SUMMARY Skeletal muscle contains a resident population of somatic stem cells capable of both self-renewal and differentiation. The signals that regulate this important decision have yet to be fully elucidated. Here we use metabolomics and mass spectrometry imaging (MSI) to identity a state of localized hyperglycaemia following skeletal muscle injury. We show that committed muscle progenitor cells exhibit an enrichment of glycolytic and TCA cycle genes and that extracellular monosaccharide availability regulates intracellular citrate levels and global histone acetylation. Muscle stem cells exposed to a reduced (or altered) monosaccharide environment demonstrate reduced global histone acetylation and transcription of myogenic determination factors (including myod1 ). Importantly, reduced monosaccharide availability was linked directly to increased rates of asymmetric division and muscle stem cell self-renewal in regenerating skeletal muscle. Our results reveal an important role for the extracellular metabolic environment in the decision to undergo self-renewal or myogenic commitment during skeletal muscle regeneration.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells.

Hematopoietic stem cell (HSC) transplantation (HSCT) represents the only curative therapy for most hematologic disease including leukemia.Recent studies suggest that "stress hematopoiesis", including that which occurs post-HSCT, is subject to distinct regulation compared steady-state hematopoiesis.Better understanding the molecular mechanisms that regulate transplanted HSCs and their engraftment is necessary to improve HSCT.Thus, we seek to identify novel molecular regulators of HSCT.Our data reveal high expression of multiple GPRASPs (G-Protein Coupled Receptor Associated Sorting Proteins) (e.g.Gprasp1 and Gprasp2) in HSCs relative to downstream progenitors.ShRNA-mediated gene knock-down transplantation-based studies revealed that both GPRASP1 and GPRASP2 act as negative regulators of HSCT, showing 4-fold increase in peripheral blood and bone marrow reconstitution and significantly enhance survival and quiescence in HSCs ex vivo and after transplant.GPRASPs regulate the post-endosomal trafficking of GPCRs to the lysosome.CXCR4, a GPCR that regulates HSCs function, contains a putative GPRASP-binding motif in its C-terminus, implicating it as a possible target of GPRASP-mediated degradation.Indeed, Gprasp1 or Gprasp2 loss increased both total CXCR4 and cell membrane-localized CXCR4 in HSPCs.Consequently, Gprasp1 or Gprasp2 loss increased HSPC migration toward SDF-1.Further, Gprasp1 or Gprasp2 loss significantly enhanced acute homing and HSCs niche retention post-transplant.These GPRASP-knockdown effects disappear when Cxcr4 is genetically deleted.In sum, we report for the first time that multiple GPRASPs function as barriers to HSCT by effecting HSC migration, homing and niche retention via regulation of CXCR4 stability and localization.

Dysregulation of cancer cell bioenergetics is one of the hallmarks of cancer.The Warburg effect is one such documented change.However, glucose metabolism is not universally increased in cancer cells.Uptake of 18F-FDG in chronic lymphocytic leukemia (CLL) fails as a marker of proliferation and whilst the underlying reason is poorly understood it suggests that CLL cells utilize energy sources other than glucose to proliferate.Using genetic, proteomic and lipidomic analyses, complemented with microscopy and nutrient uptake assays the preferred metabolic pathways of CLL cells have been identified.We measured the uptake of fluorescently labelled short, medium and long-chain fatty acids (LCFA) and the glucose analog 2-NBDG by flow cytometry and confocal microscopy.Three CLL lines (MEC1, MEC2, OSU-CLL) prefer LCFA, over short and medium chain and show a low uptake of 2-NBDG.We have also confirmed these findings in primary CLL samples.Using qPCR and western blot analysis we have identified varying levels of LCFA uptake receptors.We found an up-regulation of proteins involved in lipogenesis in quiescent peripheral CLL cells, and an increase in b-oxidation proteins in the proliferative compartment of the lymph node.Together with our morphological examination using electron and confocal microscopy we suggest that peripheral CLL cells scavenge lipids, which are stored in lipid droplets and protected from degradation by a high expression of PLIN proteins.These cells circulate back to the proliferation centres, the lipid droplets are degraded, likely by lipophagy which frees fatty acids for b-oxidation.Our results begin to unravel CLL bioenergetics and dysregulation of cellular metabolism that occurs in this disease.We are now investigating whether the manipulation of these pathways, particularly lipophagy, may represent a novel therapeutic approach in CLL.

A classical view of blood cell development is that multipotent haematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. The Lin–c-kit+Sca1+Flt3+ stage, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Through single cell RNA-sequencing, we identify heterogeneous expression of Dach1 and associated genes in this fraction where it co-expressed 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– subpopulation within LMPPs with lymphoid potential but devoid of myeloid potential - both in vivo and in vitro. Dach1 based separation is distinct from to separation by Rag1-GFP, Flt3, CD27, CD48 or VCAM. We term these ‘lymphoid-primed progenitors’, or LPPs. These findings define the earliest branch point of lymphoid development in haematopoiesis and a means for their prospective isolation.

Conventional single cell RNA-seq methods are destructive, such that a given cell cannot also then be tested for fate and function, without a time machine. Here, we develop a clonal method SIS-seq, whereby single cells are allowed to divide, and progeny cells are assayed separately in SISter conditions; some for fate, others by RNA-seq. By cross-correlating progenitor gene expression with mature cell fate within a clone, and doing this for many clones, we can identify the earliest gene expression signatures of dendritic cell subset development. SIS-seq could be used to study other populations harboring clonal heterogeneity, including stem, reprogrammed and cancer cells to reveal the transcriptional origins of fate decisions.