RESUMEN
Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Macrófagos , Pez Cebra , Pez Cebra/embriología , Animales , Macrófagos/metabolismo , Humanos , Células Madre Hematopoyéticas/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Embrión no Mamífero/metabolismo , Trasplante Heterólogo , FagocitosisRESUMEN
During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.
Asunto(s)
Desarrollo Embrionario , Hematopoyesis , Receptores de IgG , Humanos , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Desarrollo Embrionario/genética , Células Endoteliales/metabolismo , Células Endoteliales/citología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hemangioblastos/metabolismo , Hemangioblastos/citología , Hematopoyesis/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Receptores de IgG/metabolismo , Receptores de IgG/genética , TranscriptomaRESUMEN
The vascular wall is comprised of distinct layers controlling angiogenesis, blood flow, vessel anchorage within organs, and cell and molecule transit between blood and tissues. Moreover, some blood vessels are home to essential stem-like cells, a classic example being the existence in the embryo of hemogenic endothelial cells at the origin of definitive hematopoiesis. In recent years, microvascular pericytes and adventitial perivascular cells were observed to include multi-lineage progenitor cells involved not only in organ turnover and regeneration but also in pathologic remodeling, including fibrosis and atherosclerosis. These perivascular mesodermal elements were identified as native forerunners of mesenchymal stem cells. We have presented in this brief review our current knowledge on vessel wall-associated tissue remodeling cells with respect to discriminating phenotypes, functional diversity in health and disease, and potential therapeutic interest.
Asunto(s)
Células Madre Mesenquimatosas , Células Madre de Sangre Periférica , Células Endoteliales , Humanos , Células Madre Mesenquimatosas/fisiología , Pericitos , Células Madre/fisiologíaRESUMEN
Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.
Asunto(s)
Células Progenitoras de Megacariocitos , Megacariocitos , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Separación Celular/métodos , Células Madre Hematopoyéticas/citología , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , RatonesRESUMEN
The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-ß (TGF-ß) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-ß-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-ß signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.
Asunto(s)
Factor de Transcripción CDX2/genética , Leucemia Monocítica Aguda/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Antígeno CD11b/genética , Linaje de la Célula , Humanos , Leucemia Monocítica Aguda/patología , Proteínas de la Membrana/genética , Ratones , Transducción de Señal , Microambiente TumoralRESUMEN
B-cell receptor (BCR) signaling is crucial for the pathophysiology of most mature B-cell lymphomas/leukemias and has emerged as a therapeutic target whose effectiveness remains limited by the occurrence of mutations. Therefore, deciphering the cellular program activated downstream this pathway has become of paramount importance for the development of innovative therapies. Using an original ex vivo model of BCR-induced proliferation of chronic lymphocytic leukemia cells, we generated 108 temporal transcriptional and proteomic profiles from 1 h up to 4 days after BCR activation. This dataset revealed a structured temporal response composed of 13,065 transcripts and 4027 proteins, comprising a leukemic proliferative signature consisting of 430 genes and 374 proteins. Mathematical modeling of this complex cellular response further highlighted a transcriptional network driven by 14 early genes linked to proteins involved in cell proliferation. This group includes expected genes (EGR1/2, NF-kB) and genes involved in NF-kB signaling modulation (TANK, ROHF) and immune evasion (KMO, IL4I1) that have not yet been associated with leukemic cells proliferation. Our study unveils the BCR-activated proliferative genetic program in primary leukemic cells. This approach combining temporal measurements with modeling allows identifying new putative targets for innovative therapy of lymphoid malignancies and also cancers dependent on ligand-receptor interactions.
Asunto(s)
Linfocitos B/metabolismo , Proliferación Celular/genética , Leucemia Linfocítica Crónica de Células B/genética , Receptores de Antígenos de Linfocitos B/genética , Anciano , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Activación de Linfocitos/genética , Masculino , Persona de Mediana Edad , Proteoma/genética , Proteómica/métodos , Transducción de Señal/genética , Transcripción Genética/genéticaRESUMEN
Angiotensin-converting enzyme (ACE), a key element of the renin-angiotensin system (RAS), has recently been identified as a new marker of both adult and embryonic human hematopoietic stem/progenitor cells (HSPCs). However, whether a full renin-angiotensin pathway is locally present during the hematopoietic emergence is still an open question. In the present study, we show that this enzyme is expressed by hematopoietic progenitors in the developing mouse embryo. Furthermore, ACE and the other elements of RAS-namely angiotensinogen, renin, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors-are expressed in the paraaortic splanchnopleura (P-Sp) and in its derivative, the aorta-gonad-mesonephros region, both in human and mouse embryos. Their localization is compatible with the existence of a local autocrine and/or paracrine RAS in these hemogenic sites. in vitro perturbation of the RAS by administration of a specific AT1 receptor antagonist inhibits almost totally the generation of blood CD45-positive cells from dissected P-Sp, implying that angiotensin II signaling is necessary for the emergence of hematopoietic cells. Conversely, addition of exogenous angiotensin II peptide stimulates hematopoiesis in culture, with an increase in the number of immature c-Kit+ CD41+ CD31+ CD45+ hematopoietic progenitors, compared to the control. These results highlight a novel role of local-RAS during embryogenesis, suggesting that angiotensin II, via activation of AT1 receptor, promotes the emergence of undifferentiated hematopoietic progenitors.
Asunto(s)
Angiotensina II/genética , Angiotensinógeno/genética , Células Madre Hematopoyéticas/citología , Receptor de Angiotensina Tipo 1/genética , Sistema Renina-Angiotensina/genética , Animales , Aorta/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Antígenos Comunes de Leucocito/genética , Ratones , Péptidos/farmacología , Peptidil-Dipeptidasa A/genética , Receptor de Angiotensina Tipo 2/genética , Renina/genética , Transducción de Señal/efectos de los fármacos , Células Madre/citologíaRESUMEN
The continuous generation of blood cells throughout life relies on the existence of hematopoietic stem cells (HSC) generated during embryogenesis. Given the importance of HSC transplantation in cell-based therapeutic approaches, considerable efforts have been made toward understanding the developmental origins of embryonic HSC. Adult-type HSC are first generated in the aorta-gonad-mesonephros (AGM) region between days 27 and 40 of human embryonic development, but an elusive blood-forming potential is present earlier in the underlying splanchnopleura. It is relatively well accepted that the HSC emerge in the AGM through a hemogenic endothelium, but the direct precursor of this cell type remains to be clearly identified. This review is intended to summarize the recent advances made to understand the origins of hematopoietic stem cells in the early human embryo. In addition, we discuss in detail the discovery of the angiotensin-converting enzyme (ACE) as a novel marker of human HSC and of prehematopoietic precursors inside the embryo.
Asunto(s)
Desarrollo Embrionario/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Peptidil-Dipeptidasa A/genética , Adulto , Animales , Aorta/crecimiento & desarrollo , Embrión de Mamíferos , Femenino , Gónadas/crecimiento & desarrollo , Humanos , Ratones , EmbarazoRESUMEN
In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta-gonad-mesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis.
Asunto(s)
Aorta/embriología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Macrófagos/citología , Células Madre/citología , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Biología Evolutiva , Matriz Extracelular/metabolismo , Gónadas/embriología , Humanos , Macrófagos/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Mesonefro/embriología , Microscopía Fluorescente , Pez CebraRESUMEN
Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc.
Asunto(s)
Antígenos CD/metabolismo , Aorta/metabolismo , Desarrollo Embrionario , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Animales , Antígenos CD/genética , Aorta/citología , Aorta/embriología , Biomarcadores/metabolismo , Desdiferenciación Celular , Embrión de Mamíferos , Células Endoteliales/citología , Citometría de Flujo , Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , RatonesRESUMEN
Adult-type lympho-myeloid hematopoietic progenitors are first generated in the aorta-gonad-mesonephros region between days 27 and 40 of human embryonic development, but an elusive blood forming potential is present earlier in the underlying splanchnopleura. In the present study, we show that angiotensin-converting enzyme (ACE, also known as CD143), a recently identified cell-surface marker of adult human hematopoietic stem cells, is already expressed in all presumptive and developing blood-forming tissues of the human embryo and fetus: para-aortic splanchnopleura, yolk sac, aorta-gonad-mesonephros, liver, and bone marrow (BM). Fetal liver and BM-derived CD34(+)ACE(+) cells, but not CD34(+)ACE(-) cells, are endowed with long-term culture-initiating cell potential and sustain multilineage hematopoietic cell engraftment when transplanted into NOD/SCID mice. Furthermore, from 23-26 days of development, ACE expression characterizes rare CD34(-)CD45(-) cells concentrated in the hemogenic portion of the para-aortic splanchnopleura. ACE(+) cells sorted from the splanchnopleura generated colonies of hematopoietic cells more than 40 times more frequently than ACE(-) cells. These data suggest that, in addition to being a marker of adult human hematopoietic stem cells, ACE identifies embryonic mesodermal precursors responsible for definitive hematopoiesis, and we propose that this enzyme is involved in the regulation of human blood formation.
Asunto(s)
Médula Ósea/embriología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hígado/embriología , Peptidil-Dipeptidasa A/metabolismo , Animales , Antígenos CD34/metabolismo , Linfocitos B/citología , Linaje de la Célula/fisiología , Femenino , Granulocitos/citología , Trasplante de Células Madre Hematopoyéticas , Humanos , Células Asesinas Naturales/citología , Antígenos Comunes de Leucocito/metabolismo , Hígado/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Linfocitos T/citología , Trasplante HeterólogoRESUMEN
Hematopoietic stem cells (HSC) are at the origin of the adult hematopoietic system. They give rise to all blood cells through a complex series of proliferation and differentiation events that occur throughout the lifespan of the individual. Because of their potential clinical importance in transplantation, recent research has focused on the developmental origins of embryonic HSC. During development in vertebrate embryos, two independent anatomical sites generate hematopoietic cells. The yolk sac is responsible for a first ephemeral hematopoiesis, characterized by the early appearance of hematopoietic progenitors with limited development ability that rapidly differentiate toward erythro-myeloid lineages. Self-renewing, multipotent adult-type HSC that also exhibit B and T lymphoid potentials emerge autonomously in the aorta/gonad/mesonephros (AGM) region inside the embryo. In this review, we provide a brief summary of recent developments regarding the origins of hematopoietic stem cells in the early human embryo. The recent discovery that angiotensin-converting enzyme (ACE) is a novel cell surface marker of human HSC is discussed in detail.
Asunto(s)
Diferenciación Celular , Hematopoyesis , Células Madre Hematopoyéticas/citología , Sistema Hematopoyético/embriología , Linaje de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Peptidil-Dipeptidasa A/metabolismoRESUMEN
The mechanisms that determine organ identity along the digestive tract in humans are poorly understood. Here we describe the rare case of a young patient who presented with congenital gastric-type heteroplasia in the midjejunum. The lesions, located along the antimesenteric midline of the gut, were made of histologically and functionally normal gastric epithelium without inflammation or in situ/invasive carcinoma. They resembled the anatomy of the lesions developing in the mouse gut as a result of haploinsufficiency of the Cdx2 homeobox gene. The lesions were devoid of CDX2 but without mutation in the coding sequence or in a cis-regulatory element required for intestine-specific expression. Combining these data with the CDX2 expression pattern established from human embryos and cases of Meckel diverticula, we propose a scenario for this patient's presentation, in which CDX2 was missing at the site of ventral closure during gut morphogenesis, with subsequent default differentiation into gastric instead of intestinal tissue. Altogether, these observations argue in favor of a pivotal role played by CDX2 in determining intestinal identity during human embryonic development, as previously shown experimentally in mice.
Asunto(s)
Proteínas de Homeodominio/fisiología , Divertículo Ileal/etiología , Divertículo Ileal/patología , Factor de Transcripción CDX2 , Niño , Humanos , MasculinoRESUMEN
Human embryonic stem cells (hESC) have been directed to differentiate into CNS cells with clinical importance. However, for study of development and regeneration of the human PNS, and peripheral neuropathies, it would be useful to have a source of human PNS derivatives. We have demonstrated that peripheral sensory neuron-like cells (PSN) can also be derived from hESC via neural crest-like (NC) intermediates, and from neural progenitors induced from hESC using noggin. Here we report the generation of higher purity PSN from passagable neurospheres (NSP) induced by murine PA6 stromal cells. hESC were cultured with PA6, and colonies that developed a specific morphology were cut from the plates. Culture of these colonies under non-adhesive conditions yielded NSPs. Several NC marker genes were expressed in the NSP, and these were also detected in 3-5week gestation human embryos containing migrating NC. These NSPs passaged for 2-8weeks and re-plated on PA6 gave rise to many Brn3a+/peripherin+ cells, characteristic of early sensory-like neurons. Re-culturing PA6-induced NSP cells with PA6 resulted in about 25% of the human cells in the co-cultures differentiating to PSN after 1week, compared to only about 10% PSN obtained after 3 weeks when noggin-induced NSP were used. Two month adherent cultures of PA6-induced NSP cells contained neurons expressing several PSN neuropeptides, and voltage-dependent currents and action potentials were obtained from a molecularly identified PSN. hESC-derived PA6-induced NSP cells are therefore an excellent potential source of human PSN for study of differentiation and modeling of PNS disease.
Asunto(s)
Células Madre Embrionarias/fisiología , Cresta Neural/fisiología , Células Receptoras Sensoriales/fisiología , Biomarcadores , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Adhesión Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Análisis Citogenético , Electrofisiología , Células Madre Embrionarias/metabolismo , Humanos , Inmunohistoquímica , Cresta Neural/citología , Cresta Neural/metabolismo , Neuropéptidos/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/metabolismoRESUMEN
Previous studies revealed that mAb BB9 reacts with a subset of CD34(+) human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map BB9 expression throughout hematopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and surrounding endothelial cells are BB9(+). Thereafter, BB9 is expressed by primitive hematopoietic cells in fetal liver and in umbilical cord blood (UCB). BB9(+)CD34(+) UCB cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice contribute 10-fold higher numbers of multilineage blood cells than their CD34(+)BB9(-) counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34(+) population. Protein microsequencing of the 160-kDa band corresponding to the BB9 protein established its identity as that of somatic angiotensin-converting enzyme (ACE). Although the role of ACE on human HSCs remains to be determined, these studies designate ACE as a hitherto unrecognized marker of human HSCs throughout hematopoietic ontogeny and adulthood.
Asunto(s)
Feto/enzimología , Células Madre Hematopoyéticas/enzimología , Sistema Hematopoyético/enzimología , Peptidil-Dipeptidasa A/metabolismo , Adulto , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos/efectos de los fármacos , Antígenos CD34/metabolismo , Recuento de Células , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/enzimología , Femenino , Feto/efectos de los fármacos , Citometría de Flujo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Sistema Hematopoyético/embriología , Humanos , Lisinopril/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Sistema Renina-Angiotensina/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Human hematopoiesis proceeds transiently in the extraembryonic yolk sac and embryonic, then fetal liver before being stabilized in the bone marrow during the third month of gestation. In addition to this classic developmental sequence, we have previously shown that the aorta-gonad-mesonephros (AGM) embryonic territory produces stem cells for definitive hematopoiesis from 27 to 40 days of human development, through an intermediate blood-forming endothelium stage. These studies have relied on the use of traditional markers of human hematopoietic and endothelial cells. In addition, we have recently identified and characterized a novel surface molecule, BB9, which typifies the earliest founders of the human angiohematopoietic system. BB9, which was initially identified with a monoclonal antibody raised to Stro-1(+) bone marrow stromal cells, recognizes in the adult the most primitive Thy-1(+) CD133(+) Lin(-), non-obese diabetic--severe combined immunodeficiency disease (NOD-SCID) mouse engrating hematopoietic stem cells (HSCs). In the 3- to 4-week embryo, BB9 expression typifies a subset of splanchnopleural mesodermal cells that migrate dorsally and colonize the ventral aspect of the aorta where they establish a population of hemogenic endothelial cells. We have indeed confirmed that hematopoietic potential in the human embryo, as assessed by long-term culture-initiating cell (LTC-IC) and SCID mouse reconstituting cell (SRC) activities, is confined to BB9-expressing cells. We have further validated these results in the model of human embryonic stem cells (hESCs) in which we have modeled, through the development of hematopoietic embryoid bodies (EBs), primitive and definitive hematopoieses. In this setting, we have documented the emergence of BB9(+) hemangioblast-like clonogenic angiohematopoietic progenitors that currently represent the earliest known founders of the human vascular and blood systems.
Asunto(s)
Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Neovascularización Fisiológica , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Movimiento Celular , Células Endoteliales/citología , Glicoproteínas/biosíntesis , Hematopoyesis , Humanos , Hígado/embriología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Biológicos , PéptidosRESUMEN
RAG1/GFP knock-in mice were used to precisely chart the emergence and expansion of cells that give rise to the immune system. Lymphopoietic cells detectable in stromal co-cultures arose as early as E8.5, i.e. prior to establishment of the circulation within the paraaortic splanchnopleura (P-Sp). These cells were Tie2+ RAG1- CD34(Lo/-) Kit+ CD41-. While yolk sac (YS) also contained lymphopoietic cells after E9.5, CD41+ YS cells from < or =25-somite embryos produced myelo-erythroid cells but no lymphocytes. Notch receptor signaling directed P-Sp cells to T lymphocytes but did not confer lymphopoietic potential on YS cells. Thus, definitive hematopoiesis arises in at least two independent sites that differ in lymphopoietic potential. Expression of RAG1, the earliest known lymphoid event, first occurred around E10.5 within the embryos. RAG1/GFP+ cells appeared in the liver at E11.0 and progenitors with B and/or T lineage potential were enumerated at subsequent developmental stages.
Asunto(s)
Embrión de Mamíferos/citología , Linfopoyesis , Células Madre/citología , Animales , Antígenos CD34/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Linaje de la Célula , Técnicas de Cocultivo , Embrión de Mamíferos/irrigación sanguínea , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Hígado/embriología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor TIE-2/metabolismo , Circulación Esplácnica , Células del Estroma/citología , Linfocitos T/citología , Linfocitos T/inmunología , Timo/embriología , Timo/metabolismo , Saco Vitelino/citología , Saco Vitelino/embriología , Saco Vitelino/metabolismoRESUMEN
During the early weeks of human gestation, hematopoietic cells first emerge within the extraembryonic yolk sac (primitive hematopoiesis) and secondarily within the truncal arteries of the embryo. This second wave includes the stem cells giving rise to adult-type lymphohematopoiesis. In both yolk sac blood islands and embryonic aorta, hematopoietic cells arise in the immediate vicinity of vascular endothelial cells. In vitro hematopoietic differentiation of endothelial cells stringently sorted from human embryonic and fetal blood-forming tissues has demonstrated that primitive endothelium lies at the origin of incipient hematopoiesis. These anatomically and temporally localized blood-forming endothelial cells are ultimately derived from a rare subset of mesodermal angio-hematopoietic stem cells, or hemangioblasts. The evidence for an early progenitor of blood-forming cells within the walls of human embryonic blood vessels concurs with parallel data obtained from lower vertebrate, avian, and murine models. Importantly, converging results have recently been obtained with in vitro differentiated human embryonic stem cells, in which we have modeled primitive and definitive hematopoiesis via an endothelium-like developmental intermediate.