These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. development of these cells into differentiated cells and tissues. (Stage-4). CD34+CD45? putative hemogenic endothelial cells were observed in Stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34+CD45? cells generated CD34+CD45+ HSPCs that produced hematopoietic CFUs. Mid-Stage-3 CD34+CD45+ HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBP, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34?CD45+ cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBP expression were observed, with 25C65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. development of these cells into differentiated cells and tissues. Our laboratory has a longstanding interest in developing genetic and pharmacologic treatments for inherited disorders affecting the function or production of neutrophils, which can be modeled using patient derived iPSCs. Human embryonic stem cells (ESCs) or iPSCs can be differentiated to mature cells of multiple hematopoietic lineages, including erythrocytes, macrophages, B-cells, T-cells, megakaryocytes, and neutrophils [1C11], through processes recapitulating many aspects of embryonic hematopoietic development. In both mice and humans, primitive hematopoiesis is initiated in the extraembryonic yolk sac [12, 13]. After the first wave of primitive hematopoiesis, definitive hematopoietic stem/progenitor cells (HSPCs) can be detected in the embryonic aorta-gonado-mesonephros (AGM) region. Both yolk sac and AGM hematopoiesis originate from cells demonstrating hematopoietic and endothelial potential, termed hemangioblasts or hemogenic endothelium [14C16]. In human ESC differentiation studies, such cells have been found in the CD34+CD45? population  expressing Flk-1 (VEGFR-2)  and CD31 . Upon further differentiation, CD45 is expressed in hematopoietic lineages. Among both somatic cells and cells derived ML418 from human pluripotent stem cells, CD34+CD45+ cells are enriched for clonogenic HSPCs possessing the capacity to generate multiple mature hematopoietic lineages, as in methylcellulose CFU assays. Despite success in generating mature hematopoietic lineages from human pluripotent stem cells, there has been less progress towards developing techniques for generation of HSPCs that are capable of robust long-term multilineage repopulation co-culture of primate iPSC-derived CD34+ cells with human umbilical cord endothelial cells expressing Notch ligands was ML418 shown to enhance long term hematopoietic engraftment in immunodeficient mice . These studies demonstrated that human iPSCs are not intrinsically defective for production of engraftable HSPCs, depending on the conditions used for Rabbit polyclonal to ZNF483 hematopoietic differentiation, and that maneuvers such as exposure to Wnt3a or Notch ligand could improve the efficiency of HSPC differentiation and myelopoiesis from iPSCs. In order to identify additional molecular factors that are associated with the regulation or identity of human iPSC-derived hematopoietic cell lineages, we utilized a 32-day 4-stage discontinuous culture system that we previously described as supporting the generation of functionally mature neutrophils from human iPSCs , which was adapted from ML418 Yokoyamas ESC system , and which we previously utilized to demonstrate safe harbor targeted minigene correction of iPSCs from patients with chronic granulomatous disease by restoring oxidase activity in differentiated neutrophils . This culture system allows for the generation of a high percentage of mature neutrophils (25C65%) following the emergence of HSPCs. The present study delineates the kinetics of hematopoietic clonogenicity and expression of surface markers, transcription factors, and 754 microRNAs during HSPC and neutrophil differentiation in this iPSC culture system, and identifies relationships between lineage commitment, phenotype, and the expression of microRNAs and transcription factors that recapitulate features of the embryonic development of hematopoietic tissues and production of neutrophils in marrow. These analyses may provide the stem cell research community with a roadmap for developing tools to improve the efficiency and efficacy of hemogenic endothelial and hematopoietic differentiation from iPSCs. Material and Methods Human subjects All human subjects providing peripheral blood signed written informed consent allowing these studies following the Declaration of Helsinki under the National Institute of Allergy and Infectious Diseases Institutional Review Board approved NIH protocol 05-I-0213. iPSC source and maintenance Human iPSCs in this study included peripheral blood CD34+ HSPC-derived iNC-01-3, iNC-01-4, and iNC-01-12 , and fibroblast-derived iPS(IMR90)-1  (WiCell,.
These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis