Hematopoietic Differentiation Research Articles

Single-cell transcriptional mapping reveals genetic and non-genetic determinants of aberrant differentiation in AML.

In acute myeloid leukemia (AML), genetic mutations distort hematopoietic differentiation, resulting in the accumulation of leukemic blasts. Yet, it remains unclear how these mutations intersect with cellular origins and whether they converge upon similar differentiation patterns. Single-cell RNA sequencing (scRNA-seq) has enabled high-resolution mapping of the relationship between leukemia and normal cell states, yet this application is hampered by imprecise reference maps of normal hematopoiesis and small sample sizes among patient cohorts. As a first step we constructed a reference atlas of human bone marrow hematopoiesis from 263,519 single-cell transcriptomes spanning 55 cellular states, that was benchmarked against independent datasets of immunophenotypically pure hematopoietic stem and progenitor cells. Using this reference atlas, we mapped over 1.2 million single-cell transcriptomes spanning 318 AML, mixed phenotype acute leukemia (MPAL), and acute erythroid leukemia (AEL) samples. This large-scale analysis, together with systematic mapping of genotype-to-phenotype associations between driver mutations and differentiation landscapes, revealed convergence of diverse genetic alterations on twelve recurrent patterns of aberrant differentiation in AML. This included unconventional lymphoid and erythroid priming linked to RUNX1 and TP53 mutations, respectively. We also identified non-genetic determinants of AML differentiation such as two subgroups of KMT2A-rearranged AML that differ in the identity of their leukemic stem cells (LSCs), likely reflecting distinct cellular origins. Furthermore, distinct LSC-driven hierarchies can co-exist within individual patients, providing insights into AML evolution. Together, precise mapping of normal and malignant cell states provides a framework for advancing the study and disease classification of hematologic malignancies thereby informing therapy development.

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the β-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

Long-term lineage commitment in hematopoietic stem cell gene therapy.

Hematopoietic Stem Cell (HSC) gene therapy (GT) may provide lifelong reconstitution of the hematopoietic system with gene-corrected cells1. However, the effects of underlying genetic diseases, replication stress, and aging on hematopoietic reconstitution and lineage specification remain unclear. In this study, we analyzed hematopoietic reconstitution in 53 patients treated with lentiviral-HSC-GT for diverse conditions such as metachromatic leukodystrophy2,3 (MLD), Wiskott-Aldrich syndrome4,5 (WAS), and β-thalassemia6 (β-Thal) over a follow-up period of up to 8 years, using vector integration sites as markers of clonal identity. We found that long-term hematopoietic reconstitution was supported by 770 to 35,000 active HSCs. While 50% of transplanted clones demonstrated multilineage potential across all conditions, the remaining clones exhibited a disease-specific preferential lineage output and long-term commitment: myeloid for MLD, lymphoid for WAS, and erythroid for β-Thal, particularly in adult patients. Our results indicate that HSC clonogenic activity, lineage output, long-term lineage commitment, and rates of somatic mutations are influenced by the underlying disease, patient age at the time of therapy, the extent of genetic defect correction, and the hematopoietic stress imposed by the inherited disease. This suggests that HSCs adapt to the pathological condition during hematopoietic reconstitution.

Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia.

Myeloid leukemia factor 1 (Mlf1) was identified as a proto-oncoprotein that affects hematopoietic differentiation in humans. However, its cellular function remains elusive, spanning roles from cell cycle regulation to modulation of protein aggregate formation and participation in ciliogenesis. Given that structurally conserved homologs of Mlf1 can be found across the eukaryotic tree of life, we decided to characterize its cellular role underlying this phenotypic pleiotropy. Using a model of the unicellular eukaryote Giardia intestinalis, we demonstrate that its Mlf1 homolog (GiMlf) mainly localizes to two types of cytosolic foci: microtubular structures, where it interacts with Hsp40, and ubiquitin-rich, membraneless compartments, found adjacent to mitochondrion-related organelles known as mitosomes, containing the 26S proteasome regulatory subunit 4. Upon cellular stress, GiMlf either relocates to the affected compartment or disperses across the cytoplasm, subsequently accumulating into enlarged foci during the recovery phase. In vitro assays suggest that GiMlf can be recruited to membranes through its affinity for signaling phospholipids. Importantly, cytosolic foci diminish in the gimlf knockout strain, which exhibits extensive proteomic changes indicative of compromised proteostasis. Consistent with data from other cellular systems, we propose that Mlf acts in the response to proteotoxic stress by mediating the formation of function-specific foci for protein folding and degradation.

Modification of Lhx2 activity for ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells.

Hematopoietic stem cells (HSCs) obtained from patient-derived human induced pluripotent stem cells (iPSCs) are a promising tool for curing various hematological disorders. We previously demonstrated that enforced expression of the LIM-homeobox transcription factor Lhx2, which is essential for mouse embryonic hematopoiesis, leads to generation of engraftable and expandable hematopoietic stem cells (HSCs) from mouse iPSCs. However, it remained unknown whether Lhx2 can induce HSCs from human iPSCs. Here, we investigated the effect of Lhx2 overexpression on hematopoietic differentiation of human iPSCs. Unexpectedly, Lhx2 severely inhibited proliferation of human iPSC-derived hematopoietic cells. Thus, Lhx2 exhibited differential effects on mouse and human hematopoietic cells. Further studies implied that the inhibitory effect of Lhx2 on human iPSC-derived hematopoietic cells was due to insufficient transcriptional activation ability. Therefore, we modified Lhx2 to strengthen its activity as a transcriptional activator. This modified Lhx2 could induce ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells (HSPCs). We believe that these findings will facilitate the development of a method to efficiently produce HSCs from human iPSCs.

Identification of Biomarkers and Mechanisms Associated with Apoptosis in Recurrent Pregnancy Loss.

In this study, we employed bioinformatics techniques to identify genes associated with apoptosis in recurrent pregnancy loss (RPL). We retrieved the RPL expression profile datasets GSE165004 and GSE73025 from the Gene Expression Omnibus (GEO) database. We also obtained data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway of Apoptosis (hsa04210) to identify apoptosis-related genes. In addition, we performed Friends analysis to explore the interactions between differential apoptosis genes and other genes in the functional pathway. We identified six differentially expressed genes related to apoptosis, including CTSZ, BCL2, PIK3CD, KRAS, GADD45G, and CASP8, with GADD45G as the most gene. Functional fertility analysis revealed that differentially expressed genes primarily regulated protein stability, cell number homeostasis, myeloid cell homeostasis, hematopoietic progenitor cell differentiation, lytic vacuole and lysosome functions, vacuolar and lysosomal membranes, transmembrane transporter binding, protein domain-specific binding, G-protein beta-subunit binding, phospholipid binding, and were involved in pathways such as Rap1 signaling, regulation of actin cytoskeleton, and NOD-like receptor signaling. KRAS exhibited the highest mutation rate in RPL-related cancer CESC. There was also a positive correlation between differentially expressed genes and B cell memory, CD4 memory resting T cells, follicular helper T cells, naïve B cells, and resting dendritic cells. We identified six differentially expressed genes related to apoptosis in RPL, with GADD45G as the most important. NOD-like receptor signaling pathway and regulation of actin cytoskeleton could be therapeutic targets for RPL.

Processing pipelines and analytical methods for single-cell DNA methylation sequencing data.

Single-cell DNA methylation sequencing technology has seen rapid advancements in recent years, playing a crucial role in uncovering cellular heterogeneity and the mechanisms of epigenetic regulation. As sequencing technologies have progressed, the quality and quantity of single-cell methylation data have also increased, making standardized preprocessing workflows and appropriate analysis methods essential for ensuring data comparability and result reliability. However, a comprehensive data analysis pipeline to guide researchers in mining existing data has yet to be established. This review systematically summarizes the preprocessing steps and analysis methods for single-cell methylation data, introduces relevant algorithms and tools, and explores the application prospects of single-cell methylation technology in neuroscience, hematopoietic differentiation, and cancer research. The aim is to provide guidance for researchers in data analysis and to promote the development and application of single-cell methylation sequencing technology.

A bispecific nanosystem activates endogenous natural killer cells in the bone marrow for haematologic malignancies therapy.

Haematologic malignancies commonly arise from the bone marrow lesion, yet there are currently no effective targeted therapies against tumour cells in this location. Here we constructed a bone-marrow-targeting nanosystem, CSF@E-Hn, which is based on haematopoietic-stem-cell-derived nanovesicles adorned with gripper ligands (aPD-L1 and aNKG2D) and encapsulated with colony-stimulating factor (CSF) for the treatment of haematologic malignancies. CSF@E-Hn targets the bone marrow and, thanks to the gripper ligands, pulls together tumour cells and natural killer cells, activating the latter for specific tumour cell targeting and elimination. The therapeutic efficacy was validated in mice bearing acute myeloid leukaemia and multiple myeloma. The comprehensive assessment of the post-treatment bone marrow microenvironment revealed that the integration of CSF into a bone-marrow-targeted nanosystem promoted haematopoietic stem cell differentiation, boosted memory T cell generation and maintained bone homoeostasis, with long-term prevention of relapse. Our nanosystem represents a promising strategy for the treatment of haematologic malignancies.

EnhancerNet: a predictive model of cell identity dynamics through enhancer selection.

Understanding how cell identity is encoded by the genome and acquired during differentiation is a central challenge in cell biology. I have developed a theoretical framework called EnhancerNet, which models the regulation of cell identity through the lens of transcription factor-enhancer interactions. I demonstrate that autoregulation in these interactions imposes a constraint on the model, resulting in simplified dynamics that can be parameterized from observed cell identities. Despite its simplicity, EnhancerNet recapitulates a broad range of experimental observations on cell identity dynamics, including enhancer selection, cell fate induction, hierarchical differentiation through multipotent progenitor states and direct reprogramming by transcription factor overexpression. The model makes specific quantitative predictions, reproducing known reprogramming recipes and the complex haematopoietic differentiation hierarchy without fitting unobserved parameters. EnhancerNet provides insights into how new cell types could evolve and highlights the functional importance of distal regulatory elements with dynamic chromatin in multicellular evolution.

Transcription factor networks disproportionately enrich for heritability of blood cell phenotypes.

Most phenotype-associated genetic variants map to non-coding regulatory regions of the human genome. Moreover, variants associated with blood cell phenotypes are enriched in regulatory regions active during hematopoiesis. To systematically explore the nature of these regions, we developed a highly efficient strategy, Perturb-multiome, that makes it possible to simultaneously profile both chromatin accessibility and gene expression in single cells with CRISPR-mediated perturbation of a range of master transcription factors (TFs). This approach allowed us to examine the connection between TFs, accessible regions, and gene expression across the genome throughout hematopoietic differentiation. We discovered that variants within the TF-sensitive accessible chromatin regions, while representing less than 0.3% of the genome, show a ~100-fold enrichment in heritability across certain blood cell phenotypes; this enrichment is strikingly higher than for other accessible chromatin regions. Our approach facilitates large-scale mechanistic understanding of phenotype-associated genetic variants by connecting key cis-regulatory elements and their target genes within gene regulatory networks.

The Clonal Hematopoiesis-associated Gene Srcap Plays an Essential Role in Hematopoiesis.

Somatic mutations arising in hematopoietic stem cells (HSCs) may provide the latter with a fitness advantage, allowing the mutant HSC to clonally expand. Such mutations have been recurrently identified in the chromatin modifier, SRCAP, in both non-malignant and leukemic clones, suggesting that this gene plays a significant role in hematopoiesis. We generated a conditional Srcap loss of function murine model and determined the consequences of hematopoietic-specific loss of this gene. We show that Srcap is essential for normal fetal liver erythropoiesis and monocytopoiesis. In Srcap deficient fetal livers, the number of phenotypic HSCs is similar to that of controls, but these HSCs exhibit a profound repopulating defect. Likewise, conditional deletion of Srcap during adult hematopoiesis results in a rapid loss of HSCs. Loss of Srcap is associated with evidence of increased DNA damage in HSCs and lineage-restricted progenitors as assessed by y-H2AX expression. Consistent with this finding, we observed strong transcriptional upregulation of the p53 pathway in Srcap deficient erythroid precursors. Collectively our data highlight the importance of Srcap in maintaining HSC function and supporting hematopoietic differentiation and suggests that it plays an essential role in maintaining genomic integrity.

Loss of Dnajc21 leads to cytopenia and altered nucleotide metabolism in zebrafish

Mutations in the DNAJC21 gene were recently described in Shwachman–Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.

Significance of targeting DNMT3A mutations in AML.

Acute myeloid leukemia (AML) is the most prevalent form of leukemia among adults, characterized by aggressive behavior and significant genetic diversity. Despite decades of reliance on conventional chemotherapy as the mainstay treatment, patients often struggle with achieving remission, experience rapid relapses, and have limited survival prospects. While intensified induction chemotherapy and allogeneic stem cell transplantation have enhanced patient outcomes, these benefits are largely confined to younger AML patients capable of tolerating intensive treatments. DNMT3A, a crucial enzyme responsible for establishing de novo DNA methylation, plays a pivotal role in maintaining the delicate balance between hematopoietic stem cell differentiation and self-renewal, thereby influencing gene expression programs through epigenetic regulation. DNMT3A mutations are the most frequently observed genetic abnormalities in AML, predominantly in older patients, occurring in approximately 20-30% of adult AML cases and over 30% of AML with a normal karyotype. Consequently, the molecular underpinnings and potential therapeutic targets of DNMT3A mutations in AML are currently being thoroughly investigated. This article provides a comprehensive summary and the latest insights into the structure and function of DNMT3A, examines the impact of DNMT3A mutations on the progression and prognosis of AML, and explores potential therapeutic approaches for AML patients harboring DNMT3A mutations.

Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma

AbstractHematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies, being particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we characterized 48 patients treated with B-cell maturation antigen (BCMA) CAR-T cells to understand kinetics of cytopenia, identify predictive factors, and determine potential mechanisms underlying these toxicities. We observed that overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, being still present after 1 year in 4 and 3 patients, respectively. Baseline cytopenia and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms underlying cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPC differentiation, promoting more immature phenotypes, which could be prevented with a combination of interferon γ, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). These results suggest that CAR-T activation induces HSPC maturation arrest through paracrine effects and provides potential treatments to mitigate the severity of this toxicity.

In Vivo Chemical Screening in Zebrafish Embryos Identified FDA-Approved Drugs That Induce Differentiation of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy.

Structural basis of MPL activation by thrombopoietin

AbstractMyeloproliferative leukemia protein (MPL), also known as thrombopoietin (TPO) receptor, is a class I cytokine receptor that is expressed on hematopoietic progenitors, promoting growth and differentiation toward the megakaryocyte lineage and is critical for normal platelet production. Mutations in MPL, TPO, or Janus kinase 2 (JAK2) have been implicated in multiple diseases from congenital thrombocytopenias to myeloproliferative neoplasms. The ligand for MPL, TPO, stimulates platelet production by inducing MPL dimerization and results in an active conformation that allows downstream JAK2/STAT5 signaling. Despite the biological importance of this pathway, the molecular signaling mechanism remained unclear. Here, we present a 3.39-Å cryo-electron microscopy structure of the ectodomain of mouse MPL bound to TPO. The structure revealed both low and high affinity sites between MPL and TPO that contain several pathologic mutations. To better understand TPO-driven MPL signaling, we expanded upon this structure by molecular dynamic (MD) simulations to model the full-length human MPL/TPO complex, and showed that MPL D4-D4 domain interactions are functionally relevant in activity assays. To build on our understanding of downstream activation, we added JAK2 to the MPL/TPO complex by MD simulations. This ternary complex illustrates JAK2 dimerization through the pseudokinase domain, illustrates residues important for MPL interactions, and reveals the constitutive activation mechanism of patient mutant V617F. The model also suggests the mechanism of JAK2 tyrosine kinase domain transphosphorylation. Overall, our studies illuminate TPO/MPL/JAK2 signaling mechanisms and provide additional insight into the nature of receptor signaling, which will further benefit human health.

Lenalidomide and pomalidomide modulate hematopoietic cell expansion and differentiation in the presence of MSC

Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34+ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34+CD38− cells while decreasing the number of mature CD34+CD38+ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34+ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

Single-cell genomics details the maturation block in BCP-ALL and identifies therapeutic vulnerabilities in DUX4-r cases

AbstractB-cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy and is driven by multiple genetic alterations that cause maturation arrest and accumulation of abnormal progenitor B cells. Current treatment protocols with chemotherapy have led to favorable outcomes but are associated with significant toxicity and risk of side effects, highlighting the necessity for highly effective, less toxic, targeted drugs, even in subtypes with a favorable outcome. Here, we used multimodal single-cell sequencing to delineate the transcriptional, epigenetic, and immunophenotypic characteristics of 23 childhood BCP-ALLs belonging to the BCR::ABL1+, ETV6::RUNX1+, high hyperdiploid, and recently discovered DUX4-rearranged (DUX4-r) subtypes. Projection of the ALL cells along the normal hematopoietic differentiation axis revealed a diversity in the maturation pattern between the different BCP-ALL subtypes. Although the BCR::ABL1+, ETV6::RUNX1+, and high hyperdiploidy cells mainly showed similarities to normal pro-B cells, DUX4-r ALL cells also displayed transcriptional signatures resembling mature B cells. Focusing on the DUX4-r subtype, we found that the blast population displayed not only multilineage priming toward nonhematopoietic cells, myeloid, and T-cell lineages, but also an activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling that sensitized the cells to PI3K inhibition in vivo. Given the multilineage priming of DUX4-r blasts with aberrant expression of myeloid marker CD371 (CLL-1), we generated chimeric antigen receptor T cells, which effectively eliminated DUX4-r ALL cells in vivo. These results provide a detailed characterization of BCP-ALL at the single-cell level and reveal therapeutic vulnerabilities in the DUX4-r subtype, with implications for the understanding of ALL biology and new therapeutic strategies.

A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency.

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+ progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.

Histone H2A deubiquitinase BAP1 is essential for endothelial cell differentiation from human pluripotent stem cells.

Polycomb group proteins (PcGs) add repressive post translational histone modifications such as H2AK119ub1, and histone H2A deubiquitinases remove it. Mice lacking histone H2A deubiquitinases such as Usp16 and Bap1 die in embryonic stage, while mice lacking Usp3, Mysm1, Usp12, and Usp21 have been shown to be deficient in hematopoietic lineage differentiation, cell cycle regulation, and DNA repair. Thus, it is likely that histone deubiquitinases may also be required for human endothelial cell differentiation; however, there are no reports about the role of histone H2A deubiquitinase BAP1 in human endothelial cell development. We differentiated human pluripotent stem cells into the endothelial lineage which expressed stable inducible shRNA against BAP1. Our results show that BAP1 is required for human endothelial cell differentiation.