5-Fluorouracil Permits Access to a Primitive Subpopulation of Peripheral Blood Stem Cells

Alison Rice," Caroline Barbot? Francis Lacombe,"gb Nadine Dubosc-Marchenay," Ge'rald Marit,b FranFoise Hau,c Jean-Michel Boiron," Josy Reiffers".b "aboratoire de Greffe de Moelle, URA CNRS 1456, UniversitC de Bordeaux 11, Bordeaux, France; bService des Maladies du Sang, HBpital Haut Ltveque CHR Bordeaux, Pessac, France; 'Service de Cryobiologie, Centre Rtgional de Transfusion Sanguine, Bordeaux, France

Key Words. Chemotherapy mobilized PBSC 5-FU * Stem cells

Abstract. Peripheral blood stem cells (PBSC) con- tain a mixture of mature and immature hematopoi- etic progenitors. Resistance to S-Fluorouracil(5-FU) has been used to identify and characterize primi- tive quiescent stem cells among bone marrow (BM) cells. To see if the same technique could be used to isolate a similar population of cells among PBSC, low-density peripheral blood mononuclear cells (PBMNC) were collected by cytapberesis in the regenerative phase after high-dose chemotherapy from patients with hematological malignancies. These PBMNC were incubated with increasing con- centrations of 5-FU for 24 h. The viable 5-FU resis- tant cells were then cultured in semi-solid media in the presence of either single cytokines: TCM 5637, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), or a combination of cytokines: interleukin 1 (IL-1) IL-l+IL-3+5637, IL-I+IL- 3+Stem Cell Factor (SCF). Low concentrations (5-10 pglml 5-FU) eliminated mature day 7 Colony Forming Units-Granulocyte Macrophage (CFU-GM) and spared day 7 clusters while enriching for day 14 CFU-GM, irrespective of the growth factors used. Higher concentrations of 5-FU (15,20,25 pg/ml) selected for later forming clonogenic elements. A combination of synergistic growth factors was required for the development of morphologically identifiable clonogenic elements resistant to 25 pg/ml5-FU a t day 21 of culture. Further experi- mentation demonstrated that SCF could effectively replace TCM 5637 in the cytokine combination for the detection of primitive late forming clonogenic elements. The presence of SCF potentiated colony

Correspondence: Professor J. Reiffers, Laboratoire de Greffe de Moelle URA CNRS 1456 Universitt de Bordeaux 11, Rue E o Saignat, Bordeaux Cedex, France.

Received April 8, 1993; provisionally accepted May 7, 1993; accepted for publication May 24, 1993. OAlphaMed Press 1066-5099/93/$5.00/0

formation by 5-FU resistant PBMNC. It was con- firmed that GM-CSF alone was unable to support colony formation by PBMNC resistant to 25 pg/ml. These observations demonstrate that PBSC contain a heterogenous mixture of hematopoietic progenitors and that incubation with 25 pglml5-FU permits access to a quiescent primitive stem cell population that requires a combination of synergistic growth factors for the development of morphologically iden- tifiable clonogenic elements a t day 21. Taken together, these results suggest that PBSC have similar characteristics to BM derived stem cells.

Introduction

Autologous bone marrow and blood stem cell transplantation have been used as therapeu- tic alternatives for patients with hematological malignancies who cannot benefit from allogeneic bone marrow transplantation [ 1-61. Autologous bone marrow transplantation (ABMT) uses bone marrow (BM) collected following the documen- tation of complete remission. Autologous blood stem cell transplantation (ABSCT) uses blood collected by cytapheresis during the regenera- tive phase after myeloablative chemotherapy. During this period, an overshoot of hematopoietic progenitors into the blood from the BM has been demonstrated [ 1,5]. ABSCT has several advan- tages over ABMT: 1) no general anesthesia is required to collect peripheral blood stem cells (PBSC); 2) this technique is also particularly useful for patients who have received prior pelvic irradiation; and 3) the most important advantage is that patients who have undergone ABSCT seem to have a more rapid hematopoietic recon- stitution than patients who have received ABMT

STEM CELLS 1993;11:326-335

321 PBSC contain 5-FU resistant stem cells

[7-lo]. This difference may be due to the fact that steady state BM is used as the transplant product as opposed to chemotherapy-mobilized blood stem cells. Rapid, complete, and durable hematopoietic reconstitution has been observed in the majority of patients who have undergone ABSCT [ 1-61. Considerable clinical research has been carried out to determine the optimal time to collect PBSC, the different parameters that influence the yield and quality of PBSC, and the quality of hematopoietic reconstitution after PBSC transplantation [ 11-13]. However, little is known about the biology of PBSC.

Research into the human and murine sys- tems has demonstrated that BM-derived hematopoietic stem cells are quiescent; that is, they are resistant to high concentrations of cell cycle specific drugs such as 5-Fluorouracil (5-FU) [14-181, or alkylating agents such as 4-hydroperoxycyclophosphamide (4-HC) [ 15, 181. Quiescent BM stem cells have been shown to have the following characteristics: 1) they are capable of self-renewal (they produce secondary CFU-S, and immature colonies after secondary replating [ 14, 18, 191; 2) they require a synergy of growth factors (IL-I, IL-3, SCF, IL-6, Epo) for the development of primitive progenitors in semi-solid culture systems [ 16, 18, 191 or a pre- formed irradiated stromal layer for the develop- ment of primitive hematopoietic elements Long Term Culture-Initiating Cell (LTC-IC) [20,21]; and 3) they have an immature, antigenic pheno- type (CD34+, HLA-DR-, CD38-, CD33-) [22]. Thus, 5-FU and 4-HC enrich for quiescent pluripotent hematopoietic stem cells.

Until recently, it has been generally accepted that PBSC, while very efficient for rapid hematopoietic reconstitution, would seem to have a limited capacity for self-renewal as compared with marrow-derived stem cells [23,24]. These latter conclusions were drawn from experimen- tation that compared the self-renewal capacity of steady-state circulating CFU-S with that of BM-derived CFU-S [25]. Other reports suggest that mobilized PBSC are as effective as mar- row-derived stem cells in producing long-term lymphomyelocytic reconstitution [26]. During .the regenerative phase following high-dose chemotherapy, circulating CD34 positive (CD34') cells with the same light scatter prop- erties as CD34' BM cells have been identified in cytaphereses concentrates [ 10,271. Antigenic analysis of CD34+ PBSC demonstrate that these

cells express a variety of differentiation antigens and that a hierarchy of antigen expression exists within the CD34' fraction [9]. LTC of PBSC has demonstrated the presence of a pre-CFU cell among the PBSC concentrates [28]. Using a two-stage system, whereby chemotherapy-mobi- lized PBSC were cocultured with a preformed stromal layer, it was demonstrated that PBSC contain a mixture of immature stroma-dependent and mature hematopoietic progenitors [29]. With the development of the LTC-IC assay, cells with marrow repopulating ability have been identi- fied among PBSC concentrates, although at lower levels than in the BM [30]. These different stud- ies have demonstrated that PBSC have an imrna- ture antigenic phenotype and contain cells with marrow repopulating ability, suggesting that PBSC-derived stem cells are not that different from BM-derived stem cells.

To determine if chemotherapy-mobilized PBSC contain quiescent primitive stem cells with the same growth factor requirements as those found in the bone marrow, PBSC were incubated with 5-FU for 24 h, and it was found that a subpopulation of mobilized PBSC were resistant to 25 pglml 5-FU. This cell popula- tion required a combination of synergistic growth factors, known to stimulate immature stem cells [ 161, for the development of rare, late-forming clonogenic elements. These obser- vations support the concept that blood stem cells have the same characteristics as BM-derived stem cells.

Materials and Methods

Patients Peripheral blood (PB) concentrates were col-

lected by cytapheresis from patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Non Hodgkin's Lymphoma (NHL), Ewing's Sarcoma (ES), Multiple Myeloma (MM), and Hodgkin's Disease (HD). All cytaphereses were performed during the mar- row regenerative phase following high dose chemotherapy [ 1,311 without growth factor mobi- lization. Informed consent was obtained from all patients prior to study.

Cell Preparation Low-density mononuclear cells (MNC) were

obtained after a Ficoll (Poly Labo, Strasbourg,

Rice, et al. 328

France) density gradient separation and washed three times in Hank’s Buffered Salt Solution (HBSS; GIBCO BRL, Cergy-Pontoise, France) before being resuspended in Iscove’s Modification of Dulbecco’s Medium (IMDM) (GIBCO BRL) supplemented with 10% Fetal Calf Serum (FCS) (Institut Jacques Boy, Reims, France).

Elimination of Mature Cells by 5-FU 5-FU (50 pg/mi Roche, Neuilly-sur-Seine,

France) was diluted U100 in Phosphate Buffered Saline (PBS, Poly Labo) before being added to the MNC. MNC (5 x 105) in IMDM + 10% FCS, were incubated with 5, 10, 15,20, and 25 pg/ml 5-FU or without 5-FU for 24 h at 37°C in 25 cm3 tissue culture flasks (Falcon, Becton-Dickinson (BD), Poly-Labo. The cells were washed three times with HBSS after the 24 h incubation, and cell viability was assessed by Trypan Blue exclu- sion prior to direct semi-solid culture.

Cytokines Recombinant human (rh) cytokines were

used throughout the study. rhL-la: specific activ- ity 3 x lo8 unitdrng, used at 500 W m l , was a generous gift from Dr. P Lomedico, (Hoffmann- LaRoche, Nutley, NJ). rhSCF: protein concen- tration 1.33 pg/ml, used at 10 nglml, was a generous gift from Dr. K. M. Zsebo. rhIL-3: spe- cific activity 5 x lo7 U/mg, used at 15 ng/ml, was a generous gift from Dr. Seiler and Dr. Kurrle (Behringwerke, Marburg, Germany). rhGMCSF, used at 10 ng/ml, was a generous gift from Dr. Girault (Sandoz, Paris, France). The conditioned media from the bladder carcinoma cell line 5637, a generous gift from Dr. V. Praloran (CHU Limoges, France) was used at a final concentration of 10%.

Hematopoietic Progenitor Cell Assay Type I collagen (Institut Jacques Boy) or

methylcellulose (4,000 centipoise; Sigma, St. Quentin-Fallaviet, France) were used as the sup- port matrices for the semi-solid culture system. Viable MNC (1 x lo4 to 1 x lo5) were resus- pended in single strength IMDM prior to cul- ture. Collagen gels were performed according to the technique developed by Lanotte et al. [32] and modified for use in the human system by Praloran et a l . [33]. Briefly, 25% double strength IMDM, 25% Type 1 collagen, 10% FCS, 10% human serum albumin, 4% (HSA) (Centre Regional de Transfusion Sanguine,

Bordeaux, France), 20% single strength IMDM, 1 % sodium bicarbonate, 1% L-Asparagine (GIBCO BRL) and 10% cells were mixed together and kept on ice. The mixture was then plated in 35-mm petri dishes (Falcon, Subra, Toulouse, France) and allowed to set before being placed at 37°C in a fully humidified 5 % C 0 2 atmosphere. The presence of clusters (2- 39 cells) and colonies (>40 cells) were evaluated after 7, 14, and 21 days incubation. The collagen disks were then harvested onto glass slides, fixed, and stained with May-Grunwald-Giernsa (Rhone Poulenc, Villers St. Paul, France) for cytological analysis. Semi-solid cultures per- formed with methylcellulose as the support matrix employed the same reagents in the same proportions as described above. Results were expressed, where appropriate, as the number of colonies or the number of clonogenic elements (colonies and clusters) per lo5 MNC.

Statistical Analysis

using the paired Student’s t-test. Statistical significance was determined

Results

PBSC Contain a Mixture of Hematopoietic Progenitors

Mobilized PBMNC contain a mixture of mature and immature hematopoietic progenitors as demonstrated by the development of colonies and clusters at day 7, 14, and 21, in the presence of the TCM 5637 alone or with IL-I+IL-3+5637 (p = NS) (Figs. 1A and B).

Low Concentrations of 5-FU Selected for Day 14 Clonogenic Elements

With the aim of detecting a primitive qui- escent hematopoietic stem cell population, PBMNC were incubated €or 24 h with increasing concentrations of 5-FU. Direct semi-solid culture of viable cells surviving incubation with 5-FU demonstrated that low concentrations of 5-FU (5- 10 pg/ml) eliminated the majority of mature day 7 CFW-GM, sparing some day 7 clusters, and enriching for day 14 CFU-GM irrespective of the growth factors used (TCM 5637 or IL-l+IL-3+5637). It is interesting to note, how- ever, that after incubation with low concentra- tions of 5-FU (5-10 pg/ml) while day 7 colonies were eliminated and the number of colonies

329 PBSC contain 5-FU resistant stem cells

Figure 1 A Figure 1 B

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Fig. 1A Untreated PBMNC contain a mixture of day 7 and 14 colonies. Untreated PBMNC (n = 11) were stim- ulated in serni-solid media with 5637 (0) or IL-l+IL-3+5637 (W). Colonies were evaluated after 7, 14, and 21 days incubation. Fig. 1B Untreated PBMNC contain a mixture of day 7 and 14 clonogenic elements. Untreated PBMNC (n = 11) were stimulated in serni-solid media with 5637 (0) or IL-l+IL-3+5637 (W). Clonogenic elements were evaluated after 7, 14, and 21 days incubation.

peaked at day 14, no new colonies developed at day 21 of culture (Figs. 2A, B, C, and D). These observations were confirmed by cytological analysis of the clonogenic elements. Day 21 clonogenic elements derived from untreated PBMNC consisted of end stage neutrophils and were colonies that had fully proliferated by day 14 and had continued to mature until day 21 (Fig. 3A). Day 2 1 colonies and clusters derived from cultures established with MNC treated with 5 or 10 pg/ml 5-FU, consisted primarily of mature granulocytes with occasional metamyelocytes (Figs. 3B and C).

High Concentrations of 5-FU Selected for Immature Late Forming Clonogenic Elements

When the concentration of 5-FU was increased (15, 20, and 25 pg/ml), there was a “shift to the right” in the evolution of colonies and clonogenic elements, suggesting that higher concentrations of 5-FU selected for later form- ing immature clonogenic elements. Initial devel- opment of morphologically recognizable clusters occurred at day 14, with the colony appearing at day 21 (Figs. 4A, B, C, and D). The rare day 21 morphologically identifiable clonogenic elements, sr:rviving 25 pg/ml5-FU and appearing after stim- ulation with IL- 1 +IL-3+5637, were large imma- ture cells with very fine chromatin and little sign

of differentiation (Fig. 5). Of interest, the mor- phologically identifiable clusters generated at day 21 after 25 pg/ml5-FU and stimulation with IL- I+IL-3+5637 were the same as those that gener- ated at day 7 from untreated PBMNC after the same stimulation (Figs. 6A and B). The underly- ing pathology did not influence the morphology of the PBSC-derived (untreated or 5-FU resistant) clonogenic elements. These observations reflect the suppressive effect of 25 pg/ml5-FU on day 7 and day 14 clonogenic elements and illustrate the heterogeneity of the blood-derived stem cell pop- ulation which contains a subpopulation of cells capable of giving rise to very immature cells after three weeks of culture. While TCM 5637 alone stimulated very rare clonogenic elements from PBMNC resistant to 25 pg /d 5-FU, it did permit the persistence of immature blasts in culture. For these reasons, 5-Fu at 25 pg/ml was used to gain access to a primitive quiescent cell population which is induced to proliferate by a synergistic combination of cytokines.

A Synergy of Cytokines is Required for Colony Formation by PBMNC Resistant to 25 pg/ml5-FU

Preliminary studies demonstrated that each cytokine played a role in the development of clonogenic elements resistant to 25 pg/ml5-FU (data not shown). Further experimentation

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Fig. 2A Low concentrations of 5-FU eliminate the majority of day 7 CFU-GM while enriching for day 14 CFU- GM. The PBMNC (n=6) were incubated without 5-FU (HI, with 5 pg/ml5-FU (0) or 10 pg/ml5-FU (k). The number of colonies was evaluated after stimulation with IL-l+IL-3+5637. Fig. ZB The TCM 5637 is not as effective as IL-I+IL-3+5637 for the stimulation of colony formation from PBMNC treated with low concentrations of 5-FU. The PBMNC (n=6) were incubated without 5-FU (H), with 5 pg/ml 5-FU (0) or 10 pg/ml5-FU (a). The number of colonies was evaluated after stimulation with 5637. Fig. 2C Low concentrations of 5-FU eliminate the majority of day 7 CFU-GM while enriching for day 14 clono- genic elements. The PBMNC (n=6) were incubated without 5-FU (H), with 5 pg/ml 5-FU (0) or 10 pglml5-FU (L). The number of clonogenic elements was evaluated after stimulation with IL-l+IL-3+5637. Fig. 2D The TCM 5637 is not as effective as IL-I+IL-3+5637 for the stimulation of clonogenic elements from PBMNC treated with low concentrations of 5-FU. The PBMNC (n=6) were incubated without 5-FU (m), with 5 pg/ml 5-FU (0) or 10 pglml 5-FU (a). The number of clonogenic elements was evaluated after stimulation with 5637.

demonstrated that SCF could efficiently replace TCM 5637 in the cytokine combination for the detection of 5-FU resistant primitive clonogenic elements (Figs. 7A and B). Cytological analy- sis demonstrated that SCF potentiated colony formation, in that the type of clonogenic ele- ments resistant to 25 pg/ml5-FU seen at day 21 after stimulation with IL-I+IL-3+5637 were now observed at day 14 when the cytokine combina- tion IL-I+IL-3+SCF was used. IL- I+IL-3+SCF induced the formation of greater numbers of day 21 colonies from 5-FU resistant PBMNC than did IL-l+IL-3+5637 0) = 0.04).

TCM 5637 contains a variety of cytokines with the exception of IL-3 [34]. To confirm that 5-FU resistant colonies were unable to respond to a late-acting single cytokine, TCM 5637 was

replaced with rhGM-CSF. Our studies confirmed that GM-CSF alone was unable to induce the formation of day 21 colonies (day 14-25 pg/ml 5-FU, y = 0.012; day 21-25 & n l 5-FU, p = 0.024) (Fig. 8). This observation confirms the immaturity of cells resistant to 25 pg!ml5-FU and demonstrates that these cells require a combina- tion of synergistic growth factors for the devel- opment of rare, primitive, late-forming colonies.

Discussion

The use of direct semi-solid and liquid cul- ture techniques and different combinations of growth factors have confirmed that mobilized PBSC contain a mixture of mature and immature

33 I PBSC contain 5-FU resistant stem cells

B

C a

Fig. 3A Part of a day 21 co lony derived from untreated PBMNC stimulated with IL-l+IL-3+5637. May-Grunwald-Giemsa stain, original magnification x 100. Fig. 3B Part of a day 21 colony derived from 5 Nglrnl 5-FTJ treated PBMNC stimulated with IL-I +IL-3+5637. hlay-Grunwald-Giemsa stain, original magnification x 100. Fig. 3C Part of a day 21 colony derived from 10 pg/ml 5-EW treated PBMNC stimulated with IL-l+IL-3+5637. May-Griinwald-Giemsa stain, original magnification x 100.

progenitors [9, 10, 27-29, 35, 361. Recent work h a s i n ve s t i gated t h e i mmu n o p hen o t y pe of mobilized PBSC [9, 10, 271, but few studies have looked at the functional characteristics of mobilized PBSC [36]. To determine if PBSC

contained stem cells with characteristics simi- lar to those described for bone marrow pluripo- tent stem cells, the S phase specific drug 5-FU was chosen to selectively eliminate cycling cells. This drug has the advantage that cells in GI of the cell cycle will incorporate the 5-FU metabo- lites and will be killed when they progress to S phase [17]. Thus, 5-FU permits access to a qui- escent cell population. The growth factor requirements in semi-solid culture of quiescent, 5-FU resistant cells from chemotherapy-mobi- lized PBSC were assessed.

Our studies demonstrated the following: 1 ) chemotherapy-mobilized PBSC contained a heterogenous mixture of hematopoietic progeni - tors given their proliferative response to single and combinations of cytokines; 2) low doses of 5-FU enriched for day 14 CW-GM; and 3) there were very few “new” late-forming primitive colonies. These observations were made irrespec- tive of the type of growth factor used. However, when chemotherapy-mobilized PBSC were incu- bated with 25 pg/ml 5-FU, cells giving rise to mature day 7 and day 14 clonogenic elements were eliminated. The presence of a combination of syn- ergistic growth factors (IL- l+IL-3+SCF) seems to have induced the quiescent primitive progeni- tor cells to leave Go and to form primitive late- forming morphologically recognizable clonogenic elements. Further evidence for the potentializing effect of the combination of cytokines on the development of clonogenic elements after incu- bation with 25 pg/rnlS-FU was provided with the observation of the persistence of immature blasts in culture when TCM 5637 alone was used as the source of growth factors. In view of the fact that PBMNC resistant to 25 pg/ml5-FU were unable to form clonogenic elements in the presence of GM-CSF or 5637 alone, it would seem that the development of clonogenic elements by these cells, only after stimulation with multiple cytokines, is an indication of the immaturity of this cell population [37].

When these observations are compared with previous work on the characteristics of BM- derived stem cells, it becomes apparent that mobilized PBSC contain a quiescent subpopu- lation of cells that are resistant to high concen- trations of 5-FU and require the presence of a combination of synergistic “immature” cytokines to induce them to leave Go and form primitive clonogenic elements. The description of growth requirements previously thought to be unique to

Rice. et a1

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Fig. 4.4 High concentrations of 5-FU eliminate the majority of day 7 and day 14 CFU-GM and select for a late forming CFU-GM. The PBMNC (n=6) were incubated with 15 pg/ml 5-FU (m), with 20 pg/ml 5-FU (0) or 25 pglml 5-FU (B). The number of colonies was evaluated after stimulation with IL-l+IL-3+5637. Fig. 4B The TCM 5637 does not permit the detection of day 7, 14 or 21 CFU-GM after high concentrations of 5- FLJ. The PBMNC (n=6) were incubated with 15 pglml5-FU (M), with 20 pgIml5-FU (0) or 25 pg/ml 5-FU (E). The number of colonies was evaluated after stimulation with 5637. Fig. 4C High concentrations of 5-FU select for rare late forming clonogenic elements. The PBMNC (n=6) were incubated with 15 pglml5-FU (m), with 20 pglml 5-FU (0) or 25 pglml5-FU (t). The number of clonogenic ele- ments was evaluated after stimulation with IL-l+IL-3+5637. Fig. 4D The TCM 5637 alone does not facilitate the detection of rare late forming clonogenic elements after incu- bation with high concentrations of 5-FU. The PBMNC (n=6) were incubated with 15 pg/ml5-FU (R), with 20 @ml 5-FU (0) or 25 pg/ml5-FU (R). The number of clonogenic elements was evaluated after stimulation with 5637.

BM stem cells, together with the data concerning the immature antigenic phenotype [9, 10,271 and the presence of LTC-IC among PBSC [30], sug- gest that the characteristics of PB-derived stem cells are not dissimilar to those of BM-derived stem cells.

There is still considerable debate as to the true self-renewal capacity of PBSC and whether or not hematopoietic reconstitution seen after ABSCT is the result of endogenous cell recov- ery in the long term. These questions are diffi- cult to solve in the absence of an in vivo techruque to measure long-term self-renewal in the human system. Experiments with gene marking 1381 should provide some valuable information in the near future. Given the fact that PBSC have been

Fig. 5 Day 21 cluster derived from 25 pg/mI 5-FU treated P B ~ C stimulated with ~ - 1 + ~ - 3 + 5 6 3 7 . M ~ ~ - Griinwald-Giemsa stain, original magnification x 100.

333 PBSC contain 5-FU resistant stem cells

A B

Fig. 6A Day 21 cluster derived from 25 pg/ml 5-FU treated PBMNC stimulated with IL-l+IL-3+5637. May- Griinwald-Giemsa stain, original magnification x 100. Fig. 6B Part of a day 7 c.olony derived from untreated PBh4NC stimulated with IL-l+IL-3+5637. May-Griinwald- Giemsa stain, original magnification x 100.

demonstrated to possess all the characteristics of BM-derived cells, it is difficult to dismiss PBSC as a short-term transplant product or a simple

self-renewal and amplification capacity of 5-FU resistant PBSC are currently under investigation

Acknowledgments

The authors w i s h t o thank the South to bone marrow The Australian Childhood Cancer Association, the

conseil Rkgional d’Aqujtaine, and the ~ ~ ~ ~ ~ i - ation pour la ~ ~ ~ h ~ ~ ~ h ~ centre le cancer for

in our laboratory.

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IL-l+IL-3+SCF can efficiently replace IL-l+IL-3+5637 for the detection of primitive late formin colonies. Untreated PBMNC were stimulated in semi-solid media with IL- 1+IL-3+5637 (B), IL-I +IL-3+SCF (I? PBMNC resistant to 25 pg/ml5-FU were stimulated in semi-solid media with IL-l+IL-3+5637(&), IL-l+IL-3+SCF ( ). Colonies were evaluated after 7, 14, and 21 days incubation (n = 4). Fig. 7B IL- I+IL-3+SCF can efficiently replace IL-l+IL-3+5637 for the detection of primitive late forming clonogenic elements. Untreated PBMNC were stimulated in semi-solid media with IL-l+IL-3+5637 (B), IL- I+IL3+SCF (0). PBMNC resistant to 25 pglml5-FU were stimulated in semi-solid media with IL-l+IL-3+5637 (&), IL-l+IL-3+SCF ( ). Clonogenic elements were evaluated after 7, 14, and 21 days incubation (n = 4).

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Fig. 8 5-FU resistant PBSC require a combination of synergistic cytokines for the development of primitive late forming colonies. 5-FU resistant PBMNC (n = 8) were stimulated in semi-solid media with GM- CSF (0) or IL-l+IL-3+SCF (W). Colonies were eval- uated after 7, 14, and 21 days culture.

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