Effects Of Different Culture Systems On Proliferation And Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Neural Stem Cells

Objective To observe and compare the differences in cytological morphology,proliferation and differentiation of Human umbilical cord mesenchymal stem cells(hUC-MSCs)into neural stem cells,cultured with serum-free,fetal bovine serum,adult autologous serum and umbilical cord blood serum,so as to select the best hUC-MSCs culture system for proliferation and differentiation and suitable for clinical application.Method The umbilical cords of healthy newborns were isolated and cultured in vitro under aseptic conditions.The hUC-MSCs of the second generation were cultured in DMEM/F12 medium containing 10% fetal bovine serum(FBS)for subculture.The hUC-MSCs of the second generation were inoculated into 175 T culture flask,respectively,and divided into group A: serum-free culture medium;Group B: 10%FBS DMEM/F12 medium;Group C: 10%adult serum DMEM/F12 medium;Group D: 10% umbilical cord serum was cultured in DMEM/F12 medium.The cell morphology and proliferation of hUC-MSCs in P3 generation of four culture systems were observed under inverted microscope.Cell cycle and the expression of cell surface markers were detected by flow cytometry.MTT method was used to detect the absorbance values(OD values)of the P3 generation of hUC-MSCs of the four culture systems for 7 days,and the growth curve was drawn.Nerve stem induction culture system(recombinant epidermal growth factor HEGF,recombinant basic fibroblast growth factor BFGF,B27,etc.)was used to induce the directional differentiation of hUC-MSCs in the P3 generation of the four culture systems into NSCs,respectively.Cell morphology was observed under an inverted microscope,and the expression of cell surface markers in each group was detected by flow cytometry.Results(1)The morphology of hUC-MSCs in the four culture systems was long and fusiform,and the cell size was uniform,showing A swirl or irregular distribution.There was no significant change in the morphology of hUC-MSCs in the continuous culture groups.The preliminary observation showed that the cell expansion rate of group A was the fastest,and that of group D was the slowest.(2)In the P3 hUC-MSCs of the four culture systems,CD34,CD45 and HLA-DR were not expressed(P > 0.05),but CD44,CD73,CD90 and CD105 were highly expressed,and the positive rates in each group were all higher than 90%.Compared with the four groups,the positive rate in group A was the highest,followed by group B and C,and A little lower in group D(P < 0.05).(3)Cell cycle analysis: the proportion of S phase in group A was(37.26±0.47)%,group B was(42.77±3.22)%,group C was(19.05±0.98)%,and group D was(18.55±0.76)%.The proliferation index(PI)of each group was calculated.The PI of group A was(0.41±0.00),group B was(0.46±0.01),group C was(0.24±0.00),and group D was(0.21±0.00).The proliferation activity of hUC-MSCs in P3 of the four culture systems was the highest in group B,followed by group A and C,and the lowest in group D(P <0.05).(4)According to the growth curve,group A had the fastest growth rate,followed by group B and C,and group D had the slowest growth rate(P <0.05).(5)The hUC-MSCs of P3 generation of the four culture systems could be inducted into NSCs directionally.Among them,the NSCs of group D were closely connected with each other,had good light transmittance,strong three-dimensional sense of cell masses,less particulate matter released around,less cell apoptosis,and had the best growth state.(6)CD34,CD45 and HLA-DR were not expressed in NSCs of 4 groups(P > 0.05).High expression of CD29,CD44,CD73,CD105,Nestin(neuroepithelial stem cell protein),GALC(Galactosylceramidase),and NF-L(neurofilament light chain).Except for Nestin,the phenotype positive rate in groups A and C was higher than that in groups B and D.The positive rate of NESTIN in group D was the highest(P < 0.05).There was A low expression of GFAP(Glial fibrillary acidic protein),and the positive rate in group A was relatively high(P< 0.05).Conclusion(1)The morphological characteristics,phenotypic markers and differentiation ability of hUC-MSCs were well maintained in all the four culture systems.(2)The hUC-MSCs cultured in serum-free culture system were superior to FBS and adult serum culture system in terms of proliferation rate and phenotypic expression,and the umbilical cord serum culture system was inferior to the other three groups.(3)All the four culture systems could induce the differentiation of hUC-MSCs into NSCs,and the umbilical cord serum culture system was more helpful to induce the differentiation of hUC-MSCs into NSCs.