ABSTRACT
A new method for establishing ES cell lines from 129/ter. C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM) for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.
Subject(s)
Animals , Female , Mice , Rats , Alkaline Phosphatase , Metabolism , Amino Acid Sequence , Base Sequence , Cell Differentiation , Cell Division , Cell Line , Embryo, Mammalian , Cell Biology , Mice, Inbred C57BL , Molecular Sequence Data , Stem Cells , PhysiologyABSTRACT
Derivation of human embryonic stem cell and embryonic germ cell lines has widespread and far-reaching significance on human basic research and transplantation therapies. Human pluripotential stem cells provide an exciting new model for studying early human embryogenesis, understanding normal human development and abnormal development, provide a powerful system for discovering human novel genes and testing their function, offer new strategies for discovering of novel growth factors and medicines and promise a renewable source of cells for tissue transplantation, cell replacement and gene therapies. Research history of establishment of human ES and EG cell lines is reviewed. Several methods of establishment of these cell lines involving in the protocol, route, significance and possibility are discussed. Selection of the feeder layer, medium, and supplemental cytokines and their roles in establishing and maintaining human ES and EG cell lines at present are illustrated in detail systematically. Effects and used methods of several kinds of digestive en-zyme in propagations are prepared. Several methods for identifying human ES and EG cells are summarized. At the end, some key problems which are urgent to resolve in these studies at present are put forward and analyzed.