Effects Of Atra And Brain Microenviroment On The Differentiation Of MSCs Into Neuron-Like Cells

[Background] Besides hematopoletic stem cells, bone marrow contains mesenchymal stem cells (MSCs). They can self-renew and have differentiation potential. Under specific experimental conditions, they can differentiate into osteoblasts, chondroblasts, adipocytes, cardiomyocytes, hepatocytes, neural cells and so on. They are easy to get and expand in vitro and have weak immunological rejection, so MSCs become a hot topic. We have found all-trans retinoic acid (ATRA) is important for the differentiation of MSCs into neuronal cells. But the mechanism of action of ATRA is not known and there are few reports about it.[Objective] To investigate the effects of ATRA and brain microenvironment on the differentiation of MSCs into neuron-like cells.[Methods] MSCs are isolated from bone marrow of adult rat and expanded as undifferentiated cells in culture for 5-7 passages, then platedin twelve-well culture dishes. When the cells reach about 70%~80% confluence, they are cultured in the modified neuronal medium (MNM) after 24 hours of ATRA treatment. The control cells are directly cultured in MNM without ATRA pretreatment. Immunocytochemistry is used to detect the expression of nestin , neuron-specific enolase(NSE), neuron-specific nuclear protein (NeuN) , microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein(GFAP) and retinoic acid receptor beta (RARp) at different time points. And flow cytometry analysis is used to measure the change of the cell cycle before and after ATRA treatment. At the same time, MSCs are labeled with DAPI in vitro and injected into the ischemic brain of rats, which have suffered from the embolic middle cerebral artery occlusion (MACO).[Results] (1) After ATRA and MNM treatment, responsive the cells progressively assume neuronal morphological characteristics. (2)The expression of nestin , NSE, NeuN and MAP-2, is much higher than that of the control group. Nestin occurs first; and then NSE and NeuN express; the last one is MAP-2 and it's detected at 9 hours of MNM treatment. The other markers continuously express, but the expression of nestin peaks at 18 hours of MNM induction and remarkable decreases at 36 hours. (3) No expression of RARp is found in MSCs; it can be detected after 24 hours of ATRA treatment and 70.2%±5.1 % of them are positive expression at 6hours of MNM induction. (4) No changes are found in the cell cycle before and after ATRA treatment. (5) After injected into rats for 2 weeks, MSCs survive in the ischemic areas and a few cells express nestuu MAP-2 and GFAP respectively.[Conclusions] (1) ATRA can promote the differentiation of MSCs into neuron-like cells and the expression of neural-specific markers is consistent with current knowledge regarding the timepoints of markers expression in the neuronal development. Maybe this provides a good model in vitro for neuronal development research. (2) ATRA might activate gene transcription via nuclear receptors RARp and promote the neuronal differentiation of MSCs. To my knowledge, there is no such report in the world. (3) MSCs have weak immunological rejection and they can survive and differentiate in the ischemic brain.