The Establishment Of Mouse Embryonic Stem Cell Lines And The Culture System Of Human Embryonic Stem Cells

Embryonic stem cells (ESC) are pluripotent cells derived from the inner cell mass (ICM) of pre-implantation mouse embryos or primordial germ cells (PGC) of embryos. It is becoming a useful tool for studying cell differentiation, the mechanism of development, gene function, medicine selection, cell therapy and so on. Based on the establishment of embryonic stem cells lines, scientists can do their upper research work. To establish the ESC lines by excellent method and the steady culture system are our aims.Plasmid pEGFP N2 was introduced into the murine embryonic stem cell D3 by standard calcium phosphate precipitation. Transfected clones were screened out under the fluorescence microscope at the 488 nm emission light in the presence of G418. Strong fluorescent EGFP clones were picked out and further proliferated. Stable EGFP ES cells were subcultured and maintained on a feeder layer of mitomycin-C treated mouse embryonic fibroblasts (MEF) and underwent twenty passages. PCR analysis for GFP DNA showed that undifferentiated EGFP ES cells at passage 8 and 18 carried the GFP gene. Alkaline phosphatase staining, embryoid body (EB) and teratoma formation were carried to analyze the differentiation status and potential of the EGFP ES D3 cells. The cells derived from EB were able to differentiate into beating cardiomyocytes with green fluorescence clearly observable under the confocal laser scanning microscopy. 30-40% of cells from embryoid bodies were capable to differentiateinto cardiomyocyte-like cells, which appeared to be lower than the non-transfected ES cells, which could be 60-70% under the same condition. Immunocytochemistry staining indicated that the contracting cells were cardiomyocytes based on the presence of cardiac specific molecular marker cTnT. Results showed that the stable EGFP ES cell line retained the typical characteristics of ES cells. The EGFP transfected cells stably yielding bright green fluorescence in real time and in situ rendered it a powerful tool in cell transplantation and tissue engineering the researches.High quality embryos of 129Sl/SvImJ strain mouse were chosen aftersuperovulation, and directly seeded in the irradiated frozen-thaw mouse embryonic fibroblast cells. Compared with the low concentration long time digestion, the high concentration short time digestion was adopted in the study, and 4 lines of mouse embryonic stem cells were successfully established by trypsinization of 0.25%trypsin-0.04%EDTA, which is the high concentration short time digestion method. The ratio of establishment cell lines is 36.3%, and the cell strains exhibited the typical ES cell characteristic identified by different ways, including the AKP and karyotype assay, the differentiation potential in vivo and in vitro, and the examination of specific surface markers expression of Oct-4 and Nanog. The ES cell lines will be scalable culture and used as an ideal experimental material for the further research.The culture system of Human embryonic stem cells (HES) is concerned too. The culture media, serum, serum replacement and many different growth factors are adjusted effectively. Confirming the reliable MEF is important. The conditions for HES growth are good enough for HES growth. The aim to establish HES lines will carry over later for the lack of the human embryos.In the summarizing part, the process of establishment of HES lines is discussed. During the phases of establishment and maintaining, the source of human embryos, the separation of ICM, growth and passage, freezing and thawing are all difficulties. From MEF, HEF to feeder-free system, the method of nuclear transfer of human somatic nuclei into egg is the basic for cell therapy. At the same time, the molecular mechanism of ESC self-renew is talked over in order to discover the inner reason to establish the ESC line in the molecular lever. Some intrinsic ESC-specific markers, cytokines and extracelluar matrix in the microenvironment compose of a complex network to co-regulate ESC self-renewal. The molecular mechanism of signal network maintaining ESC self-renewal was investigated by studying the ESC-specific markers, such as Oct3/4 and Nonog recently. Moreover, ESC signal pathways, just like LIF-STAT3, Wnt- P -catenin, BMP-Id and bFGF were further deeply studied. In conclusion, the most important key to maintain ESC self-renewal is the balance among the content of various cytokines, extracelluar matrix, and expressing quantity of intrinsic ESC-specific markers by different signal stimulators. At last, thesource of human embryos, the research intention of ESC, and the different positions in the world to this project related to the ethical debate are advanced. Hope that ESC can be used for curing human disease as soon as possible under the protection of canonical law, the support of all the society and the effort of scientists.