A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Cerebral vasculature exhibits dose-dependent sensitivity to thrombocytopenia that is limited to fetal/neonatal life.

Whether increasing platelet counts in fetal and neonatal alloimmune thrombocytopenia (FNAIT) is effective at preventing intracerebral hemorrhage (ICH) has been a subject of debate. The crux of the matter has been whether thrombocytopenia is the major driver of ICH in diseases such as FNAIT. We recently demonstrated in mice that severe thrombocytopenia was sufficient to drive ICH in utero and in early neonatal life. It remains unclear what degree of thrombocytopenia is required to drive ICH and for how long after birth thrombocytopenia can cause ICH. By inducing a thrombocytopenic range, we demonstrate that there is a large buffer zone of mild thrombocytopenia that does not result in ICH, that ICH becomes probabilistic at 40% of the normal platelet number, and that ICH becomes fully penetrant below 10% of the normal platelet number. We also demonstrate that although the neonatal mouse is susceptible to thrombocytopenia-induced ICH, this sensitivity is rapidly lost between postnatal days 7 and 14. These findings provide important insights into the risk of in utero ICH with varying degrees of thrombocytopenia and into defining the developmental high-risk period for thrombocytopenia-driven ICH in a mouse model of FNAIT.

Severe thrombocytopenia is sufficient for fetal and neonatal intracerebral hemorrhage to occur.

Intracerebral hemorrhage (ICH) has a devastating impact on the neonatal population. Whether thrombocytopenia is sufficient to cause ICH in neonates is still being debated. In this study, we comprehensively investigated the consequences of severe thrombocytopenia on the integrity of the cerebral vasculature by using 2 orthogonal approaches: by studying embryogenesis in the Nfe2-/- mouse line and by using biologics (anti-GP1Bα antibodies) to induce severe thrombocytopenia at defined times during development. By using a mouse model, we acquired data demonstrating that platelets are required throughout fetal development and into neonatal life for maintaining the integrity of the cerebral vasculature to prevent hemorrhage and that the location of cerebral hemorrhage is dependent on when thrombocytopenia occurs during development. Importantly, this study demonstrates that fetal and neonatal thrombocytopenia-associated ICH occurs within regions of the brain which, in humans, could lead to neurologic damage.

Clonal analysis of fetal hematopoietic stem/progenitor cells reveals how post-transplantation capabilities are distributed.

It has been proposed that adult hematopoiesis is sustained by multipotent progenitors (MPPs) specified during embryogenesis. Adult-like hematopoietic stem cell (HSC) and MPP immunophenotypes are present in the fetus, but knowledge of their functional capacity is incomplete. We found that fetal MPP populations were functionally similar to adult cells, albeit with some differences in lymphoid output. Clonal assessment revealed that lineage biases arose from differences in patterns of single-/bi-lineage differentiation. Long-term (LT)- and short-term (ST)-HSC populations were distinguished from MPPs according to capacity for clonal multilineage differentiation. We discovered that a large cohort of long-term repopulating units (LT-RUs) resides within the ST-HSC population; a significant portion of these were labeled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will significantly underestimate the size and diversity of the LT-RU pool and (2) LT-RUs in the ST-HSC population have the attributes required to persist into adulthood.

TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells.

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.

Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development.

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative 'early progenitor'-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of 'emergency' haematopoiesis.

Membrane budding is a major mechanism of in vivo platelet biogenesis.

How platelets are produced by megakaryocytes in vivo remains controversial despite more than a century of investigation. Megakaryocytes readily produce proplatelet structures in vitro; however, visualization of platelet release from proplatelets in vivo has remained elusive. We show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet replacement. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. In support of this model, we demonstrate that in mice deficient for NF-E2 (the thrombopoietic master regulator), the absence of membrane budding correlates with failure of in vivo platelet production. Accordingly, we propose that membrane budding, rather than proplatelet formation, supplies the majority of the platelet biomass.