Cell differentiation is governed by dynamic changes in chromatin accessibility, and its dysregulation underlies multiple disease states. Prior to birth, development of the hematopoietic system constitutes a period of broad differentiation potential, with certain immune cells arising exclusively during ontogeny. While age is known to affect lineage bias, the underlying molecular differences driving lineage preference in fetal and adult human hematopoietic stem and progenitor cells (HSPCs) remain unclear. Through single-cell cultures of hematopoietic stem cells (HSCs), we observed that fetal cells frequently generate mixed-lineage colonies, whereas adult HSCs are biased towards myeloid output. To investigate how these lineage preferences were encoded at the chromatin level, we performed single-cell ATAC-sequencing on first-trimester HSPCs. While adult HSCs showed enrichment of lineage-specific transcription factor motifs, fetal cells lacked such enrichment, consistent with their broader differentiation potential. We additionally uncovered a developmental-specific plasticity in fetal lymphoid progenitors, manifested as a hybrid lympho-myeloid chromatin program not present in adult progenitors. Additionally, the motif and putative regulatory elements for PAX5, a master regulator of B cell development, showed markedly reduced accessibility in fetal cells, supporting a more plastic and less restricted lymphoid state. This enhanced embryonic lineage plasticity may underlie the prenatal susceptibility to mutational drivers of acute lymphoblastic leukemia.

Infant Acute Lymphoblastic Leukemia (ALL) driven by the KMT2A::AFF1 onco-fusion is an aggressive, poor prognosis disease with few co-operative mutations. The fusion originates in utero, yet the embryonic initiating steps of disease development remain poorly understood. Here, we present a novel murine KMT2A::AFF1 model, that provides key insights into KMT2A::AFF1 pre-leukemia, relevant to human disease. The model enables precise oncogene induction, and upon targeting hematopoietic stem and progenitor cells (HSPCs) a selective negative impact on proliferation of hematopoietic stem cells (HSCs) was observed, regardless of developmental state during induction. However, a unique CD24+PreProB subset expanded exclusively within the KMT2A::AFF1 embryonic context. This population was absent when targeting lymphoid progenitors, highlighting the importance of the cell of origin for leukemic development. The CD24+PreProB subset displayed key features of pre-leukemic stem cells, including lineage plasticity and aberrant engraftment ability. In line with their pre-malignant phenotype, single-cell transcriptomics revealed a signature consistent with stemness, and notable, up-regulation of Hmga2, a regulator of self-renewal. The signature was critically transferable to human KMT2A::AFF1 patients. Furthermore, given that CD24 is a potential therapeutic target, our findings uncover a distinct embryonic pre-leukemic state with direct relevance to human disease.

Cell fate decision is a tightly regulated process, highly dependent on cellular context and environmental cues, and disruption of this process in the hematopoietic system may lead to diseases such as leukemia. Changes in chromatin accessibility have been shown to be an early indicator of lineage commitment, preceding changes in gene expression. Fetal hematopoietic stem and progenitor cells (HSPCs) have been shown to differ from adult counterparts at the functional and transcriptional level. However, differences in chromatin state are largely unexplored and may play a role in the distinct molecular landscape of childhood acute lymphoblastic leukemia (ALL), in which the initiating mutation often occurs before birth. Here, we explored the chromatin landscape of first-trimester human fetal liver using single-cell assay for transposase-accessible chromatin (ATAC) sequencing, covering over 13,000 HSPCs. The fetal hematopoietic stem cell (HSC) state was mostly low-primed, whereas adult counterparts were enriched for myeloid-associated transcription factor motifs, which was supported by higher oligo-lineage output from fetal HSCs and higher unilineage myeloid output from adults.Additionally, the fetal lymphoid progenitor state had a lympho-myeloid chromatin profile not observed in the myeloid cell state. This myeloid lineage promiscuity was also not observed in corresponding adult lymphoid progenitors, revealing an ontogeny-specific phenomenon that may relate to the coexpression of lymphoid and myeloid gene programs or lineage switch capacity observed in certain subtypes of pediatric ALL. Thus, we provided a unique resource for exploring the chromatin landscape of early fetal HSPCs and insights into developmental shifts in lineage cues, giving important clues to understanding congenital blood disorders and the origin of pediatric leukemia.

Infant Acute Lymphoblastic Leukemia (ALL) driven by KMT2A::AFF1 onco-fusion can be regarded as a developmental disorder. The fusion occurs already during fetal life and gives rise to an aggressive poor prognosis B cell leukemia, however, the embryonic, initiating steps of disease development are not well studied. The disease has been difficult to recapitulate in murine models, and there is a gap in knowledge about the embryonic initiating steps of disease development. Here, we provide key insights into the KMT2A::AFF1 pre-leukemic phase by using a novel Cre inducible murine model. The oncogene induction in hematopoietic stem and progenitor cells (HSPCs) resulted in a negative effect on proliferation and a myeloid skewing in hematopoietic stem cells (HSCs) in both embryonic and adult stage. Strikingly, upon induction in the embryo an expansion of a PreProB subset marked by CD24 surface marker, was observed. The PreProB population was unique to induction in embryonic HSPCs and not observed upon postnatal induction, nor after induction in lymphoid progenitors, providing critical insight into the significance of the target cell. Additionally, the CD24+ PreProB population showed lineage plasticity, aberrant long-term engraftment capacity and pathogenic characteristics consistent with pre-leukemic stem cells. Single-cell transcriptomics revealed a distinct molecular signature with up-regulation of KMT2A::AFF1 target genes and molecular features linked to the pre-leukemic cells such as upregulation of Hmga2, a regulator of stemness, in agreement with the pre-malignant profile. This pre-leukemic signature could importantly be connected to human KMT2A::AFF1 B-ALL and notable the CD24 surface marker is interesting from a therapeutic angle. Our novel model reveals a unique pre-leukemic state, of critical importance to human disease initiation.

KMT2A::AFF1 (MLL::AF4) onco-fusion is associated with infant Acute Lymphoblastic Leukemia (ALL), an aggressive, poor prognosis disease. The mutational landscape is sparse and since the fusion originates in utero, infant ALL can be regarded as a developmental disorder. The disease has been difficult to recapitulate in murine models, and there is a gap in knowledge about the embryonic initiating steps of disease development. Here, the early stages of disease were assessed and the susceptibility of the KMT2A::AFF1 oncogene to different target cells was investigated in a novel murine KMT2A::AFF1 model. Upon induction in Hematopoietic Stem and Progenitor Cells (HSPCs) an expansion of a Pre-ProB like progenitor population was observed. The expanded population displayed lineage plasticity in vitro, generating both B and myeloid cells, in contrast to mainly B output in normal counterparts. The expansion was restricted to KMT2A::AFF1 induction in embryonic HSPCs and was not observed upon initiation in more committed lymphoid progenitors, nor after postnatal induction in HSPCs. The Pre-ProBs had self-renewal potential in vitro and upon transplantation long-term reconstitution was observed. Single cell transcriptomics of these pre-leukemic cells revealed a distinct pre-leukemic molecular program with expression of stemness genes like Hlf and Mecom, whereas gene sets associated with immune system processes were downregulated, in agreement with the pre-malignant phenotype.Thus, our novel model captures the early phase of leukemia initiation and the results highlight an embryonic susceptibility to the KMT2A::AFF1 oncogene. Furthermore, a unique population emerges in the embryo with functional and transcriptional features consistent with pre-leukemic stem cells.

ABSTRACTStroke is a leading cause of disability and the third cause of death. The immune system plays an essential role in post-stroke recovery. After an ischemic stroke, monocytes infiltrate the injured brain tissue and can exacerbate or mitigate the damage. Ischemic stroke is more prevalent in the aged population, and the aging brain exhibits an altered immune response. There are also sex disparities in ischemic stroke incidence, outcomes, and recovery, and these differences may be hormone-driven and determined by genetic and epigenetic factors. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of neuronal inflammation- and regeneration-related genes depends on age and sex. A FACS analysis of blood samples from 44 volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of natural killer cells increased in females. The proportion of B-cells decreased in both sexes with age, and subtypes of monocytes were not linked to age or sex. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly,ANXA1andCD36showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1β, S100A8, TNFα, CD64, CD33, TGFβ1, TLR8, CD91) were differentially changed in monocyte subtypes with increased aging. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes.

Aging profoundly affects the immune system leading to an increased propensity for inflammation. Age-related dysregulation of immune cells is implicated in the development and progression of numerous age-related diseases such as: cardiovascular diseases, neurodegenerative disorders, and metabolic syndromes. Monocytes and monocyte-derived macrophages, being important players in the inflammatory response, significantly influence the aging process and the associated increase in inflammatory disease risk. Ischemic stroke is among age-related diseases where inflammation, particularly monocyte-derived macrophages, plays an important deteriorating role but could also strongly promote post-stroke recovery. Also, biological sex influences the incidence, presentation, and outcomes of ischemic stroke, reflecting both biological differences between men and women. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of genes implicated in stroke-related inflammation and post-stroke tissue regeneration depends on age and sex. A flow cytometry analysis of blood samples from 44 healthy volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of NK-cells increased in females. The proportion of B-cells decreased in both sexes with age. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly, ANXA1 and CD36 showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1β, S100A8, TNFα, CD64, CD33, TGFβ1, TLR8, CD91) were differentially changed in monocyte subtypes with increasing age. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes.

Here we describe an in vitro co-culture system that can differentiate hematopoietic progenitor populations to all major hematopoietic lineages at clonal level. We present both a sensitive single-cell switch-culture system as well as a less laborious alternative barcoding protocol more convenient for larger cell numbers. Importantly, generation of all lineages from single long-term hematopoietic stem cells are described, following 21 days of culture. This protocol represents an efficient tool for validation experiments for single-cell genomics data. For complete details on the use and execution of this protocol, please refer to Safi et al. (2022).1.

Knowledge of human fetal blood development and how it differs from adult blood is highly relevant to our understanding of congenital blood and immune disorders and childhood leukemia, of which the latter can originate in utero. Blood formation occurs in waves that overlap in time and space, adding to heterogeneity, which necessitates single-cell approaches. Here, a combined single-cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), the molecular profile of established immunophenotype-gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs), such as CD90 and CD49F, were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow data set revealed that the HSC state was less frequent in FL, whereas cells with a lymphomyeloid signature were more abundant. An erythromyeloid-primed multipotent progenitor cluster was identified, potentially representing a transient, fetal-specific population. Furthermore, differentially expressed genes between fetal and adult counterparts were specifically analyzed, and a fetal core signature was identified. The core gene set could separate subgroups of acute lymphoblastic leukemia by age, suggesting that a fetal program may be partially retained in specific subgroups of pediatric leukemia. Our detailed single-cell map presented herein emphasizes molecular and immunophenotypic differences between fetal and adult blood cells, which are of significance for future studies of pediatric leukemia and blood development in general.

The emerging notion of hematopoietic stem and progenitor cells (HSPCs) as a low-primed cloud without sharply demarcated gene expression programs raises the question on how cellular-fate options emerge and at which stem-like stage lineage priming is initiated. Here, we investigate single-cell chromatin accessibility of Lineage(-), cKit(+), and Sca1(+), (LSK) HSPCs spanning the early differentiation landscape. Application of a signal-processing algorithm to detect transition points corresponding to massive alterations in accessibility of 571 transcription factor motifs reveals a population of LSK FMS-like tyrosine kinase 3 (Flt3)(int)CD9(high) cells that concurrently display stem-like and lineage-affiliated chromatin signatures, pointing to a simultaneous gain of both lympho-myeloid and megakaryocyte-erythroid programs. Molecularly and functionally, these cells position between stem cells and committed progenitors and display multi-lineage capacity in vitro and in vivo but lack self-renewal activity. This integrative molecular analysis resolves the heterogeneity of cells along hematopoietic differentiation and permits investigation of chromatin-mediated transition between multipotency and lineage restriction.

The formation of our blood system is highly relevant for our understanding of congenital immune disorders and childhood leukemia. In the embryo, blood cells emerge in waves that overlap in time and space. Due to the diversity of the system, single cell assays are important to unravel heterogeneity. Here, human first trimester primitive fetal liver (FL) cells are investigated at single cell resolution using CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing), a combined immunophenotypic and transcriptional assay. The classical immunophenotypic surface markers used to define progenitors in adult, were investigated in the embryo and the molecular profile assessed. The surface markers CD90 and CD49F, used to define Hematopoietic stem cells (HSCs), were largely preserved in the embryo. Myeloid progenitors however, identified with FLT3 or CD123, were heterogeneous, due to the ubiquitous expression of these markers during development. Using a projection approach an adult Bone Marrow (BM) data set was directly compared to the FL cells. Progenitors with a lympho-myeloid signature were found to decrease with gestational age, whereas molecularly defined HSCs were relatively enriched in adult BM. Additionally, a fetal specific multipotent progenitor with erythromyeloid signature was identified, which may represent a transient erythromyeloid progenitor originating prior to definitive HSCs. Based on differently expressed genes between fetal and adult cells, a fetal core signature was identified and found to be enriched in subtypes of pediatric leukemia, that can originate in utero. Thus, our data is of relevance for future studies of paediatric blood disorders and highlights key immunophenotypic and transcriptional differences between fetal and adult blood progenitors.

The ETV6-RUNX1 onco-fusion arises in utero , initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

Developmental hematopoiesis differs from adult and is far less described. In the developing embryo, waves of lineage-restricted blood precede the ultimate emergence of definitive hematopoietic stem cells (dHSCs) capable of maintaining hematopoiesis throughout life. During the last two decades, the advent of single-cell genomics has provided tools to circumvent previously impeding characteristics of embryonic hematopoiesis, such as cell heterogeneity and rare cell states, allowing for definition of lineage trajectories, cellular hierarchies, and cell-type specification. The field has rapidly advanced from microfluidic platforms and targeted gene expression analysis, to high throughput unbiased single-cell transcriptomic profiling, single-cell chromatin analysis, and cell tracing-offering a plethora of tools to resolve important questions within hematopoietic development. Here, we describe how these technologies have been implemented to address a wide range of aspects of embryonic hematopoiesis ranging from the gene regulatory network of dHSC formation via endothelial to hematopoietic transition (EHT) and how EHT can be recapitulated in vitro, to hematopoietic trajectories and cell fate decisions. Together, these studies have important relevance for regenerative medicine and for our understanding of genetic blood disorders and childhood leukemias.

Despite enormous progress boosted by significant technological advances of the last decade, a complete understanding of hematopoietic development has not yet been achieved. “Primitive” and “definitive” terms have been used for many years to describe the different waves of transient and persistent blood cells generated during ontogeny. However, it is now clear that this terminology is not adequate to address the surprising complexity of the developing hematopoietic system. Gaining additional insight into embryonic and fetal hematopoiesis will not only be relevant for textbook biology, but will carry important implications for the understanding of blood diseases, including genetic conditions and pediatric leukemia. This Research Topic comprises 14 articles including reviews and primary research articles focusing on different aspects on the biology of fetal blood cells, ranging from basic hematopoiesis to technological advances and translational studies.

AbstractMyelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).

The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing.