Isolation, Identification, And Nerves Of The Mouse Embryonic Stem Cells Induced Differentiation

Embryonic stem (ES) cells derived from inner cell mass of mammalian blastocyst-stage emyst-stage embryos and primordial germ cells are totipotent cells that can be cultured in virto. ES cells were established as a suitable model to study the events which occurred in early development of mammalian embryo,such as cell differentiation, mechanisegulation.It is also an ideal source of tissue engineering and model of pharmacology and clinical medicine. Kunming mice are often used as an animal model in our country. Embryonic stem cell lines of Kunniing mouse are very useful to establish transgenic mouse line for scientific research. Neural transplantation of in vitro-differentiated embryonic stem cells provides a versatile strategy for repairing central nervous system. Based on the above, it is necessary not only to isolate and understand biological characters of Kunming mouse embryonic stem cells but also to know the mechanisms on neural differentiation of mouse embryonic stem cells. The blastocysts of 3.5d from Kunming mouse were collected ,cultured on the fibroblast cell feeder layers, The ES cell-like colonies were sub-clonal cultured twice.The main characteristics of ES cells of Kunming mouse were identified. Treatment of MESPU35 ES cells with retinoic acid(RA),estrogen, 4-/4+method,induced ES cells differentiation into nerve tissues.Using immunocytochemistry, immunofluorescence and flow cytometry to identify differentiated cell type, to study the effect of inducers(retinoic acid and estrogen) in neural differentiation. Results 1.Collected 95 blastcysts of Kunniing mouse, 13 ES cell-like colonies were sub-clonal cultured twice and two stable ES clones were obtained, named KMMES 1, KMMES2. 2.KMMES 1, KMMES2 were clony-like, AK? staining positive and totipotent, but two clonies were different in some biological characters, such as the ability of differentiation both in vivo and in vitro, normal karyotype and so on. 3.By using RA treatment, the percentage of outgrowths of embryonic bodies containing neuron-like cells increased as the concentration of RA and days of differentiation increased, so as the group of RA combined with estrogen, observed by phase contrast microscope. 4.Immunocytochemistry results showing: numbers of NF200 positive cells and GFAP positive cells increased after RA treatment. It is correlated with the concentration of RA and days of differentiation. NF200 positive cells changed from spherical to multipolar in shape. Processes of GFAP positive cells become longer,connected with each other and formed a network. No Gal-c positive cell was observed in the process of differentiation. 5.Immunocytochemistry results showing: numbers of NF200 positive cells and GFAP positive cells increased in the treatment with RA+ estrogen group as the concentration of inducers and days of differentiation increased. NF200 positive cells in RA plus estrogen group are bipolar in shape, processes of GFAP positve cells become much slender than group of RA treatment. No Gal-c positive cell was observed. 6.Flow cytometry was applied to assess proportion of NF2OO~ cells and GFAP~ cells in the group of RA treatment.The results were coincide by immunocytochemistry.It was showed obvious difference as compared with the 14 days group differentiate naturely. Conclusions 1 .Two ES cell lines of Kunming mouse, KMMES 1, KMMES2, were established. The biological characters of Kunming mouse embryonic stem...