Establishment Of Mouse Embryonic Stem Cell Lines From Single Blastomere

Embryonic stem (ES) cells which do not exist in nature, usually can be derived from the inner cell mass (ICM) of the blastocyst, are considered equivalent to ICM cells possessing self-renewal and pluripotency capability. ES cells are the only stem cell type able to possess indefinite self-renewal and to differentiate into cellular derivates of ectodermal, mesodermal and endodermal lineages and are formidable models to investigate fundamental aspects of cell stemness and early embryo development. Therefore they are widely used in the areas of Development Biology, Cell Biology, Molecular Biology and Medical Research and so on..ES cell line has been considered as the most potential cell source of cell tissue therapies. But the traditional method of establishment ES cell line is surrounding by many ethical concerns. Recently success in establishing mouse ES cell lines from a single blastomere of preimplantation embryos has led to a new development in stem cell technology. To some extent this breakthrough demonstrates the possibility of personalized ES cells that would eliminate the ethical concerns surrounding therapeutic cloning. As a result, there was a strong interest in a single blastomere derived ES cells for clinical application of deriving personalized ES cells. However, a relatively low success rate in establishing ES cells from a single blastomere and whether all sister blastomeres are equally competent in deriving ES cells, or if they have distinct fate, these issues indicated that the method itself is immaturation and has many problems need to be discovered. Therefore it is crucial for ES cell clinical application to further investigate a single blastomere deriving ES cell; optimize culture system and enhance success rate in establishing ES cells from a single blastomere.The objective of this study was to optimize the condition of establishing embryonic stem cell from a single blastomere of embryo by using mice as animal models. First the study analyzed cell viability and maintence time of MEF after treatment of mitomycin C by means of MTT method, compared effects of treating time of mitomycin C and cell passages on MEF viabilities after mitomycin C treatment. The results suggest that cell viability sustains more stable after 2 hours and 3 hours of mitomycin C treatment at the concentration of 10μg/mL. Meanwhile, MEF vitalities at the first, the third and the fifth passage were proved to be the suitable condition in preparation for MEF feeder layers.Under the MEF co-culture system we compared the effect of KSOM and mES media on the establishment of ES cells. Our results suggested KSOM is better to enhance mouse single blastomere development, blastocyst development, attachment and the establishment of embryonic outgrowth. We had adopted the co-culture system of mouse ES cells and modified with sequential medium. The continuous co-culture system can promote ICM formation without change the ratio of Cdx2 positive cells (TE) and Sox-2 positive cells (ICM). In total, 71 mES-like cell lines which can be subcultured to up 5 passages have been established in KSOM and mES continuous co-culture system. Among them, there is 29 ES-like cell lines are derived from a single blastomere of two-cell embryos (72.5%). The ES-like cell lines establishment efficiency from 4CBD and 8CBD are much lower than 2CBD (30.0% and 11.3% separately).The more advanced stage embryo resulted in the lower success rate in establishing ES cells from single blastomere. According to expression pattern of stem cell markers, the formation of ICM and the derivation of ES cells, we demonstrated that sister blastomeres of two-, four- and eight-cell embryos were not identical and were not equally competent. Those suggest that the low efficiency in ES cells establishment from a single blastomere of embryos was due to the differentiation in early embryos that resulted in unequal pluripotence competent among sister blastomeres.We also compared the effect on deriveing ES cell line from a single blastomere of embryo with or without LIF. Our results indicated 2CBD, 4CBD and 8CBD can derive primary outgrowth no matter addition of LIF or not. Though the ratio of embryo development of 2CBD-LIF and 2CBD-control is similarity, the ratio of primary outgrowth of them is 83.8% and 70.0% separately. So the addition of LIF is not necessary for establishing ICM-derived ES cell-like colonies in the presence of fibroblast monolayer, and established ES cells can be maintained in an LIF-free medium. Interestingly, blockade of MEK/ERK activity in combination with increased gp130 signalling might promote the self-renewal of ES cell lines. We investigated the effect of inhibition of MEK/ERK signalling (PD98059) combined with increasing stimulation of gp130 signalling by LIF on derivation of ES cells. The result suggested the cooperation of LIF signal and MEK/ERK signalling is essential to the self-renewal of ES cell. Our finding suggested that ERK1/2 which is a target of PD98059 might involve in maternal embryonic transition (MET) and the activation of zygotic genome at the two-cell stage. Most cell lines maintained normal karyotype and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos have the potential to be converted into ES cells without any special treatment. The ES cell lines establishment efficiency from a single blastomere of embryo is inversely proportional to the embryo development stages.