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Stem Cells, Vol. 15, No. 3, 214-222, May 1997
© 1997 AlphaMed Press

Optimization of the Cycling of Clonogenic and Primitive Cord Blood Progenitors by Various Growth Factors

Mojgan Movassagh, Laurent Caillot, Claude Baillou, Martine Guigon, François M. Lemoine

Biologie et Thérapie des Pathologies Immunitaires, ERS CNRS 107 - C.E.R.V.I., CHU Pitié Salpétrière, Paris, France

Key Words. Cell cycle • CD34+ cells • Committed progenitors • Primitive progenitors • Long-term culture • Cytokines • Cord blood

Dr. F.M. Lemoine, Biologie et Thérapie des Pathologies Immunitaires, ERS CNRS 107 - C.E.R.V.I., CHU Pitié Salpétrière, 83, Bld de l'Hôpital, 75561 Paris Cedex, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cycling status of cord blood progenitors and the culture conditions triggering their activation into S-phase have been studied using flow cytometry and a 3H-thymidine suicide assay. Mononuclear cells cultured either in Iscove's modified Dulbecco's medium (IMDM) ± 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbecco's modified Eagle's medium (DMEM) ± 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimulated by various growth factors (GFs). Results showed that CD34+ cells, clonogenic progenitors (colony forming cells [CFCs]) and long-term culture initiating cells (LTC-IC) present in freshly harvested cord blood were quiescent. CFC numbers were maintained without cycling after 48-h cultures in serum-containing media without GFs. Addition of interleukin 3 (IL-3) + IL-6 + stem cell factor stimulated into S-phase ~40% of CFCs within 24-48 h, without modifying their number except in DMEM + NBS where erythroid progenitors decreased. When cells were stimulated in IMDM + FCS by these three GFs + insulin-like growth factor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S-phase and their total number was maintained. The latter culture conditions also recruited up to 66% of LTC-IC into S-phase. Our data underline the importance of the combination of GFs and culture media used for optimizing the cycling and maintenance of CFCs and LTC-IC within two days.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Umbilical cord blood (CB) is a rich source of hematopoietic stem/progenitor cells. Although their content in progenitors is relatively limited [1, 2], CB cells, which most likely represent a developmental stage intermediate between fetal liver and adult bone marrow [3], exhibit high proliferative capacities leading within a few weeks to a large expansion of cells [3-5] in response to various combinations of hematopoietic growth factors. For this reason, CB has considerable interest as a source of hematopoietic stem/progenitor cells for clinical transplantation [1, 6] as well as for gene therapy [7]. Particularly, retroviral gene transfer requires not only high viral titers and appropriate transduction protocols but also the cycling of the target cells [8, 9]. For this purpose, we have studied the cell cycle status of progenitors present in freshly harvested CB and compared the effects of various culture conditions (combinations of growth factors, serum and medium used) on their cycling. One of the consequences of the growth factor stimulation is that progenitor cells will proliferate and also differentiate into more mature cells and then lose their primitive properties [10]. For this reason, the effects of hematopoietic growth factors (GFs) such as interleukin 3 (IL-3), IL-11, IL-6, stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) preferentially acting on the G0-G1 phase [11-18] have been evaluated over a relatively short period of time (<four days) on both the cycling and maintenance of clonogenic progenitors (colony forming cells [CFCs]) and more primitive progenitor cells, namely, long-term culture initiating cells (LTC-IC) [19]. Experiments have been carried out in two serum-containing culture media currently used to generate high viral titers from various packaging cell lines [20-22]. Cell cycle was evaluated both by flow cytometry and by a 3H-thymidine (3H-Tdr) suicide assay.

Our findings show: A) that the majority of CD34+ cells, CFCs and LTC-IC present in freshly harvested CB are quiescent, and B) that it is possible using appropriate culture conditions and combination of GFs to trigger the cycling of more than 50% CFCs and LTC-IC within 48 h and to maintain their total numbers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
CB cells, collected according to institutional guidelines, were obtained during normal full-term deliveries. Mononuclear cells (MNC) were separated on Ficoll-Hypaque gradient (density 1.077 g/ml) and then resuspended in Iscove's modified Dulbecco's medium (IMDM) and counted.

Recombinant Human (rHu) Growth Factors
rHuIL-3 was obtained from Sandoz (Rueil-Malmaison, France), rHuIL-11 from British Bio Technology (Oxon, UK), rHubFGF from Sigma (St. Louis, MO) and rHuIGF-I from Boehringer (Meylan, France). rHuLIF was a generous gift from Dr. Godard (INSERM U211; Nantes, France), rHuSCF was kindly provided by Amgen (Thousand Oaks, CA) and rHuIL-6 was a kind gift from Dr. L. Aarden (Central Laboratory Blood Transfusion; Amsterdam, The Netherlands). All rHuGFs were used at concentrations that gave the highest cell proliferation in titration experiments: i.e., rHuIL-3 = 100 U/ml; rHuIL-11 = 4 ng/ml; rHubFGF = 2.5 ng/ml or 50 ng/ml; rHuIGF-I = 50 ng/ml; rHuLIF = 1000 U/ml; rHuSCF = 50 ng/ml; IL-6 = 100 U/ml.

Liquid Cultures
In order to activate the entry into S-phase of CD34+ cells, CFCs and LTC-IC, 5 x 106 CB MNC/ml were seeded in either IMDM + 10% fetal calf serum ([FCS], Dutscher; Brumath, France) or in Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn bovine serum ([NBS]; Hyclone; Cramlington, UK) in six-well flat-bottomed tissue culture plates (Costar; Cambridge, MA) and cultured with or without various combinations of GFs for one to three days. In some experiments cells were cultured without serum.

Cell Cycle Analysis of CD34+ Cells
Cell cycle analysis of CD34+ cells was performed using the DNA-binding fluorescent dye 7-amino-actinomycin D (7-AAD). The method used has been slightly modified from Rabinovitch et al. [23]. Briefly, MNC were stained with fluorescein (FITC)-conjugated CD34 (8G12-FITC) monoclonal antibody, a generous gift from Dr. P.M. Lansdorp (Terry Fox Laboratory; Vancouver, BC, Canada) using a one-step direct immunofluorescent procedure. Then, 2 x 106 cells were fixed for 15 min with 1 ml cold paraformaldehyde/phosphate-buffered saline ([PBS]; final concentration = 1%), centrifuged, and permeabilized for 30 min at 4°C in 1 ml cold 45% ethanol/PBS. Then, cells were washed twice in PBS + 2% FCS, treated at 37°C with 0.5 mg RNAse A (Sigma)/ml PBS for 10 min, stained with 0.5 ml of a solution of 7-AAD (25 mg/ml in PBS + 2% FCS) and incubated in the dark at 4°C for 90 min. Finally, cells were washed twice in PBS + 2% FCS, resuspended at 1 ml PBS + 2% FCS. This procedure has been established according to preliminary experiments indicating that under these fixation conditions the expression of the CD34 antigen was well-preserved. Before and after the fixation and permeabilization procedures, unstained cells and cells labeled with isotype-matched nonspecific mouse immunoglobulin conjugated to FITC (Coultronics; Margency, France) and 7-AAD were used as negative controls.

Cell analyses were performed on a two-laser, air-cooled, EPICS Elite cytometer (Coultronics) on the basis of light scatter properties and fluorescence intensity. Doublets were excluded by plotting peak fluorescence versus integral fluorescence of 7-AAD. At least 10,000 events gated within a lymphoid-blast gate, which normally contains the majority of CD34+ cells, were analyzed and collected on listmode files. The percentage of CD34+ cells in the different phases of cell cycle was analyzed using Multicycle AV software (Phoenix Flow Systems, Inc.; Seattle, WA).

Tritiated Thymidine (3H-Tdr) Suicide Measurements
Cells were suspended at 2 x 106 MNC/ml of IMDM. Equal volumes of cell suspension were incubated at 37°C in the presence or absence of 0.74 MBq/ml 3H-Tdr (specific activity: 925 Gbq/mmol = 25 Ci/mmol; CEA; Saclay, France) for 20 min. The amount of 3H-Tdr was chosen according to published data indicating that a plateau of killing is reached with 0.59 MBq/ml 3H-Tdr [24, 25]. Then, 5 ml of cold Tdr (Sigma) dissolved in PBS at a final concentration of 400 µg/ml were added to stop further 3H-Tdr incorporation, and cells were washed twice in the presence of excess of cold Tdr, counted and then assayed for CFCs and LTC-IC as described below. The proportion of CFCs and LTC-IC in S-phase was calculated as previously described [26, 27]:


(1)

CFC Assays
CFCs were assayed by using methylcellulose duplicate cultures as described elsewhere [28]. Briefly, cells from fresh or cultured CB and from LTC (see below) were plated at appropriate numbers in 35-mm dishes (Greiner; Freiburg, Germany) in 1.1 ml of IMDM containing 0.8% methylcellulose (Fluka 4,000; Buchs, Switzerland), 30% FCS, 1% bovine serum albumin, 100 µM 2-mercaptoethanol (Sigma), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 3 U/ml rHu erythropoietin (Boehringer; Mannhein, Germany), 100 U/ml rHuIL-3, and 200 U/ml rHuGM-CSF (Genetics Institute; Cambridge, MA). Cultures were incubated in a humidified atmosphere containing 5% CO2 in air at 37°C. BFU-E and colony forming unit-granulocyte/macrophage (CFU-GM) were counted under an inverted microscope between 16 and 18 days of culture. BFU-E were subdivided into primitive and mature subclasses according to their number of clusters [29].

LTC-IC Assays
In order to evaluate the content and cell cycle status of LTC-IC before and after GF-stimulation, MNC, either untreated or treated with 3H-Tdr, were seeded in long-term culture as follows: 1.5 x 106 cells were seeded in 35-mm tissue culture dishes (Costar) containing 2 ml of LTC medium (Stem Cell Technologies; Vancouver, BC, Canada) and a pre-established feeder layer of 2 x 105 irradiated (40 Gy) MS-5 cells, a murine stromal cell line supporting normal human hematopoiesis [30]. No exogenous growth factors or hydrocortisone were added [31]. In half of these cultures, cold Tdr (10 µg/ml) was added to the culture medium in order to exclude the possibility of 3H-Tdr reutilization occurring in the LTC assays due to the release of 3H-Tdr by dead cells. LTC were maintained at 33°C with weekly replacement of half of the medium and nonadherent cells with fresh medium plus (or minus) cold thymidine. After a five-week culture, nonadherent and adherent cells obtained after trypsinization were pooled and assayed for their CFC content as described above. The colonies obtained in these secondary methylcellulose cultures are termed LTC-IC [19].

Statistical Analysis
Results are expressed as the mean ± SE of (n) experiments. In experiments where the effects of IMDM and DMEM or NBS and FCS were compared, a nonpaired t-test was used. In experiments where the effects of the various combinations of GFs on the activation into S-phase of progenitors were compared, at least four separate experiments were analyzed using a paired t-test. Statistical significance was taken at the 5% level (p < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Cycle Status of CD34+ Cells and CFCs in Fresh CB
In a first set of experiments the cell cycle status of CD34+ cells was evaluated by flow cytometry after staining of MNC with anti-CD34 FITC-conjugated monoclonal antibody and 7-AAD. The percentage of CD34+ cells (0.75 ± 0.15%, n = 6) was not modified by the fixation and staining procedures. Cell cycle analysis indicates that 3 ± 1% of CD34+ cells were in S/G2M phase (n = 6) as shown in Figure 1A.



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  		Figure 1. Cell cycle analysis of CB CD34+ cells. MNC were labeled with HPCA2-FITC mAb, fixed, permeabilized and then stained by 7-AAD as described in the Materials and Methods section. Cell cycle analysis was performed on at least 104 CD34+ gated cells. One of six representative experiments is shown. Panel A: CD34+ cells in freshly harvested CB. Panel B: CD34+ cells cultured in IMDM + 10% FCS without GF stimulation for 48 h. Similar results were obtained when cells were cultured in DMEM + 10% NBS without GFs (not shown). Panel C: CD34+ cells cultured in DMEM + 10% NBS and IL-3 + IL-6 + SCF for 48 h. Panel D: CD34+ cells cultured in IMDM + 10% FCS and IL-3 + IL-6 + SCF for 48 h.

 
Although all progenitor cells express CD34 antigen, CD34+ cells do not exclusively contain progenitors [32]. For this reason, we next evaluated the cell cycle status of CFCs. Thus, cells were treated with high specific activity of 3H-Tdr, then plated directly in methylcellulose as described in Materials and Methods. The treatment with 3H-Tdr allows the killing of progenitors that are likely in S-phase. As shown in Table 1 , 13 ± 3% and 11 ± 3% of BFU-E and CFU-GM are in S-phase, respectively (n = 15). Subdivision of BFU-E into primitive and mature subclasses did not reveal any differences in their cell cycle status (data not shown). Thus, our findings clearly show that the majority of CD34+ cells as well as CFCs present in freshly harvested CB are quiescent.


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  		Table 1. Cell cycle and total number of CFCs in freshly harvested CB and after 48 h culture with or without serum
 
Effect of the Serum on the Activation into S-Phase and the Total Number of CFCs
Because serum of bovine origin is known to contain growth promoting components [33], the effects of NBS and FCS on the cycling of CD34+ cells and CFCs were evaluated. Thus, cells were cultured with or without serum in the absence of exogenous GFs for 48 h. The results show that when cells were cultured with serum, CD34+ cells ( Fig. 1B) and CFCs remained in a quiescent status ( Table 1): 13 ± 3% of BFU-E and 11 ± 3% of CFU-GM in DMEM + NBS and 10 ± 3% of BFU-E and 14 ± 3% of CFU-GM in IMDM + FCS in S-phase, respectively. Under these culture conditions, the number of CFCs remained stable as shown in Table 1. By contrast, when cells were cultured in the absence of serum, the total number of CFCs significantly decreased by about 40% (p < 0.05). These findings indicate that serum-containing media have no effect on the cycling of CD34+ cells and CFCs, but allow their maintenance.

Kinetics of Activation into S-Phase of CFCs
In order to study the kinetics of activation into cycle of clonogenic progenitors, MNC were first stimulated by a combination of IL-3, IL-6 and SCF for one to three days either in DMEM + NBS or in IMDM + FCS. Then, the cell cycle status of the clonogenic progenitors was measured by 3H-Tdr suicide assay. The results presented in Figure 2 show that the plateau of activation into S-phase was reached within 24 h for BFU-E and CFU-GM except for CFU-GM in DMEM + NBS. At 48 h, the percentage of progenitors in S-phase was about 40% in both culture media, a level of activation maintained for up to 72 h. These data were confirmed by flow cytometry analysis of the cell cycle of CD34+ cells: 38 ± 4% and 41 ± 2% of CD34+ cells were in S/G2M phase after 48 h of GF-stimulation in DMEM + NBS and IMDM + FCS, respectively ( Figs. 1C and 1D). Thus, our results indicate that IL-3 + IL-6 + SCF trigger the cycling of both CD34+ cells and clonogenic progenitors within 24-48 h.



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  		Figure 2. Kinetics of activation into S-phase of clonogenic progenitors with IL-3, IL-6 and SCF. Results expressed as the percentage of progenitors in S-phase (percentage of killed progenitors) are the mean ± SE of (n) experiments. *comparison to day 0 = p < 0.05 (paired t-test).

 
The total number of progenitors was evaluated under the different conditions of culture. As shown in Figure 3, the total number of CFU-GM was maintained at the input level in both media for at least 72 h, whereas the number of BFU-E was significantly reduced (p < 0.05) after a 48-h stimulation in DMEM + NBS.



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  		Figure 3. Total number of clonogenic progenitors after stimulation with IL-3, IL-6 and SCF for one to three days. Results represent the mean ± SE of (n) experiments and are expressed for 106 CB MNC. Input numbers of BFU-E and CFU-GM for 106 CB MNC are 1,620 ± 250 and 1,425 ± 215, respectively. *comparison to day 0 = p < 0.05 (paired t-test).

 
The slight differences observed between the two culture media used are likely due to the serum because when cells were cultured either in IMDM + NBS or in DMEM + FCS, the results were superimposable to those obtained in DMEM + NBS and IMDM + FCS, respectively (data not shown). Moreover, no qualitative changes concerning the morphological aspect of the colonies were observed, whatever the culture conditions.

Comparison between Various Combinations of GFs for Triggering the Cycling of CFCs
In order to improve the cycling of clonogenic progenitors, we next examined the effects of other synergistic factors after a 48-h stimulation. The factors used were IL-11, LIF, IGF-I and bFGF, the latter being used either at 2.5 ng/ml (d2.5) or at 50 ng/ml (d50) as described [11-15, 34, 35]. Table 2 shows that in both DMEM + NBS and IMDM + FCS, all combinations of GFs induced the cycling of progenitors (p < 0.05). Comparatively to IL-3 + IL-6 + SCF, the addition of bFGF and IGF-I in IMDM + FCS increased the percentage in S-phase of CFU-GM and BFU-E. However, this increase was only significant (p < 0.05) when bFGF was used at 50 ng/ml, 57 ± 4% and 49 ± 5% of CFU-GM and BFU-E in S-phase, respectively.


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  		Table 2. Cell cycle status and total number of clonogenic progenitors after 48 h of stimulation by various GF combinations
 
In all conditions, the total numbers of CFU-GM were equally maintained as compared to day 0. BFU-E numbers were always diminished, but this decrease was only significant (p < 0.05) when cells were stimulated in DMEM + NBS by IL-3 + IL-6 + SCF + IGF-I + bFGF(d50) with or without IL-11 + LIF.

These data indicate that high cycling and maintenance of clonogenic progenitors can be obtained within 48 h in DMEM + NBS in the presence of IL-3 + IL-6 + SCF. Furthermore, cultures in IMDM + FCS in the presence of IL-3 + IL-6 + SCF + IGF-I and bFGF at high concentration (50 ng/ml) appeared even more efficient to increase the cycling of CFCs.

Cell Cycle Status and Activation into S-Phase of LTC-IC
The cell cycle status and the culture conditions that might stimulate the entry into S-phase of more primitive progenitors, i.e., LTC-IC, were studied by 3H-Tdr suicide assay. Table 3 shows that only 16 ± 8% of LTC-IC (n = 3) were in S-phase. Whether these results really represent the percentage of LTC-IC in S-phase or whether they represent an overestimation due to the release of 3H-Tdr in the culture medium by dead cells that in turn might kill progenitors entering in S-phase during the culture period was tested in parallel experiments. Thus, in half of the cultures, an excess of cold Tdr was added to block further reutilization of 3H-Tdr. Interestingly, similar results were obtained under these conditions (data not shown). Thus, our data strongly suggest that the majority of LTC-IC in CB are quiescent.


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  		Table 3. Cell cycle analysis of LTC-IC before and after 48 h of GF stimulation
 
The effects of three GFs (IL-3 + IL-6 + SCF) or five GFs (IL-3 + IL-6 + SCF + IGF-I + bFGF[d50]) on the activation into S-phase of LTC-IC were then studied. Table 3 shows that more than 60% of LTC-IC (n = 3) were in S-phase after 48 h of GF-stimulation with either combination of GFs. Here again, the addition of cold Tdr to the LTC medium did not modify these results (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we have evaluated the culture conditions required to increase the cycling of clonogenic and more primitive progenitors in view of retroviral gene transfer. Cycling studies were performed both by flow cytometry and by a 3H-Tdr suicide assay. The former gives a rapid and direct evaluation of the different phases of the cell cycle of CD34+ cells. However, we have to keep in mind that immunophenotyping does not necessarily reflect the cell function, particularly after GF exposure [36]. By contrast, the latter assay, which only measures the percentage in S-phase [26, 27], allows us to study both the exit from G0/G1 phases of the cell cycle and the functionality of progenitor cells in response to various GFs. Although we showed that either method provides superimposable information concerning the cell cycle status and its activation, the technique based on the clonogenic assay only allows the evaluation of progenitor maintenance.

Thus, we have shown in agreement with previous reports [37, 38] that the majority of CB CFCs are quiescent like peripheral blood and immature bone marrow progenitors [39, 40]. This contrasts with mature bone marrow progenitors that are cycling [39, 40]. In a first set of experiments we tested the effects of serum on the cycling and maintenance of CFCs. Our data indicate that fetal and NBS or some of their components are important for the survival of hematopoietic progenitors, but are not sufficient for triggering their cycling as reported for bone marrow cells [41]. In a second set of experiments, we studied the effects of different hematopoietic GFs known to trigger the entry into cell cycle of hematopoietic progenitors [11-18]. Although the precise effects of GFs on cell cycle regulatory molecules still remain poorly understood, it turns out that GFs like IL-3, GM-CSF and SCF promote the survival of primitive dormant cells [17, 42, 43] whereas other GFs such as SCF, IL-6, IL-11 and LIF alone or in combination shorten the time for cell cycle of hematopoietic progenitor cells [17, 43, 44]. In addition, some of them can either exert permissive effects and/or upmodulate the expression of GF receptors on early hematopoietic progenitors [34, 45].

Thus, using IL-3 + IL-6 + SCF, we have shown that 30%-40% of CFCs can be activated in S-phase within 24-48 h. One can observe that the kinetics of activation of BFU-E and CFU-GM into S-phase might differ slightly depending upon the serum-containing media used. Such differences can be due to different components present in FCS or in NBS that might then interact with the GFs used in our study. Addition of LIF and IL-11 had no further significant effects. This apparent contradiction with previous reports showing that IL-11 alone or in combination with IL-3 stimulates the cycling and the proliferation of CFCs [13, 17] might be due to the fact that IL-11 was added to a combination of GFs including IL-6 that has relatively similar effects on hematopoietic progenitors and shares a common gp130 receptor [46]. Conversely, addition of IGF-I and bFGF enhanced the cycling of CFCs in IMDM + FCS only. Under this culture condition, the number of both BFU-E and CFU-GM is maintained provided bFGF is used at 50 ng/ml, confirming previous studies [15].

We have also evaluated the cycling characteristics of LTC-IC. Several reports have shown that LTC-IC in bone marrow or blood are mainly contained within the CD34+ CD38– population [30], do not retain rhodamine-123 [47] and are resistant to high concentrations of 5-fluorouracil [48]. These findings have strongly suggested that most LTC-IC are not cycling. Using the 3H-Tdr suicide assay, we have shown that in CB most LTC-IC are quiescent as reported for bone marrow LTC-IC using a slightly modified technique [49]. Interestingly, up to 60% of these cells can enter into S-phase after GF stimulation during 48 h. This high percentage in S-phase is not due to a technical drawback as reutilization of 3H-Tdr during the LTC has been prevented by the addition of an excess of cold Tdr. Thus, in our culture conditions, we have been able to trigger the cycling of CFCs and LTC-IC at similar levels. However, these results might appear surprising as primitive progenitors are expected to be less responsive to GF stimulation. In fact, the LTC-IC compartment in CB is heterogeneous and encompasses at least LTC-IC that can be assayed over a standard five-week long-term culture period and extended LTC-IC that are only revealed after 60-100 days of long-term cultures [36] and which might represent a more primitive subpopulation. Therefore, one could suggest that standard LTC-IC in CB might represent an intermediate compartment between CFCs and extended LTC-IC that can easily enter into S-phase in response to GF stimulation and then actively proliferate. Such a hypothesis is strengthened by others showing that CB progenitors, compared with bone marrow progenitors, exhibit an exquisite sensitivity in response to GFs, conferring upon them a higher ex vivo expansion potential [3-5].

Our overall results which confirm recent reports on the cycling of CB CFCs [37, 38] provide additional information concerning the culture conditions and GF stimulation for the cycling of both CFCs and more primitive progenitors. Furthermore, we have been able to trigger in a short period of time the cycling of progenitor cells without modifying their number. Using these culture conditions for optimizing retroviral gene transfer into CB progenitors, we have been able to efficiently transduce both CFCs and LTC-IC in preliminary experiments.


   Acknowledgments
 
This work was supported by a grant from Agence Nationale pour la Recherche sur le SIDA (ANRS). M. Movassagh is a recipient of a fellowship from ANRS.

We are grateful to Dr. P.M. Lansdorp (Terry Fox Laboratory; Vancouver, BC, Canada) for the kind gift of 8G12 mAb, to Dr. Godard (INSERM U211; Nantes, France) for the generous gift of purified rHuLIF, and to Dr. L. Aarden (Central Laboratory Blood Transfusion; Amsterdam, The Netherlands) for kindly providing us with rHuIL-6. We thank Amgen (Thousand Oaks, CA) for providing us with rHuSCF.

Special thanks are given to the staff of the Labor and Delivery Department, Rothschild Hospital, Paris, France, for their generous assistance in providing umbilical CB specimens.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication January 16, 1997.




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