scholarly journals Rheumatoid Arthritis Causes Hematopoietic Stem Cell Reprogramming to Maintain Functionality

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2573-2573
Author(s):  
Taylor Mills ◽  
Giovanny Hernandez ◽  
Jennifer L Rabe ◽  
Susan Kuldanek ◽  
James Chavez ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Autoimmune Disease ◽  
Mouse Model ◽  
Inflammatory Cytokines ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Differentiation Pathways

Abstract Rheumatoid arthritis (RA) is a debilitating autoimmune disease resulting from autoantibodies that cause damage to synovial joints. Joint damage causes increased systemic inflammatory cytokines which may lead to aberrant hematopoiesis. Indeed, RA is accompanied by many hematological complications including anemia, cytopenias, and suppressed bone marrow function. Hematopoietic stem cells (HSC) at root of the blood system can respond to inflammatory signals by activating the cell cycle and preferentially generating myeloid cells. However, chronic inflammation can also lead to HSC dysfunction. Previous studies using genetic mouse models of RA have identified myeloid overproduction in this context; however, HSC long-term reconstitution activity was maintained. To better understand hematopoietic alterations in RA, our group used the collagen induced arthritis (CIA) mouse model, which is inducible in adult mice and recapitulates many immunological features of the human disease, including elevated inflammatory cytokine levels in the bone marrow (BM) and peripheral blood (PB). Confirming prior reports, we found increased numbers of myeloid lineage cells in the PB and BM of CIA mice. We also found reduced erythroid and lymphoid lineage progenitor cell numbers in the BM, consistent with anemia and immunosenescence phenotypes in RA patients. Interestingly, these features were accompanied by a significant increase in the number of myeloid-biased multipotent progenitor-3 (MPP3) cells, suggesting increased activation of myeloid differentiation pathways. However, we found no changes to the number of activated (MPP1), short-term HSC (ST-HSC), or long-term HSC (LT-HSC) in CIA mice. Likewise, and in line with previous reports, long-term HSC potential was not reduced in CIA mice, as assessed by transplantation of either purified HSC or with unfractionated BM into irradiated recipient mice. While HSC from control and CIA donor mice displayed similar blood chimerism and lineage distribution over a 16-week period, we did observe increased proportions of donor-derived MPP3 in CIA recipient animals, further supporting an activation of myeloid HSC differentiation pathways. Overall, these results reveal underlying changes in the BM driving aberrant hematopoiesis, with the HSC pool remaining intact despite activation of a myeloid differentiation pathway. To better understand how HSC are impacted by arthritic inflammation, we assessed the molecular state of control and CIA HSC using RNA-seq. We found 292 genes significantly upregulated and 237 genes significantly downregulated in CIA HSC. Analysis of these genes using Ingenuity, GSEA, and DAVID tools revealed broad downregulation of inflammatory and proliferation signaling pathways including IL-1β, NFκB, MYC, and ERK. Genes for G1/S cell cycle transition, transcription, protein translation, and proliferation pathways were also significantly downregulated in CIA HSC. On the other hand, genes corresponding to cell cycle arrest and negative regulation of transcription were significantly upregulated. Notably, we find that IL-1β, which is produced in the BM of CIA mice, is sufficient to induce this molecular program. We find that HSC in CIA mice have a global downregulation of transcripts required for activation and proliferation, and a global upregulation of transcripts that would promote quiescence. Hence, HSC are forced back into a quiescent state by broad downregulation of cell growth and proliferation genes even during chronic inflammation caused by RA. Altogether, our data show that a mouse model of rheumatoid arthritis causes hematopoietic lineage skewing towards the myeloid lineage with simultaneous loss of lymphoid and erythroid lineage potential. Interestingly, in this chronic inflammatory setting HSC downregulate pathways involved in cytokine signaling, cell cycle activation, and translation. This mechanism can be triggered by chronic exposure to pro-inflammatory cytokines, and may serve to limit HSC proliferation and potential for damage in disease settings. These results may explain the relative rarity of outright bone marrow failure in autoimmune disease patients, while providing insight into mechanisms driving aberrant hematopoiesis in these individuals. Lastly, they provide functional evidence for cytokine blockade to normalize HSC function in the setting of RA and other chronic inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

VEGFR-1 (Flt-1) Tyrosine Kinase Signaling Enhances Hematopoiesis, Proliferation/Differentiation and Immunity of Monocyte/Macrophage from Bone Marrow Hematopoietic Stem Cells, and Promotes Rheumatoid Arthritis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 778-778
Author(s):  
Masato Murakami ◽  
Shinobu Iwai ◽  
Sachie Hirasuka ◽  
Yoichiro Iwakura ◽  
Yoshiro Maru ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Vascular Tissue ◽  
Systemic Disease ◽  
Bone Destruction ◽  
Inflammatory Cells ◽  
Vegf Receptor ◽  
Hematopoietic Stem

Abstract VEGF and its receptor family including VEGFR-1(Flt-1) are well known to be a crucial regulatory system for normal development and pathological angiogenesis. Rheumatoid arthritis(RA) is a chronic systemic disease characterized by an inflammatory erosive synovicitis, which show marked neovascularization, inflammatory cell infiltration and synovial hyperplasia, then produce a pannus of inflammatory vascular tissue and lead to irreversible cartilage and bone destruction. We have already shown VEGFR-1 is expressed not only in vascular endothelial cells but also in inflammatory cells, especially in monocyte/macrophage. A recent report suggests the involvement of VEGFR-1 in RA by using collagen induced RA mouse model. To examine whether the signaling from VEGFR-1 is important for the pathological process of RA, we used VEGFR-1 tyrosine kinase(−/−) mice which cannot generate the signaling from this receptor, and an arthritis mouse model system carrying Human T-cell leukemia virus(HTLV-1) pX transgene. VEGFR-1 TK(−/−) mice with pX gene clearly showed a reduction in the incidence and the degree of clinical symptom of arthritis. Furthermore, the heterozygote VEGFR-1 TK(+/−) with pX transgene showed a partial decrease in the degree of clinical as well as pathological scores. To explain the reason of reduction of clinical symptoms, we investigated involvement of VEGFR-1 TK signal in lineage of bone marrow hematopoietic stem cell(HSC) to monocyte/macrophage proliferation and differentiation and their immunity. VEGFR-1 TK activities are not associated in number of HSC in bone marrow. However, VEGFR-1 TK(−/−) HSC toward multi-lineage proliferation is suppressed in colony-formation. In addition, failures of monocyte/macrophage faculties are observed in immunological reaction, phagocytosis, cytokine secretion(IL-6, VEGF) and migration. Furthermore, expressions of hematopoiesis and inflammation related genes in VEGFR-1 TK(−/−) macrophage are downregulated by microarray analysis. Next we treated with small molecule inhibitors of VEGF receptor(VEGFR-1 and VEGFR-2) of tyrosine kinase, KRN951, in RA model(pX transgenic model and type II collagen Ab cocktail model) for treatment. Treatment with KRN951 strongly attenuated the disease symptom through inhibiting recruitment of BM hematopoietic cells into peripheral inflammatory cells. These observations indicate that VEGFR-1 signals play an important role in both RA mouse model. The tyrosine kinase activity and the signaling of VEGFR-1 enhances hematopoiesis, proliferation/differentiation and immunity of monocyte/macrophage from bone marrow HSC, and promotes rheumatoid arthritis, which may be a new possibilities for the treatment of RA in humans. Figure Figure

Cycling Marrow Stem Cells Are Lost with Purification.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2308-2308
Author(s):  
Laura R Goldberg ◽  
Mark S Dooner ◽  
Mandy Pereira ◽  
Michael DelTatto ◽  
Elaine Papa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tritiated Thymidine ◽  
Hematopoietic Stem ◽  
Cell Purification ◽  
Marrow Stem Cell ◽  
Marrow Cells

Abstract Abstract 2308 Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4354-4362 ◽  
Author(s):  
Nobuko Uchida ◽  
Annabelle M. Friera ◽  
Dongping He ◽  
Michael J. Reitsma ◽  
Ann S. Tsukamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Number ◽  
Short Term ◽  
Synthesis Inhibitor ◽  
Hematopoietic Stem ◽  
Cell Cycle Status

Abstract The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

scholarly journals IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Yang Mei ◽  
Yijie Liu ◽  
Xu Han ◽  
Jing Yang ◽  
Peng Ji
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Inflammatory Cytokines ◽  
Conflicts Of Interest ◽  
Double Knockout ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Age Related

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3596-3606 ◽  
Author(s):  
Troy D. Randall ◽  
Irving L. Weissman
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Population ◽  
Stem Cell Population ◽  
Normal Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Bone

Abstract A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

Irradiated Bone Marrow Environment Impairs Hematopoietic Stem Cells by Exposure to Tumor Necrosis Factor a.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3724-3724
Author(s):  
Sachie Suzuki ◽  
Motohito Okabe ◽  
Makoto Otsu ◽  
Hideo Ema ◽  
Hiromitsu Nakauchi
Keyword(s):  
Bone Marrow ◽  
Inflammatory Cytokines ◽  
Inhibitory Effect ◽  
Negative Effects ◽  
Hematopoietic Stem ◽  
Post Irradiation ◽  
Transplantation Outcomes ◽  
Peak Phase

Abstract Abstract 3724 Background: Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for various disorders including hematopoietic malignancies. Most HSCT requires cytoreductive conditioning such as total body irradiation (TBI) and/or chemotherapy to ensure engraftment of HSCs by emptying recipients' marrow niches. Our preliminary experiments, however, revealed that TBI could induce local inflammation peaking around days 2–3 within marrow environment. Of note is that conditioning regimens widely used in the current HSCT settings are mostly compatible with HSC exposure to inflammatory storm in post-irradiation bone marrow (BM). Although certain inflammatory cytokines have been shown to affect HSC functions in in vitro, it remains unknown whether in vivo exposure to inflammatory BM environment can alter the characteristics of transplanted HSCs. We therefore sought to investigate what effects irradiated BM environment would have on transplanted donor HSCs using murine systems. Methods and Results: We first tested whether infusion of HSCs at varying timing post TBI would affect transplantation outcomes and HSC functions. To this end, fifty HSCs (CD34-negative/low, cKit+, Sca-1+, lineage marker-negative cells) obtained from Ly5.1-B6 mice were transplanted into lethally irradiated Ly5.2-B6 mice with 1 × 106 competitor Ly5.1/5.2-B6 BM cells at day 0, 3, or 5 post irradiation. No mice survived long-term in the group that received transplants 5 days post irradiation, indicating insufficient HSC engraftment and hematopoietic reconstitution. Although hematopoiesis reconstituted in long term survivors was comparable between another two groups (d0 and d3), we found significant difference in donor HSC ability when tested in a competitive repopulation assay using Ly5.1-KSL cells sorted from primary recipients: Test HSCs obtained from mice transplanted 3 days after irradiation showed poor secondary reconstitution ability, suggesting alteration of pre-engraftment HSC functions depending on transplantation protocols. We then tested whether in vivo exposure of HSCs to irradiated BM environment would have negative effects on HSC functions. Test HSCs (400 cells) from Ly5.1-B6 mice were transplanted into Ly5.2-B6 primary recipients at varying time points (day 0, 1, 2, or 3) after lethal-dose TBI. Approximately 24 h later, BM samples were subjected to a competitive repopulation assay to test secondary reconstitution ability in test HSCs that homed to irradiated BM environment. Consequently, test HSCs that were exposed for ≂f24 h to BM environment at day 2 post TBI showed marked impairment in their long-term reconstitution ability. When “BM-homed” HSCs were enumerated 24 h after infusion, we found modestly impaired HSC homing to BM irradiated 2 days prior to transplantation, compared to BM irradiated on day 0, indicating negative effects on HSCs transplanted during a peak phase of inflammatory storm. Next we examined whether inflammatory cytokines were capable of impairing HSC functions. When tested in liquid culture using purified HSCs, both IFN-g and IL-1 had little effects on colony formation. In contrast, TNF-a inhibited colony formation in a dose-dependent manner. When in vivo HSC exposure to irradiated BM environment was tested using TNF-a KO mice as primary recipients, significant improvement of HSC ability was observed, indicating a major role for this inflammatory cytokine in the HSC-inhibitory effect observed within the irradiated host tissues. We finally sought to test if blocking of TNF-a signaling in HSCs at a peak phase of inflammation could lead to better transplantation outcomes. We utilized the peptide previously shown to block TNFR signaling, and confirmed that pre-incubation of HSCs with this molecule did suppress TNF-a induced reactive oxygen species production in HSCs. Studies are ongoing to test if HSCs transplanted in a peak-phase of TBI-mediated inflammation will benefit from this shielding measure. Conclusion: We demonstrated that inflammatory response in BM environment 2–3 days after irradiation could have a negative effect on donor HSCs regarding both homing efficiency and secondary reconstitution ability. These findings provide important implications for developing the measures that enable HSCs to escape from this inhibitory effect to achieve far more improvement in clinical HSCT outcomes. Disclosures: No relevant conflicts of interest to declare.

HDAC8 Regulates Long-Term Hematopoietic Stem Cell Quiescence and Maintenance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1468-1468
Author(s):  
Wei-Kai Hua ◽  
Jing Qi ◽  
Qi Cai ◽  
Emily Carnahan ◽  
Ling Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Poly I:C ◽  
Control Group ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Polycytidylic Acid ◽  
Deficient Mice

Abstract Long-term (LT) hematopoietic stem cells (HSC) are responsible for life-long production of mature blood cells of all lineages through tightly concerted cell fate decisions including quiescence, self-renewal, differentiation and apoptosis. Histone deacetylase 8 (HDAC8) is a member of class I HDAC enzymes that remove acetyl moieties from lysine residues on histones and a variety of non-histone proteins. Specifically, HDAC8 has been shown to modulate the acetylation cycle of cohesin complex protein SMC3. Loss-of-function mutations in HDAC8, located on the X chromosome q13, have been found in patients with Cornelia de Lange Syndrome (CdLS) and those with CdLS-like features. These HDAC8 mutations are associated with severely skewed X-inactivation (100% wild type allele) in the peripheral blood of female patients, possibly due to selection against the mutant alleles. However, the expression and function of HDAC8 in normal HSCs and hematopoiesis remain unknown. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin-cKit+Sca1+CD150+CD48-) population in adult mice. To determine the function of HDAC8 in adult hematopoiesis, we generated conditional Hdac8 deficient mice using the Mx1-Cre and a floxed Hdac8 allele (Mx1-Cre/Hdac8f/f(y)) andconfirmed that Hdac8 is successfully deleted by polyinosinic-polycytidylic acid [poly (I:C)] treatment. Phenotypic analysis of Hdac8 deficient mice showed increased LT-HSC population compared to similarly treated control mice. However, largely normal steady state hematopoietic profile was found in Hdac8 deficient mice at 6 weeks and 1 year after induction. To further track Hdac8-deleted cells, we generated Cre/Hdac8f/f(y) mice with a dual fluorescence Rosa26mT/mG (mT/mG) Cre reporter allele, which expresses dTomato prior to Cre induction and becomes GFP+ after Cre-mediated recombination. We assessed hematopoietic repopulation by transplanting bone marrow cells from Cre/Hdac8f/f(y)/mTmG+mice (2 x 105) together with wild type support cells (2 x 105) into lethally irradiated CD45.1+ congenic recipients. Hdac8 deletion was induced by treating the recipients with 7 does (14 m▢g/kg/dose) of poly (I:C). We found that Hdac8 deletion did not affect CD45.2+ or GFP+ donor-derived overall engraftment or lineage repopulation up to 16 weeks. There was also no change in the frequency or number of GFP+ donor-derived phenotypic LT-HSCs in the bone marrow. Serial transplantation was performed to further assess long-term repopulating activity of HSCs. Hdac8 deficient cells were significantly (p=0.019; n=3) compromised in multi-lineage repopulation in secondary transplant recipients. Except a modest reduction in Pre-GM, there was no change in the overall composition of Hdac8 deficient CD45.2+-derived populations. Upon tertiary transplantation, no donor engraftment was observed for Hdac8 deficient cells (0 out of 4) compared to 50% positive engraftment in control group (4 out of 8). These results indicate that HDAC8 is crucial for maintaining long-term serial-repopulating activity over time. Cell cycle analysis revealed that Hdac8 deficient LT-HSCs display reduced quiescence and increased cycling, consistent with the increased number of phenotypic LT-HSC seen in Hdac8 deleted mice. Therefore, we further tested the sensitivity of Hdac8 deficient mice to serial ablation with 5-fluorouracil (5FU), an S phase-specific cytotoxic chemotherapeutic agent. Impaired hematopoietic recovery and increased lethality (p<0.001; n=23) was seen in Hdac8 deficient mice treated with 5-FU (100 mg/kg) every 7 days, indicating that Hdac8 deletion renders hypersensitivity to serial ablation. There were significnatly less phenotypic LT-HSCs in Hdac8 deficient mice 6 days after 5-FU treatment (p<0.01; n=4). In parallel, we observed increased DNA strand beaks as indicated by γ-H2AX staining and comet assays (p<0.001; n>100 cells). Analysis of p53 activation, cell cycle regulators and DNA dmage response are ongoing. Collectively, our study indicates that HDAC8 plays a pivotal role in LT-HSC quiescence and maintenance. Disclosures No relevant conflicts of interest to declare.

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 Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3596-3606 ◽  
Author(s):  
Troy D. Randall ◽  
Irving L. Weissman
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Population ◽  
Stem Cell Population ◽  
Normal Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Bone

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

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