Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.     

Differential Sensitivity of Mouse Hematopoietic Stem Cells to Merocyanine 540

In vivo and in vitro clonal assays of immature mouse blood cells showed that diffferent populations of hematopoietic progenitor cells differ considerably with respect to their sensitivity to photodynamic damages caused by the fluorescent dye Merocyanine 540. Late erythroid progenitors were the most sensitive cells followed in order of decreasing sensitivity by pluripotent stem cells, early erythroid progenitors, and granulocyte/macrophage progenitors. Only about 2%–4% of all nucleated marrow cells were stained with Merocyanine 540 which correlated well with current frequency estimates of progenitor cells in mouse bone marrow. Our findings indicate that the expression of Merocyanine binding sites is developmentally regulated and might, therefore, provide a useful molecular marker for blood cell differentiation and a basis for an effective purification of hematopoietic progenitor cells.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.     

Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.

In this study, we have mapped the onset of hematopoietic development in the mouse embryo using colony-forming progenitor assays and PCR-based gene expression analysis. With this approach, we demonstrate that commitment of embryonic cells to hematopoietic fates begins in proximal regions of the egg cylinder at the mid-primitive streak stage (E7.0) with the simultaneous appearance of primitive erythroid and macrophage progenitors. Development of these progenitors was associated with the expression of SCL/tal-1 and GATA-1, genes known to be involved in the development and maturation of the hematopoietic system. Kinetic analysis revealed the transient nature of the primitive erythroid lineage, as progenitors increased in number in the developing yolk sac until early somite-pair stages of development (E8.25) and then declined sharply to undetectable levels by 20 somite pairs (E9.0). Primitive erythroid progenitors were not detected in any other tissue at any stage of embryonic development. The early wave of primitive erythropoiesis was followed by the appearance of definitive erythroid progenitors (BFU-E) that were first detectable at 1-7 somite pairs (E8.25) exclusively within the yolk sac. The appearance of BFU-E was followed by the development of later stage definitive erythroid (CFU-E), mast cell and bipotential granulocyte/macrophage progenitors in the yolk sac. C-myb, a gene essential for definitive hematopoiesis, was expressed at low levels in the yolk sac just prior to and during the early development of these definitive erythroid progenitors. All hematopoietic activity was localized to the yolk sac until circulation was established (E8.5) at which time progenitors from all lineages were detected in the bloodstream and subsequently in the fetal liver following its development. This pattern of development suggests that definitive hematopoietic progenitors arise in the yolk sac, migrate through the bloodstream and seed the fetal liver to rapidly initiate the first phase of intraembryonic hematopoiesis. Together, these findings demonstrate that commitment to hematopoietic fates begins in early gastrulation, that the yolk sac is the only site of primitive erythropoiesis and that the yolk sac serves as the first source of definitive hematopoietic progenitors during embryonic development.     

Colony-forming cells with high proliferative potential (HPP-CFC)

Colony-forming cells with a high proliferative potential (HPP-CFC) have been defined by their ability to form large colonies in vitro (diameters greater than 0.5 mm and containing approximately 50,000 cells) in bone marrow cell cultures. The HPP-CFC have been characterized by: 1) a relative resistance to treatment in vivo with the cytotoxic drug 5-fluorouracil, 2) a high correlation with cells capable of repopulating the bone marrow of lethally irradiated mice, 3) their multipotential ability to generate cells of the macrophage, granulocyte, megakaryocyte and erythroid lineages, and 4) their multifactor responsiveness. The HPP-CFC have been described in both mouse and human bone marrow. These properties suggest that the HPP-CFC represent an important cell type in hematopoiesis and provide a model system, particularly in the human, for studying the properties of primitive progenitor cells in vitro.     

Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

人卵黄囊造血的探讨

采用卵黄囊组织切片、涂片的形态学、细胞化学染色、造血干/祖细胞体外培养及CD_(34)单克隆抗体免疫荧光检测等方法研究表明:人卵黄囊中存在造血岛,造血岛内由于造血微环境的特点致使此期造血主要向红系分化。血岛中检测出CD_(34)~ 细胞,比例高于胎肝及成人骨髓,干/祖细胞于体外培养形成红系集落。结论:人胚胎期造血源于卵黄囊。     

Ikaros isoform x is selectively expressed in myeloid differentiation

The Ikaros gene is alternately spliced to generate multiple DNA-binding and nonbinding isoforms that have been implicated as regulators of hematopoiesis, particularly in the lymphoid lineages. Although early reports of Ikaros mutant mice focused on lymphoid defects, these mice also show significant myeloid, erythroid, and stem cell defects. However, the specific Ikaros proteins expressed in these cells have not been determined. We recently described Ikaros-x (Ikx), a new Ikaros isoform that is the predominant Ikaros protein in normal human hematopoietic cells. In this study, we report that the Ikx protein is selectively expressed in human myeloid lineage cells, while Ik1 predominates in the lymphoid and erythroid lineages. Both Ik1 and Ikx proteins are expressed in early human hematopoietic cells (Lin(-)CD34(+)). Under culture conditions that promote specific lineage differentiation, Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human Lin(-)CD34(+) cells. We show that Ikx and other novel Ikaros splice variants identified in human studies are also expressed in murine bone marrow. In mice, as in humans, the Ikx protein is selectively expressed in the myeloid lineage. Our studies suggest that Ikaros proteins function in myeloid, as well as lymphoid, differentiation and that specific Ikaros isoforms may play a role in regulating lineage commitment decisions in mice and humans.     

Comparative analysis of cell lineage differentiation during hepatogenesis in humans and mice at the single-cell transcriptome level

During embryogenesis, the liver is the site of hepatogenesis and hematopoiesis and contains many cell lineages derived from the endoderm and mesoderm. However, the characteristics and developmental programs of many of these cell lineages remain unclear, especially in humans. Here, we performed single-cell RNA sequencing of whole human and mouse fetal livers throughout development. We identified four cell lineage families of endoderm-derived, erythroid, non-erythroid hematopoietic, and mesoderm-derived non-hematopoietic cells, and defined the developmental pathways of the major cell lineage families. In both humans and mice, we identified novel markers of hepatic lineages and an ID3⁺ subpopulation of hepatoblasts as well as verified that hepatoblast differentiation follows the “default-directed” model. Additionally, we found that human but not mouse fetal hepatocytes display heterogeneity associated with expression of metabolism-related genes. We described the developmental process of erythroid progenitor cells during human and mouse hematopoiesis. Moreover, despite the general conservation of cell differentiation programs between species, we observed different cell lineage compositions during hematopoiesis in the human and mouse fetal livers. Taken together, these results reveal the dynamic cell landscape of fetal liver development and illustrate the similarities and differences in liver development between species, providing an extensive resource for inducing various liver cell lineages in vitro.Subject terms: Developmental biology, Stem-cell differentiation, Stem-cell differentiation, Developmental biology     

All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells

While it is clear that a single hematopoietic stem cell?(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on?the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in?vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.     

Cryopreservation of bovine hemopoietic progenitor cells in liquid nitrogen

Bovine marrow granulocyte/macrophage and erythroid progenitor cells maintained viability after storage in liquid nitrogen for 2 to 4 weeks. The granulocyte/macrophage progenitor cells maintained 100% viability for 4 weeks, while the erythroid progenitor cells maintained 100% viability for at least 2 weeks. The optimum concentration of either DMSO or glycerol was found to be 5–10%. DMSO was superior to glycerol as a cryopreservative of bovine granulocyte/ macrophage progenitor cells. Glycerol was found to be unable to cryopreserve bovine erythroid progenitor cells.     

Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4⁺ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34⁺ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.     

Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

During embryonic development, hematopoiesis occurs through primitive and definitive waves, giving rise to distinct blood lineages. Hematopoietic stem cells (HSCs) emerge from hemogenic endothelial (HE) cells, through endothelial‐to‐hematopoietic transition (EHT). In the adult, HSC quiescence, maintenance, and differentiation are closely linked to changes in metabolism. However, metabolic processes underlying the emergence of HSCs from HE cells remain unclear. Here, we show that the emergence of blood is regulated by multiple metabolic pathways that induce or modulate the differentiation toward specific hematopoietic lineages during human EHT. In both in vitro and in vivo settings, steering pyruvate use toward glycolysis or OXPHOS differentially skews the hematopoietic output of HE cells toward either an erythroid fate with primitive phenotype, or a definitive lymphoid fate, respectively. We demonstrate that glycolysis‐mediated differentiation of HE toward primitive erythroid hematopoiesis is dependent on the epigenetic regulator LSD1. In contrast, OXPHOS‐mediated differentiation of HE toward definitive hematopoiesis is dependent on cholesterol metabolism. Our findings reveal that during EHT, metabolism is a major regulator of primitive versus definitive hematopoietic differentiation.     

Differential proliferative effects of transforming growth factor-beta on human hematopoietic progenitor cells

Transforming growth factor-beta (TGF beta) regulates cell growth and differentiation in numerous cell systems, including several hematopoietic lineages. We used in vitro cultures of highly enriched hematopoietic progenitor cells stimulated by natural and recombinant growth factors to investigate the biologic effects of TGF beta 1 and TGF beta 2 on erythroid (CFU-E and burst-forming unit (BFU)-E), granulocyte-macrophage (CFU-GM) and multilineage (i.e., granulocyte, erythroid, macrophage, and megakaryocyte; CFU-GEMM) colony-forming cells. In the absence of exogenous CSF, neither TGF beta 1 nor TGF beta 2 supported progenitor cell growth. In the presence of recombinant or natural CSF, picomolar concentrations of TGF beta 1 inhibited growth of CFU-E, BFU-E, and CFU-GEMM and enhanced growth of day 7 CFU-GM. Inhibition of CFU-E and BFU-E by human and porcine TGF beta 1 was similar, ranging from 17 to 73% over a concentration range of 0.05 to 1.0 ng/ml, and was largely independent of the type of burst-promoting activity used (rIL-3 vs cell line 5637-conditioned medium). Inhibition of CFU-GEMM ranged from 79 to 98% over a concentration range of 0.25 to 1.0 ng/ml. The inhibitory effect of TGF beta 1 was progressively lost when its addition was delayed for 40 to 120 h, suggesting a mode of action during early cell divisions. In contrast, growth of CFU-GM stimulated by plateau concentrations of human rG-CSF, rGM-CSF, and rIL-3 was enhanced up to 154 +/- 22% by human TGF beta 1. Porcine platelet-derived TGF beta 2 was essentially without effect on the progenitor populations examined. These results support the hypothesis that TGF beta may play role in the regulation of hematopoietic progenitor cell proliferation by differentially affecting individual lineages and is apparently capable of doing so in the relative absence of marrow accessory cells.