We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs) enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2.

Natural killer (NK) cells are cytotoxic lymphocytes and play a vital role in controlling viral infections and cancer. In contrast to B and T lymphopoiesis where cellular and regulatory pathways have been extensively characterized, the cellular stages of early human NK cell commitment remain poorly understood. Here we demonstrate that a Lin(-)CD34(+)CD38(+)CD123(-)CD45RA(+)CD7(+)CD10(+)CD127(-) population represents a NK lineage-restricted progenitor (NKP) in fetal development, umbilical cord blood, and adult tissues. The newly identified NKP has robust NK cell potential both in vitro and in vivo, generates functionally cytotoxic NK cells, and lacks the ability to produce T cells, B cells, myeloid cells, and innate lymphoid-like cells (ILCs). Our findings identify an early step to human NK cell commitment and provide new insights into the human hematopoietic hierarchy.

Enterotoxigenic Escherichia coli (ETEC) cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP) on the regulation of production and secretion of heat labile (LT) enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

Hematopoietic stem cells (HSCs) are maintained in hypoxic niches in endosteal regions of bones. Here we demonstrate that Cripto and its receptor GRP78 are important regulators of HSCs in the niche. Flow cytometry analyses revealed two distinct subpopulations of CD34(-)KSL cells based on the expression of GRP78, and these populations showed different reconstitution potential in transplantation assays. GRP78(+)HSCs mainly reside in the endosteal area, are more hypoxic, and exhibit a lower mitochondrial potential, and their HSC capacity was maintained in vitro by Cripto through induction of higher glycolytic activity. Additionally, HIF-1α KO mice have decreased numbers of GRP78(+)HSCs and reduced expression of Cripto in the endosteal niche. Furthermore, blocking GRP78 induced a movement of HSCs from the endosteal to the central marrow area. These data suggest that Cripto/GRP78 signaling is an important pathway that regulates HSC quiescence and maintains HSCs in hypoxia as an intermediary of HIF-1α.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.

Retroviral overexpression of the transcription factor HOXB4 results in a rapid increase in proliferation of murine hematopoietic stem cells both in vivo and in vitro. Therefore, we asked whether transient overexpression of HOXB4 would increase proliferation of human primitive hematopoietic progenitors. Transient overexpression of HOXB4 was generated in umbilical cord blood (CB) CD34(+) cells by a recombinant adenovirus (AdHOXB4) expressing HOXB4 together with the enhanced green fluorescent protein (GFP). Transduced, GFP(+) cells were cultured in serum-free medium containing cytokines that primarily support the growth of primitive hematopoietic progenitors. In contrast to previous findings using retroviral overexpression of HOXB4, we did not observe any increase in proliferation of primitive progenitors or increased colony formation of clonogenic progenitors, including progenitor progeny from long-term culture-initiating cells following adenoviral vector overexpression of HOXB4 in CB CD34(+) cells. However, enforced expression of HOXB4 by the adenoviral vector significantly increased myeloid differentiation of primitive hematopoietic progenitors. Since retroviral vectors generate low and continuous levels of transgene expression in contrast to the high, transient levels generated by the adenoviral vector, our findings suggest that the high levels of HOXB4 expression generated by AdHOXB4 in human CB CD34(+) cells direct the cells toward a myeloid differentiation program rather than increased proliferation.

Development of the eye depends partly on the periocular mesenchyme derived from the neural crest (NC), but the fate of NC cells in mammalian eye development and the signals coordinating the formation of ocular structures are poorly understood.

Regulatory mechanisms of human hematopoiesis remain largely uncharacterized. Through expression profiling of prospectively isolated stem and primitive progenitor cells as well as committed progenitors from cord blood (CB), we identified MLLT3 as a candidate regulator of erythroid/megakaryocytic (E/Meg) lineage decisions. Through the analysis of the hematopoietic potential of primitive cord blood cells in which MLLT3 expression has been knocked down, we identify a requirement for MLLT3 in the elaboration of the erythroid and megakaryocytic lineages. Conversely, forced expression of MLLT3 promotes the output of erythroid and megakaryocytic progenitors, and analysis of MLLT3 mutants suggests that this capacity of MLLT3 depends on its transcriptional regulatory activity. Gene expression and cis-regulatory element analyses reveal crossregulatory interactions between MLLT3 and E/Meg-affiliated transcription factor GATA-1. Taken together, the data identify MLLT3 as a regulator of early erythroid and megakaryocytic cell fate in the human system.

Recent studies implicated the existence of adult lymphoid-primed multipotent progenitors (LMPPs) with little or no megakaryocyte-erythroid potential, questioning common myeloid and lymphoid progenitors as obligate intermediates in hematopoietic stem cell (HSC) lineage commitment. However, the existence of LMPPs remains contentious. Herein, global and single-cell analyses revealed a hierarchical organization of transcriptional lineage programs, with downregulation of megakaryocyte-erythroid genes from HSCs to LMPPs, sustained granulocyte-monocyte priming, and upregulation of common lymphoid (but not B and T cell-specific) genes. These biological and molecular relationships, implicating almost mutual exclusion of megakaryocyte-erythroid and lymphoid pathways, are established already in fetal hematopoiesis, as evidenced by existence of LMPPs in fetal liver. The identification of LMPPs and hierarchically ordered transcriptional activation and downregulation of distinct lineage programs is compatible with a model for HSC lineage commitment in which the probability for undergoing different lineage commitment fates changes gradually when progressing from HSCs to LMPPs.

Relapse of acute myeloid leukemia (AML) remains a significant clinical challenge due to limited therapeutic options and poor prognosis. Leukemic stem cells (LSCs) are the cellular units responsible for relapse in AML, and strategies that target LSCs are thus critical. One proposed potential strategy to this end is to break the quiescent state of LSCs, thereby sensitizing LSCs to conventional cytostatics. The hypoxia-inducible factor (HIF) pathway is a main driver of cellular quiescence and a potential therapeutic target, with precedence from both solid cancers and leukemias. Here, we used a conditional knockout Hif-1α mouse model together with a standard chemotherapy regimen to evaluate LSC targeting in AML. Contrary to expectation, our studies revealed that Hif-1α-deleted-leukemias displayed a faster disease progression after chemotherapy. Our studies thereby challenge the general notion of cancer stem cell sensitization by inhibition of the HIF pathway, and warrant caution when applying HIF inhibition in combination with chemotherapy in AML.

ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19-IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19-IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.

Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1.

Hematopoietic stem cells (HSCs) emerge during embryogenesis from hemogenic endothelium, but it remains unclear how the HSC lineage is initially established from mesoderm during ontogeny. In Xenopus, the definitive hemangioblast precursors of the HSC lineage have been identified in dorsal lateral plate (DLP) mesoderm, and a transcriptional gene regulatory network (GRN) controlling hemangioblast programming has been elucidated. Herein, we identify an essential role for microRNAs (miRNAs) in establishing the mesodermal lineage leading to both HSC emergence and vasculogenesis and determine that a single miRNA, miR-142-3p, is primarily responsible for initiation of definitive hemangioblast specification. miR-142-3p forms a double-negative gate unlocking entry into the hemangioblast program, in part by inhibiting TGFβ signaling. Our results table miR-142-3p as a master regulator of HSC lineage specification, sitting at the apex of the hierarchy programming the adult hemangioblast, thus illustrating that miRNAs can act as instructive determinants of cell fate during development.

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 .

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.

Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (GBA1), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here.

Single-cell RNAseq is a routinely used method to explore heterogeneity within cell populations. Data from these experiments are often visualized using dimension reduction methods such as UMAP and tSNE, where each cell is projected in two or three dimensional space. Three-dimensional projections can be more informative for larger and complex datasets because they are less prone to merging and flattening similar cell-types/clusters together. However, visualizing and cross-comparing 3D projections using current software on conventional flat-screen displays is far from optimal as they are still essentially 2D, and lack meaningful interaction between the user and the data. Here we present CellexalVR (www.cellexalvr.med.lu.se), a feature-rich, fully interactive virtual reality environment for the visualization and analysis of single-cell experiments that allows researchers to intuitively and collaboratively gain an understanding of their data.

Isolation of long-term hematopoietic stem cell (HSC) is possible by utilizing flow cytometry with multiple cell surface markers. However, those cell surface phenotypes do not represent functional HSCs after in vitro culture. Here we show that cultured HSCs express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSCs were divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. The result was same when HSCs were cultured in an efficient expansion medium containing polyvinyl alcohol. Chemically induced endoplasmic reticulum (ER) stress signal increased the CD244+ subpopulation, whereas ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which was correlated to improved reconstitution output. These data suggest CD244 is a potent marker to exclude non-functional HSCs after in vitro culture thereby useful to elucidate mechanism of functional decline of HSCs during ex vivo treatment.

Mouse hematopoietic stem cells (HSCs) have been extensively defined both molecularly and functionally at steady state, while regenerative stress induces immunophenotypical changes that limit high purity isolation and analysis. It is therefore important to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Here, we assessed the expression of macrophage-1 antigen (MAC-1) on HSCs during regeneration following transplantation and observed a transient increase in MAC-1 expression during the early reconstitution phase. Serial transplantation experiments demonstrated that reconstitution potential was highly enriched in the MAC-1+ portion of the HSC pool. Moreover, in contrast to previous reports, we found that MAC-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating MAC-1+ HSCs share molecular features with stem cells with low mitotic history. Taken together, our results suggest that MAC-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration.

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.