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a Human and Molecular Genetic Unit, CHUQ-H?pital Saint-Fran?ois-d’Assise, Quebec City, Quebec, Canada;
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b Department of Pediatrics, Laval University, Quebec City, Quebec, Canada
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2 K% G( L5 w' N+ p$ lKey Words. Fanconi anemia ? Hematopoietic stem cells ? Ex vivo expansion ? Self-renewal6 R: A& C$ O4 t
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Correspondence: Madeleine Carreau, Ph.D., Human and Molecular Genetic Unit, CHUQ-H?pital St-Fran?ois d’Assise, 10 rue de l’Espinay, Quebec, QC, Canada G1L 3L5. Telephone: 418-525-4402; Fax: 418-525-4195; e-mail: madeleine.carreau@crsfa.ulaval.ca
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% J s0 E8 ?' ^4 z! wABSTRACT
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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 . Nine of these disease genes have been cloned, FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, and FANCL/PHF9 . Although no specific molecular function has been attributed to their gene products, FA proteins participate in a complex involved in maintaining genomic integrity ./ F: ]6 d% \$ b- w5 y! F6 Y
3 C& N! s" |7 x1 MThe 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 . Short-term treatments include the administration of androgens and hematopoietic growth factors , 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 . 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 but carries a substantial risk with HLA-matched unrelated BM donors. 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.
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- P8 Z, _; ?; T0 }+ lMATERIALS AND METHODS
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3 y( S. y% A: p4 e# C; o2 L! WEx 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 and altered growth kinetics after culture , 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 and a reduced short-term competitive repopulating capacity of freshly isolated Fancc–/– primitive cells using 1 x 105 competitor cells (online supplemental Fig. 1 and ), 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. 1). 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. 1). These results are consistent with altered chimerism previously reported for Fancc–/– progenitors using 1 x 105 competitor cells . 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 .
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0 C, X! L" J5 J5 \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. B1 v) c4 h# x9 r% K' p s/ G8 u
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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 2, 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. 3). We found that Fancc–/– Lin–Thy1.2–Sca1 CD34 lost all short-term reconstitution potential after ex vivo culture.' X" @- l) X, ~: z1 z c' d: J+ B( i' S
! f0 @, V2 E8 FFigure 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.2 M6 y( o, V6 B Y
4 v: v% c1 i$ `) W/ _: E; W" xFigure 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.
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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. 4). 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. 5).
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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 ' o, _, |& e: |. v
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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.
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Because Fancc–/– cells were shown to have increased cytokine-mediated apoptosis , 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. 6).0 ]" E- v4 ?) b
1 C' p+ S" u5 w" ^2 u4 F. _4 FFigure 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.
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DISCUSSION6 e" M$ w! y! ?
5 N5 ?! x0 A* PWe 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.).
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发表于 2009-3-5 10:49
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