Fetal Liver Cells Research Articles

Germ line ERG haploinsufficiency defines a new syndrome with cytopenia and hematological malignancy predisposition

AbstractThe genomics era has facilitated the discovery of new genes that predispose individuals to bone marrow failure (BMF) and hematological malignancy (HM). We report the discovery of ETS-related gene (ERG), a novel, autosomal dominant BMF/HM predisposition gene. ERG is a highly constrained transcription factor that is critical for definitive hematopoiesis, stem cell function, and platelet maintenance. ERG colocalizes with other transcription factors, including RUNX family transcription factor 1 (RUNX1) and GATA binding protein 2 (GATA2), on promoters or enhancers of genes that orchestrate hematopoiesis. We identified a rare heterozygous ERG missense variant in 3 individuals with thrombocytopenia from 1 family and 14 additional ERG variants in unrelated individuals with BMF/HM, including 2 de novo cases and 3 truncating variants. Phenotypes associated with pathogenic germ line ERG variants included cytopenias (thrombocytopenia, neutropenia, and pancytopenia) and HMs (acute myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia) with onset before 40 years. Twenty ERG variants (19 missense and 1 truncating), including 3 missense population variants, were functionally characterized. Thirteen potentially pathogenic erythroblast transformation specific (ETS) domain missense variants displayed loss-of-function (LOF) characteristics, thereby disrupting transcriptional transactivation, DNA binding, and/or nuclear localization. Selected variants overexpressed in mouse fetal liver cells failed to drive myeloid differentiation and cytokine-independent growth in culture and to promote acute erythroleukemia when transplanted into mice, concordant with these being LOF variants. Four individuals displayed somatic genetic rescue by copy neutral loss of heterozygosity. Identification of predisposing germ line ERG variants has clinical implications for patient and family diagnoses, counseling, surveillance, and treatment strategies, including selection of bone marrow donors and cell or gene therapy.

Global Transcriptomic and Characteristics Comparisons between Mouse Fetal Liver and Bone Marrow Definitive Erythropoiesis.

Erythropoiesis occurs first in the yolk sac as a transit "primitive" form, then is gradually replaced by the "definitive" form in the fetal liver (FL) during fetal development and in the bone marrow (BM) postnatally. While it is well known that differences exist between primitive and definitive erythropoiesis, the similarities and differences between FL and BM definitive erythropoiesis have not been studied. Here we performed comprehensive comparisons of erythroid progenitors and precursors at all maturational stages sorted from E16.5 FL and adult BM. We found that FL cells at all maturational stages were larger than their BM counterparts. We further found that FL BFU-E cells divided at a faster rate and underwent more cell divisions than BM BFU-E. Transcriptome comparison revealed that genes with increased expression in FL BFU-Es were enriched in cell division. Interestingly, the expression levels of glucocorticoid receptor Nr3c1, Myc and Myc downstream target Ccna2 were significantly higher in FL BFU-Es, indicating the role of the Nr3c1-Myc-Ccna2 axis in the enhanced proliferation/cell division of FL BFU-E cells. At the CFU-E stage, the expression of genes associated with hemoglobin biosynthesis were much higher in FL CFU-Es, indicating more hemoglobin production. During terminal erythropoiesis, overall temporal patterns in gene expression were conserved between the FL and BM. While biological processes related to translation, the tricarboxylic acid cycle and hypoxia response were upregulated in FL erythroblasts, those related to antiviral signal pathway were upregulated in BM erythroblasts. Our findings uncovered previously unrecognized differences between FL and BM definitive erythropoiesis and provide novel insights into erythropoiesis.

Developmental Programming of the Fetal Immune System by Maternal Western-Style Diet: Mechanisms and Implications for Disease Pathways in the Offspring.

Maternal obesity and over/undernutrition can have a long-lasting impact on offspring health during critical periods in the first 1000 days of life. Children born to mothers with obesity have reduced immune responses to stimuli which increase susceptibility to infections. Recently, maternal western-style diets (WSDs), high in fat and simple sugars, have been associated with skewing neonatal immune cell development, and recent evidence suggests that dysregulation of innate immunity in early life has long-term consequences on metabolic diseases and behavioral disorders in later life. Several factors contribute to abnormal innate immune tolerance or trained immunity, including changes in gut microbiota, metabolites, and epigenetic modifications. Critical knowledge gaps remain regarding the mechanisms whereby these factors impact fetal and postnatal immune cell development, especially in precursor stem cells in bone marrow and fetal liver. Components of the maternal microbiota that are transferred from mothers consuming a WSD to their offspring are understudied and identifying cause and effect on neonatal innate and adaptive immune development needs to be refined. Tools including single-cell RNA-sequencing, epigenetic analysis, and spatial location of specific immune cells in liver and bone marrow are critical for understanding immune system programming. Considering the vital role immune function plays in offspring health, it will be important to understand how maternal diets can control developmental programming of innate and adaptive immunity.

A Bcl11bN797K variant isolated from an immunodeficient patient inhibits early thymocyte development in mice.

BCL11B is a transcription factor with six C2H2-type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11BN441K and BCL11BN807K. To elucidate the pathogenesis of the BCL11BN807K variant, we generated a mouse model of BCL11BN807K by inserting the corresponding mutation, Bcl11bN797K, into the mouse genome. In Bcl11b+/N797K mice, the proportion of immature CD4-CD8+ single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11bN797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11bN797K/N797K fetal liver cells nearly lacked CD4+CD8+ double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11bN797K/N797K fetal liver progenitors. Interestingly, Bcl11bN797K/N797K progenitors gave rise to aberrant c-Kit+ and CD44+ cells both in vivo and in vitro. The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11bN797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11bN797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4-CD8- into CD4+CD8+ thymocytes.

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells

AbstractDuring development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

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.

Trisomy 8 Remodels Chromatin and Activates Transcription of Runx1-Target Genes in Hematopoietic Stem Cell

Myelodysplastic syndrome (MDS) is a poor prognosis cancer that predominantly affects the elderly, arising from hematopoietic stem cell (HSC), resulting in bone marrow failure and predisposition to acute myelogenous leukemia (AML). It has long been known that numerical chromosome anomalies such as trisomy 8 are critical for the development of MDS. Trisomy 8 is known to be associated with relatively poor prognosis, and is a criterion for the diagnosis of MDS in patients. Systemic inflammation has been implicated in the selective advantage for clonal hematopoietic stem cells and the progression of MDS, such as trisomy 8 MDS associated with Behcet's disease. Next generation sequencing studies revealed that trisomy 8 MDS cells highly mutated RUNX1 transcriptional factor (TF) and ASXL1, an epigenetic modifier, both of which regulate HSC function and the differentiation, relative to MDS cells without trisomy 8, suggesting that those mutations may help trisomy 8 to drive the development of MDS. However, previous clinical studies failed to identify genes on the chromosome 8 commonly amplified among trisomy 8 MDS patients, which may account for the pathogenesis of MDS, the molecular mechanism of development of trisomy 8 MDS remains unclear. Thus, we hypothesized that trisomy 8 initiates the transformation by multiple malfunctions of epigenetic and transcriptional regulators in HSC other than a gene dosage effect due to the extra chromosome 8. In order to determine how trisomy 8 affects hematopoiesis, we established a new trisomy 8 model by introducing a human chromosome 8 to mimic trisomy 8 in mouse ES cells. We succeeded in generating trisomy 8 chimeric mice and confirmed the mice showing emergence of hematopoiesis in fetal liver but significantly decreased the chimerism of trisomy 8 cells than the control mice. Wecompetitively transplanted trisomy 8 fetal liver cells at E14.5 into lethally-irradiated wild-type primary recipient mice, and moreover serial transplanted hematopoietic stem and progenitor cells (HSPCs) which isolated from the primary recipient mice. We found that trisomy 8 reduced the self-renewal capacity of HSPC and impeded the differentiation, but it was not sufficient to develop myeloid malignancies in mice. To elucidate the molecular mechanism underlying the impaired hematopoiesis by trisomy 8, we performed RNA sequencing in HSCs isolated from the primary transplanted mice. Gene set enrichment analysis revealed that in comparison with the control HSCs, trisomy 8 HSCs had significantly positive enrichments in type I and type II interferon response genes, but also the canonical target genes of Runx1 and PU.1 TFs were positively enriched in trisomy 8 HSCs. In addition, ATAC-seq analysis revealed that trisomy 8 HSCs changed chromatin accessibility enriched with the binding motif of Runx TF, compared to control HSCs. Given the changes in chromatin accessibility in trisomy 8 HSC, we performed chromatin conformation capture sequencing (Hi-C) in HSPCs and found that trisomy 8 induced changes in chromatin structures such as topologically associating domain (TAD) and compartment A/B in the other chromosomes, which were partly associated with changes in transcription of genes. Since the Runx1 gene is critical for HSC integrity and differentiation, and loss-of-function mutations of RUNX1 were often found in human myeloid malignancies with trisomy 8, we attempted to determine whether Runx1 deletion cancel the impaired self-renewal capacity of trisomy 8 HSC. We found that Runx1 deletion partially canceled the impaired repopulating capacity of trisomy 8 cells in recipient mice, suggesting the selective advantage of loss of function mutation of the RUNX1 gene in trisomy 8 mosaic people and patients with trisomy 8 MDS. Furthermore, as Janus kinase (JAK) and TANK-binding kinase 1 (TBK1) are critical for interferon signalling and activation of their downstream target genes, we revealed that the inhibition of either JAK or TBK function ameliorated trisomy 8-induced chromatin remodelling in HSCs. Overall, trisomy 8 changes chromatin structure and transcription of genes on other chromosomes in HSC accompanied with the malfunction of Runx1 TF, at least in part, due to the activation of interferon signaling, and results in the impaired hematopoiesis. We demonstrated that trisomy 8 creates the basis for transformation of MDS via remodeling chromatin structures in HSC.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank < 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

Wild-Type Calreticulin Downmodulates MPL and Impacts HSC Self-Renewal

All Myeloproliferative neoplasms (MPN) driver mutations converge on the activation of thrombopoietin receptor (MPL) signaling. Somatic frameshift mutations in calreticulin (CALR), leading to a novel peptide sequence are seen in 20-30% of patients with essential thrombocythemia or myelofibrosis. CALR is a multifunctional calcium-binding protein that is primarily localized to the endoplasmic reticulum (ER) of cells. It plays important roles in calcium homeostasis, protein folding, and quality control within the ER. MPN associated CALR mutants bind to and activate MPL resulting in constitutive activation of signaling. We established a transduction-transplantation model of MPN CALR mutant (52bp deletion, referred to as CALR DEL hereafter) MPN. As expected, mice transplanted with CALR DEL cells developed thrombocytosis, this phenotype was maintained in secondary transplants. We included mice transplanted with MSCV-IRES-GFP empty vector (EV) as well as CALRWT-IRES-GFP vector as controls. Abundant GFP + cells were seen in the peripheral blood and bone marrow of all control mice (EV and CALR WT) in primary transplants. In secondary transplants GFP + cells persisted in the EV cohort, however surprisingly no GFP + cells were seen in the CALR WT cohort. These experiments were repeated three independent times with similar results. We confirmed that the inability to serially transplant was not due to a homing defect. In addition, CALR WT cells demonstrated defective serial replating in methylcellulose. The inability to serially transplant indicated that overexpression of CALR WT negatively impacts hematopoietic stem cell (HSC) self-renewal. To investigate how loss of CALR impacts hematopoiesis we utilized fetal liver (FL) cells from CALR -/- mice, the use of FL cells was required because CALR -/- is embryonic lethal. We performed competitive transplants with a 50:50 ratio of CD45.1/2 CALR -/- FL and CD45.1 CALR +/+ FL cells into CD45.2 donors. CALR -/-outcompeted CALR +/+ cells, demonstrating that loss of CALR enhances hematopoietic competitive potential. We hypothesized that WT CALR negatively modulates MPL signaling, as the defective serially transplantation is also seen with MPL -/- and MPN associated CALR mutants are known to bind MPL and enhance signaling. To interrogate interactions between WT CALR and MPL we created Ba/F3 cells co-expressing mMPL (MSCV-IRES-hCD4 vector) and CALR WT, CALR DEL, or EV (MSCV-IRES-GFP vector). Cells co-expressing CALR WT and MPL cells had inferior growth compared to cells expressing MPL alone in limiting concentrations of thrombopoietin (TPO) (Figure 1B). The negative impact of CALR WT was unique to MPL dependent growth, as CALR WT + MPL cells grew equivalently well as MPL cells in limiting concentrations of IL-3. In conclusion, we propose that CALR may serve a physiological function to modulate MPL signaling. Further studies are ongoing to interrogate direct interactions between CALR WT and MPL. Moreover, these features of CALR as a negative regulator of MPL signaling could be leveraged as a novel therapeutic approach in MPN to reduce oncogenic MPL signaling in MPN.

FLT3-Ligand Alone Enables the Identification and Maintenance in Vitro of Viable Quiescent Human Fetal Liver Hematopoietic Stem Cells with Maintenance of Their Function

Cells with sustained multi-lineage blood cell regenerative potential are referred to as hematopoietic stem cells (HSCs). Current findings indicate that they are responsible for lifelong blood production, are first detected within the first month of development in humans and are largely created prior to birth after which they expand their numbers in response to physiological demands for normal or enhanced blood cell output requirements. Interestingly, a number of the intrinsically determined functional properties of HSCs also change after birth. These include an apparent decline in their self-renewal potential. Thus, for example, in humans as in mice, cells with long-term (>6 month) HSC regenerative properties present in the developing fetal liver (FL) are a source of HSCs that possess a much higher in vivo regenerative capability than those from older donor sources, such as adult bone marrow or even cord blood (CB). Accordingly, it has been of longstanding interest to better understand the molecular regulation of this high regenerative capacity prevalent in fetal HSCs for potential future therapeutic as well as scientific exploitation. Here, we describe the results of experiments designed to answer the hypothesis that human FL HSCs with human-relevant self-renewal properties can be isolated as a quiescent CD49f+ subset of the GPI80+CD90+CD38-CD45RA-CD34+CD45+ population following their incubation in standard serum-free culture medium supplemented with FLT3-ligand (FLT3-L) alone. Initial experiments showed expression of the CD49f integrin on first trimester hFL cells selectively depleted cells able to produce colonies of granulocytes, macrophages or erythroid cells in standard 2-week methylcellulose cultures containing SCF, GM-CSF, IL6, IL3 and EPO. Conversely, expression of the CD49f integrin selectively enriched for cells with 12-week output capabilities in both growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NOD-Rag1 -/-IL2Rγc -/- W 41/41 (NRG-W) mice. Initial experiments designed to test the effect of multiple GF and small molecule additives on the maintenance over a 7-day period of this in vivo regenerative ability of the input FL HSC confirmed GPI80 expression to be a continuing positive selective phenotype. In addition, the result of transplant experiments showed that a 2-day incubation in FLT3-L alone maintained the 12-week serially transplantable activity of the HSCs (12-weeks/cycle) as fully equivalent to the unmanipulated input cells and significantly superior (P<0.05) to FLT3-L+IL3+SF (3GF) with or without addition of UM171, stemregenin, or eltrombopag. Interestingly, in vitro monitoring of GPI80+ cells showed FLT3-L alone maintained the survival of only 20% of the input GPI80+ cells compared to any of the 3GF-based conditions. In addition, the time to complete a first division and subsequent divisions of the input cells was delayed and prolonged, respectively, in the FLT3-L versus the 3GF conditions. Subsequent 7-day suspension cultures and 6-week GF-supplemented stromal co-cultures experiments have confirmed the existence of a G0 population within the HSC-enriched subset of human FL cells that best maintains their growth potential in FLT3-L alone. Together, these results demonstrate CD49f expression to be a pervasive marker of human HSCs throughout development and reveal the importance of different GF conditions to support the maintenance of viability and retention of self-renewal capability of human fetal HSCs in contrast to those required to activate/support a rapid initiation of cell division. These findings set the stage for future development of strategies to exploit human FL cells therapeutically and may also be critical to designing conditions that will best support the ex vivo maintenance of HSCs at and after birth.

AML1-ETO and CCND2 Overexpression Cooperate to Drive AML Initiation and Progression

Objectives: The translocation between chromosome 8 and 21, resulting in the AML1-ETO fusion gene, is one of the most common cytogenetic abnormalities observed in acute myeloid leukemia (AML). However, AML1-ETO alone is insufficient to cause leukemia, additional secondary events are required for leukemogenesis. CCND2 gene, a key member of the CyclinD2-CDK4/6 complex, plays a crucial role as a cell cycle regulator. In hematopoietic malignancies, CCND2 mutations (particularly with a conserved mutated hotspot on Thr280) have been found in AML, particularly in core binding factor leukemias. The mutated CCND2 protein exhibits greater stability and resistance to proteasomal degradation. Previous research had shown that CCND2 was a transcriptional target of AML1-ETO, indicating that AML1-ETO can promote CCND2 expression. However, it remains unclear whether mutated CCND2 can cooperate with the AML1-ETO to drive leukemia initiation and progression. Methods: In our previous study, a conditional AML1-ETO knock-in mouse model was developed, in which the AML1-ETO fusion gene was tagged with m-Cherry. To investigate the role of CCND2 in this model, the coding sequences of both CCND2 wt (wild-type) and CCND2 mut (mutant with Thr280Ala substitution) were cloned into the retroviral vector MSCV-IRES-EGFP. Subsequently, the fetal liver cells (embryonic day=12.5) from AML1-ETO mice were infected with the corresponding retroviruses. Results: Four months post AML1-ETO induction, the percentage of m-Cherry + GFP + cells of the mice in CCND2 wt and CCND2 mut groups began to increase. The results revealed a substantial upregulation of CCND2 mRNA and protein expression in these groups compared to that of the Vec group. Additionally, the CCND2 mut group exhibited even higher CCND2 protein expression due to impaired protein degradation. Moreover, the shortened survival time and pathological findings in mice from the CCND2 wt and CCND2 mut groups confirmed that they were succumbed to leukemia infiltration. To assess the stemness of leukemia stem cells (LSCs), extreme limiting dilution analysis (ELDA) was conducted, and it showed a significant increase in LSC frequency in the CCND2 wt and CCND2 mut groups compared to that of the Vec group. Notably, transplanting AML1-ETO cells alone didn't confer any competitive advantage in sublethally irradiated mice, while the overexpression of CCND2 provided them with the ability to survive. Transcriptome analysis indicated that compared to the Vec group, CCND2 wt and CCND2 mut groups exhibited notable enrichment in OXPHOS, E2F, and G2M gene sets. Interestingly, the mTORC1 signaling pathway was particularly enriched in the CCND2 mut group compared to the other two groups. To verify this speculation, phosphorylated S6 and total S6 protein levels were assessed through Western blotting. The results showed that compared to Vec group, the p-S6/total S6 ratio show a gradual increase in the CCND2 wt and CCND2 mut groups, indicating activation of the mTOR pathway by CCND2 protein. Everolimus, a selective inhibitor of mTOR, was administered to CCND2 wt and CCND2 mut mice at a dose of 5mg/kg via oral gavage (three times a week) for three consecutive weeks. The Everolimus-treated mice exhibited reduced leukemic burden and fewer number of LSCs compared to that of the vehicle-treated group. Additionally, Everolimus restored the non-leukemic cells and extended their survival time in CCND2 wt and CCND2 mut group mice. Conclusion: In our study, a leukemia mouse model with co-expression of AML1-ETO and CCND2 mutation was successfully established, which demonstrated that the introduction of the CCND2 gene into the existing AML1-ETO model can trigger the development of leukemia. It was also confirmed that CCND2 overexpression results in the upregulation of the mTOR pathway, which drove the progression of leukemia. Everolimus effectively improved the outcome of mice with CCND2 wt or CCND2 mut, suggesting its potential as a therapeutic agent for this subtype of leukemia.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

O-Linked N-Acetylglucosamine Transferase Ensures Survival of Mouse Fetal Liver Hematopoietic Progenitors Partly by Regulating Bcl-xL and Oxidative Phosphorylation.

O-linked N-acetylglucosamine transferase (OGT) critically regulates wide variety of biological processes such as gene expression, metabolism, stress response, signaling and proteostasis. In adult hematopoiesis, OGT is crucial for differentiation of B and T cells and the maintenance of hematopoietic stem cells (HSCs). However, a role for OGT in fetal liver (FL) hematopoiesis remains unknown. To investigate a role for OGT in FL hematopoiesis, we conditionally disrupted OGT in hematopoietic cells in developing FLs. Hematopoietic specific disruption of OGT resulted in embryonic lethality in late stage of gestation due to severe anemia and growth retardation. OGT loss led to profound reduction of differentiating erythroid cells and erythroid progenitors in FLs due to massive apoptosis. In addition, clonogenic capacity of FL cells was severely impaired by OGT loss. Interestingly, expression of BCL-XL, a well-known inhibitor of apoptosis in FL cells, dramatically decreased, and the levels of reactive oxygen species (ROS) were increased in OGT-deficient FL cells. Overexpression of Bcl-xL and reduction of ROS significantly restored the colony formation of OGT-deficient FL cells. This study revealed a novel role for OGT during embryogenesis, which ensures survival of FL hematopoietic cells partly by regulating Bcl-xL and oxidative phosphorylation.

Comprehensive Characterization and Global Transcriptome Analysis of Human Fetal Liver Terminal Erythropoiesis

The fetal liver (FL) is the key erythropoietic organ during fetal development, but knowledge on human FL erythropoiesis is very limited. In this study, we sorted primary erythroblasts from FL cells and performed RNA sequencing (RNA-seq) analyses. We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis. Notably, the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts (OrthoEs), suggesting the involvement of these pathways in enucleation. We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+ cells. Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences, indicating impacts of the culture system on gene expression. Notably, the expression of lipid metabolism-related genes was increased in cultured erythroblasts. We further immortalized erythroid cell lines from FL and cord blood (CB) CD34+ cells (FL-iEry and CB-iEry, respectively). FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs, but their enucleation ability was very low. Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity, indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate. Additionally, the expression of HBE1, HBZ, and HBG2 was up-regulated in FL-iEry compared with CB-iEry, and such up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1. Our study provides new insights into human FL erythropoiesis and rich resources for future studies.

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.

Isolation, In Vitro Differentiation, and Culture of Murine Megakaryocytes From Fetal Liver and Adult Bone Marrow

AbstractMegakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires enrichment or considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the enrichment of primary MKs directly from the murine bone marrow as well as in vitro differentiation of fetal liver– or bone marrow–derived hematopoietic stem cells into MKs. Although in vitro–differentiated MKs are not synchronized in their maturation, they can be enriched over an albumin density gradient, and one‐third to one‐half of recovered cells will typically elaborate proplatelets. Support protocols describe methods for preparing fetal liver cells, identifying mature rodent MKs by staining for flow cytometry analysis, and immunofluorescence staining of fixed MKs for confocal laser scanning microscopy. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Isolation of mature bone marrow megakaryocytes by magnetic‐activated cell sortingBasic Protocol 2: Preparation of a megakaryocyte suspension culture from murine fetal livers or lineage‐depleted adult bone marrowSupport Protocol 1: Preparation of a single‐cell suspension from murine fetal livers for megakaryocyte cultureSupport Protocol 2: Megakaryocyte culture from lineage‐depleted murine bone marrowSupport Protocol 3: Quality control of megakaryocyte culture with flow cytometrySupport Protocol 4: Immunofluorescence staining of megakaryocytes for detection with confocal laser scanning microscopy

A Refined Single Cell Landscape of Haematopoiesis in the Mouse Foetal Liver.

During prenatal life, the foetal liver is colonised by several waves of haematopoietic progenitors to act as the main haematopoietic organ. Single cell (sc) RNA-seq has been used to identify foetal liver cell types via their transcriptomic signature and to compare gene expression patterns as haematopoietic development proceeds. To obtain a refined single cell landscape of haematopoiesis in the foetal liver, we have generated a scRNA-seq dataset from a whole mouse E12.5 liver that includes a larger number of cells than prior datasets at this stage and was obtained without cell type preselection to include all liver cell populations. We combined mining of this dataset with that of previously published datasets at other developmental stages to follow transcriptional dynamics as well as the cell cycle state of developing haematopoietic lineages. Our findings corroborate several prior reports on the timing of liver colonisation by haematopoietic progenitors and the emergence of differentiated lineages and provide further molecular characterisation of each cell population. Extending these findings, we demonstrate the existence of a foetal intermediate haemoglobin profile in the mouse, similar to that previously identified in humans, and a previously unidentified population of primitive erythroid cells in the foetal liver.

Assessment of the effect of cell transplantation on DNA repair in rat hepatocytes exposed to carbon tetrachloride (CCl4) by DNA comet assay

Introduction. The effect of carbon tetrachloride (freon-10, asordin, hladon-10) is an organochlorine compound with the chemical formula CCl4 and the subsequent transplantation of fetal liver cells (FLC) on DNA degradation and repair in rat hepatocytes by means of alkaline single-cell gel electrophoresis (DNA comet assay) was assessed. Material and methods. Acute toxic damage to the rat liver was simulated by a single oral administration to female Wistar rats of CCl4 in an oil solution at a dose of 3000 mg/kg. As a protective agent, a suspension of FLC of E19 rat fetuses was used. Quantitative assessment of the degree of damage to the nuclear DNA of liver cells was performed by DNA comet assay on days 1, 3, 5, 7 and 16 of the experiment. Results. Intravenous injections of fetal liver cells 6 h after exposure to CCl4 induces DNA repair processes in rat hepatocytes in 5-7 days and led to a decrease in the intensity of nuclear DNA damage. The trend toward a decrease in the number of undamaged hepatocytes continued on the 16th day of the experiment, and, therewith, the enhancement of reparative processes after FLC injection revealed itself in in a significant decrease in the number of hepatocytes with a high intensity of nuclear DNA damage. Limitations. To prevent unwanted death of animals in the group, studies were limited to a dose of 3000 mg/kg of CCl4 in oil solution. Conclusion. The method of alkaline single-cell gel electrophoresis (DNA comet assay) allowed quantitative assessment of the degrees of genome damage and repair. The obtained positive results suggest that FLC exert a protective effect of the structure of the DNA of rat liver role after acute exposure to CCl4.