Studies Of Establishment Of Mouse And Goat Embryonic Stem Cell Lines And Differentiation Of Them Into Neurons

The successful isolation and culture of mouse embryonic stem cells (ESCs) is a milestone in development biology, and the successful isolation of human ESCs ignites the enthusiasm of schoolars to do more research on them. ESCs can be used as a gene vector to produce transgenetic animals, to uncover the function of single gene. In addition, the pluripotent potential and the infinite proliferation ability make them the proper option to be used for treating human diseases through cell transplantation, for example, differentiation of ESCs into neurons, and treatment of some neurodegenerative diseases through cell transplantation. And the success on therapeutic cloning makes the assumpation possible. However, how to efficiently isolate and culture the ESCs as well as differentiate them into adult cells are the key issues to use ESCs to treat human diseases through cell transplantation. Based on these issues, the projects of isolating and culturing mouse ESCs and goat ES-like cells and differentiating them into neurons in vitro were carried out, in order to increase the efficiency of isolating ESCs and establish a new and efficient method of differentiating ESCs into neurons.Firstly, a study of isolating and culturing C57BL/6J mouse ESCs was carried out, and one cell line was obtained from mouse 3.5 days postcoitus (dpc) blastocyst, which remains undifferentiated after more than twenty passages of culture in vitro with a normal XX karyotype and high expression of alkaline phosphatase and oct-4. When cultured in suspension, ESCs could form embryoid bodies (EBs);inoculated subcutaneously into SCID mice, they could form teratoma;after injected into ICR mouse 3.5dpc blastocysts, ESCs could incorporate into the inner cell mass of the host blastocyst and contribute to development of a chimera. The results showed that the mouse ESCs were pluripotency. And the further study indicated that the Leukemia inhibitory factor(LIF) and feeder cells are indispensable for mouse ESCs self-renewal. The experience in manipulating mouse ESCs laid the foundation for isolating and culturing ESCs of other animal species, and the mouse ESCs provided abundant materials to study treatment of some diseases through cell transplantation after induced differentiation.Secondly, just on the basis of successful isolation and culture of mouse ESCs, a study of isolating and culturing Chinese dairy goat ES-like cells was carried out. In this study, a culture system containing 40% mouse ESCs conditioned medium (ESCCM) was adopted, and in control experiment, no ESCCM was added into culture medium. In addition, the influence of different feeder cells on goat ES-like cells self-renew and the neural differentiation potential of goat ES-like cells were investigated. The results showed that only in medium with mouse ESCCM, goat ES-like cells could be isolated from 5 inner cell mass (ICM) colonies among 9, and two cell lines could be cultured for up to 8 passages without differentiation and with a normal karyotype of 60 chromosomes and high expression of alkaline phosphatase in vitro. Whereas goat ES-like cells could only be isolated from one ICM colony among 6 and cultured for 3 passages in medium without mouse ESCCM. What's more, goat ES-like cells could only form colonies on mouse embryo fibroblasts (MEFs), no colonies formed on goat embryo fibroblasts. This phenomenon can not be elucidated. Although the morphology of goat ES-like cells was similar to that of mouse ESCs, the goat ES-like cells could only form colonies when dissociated into clumps in procedure of passages. We did not obtain beating cardiomyocytes from these cells in vitro. Whereas mouse ESCs can easily differentiate into beating cardiomyocytes after EBs were cultured for several days. The results showed that the differentiation potency of the embryonic cells we obtained is limited, but the goat ES-like cells could differentiate into neurons in vitro. All results above together confirmed that some cytokines, except for LIF, secreted by mouse ESCs might promote goat ES-like cells self-renewal, especially in the process of early passages. However, which and how the cytokines play this role still need to be clarified. And the culture system adopted in this study can be used to isolate other animal species' ESCs which have different characters from those of mouse ESCs. Hopefully, this culture system canbe used to isolate and culture human ESCs, so that the projects of treatment of neurodegenerative diseases through cell transplantation can be carried out.Finally, the mouse ESCs was used to explore the new method of efficientlydifferentiating ESCs into neurons in vitro. The glial cells conditioned medium (GCM)was used to induce differentiation of mouse ESCs into neurons, and the mouse embryofibroblasts conditioned medium (MCM) was used as a control. The results showed thatneurons could be derived from mouse ESCs when cultured in GCM under attachingconditions without forming EBs within 9 days, while the generation of non-neuronalcells, such as astrocytes was limited. And the results also showed that cytokines secretedby glial cells play no specific roles in the early differentiation of ESCs, but havedeterminative effects on neural lineage commitment. These results collectively confirmedthat differentiation ESCs into neurons in GCM under attaching conditions withoutforming EBs is a simple and efficient method, through which many neurons can beobtained. It provides sufficient materials for treatment of some neurodegenerativediseases through cell transplantion. In addition, when cultured in MCM, mouse ESCscould differentiate into neural precursors in 24h, and the latter differentiated into neurons2 days later, while until 9th day, astrocytes could be generated. The sequence ofgeneration of neural precursors, neurons and astrocytes from ESCs in MCM coincideswith that in embryo development, and this provides a proper model for the study ofneural lineage determination in vitro.In summary, on the basis of successful establishment of mouse ESCs line, we carried out the project of isolating and culturing Chinese dairy goat ES-like cells using a culture system containing 40% mouse ESCCM and obtained goat ES-like cells on the basis of successful establishment of C57BL/6J mouse ESCs. And the new method adopted in our study to differentiate mouse ESCs into neurons is simple and efficient. The culture system and new differentiation method established in our study are hopeful to be used in isolation and culture of human ESCs, and their further differentiation into neurons for treatment neurodegenerative diseases through cell transplantation.