Directed hematopoietic differentiation of human pluripotent stem cells

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DESCRIPTION (provided by applicant): In the past 2 years, we and others have successfully reprogrammed human somatic cells such as adult fibroblasts into induced pluripotent stem (iPS) cells by using a few defined transcriptional factors. More recently, we and others also reprogrammed human postnatal blood cells into functional iPS cells. Although successfully reprogrammed iPS cells derived from different adult tissues exhibit pluripotency characteristics and unique molecular signatures at a global level remarkably similar to ES cells, increasing evidence also reveals differences between adult cell-derived iPS and ES cell lines. Similarities and differences in the differentiation potential between iPS and ES cells, and between various iPS cell lines from different adult cell types, remain to be determined. In the past funding cycle, we have devoted significant efforts to elucidate and modulate the Notch/HES1 signaling pathways in hematopoietic differentiation from human ES (hES) cells. We plan to continue this line of investigation to improve directed hematopoietic differentiation from both hES and iPS cells. In addition, we will apply this system to establish developmental/genetic models for human blood diseases resulting from somatic mutations that are restricted to blood cells. Based on our recent success in reprogramming human blood and marrow CD34+ cells to iPS cells, we will first develop an efficient method to reprogram un-fractionated adult leukocytes by a virus-free method (Aim 1). We will also derive paired iPS cell lines from CD45+ leukocytes and fibroblasts from the same adult donors. This approach would allow us to compare their potential in hematopoietic differentiation for mechanistic studies as outlined below. In Aim 2, we will conduct directed hematopoietic differentiation from blood-derived and fibroblast-derived human iPS cells, in comparison with that of blastocyst-derived hES cells. In this aim, we will also elucidate and modulate the role of Notch/HES1 signaling pathways in the hematopoietic differentiation from human iPS cells in order to better understand and enhance the process. In Aim 3, we will derive and conduct hematopoietic differentiation of iPS cells derived from patients with paroxysmal nocturnal hemoglobinuria (PNH), a clonal somatic disease related to the X-linked PIG-A gene mutation in hematopoietic stem cells (HSCs). In PNH patients, a PIG-A mutated HSC becomes clonally dominant and gives rise to large numbers of blood cells lacking glycosyl-phosphatidyl- inositol anchored proteins (GPI-APs). We will derive iPS cell lines from phenotypically distinguishable GPI-AP- (PIG-A null) and GPI-AP+ (PIG-A wildtype) blood cells and from marrow fibroblasts (of PIG-A wildtype) of the same PNH patients, using the best method developed in Aim 1. Then we will take the best approach established in Aim 2 to conduct directed hematopoietic differentiation from the PNH patient-derived iPS cells with or without the deficiency of PIG-A and GPI-APs. This study will help us to better understand the role of the PIG-A mutation and to identify other possible genetic mutations that may lead to HSC clonal dominance in PNH. PUBLIC HEALTH RELEVANCE: This study focuses on hematopoietic differentiation from human pluripotent stem cells. We will establish a novel prospective model using human stem cells to better understand clonal dominance and other puzzling pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH), which has not been adequately answered by existing models. This study will also help us to establish a universal method to study dozens of other somatic blood diseases as well as inherited blood diseases such as sickle cell anemia.