Studies On CD133 As A Screening Marker For Transdifferentiation Of Fibroblasts Into Neural Stem Cells

Background and Objective:Neural stem cells have the ability to differentiate into neurons,astrocytes and oligodendrocytes,can self-renew and enough to provide many brain-cells.Because neural stem cells have two basic characteristics of self-renewal and multi differentiation potential,as well as the advantages of migration function,low immunity and good tissue fusion,it becomes a transplanted material for the treatment of neurological diseases,but the key is how to obtain high purity in vitro and sorting neural stem cells.In early stage of the experiment,we found that a surface protein named CD 133 had shown a significant increasing trend in the first 5 days,and then maintained high expression state during the fibroblasts differentiated into neural stem cells.The purpose of this topic was to determine the CD 133 protein if can be used as a sorting marker in the process of fibroblasts differentiated into neural stem cells.And provide theoretical basis for the sorting of high purity neural stem cells in vitro,giving a good foundation for further research on the mechanism of transdifferentiation and the application of neural stem cells.Materials and Methods:Ear skin fibroblasts were obtained from rhesus monkey fetuses of embryonic 65 days.And the three concentrated retroviruses includingOCT4/SOX2/KLF4 were mixed to twice infect monkey fibroblasts which were passaged 24 h before at 3x 104 cells per 35 mm dish.At the 3rd day after infection(piD3),fibroblasts were harvested by trypsin digestion and replated on laminin-coated plates at 1×105 cells in one well of a 6-well-plate pre-coated with 5 mg/ml laminin in 3 ml induction medium.Y27632 and VPA were removed from the media at piD5 and piD7,respectively.And change to neural stem cell medium at piD11.During this time,immunofluorescence staining of CD 133 to observe the expression of CD33 every day.The cells were sorted by CD 133 flow,positive cells and negative cells were collected and cultured respectively at piD7,identified latter.Results:(1)We initially infected monkey embryonic fibroblasts with retroviruses constitutively expressing OCT4,SOX2 and KLF4.About 80%of fibroblast cells were infected by at least one of three viruses.(2)The expression of CD 133 was increased but there was no significant increase in the expression level due to the high level death of cells with days gone.(3)The CD 133 positive cells sorted by flow cytometry can successfully transdifferentiate into neural stem cells,while CD133 negative cells can't transdifferentiate into neural stem cells.Conclusion:CD133 can be a screening marker in the process of fibroblasts transdifferenting into neural stem cells.