Establishment Of Human IPS Cell Lines And Exploration Of ABCG2's Absence In Human Pluripotent Stem Cells

Human embryonic stem (ES) cells have the ability to grow indefinitely while maintaining pluripotency, and also could be able to differentiate into cells of all three germ layers which might be used for basic and clinical research extensively. However, ethical issues become insurmountable obstacles regarding the destruction of human fertilized eggs or blastcysts. In addition, since the functional cells for patients derived from ES cells are still allogeneic, the problem of tissue rejection following transplantation extremely prohibits their clinical application. In this circumstance, the induced pluripotent stem (iPS) cells by introducing several specific transcription factors into somatic cells were born. These cells also sustain continuously self-renewal and possess multiple differentiations potential. They displayed tremendous application potential advantages because they have exactly the same genetic background with the adult cells, but also do not involve ethical issues. However, whether human iPS cells possess the same differentiation ability and efficiency as human ES cells and can they provide functional cells for clinical transplantation are still open questions, which became the focus of attention among stem cell scientists.Side population (SP) also has been a hot topic of stem cell research. Among numbers of stem/progenitor cells, a very significant commonality is the half transporter ABCG2's expression. ABCG2 is one of members in ABC (ATP-Binding cassette) super-family of transmembrane protein structure. These transporters mediate efflux of a broad spectrum of substrates including cytotoxic drugs, steroids, peptides and phospholipids. Their ability to preferentially exclude the DNA-intercalating dye, Hoechst 33342, is regarded to be the molecular basis for the enrichment of stem cells as SP cells by the Hoechst dye efflux assay. Therefore, ABCG2 is used as specific markers of these cells. However, whether ABCG2 is also expressed in human ES cells and iPS cells? And SP sorting "golden rule" is also applicable to these cells?With these questions, we start our research projects. This dissertation includes three research projects:Project 1-"Derivation of human induced pluripotent stem (iPS) cells", Project 2-"Multiple lineages differentiation of human induced pluripotent stem (iPS) cells" and Project 3-"Dissection of the cell membrane transporter ABCG2 in human embryonic stem (ES) and iPS cells".Project 1 is the establishment of iPS technology platform. First, we transfected human fetal lung fibroblasts (IMR-90) with different combinations of transcription factors (Oct4, Sox2, c-Myc and Klf4 or Oct4, Sox2, Nonog, c-Myc, Klf4, and Lin28) by using either lentiviral or retroviral system. Then we succeed in inducing them into iPS cells which have exactly the same genetic background as fibroblasts. These iPS cells also express pluripotent markers and can form teratomas including three germ layers in mice, indicating that they have similar pluripotency and differentiation potential with human ES cells.Project 2 is an extensive study of iPS cells'pluripotency including hematopoietic, neural, and trophoblast differentiation. Based on our previous experience in human ES cell culture and differentiation, we induced iPS cells to differentiate into hematopoietic cells, neuroepithelial (NE) cells, various types of terminal neurons and trophoblast cells. Specifically, we compared human iPS cells neural differentiation efficiency as well as human ES cells. The results show that the iPS cells we derived possess the differentiation ability of the various types of cells. And neurons differentiated from both iPS cells and ES cells had similar ability to fire repetitive trains of action potentials (APs) in response to depolarizing current pulses, suggesting that these neurons are associated with feelings and conduction function, expected to be used in future clinical neurodegenerative diseases treatment. Whereas the neural differentiation efficiency among different iPS cell lines is diverse while the underlying mechanism is currently unknown.Project 3 is a discovery-driven project. We found a very strange phenomenon during human ES cells and iPS cells culture. Differ from other stem/progenitor cells, human ES cells and iPS cell nucleus could be stained by fluorescent dye Hoechst. Then we proved that this is due to the absence of ABCG2 in human ES cells and iPS cells. And on the contrary, when used conventional method to sort human ES cells by flow cytometry after Hoechst staining, we found the "SP" cells, which can exclude Hoechst, are actually differentiated cells with pluripotent makers already down-regulated. So the classic "SP" conception cannot be applied to human pluripotent stem cell (ES cells and iPS cells) for definition or selection. Interestingly, ABCG2 exists in the trophoblast cells and neural progenitor cells derived from human ES cells and therefore can resist dye Hoechst. In contrast, we detected the expression of ABCG2 and the refusal of the Hoechst dye in mouse ES cells, while lack of ABCG2 in mouse trophoblast cells. Forced expression of ABCG2 in human ES cells can significantly enhance their resistant ability to fluorescent Hoechst and chemotherapy drug mitoxantrone (MTX). They even managed to maintain their self-renewal state after removal of growth factor bFGF up to 3 days. We also found the expression of the phosphorylated AKT was significantly increased in ABCG2 expressing human ES cells, which suggesting that ABCG2 may relieve human ES cells'dependence on bFGF by activation of PI3K/AKT, a important branch of FGF signaling. However, the underlying mechanisms are still need to be further explored.In summary, we successfully derived several iPS cell lines by viral transfection, set up this platform for the future clinical application. Then we induced these established iPS cells to differentiate into hematopoietic stem/progenitor cells(HSPC), NE cells, regional specific neurons and trophoblast cells, compared the differences of their neural differentiation efficiency as well as partial functions of neurons, and explored the possibility of them to be used as candidates for cell transplantation. We also firstly systematically described the specific stem cell SP marker-ABCG2 is absent in human pluripotent stem cells (ES cells and iPS cells), this study may be very useful for us to revaluate and understand stem cell side population, relationship between ABCG2 and stem cell pluripotency, ES cell self-renewal and species differences.