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<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.

Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1799-1799
Author(s):  
Ingmar Bruns ◽  
Sebastian Büst ◽  
Akos G. Czibere ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Plasma Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3640-3640
Author(s):  
Sofieke E Klamer ◽  
Paula B van Hennik ◽  
Daphne C Thijssen-Timmer ◽  
C. Ellen Van der Schoot ◽  
Carlijn Voermans
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Cell Surface Expression ◽  
Type I ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3640 Poster Board III-576 Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population. Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation. First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources. Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34+ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34+ cells to βig-h3 is mediated by both β1- and β2- integrins. The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction. In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures: No relevant conflicts of interest to declare.

Regulation of Fetal Liver Hematopoietic Stem Cell Function By the Dpy30 Subunit of Set1/Mll Complexes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5055-5055
Author(s):  
Zhenhua Yang ◽  
Hao Jiang
Keyword(s):  
Stem Cells ◽  
Cell Function ◽  
Fetal Liver ◽  
H3k4 Methylation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Term Maintenance ◽  
Time Point

Abstract While stem cells undergo phenotypic and functional changes in development, the capacity of self-renewal and differentiation remains the defining property of stem cells throughout life, indicating certain fundamental regulatory mechanisms underlying these cardinal features of stem cells. A profound transition occurs to hematopoietic stem cells (HSCs) from embryonic to adult hematopoiesis, resulting in pronounced distinctions between fetal liver (FL) and adult bone marrow (BM) HSCs in many aspects. While many studies have documented a different dependence of fetal versus adult HSC function on epigenetic modulators including several Polycomb proteins, little is known about if Trithorax proteins play a similar or different role in fetal versus adult HSC function. More specifically, despite being a prominent epigenetic mark associated with gene activation, the role of H3K4 methylation (an activity of many Trithorax proteins) in different stages of HSCs remains unclear. As the major H3K4 methylases in mammals, the Set1/Mll family complexes play important roles in development and stem cell function, and are extensively associated with diseases including blood cancers. We have previously established a direct role of Dpy30, a core subunit in all Set1/Mll complexes, in facilitating genome-wide H3K4 methylation, and this allows an effective interrogation of the functional role of efficient H3K4 methylation through genetic studies of Dpy30. While dispensable for the self-renewal of embryonic stem cells (ESCs), Dpy30 is crucial for efficient differentiation of ESCs by facilitating the induction of many bivalently marked developmental genes (Jiang et al., Cell, 2011). We have then generated a Dpy30 conditional knockout mouse, and shown that Dpy30 plays a crucial role in the long term maintenance and differentiation of adult BM HSCs, and preferentially controls H3K4 methylation and expression of many hematopoiesis-associated genes in adult BM cells (Yang et al., J Exp Med, accepted). However, the role of Dpy30 and efficient H3K4 methylation in fetal HSCs is still unknown. To study the role of efficient H3K4 methylation in fetal HSCs, we deleted Dpy30 in fetal hematopoietic cells using VavCre line. VavCre; Dpy30F/- fetuses are anemic at E14.5 and E15.5, with reduced H3K4 methylation but significantly increased numbers of FL HSCs. However, these FL HSCs were functionally defective in colony formation and blood reconstitution following transplantation. Proliferation of the progenitors, but not HSCs, was significantly (but modestly) reduced. These results suggest a role of Dpy30 in differentiation of HSCs and progenitor proliferation in FL. We also competitively transplanted Mx1Cre; Dpy30F/- FL and deleted Dpy30 after stable engraftment. Our analysis at an early time point after deletion showed little effect on donor contribution to HSCs, but significant reduction of oligopotent progenitors. Analysis at a later time point after deletion, however, showed marked reduction of all hematopoietic cells including HSCs. These results support a cell-autonomous role of Dpy30 in the differentiation and long term maintenance of FL HSCs. The phenotypes of FL HSCs are largely similar to those of BM HSCs following Dpy30 loss, suggesting that Dpy30 and certain Dpy30 targets are fundamentally important in regulating HSCs regardless of the developmental stages. To identify these targets, we performed RNA-seq analyses for purified FL HSCs from VavCre; Dpy30F/- versus VavCre; Dpy30F/+ littermates. Among hundreds of genes that were significantly changed in FL HSCs, however, only a handful of genes were found to be co-downregulated in both FL and BM HSCs following Dpy30 loss, suggesting that Dpy30 may have different functional targets in different stages of HSCs. To identify Dpy30 targets fundamentally important to HSC regulation, we are now selectively investigating the function of a few common Dpy30 targets in HSCs by colony formation and potentially transplantation assays following their stable knockdown. The similar requirement of Dpy30 in both fetal and adult HSC differentiation as well as long-term maintenance underscores the fundamental importance of this epigenetic modulator in the central properties of stem cells, and studies of the common Dpy30 targets may identify new regulatory genes controlled by this modulator in fetal and adult HSC function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Development of adult bone marrow stem cells in H-2-compatible and -incompatible mouse fetuses.

1984 ◽  
Vol 159 (3) ◽  
pp. 731-745 ◽  
Author(s):  
R A Fleischman ◽  
B Mintz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Donor Strain ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Bone marrow of normal adult mice was found, after transplacental inoculation, to contain cells still able to seed the livers of early fetuses. The recipients' own hematopoietic stem cells, with a W-mutant defect, were at a selective disadvantage. Progression of donor strain cells to the bone marrow, long-term self-renewal, and differentiation into myeloid and lymphoid derivatives was consistent with the engraftment of totipotent hematopoietic stem cells (THSC) comparable to precursors previously identified (4) in normal fetal liver. More limited stem cells, specific for the myeloid or lymphoid cell lineages, were not detected in adult bone marrow. The bone marrow THSC, however, had a generally lower capacity for self-renewal than did fetal liver THSC. They had also embarked upon irreversible changes in gene expression, including partial histocompatibility restriction. While completely allogeneic fetal liver THSC were readily accepted by fetuses, H-2 incompatibility only occasionally resulted in engraftment of adult bone marrow cells and, in these cases, was often associated with sudden death at 3-5 mo. On the other hand, H-2 compatibility, even with histocompatibility differences at other loci, was sufficient to ensure long-term success as often as with fetal liver THSC.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Tie2Cre-mediated gene ablation defines the stem-cell leukemia gene (SCL/tal1)–dependent window during hematopoietic stem-cell development

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3871-3874 ◽  
Author(s):  
Thorsten M. Schlaeger ◽  
Hanna K. A. Mikkola ◽  
Christos Gekas ◽  
Hildur B. Helgadottir ◽  
Stuart H. Orkin
Keyword(s):  
Stem Cell ◽  
Blood Cells ◽  
Fetal Liver ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Gene Ablation ◽  
Epidermal Growth ◽  
Fetal Hematopoiesis ◽  
Stem Cell Leukemia

AbstractThe stem-cell leukemia gene (SCL/tal1) is essential for the formation of all blood lineages. SCL is first expressed in mesodermal cells that give rise to embryonic blood cells, and continues to be expressed in fetal and adult hematopoietic stem cells (HSCs). However, SCL is not required for the maintenance of established long-term repopulating (LTR) HSCs in the adult. The time point at which HSC development becomes SCL independent has not been defined. Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains–2 (Tie2) expression appears in hemogenic and vasculogenic sites shortly after SCL. We therefore used the Tie2Cre mouse to inactivate SCL early during embryonic and fetal hematopoiesis. Tie2Cre completely inactivated SCL in yolk sac, the aortagonad-mesonephros (AGM) region, and fetal liver hematopoietic cells and circulating blood cells. However, the fetal liver was colonized by functional LTR-HSCs. Yet SCL remained crucial for proper differentiation of both primitive and definitive red cells and megakaryocytes. These results indicate that the SCL-dependent phase of HSC development ends before Tie2Cre-mediated gene ablation becomes effective.

scholarly journals Single Cell-Resolution Analysis of HSC Dysfunction in Mir-146a knockout Mice

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 714-714
Author(s):  
Jennifer Grants ◽  
Joanna Wegrzyn ◽  
David Knapp ◽  
Tony Hui ◽  
Kieran O'Neill ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Transcriptome Profiling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
Resolution Analysis ◽  
Signaling Activation

Abstract MicroRNA miR-146a is frequently depleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Loss of miR-146a may be an initiating event in tumorigenesis, as miR-146a loss in mouse models is sufficient to cause features of MDS and eventual progression to AML. To define how miR-146a loss initiates tumorigenesis, we analyzed hematopoietic stem cell (HSC) function from miR-146a knockout (KO) mice prior to onset of an overt malignant phenotype. Tracking cell division kinetics, proliferation, and differentiation of single long-term HSC (LT-HSC; EPCR+CD45+CD48-CD150+) in culture, we found evidence that miR-146a KOreduces HSC quiescence and promotes differentiating cell divisions. Our data show that miR-146a KO HSC dysfunction may stem from loss of a CD150-bright EPCR-bright sub-population, which has previously been associated with robust HSC activity. In line with this, single cell DNA methylation profiling revealed a reduction in a primitive sub-population of LT-HSCs in miR-146a KO animals. In addition, single cell LT-HSC transplants revealed a myeloid repopulation bias. As reduced HSC cell cycle quiescence has been linked to impaired HSC self-renewal upon hematopoietic stress, such as serial transplantation, we assessed the frequency of serially transplantable HSCs by performing secondary transplants with limiting dilution. Serially transplantable HSC frequency was reduced in miR-146a KO compared to wild type, suggesting impaired HSC self-renewal. Transcriptome profiling of miR-146a KO hematopoietic stem and progenitor cells identified tumor necrosis factor (TNF) signaling activation as a potential driver of HSC dysfunction. LT-HSC cell cycle quiescence and the CD150-bright EPCR-bright LT-HSC sub-population were restored in miR-146a/TNF double KO mice, suggesting that aberrant TNF signaling activation drives HSC dysfunction upon loss of miR-146a. Gene expression levels in the TNF signaling network are inversely correlated with miR-146a levels in human AML, implying that TNF signaling may similarly disrupt HSC function in miR-146a- depleted myeloid malignancies. Overall, our findings suggest that miR-146a promotes HSC cell cycle quiescence and inhibits differentiation by antagonizing TNF signaling, in order to maintain a primitive sub-population of long-term self-renewing HSCs. Disclosures Eaves: Experimental Hematology: Other: Editor of journal; StemCell Technologies Inc: Other: Wife of owner.

scholarly journals Effect of Cell Cycle on Cell Surface Expression of Voltage-Gated Sodium Channels and Na+,K+-ATPase

2021 ◽  
Author(s):  
Samantha I. Edenfield ◽  
III Harry J. Gould ◽  
Dennis Paul
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Sodium Channels ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Dividing Cells ◽  
Immortalized Cell ◽  
Dapi Fluorescence

Abstract Voltage-gated sodium channels (VGSCs) are the target for many therapies. Variation in membrane potential occurs throughout the cell cycle, yet little attention has been devoted to VGSCs and Na+,K+-ATPase in the cell cycle. We hypothesized that in addition to doubling DNA and cell membrane in anticipation of cell division, there should be a doubling of VGSCs and Na+,K+-ATPase compared to non-dividing cells. We tested this hypothesis in eight immortalized cell lines by correlating immunocytofluorescent labeling of VGSCs or Na+,K+-ATPase, with propidium iodide or DAPI fluorescence using flow cytometry. Cell surface expression of VGSCs during phases S through M was double that seen during phases G0 - G1. By contrast, Na+,K+-ATPase expression increased only 1.5-fold. The increases were independent of baseline expression of channels or pumps. The variation in VGSC and Na+,K+-ATPase expression has implications for both our understanding of sodium's role in controlling the cell cycle and variability of treatments targeted at these components of the Na+ handling system.

Tet2 Uniquely Regulates Self-Renewal and Long Term Repopulating Capacity of Fetal Liver and Bone Marrow Hematopoietic Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4739-4739
Author(s):  
Hiroyoshi Kunimoto ◽  
Yumi Fukuchi ◽  
Masatoshi Sakurai ◽  
Daichi Abe ◽  
Ken Sadahira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Fetal Liver ◽  
Hematologic Malignancies ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Serial Transplantation

Abstract Abstract 4739 Ten-eleven-translocation 2 (TET2) gene is one of the frequent targets of mutation in various hematologic malignancies. These observations suggest critical roles of TET2 dysfunction in their molecular pathogenesis. To investigate physiological roles of TET2 in hematopoiesis, we previously analyzed fetal liver (FL) hematopoiesis of Tet2 gene-trap (Tet2gt) mice and showed that Tet2gt/gt FL cells displayed enhanced self-renewal and long term repopulating (LTR) capacity with expansion of Lineage(−)Sca-1(+)c-Kit(+) (LSK) and common myeloid progenitor (CMP) fractions. However, there remain several questions unanswered. First, self-renewal capacity was examined only by using bulk FL cells and therefore effects of Tet2 loss on purified cell populations such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) remain elusive. Second, because other groups have reported myeloid transformation in Tet2 conditional knockout mice, it is possible that Tet2 loss confers self-renewal capacity to non-self-renewing myeloid progenitors such as CMPs. Third, effects of Tet2 haploinsufficiency on adult hematopoiesis was not examined using purified HSCs or HPCs. To address these issues, we analyzed E14.5 FL and adult bone marrow (BM) cells from Tet2gt mice. We first performed serial replating assay of FL-LSK cells in methylcellulose containing interleukin (IL)-3, IL-6, stem cell factor (SCF) and erythropoietin (Epo). In this assay, Tet2gt/gt FL-LSK cells showed significantly higher replating capacity as compared to that of WT cells. Interestingly, Tet2gt/gt FL-LSK cells formed various types of colonies including granulocyte-macrophage (GM) and erythrocyte-megakaryocyte (EM) colonies, whereas WT FL-LSK cells generated only GM colonies at the second time of replating, showing that multipotent differentiation capacity was maintained in Tet2gt/gt cells even in the presence of lineage-acting cytokines. Next we examined the self-renewal capacity of highly purified FL-HSCs (CD34+LSK or CD150+LSK cells) by competitive repopulation assay. As expected, the recipients of Tet2gt/gt CD34+LSK cells showed significantly higher donor chimerism in peripheral blood as compared to those receiving WT cells. Furthermore, CD150+LSK cells from Tet2+/gt and Tet2gt/gt FLs demonstrated higher peripheral blood repopulation in the secondary and tertiary recipient mice as compared to that of WT recipients in serial transplantation assay. These results indicate that the enhanced self-renewal and LTR capacity of Tet2-mutant FL cells was uniquely associated with highly purified HSCs. This conclusion also applied to the BM LSK cells from adult mice, since Tet2+/gt BM LSK cells also showed significantly higher peripheral blood contribution compared to the WT cells in serial transplantation assays. This result demonstrates that Tet2 haploinsufficiency is sufficient to confer the enhanced self-renewal and LTR capacity to HSCs in adult hematopoiesis. Lastly, we examined self-renewal capacity of FL CMPs by serial replating assay. Interestingly, Tet2gt/gt FL CMP cells displayed increased replating capacity as compared to WT cells. However, in vivo repopulation assay using Tet2+/+, Tet2+/gt, and Tet2gt/gt FL CMP cells showed no significant difference in peripheral blood chimerism among these recipients. Taken together, enhanced self-renewal and LTR capacity by Tet2 ablation is uniquely associated with HSCs in FL and adult BM, but not with myeloid progenitors, indicating that Tet2 regulates self-renewal program intrinsic to HSCs. In addition, Tet2 haploinsufficiency is sufficient to enhance self-renewal and LTR capacity of HSCs, which explains pathological relation between high incidence of heterozygous TET2 mutations and hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

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