Cholinergic differentiation of neural stem cells generated from cell aggregates-derived from Human Bone marrow stromal cells

Abstract

Human Bone marrow stromal cells (hBMSCs) can differentiate under appropriate experimental conditions into neuronal and glial-like cells. This study shows a protocol for producing human neural stem cells (hNSCs) from hBMSCs and the subsequent differentiation of hNSCs into cholinergic neurons (CNs), where sequential media replaced the culturing media. hBMSCs have been used in generating cell aggregates (CAs) using bFGF, EGF and B27. The hNSCs were isolated from CAs, and the CNs differentiated from the hNSCs using sequential media, where bFGF, EGF and B27 were gradually replaced with NGF. The hNSC stemness was checked by RT-PCR of SOX2, Oct-4 and Nanog genes. Fibronectin, CD90, CD106, CD31, nestin, neurofilament 68 (NF-68), NF-200 and ChAT immunostaining evaluated the differentiation of the hBMSCs, the hNSCs and the CNs. FM1-43 was used in studying the function of the CNs. The hBMSCs were immunoreactive to fibronectin, CD90 and CD106; they were checked for lipogenic and osteogenic differentiation. The cells of the CAs were immunoreactive to nestin. The hNSCs were immunoreactive to nestin and NF-68, also, they expressed SOX2, Oct-4 and nanong. Nestin expression declined sharply following NSC differentiation into CNs, while the expression of NF-200, synapsin I, synaptophysin, MAP-2 and ChAT increased. They were stained with FM1-43, where the synaptic vesicles were released following stimulation. The present study demonstrates the conversion of hBMSCs into CASs under appropriate conditions. CAs generated hNSCs, which were induced in order to differentiate into CNs using sequential media, where the yield was 83%.