| Human embryonic stem cells (hESCs) can be potential source cells for blood cell replacement therapies and studies of the mechanisms of hematopoietic cell specification and diversification and stem cell development due to their capacities of self-renewal and pluripotency. Previous studies in Dr. Slukvin's lab revealed that hESCs differentiated in coculture with bone marrow stromal cell line OP9 generate two types of lymphomyeloid progenitors: lin -CD34+CD43+CD45- and lin-CD34+CD43+CD45+. In the present study I hypothesize that multipotent lin-CD34 +CD43+CD45+ progenitors could be expanded and differentiated into all types of mature myelomonocytic cells. I found that the best expansion of lin-CD34+CD43 +CD45+ cells could be achieved by dissociation and reaggregation of day 9 hESC/OP9 coculture cells followed by nonadherent culture with 200ng/mL GM-CSF. Using these expanded cells enriched in myeloid progenitors I developed protocols for large-scale production of mature myelomonocytic cells (neutrophils, eosinophils, macrophages, osteoclasts, dendritic and Langerhans cells) from hESCs. Morphologic, phenotypic, molecular and functional analyses revealed that hESC-derived myclomonocytic cells are comparable with their corresponding somatic counterparts. Recently, pluripotent stem cell lines have been obtained through reprogramming of somatic fibroblasts. To find out whether human induced pluripotent stem cells (hiPSCs) undergo a series of changes similar to hESCs following hematopoietc differentiation, I studied differentiation potential of seven hiPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with five hESC lines. Similar to hESCs, hiPSCs generated CD34+CD43+ hematopoietic progenitors and CD31 +CD43- endothelial cells in coculture with OP9. The hiPSC-derived CD43+ cells could be separated into the following phenotypically defined hematopoietic subsets: CD43+CD235a +CD41a+/- (erythro-megakaryopoietic), and lin -CD34+CD43+CD45- and lin CD34+CD43+CD45+ lympho-myeloid progenitors. Despite the variations in the efficiency of hematopoietic differentiation, the pattern of differentiation was very similar between hiPSCs and hESCs. In addition, I found that lin-CD34+CD43 +CD45+ generated from hiPSCs possess multilineage myeloid differentiation potential similar to the hESC-derived counterpart to generate mature myelomonocytic cells. The method developed in this work could be potentially applied to generate myeloid progenitors and mature myelomonocytic cells from hiPSCs obtained from patients with myeloid cell disorders or leukemia to address essential questions of pathogenesis of these diseases. |
