Impaired Bone Marrow Microenvironment in Fanconi Anemia Murine Model Is Responsible for the Defective Hematopoietic Stem/ Progenitor Cell Mobilization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3629-3629
Author(s):  
Yan Li ◽  
Shi Chen ◽  
Yongzheng He ◽  
Xiaohong Li ◽  
Fengchun Yang
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Stem

Abstract Abstract 3629 Poster Board III-565 Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by progressive bone marrow failure (BMF) and acquisition of malignancies. The only cure for BMF is a human leukocyte antigen (HLA)-matched BM transplantation from a family member or autologous stem cells before BMF develops. Therefore, mobilization of hematopoietic stem/progenitor cells (HSPCs) from BM into peripheral blood (PB) for collection has been a prerequisite for the therapy. However, patients with FA show a markedly decreased HSPC mobilization in response to the traditional mobilizing drug G-CSF and the mechanism(s) underlying the defect remains unknown. Mesenchymal stem/progenitor cells (MSPCs) have been known to be the common progenitor of a variety of cellular components in the bone marrow microenvironment. MSPCs express/secrete cytokines, extracellular matrix proteins and cell adhesion molecules, which regulate the homing, migration, proliferation and survival of HSPCs in vitro and in vivo. Recently, we reported that Fancg-/- MSPCs have a defect in hematopoietic supportive activity both in vitro and in vivo (Li et al. Blood, 2009). In the current studies, we show that Fancg-/- MSPCs have significant reduction in HSPC recruitment as compared to WT MSPCs in a transwell assay. Furthermore, Fancg-/- MSPCs have an alteration in the production of multiple cytokines/chemokines. Application of a neutralizing antibody to the cytokine blocked WT MSPC mediated HSPC migration in vitro. Furthermore, administration of the specific cytokine significantly increased HSPC mobilization in the Fancg-/- mice in vivo. These results demonstrated that an impaired BM microenvironment, specifically MSPCs in Fancg-/- mice, is contributory to defective HSPC mobilization. This study provides evidence of alternative clinical therapeutics for the mobilization of HSPCs in FA patients. Disclosures: No relevant conflicts of interest to declare.

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.

Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 296-296 ◽  
Author(s):  
Carine Domenech ◽  
Alix Rousseau ◽  
Laurence Petit ◽  
Sandra Sanfilippo ◽  
Jean Soulier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Embryonic Development ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Progenitor Cell ◽  
Myeloid Lineage ◽  
Hematopoietic Stem

Abstract Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion. In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT. In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism). In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data. Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mesenchymal stem/progenitor cells promote the reconstitution of exogenous hematopoietic stem cells in Fancg−/− mice in vivo

Blood ◽  
2009 ◽  
Vol 113 (10) ◽  
pp. 2342-2351 ◽  
Author(s):  
Yan Li ◽  
Shi Chen ◽  
Jin Yuan ◽  
Yanzhu Yang ◽  
Jingling Li ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Cellular Functions ◽  
Hematopoietic Stem ◽  
Quantitative Evidence ◽  
Cell Engraftment ◽  
Murine Homologue

AbstractFanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow failure and complex congenital anomalies. Although mutations in FA genes result in a characteristic phenotype in the hematopoietic stem/progenitor cells (HSPCs), little is known about the consequences of a nonfunctional FA pathway in other stem/progenitor cell compartments. Given the intense functional interactions between HSPCs and the mesenchymalmicroenvironment, we investigated the FA pathway on the cellular functions of murine mesenchymal stem/progenitor cells (MSPCs) and their interactions with HSPCs in vitro and in vivo. Here, we show that loss of the murine homologue of FANCG (Fancg) results in a defect in MSPC proliferation and in their ability to support the adhesion and engraftment of murine syngeneic HSPCs in vitro or in vivo. Transplantation of wild-type (WT) but not Fancg−/− MSPCs into the tibiae of Fancg−/− recipient mice enhances the HSPC engraftment kinetics, the BM cellularity, and the number of progenitors per tibia of WT HSPCs injected into lethally irradiated Fancg−/− recipients. Collectively, these data show that FA proteins are required in the BM microenvironment to maintain normal hematopoiesis and provide genetic and quantitative evidence that adoptive transfer of WT MSPCs enhances hematopoietic stem cell engraftment.

scholarly journals Genomic Instability in Fanconi Anemia Results from a Combination of Chromosome Mis-Segregation in Mitosis and Unresolved Interphase DNA Damage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 357-357 ◽  
Author(s):  
Donna Cerabona ◽  
Zahi Abdul Sater ◽  
Rikki Enzor ◽  
Grzegorz Nalepa
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Genomic Instability ◽  
Fanconi Anemia ◽  
Chromosomal Instability ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Biological Significance

Abstract Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow failure, multiple congenital anomalies, and genomic instability resulting in predisposition to cancer. Disruption of the FA signaling network impairs multiple genome-housekeeping processes, including DNA damage recognition and repair in interphase, DNA replication as well as high-fidelity chromosome segregation during mitosis. Recent data published by several groups, including our work (J Clin Invest 2013; 123: 3839-3847), implicated FA signaling in the control of several cell division events essential for chromosomal stability, including the spindle assembly checkpoint (SAC), centrosome maintenance, resolution of ultrafine anaphase bridges and cytokinesis. Understanding the mechanistic origins of chromosomal instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. However, the relative contribution of the interphase and mitotic events leading to genomic instability in Fanconi anemia has not been systematically evaluated. In this work, we dissected the origins and mechanistic significance of chromosomal instability in Fanconi anemia ex vivo and in vivo. We employed the cytochalasin micronucleus assay to quantify the patterns of spontaneous and chemotherapy-induced genomic lesions in FA-A patient-derived primary fibroblasts and Fancc-/- mouse embryonic fibroblasts (MEFs). In this assay, dividing cells are treated with cytochalasin to inhibit cytokinesis and generate binucleated daughter cells. The presence of micronuclei in the resulting cells is indicative of genomic instability caused by either interphase DNA damage or chromosome mis-segregation. Centromere-negative micronuclei (CNMs) represent chromosomal fragments due to unresolved ds-DNA damage. Centromere-positive micronuclei (CPMs) result from whole-chromosome mis-segregation during mitosis. The frequency of both CPMs and CNMs was significantly increased in FA-deficient human and murine cells compared to gene-corrected isogenic control cells. These results indicate that genomic instability in FA is caused by a combination of interphase DNA damage and disordered mitosis. We confirmed the biological significance of these findings by showing that FA patient cells are hypersensitive to low concentrations of taxol (a spindle checkpoint-activating chemotherapeutic) similarly to mitomycin C (a cross-linking agent). Finally, we found increased frequency of micronuclei in Fancc-/- murine red blood cells compared to age-matched wild-type mice, which indicates that spontaneous chromosome mis-segregation occurs in FA-deficient bone marrow in vivo. Our study supports the emerging model of the FA family of proteins as holistic guardians of the genome during interphase and mitosis (see figure based on F1000Prime Rep. 2014; 6: 23, modified). This model furthers our understanding of genomic instability in Fanconi anemia and FA-deficient cancers, and opens new inroads towards targeted therapeutic interventions in these diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Fanconi anemia type C–deficient hematopoietic stem/progenitor cells exhibit aberrant cell cycle control

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2081-2084 ◽  
Author(s):  
Xiaxin Li ◽  
P. Artur Plett ◽  
Yanzhu Yang ◽  
Ping Hong ◽  
Brian Freie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Cell Cycle Control ◽  
Cytokine Signaling ◽  
Compensatory Mechanism ◽  
Hematopoietic Stem ◽  
Type C

Abstract The pathogenesis of bone marrow failure in Fanconi anemia is poorly understood. Suggested mechanisms include enhanced apoptosis secondary to DNA damage and altered inhibitory cytokine signaling. Recent data determined that disrupted cell cycle control of hematopoietic stem and/or progenitor cells disrupts normal hematopoiesis with increased hematopoietic stem cell cycling resulting in diminished function and increased sensitivity to cell cycle–specific apoptotic stimuli. Here, we used Fanconi anemia complementation type C–deficient (Fancc–/–) mice to demonstrate that Fancc–/– phenotypically defined cell populations enriched for hematopoietic stem and progenitor cells exhibit increased cycling. In addition, we established that the defect in cell cycle regulation is not a compensatory mechanism from enhanced apoptosis occurring in vivo. Collectively, these data provide a previously unrecognized phenotype in Fancc–/– hematopoietic stem/progenitor cells, which may contribute to the progressive bone marrow failure in Fanconi anemia.

Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4680-4686 ◽  
Author(s):  
Kent W. Christopherson ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mouse Bone Marrow ◽  
Peptidase Activity ◽  
Hematopoietic Stem ◽  
Migratory Response ◽  
Stromal Cell Derived Factor ◽  
Marrow Cells

AbstractCXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

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.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P&lt;0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

Short-Term Foamyviral Transduction with Virions Encoding Recombinant Corrects the Mitomycin C Hypersensitivity of Fancc −/− Progenitor Cells In Vitro and Restores Long Term Repopulating Activity of Fancc −/− Stem Cells In Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3171-3171
Author(s):  
Yue Si ◽  
Cordula Leurs ◽  
Edward Srour ◽  
Samantha Ciccone ◽  
Helmut Hanenberg ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Bone Marrow Cells ◽  
Genetic Disorder ◽  
Foamy Virus ◽  
Alkylating Agents

Abstract Fanconi anemia (FA) is a complex autosomal recessive genetic disorder characterized within the hematological system by progressive bone marrow aplasia, a high propensity to develop acute myeloid leukemia, and hypersensitivity to alkylating agents including mitomycin c. The identification of individual FA genes raises the potential of using gene transfer technology to express/introduce the functional cDNA in/into deficient autologous stem cells. We have previously shown that in the absence of genetic correction with a retroviral mediated Fancc transgene, ex vivo culture of Fancc−/− stem/progenitor cells (HSPC) predisposes uncorrected Fancc−/− HSPC cells to clonal hematopoiesis (Haneline, Blood 2003). Therefore we examined the potential of a helper-free human foamy virus (HFV) derived construct that encodes both the human FANCC and EGFP transgenes to transduce murine Fancc−/− HSC in the absence of prestimulation. In initial experiments, we determined that 40–80% of progenitors were transduced following a single overnight HFV infection using a 20:1 moiety of infection. Subsequent studies demonstrated that HFV efficiently transduced primitive hematopoietic progenitors in G0 and G1 phases of the cell cycle as evidenced both by using multicolor fluorescence activated cell sorting and subsequent culture of sorted cell populations in high proliferating potential (HPP-CFC) and low proliferating potential colony forming assays. Aliquots of HFV transduced cells that were transduced with the construct encoding both Fancc and EGFP, or the reporter transgene only were transplanted into irradiated recipient mice. Four months following transplantation, bone marrow cells were isolated from the reconstituted recipients and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. In these experiments, the MMC hypersensitivity of Fancc−/− progenitors was corrected to wild-type levels. To assess quantitatively the potential of HFV expressed FANCC to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. In two replicate experiments, we determined that the repopulating activity of HFV-transduced Fancc−/− stem cells was comparable to wildtype controls six months following transplantation in primary and secondary recipients. Collectively, these data provide in vivo evidence that the HFV vector is an efficient vehicle for introducing a functional hFANCC transgene into quiescent Fancc−/− HSC in the absence of prestimulation and for complementing the cellular FA defect in vitro and in vivo.

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