Cell Cycle Regulation in Hematopoietic Stem/Progenitor Cells

Abstract CCAAT Enhancer Binding Protein b (C/EBPb) is a leucine zipper type transcription factor. While C/EBPa plays a critical role in maintaining steady-state granulopoiesis, C/EBPb is required for stress-induced granulopoiesis (Hirai et al., 2006). We have been focusing on the functions of C/EBPb in the regulation of hematopoietic stem and progenitor cells (HSPCs) especially under stressed conditions. Last year in this meeting, we have shown that 1) C/EBPb was upregulated at protein level in HSPCs after hematopoietic stresses, 2) C/EBPb was required for initial expansion of HSPCs after transplantation, and 3) C/EBPb promoted exhaustion of HSPCs under repetitive hematopoietic stresses (56th ASH, abstract #67850). Here, we further investigated the significance of C/EBPb in cell cycle regulation of HSPCs and the distinct roles of C/EBPb isoforms in HSPCs during regenerative conditions. To clarify the involvement of C/EBPb in cell cycle regulation of HSPCs, we compared the cell cycle status of wild-type (WT) and Cebpb knockout (KO) HSPCs by intracellular Ki67 staining and short-term BrdU incorporation assay in combination with multi-color flow cytometric analysis. In order to exclude the difference in the bone marrow microenvironment, CD45.2+ WT or Cebpb KO bone marrow (BM) cells were transplanted into lethally irradiated CD45.1+ WT mice. At steady state (12 weeks after the BM transplantation), the cell cycle status of Cebpb KO HSPCs was identical to that of WT HSPCs. Then cell cycle status of HSPCs was assessed at various time points during regeneration after intraperitoneal administration of 5-fluorouracil (5-FU, 150mg/kg). We found that significantly more Cebpb KO HSPCs remained in the G0 phase than WT HSPCs (in LT-HSCs on days 3-10; in MPPs on days 6-12). Significantly less Cebpb KO HSPCs were BrdU+ and were in the S/G2/M phase on day 7. These findings suggest that C/EBPb, in a cell-intrinsic manner, facilitates cell cycle entry, progression and consequent earlier expansion of HSPCs in response to hematopoietic stresses. Next, we investigated the distinct roles of C/EBPb isoforms in regulation of HSPCs. C/EBPb is a unique single exon gene and utilization of three different initiating codons result in three distinct isoforms. Liver-enriched activating protein* (LAP*) and LAP are the longer isoforms containing transactivating domains, DNA binding and dimerization domains, and liver-enriched inhibitory protein (LIP) is the shortest isoform which lacks the transactivating domains. In order to examine the expression pattern of C/EBPb isoforms in vivo in scarce populations of regenerating HSPCs, we developed a novel flow cytometric method to distinguish the cells predominantly expressing shorter isoform (LIP) from the cells expressing both LIP and the longer isoforms (LAP* and LAP) by intracellular double staining. Using this method, we found that predominantly LIP-expressing cells transiently emerged within MPP fraction in the regenerating bone marrow (on days 5-6 after administration of 5-FU, Figure below), while overall C/EBPb expression levels were significantly upregulated in most cells. To examine the roles of respective C/EBPb isoforms in regulation of HSPCs, EML cells, a murine hematopoietic stem cell line, were retrovirally transduced with one of the C/EBPb isoforms and the transduced cells were subjected to further analysis (vectors are kind gifts from Dr Watanabe-Okouchi N and Dr Kurokawa M, Tokyo Univ). LIP-expressing EML cells were more proliferative and actively cycling than EML cells transduced with a control vector, whereas the proliferation of LAP*- or LAP-expressing cells were markedly suppressed. LIP-expressing cells remained undifferentiated status (c-kithigh CD11b-) for more than 2 weeks, while LAP*- or LAP-expressing cells rapidly differentiated into c-kitlow CD11b+ myeloid cells and eventually exhausted within a week. These results indicate LIP plays quite distinct roles from LAP* and LAP in regulation of HSPCs. Collectively, our data suggest that C/EBPb isoforms distinctively and collaboratively regulate HSPCs in regenerative conditions: early transient elevation of LIP contributes to cell cycle activation and rapid expansion of HSPC population, which is in turn converted into supply of mature myeloid cells by more abundant upregulation of LAP* and LAP. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Convergence of Hypoxia and TGFβ Pathways on Cell Cycle Regulation in Human Hematopoietic Stem/Progenitor Cells

PLoS ONE ◽

10.1371/journal.pone.0093494 ◽

2014 ◽

Vol 9 (3) ◽

pp. e93494 ◽

Cited By ~ 30

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Cell Cycle Regulation ◽

Hematopoietic Stem ◽

Cycle Regulation

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Abstract 2128: Phosphorylation of Id2 at the N-terminus modulates Id2 degradation and mediates cell cycle regulation in neural progenitor cells

10.1158/1538-7445.am2015-2128 ◽

2015 ◽

Author(s):

Jaclyn Sullivan ◽

Matthew Havrda ◽

Brenton Paolella ◽

Arminja Kettenbach ◽

Scott Gerber ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Cell Cycle Regulation ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

N Terminus ◽

Cycle Regulation

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Cell cycle regulation in hematopoietic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.201102131 ◽

2011 ◽

Vol 195 (5) ◽

pp. 709-720 ◽

Cited By ~ 254

Author(s):

Eric M. Pietras ◽

Matthew R. Warr ◽

Emmanuelle Passegué

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Cycle Regulation ◽

Critical Role ◽

Cellular Damage ◽

Fetal Life ◽

Extrinsic Factors ◽

Hematopoietic Stem ◽

Cycle Regulation

Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells. Because HSCs must persist for a lifetime, the balance between their proliferation and quiescence is carefully regulated to ensure blood homeostasis while limiting cellular damage. Cell cycle regulation therefore plays a critical role in controlling HSC function during both fetal life and in the adult. The cell cycle activity of HSCs is carefully modulated by a complex interplay between cell-intrinsic mechanisms and cell-extrinsic factors produced by the microenvironment. This fine-tuned regulatory network may become altered with age, leading to aberrant HSC cell cycle regulation, degraded HSC function, and hematological malignancy.

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FLT3/ITD Expression Increases Expansion, Survival and Entry into Cell Cycle of Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.484.484 ◽

2004 ◽

Vol 104 (11) ◽

pp. 484-484

Author(s):

Li Li ◽

Obdulio Piloto ◽

Kyu-Tae Kim ◽

Zhaohui Ye ◽

Bao Nguyen ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Tyrosine Kinase ◽

Cell Cycle Regulation ◽

Cyclin D3 ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Activating Mutations ◽

Juxtamembrane Region ◽

Cycle Regulation

Abstract FMS-like tyrosine kinase-3 (FLT3) is a Class III receptor tyrosine kinase that is important for normal hematopoiesis. Activating mutations of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region are the most common molecular aberrations found in acute myeloid leukemia (AML). The contributions of FLT3 activating mutations in the leukemic transformation of normal human hematopoietic stem/progenitor cells (HSCs) have not yet been fully elucidated. In this study, using a single lentiviral vector containing two promoters, we achieved consistent and efficient coexpression of FLT3/ITD and green fluorescent protein (GFP) in transduced human CD34+ HSCs. When cultured in medium containing SCF, TPO and FLT3 ligand (FL), FLT3/ITD-transduced cells survived with enhanced self-renewal and survival potential, which was not affected by the withdrawal of FL. These cells retained a surface immunophenotype typical of HSCs (CD34+CD38−). Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSCs had a higher fraction of cells in cell cycle. Clonogenic assays showed that FLT3/ITD-transduced HSCs produced fewer CFU-GM, implying that they were at least partially blocked in their ability to differentiate along the myeloid lineage. FLT3/ITD-transduced HSCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor. In the FLT3/ITD-transduced HSCs, we detected increased expression of Pim-1, a serine/threonine kinase with an important role in cell survival, proliferation and differentiation, c-myc, a transcription factor involved in cell proliferation and cell cycle regulation, and Cyclin D3, a key factor in cell cycle regulation, each of which may contribute to the altered genetic program instituted by FLT3/ITD signaling. These results together indicate that FLT3/ITD mutations may contribute to leukemic transformation of normal HSCs by prolonging survival, promoting proliferation, and blocking differentiation. CEP-701 may act as a potent agent for AML stem cells harboring FLT3/ITD mutations.

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Cell Cycle Regulation and Cell Fate Decisions in Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.1349.1349 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1349-1349

Author(s):

Emmanuelle Passegue ◽

Amy J. Wagers ◽

Sylvie Giuriato ◽

Wade C. Anderson ◽

Irving L. Weissman

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Cell Cycle Regulation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Cycle Regulation

Abstract The blood is a perpetually renewing tissue seeded by a rare population of adult bone marrow hematopoietic stem cells (HSC). During steady-state hematopoiesis, the HSC population is relatively quiescent but constantly maintains a low numbers of cycling cells that differentiate to produce the various lineage of mature blood cells. However, in response to hematological stress, the entire HSC population can be recruited into cycle to self-renew and regenerate the blood-forming system. HSC proliferation is therefore highly adaptative and requires appropriate regulation of cell cycle progression to drive both differentiation-associated and self-renewal-associated proliferation, without depletion of the stem cell pool. Although the molecular events controlling HSC proliferation are still poorly understood, they are likely determined, at least in part, by regulated expression and/or function of components and regulators of the cell cycle machinery. Here, we demonstrate that the long-term self-renewing HSC (defined as Lin−/c-Kit+/Sca-1+/Thy1.1int/Flk2−) exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for HSC function in hematopoietic reconstitution; and a proliferative state, which represents almost a fourth of this population and is essential for HSC functions in differentiation and self-renewal. We show that when HSC exit quiescence and enter G1 as a prelude to cell division, at least two critical events occur: first, during the G1 and subsequent S-G2/M phases, they temporarily lose efficient in vivo engraftment activity, while retaining in vitro differentiation potential; and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). Together, these findings provide a direct link between HSC proliferation, cell cycle regulation and cell fate decisions that have critical implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

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Higher Vertebrate Specific Gene Ribonuclease Inhibitor (RNH1) Is Essential for Adult Hematopoietic Stem Cell Function and Cell Cycle Regulation

Blood ◽

10.1182/blood-2019-128647 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 273-273

Author(s):

Nicola Daniele Andina ◽

Mayuresh Sarangdhar ◽

Aubry Tardivel ◽

Giuseppe Bombaci ◽

Mahmoud Hallal ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Cycle Regulation ◽

Cell Function ◽

Wild Type ◽

Ribonuclease Inhibitor ◽

Hematopoietic Stem ◽

Dominant Phenotype ◽

Cycle Regulation ◽

Deficient Mice

Hematopoietic stem cells (HSC) in higher vertebrate species, especially in mammals, maintain hematopoiesis throughout adult life and require critical cell cycle regulation for their self-renewal and cell fate decisions. Although cell cycle pathways are quite conserved across animal species, it is unknown whether a higher vertebrate specific cell cycle regulation exists in adult mammalian HSCs. Recently, we have published that Ribonuclease inhibitor (RNH1) regulates erythropoiesis by controlling GATA1 mRNA translation. Here, we report that RNH1, which is present only in higher vertebrates regulates HSC cell cycle and HSC function. To study the role of RNH1 in hematopoiesis, we generated hematopoietic-specific knockout mice by backcrossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Rnh1-deficiency (Rnh1FL/FLVav1-iCre mice) resulted in hematopoietic alterations resembling emergency myelopoiesis. At 15 weeks of age Rnh1-deficient mice had reduced hemoglobin levels (144.4 ± 2.6 vs 165.0 ± 4.2 g/L, p = 0.005), decreased lymphocytes (4.1 ± 0.8 vs 9.6 ± 1.6 K/µL, p = 0.023), increased neutrophils (3.2 ± 0.6 vs 1.5 ± 0.2 K/µL, p = 0.046) and monocytes (0.65 ± 0.05 vs 0.09 ± 0.02 K/µL, p = 0.0001) in the peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type andRnh1-deficient mice, however the number of erythroid cells and lymphoid cells (T and B cells) was significantly decreased, whereas myeloid cells were significantly increased. Rnh1-deficient spleens were significantly larger than wild type controls and showed extramedullary hematopoiesis. Surprisingly, although Rnh1-deficient mice showed myeloproliferation they survived normally and did not show progression to leukemia. However, they did not tolerate even little stress, such as 35 µg LPS administration, which lead to early mortality. We analysed the progenitor populations in the BM. In line with the myelopoiesis dominant phenotype granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Cell extrinsic factors such as growth factors and the bone marrow niche play a critical role in shaping lineage choice. To exclude this, we performed bone marrow transplantation experiments (BMT) by transplanting wild type (Rnh1FL/FL) and Rnh1-deficient (Rnh1FL/FLMx1-Cre+) bone marrow into lethally irradiated CD45.1 congenic mice. After reconstitution Rnh1 was deleted by administration of polyinosinic:polycytidylic acid (polyI:C). We observed a similar myelopoiesis dominant phenotype in Rnh1-deleted mice. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-deficient mouse BM, suggesting that RNH1 could affect HSC function. Supporting this Rnh1-deficient HSCs failed to engraft lethally irradiated mice in competitive BMT experiments. Furthermore, Rnh1-deficient HSCs produced significantly less and smaller colonies in in-vitro colony forming cell (CFC) assays. Transcriptome analysis showed increased expression of genes related to cell cycle, kinetochore, DNA damage and decreased expression of genes related to stem cell function in Rnh1-deficient LT-HSCs and ST-HSCs. Corroborating this, Rnh1-deficient LT-HSCs and ST-HSCs showed increased S/G2/M phase in cell cycle analysis. In line with this, at the molecular level, we found that RNH1 directly binds to cell-cycle related proteins such as cyclin-dependent kinase 1 (CDK1), cell-division cycle protein 20 (CDC20) and mitotic checkpoint protein BUB3, suggesting direct involvement of RNH1 in cell cycle regulation. Confirming this, pharmacological inhibition of CDK1 (RO-3306, 10 µM) in Rnh1-deficinet ST-HSCs restored colony size in CFC assays, suggesting that RNH1 and CDK1 inhibition have a synergistic effect in ST-HSCs. In summary, our results demonstrate that RNH1, which is present only in higher vertebrates, is essential for HSC cell cycle regulation and steady state hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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