Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes

ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish. Disclosures No relevant conflicts of interest to declare.

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SCL/TAL1 Regulates Erythropoietin Receptor Expression.

Blood ◽

10.1182/blood.v108.11.1195.1195 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1195-1195

Author(s):

Heather M. Rogers ◽

Xiaobing Yu ◽

Constance Tom Noguchi

Keyword(s):

Erythroid Differentiation ◽

Erythropoietin Receptor ◽

Receptor Expression ◽

Binding Motif ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Mild Anemia ◽

Adult Mice ◽

High Level

Abstract The basic-helix-loop-helix transcription factor SCL/TAL1, is required for erythropoiesis during development, and conditional deletion in adult hematopoiesis results in hematopoietic stem cells with a competitive repopulation disadvantage and defective erythropoiesis in vitro. However, adult mice with a conditional SCL/TAL1 deletion survive with mild anemia, suggesting defective erythroid proliferation and indicating that SCL/TAL1 is important, but not essential in mature red blood cell production. We find that during erythroid differentiation of primary human hematopoietic CD34+ cells, SCL/TAL1 expression peaks at day 8–10 following erythropoietin (EPO) stimulation, concomitant with peak expression of GATA-1 and EKLF. Treatment with SCL/TAL1 antisense oligonucleotides during erythroid differentiation markedly decreases erythroid differentiation as indicated by decreased expression of GATA-1 and both b- and g-globin expression, along with the absence of the characteristic decrease in GATA-2. Microarray analysis of erythroid cells overexpressing SCL/TAL1 indicate increased gene expression for b- and g-globin, and other genes related to erythropoiesis including EPO receptor (EPO-R), and these results are confirmed in stable cell lines with increasing SCL/TAL1 expression. Examination of EPO-R transcription regulation indicates that E-boxes in the 5′ UTR can bind SCL/TAL1 in vitro and, in addition to the GATA-1 binding motif, provide transcription activity in reporter gene assays. These data indicate that in addition to the importance of SCL/TAL1 DNA binding for proliferation of BFU-E and expression of glycophorin A and protein 4.2, SCL/TAL1 is also necessary for high level expression of EPO-R. Reduction in EPO-R expression likely contributes to the anemia associated with the conditional adult deletion of SCL/TAL1 and to the proliferative defect of erythroid cells observed in vitro. Early expression of SCL/TAL1 in hematopoietic cells may activate expression of EPO-R prior to EPO stimulation of erythropoiesis and induction of GATA-1.

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Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.

Blood ◽

10.1182/blood.v114.22.1478.1478 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1478-1478

Author(s):

Kathryn M. Shinnick ◽

Kelly A. Barry ◽

Elizabeth A. Eklund ◽

Thomas J. McGarry

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Transcription Factors ◽

Cell Division ◽

Dna Replication ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Hematopoietic Stem ◽

Proliferation And Differentiation

Abstract Abstract 1478 Poster Board I-501 Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas. Disclosures: No relevant conflicts of interest to declare.

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Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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The bHLH Transcription Factors SCL and LYL1 Awaken Hematopoietic Stem Cells by Repression of p21

Blood ◽

10.1182/blood.v118.21.214.214 ◽

2011 ◽

Vol 118 (21) ◽

pp. 214-214

Author(s):

David J. Curtis ◽

Nhu-Y Nguyen ◽

Jessica Salmon

Keyword(s):

Cell Cycle ◽

Transcription Factors ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Short Term ◽

Hematopoietic Stem ◽

Bhlh Factors ◽

Mild Defect ◽

Bhlh Transcription Factors

Abstract Abstract 214 The basic helix-loop-helix (bHLH) transcription factors SCL (TAL1) and LYL1 are regulators of adult hematopoietic stem cell (HSC) activity with significant functional redundancy: HSCs lacking SCL (SCLδ/δ) have a mild defect in short-term repopulating activity whilst HSCs lacking LYL1 (LYL1−/−) have normal repopulating activity. In contrast, we have shown previously that HSCs lacking both SCL and LYL1 (DKO) are unable to grow in vitro and have no in vivo repopulating activity. Phenotypic and expression analyses of SCLδ/δ, LYL1−/− and DKO mice were performed to determine how bHLH factors regulate HSC activity. Consistent with the short-term repopulating defects of SCLδ/δ HSC, Lineage negative Sca-1+ c-Kit+ (LSK) bone marrow cells from SCLδ/δ mice had reduced in vitro replating activity associated with increased quiescence – 90% in G0 compared with 70% in normal LSK. Increased quiescence was associated with delayed hematopoietic recovery following treatment of mice with 5-Fluorouracil. Consistent with the increased quiescence, expression of the cell cycle inhibitor, Cdkn1a (p21) was increased three-fold in SCLδ/δ and LYL1−/− LSK. Moreover, p21 levels in LSK isolated from DKO mice were increased 50-fold. To determine the functional relevance of the elevated levels of p21 in DKO HSCs, we generated DKO mice on a p21-deficient (p21−/−) background. Remarkably, loss of p21 rescued in vitro cell growth of DKO progenitors. More importantly, primary and secondary competitive repopulation assays demonstrated multi-lineage repopulating activity of p21−/− DKO HSCs. These results suggest the bHLH factors SCL and LYL1 function as repressors of p21, allowing HSCs to enter cell cycle during stress hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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RhoA Is An Essential Regulator of Mitosis and Survival During Hematopoietic Stem Cell Differentiation to Multipotent Progenitors

Blood ◽

10.1182/blood.v118.21.1273.1273 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1273-1273

Author(s):

Xuan Zhou ◽

Jaime Meléndez ◽

Yuxin Feng ◽

Richard Lang ◽

Yi Zheng

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Intracellular Signaling ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Conditional Knockout ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Differentiated Cells ◽

Multipotent Progenitors

Abstract Abstract 1273 The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Stem Cells Are Dependent On Homologous Recombination Only During Active Cell Cycle in Nuclease-Mutant Exonuclease1mut mice

Blood ◽

10.1182/blood.v120.21.1225.1225 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1225-1225

Author(s):

Amar Desai ◽

Stanton L. Gerson ◽

Yulan Qing

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Stem Cell ◽

Homologous Recombination ◽

Conflicts Of Interest ◽

Whole Body ◽

Hematopoietic Stem ◽

Increased Sensitivity ◽

Quiescent Hscs

Abstract Abstract 1225 Hematopoietic stem cell (HSC) maintenance and self-renewal is crucial for long term tissue repopulation and immune function. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but the pathways involved and the mechanisms of regulation are still being elucidated. It has been proposed that quiescent HSCs rely on the error prone non homologous end joining pathway for DNA double strand break (DSB) repair while HSCs in cycle use both NHEJ and the high fidelity homologous recombination (HR), but functional in vivo studies have not yet been completed. Exonuclease 1 participates in homologous recombination. We used Exo1mut fibroblasts to demonstrate that loss of Exo1 function results in a defective HR response, increased sensitivity to DSB inducing agents, and aberrant DNA damage signaling. However, Exo1mut mice did not appear to require HR to maintain quiescent HSCs at steady state or to respond to DNA damage. Exo1mutmice were able to sustain long term serial repopulation, displayed no defect in competitive repopulation or quiescence maintenance, and did not display increased sensitivity to whole body ionizing radiation (IR). In contrast, when Exo1mut HSCs were pushed into cell cycle with 5-Fluorouracil, the hematopoietic population and HSCs became hypersensitive to IR stress relative to WT B6 mice, as shown by decreased bone marrow cellularity, colony forming unit defects, loss of the HSC population, and finally animal death. Thus, loss of Exo1, and in turn fully functional HR, in quiescent HSC is not critical to stem cell function, survival, or recovery after DNA damage, whereas HR mediated repair of DNA damage is essential for HSC maintenance after cell cycle entry. In HSCs, DNA damage repair response, and sensitivity is dependent on cell cycle. Disclosures: No relevant conflicts of interest to declare.

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Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽

10.1182/blood.v120.21.sci-42.sci-42 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-42-SCI-42

Author(s):

Toshio Suda

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Metabolic Regulation ◽

Conflicts Of Interest ◽

Hypoxia Inducible Factor ◽

Ros Generation ◽

Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

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Periostin Acts As An Important Cell Cycle Regulator Of Adult Hematopoietic Stem Cells Via Binding To Integrin-αvβ3

Blood ◽

10.1182/blood.v122.21.341.341 ◽

2013 ◽

Vol 122 (21) ◽

pp. 341-341 ◽

Cited By ~ 2

Author(s):

Satish Khurana ◽

Catherine M. Verfaillie

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Integrin Αvβ3 ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Cycle Regulator ◽

Blocking Antibodies

Osteoblasts are one of the important cellular components of the niche for hematopoietic stem cells (HSCs) in mammalian bone marrow (BM). Integrin receptors not only play a key role in HSC adhesion within the BM niche but also transfer regulatory signals from the microenvironment to HSCs. Periostin (Postn or osteoblast specific factor-1; OSF-1) is expressed in osteoblasts in addition to many other tissues, and acts as a ligand for Integrin-αvβ3 (ITGAV-B3). We identified POSTN as an important regulator of the cell cycle in adult murine HSCs. POSTN inhibited culture induced proliferation of HSCs thereby decreasing the total number of cells following 2-5 day culture of primitive HSCs, identified as CD150+CD48-Lin-Sca-1+c-kit+ (CD150 KLS) cells with SCF and TPO, while increasing the proportion of long-term (LT-) HSCs. Culture for 5 days with POSTN decreased the short-term (ST-) engraftment of progeny of 200 CD150 KLS cells, while significantly increasing LT- engraftment of the donor derived cells. A significant fraction of CD150 KLS cells expressed ITGAV as well as ITGB3. POSTN did not affect proliferation of HSCs in vitro following blocking of ITGAV with neutralizing antibodies. Among the important cell cycle regulators, we found an increase in p27kip1 expression in HSCs. Preliminary studies on possible signaling mechanisms involved, showed that POSTN inhibits Akt phosphorylation, known to mediate inhibition of both expression and activation of p27Kip1. Intravenous infusion of recombinant POSTN protein significantly decreased proliferation of hematopoietic progenitors as shown by Brdu incorporation and Hoechst/Pyronin staining. Interestingly, POSTN infusion also led to an increase in the number of KLS as well as CD150 KLS cells in the BM. Studies on characterization of the hematopoietic system of Postn-/- mice are underway. To further determine the role of ITGAV in HSCs, we used blocking antibodies against ITGAV and performed homing and engraftment studies. No effect on either homing potential or engraftment of ST- and LT- engraftment was seen. However, the competitive repopulation of ITGAV- CD150 KLS cells was significantly lower that that of ITGAV+ CD150 KLS cells (isolated using non-blocking antibodies). Therefore, our studies confirm the importance of ITGAV expression on primitive HSCs as well as presents POSTN as an important cell cycle regulator in the hematopoietic system. Disclosures: No relevant conflicts of interest to declare.

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ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4316.4316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4316-4316

Author(s):

Hendrik Folkerts ◽

Maria Catalina Gomez Puerto ◽

Albertus T.J. Wierenga ◽

Koen Schepers ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

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p19INK4d Modulates Human Terminal Erythroid Differentiation By Post-Transcriptionally Regulating GATA1 Expression

Blood ◽

10.1182/blood.v128.22.697.697 ◽

2016 ◽

Vol 128 (22) ◽

pp. 697-697

Author(s):

Xu Han ◽

Jieying Zhang ◽

Yuanliang Peng ◽

Huiyong Chen ◽

Xiao Chen ◽

...

Keyword(s):

Cell Cycle ◽

Late Stage ◽

Conflicts Of Interest ◽

Critical Role ◽

Ectopic Expression ◽

Erythroid Differentiation ◽

Hematopoietic Stem ◽

Erk Activation ◽

Protein Levels ◽

Terminal Erythroid Differentiation

Abstract Erythropoiesis is a process during which hematopoietic stem cell (HSCs) are first committed to erythroid progenitors, which subsequently undergo terminal erythroid differentiation to produce mature red blood cells. During terminal erythroid differentiation, proerythroblasts undergo 4-5 mitoses to sequentially generate basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts that expel their nuclei to produce enucleated reticulocytes. Terminal erythropoiesis is a tightly regulated process. The most well studied regulatory mechanisms include EPO/EPOR mediated signal transduction and transcription factors among which GATA1 plays critical role. Terminal erythroid differentiation is also tightly coordinated with cell cycle exit, which is regulated by cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain largely undefined. Our RNA-seq of human terminal erythroid differentiation shows that of seven CDKI members, only three of them, p18INK4c, p19INK4d and p27KIP1, are abundantly expressed in erythroid cells and their expressions are significantly upregulated in late stage erythroblasts, which were further confirmed by western blotting analysis. In contrast to demonstrated roles of p18INK4c and p27KIP1 in terminal erythroid differentiation, the function of p19INK4d this process has not been studied. To explore the role of p19INK4d during human erythropoiesis, we employed a shRNA-mediated knockdown approach in CD34+ cells and found that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, led to increased apoptosis and generation of abnormally nucleated late stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather it led to decreased GATA1 protein levels. Importantly, the differentiation and nucleus defects were rescued by ectopic expression of GATA1. As GATA1 protein is protected by nuclear HSP70, to explore the mechanism for the decreased GATA1 protein levels, we examined the effects of p19INK4d knockdown on HSP70 and found p19INK4d knockdown led to decreased nuclear localization of HSP70 due to reduced ERK activation. Further biochemical analysis revealed that p19INK4d directly binds to Ras kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1 which in turn led to reduced ERK activation. These results demonstrate that p19INK4d maintains GATA1 protein levels through PEBP1-pERK-HSP70-GATA1 pathway. Our findings identify previously unknown and unexpected roles for p19INK4d in human terminal erythroid differentiation via a novel pathway. Disclosures No relevant conflicts of interest to declare.

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