scholarly journals Fetal Erythropoiesis Is Defective in Rpl11 Heterozygous Mice and Increases in Severity in Young Animals

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 872-872
Author(s):  
Dolly Thomas ◽  
Cailin Joyce ◽  
Robert A. Redd ◽  
Bertil Glader ◽  
Steven Ellis ◽  
...  
Keyword(s):  
Mouse Model ◽  
Single Cell ◽  
Functional Impairment ◽  
Molecular Level ◽  
Fetal Liver ◽  
Severe Anemia ◽  
Self Renewal ◽  
Adult Mice ◽  
Single Cell Rna Sequencing ◽  
Fetal Hematopoiesis

Abstract Diamond Blackfan Anemia (DBA) is a congenital red blood cell aplasia usually caused by haploinsufficiency of selected ribosomal protein genes. Approximately 10% of DBA patients are anemic at birth, and 80% of DBA patients present by 6 months. These and other data suggest that the DBA erythroid defect is exacerbated by the switch from fetal to adult hematopoiesis. We hypothesize that unique molecular or cellular features initiate DBA pathogenesis during fetal hematopoiesis that manifests after the switch to adult erythropoiesis as severe anemia. To test this hypothesis, we developed a mouse model with constitutive Rpl11 haploinsufficiency in fetal and adult hematopoietic tissues driven by VavCre. Rpl11 is a commonly mutated ribosomal gene in DBA. In adult mice, reduced Rpl11 expression results in severe anemia by 6 weeks, with markedly reduced hemoglobin, increased red cell volume and elevated erythroid adenosine deaminase. Flow cytometric analysis of the bone marrow (BM) revealed no change in the frequency of megakaryocyte-erythroid progenitors (MEP) but a significant increase in both BFU-E and CFU-E, with a corresponding decrease in maturing Ter119+ cells. Functional progenitor assays showed reduced and delayed proliferation with an impaired ability to produce viable CD71+ Ter119+ cells. Treatment of purified Rpl11+/- BFU-E with dexamethasone rescued this proliferative defect. Thus, our data indicate that a differentiation block and functional impairment at the BFU-E stage of adult erythropoiesis contribute to Rpl11 haploinsufficient anemia. At the molecular level, Rpl11 haploinsufficiency in BM resulted in the accumulation of 32S and 12S pre-rRNA. Polysome profiling of total BM suggested reduced polysome assembly and impaired translation. Transcriptional profiling of normal and Rpl11+/- MEP, BFU-E, CFU-E and Ter119+ cells identified 13 differentially expressed genes across DBA erythropoiesis including known and novel targets such as CDKN1A, TNFSF4 and TSPAN14 . Enrichment of the p53 pathway was unique to BFU-E and CFU-E progenitors. Interestingly, TGFB1 and EGF pathways were also selectively affected within Rpl11+/- BFU-E and CFU-E progenitors, suggesting p53-independent self-renewal and proliferative defects in these populations. In contrast to adult hematopoiesis, fetal hematopoiesis defects are less severe. For example, fetal BFU-E are not significantly increased within E14 fetal livers and functionally generate a similar number of maturing erythroblasts in vitro . Lodish and colleagues have established that fetal liver BFU-E comprise at least two distinct subpopulations, termed "early" and "late". Early BFU-E have high self-renewing capacity whereas late BFU-E have low self-renewing capacity. To determine whether the balance between early and late BFU-E is affected by Rpl11 haploinsufficiency, and to identify gene expression changes in early and late fetal Rpl11+/- BFU-E, we performed single cell RNA-sequencing. Normal fetal livers contained similar proportions of early and late BFU-E, whereas normal adult bone marrows only contained late BFU-E. Surprisingly, Rpl11+/- fetal livers primarily contained late BFU-E. One explanation for this observation is that early BFU-E are depleted from Rpl11+/- fetal livers as this population compensates for reduced function of non-renewing late BFU-E. Interestingly, late BFU-E from Rpl11+/- BM have a stronger "late" signature than their normal counterparts suggesting a reduced self-renewing capacity of BM BFU-E in DBA. We are currently testing this hypothesis by overexpressing early genes associated with BFU-E self-renewal in Rpl11+/- BFU-E and reassessing erythroid phenotypes. In conclusion, our studies show that erythropoiesis is defective in Rpl11+/- fetal liver at the cellular and molecular level, although functional impairment is not as marked as in adult mice. Moreover, single cell RNA-sequencing of BM and fetal BFU-E in our Rpl11+/- mouse model has uncovered a novel hypothesis for DBA pathogenesis that the loss of early, self-renewing BFU-E in fetal liver is a harbinger of adult erythroid failure. Disclosures Glader: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Neuberg: Synta Pharmaceuticals: Other: Stock shares.

scholarly journals Single-Cell RNA Sequencing Reveals Novel Populations of Fibroblasts and Transcriptomic Changes Within Abdominal Skeletal Muscle in a Mouse Model of Aromatase-Induced Inguinal Hernia

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A816-A816
Author(s):  
Matthew Joseph Taylor ◽  
Tanvi Potluri ◽  
Hong Zhao ◽  
Serdar Ekrem Bulun
Keyword(s):  
Inguinal Hernia ◽  
Mouse Model ◽  
Single Cell ◽  
Signaling Pathways ◽  
Rna Sequencing ◽  
Marker Gene ◽  
Pathological State ◽  
Suspension Cells ◽  
Estrogen Exposure ◽  
Single Cell Rna Sequencing

Abstract Introduction: Inguinal hernia is a highly prevalent condition in men, of which the only currently available treatment is invasive surgical repair. An inguinal hernia often results from a protrusion of the intra-abdominal contents through a weakened region of the lower abdominal wall, but the etiology is unknown. One potential cause is aging-related steroid hormonal changes, which coincide with an increased incidence of hernia in aged men. Our group previously developed the first mouse model of inguinal hernia (AromHum) that is generated via the humanized expression of the enzyme aromatase, which converts androgens to estrogens. In the lower abdominal muscle (LAM), an aromatase-mediated increase in tissue estrogen causes fibroblast proliferation, fibrosis, and myocyte atrophy, resulting in hernias. However, the molecular mechanism of this phenotype remains unclear. In this study, we aimed to find genome-wide transcriptomic differences in AromHum compared to WT mice at a single-cell resolution. We hypothesized that in relation to WT mice, AromHum mice would have distinct fibroblast signatures that arise from the increased estrogen exposure to LAM tissue. Methods: LAM was harvested from 9-10-week-old male WT and AromHum mice (n=3 each) and digested into a single-cell suspension. Cells were processed via the 10X Genomics Chromium platform for single-cell RNA sequencing. The 6 samples combined yielded a total of ~63,000 cells. Data was analyzed using Cell Ranger v3, Seurat v3, Slingshot, and PROGENy R packages. Results: UMAP visualization of WT and AromHum LAM tissue revealed 22 cell clusters, which we grouped into 10 broad cell types through known marker gene expression. AromHum LAM contained a significantly higher proportion of fibroblasts than WT (44% vs. 27% of total analyzed cells), and AromHum fibroblasts expressed more pro-fibrotic genes, such as Timp1, Spon2, and Postn. In AromHum and WT combined, we found 6 clusters of fibroblast-like cells. Two of these clusters (clusters 2 and 3) were heavily represented by cells derived from AromHum mice (85-90% of cells in each cluster), which we termed “hernia-associated fibroblasts” (HAFs). Cluster 3 HAFs expressed high levels of Esr1 (gene encoding ERα), as well as estrogen-responsive genes such as Pgr and Greb1, and was enriched for estrogen, hypoxia, and TGFβ signaling pathways. Cluster 2 HAFs expressed genes associated with a pathological state, such as Lbp, Cthrc1, Mmp3, and Il33, and was enriched for the NF-κB and TNF-α signaling pathways. Conclusions: We found that LAM fibrosis in AromHum may result from the expansion of two distinct populations of HAFs - one is estrogen-responsive, and another is pathologic. Further in vitro / in vivo experiments are required to determine the relative contributions of these sub-populations of HAFs to fibrosis and inguinal hernias, leading to developing novel intervention strategies.

Abstract B34: Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature.

2015 ◽  
Author(s):  
Zohar Sachs ◽  
Rebecca S. LaRue ◽  
Klara Noble ◽  
Susan K. Rathe ◽  
Aaron L. Sarver ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Self Renewal ◽  
Single Cell Rna Sequencing

scholarly journals Integrated Single Cell Transcriptomics Defines an Engineered Niche Supporting Hematopoietic Stem Cell Development Ex Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3699-3699
Author(s):  
Brandon Hadland ◽  
Barbara Varnum-Finney ◽  
Stacey Dozono ◽  
Tessa Dignum ◽  
Cynthia Nourigat-Mckay ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Research Funding ◽  
Signal Pathways ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Cell Rna Sequencing ◽  
Equity Ownership

During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Single-Cell Analysis Reveals Molecular Mechanisms of NRAS-Mediated Leukemia Stem Cell Self-Renewal in a Murine Model of AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3060-3060
Author(s):  
Rebecca S. Larue ◽  
Klara E Noble ◽  
Conner Hansen ◽  
Ngoc Ha ◽  
David A Largaespada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Mass Cytometry ◽  
Self Renewal ◽  
Single Cell Rna Sequencing ◽  
Colony Forming Capacity ◽  
Cell Data ◽  
Group 1

Abstract Acute myeloid leukemia (AML) is a lethal malignancy because patients who initially respond to chemotherapy eventually relapse with treatment-resistant disease. Leukemia stem cells (LSCs) reestablish the disease by self-renewal: the ability of a stem cell to reproduce itself and give rise to progeny. LSC self-renewal is therefore critical to relapse. Most anticancer therapies are designed to inhibit proliferation. Yet, the mechanisms that direct hematopoietic stem cell (HSC) proliferation are distinct from the mechanisms that allow HSCs to self-renew (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to eradicate this disease. To study leukemia self-renewal, we use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). Doxycycline abolishes NRASG12V expression leading to leukemia remission. We demonstrated that expression of NRASG12V is required for self-renewal in this AML model and that NRASG12V -mediated signaling is distinct among leukemic subsets (Sachs et al. Blood 2014). We hypothesize that NRAS-activated pathways required for LSC self-renewal are limited to a subpopulation of cells with the LSC immunophenotype. Defining the mechanisms of self-renewal has been a challenge because cancer cells are highly heterogeneous and because disengaging proliferation from self-renewal can be difficult experimentally. To overcome these obstacles, we use single-cell technologies (single-cell, whole transcriptome, RNA sequencing and mass cytometry, CyTOF) to define the signaling and transcriptional profiles of individual cells. We performed single-cell RNA sequencing on unsorted leukemia cells and on a sorted, LSC-enriched population. The single-cell transcriptional profile of LSCs was distinct from the bulk population (Fig. 1A). The 100 most differentially expressed genes between these groups are involved in hematopoietic cell fate and differentiation, confirming the biological validity of this technique. Next, we sought to identify an NRASG12V -mediated self-renewing subpopulation among the LSCs. Unsupervised, two-dimensional, hierarchical clustering of LSC single-cell data identified three discrete subpopulations among the LSCs, each expressing a unique gene expression profile (Fig. 1B). Comparing the single-cell transcriptional profiles of NRASG12V -expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of the three LSC-expression profiles seen in RAS-on cells (Groups 1 and 3, Fig. 1B) are lost when NRASG12V is withdrawn (Fig. 1C). These data suggest that these two profiles (Groups 1 and 3) are NRASG12V -dependent, consistent with an earlier report that activated NRAS exerts bimodal effects on HSCs (Li et al., Nature 2013). Gene set enrichment analysis of these profiles, modified for single-cell data, revealed that Group 1 preferentially expresses genes associated with leukemia self-renewal. On the basis of this gene expression data, we identfied cell surface markers (CD36 and CD69) that delineate the two NRASG12V -responsive LSC-subpopulations (Groups 1 and 3). We sorted LSCs based on CD36 and CD69 status and found that CD36- CD69+ LSCs (consistent with Group 1 gene expression) harbor nearly all of the colony-forming capacity of the LSCs, forming an average of 13 colonies versus 0.33 colonies for CD36+CD69- LSCs (Group 3) and versus 0.11 colonies for non-LSCs (per 10,000 cells plated, p < 0.00001 for each comparison). We have previously shown that colony-forming capacity is an accurate surrogate for in vivo leukemia reconstituting ability and self-renewal in our model (Sachs, et al. Blood 2014). These experiments characterize the NRASG12V -mediated self-renewal transcriptional signature and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. Using mass cytometry to query the activation status of signaling pathways simulteneously with multiple immunophenotypic markers, we show that Ki67Low LSCs (the putative self-renewing LSCs) preferentially express increased levels of b-catenin and Myc. These data implicating AML self-renewal pathways can provide precise molecular targets for treating this deadly disease. Disclosures Largaespada: NeoClone Biotechnology, Inc.: Consultancy, Other: stockholder; Genentech Inc: Honoraria, Research Funding; Discovery Genomics Inc.: Consultancy, Other: stockholder; B-MoGen Biotechnologies Inc.: Consultancy, Other: stockholder; Orbimed Inc: Consultancy.

scholarly journals Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2852-2858 ◽  
Author(s):  
R Pawliuk ◽  
C Eaves ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fetal Liver ◽  
Complete Recovery ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Mice

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

scholarly journals YY1 Promotes SCF/c-Kit Signaling and HSC Self-Renewal in Fetal Hematopoiesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-27
Author(s):  
Deependra Kumar Singh ◽  
Zhanping Lu ◽  
Shuai Jia ◽  
Xiaona You ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Fetal Liver ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Phosphorylated Akt ◽  
Fetal Hematopoiesis

Fetal hematopoietic stem cells (HSCs) exhibit markedly different properties as compared to adult HSCs including cell surface marker expression, proliferation state and repopulation capacity. Changes of HSC activity in postnatal mice is defined by a process of decreasing in cell cycle and entering into quiescence. Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group (PcG) Protein with important functions to regulate embryonic development, lineage differentiation and cell proliferation. While homozygote deletion of YY1 in mice results in lethality at the peri-implantation stage, heterozygote deletion of YY1 causes severe developmental defects. By conditionally deleting YY1 in adult hematopoietic system, our previous study showed that YY1 is an essential regulator for adult hematopoiesis by promoting HSC long-term self-renewal and maintaining adult HSC quiescence. In contrast to adult HSCs, in which quiescence is a fundamental characteristic, over 95% of fetal HSCs are in an active cell cycle to rapidly generate homeostatic levels of blood cells for oxygen transport and immune system development in the growing organism. Herein, we assessed whether YY1 was also required for maintaining fetal HSC pool and regulating fetal HSC functions, and what was the underlying mechanism by which YY1 regulated fetal HSCs. To test how loss-of-function of YY1 impacted fetal hematopoiesis, Yy1f/f mice in which the Yy1 promoter region and exon 1 are flanked by loxP sites, were crossed to Vav-Cre mice to generate heterozygous Yy1f/+ Vav-cre mice. The Vav promoter drives Cre recombinase expression specifically in fetal liver hematopoietic cell starting at day E11.5. Yy1f/+ Vav-cre mice were then subsequently bred with Yy1f/f mice to generate homozygous Yy1f/f Vav-cre mice. Among 141 pups resulting from breeding Yy1f/fto Yy1f/+ Vav-Cre, only 7 were Yy1f/f Vav-Cre (n=7) and was significantly lower than the estimated number (n=35) according to the Mendelian ratio (P&lt;0.05). All Yy1f/f Vav-Cre pups died within 72 hours after birth, which supported essential role of YY1 in fetal hematopoiesis and survival. At E14.5 of fetal development, Yy1f/f Vav-Cre fetuses had reduced numbers of hematopoietic stem and progenitor cells in the liver. In addition, YY1 deficient fetal HSCs failed to self-renew in primary and secondary bone marrow transplantation assays. Colony formation assay showed that fetal liver cells from Yy1f/f Vav-Cre mice failed to form CFU-GEMM, CFU-GU and BFU-E compared to Vav-Cre control. While YY1 promotes SCF/c-Kit signaling in adult HSCs, it does not impact c-Kit cell surface expression in early T cell progenitors (unpublished data). To assess YY1 impact on SCF/c-Kit axle in fetal HSCs, c-Kit transcript level, c-Kit median fluorescence intensity and phosphorylated AKT were measured. Similar as its function in adult HSCs, YY1 deficient fetal HSCs had decreased Kit transcript expression, decreased c-Kit cell surface expression and decreased SCF/c-Kit signaling. Our results supported that YY1 is required for maintaining a continuous pool of HSCs in fetal liver and is critical for fetal HSC long-term self-renewal and differentiation. Similar as its function in adult HSCs, YY1 promotes SCF/c-Kit signaling in fetal HSCs. Disclosures No relevant conflicts of interest to declare.

New Candidate Regulators of Hematopoietic Stem Cells Identified from Transcriptional Comparisons of Highly Purified Populations of Longterm Repopulating Cells with Markedly Different in Vivo Self-Renewal Activities

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2460-2460
Author(s):  
David G Kent ◽  
Michael R Copley ◽  
Claudia Benz ◽  
Brad J Dykstra ◽  
Elaine Ma ◽  
...  
Keyword(s):  
Single Cell ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Culture Conditions ◽  
Molecular Signature ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Transplants

Abstract Significant advances have been made in the development of methods for purifying murine hematopoietic cells with longterm (&gt;4 months) in vivo reconstituting ability although these longterm repopulating cells (LTRCs) remain heterogeneous with regard to the self-renewal (SR) activity they display when transplanted into irradiated hosts. Furthermore our group has also identified cell culture conditions that differentially alter LTRC activity without immediate effects on their proliferation or survival. Here, we show that highly purified LTRCs with high and low SR properties can be prospectively isolated from normal adult mouse bone marrow (ABM) as 2 separate populations according to their expression of CD150 within the EPCR++CD48−CD45mid fraction of cells: 56% total LTRCs and 43% of the high SR type in the CD150+ subset vs. 39% total LTRCs and 32% of the low SR type in the CD150− subset (as determined from 62 and 28 single cell transplants, respectively). As a first test of whether these populations would likely be useful to search for new molecular differences associated with their different SR properties, we compared the level of expression in these 2 populations of a small set of genes previously reported to regulate LTRC SR activity: c-Kit, Bmi1, Gata3, Rae28, Ezh2 and Lnk by quantitative real-time PCR (Q-RT-PCR). This exercise revealed transcript levels of the first 4 of these genes to be significantly higher in the CD150+ subset that is selectively enriched in high SR LTRCs, thus validating the concept that they have a distinct molecular signature. Previous evidence shows that high SR LTRCs are present in both FL LTRCs and ABM LTRCs but they differ in some properties (i.e.: cell cycle status, regeneration kinetics). We therefore began a search for ontogeny-independent components of the SR machinery by comparing tags present in 2 LongSAGE libraries produced from CD45midlin−Rho−SP ABM cells and from lin−Sca1+CD43+Mac1+ embryonic day 14.5 fetal liver (FL) cells (each 20–30% total LTRCs and 12–20% of the high SR type, as determined by 132 (FL) and 352 (ABM) single cell transplants, respectively). From these comparisons and additional data in other publicly available datasets for primitive murine hematopoietic cells, we identified 28 genes not previously shown to have a functional role in LTRC SR control. We then compared the level of expression of these 28 genes between the CD150+ subsets of EPCR++CD48−CD45mid ABM cells and FL cells (24% total LTRCs and 12% high SR LTRCs in the FL subset) and their respective downstream lin− progeny. This comparison revealed 10 of these genes to be down-regulated in the lin− populations of both ABM and FL. Further comparison of the expression of these 10 genes between the high vs. low SR LTRCs (found in the CD150+ and CD150− subsets of EPCR++CD48−CD45mid) ABM cells showed the expression of 5 (Vwf, Rhob, Pld3, Prnp and Smarcc2) to be downregulated in the CD150− (low SR LTRC) subset. Interestingly, the first 4 of these genes, as well as 2 of the preliminary set of SR regulators (Bmi1 and Gata3), were also selectively down-regulated in EPCR++CD150+CD48−CD45mid ABM cells that had been incubated for 16 hours in 1 or 10 ng/ml Steel factor + 20 ng/ml IL-11 (conditions that decrease LTRC activity in vivo 4–5-fold before any of these divide or die). Taken together, these results point to the existence of more, although a rather small number of additional genes, including Vwf, Rhob, Pld3, and Prnp, whose products may be involved in controlling the SR potential of normal mouse LTRCs.

Early Appearance and Rapid, Selective Expansion of Lymphoid-Deficient Fetal HSCs Associated with Their Preferential Localization in the Marrow

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1271-1271
Author(s):  
Claudia Benz ◽  
Michael R. Copley ◽  
David G. Kent ◽  
Stefan Wohrer ◽  
Connie J. Eaves
Keyword(s):  
Young Adult ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Cell Transplants ◽  
Apoptotic Activity

Abstract Abstract 1271 Recent tracking of the primitive and mature progeny of more than 500 single cell transplants of highly purified hematopoietic stem cells (HSCs) in cell suspensions isolated from the bone marrow of young adult mice (8–12 weeks old) has shown that this pool is intrinsically heterogeneous. This heterogeneity is manifested as differences both in the durability (beyond 6 months) of the self-renewal activity of the HSCs detected in irradiated recipients as well as in the specific lineage contributions maintained in secondary and tertiary recipients of cells generated within each clone. Interestingly, durable HSC self-renewal activity (serial transplantability) was found to be exclusively associated with a robust and sustained ability to produce myeloid cells, regardless of their ability to produce lymphoid progeny at any stage of lymphoid progenitor development. Thus 2 subtypes of HSCs with durable self-renewal activity could be distinguished depending on whether the sustained myelopoietic activity was accompanied by an equivalent robust lymphoid differentiation activity (β-HSCs), or not (α-HSCs). Preliminary examination of fetal liver HSCs with durable self-renewal activity showed that this compartment is dominated by β-HSCs in contrast to the marrow of young adult mice where ∼30% of this HSC pool are α-HSCs. Since previous studies have documented a switch between 3 and 4 weeks after birth in several key properties of HSCs (indicative of an abrupt change from a fetal to an adult HSC program), we were interested in the question of whether the appearance of α-HSCs might be another aspect of this switch or, alternatively, whether the onset of hematopoiesis in locations other than the fetal liver (e.g., the marrow and spleen) might be a contributing factor. Accordingly, we initiated experiments to examine the proportional representation and absolute number of cells with an E-SLAM phenotype (CD45+EPCR+CD48−CD150+) and their functional activity (4-month repopulation of irradiated mice) specific for HSCs in tissues where hematopoiesis is seen during different stages of development between E14.5 and 4 weeks after birth. The number of E-SLAM cells increased ∼15 fold in the fetal liver between E14.5 and E18.5 by which time they were also present in the marrow and spleen but altogether comprising only ∼5% of the fetal liver compartment (assuming the femurs, tibiae and pelvis represent ∼30% of the total marrow). Analysis of the clonal outputs obtained in vivo from single E-SLAM cell transplants from each of these sources showed that α-HSCs are already present as a rare subet of all HSCs in the E14.5 liver and amplify equally with β-HSCs maintaining a ratio of 1:10 α-HSCs to β-HSCs in this organ. Interestingly, despite the small numbers of E-SLAM cells in the E18.5 marrow, these contained readily detectable HSCs in transplant assays and showed a ratio of α-HSCs to β-HSCs of 1:2. This ratio was mirrored in the marrow of adult mice and was also similar to that seen in the marrow and spleen of 3 and 4 week-old mice. These findings show that α-HSCs, by comparison to β-HSCs, have a greater ability to localize in the marrow which appears to then selectively promote the expansion of α-HSCs. These results also show that the initial appearance and early selective expansion of α-HSCs during fetal and early post-natal life is unlikely to be regulated by the same mechanisms that impose an adult program on murine HSCs between 3 and 4 weeks after birth. Rather these results favor a separate, intrinsically determined developmentally controlled mechanism that endows α-HSCs with a higher self-renewal probability, a more rapid turnover, a lower apoptotic activity, or a combination of these differences as compared to β-HSCs. This would explain the initial rapid and marked selective expansion of α-HSCs that occurs during the late fetal and early neonatal period of high HSC proliferation which extends to 3 weeks after birth and is then followed by a much slower rate of selective expansion of α-HSCs during adulthood when the turnover of HSCs is minimal. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunoproteasome Inhibition to Target AML with Activated RAS Pathways

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 577-577
Author(s):  
Connor Navis ◽  
Klara Noble-Orcutt ◽  
Rebecca S LaRue ◽  
Conner Hansen ◽  
Ngoc Ha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Murine Model ◽  
The Self ◽  
Cell Level ◽  
Self Renewal ◽  
Single Cell Rna Sequencing ◽  
Cd69 Expression

Abstract Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the only leukemia cells with self-renewal potential and ability to recapitulate the disease. Our goal is to develop therapeutics that target and eradicate AML LSCs. We showed that activated NRAS facilitates self-renewal in a murine model of AML; mTORC1 activation by NRAS is important for this effect (Sachs et al. Blood 2014). RAS activates mTORC1 through the PI3K/AKT/mTOR pathway which is highly upregulated in AML (Xu et al. Blood 2003, Altman et al. Oncogtarget 2011). Recently, we found that RAS-activated malignancies, including AML, induce immunoproteasome formation via mTORC1 (Yun et al. Mol Cell 2016). Pan-proteasome inhibitors, such as bortezomib and carfilzomib, are widely used in the treatment of lymphoid malignancies and have some activity in AML. We hypothesize that specific inhibition of the immunoproteasome may target NRAS-mediated self-renewal in LSCs. We use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). We investigated the possible role of the immunoproteasome in our murine model and found that abolishing NRASG12V transgene expression led to a loss of immunoproteasome component genes expression (p<0.001). We also found that the LSC-enriched subpopulation preferentially upregulated expression of immunoproteasome component genes (p<0.002) in comparison to non-stem cells from this model. Accordingly, we found that primary human AML CD34+ precursors upregulated immunoproteasomal components relative to CD34- AML cells by up to 3.7 fold. mTOR inhibition with everolimus diminished expression of immunoproteasome component genes (p=0.05) suggesting that immunoproteasome activity may be NRASG12V/mTOR-dependent in this model as well. Next, we sought to identify the self-renewing cells among immunophenotypically defined LSC-enriched subgroup in this model. We performed single-cell RNA sequencing on the LSC-enriched population from this model. We identified three discrete trasncriptional profiles among the LSCs. Comparing the single-cell transcriptional profiles of NRASG12V-expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of these profiles are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted LSCs based on CD36 and CD69 expression as well as established immunophenotypic markers, Mac1, Kit, Sca-1 (LSC-enriched supopulation in this model is Mac1LowKit+Sca1+). These sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that CD36-CD69+Mac1LowKit+Sca1+ cells (consistent with Profile 1 gene expression) rapidly transferred leukemia with high penetrance. In contrast, CD36+CD69- Mac1LowKit+Sca1+ cells (consistent with Profile 2 gene expresison) did not transfer leukemia to mice (p < 0.004). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature at the singel-cell level and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. We performed single-cell RNA sequencing on CD34+ human AML precursors obtained from a diagnostic bone marrow specimen. Analogous to our murine model, we found that these human AML cells express 2 distinct single-cell transcriptional profiles and that differentially express RAS-activated gene expression profiles. Finally, we found that, PR957, a specific immunoproteasome inhibitor, inhibits in vitro colony formation more effectively than carfilzomib (a second generation pan-proteasome inhibitor) in our murine model of AML suggesting that immunoproteasome inhibition may be specifically target self-renewal in AML. In these experiments, we use single-cell RNA sequencing to identify the self-renewing subset of RAS-driven LSCs at the single-cell level and implicate immunoproteasome inhibition as an approach to target RAS-mediated self-renewal. Disclosures No relevant conflicts of interest to declare.

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