The Function Of Small-Molecular Compounds In Caprine IPS Cell Reprogramming

Animal embryonic stem cells (ES) provide a powerful tool for studies of early embryonic development, gene targeting, cloning and regenerative medicine. However, because it is hard to obtain materials, the culture conditions are unstable and other reasons, the research on ES cells of large animals are facing many difficulties. At present, it had discovered that somatic cells could reprogramme to a pluripotent state by using exogenous pluripotent transcription factors, and the resulting cells were called induced pluripotent stem cells (iPS), whose appearance could avoid the difficulties of ES cells, and then produced pluripotent stem cells as a substitute for ES cells. Recently, iPS cells have been successfully generated from mouse, human, monkey, rat, pig, rabbit, sheep and goat.Our study mainly focused on the affect of small molecule compounds in generating iPS cells.1. The affect of small molecules on p-giPS cellsIn this study, we generated goat induced pluripotent stem cell(sgiPS cells) with viruses encoding cDNAs of Oct4, Sox2, Klf4 and c-Myc from goat embryonic fibroblasts (GEF). The induced cells which demonstrated a flattened shape as human ES cells with a neat edge, a high nuclear cytoplasmic ratio, and weak alkaline phosphatase (AP) staining could passage stably and were positive for Nanog, Sox2, TERT, and SSEA-4 genes. However, these clones failed to express SSEA-1, TRA-1-60, TRA-1-81, as well as the critical endogenous marker Oct4 and could not develop teratomas when injected into the nude mice. Then we defined them as partial goat induced pluripotent stem cells (p-giPS cells). In the process of inducing reprogramming by pluripotent facrors, due to the random process of activation of endogenous factors, so that the efficiency of reprogramming is relatively low, this will produce a large number of incomplete reprogramming cell clones. Adding of small molecules during the induction can promote the reprogramming efficiency, and will make the partial reprogrammed cells into fully reprogrammed cells. By adding small molecular compounds vitamin C (Vc) and 5-Aza-2′-Deoxycytidine (5-AzadC) to the culture medium of p-giPS cells, we found that the group cells of Vc and 5-AzadC compared to the control group without small molecules, the expression level of endogenous pluripotency factors Nanog, Sox2 and TERT were increased by 4 times, 6 times and 10 times, but still failed to activate the expression of Oct4, and the expression of exogenous factors has not been silent too. This is claimed that these two small molecules Vc and 5-AzadC can improve the endogenous expression of pluripotent factors to a certain extent, but not so completely into fully reprogrammed cells.2. The p-giPS cells differentiated to cardiac-like cellsThe p-giPS cells we obtained have a multi-differentiation potential,and they are able to differentiate into cardiac-like cells in vitro. The differentiated cells can express cardiac marker genes Nkx2.5, Gata-4,α-MHC and TNNi3, and compared with the control group cells, Vc and 5-AzadC group can also differentiate into cardiac-like cells, but the expression level of Nkx2.5, Gata-4 and TNNi3 is different in three treatments. Furthermore, immunofluorescence also showed the expression of Nkx2.5 in three treatments. These results demonstrate that even though p-giPS cells are not completely pluripotent, they still have the ability to differentiate, and the small molecules can improve their pluripotent state as well, which will provide a platform to explore goat iPS cell lines and generate gene-modified goats.