Hypoxia-preconditioned Mesenchymal Stem Cells Research Articles

Apoptotic extracellular vesicles derived from hypoxia-preconditioned mesenchymal stem cells within a modified gelatine hydrogel promote osteochondral regeneration by enhancing stem cell activity and regulating immunity

Due to its unique structure, articular cartilage has limited abilities to undergo self-repair after injury. Additionally, the repair of articular cartilage after injury has always been a difficult problem in the field of sports medicine. Previous studies have shown that the therapeutic use of mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) has great potential for promoting cartilage repair. Recent studies have demonstrated that most transplanted stem cells undergo apoptosis in vivo, and the apoptotic EVs (ApoEVs) that are subsequently generated play crucial roles in tissue repair. Additionally, MSCs are known to exist under low-oxygen conditions in the physiological environment, and these hypoxic conditions can alter the functional and secretory properties of MSCs as well as their secretomes. This study aimed to investigate whether ApoEVs that are isolated from adipose-derived MSCs cultured under hypoxic conditions (hypoxic apoptotic EVs [H-ApoEVs]) exert greater effects on cartilage repair than those that are isolated from cells cultured under normoxic conditions. Through in vitro cell proliferation and migration experiments, we demonstrated that H-ApoEVs exerted enhanced effects on stem cell proliferation, stem cell migration, and bone marrow derived macrophages (BMDMs) M2 polarization compared to ApoEVs. Furthermore, we utilized a modified gelatine matrix/3D-printed extracellular matrix (ECM) scaffold complex as a carrier to deliver H-ApoEVs into the joint cavity, thus establishing a cartilage regeneration system. The 3D-printed ECM scaffold provided mechanical support and created a microenvironment that was conducive to cartilage regeneration, and the H-ApoEVs further enhanced the regenerative capacity of endogenous stem cells and the immunomodulatory microenvironment of the joint cavity; thus, this approach significantly promoted cartilage repair. In conclusion, this study confirmed that a ApoEVs delivery system based on a modified gelatine matrix/3D-printed ECM scaffold together with hypoxic preconditioning enhances the functionality of stem cell-derived ApoEVs and represents a promising approach for promoting cartilage regeneration.Graphical

Secretome of Hypoxia-Preconditioned Mesenchymal Stem Cells Promotes Liver Regeneration and Anti-Fibrotic Effect in Liver Fibrosis Animal Model.

<b>Background and Objective:</b> Liver fibrosis (LF) is a most common pathological process characterized by the activation of hepatocytes leading to the accumulation of extracellular matrix (ECM). Hypoxia precondition treated in MSCs (H-MSCs) could enhance their immunomodulatory and regeneration capability, through expressing robust anti-inflammatory cytokines and growth factors, known as H-MSCs secretome (SH-MSCs) that are critical for the improvement of liver fibrosis. However, the study regarding the efficacy and mechanism of action of SH-MSCs in ameliorating liver fibrosis is still inconclusive. In this study, the therapeutic potential and underlying mechanism for SH-MSCs in the treatment of liver fibrosis were investigated. <b>Materials and Methods:</b> A rat model with liver fibrosis induced by CCl<sub>4</sub> was created and maintained for 8 weeks. The rats received intravenous doses of SH-MSCs and secretome derived from normoxia MSCs (SN-MSCs), filtered using a tangential flow filtration (TFF) system with different molecular weight cut-off categories, both at a dosage of 0.5 mL. The ELISA assay was employed to examine the cytokines and growth factors present in both SH-MSCs and SN-MSCs. On the ninth day, the rats were euthanized and liver tissues were collected for subsequent histological examination and analysis of mRNA expression. <b>Results:</b> The ELISA test revealed that SH-MSCs exhibited higher levels of VEGF, PDGF, bFGF, IL-10, TGF-β and IL-6 compared to SN-MSCs. <i>In vivo</i>, administration of SH-MSCs notably decreased mortality rates. It also demonstrated a reduction in liver fibrosis, collagen fiber areas, α-SMA positive staining and relative mRNA expression of TGF-β. Conversely, SN-MSCs also contributed to liver fibrosis improvement, although SH-MSCs demonstrated more favorable outcomes. <b>Conclusion:</b> Current findings suggested that SH-MSCs could improve CCl<sub>4</sub>-induced liver fibrosis and decrease α-SMA and TGF-β expression.

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs' size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.

Hypoxic-preconditioned mesenchymal stem cell-derived small extracellular vesicles promote the recovery of spinal cord injury by affecting the phenotype of astrocytes through the miR-21/JAK2/STAT3 pathway.

Secondary injury after spinal cord injury (SCI) is a major obstacle to their neurological recovery. Among them, changes in astrocyte phenotype regulate secondary injury dominated by neuroinflammation. Hypoxia-preconditioned mesenchymal stem cells (MSCs)-derived extracellular vesicle (H-EV) plays a multifaceted role in secondary injury by interacting with cellular components and signaling pathways. They possess anti-inflammatory properties, regulate oxidative stress, and modulate apoptotic pathways, promoting cell survival and reducing neuronal loss. Given the unique aspects of secondary injury, H-EV shows promise as a therapeutic approach to mitigate its devastating consequences. Our study aimed to determine whether H-EV could promote SCI repair by altering the phenotype of astrocytes. Rat bone marrow MSCs (BMSCs) and EVs secreted by them were extracted and characterized. After the SCI model was successfully constructed, EV and H-EV were administered into the tail vein of the rats, respectively, and then their motor function was evaluated by the Basso-Beattie-Bresnahan (BBB) score, Catwalk footprint analysis, and electrophysiological monitoring. The lesion size of the spinal cord was evaluated by hematoxylin-eosin (HE) staining. The key point was to use glial fibrillary acidic protein (GFAP) as a marker of reactive astrocytes to co-localize with A1-type marker complement C3 and A2-type marker S100A10, respectively, to observe phenotypic changes in astrocytes within tissues. The western blot (WB) of the spinal cord was also used to verify the results. We also compared the efficacy differences in apoptosis and inflammatory responses using terminal deoxynucleotidyl transferase dUTP terminal labeling (TUNEL) assay, WB, and enzyme-linked immunosorbent assay (ELISA). Experiments in vitro were also performed to verify the results. Subsequently, we performed microRNA (miRNA) sequencing analysis of EV and H-EV and carried out a series of knockdown and overexpression experiments to further validate the mechanism by which miRNA in H-EV plays a role in promoting astrocyte phenotypic changes, as well as the regulated signaling pathways, using WB both in vivo and in vitro. Our findings suggest that H-EV is more effective than EV in the recovery of motor function, anti-apoptosis, and anti-inflammatory effects after SCI, both in vivo and in vitro. More importantly, H-EV promoted the conversion of A1 astrocytes into A2 astrocytes more than EV. Moreover, miR-21, which was found to be highly expressed in H-EV by miRNA sequencing results, was also demonstrated to influence changes in astrocyte phenotype through a series of knockdown and overexpression experiments. At the same time, we also found that H-EV might affect astrocyte phenotypic alterations by delivering miR-21 targeting the JAK2/STAT3 signaling pathway. H-EV exerts neuroprotective effects by delivering miR-21 to promote astrocyte transformation from the A1 phenotype to the A2 phenotype, providing new targets and ideas for the treatment of SCI.

Secretome of hypoxia-preconditioned mesenchymal stem cells enhance the expression of HIF-1a and bFGF in a rotator cuff tear model.

Aim To determine the effect of secretome hypoxia mesenchymal stem cells (SH-MSCs) on the relative gene expression of hypoxia inducible factor-1a (HIF-1a) and basic fibroblast growth factor (bFGF) in accelerating histomorphometric repair of tendon to bone interface healing in rats acute rotator cuff tear (RCT) model. Methods This is experimental research with posttest control group design. Thirty-male Wistar rats were divided into five treatment groups: healthy group and rotator cuff reconstruction group included four groups: SH-MSCs W2 (the treatment group was given a SH-MSCs 0.5 mL and terminated at weeks 2), NaCl W2 (the control vehicle group was given NaCl 0.5 mL and terminated at weeks 2), SH-MSCs W8 (the treatment group was given a SH-MSCs 0.5 mL and terminated at weeks 8), and NaCl W8 (the control vehicle group was given NaCl 0.5 mL and terminated at weeks 8). On the termination day, all the rats were terminated and HIF-1a and bFGF gene expression were analysed using qRT-PCR. Results SH-MSCs significantly increased the HIF-1a and bFGF gene expression than NaCl group even in week 2 and week 8. The highest increased gene expression of HIF-1a and bFGF was on week 8. Conclusion SH-MSCs are important in the healing repair process of tendon-to-bone interface in acute RCT model rats through increasing gene expression of HIF-1α and bFGF.

Hypoxic preconditioned mesenchymal stem cells ameliorate rat brain injury after cardiopulmonary resuscitation by suppressing neuronal pyroptosis.

Cardiac arrest (CA) can result in cerebral ischaemia-reperfusion injury and poor neurological outcomes. While bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to have protective effects in brain ischaemic disease, their efficacy can be reduced by the poor oxygen environment. In this study, we investigated the neuroprotective effects of hypoxic preconditioned BMSCs (HP-BMSCs) and normoxic BMSCs (N-BMSCs) in a cardiac arrest rat model by examining their ability to ameliorate cell pyroptosis. The mechanism underlying the process was also explored. Cardiac arrest was induced in rats for 8 min and surviving rats received 1 × 106 normoxic/hypoxic BMSCs or PBS via intracerebroventricular (ICV) transplantation. Neurological function of rats was evaluated using neurological deficit scores (NDSs) and examined for brain pathology. Serum S100B and neuron-specific enolase (NSE) levels and cortical proinflammatory cytokines were measured to evaluate brain injury. Pyroptosis-related proteins in the cortex after cardiopulmonary resuscitation (CPR) were measured using western blotting and immunofluorescent staining. Transplanted BMSCs were tracked using bioluminescence imaging. Results showed significantly better neurological function and neuropathological damage after transplantation with HP-BMSCs. In addition, HP-BMSCs reduced levels of pyroptosis-related proteins in the rat cortex after CPR and significantly reduced levels of biomarkers for brain injury. Mechanistically, HP-BMSCs alleviated brain injury by reducing the expressions of HMGB1, TLR4, NF-κB p65, p38 MAPK and JNK in the cortex. Our study demonstrated that hypoxic preconditioning could enhance the efficacy of BMSCs in alleviating post-resuscitation cortical pyroptosis. This effect may be related to the regulation of the HMGB1/TLR4/NF-κB, MAPK signalling pathways.

Exosomes derived from hypoxia-preconditioned mesenchymal stem cells (hypoMSCs-Exo): advantages in disease treatment.

Mesenchymal stem cells (MSCs)-based therapy has been reported to be a potential approach to treat various diseases and the paracrine role might be the underlying mechanism. Exosomes were considered an important part of this paracrine role. It was reported that maintenance of MSCs in hypoxia conditions for a short time has shown to be beneficial for the therapeutic effect of MSCs and MSCs-derived exosomes. In this review, we summarized the recent developments on exosomes derived from hypoxia-preconditioned mesenchymal stem cells (hypoMSCs-Exo), including the characteristics of hypoMSCs-Exo in morphology and contents, diseases in which hypoMSCs-Exo showed more effective, and the cellular and molecular mechanisms that hypoMSCs-Exo showed more effective in disease treatment. Besides, we also discussed the limitations of current studies and the issues that needed to be improved in the application of hypoMSCs-Exo. This review aimed to promote a comprehensive and systematic understanding of this type of exosome with great therapeutic potential.

Secretome of Hypoxia-Preconditioned Mesenchymal Stem Cells Enhance Angiogenesis in Diabetic Rats with Peripheral Artery Disease.

Lower limb peripheral artery disease (PAD) is the main risk of diabetes mellitus which result to high mortality rate. Approximately, 50% of patients who receive several treatments have passed away or lost limbs at a year's follow-up. Secretome of hypoxia mesenchymal stem cells (S-MSCs) contains several active soluble molecules from hypoxia MSCs (H-MSCs) that capable inducing anti-inflammatory and vascular regeneration in PAD. In this study, we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats. The PAD was established by the incision from the groin to the inner thigh and distal ligation of femoral arteries in rats with diabetes. Rats were administered with 200 µL and 400 µL S-MSCs that successfully filtrated using tangential flow filtration (TFF) system based on various molecular weight cut-off categories intravenously. ELISA assay was used to analyze the cytokines and growth factors contained in S-MSCs. Tarlov score were examined at day 1, 3, 5, 7, 10 and 14. The rats were sacrificed at day 14 and muscle tissues were collected for immunohistochemistry (IHC) and gene expression analysis. ELISA assay showed that S-MSCs provides abundant level of VEGF, PDGF, bFGF, IL-10 and TGFβ. In vivo administration of S-MSCs remarkably enhanced the Tarlov score. S-MSCs improved angiogenesis through enhancing VEGF gene expression and significantly increasing CD31 positive area in muscle tissue of PAD diabetic rats. Our findings suggest that S-MSCs could improves dynamic function and angiogenesis in PAD diabetic rats.

Effects of hypoxia-preconditioned HucMSCs on neovascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice

BackgroundThe massive loss of follicles in the early stage of ovarian tissue transplantation is considered a significant restriction to the efficacy of ovarian tissue cryopreservation (OTC) and transplantation (OT). The use of mesenchymal stem cells (MSCs) before transplantation of ovarian fragments shortened the hypoxic period and boosted neovascularization. Hypoxia-preconditioned MSCs can enhance the potential of angiogenesis. Can hypoxia-preconditioned human umbilical cord mesenchymal stem cell (HucMSCs) and ovarian tissue co-xenotransplantation improve more neovascularization and subsequently more follicle survival in human ovarian tissue?MethodsFrozen-thawed cortical pieces from 4 patients were transplanted into the bilateral renal capsule of immune-deficient nude mice without HucMSCs or normoxia/hypoxia-preconditioned HucMSCs. Sixty-four mice were randomly distributed into 4 groups. In each group, the mice were euthanized for blood and/or graft retrieval on post-transplantation days 3 (n = 8) and 7 (n = 8), respectively. Non-grafted frozen-thawed ovarian fragment was taken for non-grafted control. Grafts were histologically processed and analysed for follicle density and atretic follicles by HE, neovascularization by CD34 and CD31 immunohistochemical staining, primordial follicle growth by Ki67 staining, and apoptosis of stromal cell and follicles by immunofluorescence using TUNEL. The ROS and TAC levels of grafted and non-grafted tissue were assessed. We evaluated the protein expression of HIF1α, VEGFA, pAkt, Akt, and GDF9 in grafted and non-grafted ovarian tissue. E2, Prog, AMH, and FSH levels in the plasma of mice were measured after 3 and 7 days of OT.ResultsHypoxia-preconditioned HucMSCs positively protect the grafted ovarian tissue by significantly decreasing the apoptosis and increasing higher expression of CD31, CD34, and VEGFA for earlier angiogenesis. They are crucial to preserving the resting primordial follicle pool by modulation of follicle death.ConclusionThis is the first study to demonstrate that co-transplantation of hypoxia-preconditioned HucMSC with ovarian tissue improved earlier vascularization of ovarian grafts in the early post-grafting period, which correlates with increased follicle survival and reduced apoptosis. The HIF1α/VEGFA signal pathways may play an important role in elucidating the mechanisms of action of hypoxia-preconditioned HucMSCs with regard to OT and clinical implementation.

Small extracellular vesicles derived from hypoxic mesenchymal stem cells promote vascularized bone regeneration through the miR-210-3p/EFNA3/PI3K pathway

Angiogenesis is closely coupled with osteogenesis and has equal importance. Thus, promoting angiogenesis during the bone repair process is vital for ideal bone regeneration. As important mediators of cell-cell communication and biological homeostasis, mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proved to be highly involved in bone and vascular regeneration. Because hypoxia microenvironment promotes the proangiogenic activity of MSCs, in the present study, we investigate the effect and underlying molecular mechanisms of sEVs from hypoxia-preconditioned MSCs (hypo-sEVs) on angiogenesis and develop an effective strategy to promote vascularized bone regeneration. Compared to sEVs from normoxia MSCs (nor-sEVs), hypo-sEVs promoted the proliferation, migration, and angiogenesis of HUVECs and ultimately enhanced bone regeneration and new blood vessel reconstruction in a critical-size calvarial bone defect model. miRNA sequence and the verified results showed that miR-210-3p in hypo-sEVs was increased via HIF-1α under hypoxia. The upregulated miR-210-3p in hypo-sEVs promoted angiogenesis by downregulating EFNA3 expression and enhancing the phosphorylation of the PI3K/AKT pathway. Thus, this study suggests a successful strategy to enhance vascularized bone regeneration by utilizing hypo-sEVs via the miR-210-3p/EFNA3/PI3K/AKT pathway. Statement of significanceConsidering the significance of vascularization in ideal bone regeneration, strategies to promote angiogenesis during bone repair are required. Hypoxia microenvironment can promote the proangiogenic potential of mesenchymal stem cells (MSCs). Nonetheless, the therapeutic effect of small extracellular vesicles (sEVs) from hypoxia-preconditioned MSCs on cranio-maxillofacial bone defect remains unknown, and the underlying mechanism is poorly understood. This study shows that hypo-sEVs significantly enhance the proliferation, migration, and angiogenesis of HUVECs as well as promote vascularized bone formation. Moreover, this work indicates that HIF-1α can induce overexpression of miR-210-3p under hypoxia, and miR-210-3p can hinder EFNA3 expression and subsequently activate the PI3K/AKT pathway. The application of hypo-sEVs provides a facile and promising strategy to promote vascularized bone regeneration in a critical-size bone defect model.

Secretome of Mesenchymal Stem Cells from Consecutive Hypoxic Cultures Promotes Resolution of Lung Inflammation by Reprogramming Anti-Inflammatory Macrophages.

The secretome from hypoxia-preconditioned mesenchymal stem cells (MSCs) has been shown to promote resolution of inflammation and alleviate acute lung injury (ALI) through its immunomodulatory function. However, the effects of consecutive hypoxic culture on immunomodulatory function of the MSCs secretome are largely unclarified. Here, we intend to investigate the effects of consecutive hypoxia on therapeutic efficacy of conditioned medium derived from MSCs (MSCs-CM) in alleviating ALI. Human umbilical cord-derived MSCs (UC-MSCs) were consecutively cultured in 21% O2 (Nor-MSCs) or in 1% O2 (Hypo-MSCs) from passage 0. Their conditioned medium (Nor-CM and Hypo-CM respectively) was collected and administered into ALI models. Our findings confirmed that Hypo-MSCs exhibited increased proliferation ability and decreased cell senescence compared with Nor-MSCs. Consecutive hypoxia promoted UC-MSCs to secrete immunomodulatory cytokines, such as insulin-like growth factor 1(IGF1), IL10, TNFα-stimulated gene 6(TSG6), TGFβ, and prostaglandin E2 (PGE2). Both Nor-CM and Hypo-CM could effectively limit lung inflammation, promote efferocytosis and modulate anti-inflammatory polarization of lung macrophages in ALI models. Moreover, the effects of Hypo-CM were more potent than Nor-CM. Taken together, our findings indicate that consecutive hypoxic cultures could not only promote both proliferation and quality of UC-MSCs, but also enhance the therapeutic efficacy of their secretome in mitigating lung inflammation by promoting efferocytosis and anti-inflammatory polarization of macrophages.

Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells alleviates myocardial injury by targeting thioredoxin-interacting protein-mediated hypoxia-inducible factor-1α pathway.

BACKGROUNDExtracellular vesicles (EVs) derived from hypoxia-preconditioned (HP) mesenchymal stem cells (MSCs) have better cardioprotective effects against myocardial infarction (MI) in the early stage than EVs isolated from normoxic (NC)-MSCs. However, the cardioprotective mechanisms of HP-EVs are not fully understood.AIMTo explore the cardioprotective mechanism of EVs derived from HP MSCs.METHODSWe evaluated the cardioprotective effects of HP-EVs or NC-EVs from mouse adipose-derived MSCs (ADSCs) following hypoxia in vitro or MI in vivo, in order to improve the survival of cardiomyocytes (CMs) and restore cardiac function. The degree of CM apoptosis in each group was assessed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/PI assays. MicroRNA (miRNA) sequencing was used to investigate the functional RNA diversity between HP-EVs and NC-EVs from mouse ADSCs. The molecular mechanism of EVs in mediating thioredoxin-interacting protein (TXNIP) was verified by the dual-luciferase reporter assay. Co-immunoprecipitation, western blotting, and immunofluorescence were performed to determine if TXNIP is involved in hypoxia-inducible factor-1 alpha (HIF-1α) ubiquitination and degradation via the chromosomal region maintenance-1 (CRM-1)-dependent nuclear transport pathway.RESULTSHP-EVs derived from MSCs reduced both infarct size (necrosis area) and apoptotic degree to a greater extent than NC-EVs from CMs subjected to hypoxia in vitro and mice with MI in vivo. Sequencing of EV-associated miRNAs showed the upregulation of 10 miRNAs predicted to bind TXNIP, an oxidative stress-associated protein. We showed miRNA224-5p, the most upregulated miRNA in HP-EVs, directly combined the 3’ untranslated region of TXNIP and demonstrated its critical protective role against hypoxia-mediated CM injury. Our results demonstrated that MI triggered TXNIP-mediated HIF-1α ubiquitination and degradation in the CRM-1-mediated nuclear transport pathway in CMs, which led to aggravated injury and hypoxia tolerance in CMs in the early stage of MI. CONCLUSIONThe anti-apoptotic effects of HP-EVs in alleviating MI and the hypoxic conditions of CMs until reperfusion therapy may partly result from EV miR-224-5p targeting TXNIP.

Hypoxic preconditioning promotes the immunosuppressive effects of mesenchymal stem cells in mice with colitis

Mesenchymal stem cells are promising candidates for stem cell therapy in many diseases, especially in immune-associated diseases. Inflammatory bowel disease is a chronic autoimmune disease that can lead to colorectal cancer if it is not controlled. Mesenchymal stem cells are always under a hypoxic environment in vivo, whether in bone marrow or adipose tissue, whereas researchers always culture MSCs (mesenchymal stem cells) under normoxic conditions (21%). In this study, we aimed to investigate whether hypoxia (1%) affects the therapeutic effect of MSCs. We hypothesize that hypoxia may benefit the treatment efficacy of MSCs. We used DSS to induce IBD (inflammatory bowel disease) in mice and then injected MSCs that had been preconditioned under normoxic conditions (21%) and hypoxic conditions (1%). We found that compared with normoxic-preconditioned MSCs (n-MSCs), hypoxic-preconditioned MSCs (h-MSCs) could alleviate colon inflammation to a large extent, as determined by inflammatory cytokines and CD3+ T cell activation. Mechanistic studies showed that hypoxia could promote iNOS expression in MSCs. Therefore, our data suggest that hypoxia may be more appropriate than normoxia for facilitating MSCs exertion of therapeutic functions.

Hypoxia-preconditioned mesenchymal stem cells attenuate microglial pyroptosis after intracerebral hemorrhage.

BackgroundMicroglia plays a vital role in neuroinflammation, contributing to the pathogenesis of intracerebral hemorrhage (ICH)-induced brain injury. Mesenchymal stem cells (MSCs) hold great potential for treating ICH. We previously revealed that MSCs ameliorate the microglial pyroptosis caused by an ischemic stroke. However, whether MSCs can modulate microglial pyroptosis after ICH remains unknown. This study aimed to investigate the neuroprotective effects of hypoxia-preconditioned olfactory mucosa MSCs (OM-MSCs) on ICH and the possible mechanisms.MethodsICH was induced in mice via administration of collagenase IV. At 6 h post-ICH, 2–4×105 normoxic/hypoxic OM-MSCs or saline were intracerebrally administered. To evaluate the neuroprotective effects, the behavioral outcome, apoptosis, and neuronal injury were measured. Microglia activation and pro-inflammatory cytokines were applied to detect neuroinflammation. Microglial pyroptosis was determined by western blotting, immunofluorescence staining, and transmission electron microscopy (TEM).ResultsThe two OM-MSC-transplanted groups exhibited significantly improved functional recovery and reduced neuronal injury, especially the hypoxic OM-MSCs group. Hypoxic OM-MSCs attenuated microglial activation as well as the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Moreover, we found that hypoxia-preconditioned OM-MSCs ameliorated pyroptosis by diminishing the levels of pyroptosis-associated proteins in peri-hematoma brain tissues, decreasing the expression of the microglial nod-like receptor family protein 3 (NLRP3) and caspase-1, and reducing the membrane pores on microglia post-ICH.ConclusionsOur study showed that hypoxic preconditioning augments the therapeutic efficacy of OM-MSCs, and hypoxia-preconditioned OM-MSCs alleviate microglial pyroptosis in the ICH model.

Exosomes Secreted from Hypoxia-Preconditioned Mesenchymal Stem Cells Prevent Steroid-Induced Osteonecrosis of the Femoral Head by Promoting Angiogenesis in Rats.

Recent studies have suggested that exosomes exert similar therapeutic effects to those of mesenchymal stem cells (MSCs) in regenerative medicine and MSCs-derived exosomes exhibit therapeutic effects on steroid-induced osteonecrosis of the femoral head (ONFH). Furthermore, reparative functions of exosomes from MSCs are enhanced by hypoxia treatment of the cells. However, there are no related reports about whether exosomes derived from hypoxia-preconditioned MSCs could show better therapeutic effects on steroid-induced ONFH. In vitro, we investigated the effects of hypoxia precondition on exosomes derived from bone marrow mesenchymal stem cells (BMMSCs) from rats and the proangiogenic ability of exosomes derived from hypoxia-preconditioned BMMSCs. In vivo, we investigated the role of exosomes from hypoxia-preconditioned BMMSCs on angiogenesis and protecting osteonecrosis in a rat ONFH model. We found that the potential of the proangiogenic ability of exosomes derived from hypoxia-preconditioned BMMSCs was higher than exosomes derived from BMMSCs cultured under normoxia. Exosomes derived from hypoxia-preconditioned BMMSCs significantly promoted proliferation, migration, vascular endothelial growth factor (VEGF) expression, and tube formation of human umbilical vein endothelial cells (HUVECs) compared with exosomes derived from BMMSCs cultured under normoxia. Administration of exosomes derived from hypoxia-preconditioned BMMSCs significantly prevented bone loss and increased vessel volume in the femoral head compared with exosomes derived from BMMSCs cultured under normoxia. Taken together, our data suggest that exosomes derived from hypoxia-preconditioned BMMSCs exert better therapeutic effects on steroid-induced ONFH by promoting angiogenesis and preventing bone loss.

Mesenchymal stem cells under hypoxia condition inhibit peritoneal adhesion by suppressing the prolonged release of interleukin-6.

Aim To investigate the role of hypoxia-preconditioned mesenchymal stem cells (H-MSCs) in preventing peritoneal adhesion by regulating IL-6 at days 6 and 12. Methods Twenty-four PAs rat model weighing 250 g to 300 g were randomly allocated into 4 groups: sham (Sh), control (C), H-MSCs treatment group at dose 1.5 x 106(T1) and 3 x 106(T2). To induce H-MSCs, all MSCs population were incubated under hypoxia state (5% O2 ), 5% CO2, and 37oC for 24 hours. Expression level of IL-6 was performed using ELISA. Morphological appearance of adhesion was observed by visualizing the existence of adhesion formation in intestinal. Results In this study we found that there was a trend of decrease of IL-6 level on day 6 following MSCs treatments. Interestingly, there was a significant decrease of IL-6 level on day 12 in all treatment groups. Also, no adhesion occurred in T2 group. Conclusions H-MSCs prevent PA development by suppressing the prolonged release of IL-6 at proliferation phase.

Hypoxia-preconditioned mesenchymal stem cells attenuate peritoneal adhesion through TGF-β inhibition

BackgroundPeritoneal adhesions (PAs) are generally described as fibrous bands between intra-abdominal organs following an abdominal surgical operation. The definitive treatments of PAs are currently ineffective yet. Hypoxia-mesenchymal stem cells (H-MSCs) have a higher capability to survive at the site of injury than normoxia-MSCs (N-MSCs) to repair injured tissue without fibrosis. This study aimed to analyze the effect of H-MSCs in controlling formation of PAs by reducing TGF-β level in a rat model. Methods A study of post-test only control group design was conducted, involving eighteen PA rat models weighing 250 ± 25 g that were randomly assigned into 3 groups, comprising control group (C), and groups T1 and T2 receiving H-MSC treatment at doses of 3 x 106 and 1.5 x 106, respectively. To induce H-MSCs, MSCs were incubated in hypoxic conditions at 5% O2 and 37oC for 24 hours. Expression level of TGF-β was analyzed by enzyme-linked immunosorbent assay (ELISA) at 450 nm and adhesion formation was described macroscopically. The Kruskal-Wallis variance analysis was used to analyze significant differences among the groups.ResultsThe results of this study showed that H-MSCs in group T1 inhibited TGF-β expression significantly on day 8 (p<0.001) and day 14 (p<0.05). Moreover, there was almost no adhesion apparent following H-MSC administration in group T1. ConclusionsBased on this study, we conclude that H-MSCs may attenuate PA formation following inhibition of TGF-β expression in the PA rat model.

Effect of Hypoxia Preconditioned Adipose-Derived Mesenchymal Stem Cell Conditioned Medium on Cerulein-Induced Acute Pancreatitis in Mice.

Purpose: Acute pancreatitis (AP) is an inflammatory disorder distinguished by tissue injury and inflammation of the pancreas. Using paracrine potential of mesenchymal stem cells (MSCs) provides a useful clinical approach in treating inflammatory diseases. We investigated the therapeutic effects of adipose-derived MSC conditioned medium (CM) and hypoxia preconditioned adipose-derived MSC conditioned medium (HCM) in cerulein-induced AP in mice. Methods: AP was induced in C57BL/6 mice by intraperitoneal injection of cerulein (75 μg/ kg/h × 7 times). One hour following the last injection of cerulein, mice were treated with intraperitoneal injection of CM and HCM (500 µL/mice/30 min × 3 times). Twelve hours following the treatment, serum levels of amylase and lipase were measured. In addition, pancreas pathological changes, immunohistochemical examinations for evaluation of IL-6 expression and pancreatic myeloperoxidase (MPO) enzyme activity were analyzed. Results: The in vitro results of the morphological, differentiation and immunophenotyping analyses confirmed that hypoxia preconditioned MSCs (HP-MSCs) conserve MSCs characteristics after preconditioning. However, HP-MSCs significantly expressed high mRNA level of hypoxia inducible factor 1-α and higher level of total protein. The in vivo findings of the current study showed that CM and HCM significantly reduced the amylase & lipase activity, the severity of pancreas tissue injury and the expression of IL-6 and MPO enzyme activity compared with the AP group. However, no significant difference between CM and HCM groups was demonstrated. Conclusion: Use of CM and HCM can attenuate cerulein-induced AP and decrease inflammation in the pancreas tissue in AP mice.

Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization

BackgroundSpinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism.MethodsElectron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms.ResultsOur results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p.ConclusionHypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

Intrarenal Transplantation of Hypoxic Preconditioned Mesenchymal Stem Cells Improves Glomerulonephritis through Anti-Oxidation, Anti-ER Stress, Anti-Inflammation, Anti-Apoptosis, and Anti-Autophagy.

To confer further therapeutic potential and prevent some adverse effects by the mesenchymal stem cells (MSCs) transplantation, we explored the effects of locally intrarenal arterial administration of hypoxic preconditioned MSCs in the anti-Thy1.1 induced rat glomerulonephritis. Proteinuria, histochemical staining, and western blotting were used to explore the therapeutic effects and mechanisms. Locally intrarenal arterial MSCs transplantation successfully implanted the fluorescent or CD44 labeled MSCs in the nephritic glomeruli, ameliorated proteinuria, and glomerulosclerosis in nephritic rats. Hypoxic preconditioning significantly upregulated hypoxic inducible factor-1α/VEGF (HIF-1α/VEGF) in the MSCs and was more efficient than normoxic MSCs in reducing the degree of urinary protein, glomerulosclerosis, fibrosis, macrophage/monocyte infiltration, GRP78 mediated endoplasmic reticulum stress, Beclin-1/LC3-II mediated autophagy, and Bax/Bcl-2/caspase 3 mediated apoptosis. Hypoxic MSCs could further promote intranuclear nuclear factor (erythroid-derived 2, Nrf2) and reduce nuclear factor kappa B expression in nephritic kidneys. As compared to normoxic MSCs, hypoxic MSCs transplantation significantly upregulated the renal expression of anti-oxidative response elements/enzymes including glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione peroxidase, catalase, Mn, and Cu/Zn superoxide dismutase. In summary, intrarenal hypoxic preconditioning MSCs transplantation was more effective to activate hypoxic inducible factor-1α/VEGF/Nrf2 (HIF-1α/VEGF/Nrf2) signaling, preserve anti-oxidant proteins and anti-oxidative responsive element proteins, and subsequently reduce glomerular apoptosis, autophagy, and inflammation.