The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.

The Innate Lymphoid Cell (ILC) family is a relatively recently described immune cell family involved in innate immune responses and tissue homeostasis. Lymphoid Tissue Inducer (LTi) cells are part of the type 3 (ILC3) family. The ILC3 family is the main ILC population within the embryo, in which the LTi cells are critically associated with embryonic lymph node formation. Recent studies have shown more insights in ILC origin and residency from local embryonic and tissue resident precursors. Embryonic LTi cells originating from a different hemogenic endothelial source were shown to be replaced by HSC derived progenitors in adult. This review will discuss the layered origin of the ILC3 family with an emphasis on the LTi cell lineage.

The journey of a hematopoietic stem cell (HSC) involves the passage through successive anatomical sites where HSCs are in direct contact with their surrounding microenvironment, also known as niche. These spatial and temporal cellular interactions throughout development are required for the acquisition of stem cell properties, and for maintaining the HSC pool through balancing self-renewal, quiescence and lineage commitment. Understanding the context and consequences of these interactions will be imperative for our understanding of HSC biology and will lead to the improvement of in vitro production of HSCs for clinical purposes. The aorta-gonad-mesonephros (AGM) region is in this light of particular interest since this is the cradle of HSC emergence during the embryonic development of all vertebrate species. In this review, we will focus on the developmental origin of HSCs and will discuss the novel technological approaches and recent progress made to identify the cellular composition of the HSC supportive niche and the underlying molecular events occurring in the AGM region.

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45- and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45- counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties.

Generation of the first T lymphocytes in the human embryo involves the emergence, migration, and thymus seeding of lymphoid progenitors together with concomitant thymus organogenesis, which is the initial step to establish the entire adaptive immune system. However, the cellular and molecular programs regulating this process remain unclear. We constructed a single-cell transcriptional landscape of human early T lymphopoiesis by using cells from multiple hemogenic and hematopoietic sites spanning embryonic and fetal stages. Among heterogenous early thymic progenitors, one subtype shared common features with a subset of lymphoid progenitors in fetal liver that are known as thymus-seeding progenitors. Unbiased bioinformatics analysis identified a distinct type of pre-thymic lymphoid progenitors in the aorta-gonad-mesonephros (AGM) region. In parallel, we investigated thymic epithelial cell development and potential cell-cell interactions during thymus organogenesis. Together, our data provide insights into human early T lymphopoiesis that prospectively direct T lymphocyte regeneration, which might lead to development of clinical applications.

Objective: To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC. Methods: PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1(fl/fl), PDK1(fl/+) and Vec-Cre; PDK1(fl/fl) AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31(+)c-Kit(high) cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells. Results: In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31(+)c-Kit(high) in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster. Conclusion: PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.
目的： 研究磷酸肌醇依赖性激酶1（PDK1）在内皮细胞向造血细胞转化阶段对造血干细胞（HSC）发生的影响。 方法： 应用Vec-Cre在内皮细胞中特异性敲除PDK1基因，取对照组PDK1(fl/fl)、PDK1(fl/+)小鼠及敲除组Vec-Cre；PDK1(fl/fl)小鼠胚胎的主动脉-性腺-中肾区（AGM区）细胞进行集落形成实验，检测PDK1基因对造血祖细胞功能的影响；取对照组和敲除组AGM区细胞行移植实验，检测PDK1对HSC功能的影响；取对照组和敲除组AGM区细胞，通过流式细胞术检测PDK1对能够向造血转化的CD31(+)c-Kit(high)细胞群比例、细胞周期及细胞凋亡的影响；分选对照组和敲除组AGM区CD31(+) c-Kit(high)细胞群，通过Real-time PCR检测PDK1对内皮向造血转换相关的转录因子（RUNX1、P2-RUNX1、GATA2）的影响。 结果： PDK1敲除后，造血祖细胞形成的克隆形态变小，数目减少[敲除组CFU-GM为（24±5）个/ee，对照组为（62±1）个/ee，P=0.001]；破坏了造血干细胞重建造血及多向分化的能力（敲除组移植5只，0只重建，对照组移植7只，5只重建，P=0.001）；AGM区CD31(+)c-Kit(high)比例降低[敲除组CD31(+)c-Kit(high)比例为（0.145±0.017）%，对照组比例为（0.385±0.04）%，P=0.001]；并且AGM区由内皮细胞向造血细胞转换的关键转录因子表达下降，但对CD31(+)c-Kit(high)细胞的增殖和凋亡无明显影响。 结论： 在内皮细胞中特异敲除PDK1基因，导致具有向造血转化的内皮细胞群比例降低，影响了HSC的发生，破坏了HSC重建造血的能力。.

Understanding how the blood system is formed is an ongoing fundamental research challenge. Developmental biology has provided many insights into the molecules and processes that affect the formation of the blood tissues, both in health and disease. It is of particular interest for clinical transplantation therapies to understand how hematopoietic stem cells (HSCs)-the self-renewing purveyors of the adult blood system that produce over 10 different functionally specialized cell lineages and over 10(11) cells daily-are generated during embryonic stages. Recent successes to reprogram the fate of adult differentiated cells to pluripotency and to other cell lineages now highlight the importance of identifying the cells and molecules that affect the in vivo developmental initiation of rare and robust transplantable HSCs. The close association of the developing hematopoietic and vascular system, hematopoietic cell mobility through the circulation, and the essential role of the embryonic hematopoietic system in adult hematopoietic cell development make this a formidable study. This chapter reviews the advances, controversies, and current state of our knowledge of the growing field of hematopoietic development, with a special focus on the regulation of the natural transdifferentiation of endothelial cells to HSCs within the developing embryo.

There have been few studies on human embryos describing a specific pattern of hindgut colonization by hematopoietic stem cells (HSCs) and interstitial Cajal cells (ICCs). We aimed to study CD34, CD45 and CD117/c-kit expression in late stage human embryos, to attain observational data that could be related to studies on the aorta-gonad-mesonephros (AGM)-derived HSCs, and data on hindgut ICCs. Antibodies were also applied to identify alpha-smooth muscle actin and neurofilaments. Six human embryos of 48-56 days were used. In the 48 day embryo, the hindgut was sporadically populated by c-kit+ ICCs, but, in all other embryos, a layer of myenteric ICCs had been established. Intraneural c-kit+ cells were found in pelvic nerves and vagal trunks, suggesting that the theory of Ramon y Cajal assuming that ICCs may be primitive neurons may not be so invalid. Also in the 48 day embryo, c-kit+/CD45+ perivascular cells were found along the pelvic neurovascular axes, suggesting that not only liver, but also other organs could be seeded with HSCs from the AGM region. CD45+ cells with dendritic morphologies were found in all hindgut layers, including the epithelium. This last evidence is suggestive of an AGM contribution to the tissue resident macrophages and could be related to processes of sprouting angiogenesis which, in turn, have been found to be guided by filopodia of endothelial tip cells. Further studies on human embryonic and fetal material should be performed to attempt to clarify whether the hindgut colonization with HSCs is a transitory or definitive process.

Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45(low) c-Kit(high) cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45(low) c-Kit(high) cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and β-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.