Establishment Of An In Vitro Culture System For Human Embryonic Germ Cells And Preliminary Study On Their Differentiation Into Cardiac Progenitor Cells

Human embryonic stem cells can self-renewal in vitro, and have the potential to differentiate into derivates of three embryonic germ layers. Human embryonic stem cells possess wide applications for research on cell differentiation, for testing and screening of drugs, and for transplantation of cells or tissues. Human embryonic stem cells are cell lines derived from the inner cell mass of blastocysts or the primordial germ cells (PGCs) of embryos. The human embryonic stem cells derived from PGCs are termed as human embryonic germ cells (hEG). Shamblott MJ et al. first established hEG cell lines in 1998. Research on human embryonic stem cells has become a frontier in the life sciences and medicine.To date, very few reported researches focus on the successful establishment of hEG lines. The partial reason is optimal hEG culture system has not been established. The establishment of optimal hEG culture system has become a key point in this field. At present, feeder layers are still essential for hEG culture in vitro. Domestic and overseas researchers have used some kinds of cells as feeder layers, including mouse STO fibroblast cells, primary mouse embryonic fibroblast cells (PMEF), SNL cells (mouse STO fibroblast cells transfected with LIF gene), human embryonic fibroblasts, etc. Several research groups have reported successful culture of hEG lines using mouse STO fibroblast cells or PMEF as feeder layers. This is likely to result in the contamination of hEG with heterogenous proteins or pathogens. Hence the clinical application of hEG cultured on murine-derived feeder layers is restricted. Human-derived feeder layers could theoretically avoid the limitation mentioned above. The human-derived feeder layers have been expected by researchers.In the early phase of human embryo development (before 9-week gestation), PGCs continuously migrate, proliferate and maintain undifferentiated state. The niche surround PGCs play an important role in the migration, proliferation, and undifferentiated maintainance of PGCs. The niche includes stromal cells, cytokines, and extracellular matrix. It is possible that the stromal cells adjacent PGCs are essential for the proliferation and undifferentiated maintainance. Therefore, it is postulated that the stromal cells adjacent PGCs can be used as feeder layer for the culture of hEG. In this study, the fibroblast-like cells derived from gonadal ridges and dorsal mesenteries of human early embryos were to be isolated and cultured. The fibroblast-like cells were to be used as feeder layers for in vitro culture of hEG, which simulates the in vivo niche where PGCs continously migrate, proliferate, and maintain undifferentiated state. It may promote the proliferation of PGCs and avoid the contamination of cultured hEG with heterogenous proteins or pathogens. A new method of hEG culture was to be established.Cardiovascular diseases have become a main killer threatening human being's heath in this century. Followed with the further research on stem cells, it has been known that the stem cell transplantation will open up an entirely new field of cardiovascular disease treatment. To date, the stem cells may be used to treat cardiovascular disease include human embryonic stem cells (hES), hEG, embryonic cardiomyocytes, mesenchymal stem cells, cardiac stem cells, skeletal myoblasts, etc. The"seed cells"derived from embryonic tissues include human embryonic cardiomyocytes and embryonic stem cells, which have higy capacity to proliferation, and poorly immunogenicity. These cells have wide application for the cardiovascular disease treatment.To date, only a few groups reported researches on the differentiation of hEG into cardiomyocytes. The effective method of inducement has not been found. A lot of reports demonstrate that 5-azacytidine can significantly promote the differentiation of mesenchymal stem cells into cardiomyocytes. And several reports reveal that 5-azacytidine can promote the differentiation of mouse embryonic carcinoma cells (P19 cell line) and hES into cardiomyocytes. However, the mechanism is not clear. It is postulated that 5-azacytidine results in the demethylation of some genes in the stem cells, which regulates the expression of some silent genes. Consequently, it induces or promotes the differentiation of stem cells into cardiomyocytes. Otherwise, it may be resulted from some other special functions of 5-azacytidine. Therefore, we were to research on the effect of 5-azacytidine on the differentiation of hEG into cardiomcytes. At the same time, we were to isolate and culture the human embryonic cardiomyocytes from human early embyos, and preliminarily study their biological characteristics. These reseaches lay a foundation for the future cardiovascular disease treatment by stem cells.Firstly, by using mechanically dissociation and enzyme digesting method, fibroblast-like cells were isolated from gonadal ridges and dorsal mesenteries of human embryos. These cells were cultured in high glucose-DMEM medium supplemented with serum and sodium pyruvate. The cells were uniform in morphology with a spindle shape and strong refractivity. When cultured cells reached a confluency of 90 % in culture, they were passaged with trypsin, and continuously cultured. They have been continuously grown and split beyond the 25th generation. Moreover, the proliferation rate and morphology of the frozen-thawed cells did not significantly alter. At the early stage of culture, morphology of the cells was distinct. The morphology of the cells beyond the 3th passage was comparatively homogeneous. It was detected that these cells expressed a fibroblast marker gene (prolyl 4-hydroxylaseβgene), but did not express an epithelium marker gene (cytokeratin-4 gene), which demonstrates that these cells are similar with fibroblast cells. So the cells were termed as fibroblast-like cells. The biological characteristics of the fibroblast-like cells were also detected. The growth curves show that these cells possess stable characteristics of growth and proliferation. The doubling time of these cells at passage 5, 10, 15, 20, 25 is respectively 36.2 h, 36.4 h, 36.5 h, 37.3 h and 41.2 h. It suggests that these cells grew relatively fast. During cultured in vitro, the cell cycle of these cells was analyzed. The results showed that 49.60% of cells were in G0-G1 phase, 6.53% were in S phase, and 43.87% were in G2/M phase. It suggests that most of the cells were in proliferation phase. Then reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry staining was used to examine the expression of cytokines in the fibroblast-like cells, namely basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). The results demonstrate that the fibroblast-like cells expressed both bFGF and LIF, which are essential for the in vitro growth of hEG. It is shown that these fibroblast-like cells possess potential capacity to act as feeder layers for hEG culture in vitro.In order to establish an in vitro culture condition of hEG using the human embryonic fibroblast-like cells derived from gonadal ridges and dorsal mesenteries of embryos as feeder layers, we observed the numbers of hEG-like colonies primarily cultured under the four different culture conditions for 7 d using 5 hEG cultures initiated. The results shown that: (a) In the no feeder layers and no cytokines added group, obvious hEG-like colonies were not visible under an inverted microscope. (c) In the feeder layers and no cytokines added group, the average number of hEG-like colonies was significantly more than (b) in the no feeder layers and cytokines added group. (d) In the feeder layers and cytokines added group, the average number of hEG-like colonies was significantly more than (c) in the feeder layers and no cytokines added group. It is obvious that the average number of hEG-like colonies in group c was significantly more than those in group b. So under no cytokines condition, the feeder layers can promote the proliferation of hEG. However, the average number of hEG-like colonies in group d was significantly more than those in group c, which demonstrates that the feeder layers and the cytokines can produce synergetic effect to support the growth of hEG. Therefore, the feeder layers and cytokines added culture condition was used as the better culture condition for hEG.The cuture condition mentioned above was used to culture hEG. Of 21 human primordial germ cell cultures initiated, 7 were continuously grown and split for 8～10 passages. The expression of hEG markers by cultured cell colonies was detected by immunofluorescence and alkaline phosphatase staining. The cultured hEG colonies were strongly positive for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and alkaline phosphatase, and weakly positive for SSEA-3. The hEG cultured under the culture conditions possessed normal human diploid karyotype. The hEG colonies were mechanically dissociated from the feeder layers and harvested. Then they were disaggregated into small clump. The small clumps were cultured for 14 d in suspension medium consisting of HG-DMEM supplemented with 20 % FBS, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, and 1 mM sodium pyruvate. The hEG colonies spontaneously generated embryoid bodies (EBs). The differentiated cells in EBs were detected by RT-PCR. The results showed that the differentiated cells in all EBs expressed most of marker genes of all three embryonic germ layers. The cultured hEG have capacity to differentiate into the derivatives of three embryonic germ layers. The results mentioned above demonstrated that we have successfully used the culture conditions of human embryonic fibroblast-like cells derived from gonadal ridges and dorsal mesenteries of embryos used as feeder cells and cytokines added to culture proliferative, undifferentiated, and pluripotent hEG with normal diploid karyotype. Therefore, it is feasible to culture hEG using the culture condition.Based on the researches mentioned above, we preliminarily studied whether 5-azacytidine has an effect on the differentiation of hEG into cardiac progenitor cells. Firstly, we studied the effect of different concentrations of 5-azacytidine on the differentiation of hEG into cardiac progenitor cells. From differentiation day 6 to day 8, the four groups of cells were respectively cultured in media added with different concentrations of 5-azacytidine, including 0μM,0.1μM,1μM和10μM. During this period, the cells proliferated, and further differentiated. The cells were multiform in morphology with strong refractivity. But in 10μM 5-azacytidine group, a few cells detached from the culture dishes and die. According to the characteristic of cardiac progenitor cells specially expressing GATA-4 andα-cardiac actin, we observed the influence of different concentrations of 5-azacytidine on the differentiation of hEG into cardiac progenitor cells by semi-quantitative RT-PCR.The results show that the cells expressed GATA-4 andα-cardiac actin in all groups, but the indensity was different with concentration dependent manner. There was no different indensity of GATA-4 andα-cardiac actin expression between 0μM group and 0.1μM group. The expression intensity of GATA-4 andα-cardiac actin in 1μM group was significantly stronger than that in the 0μM group or 1μM group. It is obvious that followed with the concentration of 5-azacytidine increased, the ratio of hEG differentiated into cardiac progenitor cells also increased. However, the expression indensity of GATA-4 andα-cardiac actin in 10μM group was slightly lower than that in the 1μM group, but there was no statistically significant difference between the two groups. It is thought that the high concentration of 5-azacytidine may impair the proliferation of cells, which can be preliminarily verified by the cells state observed. Moreover, we studied the effect of different days of 5-azacytidine treatment on the differentiation of hEG into cardiac progenitor cells. From 6 d, the cells were cultured in the medium added with 1μM 5-azacytidine for 0 day, 1 day, 3 days, and 5 days. Then the cells were cultured in the medium without 5-azacytidine. The cells were all cultured for 14 days. During this period, the cells were multiform in morphology with strong refractivity. But in 5 d group, a few cells detached from the culture dishes and die. The results of semi-quantitive RT-PCR demonstrate that the cells expressed GATA-4 andα-cardiac actin in all groups, but the indensity was different with time dependent manner. There was no different indensity of GATA-4 andα-cardiac actin expression between 0 d group and 1 d group. The expression intensity of GATA-4 andα-cardiac actin in 3 d group was significantly stronger than that in the 0 d group or 1 d group. The expression indensity of these two genes in 5 d group was not different from that in 0 d group and 1 d group, but significantly lower than that in 3 d group. It is thought that the too long period of 5-azacytidine treatment may impaire the cells. This can be preliminarily verified by a few cells die in the 5 d group. Based on the results mentioned above, we preliminarily verified that 5-azacytidine may have a capacity to promote the differentiation of hEG into cardiac progenitor cells. It is postulated that 5-azacytidine results in the DNA demethylation, and reactivation of some silent genes. Through regulation of some silent genes expression in the hEG, 5-azacytidine can induce or promote the differentiation of hEG into cardiomyocytes. At the same time, we isolated and cutured the embryonic cardiomyocytes derived from human early embryos (8～9 w). The human embryonic cardiomyocytes beated at the rhythm of 40～120 times/min. By immunocytochemistical staining and immunofluorescence, the cultured cells expressedα-cardiac actin and TroponinⅠ, which further verified the cultured cells were human embryonic cardiomyocytes. At present, the cells can be passed to passage 3. This study laid a foundation for the further research observation on the human embryonic cardiomyocytes.In conclusion, the culture condition of human embryonic fibroblast-like cells derived from gonadal ridges and dorsal mesenteries used as feeder layers and cytokines added has been established for the culture hEG in this study. A new effective method has been created for culturing hEG in vitro. It has laid the foundation for the further research on hEG and for the clinical application of hEG. In addition, our preliminary studies show that 5-azacytidine may have the capacity to promote the differentiation of hEG into cardiac progenitor cells. It has laid the foundation for the further research on the differentiation of hEG into cardiac progenitor cells.