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RAPID COMMUNICATION
Quantitation of the Quiescent Fraction of Long-Term Culture-Initiating
Cells in Normal Human Blood and Marrow and the Kinetics of Their
Growth Factor-Stimulated Entry Into S-Phase In Vitro
By L. Ponchio, E. Conneally, and C. Eaves
A method for quantitating the proportion of cycling longterm culture-initiatingcells (LTC-IC) in heterogeneous populations of humanhematopoietic cells is described. This procedure involves incubating the cells of interest for 16 t o 24
hours in a serum-free medium containing 100 ng/mL Steel
factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF), with or without
20 pCi/mL of high specific activity 'H-thymidine 13H-Tdr)before plating the recovered cells in standard LTC-IC assays.
The details ofthis procedure are based in part on the finding
that thenumber ofLTC-IC (regardless of their cycling status)
remains constant for at least 24 hours under these culture
conditions, as long as 3H-Tdr is notpresent. In addition, we
have determined that a 16-hour period of exposure t o the
'H-Tdr is sufficient t o maximize the discrimination of cycling
LTC-IC but not longenough t o allow a detectable redistribution of LTC-IC between noncyclingandcyclingcompartments. Finally, any isotope reutilization that may occur is
not sufficient t o affect the LTC-IC 3H-Tdrsuicide values mea-
sured. Application of this methodologyt o normally circulating LTC-IC showed these t o be a primarily quiescent population. However, within 72 hours ofincubation in a serum-free
medium containing SF, IL-3, and G-CSF, most had entered
S-phase, although there was no netchange in their numbers.
This suggests that, under certain conditions in vitro, selfrenewal divisions of LTC-IC can occur and, at least initially,
balance any losses of these cells due t o their differentiation
or death. In contrast, many of the LTC-IC in freshly aspirated
samples of normal marrow were foundt o be proliferating,
although those that were initially quiescent could also be
recruited into S-phase within 72 hours in vitro when incubated under the same conditions used t o stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should
facilitate further investigationof the mechanisms regulating
the turnoverof the most primitivecompartments of human
hematopoietic cells and howthese may bealtered in disease
states or exploited for a variety of therapeutic applications.
0 1995 by The American Societyof Hematology.
R
sults were obtained for cells that give rise to CFU-S' and
subsequentlyfor cells assayedspecifically for theirlongterm in vivo repopulatinga b i l i t ~More
. ~ recently, direct measurement of the DNA contentof highly purified populations
of long-term repopulating cells has shown that as many as
20% of those present in the marrow of adult mice are in Sphase, G?, or mitosis.' In humans, a quantitative in vivo
assay for long-term lympho-myeloid repopulating cells does
not yet exist. Nevertheless, evidence of a largequiescent
fraction within compartments of primitive (high proliferative
potential and/or multilineage) progenitor cells detectable in
standard in vitro colony assays has existed for many years."'
Recently, we have described another type of in vitro assayX
that detects a distinct populationof primitive human hematopoietic progenitors that do not, themselves, form colonies of
myeloid or erythroid cells in semisolid media (unpublished
findings, July 1995) but that, in the presence of supportive
fibroblasts, can give rise over a period of at least S weeks
to cells that do have this property. Because this latter mode
of primitive hematopoietic cell stimulation is a defining feature of the long-term marrow culture system, wehave called
the cells detected by this assay long-term culture-initiating
cells or LTC-IC.' Variable proportions of the LTC-IC present in normal human marrow or blood are small, light-density cells,that express no or low levels of HLA-DR," or
CD.38,"' do not retain rhodamine-123." and are resistant to
high concentrations of S-FU undercertainconditions
in
vitro." All of thesefindingshavesuggested
that many of
the LTC-IC in adult humans are unlikely to be cycling. In
the murine system, analogously defined LTC-IC have also
been found to exhibit a rhodamine-123 dull phenotype"."
and to show the same resistance to S-FU in vivoL5as totipotent long-term in vivo repopulating cells with which LTCIC copurify.'"'" In a previous study,we found that equivalent
numbers of human LTC-IC could be maintained over a S -
EGULATION OF hematopoiesis is a complex process
that operates at every level of hematopoietic cell differentiation and involvesavarietyofmechanisms.Potentially important playersin this processare those extracellular
factors that effect changes in the cell-cycle status of the most
primitive hematopoietic cellsresponsible for maintaining the
entire system throughout life. It is generally assumed that,
at any given time, the majority of these cells in normal adults
are in a quiescent (Go) state, although this does not exclude
the possibility thatthe population as a wholeturns over
slowly. The concept that most hematopoietic stem cells are
quiescent was initially prompted by the finding that colonyforming units-spleen (CFU-S) in the marrow of adult mice
are relatively resistant to a short exposure to high specific
activity 'H-thymidine ('H-Tdr)' or othercycle-specific drugs
like 5-fluorouracil (S-FU) or vinblastine.2 Later, similar reFronz the Terry Fox Luborutory, British Columbiu
Cuncer
Agency; and the Departments of Medical Genetics und Pathology
und Luborutoty Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Submitted July 12, 1995; accepted August 22, 1995.
Supported by the Nutional Cuncer Institute of Cunudu (NCICI
with fund., from the Terry Fox Foundation and by Sundo: Internationul. L.P. w u s U recipient of funds from the Associazione Italiana
per la Ricerca sul Cancro; E.C. held an NCIC Postdoctorul Fellow
ship; und C.E. is a Terry Fox Cuncer Research Scientist of the
NCIC.
Address reprint requests t o C. Eaves, PhD, TerryFox Laborutory,
601 W 10th Ave, Vancouver, BC, Canada V5Z IL3.
The publication costsof this articlewere defrayed in part by puge
chargepuyment.This
article must therefore be hereby marked
"advertisement" in accordance with I8 U.S.C. section 1734 solely to
indicate this j h .
0 I995 by The Americun Society of Hematology.
0006-4971/95/8609-0045$3.00/0
3314
Blood, Vol 86,No 9 (November 1). 1995:pp 3314-3321
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KINETICS OF ENTRYINTO
CYCLE OF H U M A N LTC-IC
331 5
routinely diluted to 2 X IO4 cellslml in fresh medium as soon as
they reached a concentration of 1O"lmL. To obtain a synchronized
population, Mo7e cells in the log growth phase were washed twice
andincubatedfor 24 hoursat 37°C in freshserum-free Iscove's
of this time, 10 ng/mL
DMEM without growth factors. At the end
of IL-3 and 50 ng/mL of S F were added to the cultures. Cells were
then analyzed for their DNA content
by staining with propidium
6, 12, 18, and 24 hours
iodide (PI, from Sigma) immediately and
30 minutes
later.This was performedbyincubatingthecellsfor
with SO p g of PIlmLand0.6%NP40(Calbiochem,SanDiego,
CA) in phosphate-buffered saline (PBS, from STI).
Flow cytometric
analyses were performed using the
Cellfit program on a FACSort
flow cytometer(BectonDickinsonlmmunocytometrySystems,
Mountain View, CA). Chick erythroid nuclei
(DNA-QC particles;
Becton Dickinson) stained with PI were used as a standard to optimize the instruments settings.
Tritiated "H-Tdr suicide measurements. Forexperiments with
blood and marrow progenitors, the cells were initially suspended at
5 2 X IO'lmL, or as reported, in Iscove's DMEM containing 5 X
IO-' m o l n P-mercaptoethanol and either 30% dialyzed FCS (cutoff 12,000to14,000molecularweight,
initial experimentsonly,
Table 1) or, more typically, a defined serum substitute (BIT, obtained
from ST1 and diluted in the medium to give a final concentration of
I O pg/mL human
20 mg/mL of deionized bovine serum albumin,
or without
insulin, 200 pglmL iron-saturated human transferrin) with
40 pglmL low-density lipoproteins (Sigma) and with or without 100
ng/mL SF, 20ng/mL IL-3, and 20 nglmLG-CSF, as indicated.
Equal volumes of cell suspension were then placed in 35-mm Petri
dishes(STI), in thepresence or absence of 20pCi/mLofhighspecific activity 'H-Tdr (25 Cilmmol; Amersham, Oakville, Ontario,
Canada) and incubated at 37°C in an atmosphere of S% COz in air
for the indicated period (16 hours unless noted otherwise).
The cells
were then transferred to a tube, washed twice with a solution containing excess cold Tdr (400 p g h L in the first wash, 40 pglmL in
the second wash), resuspended in Iscove's DMEM, and appropriate
quantities from each group then assayed for LTC-IC and colonyMATERIALS AND METHODS
forming cells (CFC).In the experiments with Mo7e cells, these were
initially suspended at a concentration of 2X 10' cellslmL of serumGrowthfacrors. Highly purified recombinant human interleukinfree Iscove's DMEM containing the BIT serum substitute plus
50
3 (IL-3) and recombinant human granulocyte-macrophage colonynglmL S F and 10 ng/mL IL-3 and then incubated for 18 hours in
stimulating factor (GM-CSF) werekindly provided by Sandoz Interthe presence of 'H-Tdr before being washed and then plated in a
national (Basel, Switzerland), and recombinant human G-CSF and
similar 0.9% methylcellulose medium asthat used for assays of CFC
recombinant human Steel factor (SF) by Amgen (Thousand Oaks,
in this case, the
CA). Recombinant IL-6 was obtained in the form of a crude superna- from normal human blood and marrow. However,
final mediumcontained 10% mediumconditioned by 5637cells
a human IL-6 cDNA. Highly
tant of COS cells transiently expressing
(from the American Type Culture Collection, Rockville, MD) and
purified human erythropoietin (Epo) was obtained from StemCell
2 ng/mL IL-3 to support colony formation by viable Mo7e cells.
Technologies, Inc (STI, Vancouver, British Columbia, Canada).
Progenitor assays. For assays of LTC-IC in marrow cell suspenCell separation. Human blood was obtained with informed consions, aliquots of these cells were placed
in 35-mm tissue culture
sent from normal volunteer blood donors undergoing plateletneudishes containing 2 mL of myeloid LTC medium (STI) supplemented
kapheresis at the Vancouver Health Sciences Centre. The fraction
just prior to use with IO-' m o l k freshly dissolved hydrocortisone
of mononuclear (MN) cells was isolated
by centrifugation of the
sodium hemisuccinate (Sigma), and a preestablished feeder layer of
cells on Ficoll-Hypaque (1.077 g/mL; Pharmacia Biotech, Uppsala,
3 X IO' irradiated(8,000cGy)murinefibroblastsengineeredto
T lymphocytes
Sweden) and these cells were then further depleted
of
produce human IL-3, SF, and G-CSF." These cultures were main(MNT-) byrosettingwithsheepred
blood cells" toreducethe
tained at 37°C for 6 weeks with weekly replacement of half of the
probability of a subsequent outgrowth in culture of latently Epsteinmedium and nonadherent cells with fresh LTC medium. At the end
Barr virus (EBV)-infected B cells." Human bone marrow was obtained from informed and consenting allogeneic bone marrow transof the 6 weeks, all ofthenonadherentcellswereremovedand
plant donorsatthetimeofmarrowharvest.Viablelightdensity
combined with cells harvested from the adherent fraction
by trypsinmarrow cells were isolated from both sources by centrifugation on
ization." These cells were then washed and assayed for their CFC
Ficoll-Hypaque.
content as described below. For assays of LTC-IC from peripheral
DNA analysis, synchronization. and cell-cycle analysis of Mo7e
blood, the same general procedure was followed but irradiated M2cells. Mo7ecells,ahumanfactor-dependentmegakaryocytic
leu10B4 fibroblastsz7 (not genetically engineered) were
used and the
kemic cell line,24 were maintainedin Iscove's Dulbecco's Minimum
LTC-IC assay cultures were maintained at 33°C for S weeks before
Essential Medium (DMEM) supplemented with10%fetal calf serum
procurement and assessment of their CFC content. This latter protomoll
(FCS;STI), 5 ng/mLof IL-3, 50 ng/mLof SF, and5 X
col was followed after initial studies showed that T-cell depletion
L 0-mercaptoethanol (Sigma Chemicals, St Louis, MO), and were
alone was frequently insufficient to prevent the activation of EBV-
week period under three different conditions of culture,"
although whether any of the LTC-IC present at the end of
this time had proliferated was not specifically investigated.
More recently, several examples of culture systems that
allow a net expansion of marrow LTC-IC numbers to be
obtained have been reported."^" In addition, the cycling
characteristics of highly purified populations of phenotypically defined human hematopoietic cells (that should be enriched in their LTC-IC content) have been shown to change
after the in vitro exposure of these cells to various cytokines.*'.'' Thus, there have been a number of findings to
suggest that the majority of LTC-IC in the marrowof normal
adults might be quiescent although also able to proliferate
in response to stimulation by different factors (or combinations of factors) in vitro. On the other hand, both the time
course of such responses and associated consequences o n
the biologic potentialities of the LTC-IC thus stimulated
have remained obscure.
In this report we describe the validation and initial application of a general strategy for quantitating the G, fraction of
human LTC-IC in heterogeneous cell populations that, for
the first time, has begun to allow such issues to be rigorously
investigated. The procedure involves a prolonged (16- to 24hour exposure) of the cells to high specific activity 'H-Tdr
to achieve a readily detectable differential survival of cycling
versus noncycling LTC-IC. Using this procedure we have
found that most circulating LTC-IC are normally quiescent
but that many LTC-IC in normal marrow are proliferating
and, in both cases, the absolute number of cycling LTC-IC
can be increased by incubation of the cells for 72 hours in
a defined medium containing a simple cytokine cocktail.
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PONCHIO, CONNEALLY, AND EAVES
3316
Table 1. Effects of Incubation (for 16 hours) and Exposure to High Specific Activity 'H-Tdr on CFC and LTC-IC
in Cultures of Freshly Isolated Circulating MNT- Cells
% of Input After 16 h (in
the
Progenitor
Total CFC
CFU-E
Total BFU-E
BFU-E (>l6clusters)*
CFU-GM
CFU-GEMM
LTC-IC
20% dFCS
163 2 21 (4)
162 2 61 (4)
123 2 29 (4)
186 2 53 (4)
88 i- 1 1 (41
125 t 63 (4)
129 2 29 (4)
absence of 'H-Tdr)*
SS-GF
SS
105 2 13 (6)
47 2(3)
6
117 2 18 (6)
293 5 85 (51
91 i- 1 1 (6)
83 t 8 (5)
108 -t 40 (4)
i
% Survival After 16 h (in
the
GF
103 5 4 (18)
103 f(2)
8
118 2 6 (18)
147 2 22 (18)
104 f 8 (18)
150 f 28 (12)
85 i- 15 (6)
20% dFCS
96 i- 8 (4)
64 i- (4)
24
102
84 t 12 (4)
104 2 19 (41
104 i- 13 (4)
78 i- 10 (4)
100 t 15 (4)
Presence of ? - T d r ) t
SS-GF
97 i- 10 (6)
i-(3)
6
83 2 14 (6)
80 2 18 (5)
105 i- 13 (6)
90 2 19 (5)
86 i- 20 (4)
SS
+ GF
87 2 5 (18)
79 f 46 (2)
90 i- 6 (18)
88 1: 6 118)
88 i- 7 (18)
82 t 10 (12)
78 i- 18 (6)
Cells were incubated in medium containing either dialyzed FCS (dFCS) without added growth factors (GF), or a serum substitute ( S S ) with
or without GF as shown. GF = 100 ng/mL of SF and 20 ng/mL each of IL-3 and G-CSF. Data refer to mean i- SEM (number of experiments).
* Progenitor number after 16 h in the absence of 3H-Tdr expressed as a percent of the number detected at the startof the incubation.
t Progenitor number after 16 h in the presence of 3H-Tdr expressed as a percent of the number detected after 16 h in the absence of 3H-Tdr.
BFU-E (>l6clusters) = BFU-E that produce very large colonies consisting
of more than 16 clusters of hemoglobin-containing erythroblasts.
*
infected B cells that occurred when most samples of normal peripheral blood were cultured on the cytokine-producing fibroblasts. Interestingly, this phenomenon was rarely encountered with marrow cells
even when these had not been T-cell depleted.
Cell suspensions from primary or cultured bloodandmarrow
samples, and from LTC-IC assays were assayed for erythroid colonyforming units (CFU-E), burst-forming units-erythroid (BFU-E),
granulocyte-macrophagecolony-forming units (CFU-GM), and granulocyte-erythroid-monocyte-megakaryocytecolony-forming units
(CFU-GEMM) by plating appropriate numbers of the cells in FCSsupplemented methylcellulose medium (STI) containing the following growth factors: 3 UlmL of Epo and 20 nglrnL each of IL-3,
GM-CSF, IL-6, and G-CSF, and 50 ng/mL of SF. Colonies were
then scored in situ using well-established criteria' after 16 to 18
days of incubation of the cultures at 37°C in a humidified atmosphere
of 5% CO2 in air.
RESULTS
Test of an 18-hour exposure to 'H-Tdr using M07e cells as
a model of a cycling population. When factor-dependentMo7e cells were synchronized by growth factor deprivation
for 24 hours, as described in Materials and Methods, subsequent PI staining showed that a relatively homogeneous population of viable cells blocked in G , could be obtained. The
results of a representative experiment are shown in Fig 1.
B
When 50 ng/mL of SF and 10 ng/mL of IL-3 werethen
added to these cells, most could be seen to have completed
a first mitotic cycle within the following 18 to 24 hours by
whichtimetheir
distribution between G,S and G2M had
become indistinguishable from that seen in standard exponentially growing cultures (Fig 1C). The hypothesis that incubation of a completely cycling population of cells with
'H-Tdr for a period of time equal to the length of the cell
cycle would kill all of the cells due to their passage through
S was then tested by first incubating exponentially growing
Mo7e cells for 18 hours with 'H-Tdr and thenassessing their
consequent loss of proliferative ability by measurement of
their colony-forming ability in a methylcellulose-containing
growth medium. An IS-hour exposure to 'H-Tdr consistently
killed greater than 95% of the Mo7e cells thus treated (five
experiments) and this effect could be completely blocked by
the addition of excess cold Tdr to the initial 'H-Tdr-containing incubation medium.
Cycling characteristics of normally circulating CFC and
LTC-IC. A first set of experiments was then undertaken to
investigate whether a similar procedure could beusedto
assess the cycling status of primitive hematopoietic progenitors in fresh samples of normal human blood. For this, CFC
were chosen as an initial test population because previous
C
Fig 1. Cell-cycle analysis of Mo7e cells before and after their release from G, in a representative experiment. Cells were blocked in GI after
24 hours of incubation in the absence of SF and IL-3 (A). By 12 hours after the subsequent addition of SF and IL-3 (B), some of the cells had
entered S phase but very few had reached G2 or M. By 18 hours (C), the cells showed the same distribution between the different phases of
the cell cycle as exponentially growing M07e cells. Similar results were obtained in two other experiments except that in these the type of
profile seen in (B) was notreached until 18 hours after adding SF plus IL-3 and the typeof profile seen in (C) was thenapparent after 24 hours.
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3317
KINETICS OF ENTRY INTO CYCLE OF HUMAN LTC-IC
cycling studies using the standard 'H-Tdr suicide technique
(which involves a 20-minute exposure to 'H-Tdr') had indicated that these progenitors, regardless of their differentiation or proliferative potential, did not contain a detectable
fraction of cycling cells.28 Thus, in direct contrast to the
results just described for exponentially growing Mo7e cells,
it was anticipated that circulating CFC might survive somewhat longer periods of exposure to 'H-Tdr (than 20 minutes).
As shown in Table I , when MNT" cells from a large number
of normal bloodsamples were incubated at 37°C for 16 hours
in either the presence or absence of 'H-Tdr, there was no
decrease in the number of any type of circulating CFC (or
LTC-IC), regardless of whether the medium contained 20%
dialyzed FCS or a serum substitute, or whether it also contained a mixture of SF (100 ng/mL), IL-3 (20 ng/mL), and
G-CSF (20 ng/mL). This indicates that very few circulating
progenitors of any type will die, differentiate, or enter Sphase within 16 hours of culture under the various conditions
tested, despite the fact that the growth factor cocktail used
can stimulate both circulating CFC and LTC-IC to proliferate
after longer periods of incubation (see below). The high
survival of all progenitor types seen even in a medium containing a defined serum substitute but no additional growth
factors is consistent with findings previously reported for
quiescent blast CFC isolated from normalmarrow.29 The
choice of an SF, IL-3, G-CSF cocktail at the concentrations
used here was based on previous data showing that such a
cocktail gives comparable maintenance of LTC-IC in cultures without marrow adherent layer feeders as is obtained
in cultures with such feeders (either with or without the
further addition of this combination of growth factor^).'^
The possibility that the concentration of radionucleoside
used might have been insufficient to allow the detection of
cycling LTC-IC in the peripheral blood was excluded both
by the results of 'H-Tdr dose response experiments (and by
the time course experiments described below). Comparison
of the effects of exposing cells to 20 versus 100 pCi of 'HTdr/mL showed that 20 yCi of 'H-Tdr/mL is sufficient to
achieve maximum kill under the conditions used (survival
of CFC was 99% t 1 1% and 92% 5 IS%, and of LTC-IC
was 84% t- 28%and69% ? 13%at 20 pCi/mL and 100
pCi/mL, respectively, n = 4).
Kinetics of recruitment into S-phase of initially quiescent
CFC and LTC-IC from normal blood. When MNT- cells
isolated from normal blood were incubated for as long as
96 hours in serum-free medium containing SF (100 ng/mL),
IL-3 (20 ng/mL), and G-CSF (20 ng/mL), some expansion
of total CFC numbers (2- to IO-fold) wasobserved (Fig 2A).
In contrast, the number of LTC-IC did not change. As shown
in Fig 3, the greatest expansion of CFC occurred in the more
mature compartments of erythroid progenitors (CFU-E and
those BFU-E that generate relatively small erythroid bursts,
ie, those ultimately consisting of less than 9 erythroblast
clusters). Interestingly, the increase in these progenitors was
not associated with a detectable reduction in the number of
more primitive CFC (Fig 3) or LTC-IC (Fig 2) present after
96 hours. Fig2B also shows that the persisting primitive
CFC and LTC-IC had started to become sensitive to the
presence of 'H-Tdr after between 16 and 48 hours with less
1
0.11
0724824
96
Time (hrs)
Fig 2. Population changes (A, no 'H-Tdr) and kinetics of recruitment into S phase IB) of CFC (squares) and LTC-IC (circles) when
MNT- normal blood cells were cultured over a period of 96 hours in
a medium containing a serum substitute plus SF I 1 0 0 nglmLI, IL-3
(20 nglmL1, and G-CSF 120 nglmL1. In (B), control values are those
obtained at the corresponding time point for cultures to which no
'H-Tdr was added. Eachpoint represents the mean c SEM of values
measured in three independent experiments.
than 5% of the input LTC-IC and less than 2% of the input
CFC remaining resistant to the 'H-Tdr after 72 hours. This
suggests that by the end of 72 hours under these conditions,
the majority of the cells in both of these functionally defined
progenitor populations had switched from a quiescent to a
proliferating state.
Optimization and validation of a 16-hour 'H-Tdr suicide
procedure for assessing the cycling status of LTC-IC. To
define the minimum time required to achieve a maximum
'H-Tdr-dependent inactivation of cycling LTC-IC (and
primitive CFC), freshly isolated MNT- blood cells were
incubated for 72 hours in medium containing SF, IL-3, and
G-CSF as described above to stimulate most of these progenitors to proliferate, but the addition of 'H-Tdr was delayed
until 8, 12, 16, or 24 hours before completing the 72-hour
period of incubation. In addition, unmanipulated aliquots of
MNT- cells from the same blood samples were incubated
in the same medium plus (or minus) 'H-Tdr for 16, 20, or
24 hours to obtain a more precise measurement of the maxi-
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3318
PONCHIO,CONNEALLY, AND EAVES
I"ejf 48 hr
72 hr
96 hr
-
I
103
3
T
n
c
-
i
W-
O
s 102
lo' CFU-E
BFU-E <9 cl
BFU-E >9cl
CFU-GM
mum time that quiescent progenitors can be maintained under the same conditions before an effect of exposure to 'HTdr would be seen. AS shown in Fig 4, assessment of the
LTC-IC and primitive CFC numbers in these cultures revealed that both types of progenitors remained insensitive
'
O
0
I
I
..~
...
J
0.1
0
I
1
I
8
16
24
Time of exposure to 3H-Tdr (hrs)
Fig 4. Establishment of a minimum time of exposure t o 'H-Tdr
sufficient t o obtain maximum kill of cycling
LTC-IC ( 0 )with minimal
toxicity t o quiescent LTC-IC (0).To determine the
latter, the survival
of LTC-IC was measured in cultures of freshly isolated MNT- blood
cells incubated in the presence or absence of 3H-Tdr in medium containing a serum substitute andSF (100 ng/mL), IL-3 (20 nglmL), and
shown. To determine the
former, 3HG-CSF (20 ng/mL) for the times
Tdr was added (or not) t o similarly initiated cultures only during the
last 8, 12, 16, or 24 hours of a total incubation period of 72 hours.
For comparison, the results obtained for simultaneously assessed
primitive CFC (squares) are also shown. Values shown represent the
mean 2 SEM from three t o five experiments.
CFU-GEMM
Fig 3. Changes in the numbers of different subsets of CFC when MNT- normal blood cells were
cultured for 96 hours in a medium containing a serum substitute, SF (100 ng/mL), IL-3 (20 ng/mL), and
G-CSF (20 ng/mL). A net expansion of the more mature typesof CFC was notassociated with a concomitant decrease in more primitive types ofCFC. Each
point represents the mean 2 SEM of values measured in the same three experiments shown in Fig
2.
to 'H-Tdr for up to 24 hours (open symbols) but, on activation (after a more prolonged incubation in the presence of
SF, IL-3, and G-CSF), an additional period of exposure to
'H-Tdr for I6 hours was sufficient to reduce the numbers of
both of these types of progenitors to a minimum value
(closed symbols). Taken together with the results presented
in the previous section, exposure of cycling LTC-IC (or
CFC) to 'H-Tdr for a period of I6 to 24 hours thus appeared
to be sufficient to achieve a maximum kill of the population,
but was still short enough to keep any entry of noncycling
LTC-IC (or CFC) into S-phase at undetectable levels.
The potential dependence of the progenitor kill obtained
in this 16- to 24-hour 'H-Tdr suicide protocol on the concentration of cells present during their exposure to the 'H-Tdr
was then evaluated. For these experiments, freshly isolated
MNT- peripheral blood cells were first incubated at different
cell concentrations for an initial 72 hours with SF, IL-3,
and G-CSF (but no 'H-Tdr) to obtain maximally activated
populations of LTC-IC (and CFC). They were then incubated
for an additional 16 hours in the presence (or absence) of
'H-Tdr, at the end of which progenitor assays were performed. The results of these experiments showed that exposure of cycling progenitors to 'H-Tdr at a concentration of
5 2 X IO6 cells/mL gave the most reproducible kill values
whereas at higher cell concentrations, spuriously low values
were sometimes encountered (data not shown).
The possibility of 'H-Tdr reutilization occurring in the
LTC-IC or CFC assays performed on cells obtained after
a 16- to 24-hour exposure to 'H-Tdr was thentested by
determining whether the detection of cells that had not been
exposed to 'H-Tdr was altered when these were assayed in
the presence of irradiated cells thathad previously been
allowed to incorporate 'H-Tdr. For these experiments, a population of proliferating cells was first obtained by incubating
MNT- cells (from normal blood) for 72 hours in the presence
of SF, IL-3, and G-CSF. These cells were then split into two
groups, each of which was then incubated for a further 16
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3319
KINETICS OF ENTRY INTO CYCLE OF HUMAN LTC-IC
Table 2. Lack of Evidence for Any EffectiveReutilization of 'H-Tdr
by CFC and LTC-IC After a 16-Hour Period of Exposure
Progenitor
CFC
LTC-IC
Cells Added to Assay
3H-Tdr
treated
irradiated
and
only
Irradiated
3H-Tdr treated
irradiated
and
only
Irradiated
Effect of Added Cells
on the No. of
Progenitors Detected*
70
77
27
83
-t
10
2 9
5 4
2 53
* Expressed as a % of controls containing no added cells, mean 2
SEM ( n = 3). The details of the experimental design are described in
the text.
hours with SF, IL-3, and G-CSF; one group with 3H-Tdrand
one without. At the end of this 16-hour period, aliquots of
the cells exposed to 3H-Tdr(or not, as indicated) were irradiated with 1,500cGy and combined (or not, as indicated) with
the cells that had not been exposed to 'H-Tdr (or irradiated).
Aliquots of these mixtures were then plated in LTC-IC and
CFC assays. The results, shown in Table 2, provide no evidence that the detection of LTC-IC or CFC is decreased by
the concomitant presence of dying cells that have previously
been allowed to incorporate suicidal doses of 'H-Tdr.
Measurement of the cycling status and activation kinetics
of LTC-IC from normal marrow. The 16-hour 'H-Tdr suicide procedure was then used to assess the cycling status of
the LTC-IC population present both in freshly isolated normal marrow aspirate MN cells and in cultures of these cells
that had been maintained for 72 hours in serum-free medium
containing SF (100 ng/mL), IL-3 (20 ng/mL), and G-CSF
(20 ng/mL). The results of measurements performed on fresh
marrow samples from a total of seven different individuals
are shown in comparison to results for 20 normalblood
samples in Table 3. In general, percent kill values for marrow
LTC-IC are considerably higher than those measured for
peripheral blood LTC-IC. However, both sources of LTCIC responded similarly when cultured under the same conditions. This can be seen by comparing the results for marrow
LTC-IC, shown in Fig 5 , with those described previously
for peripheral blood (Figs 2 and 4). In the absence of any
exposure to 'H-Tdr, marrow LTC-IC numbers were sustained over the total period of incubation (88 hours), but did
not change significantly (P> .05). Nevertheless, by 72 hours
many of the quiescent LTC-IC present initially had begun
to proliferate because incubating them for an additional 16
hours in the presence of 'H-Tdr further decreased their sur-
Table 3. Differences Between the Cycling Status of LTC-IC in
Individual Samples of Fresh Normal Human Blood
and Bone Marrow
% Kill Values
Source of
LTC-IC
Blood
Marrow
By Experiment
58,
53,
51,
50,
48,
44,
34,
23,
22,
19
-3, -6, -8, -11, -17, -25,
-29, -54, -118
82,
84,
85,
92,
93,
98,
58
Mean
2
SEM (n)
2 8
85
2
(20)
5 (7)
i"
'
0.1 0
24
48
72
Time (hrs)
Fig 5. Changes in marrow LTC-IC numbers over time (A, no 3HTdr) and kinetics ofrecruitment into S phase (B)of LTC-ICin cultures
of normal marrow MN cells incubated in serum-free medium containing 100 ng/mL SF,20 ng/mL 11-3. and 20 ng/mL G-CSF. In (B),
'H-Tdr was added to the test cultures for the last 16 hours of the
period shown on the abcissa and control values are those obtained
after the same total period of incubation in the absence of any 3HTdr.
viva1 below that measured when cells from the input suspensions were similarly exposed to 'H-Tdr for 16 hours (percent
kill values of the input cells = 83% 2 7% and after 72 hours
= 96% ? 2%, n = 5).
DISCUSSION
In this study we have described a procedure that allows
the cycling status of the entire LTC-IC compartment in heterogeneous populations of human cells to be quantitatively
assessed. This required the use of an indirect method because
LTC-IC cannot, as yet, be uniquely and directly identified
by any combination of morphologic or phenotypic properties. The 'H-Tdr suicide procedure offered an obvious approach. This procedure was introduced approximately 30
years ago to allow evaluation of the cycling status of murine
CFU-S' and has since been used extensively in a variety of
studies, including those designed to investigate the cycling
behavior of human CFC. However, with this procedure, only
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3320
cells in S-phase are killed because the period of exposure to
?H-Tdr is limited (typically to 20 ~ninutes).As a result, the
maximurn difference in percent kill values that cat1 be obtained between conlpletely cycling and completely quiescent
populationsisrathersmall
since thevaluesobtainedare
likely t o range anywhere from 30%)to 70% and from ---20'%
to 20%. respectively. These ranges reflect both the inaccuracies inherent in the quantitation of hematopoietic progenitors
using conventionalCFC assaya as wellasvariations
ex-.
pected in the behavior of cells conaidered to be biologically
similar. The detection of LTC-IC involves an even longer
period of culture ( 5 to 6 weeks before CFC assays are performed) and thevariability between replicate LTC-IC assays
is thus greater than that for direct CFC assays. Therefore,
we investigatedthepossibility
of obtainingtheincreased
differential kill required to assess LTC-IC by prolonging the
period of their exposure to high specitic activity 'H-Tdr.
Preliminary experiments, first using Mo7e cells and then
circulating CFC as models of cycling and quiescent populations, respectively, suggested that this approach was feasible
and likely to be useful. Conditions of exposure to 'H-Tdr
involving incubation of the test cells at a concentrationof 5 2
X IOh cells/mL for 16 to 24 hours in a serum-free medium
containing SF,IL-3, and G-CSF appeared sufficient to allow
optimal discrimination of cycling and noncycling LTC-IC
populations. Appropriate controls showedthat when the procedure described here was followed, there was no effective
reutilization of 'H-Tdr, that neither the number nor the cycling status of LTC-IC changed signiticantly during the 16to 24-hour period of exposure to 'H-Tdr, and that both the
concentration of 'H-Tdr used and the period of exposure to
'H-Tdr were sufficient to obtain a maxitnum kill value.
Application of this procedureto evaluatethe LTC-IC present in normal blood and marrow sanlples showed the former
to bea largely quiescent population and thelatter to represent
a variable mixture of quiescent and cycling cells. These tindings for marrow LTC-IC, although not anticipated, are, nevertheless, consistent with previous phenotype data that have
also suggested a larger proportion of the LTC-IC i n marrow
as compared with blood to have features of activated cells
on the basis of their size, expression of HLA-DK, ability to
retain rhodamine-l 23, and sensitivity to 4-hydroperoxycyclophosphamide.3" With the more recent introduction of
FACS technologies foranalyzing thecell-cyclestatusof
viable cells using DNA stains, it shouldsoon be possible
toobtain an independentevaluationofthe
differences in
proliferative behaviour of LTC-IC in normal blood and marrow suggested by the data presented here.
In this study we have also provided a first description of
the kinetics of human peripheral blood and marrow LTC-IC
activation by a combination of growth factorspreviously
shown to support the maintenance of marrowLTC-ICas
effectively as a marrow-derived tibroblast-containing feeder
layer." The extent of conversion of the LTC-IC population
from a noncycling to a cycling state observed without any
decrease in their numbers (Figs 2 and 5 ) implies that these
conditions also support LTC-IC self-renewal divisions. Recent experiments using single-cell cultures have confirmed
this prediction." Such tindings highlight only one of many
PONCHIO, CONNEALLY, AND EAVES
possible areas where future application of this methodology
xhould provide a powerful approachto the further elucidation
of mechanisms of heruatopoietic sten1 cell regulation i n v i v o
and in vitro. These are likely to includetheidentitication
of positive and negativeregulators of LTC-IC: cell-cycle
progression as well as the elucidation of potential abnormalities in thebe nlechanisnls in various disease states and their
controlled nlanipulation for itnprwed genet r a d e r and other
therapeutic applications.
ACKNOWLEDGMENT
Theauthors thank Dr Donna Hogge and the staff of the Cell
Separator Unit of the Vancouver Health Sciences Centre for assisused in these
tance i n making available the Ieukapheresis material
studies, and for providing the engineered fibroblast feeder lines and
the Mo7e cells. The expert secretarial assistanceof Irene Edelmann
is also acknowledged.
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1995 86: 3314-3321
Quantitation of the quiescent fraction of long-term culture-initiating
cells in normal human blood and marrow and the kinetics of their
growth factor-stimulated entry into S-phase in vitro
L Ponchio, E Conneally and C Eaves
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