Primitive Erythroid Cells Research Articles

Concise Review: Early Embryonic Erythropoiesis: Not so Primitive After All

In the developing embryo, hematopoiesis begins with the formation of primitive erythroid cells (EryP), a distinct and transient red blood cell lineage. EryP play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Progenitors for EryP arise as a cohort within the blood islands of the mammalian yolk sac at the end of gastrulation. As a strong heartbeat is established, nucleated erythroblasts begin to circulate and to mature in a stepwise, nearly synchronous manner. Until relatively recently, these cells were thought to be "primitive" in that they seemed to more closely resemble the nucleated erythroid cells of lower vertebrates than the enucleated erythrocytes of mammals. It is now known that mammalian EryP do enucleate, but not until several days after entering the bloodstream. I will summarize the common and distinguishing characteristics of primitive versus definitive (adult-type) erythroid cells, review the development of EryP from the emergence of their progenitors through maturation and enucleation, and discuss pluripotent stem cells as models for erythropoiesis. Erythroid differentiation of both mouse and human pluripotent stem cells in vitro has thus far reproduced early but not late red blood cell ontogeny. Therefore, a deeper understanding of cellular and molecular mechanisms underlying the differences and similarities between the embryonic and adult erythroid lineages will be critical to improving methods for production of red blood cells for use in the clinic.

Development of membrane mechanical function during terminal stages of primitive erythropoiesis in mice

During murine embryogenesis, primitive erythroblasts enter the circulation as immature nucleated cells and progressively mature as a semisynchronous cohort, enucleating between E12.5 and E16.5. In this report, we examine the mechanical properties of these cells to determine how their mechanical development differs from that of definitive erythroid cells, which mature extravascularly in protected marrow microenvironments. Primitive erythroid cells acquire normal membrane deformability by E12.5 (i.e., as late stage erythroblasts) and maintain the same level of surface stiffness through E17.5. During this same period, the strength of association between the membrane bilayer and the underlying skeleton increases, as indicated by an approximate doubling of the energy required to separate bilayer from skeleton. At the same time, these cells undergo dramatic changes in surface area and volume, losing 35% of their surface area and 50% of their volume from E14.5 to E17.5. Interestingly, membrane remodeling proceeded regardless of whether the cells completed enucleation. These data suggest that in primitive erythroid cells, unlike their definitive counterparts, the critical maturational processes of membrane remodeling and enucleation are uncoupled.

A Dual Role of Evi-1 During Developmental Hematopoiesis

AbstractAbstract 765The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi-1 expression with aggressiveness in myeloid leukemia. More recent studies suggest involvement of Evi-1 in developmental hematopoiesis. Here, we analyzed zebrafish embryo and human induced pluripotent stem (iPS) cells to understand how Evi-1 modulates early blood development. We found that the zebrafish homologue evi-1 co-localizes with the hematopoietic markers scl, gata1 and gata2 during embryogenesis, indicating a role in primitive hematopoiesis. To analyse the functional role of evi-1, we performed in vivo loss-of-function studies by injecting control and inhibitory morpholino oligonucleotides (MO) suppressing evi-1 pre-mRNA splicing in zebrafish zygotes. To control for off-target effects, three separate MO were designed and injected. N = 100 zebrafish embryos were analyzed per experiment in each group. In situ hybridization analyses performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells as shown by reduced pu.1, mpo and l-plastin expression and suppressed numbers of DsRed positive lyz-expressing cells in Tg(lyz:DsRed) fish. Interestingly, scl expression was not affected, suggesting that evi-1 acts at later stages, regulating specification and/or survival/proliferation of pu.1 expressing progenitor cells. Consistent with this notion, no changes were observed in primitive erythroid progenitor cells as monitored by gata1 and hbae3 expression and flow cytometry analyses of GFP-positive globin-expressing cells in Tg(globin:eGFP) fish. Furthermore, runx1+/cmyb+ cells were strongly reduced, suggesting a separate role of evi-1 in definitive hematopoiesis. Supporting this hypothesis, evi-1 morphants displayed profound suppression of ikzf1+ lymphoid progenitors and rag1+ T-lymphocytes, which was not due to impaired thymus development, since foxn1 expression remained unaltered in evi-1 morphants. Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects HSC proliferation through regulation of Gata2. Indeed, we could show that co-injection of gata2 mRNA was able to rescue the impaired myeloid as well as the HSC phenotype in evi-1 morphants. However, data generated by TUNEL assays indicate that evi-1 also confers apoptosis resistance to developing AGM-HSC. Whether this effect is also mediated by gata2 is currently being explored. We further investigated the effect of EVI-1 during differentiation of hematopoietic cells from human iPS cells. Confirming our results collected in zebrafish embryo, inhibition of EVI-1 strongly reduced the numbers of CD34+ and CD45+ blood progenitor cells and the generation of myeloid colonies in colony forming assays, while leaving SCL expression unaltered. Taken together, our data suggest that Evi-1 plays conserved roles in zebrafish and human hematopoietic development, regulating embryonic myelopoiesis and HSC formation while leaving primitive erythropoiesis unaltered. The precise molecular basis of evi-1 mediated effects on these separate phases of developmental hematopoiesis is subject of ongoing investigations. Disclosures:No relevant conflicts of interest to declare.

Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence

AbstractThe extra-embryonic yolk sac (YS) is the first hematopoietic site in the mouse embryo and is thought to generate only primitive erythroid and myeloerythroid progenitor cells before definitive HSC emergence within the embryo on E10.5. Here, we have shown the existence of T cell–restricted progenitors in the E9.5 YS that directly engraft in recipient immunodeficient mice. T-cell progenitors were also produced in vitro from both YS and para-aortic splanchnopleura hemogenic endothelial cells, and these T-cell progenitors repopulated the thymus and differentiated into mature T-cell subsets in vivo on transplantation. Our data confirm that the YS produces T-lineage–restricted progenitors that are available to colonize the thymus and provide new insight into the YS as a definitive hematopoietic site in the mouse embryo.

Characterization of a Weak Allele of Zebrafish cloche Mutant

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene.

Definitive Erythro-Myeloid Progenitors (EMP) Emerge in the Yolk Sac From Hemogenic Endothelium and Share Transcriptional Regulators with Adult Hematopoiesis

Abstract 910In the mammalian embryo, hematopoietic stem cells (HSC) emerge from vascular beds and colonize the fetal liver. The first HSC are found in the murine fetal liver by embryonic day 12.5 (E12.5). However blood function is required before HSC have formed, and two earlier waves of hematopoietic potential arise to sustain the embryo. The first wave of hematopoietic progenitors are formed in the yolk sac between E7.25 and E8.5 and are termed “primitive” because, along with megakaryocyte and macrophage potential, they differentiate into primitive erythroid cells that mature in the circulation and express embryonic globins. A second lineage of hematopoietic potential has been characterized in the murine and human yolk sac as well as in zebrafish. These cells have been termed “EMP”, erythro-myeloid progenitors, that generate definitive erythroid and myeloid lineages, including granulocytes. We find that EMP emerging in the mouse embryo express many of the markers associated with HSC emergence from hemogenic endothelium. At early stages of their emergence (E8.5), EMP express not only kit and CD41, but also VE-cadherin, CD31 and CD34. A day later, EMP constitute a robust population of over 1,000 cells in the yolk sac and display diminished expression of VE-cadherin and increased expression of CD45. However, unlike HSC, EMP do not express Sca1, but do express the myeloid progenitor marker CD16/32 (low affinity FCgamma receptor II/III). Like CD45, CD16/32 is expressed on a subset of the CD41+/kit+ cells at E8.5. Colony forming assays confirm that EMP potential is found in both CD16/32 positive and negative CD41+/kit+ cells. By E9.5, over 90% of CD41+/kit+ cells also express CD16/32 and all of the hematopoietic colony-forming potential at E9.5 is found in this triple-positive population. The transition from endothelial-associated to hematopoietic-associated genes suggests that EMP may emerge from hemogenic endothelial intermediates. Between E9.5 and E11.5, cells with the EMP immunophenotype are found in the bloodstream and become concentrated in the liver, where evidence of very robust erythro-myeloid differentiation precedes HSC colonization. In culture, EMP rapidly expand, dividing twice daily, and within 6 days generate predominately erythroid cells as well as smaller numbers of megakaryocyte, macrophage and mast cells, but only rare granulocytes. This is in contrast to lin-/kit+ ScaI- bone marrow progenitors grown in the same culture conditions that generate predominately myeloid cells, particularly granulocytes, and rarely mast cells. In agreement with data in the zebrafish, we also do not see evidence of lymphoid potential or RAG2 expression in EMP cultures or expression of the lymphoid markers Flt3 and IL7 receptor on the cell surface of EMP. In order to better understand the lineage potential of EMP, we examined the expression of known transcriptional regulators of bone marrow hematopoiesis. In the adult, relative levels of GATA1 versus Pu.1 are proposed to determine fate between megakaryocyte/erythroid progenitors (MEP- GATA1 hi) and granulocyte/macrophage progenitors (GMP-Pu.1 hi). Consistent with their erythro-myeloid potential, EMP expressed both GATA1 and Pu.1 at intermediate levels compared to adult marrow-derived MEP and GMP. In the adult, Gfi-1 and C/EBPalpha are both proposed to upregulate granulocyte versus macrophage differentiation. We found lower levels of these regulators in EMP compared to GMP, consistent with EMP cultures generating small numbers of granulocytes versus macrophages. In addition, the GATA1 expression within the Pu.1+ GMP is found to increase mast cell potential and, thus, the high GATA1 and Pu.1 expression in EMP may account for their high mast cell potential. Taken together, these data suggest that, like HSC, EMP emerge from hemogenic endothelium and their erythro-myeloid potential is governed by the action of shared regulatory networks. However, the transcription factors and markers are present in the EMP in unique combinations consistent with their specific role in providing a transient initial wave of definitive hematopoiesis in the embryo. Disclosures:No relevant conflicts of interest to declare.

STAT3 Is Differentially Activated Downstream of the Erythropoietin Receptor in Primitive Compared to Definitive Erythroblasts

Abstract 2383Erythropoietin (EPO) acting through its receptor (EPOR), is a critical regulator of definitive erythropoiesis. The EPOR signaling network has been extensively studied in cell lines overexpressing various forms of EPOR. Together with genetic models, these studies have defined an EPOR-JAK2-STAT5 pathway that is critical for the formation of red blood cells. However, the hematopoietic phenotype of mice with a truncated EPOR incapable of activating STAT5 (EPOR-HM) suggests that STAT5 activation is not the only pathway mediating the erythropoietic effects of EPO-EPOR signaling. Our recent studies indicate that EPO-EPOR signaling also regulates primitive erythroblast maturation in the mammalian embryo. To better understand EPOR signaling in the context of primary erythroid cells, we utilized primitive erythroblasts from wild-type and EPOR-HM murine embryos. Robust phosphorylation of STAT5 was detected by western blot in freshly isolated primitive erythroblasts from wild-type embryos. As expected, there is no STAT5 phosphorylation evident in primitive erythroblasts from EPOR-HM embryos. Surprisingly, EPOR-HM embryos produce nearly normal numbers of primitive erythroblasts and we detected equivalent induction of STAT target genes following EPO stimulation in wild-type and EPOR-HM primitive erythroblasts. To address the discrepancy between these data and the accepted model of EPOR-STAT5 signaling, we searched for compensatory signaling via other STAT family members. We found that STAT3, but not STAT1, was phosphorylated in primitive erythroblasts from EPOR-HM embryos, suggesting that phospho-STAT3 compensates for the loss of phospho-STAT5 in the EPOR-HM mice. We also detected a lower level of STAT3 phosphorylation in wild-type primitive erythroblasts that was increased upon in vitro EPO stimulation. These results indicate that EPOR-STAT3 signaling normally occurs in primitive erythroblasts. Furthermore, our preliminary data show that STAT3 transcript levels are higher in primitive erythroblasts compared to definitive erythroblasts from the fetal liver or bone marrow, suggesting that STAT3 activation during erythropoiesis may be lineage-dependent. To test this hypothesis, we utilized a small molecule inhibitor of STAT3 to examine the function of STAT3 signaling both in primitive and in definitive erythroblasts during in vitro maturation. The terminal maturation of definitive erythroblasts is unaffected by STAT3 inhibition. However, inhibition of STAT3 causes a 40% reduction in primitive erythroblast proliferation in a novel 2-step ex vivo culture system. We conclude that, in contrast to definitive erythroblasts, STAT3 is activated downstream of EPOR in primitive erythroid cells. EPOR-STAT3 signaling has been reported in non-erythroid cells as well as in several cancer cell types. Therefore the study of EPOR signaling in primary embryonic erythroblasts may provide novel insights regarding the function of EPO in these cells. Disclosures:No relevant conflicts of interest to declare.

Evi-1 Regulates Myelopoiesis and Hematopoietic Stem Cell Development in Zebrafish and Human Pluripotent Stem Cells

Abstract 1281The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Over the last years, Evi-1 evolved as one of the most potent oncogenes associated with human and murine myeloid leukemia. More recent studies in knockout mice suggest also involvement of Evi-1 in the regulation of developmental hematopoiesis, but the role of Evi-1 in this context is poorly understood. Here, we analyzed zebrafish embryo and human pluripotent stem cells (PSC) to understand how Evi-1 modulates early hematopoietic development.We examined the hematopoietic development in zebrafish embryo by in situ hybridization (ISH) for hematopoietic markers. The zebrafish homologue evi-1 was shown to be expressed in co-localization with scl in the posterior blood islands, indicating a role during early blood development. We also performed loss-of-function studies were by injecting morpholino oligonucleotides (MO) in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. Inhibition of evi-1 was confirmed in MO-injected versus control embryos. N=100 zebrafish embryos were analyzed per experiment in each group. To control for off-target effects, two separate MO were designed and injected. MO mediated evi-1 knockdown severely reduced numbers of circulating blood cells and induced hemorrhages. ISH performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells (measured by pu.1 expression), while no changes were observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Moreover, analyses at 36 hours and 5 days post fertilization showed strong reduction of runx1+/cmyb+ cells and rag1+ lymphoid cells, indicating a role of evi-1 in developing hematopoietic stem cells (HSC).Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects hematopoietic stem cell proliferation through regulation of Gata2. To test whether Gata2 is a putative downstream regulator of Evi-1 in our system, we performed a rescue experiment and co-injected gata2 mRNA in evi-1 MO treated fish. Indeed, ectopic gata2 rescued the impaired myeloid phenotype, as shown by re-occurrence of mpo, l-plastin as well as pu.1 expressing cells.To assess whether these molecular interactions are conserved during human developmental hematopoiesis, we surveyed in vitro differentiating human pluripotent stem cells (PSC) genetically modified to suppress EVI-1. EVI-1 expression was detected during differentiation of human PSC in embryoid bodies, especially around day 9 when hematopoietic progenitors start to emerge in this system. Treatment with EVI-1 shRNA strongly reduced the generation of myeloid colonies from human PSC in vitro as well as the numbers of emerging CD34+ and CD45+ cells. Molecularly, EVI-1 suppression inhibited the expression of PU.1 and GATA2 during the course of development, while leaving SCL and GATA1 expression unaltered.Taken together, our data suggest that, in both fish and human, Evi-1 regulates embryonic myelopoiesis through interactions with Gata2 and independently of Gata1 and embryonic erythropoiesis. Moreover, Evi-1 appears crucial for HSC development. Currently ongoing experiments in our laboratory focus on the further elucidation of the molecular mechanisms underlying the Evi-1 effects during developmental hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Transcription Factors KLF1 and KLF2 Positively Regulate Embryonic and Fetal β-Globin Genes through Direct Promoter Binding

Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ε- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ε- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ε- and γ-globin genes and locus control region in KLF1(-/-) embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos.

An embryonic stage–specific enhancer within the murine β-globin locus mediates domain-wide histone hyperacetylation

AbstractIn mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure.

Human Globin Knock-in Mice Complete Fetal-to-Adult Hemoglobin Switching in Postnatal Development

Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.

SIRT1 Deficiency Compromises Mouse Embryonic Stem Cell Differentiation, and Embryonic and Adult Hematopoiesis In the Mouse.

AbstractAbstract 1612SIRT1 is a conserved NAD-dependent deacetylase capable of deacetylating a number of protein substrates including, but not limited to, p53 and FOXO transcription factors. SIRT1 plays an important role in a variety of biological processes including stress resistance, metabolism, differentiation and aging (Rodgers et al, Nature, 2005; 434:113). SIRT1 is expressed at high levels in mouse embryos. A role for SIRT1 in mouse (m) embryonic stem cell (ESC) maintenance and differentiation is only beginning to be elucidated (Han et al, Cell Stem Cell, 2008; 2:241, Calvanese V et al, PNAS, 2010; 10713736). Here we focus on a role for SIRT1 in differentiation of mESCs into hematopoietic progenitors (HPCs), and in embryonic and adult hematopoiesis. We hypothesized that SIRT1 is involved in hematopoietic commitment within the mouse. We initially assessed the ability of WT and SIRT1-/- mESC to give rise to blast colony forming cells (BL-CFC), a transient population that is present in EBs between day 2.5 and day 3.5 of differentiation and represents the in vitro equivalent of the hemangioblast and as such, the earliest commitment step in the differentiation of mesoderm to the hematopoietic and endothelial lineages. SIRT1-/- ESCs exhibited markedly delayed formation of BL-CFC. The emergence of the Flk-1+/c-Kit- cell population pattern was also delayed, consistent with the delayed pattern of BL-CFC development in SIRT1-/- EBs. This observed delay appears to result from a slower differentiation of the SIRT1-/- ESCs as the kinetics of decline in secondary EB potential, an indication of undifferentiated ES cells, is delayed compared to that of SIRT1+/+ ES cells. When analyzed for hematopoietic and endothelial potential of individual blast colony, replated SIRT1-/- BL-CFC presented limited hematopoietic potential, whereas endothelial potential was essentially unaltered. Next, the ability of SIRT1-/- ESCs to form primitive and definitive hematopoietic cells was evaluated and we found that primitive erythroid progenitors formed from SIRT1-/- R1 cells were not only delayed but greatly decreased. Moreover, after differentiation of SIRT1 -/- mESC there were also significant decreases in granulocyte-macrophage (CFU-GM), and multipotential (CFU-GEMM) progenitors. Differences in primitive and definitive erythroid progenitors were confirmed by gene analysis of βH1 globin (embryonic hemoglobin), a marker for primitive erythroid cells, and βmajor globin (adult hemoglobin). The above delay defects were associated with delayed ability to switch off Oct4, Nanog and Fgf5, decreased β-H1 Globin, β-major globin, Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ESC commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm SIRT1 effects, we assessed embryonic and adult hematopoiesis in SIRT1+/+, +/− and -/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythorid precursors compared to wild-type and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow HPCs in 5 week and 12 month old mice, effects especially notable at lower (5%) O2 tension. Taken together, these results demonstrate that SIRT1 plays a role in mouse embryonic and adult stem cell differentiation. Disclosures:No relevant conflicts of interest to declare.

Definitive Hematopoiesis In the Mammalian Embryo Prior to HSC Formation.

AbstractAbstract 1599The ontogeny of hematopoiesis in mammalian embryos is complicated by the requirement for functional blood cells prior to the emergence of hematopoietic stem cells or the bone marrow microenvironment. In the murine embryo, transplantable HSC are first evident at embryonic day (E) 10.5 and the first few HSC are found in the fetal liver hematopoietic environment by E12.5. However, two overlapping waves of hematopoietic potential arise in the yolk sac before E10.5. The first “primitive” wave produces progenitors from E7.25 to E8.5 with primitive erythroid, megakaryocyte and macrophage potentials. The resulting primitive erythroid cells mature within the circulation and support embryonic growth past E9.5. At E8.5, a second wave of hematopoiesis begins in the yolk sac and generates definitive erythroid and multiple myeloid progenitors that are the proposed source of the hematopoietic progenitors seeding the fetal liver before HSC colonization. We have identified a cell population displaying a unique cell surface immunophenotype in the E9.5 yolk sac that contains the potential to form definitive erythroid cells, megakaryocytes, macrophages and all forms of granulocytes within days of in vitro culture. Furthermore, all definitive hematopoietic colony-forming cells (BFU-E, CFC-myeloid and HPP-CFC) in the E9.5 yolk sac have this immunophenotype. These erythro-myeloid progenitors (EMP) are lineage-negative and co-express ckit, CD41, CD16/32 and Endoglin. Interestingly, this is not an immunophenotype evident in the adult bone marrow. Other markers that have been associated with HSC formation (AA4.1, ScaI) or with lymphoid potential (IL7R, Flt3) are not present on these cells at E9.5. Consistent with the lack of lymphoid markers, we also do not observe short-term development of B-cells (CD19+B220+ expressing Rag2 RNA) in cultures of the E9.5 sorted EMP, while bone marrow Lin-/ckit+/ScaI- cells do form B-cells under the same conditions. Clonal analysis of sorted EMP cells revealed single cells with both erythroid and granulocyte potential, similar to the common myeloid progenitors in adult bone marrow. Though these EMP are enriched at E9.5 in the yolk sac, they are also found at low levels in the fetal blood, embryo proper and placenta, consistent with their entrance into the circulation. By E10.5, EMP were most highly enriched in the newly formed fetal liver. Additionally by E12.5, a time when the first few HSCs are detected in the fetal liver, we find active erythropoiesis and granulopoiesis in the liver and the first definitive red blood cells and neutrophils in the bloodstream. Therefore, we believe the yolk sac definitive progenitors' fate is to populate the fetal liver and thus provide the first definitive erythrocytes and granulocytes for the embryo. The differentiation of embryonic stem cells (ES) and induced pluripotent stem cells (iPS) cells into mature cells types offers the hope of cell-based therapies. Analysis of differentiating murine ES cells reveals overlapping waves of primitive and definitive hematopoietic colony forming potential. We demonstrate the appearance of an EMP-like (ckit+/CD41+/FcGR+) population coincident with the emergence of definitive hematopoietic progenitors during murine ES cell differentiation as embryoid bodies. We have confirmed with colony forming assays that definitive hematopoietic potential is associated with this immunophenotypic group. Our studies support the concept that blood cell emergence during ES cell differentiation closely mimics pre-HSC hematopoiesis in the yolk sac. Disclosures:No relevant conflicts of interest to declare.

Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf

AbstractThe primitive erythroid (EryP) lineage is the first to differentiate during mammalian embryogenesis. Eklf/Klf1 is a transcriptional regulator that is essential for definitive erythropoiesis in the fetal liver. Dissection of the role(s) of Eklf within the EryP compartment has been confounded by the simultaneous presence of EryP and fetal liver–derived definitive erythroid (EryD) cells in the blood. To address this problem, we have distinguished EryP from their definitive counterparts by crossing Eklf+/− mutant and ϵ-globin::histone H2B-GFP transgenic mice. Eklf-deficient EryP exhibit membrane ruffling and a failure to acquire the typical discoidal erythroid shape but they can enucleate. Flow cytometric analyses of H2B-GFP+ EryP revealed that Eklf heterozygosity results in the loss of Ter119 surface expression on EryP but not on EryD. Null mutation of Eklf resulted in abnormal expression of a range of surface proteins by EryP. In particular, several megakaryocyte markers were ectopically expressed by maturing Eklf-null EryP. Unexpectedly, the platelet tetraspanin CD9 was detected on nucleated wild-type EryP but not on mature EryD and thus provides a useful marker for purifying circulating EryP. We conclude that Eklf gene dosage is crucial for regulating the surface phenotype and molecular identity of maturing primitive erythroid cells.

CXCR7 Protein Is Not Expressed on Human or Mouse Leukocytes

Since the discovery that CXCR7 binds to CXCL12/SDF-1α, the role of CXCR7 in CXCL12-mediated biological processes has been under intensive scrutiny. However, there is no consensus in the literature on the expression of CXCR7 protein by peripheral blood cells. In this study we analyzed human and mouse leukocytes and erythrocytes for CXCR7 protein expression, using a competitive CXCL12 binding assay as well as by flow cytometry and immunohistochemistry using multiple CXCR7 Abs. CXCR7(-/-) mice were used as negative controls. Together, these methods indicate that CXCR7 protein is not expressed by human peripheral blood T cells, B cells, NK cells, or monocytes, or by mouse peripheral blood leukocytes. CXCR7 protein is, however, expressed on mouse primitive erythroid cells, which supply oxygen to the embryo during early stages of development. These studies therefore suggest that, whereas CXCR7 protein is expressed by primitive RBCs during murine embryonic development, in adult mammals CXCR7 protein is not expressed by normal peripheral blood cells.

The first trimester human placenta is a site for terminal maturation of primitive erythroid cells

AbstractEmbryonic hematopoiesis starts via the generation of primitive red blood cells (RBCs) that satisfy the embryo's immediate oxygen needs. Although primitive RBCs were thought to retain their nuclei, recent studies have shown that primitive RBCs in mice enucleate in the fetal liver. It has been unknown whether human primitive RBCs enucleate, and what hematopoietic site might support this process. Our data indicate that the terminal maturation and enucleation of human primitive RBCs occurs in first trimester placental villi. Extravascular ζ-globin+ primitive erythroid cells were found in placental villi between 5-7 weeks of development, at which time the frequency of enucleated RBCs was higher in the villous stroma than in circulation. RBC enucleation was further evidenced by the presence of primitive reticulocytes and pyrenocytes (ejected RBC nuclei) in the placenta. Extravascular RBCs were found to associate with placental macrophages, which contained ingested nuclei. Clonogenic macrophage progenitors of fetal origin were present in the chorionic plate of the placenta before the onset of fetoplacental circulation, after which macrophages had migrated to the villi. These findings indicate that placental macrophages may assist the enucleation process of primitive RBCs in placental villi, implying an unexpectedly broad role for the placenta in embryonic hematopoiesis.

Two different pathways for the transport of primitive and definitive blood cells from the yolk sac to the embryo in humans

During the early human embryonic period nutrients and blood cells are temporarily provided by the extraembryonic yolk sac (YS). The YS before week six is involved not only in primitive but also in definitive erythropoiesis. While the destiny of primitive erythroid cells that fill the blood vessels of the YS is well known, the final destination of erythrocytes present in the endodermal vesicular system is unknown. In the present study we have investigated, step by step, the destiny of the erythrocytes present in the endodermal vesicles during the embryonic period. Twelve human YSs and their corresponding yolk stalks were analyzed between weeks 4 and 7 of embryonic age by light and scanning electron microscopy. It is shown that erythrocytes (according to their size and morphological features) located within the endodermal vesicles of the YS wall are pulled out through endodermal pits into the YS cavity, from where they reach the lumen of the primitive gut of the embryo through the vitelline duct, a temporary pathway communicating both compartments. During the study period no erythrocytes were seen within the embryo's vascular network where only primitive erythroblasts were identified. Our results indicate that the vitelline duct plays an important transient role as a pathway for the transport of nutrients and blood cells between the YS and the embryo before week five of embryonic development that ends just at the time when YS-embryo circulation becomes established.

DNase I Hypersensitivity and ϵ-Globin Transcriptional Enhancement Are Separable in Locus Control Region (LCR) HS1 Mutant Human β-Globin YAC Transgenic Mice

Expression of the five β-like globin genes (ϵ, Gγ, Aγ, δ, β) in the human β-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5′HS1 through 5′HS5). We report here a novel enhancer activity in 5′HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5′HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human β-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, ϵ-, but not γ-globin, gene expression in primitive erythroid cells was severely attenuated, while adult β-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5′HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5′HS1 is critical for efficient ϵ-globin gene expression, hypersensitive site formation per se is independent of 5′HS1 function, if any, in definitive erythroid cells.

α-Thalassemia-Like Globin Gene Expression by Primitive Erythrocytes Derived from Human Embryonic Stem Cells

Under culture conditions that promote hematopoietic differentiation, human embryonic stem cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of early-stage human embryos. The globin chain distribution of these cells is similar to that seen during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of γ-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly unbalanced, with a substantial decrease of α-like globin chain synthesis in relation to that of their β-like globins, a pattern characteristically associated with α-thalassemia (α-thal). This pattern of unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize predominantly embryonic-stage globins.

Developmental niches for embryonic erythroid cells

Primitive erythroid cells (EryP) are the first differentiated cell type to be specified during mammalian embryogenesis. EryP arise from a pool of lineage-restricted progenitors in the yolk sac (YS) and then enter the newly formed embryonic circulation to mature in a stepwise, synchronous fashion. Numbering in the millions in the mid-gestation mouse embryo, EryP are the dominant circulating blood cell prior to the rapid generation of adult-type definitive erythroid (EryD) cells in the fetal liver. The identification of maturational events in this lineage presented a significant challenge, as EryD begin to outnumber EryP in the bloodstream from ∼ E14.5 onwards. We used human epsilon-globin gene regulatory elements to drive lineage-specific expression of a histone-H2B::EGFP fusion protein, allowing us to label the chromatin of EryP during their development and to track and quantify EryP nuclei following their expulsion from the cell. Using this transgenic fluorescent reporter mouse line, we have monitored primitive erythropoiesis in three distinct niches: the YS, where EryP progenitors arise; the circulation, where EryP continue to divide and mature; and the fetal liver, where EryP complete the terminal stages of their differentiation.