Hematopoietic Stem Cells from Fancc[minus] - Wiley Online Library

Stem C ells O A

®

riginal rticle

Hematopoietic Stem Cells from Fancc–/– Mice Have Lower Growth and Differentiation Potential in Response to Growth Factors MICHEL AUBÉ, MATTHIEU LAFRANCE, CHANTAL CHARBONNEAU, ISABELLE GOULET, MADELEINE CARREAU Unité de génétique humaine et moléculaire, CHUQ-Hôpital St-François d’Assise, Québec, Québec, Canada; Department of Pediatrics, Laval University, Québec, Québec, Canada Key Words. Fanconi anemia · Stem cells · CD34+ · Fancc mouse model

A BSTRACT Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow (BM) failure. We have previously shown that stem cells from the FA group C mouse model have lower long-term primary and secondary reconstitution ability, and that bone marrow of Fancc–/– mice contained fewer lineage-negative (Lin–)Thy1.2lowSca-1+c-kit+ CD34+ cells but normal levels of Lin–Thy1.2lowSca-1+c-kit+CD34– primitive cells. These data suggest that CD34+ primitive cells have either a lower growth or differentiation potential, or that these cells have greater apoptosis levels. To investigate the role Fancc might have on the growth and differentiation potentials of primitive hematopoietic stem cells, we used a single-cell culture system and monitored cell viability,

doubling potential, and apoptosis levels of Fancc–/– primitive Lin–Thy1.2–Sca-1+ (LTS)-CD34+ and LTS-CD34– stem cells. Results showed that Fancc–/– LTS-CD34– and LTS-CD34+ cells had altered growth and apoptosis responses to combinations of stimulatory cytokines, most dramatically in response to a combination of factors that included interleukin-3 (IL-3) and IL-6. In addition, Fancc–/– LTS-CD34– and LTS-CD34+ cells showed a lower differentiation potential than Fancc+/+ cells. These results support a role for Fancc in the growth and differentiation of primitive hematopoietic cells and suggest that an altered response to stimulatory cytokines may contribute to BM aplasia in FA patients. Stem Cells 2002;20:438-447

INTRODUCTION Fanconi anemia (FA) is a congenital form of aplastic anemia and is transmitted through an autosomal recessive mode [1]. FA is characterized by progressive bone marrow (BM) failure, congenital abnormalities, and a predisposition to malignancies [2, 3]. The clinical manifestation is heterogeneous, but the common outcome in the majority of patients is life-threatening hematological failure. The clinical

heterogeneity of FA is paralleled by a genetic heterogeneity, implying the role of several genes in the pathophysiology of FA [4]. Six out of 8 possible FA genes, FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG, have been cloned [5-11], and mouse models for group A, C, and G have been generated [12-15]. Cells obtained from these mutant mice have been shown to be sensitive to DNA cross-linking agents and to have an

Correspondence: Madeleine Carreau, Ph.D., Unité de génétique humaine et moléculaire, CHUQ-Hôpital St-François d’Assise, 10 rue de l’Espinay, Québec, Québec, Canada, G1L 3L5. Telephone: 418-525-4402; Fax: 418-525-4195; e-mail: [email protected] Received April 24, 2002; accepted for publication June 24, 2002. ©AlphaMed Press 1066-5099/2002/$5.00/0

STEM CELLS 2002;20:438-447

www.StemCells.com

439

abnormal G2-M progression of the cell cycle, similar to that seen in patients’ cells [14, 16, 17]. Also, hematopoietic progenitor cells from Fancc–/– mice were shown to be hypersensitive to inhibitory cytokines, including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and macrophage inhibitory protein 1 alpha (MIP-1α), showing a lower level of colony formation and greater apoptosis [13, 18, 19]. BM failure, similar to that observed in FA patients, was shown in Fancc–/– mice after treatment with a DNA-damaging agent, mitomycin C [20]. Progressive pancytopenia resulted from a reduction in BM cellularity, specifically affecting the number of both early and committed hematopoietic progenitors. We have previously shown that the stem cells from Fancc–/– mice had a profound defect, impairing their ability for long-term reconstitution in recipient mice [21, 22]. We have also demonstrated that the number of primitive Lin–Thy1.2lowc-kit+Sca-1+CD34– cells was normal, while Lin–Thy1.2lowc-kit+Sca-1+CD34+ cells were fewer in Fancc–/– mice [21]. These data suggest that stem cells from Fancc–/– mice have impaired growth and differentiation abilities or that the CD34+ cell population has greater apoptosis levels. To investigate the role Fancc might have on the growth and differentiation potentials of primitive hematopoietic stem cells, we used a single-cell culture system and monitored cell viability, doubling potential, and apoptosis levels of Fancc–/– primitive Lin–Thy1.2–Sca-1+ (LTS) CD34+ and LTS-CD34– stem cells in response to various combinations of growth factors. Results showed that Fancc–/– LTS-CD34– and LTS-CD34+ primitive cells had altered growth, apoptosis, and differentiation responses to combinations of stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand (FL), and interleukin-3 (IL-3) or IL-6 compared with Fancc+/+ cells. MATERIALS AND METHODS Hematopoietic Growth Factors Recombinant murine SCF, TPO, IL-3, and GM-CSF were purchased from StemCell Technologies, Inc. (Vancouver, BC, Canada; http://www.stemcell.com). Recombinant murine IL-6, IL-11, and FL were purchased from R&D Systems (Minneapolis, MN; http://www.rndsys tems.com). Growth factors were used at the following concentrations: SCF, 100 ng/ml; TPO, 10 ng/ml; FL, 100 ng/ml; IL-3, 25 ng/ml; IL-6, 10 ng/ml; IL-11, 100 ng/ml, and GM-CSF, 10 ng/ml. Enrichment and Purification of Subpopulations of LTS-CD34+ and LTS-CD34– Cells BM cells were obtained from 4- to 6-month-old Fancc–/– and Fancc+/+ mice (C57BL/6J, 11th generation of

Growth and Differentiation of Fancc–/– Stem Cells backcrosses). BM was collected from femurs and tibias, resuspended in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS; Life Technologies; Burlington, ON, Canada; http://www.lifetech.com), and depleted of red blood cells in ammonium chloride solution (StemCell Technologies, Inc.) for 10 minutes at 4˚C. BM cells were depleted of lineage- (CD5, CD45R, CD11b, myeloid differentiation antigen Gr-1, and TER 119) and Thy1.2-positive cells using StemSep negative-cell-selection procedure, as described by the manufacturer (StemCell Technologies, Inc.). Lin–Thy1.2– cells were labeled with fluorochrome-conjugated rat anti-mouse antibodies directed against CD34 and Sca-1 antigens (fluorescein isothiocyanate (FITC)-CD34 and phycoerythrin (PE)-Sca-1, Ly-6A/E; PharMingen; Mississauga, ON, Canada; http://www.bdbio sciences.com/pharmingen). Rat IgG2a monoclonal antibodies conjugated to FITC or PE were used as isotype standards. Cells were labeled for 30 minutes at 4˚C, washed, and resuspended in PBS supplemented with 2% FBS. Cells were sorted on an Epics Coulter cell sorter (Beckman Coulter Canada; Mississauga, ON, Canada; http://www.beckman.com). Cells were defined by forward (FSC) and side (SSC) scatter and gated for Sca-1+ and CD34+ (LTS-CD34+) or CD34– (LTS-CD34–) populations. Reanalysis of sorted Sca-1+CD34+ and Sca-1+CD34– cells showed a purity of 95%-99% in both fractions. To ensure the enrichment of colony-forming cells (CFCs) after lineage depletion, unseparated and Lin–Thy1.2– BM cells from both Fancc+/+ and Fancc–/– mice were plated in complete methylcellulose medium, as previously described [20]. Results showed that hematopoietic progenitors from Fancc+/+ and Fancc–/– mice were enriched by factors of 30 and 34, respectively. All cultures were performed in triplicate. Animal experiments were approved by the Animal Care Committee of Laval University, Québec, Canada. Single-Cell Proliferation Assay LTS-CD34+ and LTS-CD34– cells were seeded in Terasaki plates (NUNC; Roskilde, Denmark; http://www. nunc.dk) at a density of 1 cell in 10 µl of serum-free medium supplemented with 1% bovine serum albumin, 10 µg/ml bovine pancreatic insulin, 200 µg/ml human transferrin, 10–4 M 2-mercaptoethanol, 2 mM L-glutamine (StemSpan SFEM medium, StemCell Technologies, Inc.), antibiotics (penicillin, 50 U/ml, and streptomycin, 50 µg/ml; Life Technologies), and selected growth factors. Cells were incubated at 37˚C in 5% CO2, and the presence of cells was verified by light microscopy 2-12 hours after plating, to exclude wells containing more than one cell. Wells were monitored daily for cell maintenance and viability, cell division, and cell death over 7 days. Cell maintenance and

Aubé, Lafrance, Charbonneau et al. viability were established as the percentage of wells containing at least one viable cell, as previously described [23]. Viability was evaluated by characteristic morphology using phase-contrast microscopy. Doubling potential was evaluated as the percentage of wells having one or more cell division. Cloning frequency was established as the percentage of wells containing more than three viable cells after 7 days of culture, as previously described [24, 25]. Terasaki plates were numbered and coded to exclude observer bias. Each point represents the mean of three separate experiments, each representing from 30 to 45 wells. Results are expressed as the mean ± the standard error (SE), and statistical significance was done using the Student’s paired t test. Apoptosis and Differentiation Studies To evaluate apoptosis levels in response to stimulatory cytokines, sorted LTS-CD34+ and LTS-CD34– cells were plated at a density of 1,000 cells/well in serum-free medium (StemSpan; StemCell Technologies, Inc.) supplemented with various growth factors, as described above. At each time point, cells were labeled using the Annexin-V staining kit (Roche Molecular Biochemicals; Laval, Quebec, Canada; http://www.roche.com), transferred on a Teflon-printed microscope slide (Electron Microscopy Science; Washington, PA; http://www.emsdiasum.com/ems), and analyzed by fluorescence microscopy using a Nikon Eclipse TE300 epifluorescence microscope (Nikon Canada; Mississauga, ON, Canada; http://www.nikon.ca). Photographs of cells from each condition were obtained for scoring apoptotic cells using a CoolSnap charged-coupled device camera (Canberra Packard Canada; Montréal, QC, Canada; http://www.cpcan. ca/index.htm). Three separate experiments were conducted with each combination of growth factors. To evaluate the differentiation potential of primitive stem cells in response to various combinations of growth factors, sorted LTS-CD34+ and LTS-CD34– cells were plated at a density of 10,000 cells/well in 96-well plates. Cells were cultured for 7 days at 37˚C in serum-free medium supplemented with different combinations of cytokines, as described above. Cells were labeled for 30 minutes at 4˚C using a mixture of fluorochrome-conjugated rat anti-mouse antibodies directed against CD34 and lineage antigens (FITC-CD34, PE-CD5, PE-CD45R, PECD11b, and PE-myeloid differentiation antigen Gr-1; PharMingen). Cells were washed and resuspended in PBS supplemented with 2% FBS before analysis using an Epics Coulter flow cytometer (BeckmanCoulter Canada). Cells were defined by FSC and SSC and gated for CD34 and lineage populations. Two separate experiments were conducted with each combination of growth factors, and 30,000 events were scored in each experiment. Results are

440

expressed as the mean percentage of cells in each fraction ± SE, and statistical significance was tested using the Student’s paired t test. RESULTS Fancc–/– LTS-CD34– and LTS-CD34+ Cells Have Altered Growth Kinetics in Response to SCF, TPO, FL, and IL-6 Previous studies showed that stem cells from Fancc–/– mice had an impaired ability for long-term reconstitution in recipient mice, and that the BM of Fancc–/– mice had a lower number of primitive Lin–Thy1.2lowc-kit+Sca1+CD34+ cells [21, 22]. Using a single-cell culture system, we aimed to determine if the absence of the Fancc gene affected the growth potential of stem cells, impairing their ability to maintain a normal CD34+ stem cell pool, or if CD34+ cells were more susceptible to cell death. We, thus, studied the growth and apoptosis responses of Fancc–/– CD34+ and CD34– stem cells to various stimulatory cytokines. Hematopoietic stem cells from Lin–Thy1.2– BM cells were sorted into two different populations based on their CD34 and Sca-1 expression profiles, Sca-1+CD34+ (LTS-CD34+) and Sca-1+CD34– (LTS-CD34–) populations. Cells obtained from each fraction were cultured at a density of 1 cell/well in serum-free medium supplemented with cytokines. Each well, containing one single cell, was monitored daily for 7 days, and cell responsiveness was evaluated by the capacity to maintain cell viability and undergo cell division and apoptosis. Single-cytokine cultures, containing SCF, TPO, FL, IL-3, IL-6, IL-11, or GMCSF, showed poor growth kinetics, where most cells died within 3-4 days of culture (data not shown). Based on growth factor requirements reported for primitive CD34+ cell maintenance and viability [23, 25-28], LTS-CD34+ and LTS-CD34– cells were cultured in media containing at least SCF, TPO, and FL and supplemented with IL-3, IL6, IL-11, and GM-CSF, alone or in combination. No significant differences in cell viability, cell doubling, or apoptosis between Fancc–/– and Fancc+/+ cells were observed in cultures containing SCF, TPO, and FL only (data not shown). However, Fancc–/– LTS-CD34– cells showed a lower cell viability in response to SCF, TPO, FL, and IL-3 (Table 1), with a 50% lower cell viability after 4 days of culture, than Fancc+/+ cells (24% ± 6.0% versus 43% ± 7.3%, respectively, p < 0.05). Accordingly, Fancc–/– cells showed a lower doubling potential and greater apoptosis level than did Fancc+/+ cells in response to SCF, TPO, FL, and IL-3. The Fancc–/– LTS-CD34+ cell population did not show any significant differences in cell growth, cell doubling, or apoptosis when cultured in the presence of SCF, TPO, FL, and IL-3 compared with Fancc+/+ cells

Growth and Differentiation of Fancc–/– Stem Cells

441

Table 1. Growth kinetics of single CD34– cells in cultures supplemented with SCF, TPO, FL, and IL-3 Fancc+/+ Days

Cell viabilitya d

Fancc–/–

Doubling potentialb

Apoptosisc

Cell viability

Doubling potential

–

–

100

–

–

34 ± 21e

32 ± 11

Apoptosis

1

100

2

66 ± 14

43 ± 20

21 ± 0.9

71 ± 9.5

3

54 ± 8.7

31 ± 11

38 ± 3.0

47 ± 8.0e

18 ± 3.4

44 ± 9.6

4

43 ± 7.3

9.5 ± 3.0

35 ± 15

24 ± 6.0e

4.4 ± 4.4

47 ± 10

f

5

34 ± 1.1

5.4 ± 1.1

12 ± 8.4

17 ± 1.5

2.2 ± 2.2

28 ± 9.1f

7

21 ± 8.3

1.6 ± 1.6

39 ± 12

17 ± 1.5

2.2 ± 2.2

25 ± 9.1

a

Sorted single LTS-CD34– cells were plated at 1 cell/well and monitored over 7 days as described in Materials and Methods. Cell viability is expressed as the percentage of wells with at least one live cell.

a

b

Doubling potential is expressed as the percentage of wells having one or more cell division.

c

Apoptosis represents the percentage of cells staining positive for Annexin V.

d

Data represent the mean ± SE of three separate experiments each having 30-45 wells.

e

Significant difference between Fancc+/+ and Fancc–/– cells, p < 0.05.

f


Table 2. Growth kinetics of single CD34+ cells in cultures supplemented with SCF, TPO, FL, and IL-3 Fancc+/+

Fancc–/–

Cell viabilitya

Doubling potentialb

Apoptosisc

Cell viability

Doubling potential

Apoptosis

1

100d

–

–

100

–

–

2

88 ± 1.9

66 ± 3.3

8.2 ± 0.2

88 ± 1.9

67 ± 6.0

6.4 ± 1.7

3

79 ± 4.4

54 ± 5.4

3.1 ± 1.3

79 ± 11

48 ± 11

5.6 ± 0.6

4

69 ± 2.8

37 ± 3.0

8.3 ± 0.3

71 ± 2.5

24 ± 11

7.6 ± 0.1

5

60 ± 6.5

10 ± 3.9

37 ± 7.2

50 ± 13

1.0 ± 1.0

29 ± 3.1

7

41 ± 11

0.9 ± 0.9

63 ± 9.2

40 ± 13

0

51 ± 3.5

Days

a

Sorted single LTS-CD34+ cells were plated at 1 cell/well and monitored over 7 days as described in Materials and Methods. Cell viability is expressed as the percentage of wells with at least one live cell.

a

b


c


d


(Table 2). These results suggest that the Fancc–/– LTSCD34–, but not the LTS-CD34+ cell fraction, had altered growth kinetics in response to a cocktail of growth factors containing IL-3. We also investigated the growth response of Fancc–/– stem cells to a cocktail of SCF, TPO, FL, and IL-6. Fancc–/– LTS-CD34– cells showed altered growth kinetics in response to IL-6 (Table 3). Fancc–/– LTS-CD34– cell viability was significantly lower than that of Fancc+/+ cells after 4 days of culture (25% ± 2.6% compared with 39% ± 4.8%, respectively, p < 0.05), but slightly higher at days 5 and 7 (24% ± 0.7% and 22% ± 0.9% for Fancc–/– cells compared with 20% ± 0.4% and 9.5% ± 2.6% for Fancc+/+ cells, respectively). Apoptosis levels, corresponding with a lower

cell viability, were higher in the first 3 days of culture and lower at days 5 and 7. No significant differences in doubling potential were observed between Fancc–/– and Fancc+/+ LTS-CD34– cells in response to SCF, TPO, FL, and IL-6. Growth kinetics of primitive LTS-CD34+ cells in response to SCF, TPO, FL, and IL-6 showed that Fancc–/– LTS-CD34+ cells had a markedly lower cell viability (25% lower at day 4 and 40% lower at days 5 and 7) and doubling potential (50% lower at day 3) in response to a cocktail of cytokines containing IL-6 (Table 4). In addition, Fancc–/– LTS-CD34+ showed higher levels of apoptosis than Fancc+/+ cells with a significant difference at day 5 (70% ± 12% and 57% ± 12%, respectively; p < 0.01). These results suggest that both Fancc–/– primitive LTS-CD34+ and LTS-CD34– cells

Aubé, Lafrance, Charbonneau et al.

442

Table 3. Growth kinetics of single CD34– cells in cultures supplemented with SCF, TPO, FL, and IL-6 Fancc+/+ Days

Cell viabilitya d

Fancc–/–

Doubling potentialb

Apoptosisc

Cell viability

Doubling potential

–

–

100

–

–

37 ± 1.0

30 ± 10

64 ± 4.6

38 ± 7.3

40 ± 10f

Apoptosis

1

100

2

69 ± 3.2

3

49 ± 11

15 ± 4.9

34 ± 1.4

40 ± 0.1

17 ± 3.6

38 ± 0.5

4

39 ± 4.8

9.1 ± 1.1

25 ± 10

25 ± 2.6e

14 ± 0.1

23 ± 1.9

5

20 ± 0.4

0

58 ± 1.9

24 ± 0.7f

3.9 ± 0.6

7

9.5 ± 2.6

0

52 ± 31

22 ± 0.9f

0

38 ± 19 32 ± 15

a

Sorted single LTS-CD34– cells were plated at 1 cell/well and monitored over 7 days as described in Materials and Methods. Cell viability is expressed as the percentage of wells with at least one live cell.

a

b


c


d


e


f


Table 4. Growth kinetics of single CD34+ cells in cultures supplemented with SCF, TPO, FL, and IL-6 Fancc+/+ Days

Cell viabilitya d

Doubling potentialb

Fancc–/– Apoptosisc

Cell viability

Doubling potential

Apoptosis

1

100

–

–

100

–

–

2

86 ± 4.8

52 ± 2.2

19 ± 6.1

87 ± 5.2

59 ± 8.1

8.9 ± 1.4

3

67 ± 2.9

42 ± 4.3

11 ± 3.9

65 ± 8.0

20 ± 0.2f

13 ± 5.0

e

4

52 ± 6.4

11 ± 2.9

31 ± 21

39 ± 9.0

5

36 ± 4.3

3.3 ± 1.7

57 ± 12

22 ± 6.6g

0

70 ± 12f

7

25 ± 3.1

0.9 ± 0.9

81 ± 14

10 ± 2.4e

0

88 ± 6.9

10 ± 10

32 ± 9.9

a

Sorted single LTS-CD34+ cells were plated at 1 cell/well and monitored over 7 days as described in Materials and Methods. Cell viability is expressed as the percentage of wells with at least one live cell. b


c


d


e


f


g


have an altered response to IL-6. No significant differences in cell viability, doubling potential, or apoptosis were observed between Fancc+/+ and Fancc–/– LTS-CD34– and LTS-CD34+ cells in response to combinations of cytokines, including SCF, TPO, FL, IL-3, and IL-6 or SCF, TPO, FL, IL-3, and IL-6 with GM-CSF (data not shown). Fancc–/– LTS-CD34+ Cells Have a Lower Single-Cell Cloning Ability in Response to Stimulatory Cytokines To investigate the ability of Fancc–/– primitive cells to grow and give rise to cell progeny, we tested the cloning

frequency of both LTS-CD34– and LTS-CD34+ populations using the single-cell assay system. Sorted single LTS-CD34– and LTS-CD34+ cells were plated at 1 cell/well and cultured in medium supplemented with a cocktail of SCF, FL, and TPO with or without IL-3, IL-6, IL-11, or GM-CSF for 7 days, as described in Materials and Methods. The presence of a single cell per well was monitored 12 hours after plating to exclude wells containing more than 1 cell. Cloning frequency was established as wells containing more than three cells after 7 days of culture [24, 25]. The Fancc–/– LTS-CD34– population showed slightly higher levels of cloning frequency

Growth and Differentiation of Fancc–/– Stem Cells

443

A

no significant differences between Fancc–/– and Fancc+/+ LTS-CD34+ cells with a three-cytokine cocktail when compared with IL-3 alone, results from the apparent variability in the response of the Fancc+/+ cells to multicytokine cocktails. These results suggest that Fancc–/– LTS-CD34+, but not LTS-CD34–, stem cells have altered cloning ability responses to stimulatory cytokines, most dramatically to a combination of SCF, TPO, and FL, with or without IL-3, IL-6, or IL-11.

40

Fancc +/+

Cloning frequency (%)

Fancc –/– 30

20

*

10

0 Control

Cloning frequency (%)

B

IL-3

IL-6

IL-11

GM

3 + 6 + 11

3 + 6 + 11 + GM

GM

3 + 6 + 11

3 + 6 + 11 + GM

50

Fancc +/+ 40

Fancc –/–

30

20

*

10

0

†

* Control

IL-3

IL-6

† IL-11

Figure 1. Cloning frequency of single CD34+ and CD34– cells in cultures supplemented with cytokines. Sorted single (A) LTS-CD34– and (B) LTS-CD34+ cells were plated at 1 cell per well and cultured in medium supplemented with SCF, FL, and TPO plus one or several cytokines: IL-3, IL-6, IL-11, GM-CSF (GM), IL-3, IL-6, and IL-11 (3-611), or IL-3, IL-6, IL-11, and GM-CSF (3-6-11-GM) for 7 days as described in Materials and Methods. The presence of a single cell per well was monitored 12 hours after plating to exclude wells containing more than one cell. Cloning frequency was established as the percentage of wells containing three or more cells. Bars represent the mean ± SE of three separate experiments, each having 30-45 wells. The absence of SE bars represents values too low to appear in the graph. *p < 0.05, †p < 0.01.

than did the Fancc+/+ population, with significant differences only in cultures containing SCF, TPO, FL, and IL-6 (10% ± 3.2% compared with 3.7% ± 0.1% for Fancc–/– and Fancc+/+ cells, respectively, p < 0.05, Fig. 1A). Fancc–/– LTS-CD34+ cells showed a dramatically lower cloning ability in response to the combination of SCF, TPO, and FL, with or without IL3, IL-6, or IL-11, than Fancc+/+ cells (Fig. 1B). No significant differences in the cloning abilities of Fancc–/– LTS-CD34+ cells compared with Fancc+/+ cells were observed in cultures with GM-CSF or a cocktail of IL-3, IL-6, and IL-11, with or without GM-CSF. However, the cloning frequency level of Fancc–/– LTS-CD34+ cells with all three cytokines reflected the level found with IL-3 alone (15% ± 1.6% for IL-3 and 17% ± 5.7% for IL-3 + IL6 + IL-11). The fact that there were

Impaired Differentiation Potential of Primitive Fancc–/– Cells in Response to Growth Factors Previous findings, showing that Fancc–/– mice have lower numbers of Lin–Thy1.2lowSca-1+c-kit+CD34+ cells while numbers of Lin–Thy1.2lowSca-1+c-kit+CD34– cells are comparable with those of Fancc+/+ mice [21], suggest that the absence of Fancc impairs the CD34– to CD34+ transition ability in terms of differentiation and/or activation state. To evaluate the differentiation ability of primitive hematopoietic stem cells from Fancc–/– mice, sorted LTSCD34– and LTS-CD34+ cells were cultured for 7 days in the presence of different combinations of growth factors. Cell progeny were analyzed by flow cytometry for the presence of CD34 and lineage markers (Fig. 2). Results demonstrated that Fancc–/– LTS-CD34– cells had a lower differentiation ability, having fewer differentiated Lin+CD34– progeny than control Fancc+/+ cells (12% compared with 28%, respectively, p < 0.01) in response to SCF, TPO, and FL (Fig. 2A). In addition, the Fancc–/– LTS-CD34– cell fraction showed fewer differentiated Lin+CD34+ cell progeny than did Fancc+/+ cells (7% compared with 20%, respectively, p < 0.01). Similar results were obtained with both SCF, TPO, FL, and IL-3 and SCF, TPO, FL, and IL-6 culture conditions, where Fancc–/– LTS-CD34– cells gave rise to lower numbers of Lin+CD34– cell progeny (26% and 38% for Fancc–/– compared with 45% and 61% for Fancc+/+, respectively). Similarly, Fancc–/– primitive CD34+ cells had fewer differentiated, lineage-positive progeny after cultures with combinations of SCF, TPO, and FL and SCF, TPO, FL, and IL-6 (Fig. 2B). These results suggest that Fancc–/– stem cells have impaired differentiation ability in response to growth factors. DISCUSSION Hematopoietic stem cells from Fancc–/– mice were shown to have lower long-term and short-term repopulating potentials [21, 22]. Also, the fact that Lin–Thy1.2lowSca-1+ckit+CD34+ stem cells are fewer in Fancc–/– mice [21] suggests that the Fancc gene product is required for the maintenance of normal stem cell numbers and/or normal stem cell development. Using a single-cell culture system, we sought to

Aubé, Lafrance, Charbonneau et al.

444

Fancc +/+

A

Fancc –/–

B

SCF + TPO + FL

27.6

27.7

Fancc +/+

Fancc –/–

SCF + TPO + FL

19.8

25.0

11.9†

7.1†

36.4

44.6

SCF + TPO + FL + IL-3

84.1

5.2

74.4

6.8

9.6

1.2

14.4

1.5

66.6

8.8

70.7

7.9

21.3

1.2

18.9

2.5

15.1

68.8

22.6

1.3

5.1 ‡

3.8 †


12.4

25.7

18.7

21.6

20.8

23.1

32.6

Lin-PE

Lin-PE

45.3


60.7

9.6


25.4

38.3

4.4

10.1

*

40.8 **

10.9

CD34-FITC

80.5

3.2

CD34-FITC

Figure 2. Representative flow cytometric analysis of CD34 and lineage marker expression profile of Fancc–/– CD34– and CD34+ cells following cultures. Sorted (A) LTS-CD34– and (B) LTS-CD34+ cells (10,000 cells) from Fancc–/– and Fancc+/+ mice were cultured in the presence of SCF, TPO, and FL with or without IL-3 or IL-6 for 7 days. The CD34 and lineage markers expression profiles of cell progeny were analyzed by flow cytometry following cultures. Numbers in quadrants represent the mean percentages of two separate experiments with 30,000 events each. Statistical significance was tested using the Student’s paired t test. *p < 0.05, **p < 0.0005, †p < 0.01, ‡p < 0.005.

determine if the lower number of CD34+ stem cells resulted from a stem cell differentiation and/or proliferation defect, or if the CD34+ cell population was more susceptible to apoptosis. In the present study, we were able to demonstrate that both Fancc–/– CD34+ and CD34– stem cells had altered growth kinetics and differentiation responses to growth factors, most dramatically to SCF, TPO, FL, IL-3, and IL-6. We also showed that Fancc–/– LTS-CD34+ cells were more susceptible to apoptosis than Fancc+/+ cells in SCF, TPO, FL, and IL-6 cultures. In vitro single-cell culture systems have been designed to evaluate the effects of various cytokines on self-renewal and differentiation of primitive stem cells [23, 25, 28]. We considered the cytokines SCF, FL, and TPO to be generally accepted as promoting growth of primitive hematopoietic cells [23, 29, 30], and thus, we chose these as standard cytokines in all cultures. We subsequently added IL-3 and IL-6, cytokines that also have been shown to promote stem cell growth [26, 30, 31], and measured their influence on Fancc–/– stem cells. We showed that Fancc–/– stem cells had altered growth kinetics, evaluated as cell viability, doubling

potential, and cloning ability, in response to a combination of cytokines known to promote stem cell growth. This suggests that the lower stem cell pool seen in Fancc–/– mice [21] may be the result of an altered growth response to stimulatory signals from the BM microenvironment. This altered growth response of Fancc–/– stem cells supports the idea that the Fancc gene may play a role in cytokine transactivating signals required for the maintenance of stem cell numbers and stem cell development. Several lines of evidence have indicated that FA group C cells (FA-C) are hypersensitive to inhibitory cytokines such as TNF-α, IFN-γ, and MIP-1α [13, 18, 19, 32-34]. Studies on the IFN-γ signaling pathway have indicated that FANCC mutant cells are defective in signal transducer and activator of transcription (STAT) 1 activation [35]. In addition, FA-C cell lines showed constitutive overexpression of IFN-γ-inducible genes [36], suggesting that the IFN-γ pathway is aberrantly regulated in FA-C cells. The Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway is widely used by members of the cytokine receptor family and has been shown to be

445

involved in normal development and function of hematopoietic cells [37, 38]. Although studies on FA-C cells have shown IFN-γ-altered signaling responses, stimulatory cytokines, such as IL-3 and IL-6, do not share the same STAT signaling pathway as the inhibitory cytokine, IFN-γ. Of the seven known mammalian STATs (STAT1 to 6, including STAT5a and STAT5b), STAT5 and STAT3 have been shown to be specifically activated by IL-3 and the IL-6 family of cytokines, respectively [38-40]. The altered growth and differentiation responses of Fancc–/– primitive stem cells to growth factors may result from an altered STAT transactivating signal similar to the IFN-γmediated STAT1 activation defect described in FANCC mutant cells [35]. Since hematopoietic stem cells were shown to lack detectable cell-surface expression of Fas receptors, thus, being unresponsive to IFN-γ-mediated growth inhibition [41, 42], the altered IFN-γ signaling pathway may not be responsible for the altered growth and differentiation potentials of Fancc–/– stem cells. Thus, the FANCC protein may have multifunctional roles in promoting cell growth and cell survival by acting through various STAT signaling pathways. FANCC multifunctionality has been described in view of mitomycin C complementation and IFN-γ signaling function [43]. This multifunctionality may extend to signaling pathways, where FANCC could act as a regulator of STAT signaling molecules in response to specific signals in different cell types. Previous findings showed that Fancc–/– mice had lower numbers of Lin–Thy1.2lowSca-1+c-kit+CD34+ cells, while numbers of Lin–Thy1.2lowSca-1+c-kit+CD34– cells were comparable with those of Fancc+/+ mice [21]. These data indicate that the absence of Fancc impairs the ability of murine stem cells to make the transition from CD34– to CD34+ or that the CD34+ stem cell pool is not maintained properly, suggestive of altered self-renewal or greater susceptibility to cell death. Although Fancc–/– mice do not have fewer peripheral blood cells indicative of normal terminal stages of differentiation, our data, showing fewer differentiated progeny from Fancc–/– stem cells following culture, suggest that the lack of Fancc results in abnormal stem cell divisions. The lower cloning frequency, observed in Fancc–/– LTS-CD34+ cultures containing

Growth and Differentiation of Fancc–/– Stem Cells either IL-3, IL-6, or IL-11, further supports the possibility that Fancc–/– primitive cells have an aberrantly regulated differentiation process in response to growth factors. Maintenance of the stem cell pool requires self-renewal of stem cells, which is believed to result from an asymmetrical cell division giving rise to heterogeneous daughter cells with different proliferative and cell cycle properties [29, 44, 45]. Absence of Fancc may, thus, affect stem cells divisions, whereby some daughter cells, possibly at the CD34+ stage, may not differentiate properly and may be more susceptible to apoptosis. Since the murine CD34 marker has been associated not only with differentiation state but also with activation and/or cycling state [46], lower numbers of CD34+ cells in Fancc–/– mice may also reflect an altered activation state. Our results presented here, showing that Fancc–/– stem cells gave rise to lower numbers of lineage-positive cell progeny, and the fact that Fancc–/– stem/progenitor cells (Lin–ckit+Sca-1+) have been shown to be less quiescent [47] support the idea that the Fancc gene is involved in differentiation processes in response to growth factors. Together, the previous studies, reporting lower CD34+ stem cell numbers in Fancc–/– mice, and our present results demonstrate that the loss of Fancc results in a profound alteration in hematopoietic stem cell function, more specifically affecting stem cell development, suggestive of an aberrant growth factor signal transduction pathway. The delineation of the signal transduction molecular pathway in relation to the absence of Fancc may shed some light on the role of the Fancc gene in hematopoiesis and BM failure. ACKNOWLEDGMENTS We wish to thank Dr. Manuel Buchwald for providing the Fancc knockout mice and to Dr. Maurice Dufour for technical assistance in flow cytometry. This research was supported by grants from the Canadian Institutes of Health Research (CIHR), the Fanconi Anemia Research Fund Inc., the Fanconi Canada foundation, a CIHR junior investigator award (M.C.), and training awards from La Fondation de la recherche sur les maladies infantiles (M.A. and I.G.).

R EFERENCES 1 Buchwald M, Joenje H, Auerbach A. Fanconi anemia. In: Scriver C, Beaudet A, Sly W et al., eds. Metabolic and Molecular Basis of Inherited disease. New York: McGraw-Hill, 1997. CD-ROM.

4 Joenje H, Patel KJ. The emerging genetic and molecular basis of Fanconi anaemia. Nat Rev Genet 2001;2:446-457.

2 Alter BP. Fanconi’s anemia and malignancies. Am J Hematol 1996;53:99-110.

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

3 Liu JM. Fanconi’s anemia. In: Young NS, ed. Bone Marrow Failure Syndromes. Philadelphia: W.B. Saunders Company, 2000:47-68.

6 The Fanconi Anaemia/Breast Cancer Consortium. Positional cloning of the Fanconi anaemia group A gene. Nat Genet 1996;14:324-328.

Aubé, Lafrance, Charbonneau et al. 7 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. 8 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. 9 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. 10 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. 11 Timmers C, Taniguchi T, Hejna J et al. Positional cloning of a novel Fanconi anemia gene, FANCD2. Mol Cell 2001;7:241-248. 12 Chen M, Tomkins DJ, Auerbach W et al. Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. Nat Genet 1996;12:448-451. 13 Whitney MA, Royle G, Low MJ et al. Germ cell defects and hematopoietic hypersensitivity to gamma- interferon in mice with a targeted disruption of the Fanconi anemia C gene. Blood 1996;88:49-58. 14 Cheng NC, van de Vrugt HJ, van der Valk MA et al. Mice with a targeted disruption of the Fanconi anemia homolog Fanca. Hum Mol Genet 2000;9:1805-1811. 15 Yang Y, Kuang Y, De Oca RM et al. Targeted disruption of the murine Fanconi anemia gene, Fancg/Xrcc9. Blood 2001;98:3435-3440. 16 Tomkins DJ, Care M, Carreau M et al. Development and characterization of immortalized fibroblastoid cell lines from an FA(C) mouse model. Mutat Res 1998;408:27-35. 17 Otsuki T, Wang J, Demuth I et al. Assessment of mitomycin C sensitivity in Fanconi anemia complementation group C gene (Fac) knock-out mouse cells. Int J Hematol 1998;67:243-248. 18 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. 19 Rathbun RK, Faulkner GR, Ostroski MH et al. Inactivation of the Fanconi anemia group C gene augments interferongamma-induced apoptotic responses in hematopoietic cells. Blood. 1997;90:974-985. 20 Carreau, M, Gan OI, Liu L, et al. Bone marrow failure in the Fanconi anemia group C mouse model after DNA damage. Blood 1998;91:2737-2744. 21 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. 22 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.

446

23 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+Thy1+lin– cells. Blood 1998;92:4098-4107. 24 Ramsfjell V, Bryder D, Bjorgvinsdottir H et al. Distinct requirements for optimal growth and in vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells. Blood 1999;94:4093-4102. 25 Borge OJ, Ramsfjell V, Cui L et al. Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38– bone marrow cells with multilineage potential at the single-cell level: key role of thrombopoietin. Blood 1997;90:2282-2292. 26 Fujisaki T, Berger MG, Rose-John S et al. Rapid differentiation of a rare subset of adult human lin(-)CD34(-)CD38(-) cells stimulated by multiple growth factors in vitro. Blood 1999;94:1926-1932. 27 Petzer AL, Zandstra PW, Piret JM et al. Differential cytokine effects on primitive (CD34+CD38–) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin. J Exp Med 1996;183:2551-2558. 28 Zandstra PW, Conneally E, Petzer AL et al. Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proc Natl Acad Sci USA 1997;94:4698-4703. 29 Ema H, Takano H, Sudo K et al. In vitro self-renewal division of hematopoietic stem cells. J Exp Med 2000;192:1281-1288. 30 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. 31 Roβmanith T, Schroder B, Bug G et al. Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells. STEM CELLS 2001;19:313-320. 32 Rathbun RK, Christianson TA, Faulkner GR et al. Interferongamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8dependent activation of caspase 3 family members. Blood 2000;96:4204-4211. 33 Wang J, Otsuki T, Youssoufian H et al. Overexpression of the fanconi anemia group C gene (FAC) protects hematopoietic progenitors from death induced by Fas-mediated apoptosis. Cancer Res 1998;58:3538-3541. 34 Koh PS, Hughes GC, Faulkner GR et al. The Fanconi anemia group C gene product modulates apoptotic responses to tumor necrosis factor-alpha and Fas ligand but does not suppress expression of receptors of the tumor necrosis factor receptor superfamily. Exp Hematol 1999;27:1-8. 35 Pang Q, Fagerlie S, Christianson TA et al. The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. Mol Cell Biol 2000;20:4724-4735. 36 Fagerlie SR, Diaz J, Christianson TA et al. Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes. Blood 2001;97:30173024.

447

37 Ward AC, Touw I, Yoshimura A. The Jak-Stat pathway in normal and perturbed hematopoiesis. Blood 2000;95:19-29. 38 Akira S. Functional roles of STAT family proteins: lessons from knockout mice. STEM CELLS 1999;17:138-146.

Growth and Differentiation of Fancc–/– Stem Cells 43 Pang Q, Christianson TA, Keeble W et al. The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality. Blood 2001;98:1392-1401.

39 Bromberg JF. Activation of STAT proteins and growth control. Bioessays 2001;23:161-169.

44 Brummendorf TH, Dragowska W, Lansdorp PM. Asymmetric cell divisions in hematopoietic stem cells. Ann NY Acad Sci 1999;872:265-272; discussion 272-273.

40 Hirano T, Ishihara K, Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 2000;19:2548-2556.

45 Brummendorf TH, Dragowska W, Zijlmans JM et al. Asymmetric cell divisions sustain long-term hematopoiesis from single-sorted human fetal liver cells. J Exp Med 1998;188:1117-1124.

41 Bryder D, Ramsfjell V, Dybedal I et al. Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation. J Exp Med 2001;194:941-952.

46 Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hematopoietic stem cells. Blood 1999;94:25482554.

42 Aguila HL, Weissman IL. Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells. Blood 1996;87:1225-1231.

47 Haneline L, Li X, Plett A et al. Hematopoietic stem cell quiescence is dependent on the Fanconi anemia complementation type C gene product. Blood 2001;98:216a.