scholarly journals Ing4 Regulates Hematopoiesis through Suppression of NF-Kb

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 642-642
Author(s):  
Zanshe Thompson ◽  
Vera Binder ◽  
Michelle Ammerman ◽  
Ellen Durand ◽  
Leonard I. Zon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Target Gene ◽  
Target Genes ◽  
Zebrafish Embryos ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. 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, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In the absence of Ing4, there is a significant increase in NF-kB target gene expression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis, where Ing4 deficiency impairs hematopoietic stem cell (HSC) function, but enhances multipotent progenitor cell (MPP) regenerative capacity. 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. To define the role of Ing4 in zebrafish HSPCs, we designed an anti-sense morpholino oligo against Ing4 and injected into zebrafish embryos at the single cell stage. Embryos were screened using in situ hybridizations for c-myb and runx1 expression, which are highly expressed in the aorta, gonad, mesonephros (AGM) region in the developing zebrafish embryo. We found that Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the AGM, demonstrating a lack of HSPC specification. Analysis of ephrinB2 expression showed normal specification of the aorta in Ing4 morphant embryos, signifying that the step of HSPC specification is affected in the absence of Ing4. Overexpression of human Ing4 in zebrafish embryos resulted in increased HSPC marker staining suggesting that normal expression levels of Ing4 are required for HSC specification. As Ing4 is an epigenetic regulator that binds specific gene loci, we examined the chromatin occupancy of Ing4 in human peripheral blood CD34+ progenitor cells. Using ChIP-seq for Ing4 in CD34+ cells, we show that Ing4 binds to many regulators of blood development including MYB, LMO2, RUNX1, and IKAROS, and several NF-kB target genes. In other tissues, Ing4 negatively regulates NF-kB, so accordingly, loss of Ing4 results in an overabundance of NF-kB signaling. To address NF-kB target gene expression in Ing4-deficient zebrafish embryos, we performed qPCR analysis at 36hpf. These assays showed an increase in the expression of a subset of NF-kB target genes (IKBKE, IL-19, IL-1b, IL-20R). Simultaneous knockdown of both Ing4 and RelA, through combined morpholino injections against both factors, resulted in the rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the loss of Ing4. A mouse model for Ing4 deficiency was generated to further evaluate the role of Ing4 in differentiated immune cells. These mice are developmentally normal but are hypersensitive to stimulation with LPS. Interestingly, we found that Ing4-/- mice showed skewed hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. Based on these exciting findings, we hypothesize that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. 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. Disclosures No relevant conflicts of interest to declare.

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ing4 Regulates Stress Hematopoiesis and Represses Self-Renewal in Multipotent Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 724-724
Author(s):  
Zanshe Thompson ◽  
Melanie Rodriguez ◽  
Seth Gabriel ◽  
Georgina Anderson ◽  
Vera Binder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Transplant ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Multipotent Progenitor Cells ◽  
Equity Ownership

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. 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, it has been shown to promote stem-like cell characteristics in malignant cells and is a frequent target of inactivation in various cancer types. The tumor suppressive activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In zebrafish, loss of Ing4 results in loss of HSC specification and a significant increase in NF-kB target gene expression. Knockdown of NF-kB expression in Ing4 deficient zebrafish recovered HSC marker expression in the aorta suggesting that NF-kB inhibition could remediate the loss of Ing4 expression. Small molecule NF-kB pathway inhibitors with varying mechanisms were also observed to rescue of HSC marker staining in the zebrafish aorta. Ing4 deficient embryos incubated with a lower dose of inhibitor had a 31% recovery of marker staining and 82% of embryos incubated in the highest dose recovered HSC marker staining emphasizing a dose dependent rescue of HSC specification through NF-kB suppression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- bone marrow has aberrant hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with a concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Analysis of differentiation in Ing4 null bone marrow also reveals skewed hematopoiesis. We see a 14% increase in granulocytes in the null mouse marrow and observe similar skewing in CFU assays. Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. In further characterization of these cells, we found an increase in MPPs that express lower levels of CD34 (55.5% vs 67.7%). CD34 expression is a marker of HSCs. This CD34+/mid population also express CD229 (85% positive), which is barely detectable in wildtype marrow (less that 0.01%). CD229 is also an HSC marker. Based on these exciting findings, we hypothesize that we have identified a subset of CD34+/midCD229+ MPPs in Ing4 deficient mice that retain self-renewal characteristics. Our data suggest that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. 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 Zon: CAMP4: Equity Ownership; Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership.

scholarly journals Ing4 Suppresses Quiescence and Inflammation in Hematopoietic Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-16
Author(s):  
Zanshe Thompson ◽  
Melanie Rodriguez ◽  
Georgina Anderson ◽  
Seth Gabriel ◽  
Vera Binder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Target Genes ◽  
Publicly Traded ◽  
Private Company ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marker Expression

Hematopoietic stem and progenitor cell (HSPC) development and maintenance is regulated through a complex regulatory network. In a screen of epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in HSPC specification. Ing4 acts to regulate transcription through interactions with transcription factors, including HIF, NF-kB, and p53. It is often mis-expressed in many human cancers and has been shown to promote stem cell-like characteristics in malignant cells, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. The transcription factor NF-kB is a regulator of inflammatory response and serves an important role in embryonic HSPC emergence, survival, differentiation and proliferation. The Ing4 protein binds to the p65/RelA subunit of NF-kB, inhibiting DNA binding and suppressing NF-kB cytokines and inflammatory pathways. In the absence of Ing4 there is an overexpression of NF-kB target genes that have inhibitory effects on hematopoietic programming. Given the regulatory role of Ing4 in both hematopoiesis and cancer, it is likely critical to the regulation of stem cell self-renewal, maintenance and specialization. To better define the role of Ing4 on hematopoiesis we use two Ing4 loss-of-function models: zebrafish and mouse. For the zebrafish model of Ing4 deficiency, Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the aorta, gonad, mesonephros (AGM) region of the developing zebrafish embryo, demonstrating a lack of HSPC specification. 36 hours post fertilization (hpf) Ing4 morphants display increased expression of NF-kB target genes when Ing4 is absent. Genetic epistasis experiments performed to block translation of RelA, IL-1b, and additional NF-kB target gene mRNA revealed recovered HSC marker expression in the aorta. To discover small molecule inhibitors that would mimic these effects, we conducted an in vivo chemical screen of NF-kB pathway inhibitors assessing their ability to rescue HSC specification in Ing4 morphant zebrafish. Ing4 morphants treated with NF-kB inhibitors had reduced NF-kB cytokine expression, as well as a dose-dependent rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the effects of Ing4 loss on hematopoiesis. To more thoroughly profile the effects of Ing4 loss on HSC specification and the bone marrow niche, an Ing4-/-mouse model was used. These mice are developmentally normal but are hypersensitive to stimulation with LPS due to increased inflammatory signaling. Peripheral blood analysis reveals an increase in Mac-1 cells in the Ing4-/- mouse. Ing4-/- bone marrow progenitors are skewed toward granulocyte-myocyte progenitor cells (GMPs) lending to the shift in cell populations present in the peripheral blood. Ing4 loss further disrupts the mouse hematopoietic program resulting in a dramatic increase in the number of short term-HSCs (ST-HSC) (WT: 11.4%, Null: 31.7%), a modest increase in long term-HSCs (LT-HSC) (WT: 2.4%, Null: 5.52%), and a dramatic decrease in multipotent progenitors (MPPs) (WT: 47.9%, Null: 19.3%). We also found significant alterations in stress hematopoiesis following competitive HSC transplant where sorted Ing4-/- LT-HSCs failed to engraft. Following myeloablative insult, we found no significant change in Ing4-/- LT-HSC (-1.18%) when compared with ST-HSC (-14.43%) indicating reduced sensitivity to 5-FU ablation in the Ing4-/- LT-HSC group. Cell cycle analysis identified 92.9% of Ing4-/- LT-HSCs are in G0 compared to 76.2% of wildtype LT-HSCs. ST-HSCs were also more quiescent with 27% of Ing4-/- ST-HSCs in G0 compared to 11.1% of wildtype ST-HSCs. Previously published work reports hyper proliferative HSCs that exhibit loss of quiescence as a result of proinflammatory NF-kB signaling. We believe that the interaction between Ing4 and the HIF-1a pathway may play a role in the observed phenotype of Ing4-/- LT-HSCs resulting in increased quiescence and disruption of the balance between self-renewal and differentiation critical to reconstitution of the hematopoietic compartment. Overall, our findings suggest that the regulatory effects of Ing4 play a crucial role in hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures Zon: CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Cellarity: Consultancy; Celularity: Consultancy.

Intravital Imaging of Young and Aged Stem Cells in the Marrow of Long Bones: Visualizing Mammalian Stem Cell Behavior in Real-Time in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2418-2418
Author(s):  
Anja Köhler ◽  
Vince Schmithorst ◽  
Marie-Dominique Filippi ◽  
Marnie A. Ryan ◽  
Deidre Daria ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Intravital Imaging ◽  
Long Bones ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Close Vicinity ◽  
Stem And Progenitor Cells

Abstract Hematopoiesis, the process in which blood cells are generated from hematopoietic stem and progenitor cells (HSPCs) is primarily confined to the bone cavities. The interactions of hematopoietic cells with stroma cells forming niches inside the bone cavities are central to hematopoiesis, as these regulate cell proliferation, self-renewal and differentiation. Hematopoietic cell/stroma interactions have thus been, in analogy to the immunological synapse, named stem/progenitor cell synapses. So far, visualization of the behavior of somatic stem and progenitor cells in an undisturbed in vivo environment has not been reported for the mammalian system and consequently, the cellular dynamics of stem, progenitor and differentiated cells in vivo are only poorly defined. We developed and performed intravital time-lapse 2-photon microscopy in the marrow of the long bones (tibia) of mice to study the behavior and dynamics of differentiated hematopoietic cells as well as HPCs and HSCs in close vicinity to the endosteum in vivo over time. We demonstrate that HPCs as well as HSCs reside in close vicinity to the endosteum, further supporting the notion of an endosteal stem cell niche, and that they are, in contrast to differentiated macrophages and dendritic cells, solitary and immobile. Both HPCs and HSCs occupy distinct positions relative to the endosteum and show cell protrusion movement consistent with an active stem/progenitor cell synapse. Lastly, we report that aged HSCs show increased protrusion movement and localize more distantly to the endosteum compared to young HSCs. In addition, aged HSCs present with reduced adhesion to stroma as well as reduced polarity upon adhesion in vitro, implying a connection between altered stem cell dynamics in vivo and stem cell aging. The intravital imaging technology developed might establish a basis for further delineating additional important questions in stem cell biology like cellular mechanisms of hematopoietic stem cell self-renewal and differentiation in the context of the stroma/niche in vivo.

Identification of Human Hematopoietic Stem Cell-Specific STAT5 Target Genes Involved in Self-Renewal and Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 568-568
Author(s):  
Szabolcs Fatrai ◽  
Albertus T.J. Wierenga ◽  
Edo Vellenga ◽  
Simon M. G. J. Daenen ◽  
Jan Jacob Schuringa
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Target Gene ◽  
Target Genes ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 568 The transcription factor STAT5 fulfils an essential role in self-renewal of both mouse and human HSCs and persistent activation of STAT5 contributes to leukemic transformation. In patients with acute myeloid leukemia, increased STAT5 activity has been observed in over 60% of the cases. Yet, little is known about mechanisms that are involved. To gain further insight into these processes we studied whether STAT5-imposed long-term self-renewal is exclusively restricted to HSCs, or whether long-term self-renewal can also be imposed on progenitor cells. Human cord blood (CB) cells were transduced with control and STAT5-ER retroviral vectors allowing the induction of STAT5 activity by treatment of cells with 4-hydroxytamoxifen (4-OHT). Four populations were isolated: hematopoietic stem cells (HSC, CD34+CD38low), common myeloid progenitors (CMP, CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP, CD34+CD38+CD123+CD45RA+) and megakaryocyte-erythroid progenitors (MEP, CD34+CD38+CD123-CD45RA-). MS5 bone marrow stromal cocultures were initiated and STAT5 activity was induced by 4-OHT. In HSCs, STAT5 overexpression induced a long-term proliferative advantage as well as a significant increase in cobblestone formation. This coincided with elevated levels of Colony Forming Cells (CFCs) that were maintained over 5 weeks. In contrast, STAT5 was unable to induce cobblestone formation in progenitor cocultures and only a transient STAT5-induced increase in cell numbers was observed. CFC numbers dropped significantly after 2 weeks and progenitor initiated cultures could not be maintained longer than 3 weeks regardless of STAT5 activity. Myelopoiesis was blocked and an increase in erythroid differentiation in STAT5-ER-transduced HSC, CMP, and MEP populations was observed, while the differentiation potential of the GMP remained unaffected. Next, we aimed to identify HSC-specific STAT5 target genes by performing microarray analysis on HSC, CMP, GMP and MEP populations transduced with our STAT5-ER vectors. To limit STAT5 mediated effects on erythropoiesis GATA1 was downmodulated in STAT5-transduced CB cells by a lentiviral RNAi approach, which completely abrogated erythropoiesis but maintained enhanced HSC self-renewal. Microarrays were performed on GATA1 downmodulated STAT5-transduced CB cells and controls, and these data sets were compared to the HSC-specific STAT5 target gene lists. This combined approach resulted in the identification of 36 GATA1-independent STAT5 target genes in the HSC population. One of the identified genes was HIF2a. The involvement of HIF2a in STAT5 phenotypes was studied functionally by using a lentiviral HIF2a RNAi approach in STAT5 transduced CB cells. These studies revealed that expansion of STAT5/HIF2a RNAi-transduced cells on MS5 bone marrow stromal cocultures was reduced, coinciding with reduced CFC and LTC-IC frequencies, while differentiation was not affected. In summary, our data show that hematopoietic stem cells, but not progenitors are the exclusive target for STAT5-induced long-term self-renewal. Furthermore, we show that HIF2a is a novel STAT5 target gene which plays an important role in STAT5-induced stem cell phenotypes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2510-2510
Author(s):  
Lina Han ◽  
Albertus T.J. Wierenga ◽  
Marjan Rozenveld-Geugien ◽  
Kim van de Lande ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cytokine Responses ◽  
Cd34 Cells

Abstract Abstract 2510 Poster Board II-487 Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. We have observed that introduction of activated STAT5A in human stem/progenitor cells enhances long-term stem cell self-renewal, while lentiviral downmodulation of STAT5 expression in both normal as well as primary leukemic CD34+ cells impairs long-term growth and self-renewal. Many cytokines that act on the immature human stem cell compartment are known to be able to activate STAT5, such as TPO and SCF. Yet, little is known about the specificity and kinetics of STAT5 signaling in response to early-acting and lineage-restricted cytokines in specifically defined stem cell and progenitor compartments, particularly in the case of acute myeloid leukemia. We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at the single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34+CD38− hematopoietic stem cells (HSCs). EPO and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF, as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34+ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. A number of different types of responses were observed, being a) a strong STAT5 activation induced by TPO only; b) a strong STAT5 activation induced by IL3 and GM-CSF, but not TPO; c) STAT5 activation induced by various cytokines; and d) constitutive STAT5 phosphorylation that could not significantly be further induced by cytokines. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34+ cells on stroma. In most examined cases multiple cytokines acted in an additive fashion in inducing long-term growth of primary AML CD34+ cells in bone marrow stromal cocultures, and in only 1 out of 10 cases cytokine-independent growth was observed. In none of the cases only IL3 or TPO were sufficient to induced long-term expansion of primary AML CD34+ cells. In conclusion, our study demonstrates variable cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6083-6090 ◽  
Author(s):  
Ann Dahlberg ◽  
Colleen Delaney ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽  
2021 ◽  
Author(s):  
Yuqing Yang ◽  
Andrew J Kueh ◽  
Zoe Grant ◽  
Waruni Abeysekera ◽  
Alexandra L Garnham ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
High Rate ◽  
Embryonic Patterning ◽  
Self Renewal ◽  
Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

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