APLASTIC ANEMIA AND STEM CELL BIOLOGY
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS Franqoise Dieterlen-LiGvre, PhD
Although the primary object of this Clinics issue is to delineate various aspects of stem cell biology in humans that may underlie clinical problems, most specialists will readily admit that the mouse is a relevant model, experimental studies from which have brought out critical understandings and suggested tactical approaches. I hope to make clear that other models, designed in other classes of vertebrates such as birds and amphibians, have yielded important clues toward unraveling the early ontogeny of the hematopoietic system, in particular the emergence of stem cells. CELL COMMITMENT DURING DEVELOPMENT
Embryonic development proceeds by means of successive options, which progressively restrict the potential of cells until they become committed to a specific lineage. At branching points, cells usually can choose between two different pathways, after which they are unable to revert to former choices. Hematopoietic stem cells (HSC) are pluripotential in the sense that they are able to give rise to all blood cell types. Prior to their emergence, commitment of cells from the mesoderm to hematopoiesis already involves a series of restriction events. During gastrulation the mesoderm becomes established as a germ layer, then it subdivides into notochord and lateral plate. Then primordial blood islands appear in this region. Finally, paraxial mesoderm on each side of the neural tube segments into somites, while lateral plate splits into nephrogenic mesoderm, somatopleural and splanchnopleural sheets (Fig. 1). Commitment of HSC-that is, the allocation of a fraction of mesodermal cells to the blood cell-forming pathway-is thought to occur early in embryonic life and to give rise to a self-renewable pluripotential pool located in the
From the Institut d’Embryologie cellulaire et molkculaire of the Centre National de la Recherche Scientifique and the College de France, Nogent sur Mame, France ~
HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA
VOLUME 11 NUMBER 6 DECEMBER 1997
Figure 1. Evolution of the mesoderm in the chick embryo, as seen in the scanning electron microscope. The embryos (El to E3), or blastodiscs, that develop in a plane, were fractured transversely. A, Gastrulation stage; the groove indicated by an arrow is the primitive streak through which cells ingress to lay down the mesoderm (M); E, epiblast or primitive ectoderm; H = hypoblast or primitive endoderm. (Courtesy of Kessel and Fabian, Johannesburg.) Note that the mesoderm is a uniform layer of cells. 13,Neurulation stage; mesoderm has split into notochord (Ch) and lateral plate (Lp). Ec = ectoderm; En = endoderm; Np = neural plate. (Courtesy of P. Coltey, Nogent siMarne, France.) C, Somitic stage; the lateral plate has split into somatopleural mesoderm (Som. m.), associatedwith the ectoderm (Ect), and splanchnopleural mesoderm (Spl. m.) associated with the endoderm (End). Ao = aorta.
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
bone marrow, from which a few cells, normally dormant and mobilized when necessary, give rise to an abundant and diversified progeny. The embryonic site(s) and the developmental timing(s) of this commitment have been difficult to pin down. The appearance of blood cells distributed according to a reproducible pattern, in a zone located outside of the embryo-forming region, is one of the earliest events of embryonic development. In birds and mammals, this region gives rise to an appendage, the yolk sac (YS) (Fig. 2), whereas in amphibian embryos, the first developing red cells are confined to the equivalent of the YS, the so-called ventral blood island. Originally it was held that HSC developed from stromal cells within each hematopoietic organ. M a ~ i m o vfirst ~ ~ put forward the idea that HSC initially constituted a homogeneous population that became diversified within various microenvironments. This monophyletic theory proposed the existence of a single population of pluripotential stem cells, a concept validated in the adult by
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A Figure 2. Schedule of hematopoietic ontogeny in chick (A) (21 days of incubation) and quail (B) (16 days) embryos. In the case of yolk sac, spleen and bone marrow, the entry of HSC and the extent and duration of HSC are indicated by the bubbles. For thymus and bursa the timing of colonization is indicated by the stippled rectangles. The aspect of intraaortic clusters and para-aortic foci is schematized in relationship to the aorta, represented by a circle. Illustration continued on following page
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PARAAORTIC FOCI THYMUS
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6 Figure 2 (Continued).
means of the irradiation/restoration strategy.68The ontogeny of these cells remained elusive until the advent of transplantation techniques utilizing cell markers. Extrinsic HSC were then shown to colonize the stroma of all hematopoietic organ rudiments. The only recognized exception to this rule was the YS. This embryonic appendage-or its equivalent in amphibians, the ventral blood island-was then considered for a number of years as the sole provider of all HSC, according to a hypothesis of Moore and Owen.51HSC from the YS or their direct progeny were thought to colonize successively the hematopoietic organ rudiments, as they differentiated. There is now ample evidence in amphibians, birds, and mice that progenitors produced early in development by the YS are not endowed with self-renewal capacity, that is, the essential property of HSC. The experimental data that led to this conclusion and the evidence pertaining to the emergence of intraembryonic HSC are reviewed in this article. EXTRINSIC HEMATOPOIETIC STEM CELLS COLONIZE HEMATOPOIETIC ORGAN RUDIMENTS
The crux of dynamic studies involving either the grafting of labeled rudiments or vascular anastomoses between marker-bearing embryos is to carry out the experiments in the very early embryo, prior to the beginning of organ rudiment differentiation. For avian studies, the first marker system, devised by
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
Moore and Owen," relied on the sex chromosome marker. The embryos were treated for 12 hours with colchicine to arrest cells in metaphase. Unfortunately, many embryos died before analysis was possible. The quail/chick marker system38,39 brought about a remarkable advance and has been used to devise many experimental paradigms. The label relies on a distinctive difference between the distribution of heterochromatin in the nuclei of chick and quail cells. In quail cells one or several prominent heterochromatin lumps are present, whereas chick cell nuclei display only minute heterochromatin masses. All cells, rather than just the dividing ones, are available for diagnosis, and the combinations are successful because the sizes of the early embryos are comparable and because the immune system remains immature until hatching. Differentiation of quail from chick cells was first carried out using the histologic FeulgenRossenbeck staining procedure for DNA. With the advent of monoclonal antibody (moabs) technology, a number of useful markers have been obtained, including QCPN, a pan quail marker with no affinity for chicken cells (B Carlson and J Carlson, personal communication, 1993). Some other moabs recognize a lineage in one of the two species only. For example, MB163and QHl,59 raised respectively against the quail immunoglubulin p chain and quail E l 3 bone marrow cells, both recognize the "hemangioblastic" lineage, that is, hematopoietic and endothelial cells. In the mouse embryo, the T6 chromosomal marker has been used but is now replaced by alleles of antigens carried by cells of interest, such as Ly5 (CD45) for hematopoietic cells. The influx of extrinsic HSC into attractive rudiments was first recognized by Moore and Owen,5°,52 who observed a high level of chimerism in the hematopoietic organs of parabiotic chick embryos, especially when the embryos developed vascular connections at an early stage (E4), through YS rather than chorioallantoic anast0moses.4~The extrinsic colonization rule was also demonstrated through the culture of E7 chicken thymuses or El0 mouse thymic rudiments in a cell-impermeable diffusion chamber on the chorioallantoic membrane of the In these conditions the rudiments never became lymphoid. The conclusion of these two series of studies was that all hematopoietic organ rudiments except the YS were colonized by HSC, probably originating from a common source. Thus, Moore, Owen, and Metcalf elaborated an entirely new interpretation of the ontogeny of the hematopoietic system (reviewed in ref. 49), the central tenets of which were the extrinsic origin of HSC and the unique role for the YS in producing, early in development, all HSC for a lifetime. The quail/chick system confirmed the first part of this interpretation by showing that hematopoietic lineages were entirely derived from colonizing HSC and were thus distinct from stromal lineage.6,40,41 A recent claim that a common progenitor for hematopoietic and stromal cells could be isolated from bone marrow has been withdrawn.28,29 Utilization of the quail/chick marker accurately established the develop42 Furthermore, moabs mental schedule specific for each rudiment (see Fig. 2).17* as well as nuclear structure allowed the identification of cell types-for instance, dendritic and lymphoid cells in the thymus-thus demonstrating that both cell types were of hematogenous origin but had different In the case of the thymus, a remarkable cyclic pattern was detected in which attractive periods were separated by refractory periods. The bursa displayed a unique, clearly demarcated colonization period.43The initiation of bone marrow col~nization~~ could be dated also, as well as that of spleen.71 In the mouse embryo, seeding by extrinsic HSC was demonstrated for both the thymus and fetal liver. Owen and RitteP cultured thymic rudiments in
diffusion chambers on the chick chorioallantois, that is, under conditions in which the supply of extrinsic HSC is cut off. Rudiments from older embryos became fully lymphoid, whereas primordia from earlier ones failed to develop a lymphoid population. Colonization was dated to 10 and 11 days postcoitum (dpc), and the inflow was supposed to continue once full lymphopoiesis had begun. In the work of Fontaine-Pkrus et a1,2' the thymus rudiment retrieved at 10 days postcoitum, although it was still included in the third branchial arch, and was cultured either in vitro or on the chorioallantoic membrane of a chick embryo, alone or associated with various hematopoietic organs from mouse embryos or adults (YS, fetal liver thymus or bone marrow), as donors of HSC. Although the thymus failed to develop and remained epithelial when cultured alone, it became seeded by lymphoid precursors in the associations and underwent a normal histogenetic process; genetic markers made it possible to assess that the lymphoid population was of donor origin. Finally, there is some experimental evidence that the mouse thymus also receives cyclic inputs of lymphoid p r e c ~ r s o r showever, ~~; the colonization waves overlap so that they are not very clear-cut. In the case of the liver, organ culture as well as grafts to variously prepared adults have indicated that the hepatic primordium depends on extrinsic HSC to become hematopoietic, and that the first HSC enter between the stages of 28 to 32 pairs of somites.26,31 GRAFTING AN AVIAN EMBRYO ON A FOREIGN YOLK SAC: INTRAEMBRYONIC STEM CELLS IN BIRDS This paradigm brought about incontrovertible evidence that the emergence of HSC is not restricted to a unique extraembryonic location nor to an early period of development, and it is described here in some depth. The experimental scheme consisted of substituting in ovo a territory from the chick blastodisc with its quail equivalent." The informative pattern was the graft of the presumptive body of the 2-day quail on the 2-day chick extraembryonic area (Fig. 3). During days 3 to 4, formation of the extraembryonic coelom splits the three germ layers into YS and amnion, and the grafted quail embryo develops in association with a chick YS. The hematopoietic constitution of these chimeras was constant, whatever the time of grafting, between the stage of seven pairs of somites (El .5) and E3. Strikingly, the species make-up of intraembryonic organs was always entirely quail when the chimeras were analyzed between E5 and E13.I4,16, 45 These experiments led to two conclusions: (1) precursors colonizing the stromal rudiments have an intraembryonic origin; (2) in this combination, where the embryo is quail and the YS chick, precursors from the YS are entirely excluded from the colonization process. The composition of the blood was measured by the quantity of hemoglobin released by immunohemolysis, with polyclonal antibodies against quail or chick erythrocytes.' At E5,95% or more red cells were chick. From E6 onwards, quail red cells accumulated in the blood of chimeras, up to a mean of 43% at E13; however, the replacement of chick red cells by quail was irregular from one chimera to the next. To resolve this inconsistency, the quail-chick chimera study was complemented by the analysis of chick-chick chimeras constructed according to the same pattern (Fig. 4). Three independent marker systems were used: sex chromosomes, immunoglobulin allotypes, and major histocompatibility complex (MHC) class IV antigens expressed on the surface of red cells in birds. The sex chromosome study yielded strong evidence for the existence of intraembryonic HSC.35
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
Figure 3. Scheme and photograph of the “yolk sac chimera” between a quail embryonic body (a) and a chick extraembryonic area (C). The ovoid line (stippled in the scheme, white in the photogfaphj is the suture between the two components. At the time of grafting shown here, blood circulation is not yet established; blood islands are present in the outer area.
The allotype chimeras were allowed to hatch and grow to adulthood, and they always displayed the intraembryonic allotype in their immunoglobulins, whereas with MHC antigens, the replacement of YS HSC-derived erythrocytes by intraembryonic HSC-derived erythrocytes clearly appeared as both rapid and consistent (see Fig. 4), contrasting with the irregular blood evolution observed in heterospecific chimeras.%,37 Because these experiments were carried out between 1975 and 1978, quail nuclei were distinguished from chick by means of the Feulgen-Rossenbeck technique, with which single cells are difficult to diagnose among cells from the other species. In contrast, monoclonal antibodies such as MB1 and QH1, developed in the 1980s,easily detect single quail cells that belong to the hemangioblastic lineage. When these moabs were applied to the reverse experimental pattern-that is, a chick embryo grafted on a quail extraembryonic area‘O-some cells from the YS invaded the embryos. These cells were macrophages that displayed the macrophage-specific enzyme acid phosphatase, and they were quite numerous and dispersed in the whole embryo, in particular in neural tissue, where they may be precursors of microglia.1° They appeared capable of interstitial migration and they increased progressively in numbers. It is not known whether they became long-lived residents, nor whether intraembryonic HSC later contributed to this specific population, because these ”reverse” chimeras could be raised only until E5. HEMOGLOBIN SWITCHING IN QUAILKHICK YOLK SAC CHIMERAS
The red cell progeny of intraembryonic HSC begins to replace that of YS HSC from E5-6 onwards. This timing corresponds to a new phase in erythropoie-
Number of chimeras
Post hatch. 1
* % immunofluorescent cells after antiserum + FlTC antichicken IgG. NCS = normal chicken serum Figure 4. Chimeras between chick embryos differing by MHC haplotypes 82 and 815 and table of blood evolution. (From Lassila 0,Martin C, Toivanen P, et al: Erythropoiesis and lymphopoiesis in the chick yolk-sac-embryo chimeras: Contribution of yolk sac and intraembryonic stem cells. Blood 59:377,1982; with permission.)
sis, when the first definitive erythrocytes are released into the blood of normal embryos (reviewed in ref. 16). Primitive erythrocytes, present alone until that time, are characterized by their large size and round shape; they are released in the blood as erythroblasts and mature there as a cohort; they contain an assortment of specific, so-called primitive (P) and embryonic (E) hemoglobins. The first definitive erythrocytes are characterized by the presence of the two adult hemoglobins, A and D, and a transitory one, H. The evolution is parallel in the quail embryo.” With the immunohemolysis technique, it was possible to separate red cells according to species from chimeras at various stages. When the hemoglobins from each population were resolved by isoelectrofocusing, chick
INTRAEMBRYONIC HEMATOPOETIC STEM CELLS
blood appeared “younger,” because it lacked the erythroid progeny of intraembryonic HSC, whereas quail blood appeared ”older,” because it lacked the YS contribution. Nevertheless, chick blood, in these chimeras entirely of YS origin, switched its assortment of hemoglobins at E5, indicating that the YS produces not only progenitors giving rise to primitive erythropoiesis but also progenitors contributing to the first definitive series. The picture of hematopoietic ontogeny in the avian embryo emerging from this series of experiments can therefore be summarized as follows: (1)At EL5 a cohort of primitive HSC becomes committed in the extraembryonic area; it gives rise to progenitors that are released into the blood as early erythroblasts and synthesize primitive hemoglobins; (2) At E3.5 HSC becomes committed in the periaortic region in the embryo proper. They contribute to definitive erythropoiesis along with some YS progenitors, but only intraembryonic HSC colonize the rudiments of definitive blood-forming organs. This restriction could result from local attraction of nearby emerging HSC by the stroma of rudiments or from a different potential of HSC appearing at this time of development in embryos. Whatever the mechanism that excludes YS progenitors from intraembryonic rudiments, it is clear that erythropoiesis evolves according to a time-dependent program, independently from the site of emergence of HSC. SITE(S) OF PRODUCTION OF INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS IN AVIAN EMBRYOS
The next step was to pin down the sites and times where HSC are produced in the embryo proper. Cytologic aspects provided a clue. Prior to the formation of any blood-forming organ, processes of diffuse hematopoiesis occur within the dorsal aorta and later in the mesenchyme ventral to the aorta. These ”intraaortic clusters” and “para-aortic foci,” already observed early in this century, are visible in E3-4 and E6-7, respectively, in both chick and quail embryos16,1s (see Figs. 2 and 8A). Without exception, these hematopoietic displays are located in the ventral aspect of, or ventral to, the aorta. An E3 or E4 quail aorta, grafted in the dorsal mesentery of the chick embryo serving as a permissive microenvironment, gave rise to abundant QH1’ cells aggregated into a typical ”focus.”15Another blood vessel, the common cardinal vein, gave no such cells. E3-4 chick aortae dissociated into single cells, seeded into a semisolid medium with appropriate growth activities, and gave rise to colonies that were five to six times more frequent than in the bone marrow of the newly hatched The remainder of the embryonic body, after the aorta had been taken out, gave rise to no colonies. The intra-aortic clusters are tightly associated with the endothelium, a general property also true in mammals, as described subsequently. In contrast, such an association is not evident in the para-aortic foci a little later. Thus, the aorta and para-aortic region appear as the sites at E6-8 of intraembryonic HSC production. The development of the clusters and foci is synchronous with the initiating colonization of the splenic, thymic, and bursa1 rudiments; however, the foci become extinct 2 to 3 days prior to bone marrow colonization. Thus, it was not clear how these cells could retain their totipotency and where they were stored. We recently put forward the hypothesis, now supported by experimental data, that the allantois of avian embryos is also a hematopoietic organ, and that it is endowed with the crucial ability of producing HSC (Caprioli, Jaffredo, and Dieterfen-Likvre, manuscript in preparation). The existence of this novel HSC producer does not contradict the YS chimera data,
because the chimeras were constructed prior to the formation of the allantoic bud, and this bud is emitted by the anterior intestinal portal, an emanation of the embryo proper. It does modify the interpretation of the formation of the avian blood system, because the aortic region and the allantois may share a similar role. We will attempt to determine the time course of HSC production by the allantois and the respective contributions of the two sources. EVIDENCE FOR INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS IN THE MOUSE EMBRYO For many years Moore, Owen, and Metcalf's "central dogma," that all HSC originated from the YS, remained unquestioned in the case of mammalian hematopoiesis, even though the existence of intraembryonic HSC had been disclosed in birds and amphibians. A variety of experimental approaches seemed to support the YS theory. For instance, Weissman et aP9 transplanted 8 to 10 dpc YS blood island cells into the YS of 8 to 10 dpc hosts differing in H2 or Thy1 markers. Chimerism was found in the bone marrow and less frequently in the thymus of some injected embryos. The level of chimerism was admittedly low in all animals. Palacios and Imh0P7 detected lymphoid precursors in the E8-8.5 YS but not in the embryo; the tests in this case were reconstitutions of adult irradiated SCID mice or in vitro cultures on thymic epithelium for T cells, on a fetal liver stromal cell line in the presence of several cytokines, in particular I17 for B cells. Also, using both in vitro and in vivo approaches, Huang and Auerbach,z7on the other hand, detected lymphoid and myeloid potential in both YS and embryo proper at 9 dpc. The question appeared pending, because Nishikawa's group had earlier obtained experimental data in favor of a primary emergence of lymphoid progenitors in the embryonic body.55Cells dissociated from either the body of the embryo, the YS, or the fetal liver were seeded on bone marrow stromal cell line ST2 in conditions permitting B-cell differentiation. Clonable B-cell progenitors were detected in the embryonic body at approximately 9.5 dpc, and a little later in the YS.55 These data appeared as an incentive to investigate whether the rules of hematopoietic development in birds and amphibians might also apply to the mouse. Experiments were simultaneously undertaken by two groups; our group used a strategy that relied on expansion/differentiation of progenitors in culture, whereas Dzierzak's group tested hematopoietic potential by reconstituting irradiated adult mice. Of course, it would be revealing if an experimental scheme, similar to the avian one, could be devised for mouse embryos. Grafting a mouse embryo on a foreign YS does not appear feasible. Metcalf and Moore49cultured 8-dpc precirculation embryos severed from their YS. These embryos cultured for 2 days did not make blood. Applying the well-defined stringent conditionsPthat permit development of whole embryos outside of the maternal uterus, we kept the embryos in vitro for 3 to 4 days and obtained animals in which blood was present in the heart area and blood vessels (Godin, unpublished study). The poor morphology of these animals and the very low number of living animals at day 3 to 4 of culture rendered more precise analysis impossible, however. On the other hand, techniques have been devised that adequately meet the cytokine requirements of hematopoietic progenitors and permit the in vitro expansion of these cells present in a cell suspension. The technique devised by Cumano and Paigell uses stromal cell line S175as a feeder layer, and adds I17
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
to the medium (Fig. 5A).Clones totaling up to 1000 cells develop, each representing the daughter cells of one progenitor. The number of progenitors present in the original cell suspension can be evaluated. Cumano et all3 could document the presence of B-cell progenitors in YS and embryonic body from mouse fetuses, beginning at the 10-somite stage at 8.5 dpc. At this early stage, the frequency of progenitors is very low, less than one per embryonic body or YS, and it remains highly variable in both compartments until the 15-somite stage (Fig. 6). To chart the region of emergence within the embryonic body, we did the same experiment: The region of the mouse truncal aorta (Fig. 7), selected for its homology to the avian embryo hemogenic region, was dissected and assayed separately from the remainder of the embryo. Whereas in the E3 or E4 chick or quail embryo the aorta is a very large, easy-to-isolate structure, it is minute in the mouse embryo. The dissected region thus comprised the intestinal endoderm surrounded by mesoderm in which the two dorsal aortae and the omphalomesenteric artery are embedded (Fig. 7B). We called this region the para-aortic splanchnopleura (P-Sp). The P-Sp yielded increasing numbers of progenitors between the stages of 10 to 25 pairs of somites (see Fig. 6). Progenitors also could be found in the YS, their number increasing in parallel. By contrast, no progenitors were present in the remainder of the embryo, once the YS had been cut off and the P-Sp dissected out. This result, although perfectly compatible with avian data, did not permit us to conclude whether the P-Sp seeded the YS or the opposite, nor whether progenitors appeared in parallel in the two structures, because blood begins circulating at the eight pairs of somites stage. To assess the multipotentiality of precursors in the P-Sp, we grew clones from individually selected P-Sp cells enriched for the hematopoietic embryonic/ fetal antigen AA4.1, and we implemented a secondary culture in which the clones were divided into three lots; two were submitted to specific cytokine mixtures, and the third was seeded on an embryonic lymphoid-depleted thymus according to the technique devised by Jenkinson et aFO(see Fig. 5A). The three lots differentiated respectively into myeloid and lymphoid B or T cells, proving the pluripotentiality of the original progenitors. No precursors could be found before the stage of 10 pairs of somites after dissociation of the tissues; however, if the dissected structures were maintained in their three-dimensional organization and precultured for 2 days (organ culture) (see Fig. 5B) and then dissociated, clones developed and differentiated into the myeloid and lymphoid pathways during the two steps of suspension culture. Thus, progenitors, absent from 0-10 somite embryos, became committed to the hematopoietic pathway, provided cell interactions were allowed to proceed during the in toto culture step. The interesting return was that YS and splanchnopleura separated at these early stages yielded distinct progenitors, as characterized by their differentiation and maintenance potential (Table 1).YS progenitors could enter only the myeloid, but not the lymphoid, differentiationpathway, and they soon became exhausted in the expansion culture medium, whereas the Sp progenitors yielded lymphoid and myeloid progeny for up to 3 weeks. At these stages the aortae have not yet formed, hence the term Sp rather than PSp. Resorting to precirculation embryos thus provides the crucial argument for the emergence of a novel generation of HSC in the embryo proper. Simultaneously progenitors appear in the YS; however, their potential is more limited, or precluding their participation in laying the foundation of the definitive hematopoietic system. We had previously shown that cells from the P-Sp were capable of partially curing mice with a severe combined immunodeficiency (SCID). The whole dissected structure was grafted under the kidney capsule of
Fracttonrr ol don-
(8.5 to 9.5 dpc)
IN TOT0 CULTURE
II7 + KL + Ill
Figure 5. See legend on opposite page
10-25 somite embryos
II7 + KL + 113
+ + FracUons ol done.
(7.5 to 8.5 dpc)
0-8 somite embryos
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
adult SCID mice. In these conditions, cells from the P-Sp gave rise to a subpopulation of B lymphoid cellsz4;the so-called Bla cells-that is, cells that reside in the peritoneum-bear the CD5 surface antigen (normally expressed on activated T cells), differentiate during fetal life, and do not become reconstituted in irradiated adults injected with bone marrow cells. Why in this particular experiment grafted P-Sp participation was restricted to this population was not determined, although it probably results from the experimental conditions, in particular the implantation site. Solvason and Keamey& had similarly observed the restricted contribution of the omentum when implanted under the kidney capsule of SCID mice. The omentum did not reconstitute the T-cell compartment unless it was combined with an embryonic thymic epithelium in direct contact. The failure of P-Sp grafts to reconstitute T cells in SCID mice could thus be due to a deficiency in the environment immediately encountered by differentiating progenitors. Another possibility should be considered-that P-Sp cells might not have all the competence present in definitive HSC, an interpretation to be more fully considered subsequently. Simultaneously, Medvinsky et ar7,48 decided to probe the intraembryonic potential by means of in vivo reconstitution techniques. They detected a source of HSC in a region comprising the aorta, gonads, and mesonephros (AGM) at 9 to 10.5 dpc. This region is derived from the P-Sp, and the relevant rudiments which have begun organogenesis. The first colony forming units-spleen (CFUS) were detected in the AGM at the 31-33 somite stage and rose to a peak at the 38-40 somite stage with a frequency as high as in the adult bone marrow (20 to 40 per 105 cells). By 11 dpc this frequency fell rapidly. The first CFU-S appeared in the fetal liver rudiment at the 38-40 somite stage, at the time of the AGM peak.48The potential for long-term repopulation (LTR HSC) (4 to 6 months after transplantation),” the test that is admitted as detecting ”true” HSC, was found in the AGM, and only there, at late 10 dpc, in embryos with 34 to 41 pairs of somites. These LTR HSC were scarce, however, because only 3 of 96 irradiated mice were reconstituted 6 months after injection or later. After a 2- to 3-day organ culture implemented prior to dissociation and injection of cells into irradiated adults, LTR HSC were regularly detected in the AGM obtained from 10-dpc embryos (35-38 sp), whereas no reconstitution was achieved with other organ-cultured tissues from the same donors, that is, YS, liver rudiments, or body rerm1ants.4~After this preculture step, the CFU-S day-11 test detected significant activity beginning at the 32-33 pairs of somites in AGM (4.5 colonies per AGM) and YS (0.5 per YS) but not in liver, head, or heart, or body remnants. Thus, the organ culture step significantly increased the number of progenitors and made it possible to detect them earlier in both YS and AGM. But the main reward of this approach is that the AGM potential was detected 1 day earlier than the liver or YS potential. Dzerziak‘s group thus concludes that LTR HSC must originate in the AGM, a conclusion that concurs with ours. Both in vitro and in vivo assays establish
Figure 5. In vitro assays allowing detection of the hematopoietic potential of mouse embryonic structures at somitic (A) and gastrulation (6)stages. At the time of gastrulation, a preliminary step of organ culture is necessary for this potential to be present. (Adapted from Cumano A, Dieterlen-Lievre F, Godin I: Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86:907, 1996; and Godin I, Dieterlen-Lievre F, Cumano A: Emergence of multipotent hematopoietic cells in the yolk sac and para-aortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus. Proc Natl Acad Sci USA 92:773, 1995; with permission. Art by Isabelle Godin.)
Number of precursors per structure
YOLKSAC 0 PARAAORTIC REGION A EMBRYONIC BODY
SOMITES 10 GESTATION (dpc) 8.5
Figure 6. Evolution of the hernatopoietic potential in the mouse P-Sp at sornitic stages.
that secondary HSC originate in the embryo proper, from which they colonize the YS. We were able to demonstrate that these cells appear in the embryo separated from the YS prior to blood circulation. There remains an important point at issue. Dzierzak's group finds no LTR HSC in AGM prior to 30 somites even after organ culture.47We have looked for this potential in organ-cultured pre-10 somite P-Sp and have not found it either.I2It seems that the first intraembryonic HSC do not display the whole array of functions of true HSC. Thus, the question remains open whether early HSC with LTR potential are not yet able to express the potential when transferred in an adult microenvironment or whether LTR HSC emerge anew from mesodermal precursors at a later date. Table 1. EXTRAEMBRYONIC AND INTRAEMBRYONIC POTENTIAL DETERMINED BEFORE AND AFTER ESTABLISHMENT OF CIRCULATION ~
Cell Types Obtained
YS obtained from a 4-somite embryo. YS = yolk sac; P-Sp = para-aortic splanchnopleura.
Figure 7. Cross sections of mouse embryos with 5 (A) (8.5 dpc) and 15 pairs of somites, (6)(9.5 dpc), and (C) (10.5 dpc) at trunk level. This series of pictures illustrates the morphogenesis of the mouse embryo and the transformation from the Sp to P-Sp and AGM. The stippled lines indicate the structures dissected at each stage. Ao = aorta; G = genital ridge or gonad; In = intestine; M = mesonephros; OM = omphalomesenteric artery; Nt = neural tube; So = somite. Semithin sections with toluidine blue staining. Note that at the earliest stage depicted (A), the YS is continuous with the P-Sp. Arrowhead indicates the YS blood island.
THE HEMANGIOBLAST: A COMMON PROGENITOR OF HEMATOPOIETIC STEM CELLS AND ENDOTHELIAL CELLS?
The tight anatomic links between hematopoietic and endothelial cells have 70 and within the embryo.2o, 32,61 long been noticed in the early YS blood islands62, These links are striking in the intra-aortic clusters of all species where they have been looked for (Fig. 8). Hematopoietic precursors and endothelial cells have since been shown to share a large number of surface molecules, in particular adhesion molecules and growth factor receptors with tyrosine kinase activity. Furthermore, development of both hematopoietic cells and endothelial cells is defective in several knock-out mice; in particular, mice deficient for VEGF-E die between 8.5 and 9.5 dpc owing to the lack of endothelial as well as hematopoietic cells in their YS.& VEGF is the most endothelial-specific among the growth factors that are mitogenic for endothelial cells; VEGF R2 is one of its receptors, expressed early during development. All these arguments have led to the revival of an old hypothesis, according to which the two cell types may originate from a common precursor." Recent evidence strongly favors the existence of this presumptive prec~rsor.'~ A moab directed against the extracellular domain of VEGF-R2 was used to isolate VEGF R2+ cells from the chick embryo at the gastrulation stage. In semisolid cultures, VEGF-R2 cells gave rise to either hematopoietic or endothelial colonies. Although mixed colonies were never obtained, there clearly was a link between the two types of clones, because their total number developing from a definite number of sorted VEGF-R2' cells was
Figure 8. Intra-aorticclusters in birds and mammals (arrows). Cross sections at trunk level. A, E3 quail embryo. QHValkaline phosphatase. The clusters are bilateral. 6,12-mm pig embryo. (From Emmel VE: The cell clusters in the dorsal aorta of mammalian embryos. Am J Anat 19:401, 1916; with permission.) Illustration continued on opposite page
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
constant, the proportion between the two types varying with culture conditions. Only hematopoietic colonies developed in the absence of added growth factor to the serumless medium. When VEGF was added, endothelial colonies appeared and increased in number until an optimal concentration of VEGF was reached, whereas the number of hematopoietic colonies decreased. Hematopoietic differentiation occurred in the absence of VEGF; the number of these colonies was significantly reduced by the addition of soluble VEGF-R2. The authors
Figure 8 (Continued). C, 35-day human embryo CD4Ualkaline phosphatase. 0,10.5-dpc mouse embryo, recurved distal part of the aorta. Semithin section with toluidine blue. Note that, in every species depicted, the clusters are in the same position and make up the lining of the aorta. (C, From Tavian M, Coulombel L, Luton D, et al: Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 87:67, 1996; with permission. D, From Garcia-Porrero JA, Godin IE, Dieterlen-Lievre F: Potential intraernbryonic hemogenic sites at pre-liver stages in the mouse. Anat Embryo1 192:425, 1995; with permission.)
propose that the receptor titrated out from the medium a ligand other than VEGF, and that this unidentified growth factor is necessary for differentiation of the hemangioblast along the hematopoietic pathway. According to this hypothesis, the sorted VEGF-R2 population would be composed of true hemangioblasts able to follow distinct differentiation pathways, depending on two growth factors competing for the same receptor. The hemangioblast could engage along one of the two pathways, thus excluding the formation of mixed colonies. The alternative hypothesis, according to which there are two distinct precursors, is rather unlikely to be true, because the total number of colonies is constant; a tight relationship between the two is also indicated by their disappearance in several murine knock-outs. The same rules probably prevail in the aorta as in the YS, because the intraaortic clusters, when they express the panhematopoietic marker CD45, lose VEGF-R2 expression, so that the floor of the aorta appears not to be limited by bona fide endothelial cells, even though this blood vessel ensures its circulatory function (Jaffredoand Dieterlen-Lisvre, manuscript in preparation). Tracing the origins of lineages also points to the existence of developmental links between endothelial and hematopoietic cells.58, 61 Experiments consisting of orthotopic or heterotopic transplantations of rudiments from the quail to the chick uncovered two distinct mechanisms: one at work in the somatopleural layer of lateral plate mesoderm, the other in the splanchnopleural layer. Somatopleural mesoderm becomes colonized by purely endothelial precursors that originate from the somites. Splanchnopleural mesoderm provides its own precursors in situ, and these are tightly associated with hematopoiesis during the morphogenetic stages illustrated in Figure 1. At the period of organogenesis that follows, hematopoiesis in the embryo becomes restricted to the region of the aorta, and rudiments undergo different processes according to their origin. Somatopleural rudiments (body wall, limb buds, bone marrow) are colonized by extrinsic precursors for both the endothelial and the hematopoietic lineages. Splanchnopleural rudiments (visceral organs) obtain their endothelial network from their own mesoderm and receive their HSC from an extrinsic source (spleen, for example). Thus, it appears that there are two distinct endothelial lineages, a conclusion confirmed by the different behavior of these endothelial precursors when they are both grafted in a dorsal position, in the place of somites. Endothelial cells of somitic (“dorsal”) origin invade ”dorsal” structures, comprising neural tube, body wall, and kidney, as well as the roof and lateral sides of the aorta, but never its floor. Endothelial cells of splanchnopleural (or ”ventral”) origin grafted in a dorsal position invade the same structures but are also able to penetrate into visceral organs and to settle in the floor of the aorta. In this position they bear intra-aortic [email protected]
’ A COMMON PRECURSOR FOR PRIMITIVE AND DEFINITIVE HEMATOPOIESIS? It is clear from the preceding discussion that commitment of the mesoderm to hematopoiesis can occur in both the extraembryonic area and in the embryo proper, and that these events provide cells that have different fates-the first dedicated to primitive erythropoiesis, the latter to definitive hematopoiesis involving the isolation of HSC with long-term renewal potential. Three different hypotheses may be proposed about the commitment and fates of the cells from the two origins: (1) Primitive erythropoietic precursors are an independent generation, endowed with distinct properties, in particular distinct gene activi-
INTRAEMBRYONIC HEMATOPOIETIC STEM CELLS
ties; (2) The emergence of HSC during ontogeny is an extended process; the ones appearing early are not different, but the particular microenvironment they encounter restricts their fate to a unique differentiation pathway; (3) Later HSC are the progeny of the early ones, and their potential changes as mitoses occur. Recent work” appears to argue for the existence of a common precursor, which might then diverge according to the second proposal. In this study, the in vitro embryonic stem (ES) cell differentiation system was used. This system was applied previously to the study of hematopoiesis ontogeny by many investigators, without decisive progress. The method, used by Keller’s group to pin down hematopoietic progenitors, was to dissociate cells from very early embryoid bodies (3 to 3.5 days) 1 day prior to the establishment of the primitive erythroid lineage. These cells were plated in methylcellulose in the presence of VEGF and c-kit ligand (KL). Blast colonies developed within 2 to 3 days. These blast colonies were individually selected and replated in the presence of a broad spectrum of cytokines. Hematopoietic colonies with many different phenotypes developed. In particular, primitive erythroid colonies were present along with others, such as definitive erythroid, myeloid, and multilineage colonies. The use of genetic markers indicates that blast colonies were derived from a single cell, which thus had the potential to generate precursors of both primitive and definitive hematopoietic lineages. Many blast colonies expressed hematopoietic markers such as flk-1, SCL/TAL-1, CD34, and GATA-1, as well as PH1 and p major globins but not Brachyury, a mesodermal marker. As in the experiments by Eichmann et al,’9 the blast colonies required VEGF for their development. Endothelial-like cells may also be obtained in these conditions. CONCLUSION
The ontogenic emergence of the HSC destined to settle in the bone marrow as a permanent self-renewable pool is beginning to be understood. The differentiation and migration patterns worked out in the avian embryo, by means of chimeras constructed between labeled territories of the blastodisc, have been validated and extended by similar or complementary approaches in Xenopus or Rana (see ref. 72 for a review on these models) and the mouse. Clearly, the cells devolved to definitive hematopoiesis arise independently from those that engage at an early stage in primitive erythropoiesis, respectively, in the embryo proper and an extraembryonic region. In some amphibian species, however, precursors from the ventral blood island may participate in definitive hematopoiesis. In zebrafish embryos, although there are also different territories with respect to the emergence of hematopoietic precursors, their locations may overlap.n A common theme is that hematopoietic precursors become committed during an extended period of ontogeny in changing locations. The early and most peripheral ones-or most ventral depending on the anatomy of the embryo in different classes--engage entirely or mostly into primitive erythropoiesis. The later ones-more central or more dorsal-give rise to definitive, diversified hematopoiesis and display long-term renewal potential. There may be transient populations of cells that are endowed with intermediate properties. The mechanisms underlying these differences remain to be determined; they might be intrinsic to these precursors or imposed by the changing microenvironment. Finally, a principle ruling the emergence of the two classes of precursors is that they become committed in mesoderm tightly associated with endoderm. The growth factors and receptors involved in signaling between the two germ layers are beginning to be worked out, SO it should be possible in the near future,
to build a comprehensive model that will also account for the developmental relationships with the endothelial lineage.
ACKNOWLEDGMENTS I am very grateful to my collaborators and friends, without whom this article would not have been written: Isabelle Godin, Luc Pardanaud, Thierry Jaffredo, Michele Klaine, Marie-France Hallais, for their participation in the laboratory work; Marie-Franqoise Meunier for documentation and preparation of the manuscript; Franqoise Viala and Helene San Clemente for illustrations; Nicole Le Douarin and Alan Bums for reviewing the manuscript. I also want to thank Centre National de la Recherche Scientifique, Association de la Recherche sur le Cancer and Ligue Nationale contre le Cancer for their financial support.
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