Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

AbstractWe elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.

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Hematopoietic Differentiation of Human Embryonic Stem Cells in Vitro : Comparison of Three Cell Lines

Blood ◽

10.1182/blood.v112.11.4787.4787 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4787-4787

Author(s):

Marion Brenot ◽

Annelise Bennaceur-Griscelli ◽

Marc Peschanski ◽

Maria Teresa Mitjavila-Garcia

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Lines ◽

Human Embryonic Stem Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem

Abstract Human embryonic stem cells (hES) isolated from the inner cell mass of a blastocyst have the ability to self renew indefinitely while maintaining their pluripotency to differentiate into multiple cell lineages. Therefore, hES represent an important source of cells for perspective cell therapies and serve as an essential tool for fundamental research, specifically for understanding pathophysiological mechanisms of human diseases for the development of novel pharmacological drugs. The generation of hematopoietic stem cells from hES may serve as an alternative source of cells for hematopoietic reconstitution following bone marrow transplantation and an interesting approach to understand early stages of hematopoietic development which are difficult to study in human embryos. Using two different methods, we have differentiated three hES cell lines (SA01, H1 and H9) into hematopoietic cells by generating embryoid bodies and co-culturing on the murine Op9 cell line. In both experimental approaches, we obtain cells expressing CD34 and when cultured in hematopoietic conditions, SA01 and H1 cell lines differentiate into various hematopoietic lineages as demonstrated by BFU-E, CFU-GM and CFU-GEMM colony formation, whereas H9 have almost exclusively granulo-macrophage differentiation. Cells composing these hematopoietic colonies express CD45, CD11b, CD31, CD41 and CD235 and staining with May Grundwald-Giemsa demonstrate neutrophil and erythrocyte morphology. These results demonstrate the capacity of hES to differentiate into mature hematopoietic cells in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the hES cell lines used. However, we still have to evaluate their capacity to reconstitute hematopoiesis in vivo in an immune deficient mouse model. We will also be interested in developing in vitro methods to expand these hematopoietic precursor cells derived from hES which may be used as a viable source for future cell therapy.

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Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02019-5 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Selami Demirci ◽

Juan J. Haro-Mora ◽

Alexis Leonard ◽

Claire Drysdale ◽

Daniela Malide ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Close Association ◽

Embryonic Stem ◽

Precursor Cells ◽

Embryoid Bodies ◽

Confocal Imaging ◽

Hematopoietic Stem ◽

Tcr Repertoire

Abstract Background Ex vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders. Methods To derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions. Results Seven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a−, 69%, p < 0.01) and lowest number of primitive (CD34− CD235a+, 1.55%, p < 0.01) precursor cells along with the highest colony-forming units (149.8 ± 11.6, p < 0.01) in feeder-free conditions. Maximal HSPC fraction (CD34+ CD38− CD45RA− CD49f+ CD90+) was 7.6–8.9% after 10 days of hematopoietic differentiation with 14.5% adult β-globin expression following RBC differentiation. Myeloid and erythroid colonies were restricted strictly to the CD34+ CD43+ fraction (370.5 ± 65.7, p < 0.001), while the CD34− CD43+ fraction produced only a small number of colonies (21.6 ± 11.9). In addition, we differentiated the CD34+ CD43+ cells towards T-lymphocytes using the OP9/DLL1 co-culture system demonstrating double-positive T cells (CD4+ CD8+) with CD3+ expression displaying a broad T cell receptor (TCR) repertoire. Confocal imaging of organoid-like structures revealed a close association of CD31+ cells with CD34+ and CD43+ cells, suggesting a potential emergence of HSPCs through endothelial to hematopoietic transition. Furthermore, fluorescently labeled organoids exhibited the emergence of spherical non-attached cells from rare progenitors at the border of the organoid center. Conclusions In summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire.

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RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells

Blood ◽

10.1182/blood-2012-08-451641 ◽

2013 ◽

Vol 121 (15) ◽

pp. 2882-2890 ◽

Cited By ~ 81

Author(s):

Dan Ran ◽

Wei-Jong Shia ◽

Miao-Chia Lo ◽

Jun-Bao Fan ◽

David A. Knorr ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Pluripotent Stem Cells ◽

Ex Vivo ◽

Ectopic Expression ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Lineage Commitment ◽

Hematopoietic Differentiation

Abstract Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo–expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

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MEIS2 regulates endothelial to hematopoietic transition of human embryonic stem cells by targeting TAL1

Stem Cell Research & Therapy ◽

10.1186/s13287-018-1074-z ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 10

Author(s):

Mengge Wang ◽

Hongtao Wang ◽

Yuqi Wen ◽

Xiaoyuan Chen ◽

Xin Liu ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Blood Cells ◽

Integration Site ◽

Embryonic Stem ◽

Gene Profiling ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Megakaryocytic Differentiation

Abstract Background Despite considerable progress in the development of methods for hematopoietic differentiation, efficient generation of transplantable hematopoietic stem cells (HSCs) and other genuine functional blood cells from human embryonic stem cells (hESCs) is still unsuccessful. Therefore, a better understanding of the molecular mechanism underlying hematopoietic differentiation of hESCs is highly demanded. Methods In this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. We deleted MEIS2 gene in hESCs using the CRISPR/CAS9 technology and induced them to hematopoietic differentiation, megakaryocytic differentiation. Results In this study, we found that MEIS2 deletion impairs early hematopoietic differentiation from hESCs. Furthermore, MEIS2 deletion suppresses hemogenic endothelial specification and endothelial to hematopoietic transition (EHT), leading to the impairment of hematopoietic differentiation. Mechanistically, TAL1 acts as a downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 deletion exerts minimal effects on megakaryocytic differentiation and platelet generation from hESCs. Conclusions Our findings advance the understanding of human hematopoietic development and may provide new insights for large-scale generation of functional blood cells for clinical applications.

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

Blood ◽

10.1182/blood-2007-07-102087 ◽

2008 ◽

Vol 111 (4) ◽

pp. 2400-2408 ◽

Cited By ~ 103

Author(s):

Caihong Qiu ◽

Emmanuel N. Olivier ◽

Michelle Velho ◽

Eric E. Bouhassira

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Fetal Liver ◽

Embryonic Stem ◽

Fetal Hemoglobin ◽

Yolk Sac ◽

Globin Mrna ◽

Fetal Hepatocytes ◽

Cd34 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 (ζ2ϵ2) and the orthochromatic erythroblasts hemoglobin Gower II (α2ϵ2). 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 β-globin mRNA.

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Angiotensin-Converting Enzyme (ACE) Expression Defines the Earliest Primitive and Definitive Lympho-Hematopoietic Progenitors Derived from Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v108.11.1662.1662 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1662-1662

Author(s):

Elias T. Zambidis ◽

Venta J. Jokubaitis ◽

Ada Tam ◽

Lidia Sinka ◽

Curt I. Civin ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

In Vitro Assays ◽

Hematopoietic Differentiation ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

Abstract Pluripotent human embryonic stem cells (hESC) provide unprecedented opportunities for studying obscure human developmental events, such as those required for the genesis of hematopoietic stem cells (HSC). A poorly characterized aspect of embryonic and adult HSC development is the role of the renin-angiotensin system (RAS), which is implicated in regulating HSC proliferation at yolk sac (YS), fetal, and adult stages. We have recently described somatic ACE surface expression not only in adult HSC, but also at the earliest stages of emergent hemato-endotheliogenesis using a novel monoclonal antibody (BB9). ACE expression identifies primitive subsets of adult CD34+ bone marrow HSC, but more intriguingly, marks emergent hematopoietic cells from both CD34− and CD34+ areas of human YS, intraembryonic subaortic patches, and hemogenic endothelial layers of the aorta-gonad-mesonephros (AGM) region. The pattern of human embryonic ACE expression is consistent with the hypothesis of ACE+CD34− hemangioblasts emigrating dorsally from the para-aortic splanchnopleura, and subsequently colonizing the ventral aorta to give rise to CD34+ hemogenic endothelium. We tested the hypothesis that ACE expression would similarly identify emerging hemato-endothelial progenitors derived from our recently-described hESC-based hematopoietic differentiation system. This embryoid body (hEB)-based system recapitulates hemato-endothelial, primitive, and definitive stages of human embryonic blood development. ACE expression kinetics during hEB differentiation correlated well with the onset of hemato-endothelial differentiation and gene expression (e.g., SCL/tal1, CDX4, CD31, and CD34). Furthermore, using improved, novel methods of hEB hematopoietic differentiation that dramatically augment multilineage progenitors for both primitive and definitive hematopoiesis, we observed that levels of hEB ACE expression were directly correlated to increased hematopoietic potency. Subsequent FACS purification of these hEB cells demonstrated that the earliest detectable multilineage lympho-hematopoietic competency was contained entirely within both ACE+CD34−CD45− hEB and ACE+CD34+CD45− populations. These early ACE+CD45− hEB populations were heterogenous, and co-expressed abundant levels of known hemato-endothelial markers such as CD31, KDR, CD164, CD43, and CD71. Using novel in vitro assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ hEB contained common progenitors for both primitive and definitive hematopoiesis, with ACE+CD34−CD45− hEB being enriched for the highest number of progenitors. We were also able to demonstrate that further maturation of these ACE+ hEB cells in an in vitro AGM-like stromal environment produced definitive hematopoietic progenitors that resembled those obtained from cord blood CD34+ cells. The regulatory effects of angiotensin agonist/antagonist peptides on hEB-derived hematopoietic ACE+ progenitors, and their in vivo correlation to ACE+ cells obtained from early human YS and AGM tissue is currently in progress. Furthermore, single-cell analysis is underway to delineate an ACE+ hEB-derived hemangioblastic precursor of not only endothelium, but also primitive and definitive lympho-hematopoiesis.

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