The Study Of Human Foreskin Fibroblasts Biologic Character And Effect Of Human Foreskin Fibroblast As Feeder Layer For Human Embryonic Germ Cells

Human pluripotent stem cell lines, which derived from inner cell mass (ICM) of blastocysts and primordial germ cells of embryos (PGCs), are characterized by infinite proliferation potentiality and development totipotency. EG cells (embryonic germ cells) and PGCs, as the origin of stem cells implicate sufficiently theoretical and practical significance of developmental biology and abnormal germ lineage. In 1998, human embryonic stem cell (ES cells) and EG cells were successfully established from ICM and PGCs respectively, but research of human pluripotent stem cell is mainly on human ES cells, for the culture condition is harsh for EG cells in vitro and it is difficult to abtain EG cell lines for long term culture. So optimizing current culture system stands essential for promoting related research.Feeder layers are a necessary condition in-vitro culture for human EG cells, therefore suitable culture system in vitro is the key for establishing EG cell lines and clinical applications. Mouse embryonic fibroblasts (MEFs) and STO have been previously used as feeder cells to support the growth of human embryonic stem cells. But the use of animal feeder cells is associated with risks such as pathogen transmission and viral infection, which is the bottleneck for clinical applications. To overcome these problems, feeder layers originating from human fetal and adult tissues (including human fetal skin fibroblasts, fallopian-tube epithelial cells et al) have been tested. But its origin is contrary to ethics and then limited. The resource of foreskin is convenient, foreskin fibroblasts are simple and feasible for pluripotent stem cell as a culture system. Successful culture of ES cells with human foreskin fibroblasts has been reported, but for EG cells haven't. So we adopt foreskin fibroblasts as feeder layers in vitro culture, carried out following research: 1. To separate and cultivate foreskin fibroblasts, and study their biological character. 2. To isolate and cultivate PGCs on human foreskin fibroblasts. The cells have been passaged to 3 generation. Then to detect stem cell markers of the cells(alkaline phosphatase, stage-specific embryonic antigen, Oct-4).1. The study of human foreskin fibroblasts biologic character Objective : To study the biologic character of HFF and detect the concentration of fibroblast growth factor (FGF).Methods:To separate and cultivate HFF, by immunocytochemical, MTT method,flow cytometry and ELISA method, we investigate the morphology of HFF, identify the purity of HFF, study the cell growth curve and cell cycle of HFF, choose the best time and concentration of mitomycin C to inactivate the feeder layer and detect the amount of FGF.Result: HFF grew as adherence, spread like swirl, paliform and radiation, the cell form was varied. The purity of P3 passage HFF was more than 95%. There was no obvious stagnation period of cell growth curves, the rapid proliferation was in a 1～6d period, beginning at the 7th d platform stage. Most cells were in the peak corresponding to quiescent cells in G1/G0 phases, the cell cycle and cell growth curve were in line. Mitomycin C (MMC) inhibited the proliferation of HFF at 12.5μg/ml over 2.5 h of MMC. The amount of FGF was from 172.09±2.66 pg/ml to 245.25±1.66 pg/ml.2.Effect of human foreskin fibroblast as feeder layer for human embryonic germ cellsObjective:To culture human EG cells on feeder layers of HFF, observe the growth of EG cells , and detect stem cell markers of the cells.Methods:To seperate PGCs from 5～11 weeks embryos, cultivate PGCs on HFF and continuously passage, detecte alkaline phosphatase activity with alkaline phosphatase kit, stage-specific embryonic antigen SSEA-1, SSEA–4 with immunocytochemical method, transcription factor Oct-4 by RT-PCR.Result: Human EG cells had the typical colony morphology and growth characteristics. The undifferentiated state was supported by the presence of alkaline phosphatase and the expression of surface markers SSEA-1, SSEA-4 and transcription factors Oct-4 in the cells of the colonies.Conclusion: The foreskin fibroblasts as feeder layer can support human EG cell growth and maintain them in undifferentiated state. In summary, we study the biologic character of foreskin fibroblast and cultivate human EG cells in vitro using human foreskin fibroblast as feeder layer. What we have done provides useful exploration of EG cells.