Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

OBJECTIVETo explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro.

METHODSHuman cord blood samples were obtained sterilely with 20 U/ml preservative-free heparin. MSCs were isolated by lymphocyte separation medium (density 1.077 g/ml), and purified and expanded with Mesencult trade mark medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron-like cells. Specific markers and structures were detected by immunohistochemistry and histochemistry methods.

RESULTSThe number of MSCs increased two- to three-fold with each expanded passage. 6.6 x 10(5) primary MSCs were expanded ten passages to reach a number of 9.9 x 10(8), and was increased about 1.5 x 10(3)-fold. Flow cytometry showed that MSCs did not express antigens CD(34), CD(11a) and CD(11b), but expressed strongly CD(29) and weakly CD(71), which was identical to human bone marrow-derived MSCs. 70% cells exhibited typical neuron-like phenotype after induction. Immunohistochemistry staining showed that all of the induced different-passage MSCs expressed neurofilament (NF) and neuron-specific enolase (NSE). Special Nissl body was found by histochemistry.

CONCLUSIONMSCs in human umbilical cord blood can expand in vitro and differentiate into non-mesenchymal cells.

Antigens, CD ; analysis ; Antigens, Differentiation, B-Lymphocyte ; analysis ; Cell Count ; Cell Differentiation ; Cell Division ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Mesoderm ; chemistry ; cytology ; Neurofilament Proteins ; analysis ; Neurons ; cytology ; Phosphopyruvate Hydratase ; analysis ; Receptors, Transferrin ; Stem Cells ; chemistry ; cytology

Wnt signaling is implicated in the control of cell growth and differentiation during neural stem cell(CNS) development.Wnt3a, one of wnt gene family members, has effect on regeneration neurospheres and differentiation into neurons.Wnt3a inhibits regeneration of neurospheres, and promotes its differentiation. In vitro neurosphere was cultured in a serum-free defined medium DMEM/F12 supplemented with bFGF and EGF. Dissociated cells were plated onto poly-d-lysine-coated coverslips and propagated in medium containing recombined Wnt3a-adenovirus. Plenty of Nurr1 were detected by RT-PCR after 3 days. Wnt3a combined AA would improve NSC differentiation into dopaminergic (DA) neuron. The quantity of DA neuron is obviously more than the AA alone group's. Moreover, the expression of TH mRNA is 1.86 fold in Wnt3a combined AA group. Induced cells were immunostained for TH and DAT. The proportion of TH-positive was (37.42 ? 2.54) % (P

The transcriptional repressor RE1 silencer transcription factor(NRSF/REST) is an important factor that restricts some neuronal traits in neurons.Since these traits are also present in pancreatic islet cells,NRSF-regulated genes involved in islet function are searched.A NRSE-like motif was analysed in human insulin promoter.The role of NRSE was evaluated by generating a model of insulin-secreting cells that firmly express NRSF.The presence of NRSF led to a decrease in activity of human insulin promoter by stable or transient transfection with human insulin-promoter luciferase.The predicted NRSE-like motif also confers NRSF-dependent transcriptional repression in the context of a surrogate gene promoter.Specific binding activity of NRSF/REST to the NRSE-like motif was confirmed by EMSA.Moreover,the binding activity is competed by consensus NRSE sequence.These data showed that human insulin promoter is regulated by the transcriptional repressor NRSF/REST via the NRSE-like motif.