Hematopoietic Stem Cells Emerge in the Placental Vasculature in the Absence of Circulation.

Abstract The placenta was recently unveiled as an important hematopoietic organ, harboring a large pool of HSCs during midgestation. Yet, it has not been defined whether the placenta can generate HSCs de novo. By using the Runx1-LacZ and Ncx1 knockout mouse models we show that the placenta is a site of HSC generation and identify the cellular niches in which placental HSCs reside. Runx1 is essential for the emergence of definitive HSCs and remains expressed in HSCs throughout fetal development and adult life. Analysis of Runx1LacZ/+ and Runx1LacZ/LacZ placental sections nominated the large vessels of the placenta and the chorioallantoic mesenchyme as putative sites of HSC origin. Once formed, LacZ+ candidate HSCs convened in the labyrinth vessels. Co-staining of Runx1LacZ/+ placentas with an antibody specific for phosphorylated Ser 10 at histone 3, a marker of mitosis, showed mitotically active definitive hematopoietic cells in the labyrinth vessels, suggesting that the labyrinth is a microenvironmental niche capable of stimulating HSC expansion. In wild-type placentas, CD41+ nascent hematopoietic cells were found in the same vascular sites as in the Runx1-LacZ placentas but never in the mesenchyme. Instead, placental stroma was populated by F4/80+CD45+/−CD41- macrophages, suggesting that the placenta harbors two distinct hematopoietic lineages that are supported by different microenvironments. To verify that the CD41+ nascent HSCs were generated de novo in the placenta, we analyzed Ncx1−/− embryos, which lack heartbeat due to lack of the sodium-calcium exchange pump 1. In the absence of circulation, trafficking of hematopoietic cells between tissues is abolished. Strikingly, CD41+ HSCs emerge in the large vessels of the placenta in Ncx1−/− mutants. In some sections CD41+ cells formed clusters that were still connected to the vessels of the placenta and umbilical cord. These findings imply that formation of HSCs extends to a much larger anatomical area than was previously thought, including the placenta. Importantly, the placentas in both Ncx1−/− and control embryos (E8.5–9.5) generated mixed hematopoietic outgrowth including definitive progenitors in OP-9 co-culture, as verified by expression of c-kit, CD41 and CD45. When the differentiation of the definitive progenitors was assessed on methylcellulose, Ncx1−/− tissues demonstrated similar potential as Ncx1+/− hematopoietic organs (yolk sac, aorta gonad mesonephros (AGM) and placenta), yielding erythroid, myeloid and mixed colonies and B220+ lymphoid cells. These studies reveal that definitive hematopoietic cells with both myeloerythroid and lymphoid potential are generated de novo within the placental vasculature. Furthermore, the placental labyrinth provides a unique hematopoietic niche that is conducive for proliferation of hematopoietic cells and, unlike the AGM or the yolk sac, serves as a supportive niche for a large pool of HSCs prior to liver colonization.

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Placenta Is a Niche for Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.2671.2671 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2671-2671

Author(s):

Hanna K.A. Mikkola ◽

Christos Gekas ◽

Francoise Dieterlen-Lievre ◽

Stuart H. Orkin

Keyword(s):

De Novo ◽

Fetal Liver ◽

Yolk Sac ◽

Mouse Development ◽

Adult Type ◽

Cell Potential ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Hematopoietic System ◽

Hematopoietic Organs

Abstract 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 (Development2003, 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.

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Blood-forming potential of vascular endothelium in the human embryo

Development ◽

10.1242/dev.129.17.4147 ◽

2002 ◽

Vol 129 (17) ◽

pp. 4147-4157 ◽

Cited By ~ 4

Author(s):

Estelle Oberlin ◽

Manuela Tavian ◽

Istvàn Blazsek ◽

Bruno Péault

Keyword(s):

Endothelial Cells ◽

Human Embryo ◽

Hematopoietic Cells ◽

Surface Expression ◽

Yolk Sac ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Vascular Walls ◽

Developmental Age ◽

Human Ontogeny

Hematopoietic cells arise first in the third week of human ontogeny inside yolk sac developing blood vessels, then, one week later and independently, from the wall of the embryonic aorta and vitelline artery. To address the suggested derivation of emerging hematopoietic stem cells from the vessel endothelium, endothelial cells have been sorted by flow cytometry from the yolk sac and aorta and cultured in the presence of stromal cells that support human multilineage hematopoiesis. Embryonic endothelial cells were most accurately selected on CD34 or CD31 surface expression and absence of CD45, which guaranteed the absence of contaminating hematopoietic cells. Yet, rigorously selected endothelial cells yielded a progeny of myelo-lymphoid cells in culture. The frequency of hemogenic endothelial cells in the yolk sac and aorta reflected the actual blood-forming activity of these tissues, as a function of developmental age. Even less expected, a subset of endothelial cells sorted similarly from the embryonic liver and fetal bone marrow also exhibited blood-forming potential. These results suggest that a part at least of emerging hematopoietic cells in the human embryo and fetus originate in vascular walls.

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Sphingolipids in Hematopoiesis: Exploring Their Role in Lineage Commitment

Cells ◽

10.3390/cells10102507 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2507

Author(s):

Yasharah Raza ◽

Huda Salman ◽

Chiara Luberto

Keyword(s):

Stem Cells ◽

De Novo ◽

Large Body ◽

Hematopoietic Cells ◽

Lymphoid Cells ◽

Lineage Commitment ◽

Sphingolipid Metabolism ◽

Hematopoietic Stem ◽

Cell Functions ◽

Range Of Functions

Sphingolipids, associated enzymes, and the sphingolipid pathway are implicated in complex, multifaceted roles impacting several cell functions, such as cellular homeostasis, apoptosis, cell differentiation, and more through intrinsic and autocrine/paracrine mechanisms. Given this broad range of functions, it comes as no surprise that a large body of evidence points to important functions of sphingolipids in hematopoiesis. As the understanding of the processes that regulate hematopoiesis and of the specific characteristics that define each type of hematopoietic cells is being continuously refined, the understanding of the roles of sphingolipid metabolism in hematopoietic lineage commitment is also evolving. Recent findings indicate that sphingolipid alterations can modulate lineage commitment from stem cells all the way to megakaryocytic, erythroid, myeloid, and lymphoid cells. For instance, recent evidence points to the ability of de novo sphingolipids to regulate the stemness of hematopoietic stem cells while a substantial body of literature implicates various sphingolipids in specialized terminal differentiation, such as thrombopoiesis. This review provides a comprehensive discussion focused on the mechanisms that link sphingolipids to the commitment of hematopoietic cells to the different lineages, also highlighting yet to be resolved questions.

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Modeling human yolk sac hematopoiesis with pluripotent stem cells

Journal of Experimental Medicine ◽

10.1084/jem.20211924 ◽

2021 ◽

Vol 219 (3) ◽

Author(s):

Michael H. Atkins ◽

Rebecca Scarfò ◽

Kathleen E. McGrath ◽

Donghe Yang ◽

James Palis ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Immune Cell ◽

Adult Life ◽

Stem Cell Differentiation ◽

Yolk Sac ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Myeloid Progenitor

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac–derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

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DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽

10.1182/blood.v120.21.2382.2382 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2382-2382

Author(s):

Jie Xu ◽

Wei-na Zhang ◽

Tao Zhen ◽

Yang Li ◽

Jing-yi Shi ◽

...

Keyword(s):

Transcriptional Activation ◽

Dna Methyltransferase ◽

De Novo ◽

Conflicts Of Interest ◽

Epigenetic Modification ◽

Hematopoietic Cells ◽

In Vitro Assays ◽

Clinical Samples ◽

Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

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Circulation is established in a stepwise pattern in the mammalian embryo

Blood ◽

10.1182/blood-2002-08-2531 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1669-1675 ◽

Cited By ~ 166

Author(s):

Kathleen E. McGrath ◽

Anne D. Koniski ◽

Jeffrey Malik ◽

James Palis

Keyword(s):

Vascular System ◽

Fetal Liver ◽

Yolk Sac ◽

Red Cells ◽

Hematopoietic Progenitors ◽

Mammalian Embryo ◽

Hematopoietic Organ ◽

Significant Enrichment ◽

A Site ◽

The Relationship

To better understand the relationship between the embryonic hematopoietic and vascular systems, we investigated the establishment of circulation in mouse embryos by examining the redistribution of yolk sac–derived primitive erythroblasts and definitive hematopoietic progenitors. Our studies revealed that small numbers of erythroblasts first enter the embryo proper at 4 to 8 somite pairs (sp) (embryonic day 8.25 [E8.25]), concomitant with the proposed onset of cardiac function. Hours later (E8.5), most red cells remained in the yolk sac. Although the number of red cells expanded rapidly in the embryo proper, a steady state of approximately 40% red cells was not reached until 26 to 30 sp (E10). Additionally, erythroblasts were unevenly distributed within the embryo's vasculature before 35 sp. These data suggest that fully functional circulation is established after E10. This timing correlated with vascular remodeling, suggesting that vessel arborization, smooth muscle recruitment, or both are required. We also examined the distribution of committed hematopoietic progenitors during early embryogenesis. Before E8.0, all progenitors were found in the yolk sac. When normalized to circulating erythroblasts, there was a significant enrichment (20- to 5-fold) of progenitors in the yolk sac compared with the embryo proper from E9.5 to E10.5. These results indicated that the yolk sac vascular network remains a site of progenitor production and preferential adhesion even as the fetal liver becomes a hematopoietic organ. We conclude that a functional vascular system develops gradually and that specialized vascular–hematopoietic environments exist after circulation becomes fully established.

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Cul4A Is Required for Cell Viability and Its Deficiency in Hematopoietic Cells Causes Apoptosis and Is Fatal.

Blood ◽

10.1182/blood.v110.11.639.639 ◽

2007 ◽

Vol 110 (11) ◽

pp. 639-639

Author(s):

Kristin T. Chun ◽

David L. Waning ◽

Binghui Li ◽

Nan Jia ◽

Yahaira M. Naaldijk ◽

...

Keyword(s):

Bone Marrow ◽

Small Intestine ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Lymphoid Cells ◽

Conditional Knockout ◽

Hematopoietic Stem ◽

Mutant Cells ◽

Total Cellularity

Abstract Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A-deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele (Cul4Aflox) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre, were derived. When deletion of Cul4A was induced in Cul4Aflox/flox Mx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls (Cul4Aflox/flox mice without Mx1-Cre), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A-deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A-deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A-deficient hematopoietic cells, deletion was induced in vivo in Cul4Aflox/flox Mx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A-deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A-deficiency will be presented.

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Aorta-associated CD34+ hematopoietic cells in the early human embryo

Blood ◽

10.1182/blood.v87.1.67.67 ◽

1996 ◽

Vol 87 (1) ◽

pp. 67-72 ◽

Cited By ~ 290

Author(s):

M Tavian ◽

L Coulombel ◽

D Luton ◽

HS Clemente ◽

F Dieterlen-Lievre ◽

...

Keyword(s):

Stem Cells ◽

Human Embryo ◽

Hematopoietic Cells ◽

Yolk Sac ◽

Blood System ◽

In Vitro Assays ◽

Avian Embryo ◽

Hematopoietic Stem

Abstract Hematopoiesis is established from circulating blood stem cells that seed the embryonic rudiments of blood-forming tissues, a basic notion in developmental hematology. However, the assumption that these stem cells originate from the extraembryonic mesoderm, where primitive hematopoiesis is initiated by intrinsic precursors, has been reconsidered after analysis of blood cell development in avian embryo chimeras: yolk-sac-derived stem cells do not contribute significantly to the definitive blood system, whose first forerunners develop independently along the ventral aspect of the embryonic aorta. Recently, the homologous intraembryonic tissues of the mouse have been submitted to sensitive in vivo and in vitro assays, which showed that they also harbor multipotential hematopoietic stem cells. We have now identified a dense population of hematogenous cells, marked by the surface expression of the CD34 glycoprotein, associated with the ventral endothelium of the aorta in the 5-week human embryo. Therefore, we extend to the human species the growing evidence that intraembryonic hematopoietic cells developing independently of the yolk sac might be the real stem of the whole blood system.

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Engraftment of Embryonic Hematopoietic Cells in Conditioned Newborn Recipients

Blood ◽

10.1182/blood.v89.6.2176 ◽

1997 ◽

Vol 89 (6) ◽

pp. 2176-2183 ◽

Cited By ~ 95

Author(s):

Mervin C. Yoder ◽

Kelly Hiatt

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Yolk Sac ◽

Transplant Recipients ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Cell Counts ◽

Adult Recipient ◽

Yolk Sac Cells

Abstract Yolk sac hematopoiesis is characterized by restricted hematopoietic cell differentiation. Although multipotent hematopoietic progenitor cells have been identified in the early yolk sac, long-term multilineage repopulating (LTMR) hematopoietic stem cell (HSC) activity has not been demonstrable before day 11 postcoitus (PC) using standard transplantation assays. In the present study, day-10 PC yolk sac hematopoietic cells were infused into myeloablated congenic newborn pups and donor cell engraftment and multilineage reconstitution of peripheral blood cells for at least 11 months in primary recipients was observed. In contrast, transplantation of day-10 PC yolk sac cells into congenic adult recipients did not result in engraftment despite pretransplant conditioning of the recipients or use of recipients that were genetically deficient in stem cells. Although fresh yolk sac cells were incapable of reconstitution when injected into adult recipient mice, yolk sac donor-derived cells residing in the bone marrow of primary newborn transplant recipients were capable of efficient reconstitution of conditioned secondary recipient adult mice. Primary newborn and secondary adult recipient animals engrafted with yolk sac cells were observed to have normal peripheral blood white blood cell counts. Lymphocyte subsets in peripheral blood, thymus, and spleen were also similar to control animals. The distribution and frequency of lineage-restricted progenitors derived from bone marrow of secondary transplant recipients were normal. These results indicate that day-10 PC yolk sac HSCs are capable of engrafting and reconstituting the hematopoietic system of conditioned newborn but not adult recipient animals. Furthermore, the ability of the yolk sac HSCs to differentiate into all hematopoietic lineages in these recipients strongly suggests that the local cellular microenvironment plays a prominent role in regulating yolk sac HSC differentiation.

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Contributions of Embryonic HSC-Independent Hematopoiesis to Organogenesis and the Adult Hematopoietic System

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.631699 ◽

2021 ◽

Vol 9 ◽

Author(s):

Wen Hao Neo ◽

Michael Lie-A-Ling ◽

Muhammad Zaki Hidayatullah Fadlullah ◽

Georges Lacaud

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Current Knowledge ◽

Hematopoietic Cells ◽

Yolk Sac ◽

Hematopoietic Stem ◽

Hematopoietic System

During ontogeny, the establishment of the hematopoietic system takes place in several phases, separated both in time and location. The process is initiated extra-embryonically in the yolk sac (YS) and concludes in the main arteries of the embryo with the formation of hematopoietic stem cells (HSC). Initially, it was thought that HSC-independent hematopoietic YS cells were transient, and only required to bridge the gap to HSC activity. However, in recent years it has become clear that these cells also contribute to embryonic organogenesis, including the emergence of HSCs. Furthermore, some of these early HSC-independent YS cells persist into adulthood as distinct hematopoietic populations. These previously unrecognized abilities of embryonic HSC-independent hematopoietic cells constitute a new field of interest. Here, we aim to provide a succinct overview of the current knowledge regarding the contribution of YS-derived hematopoietic cells to the development of the embryo and the adult hematopoietic system.

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