Isolation And Clone Human, Murine Embryonic Stem(ES) Cells Derived From Primordial Germ Cells

Abstract Some factors influcening the efficiency of the isolation and clone human, murine embryonic stem (ES) cells derived from primordial germ cells and mouse embryonic stem cells have been investigated, inclucling feeder layer cells, gestational age, cytokines and other supplements .The results obtained were as follows: 1.Homogenuous murine embryonic fobroblast (MEF) feeder layer are optimum for the isolation and clone of murine ES cells. When day 3.5 mouse blastocysts were cultured with feeder layer cells of MEF bEF and hEF, there were significant differences among the attachment rate (91.5%, 76.2%, 64.2%), P<0.05, ICM expand rate (74.8%, 69.8%, 38.47%), P<0.05. 2. Early embryonic stem-like cell lines can be isolated and cloned from primordial germ cells derived from gonads, gonadal ridges, analogues or the surrounding tissues of 9.5-13.5 days postcoitum (dpc) mouse fetuses. ES cell-like colonies are observed at primary passage of 7.5-8.5dpc and 14.5-15.5dpc mouse fetuses and disappeared at later passage. No ES cell-like colonies are observed at primary passage of 16.5-18.5dpc mouse fetus.3. 136 fetuses were obtained and cultured. ES cell-like colonies were observed from 56 of 110 fetuses, their gestestional age was 4-13 weeks. Prinordial germ cells were passaged and cultured from 39 fetuses. H58 cell line were maintained with ES cells characteristic morphology up to 9 passages. The number of human ES-like cell lines up to 5 passages is 7. 4. 5-10 weeks fetuses are optimum for the isolation and clone of human ES-like cells, They can't be obtained from less 5 weeks or more 17 weeks fetuses. 5.Supplement with LIF (4ng/ml), bFGF(4ng/ml), SCF (20ng/ml), 2mM glutamine, 1mM sodium pyruvate, 0.1mM nonessential amino acids are advantage to the isolation and clone human, murine ES-like cells derived from PGCs. 6.MEF, bEF, hEF feeder layer cells all can promote the efficiency of the isolation and clone human ES-like cells derived from PGCs. MEF feeder layer cells are optimum for the in virtro culture of human, murine PGCs. hEF feed layer are slightly up to MEF for human PGCs. No clear discrepancy between bEF and hEF in the in vitro culture of mouse PGCs.7. The PGCs-derived colonies showed typical mouse ES-like morphology, tight, compact, well delineated colonies comprised multiple layers of cells with large nuclei and prominent nuclelci. PGCs tested stained positively with alkaline phosphatase, marker of undifferentiated. They can form embryoid bodies (ES) or differentiating into epithelia-like, fobroblast-like or neural-like cells when in virtro suspension culture, delaying passage or growing beyuned confluence of fibroblast feeder layers. Cell masses like heartbeat were observed, The rate was 60-120 times per minute. The cultured cells have been continuausly passaged. Some PGC derived colonies from human were successfully passaged and survived after frozen-thow. In conclusion, we obtain human, mouse ES-like cell lines derived from PGCs.