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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 452-461
Thrombopoietin Promotes the Survival of Murine Hematopoietic
Long-Term Reconstituting Cells: Comparison With the Effects of
FLT3/FLK-2 Ligand and Interleukin-6
By
Takuya Matsunaga,
Takashi Kato,
Hiroshi Miyazaki, and
Makio Ogawa
From the Department of Veterans Affairs Medical Center and the
Department of Medicine, Medical University of South Carolina,
Charleston, SC; and the Pharmaceutical Research Laboratory, Kirin
Brewery Co, Ltd, Takasaki, Japan.
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ABSTRACT |
The effects of thrombopoietin (TPO; c-mpl ligand),
FLT3/FLK-2 ligand (FL), and interleukin-6 (IL-6) on the survival of
murine hematopoietic long-term reconstituting cells (LTRC) were studied by using lineage-negative, Sca-1-positive, c-kit-positive
(Lin Sca-1+c-kit+) marrow
cells from 5-fluorouracil-treated mice. We tested the ability of these
cytokines to maintain the viability of LTRC by transplanting the
cultured cells to lethally irradiated Ly-5 congenic mice together with
compromised marrow cells. As a single agent, only TPO could maintain
the LTRC. Neither IL-6 nor FL was effective by itself, but they acted
synergistically to maintain the LTRC. We examined whether the
maintenance of LTRC by these cytokines was due to the survival of stem
cells or was the result of active cell divisions and self-renewal. To
monitor cell division, we used membrane dye PKH26. Enriched cells were
stained with PKH26 on day 0 and incubated in suspension culture with
TPO or with IL-6 and FL for 7 days. On day 7, PKH26low and
PKH26high cells were prepared by sorting and their in vivo
reconstituting abilities were tested by transplantation into lethally
irradiated Ly-5 congenic mice together with compromised marrow cells.
PKH26high populations cultured with both TPO alone and the
combination of IL-6 and FL showed greater reconstitution activity than
that of PKH26low populations. These data indicate that TPO
alone and the combination of IL-6 and FL can support the survival of
stem cells without stimulating their active cell proliferation.
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INTRODUCTION |
PROLIFERATION OF primitive hematopoietic
progenitors is regulated by a number of cytokines. Based primarily on
serial observations of the development of murine and human blast cell
colonies, a model of cytokine interactions regulating the proliferation
kinetics of primitive progenitors has been proposed.1 In
this model, the cytokines are classified into three functional
groups.1 Interleukin-3 (IL-3),2
IL-4,3 and granulocyte-macrophage colony-stimulating factor
(GM-CSF)4,5 form the first group of cytokines that
individually can support proliferation of multipotential progenitors
after they exit from the cell-cycle dormant state (G0
period). A group of IL-6,6 IL-11,7,8
IL-12,9 granulocyte colony-stimulating factor
(G-CSF),10 and leukemia-inhibitory factor
(LIF)11,12 forms the second group. A group consisting of
steel factor (SF; c-kit ligand)13,14 and FLT3/FLK-2 ligand (FL)15 forms the third group. Both SF and FL are ligands
for receptors with tyrosine kinase activity and they interact with the
cytokines in the first and second groups.13-15
Cytokines are also capable of supporting the survival of primitive
progenitors. In the murine system, IL-1 ,16
IL-3,17-20 IL-4,18 G-CSF,18
SF,19-21 and FL22 were shown to support the
survival of progenitors including colony-forming units-spleen (CFU-S).
IL-3,17-19 IL-4,18 and SF19,23-25
were reported also to support the survival in culture of long-term reconstituting cells (LTRC). In the human system,
IL-3,12,26 GM-CSF,12 SF,26 and
FL27 support the survival of colony-forming cells. Leary et
al12 in our laboratory observed that cell cycle dormant
progenitors in the CD34+HLA-DR human
marrow fraction require either IL-3 or GM-CSF for survival in culture.
Brandt et al26 reported that IL-3 or SF supports the
survival of human colony-forming cells by testing
CD34+HLA-DRc-kit+ bone
marrow cells. Using lineage-negative (Lin)
CD34++CD38 light-density fetal liver
cells, Muench et al27 demonstrated that FL supports the
survival of human colony-forming cells.
Despite their similarity, there appears to be some differences in the
effects of SF and FL on primitive hematopoietic
progenitors.15,28,29 Earlier, we noted that blast-like
cells persist longer in FL-containing cultures than in SF-containing
cultures.15 More recently, we reported that a combination
of FL and IL-11 supports maintenance of the in vivo reconstituting
ability of cultured cells longer than the combination of SF and
IL-11.28 Petzer et al29 reported that FL alone
stimulated a net increase in human long-term culture-initiating cells
(LTC-IC), but SF did not.
Recently, thrombopoietin (TPO) was found to possess positive effects on
primitive hematopoietic progenitors.30-32 TPO was
originally identified as a lineage-specific regulator of
megakaryocytopoiesis and thrombopoiesis.33-44 TPO acts
through a specific receptor termed c-mpl, which has been
detected in not only megakaryocytes and plateletes, but also in cell
populations containing stem cells and progenitors.45-49 As
mentioned above, Ku et al30 and Kobayashi et
al31 in our laboratory and Sitnicka et al32
observed that TPO can synergize with SF and/or IL-3 in support
of the formation of murine and human multilineage colonies.
TPO30 can trigger the cell division of dormant
(G0) multipotential progenitors in a manner similar to that
previously reported for the second group of cytokines such as
IL-6,6 IL-11,7,8 IL-12,9
G-CSF,10 and LIF.11 Injection of TPO
accelerated erythroid recovery of myelosuppressed mice50,51
and, in combination with G-CSF, neutrophil recovery.52
Megakaryocyte, erythroid, and myeloid progenitors were reduced in the
TPO- or c-mpl-deficient mice.53,54 Administration
of TPO to the TPO-deficient mice significantly increased the number of
myeloid, erythroid, and mixed progenitors.54 TPO increased
production of human LTC-IC in suspension culture.29
Together, these observations indicate that TPO may be a regulator of
the proliferation of multipotential progenitors.
In this report, we present studies of the ability of TPO to maintain
the survival of murine LTRC. Comparison was made with FL and IL-6. As a
single agent, TPO appears to be more potent than FL or IL-6 in the
maintenance of stem cell activities. In contrast, synergism between
IL-6 and FL supported the survival of LTRC. These results are in
agreement with the observations in the gene knock-out mice and indicate
that TPO has a physiological role in the maintenance of stem cell
activity. TPO may be an important factor for in vitro manipulation of
hematopoietic stem cells.
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MATERIALS AND METHODS |
Cytokines.
Purified recombinant human TPO was prepared by the Cytokine Production
Group of Kirin Brewery (Takasaki, Japan).38,39 Recombinant human FL was produced in yeast and purified as described
previously.55 Purified recombinant human IL-6 was a gift
from M. Naruto (Toray Industries, Kamakura, Japan). Purified
recombinant murine SF was obtained from Immunex (Seattle, WA). Purified
recombinant murine IL-3 was purchased from R&D Systems (Minneapolis,
MN). Purified recombinant human erythropoietin (EPO) was a gift from
F.-K. Lin (Amgen, Thousand Oaks, CA). Unless otherwise specified, the
concentration of cytokines used were as follows: 100 ng/mL TPO, 100 ng/mL FL, 100 ng/mL IL-6, 100 ng/mL SF, 10 ng/mL IL-3, and 2 U/mL EPO.
Monoclonal antibodies (MoAbs).
Hybridoma D7 (anti-Ly-6A/E [anti-Sca-1] rat IgG2a) was a gift from P. Kincade (Oklahoma Medical Research Foundation, Oklahoma City, OK). MoAb
ACK4 (anti-c-kit; rat IgG2a) was provided by S.I. Nishikawa (Kyoto
University, Kyoto, Japan). Hybridoma RB6-8C5 (antimouse granulocytes;
rat IgG2b) was provided by R.L. Coffman (DNAX, Palo Alto, CA). MoAb
TER119 (anti-erythrocytes; rat IgG2b) was a gift from T. Kina (Kyoto
University). Hybridomas 14.8 (anti-B220; rat IgG2b), M1/70.15.11.5
(anti-macrophages; rat IgG2b), GK1.5 (anti-CD4; rat IgG2b), and 53-6.72 (anti-CD8; rat IgG2a) were purchased from American Type Culture
Collection (Rockville, MD). 53-2.1 (biotin-conjugated anti-Thy-1.2;
rat IgG2a), RA3-6B2 (biotin-conjugated anti-CD45R/B220; rat IgG2a),
RB6-8C5 (biotin-conjugated anti-Gr-1; rat IgG2b), and M1/70
(biotin-conjugated anti-Mac-1; rat IgG2b) were purchased from
Pharmingen (San Diego, CA). A20-1.7 (fluorescein isothiocyanate
[FITC]-conjugated anti-Ly-5.1; mouse IgG1) was provided by H. Fleming (Emory University, Atlanta, GA).
Cell preparations.
Cells from 10- to 16-week-old female C57Bl/6 mice (Charles River
Laboratories, Raleigh, NC) were used in suspension and clonal cultures,
and cells from 10- to 16-week-old C57Bl/6 mice (Jackson Laboratories,
Bar Harbor, ME) that are congenic for Ly-5 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. Cells prepared from pooled femurs and tibiae were washed twice
and then subjected to density gradient separation by using Nycodenz
(Accurate Chemical and Scientific Corp, Westbury, NY). Cells with
densities ranging from 1.063 to 1.077 g/mL were
collected.56 Cells reacting to a cocktail of lineage-specific rat MoAbs (RB6-8C5, 14.8, M1/70.15.11.5, GK1.5, TER119, and 53-6.72) were removed twice by using immunomagnetic beads
(Dynabeads M-450 coupled to sheep antirat IgG; DYNAL, Great Neck, NY).
The resulting Lin cells were treated with normal rat
IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at 20 µg/106 cells to prevent nonspecific binding of MoAbs to
Fc receptors and then stained with FITC-conjugated rat MoAb D7
(anti-Sca-157) and biotin-conjugated rat MoAb ACK4
(anti-c-kit58). The cells were washed twice before
staining with streptavidin-conjugated R-phycoerythrin (PE) (Jackson
ImmunoResearch Laboratories). In the experiments using PKH26,
streptavidin-conjugated allophycocyanin (APC; Caltag Laboratories, San
Francisco, CA) was used instead of PE, because the emission wave length
of PKH26 is similar to that of PE. Both FITC-conjugated rat IgG2a and
biotin-labeled rat IgG2a (Caltag Laboratories) were used as isotype
controls. Sca-1+c-kit+ cells were collected by
sorting on FACS Vantage (Becton Dickinson Immunocytometry Systems, San
Jose, CA).
Clonal cell culture.
Methylcellulose cell culture was performed in 35-mm suspension culture
dishes (Falcon, Lincoln Park, NJ). One milliliter of culture contained
100 LinSca-1+c-kit+ cells,
-medium (ICN, Irvine, CA), 1.2% 1,500-cp methylcellulose (Shinetsu
Chemical, Tokyo, Japan), 30% (vol/vol) fetal calf serum (Intergen,
Purchase, NY), 1% deionized fraction V bovine serum albumin (Sigma
Chemical, St Louis, MO), 1 × 104 mol/L
2-mercaptoethanol (Sigma), and cytokine(s). Dishes were incubated at
37°C in a humidified atmosphere flashed with 5% CO2. Colony types were determined on day 14 by in situ observation on an
inverted microscope according to the criteria described previously.59 Megakaryocyte colonies were scored when the
colony contained 4 or more megakaryocytes. Abbreviations for colony
types are as follows: GM, granulocyte/macrophage colonies; GEM,
granulocyte/erythrocyte/macrophage colonies; GMM,
granulocyte/macrophage/megakaryocyte colonies; GEMM,
granulocyte/erythrocyte/macrophage/megakaryocyte
colonies59; and Meg, megakaryocyte colonies.
Survival of progenitors and reconstituting cells in suspension
culture.
One thousand LinSca-1+c-kit+
cells were incubated in each well of 24-well plate (Falcon) in
suspension culture. The culture medium consisted of -medium, 20%
(vol/vol) fetal calf serum, 1% deionized bovine serum albumin, 1 × 104 mol/L 2-mercaptoethanol, and
cytokine(s). On day 7 of incubation, aliquots of cells were analyzed
for progenitors in clonal cell culture and for the in vivo
reconstituting cells. Clonal cell culture was performed as described
above. Colonies were scored on day 8 of incubation by in situ
observation of the plates on an inverted microscope.59
In vivo reconstitution experiments.
Ten- to 12-week-old female C57Bl/6-Ly-5.2 mice were administered with a
single 850-cGy dose of total body irradiation via a 4 × 106 V linear accelerator. After irradiation, 200 freshly
sorted LinSca-1+c-kit+ cells
of male C57Bl/6-Ly-5.1 mice were injected into the tail vein of the
recipients together with 4 × 105 compromised marrow
cells of female C57Bl/6-Ly-5.2 mice. Compromised marrow cells had been
subjected to two previous rounds of transplantation and regeneration in
female mice.60 Cells cultured in suspension were also
tested for reconstituting capabilities after 7 days of incubation with
a single cytokine or combinations of cytokines. Fractions
equivalent to one fifth of a day-7 culture that had been initiated with
1,000 LinSca-1+c-kit+ cells
were injected into female C57Bl/6-Ly-5.2 mice together with compromised
marrow cells. Peripheral blood was obtained from the retro-orbital
venous plexus using heparin-coated micropipettes (Drummond Scientific
Co, Broomall, PA) 2 and 5 months after transplantation. Red blood cells
were lyzed by 0.15 mol/L NH4Cl. The samples were then used
for flow cytometric analysis of donor-derived cells by staining with
FITC-conjugated anti-Ly-5.1 (A20-1.7). Donor cells in T-cell, B-cell,
granulocyte, and monocyte/macrophage lineages at 5 months
posttransplantation were analyzed by staining with biotin-conjugated
anti-Thy-1.2, biotin-conjugated anti-CD45R/B220, biotin-conjugated
anti-Gr-1, and biotin-conjugated anti-Mac-1. For indirect staining of
cells with biotin-conjugated antibodies, cells were first incubated
with biotin-conjugated antibodies, followed by staining with
streptavidin-conjugated PE.
PKH26 experiments.
LinSca-1+c-kit+ cells of
male C57Bl/6-Ly-5.1 mice were stained with PKH26 (Sigma
ImmunoChemicals, St Louis, MO) according to the manufacturer's
instruction. Briefly, 1 × 105 cells were suspended in
1 mL of diluent (Sigma ImmunoChemicals) and immediately transferred
into a polypropylene tube containing 1 mL of 4 × 107 mol/L PKH26 in diluent. After incubation for 5 minutes at room temperature, the staining reaction was stopped by
adding 2 mL of fetal calf serum. One minute later, the total volume was
brought up to 8 mL with -medium supplemented with 10% fetal calf
serum, and the cells were washed three times by using the
serum-containing medium. After the third wash, cells were incubated
with TPO alone or the combination of IL-6 and FL in a
75-cm2 flask (Corning Coster Co, Cambridge, MA) in 100 mL
for 7 days. After washing and staining with 1 µg/mL of propidium
iodine (PI), the cells were then analyzed for PKH26 fluorescense on
FACS Vantage. Dead (PI-positive) cells were excluded from the sort
gate. The viable cells were separated into PKH26low and
PKH26high populations by sorting and their reconstituting
abilities were tested by transplanting the sorted cells into lethally
irradiated Ly-5 congenic mice together with 4 × 105
compromised marrow cells. Flow cytometric analyses of the donor-derived cells were performed as described above at 5 months after
transplantation. For indirect staining of cells with biotin-conjugated
antibodies, cells were first incubated with biotin-conjugated
antibodies, followed by staining with streptavidin-conjugated APC
instead of PE, because the emission wave length of PE is similar to
that of PKH26.
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RESULTS |
Effects of cytokines on colony formation.
Colony formation from
LinSca-1+c-kit+ cells of
5-FU-treated mice is presented in Table 1.
As a single agent, FL, IL-6, and TPO in either 100 or 1,000 ng/mL
concentrations failed to support significant colony formation. However,
IL-6 in synergy with FL supported formation of a number of colonies.
Colony types could not be determined in situ, because most colonies
supported by the combination of IL-6 and FL contained a number of
immature cells.15 Cultures containing a combination of TPO
and FL yielded only few Meg colonies, indicating that TPO does not
synergize with FL.
LinSca-1+c-kit+ cells
exhibited about 45% colony-forming efficiency when cultured in
cytokine cocktails, SF, IL-3, IL-6, FL, TPO, and EPO.
Effects of cytokines on maintenance of cells and progenitors.
We next studied the effects of cytokines on the maintenance of cells
and colony-forming cells in 7-day suspension culture. The results shown
in Table 2 are representative of three
experiments. No viable cells were present after 7 days of incubation in
medium alone. TPO was the most effective single agent to maintain the survival of progenitors, including CFU-GEMM and CFU-Meg, whereas IL-6
showed minimal activity and FL failed to support the survival of
progenitors. The maintenance of progenitors by TPO reached a plateau at
concentrations of less than 100 ng/mL. There was no synergy between TPO
and FL, because the addition of FL to TPO did not enhance the survival
of total CFU. This observation is in agreement with the study of colony
formation in methylcellulose culture (Table 1). In contrast, the
combination of IL-6 and FL significantly expanded the total population
of CFU.
Effects of cytokines on the maintenance of long-term repopulating
cells.
We then tested the in vivo reconstituting ability of the cultured
cells. Based on the observations presented in Tables 1 and 2, the
experiment was performed with 100 ng/mL TPO, 100 ng/mL FL, and 100 ng/mL IL-6. As also described in Table 2, the suspension cultures were
initiated with 1,000 Lin
Sca-1+c-kit+ C57Bl/6-Ly-5.1 cells in a final
volume of 1 mL. After 7 days of incubation, cells in each well were
harvested and 1/5 aliquots were injected individually into 5 lethally
irradiated Ly-5.2 recipients. As a control group, we transplanted 200 freshly prepared Lin
Sca-1+c-kit+ C57Bl/6-Ly-5.1 cells. The results
of analyses of peripheral blood nucleated cells are presented in
Fig 1. The numbers of cells transplanted per animal are also presented. Again, the cell population expanded only
in the group containing IL-6 and FL. Transplantation of 200 fresh
enriched cells resulted in engraftment in 10 of 10 mice, at the levels
of 46.4% ± 16.2% and 49.1% ± 24.7% donor cells at 2 and 5 months posttransplantation, respectively. As single agents, both FL and
IL-6 failed to support the survival of stem cells. In contrast, the
recipients of the cells incubated with TPO alone showed donor cell
engraftment in 8 of 8 mice at 2 months (19.7% ± 17.1%) and in 7 of 8 mice at 5 months (24.2% ± 29.8%) posttransplantation. Similar to the result of maintenance of colony-forming cells, the
addition of FL to TPO had no benefit. Recipients of the cells cultured
with IL-6 and FL showed engraftment levels of 34.3% ± 21.7% and
32.8% ± 25.3% in the peripheral blood at 2 and 5 months posttransplantation, respectively. These data indicated that TPO alone
has an ability to maintain the stem cells in culture and that IL-6 acts
synergistically with FL to promote the maintenance of LTRC.
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| Fig 1.
Long-term repopulating ability of freshly sorted bone
marrow cells and cultured cells. ( ) Two months posttransplantation. ( ) Five months posttransplantation. Results from individual animals are linked by lines. The numbers in parentheses indicate the actual number of cells transplanted per recipient.
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At 5 months posttransplantation, the proportions of donor blood
nucleated cells in each of the T-cell, B-cell, and myeloid (granulocyte
and monocyte/macrophage) compartments were determined. As shown in
Table 3, T and B lymphocytes and myeloid
cells were detected in the peripheral blood of all engrafted recipients
and the myeloid/lymphoid ratios did not differ between groups. An example of the analysis of a mouse transplanted with the cells that
were cultured with TPO alone is shown in
Fig 2.
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| Fig 2.
Hematopoietic reconstitution by cells cultured with TPO
alone. Nucleated cells of peripheral blood were analyzed by flow
cytometry 5 months after transplantation. Thy-1.2+ cells,
B220+ cells, and Gr-1+Mac-1+
cells of donor (Ly-5.1) origin are seen. The analyses of additional samples are presented in Table 4.
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Mechanisms of LTRC maintenance.
Earlier, we reported that primitive murine progenitors in
G0 are stimulated to proliferate in culture by a
combination of IL-6 and FL.15 Therefore, the maintenance of
LTRC in culture by the combination in of IL-6 and FL may be the result
of active cell division and self-renewal of stem cells rather than the
survival of LTRC in dormancy. To test this hypothesis, we performed a
reconstitution experiment by using PKH26. This dye has been used
previously to study division history of hematopoietic cells in
culture,61-64 because its intensity is reduced by roughly
one half with each cell division. Dot plots of the cells stained with
PKH26 dye before (A) and after suspension culture (B and C) are
presented in Fig 3. We made an arbitrary
separation of PKH26high from PKH26low
populations as shown in Fig 3. Before culture, all cells were PKH26high. After 7 days of culture, the majority of the
cells became PKH26low. The four cell populations defined in
Fig 3 (BI, BII, CI, and CII) were prepared by sorting, and their in
vivo reconstituting abilities were tested by transplantation into
lethally irradiated Ly-5.2 mice together with 4 × 105
compromised marrow cells of female C57Bl/6-Ly-5.2 mice. One fourth aliquots of the sorted PKH26low cells (2,720 cells) or 1/4
aliquots of the PKH26high cells (250 cells) cultured with
TPO were injected individually into each of 4 recipients. One fifth
aliquots of the sorted PKH26low cells (36,415 cells) or 1/5
aliquots of PKH26high cells (1,055 cells) cultured with
IL-6 and FL were injected individually into each of 5 recipients. Of
the cells cultured under either conditions, PKH26high
populations showed greater reconstitution activity than that of
PKH26low populations (Fig 4).
In the IL-6 plus FL group, PKH26low cells exhibited some
reconstituting capability. There may have been a limited amount of
self-renewal. The proportions of donor blood nucleated cells in each of
T-cell, B-cell, and myeloid (granulocyte and monocyte/macrophage)
compartments were determined. T and B lymphocytes and myeloid cells
were detected in the peripheral blood of all engrafted recipients
(Table 4). These data indicated that TPO
alone and combination of IL-6 and FL can support the survival of stem
cells without stimulating their active cell proliferation.
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| Fig 3.
Flow cytometric analysis and sorting windows of cells
tracked with PKH26. (A) PKH26 fluorescence of freshly sorted
LinSca-1+c-kit+ cells
stained with PKH26 on day 0. (B and C) PKH26 fluorescence of the cell
populations after 7 days of suspension culture with TPO (B) or IL-6 and
FL (C). Viable (PI-negative) cells were arbitarily divided into
PKH26low populations (I) and PKH26high
populations (II): BI, 89%; BII, 8%; CI, 94%; and CII, 2.7%.
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| Fig 4.
Hematopoietic reconstitution by PKH26 stained cells. The
four cell populations in Fig 3 (BI, BII, CI, and CII) were prepared by
sorting and their in vivo reconstituting abilities were analyzed at 5 months posttransplantation.
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DISCUSSION |
In this study, we have examined the effects of TPO, FL, and IL-6 on the
survival of murine LTRC. We used
LinSca-1+c-kit+ marrow cells
that had been prepared from 5-FU-treated mice. This cell population
represents 0.04% of the unfractionated marrow cells and has been shown
to be capable of hematopoietic reconstitution in lethally irradiated
mice.28,65 As a single agent, TPO was the best for
supporting the maintenance of colony-forming cells and survival of LTRC
in 7-day suspension culture. Earlier, Kaushansky et al32,66
had reported that TPO alone can support the survival without
proliferation of a fraction of mouse Hoescht 33342low
Rhodamine123low cells that are highly enriched for LTRC.
Together, those results are consistent with the recent observations
that megakaryocyte, erythroid, and myeloid progenitors are reduced in
TPO-deficient mice.53,54 TPO appears to be an important
cytokine in the physiological regulation of hematopoietic stem cells.
FL as a single agent failed to maintain the viability of colony-forming
cells or LTRC. Muench et al27 described that FL can support
the survival of human fetal liver colony-forming cells in suspension
culture for 7 days. Human fetal liver cell progenitors may respond to
FL differently from adult murine marrow progenitors. Veiby et
al22 reported that FL alone can support the survival of
murine bone marrow colony-forming cells in suspension culture for 40 hours. This incubation period was much shorter than ours. As summarized
in a recent review, a number of investigators have shown synergy
between FL and early-acting cytokines, such as IL-6, IL-11, and G-CSF,
on primitive hematopoietic progenitors.67 In this report,
we demonstrated that FL and IL-6 synergize to support in vitro survival
of stem cells. These observations are consistent with the report from
Mackarehtschian et al68 that bone marrow cells of
FLT3/FLK-2-deficient mice have impaired competitive long-term
repopulating ability.
We reported previously that TPO and IL-6 belong to the same group of
cytokines that trigger the dormant hematopoietic progenitors into cell
cycle.6,30 Because of their functional similarity, we
compared their effects on the survival of colony-forming cells and
LTRC. In contrast to TPO, IL-6 alone could not support the survival of
LTRC, which is in agreement with the report by Li and
Johnson.24 Although IL-6 and FL showed little
effects as single agents, they acted synergistically to promote the
survival of LTRC. Earlier, McKinstry et al69 reported that
the IL-6 receptor is expressed by the cell population highly enriched
for LTRC. These observations are consistent with the report from Bernad et al70 that absolute numbers of CFU-Sd12, pre-CFU-S and
LTRC are decreased in IL-6-deficient mice.
PKH26high populations cultured with either TPO alone or the
combination of IL-6 and FL were more efficient in their bone marrow reconstitution activity than PKH26low populations. Traycoff
et al64 documented similar results by using cells cultured
with SF, IL-1, IL-3, and IL-6. These results suggest that the
ability of cultured cells to contribute to long-term reconstitution may
be derived primarily from quiescent cells and not from de
novo-generated progeny stem cells.
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FOOTNOTES |
Submitted January 21, 1998;
accepted March 18, 1998.
Supported by National Institutes of Health Grants No. RO1 DK32294 and
RO1 DK48714 and by the Office of Research and Development, Medical
Research Service, Department of Veterans Affairs.
Address reprint requests to Makio Ogawa, MD, PhD, Ralph H. Johnson
Medical Center, 109 Bee St, Charleston, SC 29401-5799.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors thank Dr Haiqun Zeng for assistance in cell sorting, Dr
Pamela N. Pharr and Anne G. Livingston in preparation of this
manuscript, and the staff of the Radiation Oncology Department of the
Medical University of South Carolina for irradiation of mice.
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Abstract
Introduction
Abstract