scholarly journals The Mitochondrial Metabolic Checkpoint and Reversing Stem Cell Aging

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-34-SCI-34
Author(s):  
Danica Chen
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Mitochondrial Protein ◽  
Cell Aging ◽  
Quiescent State ◽  
Mitochondrial Oxidative Stress ◽  
Hematopoietic Stem

Abstract Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in hematopoietic stem cell (HSC) biology highlight a mitochondrial metabolic checkpoint that is essential for HSCs to return to the quiescent state. As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced, which is associated with increased mitochondrial protein folding stress and mitochondrial oxidative stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽  
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.

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2653-2653 ◽  
Author(s):  
Yuichiro Nakata ◽  
Norimasa Yamasaki ◽  
Takeshi Ueda ◽  
Kenichiro Ikeda ◽  
Akiko Nagamachi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Epigenetic Mark ◽  
M2 Macrophage ◽  
Leukemic Stem Cells ◽  
Cell Potential ◽  
Hematopoietic Stem

Abstract Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive. To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs. We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genetic and Epigenetic Mechanisms That Maintain Hematopoietic Stem Cell Function

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Christian Kosan ◽  
Maren Godmann
Keyword(s):  
Dna Damage ◽  
Stem Cell ◽  
Cell Fate ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Quiescent State ◽  
Ongoing Research ◽  
Hematopoietic Stem ◽  
Lineage Differentiation ◽  
Multipotent Progenitor Cells

All hematopoiesis cells develop from multipotent progenitor cells. Hematopoietic stem cells (HSC) have the ability to develop into all blood lineages but also maintain their stemness. Different molecular mechanisms have been identified that are crucial for regulating quiescence and self-renewal to maintain the stem cell pool and for inducing proliferation and lineage differentiation. The stem cell niche provides the microenvironment to keep HSC in a quiescent state. Furthermore, several transcription factors and epigenetic modifiers are involved in this process. These create modifications that regulate the cell fate in a more or less reversible and dynamic way and contribute to HSC homeostasis. In addition, HSC respond in a unique way to DNA damage. These mechanisms also contribute to the regulation of HSC function and are essential to ensure viability after DNA damage. How HSC maintain their quiescent stage during the entire life is still matter of ongoing research. Here we will focus on the molecular mechanisms that regulate HSC function.

scholarly journals Inhibition of Lysine-Specific Demethylase 1A (LSD1) Supports Human HSCs in Culture By Preserving Their Immature State

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Agatheeswaran Subramaniam ◽  
Mehrnaz Safaee Talkhoncheh ◽  
Kristijonas Zemaitis ◽  
Shubhranshu Debnath ◽  
Jun Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Division ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Enrichment Score ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract The molecular mechanisms that govern hematopoietic stem cell (HSC) fate decisions remain incompletely defined. It has been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. Lysine-specific histone demethylase 1A (LSD1), also known as lysine (K)-specific demethylase 1A (KDM1A), regulates gene expression by specifically eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues. Studies in mice have shown that, conditional knockdown of LSD1 results in an expansion of bone marrow hematopoietic stem and progenitor cells (HSPCs). However, a complete knockout of LSD1 results in pancytopenia and a dramatic reduction of HSPCs. In this study, we asked whether inhibition of LSD1 would improve the maintenance or expansion of cultured human HSCs derived from umbilical cord blood (UCB). To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) at their respective IC50 values (20µM, 16nM and 70nM) and expanded the cultures for 6 days in serum free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L). Since we (Subramaniam et. al. Haematologica 2018) and others recently have shown that EPCR is a reliable cell surface marker to track UCB derived HSCs during in vitro culture, we quantified the numbers of CD34+EPCR+ cells using flow cytometry and compared to DMSO treated control cultures. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of CD34+EPCR+ cells, compared to controls. Further, from dose response experiments we found that 2-PCPA at 1.25 µM expanded the total CD34+ cell population more efficiently than GSK-LSD1, and we therefore used 2-PCPA at this concentration for the subsequent experiments. Using carboxyfluorescein succinimidyl ester (CFSE) labeling to monitor cell division, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells compared to DMSO controls, suggesting that the expansion of CD34+EPCR+ cells is not due to increased proliferation, and that LSD1 inhibition rather may prevent differentiation of the immature HSPCs. To further explore this, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs using gene expression profiling of CD34+CD38-CD45RA-CD90+ cells following 24 hours of culture with or without 2-PCPA treatment. We found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment compared to DMSO control (Normalized Enrichment Score (NES)=1.49, q=0.05). This suggest that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Strikingly, the gene signature induced by LSD1 inhibition was highly similar to that induced by the known HSC expanding compound UM171 (NES=1.43, q=0.11). UM171 is a molecule with unknown target and has also been shown to dramatically expand the EPCR+ population in culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). LDA was performed by transplanting 4 doses (day 0 equivalents of 20000, 1000, 300 and 100 CD34+ cells) of DMSO and 2-PCPA treated cultures into sub lethally irradiated (300cGy) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human CD45+ cell engraftment in the bone marrow was analyzed 18 weeks' post transplantation. Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells per day 0 CD34+ cell equivalent compared to the DMSO control (1 in 615 vs 1 in 3041, p=0.03). Thus, the 2-PCPA treated cultures had significantly enhanced HSCs numbers. To determine the absolute expansion rate, we are currently performing LDA using uncultured cells as well. Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state. Currently we are exploring the possibilities of using LSD1 inhibitors in combination with other known modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-46-SCI-46
Author(s):  
Kristin Hope
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Self Renewal ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem

Abstract The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the "rules" by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals CDK6 Antagonizes TP53-Induced Responses during Tumorigenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-15-SCI-15
Author(s):  
Veronika Sexl ◽  
Karoline Kollmann ◽  
Florian Bellutti
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Induced Responses ◽  
Hematopoietic Stem ◽  
Hematopoietic Malignancies ◽  
Independent Functions ◽  
Oncogenic Stress

Inhibitors directed against cyclin dependent kinases (CDKs) have raised much interest as anti-cancer therapeutics over the last years. In particular, inhibitors directed against CDK4/6 have been declared as a major breakthrough in cancer therapy by the FDA. CDK4 and CDK6 bind to D-type cyclins and subsequently phosphorylate the RB protein to allow cells to progress through the G1 phase of the cell cycle. The effectiveness of CDK4/6 inhibitors was primarily assigned to their ability to block cell cycle progression. In hematopoietic malignancies high levels of CDK6, but not CDK4, are frequently found. Over the last years we have assigned a novel and unexpected role for CDK6 as global transcriptional regulator. ChIP-Seq experiments identified more than 20.000 specific CDK6 binding sites in leukemic cells with the majority located in the promoter regions. CDK6 binding to chromatin does not require kinase activity whereas transcriptional control is regulated in a kinase- dependent as well as kinase-independent manner. Overlaying ChIP-Seq and RNA-Seq experiments showed that CDK6 contributes to the induction or repression of genes. Target genes of CDK6 which are important for leukemia progression include PIM1, c-MYC, AURKA, AURKB, AKT and VEGF-A. Murine leukemia models verified the importance of CDK6 for myeloid and lymphoid tumor formation downstream of a variety of oncogenes including FLT3-ITD, NPM/ALK, MLL/AF9, BCR/ABL or JAK2V617F. CDK6 contributes to disease development by regulating proliferation, cell survival, angiogenesis and cytokine production. In hematopoietic stem cells and leukemic stem cells kinase-independent functions dominate and CDK6 controls a network of transcription factors regulating stem cell quiescence and activation. The importance of kinase-dependent transcriptional effects is pronounced under conditions of stress and transformation. Upon oncogenic stress, CDK6 induces a set of genes that counteract pro-apoptotic TP53 responses including MDM4, PRMT5, PPM1D and BCL2. This response is induced by a CDK6 - dependent phosphorylation of the transcription factors SP1 and NFYA as verified by phospho-chromatome analysis. Murine Cdk6-deficient cells only survive oncogenic stress by mutating Tp53. The link between CDK6 and TP53 is conserved in human hematopoietic malignancies. Kollmann K, Heller G, Schneckenleithner C, et al. A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis. Cancer Cell. 2013;24(2):167-181.Scheicher R, Hoelbl-Kovacic A, Bellutti F, et al. CDK6 as a key regulator of hematopoietic and leukemic stem cell activation. Blood. 2015;125(1):90-101.Uras IZ, Walter GJ, Scheicher R, et al. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6. Blood. 2016;127(23):2890-2902.Bellutti F, Tigan AS, Nebenfuehr S, et al. CDK6 antagonizes P53-induced responses during tumorigenesis. Cancer Discov. 2018;8(7):884-897.Uras IZ, Maurer B, Nivarthi H, et al. CDK6 coordinates JAK2V617F mutant MPN via NF-kB and apoptotic networks. Blood. 2019;133(15):1677-1690. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem Cells Increase Quiescence during Aging

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2484-2484 ◽  
Author(s):  
Larisa V. Kovtonyuk ◽  
Peter Ashcroft ◽  
Gianluca Spaltro ◽  
Nageswara Rao Tata ◽  
Radek C. Skoda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Hematopoietic Stem ◽  
Cfse Dilution ◽  
Turn Over ◽  
Old Mice

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs. Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011). Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions. Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging. Disclosures No relevant conflicts of interest to declare.

scholarly journals FoxM1 Haploinsufficiency Drives Clonal Hematopoiesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 726-726
Author(s):  
Chunjie Yu ◽  
Yue Sheng ◽  
Zhijian Qian
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Low Dose ◽  
Conflicts Of Interest ◽  
Clonal Expansion ◽  
Brdu Incorporation ◽  
Quiescent State ◽  
Dapi Staining ◽  
Hematopoietic Stem ◽  
Increased Risk

Hematopoiesis is an orchestrated process in which hematopoietic stem cells (HSCs) can self-renew and produce all lineages of blood cells. Majority of HSCs are in a quiescent state with a low growth rate. However, some genetic mutations that occur in HSCs impel HSCs to exit the quiescent state and to proliferate excessively, which enables mutant HSCs to outcompete normal HSCs and leads to clonal expansion of mutant HSCs. Myelodysplastic syndromes (MDSs) as a clonal disease, arise from the expansion of mutant HSCs and are characterized by morphologic dysplasia, ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia. FoxM1 is one of transcription factors in the family of Fox ('Forkhead box') proteins. Analysis of public database revealed that the expression level of FOXM1 was decreased significantly in CD34 + cells from a subset of patients with MDS as compared to healthy individuals. Thus, we sought to determine whether haploinsufficiency of FOXM1 contributes to the development of MDS in mice. Our study showed that haploinsufficiency of Foxm1 led to an expansion of hematopoietic stem/progenitor cells in mice. Since FoxM1 has previously been implicated in regulation of cell cycle, we determined the cell cycle status of Foxm1 heterozygous HSCs. By BrdU incorporation assay, we showed that Foxm1 heterozygous HSCs have an increased S phase and G2/M phase as compared to control HSCs from wildtype mice. Additional analysis with Hochest33342/Pyronin-Y staining and Ki67/DAPI staining showed a significant decrease in the number of quiescent (G0) cells in Foxm1 heterozygous HSCs as compared to control HSCs. These results suggest that FoxM1 haploinsufficiency promotes HSCs to exit quiescence and to enter cell cycle, thereby leading to exhaustion of HSCs. To further assess the function of Foxm1 heterozygous HSCs in vivo, we performed competitive repopulation assay. We found that Foxm1 haploinsufficiency HSCs exhibited competitive repopulation advantage in the first and secondary recipient mice, but displayed significantly decreased capacity of repopulation in tertiary recipient mice as compared to control HSCs, suggesting that Foxm1 haploinsufficiency promoted clonal expansion of HSCs, which leads to an exhaustion of HSCs eventually. HSC proliferation can be induced by some specific immune effectors such as Toll-like receptor 4 (TLR4). Lipopolysaccharide (LPS) stimulates HSC proliferation by activating TLR4 signaling pathway. Low dose of LPS treatment over time accelerated the development of MDS in mice. Interestingly, low dose of LPS injection chronically induced defects in hematopoiesis in Foxm1 haploinsufficient mice but not the control wildtype mice. Recipient mice transplanted with Foxm1 heterozygous BM cells but not the control BM cells developed MDS-like disease with cytopenia and a decreased number of hematopoietic stem/progenitor cells after LPS stimulation. Moreover, we found that nearly half of aged Foxm1 haploinsufficient mice (20 months) developed splenomegaly. Analysis of histologic sections in Foxm1 haploinsufficient mice showed that the mice developed hematopoietic dysplasia including dysplastic megakaryocytes with bizarre-shaped nuclei in bone marrow and extramedullary hematopoiesis with accumulation of myeloid cells in spleen. RNA-seq analysis indicated that haploinsufficiency of Foxm1 perturbed multiple stem cell-maintenance mechanisms especially in metabolic processes. Taken together, our studies suggest that Foxm1 haploinsufficiency in mice causes clonal expansion of HSCs and promotes MDS-like disease, which underscores the significant role of FOXM1 downregulation in the initiation and development of human MDS. Disclosures No relevant conflicts of interest to declare.

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.

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