alpha4-Integrin(+) endothelium derived from primate embryonic stem cells generates primitive and definitive hematopoietic cells

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. In this study, we demonstrated that VE-cadherin(+)CD45(-) endothelial cells derived from nonhuman primate embryonic stem cells are able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. Single bipotential progenitors for hematopoietic and endothelial lineages are included in this endothelial cell population. Furthermore, hemogenic activity of these endothelial cells is observed exclusively in the alpha4-integrin(+) subpopulation; bipotential progenitors are 4-fold enriched in this subpopulation. The kinetics of this hemogenic subpopulation is similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo in that they emerge for only a limited time. We suggest that VE-cadherin(+)CD45(-)alpha4-integrin(+) endothelial cells are involved in primitive and definitive hematopoiesis during primate embryogenesis, though VE-cadherin(-)CD45(-)alpha4-integrin(+) cells are the primary sources for primitive hematopoiesis.     

Development of hematopoietic cells from embryonic stem cells

Embryonic stem cells are pluripotent stem cells that can differentiate into all somatic cell lineages and germ lineage cells in vivo. In vitro differentiation capacity of the cells is rather limited compared with the in vivo pluripotency. However, differentiation into hematopoietic lineages is easily obtained, and it is a powerful tool to investigate hematopoietic development and differentiation. In this article, we describe a differentiation induction method that we established, the OP9 system, a unique method using the macrophage colony-stimulating factor-deficient stromal cell line OP9. The utility of the OP9 system includes hematopoietic development, differentiation, B-cell formation, osteoclast formation, and so on. The usefulness and limits of embryonic stem cell-derived hematopoietic cells in cell therapy are also discussed.     

Ontogenic emergence of definitive hematopoietic stem cells

Research in the past 10 years has dramatically increased our knowledge of the development of the mammalian hematopoietic system and has provided insight into the embryonic sites of hematopoietic cell generation, the variety of hematopoietic cell types produced, and some of the microenvironmental influences on the rapidly growing blood system. Indeed, within mammalian embryos, it is now widely accepted that the embryo proper produces the first adult repopulating hematopoietic stem cells. This mesodermally derived intraembryonic region, known as the aorta-gonad-mesonephros region or, at a slightly earlier developmental stage, the paraaortic splanchnopleura, produces, respectively, potent hematopoietic stem cells and multipotent progenitor cells before their appearance in the yolk sac. This review focuses on the most recent findings concerning qualitative and quantitative aspects of hematopoietic stem-cell development, the endothelium as a possible direct precursor population of hematopoietic stem cells, and the microenvironment leading to the onset and maintenance of hematopoietic stem cells in the mammalian embryo.     

The differentiation program of embryonic definitive hematopoietic stem cells is largely alpha4 integrin independent

Previous analyses of the roles of alpha4 integrins in hematopoiesis by other groups have led to conflicting evidence. alpha4 integrin mutant cells developing in [alpha4 integrin(-/-): wt] chimeric mice are not capable of completing lymphomyeloid differentiation, whereas conditional inactivation of alpha4 integrin in adult mice has only subtle effects. We show here that circumventing the fetal stage of hematopoietic stem cell (HSC) development by transplantation of embryonic alpha4 integrin(-/-) cells into the adult microenvironment results in robust and stable long-term generation of alpha4 integrin(-/-) lymphoid and myeloid cells, although colonization of Peyer patches and the peritoneal cavity is significantly impaired. We argue here that collectively, our data and the data from other groups suggest a specific requirement for alpha4 integrin during the fetal/neonatal stages of HSC development that is essential for normal execution of the lymphomyeloid differentiation program.     

Designer blood: creating hematopoietic lineages from embryonic stem cells

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.     

Comparative gene expression in hematopoietic progenitor cells derived from embryonic stem cells

OBJECTIVE: The aim of this study was to characterize at the molecular level the hematopoietic progenitor cells derived from rhesus monkey embryonic stem (ES) cell differentiation. MATERIALS AND METHODS: We purified CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cell cultures and examined the expression of a variety of genes associated with hematopoietic development, by semiquantitative polymerase chain reaction analysis. For comparison, we examined cell preparations from fresh or cultured rhesus monkey bone marrow (BM) and from mouse ES cells and BM. RESULTS: We observed a high degree of similarity in the expression patterns of these genes, with only a few exceptions. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES cell-derived CD34(+) and CD34(+)CD38(-) cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34(+) cells from cultured BM. The integrin alphaL and interleukin-6 (IL-6) receptor genes also were expressed in CD34(+)CD38(-) cells from BM, but there was little or no expression of these genes in CD34(+)CD38(-) cells derived from ES cells. Parallel analyses, using CD34(+)Lin(-) cells derived from murine ES cell cultures, showed no apparent expression of flt3, integrin alphaL, or IL-6 receptor, whereas corresponding cell preparations isolated from mouse BM expressed high levels of all of these genes. CONCLUSIONS: ES cell-derived hematopoietic progenitors, both from the rhesus monkey and from the mouse, exhibited the same alterations in gene expression compared with BM-derived cells from these animals. These observations could reflect the presence of different subpopulations in the cell fractions that were compared, or they may represent altered biologic properties of ES cell-derived hematopoietic stem cells.     

The emergence of definitive hematopoietic stem cells in the mammal

PURPOSE OF REVIEW: Hematopoietic stem cells (HSC) are the basis for blood formation during adult life. The amazing potency of HSCs has been exploited for over 30 years in regenerative therapies for patients with blood-related genetic disease and leukemia. As clinically important cells and also as the most widely studied cell differentiation system, they have been the focus of intense fundamental research. Indeed, HSC research has established many paradigms in the more general field of stem cells. Recently, the study of the embryonic origins of HSCs and their genetic program is beginning to provide unique insights into how these stem cells are formed, maintained, and expanded, and how they contribute to the complex adult hematopoietic system. Although many short-lived hematopoietic progenitors are present in early stage mammalian embryos, this review will focus on the events leading to emergence of the most potent cells of the hematopoietic system, HSCs and on their developmental lineage relationships. RECENT FINDINGS: Developmental and genetic studies further our understanding of the fate determination events occurring in several embryonic tissues leading to the generation of potent HSCs--those cells with the ability to long-term, high-level repopulate all hematopoietic lineages of the adult. SUMMARY: Several mammalian embryonic tissues contribute to the growth and/or generation of potent HSCs that are the source of blood cells throughout the lifespan of the individual. Insight into how mammalian HSC fate is determined has been provided through functional, phenotypic, and genetic studies at early developmental stages.     

Towards hematopoietic reconstitution from embryonic stem cells: a sanguine future

PURPOSE OF REVIEW: To review recent progress towards the derivation of hematopoietic stem cells (HSCs) and blood lineages from embryonic stem cells (ESCs), and to highlight the hurdles that must be overcome in order to move the field closer to a clinical application. RECENT FINDINGS: Hematopoietic repopulating cells, red blood cells, and T cells have recently been derived from both murine and human ESCs. Although these results are encouraging, several outstanding issues remain to be addressed by the field before realizing clinical applicability: the phenotype of the ESC-derived HSC must be characterized, methods to purge residual teratoma-forming cells from differentiated populations must be established, and in-vivo models of human HSC function must be optimized to better assess the functionality of putative human ESC-derived HSCs. In addition, embryonic stem-cell derived progeny often represent primitive embryonic hematopoietic cells, rather than their definitive adult counterparts; this critical issue must also be addressed. SUMMARY: The literature firmly establishes that it is possible to isolate HSCs and certain mature blood lineages from both mouse and human ESCs. Although several issues remain to be addressed, these data demonstrate the value of ESCs as a potential source of transplantable HSCs.     

In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.     

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta–gonad–mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, β-NGF (a neurotrophic factor), MIP-1γ (a C–C chemokine family member) and Bmp4 (a TGF-β family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.     

Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development

The development of vertebrate definitive hematopoiesis is featured by temporally and spatially dynamic distribution of hematopoietic stem/progenitor cells (HSPCs). It is proposed that the migration of definitive HSPCs, at least in part, accounts for this unique characteristic; however, compelling in vivo lineage evidence is still lacking. Here we present an in vivo analysis to delineate the migration route of definitive HSPCs in the early zebrafish embryo. Cell-marking analysis was able to first map definitive HSPCs to the ventral wall of dorsal aorta (DA). These cells were subsequently found to migrate to a previously unappreciated organ, posterior blood island (PBI), located between the caudal artery and caudal vein, and finally populate the kidney, the adult hematopoietic organ. These findings demonstrate that the PBI acts as an intermediate hematopoietic organ in a manner analogous to the mammalian fetal liver to sustain definitive hematopoiesis before adult kidney hematopoiesis occurs. Thus our study unambiguously documents the in vivo trafficking of definitive HSPCs among developmentally successive hematopoietic compartments and underscores the ontogenic conservation of definitive hematopoiesis between zebrafish and mammals.     

Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis

A critical issue for clinical utilization of human ES cells (hESCs) is whether they can generate terminally mature progenies with normal function. We recently developed a method for efficient production of hematopoietic progenitors from hESCs by coculture with murine fetal liver-derived stromal cells. Large numbers of hESCs-derived erythroid progenitors generated by the coculture enabled us to analyze the development of erythropoiesis at a clone level and investigate their function. The results showed that the globin expression in the erythroid cells in individual clones changed in a time-dependent manner. In particular, embryonic ε-globin-expressing erythroid cells from individual clones decreased, whereas adult-type β-globin-expressing cells increased to ≈100% in all clones we examined, indicating that the cells undergo definitive hematopoiesis. Enucleated erythrocytes also appeared among the clonal progeny. A comparison analysis showed that hESC-derived erythroid cells took a similar differentiation pathway to human cord blood CD34⁺ progenitor-derived cells when examined for the expression of glycophorin A, CD71 and CD81. Furthermore, these hESC-derived erythroid cells could function as oxygen carriers and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study should provide an experimental model for exploring early development of human erythropoiesis and hemoglobin switching and may help in the discovery of drugs for hereditary diseases in erythrocyte development.     

Characterization of a novel hematopoietic marker expressed from early embryonic hematopoietic stem cells to adult mature lineages

A novel membrane protein has been identified in the course of screening for differentially expressed cDNAs in human embryonic hematopoietic sites. This 37- to 38-kDa molecule, designated KLIP-1 (killer lineage protein), consisting of 350 amino acids and containing five transmembrane domains, is encoded by the 5093-bp KLIP-1 gene, composed of nine exons and located on chromosome 6 (6p21.1-6p21.2). We found the KLIP-1 protein to be expressed by nucleated hematopoietic cells, from early embryonic hematopoietic stem cells through mature adult blood lymphoid lineages, either as membrane or as cytoplasmic molecules. In day-30/32 human embryo sections, KLIP-1 protein expression is restricted to circulating hematopoietic cells at hematopoiesis sites. Membrane KLIP-1 is expressed by fetal and adult GP-A(+) erythroblasts, the fetal liver CD34(+) subset, fetal spleen, and adult bone marrow CD56(+) NK and CD19(+) B cells. Among mature blood cells, surface KLIP-1 expression is restricted to CD56(+) NK cells, indicating KLIP-1 to be a novel marker of this population. Altogether, these results indicate that membrane export of KLIP-1 antigen is developmentally and ontogenetically regulated. The high degree of conservation of the KLIP-1 protein sequence among mammals strongly suggests that it plays an important role during hematopoiesis and may exercise similar functions in human and mouse blood cells. The KLIP-1 molecule may therefore constitute a powerful tool for improving knowledge of both human hematopoiesis and NK cell ontogeny and immune functions.     

Endothelial and hematopoietic cell fate of human embryonic stem cells

The endothelial cells, lining the inside of blood vessels, and the blood-forming hematopoietic cells play crucial roles in vasculogenesis. The establishment of human embryonic stem cells (hESCs) provides a unique tool to study the early development of endothelial and hematopoietic cells, opening new avenues of research to explore organ vascularization and regeneration. The current study demonstrates that a population of intermediate-stage precursors, which possesses primitive endothelial properties during hESC differentiation, is capable of giving rise to endothelial and hematopoietic cells. Single cell analysis reveals that these primitive endothelial-like precursors contain rare bipotent cells with hemangioblast properties, responsible for both endothelial and hematopoietic cell fates. These findings will facilitate the further study of cellular commitment, lineage restriction, and terminal differentiation of endothelial and hematopoietic compartments and may lead to novel regenerative therapies.     

Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.     

Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5

The signal transducer Stat5 plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell function. To evaluate the effects of Stat5 signaling in the earliest hematopoietic progenitors, we have generated an embryonic stem cell line in which Stat5 signaling can be induced with doxycycline. Ectopic Stat5 activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells, suggesting a cell-intrinsic effect of Stat5 on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies, under the influence of Stat5 signaling, a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing, mainly myeloid, differentiation. When these cells are injected into lethally irradiated mice, they engraft transiently in a doxycycline-dependent manner. These results demonstrate that the hematopoietic commitment of embryonic stem cells may be augmented by a Stat5-mediated signal, and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells.     

Generation of hematopoietic colony-forming cells from embryonic stem cells: synergy between a soluble factor from NIH-3T3 cells and hematopoietic growth factors

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia- inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.