(G) Schematic showing the transplantation strategy with or knockdown FL-HSPCs. blood diseases. However, lack of HLA-matched bone marrow (BM) or cord blood (CB) donors limits their therapeutic use1. Generation of HSCs from human embryonic stem cells (hESCs) or induced pluripotent stem cells could provide alternative HSC sources. Recent studies used transcription factor reprogramming to convert fibroblasts or mature blood cells2C4 to haematopoietic cells possessing some properties of HSCs. Despite these promising approaches, clinical application of generated Ubrogepant HSCs remains unachieved. While hESCs can differentiate into most blood lineages5, efforts to produce engraftable HSCs have failed6. The molecular barriers preventing HSC generation are poorly understood due to lack of studies comparing candidate HSCs from PSC-cultures and human conceptus that match by immunophenotype and developmental stage. During embryogenesis, haematopoiesis starts in the yolk sac by the generation of two distinct waves of myelo-erythroid progenitors (primitive and transient definitive) that can be distinguished by the specific globins expressed in their progeny7. These progenitors lack self-renewal ability and robust lymphoid potential8,9. Definitive HSCs possessing these properties emerge in the third haematopoietic wave from specialized haemogenic endothelium in major arteries in the AGM (aorta-gonad-mesonephros) region, yolk sac, placenta and vitelline and umbilical vessels10. Human haemogenic endothelial cells express CD34 and CD3111 and up-regulate CD43 upon haematopoietic commitment12,13, whereas HSCs also co-express CD45 (pan-haematopoietic), CD90 (HSC, endothelium), GPI-80 (human foetal HSCs14), and typically have low CD38 expression (lineage commitment/HSC activation). Haematopoietic differentiation of mouse and human ESCs mirrors embryonic haematopoiesis8,15 and recapitulates mesoderm and haemato-vascular commitment16,17 followed by waves of primitive and definitive erythropoiesis18,19. However, hESC-derived haematopoietic cells lack reconstitution ability6,20,21 and full lymphoid and adult-type erythroid Ubrogepant potential22,23, resembling yolk sac-derived lineage-restricted progenitors24. A long-standing goal has been to identify regulatory cues and molecular landmarks that distinguish the definitive HSC fate from the short-lived embryonic progenitors. We used a two-step hESC differentiation to generate HSPCs with human foetal HSC surface phenotype (CD45+CD34+CD38?/loCD90+GPI-80+). Molecular profiling showed remarkable resemblance of hESC-HSPCs to FL-HSPCs, yet revealed distinct differences in HSC regulatory programs, Ubrogepant including the HOXA genes. Knockdown and overexpression studies revealed that medial HOXA genes, in particular (NSG) mice (Supplementary Figure 1A). Human CD45+ chimerism in BM was measured 12 weeks post-transplantation. While FL-HSPCs engrafted successfully before or after OP9-M2 culture, hESC-derived cells showed minimal engraftment (Figure 1D). Human CD45+ cells in the BM of mice transplanted with FL contained HSPCs (Supplemental Figure 1B), CD19+ B-cells, CD3+ T-cells and CD13+ or CD66+ myeloid cells, whereas the mice transplanted with hESC-derived cells only harboured rare human myeloid cells (Figure 1E). These data show that hESC-HSPCs are severely impaired functionally. hESC-HSPCs have poor proliferative potential To understand the functional defects in hESC-HSPCs, hESC- and cultured FL-HSPCs (CD34+CD38?/loCD90+CD45+) were sorted and re-plated on OP9-M2 co-culture to assess their expansion (Figure 2A). Both FL- and hESC-HSPC cultures maintained an immunophenotypic HSPC population one week later (Figure 2B, 2C), however, at three weeks, hESC-HSPCs had disappeared (Figure 2B, 2C). BrdU incorporation analysis did not reveal differences in cell cycle between FL- and hESC-HSPCs (Supplementary figure 2A), suggesting that loss of hESC-HSPCs was not due to inability to divide. Open in a separate window Figure 2 hESC-derived haematopoietic cells have limited proliferative potential etc.) were expressed in both EB-OP9-HSPCs and FL-HSPCs (Figure 3C). These data revealed that EB-OP9-HSPCs are remarkably similar to FL-HSPCs at the molecular level. Open in a separate window Figure 3 Identification of differentially expressed programs in hESC- and FL-HSPCs(A) Spearman rank correlation of HSPCs isolated at different stages of development: 3C5 week placenta (PL, CD34+CD38?/lo CD90+CD43+ n=2), hESC-HSPCs isolated from 2 week EBs (EB, CD34+CD38?/loCD90+CD43+ n=2) or after two-step differentiation (EB-OP9, CD34+CD38?/lo CD90+CD43+CD45+ n=2), and 2nd trimester FL isolated freshly MAP2K7 (FL, CD34+CD38?/lo CD90+CD45+ n=3) or after 2 or 5 weeks on OP9-M2 (FL-OP9, CD34+CD38?/loCD90+CD45) (n=3 and n=2, respectively). n represents number of tissue samples collected from separate specimens per condition. Each replicate was collected from independent experiments and analysed together. (B) Dendrogram showing hierarchical clustering of microarray samples. (C) Relative levels of haematopoietic transcription factors in different samples compared to FL-HSPCs. (D) K-means clustering of differentially expressed genes in HSPCs from different.