Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

Correction: S100A6 is a critical regulator of hematopoietic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Direct Conversion of Fibroblasts to Megakaryocyte Progenitors.

Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41+, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.