.77 structural constituent of ribosome 0.0076 2.43 ribosome 0.098 2.85 Cluster 3 2.41 intermediate filament 0.0013 7.39 type III intermediate filament 0.0013 50.97 structural molecule activity 0.034 2.91 Cluster 4 2.07 structural constituent of cytoskeleton < 0.001 7.64 microtubule-based process 0.014 7.85 cytoskeleton organization 0.022 3.77 microtubule 0.06 2.52 Cluster 5 1.91 haptoglobin-hemoglobin complex < 0.001 63.72 response to hydrogen peroxide 0.016 5.16 positive regulation of cell death 0.14 4.47 (Table 2 , 3 is here.) Notably, the blood coagulation/fibrinolytic system-related ontologies were commonly enriched as clusters 4 and 3 of Groups I and III, respectively. Cell adhesion-related ontologies were also commonly enriched as cluster 1 of Group I and as cluster 5 of Group III. These ontologies were not enriched in Group V + VII. Thus, the expression of blood coagulation/fibrinolytic system-related and cell adhesion-related proteins was altered by BPD, and Muse cell administration significantly influenced the expression of these proteins, whereas the non-Muse cell administration did not. Moreover, filament and cytoskeleton-related ontologies were commonly enriched as clusters 2 and 4 of Group III and as clusters 3 and 4 of Group V + VII. The findings suggested that the MSC may influenced the formation of respiratory cells. Particularly, the administration of Muse cells may have additional effects on the abnormalities in cell adhesion and the blood coagulation/fibrinolytic system caused by BPD. For the cell adhesion-related ontologies, eight proteins significantly affected by Muse cell administration were included. The distribution and main function of the eight proteins in the normal lungs are listed in Supplemental Table 1. The expression of all of the eight proteins was detected in the alveolar cells, bronchus, and macrophages in the normal lungs, indicating that their expression levels were altered by BPD and Muse cells administration. Lrrfip1 and Myh9 were upregulated by BPD and suppressed by Muse cell administration (Fig. 6 a). Uso1, Idh1, Ctnnb1, Axa1, and Puf60 were downregulated by BPD and enhanced by Muse cell administration (Fig. 6 a). Finally, Pkm was upregulated by BPD and further increased by Muse cell administration (Fig. 6 a). In contrast to the proteins with cell adhesion-related ontologies, almost all of proteins with blood coagulation/fibrinolytic system-related ontologies were not detected in the normal lungs (Supplemental Tables 2 and 3); hence, these proteins may be expressed and/or translocated by BPD. The expressions of Gnai3, Sod2, and Map1, which are detected in the normal lungs, were upregulated by BPD and further increased by Muse cell administration (Fig. 6 b). Kng1, Itih3, C3, and C4, which are not detected in the normal lungs, were detected in the BPD lungs and further increased by Muse cell administration (Fig. 6 b). A1i3 and Mug1 were suppressed by BPD and enhanced by Muse cell administration (Fig. 6 b). Regarding the blood coagulation/fibrinolytic system-related proteins not affected by Muse cell administration, Alox15 and Lonp1 were detected in the normal lungs, whereas Fga, Fgb, Fgg, Apoh, Crp, and Pf4 were not detected in the normal lungs. The expression of every protein, except for Pf4, was increased by BPD (Fig. 6 c). Network analysis To promote understanding of their functional association, the interaction networks among the proteins found during the functional annotation analysis were visualized using STRING and MCL clustering (Fig. 7 a, b). This network displayed the molecular interaction among the cell adhesion- and blood coagulation/fibrinolytic system-related proteins affected by BPD. These proteins were found to be closely related to each other. The nodes indicate the individual proteins, and the distance between them indicates the depth of the relationship between the proteins (the shorter the distance, the closer the relationship). The nodes of the same color have a strong relationship, indicating that they are classified in the same group. Figure 8 a illustrates the division of a network of 49 cell adhesion-related proteins affected by BPD (Table 1 cluster 1). The eight proteins (Table 2 cluster 5 and Supplemental Table 1) affected by Muse cell administration were not confined to any particular group, but they were scattered on each group. The administration of Muse cells is considered to affect various groups of cell adhesion molecules, rather than a specific group. Conversely, Fig. 8 b also demonstrates that the 17 blood coagulation/fibrinolytic system-related proteins affected by BPD (Table 1 cluster 4) are closely related. Histological analysis of Ctnnb1 and Anxa1 A network analysis using STRING was performed on the molecules selected from the statistical and annotation analyses of the protein profile. The results revealed that Ctnnb1 (β-catenin) was the most central molecule in the network (Fig. 7 a). The expression of this molecule decreased in BPD cases, and this decrease was ameliorated by the stem cell administration (Muse/non-Muse cells) (Fig. 6 a). The results of the histological analysis further demonstrated a marked upregulation of Ctnnb1 expression in cells adjacent to the Muse cells (Fig. 8 a). Contrarily, although an increase in Ctnnb1 was observed in the non-Muse group, this upregulation was detected at locations distant from the non-Muse cells (Fig. 8 a). Additionally, based on the results of the BALF (Fig. 2 g) and proteomic analyses (Supplemental Table 1), we focused on Anxa1 (Annexin A1), a molecule associated with neutrophil migration and inflammatory responses. The expression of this molecule was found to decrease in BPD cases, and its reduction was ameliorated by the administration of Muse cells (Fig. 6 a). The results of the histological analysis showed a significant increase in the number of Anxa1-positive cells in the Muse group (Fig. 8 b). Although an increase in Anxa1-positive cells was also observed in the non-Muse group, the increase was only minimal, compared to that observed in the Muse group (Fig. 8 b). Additionally, Anxa1 expression was observed in a subset of spherical GFP-positive Muse cells. Discussion Although intravenous and intratracheal administrations represent distinct delivery routes and, therefore, strictly different models, precluding a direct comparison, the present study showed that intravenous administration outperformed intratracheal administration in terms of survival, weight gain, and respiratory outcomes. Intravenously administered human Muse cells were engrafted and expressed alveolar epithelial cell markers in the BPD rat model. The Muse cells ameliorated alveolar growth impairment induced by BPD and improved the pulmonary function. Animals receiving Muse cells intravenously displayed improvement in BPD-associated pulmonary hypertension, as compared to the non-Muse and vehicle groups. A comprehensive analysis of proteins from BPD lungs revealed that Muse cells may have additional effects on the abnormalities in cell adhesion and the blood coagulation/fibrinolytic system caused by BPD. These results demonstrated the histological and functional benefits of human Muse cells in experimental BPD. A major finding of the present study is that the xenogeneic Muse cells were engrafted in the injured lung without the use of immunosuppressants and expressed alveolar epithelial cell markers (podoplanin and PE10). Given that the lungs are the first organs to trap cells following intravenous administration, a human-specific genome material was detected to some extent at 2 weeks, even in our non-Muse group, which differed from previous reports.( 9 , 24 – 28 ) However, the number of cells detected in the lung by the GFP-positive cells at 4 weeks was much higher in the Muse group than in the non-Muse group. The S1P–S1P receptor 2 axis plays an important role in the selective homing of intravenously administered Muse cells into the damaged sites.( 27 , 48 ) The S1P expression is increased at the site of injury( 24 ) and in the peripheral blood.( 49 ) The ability of intravenously administered Muse cells to migrate to the injury site is a major advantage over many other types of stem cells. In AluPCR, the Muse cells were detected in large numbers in the lungs, brain, and liver. We believe that this is because the damage caused by high oxygen exposure also occurs in the lungs, brain, and liver, causing Muse cells to migrate to the injured sites. We also believe that the Muse and non-Muse cells are detected in the spleen due to the organ’s filtering function. In BPD, the intratracheal administration of MSCs has been attempted,( 50 ) and clinical trials on the treatment of severe BPD have also been conducted( 21 ). However, the disconnection of the ventilator circuit and “fluid” administration can lead to hypoxia in patients. Therefore, we aim to avoid intratracheal administration when possible, as intravenous administration does not have this risk. Our data showed that, with intratracheal administration, whether involving solvent alone or Muse cells, the survival rate declined and weight gain was reduced compared to intravenous administration, suggesting that intravenous administration is more suitable than intratracheal administration for this treatment approach. Regarding differentiation, Muse cell differentiates into tissue-compatible cells after homing in various injury models, including stroke,( 51 ) neonatal hypoxic–ischemic encephalopathy,( 9 ) liver fibrosis,( 27 ) acute myocardial infarction,( 24 ) chronic kidney disease,( 26 ) and aortic aneurysms.( 52 ) Regarding BPD, the migration of Muse cells into the lungs and their differentiation into alveolar epithelial type 1 and 2 cells have been reported.( 53 ) In the present study, Muse cells also expressed markers for alveolar epithelial type 1 and 2 cells. The Muse cells, such as the MSCs, are also known to secrete various cytokines, producing paracrine effects.( 9 , 24 , 26 , 54 , 55 ) Although there are many reports on BPD treated with MSCs, which have demonstrated some treatment effects,( 16 ) non-Muse cells did not show sufficient efficacy in the present study. The direct effect of Muse cells to engraft and differentiate into lung cells, in addition to their paracrine effect, may have resulted in a significant therapeutic effect. To further understand the mechanisms underlying the therapeutic effects of Muse cell administration, we conducted a comprehensive proteomic analysis. The annotation of protein expression profiles highlighted the cell adhesion-related proteins as key molecules involved in this process. The pathology of BPD is characterized by alveolar simplification and abnormal pulmonary vascularization due to an interrupted pulmonary and alveolar development.( 1 ) Although the pathogenesis of BPD is multifactorial, a common pathway involves inflammatory responses.( 48 ) Inflammation triggers the upregulation of cell adhesion molecules and chemotactic proteins that recruit inflammatory cells to the lungs.( 49 ) The interactions between the adhesion molecules on the surfaces of the leukocytes and endothelial cells facilitate the migration of leukocytes from the bloodstream into the lung interstitium, mediating the inflammatory response.( 50 ) Given the evidence for subclinical pulmonary endothelial dysfunction in BPD, it may be crucial to evaluate the lung adhesion molecules—an early marker of endothelial dysfunction—in clinical specimens, such as BALF from neonates with established BPD. These adhesion molecules, which adhere to the endothelial surface, play a key role in leukocyte infiltration through the endothelial cells and into the lung parenchyma.( 48 ) Our network analysis identified Ctnnb1 (β-catenin) as the most central molecule among the cell adhesion-related proteins (Fig. 7 a). Ctnnb1 is a pivotal molecule in the Wnt/β-catenin signalling pathway, which regulates stem cell pluripotency, differentiation, proliferation, and migration during development.( 56 ) In the respiratory system, Ctnnb1 promotes cellular maturation, differentiation, and morphogenesis.( 57 ) Our findings revealed a significant reduction in Ctnnb1 expression associated with BPD, which was restored following the administration of Muse cells (Figs. 6 a and 8 ). The results of our histological analysis further demonstrated that β-catenin expression was markedly elevated in the alveolar epithelial cells adjacent to the Muse cells that had migrated into the lung tissues (Fig. 8 ). These results suggest that Muse cell administration may improve lung function by directly or indirectly inducing Wnt/β-catenin signalling in the neighbouring lung cells, either through direct cell interactions or via the paracrine factors. In addition to the cell adhesion-related proteins, the blood coagulation and fibrinolytic system-related proteins are also important to understand the effects of Muse cell administration in BPD. Among the 17 proteins analyzed, nine were significantly impacted by the Muse cell treatment (Table 2 , Cluster 3; Supplemental Table 2), whereas eight were unaffected (Supplemental Table 3). The unaffected proteins, which all promote blood coagulation, were upregulated in response to BPD (Fig. 6 c). Conversely, four out of the nine proteins influenced by Muse cell administration—Kng1, Gnai3, Map1, and Sod2—inhibit coagulation and promote vasodilation, and their expression was further upregulated following Muse cell treatment (Fig. 6 b). These findings suggest that Muse cell administration may help counteract the BPD-induced alterations in blood flow. A molecular network analysis (Fig. 7 b) indicated that most of the 17 proteins are closely interrelated, implying that Muse cells may induce compensatory changes in an alternate group of blood coagulation and fibrinolytic proteins. Indeed, the suppression of plasminogen activator-mediated fibrinolysis not only increases the fibrin deposition in the alveoli but also contributes to the recruitment and migration of inflammatory cells.( 52 ) Fibrinolytic therapies aimed at preventing pulmonary and pleural fibrin deposition, along with inflammation, show considerable promise. ( 28 , 54 ) Among the cell adhesion-related proteins, we also focused on Anxa1, a molecule known for its role in inflammation and neutrophil infiltration. According to the Human Protein Atlas database ( https://www.proteinatlas.org/ENSG00000135046-ANXA1 ). Anxa1 is strongly expressed in alveolar macrophages and bronchial epithelial cells. Additionally, Anxa1 suppresses immune cell infiltration and promotes the release of the anti-inflammatory cytokine IL-10, which helps reduce pulmonary inflammation ( 58 ). In the present study, we observed a marked upregulation of the Anxa1 expression in some Muse cells that had migrated into the lung (Fig. 8 ). This finding suggests that Muse cell administration increases the number of Anxa1-positive cells, potentially limiting excessive immune cell infiltration and the accompanying inflammation. This mechanism may underlie the greater improvement in lung function and development in comparison to the non-Muse group. These findings align with the reduction in lymphocyte counts observed in the BALF (Fig. 2 g), further supporting the role of Anxa1 in mediating the beneficial effects of Muse cells in BPD. Conclusion The intravenous administration of Muse cells demonstrated superior therapeutic effects in a BPD rat model compared to the intratracheal administration of Muse or non-Muse cells. Muse cells ameliorated respiratory impairment and pulmonary hypertension, accompanied by differentiation into alveolar epithelial cells and modulation of cell adhesion and coagulation pathways. Our study findings suggest the potential of Muse cell therapy as a promising approach for treating severe neonatal lung diseases and warrant further translational and clinical investigations to validate the results. Abbreviations BPD Bronchopulmonary Dysplasia Muse cell Multilineage-differentiating stress enduring cell MSC Mesenchymal Stromal Cell SSEA Stage-Specific Embryonic Antigen VLBW Very Low Birth Weight S1P Sphingosine-1-Phosphate HLA Human Leukocyte Antigen BM-MSC Bone Marrow- Mesenchymal Stromal Cell GFP Green Fluorescent Protein FACS Fluorescence-Activated Cell Sorter BALF Bronchoalveolar Lavage Fluid TV Tidal Volume WBP Whole Body Plethysmography TVD Tissue Volume Density qPCR quantitative Polymerase Chain Reaction PBS Phosphate-Buffered Saline RV Right Ventricle LV Left Ventricle IVS Interventricular Septum RVSP Right Ventricular Systolic Pressure LC/MS/MS Liquid Chromatography/Tandem Mass Spectrometry GO Gene Ontology iv intravenous it intratracheal Declarations Ethics approval and consent to participate The experiments were conducted with the approval of the Animal Care and Use Committee of Nagoya University School of Medicine (Nagoya, Japan; permit Nos. 26128, 27190, 28215, 29012, 30197, 31106, 20032, and M210107-003) and in compliance with the Regulations on Animal Experiments at Nagoya University. The title of the approved project was “Establishment of a neonatal rat model of BPD and development of novel therapeutic strategies”, and the date of approval were March 18,2014 (No.26126), March 16,2015 (No.27190), March 11,2016 (No.28125), March 10,2017 (No.29012), March 19,2018 (No.30197), March 8,2019 (No.31106), March 6,2020 (No.20032), March 3,2021 (No.M210107-003). Human BM-MSCs were purchased from Lonza. According to the manufacturer’s documentation and published information, Lonza obtained ethical approval for the collection of human bone marrow samples, and written informed consent was obtained from all donors(Supplemental Material 1,2). Consent for publication Not applicable Availability of data and materials All data are available from the corresponding author upon reasonable request. Raw proteomic data were submitted to the Japan Proteome Standard Repository/Database(https://repository.jpostdb.org/). The accession number of jPOST for the proteomic data used in the present study is JPST003674 (PXD061493) . Competing interests The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Y.S and S.Sh have a collaborative research and developmental agreement for perinatal disease with Life Science Institute Inc.(LSII). S.Sh is making a collaborative research and developmental agreement for the other disease, and has a contract for consulting with LSII. Y.S, T.S and S.Sh have a patent for the application of Muse cells for treatment of perinatal brain damage and the other indication. R.M, A.On, A.Ok, S.Su, K.U, Y.T and M.H declare no competing interests. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by AMED (grant No. JP 22bm0404067h0003) and Nagoya University Hospital Funding for Clinical Research. Authors' contributions R.M., A.On., and A,Ok. were involved in animal experiments. R.M., A.On., A.Ok., and Y.S. conceived and designed the study. R.M., A.On., T.S., A,Ok., S.Su., K.U., S.Sh., and Y.S. interpreted the data. R.M. drafted the initial manuscript, and. R.M., A.On., T.S., A.Ok., S.Su., K.U., S.Sh., Y.T., M.H., and Y.S. critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript and are accountable for the entire work. Acknowledgements We appreciate the technical assistance of Ms. Tomoko Yamaguchi, and acknowledge Division for Medical Research Engineering, Nagoya University Graduate School of Medicine We would also like to thank Kentaro Taki for operational support in the LC/MS/MS in the laboratory of the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine. The authors declare that they have also used AI for English proofreading in this manuscript. References Kalikkot Thekkeveedu R, Guaman MC, Shivanna B. Bronchopulmonary dysplasia: A review of pathogenesis and pathophysiology. Respir Med. 2017;132:170–7. Kusuda S, Fujimura M, Uchiyama A, Totsu S, Matsunami K. Trends in morbidity and mortality among very-low-birth-weight infants from 2003 to 2008 in Japan. Pediatr Res. 2012;72(5):531–8. Davidson L, Berkelhamer S. Bronchopulmonary Dysplasia: Chronic Lung Disease of Infancy and Long-Term Pulmonary Outcomes. J Clin Med. 2017;6(1):4. Lapcharoensap W, Lee HC, Nyberg A, Dukhovny D. Health Care and Societal Costs of Bronchopulmonary Dysplasia. Neoreviews. 2018;19(4):e211–23. Gray PH, O'Callaghan MJ, Rogers YM. Psychoeducational outcome at school age of preterm infants with bronchopulmonary dysplasia. J Paediatr Child Health. 2004;40(3):114–20. Watterberg KL. Postnatal Corticosteroids to Prevent or Treat Bronchopulmonary Dysplasia. Pediatrics. 2010;126(4):800–8. Taniguchi A, Nishida K, Suzuki T, Kataoka E, Fujishiro N, Kato E, et al. Total Hydrocortisone Dosage in the Neonatal Period May Be Related to Low Developmental Quotient in Extremely Low Birth Weight Infants: A Retrospective Cohort Study. Neonatology. 2024;121(2):195–202. Ichinohashi Y, Sato Y, Saito A, Ito M, Watanabe K, Hayakawa M, et al. Dexamethasone administration to the neonatal rat results in neurological dysfunction at the juvenile stage even at low doses. Early Hum Dev. 2013;89(5):283–8. Suzuki T, Sato Y, Kushida Y, Tsuji M, Wakao S, Ueda K, et al. Intravenously delivered multilineage-differentiating stress enduring cells dampen excessive glutamate metabolism and microglial activation in experimental perinatal hypoxic ischemic encephalopathy. J Cereb Blood Flow Metab. 2021;41(7):1707–20. Kitase Y, Sato Y, Arai S, Onoda A, Ueda K, Go S, et al. Establishment of a Novel Fetal Growth Restriction Model and Development of a Stem-Cell Therapy Using Umbilical Cord-Derived Mesenchymal Stromal Cells. Front Cell Neurosci. 2020;14(212):212. Suzuki T, Sato Y, Yamamoto H, Kato T, Kitase Y, Ueda K, et al. Mesenchymal stem/stromal cells stably transduced with an inhibitor of CC chemokine ligand 2 ameliorate bronchopulmonary dysplasia and pulmonary hypertension. Cytotherapy. 2020;22(4):180–92. Tsuji M, Taguchi A, Ohshima M, Kasahara Y, Sato Y, Tsuda H, et al. Effects of intravenous administration of umbilical cord blood CD34 cells in a mouse model of neonatal stroke. Neuroscience. 2014;263C:148–58. Ueda K, Sato Y, Shimizu S, Suzuki T, Onoda A, Miura R, et al. Systemic administration of clinical-grade multilineage-differentiating stress-enduring cells ameliorates hypoxic-ischemic brain injury in neonatal rats. Sci Rep. 2023;13(1):14958. Mimatsu H, Onoda A, Kazama T, Nishijima K, Shimoyama Y, Go S, et al. Dedifferentiated fat cells administration ameliorates abnormal expressions of fatty acids metabolism-related protein expressions and intestinal tissue damage in experimental necrotizing enterocolitis. Sci Rep. 2023;13(1):8266. Fung ME, Thebaud B. Stem cell-based therapy for neonatal lung disease: it is in the juice. Pediatr Res. 2014;75(1–1):2–7. Augustine S, Avey MT, Harrison B, Locke T, Ghannad M, Moher D, et al. Mesenchymal Stromal Cell Therapy in Bronchopulmonary Dysplasia: Systematic Review and Meta-Analysis of Preclinical Studies. Stem Cells Transl Med. 2017;6(12):2079–93. Nitkin CR, Rajasingh J, Pisano C, Besner GE, Thébaud B, Sampath V. Stem cell therapy for preventing neonatal diseases in the 21st century: Current understanding and challenges. Pediatr Res. 2020;87(2):265–76. Chang YS, Ahn SY, Yoo HS, Sung SI, Choi SJ, Oh WI, et al. Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial. J Pediatr. 2014;164(5):966–72. e6. Lim R, Malhotra A, Tan J, Chan ST, Lau S, Zhu D, et al. First-In‐Human Administration of Allogeneic Amnion Cells in Premature Infants With Bronchopulmonary Dysplasia: A Safety Study. STEM CELLS Translational Med. 2018;7(9):628–35. Powell SB, Silvestri JM. Safety of Intratracheal Administration of Human Umbilical Cord Blood Derived Mesenchymal Stromal Cells in Extremely Low Birth Weight Preterm Infants. J Pediatr. 2019;210:209 – 13.e2. Ahn SY, Chang YS, Lee MH, Sung SI, Lee BS, Kim KS, et al. Stem cells for bronchopulmonary dysplasia in preterm infants: A randomized controlled phase II trial. Stem Cells Transl Med. 2021;10(8):1129–37. Kuroda Y, Kitada M, Wakao S, Nishikawa K, Tanimura Y, Makinoshima H, et al. Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA. 2010;107(19):8639–43. Wakao S, Kitada M, Kuroda Y, Shigemoto T, Matsuse D, Akashi H et al. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proceedings of the National Academy of Sciences. 2011;108(24):9875-80. Yamada Y, Wakao S, Kushida Y, Minatoguchi S, Mikami A, Higashi K, et al. S1P–S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial InfarctionNovelty and Significance. Circul Res. 2018;122(8):1069–83. Kushida Y, Wakao S, Dezawa M. Muse Cells Are Endogenous Reparative Stem Cells. Adv Exp Med Biol. 2018;1103:43–68. Uchida N, Kushida Y, Kitada M, Wakao S, Kumagai N, Kuroda Y, et al. Beneficial Effects of Systemically Administered Human Muse Cells in Adriamycin Nephropathy. J Am Soc Nephrol. 2017;28(10):2946–60. Iseki M, Kushida Y, Wakao S, Akimoto T, Mizuma M, Motoi F, et al. Muse Cells, Nontumorigenic Pluripotent-Like Stem Cells, Have Liver Regeneration Capacity Through Specific Homing and Cell Replacement in a Mouse Model of Liver Fibrosis. Cell Transpl. 2017;26(5):821–40. Kinoshita K, Kuno S, Ishimine H, Aoi N, Mineda K, Kato H, et al. Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers. Stem Cells Transl Med. 2015;4(2):146–55. Wakao S, Oguma Y, Kushida Y, Kuroda Y, Tatsumi K, Dezawa M. Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame. Cell Mol Life Sci. 2022;79(11):542. Sato Y, Shimizu S, Ueda K, Suzuki T, Suzuki S, Miura R, et al. Safety and tolerability of a Muse cell-based product in neonatal hypoxic-ischemic encephalopathy with therapeutic hypothermia (SHIELD trial). Stem Cells Transl Med. 2024;13(11):1053–66. Matsuyama N, Shimizu S, Ueda K, Suzuki T, Suzuki S, Miura R, et al. Safety and tolerability of a multilineage-differentiating stress-enduring cell-based product in neonatal hypoxic-ischaemic encephalopathy with therapeutic hypothermia (SHIELD trial): a clinical trial protocol open-label, non-randomised, dose-escalation trial. BMJ Open. 2022;12(4):e057073. Niizuma K, Osawa SI, Endo H, Izumi SI, Ataka K, Hirakawa A et al. Randomized placebo-controlled trial of CL2020, an allogenic muse cell-based product, in subacute ischemic stroke. J Cereb Blood Flow Metab. 2023:271678X231202594. Noda T, Nishigaki K, Minatoguchi S. Safety and Efficacy of Human Muse Cell-Based Product for Acute Myocardial Infarction in a First-in-Human Trial. Circ J. 2020;84(7):1189–92. Fujita Y, Nohara T, Takashima S, Natsuga K, Adachi M, Yoshida K et al. Intravenous allogeneic multilineage-differentiating stress‐enduring cells in adults with dystrophic epidermolysis bullosa: a phase 1/2 open‐label study. J Eur Acad Dermatol Venereol. 2021;35(8). Yamashita T, Nakano Y, Sasaki R, Tadokoro K, Omote Y, Yunoki T et al. Safety and Clinical Effects of a Muse Cell-Based Product in Patients With Amyotrophic Lateral Sclerosis: Results of a Phase 2 Clinical Trial. Cell Transplant. 2023;32. Koda M, Imagama S, Nakashima H, Ito S, Segi N, Ouchida J, et al. Safety and feasibility of intravenous administration of a single dose of allogenic-Muse cells to treat human cervical traumatic spinal cord injury: a clinical trial. Stem Cell Res Ther. 2024;15(1):259. Namba F. Mesenchymal stem cells for the prevention of bronchopulmonary dysplasia. Pediatr Int. 2019;61(10):945–50. Namba F. Mesenchymal stem cells for the prevention of bronchopulmonary dysplasia. Pediatr Int. 2019;61(10):945–50. Chang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, et al. Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplant. 2009;18(8):869–86. Kuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc. 2013;8(7):1391–415. Kawai S, Takagi Y, Kaneko S, Kurosawa T. Effect of Three Types of Mixed Anesthetic Agents Alternate to Ketamine in Mice. Exp Anim. 2011;60(5):481–7. Basu RK, Donaworth E, Wheeler DS, Devarajan P, Wong HR. Antecedent acute kidney injury worsens subsequent endotoxin-induced lung inflammation in a two-hit mouse model. Am J Physiol Ren Physiol. 2011;301(3):F597–604. Zhang C, Lei GS, Shao S, Jung HW, Durant PJ, Lee CH. Accumulation of myeloid-derived suppressor cells in the lungs during Pneumocystis pneumonia. Infect Immun. 2012;80(10):3634–41. Li W-H, Gu Z, Wang H, Nekrutenko A. Evolutionary analyses of the human genome. Nature. 2001;409(6822):847–9. Pansani MC, Azevedo PS, Rafacho BP, Minicucci MF, Chiuso-Minicucci F, Zorzella-Pezavento SG, et al. Atrophic cardiac remodeling induced by taurine deficiency in Wistar rats. PLoS ONE. 2012;7(7):e41439. Jabary M, Onoda A, Kitase Y, Ueda K, Mimatsu H, Go S, et al. Fetal growth restriction followed by early catch-up growth impairs pancreatic islet morphology in male rats. Sci Rep. 2023;13(1):2732. Okuda S, Watanabe Y, Moriya Y, Kawano S, Yamamoto T, Matsumoto M, et al. jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res. 2017;45(D1):D1107–11. Yamada Y, Wakao S, Kushida Y, Minatoguchi S, Mikami A, Higashi K, et al. S1P–S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial Infarction. Circul Res. 2018;122(8):1069–83. Tanaka T, Nishigaki K, Minatoguchi S, Nawa T, Yamada Y, Kanamori H et al. Mobilized Muse Cells After Acute Myocardial Infarction Predict Cardiac Function and Remodeling in the Chronic Phase. Circ J. 2017. Chang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, et al. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Attenuate Hyperoxia-Induced Lung Injury in Neonatal Rats. Cell Transplant. 2009;18(8):869–86. Uchida H, Niizuma K, Kushida Y, Wakao S, Tominaga T, Borlongan CV, et al. Human Muse Cells Reconstruct Neuronal Circuitry in Subacute Lacunar Stroke Model. Stroke. 2017;48(2):428–35. Hosoyama K, Wakao S, Kushida Y, Ogura F, Maeda K, Adachi O, et al. Intravenously injected human multilineage-differentiating stress-enduring cells selectively engraft into mouse aortic aneurysms and attenuate dilatation by differentiating into multiple cell types. J Thorac Cardiovasc Surg. 2018;155(6):2301–e134. Win KHN, Kushida Y, Yamana K, Iwatani S, Yoshida M, Nino N, et al. Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant. Stem Cell Res Ther. 2024;15(1):147. Yabuki H, Wakao S, Kushida Y, Dezawa M, Okada Y. Human Multilineage-differentiating Stress-Enduring Cells Exert Pleiotropic Effects to Ameliorate Acute Lung Ischemia-Reperfusion Injury in a Rat Model. Cell Transplant. 2018;27(6):979–93. Fukase M, Sakata N, Kushida Y, Wakao S, Unno M, Dezawa M. Intravenous injection of human multilineage-differentiating stress-enduring cells alleviates mouse severe acute pancreatitis without immunosuppressants. Surg Today. 2021. Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell. 2012;149(6):1192–205. Harris-Johnson KS, Domyan ET, Vezina CM, Sun X. beta-Catenin promotes respiratory progenitor identity in mouse foregut. Proc Natl Acad Sci USA. 2009;106(38):16287–92. Guido BC, Zanatelli M, Tavares-de-Lima W, Oliani SM, Damazo AS. Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice. J Inflamm (Lond). 2013;10(1):10. Additional Declarations Competing interest reported. The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Y.S and S.Sh have a collaborative research and developmental agreement for perinatal disease with Life Science Institute Inc.(LSII). S.Sh is making a collaborative research and developmental agreement for the other disease, and has a contract for consulting with LSII. Y.S, T.S and S.Sh have a patent for the application of Muse cells for treatment of perinatal brain damage and the other indication. R.M, A.On, A.Ok, S.Su, K.U, Y.T and M.H declare no competing interests. Supplementary Files supplementalTable20251015.docx proteomics.xlsx Supplementarymaterial1.pdf Supplementarymaterial2.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviews received at journal 15 Dec, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers invited by journal 20 Nov, 2025 Editor assigned by journal 19 Nov, 2025 Submission checks completed at journal 04 Nov, 2025 First submitted to journal 02 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7875937","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":538693390,"identity":"2a694206-b09b-4d37-aeb7-d154f84e8cb8","order_by":0,"name":"Ryosuke Miura","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Miura","suffix":""},{"id":538693391,"identity":"7b8afdbd-248f-40ae-bece-32295e1b5f00","order_by":1,"name":"Atsuto Onoda","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Atsuto","middleName":"","lastName":"Onoda","suffix":""},{"id":538693393,"identity":"4bc626c8-6c6f-444b-b171-eeae4333eac6","order_by":2,"name":"Toshihiko Suzuki","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Toshihiko","middleName":"","lastName":"Suzuki","suffix":""},{"id":538693394,"identity":"a7b9f5fe-f144-44e6-8028-a19ca06256d7","order_by":3,"name":"Azusa Okamoto","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Azusa","middleName":"","lastName":"Okamoto","suffix":""},{"id":538693395,"identity":"8423908b-873d-42fb-93c9-2b8a5b2320e5","order_by":4,"name":"Sakiko Suzuki","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sakiko","middleName":"","lastName":"Suzuki","suffix":""},{"id":538693397,"identity":"9ebb3de5-170b-455e-a0cb-a5a02c721283","order_by":5,"name":"Kazuto Ueda","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kazuto","middleName":"","lastName":"Ueda","suffix":""},{"id":538693398,"identity":"c3060708-901d-48d6-84f8-32aaef4f56e1","order_by":6,"name":"Shinobu Shimizu","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shinobu","middleName":"","lastName":"Shimizu","suffix":""},{"id":538693399,"identity":"874231b3-c4f1-4742-8ead-6bf541a990e5","order_by":7,"name":"Yoshiyuki Takahashi","email":"","orcid":"","institution":"Nagoya University Gradate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Takahashi","suffix":""},{"id":538693402,"identity":"2e4627cd-c0c6-41ac-a1c0-f9abb20dcaaf","order_by":8,"name":"Masahiro Hayakawa","email":"","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Hayakawa","suffix":""},{"id":538693403,"identity":"2fdc2211-ca6f-4551-87ee-e561047653c3","order_by":9,"name":"Yoshiaki Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYPACGwSTsYEYDQcY0kjXcpgEFxkcYH/4+UPF+Tz+/sXHJH4A9TLPJmCNwQEeY4kDZ24XS9x4libZA9TCOOcAQS0MEgfbbic23DhjJsH7D6hlRgJBhz3+cbDtXOJ8oBbJPwxEaWEwA9pyIHHD+R4zaR5itEge5jGzOHMmOXHjDbZkaxmGdB6CfuE73v74RkWFXeK884cP3nzDYC1nSCjEFOAxIpHAIgGkeAxn4NfBIA83kv8A8wewiAQBLaNgFIyCUTDiAABet0pOTu/WggAAAABJRU5ErkJggg==","orcid":"","institution":"Nagoya University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yoshiaki","middleName":"","lastName":"Sato","suffix":""}],"badges":[],"createdAt":"2025-10-16 09:53:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7875937/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7875937/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95103974,"identity":"def30f04-f920-4c3b-bc63-70035aeb452e","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":252848,"visible":true,"origin":"","legend":"","description":"","filename":"MuseBPD1101.docx","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/e79c622fdcfe8145b8f20bb8.docx"},{"id":95103973,"identity":"2fcdecf6-3a3f-404b-b72b-dfe90974d59c","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12130,"visible":true,"origin":"","legend":"","description":"","filename":"654d18d951e84c11ad705418c36cf805.json","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/645567e104dd2d39b92ecf16.json"},{"id":95224728,"identity":"261fbb50-1a00-49ce-8b25-0d45690086c9","added_by":"auto","created_at":"2025-11-05 16:24:13","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93298,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/c999b53f4c5aa4aaed124ea8.pdf"},{"id":95103987,"identity":"06511bb7-0194-4b47-903a-91211118637d","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166938,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/708f7e38b0c8b868b5adbcdb.pdf"},{"id":95103985,"identity":"74a7dc77-927c-4bac-9e1b-1438eda56c35","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3594009,"visible":true,"origin":"","legend":"","description":"","filename":"proteomics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/42964a760bfaea06c7aabeb4.xlsx"},{"id":95224277,"identity":"35c4d1c6-2a0f-4fcf-9342-015c8eaa5a6e","added_by":"auto","created_at":"2025-11-05 16:23:34","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23532,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalTable20251015.docx","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/14637ed0ef95bf1d3bfee009.docx"},{"id":95103990,"identity":"92347e09-1f97-497b-84f8-58c65b15ccd2","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202801,"visible":true,"origin":"","legend":"","description":"","filename":"654d18d951e84c11ad705418c36cf8051enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/2b28be7f6c55b07a36910214.xml"},{"id":95103992,"identity":"606b5619-a1f3-4faf-9cdd-d7bb977b2113","added_by":"auto","created_at":"2025-11-04 10:25:35","extension":"pptx","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154516669,"visible":true,"origin":"","legend":"","description":"","filename":"MuseBPDfig20251015.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/4bf5f5653c0bec15c3dd7db7.pptx"},{"id":95226233,"identity":"95734869-5174-4a1c-9b3b-71a2b59b461d","added_by":"auto","created_at":"2025-11-05 16:30:44","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":199610,"visible":true,"origin":"","legend":"","description":"","filename":"654d18d951e84c11ad705418c36cf8051structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/eac89ca9d7a940e86f8b5f7e.xml"},{"id":95103986,"identity":"2878c46c-ed94-4183-927d-704ce1c2e85c","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":212734,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/2a707738159f7391e5bb3cca.html"},{"id":95103970,"identity":"963b7aa2-2375-4690-8166-0e9a2b255a6b","added_by":"auto","created_at":"2025-11-04 10:25:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63456,"visible":true,"origin":"","legend":"\u003cp\u003eStepwise extraction criteria and yield distribution of proteins in proteomic analysis.\u003c/p\u003e\n\u003cp\u003e(a) A flowchart illustrating the stratified analysis of 2,694 proteins detected by LC-MS/MS. First, based on the comparison between the sham and BPD-vehicle groups (p \u0026lt; 0.05, FDR \u0026lt; 0.10), proteins were classified into those significantly affected by BPD (844 proteins) and those not significantly affected by BPD (1,850 proteins). Each subset was then further divided based on whether the proteins were significantly affected by Muse cells (BPD-vehicle vs. BPD-Muse) or by non-Muse MSCs (BPD-vehicle vs. BPD-non-Muse). The resulting categories (I–XIII) cover all combinations of the “affected or unaffected by BPD” and “affected or unaffected by Muse/non-Muse cells.”\u003c/p\u003e\n\u003cp\u003e(b) A Venn diagram summarizing the classification of proteins in each subset. For example, among the 844 proteins affected by BPD, 141 were significantly affected by Muse cell administration; of which, 56 were also significantly affected by the non-Muse cells. The 1,850 proteins not significantly affected by BPD were classified in the same manner.\u003c/p\u003e\n\u003cp\u003e(c) A summary table showing the number (and percentage) of proteins in each major classification, along with the calculated enrichment ratio. The enrichment ratio is defined as “[number of proteins affected by BPD] / [number of proteins unaffected by BPD].” Abbreviations: neonatal bronchopulmonary dysplasia (BPD); multilineage-differentiating stress enduring cells (Muse cells)\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/b26f3696917a3463adc724b4.png"},{"id":95103971,"identity":"29727d79-b862-4869-96d1-5fc86ac0378a","added_by":"auto","created_at":"2025-11-04 10:25:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56919,"visible":true,"origin":"","legend":"\u003cp\u003eComparison between intravenous and intratracheal administration of Muse cells in BPD.\u003c/p\u003e\n\u003cp\u003e(a) Body weight \u0026nbsp;in the intravenous (iv) groups was no significant difference among the groups. (n = 51 for sham, 43 for vehicle, and 44 for Muse)\u003c/p\u003e\n\u003cp\u003e(b) Body weight in the intratracheal (it) groups. Regardless of whether the cells were administered or not, there was a reduction in weight gain. (n = 51 for sham, 36 for vehicle, and 32 for Muse). *p \u0026lt; 0.05; sham vs vehicle-it and Muse-it\u003c/p\u003e\n\u003cp\u003e(c) Survival rate in the iv groups. No significant difference was noted among the groups (survive on day 15; n = 51 for sham, 43 for vehicle, and 44 for Muse)\u003c/p\u003e\n\u003cp\u003e(d) Survival rate in the it group. Regardless of whether the cells were administered or not, a worsening of the survival rate was noted in the it groups. (survive on day15; n = 51 for sham, 36 for vehicle, and 32 for Muse). **p \u0026lt; 0.01; sham vs vehicle-it and Muse-it.\u003c/p\u003e\n\u003cp\u003e(e) TV on day 15 was significantly higher in the Muse-iv group than in the vehicle-iv group. (n = 51 for sham, 43 for vehicle-iv, 44 for Muse-iv, 36 for vehicle-it, and 32 for Muse-it). *p \u0026lt; 0.05, ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(f) TVD on day 29 was higher in the Muse group than in the vehicle group for both iv and it. (n = 20 for sham, 20 for vehicle-iv, 19 for Muse-iv, 16 for vehicle-it, 12 for Muse-it). ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(g) WBC counts in BALF on day 15 were significantly lower in the Muse group than in the vehicle group for both iv and it. (n = 11 for sham, 8 for vehicle-iv, 10 for Muse-iv, 10 for vehicle-it, 8 for Muse-it). **p \u0026lt; 0.01, ****p \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/3ee59b52d56295eed14f245f.png"},{"id":95103976,"identity":"fa42df00-b4ef-4162-8899-5aecf37ed9f4","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":250951,"visible":true,"origin":"","legend":"\u003cp\u003eComparison between intravenous administration of Muse and non-Muse cells in BPD.\u003c/p\u003e\n\u003cp\u003e(a) TV on day 15 was significantly lower in the vehicle group than in the sham group. However, no significant difference in TV was noted between the vehicle, non-Muse, and Muse groups. (n = 14 for sham, 12 for vehicle, 16 for non-Muse, and 13 for Muse). ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(b) Representative images of the alveolar spaces in the lung (H\u0026amp;E staining). The alveolar spaces appear enlarged in the vehicle group, whereas improvement is observed in the Muse group.\u003c/p\u003e\n\u003cp\u003eBar = 50 µm\u003c/p\u003e\n\u003cp\u003e(c) TVD on day 29 was significantly lower in the vehicle group than in the sham group and was significantly higher in the Muse group than in the vehicle and non-Muse groups. (n = 18 for sham, 14 for vehicle, 11 for non-Muse, and 12 for Muse). *p \u0026lt; 0.05, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(d) The WBC counts in BALF on day15 were significantly lower in the Muse group than in the vehicle group, but not significantly lower in the non-Muse group. (n = 5 for sham, 7 for vehicle, 5 for non-Muse, and 8 for Muse). *p \u0026lt; 0.05, **p \u0026lt; 0.01\u003c/p\u003e\n\u003cp\u003e(e) Amounts of DNA in most organs on day 15 were higher in the Muse group than in the non-Muse group. (n = 3 for non-Muse and 3 for Muse). *p \u0026lt; 0.05, **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/82241fea253ae12a5e52c9f7.png"},{"id":95226390,"identity":"ec8405b0-851b-4ff9-9efa-c99ef6d641a4","added_by":"auto","created_at":"2025-11-05 16:31:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":433976,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining\u003c/p\u003e\n\u003cp\u003e(a)\u0026nbsp; Immunofluorescence staining for GFP or STEM121\u003c/p\u003e\n\u003cp\u003eGFP- or STEM121-positive cells indicate engrafted Muse or non-Muse cells. The Muse group shows more flattened cells, whereas the non-Muse group showed more spherical cells.\u003c/p\u003e\n\u003cp\u003e(b)\u0026nbsp; Area of the GFP-positive cells per 1-mm\u003csup\u003e2 \u003c/sup\u003elung tissue\u003c/p\u003e\n\u003cp\u003eThe GFP-positive cells were detected in both the non-Muse and Muse groups, but the amount of these cells was significantly higher in the Muse group. ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(c)\u0026nbsp; Area of STEM121-positive cells per 1-mm\u003csup\u003e2 \u003c/sup\u003elung tissue\u003c/p\u003e\n\u003cp\u003eThe STEM121 positive cells were detected in both the non-Muse and Muse-treated groups, but the amount of these cells was significantly higher in the Muse group. ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(d)\u0026nbsp; Immunofluorescence staining for GFP or human podoplanin (hPdpn)\u003c/p\u003e\n\u003cp\u003eExpression of human podoplanin, a marker of human type I alveolar epithelial cells, was confirmed in the GFP-positive cells in the GFP-labeled Muse cells.\u003c/p\u003e\n\u003cp\u003e(e) Immunofluorescence staining for GFP or hPE10\u003c/p\u003e\n\u003cp\u003eExpression of human hPE10, a marker of human type2 alveolar epithelial cells, was confirmed in the GFP-positive cells in the GFP-labeled Muse cells.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/ec4b2f7606a8ab8de88f217d.png"},{"id":95223783,"identity":"abb7f4b5-9206-418f-be48-aac9de129bc6","added_by":"auto","created_at":"2025-11-05 16:22:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29322,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of pulmonary hypertension\u003c/p\u003e\n\u003cp\u003e(a) RV/LV+S on day 29 was significantly lower in the Muse group than in the vehicle and non-Muse groups, suggesting that Muse cell treatment ameliorates right ventricular hypertrophy. (n = 11 for sham, 11 for vehicle, 6 for non-Muse, and 11 for Muse). *p \u0026lt; 0.05, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e\n\u003cp\u003e(b) RVP on day 29 was significantly lower in the Muse group than in the vehicle group, suggesting that Muse cells ameliorate elevated RVSP caused by BPD. (n = 13 for sham, 11 for vehicle, 14 for non-Muse, and 10 for Muse) **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/3b3623dc3cf2efba9c16f7a1.png"},{"id":95103978,"identity":"64ba2aea-c17e-4dfb-8701-6d61999bcf38","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70938,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of proteins in the ontologies extracted by the proteomic and functional analyses.\u003c/p\u003e\n\u003cp\u003e(a) Expression levels of eight proteins included in the cell adhesion-related ontologies. Lrrfip1 and Myh9 were upregulated by BPD and suppressed by Muse cell administration. The expressions of Uso1, Idh1, Ctnnb1, Anxa1, and Puf60 were downregulated by BPD and subsequently upregulated by Muse cell administration.\u003c/p\u003e\n\u003cp\u003e(b) Expression levels of proteins in the blood coagulation/fibrinolytic system-related ontologies affected by Muse cell administration. The expressions of A1i3 and Mug1 were downregulated by BPD and upregulated by Muse cell administration, whereas the others were upregulated by BPD and further upregulated by Muse cell administration. (c) The expression levels of proteins in the blood coagulation/fibrinolytic system-related ontologies not affected by Muse cell administration. Except for Pf4, these proteins were upregulated by BPD. Dot plots show the expression levels of each protein in the four experimental groups (sham, vehicle, Muse, and non-Muse).\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/555b9f244a7e4e73385d7c19.png"},{"id":95226402,"identity":"09eca764-d0ca-4dfa-a1d0-43a87afeb2c4","added_by":"auto","created_at":"2025-11-05 16:31:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":306967,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of molecular networks in the ontologies extracted through the proteomic and functional analyses.\u003c/p\u003e\n\u003cp\u003e(a) The network of proteins included in the cell adhesion-related ontologies.\u003c/p\u003e\n\u003cp\u003e(b) The network of proteins included in the blood coagulation/fibrinolytic system-related ontologies. These networks were mapped and clustered using the Search Tool for the Retrieval of Interacting Genes/Proteins 11.0 (STRING 11.0, \u003ca href=\"https://string-db.org/\"\u003ehttps://string-db.org/\u003c/a\u003e) with Markov cluster algorithm. Each node represents a single protein, and the length of the edges reflects the closeness of the known or predicted interactions (see the legend for interaction types). Nodes with orange outlines represent the proteins affected by Muse cell administration. Nodes sharing the same color are grouped based on the stronger inter-node relationships, indicating that they share functions or regulatory pathways.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/fff84bdfd84550e48513cb88.png"},{"id":95226311,"identity":"9ed7f4db-127e-42f7-8a67-c9480686154e","added_by":"auto","created_at":"2025-11-05 16:30:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":306063,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining\u003c/p\u003e\n\u003cp\u003e(a) Immunofluorescence staining for GFP or Ctnnb1\u003c/p\u003e\n\u003cp\u003eHistological analysis demonstrated a marked upregulation of Ctnnb1 expression in the cells adjacent to the Muse cells.\u003c/p\u003e\n\u003cp\u003e(b) Immunofluorescence staining for GFP or Anxa1\u003c/p\u003e\n\u003cp\u003eHistological analysis showed a significant increase in the number of Anxa1-positive cells in the Muse group.\u003c/p\u003e","description":"","filename":"Slide8.png","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/747907d2681e64060a54e8c0.png"},{"id":95230617,"identity":"ee364880-ab09-4838-9f73-2593effa2e33","added_by":"auto","created_at":"2025-11-05 16:38:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3093226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/8ab5251c-4a3d-49eb-8fb2-7a5625a3cac7.pdf"},{"id":95224694,"identity":"7a9f7e07-8ad0-4d70-9780-265b25d4f7ae","added_by":"auto","created_at":"2025-11-05 16:24:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23532,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalTable20251015.docx","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/eff4341ae2e5e2950a4fd6d0.docx"},{"id":95103982,"identity":"cce4959e-96d0-4db1-81c9-a05eb257df78","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3594009,"visible":true,"origin":"","legend":"","description":"","filename":"proteomics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/120f16ce16650974a62c56f6.xlsx"},{"id":95224271,"identity":"b6b4e4fd-814e-49c0-84da-cb8f664541ea","added_by":"auto","created_at":"2025-11-05 16:23:33","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":93298,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/617d952df42a1b9a2a9253d4.pdf"},{"id":95103979,"identity":"9e0d1ebd-af5d-4b18-a9fb-da9dac6c2002","added_by":"auto","created_at":"2025-11-04 10:25:32","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":166938,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7875937/v1/63441d47b77ce0d246bb7450.pdf"}],"financialInterests":"Competing interest reported. The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Y.S and S.Sh have a collaborative research and developmental agreement for perinatal disease with Life Science Institute Inc.(LSII). S.Sh is making a collaborative research and developmental agreement for the other disease, and has a contract for consulting with LSII. Y.S, T.S and S.Sh have a patent for the application of Muse cells for treatment of perinatal brain damage and the other indication. R.M, A.On, A.Ok, S.Su, K.U, Y.T and M.H declare no competing interests.","formattedTitle":"Intravenously delivered Multilineage-differentiating stress enduring cells dampen in experimental bronchopulmonary dysplasia rat model","fulltext":[{"header":"Background","content":"\u003cp\u003eNeonatal bronchopulmonary dysplasia (BPD) is a lung injury caused by various factors, including intrauterine inflammation, mechanical ventilation, and oxidative stress.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) With the advancement in neonatal care, the survival rate of infants born with a birth weight of \u0026lt;\u0026thinsp;1,500 g [very low birth weight (VLBW) infants] has increased to \u0026gt;\u0026thinsp;90%.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) However, neonatal BPD remains a serious complication affecting in approximately 40% of surviving infants born at \u0026le;\u0026thinsp;28 weeks\u0026rsquo; gestation, and its incidence has been relatively stable over the last few decades.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Many infants with severe BPD die due to respiratory distress, and even those who are discharged alive require home ventilators and/or home oxygen therapy. Even after hospital discharge, BPD infants have a high likelihood of hospitalization for respiratory infections, and their medical costs are high.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Additionally, BPD can lead to impaired respiratory function persisting into adolescence(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and a low intelligence quotient score in school-aged children.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Currently, no definitive treatment exists beyond ventilator management. Systemic administration of steroids may have some short-term treatment effects, yet there are concerns about their long-term neurological outcome,(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) even with low-dose administration.(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) Therefore, the development of new therapies for BPD is an urgent issue.\u003c/p\u003e\u003cp\u003eRecently, stem cell therapy has brought \u0026ldquo;hope\u0026rdquo; to patients with various diseases for which no adequate treatment has been available. Even in perinatal and neonatal medicine, numerous animal studies across various diseases have been published.(\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) As for BPD, mesenchymal stromal cells (MSCs) are the primary cell source for stem cell therapy(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). A previous meta-analysis of preclinical studies have revealed that MSCs considerably improved alveolarization and ameliorated pulmonary hypertension, lung tissue inflammation, fibrosis, angiogenesis, and apoptosis.(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) Additionally, more than 10 clinical trials have been started worldwide,(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and the results for some of these have been already reported.(\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) However, the effect of MSC therapy in these clinical trials did not demonstrate sufficient efficacy in terms of reducing the risk for mortality or moderate/severe BPD.(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) Therefore, to enhance the treatment effects, therapy modifications and/or other strategies should be considered.\u003c/p\u003e\u003cp\u003eMultilineage-differentiating stress enduring (Muse) cells, which make up a small fraction of the total MSCs,(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) display endogenous pluripotent-like property and express pluripotent surface marker stage-specific embryonic antigen-3 (SSEA-3).(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) Muse cells express sphingosine-1-phosphate (S1P) receptor 2 and selectively home into the damaged sites by sensing S1P, induced by the injured cells.(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) Importantly, Muse cells also possess phagocytic activity that allows them to recycle transcription factors from the damaged cells and rapidly differentiate into the same cell type at the homed tissues, thereby replenishing injured cells and repairing the tissues.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) Therefore, Muse cells do not require artificial induction to a pluripotent state or artificial differentiation induction into the target cell type. Besides replacing the damaged cells, the therapy using Muse cells has paracrine effects, as it produces various trophic factors.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) Moreover, since Muse cells have an immunosuppressive property similar to that of the placenta, specifically the expressions of human leukocyte antigen (HLA)-G and express interferon gamma-induced indoleamine-2,3 dioxygenase, a mediator of immunosuppression,(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) they can survive for a long term in the homed tissue. In fact, intravenously administered allogenic Muse cells could survive for over 6 months in the host tissue without administering any immunosuppressants in preclinical studies.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) Based on these attractive properties, clinical trials for several diseases, including neonatal hypoxic\u0026ndash;ischemic encephalopathy(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), adult subacute stroke,(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) acute myocardial infarction,(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) adult epidermolysis bullosa,(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) amyotrophic lateral sclerosis,(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and cervical spinal cord injury (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), that utilized the intravenous administration of donor-derived allogeneic Muse cells without HLA matching and immunosuppressant treatment have been initiated in Japan.\u003c/p\u003e\u003cp\u003eThe effects of stem cell therapy for BPD to date were not derived from the replacement of the injured cells with exogenous MSCs, but from the paracrine effect by the trophic factors secreted from the stem cells.(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) If we can combine the replacement effect using Muse cells with the paracrine effect, we may achieve enhanced therapeutic outcomes. Even in terms of the paracrine effect, the ability of Muse cells to home into the injured sites offers a considerable advantage. In the present study, we evaluated the therapeutic effects of the intravenous administration of Muse cells in hyperoxia-induced BPD rat model and compared these outcomes with those of treatments using non-Muse MSCs (non-Muse cells).\u003c/p\u003e\u003cp\u003eIn a previous study(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) on BPD using MSCs, the dosage, timing, and route of administration were considered important factors. Moreover, it has been reported that the intratracheal administration of MSCs is more effective than their intravenous administration for the treatment of BPD.(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) Therefore, in the present study, we aimed to compare the therapeutic effects between the intravenous and intratracheal administrations of Muse cells using a BPD rat model.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e All animal experiments were approved by the Nagoya University Animal Experiment Committee (Protocol Nos. 26126, 27190, 28215, 29012, 30197, 31106, 20032, and M210107-003) and conducted in accordance with the Regulations on Animal Experiments in Nagoya University. The present study complies with the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIsolation of human Muse and non-Muse cells\u003c/h2\u003e\u003cp\u003eHuman Muse and non-Muse cells were isolated as described previously.(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) Briefly, human BM-MSCs (Lonza, PT-2501) were cultured and labelled with green fluorescent protein (GFP), then the human Muse and non-Muse cells were isolated using a fluorescence-activated cell sorter (FACS) as GFP\u003csup\u003e+\u003c/sup\u003e/SSEA-3\u003csup\u003e+\u003c/sup\u003e and GFP\u003csup\u003e+\u003c/sup\u003e/SSEA-3\u003csup\u003e\u0026minus;\u003c/sup\u003e cell fractions, respectively.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal models and experimental protocol\u003c/h3\u003e\n\u003cp\u003ePregnant Sprague\u0026ndash;Dawley rats were obtained from Japan SLC Inc. (Shizuoka, Japan). They were healthy, non-genetically modified, and had not previously undergone any procedures. Pregnant rats at 16 days\u0026rsquo; gestation were transported to the animal facility of Nagoya University and acclimatized until delivery on gestational day 21.The rats were housed under 12-h light/12-h dark cycles (9.00 A.M. to 9.00 P.M.) with the temperature controlled at 23\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe pups were placed in an incubator within 24 h of birth and exposed to hyperoxia (83%), using a high/low oxygen control system for small animal experiments (BioSpherix, Parish, NY) until postnatal day 15. Muse (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells; Muse group) or non-Muse cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells; non-Muse group) in 0.1-mL acetic acid Ringer's solution or vehicle (only 0.1-mL acetic acid Ringer's solution: vehicle group) were administered slowly \u003cem\u003evia\u003c/em\u003e the right external jugular vein or trachea on postnatal day 5. The rats in the sham group were housed under room-air conditions. The dams were rotated once every 2 days between the litters in the normoxia and hyperoxia groups to avoid excessive oxygen toxicity. Rats were sacrificed on day 15 or 29 to evaluate the treatment effect. On day 5, pups exposed to hyperoxia were allocated to the vehicle and cell-treated groups, so that the body weights were evenly balanced.\u003c/p\u003e\u003cp\u003eThe group allocations and experimental procedures were not blinded. However, the evaluations of tissues and bronchoalveolar lavage fluid (BALF) were conducted in a manner blinded to group assignment.\u003c/p\u003e\u003cp\u003eThe minimum sample for comparing the outcomes between the intravenous and intratracheal injections was calculated based on the preliminary experiments to achieve an 80% power of testing with an error rate of 1.67%, assuming a difference of 0.005 and standard deviation of 0.0065 in the tidal volume (TV) as a primary endpoint. The total sample size was calculated as n\u0026thinsp;=\u0026thinsp;36. Additionally, in our previous experiments, approximately 20% of the intratracheal rats would occasionally die, particularly during the experimental process; therefore, the number of rats for evaluations was set to 44 per group. The minimum sample for comparing the outcomes between therapies using non-Muse and Muse cells was calculated based on the preliminary experiments to achieve an 80% power of testing with an error rate of 1.25%, assuming a difference of 0.025 and a standard deviation of 0.0020 in the TV as a primary endpoint. In this case, the total sample size was calculated as n\u0026thinsp;=\u0026thinsp;15. Given that rats receiving intravenous injections rarely dies, the number of rats for evaluations was set to 15 per group. Rats with TV values of \u0026gt;\u0026thinsp;+\u0026thinsp;4 SD were considered outliers and excluded from the analysis. Two rats were excluded.\u003c/p\u003e\u003cp\u003eThe humane endpoints in this study were as follows: if severe lethargy (loss of spontaneous activity or inability to feed or drink) persisted or if the animals exhibited signs of severe distress, such as respiratory difficulty, abnormal posture, or convulsions, the experiment was terminated. Animals meeting these criteria were humanely euthanized by CO₂ inhalation. If any animals showed signs of unexpected suffering, they were immediately evaluated and euthanized according to these criteria.\u003c/p\u003e\n\u003ch3\u003eBody weight and survival rate\u003c/h3\u003e\n\u003cp\u003eThe rats\u0026rsquo; body weights were evaluated from day 5 to day 15. The cumulative survival rate in each group was also evaluated every other day from birth to day 15.\u003c/p\u003e\n\u003ch3\u003eRespiratory function test: whole body plethysmography (WBP)\u003c/h3\u003e\n\u003cp\u003eA respiratory function test was performed on day 15. TV was measured using WBP, which is an unrestrained respiratory function analyzer (IOX, emka TECHNOLOGIES Co., Ltd., Paris, France.). Each rat was housed in each animal chamber. After confirming that the respiratory parameters were stable, recording was started and continued for \u0026gt;\u0026thinsp;10 min. From the total respiratory data, a 2-min segment when the respiratory rate was stable was selected for analysis.\u003c/p\u003e\n\u003ch3\u003eTissue preparation\u003c/h3\u003e\n\u003cp\u003eThe rats were deeply anesthetized with an intraperitoneal injection of overdosed pentobarbital or an anesthetic mixture comprising medetomidine, midazolam, and butorphanol(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) on day 29. Then, these rats were transcardially perfused with saline. The lungs were fixed using 4% paraformaldehyde \u003cem\u003evia\u003c/em\u003e a tracheal catheter and kept at 20-cmH\u003csub\u003e2\u003c/sub\u003eO pressure for 20 min to be inflated, followed by immersion\u0026ndash;fixation in 4% paraformaldehyde overnight at 4\u0026deg;C. Subsequently, the right lungs were immersed in 20% and 30% sucrose solutions for 24 h each and embedded in a Tissue-Tek OCT solution (Sakura Finetek, Torrance, CA, USA) for the preparation of 30-\u0026micro;m frozen sections. Contrarily, the left lungs were dehydrated with a graded series of ethanol and xylene and embedded in paraffin for the preparation of 5-\u0026micro;m sections.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTissue morphometry\u003c/h2\u003e\u003cp\u003eAfter deparaffinization and rehydration with xylene and graded alcohols to water, the paraffin sections were stained with hematoxylin and eosin (H\u0026amp;E). To assess the alveolar maldevelopment, the tissue volume densities (TVDs) in the left lungs were evaluated.\u003c/p\u003e\u003cp\u003eThe TVD was evaluated as described previously(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). With 100 evenly spaced points (a 10 \u0026times; 10 grid), 300 \u0026micro;m apart, in each of the three random fields with six sections (a total of 18 fields), the proportion of lung tissue (alveolar ducts and sacs) in the lungs was evaluated using Stereo Investigator version 2020 stereology software (MicroBrightField, Williston, VT).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDifferential cell counts of bronchoalveolar lavage fluid\u003c/h3\u003e\n\u003cp\u003eOn day15, BALF was evaluated. The BALF was collected by instilling 0.6 mL (0.3 \u0026times; 2) saline via a tracheal tube. After staining with T\u0026uuml;rk solution, the total cell number of BALF was counted using Burker chambers. Then, a 100-\u0026micro;L aliquot was centrifuged and plated onto the glass slides. Differential cell counts were made, with at least 200 cells per animal, by staining with May\u0026ndash;Giemsa (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eQuantitative polymerase chain reaction with the Alu sequence specific primer\u003c/h3\u003e\n\u003cp\u003eTo evaluate the distribution of Muse and non-Muse cells, quantitative polymerase chain reaction (qPCR) assays were performed with Alu elements, which are primate-specific repeats and comprise 11% of the human genome,(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) as previously described.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) Briefly, genomic DNA from the brain, lung, liver and spleen was collected after the intravenous administration of Muse or non-Muse cells on day 15. PCRs were performed with a volume of 20 \u0026micro;L, containing 10-\u0026micro;L TaqMan Universal Master Mix II (Applied Biosystems, Waltham, MA), 900-nM forward and reverse primers, 250-nM TaqMan probe, and 100-ng target template. Amplification was initiated at 50\u0026deg;C for 2 min and 95\u0026deg;C for 10 min, followed by 45 cycles of an incubation step at 95\u0026deg;C for 15 s and 60\u0026deg;C for 1 min.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eAltogether, 30 \u0026micro;m-thick lung tissue sections placed into 12-well plates were used for a histological analysis. To remove the storage solution, the sections were washed three times in PBS for 10 min each. Antigen retrieval was performed by incubating the sections in a HistoVT One solution (Nacalai Tesque, Kyoto, Japan) at 70\u0026deg;C for 30 min, followed by cooling to room temperature. The sections were then blocked using a 10% normal donkey serum (IHR-8135, Immunobioscience, Mukilteo, WA) in PBS, containing 0.01% Triton X for 60 min at room temperature on a shaker. After washing three times in PBS containing 0.01% Triton X, the sections were then incubated with primary Goat anti-GFP antibody (1:50, ab6673, abcam, Cambridge, UK) diluted in the Canget Signal Solution B (TOYOBO CO., LTD., Osaka, Japan) for 24 h at 4\u0026deg;C on a shaker. The sections were washed three times with PBS containing 0.01% Triton X (MP Biomedicals, Inc., Irvine, CA) for 10 min each. The sections were then incubated with secondary Donkey anti-Goat IgG (H\u0026amp;L) Antibody conjugated DyLight\u0026trade; 488 (1:500, 605-741-002, ROCKLAND, Limerick, PA) diluted in Canget Signal Solution B for 10 h at room temperature on a shaker. To protect the fluorescent signal, all incubations were performed while shielded from light. After the first round of staining, the tissue sections were washed again three times in PBS containing 0.01% Triton X for 10 min each. A second set of primary antibodies, including Mouse anti-STEM121 (1:50, Y40410, Takara Bio Inc., Shiga, Japan), Mouse anti-hPE10 (1:50, 10375, IBL Co., Ltd., Gunma, Japan), Rat anti-hPDPN (1:50, 018-24101, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), Rabbit anti-Ctnnb1 (1:100, 51067-2-AP, Proteintech, Inc., IL), or Rabbit anti-Anxa1 (1:100, 21990-1-AP, Proteintech) were diluted in Canget Signal Solution B and applied to the sections. The sections were incubated for 24 h at 4\u0026deg;C on shaker, while again protected from light. The sections were washed three times in PBS containing 0.01% Triton X for 10 min each. Secondary antibodies including Donkey anti-mouse IgG (H\u0026amp;L) Antibody conjugated DyLight\u0026trade; 549 (1:500, 610-742-124, ROCKLAND), Rat anti-Rat IgG (H\u0026amp;L) Antibody conjugated DyLight\u0026trade; 549 (1:500, 612-442-026, ROCKLAND), or Rabbit anti-Rat IgG (H\u0026amp;L) Antibody conjugated DyLight\u0026trade; 549 (1:500, 611-142-002, ROCKLAND) were diluted in Canget Signal Solution B and applied to the sections. The sections were incubated for 10 h at room temperature on a shaker while again protected from light. Hoechst 33342 (10 \u0026micro;g/mL, Abcam, Cambridge, UK) was used for nuclear counterstaining under light-protected conditions. Finally, the tissue sections were transferred onto glass slides using a glass rod in 0.01-M phosphate buffer with 0.001% Triton X. Excess buffer was removed, and the sections were air-dried on the glass slides. The slides were mounted with a ProLong Glass mounting medium, ensuring no air bubbles or debris were trapped under the coverslip. The mounted slides were allowed to dry in a dark environment for at least 24 h to preserve the fluorescent signals.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of pulmonary hypertension\u003c/h2\u003e\u003cp\u003eTo assess for pulmonary hypertension, the dry weight ratio of the right ventricle (RV) to the left ventricle (LV) plus interventricular septum (IVS) (RV/LV\u0026thinsp;+\u0026thinsp;IVS) and right ventricular systolic pressure (RVSP) were used. The hearts obtained on day 29 were dissected, and the RV was separated from the LV and IVS. These tissues were dried up in a drying oven at 60\u0026deg;C for 48 h. The weight of each tissue was measured, and the RV/LV\u0026thinsp;+\u0026thinsp;S ratio was calculated.(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eTo measure the RVSP, the external jugular vein was exposed under isoflurane anesthesia, and a catheter (Natsume Seisakusho Co., Ltd. Tokyo, Japan) was inserted into the RV. The RVSP was measured by a pressure transducer and an amplifier system attached to the catheter (LEG-1000; Nihon Kohden Co, Tokyo, Japan) on day 29.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eProteomic analysis\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003eProtein preparation\u003c/h2\u003e\u003cp\u003eProtein lysates of lung tissues were prepared according to a previously reported method.(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) The lung tissue samples were collected from day-15 rats of each group (n\u0026thinsp;=\u0026thinsp;8/group). The lung tissues were quickly frozen by liquid nitrogen, smashed to a powdered form using the Multi-beads shocker (Yasui Kikai Co., Ltd., Osaka, Japan), and then homogenized in a T-PER protein extraction reagent (20 mL/g of tissue) (Takara Bio. Inc., Shiga, Japan) with a protease inhibitor cocktail (Complete Tablet, EDTA-free, Roche Diagnostics, Basel, Switzerland) on ice. Homogenates were centrifuged at 11,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026deg;C to separate the insoluble debris. Then, the supernatants of protein lysates were collected. A pierce detergent removal spin column (Thermo Fisher Scientific, MA) was used to remove the surfactant from the supernatants. The total protein concentrations in the supernatants were determined by the bicinchoninic acid method using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). The protein supernatants of each pup were adjusted to 100 \u0026micro;g /200 \u0026micro;L and labeled with the Tandem Mass Tag\u0026trade; system (Sixplex TMT, Thermo Fisher Scientific) for liquid chromatography/tandem mass spectrometry (LC/MS/MS).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eProteomic analysis\u003c/h2\u003e\u003cp\u003eProteomics was conducted based on our previous study.(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) The amounts of all protein types in the lung were detected and quantified by LC/MS/MS. During the LC/MS/MS, the Orbitrap Fusion mass spectrometry system (Thermo Fisher Scientific) combined with the UltiMate 3000 RSLCnano LC system (Dionex Co., Amsterdam, The Netherlands) and a nanocapillary column (150 mm \u0026times; 75 \u0026micro;m i.d., Nikkyo Technos Co., Tokyo, Japan) via a nanoelectrospray ion source was used. In reversed-phase chromatography, the flow rate of the linear gradient (0 min, 5% B; 100 min, 40% B) of 2% acetonitrile with 0.1% formic acid solvent and 95% acetonitrile with 0.1% formic acid solvent was set at 300 nL/min. Prior to tandem MS analysis, a precursor ion scan was performed at a 400\u0026ndash;1600 mass-to-charge ratio (m/z). Tandem MS was performed by quadrupole separation at 0.8 Th, HCD fragmentation at 30% normalized collision energy, and fast scan MS analysis in an ion trap. Only the precursors with charge states 2\u0026ndash;6 were sampled for tandem MS. The dynamic exclusion time was set to 15 s with a tolerance of 10 ppm. The instrument was operated at a maximum speed in 3-second cycles. After quantification of protein concentrations, the proteome software Scaffold (version Scaffold_4.4.8, Proteome Software Inc., Portland, OR) was used to validate the tandem MS-based peptide and protein identifications. The proteomic data were analyzed with Proteome Discoverer 1.4 (Thermo Fisher Scientific) and the MASCOT search engine (version 2.6.0, Matrix Science Inc., Boston, MA) to identify the proteins and peptides. The UniProt protein database (release 2021_01) was referenced for the identification, with an acceptable precursor mass range of 10 ppm and an acceptable fragment ion mass range of 0.8 Da. Raw proteomic data were submitted to the Japan Proteome Standard Repository/Database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://repository.jpostdb.org/\u003c/span\u003e\u003cspan address=\"https://repository.jpostdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). The accession number of jPOST for the proteomic data used in the present study is JPST003674 (PXD061493) .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAnalytical process and extraction criteria of the proteomic data\u003c/h2\u003e\u003cp\u003eThe expression levels of the detected proteins were statistically compared among the four groups (sham, BPD-vehicle, BPD-Muse, and BPD-non Muse) using the Steel\u0026ndash;Dwass test (nonparametric multiple test). The false discovery rate (FDR) was calculated using Storey\u0026rsquo;s method based on the p-value. To extract the significant effects, the threshold levels of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.10 were set. The sham-vehicle and BPD-vehicle groups were compared to extract proteins affected or not by BPD. The extracted proteins were divided into those showing significant effects from the Muse cell injection and those that did not, by comparing the BPD-vehicle and BPD-Muse groups. Then, by comparing the BPD-vehicle and BPD-non Muse groups, these proteins were further separated into those exhibting significant effects from the non-Muse cell injections and those that did not, by comparing the BPD-vehicle and BPD-non-Muse groups. The protein groups extracted by this stratified analysis were as follows (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea): proteins affected by BPD (I); proteins not affected by BPD (II); among the proteins affected by BPD, those showing significant effects from the Muse cell injections (III); proteins that did not receive significant effects from the Muse cell injections (IV); proteins showing significant effects from therapy with MSCs, including Muse cells (V); proteins showing significant effects from treatment with only Muse cells (VI); proteins demonstrating significant effects from treatment with MSCs, not including Muse cells (VII); and proteins not showing significant effects from treatment with all MSCs, including Muse cells (VIII); among the proteins not affected by BPD, the proteins showing significant effects from the treatment with Muse cells (IX); proteins not showing significant effects from the treatment with Muse cells (X); proteins not showing significant effects from the treatment with MSCs, including Muse cells (XI); proteins exhibiting significant effects from the treatment with only the Muse cells (XII); proteins showing significant effects from the treatment with MSCs, not including Muse cells (XIII); and Others. The enrichment ratio for the number of proteins affected by the Muse or non-Muse celll injections was defined as the number of proteins affected by BPD /the number of proteins not affected by BPD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eFunctional analysis\u003c/h2\u003e\u003cp\u003eIn the protein profiles extracted by these 13 conditions, groups I (proteins affected by BPD), III (among the proteins affected by BPD, those showing significant effects from the treatment with only Muse cell injections), and V\u0026thinsp;+\u0026thinsp;VII (among the proteins affected by BPD, those showing significant effects from the treatment with non-Muse cell injections) were used for the functional annotation analysis on the Database for Annotation, Visualization and Integrated Discovery 6.8 (DAVID 6.8, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Huang et al., 2009). The annotations database was used on Feb. 19, 2021. Functional analysis using DAVID 6.8 was performed to enrich the proteins of these 12 protein profiles into gene ontology (GO term). The flagged GO terms were then clustered to clarify the relationships among similar annotation and co-association proteins. Among the clustered GO terms, we extracted the groups with the top 5 cluster enrichment scores. Additionally, molecular networks among the proteins extracted in the clustered ontologies were visualized using the Search Tool for the Retrieval of Interacting Genes/Proteins 11.0 (STRING 11.0, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results analyzed with STRING were clustered using the Markov cluster algorithm (MCL clustering; inflation parameter is 3). The minimum required interaction score was set to 0.400. The interactions were indicated by eight criteria for linkage, including neighborhood, gene fusion, co-occurrence, co-expression, experiments, databases, text mining, and homology. Information on the distribution and main function of the extracted proteins in the lung was also investigated using the The Human Protein Atlas (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org/\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Uniprot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using the Prism 10 software (GraphPad Software, Boston, MA). The survival rate was evaluated by using the Kaplan\u0026ndash;Meier method. Proteomic analysis was evaluated by using the Steel\u0026ndash;Dwass test. The other analyses were performed using one-way analysis of variance, followed by the Holm Sidak method. All values were presented as the mean with standard error. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eComparison between the intravenous and intratracheal administrations\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003eBody weight and survival rate\u003c/h2\u003e\u003cp\u003eIn the intravenous (iv) group, the body weight on day 15 and survival rate were not significantly different among the sham, vehicle, and Muse groups.(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, c) However, in the intratracheal (it) groups, regardless of whether the cells were administered or not, a slowing in weight gain (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; sham vs vehicle-it and Muse-it) and worsening of survival rate (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; sham vs vehicle-it and Muse-it) were observed in the it groups than in the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eRespiratory function test: WBP\u003c/h2\u003e\u003cp\u003eFor intravenous administration, the TV was significantly lower in the vehicle iv group than the sham group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and significantly higher in the Muse-iv group than in the vehicle-iv group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For the intratracheal administration, although the TV was lower in the vehicle-it group than in the sham group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), the difference was not significant between the Muse-it and vehicle-it groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eImpact of Muse cells on the lung tissues\u003c/h2\u003e\u003cp\u003eThe alveolar spaces were more enlarged in the hyperoxia-loaded rats (vehicles) than in the sham group, and this enlargement was ameliorated in the Muse groups. TVD was significantly lower in the vehicle group than in the sham group. Comparing the intravenous and intratracheal injections, both injections ameliorated the lung tissue volume density in the Muse group, but not in the vehicle group (iv; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, it; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eDifferential cell counts BALF\u003c/h2\u003e\u003cp\u003eThe white blood cell (WBC) count was significantly lower in the Muse group than in the vehicle group for both iv and it injections (iv; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, it; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e\u003cp\u003eThese results show that, regardless of whether the route of administration was intravenous or intratracheal, Muse cells ameliorated the alveolar-impaired development, and also produced anti-inflammatory effects, as evidenced by lower WBC counts in the BALF of the Muse group. However, the intratracheal injection did not lead to the amelioration in respiratory function in the TV, whereas intravenous injection did. Moreover, body weight gain and survival rate were lower in the Muse-it and vehicle-it groups than in the Muse-iv and vehicle-iv groups. Based on these findings, we conducted subsequent studies on the intravenous administration.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eComparison of the intravenous Muse and non-Muse cell administrations with vehicle\u003c/h2\u003e\u003cdiv id=\"Sec26\" class=\"Section4\"\u003e\u003ch2\u003eRespiratory function test: WBP\u003c/h2\u003e\u003cp\u003eThe TV on day 15 was significantly lower in the vehicle group than in the sham (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, there is no significant difference between the vehicle, non-Muse, and Muse groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The TV in the Muse group tended to be higher than that in the non-Muse group, although the difference is not significant (p\u0026thinsp;=\u0026thinsp;0.053).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eImpact of Muse cells on lung tissue\u003c/h2\u003e\u003cp\u003eRepresentative photographs on day 29 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. The alveolar spaces of the hyperoxia-loaded rats (vehicles) were more enlarged than those of the sham group, and this enlargement was ameliorated in the Muse group, whereas, in the non-Muse group, the improvement was poor. TVD was significantly lower in the vehicle group than in the sham group, and was significantly higher in the Muse group than in the vehicle and non-Muse groups on day 29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eDifferential cell counts BALF\u003c/h2\u003e\u003cp\u003eWe evaluated the differential cell counts of BALF on day 15. The total number of cells,in BALF was increased in the vehicle group and was significantly lower in the Muse group, not in the non-Muse group, than in the vehicle group. The administration of Muse cells, not the non-Muse cells, suppressed the inflammation caused by BPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eDistribution of Muse and non-Muse cells after intravenous administration in BPD rats\u003c/h2\u003e\u003cp\u003eTo evaluate the distribution of Muse and non-Muse cells, qPCR assay with human Alu elements (Alu PCR) was performed at 10 days afte the intravenous administration (day 15). Alu PCR detected human genomic DNA in all of the organs (left lung, brain, liver, and spleen), with the highest amount being found in the lung of the Muse-treated rats. Human DNA was also detected in all of the organs of the non-Muse group. However, the amount of DNA in most organs was significantly lesser than that of the Muse group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEngraftment of Muse and non-Muse cells in the lung.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo confirm the engraftment of the Muse and non-Muse cells in the lung, immunofluorescence staining for GFP or STEM121 (human cell marker) was performed on day15. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows the representative pictures. The GFP or STEM121-positive cells indicated the presence of engrafted Muse or non-Muse cells. The Muse group showed more flattened cells, whereas the non-Muse group showed more spherical cells. Cells were detected in both the non-Muse group and Muse groups, but the number of cells was significantly higher in the Muse group in both markers.(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDifferentiation of Muse cells into type 1 and 2 alveolar epithelial cells\u003c/h3\u003e\n\u003cp\u003eExpression of human podoplanin, a marker of human type I alveolar epithelial cells, was confirmed in the GFP-positive squamous cells in the GFP-labeled Muse cells. The human podoplanin-positive cells were observed to localize in a ring-like pattern, suggesting that they may contribute to alveolar repair and formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The expression of human hPE10, a marker of human type2 alveolar epithelial cells, was also confirmed in the GFP-positive cells in the GFP-labeled Muse cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of pulmonary hypertension\u003c/h2\u003e\u003cp\u003eIn BPD, RV hypertrophy develops, evidenced by the elevated dry weight ratio of the RV to the LV plus IVS (RV/LV\u0026thinsp;+\u0026thinsp;IVS). Compared to the vehicle and non-Muse groups, the Muse group showed a significant decrease in the RV/LV\u0026thinsp;+\u0026thinsp;S on day 29 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting that Muse cell treatment ameliorates RV hypertrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn BPD, RVSP also develops. As compared to the sham group, the RVSP in the vehicle group was significantly higher at P29 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). As compared to the vehicle group, the RVSP in the Muse group was significantly lower on day 29 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that Muse cells ameliorate the elevated RVSP caused by BPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eProteomics\u003c/h2\u003e\u003cp\u003eThe protein profiles of the lungs obtained from the four experimental groups (sham, vehicle, Muse, and non-Muse groups) were analyzed to investigate the target molecules associated with the mechanisms of abnormal respiratory function and thw therapeutic effects of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). Using LC/MS/MS, 2694 proteins with high-quality signals were detected and quantified; of these, 844 proteins exhibited significant differences between the Sham and Vehicle groups. Of the 844 proteins, 141 proteins were extracted as having been affected by the Muse cell administration and 703 proteins were not significantly affected by its administration. Furthermore, among the 141 proteins extracted, 56 showed significant effects not only from the treatment with Muse cells but also from the treatment with non-Muse cells. In other words, 85 proteins showed significant effects from the treatment with only the Muse cells. Contrarily, 167 proteins were significantly affected by the treatment with only the non-Muse cells. Of the 1850 proteins, not affected by BPD, 115 and 227 proteins were significantly affected from the treatments with Muse and non-Muse cells, respectively; of these, 39 proteins were common between the Muse and non-Muse groups. According to the number of extracted proteins, the enrichment ratio of the proteins affected by Muse cell administration was 2.69, whereas that of the proteins affected by non-Muse cell administration was 2.15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003eFunctional analysis\u003c/h2\u003e\u003cp\u003eTo understand the biological significance of each extracted protein profile, we performed a functional analysis using the GO term database and its clustering analysis. First, the proteins categorized as Group I, which includes 844 proteins affected by BPD, were evaluated. As top five clusters, cell adhesion-related, ATP synthase-related, cAMP-dependent protein kinase-related, blood coagulation/fibrinolytic system-related, and lipoprotein-related ontologies were extracted through the analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTop five clusters of significantly enriched GO terms in proteins affected by BPD. Expression levels of the 844 proteins altered by BPD. Functional features of these 844 proteins were extracted using gene annotation by gene ontology and those clustering analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEnrichment\u003c/p\u003e\u003cp\u003eScore\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTerm name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFold\u003c/p\u003e\u003cp\u003eEnrichment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecadherin binding involved in cell-cell adhesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecell-cell adherens junction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecell-cell adhesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eproton-transporting ATP synthase complex, catalytic core F(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003emitochondrial proton-transporting ATP synthase complex, catalytic core F(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003emitochondrial proton-transporting ATP synthase complex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eproton-transporting ATP synthase activity, rotational mechanism\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0099\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATP synthesis coupled proton transport\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecAMP-dependent protein kinase regulator activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecAMP-dependent protein kinase complex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003enegative regulation of cAMP-dependent protein kinase activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecAMP-dependent protein kinase inhibitor activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eprotein kinase A catalytic subunit binding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecAMP binding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eplasminogen activation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epositive regulation of heterotypic cell-cell adhesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eprotein polymerization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eblood coagulation, fibrin clot formation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eplatelet alpha granule\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003efibrinogen complex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecellular protein complex assembly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.056\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epositive regulation of peptide hormone secretion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehigh-density lipoprotein particle assembly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ediscoidal high-density lipoprotein particle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ereverse cholesterol transport\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehigh-density lipoprotein particle remodeling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003elipid transporter activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e is here.)\u003c/p\u003e\u003cp\u003eThe proteins affected by BPD were classified as Group III, which includes 141 proteins affected by Muse cell administration, and as Group V\u0026thinsp;+\u0026thinsp;VII, which includes 223 proteins affected by non-Muse cell administration. The analysis identified endopeptidase and inflammation-related, filament and cytoskeleton-related, blood coagulation/fibrinolytic system-related, and cell adhesion-related ontologies as the top five clusters of Group III (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas, for Group V\u0026thinsp;+\u0026thinsp;VII, these were GTPase-related, ribosome-related, filament-related, cytoskeleton-related, and hydrogen peroxide-related ontologies (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTop five enriched GO clusters in BPD-related proteins affected by Muse cells.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEnrichment\u003c/p\u003e\u003cp\u003eScore\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTerm name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFold\u003c/p\u003e\u003cp\u003eEnrichment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003enegative regulation of endopeptidase activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eendopeptidase inhibitor activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003einflammatory response\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebrush border\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003estress fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecalmodulin binding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003emyosin complex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003emotor activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.072\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003enegative regulation of endopeptidase activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evasodilation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003enegative regulation of blood coagulation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e31.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecysteine-type endopeptidase inhibitor activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.048\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epositive regulation of cytosolic calcium ion concentration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003estress fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eactin filament binding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eactin cytoskeleton\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecadherin binding involved in cell-cell adhesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecell-cell adherens junction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0033\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecell-cell adhesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.085\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTop five enriched GO clusters in BPD-related proteins affected by non-Muse cells.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEnrichment\u003c/p\u003e\u003cp\u003eScore\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTerm name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFold\u003c/p\u003e\u003cp\u003eEnrichment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003esmall GTPase mediated signal transduction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTP binding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTPase activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGDP binding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecytosolic small ribosomal subunit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etranslation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003estructural constituent of ribosome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0076\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eribosome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.098\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eintermediate filament\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etype III intermediate filament\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e50.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003estructural molecule activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003estructural constituent of cytoskeleton\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003emicrotubule-based process\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecytoskeleton organization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003emicrotubule\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCluster 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehaptoglobin-hemoglobin complex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e63.72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eresponse to hydrogen peroxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epositive regulation of cell death\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e(Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e is here.)\u003c/p\u003e\u003cp\u003eNotably, the blood coagulation/fibrinolytic system-related ontologies were commonly enriched as clusters 4 and 3 of Groups I and III, respectively. Cell adhesion-related ontologies were also commonly enriched as cluster 1 of Group I and as cluster 5 of Group III. These ontologies were not enriched in Group V\u0026thinsp;+\u0026thinsp;VII. Thus, the expression of blood coagulation/fibrinolytic system-related and cell adhesion-related proteins was altered by BPD, and Muse cell administration significantly influenced the expression of these proteins, whereas the non-Muse cell administration did not. Moreover, filament and cytoskeleton-related ontologies were commonly enriched as clusters 2 and 4 of Group III and as clusters 3 and 4 of Group V\u0026thinsp;+\u0026thinsp;VII. The findings suggested that the MSC may influenced the formation of respiratory cells. Particularly, the administration of Muse cells may have additional effects on the abnormalities in cell adhesion and the blood coagulation/fibrinolytic system caused by BPD.\u003c/p\u003e\u003cp\u003eFor the cell adhesion-related ontologies, eight proteins significantly affected by Muse cell administration were included. The distribution and main function of the eight proteins in the normal lungs are listed in Supplemental Table\u0026nbsp;1. The expression of all of the eight proteins was detected in the alveolar cells, bronchus, and macrophages in the normal lungs, indicating that their expression levels were altered by BPD and Muse cells administration. Lrrfip1 and Myh9 were upregulated by BPD and suppressed by Muse cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Uso1, Idh1, Ctnnb1, Axa1, and Puf60 were downregulated by BPD and enhanced by Muse cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Finally, Pkm was upregulated by BPD and further increased by Muse cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast to the proteins with cell adhesion-related ontologies, almost all of proteins with blood coagulation/fibrinolytic system-related ontologies were not detected in the normal lungs (Supplemental Tables\u0026nbsp;2 and 3); hence, these proteins may be expressed and/or translocated by BPD. The expressions of Gnai3, Sod2, and Map1, which are detected in the normal lungs, were upregulated by BPD and further increased by Muse cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Kng1, Itih3, C3, and C4, which are not detected in the normal lungs, were detected in the BPD lungs and further increased by Muse cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). A1i3 and Mug1 were suppressed by BPD and enhanced by Muse cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Regarding the blood coagulation/fibrinolytic system-related proteins not affected by Muse cell administration, Alox15 and Lonp1 were detected in the normal lungs, whereas Fga, Fgb, Fgg, Apoh, Crp, and Pf4 were not detected in the normal lungs. The expression of every protein, except for Pf4, was increased by BPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eNetwork analysis\u003c/h2\u003e\u003cp\u003eTo promote understanding of their functional association, the interaction networks among the proteins found during the functional annotation analysis were visualized using STRING and MCL clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). This network displayed the molecular interaction among the cell adhesion- and blood coagulation/fibrinolytic system-related proteins affected by BPD. These proteins were found to be closely related to each other. The nodes indicate the individual proteins, and the distance between them indicates the depth of the relationship between the proteins (the shorter the distance, the closer the relationship). The nodes of the same color have a strong relationship, indicating that they are classified in the same group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea illustrates the division of a network of 49 cell adhesion-related proteins affected by BPD (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e cluster 1). The eight proteins (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e cluster 5 and Supplemental Table\u0026nbsp;1) affected by Muse cell administration were not confined to any particular group, but they were scattered on each group. The administration of Muse cells is considered to affect various groups of cell adhesion molecules, rather than a specific group. Conversely, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb also demonstrates that the 17 blood coagulation/fibrinolytic system-related proteins affected by BPD (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e cluster 4) are closely related.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eHistological analysis of Ctnnb1 and Anxa1\u003c/h3\u003e\n\u003cp\u003eA network analysis using STRING was performed on the molecules selected from the statistical and annotation analyses of the protein profile. The results revealed that Ctnnb1 (β-catenin) was the most central molecule in the network (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The expression of this molecule decreased in BPD cases, and this decrease was ameliorated by the stem cell administration (Muse/non-Muse cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The results of the histological analysis further demonstrated a marked upregulation of Ctnnb1 expression in cells adjacent to the Muse cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Contrarily, although an increase in Ctnnb1 was observed in the non-Muse group, this upregulation was detected at locations distant from the non-Muse cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eAdditionally, based on the results of the BALF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg) and proteomic analyses (Supplemental Table\u0026nbsp;1), we focused on Anxa1 (Annexin A1), a molecule associated with neutrophil migration and inflammatory responses. The expression of this molecule was found to decrease in BPD cases, and its reduction was ameliorated by the administration of Muse cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The results of the histological analysis showed a significant increase in the number of Anxa1-positive cells in the Muse group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Although an increase in Anxa1-positive cells was also observed in the non-Muse group, the increase was only minimal, compared to that observed in the Muse group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Additionally, Anxa1 expression was observed in a subset of spherical GFP-positive Muse cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough intravenous and intratracheal administrations represent distinct delivery routes and, therefore, strictly different models, precluding a direct comparison, the present study showed that intravenous administration outperformed intratracheal administration in terms of survival, weight gain, and respiratory outcomes. Intravenously administered human Muse cells were engrafted and expressed alveolar epithelial cell markers in the BPD rat model. The Muse cells ameliorated alveolar growth impairment induced by BPD and improved the pulmonary function. Animals receiving Muse cells intravenously displayed improvement in BPD-associated pulmonary hypertension, as compared to the non-Muse and vehicle groups. A comprehensive analysis of proteins from BPD lungs revealed that Muse cells may have additional effects on the abnormalities in cell adhesion and the blood coagulation/fibrinolytic system caused by BPD. These results demonstrated the histological and functional benefits of human Muse cells in experimental BPD.\u003c/p\u003e\u003cp\u003eA major finding of the present study is that the xenogeneic Muse cells were engrafted in the injured lung without the use of immunosuppressants and expressed alveolar epithelial cell markers (podoplanin and PE10). Given that the lungs are the first organs to trap cells following intravenous administration, a human-specific genome material was detected to some extent at 2 weeks, even in our non-Muse group, which differed from previous reports.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) However, the number of cells detected in the lung by the GFP-positive cells at 4 weeks was much higher in the Muse group than in the non-Muse group. The S1P\u0026ndash;S1P receptor 2 axis plays an important role in the selective homing of intravenously administered Muse cells into the damaged sites.(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) The S1P expression is increased at the site of injury(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and in the peripheral blood.(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) The ability of intravenously administered Muse cells to migrate to the injury site is a major advantage over many other types of stem cells. In AluPCR, the Muse cells were detected in large numbers in the lungs, brain, and liver. We believe that this is because the damage caused by high oxygen exposure also occurs in the lungs, brain, and liver, causing Muse cells to migrate to the injured sites. We also believe that the Muse and non-Muse cells are detected in the spleen due to the organ\u0026rsquo;s filtering function.\u003c/p\u003e\u003cp\u003eIn BPD, the intratracheal administration of MSCs has been attempted,(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) and clinical trials on the treatment of severe BPD have also been conducted(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, the disconnection of the ventilator circuit and \u0026ldquo;fluid\u0026rdquo; administration can lead to hypoxia in patients. Therefore, we aim to avoid intratracheal administration when possible, as intravenous administration does not have this risk. Our data showed that, with intratracheal administration, whether involving solvent alone or Muse cells, the survival rate declined and weight gain was reduced compared to intravenous administration, suggesting that intravenous administration is more suitable than intratracheal administration for this treatment approach.\u003c/p\u003e\u003cp\u003eRegarding differentiation, Muse cell differentiates into tissue-compatible cells after homing in various injury models, including stroke,(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) neonatal hypoxic\u0026ndash;ischemic encephalopathy,(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) liver fibrosis,(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) acute myocardial infarction,(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) chronic kidney disease,(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and aortic aneurysms.(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) Regarding BPD, the migration of Muse cells into the lungs and their differentiation into alveolar epithelial type 1 and 2 cells have been reported.(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) In the present study, Muse cells also expressed markers for alveolar epithelial type 1 and 2 cells. The Muse cells, such as the MSCs, are also known to secrete various cytokines, producing paracrine effects.(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) Although there are many reports on BPD treated with MSCs, which have demonstrated some treatment effects,(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) non-Muse cells did not show sufficient efficacy in the present study. The direct effect of Muse cells to engraft and differentiate into lung cells, in addition to their paracrine effect, may have resulted in a significant therapeutic effect.\u003c/p\u003e\u003cp\u003eTo further understand the mechanisms underlying the therapeutic effects of Muse cell administration, we conducted a comprehensive proteomic analysis. The annotation of protein expression profiles highlighted the cell adhesion-related proteins as key molecules involved in this process. The pathology of BPD is characterized by alveolar simplification and abnormal pulmonary vascularization due to an interrupted pulmonary and alveolar development.(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Although the pathogenesis of BPD is multifactorial, a common pathway involves inflammatory responses.(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) Inflammation triggers the upregulation of cell adhesion molecules and chemotactic proteins that recruit inflammatory cells to the lungs.(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) The interactions between the adhesion molecules on the surfaces of the leukocytes and endothelial cells facilitate the migration of leukocytes from the bloodstream into the lung interstitium, mediating the inflammatory response.(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) Given the evidence for subclinical pulmonary endothelial dysfunction in BPD, it may be crucial to evaluate the lung adhesion molecules\u0026mdash;an early marker of endothelial dysfunction\u0026mdash;in clinical specimens, such as BALF from neonates with established BPD. These adhesion molecules, which adhere to the endothelial surface, play a key role in leukocyte infiltration through the endothelial cells and into the lung parenchyma.(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eOur network analysis identified Ctnnb1 (β-catenin) as the most central molecule among the cell adhesion-related proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Ctnnb1 is a pivotal molecule in the Wnt/β-catenin signalling pathway, which regulates stem cell pluripotency, differentiation, proliferation, and migration during development.(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) In the respiratory system, Ctnnb1 promotes cellular maturation, differentiation, and morphogenesis.(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) Our findings revealed a significant reduction in Ctnnb1 expression associated with BPD, which was restored following the administration of Muse cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The results of our histological analysis further demonstrated that β-catenin expression was markedly elevated in the alveolar epithelial cells adjacent to the Muse cells that had migrated into the lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results suggest that Muse cell administration may improve lung function by directly or indirectly inducing Wnt/β-catenin signalling in the neighbouring lung cells, either through direct cell interactions or via the paracrine factors.\u003c/p\u003e\u003cp\u003eIn addition to the cell adhesion-related proteins, the blood coagulation and fibrinolytic system-related proteins are also important to understand the effects of Muse cell administration in BPD. Among the 17 proteins analyzed, nine were significantly impacted by the Muse cell treatment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Cluster 3; Supplemental Table\u0026nbsp;2), whereas eight were unaffected (Supplemental Table\u0026nbsp;3). The unaffected proteins, which all promote blood coagulation, were upregulated in response to BPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Conversely, four out of the nine proteins influenced by Muse cell administration\u0026mdash;Kng1, Gnai3, Map1, and Sod2\u0026mdash;inhibit coagulation and promote vasodilation, and their expression was further upregulated following Muse cell treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These findings suggest that Muse cell administration may help counteract the BPD-induced alterations in blood flow. A molecular network analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) indicated that most of the 17 proteins are closely interrelated, implying that Muse cells may induce compensatory changes in an alternate group of blood coagulation and fibrinolytic proteins. Indeed, the suppression of plasminogen activator-mediated fibrinolysis not only increases the fibrin deposition in the alveoli but also contributes to the recruitment and migration of inflammatory cells.(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) Fibrinolytic therapies aimed at preventing pulmonary and pleural fibrin deposition, along with inflammation, show considerable promise. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eAmong the cell adhesion-related proteins, we also focused on Anxa1, a molecule known for its role in inflammation and neutrophil infiltration. According to the Human Protein Atlas database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org/ENSG00000135046-ANXA1\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org/ENSG00000135046-ANXA1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Anxa1 is strongly expressed in alveolar macrophages and bronchial epithelial cells. Additionally, Anxa1 suppresses immune cell infiltration and promotes the release of the anti-inflammatory cytokine IL-10, which helps reduce pulmonary inflammation (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In the present study, we observed a marked upregulation of the Anxa1 expression in some Muse cells that had migrated into the lung (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This finding suggests that Muse cell administration increases the number of Anxa1-positive cells, potentially limiting excessive immune cell infiltration and the accompanying inflammation. This mechanism may underlie the greater improvement in lung function and development in comparison to the non-Muse group. These findings align with the reduction in lymphocyte counts observed in the BALF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), further supporting the role of Anxa1 in mediating the beneficial effects of Muse cells in BPD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe intravenous administration of Muse cells demonstrated superior therapeutic effects in a BPD rat model compared to the intratracheal administration of Muse or non-Muse cells. Muse cells ameliorated respiratory impairment and pulmonary hypertension, accompanied by differentiation into alveolar epithelial cells and modulation of cell adhesion and coagulation pathways. Our study findings suggest the potential of Muse cell therapy as a promising approach for treating severe neonatal lung diseases and warrant further translational and clinical investigations to validate the results.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBPD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBronchopulmonary Dysplasia\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMuse cell\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMultilineage-differentiating stress enduring cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMesenchymal Stromal Cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSSEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStage-Specific Embryonic Antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVLBW\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eVery Low Birth Weight\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eS1P\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSphingosine-1-Phosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHLA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuman Leukocyte Antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBM-MSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBone Marrow- Mesenchymal Stromal Cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGFP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGreen Fluorescent Protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFACS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFluorescence-Activated Cell Sorter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBALF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBronchoalveolar Lavage Fluid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTidal Volume\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWBP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWhole Body Plethysmography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTVD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTissue Volume Density\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003equantitative Polymerase Chain Reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate-Buffered Saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRight Ventricle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLeft Ventricle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIVS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterventricular Septum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRVSP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRight Ventricular Systolic Pressure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLC/MS/MS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLiquid Chromatography/Tandem Mass Spectrometry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eiv\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eintravenous\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eit\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eintratracheal\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe experiments were conducted with the approval of the Animal Care and Use Committee of Nagoya University School of Medicine (Nagoya, Japan; permit Nos. 26128, 27190, 28215, 29012, 30197, 31106, 20032, and M210107-003) and in compliance with the Regulations on Animal Experiments at Nagoya University. The title of the approved project was \u0026ldquo;Establishment of a neonatal rat model of BPD and development of novel therapeutic strategies\u0026rdquo;, and the date of approval were March 18,2014 (No.26126), March 16,2015 (No.27190), March 11,2016 (No.28125), March 10,2017 (No.29012), March 19,2018 (No.30197), March 8,2019 (No.31106), March 6,2020 (No.20032), March 3,2021 (No.M210107-003).\u0026nbsp;Human BM-MSCs were purchased from Lonza. According to the manufacturer\u0026rsquo;s documentation and published information, Lonza obtained ethical approval for the collection of human bone marrow samples, and written informed consent was obtained from all donors(Supplemental Material 1,2).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding author upon reasonable request. Raw proteomic data were submitted to the Japan Proteome Standard Repository/Database(https://repository.jpostdb.org/). The accession number of jPOST for the proteomic data used in the present study is JPST003674 (PXD061493) .\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Y.S and S.Sh have a collaborative research and developmental agreement for perinatal disease with Life Science Institute Inc.(LSII). S.Sh is making a collaborative research and developmental agreement for the other disease, and has a contract for consulting with LSII. Y.S, T.S and S.Sh have a patent for the application of Muse cells for treatment of perinatal brain damage and the other indication.\u0026nbsp;R.M, A.On, A.Ok, S.Su, K.U, Y.T and M.H declare no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by AMED (grant No. JP\u0026nbsp;22bm0404067h0003) and Nagoya University Hospital Funding for Clinical Research.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eR.M., A.On., and A,Ok. were involved in animal experiments. R.M., A.On., A.Ok., and Y.S. conceived and designed the study. R.M., A.On., T.S., A,Ok., S.Su., K.U., S.Sh., and Y.S. interpreted the data. R.M. drafted the initial manuscript, and. R.M., A.On., T.S., A.Ok., S.Su., K.U., S.Sh., Y.T., M.H., and Y.S. critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript and are accountable for the entire work.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe appreciate the technical assistance of Ms. Tomoko Yamaguchi, and acknowledge Division for Medical Research Engineering, Nagoya University Graduate School of Medicine We would also like to thank Kentaro Taki for operational support in the LC/MS/MS in the laboratory of the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have also used AI for English proofreading in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKalikkot Thekkeveedu R, Guaman MC, Shivanna B. Bronchopulmonary dysplasia: A review of pathogenesis and pathophysiology. Respir Med. 2017;132:170\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKusuda S, Fujimura M, Uchiyama A, Totsu S, Matsunami K. Trends in morbidity and mortality among very-low-birth-weight infants from 2003 to 2008 in Japan. Pediatr Res. 2012;72(5):531\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavidson L, Berkelhamer S. Bronchopulmonary Dysplasia: Chronic Lung Disease of Infancy and Long-Term Pulmonary Outcomes. J Clin Med. 2017;6(1):4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLapcharoensap W, Lee HC, Nyberg A, Dukhovny D. Health Care and Societal Costs of Bronchopulmonary Dysplasia. Neoreviews. 2018;19(4):e211\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGray PH, O'Callaghan MJ, Rogers YM. Psychoeducational outcome at school age of preterm infants with bronchopulmonary dysplasia. J Paediatr Child Health. 2004;40(3):114\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatterberg KL. Postnatal Corticosteroids to Prevent or Treat Bronchopulmonary Dysplasia. Pediatrics. 2010;126(4):800\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaniguchi A, Nishida K, Suzuki T, Kataoka E, Fujishiro N, Kato E, et al. Total Hydrocortisone Dosage in the Neonatal Period May Be Related to Low Developmental Quotient in Extremely Low Birth Weight Infants: A Retrospective Cohort Study. Neonatology. 2024;121(2):195\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIchinohashi Y, Sato Y, Saito A, Ito M, Watanabe K, Hayakawa M, et al. Dexamethasone administration to the neonatal rat results in neurological dysfunction at the juvenile stage even at low doses. Early Hum Dev. 2013;89(5):283\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuzuki T, Sato Y, Kushida Y, Tsuji M, Wakao S, Ueda K, et al. Intravenously delivered multilineage-differentiating stress enduring cells dampen excessive glutamate metabolism and microglial activation in experimental perinatal hypoxic ischemic encephalopathy. J Cereb Blood Flow Metab. 2021;41(7):1707\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKitase Y, Sato Y, Arai S, Onoda A, Ueda K, Go S, et al. Establishment of a Novel Fetal Growth Restriction Model and Development of a Stem-Cell Therapy Using Umbilical Cord-Derived Mesenchymal Stromal Cells. Front Cell Neurosci. 2020;14(212):212.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuzuki T, Sato Y, Yamamoto H, Kato T, Kitase Y, Ueda K, et al. Mesenchymal stem/stromal cells stably transduced with an inhibitor of CC chemokine ligand 2 ameliorate bronchopulmonary dysplasia and pulmonary hypertension. Cytotherapy. 2020;22(4):180\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsuji M, Taguchi A, Ohshima M, Kasahara Y, Sato Y, Tsuda H, et al. Effects of intravenous administration of umbilical cord blood CD34 cells in a mouse model of neonatal stroke. Neuroscience. 2014;263C:148\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUeda K, Sato Y, Shimizu S, Suzuki T, Onoda A, Miura R, et al. Systemic administration of clinical-grade multilineage-differentiating stress-enduring cells ameliorates hypoxic-ischemic brain injury in neonatal rats. Sci Rep. 2023;13(1):14958.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMimatsu H, Onoda A, Kazama T, Nishijima K, Shimoyama Y, Go S, et al. Dedifferentiated fat cells administration ameliorates abnormal expressions of fatty acids metabolism-related protein expressions and intestinal tissue damage in experimental necrotizing enterocolitis. Sci Rep. 2023;13(1):8266.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFung ME, Thebaud B. Stem cell-based therapy for neonatal lung disease: it is in the juice. Pediatr Res. 2014;75(1\u0026ndash;1):2\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAugustine S, Avey MT, Harrison B, Locke T, Ghannad M, Moher D, et al. Mesenchymal Stromal Cell Therapy in Bronchopulmonary Dysplasia: Systematic Review and Meta-Analysis of Preclinical Studies. Stem Cells Transl Med. 2017;6(12):2079\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNitkin CR, Rajasingh J, Pisano C, Besner GE, Th\u0026eacute;baud B, Sampath V. Stem cell therapy for preventing neonatal diseases in the 21st century: Current understanding and challenges. Pediatr Res. 2020;87(2):265\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang YS, Ahn SY, Yoo HS, Sung SI, Choi SJ, Oh WI, et al. Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial. J Pediatr. 2014;164(5):966\u0026ndash;72. e6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim R, Malhotra A, Tan J, Chan ST, Lau S, Zhu D, et al. First-In‐Human Administration of Allogeneic Amnion Cells in Premature Infants With Bronchopulmonary Dysplasia: A Safety Study. STEM CELLS Translational Med. 2018;7(9):628\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePowell SB, Silvestri JM. Safety of Intratracheal Administration of Human Umbilical Cord Blood Derived Mesenchymal Stromal Cells in Extremely Low Birth Weight Preterm Infants. J Pediatr. 2019;210:209\u0026thinsp;\u0026ndash;\u0026thinsp;13.e2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhn SY, Chang YS, Lee MH, Sung SI, Lee BS, Kim KS, et al. Stem cells for bronchopulmonary dysplasia in preterm infants: A randomized controlled phase II trial. Stem Cells Transl Med. 2021;10(8):1129\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuroda Y, Kitada M, Wakao S, Nishikawa K, Tanimura Y, Makinoshima H, et al. Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA. 2010;107(19):8639\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWakao S, Kitada M, Kuroda Y, Shigemoto T, Matsuse D, Akashi H et al. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proceedings of the National Academy of Sciences. 2011;108(24):9875-80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamada Y, Wakao S, Kushida Y, Minatoguchi S, Mikami A, Higashi K, et al. S1P\u0026ndash;S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial InfarctionNovelty and Significance. Circul Res. 2018;122(8):1069\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKushida Y, Wakao S, Dezawa M. Muse Cells Are Endogenous Reparative Stem Cells. Adv Exp Med Biol. 2018;1103:43\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUchida N, Kushida Y, Kitada M, Wakao S, Kumagai N, Kuroda Y, et al. Beneficial Effects of Systemically Administered Human Muse Cells in Adriamycin Nephropathy. J Am Soc Nephrol. 2017;28(10):2946\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIseki M, Kushida Y, Wakao S, Akimoto T, Mizuma M, Motoi F, et al. Muse Cells, Nontumorigenic Pluripotent-Like Stem Cells, Have Liver Regeneration Capacity Through Specific Homing and Cell Replacement in a Mouse Model of Liver Fibrosis. Cell Transpl. 2017;26(5):821\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKinoshita K, Kuno S, Ishimine H, Aoi N, Mineda K, Kato H, et al. Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers. Stem Cells Transl Med. 2015;4(2):146\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWakao S, Oguma Y, Kushida Y, Kuroda Y, Tatsumi K, Dezawa M. Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame. Cell Mol Life Sci. 2022;79(11):542.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSato Y, Shimizu S, Ueda K, Suzuki T, Suzuki S, Miura R, et al. Safety and tolerability of a Muse cell-based product in neonatal hypoxic-ischemic encephalopathy with therapeutic hypothermia (SHIELD trial). Stem Cells Transl Med. 2024;13(11):1053\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatsuyama N, Shimizu S, Ueda K, Suzuki T, Suzuki S, Miura R, et al. Safety and tolerability of a multilineage-differentiating stress-enduring cell-based product in neonatal hypoxic-ischaemic encephalopathy with therapeutic hypothermia (SHIELD trial): a clinical trial protocol open-label, non-randomised, dose-escalation trial. BMJ Open. 2022;12(4):e057073.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNiizuma K, Osawa SI, Endo H, Izumi SI, Ataka K, Hirakawa A et al. Randomized placebo-controlled trial of CL2020, an allogenic muse cell-based product, in subacute ischemic stroke. J Cereb Blood Flow Metab. 2023:271678X231202594.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNoda T, Nishigaki K, Minatoguchi S. Safety and Efficacy of Human Muse Cell-Based Product for Acute Myocardial Infarction in a First-in-Human Trial. Circ J. 2020;84(7):1189\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFujita Y, Nohara T, Takashima S, Natsuga K, Adachi M, Yoshida K et al. Intravenous allogeneic multilineage-differentiating stress‐enduring cells in adults with dystrophic epidermolysis bullosa: a phase 1/2 open‐label study. J Eur Acad Dermatol Venereol. 2021;35(8).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamashita T, Nakano Y, Sasaki R, Tadokoro K, Omote Y, Yunoki T et al. Safety and Clinical Effects of a Muse Cell-Based Product in Patients With Amyotrophic Lateral Sclerosis: Results of a Phase 2 Clinical Trial. Cell Transplant. 2023;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoda M, Imagama S, Nakashima H, Ito S, Segi N, Ouchida J, et al. Safety and feasibility of intravenous administration of a single dose of allogenic-Muse cells to treat human cervical traumatic spinal cord injury: a clinical trial. Stem Cell Res Ther. 2024;15(1):259.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNamba F. Mesenchymal stem cells for the prevention of bronchopulmonary dysplasia. Pediatr Int. 2019;61(10):945\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNamba F. Mesenchymal stem cells for the prevention of bronchopulmonary dysplasia. Pediatr Int. 2019;61(10):945\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, et al. Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplant. 2009;18(8):869\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc. 2013;8(7):1391\u0026ndash;415.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawai S, Takagi Y, Kaneko S, Kurosawa T. Effect of Three Types of Mixed Anesthetic Agents Alternate to Ketamine in Mice. Exp Anim. 2011;60(5):481\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBasu RK, Donaworth E, Wheeler DS, Devarajan P, Wong HR. Antecedent acute kidney injury worsens subsequent endotoxin-induced lung inflammation in a two-hit mouse model. Am J Physiol Ren Physiol. 2011;301(3):F597\u0026ndash;604.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang C, Lei GS, Shao S, Jung HW, Durant PJ, Lee CH. Accumulation of myeloid-derived suppressor cells in the lungs during Pneumocystis pneumonia. Infect Immun. 2012;80(10):3634\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi W-H, Gu Z, Wang H, Nekrutenko A. Evolutionary analyses of the human genome. Nature. 2001;409(6822):847\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePansani MC, Azevedo PS, Rafacho BP, Minicucci MF, Chiuso-Minicucci F, Zorzella-Pezavento SG, et al. Atrophic cardiac remodeling induced by taurine deficiency in Wistar rats. PLoS ONE. 2012;7(7):e41439.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJabary M, Onoda A, Kitase Y, Ueda K, Mimatsu H, Go S, et al. Fetal growth restriction followed by early catch-up growth impairs pancreatic islet morphology in male rats. Sci Rep. 2023;13(1):2732.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkuda S, Watanabe Y, Moriya Y, Kawano S, Yamamoto T, Matsumoto M, et al. jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res. 2017;45(D1):D1107\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamada Y, Wakao S, Kushida Y, Minatoguchi S, Mikami A, Higashi K, et al. S1P\u0026ndash;S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial Infarction. Circul Res. 2018;122(8):1069\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTanaka T, Nishigaki K, Minatoguchi S, Nawa T, Yamada Y, Kanamori H et al. Mobilized Muse Cells After Acute Myocardial Infarction Predict Cardiac Function and Remodeling in the Chronic Phase. Circ J. 2017.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, et al. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Attenuate Hyperoxia-Induced Lung Injury in Neonatal Rats. Cell Transplant. 2009;18(8):869\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUchida H, Niizuma K, Kushida Y, Wakao S, Tominaga T, Borlongan CV, et al. Human Muse Cells Reconstruct Neuronal Circuitry in Subacute Lacunar Stroke Model. Stroke. 2017;48(2):428\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHosoyama K, Wakao S, Kushida Y, Ogura F, Maeda K, Adachi O, et al. Intravenously injected human multilineage-differentiating stress-enduring cells selectively engraft into mouse aortic aneurysms and attenuate dilatation by differentiating into multiple cell types. J Thorac Cardiovasc Surg. 2018;155(6):2301\u0026ndash;e134.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWin KHN, Kushida Y, Yamana K, Iwatani S, Yoshida M, Nino N, et al. Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant. Stem Cell Res Ther. 2024;15(1):147.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYabuki H, Wakao S, Kushida Y, Dezawa M, Okada Y. Human Multilineage-differentiating Stress-Enduring Cells Exert Pleiotropic Effects to Ameliorate Acute Lung Ischemia-Reperfusion Injury in a Rat Model. Cell Transplant. 2018;27(6):979\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFukase M, Sakata N, Kushida Y, Wakao S, Unno M, Dezawa M. Intravenous injection of human multilineage-differentiating stress-enduring cells alleviates mouse severe acute pancreatitis without immunosuppressants. Surg Today. 2021.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell. 2012;149(6):1192\u0026ndash;205.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarris-Johnson KS, Domyan ET, Vezina CM, Sun X. beta-Catenin promotes respiratory progenitor identity in mouse foregut. Proc Natl Acad Sci USA. 2009;106(38):16287\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuido BC, Zanatelli M, Tavares-de-Lima W, Oliani SM, Damazo AS. Annexin-A1 peptide down-regulates the leukocyte recruitment and up-regulates interleukin-10 release into lung after intestinal ischemia-reperfusion in mice. J Inflamm (Lond). 2013;10(1):10.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Muse cells, BPD, Mesenchymal stromal cell","lastPublishedDoi":"10.21203/rs.3.rs-7875937/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7875937/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNeonatal bronchopulmonary dysplasia (BPD) is a lung injury caused by various factors, including intrauterine inflammation, mechanical ventilation, and oxidative stress. BPD results in serious respiratory and neurological dysfunctions and mortality. Recently, some clinical trials have commenced using intravenous delivery of donor-derived multilineage-differentiating stress enduring (Muse) cells. In the present study, we aimed to investigate the therapeutic effects of human Muse cells in a BPD rat model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eRats were put into the incubator within 24 h from birth to expose to hyperoxia (83%) until postnatal day 15. Muse and non-Muse cells, obtained from the bone marrow-mesenchymal stromal cells (MSCs) as stage-specific embryonic antigen-3 (SSEA-3)\u0026thinsp;+\u0026thinsp;and -, respectively, were administered slowly via the right external jugular vein or trachea (Muse cells only) on postnatal day 5. For the vehicle groups, only the acetic acid Ringer's solution was administered.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eRegarding the administration route, intravenous administration was superior to intratracheal administration in terms of improving survival, weight gain, and respiratory function. When administered intravenously, the Muse cells showed superior outcomes in ameliorating respiratory function impairment, lung inflammation, pulmonary hypertension, and anti-inflammatory effects, compared to the non-Muse cells. We also confirmed the engraftment of Muse cells in the lung tissues. A proteomic analysis also showed that Muse cells might have additional effects on the abnormalities in cell adhesion and the blood coagulation/fibrinolytic system caused by BPD.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings suggested that intravenously transplanted Muse cells provided functional benefits in our experimental BPD rat model.\u003c/p\u003e","manuscriptTitle":"Intravenously delivered Multilineage-differentiating stress enduring cells dampen in experimental bronchopulmonary dysplasia rat model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 10:25:27","doi":"10.21203/rs.3.rs-7875937/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T16:32:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T07:52:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T19:09:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121631849093002109635710189165877984225","date":"2025-11-20T12:10:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162112891641442121767963318436306910570","date":"2025-11-20T06:32:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-20T06:19:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T09:14:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-04T11:09:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-11-02T13:08:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a8b1193-949e-4e96-86ed-f86db5d462d0","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T06:39:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-04 10:25:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7875937","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7875937","identity":"rs-7875937","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}