Educational Session 1 Chairman: W.E. Fibbe Haematologica 1999; 84:(EHA-4 educational book):1-3 Biology of normal and neoplastic progenitor cells Emergence of the haematopoietic system in the human embryo and foetus MANUELA TAVIAN,* FERNANDO CORTÉS,* PIERRE CHARBORD,° MARIE-CLAUDE LABASTIE,* BRUNO PÉAULT* *Institut d’Embryologie Cellulaire et Moléculaire, CNRS UPR 9064, Nogent-sur-Marne; °Laboratoire d’Étude de l’Hé- matopoïèse, Etablissement de Transfusion Sanguine de Franche-Comté, Besançon, France he first haematopoietic cells are observed in es. In this setting, the recent identification in animal the third week of human development in the but also in human embryos of unique intraembryonic Textraembryonic yolk sac. Recent observations sites of haematopoietic stem cell emergence and pro- have indicated that intraembryonic haematopoiesis liferation could be of particular interest. occurs first at one month when numerous clustered We shall briefly review here the successive steps of CD34+ Lin– haematopoietic cells have been identi- human haematopoietic development, emphasising fied in the ventral aspect of the aorta and vitelline the recent progresses made in our understanding of artery. These emerging progenitors express tran- the origin and identity of human embryonic and fetal scription factors and growth factor receptors known stem cells. to be acting at the earliest stages of haematopoiesis, and display high proliferative potential in culture. Primary haematopoiesis in the human Converging results obtained in animal embryos sug- embryo and foetus gest that haematopoietic stem cells derived from the As is the case in other mammals, human haema- para-aortic mesoderm – in which presumptive endo- topoiesis starts outside the embryo, in the yolk sac, thelium and blood-forming activity could be detect- then proceeds transiently in the liver before getting ed as early as 3 weeks in the human embryo by dif- stabilised until adult life in the bone marrow. Only T ferential expression of the CD34 and Flk-1/KDR genes lymphocytes are produced in the same tissue at – play an essential role in the foundation of definitive embryonic, foetal and postnatal stages. haematopoiesis. Aorta-associated CD34+ cells also The yolk sac represent a unique localised accumulation of primi- tive haematopoietic stem cells worthy of in-depth It is at about 18.5 days of development (early head molecular characterisation. Differential screening of process) that primitive haematopoietic cells appear a cDNA library has already revealed the expression of inside forming blood vessels in the intermediate novel genes in this population, one of which appears mesodermal cell layer of the human yolk sac wall. to be involved in the development of both haema- Studies on human haematopoietic cell emergence at these early stages are scarce, but our own observa- topoietic and endothelial cells. Active blood forma- 1,2 tion is observed in the liver and bone marrow by the tions suggest that the sequence described in animal end of the first trimester. Inception of haemato- models also applies to the human yolk sac: meso- poiesis occurs earlier in the liver, where CD34+ cells derm-derived clusters of primitive haematopoietic are detected as early as 30 days, than in the marrow, stem cells – the blood islands – develop in close asso- where haematopoietic cells are not observed before ciation with the endothelium of emerging blood ves- week 11. sels, possibly from a common ancestor cell or hae- Current interest in early human blood cell ontoge- mangioblast. The coexpression of the CD34 surface ny may be partly related to the growing use of foetal molecule by haematopoietic precursor cells and stem cells for transplantation at postnatal stages, endothelial cells can be traced back to these initial stages, which may support the hypothesis of their and to emerging cell and/or gene therapies of the 1,2 3 blood system in utero, which justify a thorough char- common origin. Migliaccio et al. described several acterisation of embryonic and foetal human haema- generations of clonogenic progenitors in the human topoiesis. In addition, the prenatal haematopoietic yolk sac from 4.5 weeks of development, including system is characterised by an outstandingly high rate pluripotential (CFU-GEMM), granulomonocytic of progenitor cell expansion, migration and differ- (CFU-GM) and erythroblastic progenitors (BFU-E entiation and hence can be seen as a privileged mod- and CFU-E). The human yolk sac starts regressing at el to identify novel factors involved in these process- about 45-50 days post-ovulation and virtually all clonogenic progenitors have disappeared from that tissue by week six. Correspondence: Bruno Péault, Unité 506 INSERM, Bâtiment Lavoisier; The liver Groupe Hospitalier Paul Brousse, 12, avenue Paul Vaillant-Couturier, th 94807 Villejiuf Cedex, France. Tel. international +33-1-45595263 – The liver emerges during the 4 week of develop- Fax: international +33.1.45595268 – E-mail: [email protected] ment when the hepatic bud, an endodermal out- 2 M. Tavian et al. growth of the foregut, invades the adjacent mesoder- Concepts on the filiation of the stem cells that mal septum transversum. These two tissues con- found definitive haematopoiesis have changed in the tribute hepatocyte cords and vascular sinuses, respec- past few years with the demonstration that, in ani- tively. We have detected CD45+CD34– haematopoi- mals, these emerge inside the embryo, and not in the etic cells from day 23 of development in the liver yolk sac as previously believed. In mice and birds the anlage while the first CD34+ haematopoietic progen- original blood-forming territory develops intrinsically itors could be recognised on day 30.1 In vitro colony- in the para-aortic splanchnopleural mesoderm con- forming cells, i.e. BFU-E, CFU-GM and, slightly later, stituting the presumptive aorta-gonad-mesonephros CFU-E have been indeed detected at 4.5-5 weeks in (AGM) region of the embryo and contributes, at pre- the liver rudiment, where their frequency then increas- liver stages, multilineage haematopoietic progenitors es dramatically, paralleling their sharp decline in the and eventually long-term reconstituting true haema- yolk sac.3 At the end of the first trimester, and topoietic stem cells. The existence in the human onwards, more primitive progenitors – CFU-GEMM embryo of an equivalent site of haematopoietic stem and HPP-CFC – have also been detected in the liver. cell generation has been suggested by the identifica- Earlier studies, confirmed by more recent immuno- tion, at 4-6 weeks of development, of numerous clus- histochemical approaches, have documented the tered CD34+ haematopoietic cells on the ventral extensive erythro-myeloid haematopoiesis that takes endothelium of the aorta and vitelline artery.1,6 These place extravascularly in the human embryonic and cells express surface antigens that typify early blood foetal liver, and have stressed the prominence of ery- cell progenitors, being CD45+, CD34++, CD31+, thropoiesis therein (reviewed in ref. #4). Other CD43+, CD44+, CD164+, but display no CD38 or lin- myeloid cells present in the haematopoietic liver are eage-specific markers. In situ hybridisation on embryo granulocytes, macrophages and rare megakaryocytes. sections and screening of cDNA libraries prepared B-lymphopoiesis has been traced in the liver from from these sorted aorta-adherent progenitors have about 9 weeks of gestation by detection of surface also revealed that they express genes known to be IgM+ cells. associated with the early steps of haematopoietic development, such as Tal1/SCL, c-myb, GATA-2, The bone marrow GATA-3, flk-1/VGEFR2 and c-kit.7 When directly A cartilaginous presumptive skeleton is present in assayed in methylcellulose, human intraembryonic the 6-8-week human embryo. Bone rudiments are aorta-associated CD34+ cells exhibited negligible + then surrounded by a dense network of CD34 capil- clonogenic potential. In contrast, following a 4-10- + laries, by CD68 monocytes and by osteoblast pre- day co-culture on murine bone marrow stromal cells cursors which all invade the diaphyseal cartilage at (MS-5 cell line), they generated about six times more 8.5-9 weeks. Incoming macrophages rapidly digest progenitors, which yielded large multilineage colonies the cartilage, leaving only intact small islets of chon- in methylcellulose, than the liver rudiment.6 Of note, drocytes that soon become surrounded by osteo- the para-aortic splanchnopleura – but no other intra- blasts, from which ossification proceeds in a typical- embryonic tissue – exhibited dramatic haematopoietic ly endochondral manner. In-between ossifying tra- potential in culture as early as day 23 of development, beculae, large vascular sinuses develop leading to the i.e. several days before CD34+ stem cells can actually completion, at about 10 weeks, of bone marrow cav- be identified on the aortic wall. This result, as well as ities.5 Marrow haematopoiesis starts during the 11th provocative semi-thin section histology pictures, sug- week of development in specialised mesodermal gest that haematopoietic stem cells emerge from structures or primary logettes, constituted by a loose mesoderm in that territory, and not merely migrate network of mesenchymal cells supported by dense fib- there from another location.6 rillar material and surrounding a central artery, inside Differential screening of a cDNA library built from which CD15+ granulocytes appear first, closely fol- sorted embryonic aorta-associated CD34+ cells with lowed by erythroid cells. Haematopoiesis then devel- probes prepared from embryonic liver and foetal bone ops dramatically in rapidly enlarging logettes which by marrow CD34+ stem cells is in progress. Several dif- week 15 are densely packed with cells of the erythroid ferentially expressed genes have already been found, and granulocytic series.5 one of which encodes a serine-threonine kinase which, interestingly, is co-expressed in all developing endo- Haematopoietic stem cell emergence in thelial and haematopoietic stem cells.7 This argues early human development for the existence of haemangioblasts, i.e. common prog- As mentioned above, the emergence of the CD34 enitors for vascular and blood cells.