scholarly journals Autism-Associated Chromatin Remodeler CHD8 Governs the Survival and Differentiation of Hematopoietic Stem/Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1191-1191
Author(s):  
Zhaowei Tu ◽  
Chen Wang ◽  
Chuntao Zhao ◽  
Lei Huang ◽  
Nathan Salomonis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
P53 Gene ◽  
Autism Spectrum ◽  
Cycle Phase ◽  
Chromatin Remodeler ◽  
Hematopoietic Stem ◽  
Direct Binding

Hematopoiesis is a strictly regulated process which depends on regulated proliferation, differentiation, and survival of hematopoietic stem/progenitor cells (HSPCs). Multiple signaling pathways, transcription factors and epigenetic machineries are involved in this important process. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in stem/progenitor cell regulation. Among them, the CHD8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of autism spectrum disorders. Whether CHD8 plays a role in hematopoietic cells remains unknown. In this study, to define the role of CHD8 in hematopoiesis and HSPC regulation, we carried out conditional deletion of CHD8 in the hematopoietic lineages using an interferon inducible Mx1-Cre; Chd8F/Fmouse model. As early as one week after polyI:C injection, the CHD8 knockout mice experienced drastic pancytopenia and bone marrow failure with a reduction of bone marrow (BM) cellularity to ~1/3 of that of WT controls. In the HSPC compartment, loss of CHD8 resulted in a depletion of Lin-c-Kit+ (LK) and Lin-Sca1+c-Kit+ (LSK) progenitors and a severe reduction of the GMP, MEP, MPP and CD150+CD48-LSK HSC populations. Accompanying the loss of HSPCs were dramatically increased apoptosis rate and cell cycle arrest in Chd8-/- cells. An examination of HSPC function by transplantation found that the BM cells from Chd8-/- mice could not engraft in the recipient mice that died ~20 days post-transplantation. The chimerism of Chd8-/- BM cells also reduced drastically in a competitive BM transplantation assay when induction of Chd8 deletion occurred after Mx1-Cre; Chd8F/Fcells were pre-engrafted at 1:1 ratio with WT BM cells. These results indicate that CHD8 is essential for hematopoiesis and HSPC survival and proliferation. To elucidate the underlying mechanism of CHD8 function in HSPCs, we performed complementary biochemical, genomic and genetic analyses of the WT (Chd8F/F) and Chd8-/-HSPC cells. Firstly, we found blood progenitor cells dominantly express the short form of CHD8 containing residues 1 to 751 which retains the N-terminal P53 binding domain, rather than the long form of the CHD8 protein. Secondly, we revealed by RNA-seq and subsequent RT-PCR analyses that the P53 signaling pathway signatures including P53, P21, Bax and Noxa are aberrantly activated in Chd8-/-HSPCs. At the protein level, P53 and P21 are significantly elevated in Chd8-/- LK cells. Thirdly, ATAC-seq analysis of the LSK cells revealed increased global chromatin accessibilities after CHD8 deletion, including the promoter regions of P21 and Noxa genes. By a "Cut & Run" assay using H3K4me3 antibody in Chd8F/Fand Chd8-/- LSK cells we have also seen significantly enhanced epigenetic H3K4me3 mark on the promoter region of P21 and Noxa. An anti-CHD8 "Cut & Run" further detected direct binding sites of CHD8 on P53 and P21, but not Noxa or Bax gene. Fourthly, we observed a direct binding interaction by co-immunoprecipitation between P53 and CHD8 proteins. Finally, genetic deletion of one allele of P53 gene in Chd8-/- HSPCs (P53+/-;Chd8-/-) was able to completely suppress the Bax/Noxa expression and apoptosis phenotype and rescue most of the HSC and early progenitor defects but not the later stage of differentiation defects and BM cellularity loss. Interestingly, deletion of both alleles of P53 gene (P53-/-;Chd8-/-) was able to further remove the differentiation block between LSK and LK cells observed in the P53+/-;Chd8-/-background which is associated with a P21 mediated G0 cell cycle phase arrest. These combined results suggest a mechanism of action that CHD8 works through a direct complex with P53 to regulate P21 gene expression which mediates cell cycle control of HSPC differentiation and an indirect CHD8 mediated P53 expression and subsequent P53 regulated Noxa/Bax expression which regulates HSPC survival. Thus, we identify the autism-associated CHD8 as an essential chromatin suppressor in regulating hematopoiesis through restricting the expression of P53 and chromatin accessibility of P53 downstream targets, Bax/Noxa and P21, to enable selective control of HSPC survival and differentiation, respectively. Disclosures No relevant conflicts of interest to declare.

Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow–derived and circulating human CD34+ hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2037-2044 ◽  
Author(s):  
Ulrich Steidl ◽  
Ralf Kronenwett ◽  
Ulrich-Peter Rohr ◽  
Roland Fenk ◽  
Slawomir Kliszewski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+(BM-CD34+) or granulocyte–colony-stimulating factor–mobilized peripheral blood CD34+(PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle–initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

Fanconi anemia type C–deficient hematopoietic stem/progenitor cells exhibit aberrant cell cycle control

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2081-2084 ◽  
Author(s):  
Xiaxin Li ◽  
P. Artur Plett ◽  
Yanzhu Yang ◽  
Ping Hong ◽  
Brian Freie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Cell Cycle Control ◽  
Cytokine Signaling ◽  
Compensatory Mechanism ◽  
Hematopoietic Stem ◽  
Type C

Abstract The pathogenesis of bone marrow failure in Fanconi anemia is poorly understood. Suggested mechanisms include enhanced apoptosis secondary to DNA damage and altered inhibitory cytokine signaling. Recent data determined that disrupted cell cycle control of hematopoietic stem and/or progenitor cells disrupts normal hematopoiesis with increased hematopoietic stem cell cycling resulting in diminished function and increased sensitivity to cell cycle–specific apoptotic stimuli. Here, we used Fanconi anemia complementation type C–deficient (Fancc–/–) mice to demonstrate that Fancc–/– phenotypically defined cell populations enriched for hematopoietic stem and progenitor cells exhibit increased cycling. In addition, we established that the defect in cell cycle regulation is not a compensatory mechanism from enhanced apoptosis occurring in vivo. Collectively, these data provide a previously unrecognized phenotype in Fancc–/– hematopoietic stem/progenitor cells, which may contribute to the progressive bone marrow failure in Fanconi anemia.

The Potent Deacetylase Inhibitor Trichostatin a (TSA) Increases CXCR4 Expression in Hematopoietic Stem/Progenitor Cells by Chromatin Remodelling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3487-3487 ◽  
Author(s):  
Hilal Gul ◽  
Leah A. Marquez-Curtis ◽  
Jennifer Lo ◽  
Nadia Jahroudi ◽  
A. Robert Turner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Trichostatin A ◽  
Mrna Level ◽  
Hdac Inhibitors ◽  
Cxcr4 Expression ◽  
Chromatin Remodelling ◽  
Hematopoietic Stem ◽  
Chip Analysis

Abstract Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role in the trafficking of normal hematopoietic stem/progenitor cells (HSPC) and their homing/retention in bone marrow. Consequently, modulation of CXCR4 expression in HSPC could be applied therapeutically to improve the efficiency of HSPC transplantation. It is known that gene expression can be regulated by chromatin remodelling. Two groups of histone modifying enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) participate in the regulation of chromatin structure, and hence gene expression. Disruption of normal HAT or HDAC activities has been found in many human cancers. Recently, several structurally diverse and highly specific HDAC inhibitors (HDI) have been reported. They act as strong modulators of growth, differentiation and apoptosis in several types of cancer, particularly acute myeloid leukemia (AML). However, very little is known regarding the effects of HDI on HSPC. We have previously shown that a specific short-chain fatty acid HDI, valproic acid (VPA), enhances CXCR4 expression and function in normal HSPC (Blood2007: 110; 425a). In order to determine whether other structurally diverse classes of HDI are able to influence CXCR4 expression in HSPC through chromatin remodelling, we investigated the effect of potent hydroxamic acid HDI Trichostatin A (TSA) on CXCR4 in normal HSPC. We examined the effect of TSA on CXCR4 expression (by FACS and real-time RT-PCR), modulation of CXCR4 transcription (chromatin immunoprecipitation (X-ChIP) analysis) and on functional response towards an SDF-1α gradient (by chemotaxis assay) of HSPC (CD34+ cells from cord blood (CB) and the models of immature hematopoietic cells expressing CD34 antigen, namely AML cell lines KG-1a and KG-1). Cells were incubated for 24 h in IMDM supplemented with 20% FCS in the presence of TSA (0.1 μM). We found that TSA increases the percentage of CXCR4-expressing CB CD34+, KG-1a, KG-1 cells (2.5-, 8- and 3-fold, respectively). This effect was also confirmed at the mRNA level in CB CD34+, KG-1a and KG-1 cells (by about 2.5-, 5- and 2.5-fold up-regulation, respectively). Moreover, X-ChIP analysis showed a significant increase in association of acetyl-histone H4 binding to the CXCR4 promoter in CB CD34+ and KG-1 cells (2- and 1.7-fold, respectively). TSA was also shown to significantly increase the chemotaxis of KG-1a cells towards SDF-1α (20 ng/mL), which was inhibited by AMD3100, a potent antagonist of CXCR4. We conclude that other HDI such as TSA regulate CXCR4 expression in HSPC by chromatin remodelling and we suggest that priming of HSPC with HDI may improve their homing and engraftment into bone marrow, especially in CB transplantation.

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1505-1505
Author(s):  
Wendy W. Pang ◽  
Elizabeth A. Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Expression Profiles ◽  
Frequency Function ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1284-1284
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
James Cormier ◽  
Junling Wang ◽  
Xuejun Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Myeloid Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Stem And Progenitor Cells ◽  
Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures: No relevant conflicts of interest to declare.

Nucleophosmin Regulates Differentiation, Cell Cycle Progression, and Stress Response in Hematopoietic Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 312-312
Author(s):  
June Li ◽  
Daniel P. Sejas ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stress Response ◽  
Progenitor Cells ◽  
Cell Cycle Progression ◽  
Proliferative Potential ◽  
Hematopoietic Progenitors ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Empty Vector

Abstract Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Strong evidence indicates that NPM is involved in hematopoiesis and leukemic development. Here we report that NPM enhances the proliferative potential of hematopoietic stem/progenitor cells and increases cell survival upon stress challenge. Specifically, lin-Sca1+c-kit+ bone marrow cells transduced with retroviral vector expressing NPM exhibited higher proliferative rates in both short-term liquid culture and clonogenic progenitor cell assays, compared to the cells transduced with empty vector. Interestingly, NPM overexpression appears to inhibit differentiation of myeloid progenitors. Hematopoietic stem/progenitor cells infected with the NPM retrovirus expressed significantly lower levels of mature cell markers Gr-1 and Mac-1 compared to empty vector transduced cells, and majority of the NPM-overexpressing cells remained Sca1+C-Kit+ during the 5-day culture. Bone marrow transplantation experiments demonstrated that NPM overexpression increases long-term multi-lineage repopulating capacity of hematopoietic progenitors. We have not observed any evidence of proliferative disorders or leukemia in recipients transplanted with NPM-expressing progenitors thus far (4 months posttransplantation). Through cell-cycle profile analysis and single-cell division experiments, we showed that NPM overexpression induces rapid entry of hematopoietic progenitors into the cell cycle, probably via promoting G0/G1 to S transition. Furthermore, immunocytochemical and Western-blot analyses demonstrated that NPM-transduced cells expressed higher level of cyclin A compared to vector-transduced cells. Finally, overexpression of NPM significantly increased the survival of hematopoietic progenitors exposed to mitomycin C or hydrogen peroxide, suggesting that NPM can protect cells from DNA damage and oxidative stress. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of progenitor differentiation, cell cycle progression, and stress response.

Differential hMLH1 Gene Expression after Temozolomide Selection Linked to Microsatellite Instability in a Subset of Hematopoietic Stem/Progenitor Cells in Old Versus Young and Cancer Versus Normal Patient Samples.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 509-509
Author(s):  
Jonathan Kenyon ◽  
Emily Thomas ◽  
Karen Lingas ◽  
Stanton L. Gerson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Microsatellite Instability ◽  
Progenitor Cells ◽  
Mismatch Repair ◽  
Dna Methyltransferase ◽  
Apoptotic Cell ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract The etiology of hematologic pathologies such as leukemia, myelodysplasia, anemia, bone marrow failure, altered immune function, and how they are associated with aging, remains unclear. Our hypothesis is that these diseases are caused or aggravated by a subset of hematopoietic stem/progenitor cells (HSC) lacking effective mismatch repair (MMR) and therefore exhibiting a hypermutator phenotype. Microsatellite instability (MSI) is a marker of MMR deficiency. We used cord blood, bone marrow, and bone core samples to isolate and then clonally expand HSC for MSI analysis. Five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were analyzed for insertions and deletions. We have analyzed 38 patient samples between the ages of 0 and 86 years, including 8 cancer patients. These data show an age-dependent increase in the frequency of high grade microsatellite instability (MSI-H), i.e. those CFU with microsatellite instability at >20% of loci tested. Additionally, samples obtained from individuals older than 50 years were 6 times more likely to have a > 10% frequency of MSI-H CFU than samples obtained from younger individuals, suggesting an inflection point for the onset of hematopoietic diseases. In all instances this instability is seen only within a subset of human HSC clones. To further characterize the origin of this deficiency, a method to select for MMR deficient hematopoietic cells was developed that first selected for survival of MMR deficient HSC, and then allowed for the examination of expression status of key MMR pathway genes hMLH1 and hMSH2 and their protein products. First, CD34+ HSC were isolated from various aged patient samples. To avoid possible effects of other repair pathways, the cells were treated with O6-Benzylguanine (BG) to remove O6-methylguanine DNA-methyltransferase (MGMT) activity and prevent removal of O6-methylguanine lesions. Next, temozolomide (TMZ) at concentrations of 50–125 μM was used to induce O6-methylguanine (O6-mG) lesions that persist in the presence of BG. These O6-mG lesions mispair with cytosine and are recognized as DNA mismatches by the mismatch repair (MMR) pathway inducing apoptotic cell death. TMZ selected cells that fail to recognize the mispair due to a lack of MMR survive this selection. In these TMZ resistant clones, RT-PCR amplification of hMLH1 transcripts from total RNA isolated reveal a defect in hMLH1 but not hMSH2 expression. In the one AML sample obtained thus far HSC treated with 200 uM TMZ we have observed 0 to 30% of hMLH1 expression within TMZ resistant CFU was observed when compared to untreated controls. Together this data links MSI to MMR defects of a subpopulation of hematopoietic precursors in older individuals. This is the first examination of MMR gene expression in clones of HSC that has shown specific MMR functional deficiencies. Our study suggests that a MMR pathway deficiency in a subset of stem cells could contribute to age related hematopoietic disease processes including stem cell failure and malignant transformation.

scholarly journals Neogenin-1 distinguishes between myeloid-biased and balancedHoxb5+mouse long-term hematopoietic stem cells

2019 ◽  
Author(s):  
Gunsagar S. Gulati ◽  
Monika Zukowska ◽  
Joseph Noh ◽  
Allison Zhang ◽  
Rahul Sinha ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Surface Marker ◽  
Cycle Phase ◽  
Hematopoietic Stem ◽  
Prospective Isolation

ABSTRACTHematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single-cell transplants (1–3) and lineage tracing (4, 5) suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled withHoxb5, a specific reporter of long-term HSCs (LT-HSCs) (6). We show that NEO1+Hoxb5+LT-HSCs expand with age and respond to myeloablative stress, while NEO1−Hoxb5+LT-HSCs exhibit no significant change in number. NEO1+Hoxb5+LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1−Hoxb5+LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution, while NEO1−Hoxb5+LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression comparison reveals increased expression of cell cycle genes and evidence of lineage-priming in the NEO1+fraction. Finally, transplanted NEO1+Hoxb5+LT-HSCs rarely generate NEO1−Hoxb5+LT-HSCs, while NEO1−Hoxb5+LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced, NEO1−Hoxb5+LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+LT-HSCs.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) are rare cells that have the unique ability to regenerate themselves and produce all blood cells throughout life. However, HSCs are functionally heterogeneous and several studies have shown that HSCs can differ in their contribution to major blood lineages. In this study, we discovered that the surface marker, Neogenin-1, can divide mouse HSCs into two subpopulations—one that is more active but biased towards producing myeloid cells and another that is more dormant and capable of equally producing all blood lineages. Neogenin-1 reveals the diversity and hierarchical relationship of HSCs in the mouse bone marrow, enables the prospective isolation of myeloid-biased and balanced HSCs, and opens opportunities to do the same in humans.

scholarly journals Short-Term Fasting Induces Cell Cycle Arrest of Immature Hematopoietic Cells and Increases the Number of Naive T Cells in the Bone Marrow of Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5047-5047
Author(s):  
Teruhito Takakuwa ◽  
Yasuhiro Nakashima ◽  
Hideo Koh ◽  
Takahiko Nakane ◽  
Hirohisa Nakamae ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Progenitor Cells ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Short Term ◽  
Hematopoietic Stem ◽  
And Control

Abstract Calorie restriction has long been studied not only as a way to prolong longevity but also as an approach to improve cancer prevention and treatment. Dietary restriction may prevent senescence of the immune and hematopoietic systems. In addition, short-term fasting before chemotherapy can reduce hematological toxicity in cancer patients. We studied the influence of fasting on immune cells, hematopoietic stem cells, and progenitor cells in the bone marrow and spleen of mice. Six-to-twelve-week old C57BL/6 mice were starved for 48 hours before analysis. We collected bone marrow and splenic cells from starved and control mice. After 48 hours of fasting, the body weight significantly decreased by an average of 24.1% compared to that of normal control mice. Calorie restriction caused a significant decrease in peripheral white blood cell count by an average of 48.3%, but hemoglobin level and platelet count were less affected. The averaged total number of bone marrow cells in the fasting group was significantly lower than that in the normal control group (2.45×107 versus 3.14×107, P < 0.01). In fasted mice there was a significant reduction in the hematopoietic stem cell count, using detection based on the lineage- c-kit+ Sca-1+, compared to control mice (0.83×105 versus 1.24×105, P < 0.05). In contrast, there was no significant difference for progenitor cells detected based on the lineage- c-kit+ Sca-1- (6.81×105 versus 7.75×105, P = 0.11). We performed colony assays with bone marrow cells from fasted and control mice. There was no difference between the two groups for not only the primary colony assay but also for the secondary and tertiary assays. Annexin V and 7-AAD analysis by flow cytometry showed that the rates of early and late apoptosis were almost identical in hematopoietic stem cells and progenitor cells, on comparing fasted and control mice. Furthermore, DNA cell cycle analysis of progenitor cells showed that short-term fasting caused a significant increase in the percentage in G0/G1 phase (83.1% versus 70.7%, P < 0.05) and decreases in the S and G2/M phases. These results imply that immature bone marrow cells retained their proliferative capacity by maintaining cell cycle arrest after short-term fasting, a finding that may account for the protective effect of starvation against chemotherapy in cancer patients. Calorie restriction caused a significant decrease in B cells in bone marrow (5.38×106 versus 8.1×106, P < 0.05) and especially in the spleen (6.65×106 versus 33.0×106, P < 0.001), and also significantly decreased T cells in the spleen (3.91×106 versus 14.5×106, P < 0.01). To our surprise, we detected a remarkable increase in the number of T cells in the bone marrow of fasted mice (1.25×106 versus 0.91×106, P < 0.01). Of greatest significance CD44- naive CD8+ T cells were dramatically increased in fasted bone marrow (1.74×106 versus 0.47×106, P < 0.01), and CD44- naive CD4+ T cells were also increased (0.23×106 versus 0.07×106, P < 0.05). In contrast, the numbers of CD62L- CD44+ effector memory and CD62L+ CD44+central memory T cells were not substantially changed after starvation. The increased naive T cells had no activated markers and appear to have migrated into bone marrow in a resting state without proliferating there. Short-term fasting decreased the number of hematopoietic stem cells but progenitor cells remained in a relatively quiescent state, with a prolonged DNA cell cycle and retention of colony-forming capabilities. The number of T cells in the bone marrow of fasted mice also increased dramatically, especially naive CD8+ T cells which probably migrated in from other lymphoid tissues. These findings imply that immature hematopoietic cells and some lymphoid cells can survive starvation while maintaining their function. The mechanisms by which T lymphoid cells promptly accumulate in bone marrow during starvation are under investigation. Disclosures Koh: Pfizer: Consultancy, Honoraria. Nakane:Mundipharma KK: Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Hino:Nippon Shinyaku: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding; Alexion: Honoraria, Research Funding.

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