| ObjectiveThe purpose of the experiment is to explore the factors that influence the yields of mesenchymal stem cells(MSCs) from human umbilical cord blood(UCB) of different gestational age(GA) deliveries and to observe the biological characteristics, osteogenic and adipogenic differentiation potential of UCB-MSCs.Metrhods(1) The relationship of the yields of MSCs derived from UCB with several factors such as GA(≥40 w,37 w and≤32 w),the mononuclear cells(MNCs) count of UCB(≥2.5×10~9/L,＜2.5×10~9/L),the inoculum density of MNCs(1×10~4 cells/ml, 1×10~6 cells/ml,1×10~8 cells/ml),the concentration of fetal bovine serum(FBS)(5%, 10%,15%,20%),whether the culture flask being coated with FBS or not,and the relationship among these factors were investigated.The morphologhy and ultramicrostructure of MSCs and their growth characteristics in vitro were observed under inverted phase contrast microscope and electron microscope.The cell passage and proliferation was observed by drawing the cell growth curve,calculating the population doubling,and counting the fibroblast colony forming units(CFU-F).The surface antigen phenotype was analyzed by flow cytometry.(2) The osteogenic medium was used to induce UCB-MSCs to osteoblasts,and the osteogenic potential which were determined by the expression of alkaline phosphatase (ALP),formation of mineralized extracellular matrix were confirmed by alizarin red staining,ALP staining and the activity detecting of ALP.After UCB-MSCs being cultured in adipogenic medium for 3 weeks,oil-red O staining was used to verificated the formation of neutral lipid vacuoles.Results(1) The success rate of generating MSCs from UCB was up to 58.3%.There were some relations between the success rate and the several factors metioned above.The rate decreased as the GA becoming older,significant difference was found between the 3 GA groups(x~2=9.87,P=0.007).The success rate could be enhanced to 76.9% when the MNCs count was more than 2.5×10~9/L,significant difference was found when comparing to that of the group(36.4%) with MNCs count less than 2.5×10~9/L (x~2=8.07,P=0.005).There was a negative correlation between the MNCs count in the same UCB volume and the GA(n=20,r=-0.95,P＜0.01).In the group with the cell inoculum density of 1×10~8 cells/ml,the growth and proliferation of primary and subculturing MSCs were better than that of the groups with the cell inoculum density lower than 1×10~8 cells/ml.The adherence of MSCs in the group with 5%FBS happened much later than other 3 groups,while the purity of MSCs in the lower concentration of 5%FBS was higher.When comparing the passage rate,there was no significant difference among the 4 groups with different concentration of FBS.In the group of culture flask being coated with FBS beforehand,the purity and capability of proliferation of MSCs was higher than the groups with culture flask not being coated. In microscopic observation,UCB-MSCs were adherent with a fibroblast like morphologhy and whirlpool like growth alinement.The ultramicrostructure in electron microscopic showed that MSCs had a big cell nucleus,less cellular organelles and big karyoplasmic ratio.All of the growth curves of primary and subculturing UCB-MSCs were "S" type.The third and fifth generation of MSCs showed the greatest reproductive activity.The count of CFU-F varied with GA, significant difference was found among the 3 GA groups(F=8.53,P＜0.05),and the count of CFU-F was higher in smaller GA than the older.But there was no significant difference when comparing the 3 GA groups according to the population doubling time(F=2.68,P＞0.05).Flow cytometry showed that these cells were positive for CD29,CD44 and CD90 expression,but they failed to express hematopoictic cell surface markers,such as CD34 and CD45.(2) When the MSCs were induced to osteogenic and adipogenic differentiation for 3 weeks,there appeared positive expression of ALP,the formation of a mineral extracellular matrix and neutral lipid vacuoles detected by ALP staining,alizarin red staining and oil-red O staining respectively.Conclusion(1) UCB in different GA does contain MSCs which can be isolated from it.The generating rate of MSCs from UCB was influnenced by several factors.The success rate could be increased by choosing the fetus with relative smaller GA,collecting enough volume of UCB,inoculating cells with a higher density,choosing the medium with lower concentration of FBS,coating the culture flask with FBS beforehand,and changing the culture fluid and doing the passage in a suitable time.(2) UCB-MSCs have similar morphological character,biological characteristics and cell surface markers with MSCs derived from bone marrow,both of which have great capability of proliferation and regeneration.(3) UCB-MSCs can be induced to osteoblasts and lipoblast in a suitable condition in vitro,which was the rationale base for its use in clinical therapy. |
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