Liver Hematopoietic Stem Cells Research Articles

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.

The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

Srcap Plays an Essential Role in Normal Hematopoiesis

The incorporation of variant histones into chromatin has been revealed to be an important mechanism of epigenetic regulation. Deposition of H2A.Z, in particular, is known to mediate several essential cellular processes including chromatin organization, DNA damage response, and transcriptional regulation. Nonsense and frameshift mutations in the H2A.Z exchanger, SRCAP, have been recurrently identified in both clonal hematopoiesis and AML, suggesting the importance of this gene in both non-malignant hematopoiesis and leukemogenesis. However, the precise role of SRCAP and the impact of its dysregulation on hematopoiesis remain unknown. To investigate the role of Srcap in hematopoiesis, we generated a conditional Srcap loss of function murine model with loxP sites flanking exons 11-16. Cre-induced deletion of these exons results in a loss of the catalytic ATPase domain along with a frameshift within the remaining protein sequence. When Vav1-iCre +/-Srcap fl/+ and Srcap fl/fl mice are crossed, no Vav1-iCre +/-Srcap fl/fl pups are born, suggesting that hematopoietic-specific-loss of Srcap is embryonic lethal. At E14.5/15.5, Vav1-iCre +/-Srcap fl/fl embryos are paler than other genotypes ( Figure 1) with a reduced number of Ter119 + fetal liver cells. Interestingly, the decrease in Ter119 + cells upon Srcap loss occurs primarily in later stage erythroid progenitors, whereas the number of stage 1 erythroid progenitors is relatively well preserved, suggesting that loss of Srcap adversely impacts fetal erythroid maturation. Furthermore, Srcap loss impacts fetal liver myeloid maturation. While the number of granulocytes is unchanged in Srcap deficient embryos, they have a near complete absence of monocytes, monocyte-dendritic cell progenitors, and monocyte progenitors. In contrast to the changes observed within mature hematopoietic populations, phenotypic hematopoietic stem cells (HSCs) are maintained in Vav1-iCre +/-Srcap fl/fl fetal livers. However, their function is impaired as fetal liver HSCs fail to repopulate upon transplantation ( Figure 2). Importantly, adult HSCs isolated from Mx1-Cre +/-Srcap fl/flmice show a very similar phenotype in that they fail to maintain hematopoiesis when transplanted and treated with poly (I:C). In total, this data shows that Srcap is essential for the maintenance and proper function of both mature hematopoietic populations and hematopoietic progenitors with different lineages and developmental stages displaying varied sensitivities to Srcap loss. To investigate the cellular consequences of SRCAP loss, we knocked down SRCAP expression in hematopoietic cell lines using an inducible shRNA approach. In agreement with the results obtained from our murine model, SRCAP deficient MOLM13 cells show a baseline competitive growth disadvantage that becomes apparent within one week of culture. This is associated with increased γ-H2AX activity, suggesting enhanced DNA damage and consistent with the previously identified role of SRCAP in the cellular DNA damage response. Interestingly, murine fetal liver HSCs with loss of Srcap also exhibit increased γ-H2AX activity, and Srcap null fetal liver stage 1 erythroid progenitors display marked transcriptional upregulation of multiple p53 downstream genes. Our data demonstrate that, in hematopoietic populations, Srcap is an essential gene, with its complete loss resulting in enhanced cellular stress potentially due to increased DNA damage. In humans, nonsense and frameshift mutations in SRCAP appear to be heterozygous in nature, suggesting that the impact of SRCAP deficiency is dose dependent. Indeed, murine HSCs with heterozygous loss of Srcap maintain normal repopulating activity in both primary and secondary transplantation assays ( Figure 2). It remains to be determined how the intermediate deficiencies in SRCAP levels found in patients impact hematopoiesis and lead to clonal expansion and whether the impact of SRCAP loss on the cellular response to stress, including DNA damage, is mediated by its canonical function as a histone variant H2A.Z exchanger. These studies promise to reveal novel insights on the nature of H2A.Z-mediated epigenetic regulation in HSCs and on how its dysregulation may lead to leukemogenesis.

Dysregulated Proteostasis Restrains the Developmental HSC Expansion in Fanconi Anemia

Hematopoietic stem cells (HSCs) require precise regulation of protein synthesis, processing, and degradation. Protein homeostasis (Proteostasis) is particularly critical during development, relying on chaperone proteins to maintain folding capacity in a highly proliferative HSC pool. Even modest changes in synthesis or degradation lead to the accumulation of unfolded proteins and compromise fetal liver HSC pool formation. Fanconi anemia (FA) is a multisystem disorder that manifests with bone marrow failure (BMF) early in life. Previous reports from our lab and others suggest that pre-natal HSC depletion in FA is a critical factor for the early onset of clinical BMF. To identify the cellular and molecular mechanisms underlying prenatal HSC defects in FA, we performed single-cell transcriptome profiling in both Fancd2 +/+and Fancd2 -/- FL samples. Gene set enrichment analysis (GSEA) of differentially expressed genes revealed significantly increased expression of c-Myc target genes in Fancd2 -/ - HSCs (normalized enrichment score, NES: 2.04, p<10 -5 FDR<10 -5), matched by an increase in c-Myc in Fancd2 -/- LT-HSC (CD150 + CD48 - Lineage - Sca-1 + c-KIT +) at the protein level (1.7-fold (p<0.05). Our sc-RNA Seq data further showed an increase in ribosome biogenesis (NES: 1.8, p<10 -5, FDR<10 -3) and translation (NES: 2.2, p<10 -5, FDR<10 -5) in Fancd2 -/- HSC, functionally corroborated by increased protein synthesis rates across Fancd2 -/- FL HSPC subpopulations (1.3-fold in LT- HSCs, p<0.01) using the in vivo O-Propargyl-puromycin (OPP) assay. Not surprisingly, the increased protein synthesis rates exceed the physiologic protein folding capacity, leading to elevated levels of unfolded proteins in Fancd2 -/- FL HSPCs by Tetraphenylethene maleimide (TPE-MI) stain. The aggregated misfolded proteins (aggresomes) in Fancd2 -/- FL HSPCs were also observed by immunofluorescence microscopy. Revisiting our scRNA Seq data, the observed proteotoxic stress was also reflected in the GSEA enrichment for unfolded protein response (UPR; NES: 1.30, p=0.01, FDR=0.01) as well as heat shock stress response (NES:1.7,p<0.0001, FDR=0.09), and coincided with upregulation of response to heat shock factor-1 ( Hsf-1; NES:1.86,p<0.0001, FDR=0.02) in Fancd2 -/- FL HSCs. Because chaperone supplementation has been shown to enhance HSC protein folding capacity, we serially injected the bile acid TUDCA (a chemical chaperone) into the pregnant dam. This not only significantly reduced unfolded protein levels, but indeed rescued FL HSC numbers in Fancd2 -/- littermates. Our observations in aggregate indicate that dysregulated proteostasis is a previously unrecognized pathogenic process constraining fetal Fancd2 -/- hematopoiesis. Protection of the HSPC proteome appears to be a new, non-canonical function of FANCD2, without immediate connection to the FA pathway and DNA crosslink repair. Importantly, we demonstrate the successful pharmacological rescue of the fetal Fancd2 -/- HSPC pool.

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.

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.

HSC-independent definitive hematopoiesis persists into adult life

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Using Spatial Transcriptomics to Reveal Fetal Liver Hematopoietic Stem Cell-Niche Interactions

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E10.5, and then migrate to the fetal liver after E12.5 for expansion. After E18.5, HSCs migrate to the bone marrow where they reside for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the fetal liver HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge, there are no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we use cutting-edge spatial transcriptomics to comprehensively investigate the transcriptomics and interactions between HSCs and the niche cells in the fetal liver, and to identify the niche cells and factors for HSC expansion. To understand the spatial distribution and interactions between HSCs and niche cells in fetal liver, we introduced 2 spatial transcriptomic methods, Slide-seq (Stickels et al. Nature Biotechnology, 2021), and 10x Genomics Visium Spatial Gene Expression, in our study on E14.5 and E16.5 mouse fetal liver. By integrating with a 10x Genomics' Chromium™ Single Cell 3' Solution, we discovered the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelium cells, macrophages, megakaryocytes, and hepatic stellate cells/perivascular mesenchymal cells (PMCs) in E14.5 and E16.5 mouse fetal liver. Interestingly, we found that PMCs and hepatoblasts were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing PMCs are enriched in the portal vessel area. Importantly, the majority of fetal liver HSCs are in close proximity to N-cadherin-expressing PMCs and endothelial cells, indicating a supportive role of N-cadherin-expressing PMCs and endothelial cells in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing PMCs are major niche-signaling senders with an enriched expression of niche factors, such as CXCL12 and KITL, and stemness pathway-related ligands, such as IGFs, TGFβs, JAG2, and DLK1, indicating N-cadherin-expressing PMCs could be the major niche cells in supporting HSCs in the fetal liver. This finding is consistent with our previous finding that N-cadherin-expressing bone and marrow stromal progenitor cells can maintain reserve HSCs in the adult bone marrow. In addition, endothelium cells have enriched expression of several niche factors, including BMPs, KITL, IGFs and TGFβs. To investigate the potential role of N-cadherin-expressing cells in supporting fetal liver HSCs, we generated an N-cadCreER;Cxcl12 and an N-cadCreER;Scf mouse model to conditionally knockout the well-studied niche factors, CXCL12 and SCF, in N-cadherin-expressing cells. Interestingly, conditional knockout of either Cxcl12 or Scf in N-cadherin-expressing cells resulted in an increase in the number of HSCs. Moreover, flowcytometry analysis and scRNA-seq on conditional knockout of Cxcxl12 in N-cadherin-expressing cells showed a myeloid-biased differentiation. Subsequential slide-seq showed that fetal liver HSCs were located close to N-cadherin-expressing PMCs and endothelial cells. Intriguingly, fetal liver HSCs migrated toward and resided in close proximity to megakaryocytes after conditional knockout of Cxcxl12 in N-cadherin-expressing cells. In summary, we postulate that N-cadherin-expressing cells, especially PMCs, maintain fetal liver HSCs. The knockout of Cxcl12 in N-cadherin-expressing cells leads to the migration of HSCs towards megakaryocytes, which may induce HSC expansion and biased differentiation. In summary, by using cutting-edge spatial transcriptomics, we revealed a comprehensive spatial transcriptomics of HSCs and niche cells in E14.5 and E16.5 mouse fetal liver. The N-cadherin-expressing cells in the fetal liver is a major niche in maintaining HSCs, while other potential niches may be responsible for the expansion of HSCs. In the future, we will use multiple approaches to verify the distribution changes of HSCs in N-cadCreER;Cxcl12 mouse, and to reveal the niches in support of the expansion of HSCs.

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Using Spatial Transcriptomics to Reveal Fetal Liver Hematopoietic Stem Cell-Niche Interactions

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E11.5, and then migrate to the fetal liver after E12.5 for expansion. After E17.5, HSCs migrate to the bone marrow and reside in the bone marrow for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the fetal liver HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge, there are no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we are using cutting-edge spatial transcriptomics to comprehensively study the transcriptomics and interactions between HSCs and the niche cells in the fetal liver, and in search of the niche cells and factors for HSC expansion.To understand the spatial distribution and interactions between HSCs and niche cells in the fetal liver, we introduced 2 spatial transcriptomic methods, slide-seq, and 10x Visium, in our study on E14.5 mouse fetal liver. By integrating with a parallel single-cell sequencing analysis, we revealed the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelium cells, macrophages, megakaryocytes, and hepatic stellate cells/perivascular mesenchymal cells (PMCs) in E14.5 mouse fetal liver. Interestingly, we found that the PMCs were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing PMCs are enriched in the portal vessel area. Importantly, the majority of fetal liver HSCs are in close proximity to N-cadherin-expressing PMCs, indicating a supportive role of N-cadherin-expressing PMCs in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing PMCs are major niche-signaling senders with an enriched expression of niche factors, such as CXCL12 and KITL, and stemness pathway-related ligands, such as IGF1, IGF2, TGFβ2, TGFβ3, JAG2, and DLK1, indicating N-cadherin-expressing PMCs could be the major niche cells in supporting HSCs in the fetal liver. This finding was consistent with our previous finding that N-cadherin-expressing bone and marrow stromal progenitor cells can maintain reserve HSCs in the adult bone marrow. Moreover, CPDB analysis indicated that other potential niche cells, such as endothelium cells, macrophages, and megakaryocytes, may support HSCs in different signal transduction pathways. For example, endothelium cells have an enriched expression of KITL, IGF2, DLL1, TGFβ1, and TGFβ2; macrophages have enriched expression of KITL, IFNγ, and TGFβ1; megakaryocytes have enriched expression of PF4, JAG2 and TGFβ1. Intriguingly, our previous studies showed that megakaryocytes could promote HSC expansion under stress conditions in the bone marrow. To investigate the potential role of N-cadherin-expressing cells in supporting fetal liver HSCs, we generated an N-cad CreER;Cxcl12 and an N-cad CreER;Scf mouse model to conditionally knockout the well-studied niche factors, CXCL12 and SCF, in N-cadherin-expressing cells. Conditional knockout of either Cxcl12 or Scf in N-cadherin-expressing cells resulted in an increase in the number of HSCs. Moreover, conditional knockout of Cxcxl12 in N-cadherin-expressing cells also resulted in a myeloid-biased differentiation. We postulate that the knockout of Cxcl12 or Scf in N-cadherin-expressing cells leads to the migration of HSCs towards other potential niche cells, such as macrophages and megakaryocytes, which may induce HSC expansion and biased differentiation.In summary, by using cutting-edge spatial transcriptomics, we revealed a comprehensive spatial transcriptomics of HSCs and niche cells in E14.5 mouse fetal liver. The N-cadherin-expressing cells in the fetal liver is a major niche in maintaining HSCs, while other potential niches may be responsible for the expansion of HSCs. In the future, we will use multiple approaches, such as spatial transcriptomics and fluorescence in situ hybridization (FISH), to verify the distribution changes of HSCs in N-cad CreER;Cxcl12 mouse, and to reveal the niches in support of the expansion of HSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notch Ligand Jagged1 Is a Fetal Liver Niche Factor for the Function of Embryonic Hematopoietic Stem Cells

Embryonic hematopoietic stem cells (HSC) expand rapidly during development in the fetal liver. Notch1 is required for emergence of the definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium, and is essential for survival and function of HSCs in the fetal liver. The identity of the ligand and the ligand-presenting cell during hematopoietic development would provide valuable information of the Notch signaling mechanism in HSCs as well as the identity of key niche cells that drive the expansion and cell fate decisions of embryonic HSCs. In the present study, we have taken a comprehensive approach to determine the ligands and cells that initiate Notch signaling in the mouse fetal liver. To this end, we have performed single-cell analysis for all Notch signaling proteins and many known targets in E14.5 fetal HSCs and adult bone marrow HSCs as well as fetal liver endothelial cells. We determined that Jagged1 (Jag1) is highly expressed in both endothelial cells as well as in fetal HSCs but not in adult HSCs. We have performed conditional loss-of-function analysis of Jag1 in fetal endothelial cells as well as in fetal hematopoietic lineages, where both myeloid and megakaryocytic progenitors are shown to express high levels of Jag1. Our results indicate that while loss of endothelial Jag1 has severe effects in embryonic vascular development, loss of hematopoietic Jag1 allows for normal fetal morphology, yet severely impedes the functional ability of fetal liver HSCs to expand and differentiate. RNA-Sequencing analysis of long-term fetal HSCs in Jag1-mutant embryos (VavCreJag f/f) revealed reduced expression of Gata2, Mllt3, Hoxa7, Angpt1 and IL-12a genes in fetal HSCs, which are well-known regulators of self-renewal and expansion. Our findings indicate that Jag1 is an essential niche factor for development of HSCs in the fetal liver and for functional potential of fetal HSCs once in the bone marrow microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Self-renewal of peripheral nerve resident macrophage: does it represent a unique activation status?

Self-renewal of peripheral nerve resident macrophage: does it represent a unique activation status?

Gata2 +9.5 enhancer regulates adult hematopoietic stem cell self-renewal and T-cell development

AbstractMammalian GATA2 gene encodes a dual zinc finger transcription factor, which is essential for hematopoietic stem cell (HSC) generation in the aorta, gonad, mesonephros (AGM) region, HSC self-renewal, and specification of progenitor cell fates. Previously, we demonstrated that Gata2 expression in AGM is controlled by its intronic +9.5 enhancer. Gata2 +9.5 deficiency removes the E-box motif and the GATA site and depletes fetal liver HSCs. However, whether this enhancer has an essential role in regulating adult hematopoiesis has not been established. Here, we evaluate Gata2 +9.5 enhancer function in adult hematopoiesis. +9.5+/− bone marrow cells displayed reduced T cell reconstitution in a competitive transplant assay. Donor-derived analysis demonstrated a previously unrecognized function of the +9.5 enhancer in T cell development at the lymphoid-primed multipotent progenitor stage. Moreover, +9.5+/− adult HSCs displayed increased apoptosis and reduced long-term self-renewal capability in comparison with wild-type (WT) HSCs. These phenotypes were more moderate than those of Gata2+/− HSCs. Consistent with the phenotypic characterization, Gata2 expression in +9.5+/− LSKs was moderately higher than that in Gata2+/− LSKs, but lower than that in WT LSKs. Our data suggest that +9.5 deficiency compromises, without completely abrogating, Gata2 expression in adult HSCs.

Fetal liver hematopoietic stem cells possess the mast cell repopulation ability within a limited time window

Abstract Recent progress in developmental immunology has revealed the hematopoietic stem cell (HSC)-independent ontogeny for the various hematopoietic lineages, including mast cells (MC). Adult bone marrow (BM) HSCs fail to generate MC in both lineage tracing and transplantation assays. However, it is unclear whether fetal liver (FL) HSCs can generate MC in vivo. We investigated the MC potential of HSCs from FL and the aorta-gonad-mesonephros (AGM) in lineage tracing and transplantation assays. We employed different genetic mouse models that allowed us to identify the Tomato+ progeny of hematopoietic precursors (Runx1mer-cre-mer), endothelial cells (EC, Cdh5ERT2Cre) and HSCs (Fgd5ERT2Cre) by injecting tamoxifen (TAM) at different embryonic days starting at E7.5. TdTomato (Tom)+ HSCs and MCs were quantified after birth. In addition, we transplanted HSCs from AGM (11.5), FL (E12.5 and E15.5), and adult BM (>6 wks) into irradiated mice. Results: The EC-labeling at E7.5 efficiently generated Tomato+ MC, but not HSCs (similar results were obtained for the Runx1mer-cre-mer model). EC-labeling at E9.5 made equivalent Tom+ labeling efficiency in MC and HSCs (~85%). However, E11.5 EC mostly failed to mark MC, but still generated Tom+ HSCs (~25%). HSC-labeling at E14 or postnatal day 2 efficiently marked HSCs, but not MC. Collectively, our results indicate that active MC production terminates before E12 and that HSCs after E14 lack MC potential. Our transplantation study also shows lack of MC repopulation by HSCs after E15. However, E11.5 AGM and E12.5 FL HSCs repopulated MC, revealing a transient MC production potential of HSCs. Conclusion: MC production by hemogenic ECs terminates before E12 and early HSCs produce MC within a limited time window.

Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation

AbstractIntegrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein–coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

Zfp521 is essential for the quiescence and maintenance of adult hematopoietic stem cells under stress

SummaryZinc finger protein 521 (Zfp521), a quiescent hematopoietic stem cell (HSC)-enriched transcription factor, is involved in the self-renewal and differentiation of fetal liver HSC. However, its role in adult hematopoiesis remains elusive. Here, we found that Zfp521 deletion did not inhibit adult hematopoiesis under homeostatic conditions. In contrast, Zfp521-null chimeric mice showed significantly reduced pool size of HSC and hematopoietic progenitor cells associated with increased apoptosis and loss of quiescence. Competitive serial transplantation assays revealed that Zfp521 regulates HSC self-renewal and differentiation under regenerative stress. Mechanistically, Zfp521 transcriptionally repressed Rela expression by increasing H3K9ac and decreasing H3K9me3 levels in its promoter. Knockdown of Rela inhibited the hyper-activated NF-κB pathway and reversed the loss of quiescence in Zfp521-null HSC under stress. Thus, our results reveal a previously unrecognized role for Zfp521 as critical regulator of quiescence and self-renewal of HSC in adult hematopoiesis mediated at least partly by controlling Rela expression.

Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice.

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

Prdm16 is a critical regulator of adult long-term hematopoietic stem cell quiescence

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene Prdm16 during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. To investigate the role of Prdm16 in adult HSCs, we generated a novel conditional knockout mouse model and deleted Prdm16 in adult mouse hematopoietic system using the IFN-inducible Mx1-Cre Our results show that Prdm16 deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of Prdm16 significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which Cdkn1a and Egr1 were identified as direct targets of Prdm16 Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence.