Project description:Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.
Project description:Studying gene expression at different hematopoietic stages provides insights for understanding the genetic basis of hematopoiesis. We analyzed gene expression in human CD34(+) hematopoietic cells that represent the stem-progenitor population (CD34(+) cells). We collected >459,000 transcript signatures from CD34(+) cells, including the de novo-generated 3' ESTs and the existing sequences of full-length cDNAs, ESTs, and serial analysis of gene expression (SAGE) tags, and performed an extensive annotation on this large set of CD34(+) transcript sequences. We determined the genes expressed in CD34(+) cells, verified the known genes and identified the new genes of different functional categories involved in hematopoiesis, dissected the alternative gene expression including alternative transcription initiation, splicing, and adenylation, identified the antisense and noncoding transcripts, determined the CD34(+) cell-specific gene expression signature, and developed the CD34(+) cell-transcription map in the human genome. Our study provides a current view on gene expression in human CD34(+) cells and reveals that early hematopoiesis is an orchestrated process with the involvement of over half of the human genes distributed in various functions. The data generated from our study provide a comprehensive and uniform resource for studying hematopoiesis and stem cell biology.
Project description:The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.
Project description:Definitive hematopoiesis generates hematopoietic stem/progenitor cells (HSPCs) that give rise to all mature blood and immune cells, but remains poorly defined in human. Here, we resolve human hematopoietic populations at the earliest hematopoiesis stage by single-cell RNA-seq. We characterize the distinct molecular profiling between early primitive and definitive hematopoiesis in both human embryonic stem cell (hESC) differentiation and early embryonic development. We identify CD44 to specifically discriminate definitive hematopoiesis and generate definitive HSPCs from hESCs. The multipotency of hESCs-derived HSPCs for various blood and immune cells is validated by single-cell clonal assay. Strikingly, these hESCs-derived HSPCs give rise to blood and lymphoid lineages in vivo. Lastly, we characterize gene-expression dynamics in definitive and primitive hematopoiesis and reveal an unreported role of ROCK-inhibition in enhancing human definitive hematopoiesis. Our study provides a prospect for understanding human early hematopoiesis and a firm basis for generating blood and immune cells for clinical purposes.
Project description:Here, we present a protocol to efficiently direct human pluripotent stem cells (hPSCs) into hematopoietic stem and progenitor cells (HSPCs) under a chemically defined, albumin-free system. We describe the induction of aorta-gonad-mesonephros-like hematopoiesis from hPSCs into SOX17+ hemogenic endothelium and then into CD34+CD45+ HSPCs via application of Wnt activator and TGFβ inhibitor, respectively. The generated HSPCs, characterized by flow cytometry and colony-forming unit assay, express definitive hematopoiesis markers and exhibit multilineage differentiation potential and the capacity to expand. For complete details on the use and execution of this protocol, please refer to Chang et al. (2022a, 2022b).1,2.
Project description:A causal link between hematopoietic stem/progenitor cell (HSPC) dysfunction and DNA damage accrual has been proposed. Clinically relevant strategies to maintain genome integrity in these cells are needed. Here we report that eltrombopag, a small molecule agonist of the thrombopoietin (TPO) receptor used in the clinic, promotes DNA double-strand break (DSB) repair in human HSPCs. We found that eltrombopag specifically activates the classic nonhomologous end-joining (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Eltrombopag-mediated DNA repair results in enhanced genome stability, survival, and function of primary human HSPCs, as demonstrated in karyotyping analyses, colony-forming unit assays and after transplantation in immunodeficient NSG mice. Eltrombopag may offer a new therapeutic modality to protect human HSPCs against genome insults.
Project description:The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.
Project description:Fanconi anemia is a genetic bone marrow failure syndrome. The current treatment options are suboptimal and do not prevent the eventual onset of aplastic anemia requiring bone marrow transplantation. We previously showed that resveratrol, an antioxidant and an activator of the protein deacetylase Sirt1, enhanced hematopoiesis in Fancd2 mutant mice and improved the impaired stem cell quiescence observed in this disease. Given that Sirt1 is important for the function of hematopoietic stem cells, we hypothesized that Sirt1 activation may improve hematopoiesis. Indeed, Fancd2(-/-) mice and wild-type mice treated with the selective Sirt1 activator SRT3025 had increased numbers of hematopoietic stem and progenitor cells, platelets and white blood cells. SRT3025 was also protective against acetaldehyde-induced hematopoietic damage. Unlike resveratrol, however, SRT3025 did not affect stem cell quiescence, suggesting distinct mechanisms of action. Conditional deletion of Sirt1 in hematopoietic cells did not abrogate the beneficial effects of SRT3025, indicating that the drug did not act by directly stimulating Sirt1 in stem cells, but must be acting indirectly via extra-hematopoietic effects. RNA-Seq transcriptome analysis revealed the down-regulation of Egr1-p21 expression, providing a potential mechanism for improved hematopoiesis. Overall, our data indicate that SRT3025 or related compounds may be beneficial in Fanconi anemia and other bone marrow failure syndromes.
Project description:Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73-), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38-/CD45RA-/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT).
Project description:Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.