| Hematopoietic stem cell transplantation (HSCT) plays a vital role in various hematologic malignancies treatment. The major transplant source includes bone marrow, mobilized peripheral blood and umbilici cord blood. Currently, the large-scale application of HSCT is restricted by shortage of donor source and low success rate of matching. Therefore, it is a significant event to develop new sources. Human embryonic stem cells (hESC) may be a suitable candidate. CD34is a major marker of HSC in HSCT. In vitro study, hESC could differentiate to CD34+cells in various fashions, as coculturing with OP9or S17. However, there is no evidence that hESC-derived CD34+cells could mimic the function of umbilici cord blood (UCB)-derived CD34+cells in animal model till now. It is significant to investigate the mechanism by comparing hESC-derive CD34+cells with UCB-derived CD34+cells. It is found that HSC developed from mesoderm. The major site of producing HSC is aorta-gonad-mesonephros and fetal liver. Moreover, epithelial-mesenchymal transition (EMT) is intensively important in embryonic development, especially in fetal liver. In vitro, it is unknown about the effect of EMT during the differentiation of hESC to hESCs-CD34+cells. Hence, it will be instructive to discuss the mechanism of EMT in the above course. In addition, stem cell relevant markers are decreasing or losing gradually in the process of hESC differentiation. Is there any interaction between these markers reduction and CD34expression? There is no evident conclusion. Upon the hypothesis, this thesis investigated the change of stem cell maker NANOG and EMT relating molecules E-cadherin, N-cadherin, Snail1, Snail2, Zeb1, and Zeb2. Furthermore, we investigated the difference between the hESCs-CD34+cells and UCB-CD34+cells. 1.Objective:Culture of human embryonic stem cell and differentiation of hESC to hESCs-CD34+cellsMethods:With mouse embryonic fibroblast (MEF) as a feeder, we cocultured hESC-H9with MEF in two-dimensional culture system. Then we detected the stem cell relating markers stage specific embryonicant (SSEA-4), tumor rejection antigen (Tra-1-81) and alkaline phosphatase staining. We induced hESC-H9to differentiate to CD34+cells with two methods:changing medium and coculturing hESC-H9with OP9. During the differentiated course, we analyzed the CD34expression level by flow cytometer and Real-time PCR.Results:hESC-H9expressed stem cell markers SSEA-4and Tra-1-81. The result of alkaline phosphatase staining was positive. CD34+cells peaked during day8to day10, with the proportion of three percent to five percent. With the method of coculturing hESC-H9with OP9, CD34+cells displayed an obvious group detected by flow cytometry. Real-time PCR result was coincident with flow cytometry.2. Comparation of NANOG between hESCs-CD34+cells and UCB-CD34+cells Objective:Change of NANOG expression during the differentiation course of hESC-H9; the difference of NANOG expression between hESCs-CD34+cells and UCB-CD34+cells Methods:Firstly, after collecting cells at different time points of differentiation course, we detected the expression of NANOG by Real-time PCR. Secondly, after collecting cells of differentiation on day8and umbilical cord blood from delivery room, we sorted highly pured CD34+cells with flow cytometer and magnetic cell separation respectively. At last, we compared the expression of NANOG between hESCs-CD34+cells and UCB-CD34+cells.Results:During the differentiation course of hESC-H9to CD34"cells, the expression level of NANOG was descreasing. Compared with sorted UCB-CD34+cells. NANOG level of sorted hESCs-CD34" cells were higher.3. Comparation of EMT related molecules between hESCs-CD34+cells and UCB-CD34+cellsObjective:Change of EMT relating molecules expression during the differentiation course of hESC-H9; the difference of EMT relating molecules expression between hESCs-CD34+cells and UCB-CD34+cellsMethods:Firstly, after collecting cells at different time points of differentiation course, we detected the expression of E-Cadherin, N-Cadherin, Snaill, Snail2, Zebl> and Zeb2by Real-time PCR. Secondly, after collecting cells of differentiation on day8and umbilical cord blood from delivery room, we sorted highly pured CD34+cells with flow cytometer and magnetic cell separation respectively. At last, we compared the expression of E-Cadherin, N-Cadherin, Snaill, Snail2, Zeb1and Zeb2between hESCs-CD34+cells and UCB-CD34+cells.Results:During the differentiation course of hESC-H9to CD34+cells, the expression level of typical epithelial marker E-Cadherin was decreasing, detected by Real-time PCR. The expression of typical Mesenchymal marker N-Cadherin was increasing, detected by Real-time PCR. The EMT relating transcription factors Snail1, Snail2, Zeb1, Zeb2were increasing. Compared to sorted UCB-CD34+cells, E-cadherin level of sorted hESCs-CD34+cells was higher; N-cadherin and the EMT relating transcription factors Snail1, Snail2, Zeb1, Zeb2were lower.In summary, firstly, we tested the stem cell markers of hESC-H9. Secondly, by inducing spontaneously differentiation with medium change and facilitating differentiation with OP9coculturing, we gained CD34+cells. The expression of CD34was increasing and NANOG was decreasing in the differentiation course. EMT relating epithelial marker was decreasing; mesenchymal marker and some transcription factors were increasing. At last, we found that hESCs-CD34+cells expressed a higher level of NANOG and E-cadherin, while a lower of N-cadherin and Snaill,Snail2, Zebl and Zeb2, by comparing sorted hESCs-CD34+cells with UCB-CD34+cells. It implies that partial EMT may paly a role in hESC-H9differentiation, rather than complete EMT. |
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