Committed Progenitor Cells Research Articles

Muscle Satellite Cell Heterogeneity: Does Embryonic Origin Matter?

Muscle regeneration is an important homeostatic process of adult skeletal muscle that recapitulates many aspects of embryonic myogenesis. Satellite cells (SCs) are the main muscle stem cells responsible for skeletal muscle regeneration. SCs reside between the myofiber basal lamina and the sarcolemma of the muscle fiber in a quiescent state. However, in response to physiological stimuli or muscle trauma, activated SCs transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent evidence has stated that SCs display functional heterogeneity linked to regenerative capability with an undifferentiated subgroup that is more prone to self-renewal, as well as committed progenitor cells ready for myogenic differentiation. Several lineage tracing studies suggest that such SC heterogeneity could be associated with different embryonic origins. Although it has been established that SCs are derived from the central dermomyotome, how a small subpopulation of the SCs progeny maintain their stem cell identity while most progress through the myogenic program to construct myofibers is not well understood. In this review, we synthesize the works supporting the different developmental origins of SCs as the genesis of their functional heterogeneity.

Bone-derived sclerostin and Wnt/β-catenin signaling regulate PDGFRα+ adipoprogenitor cell differentiation.

The Wnt signaling antagonist, sclerostin, is a potent suppressor of bone acquisition that also mediates endocrine communication between bone and adipose. As a result, Sost−/− mice exhibit dramatic increases in bone formation but marked decreases in visceral and subcutaneous adipose that are secondary to alterations in lipid synthesis and utilization. While interrogating the mechanism by which sclerostin influences adipocyte metabolism, we observed paradoxical increases in the adipogenic potential and numbers of CD45−:Sca1+:PDGFRα+ adipoprogenitors in the stromal vascular compartment of fat pads isolated from male Sost−/− mice. Lineage tracing studies indicated that sclerostin deficiency blocks the differentiation of PDGFRα+ adipoprogenitors to mature adipocytes in association with increased Wnt/β‐catenin signaling. Importantly, osteoblast/osteocyte‐specific Sost gene deletion mirrors the accumulation of PDGFRα+ adipoprogenitors, reduction in fat mass, and improved glucose metabolism evident in Sost−/− mice. These data indicate that bone‐derived sclerostin regulates multiple facets of adipocyte physiology ranging from progenitor cell commitment to anabolic metabolism.

In conversation with Christine Watson.

Christine J. Watson is Professor of Cell and Cancer Biology at the University of Cambridge. Christine obtained her Bachelor's (honors) degree in Biochemistry at the University of Glasgow in 1975 and, after a soujourn in Glauco Tocchini-Valentini's lab at the Institute of Cell Biology, Consiglio Nazionale delle Ricerche in Rome, she undertook a PhD in Molecular Genetics at Imperial College London. During her PhD, she looked at differences in gene expression between differentiated and undifferentiated embryonal carcinoma stem cells, inspiring an early interest in gene expression and cell fate determination. Between 1986 and 1992, Christine undertook three postdoctoral research positions that took her from London back to Scotland, where she was first introduced to mammary gland biology through her work with John Clark at the Roslin Institute in Edinburgh. During her time in the Clark lab, Christine identified a factor - later shown to be STAT5 - that binds to the promoter of the milk protein gene β-lactoglobulin. This prompted further work identifying the key role played by the STAT family of transcription factors in mammary gland development. Shortly afterwards, Christine became a group leader at the Roslin Institute and later relocated to the University of Edinburgh to collaborate with Andrew Wyllie. This led to her recruitment to the University of Cambridge in 1998, where she has remained to date. Over the last two decades, the Watson lab has focused on elucidating the mechanisms underlying lineage commitment of mammary stem and progenitor cells and the regulation of cell death in involuting mammary gland. In this interview, Christine discusses her research highlights and provides a glimpse into her personal interests, as she moves towards retirement.

Developing Live Imaging Strategies to Analyze the Influence of Cell Cycle Rate on MEP Fate Decisions at the Single Cell Level

Elucidating the mechanisms underlying hematopoietic cell fate decision is critical to our understanding of physiological and pathological hematopoiesis. Robust response to physiological stress requires rapid commitment and expansion of mature blood cells derived from less committed progenitors. The processes dictating the directed differentiation of these progenitors are poorly understood.Preliminary data from our lab suggest that cell cycle rate influences commitment of the Megakaryocyte/Erythroid Progenitor (MEP) to the megakaryocytic and erythroid (Er) lineages. To test whether slower cycling MEPs tend to be more committed to the megakaryocytic lineage, we have developed a time-lapse microscopy imaging approach to measure the number and duration of cell cycles of single cells and their progeny as they make their cell fate decision determined by their fluorescent marker expression in real-time.CD34-selected mobilized peripheral blood cells were stained with desired antibodies and sorted using our recently published scheme: Lin-CD34+CD38midCD45RA-FLT3-MPL+CD36-CD41- (Sanada and Xavier-Ferrucio, et al. Blood 2016) that is highly enriched for bipotent MEPs than populations previously reported. MEPs were plated at low density (25 cells/15μL) in MegaCult collagen-based medium containing SCF, IL3, IL6, TPO, and EPO in the center of a MatTek plate under a coverslip to minimize focal range. After the MegaCult polymerized, 1.5mL of additional MegaCult and cytokines were added to the dish to support growth and expansion of megakaryocytes and erythroid cells for 12 days. Cells were imaged every 2 hours in DIC for the first three days, and every 20 minutes for 8 additional days. On the 10th day, fluorescently conjugated anti-CD41 (to identify Mks) and anti-CD235a (to identify Er cells) antibodies were diluted in IMDM and added to the imaged dish. The antibodies permeated the MegaCult and labeled the committed cells. Imaging continued every 20 minutes in DIC, FITC, and Cy5 channels for an additional day. Using the Baxter Algorithm to analyze time-lapse images, we traced the progeny back to the parent cell, and calculated the number of cell cycles, as well as the duration of each cycle leading to lineage commitment. We have validated this method by measuring cell cycle rates of bi-potent MEPs, Mk-restricted progenitors, and Er-restricted progenitors, and found Er-restricted progenitors to have a significantly shortened cell cycle rate compared to Mk-restricted progenitors.To further investigate the role of cell cycle rate in MEP commitment, we utilized our live imaging strategy to collect and analyze cell cycle rate data for transduced MEPs expressing shRNA against Myb. Myb is a known regulator of the G2/M transition by binding the promoter and altering expression of Cyclin B1 in human blood cells (Nakata et al., MCB, 2007). Here we show that MEPs knocked down for Myb have no difference in survival compared to scramble controls. Interestingly, the rate of cell cycle divisions is significantly longer in MEP progeny lacking Myb, and this correlates with an increase in the ratio of CFU-Mk to BFU-E and CFU-Mk/E. This data suggests that by lengthening the cell cycle rate, fate decision of the bipotent MEP can be swayed towards the Mk lineage, which has a longer cell cycle rate compared to the Er lineage. In the future, live imaging analysis of single cells can be utilized to answer a plethora of questions regarding fate decisions and lineage commitment of hematopoietic stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Clearly imaging and quantifying the kidney in 3D

For decades, measurements of kidney microanatomy using 2-dimensional sections has provided us with a detailed knowledge of kidney morphology under physiological and pathological conditions. However, the rapid development of tissue clearing methods in recent years, in combination with the development of novel 3-dimensional imaging modalities have provided new insights into kidney structure and function. This review article describes a range of novel insights into kidney development and disease obtained recently using these new methodological approaches. For example, in the developing kidney these approaches have provided new understandings of ureteric branching morphogenesis, nephron progenitor cell proliferation and commitment, interactions between ureteric tip cells and nephron progenitor cells, and the establishment of nephron segmentation. In whole adult mouse kidneys, tissue clearing combined with light sheet microscopy can image and quantify the total number of glomeruli, a major breakthrough in the field. Similar approaches have provided new insights into the structure of the renal vasculature and innervation, tubulointerstitial remodeling, podocyte loss and hypertrophy, cyst formation, the evolution of cellular crescents, and the structure of the glomerular filtration barrier. Many more advances in the understanding of kidney biology and pathology can be expected as additional clearing and imaging techniques are developed and adopted by more investigators.

FGF-2-Dependent Signaling Activated in Aged Human Skeletal Muscle Promotes Intramuscular Adipogenesis

Abstract Background: A unique feature of muscle during aging, obesity and type 2 diabetes is the appearance of adipose tissue between skeletal muscle fibers, the intramuscular adipose tissue (IMAT). IMAT is generally associated with systemic insulin resistance, decreased muscle strength and, in older adults, impaired mobility. A large body of work over the last years have addressed the origin of IMAT. Skeletal muscle contains preadipocyte progenitors termed fibro/adipogenic progenitors (FAPs) that normally do not form adipocytes, but proliferate during muscle injury to support the commitment of myogenic progenitor cells during muscle repair. However, under the conditions outlined above, FAPs differentiate to adipocytes and give rise to IMAT. The molecular cues that trigger this pathogenesis are unknown and strategies that can modulate the fate of FAPs and their propensity to differentiate to adipocytes in disease states are urgently needed. Methods: We have used molecular cloning and genome editing techniques together with a variety of biochemical, molecular, genomic, transcriptomic and proteomic analyses in pre-adipocyte and muscle cell lines, primary cells isolated from human and mouse skeletal muscle, human skeletal muscle biopsies, and animal models. Results: Using multiple screening assays upstream and downstream of a master regulator of myogenesis, microRNA (miR)-29a, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. We identified FGF-2 as an upstream regulator that increases miR-29a promoter activity and gene expression via MEK1/2/MAPK signaling and consequently decreases the conserved miR-29a target SPARC. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. CRISPR/Cas9-mediated genomic deletions of miR-29a recognition elements located in the Sparc 3′UTR of muscle cells and AAV-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of FAPs in vitro and IMAT formation in vivo. Skeletal muscle from human donors aged > 75 years versus < 55 years showed activation of FGF-2-dependent signaling and increased IMAT. Conclusion: FGF-2 not only stimulates muscle growth, but also promotes intramuscular adipogenesis via targeting the fate of FAPs. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a novel pathway, which has the potential to provide innovative diagnostic and therapeutic approaches to combat fat accumulation in aged human skeletal muscle.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Evolutionary selection of alleles in the melanophilin gene that impacts on prostate organ function and cancer risk.

Background and objectivesSeveral hundred inherited genetic variants or SNPs that alter the risk of cancer have been identified through genome-wide association studies. In populations of European ancestry, these variants are mostly present at relatively high frequencies. To gain insight into evolutionary origins, we screened a series of genes and SNPs linked to breast or prostate cancer for signatures of historical positive selection.MethodologyWe took advantage of the availability of the 1000 genome data and we performed genomic scans for positive selection in five different Caucasian populations as well as one African reference population. We then used prostate organoid cultures to provide a possible functional explanation for the interplay between the action of evolutionary forces and the disease risk association.ResultsVariants in only one gene showed genomic signatures of positive, evolutionary selection within Caucasian populations melanophilin (MLPH). Functional depletion of MLPH in prostate organoids, by CRISPR/Cas9 mutation, impacted lineage commitment of progenitor cells promoting luminal versus basal cell differentiation and on resistance to androgen deprivation.Conclusions and implicationsThe MLPH variants influencing prostate cancer risk may have been historically selected for their adaptive benefit on skin pigmentation but MLPH is highly expressed in the prostate and the derivative, positively selected, alleles decrease the risk of prostate cancer. Our study suggests a potential functional mechanism via which MLPH and its genetic variants could influence risk of prostate cancer, as a serendipitous consequence of prior evolutionary benefits to another tissue.Lay SummaryWe screened a limited series of genomic variants associated with breast and prostate cancer risk for signatures of historical positive selection. Variants within the melanophilin (MLPH) gene fell into this category. Depletion of MLPH in prostate organoid cultures, suggested a potential functional mechanism for impacting on cancer risk, as a serendipitous consequence of prior evolutionary benefits to another tissue.

Lhx6 regulates canonical Wnt signaling to control the fate of mesenchymal progenitor cells during mouse molar root patterning.

Mammalian tooth crown formation has long served as a model for investigating how patterning and morphogenesis are orchestrated during development. However, the mechanism underlying root patterning and morphogenesis remains poorly understood. In this study, we find that Lhx6 labels a subpopulation of root progenitor cells in the apical dental mesenchyme, which is closely associated with furcation development. Loss of Lhx6 leads to furcation and root number defects, indicating that Lhx6 is a key root patterning regulator. Among the multiple cellular events regulated by Lhx6 is the odontoblast fate commitment of progenitor cells, which it controls in a cell-autonomous manner. Specifically, Lhx6 loss leads to elevated expression of the Wnt antagonist Sfrp2 and down-regulation of Wnt signaling in the furcation region, while overactivation of Wnt signaling in Lhx6+ progenitor cells partially restore the furcation defects in Lhx6-/- mice. Collectively, our findings have important implications for understanding organ morphogenesis and future strategies for tooth root regeneration.

Eosinophil Lineage-Committed Progenitors as a Therapeutic Target for Asthma.

Eosinophilic asthma is the most prevalent phenotype of asthma. Although most asthmatics are adequately controlled by corticosteroid therapy, a subset (5–10%) remain uncontrolled with significant therapy-related side effects. This indicates the need for a consideration of alternative treatment strategies that target airway eosinophilia with corticosteroid-sparing benefits. A growing body of evidence shows that a balance between systemic differentiation and local tissue eosinophilopoietic processes driven by traffic and lung homing of bone marrow-derived hemopoietic progenitor cells (HPCs) are important components for the development of airway eosinophilia in asthma. Interleukin (IL)-5 is considered a critical and selective driver of terminal differentiation of eosinophils. Studies targeting IL-5 or IL-5R show that although mature and immature eosinophils are decreased within the airways, there is incomplete ablation, particularly within the bronchial tissue. Eotaxin is a chemoattractant for mature eosinophils and eosinophil-lineage committed progenitor cells (EoP), yet anti-CCR3 studies did not yield meaningful clinical outcomes. Recent studies highlight the role of epithelial cell-derived alarmin cytokines, IL-33 and TSLP, (Thymic stromal lymphopoietin) in progenitor cell traffic and local differentiative processes. This review provides an overview of the role of EoP in asthma and discusses findings from clinical trials with various therapeutic targets. We will show that targeting single mediators downstream of the inflammatory cascade may not fully attenuate tissue eosinophilia due to the multiplicity of factors that can promote tissue eosinophilia. Blocking lung homing and local eosinophilopoiesis through mediators upstream of this cascade may yield greater improvement in clinical outcomes.

A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells.

The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

Rosmarinic acid attenuates obesity and obesity-related inflammation in human adipocytes

Chronic low-grade inflammation is a hallmark of obesity and its related metabolic disorders. At the same time signaling from pro-inflammatory factors such as transforming growth factor beta (TGF-β) or interleukin 17A (IL-17A) are proposed as crucial for the commitment of fibroblast progenitor cells towards adipogenic differentiation. Modulation of inflammation during adipogenic differentiation is incompletely explored as a potential approach to prevent metabolic disorders. Rosmarinic acid (RA) is a caffeic acid derivative known for its anti-inflammatory effects. Experimental studies of its activity on adipogenic factors or in vivo obesity models are, however, controversial and hence insufficient. Here, we investigated the anti-adipogenic action of RA in human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. Gene expression levels of key players in adipogenesis and lipid metabolism were assessed. Furthermore, a molecular mechanism of action was proposed. The most prominent effect was found on the translation of C/EBPα, PPARγ and adiponectin, as well as on the modulation of TGF1B and IL17A. Interestingly, involvement of NRF2 signaling was identified upon RA treatment. In summary, our findings indicate that RA prevents inflammation and excessive lipid accumulation in human adipocytes. Data from the molecular analysis demonstrate that RA has potential for treatment of obesity and obesity-related inflammation.

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.

In response to physiological demand, the pituitary gland generates new hormone-secreting cells from committed progenitor cells throughout life. It remains unclear to what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to pituitary growth and renewal. Moreover, neither the signals that drive proliferation nor their sources have been elucidated. We have used genetic approaches in the mouse, showing that the WNT pathway is essential for proliferation of all lineages in the gland. We reveal that SOX2+ stem cells are a key source of WNT ligands. By blocking secretion of WNTs from SOX2+ PSCs in vivo, we demonstrate that proliferation of neighbouring committed progenitor cells declines, demonstrating that progenitor multiplication depends on the paracrine WNT secretion from SOX2+ PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion and homeostasis, acting as paracrine signalling centres to coordinate the proliferation of neighbouring cells.

Development of the metanephric kidney.

The kidney plays an integral role in filtering the blood-removing metabolic by-products from the body and regulating blood pressure. This requires the establishment of large numbers of efficient and specialized blood filtering units (nephrons) that incorporate a system for vascular exchange and nutrient reabsorption as well as a collecting duct system to remove waste (urine) from the body. Kidney development is a dynamic process which generates these structures through a delicately balanced program of self-renewal and commitment of nephron progenitor cells that inhabit a constantly evolving cellular niche at the tips of a branching ureteric "tree." The former cells build the nephrons and the latter the collecting duct system. Maintaining these processes across fetal development is critical for establishing the normal "endowment" of nephrons in the kidney and perturbations to this process are associated both with mutations in integral genes and with alterations to the fetal environment.

Transcriptome analysis of skeletal muscles revealed the effect of exercise on the molecular mechanisms regulating muscle growth and metabolism in patients with heart failure

Aim. Heart failure (HF) is accompanied by skeletal muscle atrophy and exercise intolerance. The aim was to study the molecular mechanisms underlying the therapeutic effect of personalized exercise in patients with HF.Material and methods. RNA sequencing obtained from skeletal muscle biopsies before and after a 12-week exercise course was used to identify changes in gene expression and signaling pathways induced by the physical rehabilitation program for patients with HF.Results. We have shown that personalized exercise program in patients with HF stimulates the activation of molecular pathways regulating the differentiation and functioning of skeletal muscles: commitment of muscle progenitor cells; mechanisms regulating the calcium release and sensitivity of myofibrillar contraction, electrical excitability of the muscle membrane, synaptic vesicle proton gradient creation, maintenance of electrochemical gradients of Na+ /K+ . Also, the analysis of differentially expressed genes revealed an increase in the expression of transcription factors MyoD and MEF2, which are responsible for the differentiation of muscle stem cells, and sarcomeric genes MYOM1, MYOM2, MYH7. Along with this, we observed activation of the CYR61 expression — a potential prognostic biomarker for HF patients.Conclusion. Our data show that the beneficial effect of personalized aerobic exercise in patients with HF depends, at least in part, on an improvement in the physiological and biochemical parameters of skeletal muscle.

EPCO-20. RELATING GLIOBLASTOMA HETEROGENEITY TO HUMAN FETAL GLIAL DEVELOPMENT THROUGH HIGH RESOLUTION SINGLE NUCLEI TRANSCRIPTOMICS

Abstract Glioblastoma (GBM) is thought to be driven by a therapy-resistant cancer stem cell population that recapitulates developmental phenotypes. Direct comparisons of GBM to glial states during human fetal development are limited due to paucity of data from late prenatal gestation, when gliogenesis is thought to occur. Here, we generated a comprehensive single nuclei RNA sequencing (snRNAseq) dataset of approximately 200,000 nuclei taken from the germinal matrix and the cortical plate of 16 fetal postmortem samples, ranging from 17 to 41 gestational weeks, enabling high spatiotemporal resolution of late neurogenesis and early-to-peak gliogenesis. We performed unbiased clustering to identify broad cell types within each sample and integrated all fetal samples to analyze evolving glial states and relationships across two regions and four developmental stages. Subclustering analysis of developing glia from the germinal matrix and cortical plate resolved developmental cell type signatures that are absent in the adult brain. Trajectory inference and pseudo-time analyses reconstructed relationships within these glial lineages and states, identifying a robust common glial progenitor population (GPC) with distinct signature, preceding both oligodendrocyte progenitor cell (OPC) and astrocyte lineage commitment during late prenatal development. We then performed snRNAseq on approximately 30,000 nuclei taken from the core and infiltrating edge of two surgically resected GBM samples with IDH-mutant and IDH-wildtype status and EGFR amplification. Uniform manifold approximation and projection (UMAP) dimensionality reduction revealed distinct neoplastic and non-neoplastic population clusters within each GBM sample. Projecting our previously defined neural stem cell / progenitor signatures onto each GBM UMAP identified notable predominance of the GPC-like developmental signature throughout both GBM tumors with focal minor contributions from the OPC-, transit amplifying-, and astrocyte-like signatures. The high spatial and temporal resolution of the generated roadmap dissolves GBM intratumoral heterogeneity into distinct developmental molecular states driven by potentially targetable regulatory networks.

Metabolomic Characterization of Red Blood Cell Differentiation

Background: Erythropoiesis is the highly coordinated multi-step process by which multipotent hematopoietic stem cells differentiate into mature enucleated red blood cells (RBCs). As erythroid cells become more terminally differentiated, they undergo changes in morphology and gene expression, start synthesizing hemoglobin, commit to an irreversible loss of proliferation, and eventually expulse their nuclei and other cytoplasmic organelles. Thus, RBCs must rely on their proteome and metabolome for proper function. The RBC proteome is estimated to contain 2,800 proteins, including a variety of receptors and transporters that allow RBCs to uptake xenobiotics or endogenous metabolites as they circulate for ∼120 days. Furthermore, they are metabolically active with glycolysis, nucleotide catabolism, and glutathione metabolism as the major pathways supporting cell survival and function. However, it is unclear how the metabolome is altered during erythropoiesis, what role metabolites play in normal erythropoiesis, and if dysregulation of metabolites contributes to diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Methods: Four models of erythropoiesis were used in this study. 1) Mice were treated with phenylhydrazine (Phz) to induce acute hemolysis followed by erythropoietic recovery, leading to an increase in circulating reticulocytes. 2) Mice were lethally irradiated and transplanted with wild-type or sickle cell bone marrow, leading to anemic profiles in sickle cell chimeras. 3) The mouse erythroleukemic (MEL) cell line was treated with DMSO to induce differentiation. 4) The human erythroleukemic (K562) cell line was treated with sodium butyrate to induce differentiation. For the in vivo mouse models, blood was collected from control and treated animals, and complete blood count (CBC) analysis was performed. For the in vitro cell culture models, the mRNA levels of β-globin were measured by Q-RT-PCR in control and differentiated cells, and the degree of hemoglobinization was determined visually and via staining for heme. In addition, metabolites were extracted from the collected RBCs and erythroleukemic cell lines, and a Snapshot LC/MS metabolomic platform was used to identify commonly altered metabolites. Results: We first validated our four models of erythropoiesis. Treatment with Phz decreased the number of total RBCs while increasing the RBC distribution width, indicating an increased number of reticulocytes (more immature RBCs) in circulation. Similar results were seen in the sickle cell chimeras. Treatment of MEL and K562 cells with DMSO and sodium butyrate, respectively, resulted in increased expression of β-globin, increased levels of heme, and increased red color. Then, using our Snapshot metabolomic platform, we identified global changes in RBC metabolism during erythropoiesis. Analyses of the commonly altered metabolites in the in vitro and in vivo models revealed an increase in amino acid, mitochondrial, and urea cycle metabolism during erythropoiesis. L-aspartate levels were particularly upregulated, especially in DMSO-treated MEL cells. We are now investigating the role of aspartate in the regulation of erythropoiesis. Conclusions: We defined how the metabolome was altered in multiple in vitro and in vivo models of erythropoiesis and identified global changes in RBC metabolism between the different models. Specifically, we found that L-aspartate was upregulated during RBC differentiation in all four models. Aspartate is an amino acid that plays a role in many processes in cells, including nucleotide biosynthesis, redox homeostasis, and amino acid biosynthesis. We hypothesize that aspartate metabolism is critical for RBC differentiation and that its dysregulation exacerbates disease of ineffective erythropoiesis, such as sickle cell anemia and β-thalassemia. We are currently testing its role in inducing hemoglobinization and in regulating the commitment of erythroid progenitor cells to an irreversible loss of proliferation. Overall, we believe that understanding the precise mechanisms by which cellular metabolism plays a role in proper RBC differentiation may lead to better therapies for diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Disclosures No relevant conflicts of interest to declare.

A Lineage-Specific Requirement for YY1 Polycomb Group Protein Function in Early T Cell Development

T cell development originates from hematopoietic stem and progenitor cells in the bone marrow, which migrate to the thymus and obtain T cell identification. Transcription factors play critical roles in regulating early T cell development. While Notch signals are critically required at the early stage of T cell development, the completion of T cell lineage commitment is far from the initial response to Notch signaling. Other transcription factors such as PU.1, Ikaros, and RUNX1 are required to enable progenitor cells to committee T cell lineage before Notch signaling. YY1 is a ubiquitous transcription factor and mammalian Polycomb Group Protein (PcG) with important functions to regulate lymphocytes development, stem cell self-renewal, cell proliferation, and survival. Previous study showed that YY1 can interact with the Notch1 receptor intracellular domain and regulate Notch1 transactivation activities in vitro. Thus, YY1 may also belong to the core T cell lineage regulatory factors and is required for progenitor cell commitment to T cell development. To test how loss-of-function of YY1 impacts early T cell development, we utilized a conditional Yy1 knockout allele Yy1f/f with loxP sites flanking the Yy1 promoter region and exon 1. Yy1f/fmice were crossed to the inducible Mx1-Cre. In Yy1f/fMx1-Cre mice, YY1 deletion was achieved after treatment with the pI-pC. Yy1-/- mice had significantly reduced numbers of lymphoid-primed multipotent progenitor, (LMPP), common lymphoid progenitor (CLP), and double-negative (DN) T cells compared to Yy1+/+ mice. YY1 deficiency resulted in an early T cell developmental blockage at the DN1 stage. In addition, Notch1 mRNA and protein expressions were significantly reduced in Yy1-/- thymocytes compared to Yy1+/+ thymocytes. In Yy1-/- thymocytes, Notch target gene Hes1 was also downregulated. Thus, YY1 is required for early T cell development and Notch1 signaling. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that catalyze histone modifications. Our previous results demonstrated that YY1 PcG function is required for Igκ chain rearrangement in early B cell development, however, it is not required for YY1 functions in promoting HSC self-renewal and maintaining HSC quiescence. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Herein, we utilized a YY1 REPO domain mutant (YY1ΔREPO). The small 25 amino acid REPO domain is necessary and sufficient for recruiting other PcG proteins to YY1-bound chromatin sites in Drosophila. While YY1ΔREPO is competent for DNA binding, transcriptional activation, transient transcriptional repression, and interaction with transcriptional coregulators such as HDACs, it is defective in all YY1 PcG functions and unable to recruit other PcG proteins to DNA. This mutant is therefore a powerful tool for dissecting mechanisms governing YY1 PcG-dependent versus -independent functions. Bone marrow cells from Yy1f/f Mx1-Cre mice were transduced retrovirally with MigR1-FlagYY1, MigR1-FlagYY1ΔREPO or MigR1 vector and transplanted into lethally irradiated CD45.1+ mice. In addition, Mx1-Cre bone marrow cells infected with MigR1 vector were used as the wild-type control and transplanted into CD45.1+ mice. While YY1 is required for DN1 to DN2 transition, YY1 PcG function/REPO domain is not required for DN1 transition. Instead, in mice lack of YY1 PcG function/REPO domain, early T cells had increased cell apoptosis and failed to survive. Interestingly, although YY1 PcG function/REPO domain is critical for early T cell survival, it is not required for YY1 regulation of Notch1 expression. We concluded that YY1 is a critical regulator for early T cell development and Notch signaling. There is a lineage-specific requirement for the YY1 PcG function/REPO domain for early T cell development. While YY1 PcG function is required for early T cell survival, it is not required for YY1 regulation of Notch1 expression. YY1 PcG and non-PcG functions promotes T cell development by unique mechanisms of promoting cell survival and Notch1 expression respectively. Disclosures No relevant conflicts of interest to declare.

Central and local controls of monocytopoiesis influence the outcome of Leishmania infection

Leishmaniases represent a complex of tropical and subtropical diseases caused by an intracellular protozoon of the genus Leishmania. The principal cells controlling the interaction between the host and the parasite Leishmania are monocytes and macrophages, as these cells play a decisive role in establishing the pathogenesis or cure. These cells are involved in controlling the growth of Leishmania and in modulating the adaptive immune responses. The heterogeneity and extensive plasticity of monocytes allow these cells to adjust their functional phenotypes in response to the pathogen-directed immunological cues. In Leishmania-infected host, the rate of myelopoiesis is augmented by enhanced monocytic lineage commitment and proliferation of myeloid progenitor cells both in the BM and at the site of infection. These newly generated monocytes play as “safe haven” for the parasite and also as the antigen-presenting cells for T cells to cause deregulated cytokine production. This altered monocytopoiesis is characterized by tissue-specific immune responses, spatiotemporal dynamics of immunoregulation and functional heterogeneity. In the presence of Th1 cytokines, monocytes exhibit a pro-inflammatory phenotype that protects the host from Leishmania. By contrast, in an environment of Th2 cytokines, monocytes display anti-inflammatory phenotype with pro-parasitic functions. In this review, we summarize the involvement of cytokines in the regulation of monocytopoiesis and differentiation of macrophages during leishmanial infection. Understanding the role of cytokines in regulating interactions between Leishmania and the host monocytes is key to developing new therapeutic interventions against leishmaniases.

The cellular origins of cancer with particular reference to the gastrointestinal tract.

Stem cells or their closely related committed progenitor cells are the likely founder cells of most neoplasms. In the continually renewing and hierarchically organized epithelia of the oesophagus, stomach and intestine, homeostatic stem cells are located at the beginning of the cell flux, in the basal layer of the oesophagus, the isthmic region of gastric oxyntic glands and at the bottom of gastric pyloric-antral glands and colonic crypts. The introduction of mutant oncogenes such as KrasG12D or loss of Tp53 or Apc to specific cell types expressing the likes of Lgr5 and Mist1 can be readily accomplished in genetically engineered mouse models to initiate tumorigenesis. Other origins of cancer are discussed including 'reserve' stem cells that may be activated by damage or through disruption of morphogen gradients along the crypt axis. In the liver and pancreas, with little cell turnover and no obvious stem cell markers, the importance of regenerative hyperplasia associated with chronic inflammation to tumour initiation is vividly apparent, though inflammatory conditions in the renewing populations are also permissive for tumour induction. In the liver, hepatocytes, biliary epithelial cells and hepatic progenitor cells are embryologically related, and all can give rise to hepatocellular carcinoma and cholangiocarcinoma. In the exocrine pancreas, both acinar and ductal cells can give rise to pancreatic ductal adenocarcinoma (PDAC), although the preceding preneoplastic states are quite different: acinar-ductal metaplasia gives rise to pancreatic intraepithelial neoplasia culminating in PDAC, while ducts give rise to PDAC via. mucinous cell metaplasia that may have a polyclonal origin.