Extracellular vesicles derived from fat-laden hepatocytes undergoing chemical hypoxia promote a pro-fibrotic phenotype in hepatic stellate cells

BackgroundThe transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression.AimTo evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2).MethodsHepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH.ResultsChemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs.ConclusionChemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.     

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O₂) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-β-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.     

Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death

Background

Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.

Methods

C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.

Results

Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.

Conclusions

These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

     

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta

Background

Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity.

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions

Background aimsMesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs).MethodsMSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs.ResultsMSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions.ConclusionsLPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.     

Independent human mesenchymal stromal cell–derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model

Background aimsExtracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application.MethodsIndependent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized.ResultsThe functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers.ConclusionsStandardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.     

Local administration of allogeneic or autologous bone marrow-derived mesenchymal stromal cells enhances bone formation similarly in distraction osteogenesis

Background aimsDistraction osteogenesis (DO) is a surgical technique to promote bone regeneration that requires a long time for bone healing. Bone marrow-derived mesenchymal stromal cells (MSCs) have been applied to accelerate bone formation in DO. Allogeneic MSCs are attractive, as they could be ready to use in clinics. Whether allogeneic MSCs would have an effect similar to autologous MSCs with regard to promoting bone formation in DO is still unknown. This study compares the effect of autologous MSCs versus allogeneic MSCs on bone formation in a rat DO model.MethodsRat bone marrow-derived MSCs were isolated, characterized and expanded in vitro. Adult rats were subjected to right tibia transverse osteotomy. On the third day of distraction, each rat received one injection of phosphate-buffered saline (PBS), autologous MSCs or allogeneic MSCs at the distraction site. Tibiae were harvested after 28 days of consolidation for micro-computed tomography examination, mechanical test and histological analysis.ResultsResults showed that treatment with both allogeneic and autologous MSCs promoted bone formation, with significantly higher bone mass, mechanical properties and mineral apposition rate as well as expression of angiogenic and bone formation markers at the regeneration sites compared with the PBS-treated group. No statistical difference in bone formation was found between the allogeneic and autologous MSC treatment groups.ConclusionsThis study indicates that allogeneic and autologous MSCs have a similar effect on promoting bone consolidation in DO. MSCs from an allogeneic source could be used off-the-shelf with DO to achieve early bone healing.     

Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy

Background aimsTo obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo.MethodsA retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo.ResultsThe cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2 ± 1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3 ± 1 passages).ConclusionsThese results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs.     

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO₂) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO₂) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.     

Chemical hypoxia induces pro-inflammatory signals in fat-laden hepatocytes and contributes to cellular crosstalk with Kupffer cells through extracellular vesicles

BackgroundObstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD.MethodsPrimary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo.ResultsHypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1β, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1.ConclusionHypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].     

CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities

Background aimsExtracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay.MethodsThe bona fide MSC antigen CD73 possesses ecto-5’-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5’-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5’-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity.ResultsThe authors showed that the ecto-5’-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5’-nucleotidase activity was resistant to EV-destroying detergent treatment.ConclusionsEcto-5’-nucleotidase activity neither reflects the potency of the authors’ MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5’-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors’ MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.     

Registered clinical trials investigating treatment with cell-derived extracellular vesicles: a scoping review

Background aimsInterest in cell-based therapy using extracellular vesicles (EVs) is intensifying, building upon promising preclinical research and a handful of published clinical studies. Registered clinical trials remain small, heterogeneous in design and underpowered to determine safety and efficacy on their own. A scoping review of registered studies can identify opportunities to pool data and perform meta-analysis.MethodsRegistered trials were identified by searching clinical trial databases (Clinicaltrials.gov, the World Health Organization International Clinical Trials Registry Platform and the Chinese Clinical Trial Registry) on June 10, 2022.ResultsSeventy-three trials were identified and included for analysis. Mesenchymal stromal cells (MSCs) were the most common cell type from which EVs were derived (49 studies, 67%). Among the 49 identified MSC-EV studies, 25 were controlled trials (51%) with a combined total of 3094 participants anticipated to receive MSC-derived EVs (2225 in controlled studies). Although EVs are being administered to treat a broad range of conditions, trials treating patients with coronavirus disease-2019 and/or acute respiratory distress syndrome were observed most commonly. Despite heterogeneity between studies, we anticipate that at least some of the studies could be combined in meaningful meta-analysis and that a combined sample size of 1000 patients would provide the ability to detect a ≥5% difference in mortality with MSC-EVs compared to controls and could be achieved by December 2023.ConclusionsThis scoping review identifies potential barriers that may stall clinical translation of EV-based treatment, and our analysis calls for more standardized product characterization, use of quantifiable product quality attributes and consistent outcome reporting in future clinical trials.     

Allogeneic Mesenchymal Stem Cells in Combination with Hyaluronic Acid for the Treatment of Osteoarthritis in Rabbits

Mesenchymal stem cell (MSC)-based therapies may aid in the repair of articular cartilage defects. The purpose of this study was to investigate the effects of intraarticular injection of allogeneic MSCs in an in vivo anterior cruciate ligament transection (ACLT) model of osteoarthritis in rabbits. Allogeneic bone marrow-derived MSCs were isolated and cultured under hypoxia (1% O₂). After 8 weeks following ACLT, MSCs suspended in hyaluronic acid (HA) were injected into the knees, and the contralateral knees were injected with HA alone. Additional controls consisted of a sham operation group as well as an untreated osteoarthritis group. The tissues were analyzed by macroscopic examination as well as histologic and immunohistochemical methods at 6 and 12 weeks post-transplantation. At 6 and 12 weeks, the joint surface showed less cartilage loss and surface abrasion after MSC injection as compared to the tissues receiving HA injection alone. Significantly better histological scores and cartilage content were observed with the MSC transplantation. Furthermore, engraftment of allogenic MSCs were evident in surface cartilage. Thus, injection of the allogeneic MSCs reduced the progression of osteoarthritis in vivo.     

In vivo therapy of myocardial infarction with mesenchymal stem cells modified with prostaglandin I synthase gene improves cardiac performance in mice

AimIntra-myocardial injection of adult bone marrow-derived stem cells (MSC) has recently been proposed as a therapy to repair damaged cardiomyocytes after acute myocardial infarction (AMI). PGI₂ has vasodilatation effects; however, the effects of combining both MSC and PGI₂ therapy on AMI have never been evaluated.Main methodsWe genetically enhanced prostaglandin I synthase (PGIS) gene expression in mouse mesenchymal stem cells (MSC) using lentiviral vector transduction (MSC_PGIS). Mice were subjected to an AMI model and injected (intra-myocardially) with either 5 × 10⁴ MSCs or MSC_PGIS before surgery. Fourteen days post AMI, mice were analyzed with echocardiography, immunohistochemistry, and apoptotic, and traditional tissue assays.Key findingsLenti-PGIS transduction did not change any characteristic of the MSCs. PGIS over-expressed MSCs secreted 6-keto-PGF1α in the culture medium and decreased free radical damage during hypoxia/re-oxygenation and H₂O₂ treatment. Furthermore, splenocyte proliferation was significantly suppressed with MSC_PGIS as compared with MSCs alone. Fourteen days post AMI, echocardiography showed more improvement in cardiac function of the MSC_PGIS group than the MSC alone group, sham-operated group, or artery ligation only group. The histology of MSC_PGIS treated hearts revealed MSCs in the infarcted region and decreased myocardial fibrosis/apoptosis with limited cardiac remodeling. Furthermore, the level of the vascular endothelial growth factor was elevated in the MSC_PGIS group as compared to the other three groups.SignificanceIn summary, our results provide both in vitro and in vivo evidence for the beneficial role of MSC_PGIS in limiting the process of detrimental cardiac remodeling in a mouse AMI model during early stages of the disease.     

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells

BackgroundMultidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.MethodsTwo pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.ResultsWe found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.ConclusionsThe determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.General significanceThis work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.     

The role of ABCG2 in maintaining the viability and proliferative activity of bone marrow mesenchymal stem cells in hypoxia

Hypoxia (5% O₂) and the basic fibroblast growth factor (bFGF) were shown to reduce the doubling time of bone marrow mesenchymal stem cells (MSCs) in vitro, indicating an increase in cell proliferation. In addition, abcg2 expression at both the mRNA and protein levels increased in MSCs that were exposed to hypoxia and bFGF. The two factors acting together potentiated the effects of hypoxia in MSCs. Blocking the functional activity of ABCG2 led to a higher production of reactive oxygen species (ROS) in MSCs. Hypoxia and bFGF were tested for effects on the MSC protein profile. These findings, combined with the published data, implicate ABCG2 expression and function in maintaining the viability and proliferative activity of MSCs that are cultured in hypoxia, with ABCG2 playing a protective role.