Embryonic hematopoiesis

Blood Cells, Molecules and Diseases 51 (2013) 226–231

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Embryonic hematopoiesis Rachel Golub a,b,⁎, Ana Cumano a a b

Unit of Lymphopoiesis, Department of Immunology, INSERM U668, Pasteur Institute, rue du Docteur Roux, 75015 Paris, France Université Paris Diderot, Sorbonne Paris Cité (Cellule Pasteur), rue du Docteur Roux ,75015 Paris, France

a r t i c l e

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Article history: Submitted 31 July 2013 Available online 13 September 2013 (Communicated by M. Lichtman, M.D., 19 August 2013) Keywords: Hematopoiesis Stem cells Embryo Fetal liver Fetal spleen

a b s t r a c t Blood cells are continually produced from a pool of progenitors that derive from hematopoietic stem cells (HSCs). In vertebrates, the hematopoietic system develops from two distinct waves or generation of precursors. The first wave occurs in the yolk sac, in mammals or equivalent embryonic structure, and produces nucleated primitive erythrocytes that provide the embryo with the first oxygen transporter and are, therefore, essential for the viability of the embryo. The yolk sac also produces myeloid cells that migrate to the central nervous system and to the skin to form the microglia and skin specific macrophages, the Langerhans cells. The second wave occurs in the dorsal aorta and produces multipotential hematopoietic progenitors. These cells are generated once in the lifetime from mesoderm derivatives closely related to endothelial cells, during a short period of embryonic development. Newly generated cells do not reconstitute the hematopoietic compartment of conventional recipients; therefore, they are designated as immature or pre-HSCs. They undergo maturation into adult HSCs in the aorta or in the fetal liver accompanied by the expression of MHC class I, CD45, CD150, Sca-1 and the absence of CD48. Differentiation of HSCs first occurs in the fetal liver, giving rise to mature blood cells. HSCs also expand in the fetal liver, and in a short time period (four days in the mouse embryo), they increase over 40-fold. HSCs and progenitor cells exit the fetal liver and colonize the spleen, where differentiation to the myeloid lineage and particular lymphoid subsets is favored. © 2013 Elsevier Inc. All rights reserved.

Introduction In mammals, blood cells are continually produced in the marrow through the expansion and differentiation of progenitors that originate from a rare cell population, the hematopoietic stem cells (HSCs). HSCs are generated once in the lifetime during embryonic development and production of mature hematopoietic cells starts in the fetal liver (FL) and continues in the bone marrow (BM). HSC transplantation is the most successful and widely used cell therapeutic approach to treat certain hematopoietic disorders. The scientific grounds that validated this approach date from the 1940s in mice and the 1960s in humans with the discovery of the existence of a population of cells in the BM with unique properties, multipotency and self-renewability. The experiments of Till and McCulloch [1,2]

Abbreviations: HSCs, hematopoietic stem cells; FL, fetal liver; FS, fetal spleen; BM, bone marrow; CFU-S, colony forming unit-spleen; LTR, long-term reconstitution; E, embryonic day; DC, dendritic cells; NK, natural killer cells; AGM, aorta gonads mesonephros; YS, yolk sac; S, somites; Rag, recombination activation gene; HIAC, intra aortic hematopoietic clusters; GFP, green fluorescence protein; FLOC, fetal liver organ culture; FSOC, fetal spleen organ culture; LTi, lymphoid tissue inducer; G-CSF, granulocyte colony-stimulating-factor; M-CSF, macrophage colony-stimulating-factor. ⁎ Corresponding author. E-mail addresses: [email protected] (R. Golub), [email protected] (A. Cumano). 1079-9796/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcmd.2013.08.004

showed that some mouse BM cells had the capacity to undergo clonal expansion in the spleen of irradiated recipients. The progenitor that founded the clone (colony) was called colony-forming-unit-spleen (CFU-S). The results also showed that a cell they designated the CFU-S could give rise to several different blood lineages and, therefore, was a multipotential progenitor. The concept of self-renewability derived from the observation that single cells could give rise to colonies that contained both differentiated blood cells and new CFU-S progenitors functionally identical to the colony founder [3,4]. These studies also established that clonal assays were the most reliable method to analyze differentiation potential [5,6]. Major efforts were concentrated in defining surface markers allowing prospective isolation of cells capable of long-term reconstitution of the hematopoietic lineage, in other words, the HSC. HSCs were initially identified in the lineage negative BM cell compartment, as determined by multiple antibodies that recognized differentiation markers on erythrocytes, myeloid, dendritic, T, B and NK cells, but that expressed high levels of c-kit and Sca-1 [7]. Negative selection for CD34 and Flk2 expression allowed their further enrichment to efficiencies of 1:3–1:5 in vivo reconstitution assays [8]. More recently, HSCs were identified as CD244−CD48−CD150+ cells [9,10]. HSCs isolated as Lin−Sca-1+c-kit+CD34−Flk2− or as CD244−CD48−CD150+ are largely overlapping. The identification of surface markers that subdivide the progenitor compartment and defines HSCs is also important to

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characterize discrete subsequent stages of differentiation that lose self-renewing capacity and are at the entry of a given pathways of lineage differentiation. In the last decades, the recognition that the properties of HSCs, if found in cells from other tissues, could allow their transplantation and consequent regeneration of these tissues, led to extensive efforts to identify stem cells in different organs. Progenitor cells were identified in many tissues. When they are injected in vivo, they give rise to cells from the tissue of origin and are therefore tissue specific. Some tissues, however, such as heart and some territories of the central nervous system do not appear to have an active adult regenerative compartment. Pluripotent cells, with broad differentiation potential, have not been reproducibly found in adult tissues. It is accepted that most, if not all, adult stem cell compartments are established during a defined period of embryonic development. The understanding of regeneration of adult tissues in steady-state conditions or after injury requires understanding the cellular and molecular processes involved in the development of the tissue. Specific delivery routes should also be envisaged. Systemic solid tissues such as skeletal muscle or peripheral nervous system will not be engrafted through circulating cells. Injection of non-hematopoietic progenitors in circulation results in their retention to more than 90% in the capillary networks of the lungs and the liver. The multiple attempts to regenerate tissues other than the cornea and hematopoiesis through circulatory stem cell injection have not yielded an improvement of the patient's conditions to our knowledge. The use of undefined cell populations administered by inappropriate routes will not improve our understanding of the physiology of a particular organ or system and solid basic knowledge is required before regenerative approaches might have a chance to be as successful as HSC transplantation has been.

Embryonic hematopoietic sites The yolk sac The first hematopoietic cells of embryonic origin are present in the yolk sac (YS), starting at mouse embryonic day (E) 7.5. The YS blood islands, generated from condensed cellular aggregates of morphologically identical cells, harbor the first endothelial and hematopoietic progenitors. Blood islands fuse to form the vascular network of the YS. This sequence of events suggests that both endothelial and hematopoietic cells share a common progenitor designated the hemangioblast. Attempts to confirm this hypothesis and isolate this cell type have not been conclusive. Recent advanced genetic approaches [11] led to the conclusion that each YS blood island is formed by more than one progenitor and contribution from single progenitors to both lineages was not consistently observed, suggesting that blood islands are formed by aggregation of distinct progenitors committed to the endothelial or hematopoietic lineages. The YS origin of HSCs was initially proposed, based on the unique early hematopoietic activity in this site. Following injection of YS cells in the embryonic circulation, T cells of donor origin were detected in the thymus of the recipient, bringing experimental evidence to the hypothesis of the YS origin of HSCs [12]. Pioneering experiments carried out in the avian model, however, indicated otherwise. Quail-chicken and congenic chicken chimeras showed that YS hematopoietic cells were only capable of transient contribution to the hematopoietic compartment [13–15]. In these chimeras, definitive hematopoiesis had an intra-embryonic origin. Additional evidence came from experiments done in Xenopus where YS (ventral blood islands) and intra-embryonic blood compartments (dorsal lateral plate) were derived from different blastomeres of 32-cell embryos and therefore independently generated [16]. Although the experiments in chicken, Xenopus and later in the mouse demonstrate that HSCs have an intra-embryonic origin, the

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multiple and sequential generation of HSCs in multiple sites is still being debated [17,18]. Initially, YS blood islands contain erythrocytes from the primitive lineage that have a large size and are nucleated [19]. In Zebrafish, a population of macrophages that originates from the ventro-lateral mesoderm and migrates to the YS, has been detected as early as the first erythrocytes. These cells bypass the conventional developmental pathway that lead to macrophage differentiation in the hematopoietic organs [20]. In the mouse, after the first nucleated erythrocytes there is, similarly to Zebrafish, a wave of macrophage progenitors (2–4 somite stage S) shortly followed by the generation of erythro-myeloid precursors (4–6S) [21,22]. Lymphocyte progenitors are not consistently detected in the YS, prior to the establishment of circulation (N 5S). After circulation is established, it is unclear whether additional waves of hematopoietic generation occur in situ and whether they contribute to adult hematopoiesis. Although suspected for a long time, the function and fate of the early YS myeloid progenitors have been recently reassessed. It was shown that they migrate via the circulation to the central nervous system where they form a stable macrophage compartment, the microglia. Although a slow turnover rate of these cells is difficult to rule out, it appears that most adult microglial cells are of embryonic YS origin [23]. YS macrophages also contribute to epidermal specific macrophage pool called the Langerhans cells, as well as to liver Kupffer cells. Their independence from the expression of the transcription factor cmyb, suggests that these particular subsets of macrophages do not follow the developmental pathway taken by the progeny of HSCs [24]. Intra-embryonic hematopoietic generation Experiments in several animal models, as previously mentioned, indicated that YS hematopoietic generation is independent from intraembryonic generation and that there is no conclusive evidence of its contribution to adult hematopoiesis. Isolating defined embryonic structures and analyzing their in vivo reconstitution capacity obtained the initial evidence that, in the mouse, progenitors of intra-embryonic origin were multipotential and capable of longterm reconstitution of the hematopoietic system. In one experimental approach, the engraftment of splanchnic mesoderm, an embryonic structure close to the aorta anlage of 10–18S embryos, under the kidney capsule of severe combined immune deficient (SCID) mice, resulted in the reconstitution of the lymphoid compartment [25]. Under the same conditions YS yielded no lymphocytes, indicating that only the intraembryonic region contained HSCs. This same embryonic structure isolated at the stage where it evolved into a territory containing the anlage of the aorta, the gonads and the mesonephros (AGM) [26] yielded the first CFU-S. These two experimental approaches relied on the precise identification and careful dissection of transient embryonic structures, at precise developmental times. Shortly thereafter, it was shown that, not only AGM cells functioned as bone fide CFU-S, but also that they were capable of long-term reconstitution (LTR) of the hematopoietic compartment, after E10 [27]. LTR activity was detected in the YS and in the FL at later stages and followed similar kinetics to that observed for CFU-S. These results supported the idea that HSCs were generated in the intra-embryonic AGM, but do not provide direct evidence for this, as cells originated elsewhere could circulate to the aorta region. Direct evidence for an AGM origin of multipotential progenitors came from experiments where short periods of organ culture allowed the development of progenitors so that they became detectable in conventional hematopoietic assays. In this work, the splanchnic mesoderm was dissected along with the YS and the remains of the embryo body from E7 to E8.5, around the time where circulation and YS hematopoietic activity are established. After a few days in organ culture, multipotential progenitors were detected exclusively in the intra-embryonic territory, but not in YS or in the remaining embryo [28]. These results firmly established the splanchnic mesoderm/AGM origin of lympho-myeloid multipotential progenitors and their

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independence from YS. Cells within any of these explants were however incapable of reconstituting the hematopoietic system of normal hosts. Immune-deficient hosts proved useful to reveal HSC activity from tissues isolated early in embryonic development. Ragγc knock-out mice (Ragγc−/−) are double mutants for the Rag 2 and for the common γ cytokine receptor and, therefore, have neither T or B lymphocytes, nor NK cell activity. When reconstituted with cells recovered from cultured explants of E8, long-term reconstitution was observed in hosts that received splanchnic mesoderm, but not YS [29,30]. The efficiency of reconstitution was however consistently lower than that obtained with E11 AGM. The requirements for FL or BM HSC colonization and differentiation might be distinct, since the FL is of endodermal origin, whereas the BM derives from mesoderm. In consequence, testing embryonic cells by their capacity to reconstitute adult organs might not be optimal to assay the properties of these cells in vivo. These results can be explained by at least two non-exclusive possibilities. Successive waves of generation result in the production of multipotential cells without reconstitution capacity and later of HSCs that establish the hematopoietic system. Alternatively, there is a unique wave of hematopoietic progenitors that need further maturation to acquire reconstitution capacity, possibly through specific interactions with a specialized environment. Several lines of evidence suggest that hematopoietic progenitors are generated in a short window of development: 1. In vivo fate mapping studies of labeled cells expressing VE-Cadherin [31] or Runx1 [17], found in endothelial and hematopoietic cells, showed that induction of Cre recombinase, between E8.5 and E9.5, results in the expression of the reporter protein in virtually 100% of adult HSCs. When induction is done before or after this short time-window, reporter expression in adult cells is either very low or undetectable, indicating that mesoderm progenitors are specified in the hematopoietic lineage, once in the lifetime, between E8.5 and E9.5–10. If there is a single short window of generation, it is unlikely that HSCs are generated in multiple sites, at different times. 2. The number of multipotential hematopoietic progenitors, present in the AGM across mid-gestation, sharply increases between E9 and E10, reaches a maximum at E10.5, and decreases thereafter, a result compatible with a single wave of hematopoietic cell production [32]. Immature or pre-HSCs The existence of a unique wave of hematopoietic progenitor generation in the AGM together with the observation that newly generated progenitors cannot efficiently reconstitute the adult hematopoietic system raises the possibility that these cells are the progenitors of adult HSCs. There is evidence that hematopoietic determination occurs prior to the acquisition of LTR activity [30,33,34]. Using a modified reaggregation organ culture, it was shown that extensive HSC generation could be obtained from a VE-Cadherin+CD45+ cell population [33]. Later, the same authors reported that maturation into adult HSCs occurred in a three-step process initiated by VE-Cadherin+CD45− CD41+ (type I) pre-HSCs that became VE-Cadherin+CD45+CD41+ (type II) pre-HSC that further acquired HSC properties [34]. Using a related approach [30], we have shown that the first progenitors capable to engraft immunodeficient recipient mice are detected in the AGM as early as at the E9 15–20S stage and in FL at the E10 30S stage. They can further mature into HSCs in the FL or in vitro with OP9 stromal cells. This maturation process involves acquisition of CD45 consistent with previous studies [34], of the LSK CD150+CD48− phenotype [9], but also MHC class I expression that confers the inhibitory properties of NK cell activity. This observation indicates that MHC class I expression is a major event conferring LTR capacity in conventional recipients to immature HSCs [30]. These aggregate results favor a single wave of generation model during which hematopoietic progenitors are specified between E8.5

and E10 [31,17] in the dorsal aorta as immature or pre-HSC. These cells mature in the aorta or after migrating to the FL through the expression of MHC class I, CD45 and likely other mechanisms that remain to be investigated. Other potential hematogenic sites. Placenta The AGM is a complex territory comprising the dorsal aorta but also the anlage of the gonads and mesonephros. Newly generated hematopoietic progenitors are concentrated in the ventral aspect of the aorta in cell formations protruding from the endothelial layer, which are hematopoietic intra-aortic clusters (HIAC). Similar to HIAC, cell aggregates are also found in the omphalomesenteric and vitelline arteries [32,35,36], and it is, therefore, possible that this process also takes place in other major blood vessels connecting the embryo to the placenta and the YS. In birds, the allantois is a diverticulum that projects in the extraembryonic compartment and opens in the terminal part of the hindgut. The allantois is lined by endoderm and covered by mesoderm. Quail allantois grafted into chicken embryos resulted in partial BM colonization by donor derived hematopoietic cells, suggesting that allantois might also contribute to hematopoiesis [37]. In mouse and man, the placenta has been suggested as a site providing a favorable environment for HSC maintenance and/or expansion [38,39]. The mouse mid-gestation placenta contaINS long-term reconstituting cells in numbers that parallel those found in the AGM. However, it has been difficult to determine whether the mammalian placenta also could be a site for HSC generation, as suggested. Immunodeficient mice are robustly engrafted with human AGM cells before LTR activity can be found in placenta. Instead, early human placenta provides long-term T cell repopulation of maternal origin [40]. This observation highlights the expansion capacity of mature lymphocytes when placed in an immunodeficient host and the need for strict evaluation of reconstitution by the analysis of the myeloid compartment. In these experiments, the recipient mice [40] did not show substantial engraftment before 5 months post-transplantation after which they were still capable of secondary engraftment, indicating the presence of human HSCs. HSC activity is detected in E9.5 placenta of mice that lack heart beat (Ncx1−/−) and where cells should not transit via circulation [41]. These embryos that die around E10 were analyzed at earlier time points (E8–E9.5) and contained lymphoid and myeloid progenitors in YS, AGM and placenta although lower number of hematopoietic cells were detected, in mutant mice. This result raises a number of questions: 1. It is possible as suggested by the authors that placenta is a site of hematopoietic cell generation; 2. the stem cell activity in these embryos was not directly assessed so the actual generation of HSCs has not been addressed but only that of lymphoid and myeloid progenitors [42,42]; 3. it has been well documented that the omphalo-mesenteric and vitelline arteries, similarly to the aorta, are a site for generation of multipotential hematopoietic progenitors. These are major vessels that connect the embryo proper to the YS and to the placenta. The extent to which the generation process expands within the vessels into the territory that is isolated as YS or placenta, is unknown. It is therefore possible that some hematogenic activity attributed to these extra-embryonic sites actually comes from the major arteries contaminating the explant. Phenotype of the hematopoietic stem cell The first marker characteristic of embryonic HSCs was identified when different subsets of FL cells were analyzed for LTR activity. The surface marker AA4.1 (CD93) was identified as labeling a population that contained all HSCs [43]. In contrast to BM, FL HSCs are also CD34+. The first attempts to characterize the progenitor compartment in AGM indicated that AA4.1 was also expressed by AGM progenitors while Sca-1 was undetectable [44]. The newly characterized phenotype CD150+CD48− that identifies LTR cells in the BM can also be found in FL

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after E12. Before that stage Sca-1 is virtually undetectable by antibody staining and the CD150+CD48− phenotype is not established before E13. All three markers are readily detected in immature HSCs derived from E9 AGM after culture with OP9 cells and thrombopoietin [30]. Considering that generation of hematopoietic progenitors is likely completed by E11 [17], these observations strengthen the idea that immature or pre-HSCs are the progenitors of adult HSCs and that maturation occurs primarily in FL. As in FL, c-kit and CD34 are markers of AGM repopulating cells [45]. In situ labeling of cells in the HIACs showed that another marker, CD41, that is only present in BM megakaryocytes, also marks AGM multipotential progenitors [34,46]. Thus c-kit and CD41 are the phenotypic markers that identify the first hematopoietic cells. These cells also co-express CD31, CD34, AA4.1, VE-Cadherin and Tie-2, all of which are also present on endothelial cells [46], and they do not have detectable levels of the pan-hematopoietic marker CD45. This finding led to the notion that both lineages are developmentally related. From the analysis of the hematopoietic activity of sorted cells, several features can be highlighted: 1. Strictly hematopoietic (c-kit and CD41) and endothelial/hematopoietic cell (CD34, CD31, VE-Cadherin, Tie-2) markers are co-expressed by cells with a unique hematopoietic potential, even in the absence of CD45. This pattern of expression created some confusion because incomplete analysis led to the wrong affiliation of hematopoietic cells to the endothelial lineage. 2. By E11, most hematopoietic cells express CD45 that is progressively acquired in c-kit+ cells. 3. The set of markers mentioned labels most colony-forming cells, irrespective of their multipotential nature and of their origin, so that erythro-myeloid YS cells that cannot generate lymphocytes express the same set of markers as AGM-derived reconstituting cells. The fetal spleen The spleen is an organ located in the posterior part of the abdomen, in close contact with the omentum and the pancreas. While the pancreas originates from a mesodermic induction of the endoderm [47], the spleen is considered of exclusive mesoderm origin [48]. The spleen has hematopoietic capacity during the embryonic and neonatal period and thereafter mainly functions as a secondary lymphoid organ. Hematopoietic progenitors in the fetal and neonatal spleen During embryogenesis and the first weeks of life, the spleen sustains hematopoietic capacity to fulfill secondary lymphoid organ function in adult mammals. The fetal spleen (FS) first develops as a splenopancreatic mesenchyme. The separation from the pancreas starts around E11.5 with the expression of Bapx1 and the Fgf10 gradient formation required for the pancreatic bud activation [49] Soon after, multiple types of hematopoietic cells colonize the splenic mesenchyme. F4/80+ monocytes and large nucleated basophilic erythroblasts were first detected in E12 FS [50]. At this stage, the splenic rudiment has not been shown to have hematopoietic potential suggesting a yolk sac origin for these subsets. Indeed, F4/80+ macrophages and erythrocytes derived from E8 YS blood islands colonize the embryo between E9.5 and E10.5 [22,51,52]. Myeloid cells derived from the YS are Myb-independent and could further be distinguished from Myb-dependent HSCs [24]. Splenic F4/80high tissue macrophages are present in Myb-deficient embryos in normal numbers whereas F4/80low are absent supporting the YS origin for these F4/80+ macrophages [24]. The expansion of F4/80high macrophages in fetal spleen just precedes the appearance of endogenous hematopoiesis. HSCs circulating in the embryo [53,54] constantly colonize the spleen. The splenic pool of HSCs is phenotypically and functionally identical to its fetal liver counterpart [9] as confirmed by long-term reconstitution assays using E13.5 FS. HSCs are infrequent in FS since no reconstitution was

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observed below 4 embryonic equivalents [53]. FS HSCs barely represent 0.1% of Lin− cells at E15.5 as observed by flow cytometry. The fetal spleen HSC frequency of engraftment was determined to be less than 5 HSCs per spleen before E15.5 considering numerous reconstitution assays. The HSC pool increases to reach more than 100 HSCs per spleen around E17.5 (R. Golub, unpublished data). The increase of the HSC number is observed between E16.5 and E18.5 [9,54]. The HSC pool is supposed to seed the spleen until 2 weeks of age during the transitional period between FL and BM hematopoiesis [54,55]. Thereafter, HSCs are found as small, barely detectable fractions in the adult spleen as well as in other extramedullary sites [56]. Compared to their BM counterpart, the adult splenic HSCs were described as identical in terms of repopulating capacity whereas cycling twice more often [57]. Hence, the HSC niches are quite different between the adult spleen and BM. Besides, the FS environment is already different from the FL since no niche for the maintenance, expansion, or survival of HSCs exists in the FS [53]. The only fate of the HSC progeny in the fetal splenic environment is myeloid. Depending on the time-point, myeloid progenitors (Lin− cKit+Mac1+) were found mixed with mature F4/80+ Mac1+/− macrophages [53]. Fetal spleen stroma cell lines were generated to analyze the in vitro differentiation of HSCs in a “splenic stromal content.” Fetal liver HSCs cultured on fetal spleen stromal cells for 7–10 days could differentiate into CD11c− F4/80− Mac1+ precursors and CD11c− F4/80+ Mac1+ cells. Transcripts specific of monocyte/macrophage development such as c/Ebpβ, PU.1 and myeloperoxidase (Mpo) are expressed by these populations. Transcripts for toll-like receptors (Tlr) 4 and 9 and M-csfr were only detected in the F4/80+ subset. Unlike their precursors, fetal spleen F4/80+ derived macrophages were capable of latex bead phagocytosis and express MHC class II and CD80 [53]. Thus, fetal splenic macrophages are partly derived from the endogenous splenic HSC pool. During embryogenesis, macrophages have been found associated with nucleated erythrocyte precursors in erythroblastic islands, suggesting a role in splenic erythropoiesis [53]. From E12 to birth, the FL is the main site for definitive erythropoiesis while the bone marrow produces the blood cells during the neonatal and adult life. A few studies have shown that the fetal and neonatal spleen serves as an additional erythropoietic site [58]. Data obtained from microscopic observations showed some rare proerythroblasts as early as E12.5. At this early stage, erythrocytes may be YS derived [19]. The kinetic analysis showed a clear population of proerythroblasts at E13 followed by the appearance of basophilic erythroblasts at E14.5 and orthochromatic erythroblasts expelling their nuclei at E15.5 [59]. From E16.5 to birth, most of splenic red cell precursors are polychromatic erythroblasts, some of them already containing ferritin. The same cell types predominate in one-week newborns, although a few proerythroblasts were still detected in spleens at birth [59]. The spleen contribution was also observed using colony-forming progenitor cells (CFC), colony-forming unit erythrocytic (CFU-E) and burst forming unit erythrocytic (BFU-E) assays [55]. Besides, erythroblastic differentiation is evident as soon as E13 by the presence of all erythrocyte progenitor stages [55,59–61]. Moreover, a kinetic study was performed by cytometry using the combination of CD71 and Ter119 markers [61]. At E15.5, clear erythroid populations are already distinguished from the immature proerythroblasts (Ter119intCD71hi), basophilic erythroblasts (Ter119hiCD71hi), late basophilic and polychromatophilic erythroblasts (Ter119hiCD71int) to the mature orthochromatophilic erythroblasts (Ter119hiCD71-/lo). However, before E15.5, orthochromatophilic erythroblasts are rare (beyond 4% of splenocytes) limiting their possible detection by microscopy. The increase of the late erythroid precursor percentage from E13.5 to E15.5 supports the role of the fetal spleen as an erythropoietic organ. Fetal and neonatal splenic erythropoietic development have been proposed to be differentially regulated since neonatal

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development required the T3 thyroid pathway and fetal environment is considered a hypothyroid milieu [62]. Hematopoietic differentiation in fetal spleen From E12.5 to E16.5, hematopoietic progenitors increase in the FS in parallel with the overall cellularity of the spleen that doubles each day. Erythromyeloid and lymphoid progenitors were observed as early as E12.5 [32,63]. Between E14.5 and E15.5, FS hematopoietic cells lack lineage specific markers (Lin−) being principally Sca-1− c-Kitlo. The splenic Lin−CD4int progenitors transiently reconstitute the B and NK compartment of alymphoid mice [63]. To assess their in situ differentiation, E15.5 fetal spleen explant organ cultures (FSOC) were reconstituted. In FSOC, Lin− progenitors could differentiate into B and NK lymphocytes as well as myeloid cells [32,63]. Ex vivo, B cell progenitors were detected in the Lin−CD4intRAG2hi subset and T/NK cell progenitors in the Lin−CD4intRAG2int subset from E14.5 fetal spleen [63]. Indeed, B cell progenitors are already present in the spleen at E12.5, since E12.5 FSOC could differentiate B cells after 15 days of culture [32]. Ex vivo, a low percentage of B cells (CD19+B220+RAG2+) was already observed at E13.5 [53] and the first IgM+ cells were identified at E17.5 [53,64]. Few immature NK cells (CD3−CD122+NK1.1+DX5+) are present in E15.5 fetal spleen, whereas mature NK cells (CD3−CD122+NK1.1+DX5+CD43+Mac-1+) arise by E16.5 [63,65]. Similarly to their FL counterpart, most of the fetal splenic NK population expresses Trail and a restricted Ly49 repertoire with reduced cytokine secretion and cytotoxic capacities [66]. Both B and NK populations expand from E17.5 to birth. In two weekold neonates the lymphopoietic activity of the spleen is interrupted and the spleen only supports B cell maturation. So far, splenic dendritic cell (DC) development has not been investigated during fetal life. Most of the experiments have been done on neonatal spleen since birth is a transitional period from the sterile intra-uterine environment to an enriched microbial environment. DCs are major players in the establishment of the immune function of the spleen, since they initiate T-cell dependent immunity. Both conventional DC (cDC) and plasmacytoid DC (pDC) can be detected in the fetal thymus by E17 and increase rapidly in number until week 5–6 of age with changes in phenotype and function, in parallel to the increase of B and T cells. At birth 0.2% of murine splenic cells are DCs. pDCs represent the major subset of splenic DC in neonates contrary to the adult. The number of pDCs in the spleen reaches adult levels by 3 weeks of age. Splenic conventional DCs identified at day 1 after birth mainly correspond to the CD4−CD8−CD11c+ DC subset. The CD4+CD8−CD11c+ DC subset is found in 7d neonates spleen in small percentages and slowly increases. In the adult, the CD4−/CD4+ ratio is inversed with CD4+ as the predominant splenic DC subset [67,68]. During the second week of life, the CD4−CD8+ DC subset appears progressively in the spleen. The double-negative subset that constitutes the majority of neonatal DCs harbors similar properties to the CD8+ DC subset and may be considered the neonatal precursor of these cells [67]. At the functional levels, ex vivo splenic cDCs express lower levels of MHC class II, CD86 and major cytokines like IL-12p70 in neonates. The splenic stroma has been shown to be involved in the differentiation and maturation of DCs [69]. Neonatal splenic stromal cells could provide a suitable environment for the maturation of DCs into regulatory DCs, which can regulate the immune response in the spleen [70]. Moreover, neonatal splenic progenitors in the Lin− ckit+ myeloid fraction are able to give rise to a DC subset after contacts with the splenic endothelium [71]. The secondary lymphoid organs start their organization after birth. As seen for the anlagen of all future secondary organs, the fetal spleen also contains the lymphoid subset of lymphoid tissue inducer (LTi) cells [72–74]. Fetal spleen LTi cells are specially found in a periarteriolar position where homeostatic chemokines can be detected in stromal and endothelial cells. These cells are situated in proximity to B cells in a zone

that corresponds to the white pulp anlagen [75]. The LTi cells originate from lymphoid-restricted FL progenitors that migrate via the mesenteric vessels between E11.5 and E15.5 to colonize the gut, the spleen and LN [74,76,77]. Thus the fetal spleen is a privileged site for the differentiation of particular subsets of hematopoietic cells.

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