study on in-vitro transdifferentiation of rat bone marrow stromal cells into neuroepithelial-like cells

ABSTRACT

Bone marrow stem cells [BMSCs] are a rich source of stem cells and may represent a valid alternative to neural or embryonic stem cells by replacing the autologous damaged tissues in neurodegenerative diseases. In this study, we attempted to devise a protocol for the induction of BMSCs into neuroepithelial-like cells [NELCs]. Rat BMSCs were isolated from the long bones of adult Sprague-Dawley rats. Their purity in the 4th passage was evaluated with fibronectin by immunocytochemistry, and the stemness marker Oct-4 was assessed by RT-PCR technique. The cells were expanded and induced in the induction stage. The BMSCs were incubated with either beta-mercaptoethanol [micro ME] [1 mM], dimethyl sulfoxide [DMSO] [2%] or biotylated hydroxyanisol or butylated hydroxyanisol [BHA] [200 micro M] in beta-MEM medium without fetal bovine serum [FBS]. They were washed with phosphate buffer saline [PBS] and proceeded to the 2nd phase of induction, where the induction medium was changed with beta-MEM and 15% FBS containing all-trans retinoic acid [RA] [1 micro M] [for 3 days]. Then, the expression of the markers was assessed with GFAP, nestin and neurofilament 68 antibodies, respectively and the expression of Oct-4 and NeuroD was evaluated by RT-PCR. The purity of the BMSCs at the 4th passage was more than 92%. The mRNA of Oct-4 was expressed in these cells. Induction of BMSCs by DMSO-RA could differentiate NELCs significantly more than beta ME-RA and BHA-RA. The transdifferentiation of NELCs was evaluated by nestin antibody and NeuroD mRNA expression; later markers expressed very low detectable level in BMSCs. But the differentiation of BMSCs into astrocytes was less in all of the experiment groups that is estimated GFAP antibody. The application of DMSO-RA can transdifferentiate BMSCs into NELCs in- vitro