Stringently purified human hematopoietic progenitors/stem cells: Analysis of cellular/molecular mechanisms underlying early hematopoiesis (original) (raw)
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Proceedings of the National Academy of Sciences, 1992
The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-
Unilineage hematopoietic differentiation in bulk and single cell culture
STEM CELLS, 2009
The rarity of hematopoietic stem and progenitor cells (HSCs, HPCs) has hampered the analysis of cellular and molecular mechanisms underlying early hematopoiesis. Methodology for HPC purification has partially offset this limitation. A further hurdle has been represented by the heterogeneity of the analyzed HPC/precursor populations: recently, development of unilineage HPC differentiation cultures has provided homogeneous populations of hematopoietic cells, particularly in the early differentiation state, i.e., populations pertaining to a single lineage and a restricted stage of differentiatiodmatu r a t i o n , b u t s u f f i c i e n t l y l a r g e f o r cellular/molecular analysis. This report focuses on the development and characterization of the unilineage HPC differentiation culture systems. A section is devoted to selected cellular and molecular mechanisms underlying hematopoiesis, which have been investigated by the HPC unilineage culture approach. Finally, recent advances in the development of HPC unilineage cultures at single cell level are discussed.
Expansion and Differentiation of Immature Mouse and Human Hematopoietic Progenitors
Developmental Hematopoiesis, 2004
A prerequisite for proper investigation of self-renewal and differentiation of hematopoietic cells is the possibility to obtain large quantities of homogenous primary progenitors under defined conditions, allowing meaningful biochemical and molecular analyses. These cells should show renewal and differentiation characteristics similar to the in vivo situation. The serum-free culture systems delineated in this chapter meet these requirements, employing primary hematopoietic cells derived from murine fetal liver and human umbilical cord blood, which show physiological self-renewal responses to cytokine/hormone combinations, which in vivo are involved in stress hematopoiesis. We describe the expansion and sustained proliferation of multipotent (mouse) and erythroid (mouse and human) progenitors, responding to physiological signals. Moreover, both mouse and human erythroid progenitors can be induced to undergo synchronous terminal differentiation by addition of high levels of erythropoietin. If fetal liver cells from p53 -/mice are used, respective multipotent and erythroid cells undergo immortalization without an obvious Hayflick crisis, but otherwise retain their primary cell characteristics. Finally, both primary and immortal mouse progenitors can be subjected to genetic manipulation via retroviral constructs with high efficiency.
Cascade transactivation of growth factor receptors in early human hematopoiesis
Blood, 1993
Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with selfrenewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,70O/cell), a 210-fold lower number of IL-6Rs (1 45/cell) and granulocyte-macrophage colonystimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/ cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and thekitligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at + 4°C. No crossreaction was observed between IL-6R. IL-3R. EpR, and the heterologous GFs, whereas the GM-CSFR showed crossreactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37%, followed by ligand-binding assay at 20°C. IL-6, IL-3. GM-CSF, and Ep induce a marked down-EMATOPOIESIS is a multistep cell proliferation/dif-H ferentiation process that is sustained by a pool of stem cells. The stem elements can self-renew and differentiate into progenitors.'-4 These progenitors are committed to specific lineages and are functionally defined as colony-or burstforming units (CFUs or BFUs), ie, progenitors ofthe erythroid series (BFU-E or CFU-E), the granulocyte-monocytic lineage (CFU-GM), and multipotent CFU for the GM, erythroid, and megakaryocytic series (CFU-GEMM).I4 The progenitors in turn differentiate into morphologically recognizable precursors that mature to terminal elements circulating in peripheral blood. A group of glycoprotein hematopoietic growth factors (HGFs), termed colony stimulating factors (CSFs) or interleukins (ILs), control the survival, proliferation, and differentiation of stem and/or progenitor cells. In addition, they affect a variety of functional activities of mature/terminal cells.'-4
Proceedings of The National Academy of Sciences, 1996
A hierarchical order of gene expression has been proposed to control developmental events in hematopoiesis, but direct demonstration of the temporal relationships between regulatory gene expression and differentiation has been difficult to achieve. We modified a single-cell PCR method to detect 2-fold changes in mRNA copies per cell (dynamic range, 250-250,000 copies͞cell) and used it to sequentially quantitate gene expression levels as single primitive (CD34 ؉ ,CD38 ؊ ) progenitor cells underwent differentiation to become erythrocytes, granulocytes, or monocyte͞macrophages. Markers of differentiation such as CD34 or cytokine receptor mRNAs and transcription factors associated with their regulation were assessed. All transcription factors tested were expressed in multipotent progenitors. During lineage-specific differentiation, however, distinct patterns of expression emerged. SCL, GATA-2, and GATA-1 expression sequentially extinguished during erythroid differentiation. PU.1, AML1B, and C͞EBP␣ expression profiles and their relationship to cytokine receptor expression in maturing granulocytes could be distinguished from similar profiles in monocytic cells. These data characterize the dynamics of gene expression accompanying blood cell development and define a signature gene expression pattern for specific stages of hematopoietic differentiation.
Cell reports, 2015
Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At embryonic day 9.5 (E9.5), we show the first murine definitive erythro-myeloid progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC)-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell po...