Bone morphogenetic protein-4 influences neural differentiation of induced mouse mesenchymal stem cells

Members of the transforming growth factor-beta superfamily, including bone morphogenetic protein 4 [BMP4], have been implicated as regulators of neural differentiation. The aim of this study was to establish whether BMP4 could influence neuronal differentiation of mesenchymal stem cells [MSCs]. Therefore, neuronal differentiation of MSCs was induced by basic fibroblast growth factor [bFGF] and epidermal growth factor [EOF] and treatment. The expression of neuronal specific markers such as Nestin, MAP2, p-Tubulin III and NKX6.1 were detected by RT-PCR, flow cytometery and/or immunostaining. While the percentage of Nestin positive cells was increased significantly during treatment, the addition of BMP4 during the first 4 days of treatment with bFGF and EGF reduced Nestin expression as showed by flow cytometry. This observation was further confirmed by relative gene expression which showed the reduction in expression of neural markers such as Nestin, MAP2 and NKX6.1 following treatment with BMP4. The results of this study suggest that BMP4 downregulates the neural fate of induced mouse MSCs

[Optimization of RT-PCR conditions for estimation of peroxisomal genes [Catalase, PEX3] expression compare with pluripotent genes [Nanog, Oct4] expression in stem cells]

Peroxisomes are ubiquitous organelles in nearly all eukaryotes that participate in the metabolism of fatty acids and other metabolites. In order to investigate peroxisome biogenesis and gene profile expression analysis during neurogenesis, we have performed to set a semi-quantitative analysis for two peroxisomal genes expression [Catalase and PEX3] during neurogenesis comparing with stem cell marker genes [Oct4 and Nanog] in P19 cells. In this project we have used P19 cells which are suitable progenitor cells for the study of neurogenesis. Moreover, two different peroxisomal genes were selected to chase both kinetics of peroxisomal matrix protein synthesis [Catalase] and peroxisomal membrane protein biogenesis [Pex3p]. Total RNA extracted from P19 cells, and was used for cDNA synthesis at the next step. Specific primers of Oct4, Nanog, PEX3 and Catalase cDNAs were amplified by RT-PCR for quantitative analysis. Various parameters were changed to optimize the PCR profiles for each gene for further assessments. Here we are going to describe the conditions which we used, e.g.: PEX3 band was detected sharply after 35 cycles and at annealing temperature of 52.5-55.1°C. For PCR of Catalase we observed a sharp band after 35 cycles and annealing temperature of 52.7-55°C. Nanog cDNA was amplified after 36 cycles and between 60.1-64.1°C. Oct4 band was observed sharply after 28 cycles of amplification and annealing temperature in range of 53.4-57°C