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Pediatric acute myeloid leukemia (pAML) is a clonal disease with recurrent genetic alterations that affect epigenetic states. However, the implications of epigenetic dysregulation in disease progression remain unclear. Here, we interrogated single-cell and clonal level chromatin accessibility of bone marrow samples from 28 pAML patients representing multiple subtypes using mtscATAC-seq, which revealed distinct differentiation hierarchies and abnormal chromatin accessibility in a subtype-specific manner. Innate immune signaling was commonly enhanced across subtypes and related to improved advantage of clonal competition and unfavorable prognosis, with further reinforcement in a relapse-associated leukemia stem cell-like population. We identified a panel of 31 innate immunity related genes to improve the risk classification of pAML patients. By comparing paired diagnosis and post-chemotherapy relapse samples, we showed that primitive cells significantly reduced MHC class II signaling, suggesting an immune evasion mechanism to facilitate their expansion at relapse. Key regulators orchestrating cell cycle dysregulation were identified to contribute to pAML relapse in drug-resistant clones. Our work establishes the single-cell chromatin accessibility landscape at clonal resolution and reveals the critical involvement of epigenetic disruption, offering insights into classification and targeted therapies of pAML patients., (Copyright © 2024 American Society of Hematology.)

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

Hematopoietic stem cells (HSCs) are a rare type of hematopoietic cell that can entirely reconstitute the blood and immune system after transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases; however, it remains a high-risk therapy because of its potential side effects, including poor graft function and graft-versus-host disease (GVHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution in low-cell dose grafts. Here, we demonstrate that the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures can be improved using physioxic culture conditions. Single-cell transcriptomic analysis helped confirm the inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GVHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, and highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Macrophages play pivotal roles in immunity, hematopoiesis, and tissue homeostasis. In mammals, macrophages have been shown to originate from yolk-sac-derived erythro-myeloid progenitors and aorta-gonad-mesonephros (AGM)-derived hematopoietic stem cells. However, whether macrophages can arise from other embryonic sites remains unclear. Here, using single-cell RNA sequencing, we profile the transcriptional landscape of mouse fetal placental hematopoiesis. We uncover and experimentally validate that a CD44 + subpopulation of placental endothelial cells (ECs) exhibits hemogenic potential. Importantly, lineage tracing using the newly generated Hoxa13 reporter line shows that Hoxa13-labeled ECs can produce placental macrophages, named Hofbauer cell (HBC)-like cells. Furthermore, we identify two subtypes of HBC-like cells, and cell-cell interaction analysis identifies their potential roles in angiogenesis and antigen presentation, separately. Our study provides a comprehensive understanding of placental hematopoiesis and highlights the placenta as a source of macrophages, which has important implications for both basic and translational research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Technological advances play a key role in furthering our understanding of stem cell biology, and advancing the prospects of regenerative therapies. Highly parallelized methods, developed in the last decade, can profile DNA, RNA, or proteins in thousands of cells and even capture data across two or more modalities (multiomics). This allows unbiased and precise definition of molecular cell states, thus allowing classification of cell types, tracking of differentiation trajectories, and discovery of underlying mechanisms. Despite being based on destructive techniques, novel experimental and bioinformatic approaches enable embedding and extraction of temporal information, which is essential for deconvolution of complex data and establishing cause and effect relationships. Here, we provide an overview of recent studies pertinent to stem cell biology, followed by an outlook on how further advances in single-cell molecular profiling and computational analysis have the potential to shape the future of both basic and translational research., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

The blood and immune systems develop in parallel during early prenatal life. Waves of hematopoiesis separated in anatomical space and time give rise to circulating and tissue-resident immune cells. Previous observations have relied on animal models, which differ from humans in both their developmental timeline and exposure to microorganisms. Decoding the composition of the human immune system is now tractable using single-cell multi-omics approaches. Large-scale single-cell genomics, imaging technologies, and the Human Cell Atlas initiative have together enabled a systems-level mapping of the developing human immune system and its emergent properties. Although the precise roles of specific immune cells during development require further investigation, the system as a whole displays malleable and responsive properties according to developmental need and environmental challenge., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing., Competing Interests: The authors have declared that no competing interests exist.

Pluripotent stem cell (PSC) differentiation in vitro represents a powerful and tractable model to study mammalian development and an unlimited source of cells for regenerative medicine. Within hematology, in vitro PSC hematopoiesis affords novel insights into blood formation and represents an exciting potential approach to generate hematopoietic and immune cell types for transplantation and transfusion. Most studies to date have focused on in vitro hematopoiesis from mouse PSCs and human PSCs. However, differences in mouse and human PSC culture protocols have complicated the translation of discoveries between these systems. We recently developed a novel chemical media formulation, expanded potential stem cell medium (EPSCM), that maintains mouse PSCs in a unique cellular state and extraembryonic differentiation capacity. Herein, we describe how EPSCM can be directly used to stably maintain human PSCs. We further demonstrate that human PSCs maintained in EPSCM can spontaneously form embryoid bodies and undergo in vitro hematopoiesis using a simple differentiation protocol, similar to mouse PSC differentiation. EPSCM-maintained human PSCs generated at least two hematopoietic cell populations, which displayed distinct transcriptional profiles by RNA-sequencing (RNA-seq) analysis. EPSCM also supports gene targeting using homologous recombination, affording generation of an SPI1 (PU.1) reporter PSC line to study and track in vitro hematopoiesis. EPSCM therefore provides a useful tool not only to study pluripotency but also hematopoietic cell specification and developmental-lineage commitment., (Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Single-cell transcriptomics has recently emerged as a powerful tool to analyze cellular heterogeneity, discover new cell types, and infer putative differentiation routes. The technique has been rapidly embraced by the hematopoiesis research community, and like other technologies before, single-cell molecular profiling is widely expected to make important contributions to our understanding of the hematopoietic hierarchy. Much of this new interpretation relies on inference of the transcriptomic landscape as a representation of existing cellular states and associated transitions among them. Here we review how this model allows, under certain assumptions, charting of time-resolved differentiation trajectories with unparalleled resolution and how the landscape of multipotent cells may be rather devoid of discrete structures, challenging our preconceptions about stem and progenitor cell types and their organization. Finally, we highlight how promising technological advances may convert static differentiation landscapes into a dynamic cell flux model and thus provide a more holistic understanding of normal hematopoiesis and blood disorders., (© 2019 by The American Society of Hematology.)

Personalized therapy is a major goal of modern oncology, as patient responses vary greatly even within a histologically defined cancer subtype. This is especially true in acute myeloid leukemia (AML), which exhibits striking heterogeneity in molecular segmentation. When calibrated to cell-specific data, executable network models can reveal subtle differences in signaling that help explain differences in drug response. Furthermore, they can suggest drug combinations to increase efficacy and combat acquired resistance. Here, we experimentally tested dynamic proteomic changes and phenotypic responses in diverse AML cell lines treated with pan-PIM kinase inhibitor and fms-related tyrosine kinase 3 ( FLT3 ) inhibitor as single agents and in combination. We constructed cell-specific executable models of the signaling axis, connecting genetic aberrations in FLT3 , tyrosine kinase 2 ( TYK2 ), platelet-derived growth factor receptor alpha ( PDGFRA ), and fibroblast growth factor receptor 1 ( FGFR1 ) to cell proliferation and apoptosis via the PIM and PI3K kinases. The models capture key differences in signaling that later enabled them to accurately predict the unique proteomic changes and phenotypic responses of each cell line. Furthermore, using cell-specific models, we tailored combination therapies to individual cell lines and successfully validated their efficacy experimentally. Specifically, we showed that cells mildly responsive to PIM inhibition exhibited increased sensitivity in combination with PIK3CA inhibition. We also used the model to infer the origin of PIM resistance engineered through prolonged drug treatment of MOLM16 cell lines and successfully validated experimentally our prediction that this resistance can be overcome with AKT1/2 inhibition. Cancer Res; 77(4); 827-38. ©2016 AACR ., (©2016 American Association for Cancer Research.)

DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AAD(dim) fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

CODEX (http://codex.stemcells.cam.ac.uk/) is a user-friendly database for the direct access and interrogation of publicly available next-generation sequencing (NGS) data, specifically aimed at experimental biologists. In an era of multi-centre genomic dataset generation, CODEX provides a single database where these samples are collected, uniformly processed and vetted. The main drive of CODEX is to provide the wider scientific community with instant access to high-quality NGS data, which, irrespective of the publishing laboratory, is directly comparable. CODEX allows users to immediately visualize or download processed datasets, or compare user-generated data against the database's cumulative knowledge-base. CODEX contains four types of NGS experiments: transcription factor chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), histone modification ChIP-Seq, DNase-Seq and RNA-Seq. These are largely encompassed within two specialized repositories, HAEMCODE and ESCODE, which are focused on haematopoiesis and embryonic stem cell samples, respectively. To date, CODEX contains over 1000 samples, including 221 unique TFs and 93 unique cell types. CODEX therefore provides one of the most complete resources of publicly available NGS data for the direct interrogation of transcriptional programmes that regulate cellular identity and fate in the context of mammalian development, homeostasis and disease., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcription factors play a key role in the development of a disease. ChIP-sequencing has become a preferred technique to investigate genome-wide binding patterns of transcription factors in vivo. Although this technology has led to many important discoveries, the rapidly increasing number of publicly available ChIP-sequencing datasets still remains a largely unexplored resource. Using a compendium of 144 publicly available murine ChIP-sequencing datasets in blood, we show that systematic bioinformatic analysis can unravel diverse aspects of transcription regulation; from genome-wide binding preferences, finding regulatory partners and assembling regulatory complexes, to identifying novel functions of transcription factors and investigating transcription dynamics during development.

Summary: Unraveling transcriptional circuits controlling embryonic stem cell maintenance and fate has great potential for improving our understanding of normal development as well as disease. To facilitate this, we have developed a novel web tool called 'TRES' that predicts the likely upstream regulators for a given gene list. This is achieved by integrating transcription factor (TF) binding events from 187 ChIP-sequencing and ChIP-on-chip datasets in murine and human embryonic stem (ES) cells with over 1000 mammalian TF sequence motifs. Using 114 TF perturbation gene sets, as well as 115 co-expression clusters in ES cells, we validate the utility of this approach., Availability and Implementation: TRES is freely available at http://www.tres.roslin.ed.ac.uk., Contact: Anagha.Joshi@roslin.ed.ac.uk or bg200@cam.ac.uk, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We recently identified 68 genomic loci where common sequence variants are associated with platelet count and volume. Platelets are formed in the bone marrow by megakaryocytes, which are derived from hematopoietic stem cells by a process mainly controlled by transcription factors. The homeobox transcription factor MEIS1 is uniquely transcribed in megakaryocytes and not in the other lineage-committed blood cells. By ChIP-seq, we show that 5 of the 68 loci pinpoint a MEIS1 binding event within a group of 252 MK-overexpressed genes. In one such locus in DNM3, regulating platelet volume, the MEIS1 binding site falls within a region acting as an alternative promoter that is solely used in megakaryocytes, where allelic variation dictates different levels of a shorter transcript. The importance of dynamin activity to the latter stages of thrombopoiesis was confirmed by the observation that the inhibitor Dynasore reduced murine proplatelet for-mation in vitro.

The endothelial ETS transcription factor Erg plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show that Erg regulates endothelial cell (EC) migration. Transcriptome profiling of Erg-deficient ECs identified ∼ 80 genes involved in cell migration as candidate Erg targets, including many regulators of Rho- GTPases. Inhibition of Erg expression in HUVECs resulted in decreased migration in vitro, while Erg overexpression using adenovirus caused increased migration. Live-cell imaging of Erg-deficient HUVECs showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient ECs, with a dramatic increase in tubulin acetylation. Among the most significant microarray hits was the cytosolic histone deacetylase 6 (HDAC6), a regulator of cell migration. Chromatin immunoprecipitation (ChIP) and transactivation studies demonstrated that Erg regulates HDAC6 expression. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization. In vivo, inhibition of Erg expression in angiogenic ECs resulted in decreased HDAC6 expression with increased tubulin acetylation. Thus, we have identified a novel function for the transcription factor Erg in regulating HDAC6 and multiple pathways essential for EC migration and angiogenesis.

The chromosome 16p13 region has been associated with several autoimmune diseases, including type 1 diabetes (T1D) and multiple sclerosis (MS). CLEC16A has been reported as the most likely candidate gene in the region, since it contains the most disease-associated single-nucleotide polymorphisms (SNPs), as well as an imunoreceptor tyrosine-based activation motif. However, here we report that intron 19 of CLEC16A, containing the most autoimmune disease-associated SNPs, appears to behave as a regulatory sequence, affecting the expression of a neighbouring gene, DEXI. The CLEC16A alleles that are protective from T1D and MS are associated with increased expression of DEXI, and no other genes in the region, in two independent monocyte gene expression data sets. Critically, using chromosome conformation capture (3C), we identified physical proximity between the DEXI promoter region and intron 19 of CLEC16A, separated by a loop of >150 kb. In reciprocal experiments, a 20 kb fragment of intron 19 of CLEC16A, containing SNPs associated with T1D and MS, as well as with DEXI expression, interacted with the promotor region of DEXI but not with candidate DNA fragments containing other potential causal genes in the region, including CLEC16A. Intron 19 of CLEC16A is highly enriched for transcription-factor-binding events and markers associated with enhancer activity. Taken together, these data indicate that although the causal variants in the 16p13 region lie within CLEC16A, DEXI is an unappreciated autoimmune disease candidate gene, and illustrate the power of the 3C approach in progressing from genome-wide association studies results to candidate causal genes., (© The Author 2011. Published by Oxford University Press.)

Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high-throughput sequencing and genomic array tiling paths. In this article, we describe a novel protocol to use real-time PCR to map DHS. Advantages of the technique reported here include the small cell numbers required for each analysis, rapid, relatively low-cost experiments with minimal need for specialist equipment. Presented examples include comparative DHS mapping of known TAL1/SCL regulatory elements between human embryonic stem cells and K562 cells.

Background: The CD34(-) murine stem cell line RM26 cloned from peripheral blood mononuclear cells has been shown to generate hematopoietic progeny in lethally irradiated animals. The peripheral blood-derived cell clones expresses a variety of mesodermal and erythroid/myeloid transcription factors suggesting a multipotent differentiation potential like the bone marrow-derived 'multipotent adult progenitor cells' (MAP-C)., Methods: SCL(+) CD34(-) RM26 cells were transfused intravenously into mice suffering from chronic hind-limb ischemia, evaluating the effect of stem cells on collateral artery growth and simultaneous skeletal muscle repair., Results: RM26 cells are capable of differentiating in vitro into endothelial cells when cultured on the appropriate collagen matrix. Activation of the SCL stem cell enhancer (SCL(+)) is mediated through the binding to two Ets and one GATA site and cells start to express milieu- and growth condition-dependent levels of the endothelial markers CD31 (PECAM) and Flt-1 (VEGF-R1). Intravenously infused RM26 cells significantly improved the collateral blood flow (arteriogenesis) and neo-angiogenesis formation in a murine hind-limb ischemia transplant model. Although transplanted RM26 cells did not integrate into the growing collateral arteries, cells were found adjacent to local arteriogenesis, but instead integrated into the ischemic skeletal muscle exclusively in the affected limb for simultaneous tissue repair., Conclusion: These data suggest that molecularly primed hem-/mesangioblast-type adult progenitor cells can circulate in the peripheral blood improving perfusion of tissues with chronic ischemia and extending beyond the vascular compartment.

Objective: Based on previous animal experiments that suggest the plasticity of peripheral blood-derived, CD34(-) stem cell lines, the aim of this study was to isolate CD34(-) stem cell lines from human peripheral blood cells and obtain evidence of their multipotency and plasticity., Materials and Methods: Adherent growing cells were isolated from peripheral blood mononuclear cells from a healthy volunteer donor and different cell clones were established after SV40 large-T-antigen-mediated immortalization. The immunophenotype of the cell lines was investigated by flow cytometry. One particular cell clone, V54/2, was stained with rhodamine 123, and the Rh123(low) and Rh123(high) subpopulations were sorted for a reverse transcriptase polymerase chain reaction gene expression survey and distinct differences in morphology and biologic behavior., Results: The peripheral blood-derived and fibroblast-like cell line V54/2 expressed high levels of CD10 and CD105 and showed only a very low level expression of CD34 (<1.0%) and CD117 (c-kit). Among the entire CD34(-)CD105(+) cell population that transcribed factors such as Myb, Tie-1, and VEGF, there was a small Rh123(low)CD34(+) subpopulation that transcribed significant levels of several members of the GATA family of transcription factors. The morphology of the Rh123(low)CD34(+) (also expressing the P-glycoprotein) was different compared to the Rh123(high)CD34(-) population. Mesenchymal differentiation into glial fibrillary acidic protein (GFAP)(+) glial cells could be shown from the entire CD34(-)CD105(+) cell population., Conclusions: The findings provide evidence that it is possible to isolate CD34(-)CD105(+) mesenchymal stem cell lines from human peripheral blood cells that contain a small subpopulation of CD34(+) and GATA-transcribing cells. Those cells are potential hematopoietic progenitors and can be recruited from the CD34(-) stem cell pool. The plasticity of stem cells seems to require essential molecular tools, such as a panel of transcription factors, to respond to the environmental demand within a biologic system.