A Study Of Mouse Embryonic Stem Cells On In Vitro Gene Labeling, Induced Differentiating And Intracranial Transplantation

Mouse embryonic stem (ES) cell lines, derived from the inner cell mass of 3.5 day blastocysts, are totipotent and have the ability to unlimitiy self-renew. ES cell can be induced to differentiated into all tissues, including germ line cells in vitro.They are the best candidate donor for cell replacement therapy. ES cell line can be maintained and proliferated permanently in vitro,so they are favorable for gene transduction. In this study, we used EGFP gene as report gene, and labeled ES cell with it randomly. EGFP expressing (green fluorescence) ES cells with nomal properties, such as viability and differentiation ability, were successfully generated. The green flurescence of EGFP expressing cells maintained in propagating the ES cells for long term and in differetiated cells. Cultured in suspension, the "green " ES cells were aggregated and formed embryoid bodies. After adding retinoic acid (RA) in the EB culture system (4-/4+), they could re-adhere to the bottom of the plastic foster, most of them spreaded and differetiated into neural cells ( such as neuron, astrocytes), and the post-differetiated cells emitted green fluorescence as well, so the EGFP expressing ES cells hold great flexibility for the study of cell differetiation, transplantation and stem cell therapy.In this study, we made a mechanical brain trauma model on rats through a free falling way. Within the 24 hrs after insult, we transplanted the EGFP expressing mouse ES cells into the point of strike. In the next 2 months we observed the transplanted cells continuously and found that the mouse ES cells mainly aggregated in the damaged area, where there were lots of astrocytes and fiber-hyperplasia, which blocked the growth of the transplanted ES cells. Under the electron microscope, we could findconjuctions between ES cells, but no synapse formation between the host s neuron and the transplanted ES cells. Among all the host rats(n=44), there were 4 cases showing subcutaneous tumor formation at the transplanted area. Cells derived from these tumors emitted strong green flurescence in the freezing slice under the fluorescent microscope. Pathological diagnosis confirmed that one of the tumors was Pilocytic astrocytoma, another was suited for RENTS (Primitive neuroectodermal tumors-Medulloblastoma), and the rest tumors were tubercle granuloma-a typical symbol of immunoreaction, beside that all of the host rats showed strongly immuno-rejection at the transplantation area.