A subset of megakaryocytes regulates development of hematopoietic stem cell precursors.

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

Murine AGM single-cell profiling identifies a continuum of hemogenic endothelium differentiation marked by ACE.

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.

Integrative transcriptomic analysis of developing hematopoietic stem cells in human and mouse at single-cell resolution.

Current understanding of hematopoietic stem cell (HSC) development comes from mouse models is considered to be evolutionarily conserved in human. However, the cross-species comparison of the transcriptomic profiles of developmental HSCs at single-cell level is still lacking. Here, we performed integrative transcriptomic analysis of a series of key cell populations during HSC development in human and mouse, including HSC-primed hemogenic endothelial cells and pre-HSCs in mid-gestational aorta-gonad-mesonephros (AGM) region, and mature HSCs in fetal liver and adult bone marrow. We demonstrated the general similarity of transcriptomic characteristics between corresponding cell populations of the two species. Of note, one of the previously transcriptomically defined hematopoietic stem progenitor cell (HSPC) populations with certain arterial characteristics in AGM region of human embryos showed close transcriptomic similarity to pre-HSCs in mouse embryos. On the other hand, the other two HSPC populations in human AGM region displayed molecular similarity with fetal liver HSPCs, suggesting the maturation in AGM before HSCs colonizing the fetal liver in human, which was different to that in mouse. Finally, we re-clustered cells based on the integrated dataset and illustrated the evolutionarily conserved molecular signatures of major cell populations. Our results revealed transcriptomic conservation of critical cell populations and molecular characteristics during HSC development between human and mouse, providing a resource and theoretic basis for future studies on mammalian HSC development and regeneration by using mouse models.

Protocol for the Generation of Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells.

This protocol offers a detailed procedure for the in vitro differentiation of human pluripotent stem cells (hPSCs) to multipotent hematopoietic progenitors that arise from SOX17+ hemogenic endothelium, mimicking intra-embryonic, HOXA-positive, aorta-gonad mesonephros (AGM) hematopoiesis. The generated endothelium displays transcriptional similarities to cells sorted from human 5-week AGM, and CD45+CD34+RUNX1C+ progenitors share an accessible chromatin profile with adult hematopoietic stem cells and multipotent progenitors. Therefore, this protocol is suitable for the mechanistic study of human multipotent progenitor development and for modeling childhood leukemias. For complete details on the use and execution of this protocol, please refer to Nafria et al. (2020).

Vast Self-Renewal Potential of Human AGM Region HSCs Dramatically Declines in the Umbilical Cord Blood.

Human hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region during Carnegie stages (CS) 14-17. Although we previously reported that these HSCs can generate no less than 300 daughter HSCs, their actual number has never been established. Here, we show that a single human AGM region HSC can generate 600-1,600 functional daughter HSCs. The presence of HSCs in the CS 17 liver in one case gave us a unique opportunity to describe a reduction of HSC self-renewal potential after liver colonization. From a clinical perspective, the efficacy of long-term hematopoietic regeneration depends on HSC self-renewal capacity. We quantitatively show that this capacity dramatically declines in the umbilical cord blood compared with HSCs in the AGM region. A full appreciation of the vast regenerative potential of the first human embryo-derived HSCs sets a new bar for generation of clinically useful HSCs from pluripotent stem cells.

Biomimetic aorta-gonad-Mesonephros-on-a-Chip to study human developmental hematopoiesis.

A fundamental limitation in the derivation of hematopoietic stem and progenitor cells is the imprecise understanding of human developmental hematopoiesis. Herein we established a multilayer microfluidic Aorta-Gonad-Mesonephros (AGM)-on-a-chip to emulate developmental hematopoiesis from pluripotent stem cells. The device consists of two layers of microchannels separated by a semipermeable membrane, which allows the co-culture of human hemogenic endothelial (HE) cells and stromal cells in a physiological relevant spatial arrangement to replicate the structure of the AGM. HE cells derived from human induced pluripotent stem cells (hiPSCs) were cultured on a layer of mesenchymal stromal cells in the top channel while vascular endothelial cells were co-cultured on the bottom side of the membrane within the microfluidic device. We show that this AGM-on-a-chip efficiently derives endothelial-to-hematopoietic transition (EHT) from hiPSCs compared with regular suspension culture. The presence of mesenchymal stroma and endothelial cells renders functional HPCs in vitro. We propose that the AGM-on-a-chip could serve as a platform to dissect the cellular and molecular mechanisms of human developmental hematopoiesis.

Blood making: learning what to put into the dish.

The generation of hematopoietic stem cells (HSCs) from pluripotent stem cell (PSC) sources is a long-standing goal that will require a comprehensive understanding of the molecular and cellular factors that determine HSC fate during embryogenesis. A precise interplay between niche components, such as the vascular, mesenchymal, primitive myeloid cells, and the nervous system provides the unique signaling milieu for the emergence of functional HSCs in the aorta-gonad-mesonephros (AGM) region. Over the last several years, the interrogation of these aspects in the embryo model and in the PSC differentiation system has provided valuable knowledge that will continue educating the design of more efficient protocols to enable the differentiation of PSCs into bona fide, functionally transplantable HSCs. Herein, we provide a synopsis of early hematopoietic development, with particular focus on the recent discoveries and remaining questions concerning AGM hematopoiesis. Moreover, we acknowledge the recent advances towards the generation of HSCs in vitro and discuss possible approaches to achieve this goal in light of the current knowledge.

The embryo of the silky shrew opossum, Caenolestes fuliginosus (Tomes, 1863): First description of the embryo of Paucituberculata.

The development of caenolestid marsupials (order Paucituberculata) is virtually unknown. We provide here the first description of Caenolestes fuliginosus embryos collected in the Colombian Central Andes. Our sample of four embryos comes from a single female caught during a fieldtrip at Río Blanco (Manizales, Caldas), in 2014. The sample was processed for macroscopic description using a Standard Event System and for histological descriptions (sectioning and staining). The grade of development of the lumbar flexure and coelomic closure differed between embryos, two of them being more advanced than the others (similar to McCrady's stages 30 and 29, respectively). The pericardial and peritoneal cavities were present, the hepatic anlage was organized in hepatic cords, the heart was in its final position, and the mesonephros was functional. Compared to other Neotropical marsupials, an early appearance of the frontonasal-maxillary fusion and the cervical growth (thickness) was observed; however, absorption of the pharyngeal arches into the body and lung development was delayed. Besides these differences, embryos were similar to equivalent stages in Didelphis virginiana and Monodelphis domestica. Previous proposals of litter size of four for C. fuliginosus are supported.

Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche.

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.

Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan.

The functional heterogeneity of hematopoietic stem cells (HSCs) has been comprehensively investigated by single-cell transplantation assay. However, the heterogeneity regarding their physiological contribution remains an open question, especially for those with life-long hematopoietic fate of rigorous self-renewing and balanced differentiation capacities. In this study, we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing Dll4 and CD44 in the mid-gestation mouse embryos, and could enrich all the HSCs of the embryonic day 11.5 (E11.5) aorta-gonad-mesonephros (AGM) region. We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-expressing cells at E9.5. Interestingly, most labeled mature HSCs in multiple sites (such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation. Furthermore, the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver (FL) and 6.9% of leukocytes in peripheral blood (PB) during one-year follow-up. Surprisingly, in aged mice of 24 months, the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages. Altogether, we demonstrated, for the first time, the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized, whose precise embryonic origin and molecular identity await further characterization.

Endothelial-to-haematopoietic transition: an update on the process of making blood.

The first definitive blood cells during embryogenesis are derived from endothelial cells in a highly conserved process known as endothelial-to-haematopoietic transition (EHT). This conversion involves activation of a haematopoietic transcriptional programme in a subset of endothelial cells in the major vasculature of the embryo, followed by major morphological changes that result in transitioning cells rounding up, breaking the tight junctions to neighbouring endothelial cells and adopting a haematopoietic fate. The whole process is co-ordinated by a complex interplay of key transcription factors and signalling pathways, with additional input from surrounding tissues. Diverse model systems, including mouse, chick and zebrafish embryos as well as differentiation of pluripotent cells in vitro, have contributed to the elucidation of the details of the EHT, which was greatly accelerated in recent years by sophisticated live imaging techniques and advances in transcriptional profiling, such as single-cell RNA-Seq. A detailed knowledge of these developmental events is required in order to be able to apply it to the generation of haematopoietic stem cells from pluripotent stem cells in vitro - an achievement which is of obvious clinical importance. The aim of this review is to summarise the latest findings and describe how these may have contributed towards achieving this goal.

Morphometric analyses and gene expression related to germ cells, gonadal ridge epithelial-like cells and granulosa cells during development of the bovine fetal ovary.

Cells on the surface of the mesonephros give rise to replicating Gonadal Ridge Epithelial-Like (GREL) cells, the first somatic cells of the gonadal ridge. Later germ cells associate with the GREL cells in the ovigerous cords, and the GREL cells subsequently give rise to the granulosa cells in follicles. To examine these events further, 27 bovine fetal ovaries of different gestational ages were collected and prepared for immunohistochemical localisation of collagen type I and Ki67 to identify regions of the ovary and cell proliferation, respectively. The non-stromal cortical areas (collagen-negative) containing GREL cells and germ cells and later in development, the follicles with oocytes and granulosa cells, were analysed morphometrically. Another set of ovaries (n = 17) were collected and the expression of genes associated with germ cell lineages and GREL/granulosa cells were quantitated by RT-PCR. The total volume of non-stromal areas in the cortex increased significantly and progressively with ovarian development, plateauing at the time the surface epithelium developed. However, the proportion of non-stromal areas in the cortex declined significantly and progressively throughout gestation, largely due to a cessation in growth of the non-stroma cells and the continued growth of stroma. The proliferation index in the non-stromal area was very high initially and then declined substantially at the time follicles formed. Thereafter, it remained low. The numerical density of the non-stromal cells was relatively constant throughout ovarian development. The expression levels of a number of genes across gestation either increased (AMH, FSHR, ESR1, INHBA), declined (CYP19A1, ESR2, ALDH1A1, DSG2, OCT4, LGR5) or showed no particular pattern (CCND2, CTNNB1, DAZL, FOXL2, GATA4, IGFBP3, KRT19, NR5A1, RARRES1, VASA, WNT2B). Many of the genes whose expression changed across gestation, were positively or negatively correlated with each other. The relationships between these genes may reflect their roles in the important events such as the transition of ovigerous cords to follicles, oogonia to oocytes or GREL cells to granulosa cells.

Analysis of Runx1 Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an In Vivo Approach.

Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis.

Adult zebrafish Langerhans cells arise from hematopoietic stem/progenitor cells.

The origin of Langerhans cells (LCs), which are skin epidermis-resident macrophages, remains unclear. Current lineage tracing of LCs largely relies on the promoter-Cre-LoxP system, which often gives rise to contradictory conclusions with different promoters. Thus, reinvestigation with an improved tracing method is necessary. Here, using a laser-mediated temporal-spatial resolved cell labeling method, we demonstrated that most adult LCs originated from the ventral wall of the dorsal aorta (VDA), an equivalent to the mouse aorta, gonads, and mesonephros (AGM), where both hematopoietic stem cells (HSCs) and non-HSC progenitors are generated. Further fine-fate mapping analysis revealed that the appearance of LCs in adult zebrafish was correlated with the development of HSCs, but not T cell progenitors. Finally, we showed that the appearance of tissue-resident macrophages in the brain, liver, heart, and gut of adult zebrafish was also correlated with HSCs. Thus, the results of our study challenged the EMP-origin theory for LCs.

Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis.

STUDY QUESTION: Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? STUDY FINDING: We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). WHAT IS KNOWN ALREADY: The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. STUDY DESIGN, SIZE, DURATION: This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. PARTICIPANTS/MATERIALS, SETTING, METHODS: We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. MAIN RESULTS AND THE ROLE OF CHANCE: We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. LARGE SCALE DATA: Non-applicable. LIMITATIONS REASONS FOR CAUTION: Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. WIDER IMPLICATIONS OF THE FINDINGS: Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro. STUDY FUNDING AND COMPETING INTEREST(S): M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011] and S.M.C.S.L. was funded by the Interuniversity Attraction Poles (IAP, P7/07) and the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). The authors declare no conflict of interest.

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

The first wave of T lymphopoiesis in zebrafish arises from aorta endothelium independent of hematopoietic stem cells.

T lymphocytes are key cellular components of the adaptive immune system and play a central role in cell-mediated immunity in vertebrates. Despite their heterogeneities, it is believed that all different types of T lymphocytes are generated exclusively via the differentiation of hematopoietic stem cells (HSCs). Using temporal-spatial resolved fate-mapping analysis and time-lapse imaging, here we show that the ventral endothelium in the zebrafish aorta-gonad-mesonephros and posterior blood island, the hematopoietic tissues previously known to generate HSCs and erythromyeloid progenitors, respectively, gives rise to a transient wave of T lymphopoiesis independent of HSCs. This HSC-independent T lymphopoiesis occurs early and generates predominantly CD4 Tαß cells in the larval but not juvenile and adult stages, whereas HSC-dependent T lymphopoiesis emerges late and produces various subtypes of T lymphocytes continuously from the larval stage to adulthood. Our study unveils the existence, origin, and ontogeny of HSC-independent T lymphopoiesis in vivo and reveals the complexity of the endothelial-hematopoietic transition of the aorta.

Multiple origins of embryonic and tadpole myeloid cells in Xenopus laevis.

Rabbit anti-serum against a myeloid-cell-specific peroxidase (Mpo) of Xenopus laevis was generated to identify myeloid cells in adult and larval animals. Smears of blood samples from adult hematopoietic organs were co-stained with Mpo and with XL-2, a mouse monoclonal antibody against a leukocyte common antigen. Lymphocytes found in the thymus and spleen were XL-2+Mpo- and granulocytes found in peripheral blood cells and the spleen were XL-2+Mpo+, indicating that double-staining with these two antibodies allowed classification of the leukocyte lineages. Immunohistochemical analysis of larval organs showed that XL-2+Mpo- cells were scattered throughout the liver, whereas XL-2+Mpo+ cells were present mainly in the cortex region. Interestingly, a cluster of XL-2+Mpo+ cells was found in the region of the larval mesonephric rudiment. The ratio of XL-2+Mpo+ cells to XL-2+ cells in the mesonephric region was approximately 80%, which was much higher than that found in other hematopoietic organs. In order to elucidate the embryonic origin of the myeloid cells in the tadpole mesonephros, grafting experiments between X. laevis and X. borealis embryos were performed to trace the X. borealis cells as donor cells. Among the embryonic tissues examined, the tailbud tissue at the early neurula stage contributed greatly to the myeloid cluster in the mesonephric region at stage 48. Therefore, at least four independent origins of the myeloid cell population can be traced in the Xenopus embryo.

Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region.

In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.