Experimental Conditions Optimization During MSCs Differentiate Into Neural Like Cells And The Detection Of Calcium Ion Dynamic Change

Objective:In order to compared the biological characteristics and proliferative capacity between bone marrow derived mesenchymal stem cells(BMSCs) and Adipose-derived mesenchymal stem cells(AMSCs) in vitro, and discussed three different methods of induction about the differentiation process of AMSCs to neuron-like cell, improve the efficiency of AMSCs differentiate into neuron-like cells, Further discussed Neuron-like cell differentiation mechanisms. Depth explore the change of induced processes intracellular calcium concentration, in order to provide a theoretical basis of AMSCs in clinical regenerative medicine.Method:The experiment was divided into three stages in order to screen the better MSCs and the best induction mothed of different three methods of AMSCs differentiation into neuron-like cells.(1) Separation of BMSCs by Percoll density gradient centrifugation in vitro and extract and amplificate AMSC by senzyme digestion. Detection of MSCs surface antigens by flow cytometry, and the estrogenic, adipogenic ability of MSC characteristics were identified.(2) Three induction methods induced AMSCs differentiation into neuron-like cells, they are chemically induced way, rat brain conditioned medium induced way and induced and differentiated into neurospheres induced way. The observed morphological changes induced in the immunofluorescence detection of the expression of neuron specific marker β-Ⅲ-Tubulin, NSE, and Nissl, To determine the most efficient methods of induction (3) Take into neurospheres induced induction AMSCs to use Fluo3/AM AMSCs and during induction of neural cells were stained, the calcium ion concentration detection AMSCs groups of cells by flow cytometry and by confocalmicroscope to determine the changes in calcium concentration in single cells, the study of intracellular free calcium concentration in AMSCs changes to the process of nerve-like cells.Result:(1) The BMSCs were obtained by Percoll density gradient centrifugationthe and the cultured AMSCs were by enzymatic digestion and they are stable to spread more than20generations. Phenotypic identification of CD13, of CD90were positive, CD34, CD45were negative, AMSCs CD106negative but BMSCs weakly positive for CD106, the expression. Induced differentiation of MSCs to osteoblasts for28d, the Von kossa staining positive to fat cell differentiation at14d, oil red O staining positive.(2) use the RA in conjunction with chemical factors induced AMSCs differentiation into neuron-like cells during the induction of cell morphology changed, the cell bodies became round, the refractive index increase. In the mature neuron marker expression of NSE induced at30d, the positive value of29.5%. Rat brain induced by conditioned medium for14d, NSE expression slightly. Adopted into neurospheres induced induced for7days as neurospheres, and then after7-9days of differentiation culture.7d, expression of NSE. Concluded into a ball induction methods do not affect the cell activity, more able to fully simulate the process of differentiation of neural stem cells into nerve cells of the adult.:Induced into a ball induced by the natural nerve ball,1-6d during the differentiation of intracellular calcium concentration before the rise, reached a peak and then falls sharply in differentiation for7d, and cell culturewhen cell apoptosis consistent with the time.Conclusion:AMSCs have higher proliferative capacity compared with BMSCs, neurospheres differentiation method has high efficiency and low damage induced AMSCs differentiate into neural cells, intracellular free calcium ions in AMSCs change to the process of nerve-like cells, the maintenance of cell differentiationsurvival have played a certain role.