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Stem Cells, Vol. 17, No. 5, 273-285, September 1999
© 1999 AlphaMed Press

The Reduction of In Vitro Radiation-Induced Fas-Related Apoptosis in CD34+ Progenitor Cells by SCF, FLT-3 Ligand, TPO, and IL-3 in Combination Resulted in CD34+ Cell Proliferation and Differentiation

Michel Droueta, Jacques Mathieua, Nancy Greniera, Eric Multona, Jean-Jacques Sottob, Francis Herodina

a Centre de Recherches du Service de Santé des Armées, La Tronche, France;
b Institut Albert Bonniot, La Tronche, France

Key Words. Apoptosis • Fas • CD34+ cells • Irradiation • Cytokines • Ex vivo expansion

Dr. Michel Drouet, Experimental Radiohematology Unit, CRSSA, 24, avenue des Maquis du Grésivaudan BP 87-38702 La Tronche Cedex, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recovery from radiation-induced (RI) bone marrow aplasia depends on appropriate cytokine support. The early effects of exogenous cytokines at the hematopoietic stem and progenitor cell (HSPC) level following irradiation are still largely unknown, especially those of survival factors such as stem cell factor (SCF) and Flt-3 ligand (FL). This study was aimed at A) clarifying Fas/Fas-Ligand (Fas-L) implication in RI apoptosis of CD34+ cells and B) assessing the capacity of a combination of cytokines to mitigate RI apoptosis in HSPCs in vitro. We showed that most of in vitro gamma-irradiated CD34+ HSPCs incubated in a medium devoid of cytokines underwent progressive apoptosis-related changes from 6 h (i.e., decreased CD34 antigen expression, Annexin V binding); then Fas/Fas-L coexpression occurred from 10 h on. A strong DNA fragmentation, as assessed by TUNEL assay and propidium iodide staining, was observed at 24 h. Within a 2.5- to 6-Gy dose range, the RI apoptotic process finally led to 97% CD34+ cell death within 48 h with a complete loss of functionality. Unirradiated cells incubated in the same conditions displayed a significantly reduced apoptotic pattern. The early addition of a combination of SCF, FL, thrombopoietin, and interleukin 3 (4F) after cell irradiation prevented 15% (2.5 Gy) and 12% (4 Gy) of HSPCs, respectively, from RI apoptosis, whereas these cytokines used as single factors were inefficient. Furthermore, irradiated HSPCs (2.5 Gy) incubated with 4F in a serum-free culture system for seven days proliferated, giving rise to an increase in the number of total cells (x 5.6-fold) and CD34+ cells (x 4.2-fold) and to megakaryocytic and granulomonocytic precursors. These results show that the prevention of apoptosis in in vitro irradiated HSPCs depends on an early combination cytokine support. These data suggest that the early therapeutic administration of anti-apoptotic cytokines may be critical for preserving functional HSPCs from in vivo radiation damage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The non-uniform radiation dose distribution observed in most of the documented nuclear accidents has resulted in heterogeneous hematopoietic damages. Therefore, the recovery from radiation-induced (RI) bone marrow aplasia depends on appropriate cytokine and/or hematopoietic stem and progenitor cell (HSPC) support, depending on the degree of myeloablation. The sparing of viable HSPCs located in underexposed bone marrow territories is the rationale for in vivo cytokine therapy of RI bone marrow (BM) aplasia [1, 2]. It has recently been proposed that ex vivo expansion of autologous peripheral blood (PB) or BM HSPCs collected from accidentally irradiated individuals may constitute grafts which would represent an additional approach to in vivo cytokine therapy or an alternative treatment in case of contraindicated administration of cytokines (i.e., internal radiocontamination) [3, 4]. We have lately shown in a baboon model of autologous mobilized PB HSPC transplantation that ex vivo expanded CD34+ cells can reduce the duration of neutropenia and thrombocytopenia in myeloablated animals in comparison with that in non-expanded grafts [5]. CD34+ cells were cultured for six days with a combination of four growth factors (4F): stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), and interleukin 3 (IL-3). This cytokine combination exerted on unirradiated cells a balanced effect on proliferation and differentiation leading to a 13.6-fold increase in CD34+ cell number as well as granulomonocytic (GM) and megakaryocytic (MK) precursors. In fact, we observed that 4F exerted a strong anti-apoptotic effect which could be extremely useful in counteracting the RI apoptosis that is the main cause of cell death mediated by ionizing radiation (IR). So far, the maintenance of long-term hematopoiesis by means of HSPC ex vivo expansion settings remains controversial. However, with respect to nuclear accidents such as the Chernobyl catastrophe, the main goal worth pursuing must be to counteract the early neutropenia and thrombocytopenia that are major causes of short-term morbidity and mortality. Actually, spared HSPCs due to heterogeneous damage account for delayed endogenous recovery. Autologous cell therapy applied to such a context should provide CD34+ cell-derived progenitors and post-progenitors capable of differentiating in vivo within a few days.

Thus, ex vivo hematopoietic cell cultures need efficient reduction of the RI apoptotic process that would develop from residual damaged cells involving putative bystander effects [6] occurring after ionizing radiation exposure. The CD95 antigen belongs to the tumor necrosis factor/nerve growth factor receptor family and induces apoptotic cell death on binding of the natural Fas-Ligand (Fas-L) or agonistic anti-Fas antibodies. CD95 is expressed on fresh fetal liver CD34+ cells but not on BM or PB CD34+ cells; its induction on the latter cells has recently been demonstrated in vitro as early as 24 h after incubation with cytokines in liquid cultures. Fas became effective between days 3 and 5 [7, 8]. The precise role of CD95-mediated apoptosis in growth control of hematopoiesis has not been described thus far.

Furthermore, functional Fas expression is also enhanced after in vitro irradiation of HSPCs which exhibit a high apoptotic ratio, especially during the first 24 h of culture [9, 10]. The aim of this study was to clarify the Fas/Fas-L implication in RI apoptosis at the CD34+ cell level to determine whether it is possible to mitigate apoptosis and expand consecutively protected cells in order to apply this strategy to autologous HSPCs collected after accidental exposure. In such situations, as well as for bone marrow transplantation in general, one of the main concerns for clinicians is the slow reconstitution of platelet count post-transplant. Therefore, we focused our research on anti-apoptotic cytokine combinations and on MK proliferation. We cultured in vitro irradiated CD34+ cells in comparison with unirradiated cells in serum-free liquid medium with cytokine combinations and assessed early anti-apoptotic effects and short-term expansion efficacy.

We show in this study that following in vitro irradiation, the majority of CD34+ cells incubated without cytokines exhibited rapid morphological changes as well as CD34 expression reduction (from 6 h) preceding Fas/Fas-L coexpression (from 10 h on) and the final strong DNA fragmentation pattern (at 24 h). In our hands, the early incubation with SCF, Flt-3 ligand, TPO, and IL-3 in combination significantly reduced apoptotic cell death, as evidenced within the first 24 h, and allowed, after seven days of culture, for a 4.2-fold CD34+ cell expansion as well as MK and GM proliferation and differentiation. The early management of irradiated hematopoietic cells with synergistic anti-apoptotic cytokines appears to be critical for cell survival and further in vitro expansion, which could have implications in vivo for the treatment of accidental irradiation casualties.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CD34+ Cells
Baboon cells were collected either by leukapheresis following mobilization with cyclophosphamide and recombinant human (rh)G-CSF or from BM after approval by the Army Ethical Committee was given. CD34+ cell purification was performed using immunoaffinity Ceprate LC columns (CellPro; Bothell, WA). Only the cell samples exhibiting the highest purity (>= 90%) were chosen for this study. The cells suspended in serum-free liquid medium made of Iscove's modified Dulbecco's medium ([IMDM] Life Technologies; Paisley, Scotland) supplemented with 1% bovine serum albumin ([BSA] Boehringer Mannheim; Meylan, France), {alpha}-thioglycerol, liposomes, iron-saturated transferrin (Sigma; St-Quentin Fallavier, France), and antibiotics were irradiated at 2.5 Gy, 4 Gy, and 6 Gy with a 60Co gamma source (dose rate of 100 cGy/min). Following irradiation, cells were washed and resuspended (1 x 105 cells/ml) in serum-free medium with 10 ng/ml matrix metalloprotease inhibitor KB8301 (Pharmingen; San Diego, CA) for membrane (mb) Fas-L evaluation, then incubated in six-well plates at 37°C in a humidified incubator with 5% CO2 in air. Unirradiated control cells and irradiated cells were analyzed after 6, 10, and 24 h of incubation relative to fresh control cells. Cell viability was assessed by trypan blue exclusion assay (TB assay) and flow cytometry (propidium iodide [PI] incorporation).

Apoptosis Evaluation
CD95, mbFAS-L, intracellular (IC) Fas-L and Bcl2, and DNA fragmentation were determined by flow cytometry (Facs Vantage, Becton Dickinson; San Jose, CA). For CD95 and mbFAS-L evaluation, double labelings were performed with 566-PE monoclonal antibody (mAb) (kindly provided by Dr. T. Egeland and Dr. G. Gaudernack) as anti-CD34 mAb and FAS (N-18) or FAS-L (Q-20) polyclonal rabbit antibodies (Tebu; Le Perray, France). Cells were incubated for 1 h (5 µg of Ab/106 cells) on ice, then for 30 min with anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated, and finally fixed. Results were expressed related to isotypic controls as percentages of non-necrotic cells exhibiting CD34 antigen. For IC Fas-L and Bcl2 evaluation, hematopoietic cells were labeled using either the polyclonal antibody previously used (fixation for 1 h in 1% formaldehyde, then overnight incubation on ice in PBS with 0.03% saponin and 1% BSA) or mAb FITC-conjugated (Dako; Trappes, France) and results related to isotypic control. DNA fragmentation was measured following A) DNA staining with PI to determine the percentage of sub-G0/G1 cells which correspond to apoptotic cells or B) UTP incorporation in the presence of exogenous terminaldeoxynucleotidyltransferase (Roche; Meylan, France). For PI staining, the cell pellet was resuspended in 70% ethanol and kept overnight at 4°C in the dark. Cells were then washed and incubated with PI (50 µg/ml, Sigma; St. Louis, MO) in phosphate buffer solution for 30 min and immediately analyzed. Moreover, a CD34-PE/Fas-FITC/UTP-Quantum Red (QR) triple labeling was performed according to three steps: cells were first labeled using 566-PE mAb and FAS polyclonal rabbit Ab; incubated with anti-rabbit IgG FITC, then cells were fixed in phosphate buffered saline 1% formaldehyde overnight; finally biotinylated-UTP (Roche) was incorporated, and staining with streptavidin-QR (Sigma) was carried out. To measure apoptotic membrane alterations, phosphatidylserine externalization was evaluated by flow cytometry using Annexin V (FITC-conjugated reagent, Pharmingen, Becton Dickinson) binding either alone or combined with PI staining.

Fas mRNA expression was semi-quantitatively assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) (Chomozinski's method) using specific human primers (FAS sense primer sequence: 5' ATG CTG GGC ATC TGG ACC CTC CTA 3' FAS antisense sequence: 5' TCT GCA CTT GGT ATT CGT GGT CCG 3') and ß-actin primers as a standard. RNA was prepared from freshly isolated or cultured CD34+ cells. Thirty cycles of PCR were performed with denaturation at 94°C for 1 min, annealing at 68°C for 45 sec (ß-actin: 55°C), and extension at 72°C for 90 sec. PCR products were electrophoresed on an ethidium bromide-stained 1.5% agarose gel. Numerized photographs were made under UV light using Kodak digital camera. Lanes were detected by Kodak digital scientific software.

Cytokine Treatment
rh cytokines were used. SCF, FL, and TPO were purchased from R & D Systems (Abbington, UK); IL-3 was provided by Novartis (Basel, Switzerland). These cytokines were tested at 10 and 50 ng/ml and added within 30 min after irradiation.

Clonogenic Assay
After 24 h of incubation, the clonogenicity of cell samples was evaluated in a short-term culture assay following cell viability assessment. Viable cells (5 x 103/ml) were plated in triplicate in a semisolid phase of 0.9% methylcellulose (Fluka) in IMDM, with 25% heat-inactivated fetal calf serum (Life Technologies), 1% BSA, antibiotics, {alpha}-thioglycerol (5 x 10-5 mol/l), with the following rh cytokines: GM-CSF and IL-3 (20 ng/ml each; Novartis; Basel), Epo (2 U/ml; Boehringer Mannheim), IL-1 (10 ng/ml; R & D Systems), SCF (50 ng/ml; R & D), IL-6 (50 ng/ml; Ares Serono; Geneva, Switzerland). Cells were incubated for 14 days at 37°C with 4.5% CO2 in air, in a fully humidified incubator. Colonies derived from granulocyte macrophage-colony-forming unit (GM-CFU), BFU-E, and granulocyte/erythroid/macrophage/megakaryocytic (GEMM)-CFU were scored, and total CFUs were expressed as percentages of the clonogenic activity of a time-matched unirradiated control sample.

Ex Vivo Expansion of CD34+ Progenitor Cells
Following irradiation, 1 x 105 BM or PB CD34+ cells/ml were incubated in multi-well plates at 37°C in serum-free medium. Unirradiated and irradiated cells were cultured for 7 or 10 days in a humidified incubator with 5% CO2 in air with two combinations of rh cytokines (SCF + FL + TPO + IL-3 or SCF + TPO) added within 30 min after irradiation. Cultures were half-renewed with fresh medium and cytokines after four days. Cells were analyzed at 7 and 10 days of culture for viable cell expansion using hemocytometer, total cell expansion, CD34+ cell amplification, clonogenic activity, and granulomonocytic and MK proliferation.

Cytologic and Phenotypic Analysis
Expanded cells were evaluated by performing cytospin preparations (Shandon; Pittsburgh, PA) that were fixed for 10 min in methanol. The slides were stained with Hemacolor (Merck; Nogent, France) for 10 min. Moreover, cells were stained with anti-CD34-PE, -Thy1-FITC (Pharmingen) and -CD41-FITC (Beckman-Coulter; Villepinte, France) mAbs and analyzed by flow cytometry. For myeloperoxidase (MPO) immunodetection, cells were fixed, then permeabilized (Harlan kit, Sera-Lab; Loughborough, UK) and stained with anti-MPO-FITC mAb (Beckman-Coulter).

In Vivo Irradiation
In vitro cell irradiations are homogeneous and constitute a model for in vivo irradiations. In fact, the documented accidental irradiations have been characterized as being heterogeneous in radiation dose distribution. In humans and large nonhuman primates, radiation exposure induces a gradient of absorbed dose, resulting in a complex coexistence of overexposed and underexposed BM territories. For radiopathological and therapeutic purposes, we therefore developed an adult baboon model of RI BM aplasia. We report here data from one irradiated animal. The anesthetized baboon, weighing 30 kg, was placed in a restraining chair and exposed to a unilateral front TBI with a 60Co gamma source to a total midline tissue dose of 6 Gy at a dose rate of 7 cGy/min-1. Dosimetry was performed using ionization chambers and thermoluminescent dosimeters.

Statistical Analysis
Statistical comparisons were performed using the Fisher's test of a non-parametric analysis of variance. Values of p of less than 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RI Apoptosis in CD34+ Cells is Related to Fas mRNA and Fas Antigen Expression
Fresh CD34+ cells did not express a significant level of Fas mRNA. Unirradiated cells exhibited a low level of expression after 6 h of incubation which became moderate at 24 h. Radiation exposure (2.5 Gy) resulted in the doubling of time-matched unirradiated cell levels at both 6 and 24 h (Fig. 1Go).



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  		Figure 1. RI Fas mRNA induction in CD34+ cells. Fas mRNAs expression is evaluated by RT-PCR using human primers. Lane A: Fas mRNA amplification products; Lane B: ß-actin mRNA amplification products. Lanes correspond to the following samples: freshly isolated unirradiated cells (1), unirradiated cells after 6 h of incubation without cytokines (2), irradiated cells at 6 h without cytokines (3) or with cytokines (4), unirradiated cells after 24 h of incubation without cytokines (5), irradiated cells at 24 h without cytokines (6) or with cytokines (7).

 
Before incubation, a very low percentage of unirradiated CD34+ cells expressed CD95 (2.1%) as evaluated by flow cytometry (Fig. 2Go). Moreover, all the Fas negative cells had a CD34high phenotype. After 3 h of incubation in culture medium, no morphological changes occurred in either unirradiated or irradiated cells (2.5 Gy). At 6 h, a reduction in cell size (i.e., forward light scatter decrease) and an increase in cell granularity (i.e., side light scatter increase) were observed, especially concerning irradiated cells. Moreover, a diminution of CD34 antigen expression in terms of mean fluorescence intensity (MFI) was observed (Fig. 3Go), and irradiation significantly enhanced this decrease. As shown in Fig. 2Go, we observed two Fasneg cell populations with different CD34 expression levels after 6 h in culture medium: cells with high expression as fresh cells (Region R1) and cells with low expression (Region R2). As described in Figure 4Go, irradiation also induced a decrease in the percentage of CD34high cells at 6 h (55.3% of CD34high cells versus 81.7% for unirradiated cells). A slight increase of Fas antigen expression was concomitantly noted (Fig. 5Go). At 24 h in culture medium, no modification of CD34 expression (i.e., MFI) was observed versus that noted at 6 h, but a strong augmentation of Fas expression was observed in control cells. Almost all the untreated irradiated cells expressed Fas antigen while exhibiting decreased CD34 expression (Fig. 6Go, Region R3). A striking decrease in CD34high/Fasneg cells was concomitantly noted. A decline of cell viability was observed at this time in both untreated samples, as evaluated by TB exclusion test (47% of reduction for irradiated cells versus 33% for unirradiated cells). Cells displayed an apoptotic pattern (44.5% hypodiploid cells in irradiated samples versus 21.8% in unirradiated ones). In fact, Fas+ cells exhibited apoptotic DNA fragmentation according to TUNEL assay, whereas Fasneg cells did not (Fig. 7AGo). The RI apoptotic process led on average to 97% CD34+ cell death within 48 h.



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  		Figure 2. RI CD34 antigen expression reduction and Fas antigen upregulation in PB CD34+ cells. CD34+ and Fas expression were evaluated by flow cytometry (double labeling with 566-PE mAb as anti-CD34 mAb and FAS [N-18] polyclonal rabbit antibody). Cells were analyzed at 6 h (Fig. 2) and 24 h (Fig. 3) of culture. Region R1: CD34+high/Fasneg cells; R2: CD34+low/Fasneg cells; R3: CD34low/Fas+ cells; R4: late apoptotic and necrotic cells characterized by low forward/side scatter and as Annexin V/IP positive cells (data not shown). T0 cells (1); unirradiated (A) or irradiated CD34+ cells (B) incubated 6 h without (2) or with (3) 4F.

 


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  		Figure 3. RI CD34 reduction of antigen expression and modulation by 4F treatment. CD34 expression was evaluated by flow cytometry as previously described in Materials and Methods. Unirradiated or irradiated PB CD34+ cells were incubated for 24 h in liquid cultures with or without 4F. Cells were analyzed at T0 and after 6 h or 24 h of culture. Each column (n = 7) represents the mean fluorescence intensity (MFI) of non-necrotic CD34+/Fasneg cells. At 24 h untreated irradiated cells significantly differ from time-matched control (p < 0.05) and from treated irradiated cells (p < 0.0001).

 


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  		Figure 4. Early RI decrease of CD34+ antigen expression and modulation by 4F treatment. Flow cytometric evaluation. Each column (n = 7) represents the percentage of CD34+high/Fasneg cells. Results are expressed after exclusion of necrotic cells as percentage of total non-necrotic CD34+ cells. At 6 h, untreated (p < 0.001) and treated (p < 0.05) irradiated cells significantly differ from time-matched control (p < 0.001 and p < 0.05 respectively). Untreated and treated cells significantly differ from each other (p < 0.01).

 


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  		Figure 5. RI Fas antigen expression in PB CD34+ cells and modulation by 4F treatment. Fas and CD34 antigen expressions were evaluated by flow cytometry at 6 h and 24 h. Cells were stained as previously described in Figure 2Go. Each column (n = 4) represents the percentage of CD34+/Fas+ cells. Results are expressed as percentage of total non-necrotic CD34+ cells. At 24 h, all samples except unirradiated treated cells (4F) significantly (p < 0.0001) differ from T0 value. Unirradiated treated cells (4F), untreated (2.5 Gy), and treated irradiated cells (2.5 Gy 4F) differ from time-matched control (p < 0.0001, p < 0.001, p < 0.05). 2.5 Gy and 2.5 Gy 4F differ from each other (p < 0.001).

 


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  		Figure 6. Unirradiated (A) or irradiated cells (B) incubated 24 h without (1) or with (2) 4F. Results are expressed as percentage of non-necrotic CD34+ cells (R1+R2+R3).

 


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  		Figure 7. Fas antigen expression and DNA fragmentation. Cells are evaluated by triple labeling as described in Materials and Methods. 1: CD34 and Fas expression (CD34-PE/Fas-FITC); 2: Fas expression and DNA fragmentation (Fas-FITC/UTP-QR); 3: CD34 expression and DNA fragmentation (CD34-PE/UTP-QR). A: irradiated cells after 24 h of incubation without cytokines; B: irradiated cells incubated 24 h with 4F. Cells from one representative baboon are expressed as percentage of total events.

 
RI Apoptosis in CD34+ Cells is Related to Fas-Ligand Antigen Expression
With respect to mb Fas-L antigen, CD34+ cells exhibited at the start a low expression (3.2% ± 1.1%) which became moderate at 10 h and stronger at 24 h; IR increased Fas-L expression at both 10 h (31.3% ± 2%) and 24 h (81.1% ± 3.3%) (Fig. 8Go). In our model, RI Fas-L and Fas overexpression exhibited no difference in kinetics. Membrane alterations measured by Annexin V binding appeared later than CD34 expression decrease but earlier than Fas/Fas-L overexpression (Fig. 9Go).



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  		Figure 8. RI mb Fas-L expression in PB CD34+ cells and modulation by 4F treatment. Unirradiated and irradiated cells were analyzed by flow cytometry at 6 h, 10 h, and 24 h of culture. Each column (n = 4) represents the percentage of mb Fas-L CD34+ cells. As indicated in Materials and Methods, results are expressed as percentage of total non-necrotic CD34+ cells at 24 h. Unirradiated treated cells (4F), untreated (2.5 Gy) and treated irradiated cells (2.5 Gy 4F) differ from time-matched control (p < 0.0001). 2.5 Gy and 2.5 Gy 4F differ from each other (p < 0.001).

 


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  		Figure 9. Annexin V binding and mb Fas-L antigen expression. Results from one representative baboon cell sample. Unirradiated or irradiated PB CD34+ cells were incubated for 6 h or 10 h in liquid medium, then analyzed as described in Materials and Methods using flow cytometry.

 
With regard to IC Fas-L, the majority of freshly isolated CD34+ cells exhibited a high IC Fas-L content (Table 1Go). At 6 h of culture following IR, no significant alteration was observed. At 24 h, a bimodal response was noted since about 25% of unirradiated cells displayed unchanged content, whereas the others exhibited a strong reduction of IC Fas-L content. Irradiation induced more cells to exhibit a reduced content (Fig. 10Go).


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  		Table 1. RI IC Fas-L downregulation in PB CD34+ cells. IC Fas-L cell content was evaluated by flow cytometry as described in Figure 7Go. IC Fas-L and IC FasLhigh cells were expressed as a percentage of total cells. At 24 h, untreated irradiated cells (2.5 Gy) significantly differ from time-matched control and from treated irradiated cells (2.5 Gy 4F) (p < 0.05) in terms of Fas-Lhigh expression (n = 3).
 


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  		Figure 10. RI IC Fas-L expression in PB CD34+ cells. IC Fas-L cell content was evaluated by flow cytometry using anti-Fas-L rabbit polyclonal Ab (Tebu) and saponin treatment: unirradiated cells before (1) and after (2) 24 h of culture without cytokines; irradiated cells after 24 h of incubation without (3) or with (4) 4F. Results from one baboon cell sample.

 
RI Apoptosis in CD34+ Cells is Related to Bcl2 Downregulation
At T0, the majority of CD34+ cells were Bcl2 positive and 85.9% ± 0.9% exhibited a high Bcl2 content. After 6 and 24 h of incubation in basal medium, 94.1% and 82.1% of CD34+ cells, respectively, remained Bcl2 positive. However at the latter time, only a minority of cells (22.7% ± 1.7%) maintained a high Bcl2 content (Table 2Go). At that time, irradiation reduced the cell population with high Bcl2 content (8.9% ± 2.8%) even further.


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  		Table 2. Irradiation downregulates Bcl2 in PB CD34+ cells. Bcl2 expression was evaluated by flow cytometry. Bcl2+ and Bcl2high cells were expressed as percentage of total CD34+ cells (n = 4)
 
Rationale for the Use of Cytokines in Combination
We first determined the best in vitro culture conditions and cytokines with unirradiated cells according to two criteria: cell survival/antiapoptotic effect and cell expansion. Serum-free cultures were associated with higher anti-apoptotic level and hematopoietic cell proliferation compared with cultures performed in the presence of serum. Based on preclinical results involving ex vivo expanded CD34+ cell grafts [5], we selected SCF and FL as cell survival factors, IL-3 as multi-CSF and cell amplification factor, and thrombopoietin as megakaryocyte growth factor. Fifty ng/ml was defined as the optimal concentration for all cytokines. Concentrations of FL higher than 100 ng/ml promote cell immaturity at cell amplification's expense. By using this 4F combination for a seven-day culture, we obtained a good compromise for CD34+ cell proliferation, colony-forming unit, granulocyte/ macrophage (CFU-GM) expansion, and MK commitment. SCF combined with TPO led to a low level of CD34+ cell amplification and especially committed cell culture to MK lineage. The three-factor combination obtained by removing IL-3 from 4F led to a notably less efficient mixture in terms of cell proliferation. Subsequent addition of other cytokines did not improve the culture system. Preliminary experiments with irradiated cells showed that single factors or combinations of two factors provided insufficient protection against apoptosis.

SCF, FL, TPO, and IL-3 in Combination Moderately Reduced the Level of RI Fas mRNA and Fas/Fas-L Coexpression in CD34+ Cells but Significantly Protected CD34high Fasneg/Fas-Lneg Cells
CD34+ cells incubated after irradiation with the 4F combination (50 ng/ml of each cytokine) displayed a reduced CD95 mRNA expression at both 6 and 24 h (Fig. 1Go).

Six hours after incubation with 4F, unirradiated PB CD34+ cells, and to a lesser extent irradiated cells, showed a partial protection of the percentage of CD34high cells (R1) and a decrease in the percentage of CD34+/low cells (R2) as shown in Figure 2Go; CD34 antigen expression (MFI) appeared slightly increased (Fig. 3Go). At 24 h, the cytokine treatment resulted in increased cell viability of irradiated cells (80% of basal viability) and unirradiated cells. Fas overexpression was reduced strongly in unirradiated cells and moderately in irradiated cells (Fig. 5Go). This resulted in a significant increase in the percentage of CD34high Fasneg cells (Fig. 6Go, R1). Morover, CD34+ Fasneg cells were preserved from apoptotic DNA fragmentation according to TUNEL assay (Fig. 7BGo). 4F preserved 30% of time-matched unirradiated cell clonogenicity. To a lesser extent, CD34+ cell protection was observed up to 6 Gy (Table 3Go). By contrast, the 4F combination tested at 10 ng/ml induced no anti-apoptotic efficacy. 4F treatment reduced mb Fas-L overexpression at 24 h (Fig. 8Go) as described for Fas expression and maintained the percentage of cells exhibiting high IC Fas-L content at this time (Table 1Go and Fig. 10Go).


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  		Table 3. 4F treatment partially protects CD34+ cells up to 6 Gy
 
SCF, FL, TPO, and IL-3 in Combination Reduced RI Bcl2 Downregulation in CD34+ Cells
4F treatment partially maintained the percentage of cells exhibiting a high Bcl2 content at 24 h (15.8% ± 6.9%; Table 2Go).

SCF, FL, TPO, and IL-3 in Combination Could Promote In Vitro CD34+ Cell Amplification and GM and MK Proliferation/ Differentiation from Irradiated CD34+ Progenitor Cells
Unirradiated PB CD34+ cells cultured for seven days with 4F combination showed an average amplification of total cells (x15.8-fold) and CD34+ cells (x13.6-fold). Expanded CD34+ cells contained two populations according to CD34 expression: the first one showed the same CD34+ MFI as fresh cells (CD34high, i.e., 24.9% of the cells), and the second one showed CD34+/low MFI. MPO+ and CD41+ cells represented respectively 39.2% ± 2.1% and 49.4% ± 9% of the expanded cells, with CD34+/ CD41+ and CD34+/MPO+ coexpression. Irradiated PB CD34+ cells (2.5 Gy) exhibited reduced amplification ratios compared with unirradiated cells, and CD34high population was decreased by 50% (Tables 4, 5GoGo). By contrast, unirradiated and irradiated CD34+ cells displayed at this time similar Fas (about 31%) and mb Fas-L (about 27%) expression. CD34high cells remained Fasneg/Fas-Lneg in both unirradiated and irradiated cells. Expanded unirradiated and irradiated cells exhibited high Bcl2 content. With respect to unirradiated BM CD34+ cells, 4F and SCF + TPO (2F) induced an overall cell expansion of 68.6-fold (4F) and 22.2-fold (2F) at 10 days (Table 4Go). Both cytokine combinations led to MK proliferation and differentiation, since after 7 to 10 days, mature MK developing proplatelets or in process of shedding platelets were seen in the cultures. Moreover, 4F induced GM differentiation from BM and PB cells. Yet 2F failed to exert anti-apoptotic effects and did not induce cell proliferation from irradiated samples. Only 4F antiapoptotic treatment allowed CD34+ cell proliferation and GM/MK differentiation from irradiated CD34+ progenitors (Table 4Go).


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  		Table 4. Anti-apoptotic 4F treatment but not 2F (TPO + SCF) amplified in vitro irradiated non-human primate CD34+ cells in a serum-free medium culture system. Overall cell expansion and MK cell expansion are related to BM CD34+ cells (n = 4); CD34+ and CD41+ cell expansion are related to PB CD34+ cells (unirradiated cells n = 8; irradiated cells n = 3).
 

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  		Table 5. Phenotypic analysis of cells cultured for seven days with 4F
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis or programmed cell death is a nonspecific physiological process which has been demonstrated for hematopoietic cells following cytokine deprivation or exposure to chemical agents and ionizing radiation. This in vitro study was aimed at assessing relevant markers of RI apoptosis at the CD34+ cell level in an attempt to counteract this process for a cell therapy purpose. Following irradiation, we reported rapid morphological and phenotypic changes leading to apoptotic cell death, whose mitigation could have implications for the early medical management of irradiated casualties. Thus, we observed a decrease of CD34 antigen expression at 6 h following in vitro irradiation. CD34 antigen is a 115 kD transmembrane glycoprotein expressed on HSPCs and is used as a comarker of immaturity, but its function remains largely unknown [11].

In our in vitro model, the CD34 expression decrease was the first event detected before Fas expression and other events occurring during RI apoptosis. This decrease could be explained either by a reduction in the number of membrane sites or by RI membrane modifications (i.e., biochemical alterations) making the class III CD34 epitope partially recognizable by the relevant antibody. Otherwise, the Fas pathway is known to be implicated in RI apoptosis of CD34+ cells [9]. The Fas/Fas-L interaction is the first known step of a complex pathway involving cascade activation of numerous proteases of the caspase family. Nagafuji's study showed that Fas induction on human CD34+ BM cells was radiation dose-dependent, and apoptosis was further induced in response to anti-Fas mAb. In our study, we confirmed Fas induction using baboon CD34+ cells after in vitro irradiation. In addition, using a triple-flow cytometric evaluation, we showed that CD34+/Fas+ cells were undergoing apoptosis, whereas CD34+/Fasneg cells were not apoptotic. These data clearly suggest the implication of Fas in the apoptotic process. Moreover, we reported the concurrent Fas-L induction within the first 24 h following cell irradiation. These data are likely to involve Fas-L in the RI apoptotic process according to either an autocrine or a paracrine modality. We report here that non-human primate CD34+ cells from mobilized PB constitutively expressed a very low level of mb Fas-L and a high content of IC Fas-L. In this model, irradiation of CD34+ cells led to a significant increase in mb Fas-L expression with a strong decrease of IC Fas-L. Such an autocrine process has been described for lymphocytes and lymphoma cells after IL-2 activation [12]. In summary, RI apoptosis occurring at the hematopoietic cell level appeared to be related to the following events: CD34 antigen downregulation and morphological alterations as early as 6 h, membrane alterations detectable in terms of Annexin V binding from 10 h and eventual Fas/Fas-L overexpression.

One cannot rule out that paracrine bystander effects take place in ex vivo cultures. This would be in agreement with Kiener's data [13], which showed paracrine and autocrine implication of the Fas/Fas-L system for unirradiated monocytes in serum-free cultures. Prolonged cultures of irradiated cells may benefit from non-apoptotic CD34+ cell sorting. In this perspective, we propose to select either CD34high cells with respect to an early sorting or CD34high/Fasneg or Fas-Lneg cells in case of later sorting.

We also report that in vitro irradiation of CD34+ cells leads to a decrease in Bcl2 content. Bcl2 is an antiapoptotic oncogene implicated in the early response to irradiation damages [14, 15]. After irradiation, p53 pro-apoptotic oncogene induction leads to cell cycle arrest and activation of repair mechanisms followed by elimination of lethally damaged cells, putatively via Bcl2 repression and subsequent apoptosis [16]. Bcl2 implication in Fas-mediated cell death has been suspected for some years. For instance, Barcena et al. [8] reported a high basal Bcl2 level in CD34+/CD95+ cells exhibiting high apoptotic levels and suggested a protective effect of Bcl2 versus Fas-induced cell death. Interestingly, Iwai [17] established earlier that the Bcl2 content of neutrophils, lymphocytes, and monocytes strongly correlates with their Fas-mediated cell death susceptibility. Our results are in agreement with recent in vivo experiments using transgenic mice which show that overexpression of Bcl2 in the hematopoietic system can increase the animal's resistance to irradiation [18].

Downregulation of Bcl2 concomitantly with Fas expression has been demonstrated physiologically in ex vivo expansion of CD34+ cells in the presence of cytokines [19]. We reported in this study that irradiation of CD34+ cells which express elevated Bcl2 content downregulated Bcl2 and concomitantly activated the Fas/Fas-L pathway. That process seemed to be implicated in RI apoptosis in CD34+ cells. This downregulation reached a significant level after 24 h of incubation, following irradiation.

Our aim was to evaluate the capacity of anti-apoptotic cytokines to rescue irradiated CD34+ cells, in an attempt to improve cell amplification level and clonogenicity. Three doses of radiation (2.5, 4, and 6 Gy) were assessed. Inhibitory effects of numerous cytokines on apoptosis have been described elsewhere. For instance, SCF and IL-3 are known to prevent unirradiated hematopoietic progenitors from apoptosis [20-23]. More recently, TPO has been demonstrated to suppress apoptosis of human quiescent primitive CD34+/CD38neg BM cells and FL to induce survival and proliferation of CD34+/CD38neg cells (with no effect on more mature CD34+/CD38+ cells) [24-26]. Moreover, RI apoptosis can be mitigated by cytokines. IL-3, for instance, inhibits RI apoptosis of BM cells, and SCF is implicated in hematopoietic recovery following lethal TBI [27-29]. The radioprotective effect of FL and the capacity of TPO to strongly enhance hematopoietic restoration after TBI have been recently demonstrated [30-32]. Among different pathways still partially known, antiapoptotic effects of FL have been shown to be correlated with Bax and not with Bcl2 upregulation [33].

In the present work, we report the in vitro anti-apoptotic efficacy on CD34+ cells of the cytokine combination SCF + FL + TPO + IL-3 used at 50 ng/ml, evaluated after 24 h of incubation. This protective effect did not appear to be specific to RI apoptosis and required the early addition of cytokines to be observed (within 6 h). In irradiated cells, a partial protection was obtained up to 6 Gy, but the addition of single cytokines or 2F was ineffective. This efficiency related at least partially to Fas and Fas-L downregulation and to Bcl2 upregulation. Actually, in our model, non-apoptotic cells rescued by the treatment exhibited high CD34 expression (i.e., MFI) and no Fas/Fas-L expression; IC levels of Fas-L and Bcl2 were concomitantly high. Whether the higher CD34 expression observed on cytokine-treated cells at 24 h could be attributed to a particular cell subpopulation remains to be determined. Further experiments are also necessary to determine at which level 4F act upstream or downstream of CPP32/Caspase3.

Anti-apoptotic treatment may improve viability of irradiated CD34+ cells, which could result in improved ex vivo expansion capacity. The engraftment capacity of expanded autologous CD34+ cells has been recently demonstrated [34], and ex vivo expanded cell infusion is proposed, with or without in vivo cytokine coadministration, as an alternative therapy for accidentally irradiated victims. Viable autologous CD34+ cells may be collected from underexposed marrow areas since heterogeneity of dose distribution has usually been observed in most documented accidents. In fact, our study pointed out that regulation of apoptosis would be crucial to obtaining a successful graft. The potential carcinogenic risk which might result from the reinfusion of damaged stem cells rescued during ex vivo management should not be excluded. However, expanded cell graft should probably not modify the endogenous risk of carcinogenesis to which the irradiated victim is exposed.

We reported previously that ex vivo expansion of unirradiated HSPCs with 4F led within six days to a significant proliferation level of CD34+ cells (13.6-fold increase with 24.9% of CD34high cells) which remained Bcl2 positive. In fact, CD34high cells did not express Fas/mb Fas-L, whereas the CD34+/low fraction partially expressed Fas and mbFas-L. Our results differ from other studies. In Nagafuji's study [19], all CD34+ cells exhibited Fas upregulation and Bcl2 downregulation after five days of culture with IL3 + SCF + G-CSF + Epo, but these differences can be related in our case to the use of a cytokine combination more active on cell survival. Terui observed reduced apoptotic ratios during MK maturation versus erythroid differentiation and related them to the increase or the downregulation of Bcl-X expression (mRNA and protein levels) with no involvement of Bcl2 [35]. In another study, erythroid colony forming cells constitutively exhibit Fas-L mRNA, high IC Fas-L level, and moderate functional mb Fas-L expression from day 5 to day 8 of culture [36]. According to Terui, mb Fas-L can interact with Fas, leading to apoptosis and clonogenicity reduction. Fas and mbFas-L expression could be related to spontaneous apoptosis, which seems to be inherent in the decline of immaturity observed in such cultures [37]. We also reported here that CD34+ cells in vitro irradiated at 2.5 Gy can be expanded within seven days (5.6-fold increase with 69% of CD34+ cells); expanded irradiated cells exhibited a lower percentage of CD34high cells than unirradiated ones but with no increase in Fas/mb Fas-L expression and with similar Bcl2 content. In fact, homogeneous doses of 2.5 Gy to 4 Gy are representative of average doses which have been estimated in underexposed BM from documented nuclear accidents.

More recently, using an adult baboon model of RI bone marrow aplasia, we were able to expand in vitro BM CD34+ cells (13.7-fold increase) collected from multiple sites (i.e., iliac crests and humerus) 3 h after a 6 Gy 60Co gamma TBI (midline tissue dose). This situation corresponds to a nonuniform IR exposure with a gradient of absorbed dose between entrance and exit sides of radiations equal to 50% inherent in the body thickness (unpublished data). Phenotypic analysis showed no significant differences from unirradiated cells except a reduced Thy1 expression in expanded irradiated cells. We performed the reinfusion of 5 x 106 CD34+ cells in a safe manner. Using this baboon model of RI BM aplasia, work is in progress A) to assess the threshold of HSPCs to be cultured or that of expanded cells capable, following reinfusion, of counteracting initial neutro-thrombopenia and B) to determine if early leukapheresis would allow the collection of the cell threshold required.

Nevertheless, ex vivo rescue of functional collected CD34+ cells with optimized cytokine cocktails and/or in vivo protection of residual HSPCs must be improved. The benefit from anticaspase treatments must also be evaluated, since it has been shown that the injection of CPP32 inhibitor peptides can protect mice against Fas-mediated fulminant liver destruction and death [38]. The early in vivo administration of various cytokines, such as TPO or fusion proteins, has been proposed to enhance hematopoietic recovery from RI BM aplasia [39]. Anti-apoptotic cytokine combinations could represent an emergency treatment to be administered early after an accidental exposure in order to counteract the dramatic BM and PB CD34+ cell decrease that is observed during the first 24 h after TBI.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication August 5, 1999.



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