Erythrocyte-mimetic oxygen carriers based on enucleated mesenchymal stem cells: A novel strategy for emergency blood substitution

Erythrocyte-mimetic oxygen carriers based on enucleated mesenchymal stem cells: A novel strategy for emergency blood substitution | 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 Erythrocyte-mimetic oxygen carriers based on enucleated mesenchymal stem cells: A novel strategy for emergency blood substitution Xinyi Chen, Zhengrong Zhou, Shuxin Chen, Zihui Zhou, Yu Guo, Jialin Ding, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8578714/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 Acute major hemorrhage triggered by a sudden reduction in blood volume, accompanied by coagulopathy, systemic hypoperfusion, and tissue hypoxia, is likely the underlying cause of death. Shortages in donor blood supplies limit the efficacy of conventional transfusion therapies, leading to heightened interest in blood substitutes as potential alternatives or supplements. To address his limitation, we developed a novel oxygen carrier based on enucleated bone marrow mesenchymal stem cells (BMSCs) in this study. A complete sequence encoding hemoglobin α/β subunits linked by a P2A sequence for tandem expression was integrated into the BMSCs genome via lentiviral transduction. Western blot and Native-PAGE analyses confirmed that these subunits were released as monomers and assembled into hemoglobin tetramers. Furthermore, ultraviolet-visible (UV-Vis) spectroscopy exhibited the characteristic dual peaks in the oxygenated state and a single peak in the deoxygenated state, indicating an oxygen-carrying capacity comparable to that of natural hemoglobin. Importantly, the resulting enucleated vesicles were comparable in size to erythrocytes, remained highly viable for up to 24 hours, and exhibited low cytotoxicity. In conclusion, this recombinant enucleated BMSC-derived hemoglobin (BMSC-Hb) shows promising oxygen transport capacity and low immunogenicity, positioning it as a potential blood substitute for emergency transfusion. Oxygen carriers BMSCs enucleation blood substitute Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights Engineered enucleated BMSCs successfully produce functional hemoglobin tetramers through self-assemble. The recombinant hemoglobin tetramers possess a promising oxygen-carrying capacity. The bio-synthetic vesicles exhibit an erythrocyte-like morphology and excellent biocompatibility. Introduction Major haemorrhage is a leading cause of mortality in trauma, obstetric emergencies, and gastrointestinal bleeding, accounting for approximately 40% of trauma-related deaths [ 1 ]. Rapid blood loss results in a critical reduction in circulating volume and systemic hypoperfusion, culminating in shock, metabolic acidosis, and coagulopathy, a pathophysiological cascade collectively termed the “lethal triad” [ 2 – 4 ]. Despite updates in clinical guidelines, transfusion remains central to the management of hemorrhagic shock [ 5 , 6 ], primarily by supplementing red blood cells (RBCs) to restore oxygen delivery. However, the inherent limitations of the transfusion cannot be overlooked. For example, the complexity of blood group systems increases risk of alloimmunization and necessitates cross-matching [ 7 ]. Furthermore, according to the WHO, global blood supply growth has consistently lagged behind demand in recent years [ 8 ], a gap expected to widen with aging populations [ 9 ]. Additional constraints include the risk of pathogen transmission, limited shelf life, and stringent storage requirements [ 10 , 11 ]. These challenges underscore an urgent clinical need for blood substitutes that eliminate the requirement for typing and cross-matching, minimize infectious risks, and offer high biocompatibility. Currently, research on blood substitutes primarily focuses on oxygen transport capabilities, encompassing perfluorocarbon-based oxygen carriers (PFOCs), hemoglobin-based oxygen carriers (HBOCs), and RBCs derived from stem cell differentiation [ 12 ]. However, each of these approaches presents considerable limitations. PFOCs leverage the high oxygen solubility of perfluorocarbons, dissolving far more oxygen than blood under normal pressure and effectively releasing oxygen under hypoxic conditions, without requiring blood typing [ 13 , 14 ]. Although second-generation PFOCs offer advantages such as improved particle size (50–100 nm) and expanded half-life (4 days) via nanoemulsion, they still carry risks of complement activation-related pseudoallergy (CARPA) and organ toxicity [ 15 ]. HBOCs stabilize hemoglobin through chemical modification or encapsulation. However, they can scavenge nitric oxide, which would lead to hypertension and kidney injury, and accumulate methemoglobin due to the absence of natural antioxidants, resulting in oxidative damage [ 16 , 17 ]. Novel nano-structured HBOCs utilizing RBCs membrane coating have demonstrated enhanced stability, reduced immunogenicity, and prolonged circulation [ 18 , 19 ], nevertheless, the presence of surface antigens still necessitates cross-matching. Stem cell-derived RBCs allow for customized blood types [ 20 ], but face challenges including limited proliferation capacity, low yield, high cost, and the unavoidable need for cross-matching due to surface antigens. Producing universal RBCs with low immunogenicity and high stability remains a significant challenge. BMSCs are multipotent progenitor cells with broad anti-inflammatory and immunomodulatory properties, positioning them as key players in regenerative medicine [ 21 ]. Owing to their intrinsic disease-targeting capability, low immunogenicity and paracrine-secretion capacities, the BMSCs have attracted considerable attentions as therapeutic delivery vehicles [ 22 ]. Notably, even after enucleation, these cells retain the ability to translate exogenous mRNAs and secrete functional proteins. Studies have demonstrated that genetic modification of BMSCs followed by enucleation enables targeted delivery of therapeutics in viv o [ 23 , 24 ], offering a controllable and effective approach for treating various diseases with an improved safety profile. To address the limitations of conventional free hemoglobin in the development of artificial carriers, including its limited supply, instability, and cytotoxicity, we established a stable mouse BMSC-Hb via lentiviral transduction. This approach enables genomic integration and sustained high-level expression of hemoglobin. Subsequent enucleation by centrifugation generated anucleated cytoplasts with dimensions comparable to those of RBCs, thereby achieving effective hemoglobin encapsulation and oxygen transport functionality. This strategy yields a blood substitute with oxygen-carrying capacity, absence of antigenic restriction, and excellent biocompatibility, offering a novel potential solution for the management of acute major hemorrhage and addressing the clinical need for rare blood types. Materials & methods Experimental animals All animal experiments were approved by the Experimental Animal Ethics Committee of Shantou Univerisity with the Animal Experimental Ethics Number: 2022506007 (Date of approval: 25/06/2025). All animal experiments were approved under the project titled “Construction and functional study of a novel oxygen carrier based on enucleated bone marrow mesenchymal stem cells”. Mice were maintained in specific pathogen-free conditions in the animal facility and housed on a 12- h light/12-h dark cycle with ad libitum access to food and water. Male C57BL/6 mice were purchased from Biocytogen Gene Biotechnology (Jiangsu, China), and were used to collect blood samples to assess of the reversible oxygen-binding capacity. The work has been reported in line with the ARRIVE guidelines 2.0. Adult male C57BL/6 mice were fasted for 12 hours prior to blood collection, with ad libitum access to water. Mice were deeply anesthetized with sodium pentobarbital, and the tails were cleansed repeatedly with 75% ethanol to induce vasodilation. Blood samples were collected into tubes containing sodium citrate as an anticoagulant. The anticoagulated blood was centrifuged at 3000 rpm for 10 minutes. Following removal of the supernatant, the RBCs were resuspended in PBS. This washing procedure consisting of centrifugation and resuspension, was repeated two to three times. Finally, the purified RBCs were resuspended in PBS. For each experiment, healthy adult mice were randomly selected, and blood was collected for the analysis of oxygen binding. At least three independent biological replicates were performed. Plasmids Plasmid Lenti-GFP served as the backbone for constructing pLenti-GFP-3Myc-Hba-3HA-Hbb (hereinafter referred to as pLenti-Hb). The target mouse hemoglobin genes, Hba (NM_008218.2) and Hbb (NM_001278161.1), fused with Myc tag and HA tag individually, were amplified via PCR. Subsequently, the amplified product was cloned into the backbone plasmid through homologous recombination. The recombinant product was then transformed into DH5α competent cells. After transformation, the recombinant plasmid was extracted and verified by sequencing. Cell culture Mouse BMSCs, HUVECs, Cos-7, HEK293 and HEK293T were maintained in high glucose DMEM medium supplemented with containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine at 37°C with 5% CO₂ and saturated humidity. Cells at ∼60–70% confluence were transfected. Plasmids were transfected into HEK293 and HEK293T cells using polyethylenimine (PEI, Sigma). Immunoblotting Immunoblotting was performed as described before [ 25 ], with the following antibodies: Anti-Myc (1:2000, MBL562), Anti-HA (1:20000, Proteintech 66006-2-Ig), and Hemoglobin β (1:500, Santa Cruz sc-sc390668). Briefly, HEK293 cells were transfected with the indicated plasmids and harvested at 24 h post-transfection. Cells were lysed in RIPA buffer supplemented with protease inhibitor. The lysates were centrifuged at 13,000 rpm for 10 min at 4 ℃ to remove the insoluble fraction. Sample loading buffer was added to the supernatant, and the mixture was boiled for 5 min. Proteins were separated on SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h and then incubated with primary antibody overnight at 4 ℃. After three washes with TBST (TBS containing 0.1% Tween-20), the membrane was incubated with HRP-conjugated secondary antibodies and visualized with enhanced chemiluminescence. Immunofluorescence Immunofluorescence staining was performed as described before [ 26 ]. Briefly, cells were fixed with 4% PFA for 30 min at room temperature, washed three times with PBS for 5 min each, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After blocking for 1 h at room temperature, cells were incubated overnight at 4°C with the indicated primary antibodies: anti-Myc (1:400, Santa Cruz SC-40) and anti-HA (1:200, Roche 12013819001). After three washes with PBS, cells were incubated with secondary antibodies and DAPI for 1 h, and then washed with PBS three times for 5 min each. Finally, the cells were mounted on slides with FluorSave reagent (Millipore), and images were acquired using a Zeiss LSM800 confocal microscope. For enucleated BMSC-Hb, the cell membrane was stained with DiI for 10–15 min at 37°C, followed by three washes with PBS. The nuclear was stained with Hoechst 33342 dyes for 15 min at 37°C, and then washed three times with PBS. The stained cells were plated onto confocal dished for imaging. Stable cell lines The construction and screening process for monoclonal stable cell lines is illustrated in Fig. 2 . Lentiviral particles were generated by cotransfecting HEK293T cells with plasmids-pLenti-Hb/pLenti-GFP, pMDL, VSVG, and REV using PEI. The culture medium was replaced 6 h and 16 h after transfection. Virus -containing supernatants were collected at 45, 54, 64, 72, and 96 h post-transfection. After each collection, the supernatant was filtered through a 0.22 µm PVDF filter (Millipore) and concentrated using PEG8000. BMSCs were plated into 96 well plates. Upon reaching 20%-30% density, lentiviral particles were added to the well, and the culture medium was replaced with fresh complete medium 24 h post-infection. Infection efficiency was verified by observation under an inverted fluorescence microscope, and the positive cells were re-plated into new 96 well plates following a serial dilution. Monoclonal stable cell lines were screened based on green fluorescence and further verified by immunoblotting and immunofluorescence staining. Native-PAGE analysis Native-PAGE was performed to evaluate the tetrameric assembly of hemoglobin. Proteins from BMSC-Hb cell lines were prepared with a non-reducing, non-denaturing sample buffer. The samples were aliquoted into two portions and resolved on a 10% non-denaturing polyacrylamide gel. Following electrophoresis, one gel was analyzed by Coomassie blue staining, and the other by Western blot. For Western blot detection, the PVDF membrane was incubated with the indicated primary antibody (Hemoglobin β/γ/δ/ε antibody, 1:500, sc-390668), followed by HRP-conjugated secondary antibody. The tetrameric assembly of hemoglobin was assessed using enhanced chemiluminescence as described in immunoblotting. Assessment of reversible oxygen binding The reversibly oxygen binding capacity of RBCs and BMSC-Hb was assessed by monitoring changes in the UV-Vis absorption spectrum (300–700 nm) between their oxygenated and deoxygenated states, following a previously established method [ 27 ]. For deoxygenation, CO₂ gas was gently bubbled through the cell suspension to displace oxygen. After at least 3 hours, sodium dithionite (Na₂S₂O₄) was added, and UV-Vis absorption spectra were recorded from 350 nm to 650 nm using a BioTek microplate reader. For reoxygenation, the cell suspension was exposed to ambient air, after which the UV-Vis absorption spectra were recorded under the same conditions. Cell enucleation Cell enucleation was performed as described before [ 28 ]. Briefly, single 24-well from 24-well plates were plated with Cos-7 cells and cultured until they reached approximately 90% confluence. The cells were then removed by treatment with ammonia hydroxide to prepare extracellular matrix (ECM)-coated 24-well. Subsequently, BMSCs were plated into these ECM-coated 24-well, and cultured until the cell density reached approximately 90%. To induce enucleation, the 24-well containing BMSCs were centrifuged at 35°C and 4500 g for 1 h in an enucleation solution containing 1 mg/mL cytochalasin B, 50 mM NaCl, 10 mg/mL colchicine, 10% sucrose, and 100 µM calcium chloride. Centrifugation was performed with the 24-well placed upside down in centrifuge tubes. After enucleation, the cells were recovered in complete medium and used for subsequent experiments. Cell viability assay Approximately 5×10⁵ HUVEC and HEK293 cells were plated into a 96-well plate and incubated overnight at 37 ℃. Enucleated cells were harvested and diluted to various concentrations. These cells were then co-cultured with the two adherent cell types for 24 hours. Cytotoxicity was evaluated using the CCK-8 assay according to the manufacturer’s instructions. Briefly, 10 µL of CCK-8 solution (Cell Counting Kit-8, C0038, Beyotime, China) was added to each well, followed by incubation for 4 hours. Absorbance at 450 nm was subsequently measured using a BioTek microplate reader. Image processing Images of cells were obtained through Zeiss 800 confocal microscope with a 63X/1.4NA oil objective. The schematic diagram in Fig. 2 was created with BioGDP [ 29 ]. Statistical analyses All statistical analyses and graphical representations were performed using GraphPad Prism 8.0.2 software. Data are presented as mean ± SEM. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001. All microscopic images were measured and analyzed using ZEN 2 software and subsequently processed with Adobe Photoshop. Results Fusion expression of hemoglobin subunits α and β The tetrameric form of hemoglobin is the only conformation capable of function as an oxygen carrier. To construct a hemoglobin-based oxygen carrier, we designed a recombinant expression system for the co-expression of the α and β subunits within the same cell (Fig. 1 A and Supplementary Fig. 1). The pLenti-GFP plasmid, which containing a CMV promoter and GFP reporter, was used as the backbone vector. Using this backbone, the α and β subunits of hemoglobin were co-expressed with Myc and HA tags fused to their N-termini, respectively. Additionally, the self-cleaving P2A peptides were inserted between the GFP reporter and two subunits to enable post-translational cleavage and release of the individual monomeric proteins. To verify whether the constructed pLenti-Hb plasmid could effectively express hemoglobin in cells, Western blot was performed to detect the target proteins following transfection of HEK293 cells. As shown in Supplementary Fig. 2, specific bands corresponding to the Myc-α-globin (~ 22 kDa) and HA-β-globin (~ 20 kDa), consistent with the expected molecular weights, were observed in the pLenti-Hb transfection group, whereas no relevant signals were detected in the control group. Furthermore, a band corresponding to the β subunit was specifically detected in the pLenti-Hb transfected group at the expected molecular weight when probed with antibody against the hemoglobin β subunit (Fig. 1 B). These results indicated successful expression of both the α and β subunits of hemoglobin. Immunofluorescence staining further supported these findings. In contrast to the control group, which exhibited only GFP signal, cells transfected with pLenti-Hb showed co-expression of Myc (α subunit) and HA (β subunit), as evidenced by red fluorescence signals (Fig. 1 C-D). In summary, the recombinant expression system is functional and enables the co-expression and subsequent release of the individual hemoglobin α and β subunits. Generation of a stable recombinant BMSC-Hb cell line by lentivirus transduction To generate a cell line stably expressing hemoglobin, we employed a lentivirus-based gene delivery method to construct stable cell lines in this study, owing to the low efficiency of chemical transfection in BMSCs. The construction process for the recombinant BMSC-Hb stable cell line is illustrated in Fig. 2 . Lentivirus particles were generated according to the manufacturer’s instruction, and the HEK293 cells were used to verify infection efficiency. As shown in Supplementary Fig. 3, strong green fluorescence signals were observed in both groups, while the plenti-Hb group also exhibited red fluorescence corresponding to hemoglobin subunits, as detected by immunofluorescence staining. These results confirmed that the packaged lentivirus was suitable for subsequent infection of BMSCs. Following infection of BMSCs, limiting dilution cloning was performed to isolate monoclonal cells based on GFP fluorescence. Several colonies were obtained and verified for successful hemoglobin expression by Western blot (Supplementary Fig. 4). Among these, clones BMSC-Hb5 and BMSC-Hb7.2 exhibited stable and high level of hemoglobin expression compared with the control group, and were therefore selected for further characterization (Fig. 3 A-B). To confirm this result, double immunofluorescence staining was performed using antibodies against GFP, HA tag, and Myc tag. The results showed co-localization of GFP with hemoglobin subunits in BMSC-Hb cells, whereas no hemoglobin signal was detected in the BMSC-GFP cells (Fig. 3 C-D). Together, these data demonstrate the successful construction of stable BMSC-Hb cell lines with integrated expression of hemoglobin α and β subunits. The α and β subunits self-assemble into tetrameric hemoglobin in BMSC-Hb cells Since the α₂β₂ tetramer is essential for hemoglobin to transport oxygen, we sought to determine whether the overexpressed α and β subunits could properly self-assemble into a functional tetramer within cells. To this end, Native‑PAGE was performed using BMSC-GFP as a negative control and bovine hemoglobin (BHb) as a positive control. As shown in Fig. 4 A, compared with the BMSC-GFP negative control, both BMSC-Hb5 and BMSC-Hb7.2 exhibited specific new bands near the position corresponding to the bovine hemoglobin tetramer (indicated by the red box). To further confirm the identity of this new band, Western blot analysis was performed. As expected, clear signals were detected in sample from BMSC-Hb cells and BHb, but not in BMSC-GFP cells (Fig. 4 B). It should be noted that the recombinant hemoglobin in BMSC-Hb cells exhibited a slightly higher molecular weight due to the presence of 3×HA tag. Moreover, only BMSC-Hb cells showed a positive HA signal, which aligned with the band detected by the hemoglobin β antibody (Fig. 4 B). Consistent with these observations, Coomassie blue staining revealed a distinct protein band at the corresponding position, marked by the black arrow (Fig. 4 A). Together, these results demonstrate that the α and β subunits can self‑assemble into tetrameric hemoglobin in BMSC‑Hb cells, suggesting their potential for oxygen transport. Recombinant BMSC-Hb cells exhibit oxygen carrier characteristics similar to natural RBCs To further confirm whether the tetrameric hemoglobin could transport the oxygen, we recorded the UV‑Vis spectrum of hemoglobin in oxygenated and deoxygenated state. Before analyzing the spectral characteristics of the recombinant hemoglobin, we first examined the RBCs from C57BL/6 mice to validate the experiment setup. As shown in Fig. 5 A, the spectrum of RBCs exhibited the typical characteristic dual peaks of hemoglobin at 540 nm and 575 nm, corresponding to the α and β subunits, along with a Soret peak at 415 nm arising from the porphyrin ring (red curve). Upon deoxygenation, the dual peaks merged into a single absorption peak at 555 nm, and the Soret peak shifted to 430 nm (blue curve). In contrast, BMSC‑GFP cells displayed no characteristic absorption between 500–600 nm in either the oxygenated or deoxygenated states, with only a weak peak near 415 nm, likely attributable to endogenous porphyrin structures (Fig. 5 B). For BMSC‑Hb cells, however, similar dual peaks appeared at 550 nm and 580 nm under oxygenated conditions, which converged into a single peak at 565 nm upon deoxygenation, while the Soret peak shifted from 410 nm to 430 nm (Fig. 5 C). These spectral recordings were considered reliable, and the shifts in absorption peaks may be attributed to the insertion of the tag and P2A sequence, which introduce additional amino acids. Importantly, when deoxygenated BMSC‑Hb cells were re‑exposed to air, the spectrum reverted to the oxygenated profile, demonstrating that oxygen binding and release in BMSC‑Hb is reversible and repeatable. Taken together, our results indicate that BMSC‑Hb cells mimics the oxygen carrier properties of natural red blood cells. Enucleated BMSC-Hb cells are comparable in size to RBCs To mimic the enucleated characteristic of RBCs, we performed enucleation treatment on BMSC-Hb in this study. The removal of genetic material also eliminates the risk of mutations and cancer. Cell morphology was visualized using Hoechst 33342 (nuclear stain) and DiI (membrane stain). Compared with non-enucleated cells, enucleated BMSC-Hb cells showed a complete absence of nuclear signals (Fig. 6 A). Moreover, the enucleated cells displayed a vesicle-like compartment, with the cell membrane fully encapsulating the cytoplasmic contents, as indicated by clearly visible GFP signals. Importantly, the enucleated cells also exhibited a smaller diameter. The diameter of non-enucleated BMSC-Hb cells ranged from 14–20 µm (Fig. 6 B), whereas that of enucleated cells was significantly reduced, with most measuring 7–9 µm in diameter, a size close to that of natural RBCs (Fig. 6 C). Thus, enucleated BMSC-Hb cells possess a size comparable to that of RBCs, suggesting their potential as promising oxygen carriers for in vivo applications. Enucleated BMSCs offer the advantages of considerable longevity and low toxicity To evaluate the application potential of the artificial oxygen carrier, the viability and cytotoxicity of enucleated BMSC‑Hb cells were analyzed. For BMSC-GFP cells, about 70% cells remained viable at 24 h post-enucleation; however, whose viability decreased markedly by 48 h, and nearly all cells were died by 72 h (Supplementary Fig. 5). A similar trend was observed for enucleated BMSC‑Hb cells, with approximately 60% remaining viable within 24 h and almost all cells dying by 48 h (Fig. 7 ). These results suggest that the oxygen carriers besed on enucleation could serve as an emergency blood substitute, exhibited considerable viability within 24 h and thereby providing critical time for patients with traumatic hemorrhage. Given that clinical translation of artificial oxygen carriers requires stringent biosafety profiles, the cytotoxicity of enucleated BMSC‑Hb toward different cell types was further evaluated. A co‑culture system was employed to simulate potential interactions between the carriers and host cells. As shown in Fig. 8 , the viability of both HUVEC and HEK293T cells remained unchanged when co‑cultured with enucleated cells across a concentration range of 0–10⁶ cells/mL. These data indicate that enucleated BMSC‑Hb cells exhibit no obvious cytotoxicity toward either HUVEC or HEK293T cells. Collectively, the enucleated BMSC‑Hb-based artificial oxygen carriers demonstrate low cytotoxicity and favorable biocompatibility, supporting their potential as a novel therapeutic strategy for acute trauma. Discussion Blood transfusion remains the primary method for patients with massive hemorrhage. Therefore, developing a convenient and efficient blood substitute has become an urgent clinical need. In this study, we focused on developing an oxygen carrier based on hemoglobin. We integrated the α- and β-globin subunits into the genome of BMSCs to establish stable cell lines. These cell lines expressed and assembled functional tetrameric hemoglobin and exhibited reversible oxygen-binding ability (Figs. 4 and 5 ). Additionally, enucleated BMSC-Hb cells exhibited a size similar to that of RBCs, and most of the enucleated cells remained viable for more than 24 h. These findings position BMSC‑Hb cells as a promising platform for the development of blood substitutes. Tetrameric hemoglobin (α₂β₂) exhibits structural stability and high oxygen-carrying capacity. In contrast, unpaired globin monomers, particularly α-globin, are prone to rapid degradation. Previous studies have demonstrated that co-expression of β-globin stabilizes α-globin and facilitates tetramer formation [ 30 – 33 ]. Optimization strategies such as adjusting subunit expression ratios (e.g., β/α/α) [ 32 ] and co-expressing α-hemoglobin stabilizing protein (AHSP) have been reported to further enhance hemoglobin assembly in yeast [ 31 , 33 ]. Additionally, overexpression of heme biosynthesis-related genes (e.g., HEM3 in yeast systems) can substantially increase yield, with some approaches achieving up to a 70‑fold improvement in hemoglobin production [ 30 ]. However, how to efficiently, stably express and maintain hemoglobin in mammalian cells remains a topic that warrants further investigation. In this study, we constructed a lentivirus-based vector to fusion express the hemoglobin α and β subunits via a 2A peptide, which ensure a 1: 1 ratio of α and β globin, and integrated the construct into the genome of BMSCs (Figs. 1 and 2 ). This strategy simplifies the expression of globin subunits and facilitates their assembly into functional tetrameric hemoglobin in mammalian cells (Fig. 4 ). HBOCs are among the most extensively studied blood substitutes. They are typically prepared by chemically modifying or encapsulating free hemoglobin to achieve effective oxygen transport. However, the source of free hemoglobin is limited, as it is predominantly derived from outdated human or bovine RBCs through multi-step processes involving cell lysis, chromatography, and sterile filtration [ 34 ]. Alternative approaches, including engineering hemoglobin expression in prokaryotic or eukaryotic systems in vitro [ 32 , 35 ], have been constrained by low yields and the need for additional separation and purification steps. To streamline the process of obtaining HBOCs, we constructed a intracellularly self-assemble system in which tetrameric hemoglobin is assembled in the cytoplasm after expression. The cytoplasmic vesicles containing hemoglobin are obtained directly via an enucleation process, requiring only 1 h of centrifugation, thereby eliminating the complex technological procedures associated with traditional methods, such as purification or modification. Consequently, this cell‑based strategy for generating artificial oxygen carriers offers a more convenient alternative to conventional preparation methods. BMSCs offer distinct clinical advantages as host cells owing to their inherently low immunogenicity, characterized by low MHC-I expression and absence of MHC-II, as well as their immunomodulatory functions [ 21 , 36 ]. Given the capability for lentiviral-mediated exogenous gene expression, employing BMSCs as a vehicle for oxygen transport constitutes a more favorable strategy. Although emerging evidence suggests the lentiviral-mediated gene therapy may carry a potential risk of oncogenesis [ 37 ], while enucleation further eliminates the risk associated with genomic integration. Moreover, the enucleated cells lose proliferative capacity, however, they retain transient translational and secretory activity [ 38 ]. After enucleation, hemoglobin-containing vesicles were obtained with a size comparable to that of natural RBCs (Fig. 7 ). In vitro cytotoxicity assays confirmed that enucleated BMSC-Hb cells exhibit negligible toxicity (Fig. 8 ), indicating that they not only approximate the size and oxygen-carrying function of erythrocytes but also preserve the low immunogenic profile of mesenchymal stem cells. Notably, BMSCs have been shown to possess anti‑inflammatory and immunomodulatory properties in injury settings [ 39 , 40 ], which could help mitigate the systemic inflammatory response often accompanying acute hemorrhage. The stable recombinant BMSC-Hb cell lines also demonstrate considerable advantages in storage stability, tolerating long‑term cryopreservation at -80°C or in liquid nitrogen while maintaining high post‑thaw viability and sustained proliferative capacity. However, the limited post‑enucleation lifespan of these cells significantly constrains their translational utility. Despite efforts to extend post‑enucleation survival, progress in this area remains limited [ 28 , 41 ]. If enucleated cells are to be used therapeutically as oxygen carriers, repeated administration may be necessary to sustain functional efficacy, posing a substantial challenge for clinical translation. Therefore, extending the in vivo half‑life of enucleated cells represents an important goal for future research. Despite the promising in vitro performance of enucleated BMSC-Hb, several key challenges remain to be addressed toward its clinical translation. First, scalable manufacturing of these vesicles in a cost-effective and reproducible manner will require further process optimization. Second, the in vivo clearance mechanisms and pharmacokinetic profiles of these vesicles are not yet fully characterized, highlighting the need for detailed biodistribution and circulation studies. Third, while preliminary biocompatibility findings are encouraging, comprehensive long-term safety evaluations, including potential immunogenicity, chronic toxicity, and organ-specific effects, are necessary in appropriate animal models before clinical consideration. Addressing these limitations will be critical for advancing this technology toward therapeutic application. Conclusion In summary, this study successfully developed a novel oxygen carrier derived from enucleated BMSCs engineered to express recombinant hemoglobin. Using lentiviral transduction, BMSC-Hb cell lines were established to stably express α- and β-globin subunits, which self-assembled into functional tetramers capable of reversible oxygen binding. Upon enucleation, these cells yielded hemoglobin‑encapsulated vesicles with a size similar to that of native erythrocytes. The resulting constructs exhibited low cytotoxicity and do not require blood‑type matching, supporting their potential as practical blood substitutes. Declarations Competing interests The authors declare no competing or financial interests. Author contributions Xinyi Chen : Methodology, Investigation, Data curation, Writing-original draft. Zhenrong Zhou : Methodology, Resources, Funding acquisition, Writing-review and editing. Shuxin Chen : Investigation, Data curation, Validation. Zihui Zhou : Methodology, Investigation, Formal analysis. Yu Guo : Investigation, Data curation. Jialin Ding : Investigation. Chiju Wei : Conceptualization, Resources. Zhong Hu : Resources, Conceptualization, Writing-review and editing. Aihua Mao :Supervision, Project administration, Funding acquisition, Writing-review and editing. Funding This work was supported by the National Natural Science Foundation of China (32100659 to M.A.), Guangdong Basic and Applied Basic Research Foundation (2023A1515012586 to M.A., 2025A1515012750 to Z.Z.), SUMC Start-up Funding (510858072 to Z.Z.). Statement on AI use No artificial intelligence was utilized in the preparation of this manuscript. Data availability All data generated in this study are available within the article. References Curry NS, Davenport R. Transfusion strategies for major haemorrhage in trauma. Brit J Haematol. 2019;184(4):508–23. Moore EE, Moore HB, Kornblith LZ, et al. Trauma-induced coagulopathy. Nat Rev Dis Primers. 2021;7(1):30. Shah A, Kerner V, Stanworth SJ, Agarwal S. Major haemorrhage: past, present and future. Anaesthesia. 2023;78(1):93–104. van Veelen MJ, Brodmann Maeder M. 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Samaja M, Malavalli A, Vandegriff KD. How nitric oxide hindered the search for hemoglobin-based oxygen carriers as human blood substitutes. Int J Mol Sci. 2023;24(19):14902. Kavdia M, Tsoukias NM, Popel AS. Model of nitric oxide diffusion in an arteriole: impact of hemoglobin-based blood substitutes. Am J Physiol Heart Circ Physiol. 2002;282(6):H2245–53. Duan L, Yan XH, Wang AH, et al. Highly loaded hemoglobin spheres as promising artificial oxygen carriers. ACS Nano. 2012;6(8):6897–904. Peng S, Liu J, Qin Y, et al. Metal-organic framework encapsulating hemoglobin as a high-stable and long-circulating oxygen carriers to treat hemorrhagic shock. ACS Appl Mater Interfaces. 2019;11(39):35604–12. Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol Med. 2018;10(6):e8454. Pittenger MF, Discher DE, Péault BM, et al. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen Med. 2019;4:22. Labusca L, Herea DD, Mashayekhi K. Stem cells as delivery vehicles for regenerative medicine-challenges and perspectives. World J Stem Cells. 2018;10(5):43–56. Wang HW, Alarcón CN, Liu B, et al. Genetically engineered and enucleated human mesenchymal stromal cells for the targeted delivery of therapeutics to diseased tissue. Nat Biomed Eng. 2022;6(7):882–97. Chen ZT, Zou Y, Sun HX et al. (2024) Engineered enucleated mesenchymal stem cells regulating immune microenvironment and promoting wound healing. Adv Mater. 36(45), e2412253. Zhou ZR, Yang XJ, Mao AH, et al. Deficiency of CAMSAP2 impairs olfaction and the morphogenesis of mitral cells. EMBO Rep. 2024;25(7):2861–77. Mao AH, Li ZY, Ning GZ, et al. Sclerotome-derived PDGF signaling functions as a niche cue responsible for primitive erythropoiesis. Development. 2023;150(22):dev201807. Jia Y, Cui Y, Fei JB, et al. Construction and evaluation of hemoglobin-based capsules as blood substitutes. Adv Funct Mater. 2012;22(7):1446–53. Chen Y, Xu LQ, Lin MJ, et al. An improved cellular enucleation method with extracellular matrix and colchicine facilitates the study of nucleocytoplasmic interaction. Eur J Cell Biol. 2019;98(5–8):151045. Jiang S, Li H, Zhang L, et al. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic Acids Res. 2025;53(D1):D1670–6. Ishchuka OP, Domenzain I, Sánchez BJ, et al. Genome-scale modeling drives 70-fold improvement of intracellular heme production in Saccharomyces cerevisiae . P Natl Acad Sci Usa. 2022;119(30):e2108245119. Ishchuk OP, Frost AT, Muñiz-Paredes F, et al. Improved production of human hemoglobin in yeast by engineering hemoglobin degradation. Metab Eng. 2021;66:259–67. Liu LF, Martinez JL, Liu ZH, et al. Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae . Metab Eng. 2014;21:9–16. Feng L, Gell DA, Zhou SP, et al. Molecular mechanism of AHSP-mediated stabilization of α-hemoglobin. Cell. 2004;119(5):629–40. Alayash AI. Setbacks in blood substitutes research and development: A biochemical perspective. Clin Lab Med. 2010;30(2):381–9. Villarreal DM, Phillips CL, Kelley AM, et al. Enhancement of recombinant hemoglobin production in Escherichia coli BL21(DE3) containing the Plesiomonas shigelloides heme transport system. Appl Environ Microbiol. 2008;74(18):5854–6. Wieczorek M, Abualrous ET, Sticht J, et al. Major histocompatibility complex (MHC) class I and MHC class II proteins: Conformational plasticity in antigen presentation. Front Immunol. 2017;8:292. Eichler F, Duncan CN, Musolino PL, et al. Lentiviral gene therapy for cerebral adrenoleukodystrophy. N Engl J Med. 2024;391(14):1302–12. Graham DM, Andersen T, Sharek L, et al. Enucleated cells reveal differential roles of the nucleus in cell migration, polarity, and mechanotransduction. J Cell Biol. 2018;217(3):895–914. Yang H, Chen J, Li J. Isolation, culture, and delivery considerations for the use of mesenchymal stem cells in potential therapies for acute liver failure. Front Immunol. 2023;14:1243220. Huang Y, He B, Wang L, et al. Bone marrow mesenchymal stem cell-derived exosomes promote rotator cuff tendon-bone healing by promoting angiogenesis and regulating M1 macrophages in rats. Stem Cell Res Ther. 2020;11(1):496. Yan W, Wu RL, Lee Y, et al. Perturbation of calcium homeostasis invokes eryptosis-like cell death in enucleated bone marrow stem cells. Biochem Cell Biol. 2025;103:1–11. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfiguresandfigurelegends.docx 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-8578714","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617031268,"identity":"7c4373d8-5aa9-4441-be7e-a19eb78d3969","order_by":0,"name":"Xinyi Chen","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Chen","suffix":""},{"id":617031269,"identity":"437442b1-de80-4b45-b0ea-c8c507b710b9","order_by":1,"name":"Zhengrong Zhou","email":"","orcid":"","institution":"Joint Shantou International Eye Center of Shantou University and The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Zhengrong","middleName":"","lastName":"Zhou","suffix":""},{"id":617031271,"identity":"506420b5-08b0-4d7f-855e-9b113f7284ff","order_by":2,"name":"Shuxin Chen","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Shuxin","middleName":"","lastName":"Chen","suffix":""},{"id":617031275,"identity":"fe7a1487-f62c-4a39-9fcd-7c17caf9cecb","order_by":3,"name":"Zihui Zhou","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Zihui","middleName":"","lastName":"Zhou","suffix":""},{"id":617031277,"identity":"cc38aee5-f22d-48f1-bf78-b18747a3fc4b","order_by":4,"name":"Yu Guo","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Guo","suffix":""},{"id":617031278,"identity":"1be1decd-183f-4555-9543-a090bfbdc55c","order_by":5,"name":"Jialin Ding","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Jialin","middleName":"","lastName":"Ding","suffix":""},{"id":617031280,"identity":"2daf8f71-601a-4269-a375-e531607cbea3","order_by":6,"name":"Chiju Wei","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Chiju","middleName":"","lastName":"Wei","suffix":""},{"id":617031282,"identity":"25fa1ea1-42a1-4103-8fbc-1a271a325a8f","order_by":7,"name":"Zhong Hu","email":"","orcid":"","institution":"Shantou University","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Hu","suffix":""},{"id":617031283,"identity":"f915fb1a-239a-45e1-bf31-682ed5271a1d","order_by":8,"name":"Aihua Mao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYDACCRBRgcQmUssZCEWCFsY2UrTwz+5O/PBxXl2dwQHmg7d5GOzyCFty5+xmyZnbDksYHGBLtuZhSC4mbM2N3A3SvNsOALXwmEnzMBxIbCCkQ/5G7ubfvHPqgFr4vxGnxeBG7jZp3gZmkC1sxGkxBGqxnHHssOTMw2zGlnMMkglrkQM67MaHmjp+vuPND2+8qbAjrAUBmMHuJF79KBgFo2AUjAI8AACZQjmn5IvLYgAAAABJRU5ErkJggg==","orcid":"","institution":"Shantou University","correspondingAuthor":true,"prefix":"","firstName":"Aihua","middleName":"","lastName":"Mao","suffix":""}],"badges":[],"createdAt":"2026-01-12 07:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8578714/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8578714/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106403921,"identity":"e544820d-2caa-4951-b4cc-b552e33a380a","added_by":"auto","created_at":"2026-04-08 09:15:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":311662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of hemoglobin α and β subunits expression. \u003c/strong\u003e(A) Schematic diagram of the construction of pLenti-GFP-Hb recombinant plasmid. Hemoglobin α and β subunits were linked by a P2A peptide. Myc tag and HA tag were fused to the N-termini of the α and β subunits, respectively. (B) Western blot analysis of hemoglobin β using an anti- hemoglobin β antibody. (C-D) Immunofluorescence staining was performed to detect the co-expression of hemoglobin α/β with GFP protein. Scla bar, 200 μm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/48a53b419018c6e961896eba.png"},{"id":106403145,"identity":"d75cf2d6-8305-4e39-83bb-f928f4c070f5","added_by":"auto","created_at":"2026-04-08 09:13:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":404996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the generation of stable monoclonal cell lines via lentiviral transduction.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/a7e61565a4f2a40a7897c4eb.png"},{"id":106246065,"identity":"39a811c2-3c18-48d1-b5ad-32ca9a0872cc","added_by":"auto","created_at":"2026-04-06 16:01:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":471396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of the target protein expression in stable recombinant BMSC-Hb cell lines.\u003c/strong\u003e(A-B) Western blot analysis of hemoglobin α and β using anti-Myc and anti-HA antibodies. (C-D) Immunofluorescence staining was performed to detect the co-expression of hemoglobin α/β with GFP protein in cells. Scla bar, 100 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/4e43ea95c9e025218656a07a.png"},{"id":106246066,"identity":"955cc614-aa12-429e-80a8-4df234469692","added_by":"auto","created_at":"2026-04-06 16:01:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHemoglobin tetramer was expressed in BMSC-Hb cells. \u003c/strong\u003e(A) Coomassie blue staining of native-PAGE gel. The red box indicates new bands that appeared near BHb in BMSC-Hb cells. The black arrow indicats the position of the verified hemoglobin tetramer. (B) Western blot analysis of hemoglobin β using anti- hemoglobin β and anti-HA antibodies.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/5c15967163f21137170a2a1e.png"},{"id":106403051,"identity":"ec187b16-d018-486c-9064-c7821f946bf4","added_by":"auto","created_at":"2026-04-08 09:13:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV-Vis absorption spectra of oxygenated and deoxygenated Hb.\u003c/strong\u003eUV-Vis spectra (350-650 nm) were obtained to detect hemoglobin in mouse red blood cell (A), BMSC-GFP cell line (B), and BMSC-Hb cell line (C). Red and blue lines represent the absorption spectra under oxygenated and deoxygenated states, respectively. The insets show the characteristic absorption peaks of\u003cstrong\u003e \u003c/strong\u003eoxy-/deoxy-Hb in detail.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/686cd3a0d43134c376c2c3b8.png"},{"id":106246069,"identity":"60d0890e-9b98-4c64-8736-ade7291bd7b9","added_by":"auto","created_at":"2026-04-06 16:01:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":264734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnucleated BMSC-Hb cells share similar characteristics with erythroid cells.\u003c/strong\u003e (A) Confocal images of BMSC-Hb cells before and after enucleation. The cell membrane was labeled with DiI, and the nucleus was labeled with Hoechst 33342. Scale bar is 20 μm. (B) Diameter of BMSC-Hb cells. (C) Diameter of enucleated BMSC-Hb cells.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/f704afb7abd2ebb5e7c3002f.png"},{"id":106246071,"identity":"9941829b-0f4b-43b0-be53-5aeb836796e7","added_by":"auto","created_at":"2026-04-06 16:01:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":310396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability of enucleated BMSC-Hb cells. \u003c/strong\u003e(A) Fluorescence signals of BMSC-Hb cells at different time points after enucleation. Scale bar, 200 μm. (B) Cell viability of enucleated BMSC-Hb cells at different time points.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/f8e890226a6ab2843222baa4.png"},{"id":106246070,"identity":"f6eb86a0-abdb-4b5d-9a11-bede6ef241e1","added_by":"auto","created_at":"2026-04-06 16:01:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":196832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity of enucleated BMSC-Hb cells on HUVEC and HEK293T cells. \u003c/strong\u003eThe cytotoxicity of different concentrations of enucleated BMSC-Hb cells was analyzed using the CCK-8 assay in (A) HUVEC and (B) HEK293T cells.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/0308379ac08eaf51bb998474.png"},{"id":106852471,"identity":"12ca7112-29f5-4c46-b849-9b49c085b7a0","added_by":"auto","created_at":"2026-04-14 06:41:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3314019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/d515cbe2-772a-4b2a-ad3b-0970f1832a3f.pdf"},{"id":106403004,"identity":"f0a9d246-53b6-4fd2-bad0-e58543bb953e","added_by":"auto","created_at":"2026-04-08 09:13:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":715906,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiguresandfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8578714/v1/3dce1d641b8f25c21aaf2685.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Erythrocyte-mimetic oxygen carriers based on enucleated mesenchymal stem cells: A novel strategy for emergency blood substitution","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eEngineered enucleated BMSCs successfully produce functional hemoglobin tetramers through self-assemble.\u003c/li\u003e\n \u003cli\u003eThe recombinant hemoglobin tetramers possess a promising oxygen-carrying capacity.\u003c/li\u003e\n \u003cli\u003eThe bio-synthetic vesicles exhibit an erythrocyte-like morphology and excellent biocompatibility.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eMajor haemorrhage is a leading cause of mortality in trauma, obstetric emergencies, and gastrointestinal bleeding, accounting for approximately 40% of trauma-related deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Rapid blood loss results in a critical reduction in circulating volume and systemic hypoperfusion, culminating in shock, metabolic acidosis, and coagulopathy, a pathophysiological cascade collectively termed the \u0026ldquo;lethal triad\u0026rdquo; [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite updates in clinical guidelines, transfusion remains central to the management of hemorrhagic shock [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], primarily by supplementing red blood cells (RBCs) to restore oxygen delivery. However, the inherent limitations of the transfusion cannot be overlooked. For example, the complexity of blood group systems increases risk of alloimmunization and necessitates cross-matching [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, according to the WHO, global blood supply growth has consistently lagged behind demand in recent years [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], a gap expected to widen with aging populations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additional constraints include the risk of pathogen transmission, limited shelf life, and stringent storage requirements [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These challenges underscore an urgent clinical need for blood substitutes that eliminate the requirement for typing and cross-matching, minimize infectious risks, and offer high biocompatibility.\u003c/p\u003e \u003cp\u003eCurrently, research on blood substitutes primarily focuses on oxygen transport capabilities, encompassing perfluorocarbon-based oxygen carriers (PFOCs), hemoglobin-based oxygen carriers (HBOCs), and RBCs derived from stem cell differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, each of these approaches presents considerable limitations. PFOCs leverage the high oxygen solubility of perfluorocarbons, dissolving far more oxygen than blood under normal pressure and effectively releasing oxygen under hypoxic conditions, without requiring blood typing [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although second-generation PFOCs offer advantages such as improved particle size (50\u0026ndash;100 nm) and expanded half-life (4 days) via nanoemulsion, they still carry risks of complement activation-related pseudoallergy (CARPA) and organ toxicity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. HBOCs stabilize hemoglobin through chemical modification or encapsulation. However, they can scavenge nitric oxide, which would lead to hypertension and kidney injury, and accumulate methemoglobin due to the absence of natural antioxidants, resulting in oxidative damage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Novel nano-structured HBOCs utilizing RBCs membrane coating have demonstrated enhanced stability, reduced immunogenicity, and prolonged circulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], nevertheless, the presence of surface antigens still necessitates cross-matching. Stem cell-derived RBCs allow for customized blood types [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], but face challenges including limited proliferation capacity, low yield, high cost, and the unavoidable need for cross-matching due to surface antigens. Producing universal RBCs with low immunogenicity and high stability remains a significant challenge.\u003c/p\u003e \u003cp\u003eBMSCs are multipotent progenitor cells with broad anti-inflammatory and immunomodulatory properties, positioning them as key players in regenerative medicine [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Owing to their intrinsic disease-targeting capability, low immunogenicity and paracrine-secretion capacities, the BMSCs have attracted considerable attentions as therapeutic delivery vehicles [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Notably, even after enucleation, these cells retain the ability to translate exogenous mRNAs and secrete functional proteins. Studies have demonstrated that genetic modification of BMSCs followed by enucleation enables targeted delivery of therapeutics \u003cem\u003ein viv\u003c/em\u003eo [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], offering a controllable and effective approach for treating various diseases with an improved safety profile.\u003c/p\u003e \u003cp\u003eTo address the limitations of conventional free hemoglobin in the development of artificial carriers, including its limited supply, instability, and cytotoxicity, we established a stable mouse BMSC-Hb via lentiviral transduction. This approach enables genomic integration and sustained high-level expression of hemoglobin. Subsequent enucleation by centrifugation generated anucleated cytoplasts with dimensions comparable to those of RBCs, thereby achieving effective hemoglobin encapsulation and oxygen transport functionality. This strategy yields a blood substitute with oxygen-carrying capacity, absence of antigenic restriction, and excellent biocompatibility, offering a novel potential solution for the management of acute major hemorrhage and addressing the clinical need for rare blood types.\u003c/p\u003e"},{"header":"Materials \u0026 methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Experimental Animal Ethics Committee of Shantou Univerisity with the Animal Experimental Ethics Number: 2022506007 (Date of approval: 25/06/2025). All animal experiments were approved under the project titled \u0026ldquo;Construction and functional study of a novel oxygen carrier based on enucleated bone marrow mesenchymal stem cells\u0026rdquo;. Mice were maintained in specific pathogen-free conditions in the animal facility and housed on a 12- h light/12-h dark cycle with ad libitum access to food and water. Male C57BL/6 mice were purchased from Biocytogen Gene Biotechnology (Jiangsu, China), and were used to collect blood samples to assess of the reversible oxygen-binding capacity. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003cp\u003eAdult male C57BL/6 mice were fasted for 12 hours prior to blood collection, with ad libitum access to water. Mice were deeply anesthetized with sodium pentobarbital, and the tails were cleansed repeatedly with 75% ethanol to induce vasodilation. Blood samples were collected into tubes containing sodium citrate as an anticoagulant. The anticoagulated blood was centrifuged at 3000 rpm for 10 minutes. Following removal of the supernatant, the RBCs were resuspended in PBS. This washing procedure consisting of centrifugation and resuspension, was repeated two to three times. Finally, the purified RBCs were resuspended in PBS. For each experiment, healthy adult mice were randomly selected, and blood was collected for the analysis of oxygen binding. At least three independent biological replicates were performed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmids\u003c/h3\u003e\n\u003cp\u003ePlasmid Lenti-GFP served as the backbone for constructing pLenti-GFP-3Myc-Hba-3HA-Hbb (hereinafter referred to as pLenti-Hb). The target mouse hemoglobin genes, Hba (NM_008218.2) and Hbb (NM_001278161.1), fused with Myc tag and HA tag individually, were amplified via PCR. Subsequently, the amplified product was cloned into the backbone plasmid through homologous recombination. The recombinant product was then transformed into DH5α competent cells. After transformation, the recombinant plasmid was extracted and verified by sequencing.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eMouse BMSCs, HUVECs, Cos-7, HEK293 and HEK293T were maintained in high glucose DMEM medium supplemented with containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine at 37\u0026deg;C with 5% CO₂ and saturated humidity. Cells at \u0026sim;60\u0026ndash;70% confluence were transfected. Plasmids were transfected into HEK293 and HEK293T cells using polyethylenimine (PEI, Sigma).\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eImmunoblotting was performed as described before [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], with the following antibodies: Anti-Myc (1:2000, MBL562), Anti-HA (1:20000, Proteintech 66006-2-Ig), and Hemoglobin β (1:500, Santa Cruz sc-sc390668). Briefly, HEK293 cells were transfected with the indicated plasmids and harvested at 24 h post-transfection. Cells were lysed in RIPA buffer supplemented with protease inhibitor. The lysates were centrifuged at 13,000 rpm for 10 min at 4 ℃ to remove the insoluble fraction. Sample loading buffer was added to the supernatant, and the mixture was boiled for 5 min. Proteins were separated on SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h and then incubated with primary antibody overnight at 4 ℃. After three washes with TBST (TBS containing 0.1% Tween-20), the membrane was incubated with HRP-conjugated secondary antibodies and visualized with enhanced chemiluminescence.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence staining was performed as described before [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, cells were fixed with 4% PFA for 30 min at room temperature, washed three times with PBS for 5 min each, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After blocking for 1 h at room temperature, cells were incubated overnight at 4\u0026deg;C with the indicated primary antibodies: anti-Myc (1:400, Santa Cruz SC-40) and anti-HA (1:200, Roche 12013819001). After three washes with PBS, cells were incubated with secondary antibodies and DAPI for 1 h, and then washed with PBS three times for 5 min each. Finally, the cells were mounted on slides with FluorSave reagent (Millipore), and images were acquired using a Zeiss LSM800 confocal microscope.\u003c/p\u003e \u003cp\u003eFor enucleated BMSC-Hb, the cell membrane was stained with DiI for 10\u0026ndash;15 min at 37\u0026deg;C, followed by three washes with PBS. The nuclear was stained with Hoechst 33342 dyes for 15 min at 37\u0026deg;C, and then washed three times with PBS. The stained cells were plated onto confocal dished for imaging.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStable cell lines\u003c/h2\u003e \u003cp\u003eThe construction and screening process for monoclonal stable cell lines is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Lentiviral particles were generated by cotransfecting HEK293T cells with plasmids-pLenti-Hb/pLenti-GFP, pMDL, VSVG, and REV using PEI. The culture medium was replaced 6 h and 16 h after transfection. Virus -containing supernatants were collected at 45, 54, 64, 72, and 96 h post-transfection. After each collection, the supernatant was filtered through a 0.22 \u0026micro;m PVDF filter (Millipore) and concentrated using PEG8000.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBMSCs were plated into 96 well plates. Upon reaching 20%-30% density, lentiviral particles were added to the well, and the culture medium was replaced with fresh complete medium 24 h post-infection. Infection efficiency was verified by observation under an inverted fluorescence microscope, and the positive cells were re-plated into new 96 well plates following a serial dilution. Monoclonal stable cell lines were screened based on green fluorescence and further verified by immunoblotting and immunofluorescence staining.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNative-PAGE analysis\u003c/h3\u003e\n\u003cp\u003eNative-PAGE was performed to evaluate the tetrameric assembly of hemoglobin. Proteins from BMSC-Hb cell lines were prepared with a non-reducing, non-denaturing sample buffer. The samples were aliquoted into two portions and resolved on a 10% non-denaturing polyacrylamide gel. Following electrophoresis, one gel was analyzed by Coomassie blue staining, and the other by Western blot. For Western blot detection, the PVDF membrane was incubated with the indicated primary antibody (Hemoglobin β/γ/δ/ε antibody, 1:500, sc-390668), followed by HRP-conjugated secondary antibody. The tetrameric assembly of hemoglobin was assessed using enhanced chemiluminescence as described in immunoblotting.\u003c/p\u003e\n\u003ch3\u003eAssessment of reversible oxygen binding\u003c/h3\u003e\n\u003cp\u003eThe reversibly oxygen binding capacity of RBCs and BMSC-Hb was assessed by monitoring changes in the UV-Vis absorption spectrum (300\u0026ndash;700 nm) between their oxygenated and deoxygenated states, following a previously established method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For deoxygenation, CO₂ gas was gently bubbled through the cell suspension to displace oxygen. After at least 3 hours, sodium dithionite (Na₂S₂O₄) was added, and UV-Vis absorption spectra were recorded from 350 nm to 650 nm using a BioTek microplate reader. For reoxygenation, the cell suspension was exposed to ambient air, after which the UV-Vis absorption spectra were recorded under the same conditions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell enucleation\u003c/h2\u003e \u003cp\u003eCell enucleation was performed as described before [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, single 24-well from 24-well plates were plated with Cos-7 cells and cultured until they reached approximately 90% confluence. The cells were then removed by treatment with ammonia hydroxide to prepare extracellular matrix (ECM)-coated 24-well. Subsequently, BMSCs were plated into these ECM-coated 24-well, and cultured until the cell density reached approximately 90%. To induce enucleation, the 24-well containing BMSCs were centrifuged at 35\u0026deg;C and 4500 g for 1 h in an enucleation solution containing 1 mg/mL cytochalasin B, 50 mM NaCl, 10 mg/mL colchicine, 10% sucrose, and 100 \u0026micro;M calcium chloride. Centrifugation was performed with the 24-well placed upside down in centrifuge tubes. After enucleation, the cells were recovered in complete medium and used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eApproximately 5\u0026times;10⁵ HUVEC and HEK293 cells were plated into a 96-well plate and incubated overnight at 37 ℃. Enucleated cells were harvested and diluted to various concentrations. These cells were then co-cultured with the two adherent cell types for 24 hours. Cytotoxicity was evaluated using the CCK-8 assay according to the manufacturer\u0026rsquo;s instructions. Briefly, 10 \u0026micro;L of CCK-8 solution (Cell Counting Kit-8, C0038, Beyotime, China) was added to each well, followed by incubation for 4 hours. Absorbance at 450 nm was subsequently measured using a BioTek microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImage processing\u003c/h2\u003e \u003cp\u003eImages of cells were obtained through Zeiss 800 confocal microscope with a 63X/1.4NA oil objective. The schematic diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e was created with BioGDP [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses and graphical representations were performed using GraphPad Prism 8.0.2 software. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was defined as *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001. All microscopic images were measured and analyzed using ZEN 2 software and subsequently processed with Adobe Photoshop.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFusion expression of hemoglobin subunits α and β\u003c/h2\u003e \u003cp\u003eThe tetrameric form of hemoglobin is the only conformation capable of function as an oxygen carrier. To construct a hemoglobin-based oxygen carrier, we designed a recombinant expression system for the co-expression of the α and β subunits within the same cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;1). The pLenti-GFP plasmid, which containing a CMV promoter and GFP reporter, was used as the backbone vector. Using this backbone, the α and β subunits of hemoglobin were co-expressed with Myc and HA tags fused to their N-termini, respectively. Additionally, the self-cleaving P2A peptides were inserted between the GFP reporter and two subunits to enable post-translational cleavage and release of the individual monomeric proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify whether the constructed pLenti-Hb plasmid could effectively express hemoglobin in cells, Western blot was performed to detect the target proteins following transfection of HEK293 cells. As shown in Supplementary Fig.\u0026nbsp;2, specific bands corresponding to the Myc-α-globin (~\u0026thinsp;22 kDa) and HA-β-globin (~\u0026thinsp;20 kDa), consistent with the expected molecular weights, were observed in the pLenti-Hb transfection group, whereas no relevant signals were detected in the control group. Furthermore, a band corresponding to the β subunit was specifically detected in the pLenti-Hb transfected group at the expected molecular weight when probed with antibody against the hemoglobin β subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results indicated successful expression of both the α and β subunits of hemoglobin. Immunofluorescence staining further supported these findings. In contrast to the control group, which exhibited only GFP signal, cells transfected with pLenti-Hb showed co-expression of Myc (α subunit) and HA (β subunit), as evidenced by red fluorescence signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). In summary, the recombinant expression system is functional and enables the co-expression and subsequent release of the individual hemoglobin α and β subunits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of a stable recombinant BMSC-Hb cell line by lentivirus transduction\u003c/h2\u003e \u003cp\u003eTo generate a cell line stably expressing hemoglobin, we employed a lentivirus-based gene delivery method to construct stable cell lines in this study, owing to the low efficiency of chemical transfection in BMSCs. The construction process for the recombinant BMSC-Hb stable cell line is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Lentivirus particles were generated according to the manufacturer\u0026rsquo;s instruction, and the HEK293 cells were used to verify infection efficiency. As shown in Supplementary Fig.\u0026nbsp;3, strong green fluorescence signals were observed in both groups, while the \u003cem\u003eplenti-Hb\u003c/em\u003e group also exhibited red fluorescence corresponding to hemoglobin subunits, as detected by immunofluorescence staining. These results confirmed that the packaged lentivirus was suitable for subsequent infection of BMSCs.\u003c/p\u003e \u003cp\u003eFollowing infection of BMSCs, limiting dilution cloning was performed to isolate monoclonal cells based on GFP fluorescence. Several colonies were obtained and verified for successful hemoglobin expression by Western blot (Supplementary Fig.\u0026nbsp;4). Among these, clones BMSC-Hb5 and BMSC-Hb7.2 exhibited stable and high level of hemoglobin expression compared with the control group, and were therefore selected for further characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). To confirm this result, double immunofluorescence staining was performed using antibodies against GFP, HA tag, and Myc tag. The results showed co-localization of GFP with hemoglobin subunits in BMSC-Hb cells, whereas no hemoglobin signal was detected in the BMSC-GFP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Together, these data demonstrate the successful construction of stable BMSC-Hb cell lines with integrated expression of hemoglobin α and β subunits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe α and β subunits self-assemble into tetrameric hemoglobin in BMSC-Hb cells\u003c/h2\u003e \u003cp\u003eSince the α₂β₂ tetramer is essential for hemoglobin to transport oxygen, we sought to determine whether the overexpressed α and β subunits could properly self-assemble into a functional tetramer within cells. To this end, Native‑PAGE was performed using BMSC-GFP as a negative control and bovine hemoglobin (BHb) as a positive control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, compared with the BMSC-GFP negative control, both BMSC-Hb5 and BMSC-Hb7.2 exhibited specific new bands near the position corresponding to the bovine hemoglobin tetramer (indicated by the red box). To further confirm the identity of this new band, Western blot analysis was performed. As expected, clear signals were detected in sample from BMSC-Hb cells and BHb, but not in BMSC-GFP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It should be noted that the recombinant hemoglobin in BMSC-Hb cells exhibited a slightly higher molecular weight due to the presence of 3\u0026times;HA tag. Moreover, only BMSC-Hb cells showed a positive HA signal, which aligned with the band detected by the hemoglobin β antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistent with these observations, Coomassie blue staining revealed a distinct protein band at the corresponding position, marked by the black arrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Together, these results demonstrate that the α and β subunits can self‑assemble into tetrameric hemoglobin in BMSC‑Hb cells, suggesting their potential for oxygen transport.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant BMSC-Hb cells exhibit oxygen carrier characteristics similar to natural RBCs\u003c/h2\u003e \u003cp\u003eTo further confirm whether the tetrameric hemoglobin could transport the oxygen, we recorded the UV‑Vis spectrum of hemoglobin in oxygenated and deoxygenated state. Before analyzing the spectral characteristics of the recombinant hemoglobin, we first examined the RBCs from C57BL/6 mice to validate the experiment setup. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the spectrum of RBCs exhibited the typical characteristic dual peaks of hemoglobin at 540 nm and 575 nm, corresponding to the α and β subunits, along with a Soret peak at 415 nm arising from the porphyrin ring (red curve). Upon deoxygenation, the dual peaks merged into a single absorption peak at 555 nm, and the Soret peak shifted to 430 nm (blue curve). In contrast, BMSC‑GFP cells displayed no characteristic absorption between 500\u0026ndash;600 nm in either the oxygenated or deoxygenated states, with only a weak peak near 415 nm, likely attributable to endogenous porphyrin structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). For BMSC‑Hb cells, however, similar dual peaks appeared at 550 nm and 580 nm under oxygenated conditions, which converged into a single peak at 565 nm upon deoxygenation, while the Soret peak shifted from 410 nm to 430 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These spectral recordings were considered reliable, and the shifts in absorption peaks may be attributed to the insertion of the tag and P2A sequence, which introduce additional amino acids. Importantly, when deoxygenated BMSC‑Hb cells were re‑exposed to air, the spectrum reverted to the oxygenated profile, demonstrating that oxygen binding and release in BMSC‑Hb is reversible and repeatable. Taken together, our results indicate that BMSC‑Hb cells mimics the oxygen carrier properties of natural red blood cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEnucleated BMSC-Hb cells are comparable in size to RBCs\u003c/h2\u003e \u003cp\u003eTo mimic the enucleated characteristic of RBCs, we performed enucleation treatment on BMSC-Hb in this study. The removal of genetic material also eliminates the risk of mutations and cancer. Cell morphology was visualized using Hoechst 33342 (nuclear stain) and DiI (membrane stain). Compared with non-enucleated cells, enucleated BMSC-Hb cells showed a complete absence of nuclear signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Moreover, the enucleated cells displayed a vesicle-like compartment, with the cell membrane fully encapsulating the cytoplasmic contents, as indicated by clearly visible GFP signals. Importantly, the enucleated cells also exhibited a smaller diameter. The diameter of non-enucleated BMSC-Hb cells ranged from 14\u0026ndash;20 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), whereas that of enucleated cells was significantly reduced, with most measuring 7\u0026ndash;9 \u0026micro;m in diameter, a size close to that of natural RBCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Thus, enucleated BMSC-Hb cells possess a size comparable to that of RBCs, suggesting their potential as promising oxygen carriers for \u003cem\u003ein vivo\u003c/em\u003e applications.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\n\u003ch2\u003eEnucleated BMSCs offer the advantages of considerable longevity and low toxicity\u003c/h2\u003eTo evaluate the application potential of the artificial oxygen carrier, the viability and cytotoxicity of enucleated BMSC‑Hb cells were analyzed. For BMSC-GFP cells, about 70% cells remained viable at 24 h post-enucleation; however, whose viability decreased markedly by 48 h, and nearly all cells were died by 72 h (Supplementary Fig. 5). A similar trend was observed for enucleated BMSC‑Hb cells, with approximately 60% remaining viable within 24 h and almost all cells dying by 48 h (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results suggest that the oxygen carriers besed on enucleation could serve as an emergency blood substitute, exhibited considerable viability within 24 h and thereby providing critical time for patients with traumatic hemorrhage.\u003cbr\u003e\n\u003cp\u003eGiven that clinical translation of artificial oxygen carriers requires stringent biosafety profiles, the cytotoxicity of enucleated BMSC‑Hb toward different cell types was further evaluated. A co‑culture system was employed to simulate potential interactions between the carriers and host cells. As shown in Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the viability of both HUVEC and HEK293T cells remained unchanged when co‑cultured with enucleated cells across a concentration range of 0\u0026ndash;10⁶ cells/mL. These data indicate that enucleated BMSC‑Hb cells exhibit no obvious cytotoxicity toward either HUVEC or HEK293T cells. Collectively, the enucleated BMSC‑Hb-based artificial oxygen carriers demonstrate low cytotoxicity and favorable biocompatibility, supporting their potential as a novel therapeutic strategy for acute trauma.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eBlood transfusion remains the primary method for patients with massive hemorrhage. Therefore, developing a convenient and efficient blood substitute has become an urgent clinical need. In this study, we focused on developing an oxygen carrier based on hemoglobin. We integrated the α- and β-globin subunits into the genome of BMSCs to establish stable cell lines. These cell lines expressed and assembled functional tetrameric hemoglobin and exhibited reversible oxygen-binding ability (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, enucleated BMSC-Hb cells exhibited a size similar to that of RBCs, and most of the enucleated cells remained viable for more than 24 h. These findings position BMSC‑Hb cells as a promising platform for the development of blood substitutes.\u003c/p\u003e \u003cp\u003eTetrameric hemoglobin (α₂β₂) exhibits structural stability and high oxygen-carrying capacity. In contrast, unpaired globin monomers, particularly α-globin, are prone to rapid degradation. Previous studies have demonstrated that co-expression of β-globin stabilizes α-globin and facilitates tetramer formation [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Optimization strategies such as adjusting subunit expression ratios (e.g., β/α/α) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and co-expressing α-hemoglobin stabilizing protein (AHSP) have been reported to further enhance hemoglobin assembly in yeast [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, overexpression of heme biosynthesis-related genes (e.g., \u003cem\u003eHEM3\u003c/em\u003e in yeast systems) can substantially increase yield, with some approaches achieving up to a 70‑fold improvement in hemoglobin production [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, how to efficiently, stably express and maintain hemoglobin in mammalian cells remains a topic that warrants further investigation. In this study, we constructed a lentivirus-based vector to fusion express the hemoglobin α and β subunits via a 2A peptide, which ensure a 1: 1 ratio of α and β globin, and integrated the construct into the genome of BMSCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This strategy simplifies the expression of globin subunits and facilitates their assembly into functional tetrameric hemoglobin in mammalian cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHBOCs are among the most extensively studied blood substitutes. They are typically prepared by chemically modifying or encapsulating free hemoglobin to achieve effective oxygen transport. However, the source of free hemoglobin is limited, as it is predominantly derived from outdated human or bovine RBCs through multi-step processes involving cell lysis, chromatography, and sterile filtration [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Alternative approaches, including engineering hemoglobin expression in prokaryotic or eukaryotic systems \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], have been constrained by low yields and the need for additional separation and purification steps. To streamline the process of obtaining HBOCs, we constructed a intracellularly self-assemble system in which tetrameric hemoglobin is assembled in the cytoplasm after expression. The cytoplasmic vesicles containing hemoglobin are obtained directly via an enucleation process, requiring only 1 h of centrifugation, thereby eliminating the complex technological procedures associated with traditional methods, such as purification or modification. Consequently, this cell‑based strategy for generating artificial oxygen carriers offers a more convenient alternative to conventional preparation methods.\u003c/p\u003e \u003cp\u003eBMSCs offer distinct clinical advantages as host cells owing to their inherently low immunogenicity, characterized by low MHC-I expression and absence of MHC-II, as well as their immunomodulatory functions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Given the capability for lentiviral-mediated exogenous gene expression, employing BMSCs as a vehicle for oxygen transport constitutes a more favorable strategy. Although emerging evidence suggests the lentiviral-mediated gene therapy may carry a potential risk of oncogenesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], while enucleation further eliminates the risk associated with genomic integration. Moreover, the enucleated cells lose proliferative capacity, however, they retain transient translational and secretory activity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. After enucleation, hemoglobin-containing vesicles were obtained with a size comparable to that of natural RBCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity assays confirmed that enucleated BMSC-Hb cells exhibit negligible toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), indicating that they not only approximate the size and oxygen-carrying function of erythrocytes but also preserve the low immunogenic profile of mesenchymal stem cells. Notably, BMSCs have been shown to possess anti‑inflammatory and immunomodulatory properties in injury settings [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which could help mitigate the systemic inflammatory response often accompanying acute hemorrhage.\u003c/p\u003e \u003cp\u003eThe stable recombinant BMSC-Hb cell lines also demonstrate considerable advantages in storage stability, tolerating long‑term cryopreservation at -80\u0026deg;C or in liquid nitrogen while maintaining high post‑thaw viability and sustained proliferative capacity. However, the limited post‑enucleation lifespan of these cells significantly constrains their translational utility. Despite efforts to extend post‑enucleation survival, progress in this area remains limited [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. If enucleated cells are to be used therapeutically as oxygen carriers, repeated administration may be necessary to sustain functional efficacy, posing a substantial challenge for clinical translation. Therefore, extending the \u003cem\u003ein vivo\u003c/em\u003e half‑life of enucleated cells represents an important goal for future research.\u003c/p\u003e \u003cp\u003eDespite the promising \u003cem\u003ein vitro\u003c/em\u003e performance of enucleated BMSC-Hb, several key challenges remain to be addressed toward its clinical translation. First, scalable manufacturing of these vesicles in a cost-effective and reproducible manner will require further process optimization. Second, the \u003cem\u003ein vivo\u003c/em\u003e clearance mechanisms and pharmacokinetic profiles of these vesicles are not yet fully characterized, highlighting the need for detailed biodistribution and circulation studies. Third, while preliminary biocompatibility findings are encouraging, comprehensive long-term safety evaluations, including potential immunogenicity, chronic toxicity, and organ-specific effects, are necessary in appropriate animal models before clinical consideration. Addressing these limitations will be critical for advancing this technology toward therapeutic application.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study successfully developed a novel oxygen carrier derived from enucleated BMSCs engineered to express recombinant hemoglobin. Using lentiviral transduction, BMSC-Hb cell lines were established to stably express α- and β-globin subunits, which self-assembled into functional tetramers capable of reversible oxygen binding. Upon enucleation, these cells yielded hemoglobin‑encapsulated vesicles with a size similar to that of native erythrocytes. The resulting constructs exhibited low cytotoxicity and do not require blood‑type matching, supporting their potential as practical blood substitutes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eThe authors declare no competing or financial interests. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXinyi Chen\u003c/strong\u003e: Methodology, Investigation, Data curation, Writing-original draft. \u003cstrong\u003eZhenrong Zhou\u003c/strong\u003e: Methodology, Resources, Funding acquisition, Writing-review and editing. \u003cstrong\u003eShuxin Chen\u003c/strong\u003e: Investigation, Data curation, Validation. \u003cstrong\u003eZihui Zhou\u003c/strong\u003e: Methodology, Investigation, Formal analysis. \u003cstrong\u003eYu Guo\u003c/strong\u003e: Investigation, Data curation. \u003cstrong\u003eJialin Ding\u003c/strong\u003e: Investigation.\u003cstrong\u003e Chiju Wei\u003c/strong\u003e: Conceptualization, Resources. \u003cstrong\u003eZhong Hu\u003c/strong\u003e: Resources, Conceptualization, Writing-review and editing. \u003cstrong\u003eAihua Mao\u003c/strong\u003e:Supervision, Project administration, Funding acquisition, Writing-review and editing.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32100659 to M.A.), Guangdong Basic and Applied Basic Research Foundation (2023A1515012586 to M.A., 2025A1515012750 to Z.Z.), SUMC Start-up Funding (510858072 to Z.Z.).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatement on AI use \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo artificial intelligence was utilized in the preparation of this manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCurry NS, Davenport R. 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Biochem Cell Biol. 2025;103:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\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":"Oxygen carriers, BMSCs, enucleation, blood substitute","lastPublishedDoi":"10.21203/rs.3.rs-8578714/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8578714/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute major hemorrhage triggered by a sudden reduction in blood volume, accompanied by coagulopathy, systemic hypoperfusion, and tissue hypoxia, is likely the underlying cause of death. Shortages in donor blood supplies limit the efficacy of conventional transfusion therapies, leading to heightened interest in blood substitutes as potential alternatives or supplements. To address his limitation, we developed a novel oxygen carrier based on enucleated bone marrow mesenchymal stem cells (BMSCs) in this study. A complete sequence encoding hemoglobin α/β subunits linked by a P2A sequence for tandem expression was integrated into the BMSCs genome via lentiviral transduction. Western blot and Native-PAGE analyses confirmed that these subunits were released as monomers and assembled into hemoglobin tetramers. Furthermore, ultraviolet-visible (UV-Vis) spectroscopy exhibited the characteristic dual peaks in the oxygenated state and a single peak in the deoxygenated state, indicating an oxygen-carrying capacity comparable to that of natural hemoglobin. Importantly, the resulting enucleated vesicles were comparable in size to erythrocytes, remained highly viable for up to 24 hours, and exhibited low cytotoxicity. In conclusion, this recombinant enucleated BMSC-derived hemoglobin (BMSC-Hb) shows promising oxygen transport capacity and low immunogenicity, positioning it as a potential blood substitute for emergency transfusion.\u003c/p\u003e","manuscriptTitle":"Erythrocyte-mimetic oxygen carriers based on enucleated mesenchymal stem cells: A novel strategy for emergency blood substitution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 16:01:14","doi":"10.21203/rs.3.rs-8578714/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":"6214e683-0dca-40fe-9ab5-df70e94e4c52","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T06:40:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 16:01:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8578714","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8578714","identity":"rs-8578714","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}