Bone Marrow Mesenchymal Stem Cells Differentiate into Neural Cells in Vitro

Abstract: Marrow stromal cells are adult stem cells from bone marrow that have the potency to differentiate into multiple nonhematopoietic lineages such as bone , cartilage, and lipocyte et al. It also can differentiate into myocytes, hepatocytes, glial cells, and neurons.Marrow stromal cell can be easily obtained and have a set of well – developed separation and culture techniques . we have tested a few antioxidants to find the relationship between the antioxidant and neural transdifferentiation of MSCs. Studies on neural differentiation is of great importance in clinic: if we can develop a good protocol , induced MSCs may be useful in the treatment of a wide variety of neurological diseases. Such as paraplegia, Parkinson's disease,and strokes et al.On seeking the inducing agent , scientist had done many works to seek the appropriate agent and protocols, Sanchez – Ramos, Wood-bury , Kohyama, Reyes and Verfaillie had their own methods and concentrate on the neuronal factors and traditional antioxidant such as beta – mercaptoethanol etc. And on the other end of the world, Chinese scientists had exciting progresses using the Chinese Herbs as effective inducing agent.In our experiment, I have tested the inducing hypothesis that antioxidant maybe one of the inducing mechanism. In my experiment,only beta – mercaptoethanol have the potency to induce MSCs to neural cells, exclude the other antioxidant such as glutathione,ascorbic acid and vitamin E( alcohol dissolved) , and using beta – mercaptoethanol and serum – free DMEM medium as the main differentiation a-gent. We can see a lot of new pictures of induced neuron – like cells, include neuron – like cells and glial – like cells, with the apparent nerve cells.Material and methodsRat bone marrow cell were collected, after sacrifice of a 3 months old WISTAR rat , from femurs and tibias by flushing the shaft with culture media(DMEM – 10% fetal bovine serum) using a syringe of 5mL. Cells were centrifuged for 5min at 1000rpm and removed supernatant. The cell peller was resuspended in 1 mL culture media and divided into two glass flask.Cell surface makers ( CD45, CD90 ) were detected by immunocy-tochemistry.Subconfluent cultures of rat were maintained in DMEM/10% FBS, 24hrs before neuronal induction, media were replaced with pre-induction media consisting of DMEM/10% FBS/lmM beta – mercaptoethanol ( BME) , to initiate neuronal differentiation, the preinduction media were removed, and the cells were washed with PBS and transferred to neuronal induction media composed of DMEM/serum – free media, 5hrs later , 40ul DMSO was given to every hole containing 2ml each.Two hours later, according to the protocol of the SABC kit of Boster Company, an immunocytochemistry was done to examinewhether there is neuron – specific proteins in cytoplasm. NSE and were examined by specific antibody from Boster Company.ResultWithin 10 minutes, the cells' shape begins to change; cytoplasm in the flat rMSCs retracted towards the nucleus, forming a contracted multipolar cell body, and leaving membranous process – like extensions peripherally. Cell exhibited increased expression of the neuronal marker NSE,this result was different from Woodbury's.DiscussionOur observations indicate that rat and human MSCs retain the capacity to differentiate into nonmesenchymal derivatives 0 Specifically neurons suggestion that the cell fate and commitment are mutable. These adult cells are self – renewing and multipotential. In experiment, we can see that beta – mercaptoethanol and SFM used in sequence can induce differentiation,each one act alone can't have same result. DMSO have the ability to prevent the cells to return to its un-differentiated state.In conclusion: GSH ascorbic acid vitamin E has not the capacity to induce MSCs to differentiate to neurons, this phenomenon suggest that not all antioxidant has the capacity to induce MSCs to neurons. AND, the beta – mercaptoethanol and SFM have many advantages such as low – toxic, highly effective, et al. Because BHA has limit…
Key words: Mesenchymal Stem Cells; Differentiation; Neuron

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