Exosomes from circ-Astn1-modified adipose-derived mesenchymal stem cells enhance wound healing through miR-138-5p/SIRT1/FOXO1 axis regulation

BACKGROUND Wound healing impairment is a dysfunction induced by hyperglycemia and its effect on endothelial precursor cells(EPCs)in type 2 diabetes mellitus.There is increasing evidence showing that exosomes(Exos)derived from adipose-derived mesenchymal stem cells(ADSCs)exhibit the potential to improve endothelial cell function along with wound healing.However,the potential therapeutic mechanism by which ADSC Exos contribute to wound healing in diabetic mice remains unclear.AIM To reveal the potential therapeutic mechanism of ADSC Exos in wound healing in diabetic mice.METHODS Exos from ADSCs and fibroblasts were used for high-throughput RNA sequencing(RNA-Seq).ADSC-Exo-mediated healing of full-thickness skin wounds in a diabetic mouse model was investigated.We employed EPCs to investigate the therapeutic function of Exos in cell damage and dysfunction caused by high glucose(HG).We utilized a luciferase reporter(LR)assay to analyze interactions among circular RNA astrotactin 1(circ-Astn1),sirtuin(SIRT)and miR-138-5p.A diabetic mouse model was used to verify the therapeutic effect of circ-Astn1 on Exo-mediated wound healing.RESULTS High-throughput RNA-Seq analysis showed that circ-Astn1 expression was increased in ADSC Exos compared with Exos from fibroblasts.Exos containing high concentrations of circ-Astn1 had enhanced therapeutic effects in restoring EPC function under HG conditions by promoting SIRT1 expression.Circ-Astn1 expression enhanced SIRT1 expression through miR-138-5p adsorption,which was validated by the LR assay along with bioinformatics analyses.Exos containing high concentrations of circ-Astn1 had better therapeutic effects on wound healing in vivo compared to wild-type ADSC Exos.Immunofluorescence and immunohistochemical investigations suggested that circ-Astn1 enhanced angiopoiesis through Exo treatment of wounded skin as well as by suppressing apoptosis through promotion of SIRT1 and decreased forkhead box O1 expression.CONCLUSION Circ-Astn1 promotes the therapeutic effect of ADSC-Exos and thus improves wound healing in diabetes via miR-138-5p absorption and SIRT1 upregulation.Based on our data,we advocate targeting the circ-Astn1/miR-138-5p/SIRT1 axis as a potential therapeutic option for the treatment of diabetic ulcers.

Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

Hollow conduits of natural or synthetic origins have shown acceptable regeneration results in short nerve gap repair;however,results are still not comparable with the current gold standard technique“autografts”.Hollow conduits do not provide a successful regeneration outcome when it comes to critical nerve gap repair.Enriching the lumen of conduits with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance.In this study,we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model.Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks.Nevertheless,the molecular assessment in the early regeneration phase(7,14,and 28 days)has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones.Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1,a growth factor that plays an important role in Schwann cell transdifferentiation.The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2,VEGF-A,BDNF,c-Jun,and ATF3.

Pericyte-like differentiation of human adipose-derived mesenchymal stem cells:An in vitro study

BACKGROUND Adipose-derived mesenchymal stem cells(ASCs)are characterized by long-term self-renewal and a high proliferation rate.Under adequate conditions,they may differentiate into cells belonging to mesodermal,endodermal or ectodermal lineages.Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level.The loss of pericytes,as occurs in diabetic retinopathy,results in a breakdown of the blood-retina barrier(BRB)and infiltration of inflammatory cells.In this context,the use of pericytelike differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage.AIM To test in vitro strategies to obtain pericyte-like differentiation of human ASCs(hASCs).METHODS Different culture conditions were tested:hASCs cultured in a basal medium supplemented with transforming growth factorβ1;and hASCs cultured in a specific pericyte medium(PM-hASCs).In a further sample,pericyte growth supplement was omitted from the PM.In addition,cultures of human retinal pericytes(hRPCs)were used for comparison.Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression ofα-smooth muscle actin(α-SMA)and neural/glial antigen 2(NG2).Interactions between human retinal endothelial cells(hRECs)and different groups of hASCs were investigated in co-culture experiments.In these cases,the expression of typical junctional proteins such as vascular endothelial-Cadherin,zonula occludens-1 and Occludin were assessed in hRECs.In an in vitro model of the BRB,values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs.The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone.Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization.RESULTS After 3-6 d of culture,α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium(PM-hASCs).In particular,α-SMA immunoreactivity,already visible at the basal level in pericytes and ASCs,was strongly increased only when transforming growth factor was added to the culture medium.NG2 expression,almost undetectable in most conditions,was substantially increased only in PMhASCs.Immunocytochemical results were confirmed by western blot analysis.The presence of pericyte growth supplement seems to increase NG2 expression rather thanα-SMA,in agreement with its role in maintaining pericytes in the proliferative state.In co-culture experiments,immunoreactivity of vascular endothelial-Cadherin,zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were also present.Supporting results were found by trans-endothelial electrical resistance measurements,gathered at 3 and 6 d of co-culture.The highest resistance values were obtained when hRECs were co-cultured with hRPCs or PM-hASCs.The pericyte-like phenotype of PM-hASCs was also confirmed in three-dimensional co-cultures in Matrigel,where PM-hASCs and hRPCs similarly localized around the tubular formations made by hRECs.CONCLUSION PM-hASCs seem able to strengthen the intercellular junctions between hRECs,likely reinforcing the BRB;thus,hASC-based therapeutic approaches may be developed to restore the integrity of retinal microcirculation.

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUND Adipose-derived stem cells(ASCs)have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability,high proliferation rate,multipotent differentiation ability and low immunogenicity.In this respect,they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease.In ocular diseases involving the retina,various cell types may be affected,such as Müller cells,astrocytes,photoreceptors and retinal pigment epithelium(RPE),which plays a fundamental role in the homeostasis of retinal tissue,by secreting a variety of growth factors that support retinal cells.AIM To test ASC neural differentiation using conditioned medium(CM)from an RPE cell line(ARPE-19).METHODS ASCs were isolated from adipose tissue,harvested from the subcutaneous region of healthy donors undergoing liposuction procedures.Four ASC culture conditions were investigated:ASCs cultured in basal Dulbecco's Modified Eagle Medium(DMEM);ASCs cultured in serum-free DMEM;ASCs cultured in serumfree DMEM/F12;and ASCs cultured in a CM from ARPE-19,a spontaneously arising cell line with a normal karyotype derived from a human RPE.Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1,4and 8 d of culture.At the same time points,ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers:Nestin,neuronal specific enolase(NSE),protein gene product(PGP)9.5,and glial fibrillary acidic protein(GFAP).RESULTS Depending on the culture medium,ASC proliferation rate and viability showed some significant differences.Overall,less dense populations were observed in serum-free cultures,except for ASCs cultured in ARPE-19 serum-free CM.Moreover,a different cell morphology was seen in these cultures after 8 d of treatment,with more elongated cells,often showing cytoplasmic ramifications.Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation.In fact,basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM.Specifically,neural marker overexpression was more marked at 8 d.The most evident increase was observed for NSE and GFAP,a modest increase was observed for nestin,and less relevant changes were observed for PGP9.5.CONCLUSION The presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

AIM To investigate the effect of adipose-derived mesenchymal stem cells(ADMSCs)and their conditioned media(CM) on hepatocellular carcinoma(HCC) cell tumorigenesis.METHODS The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit8(commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTS Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSION These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

Bone marrow-derived mesenchymal stem cells versus adipose-derived mesenchymal stem cells for peripheral nerve regeneration

Studies have confirmed that bone marrow-derived mesenchymal stem cells(MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs(BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs(ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham(only sciatic nerve exposed), Matrigel(MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs(sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs(sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios(axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.

Adipose-derived mesenchymal stem cells accelerate nerve regeneration and functional recovery in a rat model of recurrent laryngeal nerve injury

Medialization thyroplasty or injection laryngoplasty for unilateral vocal fold paralysis cannot restore mobility of the vocal fold. Recent studies have shown that transplantation of mesenchymal stem cells is effective in the repair of nerve injuries. This study investigated whether adipose-derived stem cell transplantation could repair recurrent laryngeal nerve injury. Rat models of recurrent laryngeal nerve injury were established by crushing with micro forceps. Adipose-derived mesenchymal stem cells(ADSCs; 8 × 105) or differentiated Schwann-like adipose-derived mesenchymal stem cells(d ADSCs; 8 × 105) or extracellular matrix were injected at the site of injury. At 2, 4 and 6 weeks post-surgery, a higher density of myelinated nerve fiber, thicker myelin sheath, improved vocal fold movement, better recovery of nerve conduction capacity and reduced thyroarytenoid muscle atrophy were found in ADSCs and d ADSCs groups compared with the extracellular matrix group. The effects were more pronounced in the ADSCs group than in the d ADSCs group. These experimental results indicated that ADSCs transplantation could be an early interventional strategy to promote regeneration after recurrent laryngeal nerve injury.

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease

Adipose-derived mesenchymal stem cells(ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient's own subcutaneous adipose tissue, a high cell yield(i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, antiapoptotic effects, and control of immune cells via cellcell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.

Gene expression profiles of human adipose-derived mesenchymal stem cells dynamically seeded on clinically available processed nerve allografts and collagen nerve guides

It was hypothesized that mesenchymal stem cells(MSCs) could provide necessary trophic factors when seeded onto the surfaces of commonly used nerve graft substitutes. We aimed to determine the gene expression of MSCs when influenced by Avance■ Nerve Grafts or Neura Gen■ Nerve Guides. Human adipose-derived MSCs were cultured and dynamically seeded onto 30 Avance■ Nerve Grafts and 30 Neura Gen■ Nerve Guides for 12 hours. At six time points after seeding, quantitative polymerase chain reaction analyses were performed for five samples per group. Neurotrophic [nerve growth factor(NGF), glial cell line-derived neurotrophic factor(GDNF), pleiotrophin(PTN), growth associated protein 43(GAP43) and brain-derived neurotrophic factor(BDNF)], myelination [peripheral myelin protein 22(PMP22) and myelin protein zero(MPZ)], angiogenic [platelet endothelial cell adhesion molecule 1(PECAM1/CD31) and vascular endothelial cell growth factor alpha(VEGFA)], extracellular matrix(ECM) [collagen type alpha I(COL1A1), collagen type alpha III(COL3A1), Fibulin 1(FBLN1) and laminin subunit beta 2(LAMB2)] and cell surface marker cluster of differentiation 96(CD96) gene expression was quantified. Unseeded Avance■ Nerve Grafts and Neura Gen■ Nerve Guides were used to evaluate the baseline gene expression, and unseeded MSCs provided the baseline gene expression of MSCs. The interaction of MSCs with the Avance■ Nerve Grafts led to a short-term upregulation of neurotrophic(NGF, GDNF and BDNF), myelination(PMP22 and MPZ) and angiogenic genes(CD31 and VEGFA) and a long-term upregulation of BDNF, VEGFA and COL1A1. The interaction between MSCs and the Neura Gen■ Nerve Guide led to short term upregulation of neurotrophic(NGF, GDNF and BDNF) myelination(PMP22 and MPZ), angiogenic(CD31 and VEGFA), ECM(COL1A1) and cell surface(CD96) genes and long-term upregulation of neurotrophic(GDNF and BDNF), angiogenic(CD31 and VEGFA), ECM genes(COL1A1, COL3A1, and FBLN1) and cell surface(CD96) genes. Analysis demonstrated MSCs seeded onto Neura Gen■ Nerve Guides expressed significantly higher levels of neurotrophic(PTN), angiogenic(VEGFA) and ECM(COL3A1, FBLN1) genes in the long term period compared to MSCs seeded onto Avance■ Nerve Grafts. Overall, the interaction between human MSCs and both nerve graft substitutes resulted in a significant upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the Neura Gen■ Nerve Guide was more pronounced, particularly in the long term period(> 14 days after seeding). These results suggest that MSC-seeding has potential to be applied in a clinical setting, which needs to be confirmed in future in vitro and in vivo research.

Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

AIM:To investigate the impact of adipose-derived mesenchymal stem cells(ADSCs) on cell viability and extracellular matrix(ECM) synthesis of corneal stromal cells(CSCs). METHODS:ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly cocultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate(FITC)/proliferation indices(PI) assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase(MMP), such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS:ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis(early and late) between the negative control group(6.34% and 2.06%) and the ADCSs-treated group(4.69% and 1.59%). The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type(I, II, III, IV, and V) in CSCs were observed in CSCs/ADSCs group, 3.47-fold,4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models.CONCLUSION:ADSCs play a promotive role in CSCs' growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synthesis of CSCs. This provided a new insight into the extensive role of ADSCs in CSCs and a potential molecular target for corneal therapy.

Adipose-derived mesenchymal stem cells alleviate TNBS-induced colitis in rats by influencing intestinal epithelial cell regeneration, Wnt signaling, and T cell immunity

BACKGROUND Conventional Crohn’s disease(CD)treatments are supportive rather than curative and have serious side effects.Adipose-derived mesenchymal stem cells(ADSCs)have been gradually applied to treat various diseases.The therapeutic effect and underlying mechanism of ADSCs on CD are still not clear.AIM To investigate the effect of ADSC administration on CD and explore the potential mechanisms.METHODS Wistar rats were administered with 2,4,6-trinitrobenzene sulfonic acid(TNBS)to establish a rat model of CD,followed by tail injections of green fluorescent protein(GFP)-modified ADSCs.Flow cytometry,qRT-PCR,and Western blot were used to detect changes in the Wnt signaling pathway,T cell subtypes,and their related cytokines.RESULTS The isolated cells showed the characteristics of ADSCs,including spindle-shaped morphology,high expression of CD29,CD44,and CD90,low expression of CD34 and CD45,and osteogenic/adipogenic ability.ADSC therapy markedly reduced disease activity index and ameliorated colitis severity in the TNBS-induced rat model of CD.Furthermore,serum anti-sacchromyces cerevisiae antibody and panti-neutrophil cytoplasmic antibody levels were significantly reduced in ADSCtreated rats.Mechanistically,the GFP-ADSCs were colocalized with intestinal epithelial cells(IECs)in the CD rat model.GFP-ADSC delivery significantly antagonized TNBS-induced increased canonical Wnt pathway expression,decreased noncanonical Wnt signaling pathway expression,and increased apoptosis rates and protein level of cleaved caspase-3 in rats.In addition,ADSCs attenuated TNBS-induced abnormal inflammatory cytokine production,disturbed T cell subtypes,and their related markers in rats.CONCLUSION Successfully isolated ADSCs show therapeutic effects in CD by regulating IEC proliferation,the Wnt signaling pathway,and T cell immunity.

Autologous Adipose-Derived Mesenchymal Stem Cells Embedded in Platelet-Rich Fibrin in Diabetic Foot Ulcers

Context and Aim: Mesenchymal stem cells (MSCs) and platelet-rich fibrin (PRF) have emerged as ideal candidates for advanced therapies of various therapeutically-challenging diseases;however, their regenerative potential in diabetic foot ulcers (DFU) has not been well determined. In this study, we reviewed our clinical experience in mitigating chronic ulcer complications of diabetic foot through a conventional treatment of autologous adipose-derived MSCs embedded in PRF with pure PRF injections. Materials and Methods: The present study was carried out in 10 patients with an open DFU wound selected over a period of 1 year starting from April 2019. Patients were either injected with PRF alone (Group A) or injected with MSCs derived from adipose tissue (ADMSC) embedded in (PRF (Group B). Results: Patients in Group B had a better healing index when compared to Group A. Conclusion: Use of ADMSC embedded in PRF showed promising results to treat DFU.

ISOLATION AND INDUCTION OF DIFFERENTIATION OF SEINE ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

Objectives To establish a method for high yield mesenchymal stem cells collection,as well as a culture method for identifying mesenchymal stem cells from the swine adipose-derived mesenchymal stem cell(ADMSC).Methods Swine ADMSCs were isolated from fat tissue with collagenase,followed by induction of differentiation to osteogenic,adipogenic and chondrogrnic cells.The survival curve of the ADMSC at the 37oC and 38oC were measured using WST-1Cell Proliferation Assay Reagent.Result ADMSCs isolated with collagenase from swine neck fat tissue generated a stable uniform appearance after the second generation.The passage period was five days.ADMSC could differentiate into osteogenic,adipogenic or chondrogrnic cells under different culture conditions.The highest growth rate was achieved at 38oC in this study.Conclusion Swine ADMSCs have the potential to differentiate into osteogenic,adipogenic or chondrogrnic cells,and they may be appropriate for transplantation for both research and clinical purpose.

Advances in the research of basic study and clinical application of adipose-derived mesenchymal stem cells

Since the discovery of adipose-derived mesenchymal stem cell (ADSC) in more than ten years, a great progress has been made from its basic research to clinical application. Compared with bone marrow mesenchymal stem cells, ADSC is more abundant in reserve, easier to obtain with fewer injuries and less complications. These cells have multiple differentiation potential and can differentiate into adipocytes, chondrocytes and osteoblasts with the influence of different inducing factors. Early studies of ADSC mainly focused on the ability of multi-directional differentiation, especially on the regeneration of bone defects and cartilage tissue. At present, the researches mainly focus on immunoregulation and paracrine function of ADSC. Although ADSC has made a great progress in clinical application, the cell preparation, use pattern, and mechanisms in clinical treatment are not clear. This paper elaborates on these issues.

Immunomodulation of adipose-derived mesenchymal stem cells on peripheral blood mononuclear cells in colorectal cancer patients with COVID-19

BACKGROUND Accumulating evidence has shown that adipose tissue-derived mesenchymal stem cells(ADSCs)are an effective therapeutic approach for managing coronavirus disease 2019(COVID-19);however,further elucidation is required to determine their underlying immunomodulatory effect on the mRNA expression of T helper cell-related transcription factors(TFs)and cytokine release in peripheral blood mononuclear cells(PBMCs).AIM To investigate the impact of ADSCs on the mRNA expression of TFs and cytokine release in PBMCs from colorectal cancer(CRC)patients with severe COVID-19(CRC^(+)patients).METHODS PBMCs from CRC^(+)patients(PBMCs-C+)and age-matched CRC patients(PBMCs-C)were stimulated and cultured in the presence/absence of ADSCs.The mRNA levels of T-box TF TBX21(T-bet),GATA binding protein 3(GATA-3),RAR-related orphan receptor C(RORC),and forkhead box P3(FoxP3)in the PBMCs were determined by reverse transcriptase-polymerase chain reaction.Culture supernatants were evaluated for levels of interferon gamma(IFN-γ),interleukin 4(IL-4),IL-17A,and transforming growth factor beta 1(TGF-β1)using an enzyme-linked immunosorbent assay.RESULTS Compared with PBMCs-C,PBMCs-C+exhibited higher mRNA levels of T-bet and RORC,and increased levels of IFN-γ and IL-17A.Additionally,a significant decrease in FoxP3 mRNA and TGF-β1,as well as an increase in Tbet/GATA-3,RORC/FoxP3,IFN-γ/IL-4,and IL-17A/TGF-β1 ratios were observed in PBMCs-C+.Furthermore,ADSCs significantly induced a functional regulatory T cell(Treg)subset,as evidenced by an increase in FoxP3 mRNA and TGF-β1 release levels.This was accompanied by a significant decrease in the mRNA levels of T-bet and RORC,release of IFN-γ and IL-17A,and T-bet/GATA-3,RORC/FoxP3,IFN-γ/IL-4,and IL-17A/TGF-β1 ratios,compared with the PBMCs-C+alone.CONCLUSION The present in vitro studies showed that ADSCs contributed to the immunosuppressive effects on PBMCs-C+,favoring Treg responses.Thus,ADSC-based cell therapy could be a beneficial approach for patients with severe COVID-19 who fail to respond to conventional therapies.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice

AIM:To investigate the effects of Sonic hedgehog(Shh)gene-modified bone marrow mesenchymal stem cells(MSCs)on graft-induced retinal gliosis and retinal ganglion cells(RGCs)survival in diabetic mice.METHODS:Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs(MSC-Shh).Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs(MSC-Gfp;control)were administered in diabetic mice.After 4wk,the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography,and markers of gliosis were examined by immunofluorescence and Western blotting.The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence.The mechanisms underlying the effects of MSC-Shh was investigated.RESULTS:A significant reduction of proliferative vitreoretinopathy(PVR)was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp.Significant downregulation of glial fibrillary acidic protein(GFAP)was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp.The extracellular signal-regulated kinase 1/2(ERK1/2),protein kinase B(AKT)and phosphatidylin-ositol-3-kinase(PI3K)pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp.Brain-derived neurotrophic factor(BDNF)and ciliary neurotrophic factor(CNTF)levels were significantly increased in the host retina,and RGCs loss was significantly prevented after MSC-Shh administration.CONCLUSION:MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice.The ERK1/2,AKT and PI3K pathways are involved in this process.MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

Extracellular vesicles derived from mesenchymal stem cells mediate extracellular matrix remodeling in osteoarthritis through the transport of microRNA-29a

BACKGROUND Knee osteoarthritis(KOA)is a common orthopedic condition with an uncertain etiology,possibly involving genetics and biomechanics.Factors like changes in chondrocyte microenvironment,oxidative stress,inflammation,and immune responses affect KOA development.Early-stage treatment options primarily target symptom relief.Mesenchymal stem cells(MSCs)show promise for treatment,despite challenges.Recent research highlights microRNAs(miRNAs)within MSC-released extracellular vesicles that can potentially promote cartilage regeneration and hinder KOA progression.This suggests exosomes(Exos)as a promising avenue for future treatment.While these findings emphasize the need for effective KOA progression management,further safety and efficacy validation for Exos is essential.AIM To explore miR-29a’s role in KOA,we’ll create miR-29a-loaded vesicles,testing for early treatment in rat models.METHODS Extraction of bone marrow MSC-derived extracellular vesicles,preparation of engineered vesicles loaded with miR-29a using ultrasonication,and identification using quantitative reverse transcription polymerase chain reaction;after establi-shing a rat model of KOA,rats were randomly divided into three groups:Blank control group injected with saline,normal extracellular vesicle group injected with normal extracellular vesicle suspension,and engineered extrace-llular vesicle group injected with engineered extracellular vesicle suspension.The three groups evaluation,histological detection,and immunohistochemical detection to compare and evaluate the progress of various forms of arthritis.RESULTS General behavioral observation results showed that the extracellular vesicle group and engineered extracellular vesicle group had better performance in all four indicators of pain,gait,joint mobility,and swelling compared to the blank control group.Additionally,the engineered extracellular vesicle group had better pain relief at 4 wk and better knee joint mobility at 8 wk compared to the normal extracellular vesicle group.Imaging examination results showed that the blank control group had the fastest progression of arthritis,the normal extracellular vesicle group had a relatively slower progression,and the engineered extracellular vesicle group had the slowest progression.Gross histological observation results showed that the blank control group had the most obvious signs of arthritis,the normal extracellular vesicle group showed signs of arthritis,and the engineered extracellular vesicle group showed no significant signs of arthritis.Using the Pelletier gross score evaluation,the engineered extracellular vesicle group had the slowest progression of arthritis.Results from two types of staining showed that the articular cartilage of rats in the normal extracellular vesicle and engineered extracellular vesicle groups was significantly better than that of the blank control group,and the engineered extracellular vesicle group had the best cartilage cell and joint surface condition.Immunohistochemical detection of type II collagen and proteoglycan showed that the extracellular matrix of cartilage cells in the normal extracellular vesicle and engineered extracellular vesicle groups was better than that of the blank control group.Compared to the normal extracellular vesicle group,the engineered extracellular vesicle group had a better regulatory effect on the extracellular matrix of cartilage cells.CONCLUSION Engineered Exos loaded with miR-29a can exert anti-inflammatory effects and maintain extracellular matrix stability,thereby protecting articular cartilage,and slowing the progression of KOA.

Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing

BACKGROUND Mesenchymal stem cells(MSCs)modulated by various exogenous signals have been applied extensively in regenerative medicine research.Notably,nanosecond pulsed electric fields(nsPEFs),characterized by short duration and high strength,significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways.Consequently,we used transcriptomics to study changes in messenger RNA(mRNA),long noncoding RNA(lncRNA),microRNA(miRNA),and circular RNA expression during nsPEFs application.AIM To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs.METHODS The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing.MSCs were pretreated with 5-pulse nsPEFs(100 ns at 10 kV/cm,1 Hz),followed by total RNA isolation.Each transcript was normalized by fragments per kilobase per million.Fold change and difference significance were applied to screen the differentially expressed genes(DEGs).Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions,complemented by quantitative polymerase chain reaction verification.RESULTS In total,263 DEGs were discovered,with 92 upregulated and 171 downregulated.DEGs were predominantly enriched in epithelial cell proliferation,osteoblast differentiation,mesenchymal cell differentiation,nuclear division,and wound healing.Regarding cellular components,DEGs are primarily involved in condensed chromosome,chromosomal region,actin cytoskeleton,and kinetochore.From aspect of molecular functions,DEGs are mainly involved in glycosaminoglycan binding,integrin binding,nuclear steroid receptor activity,cytoskeletal motor activity,and steroid binding.Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation.CONCLUSION Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs,2 miRNAs,and 65 lncRNAs.Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways,which are involved in vesicular transport,calcium ion transport,cytoskeleton,and cell differentiation.