Modulation in the Expression of p70S6 Kinase Impairs the Engraftment and Self-Renewal of Hematopoietic Stem Cells

Abstract Regulation of hematopoietic stem cell (HSC) function(s) via the mammalian target of rapamycin complex1 (mTORC1) and its upstream regulators including PI3K and Akt has been described before. To this end, we and others have shown that hyperactivation and deficiency of the PI3K-mTORC1 pathway results in altered development, maintenance and function(s) of HSCs. However, the role of downstream effector of mTORC1, p70S6 kinase (S6K1), in HSC development and functions is unknown. Previous studies have implicated S6K1 as a regulator of ageing, by virtue of its ability to regulate cellular metabolic processes as well as protein translation. In certain cells, however S6K1 regulates cell survival and also acts as a negative regulator of PI3K-mTORC1 pathway, thus creating a negative feedback loop. Thus, how S6K1 impacts HSC ageing and stem cell functions remains an enigma. We have assessed the role of S6K1 in HSC development and function under steady-state as well as during recovery of hematopoietic system following myelosuppressive stress. We used a genetic model of S6K1 knockout mice (S6K1-/-). S6K1 deficiency in bone marrow hematopoietic cells resulted in decrease of absolute number of bone marrow hematopoietic progenitor cells as well as HSCs (Lin- Sca1+ c-Kit+; LSK) were significantly reduced relative to controls (n=14 in each group, p<0.04). Interestingly, in vitro, hematopoietic progenitor cells from S6K1-/- mice showed increased colony forming ability in response to cytokines which was associated with hyperactivation of Akt and ERK MAP kinase. To determine whether the reduced number of HSCs in S6K1-/- mice was due to deficiency of S6K1 in bone marrow microenvironment, we transplanted WT hematopoietic bone marrow cells into lethally irradiated WT or S6K1-/- mice. S6K1-/- mice transplanted with WT hematopoietic cells showed similar bone marrow cellularity and HSC numbers compared to controls suggesting that the bone marrow hypocellularity and reduced HSCs numbers in S6K1-/- mice were due to a cell intrinsic defect. To assess whether the reduced HSC number in S6K1-/- mice impacted the recovery of hematopoietic system following stress, WT and S6K1-/- mice were treated with a single dose of 5-fluorouracil (5-FU). In response to myelosuppressive stress, S6K1 deficiency resulted in increased frequency of HSCs in bone marrow despite a significant reduction in overall cellularity (n=12 in each group, p<0.02). Following administration of 5-FU, S6K1 deficiency resulted in increased cell cycle progression of HSCs in bone marrow and showed increased expression of CDK4 and CDK6 as compared to control suggesting that 5-FU administration results in upregulation of cell cycle regulatory genes in S6K1 deficient HSCs. Moreover, S6K1-/- mice showed more sensitivity to repeated injections of 5-FU (n=11 WT, 15 S6K1-/-, p<0.01). Given the differential role of S6K1 in HSCs and mature progenitors, we assessed the effect of S6K1 deficiency in HSC function. We performed competitive repopulation assay using S6K1 deficient HSCs. When transplanted into lethally irradiated primary and secondary recipients, S6K1 deficient HSCs show significantly reduced engraftment relative to controls (n=11-13 in each group; p<0.05). Interestingly, overexpression of S6K1 in wild type HSCs also resulted in reduced engraftment of HSCs in primary and secondary transplant recipients, suggesting that S6K1 overexpression in HSCs leads to decreased self-renewal. In summary, our study identifies S6K1 as a critical regulator of hematopoietic stem cell development and functions both under steady-state conditions as well as under conditions of genotoxic stress. Using both gain of function and loss of function approaches, we demonstrate that the level of expression and activation of S6K1in HSCs plays a critical role in the maintenance of HSC self-renewal and engraftment. Disclosures No relevant conflicts of interest to declare.

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The Side Population Contains Functionally Distinct Hematopoietic Stem Cell Subpopulations

Blood ◽

10.1182/blood.v112.11.2464.2464 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2464-2464

Author(s):

Grant Anthony Challen ◽

Margaret A Goodell

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Side Population ◽

Clonal Diversity ◽

Stem Cell Markers ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System

Abstract Over the decades since hematopoietic stem cells (HSCs) were first identified, the traditional view has been that the hematopoietic system is regenerated by a single pool of multipotent, quiescent HSCs that are sequentially recruited into cell cycle and which then progressively divide and differentiate until they are exhausted and ultimately replaced by the next cohort of stem cells. However, recent evidence has challenged this classical clonal succession model of HSC hierarchy by suggesting that the hematopoietic system is maintained by a pool of different HSC subtypes, with distinct self-renewal and differentiation potentials (the clonal diversity model, Figure 1). The side population (SP), characterized by Hoechst dye efflux, has been used as a method for isolating HSCs for over a decade and the SP has been shown to be highly enriched for HSC activity. While the entire SP is strikingly homogeneous with respect to expression of canonical stem cell markers such as Sca-1 and c-Kit, we recently observed heterogeneous expression for the SLAM family molecule CD150 within the SP, with CD150+ cells more prevalent in the lower SP and CD150− cell more prevalent in the upper SP. We decided to examine this observation further by investigating the properties of cells from different regions of the SP. Functional capacity was assessed by competitive bone marrow transplantation of upper SP cells, lower SP cells, and a combination of the two populations. Lower SP cells showed better engraftment than upper SP cells in recipient mice, a trend that continued when donor HSCs were isolated from primary recipients and re-transplanted into secondary hosts. Lower SP cells showed 3-fold better engraftment than upper SP cells in secondary transplants, suggesting better self-renewal capacity. However, analysis of the hematopoietic lineages formed by donor cells in recipient mice demonstrated that while both upper and lower SP cells were capable of forming all mature lineages, lower SP cells were biased towards myeloid differentiation while upper SP cells were biased towards lymphoid differentiation. The lineage biases observed from transplantation of one cell population alone were exacerbated when both upper and lower SP cells were co-transplanted into the same recipient mouse, suggesting that while both populations are capable of forming all hematopoietic lineages, in the presence of the other stem cell type (as would be the case in normal homeostasis) that the majority of the output from each HSC subtype is almost exclusively lymphoid or myeloid. The lineage contribution trends observed in the peripheral blood were also reproduced when bone marrow of transplanted mice was analyzed, including at the level of progenitors with lower SP cells showing greater ability to make myeloid progenitors (megakaryocyte-erythrocyte progenitors and granulocyte-macrophage progenitors) and upper SP cells producing proportionately more common lymphoid progenitors. Microarray analysis of upper and lower SP cells to determine the molecular signatures underlying these functional differences found many genes critical for long-term HSC self-renewal to be highly expressed in lower SP cells including Rb1, Meis1, Pbx1 and TGFbr2 while upper SP cells showed higher expression of cell cycle and activation genes. Cell cycle analysis showed upper SP cells to be approximately 2-fold more proliferative than lower SP cells (18.9% to 8.3% Ki-67+, 39.4% to 20.1% BrdU+ 3-days post-BrdU administration). The clonal diversity model which proposes the adult HSC compartment consists of a fixed number of different HSC subtypes each with pre-programmed behavior has important implications for using HSCs in experimental and clinical settings. While other studies have provided functional evidence for the clonal diversity model, this is the first study to prospectively isolate the functionally distinct HSC subtypes prior to transplantation. Figure Figure

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Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.797.797 ◽

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.

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Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽

10.1182/blood.v124.21.1591.1591 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1591-1591

Author(s):

Juliana M. Xavier ◽

Lauremilia Ricon ◽

Karla Priscila Vieira ◽

Longhini Ana Leda ◽

Carolina Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Progenitor Cells ◽

Bone Marrow Niche ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Short Term ◽

Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (meanÂ± SD) (2.07 Â± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 Â± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 Â±1.39; Arhgap21+/-: 2.17 Â± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 Â± 3.85; Arhgap21+/-: 14.07 Â±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 Â±0.03; Arhgap21+/-0.12Â±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 Â±1.1; Arhgap21+/-94.3Â±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

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Ilomastat contributes to the survival of mouse after irradiation via promoting the recovery of hematopoietic system

10.1101/2020.08.13.249235 ◽

2020 ◽

Author(s):

Baoquan Zhao ◽

Xiaoman Li ◽

Xingzhou Li ◽

Dongqin Quan ◽

Fang Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Tnf Α ◽

Γ Rays ◽

Total Body ◽

Tgf Β1

AbstractIlomastat, a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), has drawn attentions for its function in alleviating radiation damage. However, the detailed mechanisms of Ilomastat’s protection from animal model remain not fully clear. In this study, the C57BL/6 mice were pre-administrated with Ilomastat or vihicle for 2 h, and then total body of mice were exposed to 6 Gy of γ-rays. The protective effect of Ilomastat on the hematopoietic system in the irradiated mice were investigated. We found that pretreatment with Ilomastat significantly reduced the level of TGF-β1 and TNF-α, and elevated the number of bone marrow (BM) mononuclear cells in the irradiated mice. Ilomastat pretreatment also increased the fraction of BM hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) at day 30 after irradiation, and protected the spleen of mouse from irradiation. These results suggest that Ilomastat promotes the recovery of hematopoietic injury in the irradiated mice, and thus contributes to the survival of mouse after irradiation.

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

Blood ◽

10.1182/blood-2019-129054 ◽

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.

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N-Cadherin Induces Hematopoietic Stem Cells in a Quiescent State in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v106.11.470.470 ◽

2005 ◽

Vol 106 (11) ◽

pp. 470-470 ◽

Cited By ~ 1

Author(s):

Kentaro Hosokawa ◽

Fumio Arai ◽

Toshio Suda

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Adhesion ◽

Stromal Cells ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Quiescent Hscs

Abstract Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout the lifetime of individuals. Interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining the stem cell properties, including self-renewal capacity and the ability of differentiation into single or multiple lineages. The niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules, and regulate stem cell fates. Recently, it was clarified that long-term bone marrow (BM) repopulating (LTR) HSCs exist frequently in BM trabecular bone surface, and that N-cadherin + spindle-shaped osteoblasts (OBs) are identified as a major niche component. We found that side-population (SP) in c-Kit +Sca-1 +Lin −(KSL) fraction, which is the quiescent HSC in the OB niche, expressed N-cadherin. Expression of N-cadherin in both of the quiescent HSCs and OBs thought to be essential for an adherens junction between HSCs and OBs in the niche. However, the role of N-cadherin in hematopoiesis is still unclear. In this study, we focused on the function of N-cadherin in the maintenance of the stem cell specific property, such as cell adhesion, quiescence, and LTR-activity. To clarify the function of N-cadherin in hematopoiesis, we prepared the retroviruses expressing wild-type N-cadherin, transfected retroviruses into OP9 stromal cell line and KSL cells, and performed the coculture. After coculture of KSL cells with OP9 cells, long-term culture-initiating cells (LTC-ICs) were maintained on OP9 cells overexpressing WT-N-cadherin (OP9/WT-NCAD). In addition, overexpression of WT-N-cadherin in both of the KSL cells and stromal cells enhanced cobblestone formation. N-cadherin overexpressing KSL cell showed slow-cell division from the single cell, when they cultured on OP9/WT-N-cedherin or N-cadherin-Fc protein coated plates, suggesting that N-cadherin-mediated cell-cell adhesion between HSCs and stromal cells enhances the quiescence of HSCs and keeps HSCs in immature state in in vitro. To clarify the role of N-cadherin in the BM reconstitution ability of HSC, we transfected control-IRES-GFP, WT-N-cadherin-IRES-GFP and N-cedherin/390Δ-IRES-GFP retrovirus into the Ly5.1 BM mononuclear cells and transplanted into lethally irradiated Ly5.2 mice. N-cedherin/390Δ, which is a mutant N-cadherin with a deletion at the extracellular domain, exhibits a dominant negative effect on the activity of endogenous cadherins. Control and WT-N-cadherin expressing cell reconstitute the recipient mice BM, while N-cadherin/390Δ expressing cells did not. It suggests that the adhesion between HSCs and BM niche cell is indispensable for the LTR-activity. In addition, we found that WT-N-cadherin overexpressing HSCs were enriched in the SP fraction after 4 months of BM transplantation, indicating that N-cadherin-mediated cell adhesion induced HSCs in the quiescent and kept quiescent HSCs in the niche. Altogether, these observations suggest that N-cadherin is a critical niche factor for the maintenance of the quiescence and self-renewal activity of HSCs. N-cadherin promotes tight adhesion of HSCs to the niche and keeps HSCs in the quiescent state

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Kit Deficiency Regulates Stable Human Hematopoietic Stem Cell Engraftment in Mice

Blood ◽

10.1182/blood.v124.21.653.653 ◽

2014 ◽

Vol 124 (21) ◽

pp. 653-653

Author(s):

Claudia Waskow ◽

Susann Rahmig ◽

Nehir Cosgun

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Myeloid Cell ◽

Cell Types ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Kit Receptor ◽

Human Hscs

Abstract Humanized mice are required for in-depth analysis of human hematopoietic stem cell (HSC) function and immunobiology. The currently available options are problematic because stable engraftment of substantial numbers of human HSCs and the continuous generation of human myeloid cell types remain difficult to achieve. We generated three novel recipient mouse strains that combine immune deficiency with a functionally impaired endogenous HSC compartment mediated by a defective Kit receptor: BALB/c Rag2- Il2rg-KitWv/Wv (BRgWv), NOD/SCID Il2rg- (NSG) KitWv/Wv (NSGWv) and NSG KitW41/W41 (NSGW41). We find that the mutant Kit receptor opens up stem cell niches across species barriers and allows for robust and sustained engraftment of human HSCs after transfer into adult mice without the necessity for irradiation conditioning prior to transplantation. Following stable engraftment in the mouse bone marrow niches, human HSCs give rise to lymphoid cells and to robust numbers of erythroid and myeloid lineage cells over extended periods of time in primary and secondary recipient mice. Particularly in NSGW41 mice, we observe improved reconstitution of human myeloid cell types compared to control irradiated NSG mice. In the bone marrow, endogenous hematopoietic progenitor cells with a defective Kit receptor are largely replaced by human Kit-proficient hematopoietic progenitor cells because progenitor cell expansion requires normal signaling by Kit. Thus, human Kit-proficient donor cells have an advantage over endogenous murine Kit-mutant cells. Increased numbers of myeloid cells are found in the bone marrow and spleen of NSGW41 transplanted mice compared with grafts established in irradiated NSG mice. We conclude that Kit-signaling regulates HSC engraftment across the human-mouse species barrier and that Kit deficient mice show great potential for the study of human HSC functions including self-renewal, differentiation and mechanisms of innate immunity. Disclosures No relevant conflicts of interest to declare.

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Ing4 Suppresses Quiescence and Inflammation in Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2020-143388 ◽

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.

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p16INK4a Is a Key Downstream Mediator of the Deleterious Effects of FoxO Deficiency on Maintenance of the Hematopoietic Stem Cell Compartment.

Blood ◽

10.1182/blood.v112.11.1405.1405 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1405-1405

Author(s):

Zuzana Tothova ◽

Stephen M Sykes ◽

Dena S Leeman ◽

James W Horner ◽

Norman Sharpless ◽

...

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Accelerated Aging ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Downstream Mediator

Abstract Regulation of oxidative stress in the hematopoietic stem cell (HSC) compartment is critical for the maintenance of HSC self-renewal. A number of reports have previously implicated p16 in aging of HSCs, pancreatic β-islet cells and subventricular zone progenitors in the brain [1–3]. In the context of the hematopoietic system, p16INK4a expression in HSCs increases with age, and correlates with decreased HSC repopulating ability, decreased self-renewal, and increased apoptosis with stress [1]. We and others have recently reported that FoxO play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment [4, 5]. Young mice deficient in FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system, with striking similarity to aging wild-type mice, show a defect in bone marrow repopulating ability, decrease in self-renewal, myeloid skewing in differentiation and increased levels of apoptosis. Furthermore, young FoxO-deficient HSC show increased levels of p16 when compared to their wildtype counterparts. These collective findings suggested the possibility that FoxO loss could result in accelerated aging of HSC due to increased expression of p16 as a consequence of increased ROS. To test the hypothesis that p16 is one of the key mediators of FoxO loss responsible for accelerated aging of HSC, we deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system of mice deficient in p16INK4a. Young mice deficient in FoxO and p16 shared the same characteristics of their HSC(Lin−Sca1+c-kit+) compartment as mice deficient in FoxO only, including decreased number of HSC, increased percentage of HSC entering S/G2/M and apoptosis, and increased levels of ROS as compared to their wildtype counterparts. However, in a setting of long-term repopulation studies, bone marrow isolated from mice deficient in p16 and FoxO demonstrated a rescue of long-term repopulation for up to 20 weeks, as compared to FoxO deficient bone marrow that showed a severe defect in long-term repopulation. p16 deficiency in the setting of FoxO deficiency did not result in reduction of ROS levels in the HSC compartment. Taken together, these findings indicate that p16 is a critical downstream mediator of FoxO in the maintenance of the HSC compartment, and that it can dissociate the detrimental effects of ROS on HSC self-renewal in a setting of FoxO deficiency.

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Loss of MiR-143 and MiR-145 Inhibits Hematopoietic Stem Cell Self-Renewal through Dysregulated TGFβ Signaling

Blood ◽

10.1182/blood.v124.21.527.527 ◽

2014 ◽

Vol 124 (21) ◽

pp. 527-527 ◽

Cited By ~ 1

Author(s):

Jeffrey C Lam ◽

Joanna Wegrzyn-Woltosz ◽

Rawa Ibrahim ◽

Kate Slowski ◽

Patricia Umlandt ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Signaling Pathway ◽

Myelodysplastic Syndromes ◽

Interstitial Deletion ◽

Tgfβ Signaling ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Hematopoietic Stem ◽

Limiting Dilution

Abstract Myelodysplastic syndromes (MDS) are a collection of hematopoietic malignancies in which genomic abnormalities within the hematopoietic stem cell (HSC) compartment results in dysplasia of the marrow cells and ineffective hematopoiesis. As a result, the primary cause of mortality in these patients is eventual bone marrow failure although MDS patients also have a significantly increased risk of transformation to acute myeloid leukemia (AML). The most common karyotypic change in MDS is an interstitial deletion of the long arm of chromosome 5, del(5q) MDS. Patients with an isolated interstitial deletion of chromosome 5q are referred to as having 5q- syndrome. Mapping of the commonly deleted region (CDR) within 5q- syndrome has identified a 1.5-megabase region on band 5q32. MicroRNA (miRNA) -143 and -145 are located within the CDR of del(5q) MDS and have been implicated in the pathogenesis of the disease. However, their functional role in myelodysplastic syndromes has not been well studied. To investigate the role of miR-143 and miR-145, we utilized a gene-targeted mouse model containing deletion of miR-143 and miR-145. Here we show that mouse marrow lacking miR-143 and miR-145 have a decrease in short-term repopulating HSC and progenitors of the myeloid lineage by flow cytometry, as well as of hematopoietic progenitor activity using colony forming assays. Additionally, we performed a limiting dilution assay of miR-143-/-145-/- bone marrow and observed significantly fewer functional HSCs compared to wildtype marrow. To explore the molecular mechanism behind this defect, we performed Ingenuity Pathway Analysis of the predicted targets of miR-143 and miR-145. We identified the transforming growth factor-beta (TGFβ)-signaling pathway as a common target of these two miRNAs. Gene Set Enrichment Analysis of del(5q) using mRNA expression of MDS patient CD34+ marrow cells show an enriched TGFβ-signature compared to healthy controls. In addition, the defect in hematopoietic progenitor activity in miR-143-/-145-/- marrow can be rescued by inhibiting Smad3 using the chemical inhibitor SIS3. We validated the TGFβ pathway adaptor protein, Disabled-2 (DAB2), as a target of miR-145 and show that TGFβ signaling is activated upon loss of miR-145 or enforced expression of DAB2. Enforced expression of DAB2 in mouse marrow is able to recapitulate many of the features of miR-143-/-145-/- mice. DAB2 overexpressing marrow formed significantly fewer colonies in progenitor assays, and in competitive transplants, vector-transduced marrow was able to out compete DAB2-overexpressing marrow in both primary transplants as well as in secondary limiting dilution assays. Interestingly, compared to wildtype mice, aged miR-143-/-145-/- mice showed decreased hemoglobin and platelet counts with elevated white blood cell counts. This phenotype was also observed in a subset of mice with enforced DAB2 expression where a proportion of mice developed a transplantable myeloproliferative disorder. Together, our data identifies a role for miR-143 and miR-145 in the pathogenesis of del(5q) MDS where their loss results in a defect in HSC activity. We observe that the TGFβ signaling pathway is activated in patient marrow and we validate DAB2 as a direct target of miR-145. We provide evidence that the defect observed in miR-143-/-145-/- marrow is mediated in part by DAB2 where its enforced expression leads to a defect in HSC self-renewal but contributes to myeloproliferation. Disclosures Karsan: Celgene: Research Funding.

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