The effects of FLT3/FLK-2 ligand (FL) and KIT ligand (KL) on in vitro expansion of hematopoietic stem cells were studied using lineage-negative (Lin)Sca-1–positive (Sca-1+) c-kit–positive (c-kit+) marrow cells from 5-fluorouracil (5-FU)–treated mice. As single agents, neither FL nor KL could effectively support the proliferation of enriched cells in suspension culture. However, in combination with interleukin-11 (IL-11), both FL and KL enhanced the production of nucleated cells and progenitors. The kinetics of stimulation by FL was different from that by KL in that the maximal expansion by FL of the nucleated cell and progenitor pools required a longer incubation than with KL. We then tested the reconstituting abilities of cells cultured for 1, 2, and 3 weeks by transplanting the expanded Ly5.1 cells together with “compromised” marrow cells into lethally irradiated Ly5.2 mice. Cells that had been expanded with either cytokine combination were able to maintain the reconstituting ability of the original cells. Only cells that had been incubated with KL and IL-11 for 21 days had less reconstituting ability than fresh marrow cells. These results indicate that there can be significant expansion of progenitors in vitro without compromising the reconstituting ability of stem cells. Addition of IL-3 to permissive cytokine combinations significantly reduced the ability of cultured cells to reconstitute the hematopoiesis of irradiated hosts. These observations should provide a basis for a rational approach to designing cytokine combinations for in vitro expansion of hematopoietic stem cells.

BOTH FLT3/FLK-2 and KIT receptor tyrosine kinases belong to the type III receptor tyrosine kinase family that includes FMS and platelet-derived growth factor receptors.1-9 The ligands for several of these receptors stimulate the proliferation of hematopoietic cells.7,8,10-12 FLT3/FLK-2 transcripts have been detected in murine and human cell populations enriched for hematopoietic stem cells and progenitors and are absent in more mature cells.1-4 In addition, targeted disruption of the flt3/flk-2 gene led to deficiencies in primitive hematopoietic progenitors.13 The FLT3/FLK-2 ligand (FL) is similar to KIT ligand (KL) in that both proteins stimulate the proliferation of primitive hematopoietic progenitors.14-24 Neither factor has much stimulatory activity on its own, but each factor synergizes with other early-acting cytokines such as interleukin-6 (IL-6), IL-11, IL-12, and granulocyte colony-stimulating factor (G-CSF ).17,18,25 26 

Currently, there is significant interest in hematology in the in vitro (ex vivo) expansion of hematopoietic stem cells and progenitors.27-40 A number of investigators have already shown that it is possible to increase the number of hematopoietic progenitors in culture by using combinations of early-acting cytokines.41 In studies of murine lymphohematopoietic progenitors in culture, we observed that a combination of FL and IL-11 stimulates production of cells with a blast-like appearance in suspension culture for a longer time than KL-containing cytokine combinations.17 We report here the results of our studies of the effects of FL or KL on the long-term engrafting capability of stem cells. Although kinetic differences exist, both cytokines are capable of yielding committed and uncommitted progenitors without compromising stem cell reconstituting capability in lethally irradiated hosts.

Growth factors.Recombinant soluble human FL was produced in yeast and purified as previously described.14 Purified recombinant murine KL was provided by Kirin Brewery Co (Tokyo, Japan). Medium conditioned by Chinese hamster ovary cells that had been genetically engineered to produce murine IL-3 at a high titer (70,000 U/mL) was a gift from T. Sudo of the Biomaterial Institute (Yokohama, Japan). Purified recombinant human IL-6 was a gift from M. Naruto of Toray Industries (Kamakura, Japan). Purified recombinant human IL-11 was a gift from P. Schendel of the Genetics Institute (Cambridge, MA). Purified recombinant erythropoietin (Ep) was a gift from F.-K. Lin of Amgen (Thousand Oaks, CA). Unless otherwise specified, concentrations of the cytokines used were as follows: FL 100 ng/mL, KL 100 ng/mL, IL-3 200 U/mL, IL-6 100 ng/mL, IL-11 20 ng/mL, and Ep 2 U/mL.

Cell preparations.Cells from 2- to 5-month-old BDF1 and C57B1/6 mice (Charles River Laboratories, Raleigh, NC) were used in suspension culture, and cells from 2- to 3-month-old C57B1/6 mice (Jackson Laboratories, Bar Harbor, ME) that are congenic for Ly5 allotypes were used in transplantation experiments. 5-Fluorouracil (5-FU; Adria Laboratories, Columbus, OH) was administered intravenously through the tail vein at 150 mg/kg body weight, and bone marrow cells were harvested 2 days later. Single cell suspensions were prepared from pooled femurs and tibiae, and the cells with densities between 1.063 and 1.077 g/mL were collected with gradients of Metrizamide (Accurate Chemical & Scientific Corp, Westburg, NY). The cells were further enriched for progenitors by negative immunomagnetic selection with a mixture of lineage-specific antibodies.42 Lineage-negative (Lin) cells were incubated with fluorescein isothiocyanate–conjugated monoclonal antibody D7 (anti-Sca-1)43 and biotin-conjugated monoclonal ACK4 (anti-c-kit)44 for 15 minutes on ice. Isotype controls were fluorescein isothiocyanate–conjugated rat IgG2a and biotin-conjugated rat IgG2a. The cells were washed twice with Ca2+, Mg2+-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA; Fraction V; Sigma Chemical Co, St Louis, MO) and incubated with R-phycoerythrin–conjugated Streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) for 15 minutes on ice. The cells were washed twice, resuspended in PBS/BSA, and kept on ice until cell sorting. Flow cytometric analysis and cell sorting were performed on a FACStarPlus (Becton Dickinson, San Jose, CA).

Suspension and clonal cell cultures.One thousand Lin Sca-1–positive (Sca-1+) c-kit–positive (c-kit+) cells were incubated in each well of a 24-well plate (Falcon, Lincoln Park, NJ) in suspension culture. The culture medium contained α-medium, 20% (vol/vol) fetal calf serum (Intergen, Purchase, NY), 1% deionized Fraction V BSA, 1 × 10−4 mol/L 2-mercaptoethanol (Sigma), and combinations of cytokines. On day 7 of incubation, the cultured cells were diluted, replated in freshly prepared media in 24-well plates at 2,000 or 5,000 cells/mL, and incubated for 7 more days. On day 14, the cultured cells were diluted, replated in freshly prepared media in 24-well plates at 5 or 10 × 104 cells/mL, and incubated for 7 more days. At each time of replating, aliquots were analyzed for colony formation in 35-mm suspension culture dishes (Falcon) containing α-medium, 1.2% 1,500-cp methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% fetal calf serum, 1% BSA, 1 × 10−4 mol/L 2-mercaptoethanol, KL, IL-3, IL-11, and Ep. Total colony-forming units in culture (CFU-C) and the progenitors for granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) were determined on day 8 of incubation by in situ observation of the plates on an inverted microscope.45 46 

Table 1.

Expansion of Cell and Progenitor Populations in Suspension Culture

CytokinesDay 7Day 14
TNCCFU-CCFU-GEMMTNCCFU-CCFU-GEMM
FL <1,000 ND ND <1,000 ND ND 
KL 3.4 × 103 400 50 3.2 × 103 110 10 
IL-11 
FL,KL 5.6 × 103 580 30 1.3 × 104 110 10 
FL, IL-11 5.6 × 104 7,800 180 2.2 × 107 228,000 2,500 
KL, IL-11 2.2 × 105 33,700 1,050 1.1 × 108 706,000 12,000 
CytokinesDay 7Day 14
TNCCFU-CCFU-GEMMTNCCFU-CCFU-GEMM
FL <1,000 ND ND <1,000 ND ND 
KL 3.4 × 103 400 50 3.2 × 103 110 10 
IL-11 
FL,KL 5.6 × 103 580 30 1.3 × 104 110 10 
FL, IL-11 5.6 × 104 7,800 180 2.2 × 107 228,000 2,500 
KL, IL-11 2.2 × 105 33,700 1,050 1.1 × 108 706,000 12,000 

One thousand LinSca-1+c-kit+ cells were cultured in the presence of the designated cytokines. On day 7, the cells were washed and then diluted in freshly prepared media, and the incubation continued for 7 more days. On both day 7 and day 14 of culture, cells were analyzed for CFU-C and CFU-GEMM in methylcellulose culture.

Abbreviation: ND, not determined.

Table 2.

Time Course Analysis of Expansion of TNC and CFU-C in Suspension Culture

CytokinesDay 7Day 14Day 21
TNCCFU-CTNCCFU-CTNCCFU-C
FL, KL 3.8 × 103 970 1.6 × 104 180 1.5 × 104 30 
FL, IL-11 5.6 × 104 8,600 4.5 × 106 53,000 4.0 × 107 232,200 
KL, IL-11 8.6 × 105 105,300 2.2 × 108 453,800 5.2 × 108 36,300 
CytokinesDay 7Day 14Day 21
TNCCFU-CTNCCFU-CTNCCFU-C
FL, KL 3.8 × 103 970 1.6 × 104 180 1.5 × 104 30 
FL, IL-11 5.6 × 104 8,600 4.5 × 106 53,000 4.0 × 107 232,200 
KL, IL-11 8.6 × 105 105,300 2.2 × 108 453,800 5.2 × 108 36,300 

One thousand LinSca-1+c-kit+ cells were cultured in the presence of the designated cytokines. On day 7 and day 14 of culture, the cells were washed and then diluted in freshly prepared media, and the incubation continued for 7 more days. At each time of replating, aliquots were analyzed for colony formation.

In vivo reconstitution experiments.Female C57B1/6-Ly5.2 mice were administered single 850-cGy total-body irradiation via a 4 × 106 V linear accelerator. After total-body irradiation of the recipient mice, sorted fresh marrow cells of male C57B1/6-Ly5.1 mice were injected into the tail vein of the recipients together with 4 × 105 “compromised” marrow cells of female C57B1/6-Ly5.2 mice in a final volume of 0.2 mL PBS containing 0.1% BSA. “Compromised” cells had been subjected to two previous rounds of transplantation and regeneration in female mice.47 After 1 to 3 weeks' incubation, fractions of cultured cells were injected into female C57B1/6-Ly5.2 mice together with “compromised” cells. Peripheral blood was obtained from the retro-orbital venous plexus using heparin-coated micropipettes (Drummond Scientific Co, Broomall, PA) 2, 4, and/or 6 months after transplantation. Red blood cells were lysed by 0.15 mol/L NH4Cl. The samples were then used for flow cytometric analysis of donor-derived cells by staining with anti-Ly5.1 (A20-1.7). In all experiments, cultures were initiated with the same number of cells as the “fresh sample.” In designated experiments, duplicate cultures were performed so that the entire sample and fractions equivalent to 1/10 and 1/100 of the sample were injected.

Effects of FL on the expansion of progenitor cells.First, we determined the optimal concentrations of FL and KL for stimulation of growth of LinSca-1+c-kit+ marrow cells of 5-FU–treated mice. Varying concentrations of FL and KL were added to suspension cultures containing 20 ng/mL IL-11. The optimal doses of both FL and KL were determined to be 100 ng/mL.

Next, we studied the effects of FL as a single factor and in combination with KL and/or IL-11 on the proliferation of LinSca-1+ c-kit+ cells in suspension culture. The results presented in Table 1 are representative of two experiments. As single factors, FL, KL, and IL-11 had little or no effect on the production of total nucleated cells (TNC). When FL or KL were combined with IL-11, significant expansion of both cells and progenitors was observed. However, direct comparison of FL and KL is not meaningful, since the initial cells that were expanded were c-kit+ cells and thus may not represent the physiologic composition of primitive progenitors. KL did not synergize with FL.

Later effects of FL on the expansion of progenitor cells.We have recently observed that many blast cells persist on day 13 of methylcellulose culture containing FL and IL-11.17 Therefore, in the next set of experiments, we extended suspension culture to 3 weeks and analyzed the effects of two-factor combinations (FL and KL, FL and IL-11, and KL and IL-11) on the expansion of cells and progenitors. The results of one of two similar experiments are presented in Table 2. Again, both the combination of FL and IL-11 and the combination of KL and IL-11 significantly increased the number of cells and CFU-C, whereas the combination of FL and KL showed little synergy. Proliferation of cells and progenitors appeared slower in culture with FL and IL-11 than with KL and IL-11. However, by day 21 of incubation, the number of CFU-C in FL-containing cultures was significantly higher than in KL-containing cultures, even though there were fewer TNC in FL-containing cultures than in KL-containing cultures.

Table 3.

Reconstituting Ability of Fresh Bone Marrow Cells and Day-14 Cultured Cells

A. Quantitation of Donor Cells
Cytokines in CultureNo. of Cells InjectedNo. of CFU-C Injected% Ly5.1 (donor) PB Cells3-150
2 Months6 Months
Summary Data 
 
     
Fresh marrow 100 41 45.1 ± 7.0 41.1 ± 9.8 
 10  11.0 ± 13.6 7.1 ± 8.9 
  2.5 ± 3.2 2.7 ± 4.1 
FL, KL 660 ND 0.5 ± 0.1 0.6 ± 0.1 
FL, IL-11 5.0 × 105 4,630 59.0 ± 15.8 72.3 ± 23.9 
 5.0 × 104  11.8 ± 15.7 18.5 ± 26.9 
 5.0 × 103  1.1 ± 0.9 1.1 ± 0.6 
KL, IL-11 2.0 × 106 15,300 57.1 ± 19.6 50.0 ± 27.2 
 2.0 × 105  4.6 ± 2.4 1.9 ± 0.7 
 2.0 × 104  0.3 ± 0.1 0.6 ± 0.1 
Individual Recipient Data 
 
     
Fresh marrow 100  35.6 27.9 
   55.2 57.2 
   41.2 40.3 
   50.7 35.1 
   42.8 44.8 
FL, IL-11 5.0 × 105  79.3 92.8 
   69.3 84.0 
   32.8 28.8 
   60.7 90.8 
   53.0 65.0 
KL, IL-11 2.0 × 106  82.1 80.2 
   42.2 15.4 
   78.1 80.1 
   32.8 23.6 
   50.2 50.5 
 
     
B. Surface Phenotype of Donor Cells 
     
Cytokines in Culture  % Mac-1+Gr-1+ Cells % B 220+ Cells % Thy-1+ Cells 
 
     
Fresh marrow  3.1 30.5 20.8 
  2.5 22.3 13.2 
FL, IL-11  8.5 42.0 28.5 
  6.0 48.0 25.2 
KL, IL-11  2.1 32.5 26.5 
  4.0 25.3 21.5 
A. Quantitation of Donor Cells
Cytokines in CultureNo. of Cells InjectedNo. of CFU-C Injected% Ly5.1 (donor) PB Cells3-150
2 Months6 Months
Summary Data 
 
     
Fresh marrow 100 41 45.1 ± 7.0 41.1 ± 9.8 
 10  11.0 ± 13.6 7.1 ± 8.9 
  2.5 ± 3.2 2.7 ± 4.1 
FL, KL 660 ND 0.5 ± 0.1 0.6 ± 0.1 
FL, IL-11 5.0 × 105 4,630 59.0 ± 15.8 72.3 ± 23.9 
 5.0 × 104  11.8 ± 15.7 18.5 ± 26.9 
 5.0 × 103  1.1 ± 0.9 1.1 ± 0.6 
KL, IL-11 2.0 × 106 15,300 57.1 ± 19.6 50.0 ± 27.2 
 2.0 × 105  4.6 ± 2.4 1.9 ± 0.7 
 2.0 × 104  0.3 ± 0.1 0.6 ± 0.1 
Individual Recipient Data 
 
     
Fresh marrow 100  35.6 27.9 
   55.2 57.2 
   41.2 40.3 
   50.7 35.1 
   42.8 44.8 
FL, IL-11 5.0 × 105  79.3 92.8 
   69.3 84.0 
   32.8 28.8 
   60.7 90.8 
   53.0 65.0 
KL, IL-11 2.0 × 106  82.1 80.2 
   42.2 15.4 
   78.1 80.1 
   32.8 23.6 
   50.2 50.5 
 
     
B. Surface Phenotype of Donor Cells 
     
Cytokines in Culture  % Mac-1+Gr-1+ Cells % B 220+ Cells % Thy-1+ Cells 
 
     
Fresh marrow  3.1 30.5 20.8 
  2.5 22.3 13.2 
FL, IL-11  8.5 42.0 28.5 
  6.0 48.0 25.2 
KL, IL-11  2.1 32.5 26.5 
  4.0 25.3 21.5 

Irradiated recipients (5 mice per group) were transplanted with 100, 10, or one cell equivalents of fresh LinSca-1+c-kit+ cells or with whole or fractions of expanded cultures initiated with 100 LinSca-1+c-kit+. Levels of engraftment at 2 and 6 months are shown both as a summary of groups and as individual data. Lineage phenotypes of the donor cells were analyzed at 6 months in 2 mice per group.

F3-150

Mean ± SD % donor-derived nucleated peripheral blood cells identified by anti-Ly5.1. Background (peripheral blood cells of nontransplanted female mice) staining was <1.0%.

Reconstituting ability of expanded cells.We then tested the in vivo reconstituting ability of cells expanded with combinations of cytokines. The suspension cultures were initiated with LinSca-1+c-kit+ C57B1/6-Ly5.1 cells. The cultures were serially transferred to new flasks containing fresh media every 7 days to keep cell concentrations at less than 1 × 106/mL as described in the methods. We then transplanted the whole sample or fractions of the sample of cells expanded from 100 enriched cells into lethally irradiated C57B1/6-Ly5.2 mice. As controls, we also transplanted 100, 10, and one enriched marrow cells. The results of analyses of day 14 expanded cells are presented in Table 3. Incubation with the combination of FL and KL barely supported cell proliferation and failed to maintain the population of stem cells. Both the combination of FL and IL-11 and the combination of KL and IL-11 maintained the reconstituting ability of the expanded cells. Monocytes, granulocytes, and T and B lymphocytes of donor cell type were detected in the peripheral blood of mice injected with the expanded cells (Fig 1 and Table 3). Because of the observation (Table 2) that the combination of FL and IL-11 supports maintenance of progenitor cells longer than the combination of KL and IL-11, we next compared the reconstitution ability of cells that were expanded for 7 or 21 days under two different cytokine conditions (Table 4). The cells expanded with FL or KL and IL-11 for 7 days had almost the same reconstituting ability as fresh cells. However, on day 21 of culture, the combination of FL and IL-11 maintained the reconstituting ability of cultured cells much better than the combination of KL and IL-11.

Fig. 1.

Hematopoietic reconstitution by cells cultured for 14 days in the presence of FL and IL-11. Nucleated cells of the peripheral blood were analyzed using flow cytometry 6 months after transplantation. Thy-1+ cells, B220+ cells, and Mac-1+, Gr-1+ cells of donor (Ly5.1) origin are seen in the peripheral blood of the recipient. Analyses of additional samples are presented in Table 3.

Fig. 1.

Hematopoietic reconstitution by cells cultured for 14 days in the presence of FL and IL-11. Nucleated cells of the peripheral blood were analyzed using flow cytometry 6 months after transplantation. Thy-1+ cells, B220+ cells, and Mac-1+, Gr-1+ cells of donor (Ly5.1) origin are seen in the peripheral blood of the recipient. Analyses of additional samples are presented in Table 3.

Close modal
Table 4.

Reconstituting Ability of Fresh Bone Marrow and Day 7 or Day 21 Cultured Cells

CytokinesDays of IncubationNo. of Cells InjectedNo. of CFU-C Injected% Ly5.1 (donor) PB Cells4-150
2 mo4 mo
Fresh marrow  100 42 51.3 ± 14.1 52.3 ± 20.8 
FL, IL-11 1.6 × 104 ND 50.5 ± 8.3 40.1 ± 8.60 
KL, IL-11 2.0 × 105 ND 63.4 ± 12.2 44.7 ± 16.9 
FL, IL-11 21 3.4 × 107 34,100 47.3 ± 8.5 36.2 ± 16.8 
KL, IL-11 21 3.2 × 107 9,200 16.3 ± 2.5 11.7 ± 2.2 
CytokinesDays of IncubationNo. of Cells InjectedNo. of CFU-C Injected% Ly5.1 (donor) PB Cells4-150
2 mo4 mo
Fresh marrow  100 42 51.3 ± 14.1 52.3 ± 20.8 
FL, IL-11 1.6 × 104 ND 50.5 ± 8.3 40.1 ± 8.60 
KL, IL-11 2.0 × 105 ND 63.4 ± 12.2 44.7 ± 16.9 
FL, IL-11 21 3.4 × 107 34,100 47.3 ± 8.5 36.2 ± 16.8 
KL, IL-11 21 3.2 × 107 9,200 16.3 ± 2.5 11.7 ± 2.2 

Irradiated recipients (5 mice per group) were transplanted with 100 fresh LinSca-1+c-kit+ cells or with the whole fractions of day 7 or day 21 cultured cells that had been initiated with 100 LinSca-1+c-kit+ cells.

F4-150

Mean ± SD % donor-derived nucleated peripheral blood cells identified by anti-Ly5.1. Background (peripheral blood cells of nontransplanted female mice) staining was <1.0%.

Finally, we examined the in vivo reconstituting ability of cells expanded in the presence of multiple cytokines. The four cytokine combinations with FL or KL did not enhance the in vivo reconstituting ability of cultured cells versus fresh cells (Table 5, experiment 1). We previously reported that IL-3 and IL-1 possess negative regulatory effects on early B lymphopoiesis,48 T lymphopoiesis,49 and in vitro expansion of stem cells with long-term reconstitution capabilities.50 Therefore, in one experiment, we examined the addition of IL-3 to multiple cytokines. When added to four-cytokine combinations consisting of KL, IL-6, IL-11, and Ep, IL-3 significantly reduced the in vivo reconstituting ability of cultured cells, whereas FL did not (Table 5, experiment 2).

Table 5.

Effects of Multiple Cytokines on the Reconstituting Ability of Cultured Cells

Cytokines in CultureNo. of Cells InjectedNo. of CFU-C Injected% Ly5.1 (donor) PB Cells5-150
2 mo6 mo
Experiment 1 
Fresh marrow 100 48 47.1 ± 22.8 54.6 ± 29.8 
FL,IL-6,IL-11,Ep 2.7 × 104 4,500 58.7 ± 19.1 42.0 ± 18.7 
KL,IL-6,IL-11,Ep 3.3 × 105 30,000 72.9 ± 11.2 48.3 ± 26.2 
Experiment 2 
Fresh marrow 500 240 71.3 ± 17.8 65.1 ± 28.3 
KL,IL-6,IL-11,Ep 8.0 × 105 182,000 69.5 ± 17.5 63.5 ± 30.6 
FL,KL,IL-6,IL-11,Ep 1.5 × 106 281,000 80.5 ± 16.2 73.8 ± 22.6 
IL-3,KL,IL-6,IL-11,Ep 3.3 × 106 35,000 19.3 ± 11.1 14.5 ± 11.5 
Cytokines in CultureNo. of Cells InjectedNo. of CFU-C Injected% Ly5.1 (donor) PB Cells5-150
2 mo6 mo
Experiment 1 
Fresh marrow 100 48 47.1 ± 22.8 54.6 ± 29.8 
FL,IL-6,IL-11,Ep 2.7 × 104 4,500 58.7 ± 19.1 42.0 ± 18.7 
KL,IL-6,IL-11,Ep 3.3 × 105 30,000 72.9 ± 11.2 48.3 ± 26.2 
Experiment 2 
Fresh marrow 500 240 71.3 ± 17.8 65.1 ± 28.3 
KL,IL-6,IL-11,Ep 8.0 × 105 182,000 69.5 ± 17.5 63.5 ± 30.6 
FL,KL,IL-6,IL-11,Ep 1.5 × 106 281,000 80.5 ± 16.2 73.8 ± 22.6 
IL-3,KL,IL-6,IL-11,Ep 3.3 × 106 35,000 19.3 ± 11.1 14.5 ± 11.5 

Irradiated recipients (5 mice per group) were transplanted with 100 (experiment 1) or 500 (experiment 2) freshly prepared LinSca-1+c-kit+ cells or with the whole fractions of day 7 cultured cells that had been initiated with 100 or 500 LinSca-1+c-kit+ cells.

F5-150

Mean ± SD % donor-derived nucleated peripheral blood cells identified by anti-Ly5.1. Background (peripheral blood cells of nontransplanted female mice) staining was <1.0%.

Previously, we reported that combinations of FL and either IL-6, IL-11, or G-CSF support proliferation of primitive hematopoietic progenitors including lymphohematopoietic progenitors.17 In this regard, FL and KL are similar in hematopoietic function, although the number and size of the colonies supported by FL-containing cytokine combinations were smaller than those supported by KL-containing cytokine combinations.17 During the same study, we noted that blast-like cells persist longer in incubation with FL than with KL. FL and KL signal through different but related tyrosine kinase receptors.1,2 14 Because of their similarities and dissimilarities, we compared their effects on the in vitro expansion of progenitors and hematopoietic stem cells with reconstituting capability. Proliferation of the progenitors appeared slower but persisted longer in suspension culture with FL and IL-11 than in culture with KL and IL-11.

We then compared the ability of FL and KL to support in suspension culture the in vivo long-term reconstituting cells. In the presence of IL-11, both factors maintained the reconstituting cells in culture for 14 days. Although there was no apparent expansion of the stem cells, the standard deviations of the means of the results were large. Neither were there apparent differences between the combination of FL and IL-11 and the combination of KL and IL-11. However, there were kinetic differences in the peak production of CFU-C in that for KL it occurred at day 14 and for FL at day 21 or longer. This time course of the reconstituting cells appears to be consistent with that of the progenitors. In our previous studies, we noted a gradual decline of stem cell functions in 2-week suspension cultures with KL and IL-11.50 However, in that series of experiments, we sorted the cultured cells on day 7 for cells with stem cell phenotypes and recultured the enriched cells for 7 more days. The serial dilution technique used in the current studies allowed maintenance of stem cell functions and appeared to be better than the resorting strategies we used previously. It is possible that stem cells change phenotypes during suspension culture. Alternatively, the resorting of cultured cells may have traumatized the stem cells.

As stated in the introduction, many investigators have already shown that it is possible to expand the population of cells and colony-forming cells in suspension culture in the presence of combinations of early-acting cytokines. However, attempts to expand the population of cells that are capable of long-term reconstitution have met with variable success.28,31-33 Recently, Peters et al51 reported that a 48-hour suspension culture of murine marrow cells in the presence of IL-3, IL-6, IL-11, and KL results in impairment of the engrafting capability of the cultured cells. They proposed that expansion of the progenitors may produce a defective long-term repopulating capability of the stem cells. We believe that the reason for their observation is that IL-3 abrogates the long-term reconstitution capability of cultured cells. Results presented herein and in a previous publication50 indicate that cells expanded in the absence of IL-3 or IL-1 maintain reconstituting ability.

In vitro expansion of hematopoietic stem cells promises to be important in clinical stem cell transplantation. We have shown that both FL and KL are capable of expanding the progenitor cell pool without compromising engraftment. Significant kinetic differences appear to exist between the two cytokines, but we have not determined which factor combination is superior. Again, we noted negative effects of IL-3 on the ability of cultured cells to engraft the marrow of recipient mice. Although it is not wise to translate findings in murine systems directly to human cells, cytokine combinations containing IL-3 need to be evaluated carefully for in vitro expansion of human stem cells.

We thank P.N. Pharr, A.G. Leary, and N.D. Grant for assistance in preparing the manuscript and H.Q. Zeng in FACS cell sorting.

Supported by National Institutes of Health grants no. DK/HL48714 and DK32294, the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and a grant from Amgen Inc.

Address reprint requests to Makio Ogawa, MD, PhD, VA Medical Center, 109 Bee St, Charleston, SC 29401-5799.

1
Rosnet
O
Marchetto
S
deLapeyriere
O
Birnbaum
D
Murine Flt3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family.
Oncogene
6
1991
1641
2
Matthews
W
Jordan
CT
Wiegand
GW
Pardoll
D
Lemischka
IR
A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell–enriched populations.
Cell
65
1991
1143
3
Rosnet
O
Schiff
C
Pebusque
M-J
Marchetto
S
Tonnelle
C
Toiron
Y
Birg
F
Birnbaum
D
Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells.
Blood
82
1993
1110
4
Small
D
Levenstein
M
Kim
E
Carow
C
Amin
A
Rockwell
P
Witte
L
Burrow
C
Ratajczak
MZ
Gewirtz
AM
Civin
CI
STK-1, the human homologue of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.
Proc Natl Acad Sci USA
91
1994
459
5
Sherr
CJ
Rettenmier
CW
Sacca
R
Roussel
MF
Look
AT
Stanley
ER
The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1.
Cell
41
1985
665
6
Williams
DE
Eisenman
J
Baird
A
Rauch
C
Van Ness
K
March
CJ
Park
LS
Martin
U
Mochizuki
DY
Boswell
HS
Burgess
GS
Cosman
D
Lyman
SD
Identification of a ligand for the c-kit proto-oncogene.
Cell
63
1990
167
7
Zsebo
KM
Wypych
J
McNiece
IK
Lu
HS
Smith
KA
Karkare
SB
Sachdev
RK
Yuschenkoff
VN
Birkett
NC
Williams
LR
Satyagal
VN
Tung
W
Bosselman
RA
Mendiaz
EA
Langley
KE
Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium.
Cell
63
1990
195
8
Huang
E
Nocka
K
Beier
DR
Chu
T-Y
Buck
J
Lahm
H-W
Wellner
D
Leder
P
Besmer
P
The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus.
Cell
63
1990
225
9
Ullrich
A
Schlessinger
J
Signal transduction by receptors with tyrosine kinase activity.
Cell
61
1990
203
10
Stanley
ER
Chen
DM
Lin
H-S
Induction of macrophage production and proliferation by a purified colony stimulating factor.
Nature
274
1978
168
11
Stanley
ER
Guilbert
LJ
Tushinski
RJ
Bartelmez
SH
CSF-1 — A mononuclear phagocyte lineage–specific hemopoietic growth factor.
J Cell Biochem
21
1983
151
12
Metcalf
D
Nicola
NA
Direct proliferative actions of stem cell factor on murine bone marrow cells in vitro: Effects of combination with colony-stimulating factors.
Proc Natl Acad Sci USA
88
1991
6239
13
Mackarehschian
K
Hardin
JD
Moore
KA
Boast
S
Goff
SP
Lemischka
IR
Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors.
Immunity
3
1995
147
14
Lyman
SD
James
L
Vanden Bos
T
de Vries
P
Brasel
K
Gliniak
B
Hollingsworth
LT
Picha
KS
McKenna
HJ
Splett
RR
Fletcher
FF
Maraskovsky
E
Farrah
T
Foxworthe
D
Williams
DE
Beckmann
MP
Molecular cloning of a ligand for the flt3/flk2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells.
Cell
75
1993
1157
15
Hannum
C
Culpepper
J
Campbell
D
McClanahan
T
Zurawski
S
Bazan
JF
Kastelein
R
Hudak
S
Wagner
J
Mattson
J
Luh
J
Duda
G
Martina
N
Peterson
D
Menon
S
Scanafelt
A
Muench
M
Kelner
G
Namikawa
R
Rennick
D
Roncarolo
M-G
Zlotnik
A
Rosnet
O
Dubreuil
P
Birnbaum
D
Lee
F
Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs.
Nature
368
1994
643
16
Muench
MO
Roncarolo
MG
Menon
S
Xu
Y
Kastelein
R
Zurawski
S
Hannum
CH
Culpepper
J
Lee
F
Namikawa
R
FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver.
Blood
85
1995
963
17
Hirayama
F
Lyman
SD
Clark
SC
Ogawa
M
The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors.
Blood
85
1995
1762
18
Jacobsen
SEW
Okkenhaug
C
Myklebust
J
Veiby
OP
The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: Synergistic interactions with interleukin (IL)-11, IL-12, and other hematopoietic growth factors.
J Exp Med
181
1995
1357
19
Broxmeyer
HE
Lu
L
Cooper
S
Ruggieri
L
Li
Z-H
Lyman
SD
Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells.
Exp Hematol
23
1995
1121
20
Hudak
S
Hunte
B
Culpepper
J
Menon
S
Hannum
C
Thompson-Snipes
LA
Rennick
D
FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units.
Blood
85
1995
2747
21
Gabbianelli
M
Pelosi
E
Montesoro
E
Valtieri
M
Luchetti
P
Samoggia
P
Vitelli
L
Barberi
T
Testa
U
Lyman
SD
Peschle
C
Multi-level effects of flt3 ligand on human hematopoiesis: Expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors.
Blood
86
1995
1661
22
Tsuji
K
Zsebo
KM
Ogawa
M
Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit.
Blood
78
1991
1223
23
Hirayama
F
Shih
JP
Awgulewitsch
A
Warr
GW
Clark
SC
Ogawa
M
Clonal proliferation of murine lymphohemopoietic progenitors in culture.
Proc Natl Acad Sci USA
89
1992
5907
24
Katayama
N
Clark
SC
Ogawa
M
Growth factor requirement for survival in cell-cycle dormancy of primitive murine lymphohematopoietic progenitors.
Blood
81
1992
610
25
Tsuji
K
Lyman
SD
Sudo
T
Clark
SC
Ogawa
M
Enhancement of murine hemopoiesis by synergistic interactions between Steel factor (ligand for c-kit ), interleukin-11 and other early-acting factors in culture.
Blood
79
1992
2855
26
Hirayama
F
Katayama
N
Neben
S
Donaldson
D
Nickbarg
EB
Clark
SC
Ogawa
M
Synergistic interaction between interleukin-12 and Steel factor in support of proliferation of murine lymphohemopoietic progenitors in culture.
Blood
83
1994
92
27
Heimfeld
S
Hudak
S
Weissman
I
Rennick
D
The in vitro response of phenotypically defined mouse stem cells and myeloerythroid progenitors to single or multiple growth factors.
Proc Natl Acad Sci USA
88
1991
9902
28
Bodine
DM
Crosier
PS
Clark
SC
Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture.
Blood
78
1991
914
29
Miura
N
Okada
S
Zsebo
KM
Miura
Y
Suda
T
Rat stem cell factor and IL-6 preferentially support the proliferation of c-kit–positive murine hemopoietic cells rather than their differentiation.
Exp Hematol
21
1993
143
30
Muench
MO
Firpo
MT
Moore
MA
Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential.
Blood
81
1993
3463
31
Rebel
VI
Dragowska
W
Eaves
CJ
Humphries
RK
Lansdorp
PM
Amplification of Sca-1+Lin−WGA+ cells in serum-free cultures containing Steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential.
Blood
83
1994
128
32
Knobel
KM
McNally
MA
Berson
AE
Rood
D
Chen
K
Kilinski
L
Tran
K
Okarma
TB
Lebkowski
JS
Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: Differential activity of cultured bone marrow and enriched stem cell populations.
Exp Hematol
22
1994
1227
33
Peters
SO
Kittler
ELW
Ramshaw
HS
Quesenberry
PJ
Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts.
Exp Hematol
23
1995
461
34
Brandt
J
Briddell
RA
Srour
EF
Leemhuis
TB
Hoffman
R
Role of c-kit ligand in the expansion of human hematopoietic progenitor cells.
Blood
79
1992
634
35
Haylock
DN
To
LB
Dowse
TL
Juttner
CA
Simmons
PJ
Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage.
Blood
80
1992
1405
36
Sato
N
Sawada
K
Koizumi
K
Tarumi
T
Ieko
M
Yasukouchi
T
Yamaguchi
M
Takahashi
TA
Sekiguchi
S
Koike
T
In vitro expansion of human peripheral blood CD34+ cells.
Blood
82
1993
3600
37
Lansdorp
PM
Dragowska
W
Mayani
H
Ontogeny-related changes in proliferative potential of human hematopoietic cells.
J Exp Med
178
1993
787
38
Brugger
W
Möcklin
W
Heimfeld
S
Berenson
RJ
Mertelsmann
R
Kanz
L
Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1β (IL-1β), IL-6, IL-3, interferon-γ, and erythropoietin.
Blood
81
1993
2579
39
Flasshove
M
Banerjee
D
Mineishi
S
Li
MX
Bertino
JR
Moore
MAS
Ex vivo expansion and selection of human CD34 peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer.
Blood
85
1995
566
40
Rice
A
Boiron
JM
Barbot
C
Dupouy
M
Dubosc-Marchenay
N
Dumain
P
Lacombe
F
Reiffers
J
Cytokine-mediated expansion of 5-FU–resistant peripheral blood cells.
Exp Hematol
23
1995
303
41
Ogawa
M
Differentiation and proliferation of hematopoietic stem cells.
Blood
81
1993
2844
42
Shih
JP
Zeng
HQ
Ogawa
M
Enrichment of murine marrow cells for progenitors of multilineage hematopoietic colonies.
Leukemia
6
1992
193
43
Ortega
G
Korty
PE
Shevach
EM
Malek
TR
Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity.
J Immunol
137
1986
3240
44
Nishikawa
S
Kusakabe
M
Yoshinaga
K
Ogawa
M
Hayashi
S
Kunisada
T
Era
T
Sakakura
T
Nishikawa
S-I
In utero manipulation of coat color formation by a monoclonal anti–c-kit antibody: Two distinct waves of c-kit–dependency during melanocyte development.
EMBO J
10
1991
2111
45
Nakahata
T
Ogawa
M
Clonal origin of murine hemopoietic colonies with apparent restriction to granulocyte-macrophage-megakaryocyte (GMM) differentiation.
J Cell Physiol
111
1982
239
46
Nakahata
T
Ogawa
M
Identification in culture of a class of hemopoietic colony-forming units with extensive capability to self-renew and generate multipotential hemopoietic colonies.
Proc Natl Acad Sci USA
79
1982
3843
47
Harrison
DE
Astle
CM
Delaittre
JA
Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging.
J Exp Med
147
1978
1526
48
Hirayama
F
Clark
SC
Ogawa
M
Negative regulation of early B lymphopoiesis by interleukin 3 and interleukin 1α.
Proc Natl Acad Sci USA
91
1994
469
49
Hirayama
F
Ogawa
M
Negative regulation of early T lymphopoiesis by interleukin-3 and interleukin-1α.
Blood
86
1995
4527
50
Yonemura
Y
Ku
H
Hirayama
F
Souza
LM
Ogawa
M
Interleukin-3 or interleukin-1 abrogates the reconstituting ability of hematopoietic stem cells.
Proc Natl Acad Sci USA
93
1996
4040
51
Peters
SO
Kittler
ELW
Ramshaw
HS
Quesenberry
PJ
Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts.
Blood
87
1996
30
Sign in via your Institution