scholarly journals Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice

2016 ◽  
Vol 37 (2) ◽  
Author(s):  
Mikko N. M. Myllymäki ◽  
Jenni Määttä ◽  
Elitsa Y. Dimova ◽  
Valerio Izzi ◽  
Timo Väisänen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Notch Signaling ◽  
Hypoxia Inducible Factor ◽  
Primary Site ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Notch Ligand ◽  
Extramedullary Erythropoiesis ◽  
Deficient Mice

ABSTRACT Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2 gt/gt ) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2221-2221
Author(s):  
Cyrus Khandanpour ◽  
Ulrich Duehrsen ◽  
Tarik Möröy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

Dual functions of cell-autonomous and non–cell-autonomous ADAM10 activity in granulopoiesis

Blood ◽  
2011 ◽  
Vol 118 (26) ◽  
pp. 6939-6942 ◽  
Author(s):  
Masaki Yoda ◽  
Tokuhiro Kimura ◽  
Takahide Tohmonda ◽  
Shinichi Uchikawa ◽  
Takeshi Koba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Notch Signaling ◽  
Bone Marrow Cells ◽  
Myeloproliferative Disorder ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Membrane Bound ◽  
Reciprocal Transfer ◽  
Dual Functions

Abstract Previous studies have revealed various extrinsic stimuli and factors involved in the regulation of hematopoiesis. Among these, Notch-mediated signaling has been suggested to be critically involved in this process. Herein, we show that conditional inactivation of ADAM10, a membrane-bound protease with a crucial role in Notch signaling (S2 cleavage), results in myeloproliferative disorder (MPD) highlighted by severe splenomegaly and increased populations of myeloid cells and hematopoietic stem cells. Reciprocal transfer of bone marrow cells between wild-type and ADAM10 mutant mice revealed that ADAM10 activity in both hematopoietic and nonhematopoietic cells is involved in the development of MPD. Notably, we found that MPD caused by lack of ADAM10 in nonhematopoietic cells was mediated by G-CSF, whereas MPD caused by ADAM10-deficient hematopoietic cells was not. Taken together, the present findings reveal previously undescribed nonredundant roles of cell-autonomous and non–cell-autonomous ADAM10 activity in the maintenance of hematopoiesis.

Irgm1 Is a Negative Regulator of Interferon-Gamma Signaling in Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 382-382 ◽  
Author(s):  
Katherine Y King ◽  
Megan T Baldridge ◽  
David C Weksberg ◽  
Margaret A Goodell
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Interferon Gamma ◽  
Negative Regulator ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Deficient Mice

Abstract Abstract 382 Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that give rise to all of the cellular elements of the blood and retain enormous proliferative potential in vivo. We have a growing understanding that the controls on HSC proliferation are tied in part to regulation by the immune system—specifically, that HSC proliferation and mobilization can be stimulated by the immune cytokines interferon-alpha and interferon-gamma (IFNg). Our previous work has demonstrated that HSC quiescence and function are aberrant in mice lacking the immunity-related GTPase Irgm1 (also Lrg47). Indeed, the bone marrow of Irgm1-deficient animals at baseline mimics the bone marrow of wild type animals that have been stimulated with IFNg. We hypothesized that the HSC defects in Irgm1-deficient animals are due to overabundant IFNg signaling, and that Irgm1 normally serves to dampen the stimulatory effects of IFNg on HSCs. To test this hypothesis, we used RNA expression profiling to compare gene expression in wild type versus Irgm1-deficient mice. We found that interferon-dependent signaling is globally upregulated in the HSCs of Irgm1-deficient mice. Next we generated Irgm1-/-IFNgR1-/- and Irgm1-/-Stat1-/- double knock out animals. In contrast to the phenotype of Irgm1 single knock out mutants, the hyperproliferation and self-renewal defects in HSCs were both rescued in the double knock out animals, indicating that IFNg signaling is required for manifestation of the Irgm1-deficient phenotype. Futhermore, we found that Irgm1 is expressed in HSCs in a Stat1- and IFNgR-dependent fashion, suggesting that it forms a negative feedback loop for IFNg signaling in the HSC population. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFNg-dependent stimulation in HSCs. These findings demonstrate that IFNg provides a significant stimulus for HSC proliferation even in the absence of infection, and that IFNg-dependent signaling must be tightly regulated to preserve HSC self-renewal capacity. This study provides evidence that the Irgm1 protein can serve as a link between immunity and regulation of hematopoiesis at the level of the stem cell. We speculate that utilization of Irgm1 for its immune functions may detract from its ability to regulate HSC self-renewal capacity, thus ultimately contributing to myelosuppression and increased risk of death from chronic infections such as tuberculosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Extramedullary Erythropoiesis in Spleen of HIF Prolyl 4-Hydroxylase-2 Deficient Mice Is Mediated By Notch Signaling Downregulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2656-2656
Author(s):  
Mikko Myllymäki ◽  
Jenni Määttä ◽  
Elitsa Dimova ◽  
Valerio Izzi ◽  
Timo Väisänen ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Mass ◽  
Hypoxia Inducible Factor ◽  
Premature Death ◽  
Hematopoietic Stem ◽  
Extramedullary Erythropoiesis ◽  
Equity Ownership ◽  
The Stability ◽  
Deficient Mice ◽  
Notch Ligands

Abstract Erythrocytosis, an increase in absolute red cell mass, is mainly driven by erythropoietin, while hypoxia-inducible factor (HIF) regulates the expression of a number of genes involved in it, including erythropoietin. Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2/PHD2/EGLN1), the major regulator of the stability of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis and premature death. Although bone marrow is the primary site for erythropoiesis, spleen retains a capability for extramedullary erythropoiesis. We studied HIF-P4H-2 hypomorphic mice (Hif-p4h-2gt/gt) which show slightly induced erythropoiesis only upon aging despite no increased erythropoietin levels. Spleen was identified as the site of extramedullary erythropoiesis in these mice. Hematopoietic stem cells (HSCs) from spleens of the Hif-p4h-2gt/gt mice showed increased growth of BFU-Es and the mice were protected against anemia by induced extramedullary erythropoiesis. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligands and target Jag1, Jag2, Dll1 and Hes1 became downregulated upon aging dependent on HIF-2α. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased growth of BFU-Es in the Hif-p4h-2gt/gt mice. We conclude that HIFα stabilization can mediate non-erythropoietin-driven extramedullary erythropoiesis in the spleen via altered Notch signaling. Disclosures Myllyharju: FibroGen Inc.: Equity Ownership, Research Funding.

The Notch Ligand Jagged1 Causes a Myeloproliferative Disorder in Mice Lacking IκBα.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1226-1226
Author(s):  
Franziska Jundt ◽  
Rudolf A. Rupec ◽  
Bernd Rebholz ◽  
Bernd Doerken ◽  
Irmgard Foerster ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Notch Signaling ◽  
Cell Fate ◽  
Hematopoietic Cells ◽  
Myeloproliferative Disorder ◽  
Newborn Mice ◽  
Notch Ligand ◽  
Cell Fate Decisions ◽  
Deficient Mice

Abstract Hematopoiesis occurs in the liver and the bone marrow during murine development. Newborn mice with a ubiquitous deletion of IκBα develop a severe hematological disorder characterized by an increase of granulocyte/erythroid/monocyte/macrophage colony-forming units (CFU-GEMM) and hypergranulopoiesis. Here, we provide evidence that this particular myeloproliferative disturbance is mediated by continuously deregulated perinatal expression of the Notch ligand Jagged1 in IκBα-deficient hepatocytes. Signaling through Notch-family cell surface receptors and their ligands has been shown to be involved in cell fate decisions of stem cells during hematopoietic/mesenchymal differentiation. However, the role of Notch signaling in myelopoiesis is still under discussion as results gained using different experimental conditions are contradictory. Due to embryonic lethality of Notch1- and Jagged1-deficient mice, alterations of myelopoiesis are difficult to be adressed. In this study, we investigated the function of IκBα and its role within the Jagged/Notch signaling pathway during myelopoiesis. Therefore, a novel mouse line with a conditional (floxed) allele of ikba was established. Ubiquitous deletion of IκBα after cross-breeding with Deleter-Cre mice results in hypergranulopoiesis comparable to the conventional deletion of the allele. A detailed analysis revealed a myeloproliferative syndrome with increased numbers of cycling progenitor cells. The morphological analysis of liver and bone marrow of IκBα-deficient mice showed hypercellularity. The cellular components were dominated by myeloid lineages and represented mostly granulocyts with dysplastic features, characterized by pseudo-Pelger-Huet formation. Myelodysplasia could also be detected in megakaryopoiesis by the presence of micromegakaryocytes. Alterations in erythropoiesis were detectable by condensed chromatin and an asychrony of the nucleocytoplasmic ratio in the red cell precursor population. Together, our results indicate that ubiquitous loss of IκBα results in hypergranulopoiesis progressing to a myelodysplastic syndrome. Systematic analysis of transcription factors, growth factor receptors and NF-κB-regulated cell-survival genes was performed to determine molecular mechanisms underlying hypergranulopoiesis. Our data suggested that Notch1-dependent signals were responsible for the myeloproliferative disorder as Notch1 was upregulated in neutrophils and the Notch ligand Jagged1 in non-hematopoietic cells, namly hepatocytes. Myeloproliferation could be inhibited by blocking the Notch1 ligand Jagged1. Interestingly, deletion of IκBα in neutrophils and macrophages or hematopoietic stem cells did not result in dysregulation of myelopoiesis despite constitutive NF-κB activation in these cells. This establishes the relevance of non-hematopoietic expression of Jagged1 for the control and regulation of myelopoiesis. In summary, we show that cell-fate decisions leading to a premalignant hematopoietic disorder can be initiated by non-hematopoietic cells with inactive IκBα.

scholarly journals p38α Is Required for Proper Proliferation of Hematopoietic Stem Cells during Stress

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4317-4317
Author(s):  
Daiki Karigane ◽  
Keiyo Takubo ◽  
Shinichiro Okamoto ◽  
Toshio Suda
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Cellular Senescence ◽  
Expression Level ◽  
Wild Type ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Hematopoietic stem cells (HSCs) are capable of self-renewal and multilineage-differentiation during lifespan. HSCs are maintained in a quiescent state to avoid cellular senescence. Previous reports utilizing pharmacological inhibitors or shRNAs against p38MAPK suggest a pivotal role of p38MAPK-pRb-Ink4a signaling in induction of HSC senescence by hematological stress or chronological aging. However, no genetic evidence exists for p38MAPK-mediated cellular senescence in vivo. Here we report unexpected roles of the dominant isoform of p38MAPK family, p38α, in adult hematological system. p38MAPK has four isozymes, α, β, γ and δ. Among them, p38α isozyme was highly expressed in various bone marrow hematopoietic cells, and the expression level of p38α in HSCs was higher than differentiated cells (p<0.01). Phosphorylation of p38MAPK was mainly observed in multipotent progenitors but not in HSCs in steady-state hematopoiesis, in addition, physiological aging (1 year old mouse bone marrow) did not affect phosphorylation status of p38MAPK in steady state. In contrast, p38MAPK was phosphorylated in HSCs after transplantation or 5-FU treatment. Mean fluorescence index (MFI) of phosphorylation of p38MAPK in HSCs is significantly higher at day 3 post 5-FU treatment (250 mg/kg) than steady-state. MFI of phosphorylation of p38MAPK in HSCs was higher at day 1 post transplantation than steady state, and returned to normal at day 7 post transplantation. These results showed phosphorylation of p38α was immediately induced after hematopoietic demand. p38α-deficient embryos die due to defective erythropoiesis in a non-cell-autonomous manner. Thus, we used a conditional knockout model; CAG-CreERT2:p38αfl/fl mouse to analyze the effects of p38α on adult hematopoiesis and HSCs. Expression level of p16Ink4a, one of the cellular senescence markers, was not significantly different between p38α-deficient mice and wild-type mice. Treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type, N=6, p<0.05). Loss of p38α in HSCs showed a defective transplantation capacity. Inducible loss of p38α in bone marrow chimera resulted in a gradual loss of peripheral blood chimerism of p38α-deficient cells. In addition, short-term BrdU incorporation assay showed that the cell cycle progression of p38α-deficient HSCs was suppressed (BrdU positive rate; 3.5±2.2%, N=9, p38α-deficient cells vs 6.5±2.6%, N=5, wild-type, p<0.05). Therefore, hematopoietic function was obviously lowered in p38α-deficient HSCs during hematopoietic stresses. These observations collectively support the requirement of p38α for proper proliferation of HSCs during stress hematopoiesis. Disclosures No relevant conflicts of interest to declare.

The Mll-AF9 Fusion Gene Results in Overexpression of Homeobox Genes and Deregulation of Granulocyte-Monocyte Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 645-645
Author(s):  
Ashish Kumar ◽  
Weili Chen ◽  
John H. Kersey
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Young Adult ◽  
Hematopoietic Stem Cells ◽  
Fusion Gene ◽  
Homeobox Genes ◽  
Specific Cell ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Over Expression

Abstract Our understanding of the biology of MLL fusion gene leukemias is limited by the lack of knowledge of the effects of the different MLL fusion genes on expression of specific homoebox genes and the specific cell compartment(s) that are subsequently deregulated. In this study we investigated whether cellular deregulation was present in committed myeloid precursors and/or the multi-potent hematopoietic stem cells derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll fusion gene under the control of the endogenous promoter that is present in every hematopoietic stem/progenitor cell. The Mll-AF9 knock-in mice display expansion of the myeloid compartment as early as 6 weeks of age (young adult) and develop myeloid leukemia at approximately 6 months. We purified hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitors (GMPs) from wild type and Mll-AF9 young adult bone marrow. We depleted lineage positive cells using a magnetic separation system and purified the respective populations using fluorescence activated cell sorting with specific panels of antibodies (HSC=Li−/Thy1.1lo/IL-7R−/C-kit+/Sca-1+; GMP=Lin−/IL-7R−/Sca-1+/C-kit+/CD34+/CD16/32hi). We cultured these cells in methylcellulose supplemented with GM-CSF, IL-3, SCF and IL-6, conditions that promote the growth of myeloid colonies. We assessed growth deregulation by increased colony numbers at the end of 7 days of culture and by the predominance of dense, compact colony morphology, the latter comprised of immature myeloid cells. Culture of HSCs from Mll-AF9 and wild type mice yielded an identical number of colonies (1102 and 1315 colonies per 104 cells respectively, average). In contrast, GMPs from Mll-AF9 mice yielded almost four times the number of colonies compared to wild type GMPs (3331 and 920 colonies per 104 cells respectively, average). Additionally, Mll-AF9 GMPs formed a higher number of dense, compact colonies compared to Mll-AF9 HSCs (1314 and 352 colonies per 104 cells respectively, average). Neither HSCs nor GMPs from wild type mice formed dense, compact colonies. These results indicate a greater deregulation of GMPs compared to HSCs in Mll-AF9 mice. MLL fusion gene leukemias are characterized by over-expression of specific homeobox genes, and we have previously shown that Mll-AF9 bone marrow cells display increased expression of 5′ Hox-a genes and of the Hox co-factor Meis1 compared to wild type counterparts. We hypothesized that these genes are over-expressed in Mll-AF9 GMPs compared to wild type GMPs. Real time quantitative RT-PCR showed that expression levels of Hoxa7, Hoxa9 and Meis1 were increased in Mll-AF9 GMPs compared to wild type (2.7 ± 0.8, 11.7 ± 7.8 and 19 ± 11.3 fold respectively, mean ± SEM). Overall, these data support the hypothesis that the Mll-AF9 gene is “instructive” at the molecular level at least in part via specific homeobox gene over-expression, resulting in deregulation and expansion of specific progenitor/stem cells such as the GMP population. This expanded GMP population then becomes a target for secondary mutations and later development of leukemia. Future studies focused on understanding the biology of this compartment in Mll-AF9 mice will help in our understanding of the pathogenesis of leukemia and aid in the development of newer, more effective therapies.

Notch Signaling Pathway in Hematopoietic Stem Cells Is Activated through Interactive Communication with Mesenchymal Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1408-1408
Author(s):  
Yuji Kikuchi ◽  
Akihiro Kume ◽  
Masashi Urabe ◽  
Hiroaki Mizukami ◽  
Takahiro Suzuki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Notch Signaling ◽  
Notch Signaling Pathway ◽  
Cell Contact ◽  
Rt Pcr ◽  
Interactive Communication ◽  
Hematopoietic Stem ◽  
Notch Ligands

Abstract Mesenchymal stem cells (MSCs), which are key elements of hematopoietic microenvironment in bone marrow, are known to play a critical role in supporting hematopoiesis. A variety of hematopoietic growth factors are produced from MSCs, and cell-to-cell contact is also believed to be crucial in the interaction between hematopoietic stem cells (HSCs) and MSCs. However, the molecular mechanisms of hematopoiesis-supporting ability of MSCs are still unclear. In particular, there is little information regarding the effects of HSCs on MSC function. In the present study, we investigated the cellular and molecular events in the interactive communication between HSCs and MSCs using a differentiation-inducible MSC model; i.e. parent C3H10T1/2 cells and 10T1/2-derived cell lines, A54 preadipocytes and M1601 myoblasts. These cells were co-cultured with murine HSCs (Lin-Sca1+) isolated from bone marrow. There was 9-fold increase in the number of hematopoietic progenitors after co-culture with A54 preadipocytes, whereas there was no increase when co-cultured with parent 10T1/2 or M1601 cells. More intriguingly, cobblestone areas were observed only when HSCs were co-cultured with A54 cells. Quantitative RT-PCR showed that A54 cells express significantly higher levels of SCF, SDF-1, and angiopoietin-1 (Ang-1) compared with parent 10T1/2 cells and M1601 cells, although these cytokines were not up-regulated when co-cultured with HSCs. To search for the genes involved in the interaction between HSCs and MSCs, we compared gene expression profiles before and after the co-culture by using a microarray analysis. Among the candidate genes with up-regulation after the co-culture, we paid attention to the Notch system, because Notch ligands are considered to play an important role in nurturing HSCs within the hematopoietic microenvironment. As a result, the expression of Notch ligands, Jagged1 and Dll3, increased in A54 cells after the coculture with HSCs. On the other hand, the expression of Notch1 and Hes-1 also increased in HSCs upon co-culture with A54 cells. These data were confirmed by quantitative RT-PCR. Moreover, when HSCs were co-cultured with A54 cells without cell-to-cell contact using Transwell permeable supports, there was neither increase in the number of progenitors in the upper wells, nor the up-regulation of Notch ligands in A54 cells in the lower wells. These findings support the idea that HSCs act on MSCs to induce the expression of Notch ligands via direct cell-to-cell contact and that the Notch ligands derived from MSCs act on HSCs in turn to activate Notch signaling pathway, possibly leading to the cobblestone formation with the maintenance of immature state of HSCs. The Notch system may be one of the critical elements in the interactive communication between HSCs and MSCs.

Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4314-4314
Author(s):  
Akm Khyrul Wara ◽  
Kevin Croce ◽  
ShiYin Foo ◽  
Xinghui Sun ◽  
Basak Icli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Hindlimb Ischemia ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Deficient Mice ◽  
Tgf Β1 ◽  
Impaired Angiogenesis

Abstract Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P&lt;0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P&lt;0.0001; GMPs: WT 32.8% vs. KO 8.97%, P&lt;0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P&lt; 0.0004; GMPs: WT 275.25 vs. KO 108.75, P &lt;0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P&lt;0.00004; GMPs: WT 635.66 vs. KO 263.66, P&lt;0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P &lt;0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P&lt;0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects. Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

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