Growth Of Hematopoietic Stem Cells Research Articles

Human yolk sac-derived innate lymphoid-biased multipotent progenitors emerge prior to hematopoietic stem cell formation.

Hematopoietic stem cell (HSC)-independent lymphopoiesis has been elucidated in murine embryos. However,our understanding regarding human embryonic counterparts remains limited. Here, we demonstrated the presence of human yolk sac-derived lymphoid-biased progenitors (YSLPs) expressing CD34, IL7R, LTB, and IRF8 at Carnegie stage 10, much earlier than the first HSC emergence. The number and lymphopoietic potential of these progenitors were both significantly higher in the yolk sac than the embryo proper at this early stage. Importantly, single-cell/bulk culture and CITE-seq have elucidated the tendency of YSLP to differentiate into innate lymphoid cells and dendritic cells. Notably, lymphoid progenitors in fetal liver before and after HSC seeding displayed distinct transcriptional features, with the former closely resembling those of YSLPs. Overall, our data identified the origin, potential, and migratory dynamics of innate lymphoid-biased multipotent progenitors in human yolk sac before HSC emergence, providing insights for understanding the stepwise establishment of innate immune system in humans.

ADA2 regulates inflammation and hematopoietic stem cell emergence via the A2bR pathway in zebrafish

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.

Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

Despite the identification of driver mutations leading to the initiation of myeloproliferative neoplasms (MPNs), the molecular pathogenesis of MPNs remains incompletely understood. Here, we demonstrate that growth arrest and DNA damage inducible gamma (GADD45g) is expressed at significantly lower levels in patients with MPNs, and JAK2V617F mutation and histone deacetylation contribute to its reduced expression. Downregulation of GADD45g plays a tumor-promoting role in human MPN cells. Gadd45g insufficiency in the murine hematopoietic system alone leads to significantly enhanced growth and self-renewal capacity of myeloid-biased hematopoietic stem cells, and the development of phenotypes resembling MPNs. Mechanistically, the pathogenic role of GADD45g insufficiency is mediated through a cascade of activations of RAC2, PAK1 and PI3K-AKT signaling pathways. These data characterize GADD45g deficiency as a novel pathogenic factor in MPNs.

Nlrc3 signaling is indispensable for hematopoietic stem cell emergence via Notch signaling in vertebrates

Hematopoietic stem and progenitor cells generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of hematopoietic stem and progenitor cells is finely tuned by a variety of signaling pathways. Previous studies have revealed the roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in hematopoiesis. In this study, we find that Nlrc3, a nucleotide-binding domain leucine-rich repeat containing family gene, is highly expressed in hematopoietic differentiation stages in vivo and vitro and is required in hematopoiesis in zebrafish. Mechanistically, nlrc3 activates the Notch pathway and the downstream gene of Notch hey1. Furthermore, NF-kB signaling acts upstream of nlrc3 to enhance its transcriptional activity. Finally, we find that Nlrc3 signaling is conserved in the regulation of murine embryonic hematopoiesis. Taken together, our findings uncover an indispensable role of Nlrc3 signaling in hematopoietic stem and progenitor cell emergence and provide insights into inflammation-related hematopoietic ontogeny and the in vitro expansion of hematopoietic stem and progenitor cells.

Post-transcriptional (re)programming of B lymphocyte development: From bench to bedside?

Hematopoiesis, a process which generates blood and immune cells, changes significantly during mammalian development. Definitive hematopoiesis is marked by the emergence of long-term hematopoietic stem cells (HSCs). Here, we will focus on the post-transcriptional differences between fetal liver (FL) and adult bone marrow (ABM) HSCs. It remains unclear how or why exactly FL HSCs transition to ABM HSCs, but we aim to leverage their differences to revive an old idea: in utero HSC transplantation. Unexpectedly, the expression of certain RNA-binding proteins (RBPs) play an important role in HSC specification, and can be employed to convert or reprogram adult HSCs back to a fetal-like state. Among other features, FL HSCs have a broad differentiation capacity that includes the ability to regenerate both conventional B and T cells, as well as innate-like or unconventional lymphocytes such as B-1a and marginal zone B (MzB) cells. This chapter will focus on RNA binding proteins, namely LIN28B and IGF2BP3, that are expressed during fetal life and how they promote B-1a cell development. Furthermore, this chapter considers a potential clinical application of synthetic co-expression of LIN28B and IGF2BP3 in HSCs.

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Tagraxofusp Shows Promising Anti-Tumoral Efficacy in Preclinical in Vitro Models of Myelofibrosis, Both As a Single Agent and in Combination with Janus Kinase Inhibitors

BACKGROUND: Myelofibrosis (MF) is an aggressive myeloproliferative neoplasm. Abnormal growth of hematopoietic stem and progenitor cells in MF is driven by somatic mutations affecting the Janus Kinase (JAK) signaling pathway with JAK2 V617F occurring in 50-60% of patients (Cardoso et al, Plos One 2015). The role of the JAK/Signal transducer and activator of transcriptions (STAT) pathway in MF has led to the recent approval of three different JAK2 inhibitors (ruxolitinib, pacritinib, and fedratinib) for MF treatment. However, despite limiting the symptoms of the disease, JAK2 inhibitors do not result in remission due to persistence of JAK2 activation and MF-driving cells (Pandey et al, Blood Cancer J 2022). Thus, there remains a high unmet need for treatment strategies for patients with MF. Tagraxofusp, a first-in-class CD123-directed therapy, is a recombinant fusion protein consisting of human interleukin-3 conjugated to a truncated diphtheria toxin payload approved (US/EU) for the treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm. In MF, CD123 is expressed on malignant cells and bone marrow (BM) accessory cells that support the proliferation of neoplastic cells (Bao et al, Hematol Oncol Stem Cell Ther 2019; Lasho et al, Blood 2014). Based on CD123 expression, tagraxofusp is a potential, novel approach for MF treatment either alone or in combination. Initial results from a two-stage multicenter, phase 2 study (STML-0401-0314; NCT02268253) of single-agent tagraxofusp in pts with relapsed or refractory (R/R) MF demonstrated encouraging clinical efficacy and a manageable safety profile (Yacoub et al. ASH 2021). We aim to further investigate the antitumoral efficacy of tagraxofusp in MF preclinical models and the potential improvement of antitumoral efficacy by combining tagraxofusp with the JAK inhibitors, ruxolitinib, and pacritinib. METHODS: MF cell lines (SET2, UKE1, HEL: JAK2 V617F; UT7: JAK2 wild type [wt]) and bone marrow mononuclear cell (BMMC) samples from healthy donors (HD) were characterized for the extracellular expression of CD123 by flow cytometry staining. In vitro cytotoxicity experiments in CD123-positive MF cell lines (SET2, UT7, UKE-1) tested tagraxofusp and ruxolitinib or pacritinib either as single agents or in combination (MTS assay; 72 h). The combination index (CI) was calculated according to the Compusyn-Chou method (Chou TC., Pharmacol Rev, 2006) and Combenefit platform. In vitro co-culture experiments were performed with MF cell lines incubated in the presence of human primary HD-BMMCs. Cell death, as measured by LiveDead/AnnV staining, was monitored by FACS analysis through differential staining of MF cells and BMMCs with Cell Trace Violet dye (CTV). A signaling pathway analysis (JAK/STAT, S6, Mcl1) was performed through immunocapillary electrophoresis on MF cell lines treated with single agents or in combination. RESULTS: The MF cell lines showed different levels of CD123 expression. Except for the CD123 negative cell line (HEL), CD123 positive cell lines were all sensitive to tagraxofusp independent of CD123 expression intensity. As expected, both ruxolitinib and pacritinib were active in the presence of JAK2 wt pathway. A synergistic drug interaction was observed (mean CI range: 0.59-1.12) in most combinations of tagraxofusp with JAK inhibitors, as shown in a representative CD123+ cell line in Figure 1. When BMMCs, which are a subpopulation of BM Accessory Cells partially expressing CD123, were co-cultured with MF cell lines, the cytotoxic effect induced by tagraxofusp was enhanced, and this improvement was maintained in combination with JAK2 inhibition. Molecular biomarkers, such as JAK/STAT, S6, and Mcl1, were evaluated in MF cell lines treated with tagraxofusp and JAK inhibitors and supported the MoA of this combination. CONCLUSIONS: Our studies showed high sensitivity of MF cell lines to tagraxofusp as a single agent, which is expected given phase 2 results demonstrated clinical efficacy of single-agent tagraxofusp in R/R MF (Yacoub et al. ASH 2021). In addition, synergism was observed in combination with JAK inhibitors. The in vitro antitumoral effect was more pronounced in the presence of BM accessory cells, suggesting an extra targeting of CD123-positive BM cells by tagraxofusp in vivo. These results support potential clinical development of TAG in combination with JAK inhibitors for the treatment of MF.

Notch Signaling Is Required for Extramedullary Hematopoiesis in the Adult Liver

During embryonic development, hematopoietic stem cells (HSC) in the fetal liver undergo expansion and self-renewal. Fetal HSCs migrate to the bone marrow (BM) niche towards birth and remain there during adulthood. Extramedullary hematopoiesis (EMH), or hematopoiesis outside of the BM, predominantly occurs in the fetal liver or when the adult BM niche ceases to be a site for functional hematopoiesis. The mechanism by which the adult liver becomes reactivated as a site of EMH for itinerant HSCs is unknown. Our project is investigating EMH in the adult liver by inducing acute hemolytic anemia in adult mice that induces egress and homing of hematopoietic progenitors to the liver and spleen. Our work demonstrates that long-term HSCs isolated from the portal vein in the adult liver can serially reconstitute lethally irradiated mice. Thus, the adult liver serves as a potential reservoir for long-term, functional HSCs in the presence of a damaged BM niche. In pursuit of a mechanism, we focused on Notch-signaling which is an evolutionarily conserved pathway that has been shown to play a critical role in HSC emergence and fetal liver development. When comparing WT liver donors to Notch-deficient (Notch1 +/ΔTAD) or Notch knockout (Notch1 ffVavCre +) liver donors, hematopoietic transplants reveal that Notch mutant progenitors fail to efficiently reconstitute irradiated recipients. Using an HSC-specific a-catulin-GFPmouse model, we are able to track the expression of GFP + liver-originating HSCs after hematopoietic reconstitution. We show that mice reconstituted by EMH-activated a-catulin-GFP liver HSCs are able to provide long-term reconstitution in lethally irradiated recipients and resume residence in the bone marrow niche of recipients. Additionally, we see distinct localization of GFP + HSCs localized near sinusoids throughout the EMH liver niche using whole-mount imaging. We have validated receptor-ligand binding interactions of the Notch1 receptor with ligand-expressing hematopoietic cells in the adult liver. Using intracellular flow cytometry after EMH in the adult liver, we see a 3-fold increase in mean fluorescent intensity of GATA2 in HSPCs (hematopoietic stem cell progenitors) when compared to our bone marrow and isotype control mice. We demonstrate that Notch-driven GATA2 expression is a crucial regulator of self-renewal and functional maintenance of stemness during EMH of HSCs in the adult liver. Our findings reveal a developmentally conserved Notch signaling mechanism is reactivated in the adult liver to retain the functional potential and maintenance of HSCs as they adapt to inhospitable conditions in the bone marrow niche.

Establishment of Human Bone Marrow-on-Chip As a Preclinical Model to Evaluate Drug-Induced Toxicities and Myelofibrosis Patient-Specific Pathophysiology

Hematopoiesis takes place in the bone marrow (BM) and is supported by a complex cellular and molecular network in the BM microenvironment. The inaccessibility of living human BM hampers the study of its pathophysiology under stress induced by drugs, radiation, or genetic mutations. Commonly used models of the human BM microenvironment rely on mouse models and 2D or 3D static tissue cultures. Although existing in vitro hematopoiesis models have led to advances in the field, they fail to recapitulate the biology of cellular interactions, cell extrinsic factors, and complex functions of living human BM hematopoiesis. This has also limited our understanding of human BM biology and the influence of BM niche cells in BM defects in diseases such as myeloproliferative neoplasms (MPNs) and leukemias. Consequently, there is an unmet need to develop a preclinical human BM hematopoiesis model that supports growth and differentiation of multilineage hematopoietic stem cells to enable evaluation of investigational compounds for myeloerythroid toxicity and efficacy. Microphysiologic systems (MPS), such as organ-on-chips, emulate the complex interplay between intrinsic and extrinsic cell factors in the BM microenvironment, the 3D tissue architecture, and are more clinically relevant compared with static co-culture approaches (PMID: 31988457; PMID: 37359774). Here we present the development of a human BM-on-chip MPS model to recapitulate both healthy and MPN disease and evaluate the alterations in the BM niche environment. We utilized a commercially available organ-on-chip microfluidic platform (Emulate, Boston, MA) to develop a living human BM with a functional hematopoietic niche in vitro by perfusion with culture medium. This fluidic-controlled modular chip comprises an osteogenic apical channel separated from a vascular basal channel by a semiporous membrane, which allows for media perfusion, cellular crosstalk, and intercellular signaling. The osteogenic apical channel is filled with a fibrin gel that allows the co-culture of all critical cells of the BM niche, including osteoblasts, endothelial cells, mesenchymal stem cells, hematopoietic stem and progenitor cells. The parallel basal channel is lined by human vascular endothelium and is perfused with culture medium optimized to allow survival and development of functional hematopoietic lineage niche cell populations. This unidirectional fluidic MPS model allows for collection of cell effluents for evaluation of cell-extrinsic factors such as cytokines, extracellular vesicles, and metabolites. The modular and accessible nature of this system provides an advantage to investigate the effects of the microenvironment and drug candidates individually on the osteogenic and vascular compartments. This human BM-on-chip has been tested with cells from several healthy and myelofibrosis donors. Utilizing imaging, flow cytometry, cytokine profiling, and transcriptomics, we demonstrate that the vascularized human BM-on-chip recapitulates the living human hematopoietic niche, with BM relevant cytokine levels, retains hematopoietic stem and progenitor cells in in vivo-like proportions for over 3 weeks in culture, allows for the differentiation of BM niche lineage cell populations similar to human BM aspirate, and captures human donor variabilities. We also show that BM-on-chips derived from MPN patients recapitulate the hematopoietic defects to understand the biology and disease response in myelofibrosis. Our robust experimental workflow and bioinformatics platform will be utilized to evaluate candidate compounds for BM myelotoxicities, drug mechanisms of action, and biomarkers in a multifactorial manner to support preclinical programs. This biomimetic platform offers a new approach for analysis of drug responses and toxicities in BM as well as for study of hematopoiesis and hematologic diseases such as myelofibrosis.

Dermomyotome-derived endothelial cells migrate to the dorsal aorta to support hematopoietic stem cell emergence.

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Here, we profile the transcriptome of the earliest detectable endothelial cells (ECs) during zebrafish embryogenesis to demonstrate that tissue-specific EC programs initiate much earlier than previously appreciated, by the end of gastrulation. Classic studies in the chick embryo showed that paraxial mesoderm generates a subset of somite-derived endothelial cells (SDECs) that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. We describe a conserved program in the zebrafish, where a rare population of endothelial precursors delaminates from the dermomyotome to incorporate exclusively into the developing dorsal aorta. Although SDECs lack hematopoietic potential, they act as a local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of ECs contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Different niches for stem cells carrying the same oncogenic driver affect pathogenesis and therapy response in myeloproliferative neoplasms

Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK–STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver.

Minichromosome maintenance protein 10 (mcm10) regulates hematopoietic stem cell emergence in the zebrafish embryo.

Hematopoietic stem cells (HSCs) guarantee the continuous supply of all blood lineages during life. In response to stress, HSCs are capable of extensive proliferative expansion, whereas in steady state, HSCs largely remain in a quiescent state to prevent their exhaustion. DNA replication is a very complex process, where many factors need to exert their functions in a perfectly concerted manner. Mini-chromosome-maintenance protein 10 (Mcm10) is an important replication factor, required for proper assembly of the eukaryotic replication fork. In this report, we use zebrafish to study the role of mcm10 during embryonic development, and we show that mcm10 specifically regulates HSC emergence from the hemogenic endothelium. We demonstrate that mcm10-deficient embryos present an accumulation of DNA damages in nascent HSCs, inducing their apoptosis. This phenotype can be rescued by knocking down p53. Taken all together, our results show that mcm10 plays an important role in the emergence of definitive hematopoiesis.

Similar but distinct: The impact of biomechanical forces and culture age on the production, cargo loading, and biological efficacy of human megakaryocytic extracellular vesicles for applications in cell and gene therapies.

Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR-486-5p and miR-22-3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude-dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR-486-5p and miR-22-3p cargo. However, while the shake flask-derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump-derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by "older" Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late-differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

Modulation of WNT, Activin/Nodal, and MAPK Signaling Pathways Increases Arterial Hemogenic Endothelium and Hematopoietic Stem/Progenitor Cell Formation During Human iPSC Differentiation.

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human-induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation, and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal, and MAPK signaling pathways by stage-specific addition of small-molecule regulators CHIR99021, SB431542, and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo.

Hematopoietic Jagged1 is a fetal liver niche factor required for functional maturation and engraftment of fetal hematopoietic stem cells

Notch signaling is essential for the emergence of definitive hematopoietic stem cells (HSCs) in the embryo and their development in the fetal liver niche. However, how Notch signaling is activated and which fetal liver cell type provides the ligand for receptor activation in HSCs is unknown. Here we provide evidence that endothelial Jagged1 (Jag1) has a critical early role in fetal liver vascular development but is not required for hematopoietic function during fetal HSC expansion. We demonstrate that Jag1 is expressed in many hematopoietic cells in the fetal liver, including HSCs, and that its expression is lost in adult bone marrow HSCs. Deletion of hematopoietic Jag1 does not affect fetal liver development; however, Jag1-deficient fetal liver HSCs exhibit a significant transplantation defect. Bulk and single-cell transcriptomic analysis of HSCs during peak expansion in the fetal liver indicates that loss of hematopoietic Jag1 leads to the downregulation of critical hematopoietic factors such as GATA2, Mllt3, and HoxA7, but does not perturb Notch receptor expression. Exvivo activation of Notch signaling in Jag1-deficient fetal HSCs partially rescues the functional defect in a transplant setting. These findings indicate a new fetal-specific niche that is based on juxtracrine hematopoietic Notch signaling and reveal Jag1 as a fetal-specific niche factor essential for HSC function.

Timing and regulation of hematopoietic stem cell colonization of the human fetal bone marrow by endothelial and CAR stromal cells during pregnancy

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

Lymphoid cell development from fetal hematopoietic progenitors and human pluripotent stem cells.

Lymphoid cells encompass the adaptive immune system, including T and B cells and Natural killer T cells (NKT), and innate immune cells (ILCs), including Natural Killer (NK) cells. During adult life, these lineages are thought to derive from the differentiation of long-term hematopoietic stem cells (HSCs) residing in the bone marrow. However, during embryogenesis and fetal development, the ontogeny of lymphoid cells is both complex and multifaceted, with a large body of evidence suggesting that lymphoid lineages arise from progenitor cell populations antedating the emergence of HSCs. Recently, the application of single cell RNA-sequencing technologies and pluripotent stem cell-based developmental models has provided new insights into lymphoid ontogeny during embryogenesis. Indeed, PSC differentiation platforms have enabled de novo generation of lymphoid immune cells independently of HSCs, supporting conclusions drawn from the study of hematopoiesis in vivo. Here, we examine lymphoid development from non-HSC progenitor cells and technological advances in the differentiation of human lymphoid cells from pluripotent stem cells for clinical translation.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.