Polycomb protein Bmi1 in ES cell hematopoiesis and generation of iPSC from Flt3+ hematopoietic stem cells

During last decades studies on embryonic stem cell (ESC) differentiation have led to a better understanding of tissue homeostasis, including hematopoietic cell development. It is also generally accepted that ESC-derived progenitors or somatic cells hold great potential as novel cell sources for cell or tissue replacement therapy. This thesis focuses on establishing ESC culture and ESC differentiation into hematopoietic cells. Hematopoietic cells were derived from ESC following established protocols by OP9 stroma cell co-culture and embryoid body (EB) formation. To enhance hematopoietic cell generation from ESC, we assessed the impact of Bmi1 (B lymphoma Mo-MLV insertion region 1 homolog). Bmi1 is a Polycomb group (PcG) protein and a key epigenetic regulator during development. It plays an essential role in maintaining adult stem cell self-renewal as revealed by loss of function and over-expression studies. However, the role of Bmi1 on pluripotent stem cells has not been explored so far. Here we found that Bmi1 is not expressed in pluripotent ESC. To examine the impact of Bmi1 on self-renewal and differentiation of pluripotent stem cells, we introduced Bmi1 into ESC by lentivirus vector. Bmi1-transduced ESC (Bmi1-ESC) could be maintained in an undifferentiated state during culture similar to control ESC. Upon differentiation, Bmi1-ESC showed similar pattern of mesoderm hemangioblasts formation, yielding Flk1+ cells at frequencies similar as controls. However, primitive hematopoietic cell generation from mesoderm was highly enhanced, as determined by colony forming assay. Global transcriptional profiling by DNA microarrays identified a panel of genes that were distinctly regulated by Bmi1 during differentiation. Several homeotic genes were repressed, but GATA-2 expression was induced. When we studied EB-derived Bmi1-ESC in serum-free medium with hematopoietic cytokines, Bmi1 led to more than 100-fold expansion of hematopoietic stem/progenitor cells within 2-3 weeks of culture versus control ESC. Additionally, Ink4a/Arf locus appeared being silenced in Bmi1-ESC derived hematopoietic progenitor cells, which relates to their high proliferation capacity. Taken together, these data demonstrate distinct activities of Bmi1 on ESC and ESC-derived hematopoietic progenitor cells. The activity of Bmi1 described here suggests how aberrant Bmi1 expression might contribute to leukemia formation in adult hematopoietic cells. Finally, Bmi1 might be a candidate gene for facilitating adult stem cell derivation from ESC. The better understanding of the transcriptional circuities that maintain ESC identity has allowed novel approaches of generating pluripotent stem cells from somatic cells. For a variety of reasons, hematopoietic stem/progenitor cells (HSC/HPC) represent an advantage cell source for reprogramming. For example, they are easily accessible and can be readily isolated. They can be rapidly expanded to a large cell number with hematopoietic cytokines. Therefore, the second part of this thesis focuses on reprogramming of ex vivo expanded Flt3+ HSC into pluripotent ESC-like cells. Flt3+ HSC were reprogrammed into pluripotent state by different approaches, including ESC fusion and pluripotent factor transduction. Initially, we found that reprogrammed Flt3 ESC hybrids obtained by cell fusion of Flt3+ HSC with ESC, contained donor cell memory, both in epigenetic information and in differentiation behavior. To examine whether somatic memory is also retained in Flt3 iPSC, we compared gene expression profiles of reprogrammed Flt3 ESC hybrids, Flt3 iPSC, ESC and Flt3+ HSC by DNA microarray. We found that expression profiles of pluripotent stem cells from different origins clustered together. This indicates that pluripotent stem cells from different origins maintain their specific epigenetic and gene expression profiles. Further analysis of Flt3+ HSC-derived pluripotent stem cells is ongoing in our lab. These studies will be helpful for understanding the molecular mechanisms underlying reprogramming, also will shed light on the understanding of the ground state of pluripotency.