| Objective:Human Embryonic Stem Cells(hESCs)are recognized as pluripotent stem cells,human induced Pluripotent Stem Cells(hiPSCs)have the same potential for allogeneic differentiation in vitro as hESCs in theory.They have the ability to synthesize all kinds of cells of human and animal individuals,and even differentiated into new individuals.hiPSCs/hESCs unique self-renewal and all-round differentiation potential in the clinical medicine and regenerative medicine research has a wide range of applications,become the application prospects"seed"cells.However,the regulatory mechanisms of pluripotent molecules are the same or not is still a cause for concern;whether the potential for differentiated into cells of different tissues is the same or not still need to pay attention to.There are many ways to study the differentiation of stem cells in vitro,but safety effect is a necessary condition for the clinical transformation.This study induce hiPSCs and hESCs to form embryoid bodies(EB),and compare the expression changes of the pluripotent marker gene,three-layer differentiation related genes,hematopoietic marker gene and the ratio of hematopoietic stem cell in the early process of spontaneous differentiation of EB derived from hiPSCs and hESCs.And to compare the similarities and differences of them in molecular regulation mechanism of pluripotent in the process of differentiation in vitroe and the potentia of differentiation into different tissue cells,especially in the difference of potential in hematopoietic differentiation of hiPSCs/hESCs.Methods:In this study,hiPSCs/hESCs were established by our laboratory.The hiPSCs were constructed from amniotic cells-derived cells and Urine cells-derived cells.The hESCs were obtained from the inner cell mass(ICM)in the blastocyst.Cut hiPSCs/hESCs clones which were cultured with m Te SR into small groups by mechanical method,after 24 hours of suspension culture in low adhesion plateto form the EB.Collect the EB of d4,d8,d12,d16,the hiPSCs/hESCs of d0 were considered as control groups.Changes in the expression of absolute values of pluripotency gene Oct4/Sox2/Nanog,endoderm/mesoderm/ectoderm differentiation related gene GATA4/MSX1/PAX6 and hematopoietic marker gene CD34/CD43 were detected by RT-PCR;Used Flow cytometry to detect the ratio of CD34+-CD43+cells of d4,d8,d12,d16 EB.Resultes:Both hiPSCs and hESCs could form EB after been induced for 24 h.Both Both hiPSCs and hESCs derived EB in d4 can be developed to form a cystic structure,and differentiation to d16 can be differentiated into different types of cells.The expressions trend of pluripotency marker gene of OCT-4,SOX2 and NANOG in EB derived from amniotic fluid cells induced hiPSCs were the same as those derived from hESCs,the expression of pluripotent gene in EB gradually decreased with the extension of the culture time,the expression of OCT-4,SOX2 and NANOG in EB derived from hiPSCs was almost undetectable at d8,which was different from that of hESCs,and almost sustained expression of d12-d16.The expression level of OCT-4,SOX2 and NANOG in EB derived from urine cells induced hiPSCs increased first and then decreased,can be maintained to d16,and the expression level was significantly higher than that of hiPSCs which derived from amniotic fluid cells and hESCs.In the EB which derived from hiPSCs and hESCs,the endosperm marker gene GATA4,mesodermal marker gene MSX1,ectodermal marker gene PAX6 can be detected in d4.and the expression of GATA4,MSX1 and PAX6 showing increased first and then decreased stage changes with the extension of the incubation time.However,the expression duration and peak time of three germline marker genes in EB derived from amniotic fluid cells induced hiPSCs were different from those of urine cells induced hiPSCs and hESCs.The peak expression of GATA4,MSX1 and PAX6 in the EB derived from amniotic fluid cells induced hiPSCs appeared at d4 and sustained expression to d8-d12,while the peak in the EB derived from urine cells induced hiPSCs and hESCs appeared at d4 or d8 and which csustained expression to d12-d16.The expression level of three germ layer marker gene in EB derived from urine cells induced hiPSCs were significantly higher than in hiPSCs which derived from amniotic fluid cells and hESCs..Both CD34 and CD43 of hematopoietic stem cell marker genein were detected both in EB derived from hiPSCs and hESCs,and the expression of CD34 and CD43 showed the trend of increasing first and then decreasing,have the peak of expression.CD34,CD43 began to show a small amount of expression in the EB derived from hiPSCs derived from amniotic fluid cells at d4,expression reached the peak value at d8,the expression disappeared at day 12.In EB derived from urine cells induced hiPSCs,the peak of expression level appears at d4 or d8,and sustainable expression to d16.It was different from that of hESCs,the expression of d4 in EB derived from hESCs was significantly higher than that in hiPSCs,and the expression of d4-d8 has a little change,expression reached the peak value at d4 or d12 and can be sustained hematopoietic expression d12-d16.Use Flow cytometry to detecte the percentage of hematopoietic stem progenitor cells marker cells CD34+</sup>CD43+cells in EB(d4,d8,d12,d16)in different differentiation periods of EB.The percentage of hematopoietic stem progenitor cells of CD34+</sup>CD43+cells in EB derived from hiPSCs were(0.014±0.000)%?(0.018±0.018)%?(0.021±0.013)%?(0.057±0.057%)%,and the percentage of CD34+</sup>CD43+cells in EB which derived from hESCs were(0.035±0.009)%?(0.102±0.099)%?(0.027±0.012)%?(0.039±0.013)%?CD34+-CD43+cells were detected in d4 and d12 of amniotic fluid cells induced hiPSCs derived EB,but can be detectedt in d4,d8,d12 and d16 in EB derived from urine cells induced hiPSCs,the hematopoietic stem cell ratio was the highest in d12 in EB derived both fromamniotic fluid cells induced hiPSCs and urine cells induced hiPSCs,.CD34+</sup>CD43+cells in d4,d8,d12 and d16 in EB derived from hESCs can be detected,in d4 the ratio of CD34+</sup>CD43+cells were the highest,but the ratio of positive hematopoietic progenitor cells in d8-d16 was gradually increased.Conclusion:Established a EB culturing system to induce the differentiation of hiPSCs and hESCs.Hi PSCs and hESCs have similar differentiation patterns,hiPSCs have the potential of development and differentiation in vitro as hESCs and have the ability to be induced into EB and also have the same ability to differentiate into three germ layers and to differentiate into hematopoietic stem progenitor cells.But different cell-derived hiPSCs has differentiated differences from hESCs.In the case of pluripotent gene expression,amniotic cells induced hiPSCs was weaker than hESCs during EB spontaneous differentiation;urine-induced hiPSCs was stronger than amniotic cells induced hiPSCs and hESCs during the progress of EB spontaneous differentiation.In the aspect of embryo differentiation potential,Peak of expression level of the three-layer marker gene in EB derived from amniotic cells induced hiPSCs and urine-induced hiPSCs was higher than in EB derived from hESCs,but the persistence expression time in amniotic cells induced hiPSCs derived EB was shorter than in hESCs derived EB.In terms of hematopoietic potential,urine cell-induced hiPSCs derived EB hematopoietic stem progenitor cells ratio were higher than the corresponding period of amniotic fluid cells induced hiPSCs and hESCs derived from EB.The expression time of hematopoietic stem progenitor cells in EB derived from amniotic cells induced hiPSCs was shorter thanin hESCs derived EB,but the peak of the ratio of hematopoietic progenitor cells was higher than in EB derived from hESCs. |
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