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Stem Cells, Vol. 18, No. 6, 444-452, November 2000
© 2000 AlphaMed Press

Signal through gp130 Activated by Soluble Interleukin (IL)-6 Receptor (R) and IL-6 or IL-6R/IL-6 Fusion Protein Enhances Ex Vivo Expansion of Human Peripheral Blood-Derived Hematopoietic Progenitors

Takafumi Kimuraa, Jianfeng Wanga, Hitoshi Minamiguchia,d, Hiroshi Fujikia,b, Sachio Haradaa,b, Keiko Okudaa, Hiroto Kanekoc, Shouhei Yokotac, Kiyoshi Yasukawae, Tatsuo Abea, Yoshiaki Sonodaa

a Department of Hygiene, the
b Department of Gastroenterological Surgery, and the
c Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan;
d Second Department of Internal Medicine, Shiga University of Medical Science, Shiga, Japan; the
e Tokyo Research Center, Tosoh Corporation, Kanagawa, Japan

Key Words. IL-6R/IL-6 fusion protein • Soluble IL-6R • gp130 • PB-derived CD34+IL-6R- cells • Ex vivo expansion

Yoshiaki Sonoda, M.D., Department of Hygiene, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602, Japan. Telephone: 075-251-5335; Fax: 075-251-5334; e-mail: sonoda{at}basic.kpu-m.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was designed to investigate the effects of a combination of soluble interleukin (sIL)-6 receptor (R) and IL-6 on the ex vivo expansion of human peripheral blood (PB)-derived hematopoietic progenitor cells in a short-term serum-free liquid suspension culture system, using PB-derived CD34+IL-6R+/– cells as a target. In combination with stem cell factor (SCF), IL-3, and sIL-6R/IL-6, the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU-E) and mixed colony-forming unit (CFU-Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL-3 + sIL-6R/IL-6 + flt3 ligand (FL) most effectively expanded CFU-GM and CFU-Mix. The maximum EEs for CFU-GM and CFU-Mix were 208-fold and 42-fold, respectively. While the EE for BFU-E was 70-90-fold in the presence of SCF + IL-3 + sIL-6R/IL-6, FL significantly augmented the EE for CFU-GM and CFU-Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU-Mix. Interestingly, in combination with IL-3 and SCF, newly generated IL-6R/IL-6 fusion protein (FP) expanded PB-derived BFU-E and CFU-Mix twice more effectively than a combination of sIL-6R and IL-6. These results demonstrated that human PB-derived committed progenitors were effectively expanded in vitro using sIL-6R/IL-6 or FP, in combination with IL-3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL-6R and IL-6.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recently, we reported that simultaneous activation of signals through gp130, c-kit, and interleukin (IL)-3 receptor (R) dramatically promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors, such as G-CSF, erythropoietin (Epo), and thrombopoietin (TPO) [1]. Therefore, we intend to investigate the effects of these three signals on the ex vivo expansion of peripheral blood (PB)-derived hematopoietic progenitors. As we reported previously [2], a combination of stem cell factor (SCF) plus IL-3 most effectively expanded cord blood (CB)-derived committed progenitors among two-factor combinations tested. IL-6 significantly augmented the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) in combination with SCF + IL-3 or flt3 ligand (FL) + IL-3. It is well documented that signals activated by IL-6 are transmitted through a signal-transducing gp130 [3]. Therefore, we used a combination of soluble IL-6 receptor (sIL-6R) and IL-6, or newly generated IL-6R/IL-6 fusion protein (FP) instead of IL-6 in order to activate signal through gp130 in PB-derived CD34+IL-6R– cells in this experiment.

The IL-6/sIL-6R FP covalently linked by the flexible polypeptide linker, named Hyper-IL-6 (HIL-6, approximately 60 kDa), was previously reported [4, 5] and that HIL-6 was shown to be active on gp130-expressing cells at 100- to 1,000-fold lower concentration than the combination of IL-6 and sIL-6R. It was recently reported that HIL-6 improved retroviral-mediated gene transfer to CB-derived severe-combined immunodeficiency-repopulating cell (SRC) [6]. On the other hand, Kollet et al. reported that IL-6R/IL-6 chimeric protein was useful to expand CB-derived hematopoietic progenitors and SRC after short-term liquid suspension cultures [7].

In this paper, we have investigated the effect of a combination of IL-6 and sIL-6R or FP on the expansion of PB-derived committed progenitors, including CFU-mix. In contrast to a recent report [8], the combination of SCF + sIL-6R/IL-6 showed much weaker activity on the hematopoietic colony formation, as well as the expansion of committed progenitors than that of SCF + IL-3 or SCF + IL-3 + sIL-6R/IL-6 or SCF + IL-3 + FP.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of PB Cells
After informed consent was obtained, PB mononuclear (MN) cells were collected from patients with testicular tumors by leukapheresis using a Fenwall CS-3000 (Fenwall Laboratories, Inc.; Deerfield, IL), as reported elsewhere [1, 9]. First, the patients were treated with high-dose etoposide. Administration of G-CSF was started after the nadir state and MN cells were collected during the recovery phase from myelosuppression. Low-density PB cells were separated by Ficoll-Paque (Pharmacia; Piscataway, NJ; http://www.pnu.com) density gradient centrifugation at 400 g for 30 min. Nonadherent mononuclear (NAMN) cells were recovered by overnight adherence to plastic dishes. The NAMN cell fraction was further enriched by negative depletion of lineage-positive cells using a StemSepTM device (StemCell Technologies; Vancouver, BC, Canada; http://www.stemcell.com) as reported elsewhere [10]. At the end of the negative depletion procedure, lineage-negative cells were recovered in a sterile tube containing phosphate-buffered saline (PBS) with 2% fetal calf serum (FCS) (Hyclone Laboratories, Inc.; Logan, UT; http://www.hyclone.com). PB-derived lineage-negative cells had an approximately 80%-90% content CD34+ cells by flow cytometric analysis (data not shown).

Recombinant Factors
Purified bacterially derived recombinant human (rHu)IL-3, GM-CSF, G-CSF, SCF, and purified Chinese hamster ovary cell-derived rHuEpo were generous gifts from Kirin Brewery Co., Ltd. (Tokyo, Japan; http://www1.kirin.co.jp/english/r_d/pha/index.html). Purified E. coli-derived rHuIL-6 was kindly provided by Dr. Akira Okano (Ajinomoto Co., Inc.; Yokohama, Japan) and had a specific activity of 6 x 106 U/mg protein. Purified rHuTPO prepared by the TPO Production Group (Kirin Brewery Co., Ltd.; Takasaki, Japan) and was provided by Dr. Hiroshi Miyazaki (Kirin Brewery; Tokyo, Japan). Recombinant sIL-6R [11] and FP were kind gifts from Tosoh Corp. (Kanagawa, Japan; http://www.tosoh.com). The FP in which Ala (333) of IL-6R residue was linked to Ala (28) of IL-6 was expressed in Pichia pastoris. Purified FP exerted the highest activity at the concentration of 20 ng/ml on the growth of BAF130 cells carrying human gp130 cDNA (Yasukawa et al., manuscript in preparation). A recombinant soluble form of the human FL was produced in yeast as reported elsewhere [12] and kindly provided by Dr. Stewart D. Lyman (Immunex Research and Developmental Corp.; Seattle, WA; http://www.immunex.com).

Immunostaining and Cell Sorting
First, PB-derived lineage-negative cells were washed twice with PBS containing 2% FCS (staining medium) and were pelleted before staining with the following monoclonal antibodies (mAbs): fluorescein isothiocyanate (FITC)-conjugated HPCA-2 (mouse IgG1 CD34 mAb, Becton Dickinson [BD] Immunocytometry Systems; San Jose, CA; http://www.bd.com) and purified antihuman IL-6R mAb (MT18, mouse IgG1) [13]. For staining of CD34 and IL-6R, incubation was performed with 5 µg of biotinylated MT18 per 106 cells for 30 min at room temperature [1]. After washing, cells were incubated with streptavidin-phycoerythrin (PE) (BD) for 30 min on ice. The cells were then washed twice with staining medium and incubated with 20 µl of FITC-conjugated HPCA-2 per 106 cells on ice for 30 min. Subsequently, all cells were washed twice with staining medium and were kept on ice for cell sorting. Negative controls included unstained cells and cells stained only with an FITC-conjugated isotype control IgG or the streptavidin-PE.

PB-derived CD34+IL-6R+/– cells were sorted using a fluorescence-activated cell sorter Vantage (BD) equipped with an argon laser tuned at 488 nm as reported elsewhere [1, 9, 10]. Sorting gates were first established for both intermediate forward scatter (FSC) and low side scatter (SSC). A dual-parameter dot diagram displaying FITC fluorescence (CD34) and PE fluorescence (IL-6R) was then generated from the gated events. Using gated dot diagrams, CD34+IL-6R+/– cells were sorted for clonal cell culture and ex vivo expansion.

As we reported elsewhere [1, 9, 10], PB-derived CD34+ cells ubiquitously expressed gp130 and IL-3R and approximately 20% of them expressed c-kit. However, most of CD34+IL-6R– cells expressed a high level of c-kit [10].

Short-Term Serum-Free Liquid Suspension Culture
PB-derived CD34+IL-6R+/– cells were cultured at 5 or 2 x 103/ml in 1 ml of Iscove's modified Dulbecco's medium supplemented with 5 x 10–5 M 2-mercaptoethanol ([2-ME], Sigma Chemical Co.; St Louis, MO; http://www.sigma-aldrich.com), 10 µg/ml lecithin (Sigma), 6 µg/ml cholesterol (Sigma), 300 µg/ml fully iron-saturated human transferrin (95% pure, Sigma), 1% deionized fraction V bovine serum albumin ([BSA], Sigma), 1 µg/ml bovine pancreas insulin (Sigma), and various growth factors as reported [1, 2]. The concentration of each colony-stimulating factor (CSF) used in the culture was determined by titration experiments (data not shown) and was as follows: IL-3, 10 ng/ml; IL-6, 100 U/ml; TPO, 100 ng/ml; SCF, 20 ng/ml; FL, 20 ng/ml. The concentration of sIL-6R or FP was 1,000 ng/ml or 200 ng/ml, respectively. Over 1,000 ng/ml of sIL-6R or 100 ng/ml of FP supported maximum colony formation in the presence of SCF + IL-3 (data not shown). Dishes were incubated at 37°C in a fully humidified atmosphere flushed with a combination of 5% CO2, 5% O2, and 90% N2 for up to 21 days. Half of the medium was replaced weekly with fresh medium. We have found that low (5%) oxygen culture was superior to culture in ambient oxygen tension in preliminary experiments (data not shown). At specified intervals, the cells were collected by vigorous pipetting and counted using a counting chamber. Aliquots of cells were used for the colony assay to measure the EE of colony-forming cell (CFC).

Clonal Cell Culture
Serum-containing methylcellulose culture was carried out in 35-mm Lux suspension culture dishes (No. 171099, Nunc Inc.; Naperville, IL; http://www.nalgenunc.com) as we reported elsewhere [1, 2, 9, 10]. One ml of culture contained 200 CD34+IL-6R+/– cells or 0.2 to 2 x 103 expanded cells, 1.2% of 1,500 centipoise methylcellulose (Shinetsu Chemical; Tokyo, Japan), 30% FCS (Hyclone), 1% deionized fraction V BSA (Sigma), 5 x 10–5 mol/l 2-ME (Sigma), and various combination of CSFs. The final concentration of each CSF was as follows: IL-3, 10 ng/ml; GM-CSF, 10 ng/ml; G-CSF, 20 ng/ml; Epo, 2 U/ml; SCF, 20 ng/ml. These concentrations supported maximum total colony formation in preliminary titration experiments (data not shown).

Dishes were incubated at 37°C in a fully humidified atmosphere flushed with a combination of 5% CO2, 5% O2, and 90% N2 [1, 2, 9, 10]. On days 12-14 of culture, all colonies were scored on an inverted microscope according to their typical morphologic appearance as reported elsewhere [1, 2, 9, 10]. Colony types identified in situ were granulocyte colonies (CFU-G), macrophage colonies (CFU-M), CFU-GM, erythroid burst-forming units (BFU-E), eosinophil colonies (CFU-Eo), and erythrocyte-containing mixed colonies (CFU-Mix).

The average plating efficiencies (PEs) of PB-derived CD34+IL-6R+ and CD34+IL-6R– cells before liquid suspension culture were 40%-80% in the presence of a cocktail of CSFs, including SCF, IL-3, GM-CSF, G-CSF, and Epo (five CSFs). However, BFU-E and CFU-Mix were highly enriched in the CD34+IL-6R– cell population, whereas the CD34+IL-6R+ cell population contained largely CFU-G as reported [1]. On the other hand, PEs of expanded cells varied widely between different cytokine combinations used in liquid suspension cultures. The PEs of expanded cells on days 7 and 10 of culture were 15%-60% and 10%-30%, respectively.

The absolute number and fold increase of committed progenitors including CFU-GM (the sum of CFU-G, CFU-M, CFU-GM, and CFU-Eo), BFU-E, and CFU-Mix were calculated as follows: A) absolute number of colonies = (number of colonies/number of seeded cells) x number of viable nucleated cells; B) fold increase = absolute number of colonies on day 7 or day 10/absolute number of colonies on day 0.

Statistical Analysis
Results expressed as the mean ± standard deviation (SD) were obtained from three to six independent experiments. The significance of differences in means was determined using the two-tailed Student's t-test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serial Changes of the Absolute Numbers of Nucleated Cells (NCs) and CFCs in the Presence of SCF + IL-3 + IL-6 or SCF + IL-3 + sIL-6R/IL-6
First, we serially examined the increase of the total NC and absolute numbers of committed progenitors, such as CFU-GM, BFU-E, and CFU-Mix, from PB-derived CD34+IL-6R+/– cells in the presence of SCF + IL-3 + IL-6 or SCF + IL-3 + sIL-6R/IL-6. The numbers of viable NCs and absolute numbers of CFU-GM, BFU-E, and CFU-Mix were examined over time for 21 days. Representative data of three similar experiments were shown in Figure 1Go. Total NCs increased continuously for 14 days and consistently reached approximately 200-fold the input level by day 14 for CD34+IL-6R+ cells (Fig. 1AGo), and 500-fold for CD34+IL-6R– cells (Fig. 1BGo), respectively. The numbers of NCs in both populations remained at this peak level until day 21.



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  		Figure 1. Total numbers of viable NCs and the absolute numbers of committed progenitors were serially analyzed on day 7, 10, 14, and 21 of culture in the presence of SCF + IL-3 + IL-6 (A) or SCF + IL-3 + sIL-6R/IL-6 (B). Bold, solid, dotted, and shaded lines, respectively, show NC, CFU-GM, BFU-E, and CFU-Mix. Cultures were initiated with 5 x 103 PB-derived CD34+IL-6R+ cells or 2 x 103 PB-derived CD34+IL-6R– cells per dish, since the proliferative ability of CD34+IL-6R– cells was much higher than that of CD34+IL-6R+ cells. In fact, both cultures reached the similar levels of cell concentration (1 x 106/ml) on day 14 of cultures.

 
Serial changes of the absolute numbers of different committed progenitors expanded from CD34+IL-6R+ cells in the presence of SCF + IL-3 + IL-6 or those from CD34+IL-6R– cells in the presence of SCF + IL-3 + sIL-6R/IL-6 were also analyzed. When 5 x 103 CD34+IL-6R+ cells were cultured, the numbers of CFU-GM, BFU-E, and CFU-Mix increased continuously and reached 32-fold (1.2 x 105), 57-fold (2.7 x 104), and 9-fold (1.2 x 103) the initial levels by day 10. In case of cultures containing 2 x 103 CD34+IL-6R– cells, the numbers of BFU-E and CFU-Mix reached a peak level by day 7 and the extents of expansion were 60-fold (7.5 x 104) and 15-fold (2.0 x 103), respectively. In contrast, the number of CFU-GM reached a peak level on day 10 of incubation and the extent of expansion was 84-fold (9.2 x 103).

Based on these data, we cultured PB-derived CD34+IL-6R– cells for 10 days and examined the effects of various cytokine combinations on the expansion of committed hematopoietic progenitors on days 7 and 10 in the following experiments.

Effects of Combinations of SCF + IL-3 + sIL-6R/IL-6 on the Expansion of PB-Derived CD34+IL-6R– Cells in the Presence of TPO or FL
We next examined the effects of combinations of SCF + IL-3 + sIL-6R/IL-6 on the expansion of various types of committed progenitors in serum-free culture. Figure 2Go (A and B) shows the mean ± SD of fold increases of each progenitor in the presence of various cytokine combinations obtained from three to four independent experiments. Among the various two-factor combinations tested, SCF plus IL-3 most effectively expanded CFC as reported previously [2]. The number of BFU-E supported by SCF + IL-3 + sIL-6R/IL-6 showed a 70-fold increase on day 7 (Fig. 2AGo). And the EE was significantly increased to 90-fold on day 10 (p < 0.05) compared with approximately 30-fold of SCF + IL-3 (Fig. 2AGo). With the addition of TPO or FL to these three signals, the number of CFU-Mix increased more significantly than that supported by SCF + IL-3 and showed an approximately 20-fold increase on day 7 of culture (Fig. 2AGo). The number of CFU-GM increased 200-fold on day 10 of culture in the presence of SCF + IL-3 + sIL-6R/IL-6 + FL (Fig. 2BGo). In contrast to previous research [8], SCF + sIL-6R/IL-6 did not effectively expand CFCs compared with that obtained by SCF + IL-3. These results indicated that addition of sIL-6R/IL-6 to the cultures containing SCF + IL-3 is more effective for expanding BFU-E, and that the combination of TPO or FL with these three signals is effective for expansion of CFU-Mix. On day 10 of culture, FL specifically expanded CFU-GM.




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  		Figure 2. The fold increase of committed progenitors (including CFU-GM, BFU-E, and CFU-Mix) derived from 2 x 103 PB CD34+IL-6R– cells after 7 days (A) or 10 days (B) of serum-free liquid suspension culture in the presence of SCF and various other cytokines. Data represent the mean ± SD of three to four independent experiments. Addition of TPO or FL to the cultures containing SCF + IL-3 + sIL-6R/IL-6 significantly augmented the EE for CFU-Mix compared to SCF + IL-3 (***p < 0.01 and *p < 0.05). Open, dotted, and closed bars show the fold increase of CFU-GM, BFU-E, and CFU-Mix, respectively. The cultures contained 132 ± 40 CFU-GM, 1,192 ± 150 BFU-E, and 105 ± 67 CFU-Mix at the initial day.

 
Effects of Combinations of FL + IL-3 + sIL-6R/IL-6 on the Expansion of PB-Derived CD34+IL-6R– Cells in the Presence of TPO
We subsequently examined the effects of a ligand for FL [12] on the expansion of PB-derived CD34+IL-6R– cells in the presence of IL-3, sIL-6R/IL-6, and also compared its effect with that of SCF. Figure 3Go (A and B) shows the mean ± SD of three independent experiments. Among the two-factor combinations tested, the combination of FL and IL-3 most effectively expanded CFC (including CFU-GM, BFU-E, and CFU-Mix), as was the case with SCF. However, the EE for each progenitor was under 15-fold. When FL, IL-3, and sIL-6R/IL-6 were combined, CFU-GM, BFU-E, and CFU-Mix showed an 18-fold, 16-fold, and 11-fold increase on day 7, respectively (Fig. 3AGo). With the addition of TPO to the culture containing FL + IL-3 + sIL-6R/IL-6, the number of BFU-E significantly increased compared with that of FL + IL-3, and showed an approximate 20-fold increase (Figs. 3AGo, B). In a series of experiments using FL as one of the stimuli, CFU-GM was most effectively expanded both on days 7 and 10 of cultures compared with BFU-E and CFU-Mix. These results suggest that FL may mainly act on myeloid progenitors. However, the EE for each progenitor was much lower than that of SCF.




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  		Figure 3. The fold increase of committed progenitors (including CFU-GM, BFU-E, and CFU-Mix) derived from 2 x 103 PB CD34+IL-6R– cells after 7 days (A) or 10 days (B) of serum-free liquid suspension culture in the presence of FL and various other cytokines. Data represent the mean ± SD of three independent experiments. The EEs for CFU-GM was superior to the EEs for BFU-E and CFU-Mix in these experiments. Addition of TPO to the cultures containing FL + IL-3 + sIL-6R/IL-6 significantly augmented the EE for BFU-E compared to SCF + IL-3 (**p < 0.02). Open, dotted, and closed bars show the fold increase of CFU-GM, BFU-E, and CFU-Mix, respectively. The cultures contained 160 ± 86 CFU-GM, 1,335 ± 368 BFU-E, and 128 ± 23 CFU-Mix at the initial day.

 
Effects of IL-6R/IL-6 FP or sIL-6R/IL-6 on Hematopoietic Colony Formation by PB-Derived CD34+IL-6R– Cells
Next, we studied the effects of FP on hematopoietic colony formation in the presence of SCF with or without IL-3 and compared its effect with that of sIL-6R/IL-6. The representative data of three experiments are shown in Table 1Go. As we reported elsewhere [1], the number and type of colonies supported by a combination of SCF + IL-3 + sIL-6R/IL-6 were almost identical both in serum-free cultures and cultures with 30% FCS. Therefore, this experiment was performed in the presence of 30% FCS. Again, a combination of SCF and sIL-6R/IL-6 failed to support hematopoietic colony formation. In the presence of IL-3, a significant number of colonies derived from CFU-G, BFU-E, and CFU-Mix was identified. Interestingly, a combination of SCF plus 200 to 600 ng/ml of FP supported significant numbers of granulocyte, erythroid, and erythrocyte-containing mixed colonies. However, sizes of colonies were smaller than those supported by SCF + IL-3 + FP. A combination of SCF + IL-3 + FP (either 200 or 600 ng/ml) supported maximum colony formation including CFU-G, BFU-E, and CFU-Mix, which was comparable to the number of colonies supported by five CSFs. FP supported a significantly higher number of CFU-Mix-derived colonies than sIL-6R/IL-6. These results suggested that FP acts twice more potently on PB-derived progenitors than sIL-6R/IL-6 complex, and that signal through gp130 more efficiently acts on BFU-E and CFU-Mix.


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  		Table 1. Effect of IL-6R/IL-6 fusion protein on colony formation by PB-derived CD34+IL-6R– cells: comparison with effect of sIL-6R/IL-6

 
Effects of IL-6R/IL-6 FP on the Expansion of PB-Derived CD34+IL-6R– Cells in the Presence of SCF and IL-3: Comparison with the Effect of sIL-6R/IL-6
Finally, we examined the effect of FP on the expansion of PB-derived CD34+IL-6R– cells in the presence of SCF and IL-3, and compared its effect with that of sIL-6R/IL-6. Figure 4Go shows data that represent the mean ± SD of five independent experiments. As previously shown [2], the combination of SCF plus IL-3 most effectively expanded CFCs among all the two-factor combinations tested. A combination of SCF + IL-3 + FP showed a significant increase of the numbers of BFU-E and CFU-Mix on day 7 of cultures compared with that obtained by a combination of SCF + IL-3 + sIL-6R/IL-6 (Fig. 4AGo). However, the number of CFU-GM did not significantly change with the addition of FP instead of sIL-6R/IL-6. These results suggest that FP exerts a more potent effect on the expansion of BFU-E and CFU-Mix than the sIL-6R/IL-6 complex.



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  		Figure 4. The fold increase of committed progenitors (including CFU-GM, BFU-E, and CFU-Mix) derived from 2 x 103 PB CD34+IL-6R– cells after 7 days (A) and 10 days (B) of serum-free liquid suspension culture in the presence of SCF + IL-3 + sIL-6R/IL-6 or SCF + IL-3 + FP. Data represent the mean ± SD of five independent experiments. The EEs for BFU-E and CFU-Mix supported by SCF + IL-3 + FP were significantly augmented compared with those obtained by SCF + IL-3 + sIL-6R/IL-6 on day 7 of culture (**p < 0.02 and *p < 0.05). Open and closed bars show the fold increase of progenitors supported by SCF + IL-3 + sIL-6R/IL-6 or SCF + IL-3 + FP, respectively. The cultures contained 221 ± 149 CFU-GM, 1,108 ± 184 BFU-E, and 40 ± 30 CFU-Mix at the initial day.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of the present study was to evaluate the effectiveness of signal through gp130 activated by sIL-6R/IL-6 or IL-6R/IL-6 FP on the expansion of PB-derived hematopoietic progenitors. We previously reported that SCF plus either IL-6 or IL-11 most effectively expands murine hematopoietic progenitors, including high-proliferative potential colony-forming cells (HPP-CFC) derived from day-2 post-5-fluorouracil bone marrow (BM) cells in vitro [14]. However, in contrast to murine hematopoietic progenitors, SCF + sIL-6R/IL-6 did not effectively expand human hematopoietic progenitors derived from PB CD34+IL-6R– cells as shown in this study. This discrepancy may be explained by differences of human and murine hematopoietic progenitor cells. The present study clearly demonstrated that a sIL-6R/IL-6 complex or FP only effectively expanded PB-derived committed progenitors in combination with IL-3 and SCF or FL. Recently we reported that a combination of three signals through gp130, c-kit, and IL-3R exerted a dramatic synergistic action on the proliferation and differentiation of hematopoietic progenitors [1]. This study suggested that IL-3 support was important for survival and initial proliferation, whereas SCF was important for augmentation of the proliferation of progenitors. The signal through gp130 activated by sIL-6R/IL-6 complex may mainly contribute to the maturation of progenitors in the presence of SCF + IL-3. Together, IL-3 is the pivotal cytokine for the expansion of human hematopoietic progenitor cells in the presence of SCF. Our interpretation of the role of these three signals is consistent with a number of other reports [15-19]. However, Sui et al. reported that a combination of SCF and sIL-6R/IL-6 synergistically acted on the ex vivo expansion of CB-derived human hematopoietic progenitors [8]. As shown in Results, we could not induce such a potent activity on the expansion of PB-derived hematopoietic progenitors by SCF + sIL-6R/IL-6. Our data are consistent with the previous report [19] in which a combination of SCF + IL-6 failed to effectively expand CB-derived progenitors. These discrepancies between studies may reflect differences in culture condition as well as the target population. In fact, the expression of IL-6R on PB-derived CD34+ cells was approximately 80% as reported previously [1]. Conversely, its expression on CB-derived CD34+ cells was only 20% [1, 10].

An important finding of the present study is that signal through gp130 most effectively expanded BFU-E derived from PB CD34+IL-6R– cells in the presence of SCF + IL-3. In the copresence of TPO or FL, these three signals greatly facilitated the EE for CFU-Mix. These results were supported by the fact that TPO and FL both affect the proliferation and differentiation of primitive hematopoietic stem/progenitor cells in vitro and in vivo [12, 20-27]. On the other hand, signal through flt3 TK receptor also had potent action on myeloid progenitors [12, 20, 24-27]. Our data also demonstrated that newly generated FP more potently expanded BFU-E and CFU-Mix than the sIL-6R/IL-6 complex. An association constant of IL-6 and sIL-6R was observed to be 5 x 10–9 mol/l [11]. This indicates that in the solution containing 200 ng/ml (1 x 10–8 mol/l) of IL-6 (20 kDa) and 500 ng/ml (1 x 10–8 mol/l) of sIL-6R (50 kDa), 50% of the molecules exist as a monomer. Indeed, more than 1,000 ng/ml of sIL-6R are required for the full stimulation of CD34+IL-6R– cells [1]. We presume that our newly generated FP, as well as the previously reported ones, did not dissociate even at lower protein concentration. Collectively, the FP may more efficiently bind to gp130 and initiate stronger intracellular signaling than the sIL-6R/IL-6 complex, resulting in enhancement of in vitro colony formation as well as ex vivo expansion efficiency.

It was recently reported that IL-3 and IL-1 can suppress the B- and T-lymphoid potential of murine primitive progenitors [28, 29]. Moreover, IL-3 or IL-1 abrogates the long-term reconstituting ability of murine [30] and human [31] hematopoietic stem cells. These observations raise the possibility that IL-3 and IL-1 may suppress the earliest process of hematopoiesis. However, there are a number of conflicting reports [32-42]. Namely, many investigators reported that ex vivo cultures of subpopulations of BM-, PB-, or CB-derived CD34+ cells significantly expanded CFC, HPP-CFC, long-term culture-initiating cell (LTC-IC), and SRC in the presence of various cytokines including IL-3 [32-37]. In addition, Nolta et al. recently demonstrated that pluripotent human hematopoietic stem cells give rise to lymphomyeloid cells in immune-deficient mice after cotransplantation of transduced human progenitors and human IL-3-producing stromal cells [38]. Their result was consistent with a previous report [39] that cotransplantation of a rat fibroblast cell line carrying the human IL-3 gene significantly accelerated the process of engraftment of the human PB-derived stem cell.

In contrast, Verfaillie et al. reported important findings, that addition of IL-3 results in exhaustion of human LTBMC-IC after eight weeks of culture, possibly as a result of their terminal differentiation [40]. However, LTBMC-IC can be maintained for at least eight weeks when cultured in the presence of both macrophage inflammatory protein-1{alpha} and IL-3. It was also reported that stroma-contact culture of mobilized PB-derived hematopoietic stem cells in the presence of IL-3, SCF, and anti-transforming growth factor ß prevents loss of hematopoietic stem cell quality evaluated by LTC-IC and cobblestone area-forming cell assays [41]. These results may suggest that chemokine or some unidentified factor (signal) derived from stromal cells abrogates or reverses the inhibitory effects of IL-3 on early hematopoiesis, as did IL-4 in murine culture system [42].

These inconsistencies may reflect differences in experimental methods as well as different stem cell characteristics in humans and mice, and suggest that use of IL-3 for the expansion of human hematopoietic progenitor or stem cells may need to be cautiously reevaluated. Ex vivo-expanded hematopoietic stem/progenitor cells were already transplanted in patients with hematological and nonhematological malignancies after high-dose chemotherapy [43-45]. Therefore, further studies are needed to elucidate whether ex vivo-expanded hematopoietic cells contain truly long-term repopulating lympho-myeloid stem cells and whether they can be maintained, expanded or inhibited in vitro in the presence of IL-3.

In conclusion, the present study provides evidence that the combination of SCF + IL-3 + sIL-6R/IL-6 effectively expands human hematopoietic progenitors derived from PB lin–CD34+gp130+IL-6R– cells. Moreover, newly generated IL-6R/IL-6 FP more potently expanded BFU-E and CFU-Mix from this population. Our culture system, which can expand PB-derived hematopoietic progenitors as well as mature blood cells, may provide a useful tool for future cellular therapeutics in a variety of clinical settings.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors are grateful to Kirin Brewery Co., Ltd. for providing the various growth factors used in this study. The authors also thank Dr. Zhaozhu Zeng for his excellent technical assistance.

Supported in part by Grants-in-Aid for Scientific Research B (Grant No. 09470232) from the Ministry of Education, Science and Culture of Japan.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received July 10, 2000; accepted for publication September 13, 2000.




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