The development of skeletal muscle is a highly regulated, multi-step process in which pluripotent mesodermal cells give rise to myoblasts that subsequently withdraw from the cell cycle and differentiate into myotubes as well as myofibers. The plasticity of myoblasts plays a critical role in maintaining skeletal muscle structure and function by myoblast activation, migration, adhesion, membrane reorganization, nuclear fusion, finally forming myotubes/myofibers. Our studies demonstrate that the local hypoxic microenvironment, a great diversity of regulatory factors such as IL-6 superfamily factors (IL-6, LIF, CNTF) and TGF-beta1 could regulate the myoblast plasticity. The aim of this paper is to review the previous studies focused on the regulation of myoblast plasticity and its mechanism in our laboratory. Knowledge about the microenvironment or factors involved in regulating the myoblast plasticity will help develop the prevention and cure measures of skeletal muscle diseases.
Cell Differentiation ; Cellular Microenvironment ; Humans ; Hypoxia ; Muscle Fibers, Skeletal ; cytology ; Muscle, Skeletal ; cytology ; Myoblasts ; cytology

OBJECTIVETo explore the way of stably inducing canine bone marrow mesenchymal stem cells (BMSCs) to differentiate into fibroblasts and myofibroblasts in vitro, and provide seed cells for fabricating tissue engineering heart valves (TEHV).

METHODSAdult canine BMSCs were separated by a gradient centrifugation on Percoll (density 1.073 g/ml), then the cells were incubated in low-glucose Dulbecco Eagle's minimum essential medium (LG-DMEM) with 10% bovine calf serum. Cell phenotype were identified by immunohistochemistry staining. The second and third generation of BMSCs were committedly induced by conditioning culture medium, which were detected by immunohistochemistry staining. The induced-BMSCs were freezed, preserved and resuscitated after 7 d to observe the cell growth, proliferation and function.

RESULTSBMSCs deriving from the bone marrow mononuclear cells separated by a Percoll gradient were positive expression of alpha-smooth muscle antibody, vimentin and negative expression of CD34, laminin. About (50 +/- 3)% induced-BMSCs were positive expression of laminin. Approximately (85 +/- 3)% freezed induced-BMSCs could be resuscitated. And the growth, proliferation and function were well.

CONCLUSIONBMSCs could be committedly induced to differentiate into fibroblasts and myofibroblasts in vitro. It is suitable to be the seed cells.

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Dogs ; Fibroblasts ; cytology ; Mesenchymal Stromal Cells ; cytology ; Monocytes ; cytology ; Myoblasts ; cytology

Animals ; Cell Differentiation ; physiology ; Cell Hypoxia ; Cell Line ; Mice ; Myoblasts ; cytology ; Transcription Factors ; metabolism

Duchenne muscular dystrophy (DMD) is a fatal, genetic neuromuscular disorders that manifests as progressive muscle wasting. Although there has been enormous progress in the studies of the molecular mechanism of muscular dystrophy, there is still no cure. Cell-based therapy is a promiseful option. This review will focus on the present status of cell-based therapy. Myoblast transfer therapy is hindered by minimal distribution of cells after injection, immune rejection, and poor cell survival. The drawback of bone marrow-derived stem cell transplantation is the low efficiency of transdifferentiation. Compared with them, the injection of postnatal muscle-derived stem cells (MDSC) results in a superior regeneration of dystrophin-expressing myofibers.
Animals ; Bone Marrow Cells ; cytology ; Humans ; Muscle, Skeletal ; cytology ; Muscular Dystrophy, Duchenne ; therapy ; Myoblasts, Skeletal ; transplantation ; Stem Cell Transplantation ; methods

OBJECTIVETo isolate human adipose tissue-derived stromal cells and study the potential of osteogenic differentiation after inductive culture.

METHODSLiposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were plated in BGJb medium as primary culture for ten days. The second passage cells were harvested and plated in DMEM/F12 medium supplemented with 10% FBS, 5% horse serum and 50 micromol/L hydrocortisone for myogenic induction culture. The cell-anchored slips were removed and fixed in 4% formaldehydam polymerisatum. Toluidine blue, Mallory's phosphotungstic hematoxylin staining and monoclonal antibody to human skeletal muscle myosin heavy chain immunocytochemical methods were used to assay the differentiation of cells.

RESULTSIt was observed that the size and shape of induced cells were much different from those of non-induced cells. Toluidine blue, Mallory's phosphotungstic hematoxylin staining demonstrated there were many basophilic striations within cytoplasm and multinucleated myotubes were formed. Immunocytochemical stain indicated that characterastic skeletal myosin heavy chain was positive in myogenic induced cells.

CONCLUSIONIt seems that human adipose tissue represents an abundant reservoir of adult stem cells that have multi-germline potential to differentiate into myoblasts. Adipose tissue derived stromal cells will be another alternative source for cell-based tissue engineering in skeletal muscle reconstruction.

Adipose Tissue ; cytology ; Adult Stem Cells ; cytology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Culture Media ; Humans ; Myoblasts ; cytology ; Myosin Heavy Chains ; metabolism ; Stromal Cells ; cytology

OBJECTIVETo study the growth and differentiation of adult canine autologous skeletal myoblasts after being transplanted into acute myocardial infarction (AMI) region by intramyocardium injection (IMI) and intracoronary infusion (ICI).

METHODSAutologous skeletal myoblasts were procured by a modified method. AMI model of adult canine was obtained through left anterior descending branch ligation and was divided into 4 groups (n = 5 for each group). Autologous skeletal myoblasts (1.0 - 1.4 x 10(8) cells) were injected locally into AMI region or infused into infarction-related coronary artery. Specimens were harvested 4 weeks after cellular transplantation for histological study including HE, PTH, immunochemical stain and transmission electronmicroscope.

RESULTSIn both two transplantation groups, newborn muscle-derived cells, striated muscle tissue and mature skeletal myofibril were demonstrated existing in MI region by electronmicroscope, PTH stain or anti-skeletal myosin heavy chain (slow) immunochemical stain, respectively. Newborn striated muscle tissues arranged in order of consistency with host myocardial fibers in two treatment groups. Newborn striated muscle tissue was more inclined to gather in MI region in the local injection group but distracted from each other in the intracoronary infusion group.

CONCLUSIONAutologous skeletal myoblasts appears to live and differentiate into mature striated muscle tissue after transplanting into AMI region by IMI or ICI routes.

Animals ; Cell Transplantation ; methods ; Cells, Cultured ; Dogs ; Female ; Male ; Myoblasts, Skeletal ; cytology ; transplantation ; Myocardial Infarction ; surgery ; Transplantation, Autologous

The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism and cell differentiation. However, its role in myoblast differentiation is unclear. To study the biological function of FoxO1 during differentiation in porcine primary myoblast, we constructed stably FoxO1 over-expressed porcine myoblast mediated by liposome and adopted morphological observation, quantitative real-time RT-PCR and Western blotting methods to analyze FoxO1 and early and late myogenic regulation factors MyoD and myogenin expression. During differentiation the mRNA level of FoxO1 was significantly increased. However, the total protein did not change but the phosphorylation of FoxO1 was upregulated. Furthermore, overexpression of FoxO1 in porcine myoblast decreased MyoD and myogenin mRNA, whereas MyoD protein changed little and myogenin was significantly suppressed (P < 0.05). These results indicated that FoxO1 delays and negatively regulates the porcine myoblast differentiation. Moreover, FoxO1 may play a critical role in muscle fiber-type specification through the inhibition of myogenic regulation factors.
Animals ; Animals, Newborn ; Cell Differentiation ; genetics ; Cells, Cultured ; Forkhead Transcription Factors ; biosynthesis ; genetics ; Muscle, Skeletal ; cytology ; metabolism ; Myoblasts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Swine

AIMTo investigate whether AcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

METHODSNeonatal rat cardiac fibroblasts were isolated. The cell proliferation was observed by 3H-proline incorporation assay.

RESULTSOn the culture of 0.4% FBS, PDGF stimulated cardiac fibroblasts proliferation and collagen synthesis with a dose-dependent manner at the concentrations from 1 ng/ml to 20 ng/ml, in which 10 ng/ml PDGF reached its peak. AcSDKP at the concentration from 10(-10) mol/L to 10(-8) mol/L could inhibit cardiac fibroblasts proliferation and collagen synthesis mediated by PDGF. 10(-9) mol/L AcSDKP attained its peak on inhibiting cardiac fibroblasts proliferation and collagen synthesis.

CONCLUSIONAcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

Animals ; Cell Proliferation ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Myoblasts, Cardiac ; cytology ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Rats, Wistar

To determine the role of recombinant human ciliary neurotrophic factor (rhCNTF) in myogenesis, we observed the effects of rhCNTF (0 10 ng/ml) on myoblast differentiation of adult human in vitro. The results showed that compared with the control group, the groups of 2.5 10 ng/ml rhCNTF treatment significantly inhibited myoblast differentiation (P<0.01), and the inhibition was dose-dependent and reversible. Western blots also indicated that compared with the control group, the expression of myogenin and p21, markers of myoblast differentiation phase, was significantly reduced (P<0.01), while Myf5 and desmin, markers of myoblast proliferative phase, significantly increased (P<0.01) in the groups of 2.5 10 ng/ml rhCNTF treatment. These findings demonstrate that exogenous rhCNTF can reversibly inhibit differentiation but permits proliferation of adult human myoblasts in vitro.
Adult ; Cell Differentiation ; drug effects ; Cell Proliferation ; Cells, Cultured ; Ciliary Neurotrophic Factor ; pharmacology ; Humans ; Middle Aged ; Muscle, Skeletal ; cytology ; Myoblasts ; cytology ; Recombinant Proteins ; pharmacology ; Young Adult

OBJECTIVE: To clarify the effect of nucleolin on the proliferation and apoptosis in C2C12 cells. METHODS: After inhibiting the expression of nucleolin using antisense oligonucleotides, the cellular proliferation was determined by MTT, and the apoptosis was detected by flow cytometry (FCM) assays and DNA ladder assays. RESULTS: After being transfected with antisense oligonucleotides for 24 hours, Western blotting showed that the expression of nucleolin was repressed significantly. In cells treated with antisense oligonucleotides, the cellular proliferation was obviously inhibited; the apoptotic cell increased significantly; and the "DNA ladder" was clearly observed. But the sense and random oligonucleotides had no effect on the cellular proliferation and apoptosis. CONCLUSION The down-regulation of nucleolin can inhibit the cellular proliferation and initiate the apoptosis in C2C12cells.
Animals ; Apoptosis ; physiology ; Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Mice ; Myoblasts ; cytology ; Myocytes, Cardiac ; cytology ; Oligonucleotides, Antisense ; Phosphoproteins ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; Transfection