Autologous multipotent stem cells are most relevant cells for regenerative medicine and show prosperous future in the treatment of human diseases. Previous reports have indicated that multipotent stem cells (MSCs) can be obtained from bone marrow and adipose tissues. In this study, we proved that dermis may be another source of these cells. MSCs were isolated from the dermis of newborn rats one day old by adhesion competition and successive culture. These cells conserved the ability to differentiate to osteoblasts, chondrocytes and adipocytes by induction media containing dexamethasone. After long term of more than 6 months, till 25th generation, the cells still maintained the characteristics of stem cells: high activity of self-renewal and multipotency. Mixed collagen matrix from dermis could promote the growth of dermis-derived multipotent stem cells and collagen sponge stent could promote their three-dimensional growth in vitro.
Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; methods ; Cells, Cultured ; Collagen ; pharmacology ; Dermis ; cytology ; Multipotent Stem Cells ; cytology ; drug effects ; physiology ; Rats ; Rats, Wistar

OBJECTIVE: To explore the differentiation potential of QY1 bone marrow mesenchymal stem cell (MSCs) line cells into cardiacmyocytes and vascular endothelial cells in vitro, to optimize the suitable conditions of MSCs differentiating into cardiomyocytes in vitro, and to examine the potentials of MSCs differentiating into cardiomyogenesis and vasculogenesis. METHODS: Specifically committed differentiation inductive medium was employed, including 5-azacytidine for cardiomyogenesis and vascular endothelial growth factor for vasculogenesis in culture respectively in vitro. The differentiated cells were identified by immunohistochemistry and molecular biology. RESULTS: MSCs line cells had been cultured in the normal culture medium for 72 hours, then the differentiation inductive medium including 10 micromol/L 5-azacytidine was added into the normal culture dishes for 24 hours only. After that the culture medium was changed back to the normal culture medium. Normal culture medium was changed every 7 days. The second induction was performed after 14 days. The differentiated cells treated with 5-azacytidine could beat spontaneously and formed myotube structures in the optimal induction conditions, and the differentiation rate was (39.47+/-0.56)%. The differentiated cells expressed specific cardiomyocytic proteins identified by the positive immunohistochemistry staining with anti-alpha-sarcomeric antibody and anti-Cx-43 antibody, and also expressed the alpha-myosin heavy chain examined by RT-PCR. The differentiated cells began to appear as the lined up vascular endothelial cells after 48 hour treatment with vascular endothelial growth factor. Some of the differentiated cells connected each other to form vascular endothelial web-like structure after 7 day treatment with vascular endothelial growth factor. On 14 d after treating with vascular endothelial growth factor, the differentiated cells were identified by immunohistochemistry staining. The expressions of both specific surface antibody CD31 and factor VIII for vascular endothelial cells were positive. CONCLUSION The cells of QY1 bone marrow mesenchymal stem cell line may differentiate into cardiomyocytes or vascular endothelial cells in vitro under specific condition.
Azacitidine ; pharmacology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; metabolism ; Factor VIII ; biosynthesis ; Humans ; Immunohistochemistry ; Mesenchymal Stem Cells ; cytology ; Multipotent Stem Cells ; cytology ; Myocytes, Cardiac ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; biosynthesis

The objective of present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural stem cells NSCs differentiation in vitro, Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. Immunofluorescence method was used to detect the expression of NSCs marker-Nestin within these cells. Cultured in 10% FBS, NSCs began to differentiate. On the onset of the differentiation, HCMV AD169 (MOI=5) was added into the differentiation medium. After 7 days differentiation, the effect of HCMV infection on NSCs differentiation was observed by detecting the rate of nestin, GFAP and HCMV immediate-early (IE) positive cells with confocal microscopy and immunofluorescence method. The resucts showed most of the cells (passage 4-6 ) were Nestin positive and could differentiate into NSE-positive neurons and GFAP-positive astrocytes. On day 7 postinfection, 86% +/- 12% of infected cells were IE positive. The percentage of Nestin-positive cells was 50% +/- 19% and 93% +/- 10% (t= 6.03, P<0.01)and those of GFAP-positive cells was 81% +/- 11% and 55 +/- 17% (t=3.77, P<0.01) in uninfected and infected cells respectively. These findings indicated that NSCs were HCMV permissive cell and HCMV AD 169 infection suppressed the differentiation of Hippocampus-genetic human neural stem cells into astrocytes.
Astrocytes ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Cytomegalovirus ; growth & development ; physiology ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Hippocampus ; cytology ; Humans ; Intermediate Filament Proteins ; metabolism ; Microscopy, Fluorescence ; Multipotent Stem Cells ; cytology ; drug effects ; metabolism ; virology ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals.
Animals ; Brain/*cytology ; Buthionine Sulfoximine/pharmacology ; Cell Differentiation ; Cell Line ; Cell Lineage ; Cytokines/pharmacology ; Enzyme Inhibitors/pharmacology ; Excitatory Amino Acid Agonists/pharmacology ; Glutamic Acid/pharmacology ; Humans ; Hydrogen Peroxide/pharmacology ; Intercellular Signaling Peptides and Proteins/pharmacology ; Kainic Acid/pharmacology ; Mice ; Mice, Inbred BALB C ; Multipotent Stem Cells/cytology/*drug effects/physiology ; Neuroglia/cytology/drug effects/physiology ; Neurons/cytology/*drug effects/physiology ; Neurotoxins/*pharmacology ; Oxidants/pharmacology ; Phenotype ; Reactive Oxygen Species/metabolism