Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular ...state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f + cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell—based therapeutics.
The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. The validity of this model in the mouse has been questioned; however, ...little is known about the lineage potential of human progenitors. Here we provide a comprehensive analysis of the human hematopoietic hierarchy by clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow. Human multilymphoid progenitors, identified as a distinct population of Thy-1(neg-lo)CD45RA(+) cells in the CD34(+)CD38(-) stem cell compartment, gave rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells, which indicated that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation.
Understanding how differentiation programs originate from the gene-expression 'landscape' of hematopoietic stem cells (HSCs) is crucial for the development of new clinical therapies. We mapped the ...transcriptional dynamics underlying the first steps of commitment by tracking transcriptome changes in human HSCs and eight early progenitor populations. We found that transcriptional programs were extensively shared, extended across lineage-potential boundaries and were not strictly lineage affiliated. Elements of stem, lymphoid and myeloid programs were retained in multilymphoid progenitors (MLPs), which reflected a hybrid transcriptional state. By functional single cell analysis, we found that the transcription factors Bcl-11A, Sox4 and TEAD1 (TEF1) governed transcriptional networks in MLPs, which led to B cell specification. Overall, we found that integrated transcriptome approaches can be used to identify previously unknown regulators of multipotency and show additional complexity in lymphoid commitment.
Repopulation of immunodeficient mice remains the primary method to assay human hematopoietic stem cells (HSCs). Here we report that female NOD/SCID/IL-2Rgc-null mice are far superior in detecting ...human HSCs (Lin−CD34+CD38−CD90+CD45RA−) compared with male recipients. When multiple HSCs were transplanted, female recipients displayed a trend (1.4-fold) toward higher levels of human chimerism (female vs male: injected femur, 44.4 ± 9.3 vs 32.2 ± 6.2; n = 12 females, n = 24 males; P = .1). Strikingly, this effect was dramatically amplified at limiting cell doses where female recipients had an approximately 11-fold higher chimerism from single HSCs (female vs male: injected femur, 8.1 ± 2.7 vs 0.7 ± 0.7; n = 28 females, n = 20 males; P < .001). Secondary transplantations from primary recipients indicate that females more efficiently support the self-renewal of human HSCs. Therefore, sex-associated factors play a pivotal role in the survival, proliferation, and self-renewal of human HSCs in the xenograft model, and recipient sex must be carefully monitored in the future design of experiments requiring human HSC assays.
Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable ...human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.
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•Respecification is a strategy to generate HSCs from committed progenitors•ERG, HOXA9, and RORA induce multipotent progenitors from human IPSCs•Addition of SOX4 and MYB enables robust short-term erythro-myeloid transplant•Engrafted human erythroblasts mature and undergo hemoglobin switching
A strategy for making transplantable hematopoietic cells from human pluripotent cells involving transcription-factor-based induction of stem-cell-like properties in differentiated progenitors.
SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells ...with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5′ UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non–rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.
•Induced pluripotent stem cell model of SF3B1-mutant MDS develops RS.•Coordinated mis-splicing of TMEM14C and ABCB7 causes RS formation.
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Myeloid neoplasms, including myelodysplastic syndromes (MDS), are genetically heterogeneous disorders driven by clonal acquisition of somatic mutations in hematopoietic stem and progenitor cells ...(HPCs). The order of premalignant mutations and their impact on HPC self-renewal and differentiation remain poorly understood. We show that episomal reprogramming of MDS patient samples generates induced pluripotent stem cells from single premalignant cells with a partial complement of mutations, directly informing the temporal order of mutations in the individual patient. Reprogramming preferentially captured early subclones with fewer mutations, which were rare among single patient cells. To evaluate the functional impact of clonal evolution in individual patients, we differentiated isogenic MDS induced pluripotent stem cells harboring up to 4 successive clonal abnormalities recapitulating a progressive decrease in hematopoietic differentiation potential. SF3B1, in concert with epigenetic mutations, perturbed mitochondrial function leading to accumulation of damaged mitochondria during disease progression, resulting in apoptosis and ineffective erythropoiesis. Reprogramming also informed the order of premalignant mutations in patients with complex karyotype and identified 5q deletion as an early cytogenetic anomaly. The loss of chromosome 5q cooperated with TP53 mutations to perturb genome stability, promoting acquisition of structural and karyotypic abnormalities. Reprogramming thus enables molecular and functional interrogation of preleukemic clonal evolution, identifying mitochondrial function and chromosome stability as key pathways affected by acquisition of somatic mutations in MDS.
•Reprogramming identifies clonal history and recapitulates disease progression in individual patients with MDS.•Mitochondrial function and chromosome stability are key pathways affected by acquisition of mutations in MDS.
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All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, ...lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.
Congenital or acquired cellular deficiencies in humans have the potential to reveal much about normal hematopoiesis and immune function. We show that a recently described syndrome of monocytopenia, B ...and NK lymphoid deficiency additionally includes the near absence of dendritic cells. Four subjects showed severe depletion of the peripheral blood HLA-DR(+) lineage(-) compartment, with virtually no CD123(+) or CD11c(+) dendritic cells (DCs) and very few CD14(+) or CD16(+) monocytes. The only remaining HLA-DR(+) lineage(-) cells were circulating CD34(+) progenitor cells. Dermal CD14(+) and CD1a(+) DC were also absent, consistent with their dependence on blood-derived precursors. In contrast, epidermal Langerhans cells and tissue macrophages were largely preserved. Combined loss of peripheral DCs, monocytes, and B and NK lymphocytes was mirrored in the bone marrow by complete absence of multilymphoid progenitors and depletion of granulocyte-macrophage progenitors. Depletion of the HLA-DR(+) peripheral blood compartment was associated with elevated serum fms-like tyrosine kinase ligand and reduced circulating CD4(+)CD25(hi)FoxP3(+) T cells, supporting a role for DC in T reg cell homeostasis.
A Stem Cell Perspective on Cellular Engineering Doulatov, Sergei; Daley, George Q.
Science (American Association for the Advancement of Science),
11/2013, Letnik:
342, Številka:
6159
Journal Article
Recenzirano
Alternative ways of engineering cells in vitro are being tested to realize their therapeutic potential.
A fundamental enigma in modern biology concerns the molecular rules that govern how cells ...establish and maintain identity during development. These rules are the key to generating therapeutic cell types in vitro and the foundation of regenerative medicine. The isolation of embryonic stem cells (ESCs) has enabled scientists to recapitulate the process of embryonic development in a dish, by directing differentiation of ESCs with combinations of morphogens and growth factors to mimic embryonic development. These experiments assume that cell and tissue fates evolve along specific paths, and once established, remain fixed. But the advent of cellular reprogramming has fundamentally altered our view of the stability of cell identity, and dramatic demonstrations of the interconversions of mature cell types have introduced the provocative idea that cell identity can be engineered to play beneficial therapeutic roles.
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