Hypoxic Mesenchymal Stem Cells Research Articles

The Effect of Hypoxic Mesenchymal Stem Cells (S-HypMSCs) Secretome Creamon AP1S3 Gene Expression (In Vivo Study on Psoriasis-like Rats Model Induced by Imiquimod)

Psoriasis is known as a complex disease with interactions between genes, the body's immune system and environmental factors, which is a chronic inflammation characterized by scaly red plaques. Secretome Hypoxic Mesenchymal Stem Cells (S-HypMSCs) contain various components released by MSCs consisting of chemokines, growth factors and anti-inflammatory cytokines, making it possible as a therapeutic option. This study aims to investigate the effect of S-HypMSCs on psoriasis improvement. MSCs were isolated from the umbilical cord of rats, Secretome which could be mixed with cream and applied to the wounds of psoriasis-like animal models at a dose of 100 μL/kgBW in 100 mg cream for group P1 and 200 μL/kgBW in 100 mg cream for group P2. The control group was treated with a cream base. The mRNA expression of AP1S3 was analyzed using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). All examinations were assessed on day 14. There was a significant difference in the healthy group against the psoriasis-like group, psoriasis-like treatment 1 and treatment 2 groups. administration of S-HypMSCs at a dose of 100 μL/kgBW in 100 mg cream and a dose of 200 μL/kgBW in 100 mg cream significantly decreased AP1S3 expression in Psoriasis-like male white rats of the Wistar strain. The administration of S-HypMSCs cream at a dose of 100 μL/kgBW in 100 mg cream and a dose of 200 μL/kgBW in 100 mg cream had an effect on decreasing AP1S3 gene expression between treatment groups compared to controls in psoriasis-like male Wistar rats. imiquimod induced.

Secretome of Hypoxia-Preconditioned Mesenchymal Stem Cells Ameliorates Hyperglycemia in Type 2 Diabetes Mellitus Rats

Introduction: Type 2 diabetes mellitus (T2DM) is a prevalent form of diabetes that affects 90 - 95 % of all diabetic patients. Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia; however, they are accompanied by side effects. As a result, new approaches are required to address insulin resistance and regenerate beta cells simultaneously. The secretome of hypoxic mesenchymal stem cells (SH-MSCs) contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function. Objectives: In this study, we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T2DM rats. Methods: We created a T2DM rat model using a combination of a high-fat diet (HFD) and streptozotocin (STZ) administration. Then, we administered SH-MSCs injection at doses of 250 and 500 µL and assessed the therapeutic effects of SH-MSCs. We also investigated the potential underlying mechanisms involved. Results: The administration of SH-MSCs improved hyperglycemia in rats with T2DM. Infusion of SH-MSCs at 500 µL dose decreased homeostatic model assessment for insulin resistance (HOMA-IR). Histological analysis revealed that injection of SH-MSCs alleviated morphological damage of pancreas. SH-MSCs administration also inhibit the level of IL-6 and promote the expression of CD163 type 2 macrophage. Conclusion: The results of our study indicate that SH-MSCs have the potential to improve hyperglycemia and exert a protective effect on T2DM rats. HIGHLIGHTS Administration of SH-MSCs effectively improved hyperglycemia and decreased insulin resistance in TD2M rats through modulation of IL-6 levels and promotes the expression of CD163 type 2 macrophage Histological analysis demonstrated the protective effect of SH-MSCs on pancreatic morphology SH-MSCs hold promise for improving hyperglycemia, insulin resistance, and providing a protective effect in TD2M GRAPHICAL ABSTRACT

Hypoxic mesenchymal stem cells (MSCS)-induced interleukin (IL)-10 alleviate systemic lupus erythematosus (SLE) infl ammation through inhibiting interferon (IFN)-gamma production

Hypoxic mesenchymal stem cells (MSCS)-induced interleukin (IL)-10 alleviate systemic lupus erythematosus (SLE) infl ammation through inhibiting interferon (IFN)-gamma production

The Effect of Topical Gel Secretome Hypoxic Mesenchymal Stem Cells (SH-MSCs) on IL-6 and TGF- β Gene Expression (Experimental Study in Male Wistar Alopecia-Like Rat Models Induced by Fluconazole)

Alopecia induced by the accumulation of reactive oxygen species (ROS) stimulates changes in the immune and neuroendocrine systems. Hair follicles have dermal papillary cells (DPCs) that bind to corticotropin-releasing factor (CRF) receptors. Research shows that transforming growth factor (TGF-β) is one of the expressions that can be an indication of the influence of CRF. The effect of CRF itself is to cause hair loss. The purpose of this study was to determine the Secretome of Hypoxia Mesenchymal Stem Cells (SH-MSCs) against IL-6 expression and TGF-β expression. This study used experimentally in vivo with the Post Test Only Control Group Design method. The subjects of this study were male rats of Wistar strain which were divided into 4 treatments consisting of 1 healthy group, 1 negative control group, 1 treatment group with SH-MSCs dose 10% in a dose of 200 mg/rat/day gel, and 1 more treatment group with SH-MSCs dose 20% in a dose of 200 mg/rat/day. On the 21st day, IL-6 expression and TGF-β expression were examined. Data were analyzed using the One-Way ANOVA Test and the Kruskall-Wallis Test to determine the effect of each group. The One-Way ANOVA test showed the results of IL-6 expression (p = 0.000) showed a significant difference (p < 0.05) and the Kruskall-Wallis test of TGF-β expression (p = 0.000) with SH-MSCs dose 200 was a significant difference (p < 0.05). The administration of 10% and 20% doses of SH-MSCs in 200 mg gel may decrease IL-6 expression. Dosed SH-MSCs 20%increased TGF-β expression in alopecia-like rats. The development of the use of SH-MSCs is expected to be an alternative to alopecia therapy that is more beneficial and safe for the body.

The Effect of Secretome Hypoxic Mesenchymal Stem Cells on Pro-Inflammatory and Anti-Inflammatory Markers Expression (in Vivo Experimental Study on the Macrophage Cell Polarization of Wistar Male Mice Type 1 DM Model)

The autoreactivity of T cells in DMT1 patients causes activation of M1 and M2 macrophages as proinflammatory and anti-inflammatory markers which promote pancreatic cell damage. Secretome Hypoxic Mesenchymal Stem Cells (SH-MSCs) therapy methods play a role in repairing damaged tissue and regenerating pancreatic islet cells. This study aims to examine the effect of SH-MSCs on macrophage cell polarization through the expression of CD68 as a marker of M1 macrophages and CD163 as a marker of M2 macrophages in DMT1 rats. The sample of this study were 5 Wistar rats which were divided into 4 groups; normal group (no treatment), negative control group, and 2 treatment groups with 0.5cc of SH-MSCs (P1 group) and 1cc of SH-MSCs (P2 group). STZ 60 mg/kgBW induced DMT1 in the negative control, P1, and P2 groups. Data were tested parametrically using SPSS 22. The mean of CD68 expression in normal group was 3.06±0.35%, negative control: 21.67±0.93%, P1: 10.98±0.68%, P2: 5.85±0.55%. The mean of CD163 expression in normal group: 22.83±0.51%, negative control group: 3.56±0.50%, P1:15.81±1.80%, P2: 20.80±2.24%. The results of the normality and homogeneity test were p> 0.05. Anova test results p < 0.05. Post Hoc LSD test results p < 0.05. The administration of SH-MSCS was shown to decrease CD68 and increase the CD163 expression significantly in DMT1 rats.

Hypoxic Mesenchymal Stem Cells Promote Diabetic Wound Healing in Rats by Increasing VEGF Secretion

Hypoxic Mesenchymal Stem Cells Promote Diabetic Wound Healing in Rats by Increasing VEGF Secretion

The Comparison of Normoxic and Hypoxic Mesenchymal Stem Cells in Regulating Platelet-derived Growth Factors and Collagen Serial Levels in Skin Excision Animal Models

BACKGROUND: The healing process of a skin excisions involves a complex cascade of cellular responses to reverse skin integrity formation. These processes require growth factors particularly platelet-derived growth factors (PDGF). On the other hand, hypoxia- preconditioned mesenchymal stem cells (MSCs) could secrete growth factors that notably contribute to wound healing acceleration, characterized by the enhancement of collagen density. AIM: This study was aimed to investigate the role of hypoxia-preconditioned MSCs in regulating the serial levels of PDGF associated with the enhancement of collagen density in the skin excision animal models. METHODS: Twenty-seven male Wistar rats of skin excision were created as animal models. The animals were randomly assigned into four groups consisting of two treatment groups (treated by normoxia-preconditioned MSCs as T1 and hypoxia-preconditioned MSCs as T2), positive control (treated with phosphate-buffered saline) and sham (non-treated and healthy rats). PDGF levels were examined by ELISA. The collagen density was determined using Masson‘s trichrome staining. RESULTS: This study showed that there was a significant increase in PDGF levels on days 3 and 6 after hypoxia- preconditioned MSCs treatment. In line with these findings, the collagen density was also increased significantly after hypoxia-preconditioned MSCs treatment on days 3, 6, and 9. CONCLUSION: Hypoxia-preconditioned MSCs could regulate the serial PDGF levels that lead to the enhancement of collagen density in the skin excision rat’s model.

Secretome Hypoxia Mesenchymal Stem Cells Inhibited Ultraviolet Radiation by Inhibiting Interleukin-6 through Nuclear Factor-Kappa Beta Pathway in Hyperpigmentation Animal Models

UVB radiation is the main factor causing hyperpigmentation. Secretome hypoxic mesenchymal stem cells (S-HMSCs) contain bioactive soluble molecules such as growth factors and anti-inflammatory cytokines that can prevent melanin synthesis and induce collagen formation. However, the role of S-HMSCSs on IL-6, p50, and p65 gene expression in hyperpigmentation is still unclear. This study aimed to determine the effect of administration of S-HMSCSs gel on the expression of IL-6, p50, and p65 in a hyperpigmented rat skin model induced by UVB light exposure. Twenty-five male Wistar rats of hyperpigmented were created as an animal model under exposed to UVB 6 times in 14 days at 302 nm with a MED of 390 mJ/cm2. The animal was randomly assigned into five groups consisting of two treatment groups (treated by S-HMSCs at a 100µL as T1 and 200µL as T2 on bases gel) for 14 days, control groups (UVB-irradiation), sham (negative control), and base gel groups. On the 14th day, IL-6, p50, and p65 were terminated and analyzed using qRT-PCR. Statistical analysis will perform using one way ANOVA followed with post hoc LSD test. Analysis of IL-6 (8.59± 3.32), p50 (4.35±2.27), and p65 (4.09±1.82) gene expression in the treatment group decreased along with the increase in the concentration of S-MSCs compared to the control group. In conclusion, the administration of S-HMSCs gel is expected to affect the speed of decreasing the hyperpigmentation process significantly.

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.

Hypoxia-induced mesenchymal stem cells inhibit corneal fibroblast proliferation by regulating the WWP2/Notch1 axis.

Aim: This study aimed to explore the role of hypoxic mesenchymal stem cells (MSCs) in corneal alkali burns and the underlying mechanism. Materials & methods: Rat corneal fibroblasts were incubated with IL-6, followed by treatment with hypoxic MSC supernatant. A rat corneal alkali burn model was implemented and processed with hypoxic MSCs. The associated factors were detected by corresponding methods. Results: Hypoxic MSCs reduced the Notch1 level and the proliferation of rat corneal fibroblasts. Hypoxic MSCs or WWP2 overexpression in MSCs enhanced ubiquitination of Notch1. WWP2 interacted with Notch1, and WWP2 silencing reversed the effects of the hypoxic MSCs. Hypoxic MSC treatment in vivo decreased the corneal neovascularization scores and opacity scores. Conclusion: Hypoxic MSCs inhibited inflammation and alleviated corneal injury in alkali burns via the WWP2/Notch1 axis.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. Statement of significanceIntervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

Extracellular vesicles derived from hypoxia-preconditioned olfactory mucosa mesenchymal stem cells enhance angiogenesis via miR-612

Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3′-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer.Graphical

Mkx mediates tenogenic differentiation but incompletely inhibits the proliferation of hypoxic MSCs

BackgroundHypoxia has been shown to be able to induce tenogenic differentiation and proliferation of mesenchymal stem cells (MSCs) which lead hypoxia-induced MSCs to be a potential treatment for tendon injury. However, little is known about the mechanism underlying the tenogenic differentiation and proliferation process of hypoxic MSCs, which limited the application of differentiation-inducing therapies in tendon repair. This study was designed to investigate the role of Mohawk homeobox (Mkx) in tenogenic differentiation and proliferation of hypoxic MSCs.MethodsqRT-PCR, western blot, and immunofluorescence staining were performed to evaluate the expression of Mkx and other tendon-associated markers in adipose-derived MSCs (AMSCs) and bone marrow-derived MSCs (BMSCs) under hypoxia condition. Small interfering RNA technique was applied to observe the effect of Mkx levels on the expression of tendon-associated markers in normoxic and hypoxic BMSCs. Hypoxic BMSCs infected with Mkx-specific short hair RNA (shRNA) or scramble were implanted into the wound gaps of injured patellar tendons to assess the effect of Mkx levels on tendon repair. In addition, cell counting kit-8 assay, colony formation unit assay, cell cycle analysis, and EdU assay were adopted to determine the proliferation capacity of normoxic or hypoxic BMSCs infected with or without Mkx-specific shRNA.ResultsOur data showed that the expression of Mkx significantly increased in hypoxic AMSCs and increased much higher in hypoxic BMSCs. Our results also detected that the expression of tenogenic differentiation markers after downregulation of Mkx were significantly decreased not only in normoxic BMSCs, but also in hypoxic BMSCs which paralleled the inferior histological evidences, worse biomechanical properties, and smaller diameters of collagen fibrils in vivo. In addition, our in vitro data demonstrated that the optical density values, the clone numbers, the percentage of cells in S phage, and cell proliferation potential of both normoxic and hypoxic BMSCs were all significantly increased after knockdown of Mkx and were also significantly enhanced in both AMSCs and BMSCs in hypoxia condition under which the expression of Mkx was upregulated.ConclusionsThese findings strongly suggested that Mkx mediated hypoxia-induced tenogenic differentiation of MSCs but could not completely repress the proliferation of hypoxic MSCs.

Hypoxic preconditioning rejuvenates mesenchymal stem cells and enhances neuroprotection following intracerebral hemorrhage via the miR-326-mediated autophagy

BackgroundIntracerebral hemorrhage (ICH) is a major public health concern, and mesenchymal stem cells (MSCs) hold great potential for treating ICH. However, the quantity and quality of MSCs decline in the cerebral niche, limiting the potential efficacy of MSCs. Hypoxic preconditioning is suggested to enhance the survival of MSCs and augment the therapeutic efficacy of MSCs in ICH. MicroRNAs (miRNAs) are known to mediate cellular senescence. However, the precise mechanism by which miRNAs regulate the senescence of hypoxic MSCs remains to be further studied. In the present study, we evaluated whether hypoxic preconditioning enhances the survival and therapeutic effects of olfactory mucosa MSC (OM-MSC) survival and therapeutic effects in ICH and investigated the mechanisms by which miRNA ameliorates hypoxic OM-MSC senescence.MethodsIn the in vivo model, ICH was induced in mice by administration of collagenase IV. At 24 h post-ICH, 5 × 105 normoxia or hypoxia OM-MSCs or saline was administered intracerebrally. The behavioral outcome, neuronal apoptosis, and OM-MSC survival were evaluated. In the in vitro model, OM-MSCs were exposed to hemin. Cellular senescence was examined by evaluating the expressions of P16INK4A, P21, P53, and by β-galactosidase staining. Microarray and bioinformatic analyses were performed to investigate the differences in the miRNA expression profiles between the normoxia and hypoxia OM-MSCs. Autophagy was confirmed using the protein expression levels of LC3, P62, and Beclin-1.ResultsIn the in vivo model, transplanted OM-MSCs with hypoxic preconditioning exhibited increased survival and tissue-protective capability. In the in vitro model, hypoxia preconditioning decreased the senescence of OM-MSCs exposed to hemin. Bioinformatic analysis identified that microRNA-326 (miR-326) expression was significantly increased in the hypoxia OM-MSCs compared with that of normoxia OM-MSCs. Upregulation of miR-326 alleviated normoxia OM-MSC senescence, whereas miR-326 downregulation increased hypoxia OM-MSC senescence. Furthermore, we showed that miR-326 alleviated cellular senescence by upregulating autophagy. Mechanistically, miR-326 promoted the autophagy of OM-MSCs via the PI3K signaling pathway by targeting polypyrimidine tract-binding protein 1 (PTBP1).ConclusionsOur study shows that hypoxic preconditioning delays OM-MSC senescence and augments the therapeutic efficacy of OM-MSCs in ICH by upregulating the miR-326/PTBP1/PI3K-mediated autophagy.

Hypoxic mesenchymal stem cells ameliorate acute kidney ischemia-reperfusion injury via enhancing renal tubular autophagy

BackgroundAcute kidney injury (AKI) is an emerging global healthcare issue without effective therapy yet. Autophagy recycles damaged organelles and helps maintain tissue homeostasis in acute renal ischemia-reperfusion (I/R) injury. Hypoxic mesenchymal stem cells (HMSCs) represent an innovative cell-based therapy in AKI. Moreover, the conditioned medium of HMSCs (HMSC-CM) rich in beneficial trophic factors may serve as a cell-free alternative therapy. Nonetheless, whether HMSCs or HMSC-CM mitigate renal I/R injury via modulating tubular autophagy remains unclear.MethodsRenal I/R injury was induced by clamping of the left renal artery with right nephrectomy in male Sprague-Dawley rats. The rats were injected with either PBS, HMSCs, or HMSC-CM immediately after the surgery and sacrificed 48 h later. Renal tubular NRK-52E cells subjected to hypoxia-reoxygenation (H/R) injury were co-cultured with HMSCs or treated with HMSC-CM to assess the regulatory effects of HSMCs on tubular autophagy and apoptosis. The association of tubular autophagy gene expression and renal recovery was also investigated in patients with ischemic AKI.ResultHMSCs had a superior anti-oxidative effect in I/R-injured rat kidneys as compared to normoxia-cultured mesenchymal stem cells. HMSCs further attenuated renal macrophage infiltration and inflammation, reduced tubular apoptosis, enhanced tubular proliferation, and improved kidney function decline in rats with renal I/R injury. Moreover, HMSCs suppressed superoxide formation, reduced DNA damage and lipid peroxidation, and increased anti-oxidants expression in renal tubular epithelial cells during I/R injury. Co-culture of HMSCs with H/R-injured NRK-52E cells also lessened tubular cell death. Mechanistically, HMSCs downregulated the expression of pro-inflammatory interleukin-1β, proapoptotic Bax, and caspase 3. Notably, HMSCs also upregulated the expression of autophagy-related LC3B, Atg5 and Beclin 1 in renal tubular cells both in vivo and in vitro. Addition of 3-methyladenine suppressed the activity of autophagy and abrogated the renoprotective effects of HMSCs. The renoprotective effect of tubular autophagy was further validated in patients with ischemic AKI. AKI patients with higher renal LC3B expression were associated with better renal recovery.ConclusionThe present study describes that the enhancing effect of MSCs, and especially of HMCSs, on tissue autophagy can be applied to suppress renal tubular apoptosis and attenuate renal impairment during renal I/R injury in the rat. Our findings provide further mechanistic support to HMSCs therapy and its investigation in clinical trials of ischemic AKI.

Hypoxia Mesenchymal Stem Cells Accelerate Wound Closure Improvement by Controlling α-smooth Muscle actin Expression in the Full-thickness Animal Model

BACKGROUND: The active myofibroblast producing extracellular matrix deposition regarding wound closure is characterized by alpha-smooth muscle actin (α-SMA) expression. However, the persistence of α-SMA expression due to prolonged inflammation may trigger scar formation. A new strategy to control α-SMA expression in line with wound closure improvement uses hypoxic mesenchymal stem cells (HMSCs) due to their ability to firmly control inflammation for early initiating cell proliferation, including the regulation of α-SMA expression associated with wound closure acceleration. AIM: This study aimed to explore the role of HMSCs in accelerating the optimum wound closure percentages through controlling the α-SMA expression. MATERIALS AND METHODS: Twenty-four full-thickness rats wound model were randomly divided into four groups: Sham (Sh), Control (C) by NaCl administration only, and two treatment groups by HMSCs at doses of 1.5×106 cells (T1) and HMSCs at doses of 3×106 cells (T2). HMSCs were incubated under hypoxic conditions. The α-SMA expression was analyzed under immunohistochemistry staining assay, and the wound closure percentage was analyzed by ImageJ software. RESULTS: This study showed a significant increase in wound closure percentage in all treatment groups that gradually initiated on days 6 and 9 (p < 0.05). In line with the increase of wound closure percentages on day 9, there was also a significant decrease in α-SMA expression in all treatment groups (p < 0.05), indicating the optimum wound healing has preceded. CONCLUSION: HMSCs have a robust ability to accelerated wound closure improvement to the optimum wound healing by controlling α-SMA expression depending on wound healing phases.

Topical application of adipose tissue-derived mesenchymal stem cells (ADMSCs) reduced cerebral edema in experimental traumatic brain injury (TBI)\u2014a preliminary study

BackgroundOur previous studies showed that topical application of mesenchymal stem cells (MSCs) improved functional recovery in rat traumatic brain injury (TBI) model, and hypoxic precondition further enhanced the therapeutic effects of MSCs. There was no previous study on the attenuation of cerebral edema by MSCs.We investigated whether topical application of normoxic and hypoxic MSCs could reduce cerebral edema in an experimental TBI model.MethodsTwo million normoxic (N = 24) and hypoxic (N = 24) MSCs were applied topically to exposed the cerebral cortex in a controlled cortical impact (CCI) model. The MSCs were fixed in position with fibrin glue. No treatment was given to control animals (TBI only: n = 24). After surgery, four animals in each group were sacrificed daily (day 1 to day 6) for edema evaluation. Normal animals without TBI were used as reference (n = 4). The expressions of GFAP, AQP4, and MMP9 were also investigated by immunofluorescence staining and RT-PCR at day 3.ResultsThe edema peaked within 3 days after TBI. Compared with the control, hypoxic MSCs reduced brain water content significantly (p < 0.05). Both hypoxic and normoxic MSCs downregulated the expression of MMP9 and normalized AQP4 distribution to astrocyte end feet.ConclusionOur preliminary study showed that topical application of hypoxic MSCs suppressed both vasogenic and cytotoxic edema formation.

The Role of Hypoxic Mesenchymal Stem Cells Conditioned Medium in Increasing Vascular Endothelial Growth Factors (VEGF) Levels and Collagen Synthesis to Accelerate Wound Healing

Full-thickness wound are areas damage of skin associated with loss of epidermis and dermis. The wound healing mechanism consists proliferation, migration and remodeling. Hypoxic conditional medium of mesenchymal stem cells (HMSCs-CM) contains lots of soluble molecules, such as protein growth factor and cytokine anti-inflammation. The soluble molecule of HMSCs-CM plays a critical role in wound healing by upregulation of VEGF and collagen synthesis. The objective of this study was to evaluate the effect of HMSCs-CM on VEGF and collagen concentrations in rats with incised wounds. The methods of this study were an experimental animal study with post-test only control group design was performed involving 24 Wistar rats. The rats were randomized into four groups consisting of sham, control and two treatment groups (gel of HMSCs-CM at doses of 200 μL and 400 μL). The VEGF levels and collagen density were analyses using ELISA assay and Masson-trichome specific staining, respectively. One-way ANOVA and Post Hoc LSD were used to analyses the data. The results of this study showed that a VEGF levels was significant increased on day 6 with doses-dependent manner. Interestingly, the VEGF levels gradual decrease on day 9. In addition, the decreased of VEGF levels on day 9 in this study in line with our findings in which we found there was a trend in the decreased of collagen density, it indicated the completion of remodeling phase and there has been an acceleration in wound healing. This study demonstrated that HMSCs-CM were able to regulate VEGF levels and collagen synthesis in accelerate wound healing. The role of HMSCs-CM stimulate cutaneous wound healing should be clarified further.Keywords: hypoxic conditional medium of mesenchymal stem cells (HMSCs-CM), vascular endothelial growth factor, collagen synthesis, paracrine factors

Hypoxia-induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.

Hypoxia-induced increase in Sug1 leads to poor post-transplantation survival of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are immunoprivileged and have the potential to treat numerous degenerative diseases. However, recent reviews of clinical trials report poor long-term survival of transplanted cells in the recipient that turned down the enthusiasm regarding MSC therapies. Increasing evidence now confirm that though initially immunoprivileged, MSCs eventually become immunogenic after transplantation in the ischemic or hypoxic environment of diseased tissues and are rejected by the host immune system. We performed in vitro (in rat and human cells) and in vivo (in a rat model) investigations to understand the mechanisms of the immune switch in the phenotype of MSCs. The immunoprivilege of MSCs is preserved by the absence of cell surface immune antigen, major histocompatibility complex II (MHC-II) molecule. We found that the ATPase subunit of 19S proteasome "Sug1" regulates MHC-II biosynthesis in MSCs. Exposure to hypoxia upregulates Sug1 in MSCs and its binding to class II transactivator (CIITA), a coactivator of MHC-II transcription. Sug1 binding to CIITA in hypoxic MSCs promotes the acetylation and K63 ubiquitination of CIITA leading to its activation and translocation to the nucleus, and ultimately MHC-II upregulation. In both rat and human MSCs, knocking down Sug1 inactivated MHC-II and preserved immunoprivilege even following hypoxia. In a rat model of myocardial infarction, transplantation of Sug1-knockdown MSCs in ischemic heart preserved immunoprivilege and improved the survival of transplanted cells. Therefore, the current study provides novel mechanisms of post-transplantation loss of immunoprivilege of MSCs. This study may help in facilitating better planning for future clinical trials.