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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4093-4102
Distinct Requirements for Optimal Growth and In Vitro Expansion of
Human CD34+CD38 Bone Marrow Long-Term
Culture-Initiating Cells (LTC-IC), Extended LTC-IC, and Murine In Vivo
Long-Term Reconstituting Stem Cells
By
Veslemøy Ramsfjell,
David Bryder,
Helga Björgvinsdóttir,
Sten Kornfält,
Lars Nilsson,
Ole J. Borge, and
Sten E.W. Jacobsen
From the Stem Cell Laboratory, Department of Molecular Medicine and
Gene Therapy, Department of Internal Medicine, Institute for Laboratory
Medicine, University Hospital of Lund, Lund, Sweden.
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ABSTRACT |
Recently, primitive human bone marrow (BM) progenitors supporting
hematopoiesis in extended (>60 days) long-term BM cultures were
identified. Such extended long-term culture-initiating cells (ELTC-IC)
are of the CD34+CD38 phenotype, are
quiescent, and are difficult to recruit into proliferation, implicating
ELTC-IC as the most primitive human progenitor cells detectable in
vitro. However, it remains to be established whether ELTC-IC can
proliferate and potentially expand in response to early acting
cytokines. Here, CD34+CD38 BM ELTC-IC
(12-week) were efficiently recruited into proliferation and expanded in
vitro in response to early acting cytokines, but conditions for
expansion of ELTC-IC activity were distinct from those of traditional
(5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit
ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and
maintenance or expansion of murine long-term reconstituting activity
and human LTC-IC, they dramatically depleted ELTC-IC activity. In
contrast, KL, flt3 ligand (FL), and megakaryocyte growth and
development factor (MGDF) (and KL + FL + IL-3) expanded murine
long-term reconstituting activity as well as human LTC-IC and ELTC-IC.
Expansion of LTC-IC was most optimal after 7 days of culture, whereas
optimal expansion of ELTC-IC activity required 12 days, most likely
reflecting the delayed recruitment of quiescent
CD34+CD38 progenitors. The need for high
concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free
conditions was more critical for expansion of ELTC-IC than of LTC-IC.
The distinct requirements for expansion of ELTC-IC activity when
compared with traditional LTC-IC suggest that the ELTC-IC could prove
more reliable as a predictor for true human stem cell activity after in
vitro stem cell manipulation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE HALLMARK OF TRUE hematopoietic stem
cells is their ability to long-term reconstitute large numbers of all
blood cell lineages.1 Considerable efforts have been
devoted towards promoting in vitro proliferation and potential
expansion of human stem cells for applications in stem cell
transplantation and gene therapy.2-4 A requisite for
successful stem cell expansion and gene transfer would be to
efficiently promote proliferation of true stem cells without a
concomitant loss of long-term reconstituting ability. However, so far
results from clinical gene marking protocols have been disappointing,
in particular in adults, suggesting little or no ability to mimic the
self-renewal process in vitro.2,5,6
A major obstacle to development of successful clinical stem cell
expansion and gene marking has been lack of easy, accessible, and
optimal human stem cell assays. Although ultimately clinical gene
marking protocols will be needed to unequivocally demonstrate successful ex vivo expansion and retroviral marking, there is an
obvious need for further development and evaluation of the ability of
surrogate human stem cell assays to predict long-term reconstituting
potential.7-10 Over the last 2 decades, in vitro stromal
cell-based surrogate human stem cell assays have been developed,7,11-14 taking advantage of the ability of an
infrequent and primitive hematopoietic cell population to produce
myeloid progenitor cells for prolonged time (long-term
culture-initiating cells [LTC-IC]).15,16 This assay was
originally developed for stem cells in mice,17 in which
LTC-IC and long-term in vivo reconstituting stem cells (LTRC) were
demonstrated to be present at similar frequencies and to have similar
phenotypes.18-21 Although closely overlapping, more recent
studies have suggested that the requirements for maintenance and
expansion of murine LTC-IC and LTRC might differ.22,23 More
importantly, whereas high efficiency retroviral-mediated transduction
of human 5- to 8-week LTC-IC has been obtained through short-term
stimulation of human adult bone marrow (BM) cells with c-kit ligand
(KL), interleukin-3 (IL-3), and IL-6,24-27 this has
primarily translated into retroviral marking of short-term repopulating
cells in transplanted patients.2,5,6,28,29 Thus, the
traditional LTC-IC assay might predominantly reflect the presence of
primitive hematopoietic cells with short-term rather than long-term in
vivo reconstituting activity, thus limiting its potential
utility in predicting gene transfer and expansion of true stem cells.
Interestingly, Hao et al27,30 recently identified a small
population of primitive human BM cells capable of producing myeloid
progenitors through more extended long-term cultures (beyond
60 days). The finding that such extended LTC-IC (ELTC-IC) appear to be
quiescent, more exclusively of CD34+CD38
phenotype, and difficult to recruit into proliferation27,30 suggested that ELTC-IC might be a better predictor of long-term repopulating stem cells than standard LTC-IC. Most importantly, conditions that promoted efficient retroviral marking of standard LTC-IC failed to mark ELTC-IC,27,31 as they had
previously failed to mark long- term repopulating stem cells in
clinical gene marking protocols2,5,6,28,29 and NOD/SCID
repopulating cells (SRC).32
The studies of Shah et al33 suggested that the cloning
efficiency of BM CD34+CD38 cells
supplemented with KL + IL-3 + IL-6 or KL + IL-3 + IL-6 + flt3 ligand
(FL) was rather low (0.5% and 11.7%, respectively). It is noteworthy
that most of these clones, in particular those thought to reflect
ELTC-IC, could only be detected after as much as 35 days of incubation
or more.33 Collectively, these findings raised a question
as to whether ELTC-IC, although likely to be attractive targets for ex
vivo expansion and retroviral gene transduction, might prove difficult
to recruit into proliferation. However, the cloning efficiency was only
investigated in long-term stroma-supported cultures, and only
CD34+CD38 cells producing a minimum of
100 cells were defined as clones.33 This might be of
importance, because others have demonstrated efficient recruitment and
expansion of BM CD34+CD38 LTC-IC in the
absence of stroma and that such expanded LTC-IC are predominantly
produced after a limited number of cell divisions.34,35 Thus, the present studies were designed to investigate to what degree
ELTC-IC derived from CD34+CD38 BM cells
could be induced to proliferate, in the absence of stromal cells,
without detrimental effects on their ability to long-term reconstitute
in vitro.
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MATERIALS AND METHODS |
Hematopoietic growth factors.
Recombinant human megakaryocyte growth and development factor (rhMGDF),
recombinant human granulocyte colony-stimulating factor (rhG-CSF),
recombinant human KL (rhKL), recombinant rat KL (rrKL; stem cell
factor), rhIL-3, and recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) were generously provided by Amgen
Corp (Thousand Oaks, CA). Recombinant human erythropoietin (rhEpo) was
kindly supplied by Boehringer Mannheim Corp (Mannheim, Germany) and
rmIL-3 was from PeproTech Inc (Rocky Hill, NJ). rhIL-6 was a generous
gift from Genetics Institute (Cambridge, MA). rhFL was kindly supplied
by Immunex (Seattle, WA). Unless otherwise indicated, all growth
factors were used at the following predetermined optimal
concentrations: rhMGDF, rhKL, rhFL, and rhIL-6: all at 250 ng/mL;
rhIL-3 and rrKL: 50 ng/mL; rhEpo: 5 U/mL; rhG-CSF: 25 ng/mL; and rmIL-3
and rmGM-CSF: 20 ng/mL.
Enrichment and purification of
CD34+CD38 and
CD34+CD38+ human BM cells.
After informed consent was given by healthy adults, and with the
approval of the Ethics Committee at the Medical Faculty at the
University Hospital of Lund, BM cells were obtained from the posterior
iliac crest and collected in syringes containing preservative-free heparin (Pharmacia, Stockholm, Sweden). Mononuclear cells (MNC) were
isolated as previously described.36 Positive selection of
CD34+ cells was performed using a magnetically activated
cell sorting (MACS) CD34 progenitor cell isolation kit (Miltenyi
Biotec, Bergisch Gladbach, Germany) according to the manufacturer's
instructions. The purity of CD34+ cells was between 56%
and 91% (average 77%), as determined by flow cytometric analysis.
CD34+CD38 and
CD34+CD38+ cells were obtained by incubating
enriched CD34+ cells with a phycoerythrin (PE)-conjugated
mouse antihuman CD38 monoclonal antibody (MoAb; 2.5 µg/mL) and a
fluorescein isothiocyanate (FITC)-conjugated mouse antihuman CD34 MoAb
(5 µg/mL; or isotype-matched irrelevant control antibodies; all from
Becton Dickinson [BD], San Jose, CA) for 15 minutes at 6°C to
12°C. Subsequently, the 75% CD34+ cells expressing the
highest levels of CD38 (CD34+CD38+) and those
lacking detectable CD38 expression
(CD34+CD38) were sorted on a FACSVantage
(BD). As previously described,30 a conservative approach
was taken to only sort the 3% lowest
CD34+CD38 cells, in an effort to obtain
a highly enriched population of primitive progenitors.
Enrichment and purification of
LinSca1+c-kit+
murine BM cells.
Lineage-depleted (Lin) BM cells were isolated from
normal 6- to 10-week-old female C57BL/6 mice (Ly5.2) according to
previously described protocols.37-39 Lin
cells were incubated for 30 minutes on ice with a PE-conjugated goat
antirat antibody (Southern Biotechnology, Birmingham, AL). Subsequently, cells were washed and stained with a Sca1-FITC-conjugated antibody and a c-kit-Allophycocyanin (APC)-conjugated antibody (or
isotype-matched control antibodies; all from PharMingen, San Diego,
CA). LinSca1+c-kit+ cells
(purity of 96% to 99%) were sorted on a FACSVantage.
Limiting dilution assays.
CD34+CD38 (180 cells per group) and
LinSca1+c-kit+cells (120 cells per group) were seeded in Terasaki plates (Nunc, Kamstrup, Denmark) at a density of 1 cell per well in 20 µL X-vivo 15 (BioWhittaker, Walkersville, MD) supplemented with 1% detoxified
bovine serum albumin (BSA; StemCell Technologies, Vancouver,
British Columbia, Canada) containing 100 U/mL penicillin
(BioWhittaker), 100 U/mL streptomycin (BioWhittaker), 2 mmol/L L-glutamine (BioWhittaker), 104 mol/L
2-mercaptoethanol (Sigma, St Louis, MO; serum-free [SF] medium), and
various cytokines. In some experiments, Iscove's Modified Dulbecco's
Medium (IMDM) supplemented with 20% fetal calf serum (FCS; both from
BioWhittaker) was used as well. Wells were scored for cell growth after
10 to 12 days of incubation at 37°C in a humidified atmosphere with
5% CO2 in air. Because the statistical probability (based
on Poisson probability distribution) of a well not receiving any cell
is 37% by this method, the maximum expected clones were 76 for
LinSca1+c-kit+ cells and 113 for CD34+CD38 cells. In some
experiments, cells were deposited by a single-cell depositor coupled to
a FACSVantage and subsequently carefully visualized by microscopy to
only include wells containing a single cell. The cytokine response
pattern was similar with these 2 methods.
High-resolution cell division tracking of candidate murine and human
stem cells.
Staining and flow procedures for high-resolution cell division tracking
of CD34+CD38 and
LinSca1+c-kit+ cells using
5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE;
Molecular Probes, Eugene, OR) were performed based on previously
described procedures.40 Briefly, CFSE was added to cells at
5 × 106 cells/mL in Dulbecco's
phosphate-buffered saline (DPBS; BioWhittaker) to give a final
concentration of 1 µmol/L. Murine Lin or human
CD34+ cells were incubated at 37°C for 10 minutes, and
the staining reaction was stopped by adding a 10-fold excess of DPBS
with 10% to 20% FCS. Subsequently, cells were washed 2 to 3 times in
DPBS with 1% FCS. To allow any unbound dye to diffuse out of the
cells, CFSE-labeled cells were incubated overnight (12 to 15 hours) at 37°C in SF-medium supplemented with either KL + FL + MGDF (250 ng/mL each; human cells) or KL (50 ng/mL; murine cells). These cytokines induced proliferation of only a minor fraction of cells within the 12- to 15-hour incubation period (Bryder and Jacobsen, unpublished observations, May 1998). Murine cells were
subsequently stained with rat antimouse Sca1-PE and rat antimouse
c-kit-APC or appropriate isotype control antibodies (all PharMingen).
Cells were sorted on a FACSVantage based on dual expression of Sca1 and
c-kit and a 40 to 50 channel wide sorting gate set around the mean
fluorescence channel for CFSE. After several days of culture, cells
were analyzed with identical instrument settings and sorted based on
number of cell divisions.
LinSca1+c-kit+ CFSE-stained
cells were run on the FACSVantage before culture as a control for
undivided cells. However, because some CFSE diffusion appeared to occur
also after the 12- to 15-hour preincubation, sort gates were also set
based on preliminary studies in which cells were analyzed every 12 hours throughout the 7 days of culture. This allowed a careful and
accurate tracking of cell divisions and demonstrated, in agreement with
others,40 that a maximum number of 7 cell divisions could
reproducibly be detected with high resolution (Bryder and Jacobsen,
unpublished observations, May 1998).
Human cells preincubated in CFSE for 12 to 15 hours were stained with
mouse antihuman CD34-PE and mouse antihuman CD38-APC, or appropriate
isotype control antibodies (all BD), and sort regions were then set to
allow sorting of CD34+CD38 cells with a
defined gate of 40 to 50 channels based on CFSE-fluorescence.
Long-term and extended long-term culture-initiating cell (LTC-IC and
ELTC-IC) assay.
Long-term cultures were established and maintained as previously
described.41 Briefly, stroma layers were initiated with human BM MNC by seeding 20 to 30 × 106 cells in a
80-cm2 culture flask in 15 mL myeloid long-term culture
medium (Myelocult; StemCell) supplemented with 106
mol/L hydrocortisone 21-hemisuccinate (Sigma). Half of the medium was
changed weekly, and at confluency (usually after 4 to 6 weeks), cells
were irradiated with an absorbed dose of 15 Gy with 6 MV x-rays from a
medical linear accelerator (Philips SL25; Philips Medical Systems,
Crawley, West Sussex, UK). After irradiation, cells were trypzinated
(Trypsin Versene; BioWhittaker) and transferred to 24-well microtiter
plates (Nunc; each flask sufficient for 35 wells). Freshly isolated and
in vitro cultured cells were added in triplicates (1 well for each
replicate of cultured cells was transferred to 1 well of stroma after
counting) to irradiated allogeneic BM stroma layer, always using the
same stroma for fresh and expanded cells. Cocultures were maintained by
half medium changes weekly.
For ELTC-IC experiments, nonadherent and adherent cells were
transferred to new irradiated stroma every 4 weeks throughout the 12 weeks of stromal cocultivation. We chose 12 weeks (84 days) as the
endpoint of our ELTC-IC assay, because it has been demonstrated that
stromal cocultivation for a period of 60 to 100 days detects a distinct
and more primitive population of hematopoietic cells than the standard
5-week LTC-IC.27,30
After 5 weeks (LTC-IC) and 12 weeks (ELTC-IC), nonadherent and adherent
cells were transferred to methylcellulose cultures containing
predetermined optimal concentrations of MGDF, G-CSF, KL, FL (all at 25 ng/mL), Epo (5 U/mL), and IL-3 (10 ng/mL). To ensure formation of a
reliable number of colonies from the long-term cultures, the content of
each stroma coculture well was transferred to methylcellulose cultures
at both a low and a high cell concentration. Colony-forming cells (CFC;
read out of LTC-IC and ELTC-IC) were scored after an additional 10 to
12 days in culture.
Competitive repopulating assay for murine BM stem cells.
C57BL/6 recipient mice (Ly5.2) were lethally irradiated by a single
exposure to 9.5 Gy of gamma irradiation from a 137Cs source
(Instrument AB Scanditronix; Husbyborg, Uppsala, Sweden). Irradiated
recipients were transplanted intravenously (0.5 mL/mouse) by tail
injection with 750 CFSE-stained
LinSca1+c-kit+ cells (from
B6SJL mice, Ly5.1) cultured for 12 to 15 hours in KL (control) or 750 expansion equivalents (EE) of CFSE-stained LinSca1+c-kit+ cells. Donor
cells were cotransplanted with 200,000 unfractionated congenic (Ly5.2)
BM cells as a competitor and survival population. All mice (4 mice/group) were kept in individually ventilated cages throughout the
experiment and given sterile food and autoclaved acidified water. Mice
were bled from the retroorbital sinus and analyzed for donor
reconstitution on a FACSCalibur (BD), after staining with antibodies
against Ly5.1, Ly5.2, and lineage-specific antigens (all from PharMingen).
Statistics.
The statistical significance of differences between groups were
determined using the paired Student's t-test. For LTC-IC and ELTC-IC data, the Student's t-test was performed with log
transformation of the data.
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RESULTS |
KL + IL-3 + IL-6 efficiently promote proliferation of murine
LinSca1+c-kit+
LTRC.
A main rationale for using KL + IL-3 + IL-6 (K36) to promote
stem cell cycling in clinical gene marking protocols was their ability
to efficiently promote retroviral-mediated gene transfer to LTRC from
mice primed with 5-fluorouracil (5-FU) in vivo.42,43 However, previous and recent studies have suggested that cytokine combinations containing K36 are inefficient at promoting gene transfer
to unprimed murine BM LTRC.44,45 Thus, it remains to be
established whether K36 can efficiently promote cycling and
proliferation of unprimed murine BM stem cells without compromising their long-term reconstituting ability.
K36 recruited most if not all clonable candidate
(LinSca1+c-kit+) BM stem
cells into proliferation under SF-conditions
(Fig 1). K36 were equally efficient at
promoting recruitment of
LinSca1+c-kit+
progenitors into proliferation as KL + FL + IL-3 (KF3) and KL + FL + MGDF (KFM) (Fig 1), 2 cytokine combinations that recently have been
suggested to be particularly promising at promoting growth of candidate
human BM stem cells.35,36,46 In fact, stimulating
LinSca1+c-kit+ cells with a
cocktail of 7 cytokines resulted in recruitment of only a few
additional progenitors, indicating that K36, KF3, and KFM optimally
induce in vitro growth of candidate murine stem cells. Whereas K36,
KF3, and the cocktail resulted in formation of predominantly large
colonies, most of the colonies formed in response to KFM were small
(Fig 1).
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| Fig 1.
Recruitment of candidate murine stem cells into
proliferation.
LinSca1+c-kit+ BM cells were
plated at 1 cell per well in SF-medium (X-vivo 15 with 0.5% detoxified
BSA) supplemented with indicated cytokines at 50 ng/mL each, except for
IL-3 (20 ng/mL). The cocktail contained the following 7 cytokines:
GM-CSF, KL, IL-3, IL-6, MGDF, FL, and G-CSF. The total number of clones
(containing 2 or more cells) and large colonies (clones covering more
than 50% of well) were scored after 10 days of incubation. 0 = no
clones. Results are presented as the means (±SEM) of 4 separate
experiments. Paired Student's t-test was performed comparing
KFM with the other cytokine combinations presented. *P < .05;
**P < .005.
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LinSca1+c-kit+ cells
cultured for 5 to 7 days in the presence of both K36 or KFM
demonstrated a preserved ability to short- and long-term in vivo
reconstitute (Bryder and Jacobsen, unpublished observations, January
1999). However, to unequivocally demonstrate that the reconstituting
activities of the in vitro cultured stem cell populations were derived
from cells that had undergone proliferation, high-resolution cell
division tracking was performed to allow distinction and purification
of cells that had undergone cell divisions. These studies demonstrated
that almost all
LinSca1+c-kit+ cells had
undergone multiple cell divisions after 7 days of culture in response
to K36 and KFM (Fig 2A and B) and that most
if not all of the reconstituting ability was derived from cells that had proliferated (Fig 2C). Furthermore, the long-term repopulating activities of the expanded cells were enhanced when compared with that
of unexpanded CFSE-sorted cells (Fig 2C) and were comparable with that
of freshly isolated
LinSca1+c-kit+ cells (Bryder
and Jacobsen, unpublished observations, April 1999).
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| Fig 2.
Most in vivo reconstituting murine stem cells proliferate
in response to KL + IL-3 + IL-6 as well as KL + FL + MGDF.
CFSE-stained LinSca1+c-kit+
(Ly5.1) BM cells were cultured in SF-medium supplemented with either
K36 (A) or KFM (B) (all at 50 ng/mL, except for IL-3 at 20 ng/mL). (A)
and (B) show the proliferation history (number of cell divisions) after
7 days of incubation. Cultured CFSE-stained cells were resorted to
include either all cells (total) or only cells that had proliferated (a
conservative approach was taken to only include cells that had
undergone  2 divisions). Both cell populations, as well as the
unexpanded CFSE-stained cells (control), were transplanted into
lethally irradiated mice (C) together with unfractionated BM cells
(Ly5.2). Analysis of the percentage of total donor reconstitution in
peripheral blood was performed 6 and 16 weeks posttransplantation.
Control and expanded cell populations showed comparable repopulation of
all (myeloid, B, and T) lineages. All data are the means (±SEM) of 4 mice, except for KFM (total) 16 weeks posttransplantation, where n = 3. Data are from 1 of 2 experiments with similar results.
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KL + IL-3 + IL-6 only recruit a minor subpopulation of
candidate human BM CD34+CD38 stem
cells into proliferation.
K36 have frequently, but with limited success, been used to promote
retroviral-mediated gene transfer into human stem cells in clinical
gene marking protocols.2,5,6,28,29 Surprisingly, in a
SF-medium K36 only recruited approximately 10% of
CD34+CD38 cells into proliferation
(Fig 3). Expansion of stem cells has been
suggested to depend on high concentrations of cytokines47; however, the growth response to K36 was not altered by enhancing the
concentration of each of the 3 cytokines 5-fold (250 ng/mL each;
Ramsfjell and Jacobsen, unpublished observations, August 1998).
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| Fig 3.
Recruitment of candidate human stem cells into
proliferation. CD34+CD38 BM cells were
plated at 1 cell per well in SF-medium and supplemented with indicated
cytokines at 250 ng/mL, except for IL-3, which was used at 50 ng/mL.
#KFM were compared at high (250 ng/mL each) and low (50 ng/mL each)
concentrations. The cocktail consisted of the following 6 cytokines:
KL, IL-3, IL-6, MGDF, FL, and G-CSF. The total number of clones and
colonies (>50 cells) were scored after 10 to 12 days of incubation. 0 = no clones. Results are presented as the means (±SEM) of 4 separate experiments. Paired Student's t-test was performed
comparing KFM high with the other cytokine conditions presented.
*P < .05; **P < .005.
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As previously demonstrated, KFM efficiently recruited
CD34+CD38 BM cells into
proliferation,36 resulting in 62% of the wells containing
proliferative clones (Fig 3). The addition of 3 additional cytokines
(IL-3, IL-6, and G-CSF) did not further enhance the recruitment seen in
response to KFM (P = .56; Fig 3), and the cloning frequency and
size of clones in KFM-stimulated cultures were similar at low and high
cytokine concentrations (Fig 3).
Because KF3 have been demonstrated to efficiently expand
CD34+CD38 BM LTC-IC,35 it
was surprising that this cytokine combination only promoted growth of
approximately 15% of CD34+CD38 cells,
similar to K36 (Fig 3). Again, a 5-fold increase in the concentration
of KL, FL, and IL-3 did not affect the number or size of clones formed
(Ramsfjell and Jacobsen, unpublished observations, August
1998). Because KFM were much more efficient than K36
(P < .005) and KF3 (P < .005) at promoting growth
of CD34+CD38 cells under SF-conditions,
we also compared the effects of these 3 cytokine combinations in
FCS-containing medium (IMDM + 20% FCS). Interestingly, although the
cloning frequency seen in response to K36 and KF3 was considerably
higher in FCS-containing medium than in SF-medium, resulting in the
growth of 31% and 52% of CD34+CD38
cells, respectively, KFM remained most efficient at promoting recruitment of CD34+CD38 cells (68% of
the cells; means of 2 experiments).
KL + IL-3 + IL-6 efficiently promote expansion of
CD34+CD38 LTC-CFC but deplete the
more primitive ELTC-CFC.
Next, we investigated whether the different levels of recruitment of
CD34+CD38 cells into cycling by the
cytokine combinations investigated might reflect activation and
potential expansion of functionally distinct subpopulations within the
CD34+CD38 cell compartment. Of
particular relevance, we evaluated and compared the usefulness of the
standard (5-week) and extended (12-week) LTC-IC for predicting
expansion of candidate human stem cells. Standard LTC-IC cultures
initiated with 10 times as many CD34+CD38+ as
CD34+CD38 cells resulted in a similar
level of CFC production, in that 73 (experiment no. 1) to 148 (experiment no. 2) LTC-CFC were produced from 1,500 CD34+CD38+ cells and 27 (experiment no. 1) to
113 (experiment no. 2) LTC-CFC from 150 CD34+CD38 cells (means of triplicates
from 2 experiments), demonstrating that LTC-IC were highly enriched in
the CD34+CD38 (lowest 3%) cell
population, but were also present in the less primitive
CD34+CD38+ population (highest 75%). In
contrast, and in agreement with others,30 we found ELTC-IC
exclusively in the CD34+CD38 population.
Specifically, 1,000 CD34+CD38 cells
generated 14 to 150 12-week LTC-CFC (ELTC-CFC), whereas no CFC was
detected from 10,000 CD34+CD38+ cells in
ELTC-IC cultures (n = 2).
The abilities of K36, KF3, and KFM (all cytokines at 250 ng/mL; except
for IL-3 at 50 ng/mL) to expand
CD34+CD38-derived 5-week LTC-IC were
investigated (for experimental set up, see
Fig 4). A number of previous studies have
shown that the average number of CFC produced per LTC-IC remains
constant when comparing fresh and cultured LTC-IC,34,47,48
thus supporting the validity of using the number of LTC-CFC generated
under various conditions as an indication for differences in LTC-IC
activity. K36 increased the number of CFC produced after 5 weeks of
long-term culture (5-week LTC-CFC) from
CD34+CD38 cells many-fold when cultured
for 7 as well as 12 days (Table 1). KF3
expanded 5-week LTC-CFC more than K36 (P < .05). However, KFM
were most efficient (both at day 7 and 12; P < .05 for KFM v KF3 and KFM v K36) at expanding 5-week LTC-CFC (up to
244-fold). Noteworthy, for all 3 cytokine combinations (except for KF3
in experiment no. 2), the expansion of 5-week LTC-CFC was higher at day
7 than after 12 days of ex vivo culture (Table 1), although only K36
reached statistical significance (P < .05).
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| Fig 4.
Experimental design of ex vivo expansion of LTC-IC and
ELTC-IC. CD34+CD38 BM cells (BMC; maximum
1,000 cells/mL) were cultured in SF medium supplemented with cytokines.
After 7 or 12 days of expansion culture, cells were counted and
transferred to either 5- or 12-week LTC. At the end of the 5 or 12 weeks of stroma coculture (no cytokines added), LTC-IC-derived CFC
(LTC-CFC) were detected in a CFC assay.
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Experiments in which CD34+CD38 cells
were stained with CFSE before culture in SF-medium supplemented with
KFM demonstrated that most if not all cells had undergone cell
division(s) in response to KFM after 7 days of incubation, with the
majority of the cells having divided 3 to 5 times. In agreement with
this, cells sorted into CFSE-high (undivided) and CFSE-low (divided)
cells demonstrated that the majority of BM
CD34+CD38 LTC-IC had undergone
proliferation after 7 days of incubation in KFM (Kornfält and
Jacobsen, unpublished observations, November 1998).
Whereas the LTC-IC assay has been used extensively to evaluate ex vivo
expansion of primitive human progenitors, the ELTC-IC assay has not yet
been used for this purpose. Interestingly, whereas K36 expanded
CD34+CD38-derived 5-week LTC-CFC
many-fold (Table 1), the number of 12-week LTC-CFC was consistently and
dramatically reduced after 7 as well as 12 days of culture
(Table 2). In contrast, both KF3 and KFM expanded 12-week LTC-CFC, although less efficiently than 5-week LTC-CFC. Noteworthy, and in contrast to 5-week LTC-CFC, 12-week LTC-CFC
were more efficiently expanded after 12 than 7 days of ex vivo
expansion in the presence of KFM (P < .05). Furthermore, after 12 days of culture, KFM-stimulated cultures contained
consistently more 12-week LTC-CFC (3- to 30-fold expansion) than
KF3-stimulated cultures (Table 2; P < .05).
In agreement with Zandstra et al,47 we found that lowering
the concentrations of KFM to 50 ng/mL each resulted in production of
54% less 5-week LTC-CFC (Fig 5).
Interestingly, the difference in expansion was more evident with
12-week LTC-CFC, which were expanded as much as 20-fold more with the
high than low cytokine concentrations (Fig 5).
View larger version (19K):
[in this window]
[in a new window]
| Fig 5.
Efficient expansion of ELTC-CFC requires very high
concentrations of early acting cytokines. Fifty (LTC-IC assay) and
1,000 (ELTC-IC assay) human CD34+CD38
cells were cultured in SF-medium in the presence of KFM at low (50 ng/mL) and high (250 ng/mL) concentrations. After 12 days of
stroma-free culture, expanded cells were transferred to irradiated
stroma cocultures. After either 5 or 12 weeks, the number of CFC
produced was evaluated in methylcellulose. Results are the means of
triplicate wells per group from 4 separate experiments.
|
|
Serum-containing medium negatively affects prolonged ex vivo
expansion of ELTC-CFC.
KFM were equally efficient at promoting recruitment of
CD34+CD38 cells into proliferation in
FCS-containing medium as in SF-medium (114 ± 9 and 117 ± 5 clones derived from 180 CD34+CD38 cells, respectively; n = 4).
Despite this, expansion of ELTC-CFC in SF-medium was 17-fold higher
than in FCS-containing medium (Table 3;
P < .05). Again, the effect was more pronounced on ELTC-CFC than LTC-CFC, because both SF- and FCS-containing medium supported expansion of LTC-CFC (experiment no. 1: 81-fold [SF] and
71-fold [FCS]; and experiment no. 2: 183-fold [SF] and 24-fold [FCS]). Although its predictive value has been questioned, the potential usefulness of a phenotypic quantification of candidate stem
cells is obvious, because functional surrogate human stem cell assays
are tedious and require multiple weeks before a final read out. In good
correlation with the 12-week LTC-CFC data, as much as 52% of
KFM-derived cells maintained a
CD34+CD38 phenotype after 12 days of
SF-culture, whereas virtually no (0.2%) CD34+CD38 cells could be recovered from
FCS-supported cultures (Fig 6; P < .05). Further phenotypic analysis showed that more than 80% of day
12 KFM-derived CD34+CD38 cells also
remained negative for lineage markers (CD2, CD4, CD8, CD19, CD14, CD15,
CD16, CD56, and glycophorin A; n = 2).
View larger version (21K):
[in this window]
[in a new window]
| Fig 6.
Phenotypic characterization of
CD34+CD38 cells expanded under serum-free
and serum-containing conditions. CD34+CD38
BM cells were cultured in 1.5 mL SF-medium or FCS-containing medium
supplemented with KFM. After 12 days of incubation, cell numbers were
counted and cells were analyzed for CD34 and CD38 expression by flow
cytometry. Results are presented as the means (±SEM) of 3 separate
experiments. Paired Student's t-test was performed comparing
the 2 different growth conditions. *P < .05.
|
|
| |
DISCUSSION |
The potential utility of ex vivo expanded stem cells for clinical
transplantation and gene therapy purposes is obvious.2-4 A
successful approach to ex vivo expand HSC would also provide a
means of addressing the molecular regulation of stem cell self-renewal and cell cycle status of reconstituting stem cells. For transplantation purposes, ex vivo expanded grafts must contain a minimum number of stem
cells to promote efficient long-term reconstitution. Although it has
been postulated that ex vivo expanded committed progenitor cells could
facilitate short-term engraftment,3,4,49-51 studies in
animal models suggest that LTRC might also be the best short-term repopulating cells.52,53 Thus, a primary objective of all
ex vivo expansion protocols designed to improve engraftment or
promote retroviral gene transfer must be to preserve and/or expand the number of LTRC.
Because of the absence of optimal preclinical human stem cell assays,
studies of murine long-term reconstituting stem cells continue to play
an important role for preclinical development of stem cell and gene
therapy. Murine BM LTRC primed with 5-FU in vivo have been demonstrated
to be excellent targets for retroviral-mediated gene transfer if
prestimulated with K36, suggesting that they can self-renew in response
to these cytokines in vitro.42,43 Thus, the ability of this
cytokine combination to promote gene transfer into murine BM stem cells
in vitro, combined with its ability to transduce human
LTC-IC,24-27 provided a rationale for using K36 to promote
gene transfer into human stem cells. However, clinical studies have
failed to demonstrate efficient transduction of long-term
reconstituting human BM stem cells using this
approach.2,5,6,28,29 Furthermore, retroviral gene targeting
of unprimed murine BM stem cells (more closely resembling human BM
target cells in previous gene marking protocols) has also been
inefficient.44,45 One obvious reason could be that K36 do
not efficiently promote self-renewal divisions of unprimed BM stem
cells. However, in the present studies, we demonstrate that the LTRC
activity is retained in the fraction of cells that has undergone 2 or
more cell divisions, unequivocally demonstrating that K36 (and KFM)
promote proliferation and potential expansion of murine LTRC. Although
a quantification of stem cell numbers was not performed, K36 and KFM
expanded stem cells showed enhanced long-term reconstituting activity
when compared with unexpanded CFSE-stained
LinSca1+c-kit+ cells. Thus,
K36 and KFM efficiently promote proliferation of murine BM stem cells
without compromising their long-term reconstituting ability.
Similarly to what we observed with candidate
(LinSca1+c-kit+) murine stem
cells, KFM efficiently induced recruitment of candidate (CD34+CD38) human BM stem cells into
proliferation. Actually, KFM were as efficient at stimulating
recruitment of CD34+CD38 cells as a
cocktail containing 3 additional cytokines. In contrast, a much smaller
fraction of CD34+CD38 BM cells
proliferated in response to K36. Surprisingly, also KF3, shown to
potently expand BM LTC-IC,35 were rather inefficient (when
compared with KFM) at promoting recruitment of
CD34+CD38 BM cells in the present
studies. We cannot rule out that our specific SF-conditions might
selectively support KFM- and not K36- or KF3-stimulated proliferation
of CD34+CD38 cells. However, using the
same SF-conditions, K36 and KF3 induced proliferation and cellular
expansion of total CD34+ cells more efficiently than KFM
(Ramsfjell and Jacobsen, unpublished observations, August 1998).
In the absence of an optimal human stem cell assay, the LTC-IC assay
has played an instrumental role in the identification and
characterization of candidate stem cells.9,14-16 However, in support of such standard (5- to 8-week) LTC-IC not necessarily representing the most primitive hematopoietic progenitor/stem cells,
recent studies of freshly isolated BM and cord blood cells suggested
that an extension of the long-term culture beyond 60 days selects for
detection of a distinct and more infrequent progenitor, with unique
phenotypic and cell cycle characteristics thought to be associated with
true hematopoietic stem cells.27,30 Furthermore, previous
studies had implicated a low cloning efficiency, delayed onset of
proliferation, and in particular resistance to retroviral marking of
ELTC-IC (unlike LTC-IC).27,30 However, in the present studies, we provide new data supporting that ELTC-IC can be recruited into proliferation and expanded in response to early acting cytokines.
Because our ELTC-IC assay, in contrast to previous
studies,30 was performed without addition of cytokines, we
cannot conclude that we are evaluating the same cells. However, in
agreement with the studies of Hao et al,30 we found
ELTC-CFC to be derived from CD34+CD38
cells and never from the sorted CD34+CD38+
population. However, our findings do not rule out that
CD34+CD38low cells might also contain ELTC-IC,
because our CD34+CD38+ population only
contained the 75% highest CD38+ cells.
Through a direct comparison of the conditions required for expansion of
LTC-CFC and ELTC-CFC, we observed a number of distinct differences, all
supporting the more primitive and quiescent nature of ELTC-IC. After 7 as well as 12 days of incubation, K36 expanded 5-week LTC-CFC derived
from CD34+CD38 BM cells many-fold, while
dramatically depleting ELTC-CFC. This finding provides one plausible
explanation as for why Hao et al27 and Case et
al31 were unable to retrovirally transduce ELTC-IC using
the same cytokine combination, while obtaining efficient gene marking
of LTC-IC. These data might (at least in part) also explain the
inefficient stem cell gene marking observed in previous clinical gene
therapy/marking protocols.2,6,28,29 It is also noteworthy
that the inability of K36 to promote proliferation of ELTC-IC could not
be predicted from our parallel studies on murine stem cells. Thus, the
present studies implicate an important qualitative difference in the in
vitro cytokine responsiveness of candidate murine and human BM stem cells.
Because limiting dilution was not performed, we cannot conclude to what
degree the observed increase in ELTC-CFC activity results from an
increased number of ELTC-IC with sustained or reduced level of CFC
production. The ability of KFM and KF3 to efficiently expand not only
LTC-CFC but also ELTC-CFC agrees with previous studies suggesting that
these 2 cytokine combinations might be particularly useful at promoting
expansion of candidate adult human stem cells.35,36,46 Our
studies also further implicate MGDF as an essential and potent cytokine
for optimal recruitment of BM CD34+CD38
cells into proliferation, because KFM were both required and sufficient
for optimal recruitment. Although KFM were somewhat more efficient than
KF3 at expanding ELTC-CFC after 12 days of culture, the ELTC-CFC
activity derived per clonable CD34+CD38
cell was comparable. Furthermore, because others have demonstrated a
higher cloning efficiency of CD34+CD38
BM cells in response to KF3,47 the abilities of these 2 cytokine combinations to expand candidate BM stem cells are likely to
be comparable.
Optimal expansion of LTC-CFC was observed after 7 rather than 12 days
of culture, whereas expansion of ELTC-CFC was most pronounced after
more prolonged (12-day) culture. This observation is likely to be
explained by a quiescent/primitive fraction of
CD34+CD38 cells being recruited into the
first cell division after prolonged incubation.34,36 Thus,
previous retroviral-mediated gene marking protocols, including those
seeking to transduce BM ELTC-IC,27,31 might have proved
more efficient if the period of prestimulation had been significantly extended.
Previous studies have convincingly demonstrated that very high
concentrations of early acting cytokines (250 to 300 ng/mL) might be
required to preferentially promote stem cell self-renewal, rather than
cell divisions associated with commitment and loss of stem cell
function.47 Our studies support that this effect is
strictly associated with preservation of stem cell function and not
proliferation, because the level of recruitment and expansion of
CD34+CD38 BM cells were not affected by
increasing the cytokine concentrations. Of particular interest is our
finding that increasing the concentration of KFM from 50 ng/mL to 250 ng/mL preferentially expanded ELTC-CFC.
Hogge et al54 have demonstrated that the number of LTC-IC
detectable in normal BM, the average number of CFC produced per LTC-IC,
as well as the maintenance of LTC-IC are all increased when using
engineered murine fibroblast feeders as compared with normal BM feeders
used in the present studies. Thus, obviously normal BM feeders only
detect a fraction of the LTC-IC (and ELTC-IC) supported by engineered
murine fibroblast cell lines. Whether the LTC-IC detected in these
different assays are also qualitatively different remains to be established.
Another intriguing finding in the present studies was the observation
that culturing of CD34+CD38 BM cells in
FCS-supplemented medium negatively affected expansion of ELTC-CFC when
compared with cells cultured under SF-conditions. The negative effect
of the FCS-containing medium was much more evident on expansion of
12-week than of 5-week LTC-CFC. Similar to the cytokine concentration
effect discussed above, the difference observed between the
FCS-containing and SF-medium appeared to be unrelated to cell
proliferation. However, whereas more than 50% of SF-expanded cells
remained CD34+CD38, less than 1%
maintained this phenotype in the FCS-containing medium. Although our
observations do not definitely prove that FCS has detrimental effects
on stem cell self-renewal, they argue caution when exposing stem cells
to FCS ex vivo.
In conclusion, we have demonstrated that human
CD34+CD38 BM ELTC-CFC can be expanded
after prolonged exposure to high concentrations of early acting
cytokines (KF3 and KFM) under SF-conditions. The distinct and stringent
requirements shown for expansion of BM ELTC-CFC when compared with
LTC-IC support that the ELTC-IC assay detects a more primitive stem
cell, which could prove to be a more reliable predictor for LTRC.
Ongoing efforts are devoted towards comparing the identity, expansion
potential, and retroviral-mediated transduction of ELTC-IC and SRC,
because the SRC assay has also been implicated to detect a more
primitive progenitor than the traditional LTC-IC.10,32 The
ELTC-IC and SRC assays might prove to play a complimentary role towards
development of more efficient ex vivo stem cell expansion and
gene therapy.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge the Department of Hematology (Lund
University Hospital) for performing the human BM aspirations and thank
our BM volunteers for their donations. We are indebted to Dr Per
Nilsson and Stefan Johnsson for performing the stroma irradiations and
to Per Anders Bertilsson and Sverker Segrén for expert assistance
with the cell sorting. We thank Ingbritt Åstrand-Grundström, Eva
Gynnstam, Irene Persson, and Lilian Wittman for technical assistance
and animal care. We are also grateful to Drs Ian K. McNiece, Janet
Nichol, and Graham Molineux for generously supplying MGDF, stem cell
factor, and other cytokines and to Dr Stewart Lyman for providing FL
for these studies. We also thank Dr Stefan Karlsson for helpful
discussions and reviewing the manuscript.
| |
FOOTNOTES |
Submitted April 7, 1999; accepted August 24, 1999.
Supported by grants from the Berta Kamprad Foundation; the Crafoord
Foundation; the Georg Danielsson Foundation; the Gunnar, Arvid and
Elisabeth Nilsson Foundation; the John and Augusta Persson Foundation;
the Medical Faculty, University of Lund; the O and E and Edla Johansson
Foundation; the Royal Physiographic Society in Lund; the Swedish Cancer
Society; the Swedish Child Cancer Fund; the Harald and Greta
Jeansson's Foundation; the Syskonon Svensson's Foundation; the Thelma
Zoega's Foundation; and the Tobias Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sten E.W. Jacobsen, MD, PhD, Stem Cell
Laboratory, Department of Molecular Medicine and Gene Therapy,
Department of Internal Medicine, Institute for Laboratory Medicine,
University Hospital of Lund, 221 85 Lund, Sweden; e-mail:
sten.jacobsen{at}molmed.lu.se.
| |
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INTRODUCTION