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First published online July 14, 2005
Stem Cells Vol. 23 No. 8 September 2005, pp. 1135 -1141
doi:10.1634/stemcells.2004-0356; www.StemCells.com
© 2005 AlphaMed Press

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Lack of Self-Renewal Capacity in Fancc–/– Stem Cells After Ex Vivo Expansion

Ouassila Habia, Marie-Chantal Delislea, Nancy Messiera, Madeleine Carreaua,b

a Human and Molecular Genetic Unit, CHUQ-Hôpital Saint-François-d’Assise, Quebec City, Quebec, Canada;
b Department of Pediatrics, Laval University, Quebec City, Quebec, Canada

Key Words. Fanconi anemia • Hematopoietic stem cells • Ex vivo expansion • Self-renewal

Correspondence: Madeleine Carreau, Ph.D., Human and Molecular Genetic Unit, CHUQ-Hôpital St-François d’Assise, 10 rue de l’Espinay, Quebec, QC, Canada G1L 3L5. Telephone: 418-525-4402; Fax: 418-525-4195; e-mail: madeleine.carreau{at}crsfa.ulaval.ca


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatments of the hematological manifestation in Fanconi anemia (FA) are first supported by attempts to stimulate hematopoiesis with androgens or hematopoietic growth factors. However, the long-term curative treatment of the hematological manifestation in FA patients is bone marrow (BM) or cord blood stem cell transplantation. The success rate for BM transplantation is fairly high with HLA-matched sibling donors but is, unfortunately, low with HLA-matched unrelated donors. An alternative curative treatment for those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Because FA patients have reduced numbers of stem/progenitor cells, ex vivo expansion of hematopoietic stem cells would be a crucial step in gene transfer protocols. Using the FA mouse model, Fancc–/–, we tested the ability of CD34– hematopoietic stem cells to support ex vivo expansion. We determined that Fancc–/– CD34– stem cells have reduced reconstitution ability and markedly reduced self-renewal ability after culture, as shown by secondary transplants. These results indicate that FA stem cells may not be well suited for ex vivo expansion before gene transfer or transplantation protocols.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fanconi anemia (FA) is a severe bone marrow (BM) failure syndrome affecting children at an early age. Somatic cell fusion studies resulted in the classification of FA patients into11complementation groups, each corresponding to a separate gene [1]. Nine of these disease genes have been cloned, FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, and FANCL/PHF9 [2–11]. Although no specific molecular function has been attributed to their gene products, FA proteins participate in a complex involved in maintaining genomic integrity [12–17].

The clinical manifestation of FA is defined by a progressive BM failure and, in most cases, a multitude of congenital malformations and an increased risk of developing cancers [18, 19]. Short-term treatments include the administration of androgens and hematopoietic growth factors [20–23], which may transiently improve peripheral blood counts. However, studies using the Fancc mouse model have shown that long-term administration of growth factors did not prevent eventual BM failure and may even accelerate BM hypoplasia [24]. The long-term curative treatment of the hematological manifestation of the disease is BM or peripheral blood stem cell transplantation. This procedure offers a significant chance of cure if a sibling HLA-matched donor is available [25, 26] but carries a substantial risk with HLA-matched unrelated BM donors[27]. An alternative curative treatment of those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Stem and progenitor cells can easily be harvested from the BM or peripheral blood and transduced ex vivo using viral vectors as a delivery system. In view of their self-renewal capacity, corrected stem cells, in theory, could replenish the hematopoietic compartment and sustain long-term hematopoiesis. However, FA patients were shown to have reduced numbers of stem/progenitor cells, which may represent a significant obstacle. Therefore, ex vivo expansion of hematopoietic stem cells would be of interest for gene therapy in FA. We tested the ability of Fancc–/– CD34– stem cells to support ex vivo culture. We demonstrate that Fancc–/– CD34– stem cells not only had reduced reconstitution ability but had a dramatically reduced self-renewal capacity after ex vivo culture, as shown in secondary transplant experiments.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification and Transplantation of Lin–Thy1.2–Sca1+CD34– Hematopoietic Cells
BM cells obtained from 4- to 6-month-old Fancc–/– and Fancc+/+ littermates (C57BL/6J, 11th generation of back-crosses; CD45.2+) were collected from femurs and tibias. Stem cells, Lin–Thy1.2–Sca1+CD34–, were purified as described previously [28].

Lin–Thy1.2–Sca1+CD34–stemcellsfromCD45.2+donormice were transplanted into lethally irradiated (950-cGy) CD45.1+ recipient mice (B6.SJL-PtrcaPep3b/BoyJ) 5–6 weeks of age. Five to 10 recipient mice were used per point, each transplanted with 2,500 Lin–Thy1.2–Sca1+CD34– cells from either Fancc–/– or Fancc+/+ donor mice along with 2 x 104 Lin– cells of recipient origin (CD45.1+) for competition and radioprotection.

To evaluate percent engraftment, mice were bled from the saphen vein once per month for 4 months and cells of donor origin were detected by multiparameter flow cytometry. Briefly, blood samples were depleted of red blood cells in ammonium chloride solution for 10 minutes at 4°C, washed in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS), and stained with CD45.2–fluorescein isothiocyanate (donor origin), CD45.1-phycoerythrin (recipient origin), CD11b–peridinin chlorophyll protein (PerCP) (monocytes) or CD45R-PerCP (B lymphocytes), and Ly-6G–allophycocyanin (APC) (granulocytes) or CD5-APC (T lymphocytes) for 30 minutes at 4°C. Samples were washed and resuspended in PBS supplemented with 2% FBS. Samples were analyzed by flow cytometry using a fluorescence-activated cell sorter (FACS) Calibur cytometer (BD Biosciences, Mississauga, Ontario, Canada, http://www.bd.com) using the CellQuest Pro software.

For secondary transplants, BM cells from primary recipient mice were collected at 4 months after transplant and depleted of CD45.1+ cells (recipient origin) with the StemSep-negative cell selection procedure using biotinylated anti-CD45.1 antibodies. Flow cytometric analysis of CD45.1-depleted cells showed a purity of 99% CD45.2+ cells (data not shown). Lethally irradiated secondary recipient mice (B6.SJL-PtrcaPep3b/BoyJ;CD45.1+) were transplanted with 1 x 106 CD45.2+ BM cells from Fancc–/– or Fancc+/+ primary recipients. Reconstitution ability of donor cells was monitored as previously described for primary transplants. Five to seven mice were used per point. Recipient mice received antibiotics 1 week before irradiation and transplantation. Animal experiments were approved by the Animal Care Committee of Laval University.

Ex Vivo Culture Conditions
A total of 10,000 to 12,000 purified Lin–Thy1.2–Sca1+CD34– cells from both Fancc–/– and Fancc+/+ mice were seeded in 96-well plates in 100 µl of serum-free essential media (SFEM) (StemCell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com) supplemented with 100 ng/ml recombinant murine stem cell factor (SCF) (StemCell Technologies), 10 ng/ml thrombopoietin (TPO) (StemCell Technologies), and 100 ng/ml recombinant murine FTL-3 ligand (FL) (R&D Systems, Minneapolis, http://www.rndsystems.com). Cells were cultured for 5 days at 37°C with 5% CO2 before transplants. Recipient mice were transplanted with equivalent 2,500 Lin–Thy1.2–Sca1+CD34– cells as described above. Reconstitution ability of donor cells was monitored as described above.

Cell Division Tracking
Lin–Thy1.2–Sca1+CD34– stem cells (2 x 105 cells/ml) were labeled in the dark with carboxyfluorescein-diacetate succinimidyl ester (CFDA-SE) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) at a dye concentration of 2 µM in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 20% BIT (bovine serum albumin, insulin, transferrin) (StemCell Technologies) for 10 minutes at room temperature with occasional mixing. Cells were washed twice with PBS 2% FBS, resuspended in SFEM, and incubated overnight at 37°C without cytokines to allow unbound dye to leak out of the cells (quenching) and to obtain the day-0 fluorescence profile. Cells were then washed in cold IMDM, resuspended in SFEM with cytokines (100 ng/ml SCF, 10 ng/ml TPO, and 100 ng/ml FL), and cultured for 4 days at 37°C. At the end of the culture period, cells were collected and visualized by flow cytometry using a FACS Calibur cytometer with CellQuest Pro software. Analysis was carried out using the cell proliferation model based on the ModFit LT analysis software (BD Biosciences). CFDA-SE–labeled cells were also analyzed each day by fluorescence microscopy. Briefly, after CFDA-SE labeling, Lin–Thy1.2–Sca1+CD34– stem cells were seeded at a concentration of 2 x 105 cells per ml in Terasaki plates and cultured in SFEM with SCF, TPO, and FL. Before analysis, propidium iodine (PI) (Sigma, St. Louis, http://www.sigmaaldrich.com) was added at a final concentration of 50 ng/ml to detect apoptotic nuclei. Cells were visualized by fluorescence microscopy using an inverted fluorescence microscope (Nikon TE300; Nikon Canada, Mississauga, Ontario, Canada, http://www.nikon.ca) equipped with a CCD camera (Hamamastu, Orca ER; Nikon Canada). Percent PI-positive cells were estimated from 100 to 500 cells in two separate experiments.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ex vivo expansion of long-term repopulating cells is of great interest for clinical application for FA patients, including stem cell transplantation and gene therapy. Because Fancc–/– primitive hematopoietic cells were shown to have altered reconstitution ability [29, 30] and altered growth kinetics after culture [28], FA mutant cells may not respond properly to ex vivo expansion. We therefore tested the reconstitution ability and self-renewal potential of Fancc–/– Lin–Thy1.2–Sca1+CD34– stem cells after ex vivo culture. A total of 2,500 purified stem cells were cultured ex vivo for 5 days and transplanted into wild-type recipients along with 2 x 104 freshly isolated Lin– competitor cells. Because of the reported defect in long-term and secondary reconstitution potential [29] and a reduced short-term competitive repopulating capacity of freshly isolated Fancc–/– primitive cells using 1 x 105 competitor cells (online supplemental Fig. 1Go and [30]), 2 x 104 Lin– cells were used for radioprotection/competition. Using these transplantation conditions, we found that whereas normal wild-type cells conserved their repopulating ability after ex vivo culture, Fancc–/– stem cells showed a dramatic reduction in repopulating activity (Fig. 1Go). The decrease in donor cells was reflected by a reduction in all blood cell lineages but, more dramatically, T and B lymphocytes. Freshly isolated Fancc–/– Lin–Thy1.2–Sca1+CD34– stem cells showed normal levels of short-term reconstitution ability using 2 x 104 Lin– competitor cells compared with 1 x 105 competitors, where reduced repopulating ability was observed (online Fig. 1Go). These results are consistent with altered chimerism previously reported for Fancc–/– progenitors using 1 x 105 competitor cells [30]. Our results are also in agreement with previous studies in which normal levels of reconstitution potential (without competitor cells) were observed at 4 months while altered long-term repopulating potential in Fancc–/– progenitor cells was observed 6 to 9 months after transplants [29].



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  		Figure 1. Fancc–/– CD34– stem cell reconstitution potential after ex vivo culture. Percent reconstitution ability of Lin–Thy1.2–Sca1+CD34– (CD45.2+) cells after ex vivo culture. A total of 2,500 cells were cultured for 5 days before transplants. Total amount of cells after culture was transplanted in each animal together with 2 x 104 Lin– competitive cells. Reconstitution ability was evaluated by fluorescence-activated cell sorting analysis of peripheral blood cells as a function of time. Percent CD45.2+CD45R+, CD45.2+CD5+, CD45.2+CD11b+, and CD45.2+CDLy–6G+ was also evaluated for each transplanted mouse. Each point represents the mean ±SEM of four to seven individual recipients. Fancc–/– and wild-type WT controls indicate the reconstitution ability of 2,500 Lin–Thy1.2–Sca1+CD34– transplanted cells without culture. Absence of SEM bars represents values too low to appear. Abbreviation: WT, wild-type.

 
To evaluate the self-renewal potential of Fancc–/– stem cells after ex vivo culture, we performed secondary transplants in which donor cells (CD45.2+) from primary recipients at 4 months after transplant were harvested and purified to transplant equal cell numbers into secondary recipients. Secondary transplants were performed with 1 x 106 CD45.2+ cells (donor origin). As shown in Figure 2Go, Fancc–/– Lin–Thy1.2–Sca1+CD34– stem cells have a dramatically reduced self-renewal potential after ex vivo culture (less than 5% secondary reconstitution levels). Consequently, all blood cell lineages originating from primary donors were reduced to less than 5%. We also tested the reconstitution potential of freshly isolated Lin–Thy1.2–Sca1+CD34+ cells, known as short-term reconstituting cells, before and after ex vivo culture (Fig. 3Go). We found that Fancc–/– Lin–Thy1.2–Sca1+CD34+ lost all short-term reconstitution potential after ex vivo culture.



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  		Figure 2. Fancc–/– CD34– stem cell self-renewal ability after ex vivo culture. Bone marrow cells from Lin–Thy1.2–Sca1+CD34– primary recipients (CD45.2+ cells) were pooled and depleted from cells of primary recipient’s origin (CD45.1). One million CD45.1-depleted bone marrow cells of primary recipient’s origin were injected into secondary recipient mice. Reconstitution ability was evaluated by fluorescence-activated cell sorting analysis of peripheral blood cells as a function of time. Percent CD45.2+CD45R+, CD45.2+CD5+, CD45.2+CD11b+, and CD45.2+CDLy-6G+ was also evaluated for each transplanted mouse. Each point represents mean ± SEM of 7 to 10 individual recipients. Fancc–/– and wild-type (WT) controls indicate the reconstitution ability of 1 x 106 CD45.1-depleted bone marrow cells from primary recipients transplanted with Lin–Thy1.2–Sca1+CD34– cells without culture. Absence of SEM bars represents values too low to appear.

 


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  		Figure 3. Fancc–/– CD34+ cell reconstitution ability after ex vivo culture. Five thousand Lin–Thy1.2–Sca1+CD34+ cells (CD45.2+) were cultured for 5 days before transplants. Total amount of cells after culture was transplanted in each animal together with 2 x 104 Lin– competitive cells. Reconstitution ability was evaluated by fluorescence-activated cell sorting analysis of peripheral blood cells as a function of time. Percent CD45.2+CD45R+, CD45.2+CD5+, CD45.2+CD11b+, and CD45.2+CDLy-6G+ was also evaluated for each transplanted mouse. Each point represents the mean ± SEM of 7 to 10 individual recipients. Fancc–/– and wild-type (WT) controls indicate the reconstitution ability of 5,000 Lin–Thy1.2–Sca1+CD34+ transplanted cells without culture. Absence of SEM bars represents values too low to appear.

 
Several possibilities that could explain why Fancc–/– stem cells do not respond properly to ex vivo expansion are reduced expression of specific growth-factor receptors, altered growth-factor proliferative responses, or increased cell death during culture. First, we measured the expression levels of SCF, TPO, and FL growth-factor receptors, notably, the c-Kit receptor, TPO-R/c-Mpl, and CD135 by flow cytometry. We found no difference in expression levels of all cytokine receptors in primitive Lin–Thy1.2–Sca1+CD34– stem cells from either Fancc–/– or Fancc+/+ mice (data not shown). We next analyzed cellular division of sorted Lin–Thy1.2–Sca1+CD34– stem cells in response to ex vivo culture using the tracking dye CFDA-SE. We found no significant differences between Fancc–/– and wild-type lineage-depleted, Lin–Thy1.2–Sca1+CD34+ cells or Lin–Thy1.2–Sca1+CD34– stem cells (Fig. 4Go). However, Fancc–/– Lin–Thy1.2–Sca1+CD34– fast-dividing cells (CFDA-SEdim cells) showed a significant delay in cellular division, with increased cells in the fourth generation and reduced cell numbers in the fifth generation compared with wild-type cells. To determine if the reduced reconstitution ability was associated with reduced numbers of cells after ex vivo expansion, purified Lin–Thy1.2–Sca1+CD34– stem cells from both Fancc–/– and wild-type mice were seeded in Terasaki plates and counted during culture. Whereas wild-type stem cells showed an average of 4.5-fold increase in cell numbers, Fancc–/– stem cells showed a slight reduction in cellular expansion during ex vivo culture (mean of 3.6 ± 1.3-fold; Fig. 5Go).



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  		Figure 4. Cell division tracking of Fancc–/– stem cells after ex vivo culture. (A): Representative flow cytometry profile of carboxyfluorescein-diacetate succinimidyl ester (CFDA-SE) fluorescence intensity as a function of cell number of both Fancc+/+ and Fancc–/– lineage-depleted cells (Lin–Thy1.2–), Lin–Thy1.2–Sca1+CD34+, and Lin–Thy1.2–Sca1+CD34– stem cells after ex vivo expansion. (B): Mean percent number of cells per generation as establish by CFDA-SE cell division tracking from three separate experiments. Significant differences (*p < .01) were observed between Fancc–/– Lin–Thy1.2–Sca1+CD34– and wild-type (WT) stem cells. Generations were established using the ModFit LT Proliferation wizard software (BD Biosciences).

 


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  		Figure 5. Expansion of Lin–Thy1.2–Sca1+CD34– stem cells during culture. Purified Lin–Thy1.2–Sca1+CD34– stem cells from both Fancc+/+ and Fancc–/– mice were seeded in Terasaki plates and counted during culture. Fold increase in cell number was determined as the number of cells counted on each day divided by the number of cells seeded in each respective well at day 0. Each point represents the mean ± SEM from three separate experiments. Absence of SEM bars represents values too low to appear. Abbreviation: WT, wild-type.

 
Because Fancc–/– cells were shown to have increased cytokine-mediated apoptosis [31–33], we tested the possibility that reduced expansion and loss of self-renewal potential in primitive CD34– cells results from increased apoptosis during ex vivo culture. Whereas Fancc–/– Lin–Thy1.2–Sca1+CD34– stem cells were able to divide and differentiate, these cells showed twice the apoptosis during culture that wild-type stem cells did (Fig. 6Go).



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  		Figure 6. Apoptosis of Fancc–/– stem cells after ex vivo culture. Fluorescence microscopy of propidium iodine–labeled (red) CD34– stem cells after ex vivo culture in Terasaki plates. Apoptosis was evaluated every day during culture. Numbers represent the mean percentage of propidium iodine–positive cells (from 100 to 500 cells counted) from two separate experiments.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Different combinations of cytokines have been tested to support ex vivo expansion of hematopoietic stem cells. In our experiments, we used a combination of SCF, FL, and TPO, which is generally accepted as promoting growth of primitive hematopoietic cells [34–36] and, in fact, did promote growth and maintenance of wild-type hematopoietic stem cells. However, using the same culture condition, Fancc–/– stem cells were not able to maintain their reconstitution ability or their self-renewal potential. This lack of stem cell maintenance in ex vivo culture does not result from altered cytokine receptor expression but may be a consequence of aberrant cytokine-signaling responses, consistent with a delay in cellular division, slightly reduced expansion, and increased apoptosis. Fancc–/– cells have been previously shown to have aberrant signaling responses and increased apoptosis in response to inhibitory cytokines and DNA-damaging agents [31, 37, 38]. Therefore, it is conceivable that Fancc–/– stem cells do not respond properly to culture conditions and fail to divide correctly. We thus propose two possible scenarios explaining the markedly reduced self-renewal ability of Fancc–/– hematopoietic stem cells (Fig. 7Go); first, Fancc–/– stem cells cannot maintain their self-renewal potential because both daughter cells are committed to differentiate (Fig. 6BGo), or, second, Fancc–/– daughter stem cells, those programmed to self-renew, die of apoptosis (Fig. 6CGo). Based on our results showing a slight reduction in cell numbers and increased apoptosis of Fancc–/– stem cells after ex vivo culture, we favor the second scenario.



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  		Figure 7. Proposed model of Fancc–/– stem cell division and maintenance. (A): Schematic representation of wild-type stem cell (SC) division and maintenance during ex vivo expansion. Upon division, stem cells give rise to one daughter stem cell with identical biological properties (self-renewal) and one committed daughter. In Fancc–/– mice, either the stem cell does not divide properly and both daughter cells are committed to differentiate (B) or the self-renewing daughter stem cell dies of apoptosis due to aberrant signaling responses (C). Both scenarios will lead to a dramatically reduced self-renewal ability of Fancc–/– stem cells.

 
Taken together, the results presented here clearly show that ex vivo culture impaired the reconstitution potential and self-renewal capacity of Fancc–/– CD34– stem cells having fewer cellular divisions, less expansion, and increased apoptosis, thus suggesting that FA cells do not respond properly to ex vivo expansion using SCF, TPO, and FL growth factors.

The feasibility of gene therapy for FA has been well established with FA mouse models [39–41]; however, the availability of hematopoietic stem cells in FA mice has never been an issue because FA mice have normal hematopoiesis. In the context of reduced stem/progenitor cells, FA patients may not be well suited for gene transfer protocols unless their stem cells can be increased. Our work shows that Fancc–/– CD34– stem cells have reduced reconstitution ability and increased apoptosis after ex vivo culture. Haneline et al. also determined that ex vivo culture of Fancc–/– low-density progenitor cells using SCF and interleukin 6 growth factors compromised their repopulation ability [42]. In addition, our work is the first to establish that the most primitive stem cells had lost all self-renewal potential after ex vivo culture. Consequently, FA mutant cells may not be suited for ex vivo expansion before gene transfer. Other means, or possibly other culture conditions for hematopoietic stem cell expansion, will have to be assessed for FA cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Manuel Buchwald (Hospital for Sick Children) for providing the Fancc+/– mice. We also thank Dr. Maurice Dufour for his excellent expertise in cell sorting. This study was supported by a grant from the Canadian Institutes of Health Research (CIHR), a CIHR junior investigator award (to M.C.), and a training award from La Fondation pour la recherche sur les maladies infantiles (to O.H.).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Joenje H, Patel KJ. The emerging genetic and molecular basis of Fanconi anaemia. Nat Rev Genet 2001;2:446–457.[CrossRef][Medline]

  2. Strathdee CA, Gavish H, Shannon WR et al. Cloning of cDNAs for Fanconi’s anaemia by functional complementation. Nature 1992;358:434.[Medline]

  3. Lo Ten Foe JR, Rooimans MA, Bosnoyan-Collins L et al. Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA. Nat Genet 1996;14:320–323.[CrossRef][Medline]

  4. de Winter JP, Waisfisz Q, Rooimans MA et al. The Fanconi anaemia group G gene FANCG is identical with XRCC9. Nat Genet 1998;20:281–283.[CrossRef][Medline]

  5. de Winter JP, Rooimans MA, van Der Weel L et al. The Fanconi anaemia gene FANCF encodes a novel protein with homology to ROM. Nat Genet 2000;24:15–16.[CrossRef][Medline]

  6. de Winter JP, Leveille F, van Berkel CG et al. Isolation of a cDNA representing the Fanconi anemia complementation group E gene. Am J Hum Genet 2000;67:1306–1308.[Medline]

  7. Timmers C, Taniguchi T, Hejna J et al. Positional cloning of a novel Fanconi anemia gene, FANCD2. Mol Cell 2001;7:241–248.[CrossRef][Medline]

  8. The Fanconi Anaemia Breast Cancer Consortium. Positional cloning of the Fanconi anaemia group A gene. Nat Genet 1996;14:324–328.[CrossRef][Medline]

  9. Howlett NG, Taniguchi T, Olson S et al. Biallelic inactivation of BRCA2 in Fanconi anemia. Science 2002;297:606–609.[Abstract/Free Full Text]

  10. Meetei AR, de Winter JP, Medhurst AL et al. A novel ubiquitin ligase is deficient in Fanconi anemia. Nat Genet 2003;35:165–170.[CrossRef][Medline]

  11. Meetei AR, Levitus M, Xue Y et al. X-linked inheritance of Fanconi anemia complementation group B. Nat Genet 2004;36:1219–1224.[CrossRef][Medline]

  12. de Winter JP, van der Weel L, de Groot J et al. The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG. Hum Mol Genet 2000;9:2665–2674.[Abstract/Free Full Text]

  13. Garcia-Higuera I, Kuang Y, Naf D et al. Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. Mol Cell Biol 1999;19:4866–4873.[Abstract/Free Full Text]

  14. Garcia-Higuera I, Taniguchi T, Ganesan S et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. MolCell 2001;7:249–262.[CrossRef][Medline]

  15. Hussain S, Wilson JB, Medhurst AL et al. Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways. Hum Mol Genet 2004;13:1241–1248.[Abstract/Free Full Text]

  16. Niedzwiedz W, Mosedale G, Johnson M et al. The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair. Mol Cell 2004;15:607–620.[CrossRef][Medline]

  17. Hussain S, Witt E, Huber PA et al. Direct interaction of the Fanconi anaemia protein FANCG with BRCA2/FANCD1. Hum Mol Genet 2003;12:2503–2510.[Abstract/Free Full Text]

  18. Young N. Bone Marrow Failure Syndromes. Philadelphia: WB Saunders, 2000.

  19. Alter BP. Leukemia and preleukemia in Fanconi’s anemia. Cancer Genet Cytogenet 1992;58:206–209.[CrossRef][Medline]

  20. Kemahli S, Canatan D, Uysal Z et al. GM-CSF in the treatment of Fanconi’s anaemia. Br J Haematol 1994;87:871–872.[Medline]

  21. Guinan EC, Lopez KD, Huhn RD et al. Evaluation of granulocyte-macrophage colony-stimulating factor for treatment of pancytopenia in children with fanconi anemia. J Pediatr 1994;124:144–150.[CrossRef][Medline]

  22. Rackoff WR, Orazi A, Robinson CA et al. Prolonged administration of granulocyte colony-stimulating factor (filgrastim) to patients with Fanconi anemia: a pilot study. Blood 1996;88:1588–1593.[Abstract/Free Full Text]

  23. Scagni P, Saracco P, Timeus F et al. Use of recombinant granulocyte colony-stimulating factor in Fanconi’s anemia. Haematologica 1998;83:432–437.[Abstract/Free Full Text]

  24. Carreau M, Liu L, Gan OI et al. Short-term granulocyte colony-stimulating factor and erythropoietin treatment enhances hematopoiesis and survival in the mitomycin C-conditioned Fancc–/– mouse model, while long-term treatment is ineffective. Blood 2002;100:1499–1501.[Abstract/Free Full Text]

  25. Gluckman E. Bone marrow transplantation in Fanconi’s anemia. STEM CELLS 1993;11(suppl 2):180–183.

  26. Kohli-Kumar M, Morris C, DeLaat C et al. Bone marrow transplantation in Fanconi anemia using matched sibling donors. Blood 1994;84:2050–2054.[Abstract/Free Full Text]

  27. Guardiola P, Pasquini R, Dokal I et al. Outcome of 69 allogeneic stem cell transplantations for Fanconi anemia using HLA-matched unrelated donors: a study on behalf of the European Group for Blood and Marrow Transplantation. Blood 2000;95:422–429.[Abstract/Free Full Text]

  28. Aube M, Lafrance M, Charbonneau C et al. Hematopoietic stem cells from fancc–/– mice have lower growth and differentiation potential in response to growth factors. STEM CELLS 2002;20:438–447.[Abstract/Free Full Text]

  29. Carreau M, Gan OI, Liu L et al. Hematopoietic compartment of Fanconi anemia group C null mice contains fewer lineage-negative CD34+ primitive hematopoietic cells and shows reduced reconstruction ability. Exp Hematol 1999;27:1667–1674.[CrossRef][Medline]

  30. Haneline LS, Gobbett TA, Ramani R et al. Loss of FancC function results in decreased hematopoietic stem cell repopulating ability. Blood 1999;94:1–8.[Abstract/Free Full Text]

  31. Haneline LS, Broxmeyer HE, Cooper S et al. Multiple inhibitory cytokines induce deregulated progenitor growth and apoptosis in hematopoietic cells from Fac–/– mice. Blood 1998;91:4092–4098.[Abstract/Free Full Text]

  32. Cumming RC, Lightfoot J, Beard K et al. Fanconi anemia group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1. Nat Med 2001;7:814–820.[CrossRef][Medline]

  33. Rathbun RK, Christianson TA, Faulkner GR et al. Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members. Blood 2000;96:4204–4211.[Abstract/Free Full Text]

  34. Ema H, Takano H, Sudo K et al. In vitro self-renewal division of hematopoietic stem cells. J Exp Med 2000;192:1281–1288.[Abstract/Free Full Text]

  35. Goff JP, Shields DS, Greenberger JS. Influence of cytokines on the growth kinetics and immunophenotype of daughter cells resulting from the first division of single CD34+Thy-1+lin– cells. Blood 1998;92:4098–4107.[Abstract/Free Full Text]

  36. Piacibello W, Sanavio F, Garetto L et al. Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation. Leukemia 1998;12:718–727.[CrossRef][Medline]

  37. Otsuki T, Nagakura S, Wang J et al. Tumor necrosis factor-alpha and CD95 ligation suppress erythropoiesis in Fanconi anemia C gene knockout mice. J Cell Physiol 1999;179:79–86.[CrossRef][Medline]

  38. Rathbun RK, Faulkner GR, Ostroski MH et al. Inactivation of the Fanconi anemia group C gene augments interferon- gamma-induced apoptotic responses in hematopoietic cells. Blood 1997;90:974–985.[Abstract/Free Full Text]

  39. Gush KA, Fu KL, Grompe M et al. Phenotypic correction of Fanconi anemia group C knockout mice. Blood 2000;95:700–704.[Abstract/Free Full Text]

  40. Galimi F, Noll M, Kanazawa Y et al. Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors. Blood 2002;100:2732–2736.[Abstract/Free Full Text]

  41. Otsuki T, Young DB, Sasaki DT et al. Fanconi anemia protein complex is a novel target of the IKK signalsome. J Cell Biochem 2002;86:613–623.[CrossRef][Medline]

  42. Haneline LS, Li X, Ciccone SL et al. Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc–/– hematopoietic stem cells and decreases the risk of clonal evolution. Blood 2003;101:1299–1307.[Abstract/Free Full Text]

Received December 14, 2004; accepted for publication March 22, 2005.




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