Generation Of Erythroid Cells Research Articles

Placenta Is a Niche for Hematopoietic Stem Cells.

The hematopoietic system in the embryo develops in anatomically distinct sites, facilitating rapid generation of erythroid cells and formation of a pool of pluripotent HSCs. The origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct the genesis, maturation, expansion and differentiation of HSCs. In avians, de novo hematopoiesis occurs not only in the yolk sac and the AGM but also in another mesodermal appendage, the allantois. In mammals, the allantois forms the umbilical cord and fetal placenta upon fusion with the chorion. The placenta has not been recognized as a hematopoietic organ, although Melchers reported fetal B-cell potential in murine placenta 25 years ago (Nature 1979, 277:219). Recently, Alvarez-Silva et al. showed that the placenta is a rich source for multipotential hematopoietic progenitors prior to the fetal liver (Development 2003, 130:5437).We have performed spatial and temporal analysis of HSCs during mouse development and for the first time assessed HSC activity in the placenta. Hematopoietic organs from E10.5-18.5 embryos (CD45.1/CD45.2) were treated with collagenase and transplanted in limiting dilutions (3–1/1000 embryo equivalents, ee) into irradiated CD45.2+ adult hosts with CD45.1+ support BM cells. Reconstitution was analyzed by FACS and HSCs were quantified as repopulating units (RUs/ee = ([reconstituted recipients] /[total recipients]) /[transplanted dose]).Our data show that the placenta functions as a hematopoietic organ that during midgestation harbors a large pool of pluripotent HSCs. The onset of HSC activity in the placenta parallels that of the AGM starting at E10.5–11.0. However, the placenta HSC pool expands until E12.5–13.5 (>50 RUs) contrasting lack of HSC expansion in the AGM. The expansion of CD34+c-kit+ HSCs in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver and is not accompanied with myeloerythroid differentiation. A far greater expansion of placenta HSCs compared to that of clonogenic progenitors (17-fold vs. 2-fold at E11.5–12.5) suggests that the placenta provides a favorable niche for HSCs. Indeed, placenta HSCs possess functional properties of authentic adult-type HSCs by providing high level multilineage reconstitution for >5 months and exhibiting self-renewal capacity upon serial transplantation. Importantly, placenta HSCs are distinct from circulating HSCs that appear in low numbers after E11.5. HSC activity in the placenta declines towards the end of gestation while HSCs in the fetal liver and blood continue to increase, possibly reflecting mobilization of placenta HSCs to the fetal liver and other developing hematopoietic organs.The early onset of HSC activity in the placenta suggests that the allantois and its derivatives may participate in de novo genesis and maturation of HSCs together with the AGM and possibly the yolk sac. As the main blood volume from the dorsal aorta reaches the fetal liver via umbilical vessels and the placenta, placenta may also provide a niche where nascent HSCs, or pre-HSCs, from the AGM colonize for maturation and expansion prior to seeding fetal liver. While further studies are needed to define the precise origin of placenta HSCs and the function of placenta microenvironment as an HSC supportive niche, the unique kinetics and magnitude of HSC activity suggest an important, previously unappreciated role for the placenta in establishing the definitive hematopoietic system.

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.

Stem Cell Factor and Interleukin-3 Induce Stepwise Generation of Erythroid Precursor Cells from a Basic Fibroblast Growth Factor-Dependent Hematopoietic Stem Cell Line, A-6

A multipotent immature myeloid cell population was produced from a basic fibroblast growth factor (bFGF)-dependent hematopoietic stem cell line, A-6, when cultured with stem cell factor (SCF) replacing bFGF. Those cells were positive for stem cell markers, c-kit and CD34, and a myeloid cell marker, F4/80. Some cell fractions were also positive for Mac-1, a macrophage marker or Gr-1, a granulocytic maker, but negative for an erythroid marker TER119. They also showed the expression of mRNA for the myeloid-specific PU.1 but did not that for the erythroid-specific GATA-1. Among various cytokines, interleukin-3 (IL-3) induced erythroid precursor cells that expressed the erythroid-specific GATA-1 and β-major globin. The quantitative analysis showed that erythroid precursor cells were newly produced from the immature myeloid cells by cultivation with IL-3. SCF and IL-3 induced stepwise generation of erythroid precursor cells from an A-6 hematopoietic stem cell line.

Disruption of the homeobox gene Hoxb-6 in mice results in increased numbers of early erythrocyte progenitors.

Hox genes encode transcription factors that are required for proper development of certain tissues and for patterning of the hindbrain, the limbs, and skeleton. They are also expressed in the hematopoietic system with a preference for specific cell lineages. To determine the role of Hoxb-6 in normal hematopoiesis, mice with a targeted disruption in the Hoxb-6 gene were generated. Mature hematopoietic cell types and immune responses are normal in homozygous Hoxb-6 mutants. Clonogenic progenitor cell assays demonstrate an increased number of early erythroid progenitor cells in the bone marrow and fetal liver of mutants, while differentiation of other cell lineages is unaffected. These results suggest that Hoxb-6 controls the generation, proliferation, or survival of erythroid progenitor cells.

Prospective identification of erythroid elements in cultured peripheral blood

We have developed a prospective approach to identify the generation of erythroid cells derived from cultured peripheral blood mononuclear cells (PBMC) by monitoring the expression of the cell surface protein CD48. Unpurified populations of PBMC obtained from the buffy coats of normal volunteers were grown in suspension culture in the absence or presence of erythropoietin. A profile of surface CD48 expression permitted a flow cytometric identification of erythropoietin responsive populations at various stages of their maturation. In the absence of erythropoietin (EPO) supplemented media, the CD48 − cells represented <5% of the total population of PBMC remaining in culture. In cultures supplemented with 1 U/mL EPO, the mean percentage of CD48 − cells increased to 34.7 + 14.9% ( p < 0.01) after 14 days in culture. Coordinated CD34 and CD71 (transferrin receptor) expression, morphology, gamma-globin transcription, and colony formation in methylcelluose were observed during the 14-day culture period. Flow cytometric monitoring of bulk cultured PBMC provides a simple and reliable means for the prospective or real-time study of human erythropoiesis.

Abnormalities of Cytokine Receptor Signalling Contributing to Diseases of Red Blood Cell Production

The production of erythroid cells is a dynamic and exquisitely regulated process. The mature red cell is only the final phase of a complex but orderly series of genetic events that are initiated at the time a multipotent stem cell becomes committed to expressing the erythroid programme. Aberrations either in the intrinsic generation and/or amplification of functional erythroid cells or in the regulatory influences of microenvironment or cytokines form the basis for a number of blood diseases. In this review we focus upon abnormalities in red blood cell production and discuss how alterations in cytokine regulation of red blood cell production may contribute to these disease processes. We discuss clinical states in which blood red cell numbers are altered, including primary familial and congenital polycythaemia, the myeloproliferative disorder polycythaemia vera, erythroleukaemia, and Diamond—Blackfan anaemia. These disorders are briefly described and evidence supporting a potential role of specific cytokine receptor signalling defects as contributing to these phenotypes is discussed.

CD98: A Type II Transmembrane Glycoprotein Expressed From the Beginning of Primitive and Definitive Hematopoiesis May Play a Critical Role in the Development of Hematopoietic Cells

In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.

Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

Do the preclinical effects of thrombopoietin correlate with its in vitro properties?

In the short time since its cloning, much has been learned of the in vitro properties of thrombopoietin (TPO). In addition to effects on the differentiation of megakaryocytes, TPO has also been shown to stimulate the proliferation of megakaryocytic progenitor cells, colony-forming units-megakaryocytes (CFU-MK), to act in synergy with interleukin 3 or c-kit ligand and erythropoietin (Epo) to stimulate the development of early and the generation of late erythroid progenitor cells, and to affect the rate of entry into the cell cycle and proliferative capacity of hematopoietic stem cells. An important question posed by these observations, for both TPO and for hematopoietic research in general, is whether the in vitro effects of a cytokine are mirrored by its preclinical and clinical biology. The results of recent studies in mice and nonhuman primates will be presented which have attempted to address this issue. In normal animals, TPO increases the numbers of marrow and spleen CFU-granulocyte/erythroid/macrophage/megakaryocyte, CFU-MK, CFU-GM and BFU-E, but its effects in the peripheral blood are limited to marked increases in the platelet count. The reason for widespread progenitor cell effects, yet stable leukocyte and erythrocyte blood counts, is likely the predominant regulatory effects of G-CSF and Epo; in the absence of elevated levels of the lineage-dominant regulator of each of these cell types, expanded progenitor cell numbers are not translated into increased peripheral blood counts. However, in states of increased blood cell demand such as follows myelosuppressive therapy, elevated levels of Epo and G-CSF allow the effects of TPO on erythropoiesis and myelopoiesis to become manifest. The administration of TPO to myelosuppressed animals is associated with not only greatly expanded hematopoietic progenitor cell recovery, but also improvement in platelet, red cell and leukocyte nadir levels and greatly accelerated recovery of all three cell lineages. These results indicate that the panhematopoietic properties of TPO identified by in vitro culture techniques correlate well with its effects in animals. The results of ongoing clinical trials should soon establish whether these conclusions can be extended to patient care.

Induction of the murine "W phenotype" in long-term cultures of human cord blood cells by c-kit antisense oligomers.

The murine white (W) spotting locus is the site of the c-kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) locus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c-kit antisense oligonucleotides to long-term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c-kit gene expression and the preferential suppression of the generation of erythroid burst-forming cells (BFU-E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the "W phenotype" in human hematopoiesis and confirm the importance of c-kit gene function in early erythropoiesis. Because the generation of BFU-E was suppressed even after c-kit gene expression had recovered, this gene product may be critical to the erythroid commitment process.