Isolation, cultivation and identification of skeletal muscle stem cells derived from human orbicularis oculi muscle / 中华整形外科杂志

Objective@#To establish the isolation and culture methods of skeletal muscle stem cells, derived from human orbicularis oculi muscle (OOMSCs), and to identify their multi-directional differentiation potential in vitro.@*Methods@#The cellswere isolated from pretarsal orbicularis oculi muscle (OOM), obtained in routine blepharoplasty surgery.The tissue was digested using collagenase type I combined with re-plating methods. Specific cell surface antigen markers were detected using flow cytometry analysis. OOMSCs were cultured under different inductive conditions, to identify their pluripotent differentiation ability.@*Results@#OOMSCs exhibited similar fibroblast-like morphology as mesenchymal stem cells with high expression of skeletal muscle-derived stem cell surface markers. OOMSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes in vitro, in the presence of lineage-specific inductive media. Moreover, after myogenic induction, the differentiated cells were fused into multinucleated myotube-like structure, and positive for myogenic-related marks, Pax3, Pax7, Myf5 and MyoD.@*Conclusions@#Muscle-derived stem cells can be isolated from human OOM with myogenic differentiation properties, showing further applications potential intissue regeneration and medical therapies of muscle diseases.

Construction of vascular endothelial growth factor modified muscle-derived stem cells and its expression / 实用医学杂志

Objective To construct the lentiviral vector encoding vascular endothelial growth factor gene and detect the vascular endothelial growth factor (VEGF) expression in muscle-derived stem cells (MDSCs). Methods To culture MDSCs and detect the CD34,CD45,Bcl-2 and Desmin expression in MDSCs by immunofluorescence. A cDNA encoding VEGF gene was amplified by PCR. This fragment was cut with EcoRI and BamHI and ligated with an EcoRI- and BamHI-reated lentiviral vector pCDH-CMV-MCS-EF1-copGFP. Then DNA sequencing analysis was performed to confirm successful construction of pCDH-CMV-MCS-EF1-copGFP -VEGF. The expression of VEGF was confirmed using enzyme-linked immunosorbent assay (ELISA), Western blot, and Real-time PCR analyses. Results The pCDH-CMV-MCS-EF1-copGFP-VEGF lentiviral vector was constructed successfully. When MOI values in the transfection efficiency MDSCs by FCM. were 1,5,15, the transfection rate reached to 16.7%, 45.6%, 66.3% and 85.6% respectively. When MOI value was of 20, the rate was up to 90.1%. Real-time PCR, Western blot and ELISA showed stable expression of VEGF in MDSCs. Conclusion We successfully constructed lentiviral vector carrying the VEGF and stable expression in MDSCs.

Role of TGF-β1 in the process of differentiation of denervated skeletal muscle-derived stem cells in vitro / 中华显微外科杂志

Objective To investigate the effect on differentiation of denervated skeletal muscle-derived stem cells (MDSCs) induced by TGF-β1 in vitro.Methods MDSCs were obtained from the rat denervated skeletal muscle by preplate technique,with TGF-β1 adding on medium.Cultured cells were divided into two groups.A,control group; B,10 ng/ml TGF-β1 group.Cell growth was observed with phase contrast microscope.lmmunocytochemistry,quantitative RT-PCR and Western blot was used to detect the expression of Sca-1,COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin in denervated MDSCs.Results The synthesis of COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin by denervated MDSCs was extremely low at protein level in vitro,while Sca-1 level was really high.Belong to the treatment with TGF-β1,COL-Ⅰ,COL-Ⅲ,oα-SMA and vimentin in the denervated MDSCs had strong expression,but Sca-1 in which had a weak expression.Under the stimulation of TGF-β1,COL-Ⅰ expression reached peak at the 2nd day (12.5591 ± 0.3389),which was about 3 times as control group.COL-Ⅲ reached highest value at the 5th day (0.8956 ± 0.0438),which was about 23 times as control group.α-SMA topped out to 18 times at the 5th day (1.1090 ± 0.0018).Vimentin expression rose by 8.5 times and peaked at the 5th day (0.1794 ± 0.0019).The expression of Sca-1 began to decline at the 2nd day,with a remarkable reduction at the 5th day (0.0636 ± 0.0015).Conclusion TGF-β1 could induce differentiation of the denervated MDSCs to myofibroblasts in vitro,and promote the synthesis and excretion of extracellular matrix.

Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Skeletal Muscle-derived Stem Cells Exhibit Cardiocyte Competences / 华中科技大学学报（医学）（英德文版）

Adult stem cells from skeletal muscle cells were induced to differentiate into cardiocytes to see if stem cells from another different but histologically-comparable tissues can differentiate to the target cells. Skeletal muscles-derived stem cells (MDSCs) were isolated from adult skeleton muscle tissues by differential adhesion,and immunocytochemically identified by using Sca-1. In order to induce the proliferation but not differentiation of MDSCs,the cells were cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) supplemented with 1:50 B27,20 ng/mL basic fibroblast growth factor (bFGF),20 ng/mL epidermal growth factor (EGF) in a suspension for 6 days. Then these stem cells were treated with 5 μmol/L 5-azacytidine for 24 h in an adherence culture. The characteristics of induced cells were examined by immunocytochemistry,quantitative real time RT-PCR and morphological observation of cell phenotype. Our results showed that the appearance of some cells gradually changed from spindle-shape into polygonal or short-column-shape. Some of these post-treated cells could contract spontaneously and rhythmically. The expression of GATA-4 and cTnT was increased 1 and 2 week(s) after the treatment. And about 16.6% of post-treated cells were cTnT-positive. Therefore,we are led to conclude that skeletal muscle-derived stem cells could differentiate into cardiocyte-like cells,which exhibited some characteristics of cardiocytes.

Transdifferentiation of rat muscle derived stem cells into insulin-secreting cells in vitro / 基础医学与临床

Objective To explore the potential diferentiation of insulin-secreting cells from muscle derived stem cells.Methods SD rat muscle derived stem cells(MDSCs)were isolated and then induced for 7～12 days with combination inductor.Morphological changes of the induced cells were observed under an inveted microscope.DTZ staining,immunocytochemical staining were conducted to examine the production of insulin.Glucose challenge and radio immunoassay were perfomed to evaluate the function of the aggregates,and RT-PCR was conducted to detect the expression of endocrine gene.Results Typical insulin-producing cells were observed at the end of the protocol(12 days).Ditizone staining and immunohistochemistry for insulin were both positive.Insulin was secreted by these cells in response to different concentrations of glucose stimulation(P

Recent Progress in Research on Muscle-derived Stem Cells / 中国生物工程杂志

Muscle recently has been identified as a good source of adult stem cells that can differentiate into cells of different lineages.Researchers have identified two types of stem cells in skeletal muscle.Further research is necessary to delineate the relationship between different populations of musclederived stem cells(MDSCs)and between MDSCs and other adult stem cells.The methods used to isolate these cells appear to influence the stem cell characteristics.As these efforts continue,the potential for MDSCsbased therapy for other musculoskeletal injuries,as well as for cardiac and smooth muscle injuries,is currently being explored.The behavior,biocharacteristic,isolation,differentiation and the probability of application to regenerate lost or diseased tissue of MDSCs were summarized.

Application of tissue engineering in treatment of stress urinary incontinence / 上海第二医科大学学报

Stress urinary incontinence is one of the most common diseases in urinary system.At present,the major methods for treating stress urinary incontinence include medication,physico-behavior therapy and operation.However,for various reasons,the current methods do not yield satisfactory results.As a newly emerging technique,tissue engineering provides a new concept and method to treat stress urinary incontinence.The application of tissue engineering in the treatment of stress urinary incontinence is reviewed in this article.