Summary Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential to enable the investigation of the molecular determinants of HSC specification. Here we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by VE-cadherin+CD73−CD235a/CD43− phenotype. This phenotype discriminates true HEPs from VE-cadherin+CD73+ non-HEPs, and VE-cadherin+CD235a+CD41a− early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post primitive streak stage of differentiation directly from VE-cadherin-negative KDRbrightAPLNR+PDGFRαlow/− hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR+PDGFRα+ mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal to facilitate generation of HSCs from hPSCs.

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34 + CD43 + hematopoietic progenitors and CD31 + CD43 − endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM-CFCs. hiPSCderived CD43 + cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43 + CD235a + CD41a +/− (erythro-megakaryopoietic), lin − CD34 + CD43 + CD45 − (multi-potent) and lin − CD34 + CD43 + CD45 + (myeloid-skewed) cells. While we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks. Materials and methods Cell linesH1 (NIH code WA01), H7 (WA07), H9 (WA09), H13 (WA13), and H14 (WA14) hES cells (passages 27-45; WiCell Research Institute, Madison, WI) were maintained as undifferentiated cells in cocultures with mouse embryonic fibroblasts (MEFs) [11]. iPS(IMR90)-4 and iPS (foreskin)-1 hiPSC lines were obtained by reprogramming IMR90 fetal and newborn foreskin fibroblasts with POU5F1, SOX2, and NANOG; iPS(IMR90)-1, iPS(foreskin)-2, were obtained by reprogramming of fetal and newborn foreskin fibroblasts (ATCC) with POU5F1, SOX2, NANOG, and LIN28 as described [7]. iPS(SK46)-M3-6, iPS(SK46)-M4-8, and iPS(SK46)-M4-10 hiPSC lines were obtained by reprogramming adult skin fibroblasts with POU5F1, SOX2, and NANOG (M3-6) or POU5F1, SOX2, NANOG, Hematopoietic and endothelial differentiation of hiPSCs and hESCs was induced by transferring the cells onto OP9 feeders, as we previously described in details [12,13]. Briefly, undifferentiated hESC/iPSCs were harvested by treatment with 1 mg/ml collagenase IV (Invitrogen) and added to OP9 cultures at an approximate density of 1.2×10 6 /20 ml per 10 cm dish in α-MEM supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah) and 100 µM monothioglycerol (...

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Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells (DCs), Langerhans cells (LCs), macrophages, and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 days of culture, and an additional 2 days are needed to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 days of culture with cytokines, depending on the cell type.