Efficient generation of human NOTCH ligand-expressing haemogenic endothelial cells as infrastructure for in vitro haematopoiesis and lymphopoiesis.

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.

Ribosome biogenesis is essential for hemogenic endothelial cells to generate hematopoietic stem cells.

Undergoing endothelial-to-hematopoietic transition, a small fraction of embryonic aortic endothelial cells specializes into hemogenic endothelial cells (HECs) and eventually gives rise to hematopoietic stem cells (HSCs). Previously, we found that the activity of ribosome biogenesis (RiBi) is highly enriched in the HSC-primed HECs compared with adjacent arterial endothelial cells; however, whether RiBi is required in HECs for the generation of HSCs remains to be determined. Here, we have found that robust RiBi is markedly augmented during the endothelial-to-hematopoietic transition in mouse. Pharmacological inhibition of RiBi completely impeded the generation of HSCs in explant cultures. Moreover, disrupting RiBi selectively interrupted the HSC generation potential of HECs rather than T1 pre-HSCs, which was in line with its influence on cell cycle activity. Further investigation revealed that, upon HEC specification, the master transcription factor Runx1 dramatically bound to the loci of genes involved in RiBi, thereby facilitating this biological process. Taken together, our study provides functional evidence showing the indispensable role of RiBi in generating HSCs from HECs, providing previously unreported insights that may contribute to the improvement of HSC regeneration strategies.

Tropomyosin 1 deficiency facilitates cell state transitions and enhances hemogenic endothelial cell specification during hematopoiesis.

Tropomyosins coat actin filaments to impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. TPM1 has been shown to regulate blood cell formation in vitro, but it remains unclear how or when TPM1 affects hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, we found that TPM1 knockout augmented developmental cell state transitions and key signaling pathways, including tumor necrosis factor alpha (TNF-α) signaling, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses revealed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced HE formation during embryogenesis, without increasing the number of hematopoietic stem cells. These findings illuminate novel effects of TPM1 on developmental hematopoiesis.

Generation of functionally distinct hemogenic endothelial cell populations from pluripotent stem cells.

A diverse array of protocols have been established for the directed differentiation of human pluripotent stem cells (hPSCs) into a variety of cell types, including blood cells, for modeling development and disease, and for the development of cell-based therapeutics. These protocols recapitulate various signaling requirements essential for the establishment of the hematopoietic systems during embryonic development. However, in many instances, the functional properties of those progenitors, and their relevance to human development, remains unclear. The human embryo, much like other vertebrate model organisms, generates hematopoietic cells via successive anatomical location- and time-specific waves, each yielding cells with distinct functional and molecular characteristics. Each of these progenitor "waves" is characterized at the time of emergence of the direct hematopoietic progenitor in the vasculature, the hemogenic endothelial cell (HEC). Critically, despite decades of study in model organisms, the origins of each of these HEC populations remain unclear. Fortunately, through the directed differentiation of hPSCs, recent insights have been made into the earliest origins of each HEC population, revealing that each arises from transcriptionally and phenotypically distinct subsets of nascent mesoderm. Here, we outline the protocols to generate each mesodermal and HEC population via the formation of embryoid bodies and subsequent stage-specific signal manipulation. Through implementation of these discrete signal manipulations, it is possible to obtain human HEC populations that are exclusively extraembryonic-like or exclusively intraembryonic-like, enabling comparative developmental biology studies or specific translational applications.

Transition of signal requirement in hematopoietic stem cell development from hemogenic endothelial cells.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.

Tuning apicobasal polarity and junctional recycling in the hemogenic endothelium orchestrates the morphodynamic complexity of emerging pre-hematopoietic stem cells.

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba - one of the four Pard3 proteins expressed in the zebrafish - is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate.

In mammals and other animals with backbones, the cells that will make up blood and immune cells are generated during a very narrow timeframe in embryonic development. These cells, called hematopoietic stem cells and progenitors (or HSPCs for short), emerge from tissue known as hemogenic endothelium that makes up the floor of early blood vessels. For HPSCs to eventually specialise into different types of blood and immune cells, they require diverse migratory and homing properties that, ultimately, will determine the specific type of functions they exert. An important question for scientists studying the development of different blood and immune cell types is when this commitment to functional diversity is established. It could, for example, arise due to cells in the hemogenic endothelium having different origins. Alternatively, the signals that generate hemogenic endothelium cells could be responsible. It is also possible that both explanations are true, and that having different mechanisms involved ensures diversity in populations of HSPCs. To investigate differences between the HSPCs emerging from the hemogenic endothelium, Torcq et al. studied zebrafish embryos that had been modified so that one of the proteins involved in sensing cell polarity ­ where the top and bottom of the cell are located ­ was fluorescent. Live imaging of the embryos showed that two types of cells, with striking differences in morphology, emerge from the hemogenic tissue. In addition, one cell type displays the same polarity as the other vessel cells, whereas the other does not. Torcq et al. also present evidence suggesting that the signals responsible for controlling this cell polarity are provided by surrounding blood vessel cells, supporting the idea of an interplay between the different cell types. The finding that two different cell types emerge from the hemogenic endothelium, reveals a potential new source of diversity in HSPCs. Ultimately, this is expected to contribute to their functional complexity, resulting in both long-term stem cells that retain their full regenerative potential into adulthood and more specialized blood and immune cells.

The Onset of Intussusceptive Angiogenesis in COVID-19 Patients Might Come from the Mobilization of Stem Cell Sub-Populations Expressing the Hemangioblast Marker CD143.

COVID-19 and infectious diseases have been included in strategic development goals (SDG) of United Nations (UN). The SARS-CoV-2 pandemic has unveiled complex pathophysiological mechanisms underpinning COVID-19, notably inducing a systemic acquired vascular hemopathy characterized by endothelial dysfunction and intussusceptive angiogenesis, a rapid vascular remodeling process identified as a hallmark in severe COVID-19 cases affecting pulmonary and cardiac tissues. Stem cell migration have been proposed as significant regulators of this neoangiogenic process. In a monocentric cross-sectional study, through spectral flow cytometry analysis of peripheral blood mononuclear cells, we identified a distinct stem cell subpopulation mobilized in critical COVID-19. Indeed, by an unsupervised analysis generating a UMAP representation we highlighted eleven different clusters in critical and non-critical COVID-19 patients. Only one cluster was significantly associated to critical COVID-19 compared to non-critical patients. This cluster expressed the markers: CD45dim, CD34+, CD117+, CD147+, and CD143+, and were negative for CD133. Higher level of expression of hemangioblast markers CD143 were found in critical COVID-19 patients. This population, indicative of hemangioblast-like cells, suggests a key role in COVID-19-related neoangiogenesis, potentially driving the severe vascular complications observed. Our findings underscore the need for further investigation into the contributions of adult stem cells in COVID-19 pathology, offering new insights into therapeutic targets and interventions.

CD32 captures committed haemogenic endothelial cells during human embryonic development.

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.

The RNA-binding protein CSDE1 promotes hematopoietic stem and progenitor cell generation via translational control of Wnt signaling.

In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/ß-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding ß-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances ß-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/ß-catenin signaling is precisely regulated at the post-transcriptional level.

Aggf1 Specifies Hemangioblasts at the Top of Regulatory Hierarchy via Npas4l and mTOR-S6K-Emp2-ERK Signaling.

BACKGROUND: Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. METHODS: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. RESULTS: Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. CONCLUSIONS: These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.

De novo hematopoiesis from the fetal lung.

Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial-to-hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta-gonad-mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here, we demonstrate that the fetal lung is a potential source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells, accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal-lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43, and CD44. scRNA-seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFß/BMP, Notch, and YAP. Collectively, these data support the possibility that post-AGM development, functional HECs are present in the fetal lung, establishing this location as a potential extramedullary site of de novo hematopoiesis.

Divergent expression of Neurl3 from hemogenic endothelial cells to hematopoietic stem progenitor cells during development.

Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.

Dorsal aorta polarization and haematopoietic stem cell emergence.

Recent studies have highlighted the crucial role of the aorta microenvironment in the generation of the first haematopoietic stem cells (HSCs) from specialized haemogenic endothelial cells (HECs). Despite more than two decades of investigations, we require a better understanding of the cellular and molecular events driving aorta formation and polarization, which will be pivotal to establish the mechanisms that operate during HEC specification and HSC competency. Here, we outline the early mechanisms involved in vertebrate aorta formation by comparing four different species: zebrafish, chicken, mouse and human. We highlight how this process, which is tightly controlled in time and space, requires a coordinated specification of several cell types, in particular endothelial cells originating from distinct mesodermal tissues. We also discuss how molecular signals originating from the aorta environment result in its polarization, creating a unique entity for HSC generation.

Targeting Redundant ROBO1 and SDF-1 Pathways Prevents Adult Hemangioblast Derived-EPC and CEC Activity Effectively Blocking Tumor Neovascularization.

Neovascularization is a key therapeutic target for cancer treatment. However, anti-angiogenic therapies have shown modest success, as tumors develop rapid resistance to treatment owing to activation of redundant pathways that aid vascularization. We hypothesized that simultaneously targeting different pathways of neovascularization will circumvent the current issue of drug resistance and offer enhanced therapeutic benefits. To test this hypothesis, we made use of two distinct models of tumor-neovascularization, which exhibit equally dense microvasculature but show disparate sensitivity to anti-SDF-1 treatment. Lewis lung carcinoma (LLC) is primarily a vasculogenic-tumor that is associated with HSC functioning as a hemangioblast to generate circulating Endothelial Progenitor Cells contributing to formation of new blood vessels, and responds to anti-SDF-1 treatment. B16F0 melanoma is an angiogenic-tumor that derives new blood vessels from existing vasculature and is resistant to anti-SDF-1 therapy. In this study, we observed increased expression of the angiogenic-factor, Robo1 predominantly expressed on the blood vessels of B16F0 tumor. Blockade of Robo1 by the decoy receptor, RoboN, resulted in reduced microvascular-density and tumor-growth. However, this was associated with mobilization of BM-cells into the B16F0 tumor, thus switching the mode of neovascularization from angiogenic to vasculogenic. The use of a combinatorial treatment of RoboN and the monoclonal anti-SDF-1 antibody effectively attenuated tumor-growth and inhibited both angiogenic and BM-derived microvessels.

CD34dim cells identified as pluripotent stem cell-derived definitive hemogenic endothelium purified using bone morphogenetic protein 4.

Hemogenic endothelium (HE) plays a pivotal and inevitable role in haematopoiesis and can generate all blood and endothelial lineage cells in the aorta-gonad-mesonephros of mouse embryos. Whether definitive HE can prospectively isolate pure HE from human pluripotent stem cells that can spontaneously differentiate into heterogeneous cells remains unknown. Here, we identified and validated a CD34dim subpopulation with hemogenic potential. We also purified CD34 cells with a CXCR4- CD73- phenotype as a definitive HE population that generated haematopoietic stem cells and lymphocytes. The frequency of CXCR4- CD73- CD34dim was evidently increased by bone morphogenetic protein 4, and purified HE cells differentiated into haematopoietic cells with myeloid and T lymphoid lineages including Vδ2+ subset of γ/δ T cells. We developed a simple method to purify HE cells that were enriched in CD34dim cells. We uncovered an initial step in differentiating haematopoietic lineage cells that could be applied to basic and translational investigations into regenerative medicine.

Tmem88 confines ectodermal Wnt2bb signaling in pharyngeal arch artery progenitors for balancing cell cycle progression and cell fate decision.

Pharyngeal arch artery (PAA) progenitors undergo proliferative expansion and angioblast differentiation to build vessels connecting the heart with the dorsal aortae. However, it remains unclear whether and how these two processes are orchestrated. Here we demonstrate that Tmem88 is required to fine-tune PAA progenitor proliferation and differentiation. Loss of zebrafish tmem88a/b leads to an excessive expansion and a failure of differentiation of PAA progenitors. Moreover, tmem88a/b deficiency enhances cyclin D1 expression in PAA progenitors via aberrant Wnt signal activation. Mechanistically, cyclin D1-CDK4/6 promotes progenitor proliferation through accelerating the G1/S transition while suppressing angioblast differentiation by phosphorylating Nkx2.5/Smad3. Ectodermal Wnt2bb signaling is confined by Tmem88 in PAA progenitors to ensure a balance between proliferation and differentiation. Therefore, the proliferation and angioblast differentiation of PAA progenitors manifest an inverse relationship and are delicately regulated by cell cycle machinery downstream of the Tmem88-Wnt pathway.

The ETS transcription factor Spi2 regulates hematopoietic cell development in zebrafish.

The E26 transformation-specific or E-twenty-six (ETS) genes encode a superfamily of transcription factors involved in diverse biological processes. Here, we report the identification and characterization of a previously unidentified member of the ETS transcription factors, Spi2, that is found exclusively in the ray-finned fish kingdom. We show that the expression of spi2 is restricted to hemogenic endothelial cells (HECs) and to hematopoietic stem and progenitor cells (HSPCs) in zebrafish. Using bacteria artificial chromosome transgenesis, we generate a spi2 reporter line, TgBAC(spi2:P2a-GFP), which manifests the GFP pattern recapitulating the endogenous spi2 expression. Genetic ablation of spi2 has little effect on HEC formation and the endothelial-to-hematopoietic transition, but results in compromised proliferation of HSPCs in the caudal hematopoietic tissue (CHT) during early development and in severe myeloid lineage defect in adulthood. Epistatic analysis shows that spi2 acts downstream of runx1 in regulating HSPC development in the CHT. Our study identifies Spi2 as an essential regulator for definitive hematopoietic cell development and creates a TgBAC(spi2:P2a-GFP) reporter line for tracking HECs, HSPCs, myeloid cells and thrombocytes from early development to adulthood.

Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression.

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a "hemogenic endothelium" (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45-) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential.

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.

Embryonic vascular establishment requires protein C receptor-expressing endothelial progenitors.

Vascular establishment is one of the early events in embryogenesis. It is believed that vessel-initiating endothelial progenitors cluster to form the first primitive vessel. Understanding the molecular identity of these progenitors is crucial in order to elucidate lineage hierarchy. In this study, we identify protein C receptor (Procr) as an endothelial progenitor marker and investigate the role of Procr+ progenitors during embryonic vascular development. Using a ProcrmGFP-2A-lacZ reporter, we reveal a much earlier Procr expression (embryonic day 7.5) than previously acknowledged (embryonic day 13.5). Genetic fate-mapping experiments using ProcrCre and ProcrCreER demonstrate that Procr+ cells give rise to blood vessels throughout the entire embryo proper. Single-cell RNA-sequencing analyses place Procr+ cells at the start of endothelial commitment and maturation. Furthermore, targeted ablation of Procr+ cells results in failure of vessel formation and early embryonic lethality. Notably, genetic fate mapping and scRNA-seq pseudotime analysis support the view that Procr+ progenitors can give rise to hemogenic endothelium. In this study, we establish a Procr expression timeline and identify Procr+ vessel-initiating progenitors, and demonstrate their indispensable role in establishment of the vasculature during embryo development.