Abstract 577: Origin and Therapeutic Efficacy of Human Cardiac Progenitor Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Federico Mosna ◽  
Claudia Bearzi ◽  
Marcello Rota ◽  
Toru Hosoda ◽  
Jochen Tillmanns ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Connexin 43 ◽  
Human Heart ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Functional Integration ◽  
Phenotypic Properties ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice

To establish whether progenitor cells are present in the human heart (hCPCs) and have phenotypic properties distinct from human hematopoietic stem cells (hHSCs), samples of human myocardium were enzymatically dissociated and c-kit-positive-cells were sorted and plated immediately to obtain clones derived from single founder cells. By FACS analysis, freshly isolated hCPCs comprised 1.1±1.0% of the population and were negative for HSC markers, CD34 and CD133, and KDR. They were also negative for epitopes of monocytes, CD14 and CD16, mast cells, CD45, and lymphocytes, CD3 and CD20. The phenotype of hCPCs was completely different from that of human bone marrow cells which were positive for these surface antigens. Only small fractions of hCPCs expressed GATA4 and Nkx2.5. Of 1,530 seeded hCPCs, 11 clones were generated accounting for 0.7% cloning efficiency. Subsequently, these cells were injected in immunodeficient mice and rats at the time of coronary occlusion and 5 days after infarction. This was done to assess whether hCPCs differentiated into myocytes and coronary vessels immediately after ischemic injury and in the presence of a well-developed infarct. In both cases, hCPCs regenerated the infarcted myocardium. Connexin 43 and N-cadherin were detected between recipient rodent myocytes and newly formed human myocytes. The problem was then whether the structural integration of these two myocyte populations had a physiological counterpart. Studies were performed using an ex vivo preparation together with two-photon microscopy and laser line-scan imaging. EGFP-positive-hCPCs were injected in infarcted mice and the heart was studied 2-weeks later. The heart was perfused with an oxygenated Tyrode solution containing the calcium indicator Rhod-2 and stimulated at 1 Hz. Calcium transients was recorded in EGFP positive human myocytes and EGFP negative mouse myocytes. The synchronicity in calcium tracings between these two distinct cell pools was apparent, pointing to the functional integration of newly formed EGFP-positive human myocytes with the surrounding EGFP-negative mouse myocytes. Thus, the human heart contains hCPCs which are not of bone marrow origin and possess the ability to acquire the cardiomyocyte and coronary vascular cell lineages.

Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 477-477
Author(s):  
Christopher B Cole ◽  
Angela M. Verdoni ◽  
David H Spencer ◽  
Timothy J. Ley
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

Leukemic Predisposition of Mice Transplanted With Gene-Modified Hematopoietic Precursors Expressing flt3 Ligand

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2003-2011 ◽  
Author(s):  
Teresa S. Hawley ◽  
Andrew Z.C. Fong ◽  
Henrik Griesser ◽  
Stewart D. Lyman ◽  
Robert G. Hawley
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Transmembrane Protein ◽  
Macrocytic Anemia ◽  
Leukemic Cell ◽  
Cell Types ◽  
Soluble Form ◽  
Hematopoietic Stem

Abstract flt3/flk-2 ligand (FL) is a cytokine that exhibits synergistic activities in combination with other early acting factors on subpopulations of hematopoietic stem/progenitor cells. In addition to normal hematopoietic precursors, expression of the FL receptor, flt3R, has been frequently demonstrated on the blast cells from patients with acute B-lineage lymphoblastic, myeloid, and biphenotypic (also known as hybrid or mixed) leukemias. Because many of these leukemic cell types express FL, the possibility has been raised that altered regulation of FL-mediated signaling might contribute to malignant transformation or expansion of the leukemic clone. In humans, FL is predominantly synthesized as a transmembrane protein that must undergo proteolytic cleavage to generate a soluble form. To investigate the consequences of constitutively expressing the analogous murine FL isoform in murine hematopoietic stem/progenitor cells, lethally irradiated syngeneic mice (18 total) were engrafted with post–5-fluorouracil–treated bone marrow cells transduced ex vivo with a recombinant retroviral vector (MSCV-FL) encoding murine transmembrane FL. Compared with control mice (8 total), MSCV-FL mice presented with a mild macrocytic anemia but were otherwise healthy for more than 5 months posttransplant (until 22 weeks). Subsequently, all primary MSCV-FL recipients observed for up to 1 year plus 83% (20 of 24) of secondary MSCV-FL animals that had received bone marrow from asymptomatic primary hosts reconstituted for 4 to 5 months developed transplantable hematologic malignancies (with mean latency periods of 30 and 23 weeks, respectively). Phenotypic and molecular analyses indicated that the tumor cells expressed flt3R and displayed B-cell and/or myeloid markers. These data, establishing that dysregulated expression of FL in primitive hematopoietic cells predisposes flt3R+ precursors to leukemic transformation, underscore a potential role of this cytokine/receptor combination in certain human leukemias. © 1998 by The American Society of Hematology.

Identification of Cytogenetically Normal Human CD34+CD38+ Hematopoietic Stem/Progenitor Cells from Inv(16)+ Leukemic Bone Marrow

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1059-1059
Author(s):  
Jason N. LeGrand ◽  
Stephanie C. Heidemann ◽  
C. Scott Swindle ◽  
Christopher A. Klug
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Cell Subset ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Heterogeneous Expression

Abstract For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34+ CD38- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34+ CD38- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34+ CD38- population as single TIM3-, CLL1-, or CD33- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34+ CD38- subsets. Results from methylcellulose assays showed that the TIM3- CLL1- CD33- subset of CD34+CD38- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3- CLL1- CD33- subset of CD34+CD38- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)+ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples. Disclosures No relevant conflicts of interest to declare.

Leukemic Predisposition of Mice Transplanted With Gene-Modified Hematopoietic Precursors Expressing flt3 Ligand

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 2003-2011 ◽  
Author(s):  
Teresa S. Hawley ◽  
Andrew Z.C. Fong ◽  
Henrik Griesser ◽  
Stewart D. Lyman ◽  
Robert G. Hawley
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Transmembrane Protein ◽  
Macrocytic Anemia ◽  
Leukemic Cell ◽  
Cell Types ◽  
Soluble Form ◽  
Hematopoietic Stem

flt3/flk-2 ligand (FL) is a cytokine that exhibits synergistic activities in combination with other early acting factors on subpopulations of hematopoietic stem/progenitor cells. In addition to normal hematopoietic precursors, expression of the FL receptor, flt3R, has been frequently demonstrated on the blast cells from patients with acute B-lineage lymphoblastic, myeloid, and biphenotypic (also known as hybrid or mixed) leukemias. Because many of these leukemic cell types express FL, the possibility has been raised that altered regulation of FL-mediated signaling might contribute to malignant transformation or expansion of the leukemic clone. In humans, FL is predominantly synthesized as a transmembrane protein that must undergo proteolytic cleavage to generate a soluble form. To investigate the consequences of constitutively expressing the analogous murine FL isoform in murine hematopoietic stem/progenitor cells, lethally irradiated syngeneic mice (18 total) were engrafted with post–5-fluorouracil–treated bone marrow cells transduced ex vivo with a recombinant retroviral vector (MSCV-FL) encoding murine transmembrane FL. Compared with control mice (8 total), MSCV-FL mice presented with a mild macrocytic anemia but were otherwise healthy for more than 5 months posttransplant (until 22 weeks). Subsequently, all primary MSCV-FL recipients observed for up to 1 year plus 83% (20 of 24) of secondary MSCV-FL animals that had received bone marrow from asymptomatic primary hosts reconstituted for 4 to 5 months developed transplantable hematologic malignancies (with mean latency periods of 30 and 23 weeks, respectively). Phenotypic and molecular analyses indicated that the tumor cells expressed flt3R and displayed B-cell and/or myeloid markers. These data, establishing that dysregulated expression of FL in primitive hematopoietic cells predisposes flt3R+ precursors to leukemic transformation, underscore a potential role of this cytokine/receptor combination in certain human leukemias. © 1998 by The American Society of Hematology.

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.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

scholarly journals Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone

2020 ◽  
Vol 17 (16) ◽  
pp. 5865
Author(s):  
Ramya Dewi Mathialagan ◽  
Zariyantey Abd Hamid ◽  
Qing Min Ng ◽  
Nor Fadilah Rajab ◽  
Salwati Shuib ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Dna Damage ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Protein Carbonyls ◽  
Mouse Bone Marrow ◽  
Tail Moment ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1783-1783
Author(s):  
Mariela Sivina ◽  
Takeshi Yamada ◽  
Natalie Dang ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

Requirement for p85α Regulatory Subunit of Class IA PI3Kinase and Rac2 GTPase in Myeloproliferative Disease Driven by Activation Loop Mutant of KIT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 89-89
Author(s):  
Veerendra Munugalavadla ◽  
Emily C. Sims ◽  
Stephen D. Lenz ◽  
Reuben Kapur
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Regulatory Subunit ◽  
Activation Loop ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Oncogenic activation-loop mutants of KIT, the receptor for stem cell factor (SCF), are commonly observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants (found in patients with gastrointestinal stromal tumors [GISTs]), the activation-loop mutants are commonly insensitive to inhibition by tyrosine kinase inhibitors. Furthermore, little is known about the signaling pathways that contribute to oncogenic KIT-induced transformation in SM or AML. We demonstrate that expression of KITD814V (KIT activation-loop mutant) in primary hematopoietic stem and progenitor cells induces constitutive KIT autophosphorylation, promotes ligand-independent hyperproliferation, skews myeloid differentiation towards the granulocytic lineage, and promotes promiscuous cooperation with multiple cytokines, including G-CSF, M-CSF and IL-3. KITD814V expressing primary mast cells also demonstrated hyperproliferation in response to SCF, IL-3, IL-4 and IL-10. Biochemical analyses of KITD814V expressing cells revealed constitutively elevated levels of phosphatidylinositol-3-kinase (PI3K) and its downstream substrate, the Rho family GTPase Rac. Genetic disruption of p85a, the regulatory subunit of class IA PI-3Kinase, but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalized KITD814V-induced ligand independent hyperproliferation in vitro. Additionally, deficiency of p85α or Rac2 corrected the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V expressing stem/progenitor cells as well as mast cells in vitro. Although p85α is hyperphosphorylated and constitutively bound to KITD814V in bone marrow cells in vitro; its physiologic role in transformation in vivo is not known. To address this, we generated a new mouse model to study KITD814V induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of phenotypes resembling acute lymphocytic leukemia via this mutation. Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal myeloproliferative disease (MPD) characterized by leukocytosis, splenomegaly, disruption of the splenic architecture as well as myeloid cell infiltration in the lung and liver. Importantly, in this model, transplantation of KITD814V expressing p85α deficient bone marrow cells rescued the MPD phenotype, including splenomegaly, peripheral blood leukocytosis and the reduced life span associated with the transplantation of KITD814V expressing wildtype bone marrow cells. Treatment of KITD814V-expressing hematopoietic progenitors with either a Rac inhibitor (NC23766) or rapamycin showed a dose-dependent suppression in KITD814V induced growth. Taken together, our results describe the generation of a new murine transplant model to study KITD814V induced transformation and identify p85a and Rac2 as potential novel therapeutic target for the treatment of KITD814V-bearing diseases including SM and AML.

Sign in / Sign up

Export Citation Format

Share Document