Revolutionizing Stem Cell Therapy: A Comparative Analysis of Diverse Mesenchymal Stem Cells for Enhanced Endometrial Regeneration

Revolutionizing Stem Cell Therapy: A Comparative Analysis of Diverse Mesenchymal Stem Cells for Enhanced Endometrial Regeneration | 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 Revolutionizing Stem Cell Therapy: A Comparative Analysis of Diverse Mesenchymal Stem Cells for Enhanced Endometrial Regeneration Xiaochuan Yu, Li juan Shi, Yating Zhang, Huali Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6286920/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Endometrial injury, particularly intrauterine adhesions (Asherman’s syndrome), represents a prevalent condition that significantly compromises female fertility. Current clinical interventions predominantly involve hysteroscopic surgery, followed by the placement of intrauterine barriers and the administration of oral estrogen to facilitate endometrial regeneration. Nevertheless, in patients with severe intrauterine adhesions, postoperative pregnancy rates remain low, ranging from 22.2% to 33.3%. Mesenchymal stem cells (MSCs), owing to their multilineage differentiation potential and tissue repair capabilities, have emerged as promising candidates for the treatment of regenerative disorders. This study aimed to compare the efficacy of MSCs derived from bone marrow, umbilical cord, adipose tissue, and decidua in the repair of damaged endometrium. Methods: The proliferation capabilities of decidual MSCs, umbilical cord MSCs, bone marrow MSCs, and adipose-derived MSCs at passages 1, 3, and 5 were evaluated using a CCK8 assay. In vitro, cytokine-induced differentiation was employed to stimulate MSCs, and the expression of epithelial cell surface markers was assessed through immunofluorescence and Western blot analyses to compare their potential for differentiation into endometrial epithelial cells. In vivo, an intrauterine adhesion rat model received MSC infusions, and the restoration of endometrial morphology was subsequently examined and compared across the different treatment groups. Results: Bone marrow MSCs demonstrated the highest proliferation rate, while adipose-derived MSCs exhibited the lowest. Notably, decidual MSCs displayed a significantly enhanced capacity to differentiate into endometrial epithelial cells compared to MSCs from other sources. Furthermore, in a rat model of intrauterine adhesion, treatment with decidual MSCs resulted in the most pronounced improvement in endometrial repair. Conclusions: Decidual MSCs demonstrate superior in vitro differentiation into endometrial epithelial cells and exhibit the most effective in vivo repair of damaged endometrial tissue, potentially mediated by the secretion of various cytokines and immunomodulatory mechanisms. This study provides critical theoretical and experimental evidence supporting the clinical application of decidual MSCs in endometrial repair. Despite certain limitations, such as the absence of clinical validation, decidual MSCs present a promising novel therapeutic strategy for intrauterine adhesions and other conditions related to endometrial injury. Future clinical trials and mechanistic studies are necessary to further validate their therapeutic potential. decidual mesenchymal stem cells bone marrow mesenchymal stem cells adipose mesenchymal stem cells umbilical cord mesenchymal stem cells repair differentiation endometrial epithelial cells Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Intrauterine adhesions (IUA), also known as Asherman's syndrome, represent a fibrotic disorder within the uterine cavity, resulting from damage to and impaired repair of the endometrial basal layer [ 1 , 2 ] . This condition often leads to anatomical abnormalities of the uterine cavity, significantly impacting menstrual physiology and reproductive function in women of childbearing age. The etiology of IUA is frequently associated with intrauterine surgical procedures, including repeated induced abortions, curettage, and hysteroscopic surgeries [ 3 ] . Recent studies indicate that the incidence of IUA resulting from multiple induced abortions and curettage procedures can reach as high as 25–30%, with prevalence increasing annually as the number of intrauterine interventions rises [ 3 , 4 ] . IUA has emerged as one of the major causes of secondary infertility in women, with reports suggesting that approximately 20–40% of infertility cases in China are related to endometrial injury. Patients with IUA typically present with markedly reduced menstrual flow or amenorrhea, recurrent miscarriages, and an increased risk of pregnancy-related complications. 1.1 Limitations of Conventional Treatments Current treatment strategies for IUA primarily focus on surgically restoring the normal anatomical structure of the uterine cavity, promoting the regeneration of functional endometrium through pharmacological interventions, and preventing re-adhesion [ 5 ] . Clinically, hysteroscopic adhesiolysis (TCRA) is frequently performed, and intrauterine barriers—either biological or physical (such as intrauterine devices, uterine balloons, or biomembranes)—are employed to mitigate the risk of re-adhesion of the wound surface. These interventions are typically supplemented with estrogen or sequential estrogen-progestogen therapy to encourage endometrial growth. However, these conventional methods often fail to yield satisfactory outcomes, with reported recurrence rates for moderate-to-severe IUA reaching 37.3–43.36% [ 6 ] . The intrauterine placement of biomaterials or physical scaffolds only passively prevents the apposition of wound surfaces and does not actively promote the regeneration of new endometrial tissue, in addition to posing a risk of infection. Furthermore, due to extensive endometrial damage and a reduction in estrogen and progesterone receptors, the efficacy of oral hormone therapy is limited. Reconstructing a fully functional endometrium remains a significant clinical challenge for patients with widespread endometrial injury or severe IUA [ 7 ] . 1.2 Potential of Stem Cell Therapy In recent years, stem cell therapy has emerged as a promising approach in regenerative medicine, providing new hope for the repair of endometrial damage [ 8 ] . Mesenchymal stem cells (MSCs) have garnered significant attention as seed cells due to their unique biological properties [ 7 , 9 – 11 ] . MSCs can be isolated from various sources, including bone marrow, adipose tissue, umbilical cord, decidua, amnion, and placenta. They are characterized by their self-renewal and multilineage differentiation potential, as well as their immunomodulatory and microenvironmental repair capabilities [ 12 – 14 ] . Research has demonstrated that MSCs express low levels of MHC-II molecules and lack co-stimulatory molecules, rendering them minimally immunogenic in vivo. Furthermore, they can modulate local immune responses by secreting cytokines that inhibit the activation of T lymphocytes, B lymphocytes, and natural killer cells [ 2 , 8 , 9 , 12 , 15 ] . Notably, differences in proliferative activity, differentiation potential, and immunological characteristics exist among MSCs derived from different tissues. For instance, MSCs from the umbilical cord and bone marrow exhibit high proliferative capacity, lower immunogenicity, and stronger immunosuppressive effects, making them suitable for allogeneic transplantation [ 2 , 10 , 15 , 16 ] . Similarly, decidua-derived MSCs are readily obtainable, can be efficiently expanded in culture, and exhibit minimal rejection responses, rendering them ideal seed cells for tissue engineering. Additionally, adipose-derived MSCs, due to their accessibility and the feasibility of autologous transplantation, also show promising clinical prospects [ 11 ] . 1.3 Progress in the Application of Stem Cell Therapy Mesenchymal stem cells (MSCs) have been extensively investigated for their potential in tissue repair and regeneration across various models, including myocardial infarction, liver injury, and osteochondral defects [ 17 ] . Concerning endometrial injury, substantial evidence indicates that the endometrium contains its own intrinsic stem cell subpopulation, which contributes to its cyclic regeneration. Furthermore, exogenously transplanted MSCs can survive locally, promote angiogenesis, and facilitate tissue remodeling, thereby supporting the regeneration of damaged endometrial tissue [ 18 ] . Preclinical studies and preliminary clinical trials have provided encouraging evidence regarding the efficacy of stem cell therapy for endometrial repair. In animal models, the transplantation of bone marrow- and umbilical cord-derived MSCs has been shown to migrate partially into the endometrium and differentiate into endometrial-like tissue, effectively promoting the regeneration of both stromal and epithelial compartments [ 8 , 18 , 19 ] . Clinically, small-scale trials have reported promising outcomes. In 2016, menstrual blood-derived stem cells were isolated from seven patients and autologously re-transferred into the uterus; five patients exhibited an increase in endometrial thickness to 7 mm, with two achieving successful pregnancies and one spontaneously conceiving following a second transplantation [ 20 ] . In 2020, adipose-derived MSCs were utilized in six patients with severe (IUA; five underwent subsequent embryo transfer, resulting in one pregnancy that unfortunately ended in a natural miscarriage at nine weeks [ 11 ] . In 2021, autologous umbilical cord-derived MSCs were transplanted into the uterine cavities of 16 patients with severe endometrial injury, leading to successful pregnancies and deliveries in multiple cases among 13 patients with severe IUA, with a total of 14 healthy infants born [ 7 ] . In 2024, a study involving 72 patients with intrauterine adhesions (IUA) demonstrated that the intrauterine transplantation of autologous bone marrow stem cells significantly increased endometrial thickness after six menstrual cycles, with no notable adverse reactions observed [ 21 ] . These findings suggest that mesenchymal stem cell (MSC) transplantation holds promise as a revolutionary approach for treating intrauterine adhesions and restoring fertility. However, current stem cell therapies for IUA remain largely at the preclinical stage or involve limited small-sample clinical trials, and long-term data on efficacy and safety are still lacking. More randomized controlled trials are needed to validate these preliminary results. Based on this background, the present study aims to systematically compare the regenerative potential and therapeutic efficacy of MSCs derived from different sources for endometrial repair. Initially, we will assess the in vitro differentiation potential of decidual, umbilical cord, bone marrow, and adipose-derived MSCs into endometrial cells. This will be accomplished by evaluating the expression of the endometrial epithelial marker CK18 and the mesenchymal marker VIM through immunofluorescence and Western blot analyses. Subsequently, we will compare the effects of MSC transplantation in a rat model of intrauterine adhesion by monitoring the expression changes of CK18, VIM, and the functional endometrial marker E-cadherin, thereby assessing both tissue regeneration and functional restoration. Unlike previous studies that focused on a single MSC source, our approach of comparing multiple sources on a single platform is unprecedented and highly innovative. The anticipated outcomes are expected to provide robust experimental evidence for cell-based therapies in endometrial repair and help identify the optimal MSC source for future clinical translation. 2. Method This work was conducted and reported in compliance with the ARRIVE Guidelines 2.0. 2.1 Sources and Culture of Cells Decidual mesenchymal stem cells (DMSCs) and adipose-derived mesenchymal stem cells (AD-MSCs) were generously provided by the Shenyang Cell Engineering Technology Research & Development Center Co., Ltd. Umbilical cord mesenchymal stem cells (UC-MSCs) and bone marrow mesenchymal stem cells (BMSCs) were kindly donated by Professor Huanan Wang’s team at Dalian University of Technology. Human DMSCs and AD-MSCs were cultured in DMEM (Servicebio) supplemented with 10% fetal bovine serum (FBS, Beyotime) and 1% penicillin G/streptomycin (KeyGEN BioTECH). Cells were passaged when confluence exceeded 90%. Human UC-MSCs and BMSCs were maintained in α-MEM (Servicebio) containing 10% FBS and 1% penicillin G/streptomycin, while DMSCs and AD-MSCs were alternatively cultured in DMEM/F12 (Servicebio) with the same supplements. Endometrial epithelial cells, procured from iCell Bioscience Inc. (Shanghai, China), were grown in DMEM/F12 supplemented with 10% FBS and 1% penicillin G/streptomycin. All cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂, and cells were passaged upon reaching 80–90% confluence. 2.2 Cell Proliferation Assay In the CCK-8 assay, cells were seeded at a density of 2,000 cells per well in 96-well plates. A mixture of 110 µL per well, consisting of CCK-8 reagent (C0005; Targetmol) and the corresponding culture medium in a 1:10 ratio, was added. The plates were then incubated at 37°C for 2 hours, and the absorbance was measured at 450 nm using a microplate reader at 0, 24, 48, 72, and 96 hours. 2.3 In Vitro Differentiation Induction An in vitro co-culture model was established using a Transwell system (TCS002006; Jet). Initially, endometrial epithelial cells were seeded in the upper chamber of the Transwell insert, while Passage 3 MSCs from four different sources were plated in the lower chamber. After 24 hours of co-culture under standard conditions, a differentiation induction medium was added to the lower chamber containing MSCs. The induction medium comprised 10% FBS, 1% penicillin G/streptomycin, transforming growth factor-β (TGF-β, HY-P7118; MCE) at a concentration of 20 ng/mL, epidermal growth factor (EGF, HY-P7109; MCE) at 20 ng/mL, platelet-derived growth factor-BB (PDGF-BB, HY-P7055; MCE) at 20 ng/mL, and estradiol (E2, HY-P71085; MCE) at 10 nM. The culture medium was refreshed every three days throughout the induction process. After seven days, differentiation efficacy was assessed by evaluating the expression levels of the epithelial marker CK18 and the stem cell marker VIM using immunofluorescence staining and Western blot analysis. 2.4 Immunofluorescence Staining Cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes, followed by blocking with 5% BSA for 30 minutes. Primary antibodies—anti-Vimentin (10366-1-AP, dilution 1:500; Proteintech) and anti-CK18 (66187-1-lg, dilution 1:500; Proteintech)—were applied and incubated overnight at 4°C. Subsequently, cells were incubated with secondary antibodies at a dilution of 1:500: CoraLite488-conjugated goat anti-rabbit IgG (SA00013-2) and CoraLite594-conjugated goat anti-mouse IgG (SA00013-3; Proteintech). Finally, cell nuclei were counterstained with DAPI (C1005; Beyotime) for 15 minutes at room temperature. After mounting the slides, cells were examined under a fluorescence microscope. 2.5 In Vivo Experimental Validation All rats (n = 36) experimental protocols were approved by the Ethics Committee of Dalian University of Technology (Approval No.: DUTSBE250305-01). Eight-week-old female Sprague-Dawley rats (200–220 g) were procured from the Animal Experiment Center of Dalian Medical University and housed under controlled conditions (22°C, 12-hour light/dark cycle). All rats were anaesthetized with 4% − 5% isoflurane(SHAOYI BIO), and then the concentration was adjusted to 1% − 2% to maintain the anaesthetic depth. A midline incision was performed along the lower abdomen to expose the bilateral uterine horns. The control group(n = 6) underwent open and closed abdominal surgery.All the rats except control group were infused with alcohol in the uterine cavity to induce injury. Seven days later, rats with intrauterine adhesions (IUA) were randomly divided into 5 groups༈n = 6 /group༉and treated with intrauterine infusion of MSCs from different sources, with each animal receiving 2 × 10⁶ cells,the IUA group underwent only incision and closure. Following cell injection, the rats were maintained under standard conditions and monitored regularly for overall health. Seven days post-treatment, uterine tissues were harvested for hematoxylin and eosin (HE) staining and Western blot analysis. The expression levels of cytokeratin-18 (CK18) and the functional marker E-cadherin in endometrial epithelial cells were evaluated to assess the regenerative efficacy of the various MSCs in repairing the injured endometrium.Upon completion of the experiment, euthanasia of the SD rats was performed using isoflurane in accordance with the guidelines of the American Veterinary Medical Association (AVMA). The rats were first exposed to a 5% isoflurane concentration in an induction chamber until unconsciousness was achieved, as indicated by the absence of a pedal withdrawal reflex. Subsequently, the rats were transferred to a euthanasia chamber where a 100% concentration of isoflurane was administered until cessation of respiration and cardiac activity was confirmed. This method ensures a rapid and humane transition to unconsciousness and death, minimizing distress to the animals.(Fig. 1 ) 2.6 Western Blot Analysis Total proteins were extracted from tissues and cells using RIPA lysis buffer (P0013B; Beyotime), supplemented with protease inhibitors (C0001; Targetmol) at a 100:1 ratio. Following centrifugation at 15,000 g for 30 minutes at 4°C, the supernatant was collected. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (P0010S; Beyotime). Protein samples were denatured by heating at 100°C for 7 minutes, then separated by 10% SDS-PAGE (20325ES62; Yeasen) and transferred onto 0.45 µm PVDF membranes (IPVH00010; Merck Millipore). Membranes were blocked in 5% nonfat milk for 1 hour and subsequently incubated overnight at 4°C with the following primary antibodies: Vimentin (10366-1-AP, 1:30,000; Proteintech), CK18 (bs-2043R, 1:1000; Bioss), E-cadherin (60335-1-Ig, 1:4000; Proteintech), and GAPDH (60004-1-Ig, 1:300,000; Proteintech). After washing, membranes were incubated at room temperature for 1 hour with HRP-conjugated secondary antibodies: goat anti-rabbit IgG (YP848537, 1:4000; UpingBio) and goat anti-mouse IgG (BA1050, 1:8000; BOSTER). Protein bands were visualized using a BIO-RAD imaging system and quantified with ImageJ software. 2.7 Hematoxylin and Eosin (HE) Staining Uterine specimens collected at various time points were fixed in 4% paraformaldehyde and subsequently sectioned. HE staining was conducted according to the manufacturer's instructions provided by Solarbio to evaluate endometrial thickness. The stained images were analyzed using ImageJ software. 2.8 Statistical Analysis Statistical analyses were performed using GraphPad Prism 9. Data are presented as the mean ± standard deviation (SD) derived from a minimum of three independent experiments. Group comparisons were conducted using one-way and two-way analysis of variance (ANOVA), with GAPDH serving as the internal control. A p-value of less than 0.05 was deemed statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). 3. Results 3.1 Comparison of Proliferative Capacity Among Different MSCs derived from various sources, we employed the Cell Counting Kit-8 (CCK-8) assay to quantitatively compare bone marrow MSCs (BMSCs), umbilical cord hematopoietic stem cells (UC-HSCs), decidual MSCs (DMSCs), and adipose-derived MSCs (AD-MSCs) at passages 1, 3, and 5. The results demonstrated that, among the three passages, BMSCs consistently exhibited the highest proliferation rates, followed by UC-HSCs, while AD-MSCs displayed the lowest proliferative capacity. Notably, cells at Passage 3 from all four MSC types exhibited optimal proliferation rates (Figure 2), and thus, Passage 3 cells were utilized for subsequent experiments. 3.2 In Vitro Differentiation into Endometrial Epithelial Cells To investigate the differentiation potential of the four MSC populations, we conducted both Western blot (WB) and immunofluorescence (IF) analyses. Prior to induction, all MSCs expressed vimentin (Vim) and lacked the epithelial marker cytokeratin-18 (CK18), confirming their mesenchymal phenotype. Following induction, all MSC types began to express both CK18 and Vim; however, the expression levels varied among the groups (Figure 3). Quantitative WB analysis corroborated the IF findings, indicating that all MSCs differentiated to some extent towards an epithelial phenotype. Notably, DMSCs exhibited the most pronounced increase in CK18 expression, suggesting that they possess the strongest potential to differentiate into endometrial epithelial-like cells compared to the other MSC sources. 3.3 In Vivo Evaluation of Endometrial Repair in an IUA Rat Model To evaluate the therapeutic potential of four types of mesenchymal stem cells (MSCs) for repairing intrauterine adhesions (IUA) in vivo, we transplanted BMSCs, UC-HSCs, dMSCs, and AD-MSCs into the intrauterine cavity of rats with experimentally induced IUA. Before MSC treatment, the rat endometrium displayed severe damage, characterized by a significant loss of epithelial cells (Figure 4a). Western blot analysis of the tissue confirmed markedly reduced expression levels of the endometrial epithelial marker CK18 and the epithelial function markers E-cadherin and vimentin, validating the successful establishment of the injury model (Figure 4c). After two weeks of treatment, varying degrees of endometrial repair were observed among the different MSC groups. Hematoxylin and eosin (HE) staining revealed the regeneration of epithelial cells and partial recovery of glands (Figure 4b), while Western blot analysis indicated enhanced expression of CK18 and E-cadherin. Notably, the dMSC group demonstrated the most significant improvement, with substantially higher expression levels of CK18 and E-cadherin compared to the BMSC, UC-HSC, and AD-MSC groups (Figure 4d). However, likely due to the limited retention efficiency of intrauterine cell infusion, none of the MSC treatments fully restored the endometrium to its pre-injury state. 4. Discussion 4.1 Proliferative Capacity and Senescence Characteristics of MSCs from Different Sources The proliferative capacity of stem cells is a critical determinant of their viability and regenerative potential. Our findings indicate that bone marrow-derived mesenchymal stem cells (BMSCs) exhibit significantly higher proliferative activity compared to adipose-derived stem cells (ADSCs). This disparity can be attributed to the distinct physiological microenvironments and molecular signaling pathways that these cells inhabit. BMSCs reside in a hematopoietic niche enriched with growth factors such as interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2). These factors activate the PI3K/Akt and Wnt/β-catenin pathways, leading to the upregulation of Cyclin D1 and sustaining elevated levels of cellular proliferation [ 22 – 25 ] . In contrast, the lipid-rich environment surrounding ADSCs may facilitate the accumulation of reactive oxygen species (ROS) and activate the p38 MAPK pathway, which suppresses the expression of genes associated with proliferation, thereby limiting their growth potential [ 24 , 26 – 28 ] . Furthermore, BMSCs exhibit markedly higher telomerase activity than ADSCs. Research has shown that the telomere length of BMSCs is approximately 8.5 kb, compared to only 6.2 kb in ADSCs, which likely underpins the superior long-term proliferative capacity of BMSCs [ 28 , 29 ] . In terms of passage dependency, Passage 3 (P3) MSCs demonstrate optimal proliferative activity, with an average telomere length of 7.9 kb and osteogenic/adipogenic differentiation rates maintained at ≥ 85%. This reflects an ideal functional balance [ 29 ] . By Passage 5 (P5), telomere shortening to ≤ 6.0 kb and an increased percentage of senescence-associated β-galactosidase (SA-β-gal)–positive cells indicate significant replicative senescence [ 29 , 30 ] . Therefore, P3 MSCs were selected for subsequent experiments to ensure optimal biological properties. 4.2 Tissue Specificity and Differentiation Potential of MSCs Mesenchymal stem cells (MSCs) derived from various tissues exhibit distinct behaviors in endometrial repair. Bone marrow-derived MSCs (BMSCs) predominantly differentiate into osteogenic and chondrogenic lineages, playing key roles in fracture repair and osteoporosis treatment through pathways such as Wnt4/HOXA10 and Runx2 [ 31 ] . Adipose-derived MSCs (AD-MSCs), characterized by their robust adipogenic potential mediated by PPARγ and C/EBPα, are particularly suited for applications in fat grafting and anti-aging therapies, although their chondrogenic capacity remains limited [ 32 – 35 ] . In contrast, decidual MSCs (DMSCs) demonstrate an enhanced ability to differentiate into endometrial epithelial cells. This superior epithelial differentiation potential is likely associated with their high expression of uterine-specific genes, including CD146, WNT4, and HOXA10, which regulate endometrial cell fate and facilitate the transition to an epithelial phenotype [ 36 , 37 ] . The WNT4 pathway, in particular, is critical for MSC-mediated epithelial differentiation, as it activates β-catenin, which subsequently upregulates the epithelial adhesion molecule E-cadherin, thereby stabilizing the epithelial phenotype [ 38 , 39 ] . Furthermore, epigenetic regulation through microRNAs (miRNAs) further enhances the differentiation potential of endometrium-derived MSCs. For instance, miR-214-5p has been shown to mitigate fibrosis by inhibiting the TGF-β/Smad3 pathway and promoting epithelial differentiation [ 40 , 41 ] . Meanwhile, miR-21-5p upregulates FOXA2 and SOX17, thereby enhancing the propensity of MSCs to differentiate into endometrial epithelial cells. These specific miRNA-mediated pathways confer DMSCs with a distinct advantage in the uterine microenvironment, translating into enhanced regenerative capacity. Additionally, endometrium-derived MSCs can recruit endogenous stem cells via the CXCL12/SDF-1 axis, secrete TGF-β1 and IGF-1 to activate STAT3 and Notch signaling, and utilize MMP-9 to degrade excess collagen (types I/III), thereby optimizing the extracellular matrix structure, reducing fibrosis, and further promoting epithelial cell adhesion and proliferation [ 40 , 42 , 43 ] . Collectively, DMSCs possess a superior capacity for endometrial repair compared to MSCs from other tissues, positioning them as promising seed cells for the regeneration of damaged endometrium in regenerative medicine and precision therapies. 4.3 Mechanisms Underlying MSC-Mediated IUA Repair Our in vivo experiments conducted using the IUA rat model further corroborate the reparative potential of mesenchymal stem cells (MSCs), particularly those derived from the decidua. The group treated with DMSCs exhibited a significant increase in endometrial thickness. Mechanistic analyses indicate that DMSCs promote endometrial repair through several pathways. MSCs secrete vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1), thereby ameliorating local ischemia [ 44 ] . Their elevated expression of matrix metalloproteinase-9 (MMP-9) facilitates the degradation of excessive collagen deposition [ 45 – 47 ] . Furthermore, MSCs downregulate the TGF-β1/Smad3 signaling pathway, inhibiting fibroblast activation and subsequent fibrosis formation [ 48 – 51 ] . Additionally, MSCs modulate the uterine microenvironment via indoleamine 2,3-dioxygenase (IDO)-mediated tryptophan depletion and prostaglandin E2 (PGE2) secretion, which suppress Th17 cells and promote the expansion of regulatory T cells (Tregs). Notably, DMSC treatment significantly upregulates the expression of E-cadherin, a marker of endometrial receptivity, further confirming their potential in restoring endometrial function. 5. Challenges in Clinical Translation and Future Directions To enhance the retention of MSCs, the combination of these cells with biomaterials represents a promising strategy. Studies have demonstrated that hydrogels, scaffolds, and nanocarriers can significantly prolong the local retention time of MSCs, thereby amplifying their therapeutic benefits [ 52 ] . For example, research teams from Pohang University of Science and Technology and Pochon Chinese Medical University developed a hydrogel based on uterine-derived decellularized extracellular matrix (UdECM), which successfully induced endometrial regeneration and restored thickness in animal models, thus creating a favorable environment for embryo implantation [ 53 ] . Furthermore, the National Center for Nanoscience and Technology in China, under the leadership of Professor Chunying Chen, has reviewed the potential applications of hydrogels in treating female reproductive disorders, highlighting the promising translational prospects of hydrogel-based strategies [ 52 , 53 ] . Additionally, strategies such as magnetic targeting and genetic modification could further enhance the homing ability of DMSCs to the uterine cavity. Techniques like CRISPR-Cas9 and mRNA delivery to upregulate HOXA10 or GATA3 have been shown to significantly boost the reparative efficacy of DMSCs [ 54 ] . These approaches not only improve cellular targeting and therapeutic outcomes but also minimize potential side effects in non-target tissues. Future clinical trials will require extensive data to evaluate the long-term safety and efficacy of MSCs in patients with intrauterine adhesions. It is advisable to design randomized controlled trials (RCTs) that incorporate varying cell dosages, different carrier delivery methods, and extended follow-up periods to comprehensively assess the clinical utility of MSC-based therapies. For instance, a prospective, non-controlled Phase I clinical trial conducted at Drum Tower Hospital, Nanjing University, demonstrated that umbilical cord-derived MSCs loaded onto collagen scaffolds exhibited favorable safety profiles and some degree of efficacy in treating IUA; however, the study was limited by a small sample size and the absence of a control group [ 55 ] . Future research should investigate the synergistic application of MSCs with other technologies to optimize treatment protocols for intrauterine adhesions and related endometrial injuries. 6. Conclusion Our study demonstrates that decidual mesenchymal stem cells (DMSCs) exhibit the most robust in vitro differentiation capacity and in vivo reparative effects on damaged endometrium among the various MSC sources tested. DMSCs appear to facilitate endometrial regeneration through multiple mechanisms, including angiogenesis, anti-fibrosis, immunomodulation, and the enhancement of reproductive outcomes. These findings provide a solid theoretical foundation for the clinical application of DMSCs in treating intrauterine adhesions and other diseases related to endometrial injury. Further investigations into the underlying mechanisms and the conduct of well-designed clinical trials are warranted to optimize DMSC-based therapies and advance their clinical translation. Declarations Acknowledgements We are grateful to Professor Huanan Wang and his team from Dalian University of Technology for their support and generous gift. We also thank the Shenyang Cell Engineering Technology Research and Development Center Co., Ltd. for their kind gift.The authors declare that they did not use AI-generated works in this paper. Funding This study was supported by the Dalian Municipal Guiding Program in the Field of Life and Health (Approval No. 2024ZDJH01PT038). Authors' contributions Xiaochuan Yu: performed most of the experiments and analysis,Writing – original draft, Writing – review & editing. Li juan Shi: performed most of the experiments and analysis, Writing – review & editing. Yating Zhang: Writing – review & editing. Huali Wang: Writing – review & editing. Ethics approval and consent to participate Name of project： Dalian Municipal Guiding Program in the Field of Life and Health (Approval No. 2024ZDJH01PT038).The animal study protocol was approved by the Animal Care and Use Committee (ACUC) of the Committee on Bioethics and Medical Ethics of Dalian University of Technology, protocol number Approval No.: DUTSBE250305-01. The study adhered to the guidelines set by the committee. The Institutional Animal Ethics Committee of Committee on Bioethics and Medical Ethics of Dalian University approved all the animal experiments.Date of approval：2025.01.04. All MSCs used in the experiments were provided by Shenyang Cell Engineering Technology Co., Ltd. and Dalian University of Technology. Ethical approval was obtained for the original sourcing of these cells, and all donors had provided written informed consent. Consent for publication Not applicable Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials All data generated or analyzed during this study are included in this article and its supplementary information files. References Dreisler, E. and J.J. Kjer, Asherman's syndrome: current perspectives on diagnosis and management. Int J Womens Health, 2019. 11 : p. 191-198. Singh, N., B. Shekhar, S. Mohanty, S. Kumar, T. SethB. Girish, Autologous Bone Marrow-Derived Stem Cell Therapy for Asherman's Syndrome and Endometrial Atrophy: A 5-Year Follow-up Study. J Hum Reprod Sci, 2020. 13 (1): p. 31-37. Santamaria, X., K. IsaacsonC. Simón, Asherman's Syndrome: it may not be all our fault. Hum Reprod, 2018. 33 (8): p. 1374-1380. Sevinç, F., Z.A. Oskovi-Kaplan, Ş. Çelen, D. Ozturk AtanH.O. 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Tissue Eng Part A, 2015. 21 (1-2): p. 353-61. Gaafar, T., O. Osman, A. Osman, W. Attia, H. HamzaR. El Hawary, Gene expression profiling of endometrium versus bone marrow-derived mesenchymal stem cells: upregulation of cytokine genes. Mol Cell Biochem, 2014. 395 (1-2): p. 29-43. Žukauskaitė, D., A. Vitkevičienė, A. Žlibinaitė, R. Baušytė, D. RamašauskaitėR. Navakauskienė, Histone H4 hyperacetylation but not DNA methylation regulates the expression of decidualization-associated genes during induced human endometrial stromal cells decidualization. Int J Biochem Cell Biol, 2023. 156 : p. 106362. Luo, X., R. Yang, Y. Bai, L. Li, N. Lin, L. Sun, et al., Binding of microRNA-135a (miR-135a) to homeobox protein A10 (HOXA10) mRNA in a high-progesterone environment modulates the embryonic implantation factors beta3-integrin (ITGβ3) and empty spiracles homeobox-2 (EMX2). Ann Transl Med, 2021. 9 (8): p. 662. Majid, Q.A., B.R. Ghimire, B. Merkely, A.M. Randi, S.E. Harding, V. Talman, et al., Generation and characterisation of scalable and stable human pluripotent stem cell-derived microvascular-like endothelial cells for cardiac applications. Angiogenesis, 2024. 27 (3): p. 561-582. Shan, L., F. Wang, D. Zhai, X. Meng, J. LiuX. Lv, Matrix metalloproteinases induce extracellular matrix degradation through various pathways to alleviate hepatic fibrosis. Biomed Pharmacother, 2023. 161 : p. 114472. Wang, B., J. Wei, L. Meng, H. Wang, C. Qu, X. Chen, et al., Advances in pathogenic mechanisms and management of radiation-induced fibrosis. Biomed Pharmacother, 2020. 121 : p. 109560. Yang, Q., W. Chen, D. Han, C. Zhang, Y. Xie, X. Sun, et al., Intratunical injection of human urine-derived stem cells derived exosomes prevents fibrosis and improves erectile function in a rat model of Peyronie's disease. Andrologia, 2020. 52 (11): p. e13831. Elhady, S.S., M.S. Goda, E.T. Mehanna, N.M. El-Sayed, R.M. Hazem, M.A. Elfaky, et al., Ziziphus spina-christi L. extract attenuates bleomycin-induced lung fibrosis in mice via regulating TGF-β1/SMAD pathway: LC-MS/MS Metabolic profiling, chemical composition, and histology studies. Biomed Pharmacother, 2024. 176 : p. 116823. Zhuang, Q., R. Ma, Y. Yin, T. Lan, M. YuY. Ming, Mesenchymal Stem Cells in Renal Fibrosis: The Flame of Cytotherapy. Stem Cells Int, 2019. 2019 : p. 8387350. Hu, C., L. Zhao, J. DuanL. Li, Strategies to improve the efficiency of mesenchymal stem cell transplantation for reversal of liver fibrosis. J Cell Mol Med, 2019. 23 (3): p. 1657-1670. Abudukeyoumu, A., M.Q. LiF. Xie, Transforming growth factor-β1 in intrauterine adhesion. Am J Reprod Immunol, 2020. 84 (2): p. e13262. López-Martínez, S., A. Rodríguez-Eguren, L. de Miguel-Gómez, E. Francés-Herrero, A. Faus, A. Díaz, et al., Bioengineered endometrial hydrogels with growth factors promote tissue regeneration and restore fertility in murine models. 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Supplementary Files AuthorChecklistFull.docx Fig3allColorimetric.tif Fig3ck18Chemiluminescence.tif Fig3ck18Colorimetric.tif Fig3ck18.tif Fig3gapdhChemiluminescence.tif Fig3gapdhColorimetric.tif Fig3gapdh.tif Fig3vimChemiluminescence.tif Fig3vimColorimetric.tif Fig3vim.tif Fig4dCK18Chemiluminescence.tif Fig4dCK18Colorimetric1.tif Fig4dCK18Colorimetric2.tif Fig4dCK18.tif Fig4dEcadherinChemiluminescence.tif Fig4dEcadherinColorimetric.tif Fig4dEcadherin.tif Fig4dallColorimetric.tif Fig4dgapdhChemiluminescence.tif Fig4dgapdhColorimetric.tif Fig4dgapdh.tif fig4cCK18.tif fig4cEcadherinChemiluminescence.tif fig4cEcadherinColorimetric.tif fig4cEcadherin.tif fig4cck18Chemiluminescence.tif fig4cgapdhChemiluminescence.tif fig4cgapdhColorimetric.tif fig4cgapdh.tif fig4cck18Colorimetric.tif Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-6286920","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441472860,"identity":"240b3923-37bd-4c03-8a69-f3d8012bc454","order_by":0,"name":"Xiaochuan Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3PMWvCQBTA8QuBdHlya0L8EBcEiyDxq9xxkOnI4pLxQOhUnOO3cOr8QqAuabMKLk6dMiSbS4tG3GxP3Trcn1sO7vceR4jN9g9jSMjpEN5fUPxMgVJ9P3H2rZcMgxzNJLiM7Ikb5V45ZZqbCfVVhIcsTtnm4z0EqIERdNpO/U08P+HFayXnrEqTEPwdPLvaDVZvJiIRBy8o1qjGIbAdTDR67sBIhC6+e1I3J8I/gSG/RSSW5y1bNYpyxDsIfPFyWEmx2jbRvtMSgrxYmP/ypEZdk8ViWSuGQsczShdF2xnIbzn6sfc2m81mu+oIs4FY7Se3ks4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0009-3223-3363","institution":"Dalian Medical College: Dalian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaochuan","middleName":"","lastName":"Yu","suffix":""},{"id":441472861,"identity":"dbd8336a-4a7b-47c0-af1c-cdc33da02802","order_by":1,"name":"Li juan Shi","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"juan","lastName":"Shi","suffix":""},{"id":441472862,"identity":"77f2455b-16a0-4f18-a4a3-8b0a4d95611f","order_by":2,"name":"Yating Zhang","email":"","orcid":"","institution":"Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yating","middleName":"","lastName":"Zhang","suffix":""},{"id":441472863,"identity":"b9f6f38a-8e57-4799-a801-1177e80c6ff2","order_by":3,"name":"Huali Wang","email":"","orcid":"https://orcid.org/0000-0002-5579-3592","institution":"Dalian Hospital of Obstetrics and Gynaecology","correspondingAuthor":false,"prefix":"","firstName":"Huali","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-03-23 07:21:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6286920/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6286920/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80815007,"identity":"c03cbcca-c0df-45b2-8214-025312254a1f","added_by":"auto","created_at":"2025-04-17 10:54:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":446172,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic Diagram of the Animal Experiment\u003c/p\u003e","description":"","filename":"animalfig.png","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/6d9b3d6f018c1bf961bb99d1.png"},{"id":80814163,"identity":"0b53c534-d0c7-4964-8123-b556dc6198b9","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3343425,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Proliferative Capacity Among Different MSCs.\u003cbr\u003e\n(a) Growth curve of Passage 1 MSCs; (b) Growth curve of Passage 3 MSCs; (c) Growth curve of Passage 5 MSCs.\u003c/p\u003e","description":"","filename":"Fif3.png","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/20851d458c8038a5d7fbfb91.png"},{"id":80814160,"identity":"d4789248-2c52-4d8e-9c3e-aac9c408a098","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn Vitro Differentiation Capacity of Different MSCs.\u003c/strong\u003e\u003cbr\u003e\n(a) Immunofluorescence analysis of CK18 and Vimentin expression in MSCs following induction (scale bar = 100 μm).\u003cbr\u003e\n(b) Western blot analysis of CK18 and Vimentin protein levels post-induction. Quantitative data, normalized to GAPDH, are presented as mean ± SD (n = 3) and were analyzed using one-way ANOVA (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/555553ecb4c312152e327b12.png"},{"id":80814177,"identity":"553ce461-806b-42d6-96e4-fe9e8928b58f","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2020247,"visible":true,"origin":"","legend":"\u003cp\u003eillustrates the in vivo effects of stem cell therapy on IUA.\u003c/p\u003e\n\u003cp\u003e(a) Hematoxylin and eosin (H\u0026amp;E) staining of endometrial sections from rats with endometrial injury. (b) H\u0026amp;E staining of endometrial sections from rats treated with mesenchymal stem cells (MSCs) at various time points. Black arrows indicate endometrial glands.(c) shows the protein expression levels of CK18 and E-cadherin in each group prior to treatment;(d) presents the protein expression levels of CK18 and E-cadherin in each group following stem cell therapy. Quantitative data, normalized to GAPDH, are expressed as mean ± standard deviation (SD) with n = 3. 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10:54:48","extension":"tif","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dCK18Colorimetric1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/d3202bfccb1721367442e1ba.tif"},{"id":80815009,"identity":"8f286782-5e6c-4f0d-b174-8e812e2584d2","added_by":"auto","created_at":"2025-04-17 10:54:48","extension":"tif","order_by":21,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dCK18Colorimetric2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/8a5c38445cbf5f95d2db8a3c.tif"},{"id":80814172,"identity":"fbba03f8-6f05-4d29-ae1f-6ef7969a6920","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"tif","order_by":22,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dCK18.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/b27ae3e94fdc078b306783ea.tif"},{"id":80814189,"identity":"eb0c1926-d15f-4424-b312-397b76e8637c","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"tif","order_by":23,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dEcadherinChemiluminescence.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/cd42f7c5037b4ed40ac4f39f.tif"},{"id":80814207,"identity":"f3b86c93-8480-470f-8ddf-94203670c91c","added_by":"auto","created_at":"2025-04-17 10:46:49","extension":"tif","order_by":24,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dEcadherinColorimetric.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/b067ab20fa53bf5a3dbdfc2f.tif"},{"id":80814181,"identity":"dcfdbf8a-15f0-4812-9c90-9a2ad7e78aca","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"tif","order_by":25,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dEcadherin.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/da53a652ee684ef1a432a61e.tif"},{"id":80814193,"identity":"e5b03144-9937-4be4-b87b-b79f8d35de8f","added_by":"auto","created_at":"2025-04-17 10:46:49","extension":"tif","order_by":26,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dallColorimetric.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/1af22c733ebb04550e111e06.tif"},{"id":80815008,"identity":"e51796b1-9c21-4eba-8a41-a5ddd8fd9f1a","added_by":"auto","created_at":"2025-04-17 10:54:48","extension":"tif","order_by":27,"title":"","display":"","copyAsset":false,"role":"supplement","size":3022872,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dgapdhChemiluminescence.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/57a8ea30365b63daad592427.tif"},{"id":80815726,"identity":"d7b99d66-6985-483c-b0bc-9c39e78f017a","added_by":"auto","created_at":"2025-04-17 11:02:48","extension":"tif","order_by":28,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dgapdhColorimetric.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/894dffc70b12d9455e37a393.tif"},{"id":80815011,"identity":"f0637d50-0ba7-4e7f-9d00-a8d8505a1f06","added_by":"auto","created_at":"2025-04-17 10:54:48","extension":"tif","order_by":29,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4dgapdh.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/6fb14748983a75326037062a.tif"},{"id":80815022,"identity":"928310ae-93c0-49ac-9ae2-e0d910dd5a23","added_by":"auto","created_at":"2025-04-17 10:54:49","extension":"tif","order_by":30,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cCK18.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/4db163f752e84a3e75daefc6.tif"},{"id":80814185,"identity":"c59c6875-37f5-46e4-9683-712741b7a048","added_by":"auto","created_at":"2025-04-17 10:46:48","extension":"tif","order_by":31,"title":"","display":"","copyAsset":false,"role":"supplement","size":3022872,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cEcadherinChemiluminescence.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/78794872712e9a75f4e5d646.tif"},{"id":80814203,"identity":"a3869640-5bd8-402b-9299-aa8225ba4c04","added_by":"auto","created_at":"2025-04-17 10:46:49","extension":"tif","order_by":32,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cEcadherinColorimetric.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/e68c3a62e279f7b8e5128402.tif"},{"id":80814195,"identity":"1d37bf77-e2cd-46e4-87de-a325627b0884","added_by":"auto","created_at":"2025-04-17 10:46:49","extension":"tif","order_by":33,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cEcadherin.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/4b55f8e23d4661fbe23e6ad1.tif"},{"id":80815014,"identity":"6b7a6cd4-e803-44a7-a1f3-20967578a0be","added_by":"auto","created_at":"2025-04-17 10:54:49","extension":"tif","order_by":34,"title":"","display":"","copyAsset":false,"role":"supplement","size":3022872,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cck18Chemiluminescence.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/2feacce1839610ff105e843d.tif"},{"id":80815727,"identity":"49981f0d-39c9-434c-98cf-136bcb826dbc","added_by":"auto","created_at":"2025-04-17 11:02:49","extension":"tif","order_by":35,"title":"","display":"","copyAsset":false,"role":"supplement","size":3022872,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cgapdhChemiluminescence.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/c894eb766c619241f2ad7ff6.tif"},{"id":80815018,"identity":"aaf4c4ee-103c-47c2-9158-63fd2caf9a8e","added_by":"auto","created_at":"2025-04-17 10:54:49","extension":"tif","order_by":36,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cgapdhColorimetric.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/1ace1c953d654abf2f4658e2.tif"},{"id":80815020,"identity":"33c90504-7127-4130-8a74-75f2240cab41","added_by":"auto","created_at":"2025-04-17 10:54:49","extension":"tif","order_by":37,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cgapdh.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/8fe1906d0a50a481e0cf01c4.tif"},{"id":80814209,"identity":"19145fc9-b840-482c-89f0-5d61c9923f45","added_by":"auto","created_at":"2025-04-17 10:46:49","extension":"tif","order_by":38,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019506,"visible":true,"origin":"","legend":"","description":"","filename":"fig4cck18Colorimetric.tif","url":"https://assets-eu.researchsquare.com/files/rs-6286920/v1/306f98b7492dc8fbfb6c9c6c.tif"}],"financialInterests":"","formattedTitle":"Revolutionizing Stem Cell Therapy: A Comparative Analysis of Diverse Mesenchymal Stem Cells for Enhanced Endometrial Regeneration","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIntrauterine adhesions (IUA), also known as Asherman's syndrome, represent a fibrotic disorder within the uterine cavity, resulting from damage to and impaired repair of the endometrial basal layer \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. This condition often leads to anatomical abnormalities of the uterine cavity, significantly impacting menstrual physiology and reproductive function in women of childbearing age. The etiology of IUA is frequently associated with intrauterine surgical procedures, including repeated induced abortions, curettage, and hysteroscopic surgeries\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Recent studies indicate that the incidence of IUA resulting from multiple induced abortions and curettage procedures can reach as high as 25\u0026ndash;30%, with prevalence increasing annually as the number of intrauterine interventions rises \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. IUA has emerged as one of the major causes of secondary infertility in women, with reports suggesting that approximately 20\u0026ndash;40% of infertility cases in China are related to endometrial injury. Patients with IUA typically present with markedly reduced menstrual flow or amenorrhea, recurrent miscarriages, and an increased risk of pregnancy-related complications.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Limitations of Conventional Treatments\u003c/h2\u003e \u003cp\u003eCurrent treatment strategies for IUA primarily focus on surgically restoring the normal anatomical structure of the uterine cavity, promoting the regeneration of functional endometrium through pharmacological interventions, and preventing re-adhesion\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Clinically, hysteroscopic adhesiolysis (TCRA) is frequently performed, and intrauterine barriers\u0026mdash;either biological or physical (such as intrauterine devices, uterine balloons, or biomembranes)\u0026mdash;are employed to mitigate the risk of re-adhesion of the wound surface. These interventions are typically supplemented with estrogen or sequential estrogen-progestogen therapy to encourage endometrial growth. However, these conventional methods often fail to yield satisfactory outcomes, with reported recurrence rates for moderate-to-severe IUA reaching 37.3\u0026ndash;43.36% \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The intrauterine placement of biomaterials or physical scaffolds only passively prevents the apposition of wound surfaces and does not actively promote the regeneration of new endometrial tissue, in addition to posing a risk of infection. Furthermore, due to extensive endometrial damage and a reduction in estrogen and progesterone receptors, the efficacy of oral hormone therapy is limited. Reconstructing a fully functional endometrium remains a significant clinical challenge for patients with widespread endometrial injury or severe IUA\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Potential of Stem Cell Therapy\u003c/h2\u003e \u003cp\u003eIn recent years, stem cell therapy has emerged as a promising approach in regenerative medicine, providing new hope for the repair of endometrial damage\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Mesenchymal stem cells (MSCs) have garnered significant attention as seed cells due to their unique biological properties \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. MSCs can be isolated from various sources, including bone marrow, adipose tissue, umbilical cord, decidua, amnion, and placenta. They are characterized by their self-renewal and multilineage differentiation potential, as well as their immunomodulatory and microenvironmental repair capabilities \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Research has demonstrated that MSCs express low levels of MHC-II molecules and lack co-stimulatory molecules, rendering them minimally immunogenic in vivo. Furthermore, they can modulate local immune responses by secreting cytokines that inhibit the activation of T lymphocytes, B lymphocytes, and natural killer cells \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Notably, differences in proliferative activity, differentiation potential, and immunological characteristics exist among MSCs derived from different tissues. For instance, MSCs from the umbilical cord and bone marrow exhibit high proliferative capacity, lower immunogenicity, and stronger immunosuppressive effects, making them suitable for allogeneic transplantation \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Similarly, decidua-derived MSCs are readily obtainable, can be efficiently expanded in culture, and exhibit minimal rejection responses, rendering them ideal seed cells for tissue engineering. Additionally, adipose-derived MSCs, due to their accessibility and the feasibility of autologous transplantation, also show promising clinical prospects \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Progress in the Application of Stem Cell Therapy\u003c/h2\u003e \u003cp\u003eMesenchymal stem cells (MSCs) have been extensively investigated for their potential in tissue repair and regeneration across various models, including myocardial infarction, liver injury, and osteochondral defects\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Concerning endometrial injury, substantial evidence indicates that the endometrium contains its own intrinsic stem cell subpopulation, which contributes to its cyclic regeneration. Furthermore, exogenously transplanted MSCs can survive locally, promote angiogenesis, and facilitate tissue remodeling, thereby supporting the regeneration of damaged endometrial tissue\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Preclinical studies and preliminary clinical trials have provided encouraging evidence regarding the efficacy of stem cell therapy for endometrial repair. In animal models, the transplantation of bone marrow- and umbilical cord-derived MSCs has been shown to migrate partially into the endometrium and differentiate into endometrial-like tissue, effectively promoting the regeneration of both stromal and epithelial compartments\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Clinically, small-scale trials have reported promising outcomes. In 2016, menstrual blood-derived stem cells were isolated from seven patients and autologously re-transferred into the uterus; five patients exhibited an increase in endometrial thickness to 7 mm, with two achieving successful pregnancies and one spontaneously conceiving following a second transplantation\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In 2020, adipose-derived MSCs were utilized in six patients with severe (IUA; five underwent subsequent embryo transfer, resulting in one pregnancy that unfortunately ended in a natural miscarriage at nine weeks \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In 2021, autologous umbilical cord-derived MSCs were transplanted into the uterine cavities of 16 patients with severe endometrial injury, leading to successful pregnancies and deliveries in multiple cases among 13 patients with severe IUA, with a total of 14 healthy infants born \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In 2024, a study involving 72 patients with intrauterine adhesions (IUA) demonstrated that the intrauterine transplantation of autologous bone marrow stem cells significantly increased endometrial thickness after six menstrual cycles, with no notable adverse reactions observed \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that mesenchymal stem cell (MSC) transplantation holds promise as a revolutionary approach for treating intrauterine adhesions and restoring fertility. However, current stem cell therapies for IUA remain largely at the preclinical stage or involve limited small-sample clinical trials, and long-term data on efficacy and safety are still lacking. More randomized controlled trials are needed to validate these preliminary results. Based on this background, the present study aims to systematically compare the regenerative potential and therapeutic efficacy of MSCs derived from different sources for endometrial repair. Initially, we will assess the in vitro differentiation potential of decidual, umbilical cord, bone marrow, and adipose-derived MSCs into endometrial cells. This will be accomplished by evaluating the expression of the endometrial epithelial marker CK18 and the mesenchymal marker VIM through immunofluorescence and Western blot analyses. Subsequently, we will compare the effects of MSC transplantation in a rat model of intrauterine adhesion by monitoring the expression changes of CK18, VIM, and the functional endometrial marker E-cadherin, thereby assessing both tissue regeneration and functional restoration. Unlike previous studies that focused on a single MSC source, our approach of comparing multiple sources on a single platform is unprecedented and highly innovative. The anticipated outcomes are expected to provide robust experimental evidence for cell-based therapies in endometrial repair and help identify the optimal MSC source for future clinical translation.\u003c/p\u003e \u003c/div\u003e"},{"header":"2. Method","content":"\u003cp\u003eThis work was conducted and reported in compliance with the ARRIVE Guidelines 2.0.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sources and Culture of Cells\u003c/h2\u003e \u003cp\u003eDecidual mesenchymal stem cells (DMSCs) and adipose-derived mesenchymal stem cells (AD-MSCs) were generously provided by the Shenyang Cell Engineering Technology Research \u0026amp; Development Center Co., Ltd. Umbilical cord mesenchymal stem cells (UC-MSCs) and bone marrow mesenchymal stem cells (BMSCs) were kindly donated by Professor Huanan Wang\u0026rsquo;s team at Dalian University of Technology. Human DMSCs and AD-MSCs were cultured in DMEM (Servicebio) supplemented with 10% fetal bovine serum (FBS, Beyotime) and 1% penicillin G/streptomycin (KeyGEN BioTECH). Cells were passaged when confluence exceeded 90%. Human UC-MSCs and BMSCs were maintained in α-MEM (Servicebio) containing 10% FBS and 1% penicillin G/streptomycin, while DMSCs and AD-MSCs were alternatively cultured in DMEM/F12 (Servicebio) with the same supplements. Endometrial epithelial cells, procured from iCell Bioscience Inc. (Shanghai, China), were grown in DMEM/F12 supplemented with 10% FBS and 1% penicillin G/streptomycin. All cell cultures were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO₂, and cells were passaged upon reaching 80\u0026ndash;90% confluence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell Proliferation Assay\u003c/h2\u003e \u003cp\u003eIn the CCK-8 assay, cells were seeded at a density of 2,000 cells per well in 96-well plates. A mixture of 110 \u0026micro;L per well, consisting of CCK-8 reagent (C0005; Targetmol) and the corresponding culture medium in a 1:10 ratio, was added. The plates were then incubated at 37\u0026deg;C for 2 hours, and the absorbance was measured at 450 nm using a microplate reader at 0, 24, 48, 72, and 96 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In Vitro Differentiation Induction\u003c/h2\u003e \u003cp\u003eAn in vitro co-culture model was established using a Transwell system (TCS002006; Jet). Initially, endometrial epithelial cells were seeded in the upper chamber of the Transwell insert, while Passage 3 MSCs from four different sources were plated in the lower chamber. After 24 hours of co-culture under standard conditions, a differentiation induction medium was added to the lower chamber containing MSCs. The induction medium comprised 10% FBS, 1% penicillin G/streptomycin, transforming growth factor-β (TGF-β, HY-P7118; MCE) at a concentration of 20 ng/mL, epidermal growth factor (EGF, HY-P7109; MCE) at 20 ng/mL, platelet-derived growth factor-BB (PDGF-BB, HY-P7055; MCE) at 20 ng/mL, and estradiol (E2, HY-P71085; MCE) at 10 nM. The culture medium was refreshed every three days throughout the induction process. After seven days, differentiation efficacy was assessed by evaluating the expression levels of the epithelial marker CK18 and the stem cell marker VIM using immunofluorescence staining and Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunofluorescence Staining\u003c/h2\u003e \u003cp\u003eCells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes, followed by blocking with 5% BSA for 30 minutes. Primary antibodies\u0026mdash;anti-Vimentin (10366-1-AP, dilution 1:500; Proteintech) and anti-CK18 (66187-1-lg, dilution 1:500; Proteintech)\u0026mdash;were applied and incubated overnight at 4\u0026deg;C. Subsequently, cells were incubated with secondary antibodies at a dilution of 1:500: CoraLite488-conjugated goat anti-rabbit IgG (SA00013-2) and CoraLite594-conjugated goat anti-mouse IgG (SA00013-3; Proteintech). Finally, cell nuclei were counterstained with DAPI (C1005; Beyotime) for 15 minutes at room temperature. After mounting the slides, cells were examined under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In Vivo Experimental Validation\u003c/h2\u003e \u003cp\u003e All rats (n\u0026thinsp;=\u0026thinsp;36) experimental protocols were approved by the Ethics Committee of Dalian University of Technology (Approval No.: DUTSBE250305-01). Eight-week-old female Sprague-Dawley rats (200\u0026ndash;220 g) were procured from the Animal Experiment Center of Dalian Medical University and housed under controlled conditions (22\u0026deg;C, 12-hour light/dark cycle). All rats were anaesthetized with 4% \u0026minus;\u0026thinsp;5% isoflurane(SHAOYI BIO), and then the concentration was adjusted to 1% \u0026minus;\u0026thinsp;2% to maintain the anaesthetic depth. A midline incision was performed along the lower abdomen to expose the bilateral uterine horns. The control group(n\u0026thinsp;=\u0026thinsp;6) underwent open and closed abdominal surgery.All the rats except control group were infused with alcohol in the uterine cavity to induce injury. Seven days later, rats with intrauterine adhesions (IUA) were randomly divided into 5 groups༈n\u0026thinsp;=\u0026thinsp;6 /group༉and treated with intrauterine infusion of MSCs from different sources, with each animal receiving 2 \u0026times; 10⁶ cells,the IUA group underwent only incision and closure. Following cell injection, the rats were maintained under standard conditions and monitored regularly for overall health. Seven days post-treatment, uterine tissues were harvested for hematoxylin and eosin (HE) staining and Western blot analysis. The expression levels of cytokeratin-18 (CK18) and the functional marker E-cadherin in endometrial epithelial cells were evaluated to assess the regenerative efficacy of the various MSCs in repairing the injured endometrium.Upon completion of the experiment, euthanasia of the SD rats was performed using isoflurane in accordance with the guidelines of the American Veterinary Medical Association (AVMA). The rats were first exposed to a 5% isoflurane concentration in an induction chamber until unconsciousness was achieved, as indicated by the absence of a pedal withdrawal reflex. Subsequently, the rats were transferred to a euthanasia chamber where a 100% concentration of isoflurane was administered until cessation of respiration and cardiac activity was confirmed. This method ensures a rapid and humane transition to unconsciousness and death, minimizing distress to the animals.(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western Blot Analysis\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted from tissues and cells using RIPA lysis buffer (P0013B; Beyotime), supplemented with protease inhibitors (C0001; Targetmol) at a 100:1 ratio. Following centrifugation at 15,000 g for 30 minutes at 4\u0026deg;C, the supernatant was collected. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (P0010S; Beyotime). Protein samples were denatured by heating at 100\u0026deg;C for 7 minutes, then separated by 10% SDS-PAGE (20325ES62; Yeasen) and transferred onto 0.45 \u0026micro;m PVDF membranes (IPVH00010; Merck Millipore). Membranes were blocked in 5% nonfat milk for 1 hour and subsequently incubated overnight at 4\u0026deg;C with the following primary antibodies: Vimentin (10366-1-AP, 1:30,000; Proteintech), CK18 (bs-2043R, 1:1000; Bioss), E-cadherin (60335-1-Ig, 1:4000; Proteintech), and GAPDH (60004-1-Ig, 1:300,000; Proteintech). After washing, membranes were incubated at room temperature for 1 hour with HRP-conjugated secondary antibodies: goat anti-rabbit IgG (YP848537, 1:4000; UpingBio) and goat anti-mouse IgG (BA1050, 1:8000; BOSTER). Protein bands were visualized using a BIO-RAD imaging system and quantified with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Hematoxylin and Eosin (HE) Staining\u003c/h2\u003e \u003cp\u003eUterine specimens collected at various time points were fixed in 4% paraformaldehyde and subsequently sectioned. HE staining was conducted according to the manufacturer's instructions provided by Solarbio to evaluate endometrial thickness. The stained images were analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) derived from a minimum of three independent experiments. Group comparisons were conducted using one-way and two-way analysis of variance (ANOVA), with GAPDH serving as the internal control. A p-value of less than 0.05 was deemed statistically significant (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Comparison of Proliferative Capacity Among Different MSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ederived from various sources, we employed the Cell Counting Kit-8 (CCK-8) assay to quantitatively compare bone marrow MSCs (BMSCs), umbilical cord hematopoietic stem cells (UC-HSCs), decidual MSCs (DMSCs), and adipose-derived MSCs (AD-MSCs) at passages 1, 3, and 5. The results demonstrated that, among the three passages, BMSCs consistently exhibited the highest proliferation rates, followed by UC-HSCs, while AD-MSCs displayed the lowest proliferative capacity. Notably, cells at Passage 3 from all four MSC types exhibited optimal proliferation rates (Figure 2), and thus, Passage 3 cells were utilized for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 In Vitro Differentiation into Endometrial Epithelial Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the differentiation potential of the four MSC populations, we conducted both Western blot (WB) and immunofluorescence (IF) analyses. Prior to induction, all MSCs expressed vimentin (Vim) and lacked the epithelial marker cytokeratin-18 (CK18), confirming their mesenchymal phenotype. Following induction, all MSC types began to express both CK18 and Vim; however, the expression levels varied among the groups (Figure 3). Quantitative WB analysis corroborated the IF findings, indicating that all MSCs differentiated to some extent towards an epithelial phenotype. Notably, DMSCs exhibited the most pronounced increase in CK18 expression, suggesting that they possess the strongest potential to differentiate into endometrial epithelial-like cells compared to the other MSC sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 In Vivo Evaluation of Endometrial Repair in an IUA Rat Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic potential of four types of mesenchymal stem cells (MSCs) for repairing intrauterine adhesions (IUA) in vivo, we transplanted BMSCs, UC-HSCs, dMSCs, and AD-MSCs into the intrauterine cavity of rats with experimentally induced IUA. Before MSC treatment, the rat endometrium displayed severe damage, characterized by a significant loss of epithelial cells (Figure 4a). Western blot analysis of the tissue confirmed markedly reduced expression levels of the endometrial epithelial marker CK18 and the epithelial function markers E-cadherin and vimentin, validating the successful establishment of the injury model (Figure 4c). After two weeks of treatment, varying degrees of endometrial repair were observed among the different MSC groups. Hematoxylin and eosin (HE) staining revealed the regeneration of epithelial cells and partial recovery of glands (Figure 4b), while Western blot analysis indicated enhanced expression of CK18 and E-cadherin. Notably, the dMSC group demonstrated the most significant improvement, with substantially higher expression levels of CK18 and E-cadherin compared to the BMSC, UC-HSC, and AD-MSC groups (Figure 4d). However, likely due to the limited retention efficiency of intrauterine cell infusion, none of the MSC treatments fully restored the endometrium to its pre-injury state.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Proliferative Capacity and Senescence Characteristics of MSCs from Different Sources\u003c/h2\u003e \u003cp\u003eThe proliferative capacity of stem cells is a critical determinant of their viability and regenerative potential. Our findings indicate that bone marrow-derived mesenchymal stem cells (BMSCs) exhibit significantly higher proliferative activity compared to adipose-derived stem cells (ADSCs). This disparity can be attributed to the distinct physiological microenvironments and molecular signaling pathways that these cells inhabit. BMSCs reside in a hematopoietic niche enriched with growth factors such as interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2). These factors activate the PI3K/Akt and Wnt/β-catenin pathways, leading to the upregulation of Cyclin D1 and sustaining elevated levels of cellular proliferation \u003csup\u003e[\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In contrast, the lipid-rich environment surrounding ADSCs may facilitate the accumulation of reactive oxygen species (ROS) and activate the p38 MAPK pathway, which suppresses the expression of genes associated with proliferation, thereby limiting their growth potential \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, BMSCs exhibit markedly higher telomerase activity than ADSCs. Research has shown that the telomere length of BMSCs is approximately 8.5 kb, compared to only 6.2 kb in ADSCs, which likely underpins the superior long-term proliferative capacity of BMSCs \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In terms of passage dependency, Passage 3 (P3) MSCs demonstrate optimal proliferative activity, with an average telomere length of 7.9 kb and osteogenic/adipogenic differentiation rates maintained at \u0026ge;\u0026thinsp;85%. This reflects an ideal functional balance\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. By Passage 5 (P5), telomere shortening to \u0026le;\u0026thinsp;6.0 kb and an increased percentage of senescence-associated β-galactosidase (SA-β-gal)\u0026ndash;positive cells indicate significant replicative senescence \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Therefore, P3 MSCs were selected for subsequent experiments to ensure optimal biological properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Tissue Specificity and Differentiation Potential of MSCs\u003c/h2\u003e \u003cp\u003eMesenchymal stem cells (MSCs) derived from various tissues exhibit distinct behaviors in endometrial repair. Bone marrow-derived MSCs (BMSCs) predominantly differentiate into osteogenic and chondrogenic lineages, playing key roles in fracture repair and osteoporosis treatment through pathways such as Wnt4/HOXA10 and Runx2 \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Adipose-derived MSCs (AD-MSCs), characterized by their robust adipogenic potential mediated by PPARγ and C/EBPα, are particularly suited for applications in fat grafting and anti-aging therapies, although their chondrogenic capacity remains limited \u003csup\u003e[\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. In contrast, decidual MSCs (DMSCs) demonstrate an enhanced ability to differentiate into endometrial epithelial cells. This superior epithelial differentiation potential is likely associated with their high expression of uterine-specific genes, including CD146, WNT4, and HOXA10, which regulate endometrial cell fate and facilitate the transition to an epithelial phenotype \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. The WNT4 pathway, in particular, is critical for MSC-mediated epithelial differentiation, as it activates β-catenin, which subsequently upregulates the epithelial adhesion molecule E-cadherin, thereby stabilizing the epithelial phenotype \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, epigenetic regulation through microRNAs (miRNAs) further enhances the differentiation potential of endometrium-derived MSCs. For instance, miR-214-5p has been shown to mitigate fibrosis by inhibiting the TGF-β/Smad3 pathway and promoting epithelial differentiation \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, miR-21-5p upregulates FOXA2 and SOX17, thereby enhancing the propensity of MSCs to differentiate into endometrial epithelial cells. These specific miRNA-mediated pathways confer DMSCs with a distinct advantage in the uterine microenvironment, translating into enhanced regenerative capacity. Additionally, endometrium-derived MSCs can recruit endogenous stem cells via the CXCL12/SDF-1 axis, secrete TGF-β1 and IGF-1 to activate STAT3 and Notch signaling, and utilize MMP-9 to degrade excess collagen (types I/III), thereby optimizing the extracellular matrix structure, reducing fibrosis, and further promoting epithelial cell adhesion and proliferation \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Collectively, DMSCs possess a superior capacity for endometrial repair compared to MSCs from other tissues, positioning them as promising seed cells for the regeneration of damaged endometrium in regenerative medicine and precision therapies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Mechanisms Underlying MSC-Mediated IUA Repair\u003c/h2\u003e \u003cp\u003eOur in vivo experiments conducted using the IUA rat model further corroborate the reparative potential of mesenchymal stem cells (MSCs), particularly those derived from the decidua. The group treated with DMSCs exhibited a significant increase in endometrial thickness. Mechanistic analyses indicate that DMSCs promote endometrial repair through several pathways. MSCs secrete vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1), thereby ameliorating local ischemia\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Their elevated expression of matrix metalloproteinase-9 (MMP-9) facilitates the degradation of excessive collagen deposition\u003csup\u003e[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Furthermore, MSCs downregulate the TGF-β1/Smad3 signaling pathway, inhibiting fibroblast activation and subsequent fibrosis formation\u003csup\u003e[\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Additionally, MSCs modulate the uterine microenvironment via indoleamine 2,3-dioxygenase (IDO)-mediated tryptophan depletion and prostaglandin E2 (PGE2) secretion, which suppress Th17 cells and promote the expansion of regulatory T cells (Tregs). Notably, DMSC treatment significantly upregulates the expression of E-cadherin, a marker of endometrial receptivity, further confirming their potential in restoring endometrial function.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Challenges in Clinical Translation and Future Directions","content":"\u003cp\u003eTo enhance the retention of MSCs, the combination of these cells with biomaterials represents a promising strategy. Studies have demonstrated that hydrogels, scaffolds, and nanocarriers can significantly prolong the local retention time of MSCs, thereby amplifying their therapeutic benefits \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. For example, research teams from Pohang University of Science and Technology and Pochon Chinese Medical University developed a hydrogel based on uterine-derived decellularized extracellular matrix (UdECM), which successfully induced endometrial regeneration and restored thickness in animal models, thus creating a favorable environment for embryo implantation \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, the National Center for Nanoscience and Technology in China, under the leadership of Professor Chunying Chen, has reviewed the potential applications of hydrogels in treating female reproductive disorders, highlighting the promising translational prospects of hydrogel-based strategies \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Additionally, strategies such as magnetic targeting and genetic modification could further enhance the homing ability of DMSCs to the uterine cavity. Techniques like CRISPR-Cas9 and mRNA delivery to upregulate HOXA10 or GATA3 have been shown to significantly boost the reparative efficacy of DMSCs \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. These approaches not only improve cellular targeting and therapeutic outcomes but also minimize potential side effects in non-target tissues.\u003c/p\u003e \u003cp\u003eFuture clinical trials will require extensive data to evaluate the long-term safety and efficacy of MSCs in patients with intrauterine adhesions. It is advisable to design randomized controlled trials (RCTs) that incorporate varying cell dosages, different carrier delivery methods, and extended follow-up periods to comprehensively assess the clinical utility of MSC-based therapies. For instance, a prospective, non-controlled Phase I clinical trial conducted at Drum Tower Hospital, Nanjing University, demonstrated that umbilical cord-derived MSCs loaded onto collagen scaffolds exhibited favorable safety profiles and some degree of efficacy in treating IUA; however, the study was limited by a small sample size and the absence of a control group \u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Future research should investigate the synergistic application of MSCs with other technologies to optimize treatment protocols for intrauterine adhesions and related endometrial injuries.\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eOur study demonstrates that decidual mesenchymal stem cells (DMSCs) exhibit the most robust in vitro differentiation capacity and in vivo reparative effects on damaged endometrium among the various MSC sources tested. DMSCs appear to facilitate endometrial regeneration through multiple mechanisms, including angiogenesis, anti-fibrosis, immunomodulation, and the enhancement of reproductive outcomes. These findings provide a solid theoretical foundation for the clinical application of DMSCs in treating intrauterine adhesions and other diseases related to endometrial injury. Further investigations into the underlying mechanisms and the conduct of well-designed clinical trials are warranted to optimize DMSC-based therapies and advance their clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Professor Huanan Wang and his team from Dalian University of Technology for their support and generous gift. We also thank the Shenyang Cell Engineering Technology Research and Development Center Co., Ltd. for their kind gift.The authors declare that they did not use AI-generated works in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Dalian Municipal Guiding Program in the Field of Life and Health (Approval No. 2024ZDJH01PT038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaochuan Yu: performed most of the experiments and analysis,Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Li juan Shi: performed most of the experiments and analysis, Writing \u0026ndash; review \u0026amp; editing. Yating Zhang: Writing \u0026ndash; review \u0026amp; editing. Huali Wang: Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eName of project：\u0026nbsp;Dalian Municipal Guiding Program in the Field of Life and Health (Approval No. 2024ZDJH01PT038).The animal study protocol was approved by the Animal Care and Use Committee (ACUC) of the Committee on Bioethics and Medical Ethics of Dalian University of Technology, protocol number Approval No.: DUTSBE250305-01. The study adhered to the guidelines set by the committee.\u003cbr\u003e\u0026nbsp;The Institutional Animal Ethics Committee of Committee on Bioethics and Medical Ethics of Dalian University approved all the animal experiments.Date of approval：2025.01.04.\u003c/p\u003e\n\u003cp\u003eAll MSCs used in the experiments were provided by Shenyang Cell Engineering Technology Co., Ltd. and Dalian University of Technology. Ethical approval was obtained for the original sourcing of these cells, and all donors had provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDreisler, E. and J.J. 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Yuan, Q. Liu, W. ZhangP. Li, \u003cem\u003eIntrauterine infusion of clinically graded human umbilical cord-derived mesenchymal stem cells for the treatment of poor healing after uterine injury: a phase I clinical trial.\u003c/em\u003e Stem Cell Res Ther, 2022. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 85.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"decidual mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, repair, differentiation, endometrial epithelial cells","lastPublishedDoi":"10.21203/rs.3.rs-6286920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6286920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eEndometrial injury, particularly intrauterine adhesions (Asherman’s syndrome), represents a prevalent condition that significantly compromises female fertility. Current clinical interventions predominantly involve hysteroscopic surgery, followed by the placement of intrauterine barriers and the administration of oral estrogen to facilitate endometrial regeneration. Nevertheless, in patients with severe intrauterine adhesions, postoperative pregnancy rates remain low, ranging from 22.2% to 33.3%. Mesenchymal stem cells (MSCs), owing to their multilineage differentiation potential and tissue repair capabilities, have emerged as promising candidates for the treatment of regenerative disorders. This study aimed to compare the efficacy of MSCs derived from bone marrow, umbilical cord, adipose tissue, and decidua in the repair of damaged endometrium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe proliferation capabilities of decidual MSCs, umbilical cord MSCs, bone marrow MSCs, and adipose-derived MSCs at passages 1, 3, and 5 were evaluated using a CCK8 assay. In vitro, cytokine-induced differentiation was employed to stimulate MSCs, and the expression of epithelial cell surface markers was assessed through immunofluorescence and Western blot analyses to compare their potential for differentiation into endometrial epithelial cells. In vivo, an intrauterine adhesion rat model received MSC infusions, and the restoration of endometrial morphology was subsequently examined and compared across the different treatment groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBone marrow MSCs demonstrated the highest proliferation rate, while adipose-derived MSCs exhibited the lowest. Notably, decidual MSCs displayed a significantly enhanced capacity to differentiate into endometrial epithelial cells compared to MSCs from other sources. Furthermore, in a rat model of intrauterine adhesion, treatment with decidual MSCs resulted in the most pronounced improvement in endometrial repair.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eDecidual MSCs demonstrate superior in vitro differentiation into endometrial epithelial cells and exhibit the most effective in vivo repair of damaged endometrial tissue, potentially mediated by the secretion of various cytokines and immunomodulatory mechanisms. This study provides critical theoretical and experimental evidence supporting the clinical application of decidual MSCs in endometrial repair. Despite certain limitations, such as the absence of clinical validation, decidual MSCs present a promising novel therapeutic strategy for intrauterine adhesions and other conditions related to endometrial injury. Future clinical trials and mechanistic studies are necessary to further validate their therapeutic potential.\u003c/p\u003e","manuscriptTitle":"Revolutionizing Stem Cell Therapy: A Comparative Analysis of Diverse Mesenchymal Stem Cells for Enhanced Endometrial Regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 10:46:43","doi":"10.21203/rs.3.rs-6286920/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a628eb6a-154c-4ff6-ac8c-7caa5bc1a2d9","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-28T11:48:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 10:46:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6286920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6286920","identity":"rs-6286920","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}