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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 855-862
HEMATOPOIESIS
High-resolution tracking of cell division suggests similar cell
cycle kinetics of hematopoietic stem cells stimulated in vitro and
in vivo
Robert A. J. Oostendorp,
Julie Audet, and
Connie
J. Eaves
From the Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver, BC, Canada; the Department of Medical Genetics and the
Biotechnology Laboratory, University of British Columbia, Vancouver,
BC, Canada; and the Department of Cell Biology and Genetics, Erasmus
University, Rotterdam, the Netherlands.
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Abstract |
The kinetics of proliferation of primitive murine bone marrow (BM)
cells stimulated either in vitro with growth factors (fetal liver
tyrosine kinase ligand 3 [FL], Steel factor [SF], and
interleukin-11 [IL-11], or hyper-IL-6) or in vivo by factors active
in myeloablated recipients were examined. Cells were first labeled with
5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and
then incubated overnight prior to isolating CFSE+ cells.
After 2 more days in culture, more than 90% of the in vivo
lymphomyeloid repopulating activity was associated with the most
fluorescent CFSE+ cells (ie, cells that had not yet
divided), although this accounted for only 25% of the repopulating
stem cells measured in the CFSE+ "start"
population. After a total of 4 days in culture (1 day later), 15-fold
more stem cells were detected (ie, 4-fold more than the day 1 input
number), and these had become (and thereafter remained) exclusively
associated with cells that had divided at least once in vitro. Flow
cytometric analysis of CFSE+ cells recovered from the BM
of transplanted mice indicated that these cells proliferated slightly
faster (up to 5 divisions completed within 2 days and up to 8 divisions
completed within 3 days in vivo versus 5 and 7 divisions, respectively,
in vitro). FL, SF, and ligands which activate gp130 are thus efficient
stimulators of transplantable stem cell self-renewal divisions in
vitro. The accompanying failure of these cells to accumulate rapidly
indicates important changes in their engraftment potential independent
of accompanying changes in their differentiation status.
(Blood. 2000;95:855-862)
© 2000 by The American Society of Hematology.
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Introduction |
Over the last few years, many studies have described
culture conditions (including a variety of growth factor combinations) that support the production of large numbers of murine and human hematopoietic progenitors which are capable of further proliferation and terminal differentiation in semisolid media.1 It is now widely appreciated that regardless of their origin, very few if any of
these cells have long-term multilineage in vivo engrafting potential.
Moreover, a surface phenotype that can be uniquely associated with
transplantable hematopoietic stem cell activity, even when the cells
are variably stimulated in vitro or in vivo, has not yet been
identified. Thus, progress in defining factors that regulate the
maintenance or loss of hematopoietic stem cell integrity has had to
depend on studies that use quantitative methods to measure changes in
cells which can express long-term multilineage repopulating activity in
transplanted recipients.2-4 The competitive repopulation
unit (CRU) assay3,5,6 is 1 example of such a method, in
which stem cells are defined by their ability to sustain circulating
levels of at least 105 lymphoid and myeloid cells for more
than 4 months. The period of 4 months appears to be of sufficient
duration to distinguish stem cells that can remain active for much
longer periods from those that may not.2,7 CRU frequencies
are determined using the technique of limiting dilution analysis in
combination with one of several strategies for ensuring the survival of
all of the recipients. This is accomplished independent of
whether or not the mice are injected with any CRU in the
population being assessed.
In the last 10 years, several cytokines expressed by stromal cells have
been cloned. The fact that stromal cells are the presumed source of
factors that stimulate stem cell proliferation both in
vivo8 and in the long-term culture (LTC)
system,9 prompted the investigation of the abilities of
these cytokines to stimulate murine stem cell amplification in
stroma-free cultures. From such studies, it has been found that Steel
factor (SF) or c-kit-ligand in combination with interleukin-6
(IL-6)5 or IL-11, either without10,11 or with
fetal liver tyrosine kinase ligand 3 (FL)6 can stimulate a
significant, albeit modest, net expansion of murine stem cell numbers
in short-term cultures. More recently, evidence of cyclic oscillations
in stem cell activity within such cultures has suggested that these
cells may transiently (ie, reversibly) lose their engrafting
ability.12 The latter observations have complicated the
definition of what constitutes a stem cell self-renewal division and
have raised new questions about the contribution of proliferation to
the stem cell populations obtained from cytokine-stimulated cultures.
In the present study, we used a high-resolution cell tracking procedure
to monitor the stem cell activity of cultured cells with different
proliferative histories. This procedure relies on the ability of 5- and
6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to
stably label a population of cells such that their fluorescence is
precisely halved at each successive cell generation.13 By
preselection of a starting population with a minimal variation in
fluorescence, we have shown that up to 7 successive generations of
human hematopoietic cells can be subsequently
resolved.14,15 We have used this technology here to analyze
the kinetics of murine CRU proliferation in short-term cultures of
CRU-enriched suspensions of normal adult mouse bone marrow (BM) cells
stimulated with SF, FL, and either IL-11 or hyper-IL-6 (H-IL-6, a
recombinant fusion protein of IL-6 and its soluble receptor
16). We then compared these results with the initial cell
division kinetics seen in vivo when freshly isolated CFSE+
cells were transplanted into myeloablated congenic recipients. The
results indicate that currently available culture conditions can
closely mimic the potent and rapidly mitogenic environment of the
myeloablated host. This suggests that other mechanisms may be
responsible for the general inability to obtain large amplifications of
hematopoietic stem cell populations in vitro.
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Materials and methods |
Animals
Breeding pairs of mice with the following genetic codes were
purchased: C57BL/6J (B6, Ly-5.2, Gpi-1a); B6-Pep3b
(Pep3b, Ly-5.1); (B6 × C3H)F1 (B6C3, Ly-5.2/5.2); and
(Pep3b × C3)F1 (PepC3, Ly-5.1/5.2) (Jackson Laboratory, Bar Harbor, MN). Breeding pairs of mice also included B6-Gpi-1b
(Gpi-1b) and
B6-W41/W41 (W41)
(Dr J. Barker, Jackson Laboratory). All mice were subsequently bred and
maintained at the Joint Animal Facility of the British Columbia Cancer
Research Center in microisolator units provided with sterilized food,
water, and bedding. Irradiated animals were additionally provided with
acidified water (pH 3.0).
Isolation of lineage marker BM cells
Suspensions of mouse femoral and tibial BM cells were depleted of
cells expressing CD5, CD11b, CD45RA, Ly-6G, and the antigen recognized
by Ter119 using a murine lineage depletion kit (StemSep; StemCell
Technologies, Vancouver, BC, Canada) as described.17 In
brief, cells were first incubated with 5% normal rat serum in Hanks'
balanced salt solution containing 2% fetal calf serum (HF/2, StemCell)
on ice, and then biotinylated monoclonal antibodies (mAbs) against the
above-named markers were added. After 15 minutes at 4°C, the cells
were washed once in HF/2 and resuspended at 5 × 107
mL in HF/2. After further addition and mixing with tetrameric antibiotin/antidextran complexes, the cells were incubated for another
15 minutes at 4°C. The magnetic colloid was added, and 15 minutes
later the cells were applied to a primed column (StemSep, StemCell
Technologies) of an appropriate size placed inside the magnet. Lineage
marker (lin) BM cells were
collected using a peristaltic pump.
CFSE labeling
BM cells (either lin or unmanipulated) to be
labeled with CFSE (Molecular Probes, Eugene, OR) were first centrifuged
at 350g for 5 minutes, and the cell pellet was resuspended in
phosphate-buffered saline (PBS, StemCell). Samples were set aside for
flow cytometric analysis and functional assays prior to CFSE labeling.
CFSE was then added to the remainder at a final concentration of 10 or 20 µmol/L. After 10 minutes at 37°C, further dye uptake was
prevented by the addition of a quarter volume of cold FCS. Cells were
washed once in a serum-free medium (SFM) consisting of Iscove's medium with 1% wt/vol bovine serum albumin, 10 µg/mL insulin, 200 µg/mL iron-saturated transferrin (BIT, StemCell), 40 µg/mL low-density lipoproteins (Sigma Chemicals, St. Louis, MO), 100 µmol/L
2-mercaptoethanol, and 2 mmol/L L-glutamine. Cells were then
transferred to 35-mm petri dishes (StemCell), and the following growth
factors were added: 100 ng/mL human FL (Immunex Corporation, Seattle,
WA); 50 ng/mL murine SF (expressed in COS cells and
purified in the Terry Fox Laboratory); and either 100 ng/mL human IL-11
(Genetics Institute, Cambridge, MA) or 20 ng/mL H-IL-616
(provided by S. Rose-John, University of Mainz, Mainz, Germany). The
CFSE-labeled cells were then cultured overnight at 37°C and
harvested from the dishes (including cells obtained by rinsing each
dish twice with HF/2).
Labeled cells that had not been depleted of lin+ cells
prior to the CFSE labeling and overnight incubation step were
centrifuged at 1000g for 20 minutes on a 1.087 g/mL solution
(Lympholyte-M; CedarLane Laboratories, Hornby, ON, Canada) to isolate
the low-density (LD) fraction. These and the CFSE-labeled
lin cells were then washed once in HF/2 at
600g for 10 minutes and sorted using a fluorescence-activated
cell sorter (FACStar Plus; Becton Dickinson, San Jose, CA) equipped
with an argon (excitation 488 nm) and a helium-neon (excitation 633 nm)
laser using a narrow gate (30-36 channels wide with a 1024 channel log
amplifier) for collecting CFSE+ cells (Figure
1), as previously described.14
This narrow gating interval was chosen slightly to the left of the
median CFSE fluorescence to include the majority of cells with low-side
and forward light scattering characteristics. In 2 experiments the
harvested CFSE-labeled lin cells were also stained
with an R-phycoerythrin-conjugated (R-PE-conjugated) anti-Sca-1
antibody (PharMingen, San Diego, CA) prior to sorting to allow
isolation of linSca-1+ CFSE+
cells as described.6
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| Fig 1.
Fluorescence profiles of CFSE-labeled mouse
lin BM cells.
Profiles shown are of cells before (left panel) and after (middle
panel) FACS selection of the most brightly labeled subpopulation using
a 30 channel-wide gate. The right panel shows the fluorescence profile
of the cells recovered another 2 days later, after placing the selected
CFSE+ cells (middle panel) into serum-free cultures
containing FL, SF, and IL-11 either with (open peak) or without (solid
peaks) colcemid. The distribution of cells according to the number of
divisions they completed in the 2 days in culture after being labeled
is shown by the different sized peaks corresponding to serial 2-fold
decreases of fluorescence intensity. The fluorescence intensity of the
cells incubated with colcemid confirms the position on a similar plot
of the cells that had not divided.
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Suspension cultures
Sorted CFSE+ cells were suspended at
 4 × 104 cells/mL in SFM plus FL, SF and IL-11,
or H-IL-6 (at the same final concentrations used during the CFSE
labeling procedure). Aliquots of 2.5 mL were incubated at 33°C in
35-mm petri dishes for 3 or 4 days and in 50-mL volumes in 150 cm2 tissue culture flasks for 10 days. In each experiment,
0.1 µg/mL of colcemid (Karyomax, Gibco BRL Life
Technologies, Grand Island, NY) was added to a separate culture
containing 105 cells. The colcemid-treated cells were used
to calibrate the fluorescence intensity of cells that did not undergo a
single division in each experiment because there is a slow but
continuous loss of fluorescence from CFSE-labeled cells over time. The
cells to be incubated for 10 days were given new colcemid on day 7. Cells were harvested by removing the suspended cells and then rinsing
the dishes (or flasks) twice with Iscove's medium. When present,
adherent cells were removed by incubating them for 2-5 minutes in a
Trypsin/EDTA (ethylenediaminetetraacetic acid) solution (Gibco BRL),
scraping any remaining cells off the bottom of the flask with a rubber
policeman (Falcon), and rinsing two times with HF/2. Cells were then
pooled, centrifuged at 350g for 5 minutes, resuspended in HF/2
with 1 µg/mL propidium iodide (PI) (Sigma), centrifuged again, and
finally resuspended in HF/2 for sorting into fractions of divided and
undivided cells as described below.
FACS analysis and isolation of divided and undivided cells
Gates were set using a 1024 channel scale. Undivided cells
were collected between a gate set at equal to the median intensity of
the colcemid-treated CFSE+ cells and extending to higher
fluorescence levels. To minimize cross-contamination, the upper gate
for collecting cells that had divided was set 60 channels beneath the
lower gate used to define the fraction of undivided cells (ie,
approximately twice the channel width originally used to isolate the
CFSE+ cells; Figure 1, middle panel). Cells that had
divided were collected between this upper channel and channel 350 to
provide a gate width such that the maximum fluorescence of unstained BM
cells corresponded to the minimal fluorescence level (channel 350) used
to identify cells that had divided. Cells that had divided 8 times
could then be reliably resolved as distinct from unstained cells.
Comparison of viable (trypan blue dye excluding) presort and postsort
cell numbers showed that 75%-89% of the presort cells were recovered using these 4 collection gates. For the calculations of numbers of
recovered cells, the median recovery value of 83% was used. Sorted
cells were collected in HF/2, centrifuged, counted, and injected into
irradiated recipients for CRU assays, as indicated.
Assays for colony-forming cells
Numbers of committed progenitors (burst-forming units erythroid
[BFU-E], colony-forming unitsgranulocyte/macrophage [CFU-MG], CFU-granulocyte, erythroid, macrophage, megakaryocyte [CFU-GEMM]) were determined by plating suitable aliquots of test cells in methylcellulose medium supplemented with 10 ng/mL murine IL-3, 10 ng/mL
human IL-6, 50 ng/mL murine SF, and 3 units/mL human erythropoietin
(HCC-3434, StemCell) and scoring the corresponding types of colonies
(containing more than 30 cells each) present after 12 days of
incubation at 37°C.
Competitive repopulation unit assays
Various doses of Pep3b (Ly-5.1) or PepC3 (Ly-5.1/5.2) BM
cells were injected intravenously into irradiated congenic Ly-5.2 recipients as previously described.5,6 Recipients were
either W41 mice given a sublethal dose of 450 cGy, B6 mice given a lethal dose of 900 cGy, or B6C3 mice
given a lethal dose of 950 cGy. In the latter 2 cases, 105
normal marrow cells of the same genotype as the recipient were coinjected with the test cells to ensure the survival of all recipients but not to interfere with the detection of engrafted test
cells.5 At the subsequent times indicated, cells obtained
from tail blood samples were stained first with a combination of
biotinylated antibodies (PharMingen) against either myeloid
(CD11b, Mac-1 [clone M1/70] and Ly-6G, Gr-1 [clone
RB6-8C5]) or lymphoid (CD5, Ly-1 [clone 53-7.3] and CD45R, B220
[clone RA3-6B2]) markers. Cells were then washed twice in HF/2 and
finally stained with a fluorescein isothiocyanate-labeled
(FITC-labeled) anti-CD45.1 (Ly-5.1, [clone A20]) antibody (prepared
in the Terry Fox Laboratory) and streptavidin-conjugated R-PE (Southern
Biotechnologies, Birmingham, AL). After 2 washes in HF/2, with 1 µg/mL PI in the last wash, a minimum of 5000 PI
cells were analyzed (FACSort, Becton Dickinson). Positively-stained cells were defined by gates that excluded more than
99.9% of cells stained with an appropriate isotype control antibody. A
CRU assay recipient was considered positive only if its
Mac-1/Gr-1+ and Ly-1/B220+ populations each
contained at least 1% Ly-5.1+ (donor-derived) cells.
In experiment No. 4 (Table 1), various
doses of Gpi-1b BM cells were injected
intravenously into lethally irradiated (900 cGy) congenic
B6-Gpi-1a mice. Tail blood samples in this
experiment were divided into 2 aliquots. In 1 aliquot, the red blood
cells were first lysed by a brief exposure to NH4Cl. The
cells of both aliquots were then centrifuged, frozen, and thawed 3 times, and the 2 Gpi-1 isoenzymes were separated by
electrophoresis in cellulose acetate gels (Helena Laboratories,
Bearmont, TX), as described earlier.2 In this case a CRU
assay recipient was considered positive only if both the red and white
blood cell lysates each contained 1% Gpi-1b
isoenzyme. Estimates of CRU frequencies were obtained from the proportions of negative mice in each of the various cell dose groups
using the method of maximum likelihood18 (L-Calc software, StemCell). Previous experiments have shown that CRU frequencies determined using either Gpi-1 isoenzyme differences or the
Ly-5.1/5.2 system to distinguish regenerated donor and recipient
granulocytes and lymphocytes are the same.
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Results |
Kinetics of expansion of mouse BM cells in culture
A total of 8 experiments were performed. In 7 of these, CFSE
labeling was used to analyze the proliferative activity of adult mouse
BM cells in serum-free cultures containing 100 ng/mL FL, 50 ng/mL SF,
and either 100 ng/mL IL-11 or 20 ng/mL H-IL-6. The associated change
over time in the distribution of CRU between the fractions of cells
that had and had not yet begun to divide under these conditions was
also determined. A summary of these experiments is given in Table 1.
Cultures were initiated with either LD, lin, or
lin Sca-1+ BM cells obtained from Pep3b,
PepC3, or Gpi mice. In 4 experiments, most of the
CFSE+ cells were injected directly into congenic
myeloablated mice. These were given 900 or 950 cGy, as with the CRU
assays, followed by 3 × 106 LD CFSE+
cells or 1.8 × 105 to 1.5 × 106
lin CFSE+ cells per mouse (4 mice per
group). This allowed concomitant comparison of the in vivo and in vitro
proliferation kinetics of the same starting populations (see below).
Thus in most experiments, sufficient cells to examine only a single
culture time point were available. Nevertheless, the consistency of the
cell count (Table 1) and CRU data (Tables 2 and
3) between like experiments, regardless of the cytokine combination used, support their
consideration as single groups. The ability of these 2 cytokine
combinations to give similar CRU expansions in vitro has also been
confirmed in multifactorial design experiments (manuscript in
preparation). Because the isolation of an appropriately CFSE-labeled
population required an initial overnight culture procedure, this was
carried out in the presence of the same combination of growth factors that the cells were subsequently exposed to. Thus, the total period of
culture included 1 day prior to the initiation of CFSE cell division tracking.
Between the end of the first and third days in culture of
lin cells maintained in SFM with FL, SF, and IL-11,
the total number of cells present remained constant (Table 1).
Nevertheless, during this interval, analysis of the distribution of
cell numbers according to their persisting fluorescence revealed that
some cells had already completed up to 5 divisions (Figure 1, right
panel). Thus, during this short 2-day period of incubation, there must
have been a substantial loss of cells to offset the considerable
amplification that would have been anticipated from the extent of
proliferative activity indicated by the loss of the CFSE label. Similar
analysis of cultures harvested 1 day later showed that within a 3-day
tracking period (but 4 days of growth factor stimulation), up to 7 cell cycles were completed by some of the initially labeled cells (Figure 2, bottom panel). This was associated with
an average 3-fold net expansion in total cell numbers in these
cultures. Interestingly, in the absence of added growth factors, we did
not detect any cells that had completed more than 3 divisions (Figure
2, top panel), and the yield of cells was 4-fold lower. After 9 days of
tracking (10 days of culture), most cells had divided more than 7 times, and the proportion classified as still undivided was 0.005% of
the total present at that time (Table 1). The total cell number was
increased approximately 60-fold. However, this number cannot be readily
compared with the total cell expansions measured at earlier times,
since the 10-day cultures were initiated with more highly purified stem
cell populations (ie, linSca-1+ cells).
Accordingly, the expansions observed would be correspondingly increased, (see, for example, the approximately 2-fold greater expansion of total cells already seen after 4 days of culture of
lin versus LD cells; Table 1, experiment No. 8 versus experiment Nos. 2, 5, and 7).
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| Fig 2.
Distribution of cells according to their CFSE
fluorescence after being cultured.
Distribution is shown after 3 days of incubation in serum-free culture
with (B) or without (A) exogenous growth factors (as shown) in addition
to an initial period of overnight culture prior to selection of a LD
CFSE+ population. Open peaks show the data for control
(undivided) cells incubated under the same conditions but in the
presence of colcemid to block cell division. The presence of FL, SF,
and H-IL-6 stimulated the cells in this experiment to complete an extra
4 cycles (7 divisions shown in B versus 3 divisions in A).
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Kinetics of recruitment of CRU into division in culture
Cultured cells from the experiments described in Table 1 were also
sorted according to their level of fluorescence to isolate those that
had not yet divided as a separate population from those that had, as
outlined previously. Essentially all of the cells recovered were used
to estimate the distribution of CRU between the 2 fractions. The CRU
frequency values obtained (and their 95% confidence limits) are shown
in Table 2. From these values and the total cell recoveries in each
fraction, the total absolute number of CRU in each fraction was then
calculated. These values are shown in Table 3. Four of the 5 mice we
injected with 300 undivided cells from day 3 cultures (2 days of
tracking) showed stable levels of lymphoid and myeloid engraftment from
the cultured cells for at least 6 months (at which time the average
total repopulation was 18%). In contrast, only 1 of the 20 mice
injected with up to 7500 cells that had divided within 3 days of
exposure to FL, SF, and IL-11 showed multilineage engraftment (3%).
Curiously, although more than 90% of the CRU detected in these
cultures had not yet divided, their absolute numbers had decreased 3- to 5-fold relative to the input lin
CFSE+ population (Table 3).
Cultures analyzed 1 day later showed the opposite picture. In these
later cultures, all of the repopulating stem cell activity was detected
in the fraction of cells which had, by that time, divided. Moreover,
the CRU assays of these cells indicated the number of CRU detectable to
be 3- to 5-fold higher than the number of CRU in the CFSE+
input population (Table 3) and 15-fold higher than the number of CRU
detected in the day 3 cultures. The lack of any positive mice among the
12 recipients of cells that had not divided within 4 days in vitro
indicates that any persisting quiescent CRU in these cultures must have
been present at a frequency of less than 1 per
5 × 104 undivided cells. This upper limit is the
same as the measured frequency in the input CFSE+
population. Thus the possibility of persisting quiescent CRU in the day
4 cultures cannot be excluded, although it is clear that they would no
longer represent a significant proportion of the total CRU present. In
vitro assays were performed on small aliquots of cells removed from
both fractions of the day 4 cultures in 2 experiments (Nos. 5 and 7).
These showed an overall 9-fold expansion of the CFC compartment in both
experiments between day 1 and day 4 of culture. These CFC were also
detected only among the cells that had divided at least once.
Analysis of cultures that had been maintained for 10 days also showed
CRU activity to be present only in the fraction of cells that had
divided. However, in these experiments, the total CRU population
detected after 10 days was 3- to 5-fold smaller than the input (day 1)
value. CRU were not identified among the even smaller population of
undivided cells that were still present after 10 days. However, because
of the very small number of these cells, it was again possible only to
exclude the persistence of CRU in this fraction at or above the
frequency of CRU measured for the input lin
Sca-1+ CFSE+ cells.
Secondary transplants of cells from primary recipients of
cultured cells
Some of the mice that were injected with cells which had divided (or
had not divided) in culture were sacrificed from 4-12 months after
transplantation, and CRU assays were performed on the cells that had
been regenerated in their BM. As can be seen in Table
4, when primary mice had initially been
injected with at least 1 cultured CRU, regenerated CRU capable of
producing multilineage engraftment in secondary recipients could be
consistently demonstrated in the BM of the primary mice, regardless of
the time after transplantation when the primary mice were killed. In
addition, there was no evidence of reactivation of "silenced" CRU
in primary recipients of cultured cells that had divided within 3 days
in vitro or of cells that had remained quiescent after 4 to 10 days
in vitro.
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Table 4.
Results of secondary transplants of cells obtained from
primary mice originally transplanted with cultured
cells
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Kinetics of division of donor cells after transplantation
To analyze the rate at which murine BM cells are stimulated to
proliferate in vivo when transplanted into myeloablated mice, 1.5-18 × 105 lin BM cells or
3 × 106 LD BM cells isolated at the end of the
overnight CFSE-labeling procedure were injected directly into
irradiated (900 or 950 cGy) congenic mice (with at least 4 mice per
experiment), and these were then sacrificed 2 or 3 days later.
Detectable levels of donor (CFSE+) cells could be found in
the BM, spleen, blood, liver, and thymus of mice at both time points,
as reported by others.19-21 However, only in BM, blood, and
spleen were sufficient numbers of these recovered to permit examination
of their in vivo proliferative history. Representative profiles for
CFSE+ cells recovered 2 and 3 days after transplantation
showed that these cells underwent up to 5 divisions after 2 days in
vivo and up to 8 divisions after 3 days (Figures
3 and 4).
This is, at most, 1 cell generation more than what was seen when
aliquots of the same cells were stimulated in vitro with FL, SF, and
IL-11 or H-IL-6. Interestingly, in 3 out of 4 evaluable experiments, the proportion of undivided CFSE+ cells recovered from the
BM of mice within 2 or 3 days after transplantation was also slightly
lower than that seen when aliquots of the same original
lin or LD CFSE+ cells were maintained
for the same time in vitro with FL, SF, and IL-11 or H-IL-6 (data not
shown).
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| Fig 3.
Comparison of the fluorescence profiles of
CFSE+ BM cells after maintenance in culture or in vivo in
different tissues.
LD BM cells were labeled overnight, and then CFSE+ cells
were analyzed after 3 further days of culture (labeled BM cells
cultured with FL, SF, and H-IL-6 growth factors, top left panel) or
after 3 days after transplantation of
3 × 106-labeled LD BM cells. Results are shown for
cells harvested from the BM (top right panel), spleen (bottom left
panel), and blood (bottom right panel) of the injected mice. The
distribution of cells among different subpopulations with different
proliferative histories is similar for the cells expanding in vitro and
in vivo, although there is a slight but obvious shift to the left of
the cells obtained from the mice (up to 8 cell divisions in the spleen)
compared with the cells maintained in culture for the same period prior
to analysis (just a few cells entering the seventh progeny generation).
The open peak indicates the fluorescence profile of the same cells
maintained in vitro with colcemid to fix the position of the undivided
cells.
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| Fig 4.
Comparison of the fluorescence profiles of lin
CFSE+ BM cells after maintenance in culture or in vivo
(in the marrow) for 3 and 4 days.
Lin BM cells were labeled overnight, and then
CFSE+ cells were analyzed after being maintained in vitro
or for another 2 or 3 days with FL, SF, and IL-11 (left panels) or for
2 or 3 days in vivo (after 1 day in culture) prior to being
recovered from the BM of mice injected with cells taken immediately
after they were labeled (right panel). The open peak indicates the
fluorescence profile of cells maintained in vitro with colcemid to
block cell division and analyzed at the same time. In the top right
graph, the recipient was injected with 1.8 × 105
lin CFSE+ cells, and in the bottom
right graph the recipient was injected with
1.5 × 106 lin
CFSE+ cells. The 2 right-hand graphs show the results
obtained from 1 of 2 mice sacrificed at each time point. The results
show the same slight shift to the left in the profiles obtained for the
transplanted cells as seen in Figure 3.
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Discussion |
In the present studies we used a high-resolution tracking procedure
to document the proliferative history of CFSE-labeled mouse BM cells
stimulated in short-term cultures with FL, SF, and IL-11 or H-IL-6. The
results provide new evidence that stem cells with long-term
lymphomyeloid reconstituting activity do not complete a first division
for at least 3 days in vitro, even under conditions that will stimulate
their subsequent proliferation without loss of their original stem cell
integrity. Alternative evidence that this is likely to be the case has
been inferred from the direct observation or FACS-based detection of
the cell cycle progression of individual cells in cultures of highly
enriched stem cell populations.22-27 Delayed effects of
cell cycle-active agents on stimulated populations of primitive
hematopoietic cells have also been observed, 28-31 and the
inability to retrovirally mark hematopoietic stem cells, unless they
are exposed to active virus after more than 24 hours in
culture,32 further supports this kinetic behavior of
quiescent stem cells following their exposure to growth factor
combinations with mitogenic activity.
The present studies also provide indirect evidence that hematopoietic
stem cells can be stimulated to execute self-renewal divisions in
vitro. We observed that after 4 days of exposure to FL, SF, and IL-11
or H-IL-6, all of the detected stem cell activity became associated
with cells that had completed at least 1 division. Moreover, this
association did not alter during the following week of culture. Such
findings confirm and extend previous observations of modest net
increases in murine stem cell numbers in these cultures.6
Recently, analogous results from studies of transplantable CFSE-labeled
human stem cells stimulated in vitro have been
reported.15,33 Taken together, these findings suggest that
failure to obtain larger expansions of stem cells in vitro is not due
to inadequate mitogenic stimulation within a relatively short period.
Our data, in fact, indicate that at least 90% of the CRU detectable
after 4 days in vitro are the progeny of cells that have undergone at
least 1 division during the previous 3 days and most likely during the
previous 24 hours. These findings are further reinforced by the results
of secondary transplant experiments. These confirmed a lack of stem
cells in cultured cell populations that did not regenerate mature cells in primary mice. They also showed that CRU proliferation in vitro does
not reduce their subsequent in vivo self-renewal capacity, as reported
previously.6
Proliferation of primitive hematopoietic cells has been previously
studied in cultures of cells with membrane-labeling fluorescent dyes
such as PKH-2,34,35 PKH-26,36-38
and PKH-67.39 All of these labels confer a measurable and
relatively stable level of fluorescence on the cells initially stained,
and this has allowed the detection of a significantly decreased level
of fluorescence to identify populations that have subsequently
proliferated extensively. Conversely, maintenance of a highly
fluorescent phenotype by PKH-2- or PKH-26-labeled cells has been used
to identify cells that have divided little or not at all. However, the
heterogeneity in staining intensity obtained with these dyes does not
allow small numbers of divisions to be resolved. The high-precision
tracking possible with CFSE-labeling, particularly as modified by
Nordon et al14 obviates this problem and thus allows
definitive and unique information about the initial proliferative
behavior of functionally defined stem cells to be obtained. For
example, a predominance of G1/G0 cells in the PKH-26bright population recovered from the
marrow of mice injected 48 hours previously with PKH-26-labeled R/O or
FR25lin cells20 does not preclude the
possibility that some of these cells may have already divided during
the previous 48 hours after transplantation, as suggested by the data
presented here.
Our analysis of the total CFSE+ cell population recovered
from the BM of myeloablated recipients indicated that the injected cells, as a whole, began to proliferate only slightly sooner than was
achieved in vitro through activation of the flt-3, c-kit, and gp130
receptors by soluble ligand binding. Thus it may be predicted that most
of the long-term repopulating stem cells, which are highly enriched in
the FR25lin population but still represent a minor
subset, would not begin to divide within the first 2 days after
transplantation, in contrast to other FR25lin cells.
Further investigation of this question must await functional studies of the undivided and divided fractions of in vivo
harvested donor-derived cells.
It is now clear, however, that CRU numbers can be amplified many-fold
over prolonged periods in vivo,6,40 whereas a comparable net expansion in vitro has not yet been reported. Although modest increases in CRU numbers can be documented,6 these must now be reconciled with the large and rapid fluctuations in their numbers that are detectable just before and immediately following completion of
a first division, as noted in the present study (Table 3) and suggested
by others.12 The magnitude of these fluctuations points to
the operation of a cell cycle and/or time-dependent mechanism that may
reversibly affect the ability of stem cells to either home into the
extravascular space of the marrow or to respond to stimuli that recruit
them to proliferate in this tissue. Such a model is further supported
by evidence that the exit of human CD34+ cells from
G0/G1 is also associated with a decrease in
their ability to repopulate irradiated nonobese diabetic
severe combined immunodeficiency (NOD/SCID)
mice.25 Conversely, using the same xenotransplant model,
Peled et al41 showed that up-regulation of
CXCR4 expression on human CD34+ cells
following their exposure to SF and IL-6 increased the effective repopulating activity of these cells within a time frame too short to
be readily accounted for by stem cell divisions. Thus transplantability and control of the "undifferentiated" status of cells with
hematopoietic stem cell potential may be subject to independent
regulation. Such a possibility underscores the need for definitive
molecular indicators of each of these functions so that their degree of overlap and extrinsic control can be further delineated.
| |
Acknowledgments |
We thank C. Miller (StemCell Technologies, Vancouver, BC, Canada) and
T. Holyoake (University of Glasgow, Glasgow, Scotland) for critical
discussions; J. Barker (Jackson Laboratory, Bar Harbor, MN) for
breeding pairs of B6-Gpi-1b and
B6-W41/W41 mice; S. Rose-John
(University of Mainz, Mainz, Germany) for H-IL-6; M. Sinclaire and N. Raymond for technical assistance; G. Cameron, G. Thornbury, and R. Zapf
for operating the FACS; Y. Yang for typing the manuscript; and Cangene,
Genetics Institute, Immunex, and StemCell Technologies for reagent gifts.
| |
Footnotes |
Submitted July 14, 1999; accepted September 29, 1999.
Supported by the National Cancer Institute of Canada
(NCIC), with funds from the Terry Fox Run (Toronto, ON,
Canada), and by grant PO1-HL55435 from the National Institutes of
Health (Bethesda, MD).
J. A. holds a studentship from the Natural Sciences and Engineering
Research Council of Canada (Ottawa, ON, Canada) and a scholarship from
the Science Council of British Columbia (Vancouver, BC, Canada). C. E. is a Terry Fox Research Scientist of the NCIC.
Reprints: Connie J. Eaves, Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada; e-mail:
connie{at}terryfox.ubc.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction