Transcription Factor Bach1 and Bach2 Operate Erythro-Myeloid Competitive Differentiation By Responding to Environmental Changes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2649-2649
Author(s):  
Hiroki Kato ◽  
Ari Itoh-Nakadai ◽  
Mitsuyo Matsumoto ◽  
Risa Ebina-Shibuya ◽  
Yuki Sato ◽  
...  
Keyword(s):  
Target Genes ◽  
Environmental Changes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Myeloid Differentiation ◽  
Dependent Manner ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Expression Levels

Abstract Hematopoietic system is maintained by the differentiation and proliferation of hematopoietic stem/progenitor cells (HSPCs) and their commitment to the mature blood cells should be tightly controlled by gene regulatory networks (GRNs) governed by transcription factors (TFs). To keep the homeostasis, GRNs should respond to the environmental changes, such as infection. However, the precise mechanism of such a system remains to be elucidated. TFs Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements containing AP-1 site (Oyake et al., 1996). We have previously shown that Bach1-/-Bach2-/-(DKO; double knockout) mice show erythropoiesis disorders with increased myelopoiesis from common myeloid progenitors (CMPs), which is an erythro-myeloid bifurcation point (ASH2015; Abstract ID# 81562) (Akashi et al., 2000). Since this phenotype is similar to that of LPS treated mice (O'Connell et al., 2008), we hypothesized that Bach factors work as sensors for infection. First, to evaluate the cell-intrinsic function of Bach factors, WT or DKO bone marrow cells were depleted of mature differentiated cells and transplanted to lethally irradiated WT mice. After 8 weeks, DKO donor cells showed greater myelopoiesis and lesser lymphogenesis compared to WT, suggesting Bach factors are necessary to suppress myelopoiesis to the appropriate level in regenerating hematopoiesis. To reveals the function of Bach factors in HSPCs from other aspect, LSKs (Lin-Sca1+c-kit+) were infected with retroviruses expressing Bach1-IRES-eGFP or Bach2-IRES-eGFP and transplanted to lethally irradiated WT mice. Cells derived from transgene induced LSKs were monitored by GFP fluorescence. After 2 weeks, Bach1 overexpressing LSKs did not show any difference in erythropoiesis and myelopoiesis. This might be explained by the high Bach1 expression levels in HSPCs according to the previous report (Lara-Astiaso et al., 2014). On the other hand, Bach2 overexpressing LSKs showed increased erythropoiesis and decreased myelopoiesis, suggesting that Bach2 regulates the erythro-myeloid lineage specification as expected by the observations of DKO mice. To assess the function of Bach factors under infection, we used M1 murine myeloid leukemia cells that differentiate to macrophage-like cells by LPS stimulation. LPS stimulation reduced expressions of Bach1, Bach2 and erythroid gene Gata1, and induced those of myeloid genes such as Cebpb and Csf1rin a dose-dependent manner. To determine if down-regulation of Bach factors is necessary for myeloid differentiation, Bach1 or Bach2 were transgenically overexpressed in M1 cells. Both of the M1 cells overexpressing Bach1 or Bach2 showed lower expression levels of myeloid marker CD11b compared to control under LPS stimulation. Thus, reductions of the expression of Bach factors in response to LPS were necessary for appropriate myeloid differentiation. To identify the direct target genes of Bach factors, Bach1 or Bach2 ChIP-seq data of M1 cells (Ebina-Shibuya et al., 2016) were merged with results of expression profile of WT and DKO CMPs. Several myeloid or inflammatory genes such as Cebpb, Fcgr1 and Tlr4 were identified as putative repressed target genes and several erythroid or lymphoid genes such as Klf1, Rag1 and Rag2 were identified as putative activated target genes. In addition, when Bach1 or Bach2 ChIP-seq data were merged by that of C/EBPb, which also possesses AP-1 site as its target motif, obtained from ENCODE database (ENCSR000AIB), we found that there were several co-localized regions of Bach and C/EBPb near the myeloid genes such as Cebpa, Il6 and Fcgr1. These observations suggest that Bach factors repress myeloid genes by competitively working with C/EBPb at same genomic regions. This is particularly interesting in the light of the latest findings showing the Bach2 function on AP-1 site in lymphoid cells (Sidwell et al., 2016). These results reveal a novel mechanism by which how the differentiation of erythro-myeloid bifurcation is controlled by responding to environmental changes. Bach factors regulate erythro-myeloid competitive differentiation by promoting and repressing erythroid and myeloid differentiation, respectively. We suggest that infection promote myelopoiesis at the expense of erythropoiesis by reducing the expression of Bach factors. Therefore, Bach factors may function as sensors for environmental changes. Disclosures No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Myosin-II Is a Major Modulator of Human Hematopoietic Stem Cell Proliferation and Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2343-2343
Author(s):  
Jae-Won Shin ◽  
Amnon Buxboim ◽  
Dennis E Discher
Keyword(s):  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myosin Ii ◽  
Dependent Manner ◽  
Specific Flow ◽  
Hematopoietic Stem ◽  
Matrix Elasticity ◽  
Proliferation And Differentiation

Abstract Abstract 2343 Non-muscle myosin-II (NMM-II) promotes cell division, membrane rigidity and adhesion to a rigid matrix, and so NMM-II activity might be predicted to be low in dormant hematopoietic stem cells (HSCs) and to increase with differentiation. Deletion of NMM-II is known to be embryonic lethal, but its role in adult HSC differentiation is not known. Recently, we showed that sustained pharmacological inhibition of NMM-II together with soft 2D matrices like the perivascular niches in marrow, rather than rigid like bone, maximizes both MK maturation and platelet generation (Shin et al., PNAS, 2011; 108:11458-63). HSCs exhibit some similarities to mature MKs in that long-term HSCs remain undivided in vivo while various progenitors and maturing cells rapidly expand in number. Here, reversible inhibition of NMM-II sustained over several cell cycles enriches long-term HSCs up to 20 fold by selective elimination of proliferating progenitors. CFSE dilution analysis indicates that inhibition of NMM-II eliminates the accumulation phase of hematopoietic progenitors and accelerates natural cell death rate by apoptosis. Interestingly, supplementation of G-CSF significantly enhances HSC survival under NMM-II inhibition and further accelerates progenitor elimination. Molecular profiling and functional analyses indicate that NMM-II isoforms play distinct roles during HSC differentiation. NMM-IIA is a marker for differentiation with significantly lower expression in HSCs than committed progenitors, which is consistent with greater membrane flexibility of HSCs measured by micropipette aspiration. In contrast, NMM-IIB is 5 fold higher in HSCs and progenitors than differentiated CD34− cells. HSC and progenitor numbers are also sensitive to matrix elasticity in a NMM-II dependent manner, with maximal expansion on soft and high-density fibronectin matrices (not collagen). However, upon NMM-II inhibition, the extent of HSC enrichment relative to multipotent progenitors is more sensitive to matrix ligand density than matrix elasticity. To identify physiological mechanisms of regulating NMM-II activity during early HSC differentiation, we investigated post-translational modifications of NMM-IIA, specifically the de-activating and isoform-specific phosphorylation at myosin Ser1943 (pS1943) in HSC and progenitors. In a phospho-specific flow cytometry approach, pS1943 level proves highest in HSCs and decreases during differentiation with Tpo and G-CSF but not SCF alone. TGF-beta inhibits the reduction of pS1943 level, consistent with TGF-beta's known role in HSC hibernation. Therefore, pS1943 level dictates HSC enrichment and parallels the dose-response to pharmacological NMM-II inhibitors. Furthermore, phospho-mimetic mutation of NMM-IIA at Ser1943 decreases cytoskeletal integrity, increases membrane flexibility, and limits matrix elasticity sensing, indicating that biophysical properties of HSCs can also be regulated by HSC-specific signaling via NMM-IIA heavy chain phosphorylation. Myosin-inhibited CD34+-derived bone marrow cells show reduced colony-forming unit progenitors in vitro, but maintain functional long-term HSCs in vivo in the marrows of xenografted mice with an added benefit to increase platelet circulation simultaneously. Therefore, myosin-II inhibition and soft, high ligand fibronectin constitutes an important ‘microenvironment mimetic’ approach to enrichment of long-term HSCs. Myosin-II is clearly a central, matrix-regulated node for HSC proliferation and differentiation. Disclosures: No relevant conflicts of interest to declare.

JAK2V617F Promotes Stem Cell Amplification Driving MPN Clonal Dominance in Mice and Treatment by IFNa Prevents This Effect

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 616-616 ◽  
Author(s):  
Caroline Marty ◽  
Catherine Lacout ◽  
Marie Cuingnet ◽  
Salma Hasan ◽  
Eric Solary ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Endogenous Expression ◽  
Long Term Treatment

Abstract Abstract 616 JAK2V617F is the major mutation involved in classic myeloproliferative neoplasm (MPN). It promotes growth factor independent cell growth and is able to recapitulate MPN features in retroviral, transgenic (TG) or knock-in (KI) mouse models. Several mutations implicated in epigenetic modifications or leukemic transformations have been also identified in MPN and several reports have questioned the particular role of JAK2V617F on hematopoietic stem cells (HSC) proliferation thus as a driver of MPN emergence. Therefore, we investigated the in vivo effect of an endogenous expression of JAK2V617F on early stages of differentiation and their ability to compete for normal cells in a repopulation assay. For this study, we develop a novel mouse conditional JAK2V617F KI model based on the “FLEX switch” strategy. These KI mice were crossed with TG mice expressing the Cre recombinase under the control of the vav promoter in order to restrict JAK2V617F expression to hematopoietic and some endothelial tissues. VavCre/JAK2+/V617F KI mice developed high hematocrit (70 ± 2 %, control values 49 ± 1 % n=13), platelet (2.3 ± 0.1 × 109 / mL, control values 0.84 ± 0.04 × 109 / mL n=20) and white blood cell (20-40 × 106/mL, control values between 6–10 × 106 / mL) values and a splenomegaly at 2–3 months of age but after 6 months of age an anemia and a thrombocytopenia appeared. This model mimics human polycythemia vera with secondary myelofibrosis. At 2–3 months of age, cumulative numbers in bone marrow (BM) and spleen of CFU-E, BFU-E and GM-CFC were increased 15-, 3-, 1.2–fold, respectively, compared to control. Most CFU-E grew without the addition of erythropoietin. A 6-fold amplification of total early progenitors LSK and a tendency toward SLAM (LSK/CD48−/CD150+) cell amplification, mainly due to a significant 9-fold increase in the spleen, were also observed. Competitive repopulation assays using 30% KI and 70% WT bone marrow cells demonstrated 17 weeks after BM transplantation (BMT) a rapid and strong amplification, from 30% to > 80%, of blood myeloid cells (Gr-1+/Mac1+) from KI origin. Late after transplantation (35 weeks), Lin-, LSK and SLAM cell compartments from KI origin raised from the initial 30% to almost 100% in the BM and even KI blood lymphoid cells (B220+ and CD3+) demonstrated a significant amplification compared to control. This shows that endogenous expression of JAK2V617F gives an advantage to HSC, promoting clonal dominance in mice. Then, we analyzed at which levels of differentiation acts IFNα, a drug promoting cycling of dormant cells and proven efficacious in PV treatment in human. In a chimeric model, we demonstrated that IFNα could prevent the development of MPN induced in vavCre/JAK2+/V617F KI recipient mice by inhibiting the amplification of KI cells. Secondary BMT from treated animals demonstrated the eradication of disease-initiating cells after long-term treatment. This study shows that IFNα acts at the level of the disease-initating cell by reverting the HSC promoting clonal dominance induced by JAK2V617F. Disclosures: No relevant conflicts of interest to declare.

Alda-1 Constrains TLR-Induced Cytokine Overproduction By Fanconi Anemia Macrophages By Enhancing ALDH1A1 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1000-1000
Author(s):  
Michael Garbati ◽  
Winifred Keeble ◽  
Wenjin Yang ◽  
Grover C. Bagby
Keyword(s):  
Inflammatory Cytokines ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Monocytic Leukemia ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Suppressive Function

Abstract Replication and survival of FA-deficient hematopoietic stem and progenitor cells (HSPC) are highly suppressed by exposure to inflammatory cytokines including TNFα, IL-1β, IFNγ, and MIP-1α. Additionally, FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce the very inflammatory cytokines that exhaust the stem cell pool. Recent evidence using double knockout mice suggests that aldehyde dehydrogenases (ALDH) may play a role in protecting FA HSPC but the mechanisms involved are unclear. We tested the hypothesis that the TLR-dependent overproduction of inflammatory cytokines in FA macrophages results from a loss of FA protein-dependent ALDH function. Control (T-shNT) or FANCC-deficient (T-shFC) THP-1 human monocytic leukemia cells were treated with Alda-1 (a small molecule ALDH agonist known to enhance the activity of both ALDH1A1 and ALDH2), before exposing them to the TLR-7/8 agonist R848 and quantifying the production of TNFα (ELISA of 24- culture supernatants). While R848 alone induced a 6-fold increase in TNFα production by the FANCC-deficient cells, Alda-1 suppressed TLR-induced TNFα production in a dose-dependent manner, by both T-shNT and T-shFC cells (Fig. 1). To identify the ALDH isoform that played a role in the suppression of TNFα production, we ectopically expressed ALDH1A1 or ALDH2 in T-shNT and T-shFC cells. Overexpression of ALDH2 had little effect on R848-induced TNFα production, but ALDH1A1 overexpression completely suppressed TNFα production by FANCC-deficient cells and suppressed (by approximately 50%) the production of TNFα by control cells (Fig. 2). Likewise, in loss-of-function studies, siRNA knockdown of ALDH1A1 (but not ALDH2) enhanced R848-induced production of TNFα (1.8-fold) in T-shNT cells but did not enhance TNFα overproduction in either FANCC-deficient (T-shFC) or FANCA-deficient (T-shFA [FANCA knockdown]) cells. Additionally, ALDH1A1 (but not ALDH2) mRNA was highly inducible in both THP-1 cells (by R848) and in Lin- Sca-1+ Kit+ murine marrow cells (by TNFα). While ALDH1A1 mRNA is expressed similarly in both T-shNT and T-shFC cells, our results indicate that in normal macrophages ALDH1A1 plays a role in the constraining TLR-induced TNF production but is significantly less functional in FANCC-deficient macrophages. In summary, (1) either pharmacological enhancement of ALDH function with Alda-1 or overexpression of ALDH1A1 is sufficient to restore the TLR-suppressive function of ALDH1A1 in FANCC-deficient macrophages, and (2) specific suppression of ALDH1A1, (but not ALDH2) induces an FA-like phenotype in control macrophages. We conclude that (1) optimal function of ALDH1A1 is FANCC- and FANCA-dependent in normal macrophages, (2) TLR-dependent overproduction of inflammatory cytokines by FA-deficient macrophages may result either from an increase in aldehyde load or the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3) enhancement of ALDH activity using small molecule agonists such as Alda-1 may alleviate the FA macrophage/cytokine phenotype and thereby protect HSC from inflammation-induced exhaustion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ing4-Deficiency Enhances Hematopoietic Stem Cell Quiescence and Confers Resistance to Inflammatory Stress

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1095-1095
Author(s):  
Zanshé Thompson ◽  
Georgina A Anderson ◽  
Seth Gabriel ◽  
Melanie Rodriguez ◽  
Vera Binder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Loss Of Function ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tyrosine Kinase-Directed Ubiquitin Ligases Cbl and Cbl-b Enforce Hematopoietic Stem Cell Quiescence By Negatively Regulating c-Kit and FLT3

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4313-4313
Author(s):  
Wei An ◽  
Scott Nadeau ◽  
Bhopal Mohapatra ◽  
Dan Feng ◽  
Neha Zutshi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Ubiquitin Ligases ◽  
Fine Tuning ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Mechanistic Basis

Abstract Maintenance of quiescent hematopoietic stem cells (HSCs) is essential for life-long hematopoiesis. A prominent category of HSC quiescence regulators that determine HSC fate by directly interacting with niche-derived growth factors are receptor tyrosine kinases (RTKs). Cbl and Cbl-b are E3 ubiquitin ligases that are directed to activated tyrosine kinases and negatively regulate a number of cellular activation pathways. Previously, we established a conditional Cbl and Cbl-b double knockout (DKO) mouse strain using MMTV-Cre to delete floxed Cbl on a Cbl-b-null background, and identified that Cbl and Cbl-b function redundantly in controlling the growth factor-induced proliferation of HSCs. These DKO mice developed a rapidly fatal myeloproliferative disorder (MPD) accompanied by expansion of the HSC compartment. However, how the negative regulatory functions of Cbl-family proteins are integrated into HSC homeostatic program and the mechanistic basis for their role remain unknown. Here, we utilize mouse models to examine the functional role of Cbl and Cbl-b in regulating HSCs and its potential mechanistic basis. Transplant analyses revealed that DKO HSCs are the disease-initiating cells. However, in vitro serial colony-forming assays showed that DKO HSCs possess a reduced colony-forming ability despite their hyper-proliferative status. Cell cycle analyses demonstrated a smaller pool of quiescent long-term HSCs in DKO mice. Non-responder assays in vivo showed a reduced frequency of functional HSCs in the DKO LSK compartment and serial HSC transplantation demonstrated compromised reconstitution ability of DKO HSCs, especially at the 2nd round of transplantation. Mechanistically, DKO HSCs exhibit sustained signaling in response to c-Kit and FLT3 ligands, especially via p-Akt but also p-Erk and p-S6. The ligand-induced cell surface c-Kit and FLT3 receptor downregulation was slower in DKO HSCs. Furthermore, while c-Kit and FLT3 ligands promote higher proliferation of DKO bone marrow cells, this hyper-proliferation leads to loss of colony-forming potential that is significantly reversed by respective kinase inhibitors. Together, our data reveal a novel and physiologically essential role of Cbl and Cbl-b in the enforcement of HSC quiescence and protection against exhaustion by fine-tuning the signaling pathways downstream of tyrosine kinase-coupled receptors such as c-Kit and FLT3. These findings could have significant implications for hematological neoplasms associated with mutations of Cbl. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2641-2641
Author(s):  
Lorena Lobo Figueiredo-Pontes ◽  
Robert S. Welner ◽  
Miroslava Kardosova ◽  
Hong Zhang ◽  
Meritxell Alberich-Jorda ◽  
...  
Keyword(s):  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cytokine Production ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Nk Cytotoxicity

Abstract Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function. By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group. In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production. Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment. Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mutations in the Telomerase Complex and Expression Levels of the TERT Gene Determine Severity and Outcome of Disease in Aplastic Anemia Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1502-1502 ◽  
Author(s):  
Arati Khanna-Gupta ◽  
Durga Sarvepalli ◽  
Snigdha Majumder ◽  
Coral Karunakaran ◽  
Malini Manoharan ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Disease Severity ◽  
Poor Prognosis ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Response To Therapy ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Shelterin Complex ◽  
Tert Expression

Abstract Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complexes have been found in both congenital and idiopathic AA. In particular, several TERT and TERC mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo. Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have recently demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012, 14, 1270). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia. In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient samples were collected at a tertiary medical center in Bangalore, India. Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TERT expression compared to that of normal controls (n=6). An 8 fold reduction in TERT expression was observed in 17/24 patients, while 7/24 patients maintained normal TERT expression. In general, TERT-low patients were younger in age (mean age 29y) compared with the TERT-normal patients (mean age 40y). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy. Targeted panel sequencing of the 24 samples on an Illumina platform revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients carried predicted pathogenic variants in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes, all associated with the telomerase complex. Somatic gene variants were also identified in other AA associated genes, PRF1 and CDAN1, in the TERT-low cohort. In addition, novel predicted pathogenic mutations associated with the shelterin complex were found in two TERT-low patients in the TNKS gene. We also detected mutations in TET2, BCORL1, FLT-3, MLP and BRAF genes in TERT-low patients. Mutations in these genes are associated with clonal evolution, disease progression and poor prognosis. Our observations were further illustrated in a single patient where normal TERT expression was noted at initial clinical presentation. ATG therapy led to CR, but the patient returned within a year and succumbed to E.coli related sepsis. At that stage he had low TERT expression, suggesting that TERT expression can change as the disease progresses. Taken together, our data support the hypothesis that loss of TERT expression correlates with disease severity and poor prognosis. Our observations further suggest that preliminary and periodic evaluation of TERT expression levels in AA patients is likely to serve as a predictor of disease severity and influence the choice of therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

2017 ◽  
Author(s):  
Philippe E. Mangeot ◽  
Valérie Risson ◽  
Floriane Fusil ◽  
Aline Marnef ◽  
Emilie Laurent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Target Cells ◽  
Mouse Bone Marrow ◽  
Primary Cells ◽  
Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

scholarly journals Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels

2008 ◽  
Vol 28 (21) ◽  
pp. 6668-6680 ◽  
Author(s):  
Albertus T. J. Wierenga ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Target Genes ◽  
Expression Patterns ◽  
Activity Levels ◽  
Dependent Manner ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions

ABSTRACT The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.

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