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.

ABSTRACT:The BCR::ABL1 tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) represent a paradigm for molecularly targeted therapy. However, clinical outcomes (rate/depth of response, treatment-free remission [TFR], progression to blast crisis [BC]) and adverse events vary among patients. While additional somatic mutations have been invoked to explain varying clinical outcomes, we here propose a complementary perspective based on single-cell omics (sc-omics) approaches that have enabled unprecedented resolution of the cellular ecosystems, including their composition, interactions, and activity. In patients who were treatment-naïve and in chronic phase (CP), this has revealed differences in the growth-rate of BCR::ABL1+ clones, ratio of TKI-insensitive leukemic stem cells (LSC) to residual hematopoietic stem cells (HSC), and immune cell composition, factors that collectively contribute to variability in therapy efficacy. Together these findings suggest that cellular heterogeneity serves as a foundation of clinical outcome in CML. Patients who remain in CP exhibit an erythroid signature in LSC, while those progressing to BC manifest an inflammatory profile, additional mutations, and expansion of early progenitors. Deep responders with active natural killer, and regulatory T cells are more likely to sustain TFR. Similarly, the outcomes of donor lymphocyte infusion after allogeneic stem cell transplant are heterogeneous, and reflect differences in preexisting T-cell clonotypes, their expansion, and interaction with leukemic cells in responders vs nonresponders. Here, we summarize key insights from sc-omics in CML, and propose an actionable road map to further leverage these technologies. This includes mechanistically explaining heterogeneity, predicting therapy response and BC, tracking leukemogenic clones longitudinally, targeting TKI-insensitive LSC, and restoring hematopoiesis from residual HSCs.

Cell type annotation remains a critical bottleneck, with current methods often inaccurate and requiring extensive manual validation, particularly in disease contexts. While large language models (LLMs) show promise, they can be unreliable due to hallucinations. We developed CyteType, a multi-agent framework that generates competing hypotheses grounded in full expression data and study context, validates against external databases, and iteratively self-evaluates. Comprehensive benchmarking demonstrates that CyteType substantially outperforms reference-based and LLM-based methods, with self-generated confidence scores reliably identifying trustworthy annotations. CyteType transforms cell type annotation from label assignment into evidence-grounded biological discovery. Python (AnnData compatible): R (Seurat compatible): ### Competing Interest Statement G.A., Y.S., S.B., P.D., G.K., and G.M.D. are employees of, hold equity in, or consult for Nygen Analytics AB, which develops commercial solutions based on this work. A.A. declares no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Swedish Research Council, https://ror.org/03zttf063 Swedish Cancer Society, https://ror.org/0527jb766 Knut and Alice Wallenberg Foundation, https://ror.org/004hzzk67 HALRIC

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.

Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαβ-seq) from patients with CML ( n = 13, N = 25), other cancers ( n = 28), and healthy ( n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56 dim cluster with high expression of GZMA/B, PRF1, CCL3/4 , and IFNG , with interactions with leukemic cells via inhibitory LGALS9 – TIM3 and PVR – TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRβ-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + T EMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.

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.

Aging negatively affects hematopoiesis, with consequences for immunity and acquired blood cell disorders. Although impairments in hematopoietic stem cell (HSC) function contribute to this, the in vivo dynamics of such changes remain obscure. Here, we integrate extensive longitudinal functional assessments of HSC-specific lineage tracing with single-cell transcriptome and epitope profiling. In contrast to recent suggestions from single-cell RNA sequencing alone, our data favor a defined structure of HSC/progenitor differentiation that deviates substantially from HSC-derived hematopoiesis following transplantation. Native age -depen-dent attrition in HSC differentiation manifests as drastically reduced lymphoid output through an early lymphoid-primed progenitor (MPP Ly-I). While in vitro activation fails to rescue lymphoid differentiation from most aged HSCs, robust lymphopoiesis can be achieved by culturing elevated numbers of candidate HSCs. Therefore, our data position rare chronologically aged HSC clones, fully competent at producing lymphoid offspring, as a prime target for approaches aimed to improve lymphopoiesis in the elderly.

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.

Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing (scRNAseq), we systematically characterized the human non-hematopoietic BM stromal compartment and we investigated stromal cell regulation principles based on the RNA velocity analysis using scVelo and studied the interactions between the human BM stromal cells and hematopoietic cells based on ligand-receptor (LR) expression using CellPhoneDB. scRNAseq led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis and in vitro proliferation capacities and differentiation potentials. Potential key factors that might govern the transition from stem and progenitor cells to fate-committed cells were identified. In situ localization analysis demonstrated that different stromal cells were localized in different niches in the bone marrow. In silico cell-cell communication analysis further predicted that different stromal cell types might regulate hematopoiesis through distinct mechanisms. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on human hematopoietic niche organization.

Rare hematopoietic stem cells make up an infrequent but critical population in the bone marrow (BM), maintaining and replenishing the entire hematopoietic system. Importantly, despite sharing the unique stem cell properties of multilineage differentiation and self-renewal, individual HSCs are functionally heterogeneous, and this heterogeneity increases during aging. While HSCs in young mice are qualitatively more similar, ageing is marked by an increased size of the HSC pool and substantial functional variation of individual HSCs. CD9 is a cell surface marker that is highly expressed in HSCs in mice, while CD9 expression within the human HSC population has been reported to be low during neonatal hematopoiesis. Here, we have investigated CD9 expression levels in the human HSPC population over time and identified that early in life; CD9 is infrequent in HSCs, but marks progenitor populations with low engraftment potential and high proliferation capacity. However, during situations of myeloid/Megakaryocyte-erythoid (MegE) biased hematopoiesis, such as during ageing or in leukemia, there is a substantial increase of CD9 expression in HSPCs. Thus, CD9 represents an HSC marker for myeloid/MegE-biased hematopoiesis.

While hematopoietic decline is a well-established feature of aging, the in vivo dynamics of such changes remains obscure. Here, using an inducible lineage tracing model, single cell transcriptome and epitope profiling, and an in vitro hematopoietic stem cell (HSC) self-renewal system, we dissected the contribution of HSCs during chronological aging. Age-related attrition in differentiation decreased the HSC output to multipotent progenitors (MPPs), which associated with a pronounced reduction in lymphopoiesis. Combined single-cell molecular profiling and linage tracing revealed that aging associated with a specific depletion of an hitherto uncharacterized early lymphoid-primed progenitor, that we denoted as MPP Ly-I. In native hematopoiesis, MPP Ly-I cells are characterized by a high degree of dormancy and share several phenotypic attributes with HSCs. In a transplantation setting, MPP Ly-I cells generated lymphoid output faster than HSCs and with minimal myeloid differentiation, suggesting that MPP Ly-I cells represent the earliest lymphoid-primed progenitor downstream of HSCs. The apparent reduction of MPP Ly-I cells during aging may underlie defective age-associated lymphopoiesis. Finally, when applying a novel HSC self-renewal promoting cell culture system, we found that while some of the aging phenotypes could be rescued by an in vitro activation approach, most aged HSCs retained a compromised lymphoid differentiation capacity after culture. When increasing the numbers of input HSCs to such cultures, we observed a robust lymphoid output following transplantation, thereby unequivocally demonstrating the existence of rare aged HSC clones fully competent in producing lymphoid offspring.

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.

Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.

Stem cell transplantation is a cornerstone in the treatment of blood malignancies. The most common method to harvest stem cells for transplantation is by leukapheresis, requiring mobilization of CD341 hematopoietic stem and progenitor cells (HSPCs) from the bone marrow into the blood. Identifying the genetic factors that control blood CD341 cell levels could reveal new drug targets for HSPC mobilization. Here we report the first large-scale, genome-wide association study on blood CD341 cell levels. Across 13167 individuals, we identify 9 significant and 2 suggestive associations, accounted for by 8 loci (PPM1H, CXCR4, ENO1-RERE, ITGA9, ARHGAP45, CEBPA, TERT, and MYC). Notably, 4 of the identified associations map to CXCR4, showing that bona fide regulators of blood CD341 cell levels can be identified through genetic variation. Further, the most significant association maps to PPM1H, encoding a serine/threonine phosphatase never previously implicated in HSPC biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD341 cell levels downregulates PPM1H. Through functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates an MYB transcription factor-binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, PPM1H knockdown increases the proportion of CD341 and CD341901 cells in cord blood assays. Our results provide the first large-scale analysis of the genetic architecture of blood CD341 cell levels and warrant further investigation of PPM1H as a potential inhibition target for stem cell mobilization.

As the scale of single-cell genomics experiments grows into the millions, the computational requirements to process this data are beyond the reach of many. Herein we present Scarf, a modularly designed Python package that seamlessly interoperates with other single-cell toolkits and allows for memory-efficient single-cell analysis of millions of cells on a laptop or low-cost devices like single-board computers. We demonstrate Scarf’s memory and compute-time efficiency by applying it to the largest existing single-cell RNA-Seq and ATAC-Seq datasets. Scarf wraps memory-efficient implementations of a graph-based t-stochastic neighbour embedding and hierarchical clustering algorithm. Moreover, Scarf performs accurate reference-anchored mapping of datasets while maintaining memory efficiency. By implementing a subsampling algorithm, Scarf additionally has the capacity to generate representative sampling of cells from a given dataset wherein rare cell populations and lineage differentiation trajectories are conserved. Together, Scarf provides a framework wherein any researcher can perform advanced processing, subsampling, reanalysis, and integration of atlas-scale datasets on standard laptop computers. Scarf is available on Github: https://github.com/parashardhapola/scarf .

ABSTRACTUnderstanding how hematopoietic stem and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and stem cell transplantation. To date, HSPC regulation has been extensively studiedin vitroand in animal models, but less is known about the mechanismsin vivoin humans. Here, in a genome-wide association study on 13,167 individuals, we identify 9 significant and 2 suggestive DNA sequence variants that influence HSPC (CD34+) levels in human blood. The identified loci associate with blood disorders, harbor known and novel HSPC genes, and affect gene expression in HSPCs. Interestingly, our strongest association maps to thePPM1Hgene, encoding an evolutionarily conserved serine/threonine phosphatase never previously implicated in stem cell biology.PPM1His expressed in HSPCs, and the allele that confers higher blood CD34+cell levels downregulatesPPM1H. By functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates a MYB transcription factor binding site inPPM1Hintron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, rs772557-A selectively increases HSPC subpopulations in which the MYB site is active, andPPM1HshRNA- knockdown increased CD34+and CD34+90+cell proportions in umbilical cord blood cultures. Our findings represent the first large-scale association study on a stem cell trait, illuminating HSPC regulationin vivoin humans, and identifyingPPM1Has a novel inhibition target that can potentially be utilized clinically to facilitate stem cell harvesting for transplantation.

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.

ABSTRACT The increasing capacity to perform large-scale single-cell genomic experiments continues to outpace the computational requirements to efficiently handle growing datasets. Herein we present Scarf, a modularly designed Python package that seamlessly interoperates with other single-cell toolkits and allows for memory-efficient single-cell analysis of millions of cells on a laptop or low-cost devices like single board computers. We demonstrate Scarf’s memory and compute-time efficiency by applying it to the largest existing single-cell RNA-Seq and ATAC-Seq datasets. Scarf wraps memory-efficient implementations of a graph-based t-stochastic neighbour embedding and hierarchical clustering algorithm. Moreover, Scarf performs accurate reference-anchored mapping of datasets while maintaining memory efficiency. By implementing a novel data downsampling algorithm, Scarf additionally can generate representative sampling of cells from a given dataset wherein rare cell populations and lineage differentiation trajectories are conserved. Together, Scarf provides a framework wherein any researcher can perform advanced processing, downsampling, reanalysis, and integration of atlas-scale datasets on standard laptop computers.