MSC-derived exosomes protect auditory hair cells from neomycin-induced damage via autophagy regulation

BACKGROUND: Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear. RESULTS: Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity. CONCLUSIONS: In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.

Stability study of umbilical cord mesenchymal stem cells formulation in large-scale production / 药学学报

Umbilical cord mesenchymal stem cells (UC-MSCs) have been widely used in regenerative medicine, but there is limited research on the stability of UC-MSCs formulation during production. This study aims to assess the stability of the cell stock solution and intermediate product throughout the production process, as well as the final product following reconstitution, in order to offer guidance for the manufacturing process and serve as a reference for formulation reconstitution methods. Three batches of cell formulation were produced and stored under low temperature (2-8 ℃) and room temperature (20-26 ℃) during cell stock solution and intermediate product stages. The storage time intervals for cell stock solution were 0, 2, 4, and 6 h, while for intermediate products, the intervals were 0, 1, 2, and 3 h. The evaluation items included visual inspection, viable cell concentration, cell viability, cell surface markers, lymphocyte proliferation inhibition rate, and sterility. Additionally, dilution and culture stability studies were performed after reconstitution of the cell product. The reconstitution diluents included 0.9% sodium chloride injection, 0.9% sodium chloride injection + 1% human serum albumin, and 0.9% sodium chloride injection + 2% human serum albumin, with dilution ratios of 10-fold and 40-fold. The storage time intervals after dilution were 0, 1, 2, 3, and 4 h. The reconstitution culture media included DMEM medium, DMEM + 2% platelet lysate, 0.9% sodium chloride injection, and 0.9% sodium chloride injection + 1% human serum albumin, and the culture duration was 24 h. The evaluation items were viable cell concentration and cell viability. The results showed that the cell stock solution remained stable for up to 6 h under both low temperature (2-8 ℃) and room temperature (20-26 ℃) conditions, while the intermediate product remained stable for up to 3 h under the same conditions. After formulation reconstitution, using sodium chloride injection diluted with 1% or 2% human serum albumin maintained a viability of over 80% within 4 h. It was observed that different dilution factors had an impact on cell viability. After formulation reconstitution, cultivation in medium with 2% platelet lysate resulted in a cell viability of over 80% after 24 h. In conclusion, the stability of cell stock solution within 6 h and intermediate product within 3 h meets the requirements. The addition of 1% or 2% human serum albumin in the reconstitution diluent can better protect the post-reconstitution cell viability.

BMSCs promote M2 macrophage polarization to attenuate acute radiation-induced lung injury / 中国辐射卫生

Objective To investigate the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) for radiation-induced lung injury (RILI) and the underlying mechanism. Methods Forty-five healthy adult male C57BL/6 mice were randomly divided into control, model, and BMSCs groups. The model and BMSCs groups received a single irradiation dose of 20 Gy to the chest, while the control group did not receive X-ray irradiation. For the BMSCs group, an injection of 1 × 106 BMSCs cells was administered via the tail vein within 6 h after irradiation. In the 5th week, the lung tissue was taken to observe pathological changes with HE staining; examine the expression of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) with immunohistochemical staining; observe the polarization of macrophages with immunofluorescence staining; and measure the expression of the epithelial-mesenchymal transition markers E-cadherin, N-cadherin, and vimentin proteins by Western blot. Results After radiation, the model group developed pulmonary vasodilation and congestion with septal thickening and inflammatory cell infiltration, and these changes were markedly reduced in the BMSCs group. The model group showed significantly down-regulated expression of IL-6 and TNF-α compared with significantly increased levels in the model group (P < 0.01, P < 0.05). Treatment with BMSCs significantly increased the polarization of lung macrophages towards the M2 type, while significantly decreasing the abnormally increased N-cadherin and vimentin levels in RILI mice (P < 0.05, P < 0.01). Conclusion BMSCs have therapeutic effects for RILI mice, which may be through promoting macrophage polarization from M1 to M2.

Application progress of mesenchymal stem cells in islet transplantation for type 1 diabetes mellitus / 器官移植

Islet transplantation is considered as one of the most effective approach for type 1 diabetes mellitus, although its efficacy is limited by several factors. Anoxia, stress and rejection occurring during the isolation, culturing and transplantation of islets may have impact on the outcome of the islet transplantation. Due to the biological properties such as anti-inflammation, angiogenetic promotion and immune regulation, mesenchymal stem cells (MSCs) are all the way focused by researchers. Additionally, exosome, a derivative of MSC, also plays an import role in regulating anoxia-induced oxidative stress modulation, angiogenetic promotion, and immune regulation. MSC-based islet transplantation may be a useful therapeutic tool in treating type 1 diabetes. Therefore, in this review, the potential effect of MSC prior and posterior to the operation of the islet transplantation, its clinical application as well as its limitations were reviewed, aiming to offer insights into the future application of islet transplantation in treating type 1 diabetes.

Isolation of bone marrow mesenchymal stem cells in transgenic pigs and co-culture with porcine islets / 器官移植

Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells.

Mesenchymal cells in rotator cuff repair - technique description and case reports / Células mesenquimais no reparo da rotura do manguito rotador - descrição de técnica e relato de casos

ABSTRACT Objective: To describe a protocol of obtention of mesenchymal stem cells and to report their use as a biological adjuvant in three patients undergoing arthroscopic rotator cuff repair. Methods: Case series of patients who underwent arthroscopic repair of isolated full-thickness supraspinatus tear using mesenchymal stem cells obtained from the bone marrow as a biological adjuvant. All patients were operated on at the same institution, by a surgeon with 13 years of experience. The cells were applied at the end of the procedure, at the tendon-bone interface, at an approximate concentration of 2,000,000 mesenchymal cells/mm3 and a total volume of 5 ml. Results: All patients improved with the procedure, with one excellent and two good results. All cases overcame the minimally important clinical difference. All cases reached tendon healing, without partial or complete re-tears. We observed no complications. Conclusion: Arthroscopic rotator cuff repair with added mesenchymal cells obtained from bone marrow and submitted to a cell expansion process led to good functional results and healing in all cases in the sample, with no complications. Level of Evidence IV, Case Series.

RESUMO Objetivo: Descrever o protocolo de obtenção de células mesenquimais e relatar seu uso como adjuvante biológico em três pacientes submetidos ao reparo artroscópico do manguito rotador. Métodos: Série de casos de pacientes submetidos ao reparo artroscópico de rotura transfixante do músculo supraespinal utilizando como adjuvante biológico células mesenquimais obtidas da medula óssea. Todos ospacientes foram operados na mesma instituição por um cirurgião com 13 anos de experiência. As células foram aplicadas ao final do procedimento, na interface do tendão com o osso, na concentração aproximada de 2 milhões de células mesenquimais/mm3 e volume total de 5 ml. Resultados: Todos os pacientes melhoraram após o procedimento, havendo um resultado excelente e dois bons. Todos superaram a diferença clínica minimamente importante. Em todos os casos ocorreu cicatrização tendínea, sem a presença de rerroturas parciais ou completas. Não observamos complicações. Conclusão: O reparo do manguito rotador artroscópico com adição de células mesenquimais obtidas da medula óssea e submetidas a processo de expansão celular levou a bons resultados funcionais e cicatrização, sem complicações, em todos os casos da amostra. Nível de Evidência IV, Série de Casos.

Research progress of periodontal tissue regeneration by combined biomaterials of dental derived mesenchymal stem cells / 口腔医学

@#The ultimate treatment goal of periodontitis is the structural and functional regeneration of periodontium. However, existing methods for periodontal regeneration have difficulties in regenerating the hierarchical structure. Therefore, stem cell-based tissue engineering has attracted more and more attention for its advantages of self-renewal and multi-lineage differentiation potential. This review summarized the progress of research on periodontal tissue regeneration by combined biomaterials of dental-derived stem cells. It is pointed out that the application of autologous stem cell transplantation is limited by the donor source, and the subsequent research should focus on the development of multi-phase scaffold materials and the attempt to establish a stem cell bank.

Cross-regulation of Wnt/β-catenin and NF-κB signaling pathway and their involvement in the differentiation of mesenchymal stem cells / 口腔医学

@#Both Wnt/β-catenin signaling pathway and NF-κB signaling pathway are highly conservative pathways that regulate a variety of biological processes, and their cross-regulation have attracted attention in many biological and medical research fields. In this review, we summarize the cross-regulation between Wnt/β-catenin signaling pathway and NF-κB signaling pathway and discuss their involvement in the multi-directional differentiation of mesenchymal stem cells.

Potential value of mesenchymal stem cells and their derivatives in repairing donor liver / 器官移植

In recent years, organ transplantation has developed rapidly in China, whereas the proportion of supply and demand of organs for donation is severely unbalanced. To resolve the shortage of donor livers, repairing extended criteria donor liver and improving the quality of donor liver are critical research directions. Mesenchymal stem cell (MSC) is a category of stem cells with self-renewal and differentiation potential, which possess the functions of immunomodulation and tissue repair. The derivatives of MSC have the advantages of low immunogenicity and high biocompatibility, which have been widely applied in the treatment of multiple diseases. In this article, research progress on the role of MSC, exosomes and extracellular vesicles in alleviating liver steatosis, repairing ischemia-reperfusion injury and promoting the regeneration of small-for-size liver allograft was reviewed, and the feasibility and safety of MSC and the derivatives in repairing donor liver were summarized, aiming provide novel ideas for repairing marginal donor liver and enhancing the quality of liver allograft.

In vitro research of mesenchymal stem cell-coated human islets to alleviate instant blood-mediated inflammatory reaction / 器官移植

Objective To evaluate the effect of mesenchymal stem cell (MSC) coated-islets on instant blood-mediated inflammatory reaction (IBMIR) after islet transplantation. Methods MSC labeled with tracer and human islets were placed into an ultra-low adsorption cell culture dish, shaken and mixed twice at an interval of 0.5 h, and then incubated at 37 ℃ and 5% CO2 for 24 h to obtain MSC-coated islets. The coating effect of MSC and in vitro function of the islets were assessed. A blood circulation tube-shaped model was established in vitro. In the blank control group, 0.2 mL of islet culture solution was added. In the islet group, 800 islet equivalent quantity (IEQ) of uncoated islets were supplemented. In the MSC-coated islets group, 800 IEQ of MSC-coated islets were added, and circulated for 60 min at 37 ℃. A portion of 0.5 mL blood sample was taken for routine blood test at 0, 30 and 60 min, respectively. After 60 min circulation, the blood sample was filtered with a 70 μm filter to collect plasma, blood clots and islets. Blood clots and islets were subject to hematoxylin-eosin (HE) staining and immunohistochemical staining. Morphological changes and the aggregation of CD11b-positive cells surrounding the islets were observed. The contents of plasma thrombin-antithrombin complex (TAT), tissue factor (TF), C3a, C5b-9, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1 and IL-8 were determined by enzyme-linked immune absorbent assay. Results After 24 h co-incubation, the islets were coated by MSC, with a coating degree of approximately 80%. In the islet and MSC-coated islet group, a large quantity of neutrophils and monocytes were observed surrounding the blood clots and islets, and the quantity of CD11b-positive cells in the MSC-coated islet group was less compared with that in the islet group. After co-incubation with the whole blood for 0, 30 and 60 min, the quantity of platelets, neutrophils and monocytes was declined in the MSC-coated and islet groups, and gradually decreased over time. Compared with the blank control group, the quantity of platelets, monocytes and neutrophils was lower, whereas the TF content was higher in the MSC-coated islet group. Compared with the islet group, the quantity of platelets, monocytes and neutrophils was higher, whereas the TAT and TF contents were less in the MSC-coated islet group, the differences were statistically significant (all P < 0.05). Compared with the blank control group, the expression levels of C3a, C5b-9, IL-6, TNF-α and IL-8 were up-regulated in the MSC-coated islet group. Compared with the islet group, the expression levels of C3a, C5b-9, IL-1β, IL-6, TNF-α, IL-8 and MCP-1 were down-regulated in the MSC-coated islet group, and the differences were statistically significant (all P < 0.05). Conclusions MSC-coated islets may reduce the exposure of islet TF in the blood and prevent the incidence of IBMIR during the coagulation response stage, thereby mitigating the injury and loss of islet allograft in the early stage of islet transplantation.

Comparison of Exosomes Derived from Fibroblasts and Mesenchymal Stem Cells for the Treatment of Acute Wound Healing / 中山大学学报(医学科学版)

ObjectiveTo compare the therapeutical effect of exosomes derived from fibroblasts and mesenchymal stem cells on acute wound healing. MethodsPrimary human dermal fibroblasts （hDF） were isolated， cultured and identified. Human bone marrow mesenchymal stem cell exosomes （hMSC-EXO） and hDF exosomes （hDF-EXO） were extracted by ultracentrifuga tion. After 24 h of coincubation with hDF-EXO or hMSC-EXO， hDFs proliferation and migratory capacity were evaluated by cell counting kit-8 （CCK8） assay and scratch test. Full-thickness cutaneous wounds were created on 8-week-old female C57BL/6 mice， and topically applied with PBS （control）， hDF-EXO or hMSC-EXO. Wounds were measured at day 0， 2， 4， 7， and the uptake of exosomes in wound was observed at day 1. Quantitative PCR （qPCR） analysis was performed to detect the mRNA expression levels of TNF-α， IL-6， IL-1β， IL-10 in wound at day 1. HE staining was conducted to analyze the histological structure of wounds at day 7， while immunofluorescence staining was used to examine expression of PDGFR-α、α-SMA、Ki67. ResultshDF exhibited certain fibrolast-like characteristics with respect to expression of cell surface markers and specific proteins. hDF-EXO and hMSC-EXO presented exosomal morphology， size， and markers， and both concentrations were not statistically different （P>0.05）； CCK8 assay showed that both exosomes promoted hDF cell viability， compared with the negative control （P<0.01）， and hDF-EXO group had greater cell viability than hMSC-EXO group （P<0.01）. Scratch test indicated that hDF-EXO induced a significant increase in scratch healing rate versus the negative control （P<0.01）， hMSC-EXO （P<0.05）. In vivo experiments showed wound tissues took up exosomes at day 1. qPCR detected TNF-α， IL-6， IL-1β expression levels in wound at day 1 were lower in exosomes group than in the control group， and were the lowest in hMSC-EXO group （all P<0.01）. Wound areas were measured smaller at day 7 in exosomes group than in the control group （all P<0.01） and hDF-EXO group had better closure than hMSC-EXO group （P<0.05）. HE staining revealed that compared with control group， scar， incomplete epidermis and few collagen deposition remained in the hMSC-EXO group， whereas hDF-EXO group showed re-epithelialization， continuous neo-epidermis and regenerated dermis. Immunofluorescence staining suggested that the number of fibroblasts， myofibroblasts， proliferating cells was higher in both exosomes group than that in the control group， especially the highest in hDF-EXO group. ConclusionOur study shows both exosomes accelerate wound healing， whereas hDF-EXO is more effective in promoting fibroblasts proliferation， migration， transition to myofibroblasts， and hMSC-EXO may play a role in inhibiting inflammatory reaction during early stage of wound healing.

Mechanism of regulatory effect of recombinant human HMGB1 on endothelial cell angiogenesis / 器官移植

Objective To unravel the possible mechanism of the role of recombinant human high mobility group box 1 (rhHMGB1) protein in regulating the angiogenesis of endothelial cells. Methods Endothelial cells were divided into the control group, bone marrow mesenchymal stem cells (MSC) supernatant group and rhHMGB1 group. The proliferation and survival of endothelial cells were detected by cell counting kit(CCK)-8 assay. The relative expression levels of vascular endothelial growth factor (VEGF), Yes-associated protein (YAP), CD31 and hypoxia inducible factor (HIF)-1α proteins were determined by Western blot. The relative expression levels of VEGF, YAP, CD31 and HIF-1α messenger RNA (mRNA) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The migration ability of endothelial cells was assessed by Transwell chamber test. The localization of YAP was detected by immunofluorescence staining. Results Compared with the control group, the migration rate of endothelial cells was increased in the rhHMGB1 group (P < 0.05), and the cell migration rate was enhanced over time. Compared with the control group, the relative expression levels of VEGF and p-YAP proteins were up-regulated in the MSC supernatant group, and the differences were statistically significant (both P < 0.05). Compared with the control group, the relative expression levels of VEGF and HIF-1α proteins, VEGF and CD31 mRNA and YAP and p-YAP proteins were up-regulated, and YAP/p-YAP ratio was increased in the rhHMGB1 group, and the differences were statistically significant (all P < 0.05). Compared with the MSC supernatant group, the relative expression levels of CD31 mRNA and YAP protein were up-regulated, and the YAP/p-YAP ratio was increased in the rhHMGB1 group, and the differences were statistically significant (all P < 0.05). Conclusions Exogenous high-concentration rhHMGB1 may promote the migration ability of endothelial cells and up-regulate the expression levels of angiogenesis-related proteins by regulating the recruitment of YAP to the nucleus.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles enhance the regenerative capability of fibrotic liver / 器官移植

Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl4+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl4+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl4+PHx group. Compared with the CCl4+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl4+PHx group. Compared with the CCl4+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl4+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl4+PHx+MSC-EV group was higher than that in the CCl4+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.

Protective role and mechanism of human umbilical cord mesenchymal stem cell-derived exosome in renal ischemia-reperfusion injury / 器官移植

Objective To investigate the protective effect of human umbilical cord mesenchymal stem cell-derived exosome (hucMSC-Exo) on renal ischemia-reperfusion injury (IRI), and to clarify the critical role and regulating mechanism of transient receptor potential canonical (TRPC) 6/poly adenosine-diphosphate-ribose polymerase (PARP) 1 signaling pathway during this process. Methods The hucMSC-Exo was extracted by ultracentrifugation, and identified by transmission electron microscope (TEM), nanoparticle tracing analysis and Western blot. SD rats were randomly divided into the sham operation group (group S), sham operation+TRPC6 inhibitor SKF96365 group (group SS), renal IRI group (group IRI), exosome treatment group (group EXO) and exosome +TRPC6 inhibitor SKF96365 group (group ES), with 6 rats in each group. Serum creatinine and blood urea nitrogen levels were detected. Pathological changes of renal tissues were observed by hematoxylin-eosin (HE) staining and Paller score was calculated. The expression levels of key molecules of necroptosis in rat renal tissues, including receptor-interacting protein kinase (RIPK)1, RIPK3 and mixed-lineage kinase domain-like protein (MLKL), TRPC6 and PARP1, were detected by Western blot. Results Typical saucer-like structure was observed under TEM. Nanoparticle tracing analysis showed that the average diameter of the extracted substance was 125.9 nm. Western blot revealed that the surface markers of CD9, CD63 and CD81 were positively expressed, confirmed that the extracted substance was exosome. Compared with group S, the serum creatinine and blood urea nitrogen levels were up-regulated, the pathological damage of renal tissues was worsened, Paller score was elevated, the relative expression levels of TRPC6 and PARP1 proteins were down-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were up-regulated in group IRI (all P < 0.05). Compared with group IRI, the serum creatinine and blood urea nitrogen levels were down-regulated, the pathological damage of renal tissues was mitigated, Paller score was decreased, the relative expression levels of TRPC6 and PARP1 proteins were up-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were down-regulated in group EXO (all P < 0.05). Compared with group EXO, the serum creatinine and blood urea nitrogen levels were up-regulated, the pathological damage of renal tissues was aggravated, Paller score was increased, the relative expression levels of TRPC6 and PARP1 proteins were down-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were up-regulated in group ES (all P < 0.05). Conclusions hucMSC-Exo may alleviate the necroptosis induced by renal IRI in rat models, which is related to the activation of TRPC6/PARP1 signaling pathway.

Prevention and treatment of chronic graft-versus-host disease by mesenchymal stem cells: advances and challenges / 器官移植

Chronic graft-versus-host disease (cGVHD) is the main complication after allogeneic hematopoietic stem cell transplantation, which is also the major cause of non-relapse -related death. Due to its complex pathophysiological process, the response rate of conventional glucocorticoids combined with immunosuppressants is less than 50%. Second-line therapy should be given for patients with glucocorticoid-resistant cGVHD. Nevertheless, no consensus has been reached on current second-line therapy and the therapeutic effect is relatively poor. Mesenchymal stem cell (MSC) is one of the most common adult stem cells. Due to multi-dimensional and multi-target immune regulating function, MSC has been widely applied in the prevention and treatment of cGVHD. In addition, accumulated studies have confirmed the safety and efficacy of MSC in the treatment of cGVHD, which is expected to become a novel strategy for the prevention and management of cGVHD. In this article, research progress, mechanism and existing problems of prevention and treatment of cGVHD by MSC were reviewed, aiming to provide novel ideas for optimizing therapeutic regimens of MSC and enhancing the prevention and treatment effect of cGVHD in subsequent research.

Current status and reflections on stem cell transplantation in the treatment of testicular aging / 器官移植

Testicular aging is mainly characterized by a gradual decline in the capability of testosterone synthesis and spermatogenesis, which not only affects male fertility, but also correlates with aging-related chronic diseases intimately. Therefore, delaying testicular aging plays a significant role in improving the health and quality of life of middle-aged and elderly men. Stem cells are a cell group with potent self-renewal capability and multi-directional differentiation potential. In recent years, the research of stem cells in basic and clinical application has been carried out in-depth, which has accelerated the development of cell therapy. Currently, stem cell transplantation has been employed to treat multiple diseases, which has captivated widespread attention in the field of aging and regenerative medicine. Stem cell transplantation has demonstrated promising prospects in the treatment of testicular aging. In this article, research profile and progress of stem cell transplantation in the treatment of testicular aging were reviewed, and bottleneck issues encountered in clinical translation and strategies for optimizing clinical efficacy were discussed, aiming to provide novel ideas for the research and development and clinical translation of stem cell therapy for testicular aging.

Recent Progress on Pharmaceutical Properties of Extracellular Vesicles from Mesenchymal Stem Cells--Review / 中国实验血液学杂志

Mesenchymal stem cells (MSCs) have been officially approved in many countries to treat graft-versus-host disease, autoimmune disorders and those associated with tissue regeneration after hematopoietic stem cell transplantation. Studies in recent years have confirmed that MSC acts mainly through paracrine mechanism, in which extracellular vesicles secreted by MSC (MSC-EV) play a central role. MSC-EV has overwhelming advantages over MSC itself in the setting of adverse effects in clinical application, indicating that MSC-EV might take the place of its parent cells to be a potentially therapeutic tool for "cell-free therapy". The pharmaceutical properties of MSC-EV largely depend upon the practical and optimal techniques including large-scale expansion of MSC, the modification of MSC based on the indications and the in vivo dynamic features of MSC-EV, and the methods for preparing and harvesting large amounts of MSC-EV. The recent progresses on the issues above will be briefly reviewed.

Hydrogel loaded with exosomes from Wharton 's Jelly-derived mesenchymal stem cells enhances wound healing in mice / 浙江大学学报·医学版

OBJECTIVES@#To explore the effect of hydrogel loaded with exosomes from Wharton's Jelly-derived mesenchymal stem cell (WJMSC) on wound healing.@*METHODS@#Exosomes were extracted from WJMSC, and the morphology and size of WJMSC-derived exosomes (WEX) were analyzed by transmission electron microscopy and nanoparticle size analyzer, respectively. The surface markers CD9, CD81, and Calnexin of WEX were detected by Western blotting. Exosome-loaded alginate hydrogel (WEX-gel) was prepared; its morphology was studied by scanning electron microscope, and its rheological behavior was examined by a rheometer. The in vitro drug release performance of WEX-gel was investigated by BCA method. RAW264.7 cells were treated with alginate hydrogel, WEX and WEX-gel, respectively; and the expression of CD86 and CD206 in macrophages was detected by flow cytometry. A full-thickness skin wound model was established in mice; the model mice were randomly divided into blank control group, WEX control group and WEX-gel group, and PBS, WEX and WEX-gel were applied to the wound area of mice, respectively. On day 3, the skin tissue of mice was excised, and the antibacterial effect of WEX hydrogel was evaluated by plate counting. On day 15, the mice were euthanized and the percentage of residual wounds was calculated. The histological changes of the skin wound were observed after hematoxylin and eosin (HE) and Masson stainings. The expression of CD86, CD206, CD31 and vascular endothelial growth factor (VEGF) in the skin wound tissue was detected by immunohistochemistry.@*RESULTS@#Exosomes were successfully extracted from WJMSC. WEX-gel presented a regular three-dimensional network structure, good rheology and controlled drug release performance. WEX-gel promoted the polarization of RAW264.7 cells from the M1 phenotype to M2 phenotype in vitro. The residual wound percentage in blank control group, WEX control group and WEX-gel group were (27.5±3.4)%, (15.3±1.2)% and (7.6±1.1)%, respectively (P<0.05). The antibacterial property of WEX-gel is better than that of WEX (P<0.05). The dermis thickness, the number of new hair follicles, and the rate of collagen deposition in the WEX-gel group were significantly higher than those in the other two groups (all P<0.05). The expression of CD206, CD31 and VEGF in skin wound tissue was higher and the expression of CD86 was lower in WEX-gel group than those in other two groups (all P<0.05).@*CONCLUSIONS@#WEX-gel can significantly promote wound healing in mice by regulating the polarization of macrophages.

A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy

Currently the main treatment of acute myeloid leukemia (AML) is chemotherapy combining hematopoietic stem cell transplantation. However, the unbearable side effect of chemotherapy and the high risk of life-threatening infections and disease relapse following hematopoietic stem cell transplantation restrict its application in clinical practice. Thus, there is an urgent need to develop alternative therapeutic tactics with significant efficacy and attenuated adverse effects. Here, we revealed that umbilical cord-derived mesenchymal stem cells (UC-MSC) efficiently induced AML cell differentiation by shuttling the neutrophil elastase (NE)-packaged extracellular vesicles (EVs) into AML cells. Interestingly, the generation and release of NE-packaged EVs could be dramatically increased by vitamin D receptor (VDR) activation in UC-MSC. Chemical activation of VDR by using its agonist 1α,25-dihydroxyvitamin D3 efficiently enhanced the pro-differentiation capacity of UC-MSC and then alleviated malignant burden in AML mouse model. Based on these discoveries, to evade the risk of hypercalcemia, we synthetized and identified sw-22, a novel non-steroidal VDR agonist, which exerted a synergistic pro-differentiation function with UC-MSC on mitigating the progress of AML. Collectively, our findings provided a non-gene editing MSC-based therapeutic regimen to overcome the differentiation blockade in AML.

MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell / 中国医学科学院学报

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.