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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4056-4064
Quantitative Long-Term Culture-Initiating Cell Assays Require
Accessory Cell Depletion That Can Be Achieved by
CD34-Enrichment or 5-Fluorouracil Exposure
By
Manfred R. Koller,
Ilana Manchel, and
Alan K. Smith
From Aastrom Biosciences, Inc, Ann Arbor, MI.
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ABSTRACT |
Characterization of hematopoietic cells and measurement of their
proliferative potential is critical in many research and clinical
applications. Because in vivo assay of human cells is not possible and
xenogeneic assays are not yet routine, in vitro assays such as the
long-term culture-initiating cell (LTC-IC) assay have been widely
adopted. This study investigated LTC-IC assay linearity and
reproducibility and resulting implications with respect to quantitation
of primitive cell expansion. Measurement of secondary colony-forming
cells (2° CFCs) from 5-week cultures of bone marrow (BM)
mononuclear cells (MNCs) showed that 2° CFC frequency varied with
assay plating density in a nonlinear fashion. The measured 2° CFC
frequency increased from 4.6 to 63.8 (per 105 MNCs) as
assay plating density was decreased from 5 × 105 to 2 × 104 MNCs per well (P < 10 6, n = 37). In contrast, assay of CD34-enriched cells was linear within the
range studied. Assays of cells obtained from expansion cultures
initiated with either MNCs or CD34-enriched cells were also nonlinear.
Consequently, calculated 2° CFC expansion ratios were ambiguous and
dependent on the assay plating densities used. Limiting dilution
analysis (LDA) results were also nonlinear, with LTC-IC frequency
increasing from 8.2 to 22.4 per 105 MNCs (P < 104, n = 100) as assay plating densities were
decreased. Despite the nonlinearity, 2° CFC and LTC-IC assay
results were consistent and reproducible over time with different
samples and techniques and gave a semiquantitative indication of
relative primitive cell frequency. Although CD34-enriched cells gave
linear assay output, purification of cells for every assay is
impractical. Therefore, exposure of cells to 5-fluorouracil (5-FU) was
explored for improving assay linearity. Incubation of MNCs in 250 µg/mL 5-FU for 1 to 2 hours depleted accessory cells and resulted in
a cell population that gave linear 2° CFC readout. The
5-FU-resistant LTC-ICs accounted for 49% of the total LTC-IC
population, adding the potential benefit of restricting assay
measurement to more primitive noncycling LTC-ICs. Consequently, similar
linear assay results can be obtained with either the bulk 2° CFC or
LDA LTC-IC methods after 5-FU, but multiple plating densities are
nevertheless still required in both methods due to the greater than
100-fold range in primitive cell frequency present in normal human
donor BM.
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INTRODUCTION |
THE HEMATOPOIETIC system is composed of
many different cell types at various stages of maturity. The
characterization of these cells and measurement of their proliferative
potential is critical in a number of research and clinical
applications. The current definition of a hematopoietic stem cell
includes the ability to confer long-term repopulation of the myeloid
and lymphoid lineages of an ablated host. This activity can be greatly
enriched in certain purified murine cell populations, supporting the
hypothesis of pluripotent stem cells.1,2 Subsequent genetic
marking experiments in mice have demonstrated that long-term
engraftment of both lymphoid and myeloid lineages can indeed be
achieved by the progeny of a single cell,3 thereby
confirming the existence of true hematopoietic stem cells. Analogous in
vivo experimental evidence for clonal, pluripotent human hematopoietic
stem cells is thus far lacking. Xenogeneic transplant models have
suggested that human stem cells with long-term repopulating ability
exist, but these in vivo assays are not yet routine and suffer from low
and variable levels of human chimerism.4-7
Although human hematopoietic stem cell transplantation is widely used
to rescue patients after cytoablative therapies, quantitative in vivo
human assays for hematopoietic cells are neither ethical nor practical.
In an attempt to predict long-term in vivo repopulating ability, human
cells have been cultured to assess their longevity in vitro. For
example, the high proliferative potential colony-forming cell (HPP-CFC)
assay requires 4 weeks of culture and identifies a cell that is more
primitive than the colony-forming unit-granulocyte-macrophage (CFU-GM).8 Cells more primitive than the
HPP-CFC are measured in the long-term culture-initiating cell (LTC-IC)
assay that requires from 7 to 10 weeks of culture.8,9 This
concept has been carried even further in the extended (E)LTC-IC assay,
in which even more primitive cells are measured after as long as 16 weeks in vitro.10 It is uncertain whether these in vitro
assays are truly measuring human long-term in vivo repopulating cells.
Fortunately, a correlation between long-term in vitro and long-term in
vivo repopulating ability has been demonstrated for different murine
cell populations,11-13 suggesting that the same may be true
for human cells. Consequently, the LTC-IC assay concept has been
adopted by many investigators, using either 2° CFC measurement from
bulk cultures14-20 or limiting dilution analysis
(LDA)9,21-25 conditions to quantitate primitive human
cells.
Original reports describing the use of a long-term culture system for
the quantitation of primitive human hematopoietic cells stated that the
assay readout of 2° CFC was linearly related to the input number of
cells over a wide range tested,9,26 and this assumption is
implicit in many studies using this assay. However, recent studies
focusing on the effects of bone marrow (BM) cell inoculum density in
2-week cultures on irradiated stroma with growth factor supplementation
showed that the output of cells, CFU-GM, and LTC-IC were not linear
over any of the range of inoculum densities tested.27 These
observations prompted a detailed study into the linearity of the LTC-IC
assay using different fresh and expanded BM cell populations, and the
implications with respect to measurement of primitive cell expansion
were addressed. The quantitative measure of 2° CFC or LTC-IC was
found to be critically dependent on the removal of accessory
cells through either CD34-enrichment or 5-fluorouracil (5-FU)
exposure.
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MATERIALS AND METHODS |
Medium and cytokines.
Medium for LTC-IC assays was prepared by supplementing Iscove's
modified Dulbecco's medium (IMDM) with 10% horse serum, 10% fetal bovine serum (FBS), 4 mmol/L L-glutamine, 100 U/mL
penicillin and 100 µg/mL streptomycin (all from GIBCO, Grand Island,
NY), and 5 µmol/L hydrocortisone (Sigma, St Louis, MO). Medium for ex
vivo expansion cultures was prepared by supplementing LTC-IC assay
medium with 5 ng/mL PIXY321 (Immunex, Seattle, WA), 0.1 U/mL
erythropoietin (Epo; Amgen, Thousand Oaks, CA), and 10 ng/mL c-kit
ligand (KL; Immunex), as previously described.28
Cells and cell separation procedure.
Human BM cells were obtained with informed consent from iliac crest
aspirates or from BM processing screens (Baxter Fenwal, Deerfield, IL)
obtained after the harvest of BM from normal donors. Mononuclear cells
(MNCs) were collected by Ficoll (1.077 g/mL; Pharmacia, Uppsala,
Sweden) separation and CD34-enriched cells were collected with a MACS
laboratory separation system (Miltenyi Biotec, Auburn, CA), as
previously described.29
Flow cytometry analysis.
Cells to be analyzed were washed and resuspended in phosphate-buffered
saline (PBS; GIBCO) containing 1% bovine serum albumin (BSA; Intergen,
Purchase, NY). Tubes containing 106 cells in 0.5 mL were
stained with either phycoerythrin (PE)-HPCA-2 (anti-CD34)
or PE-IgG (control) monoclonal antibodies (Becton Dickinson, San Jose,
CA) along with a cocktail of lineage (lin)-specific antibodies:
fluorescein isothiocyanate (FITC)-Leu4 (anti-CD3), FITC-Leu12 (anti-CD20), FITC-LeuM3 (anti-CD15; all from Becton Dickinson), FITC-anti-CD11b (Serotec, Indianapolis, IN), and
FITC-anti-glycophorin A (Dako, Carpinteria, CA). After 15 minutes,
cells were washed and resuspended in 0.5 mL PBS/BSA for analysis on
either a FACS Vantage or FACScan (Becton Dickinson) flow cytometer.
Bulk 2° CFC long-term culture assay.
Five-week 2° CFCs were determined by culture on irradiated
preformed stroma using a modification29 of a previously
described procedure.9 Briefly, preformed stroma was
prepared by trypsinizing adherent stromal cells from 2-week-old primary
human BM cultures in LTC-IC medium. Cells were irradiated with 20 cGy
from a 137Cs source and were immediately plated in 24-well
plates in LTC-IC medium. Preliminary experiments determined that 5 × 104 stromal cells per well were sufficient and
maintained a nearly confluent layer of stroma for the duration of the
assay (not shown). Test cells were added to these wells at the
concentrations indicated using three to six replicates each. Plates
were maintained at 33°C in a fully humidified atmosphere of 5%
CO2 in air, and cultures were fed weekly by replacing 0.5 mL LTC-IC medium per well. At week 5, adherent and nonadherent cells
were harvested from each well as previously described.29
Cells from each well were transferred into a non-tissue culture-treated
35-mm dish (Nunc, Naperville, IL) containing methylcellulose colony
assay medium, composed of 0.9% methylcellulose (Sigma), 30% FBS, 1%
BSA, 100 µmol/L 2-mercaptoethanol (Sigma), 2 mmol/L L-glutamine
(GIBCO), 5 ng/mL PIXY321, 5 ng/mL granulocyte colony-stimulating factor
(G-CSF; Amgen), and 10 U/mL Epo. Cultures were maintained for 14 days
and were then scored as previously described.29 For each
sample, the total number of secondary colonies was enumerated and used
to calculate the frequency of 5-week 2° CFCs per 105
cells used to initiate the culture assay.
LTC-IC assay by LDA.
LTC-IC were determined by LDA of cultures on irradiated preformed
stroma using a modification28 of a previously described technique.9 Briefly, irradiated stromal cells were prepared as described above and were added to 96-well plates at 104
per well in 100 µL LTC-IC medium. Test cells were then added to these
irradiated stromal layers at four concentrations in 100 µL LTC-IC
medium per well (20 replicates each). The plates were then placed at
33°C in a fully humidified atmosphere of 5% CO2 in
air, and cultures were fed weekly by replacing 100 µL LTC-IC medium
per well. At week 5, adherent and nonadherent cells were harvested from
each well as previously described.28 Cells from each well
were added directly to 0.25 mL of colony assay medium in non-tissue
culture-treated 24-well plates (Falcon, Lincoln Park, NJ). After 14 days, wells were scored for colonies as described above. For each
sample, the number of LTC-IC was determined through an iterative
calculation procedure30 based on the maximum likelihood solution method.31
In some experiments, the murine BM-derived stromal cell line
M2-10B432 (American Type Culture Collection, Rockville, MD)
was compared with human stroma for use in the assay. The LDA readout on
primary human stroma was fivefold higher than on M2-10B4 at week 5 and eightfold higher at week 8 (P < 104, not
shown). This result, along with the previous observation that there was
little donor-to-donor variability in the ability of irradiated stroma
generated from different human BM samples to support CD34-enriched cell
growth,33 supported the use of human stroma in all assays.
5-FU exposure.
Because the observed nonlinearity in the 2° CFC and LDA LTC-IC
assays appeared to be due to accessory cells, the use of the chemical
purging agent, 5-FU, was explored. BM MNCs were suspended at 3 × 106 cells/mL in LTC-IC medium containing 20 to 1,000 µg/mL 5-FU (Sigma). After 0.5 to 24 hours of incubation at 37°C,
the cells were washed and then used to set up progenitor,29
CFU-F,34 and LDA assays. The percent kill of each
population was determined with respect to a control that was incubated
for the same period of time without 5-FU.
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RESULTS |
Linearity of the Bulk 2° CFC long-term culture assay.
Original reports describing the use of a long-term culture system for
the quantitation of primitive human hematopoietic cells stated that the
assay readout of 2° CFC was linearly related to the input number of
cells over a wide range tested.9,26 Consequently, bulk
measurement of 2° CFC generated from a test population gave results
that were similar to those obtained by LDA. The linear range for these
bulk 2° CFC assays was reported to be up to 105
low-density BM cells per well in 96-well plates and up to
106 cells per well in 24-well plates.9,26 Based
on these published results, our laboratory had performed the bulk
2° CFC assay using a single density of 5 × 105
MNCs per well in 24-well plates. However, a subsequent unrelated study
on the effect of inoculum density in 2-week MNC expansion cultures27 raised questions about the linearity of the bulk 2° CFC assay. Importantly, that study showed that cell and CFU-GM output from 2-week MNC expansion cultures changed little over a large
range of inoculum densities.27 Furthermore, the addition of
preformed stroma, which is used in 2° CFC and LTC-IC assays, caused
the cell and CFU-GM output to be flat over an even larger range of MNC
inoculum densities.27 These results showed that CFU-GM
output from 2-week MNC expansion cultures on preformed stroma was not
linearly related to the input number of cells. The implications of
these results precipitated a study on the effect of plating density in
the 5-week bulk 2° CFC assay. The first experiments were performed
using plating densities of 2.5 × 105 and 7.5 × 105 cells per well in addition to our then standard 5 × 105 cells per well (Fig
1A). In four experiments, the measured frequency of 2° CFC
(expressed per 105 cells) was significantly greater when
the assay was plated at 2.5 × 105 per well as
compared with 5 × 105 per well. The 2° CFC
frequency was on average 2.8-fold greater (P < .01) when
measured at the lower plating density.
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| Fig 1.
Measurement of 2° CFC frequencies (expressed as
number per 105 MNCs) from a series of BM MNC samples
assayed over three ranges of plating densities. Bulk long-term culture
assays were inoculated in 24-well plates at (A) 2.5 × 105, 5 × 105, and 7.5 × 105
cells per well (n = 4); (B) 3 × 104, 105,
and 2.5 × 105 cells per well (n = 7); and (C) 2 × 104, 5 × 104, and 105 cells per
well (n = 22). Each point represents the mean 2° CFC frequency
from three to six replicate cultures, and lines connect the points
performed with the same BM sample. A flat line with zero slope would
indicate a linear assay response that is unaffected by the assay
plating density.
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The results above prompted study over a wider range of plating
densities. Seven BM samples were assayed at 3 × 104,
105, and 2.5 × 105 cells per well (Fig
1B). Again, the measured 2° CFC frequency was consistently higher
from assays plated at lower densities. As compared with the values
obtained at 2.5 × 105 cells per well, 105
cells per well gave 2.0-fold higher 2° CFC frequencies (P < .001) and 3 × 104 cells per well gave 4.0-fold
higher 2° CFC frequencies (P < .001). Another set of 22 BM samples was assayed at 2 × 104, 5 × 104, and 105 cells per well (Fig 1C), and once
again, the measured 2° CFC frequencies were significantly higher at
the lower plating densities. In this case, as compared with the values
obtained at 105 cells per well, 5 × 104
cells per well gave 1.4-fold higher 2° CFC frequencies (P < .01) and 2 × 104 cells per well gave 2.0-fold
higher 2° CFC frequencies (P < .001).
Therefore, as the assay plating density was decreased, the measured
5-week 2° CFC frequency values increased to surprisingly high
levels (Table 1). The average 2° CFC
frequency measured at 5 × 105 cells per well was 4.2 per 105 MNCs, whereas the average 2° CFC frequency
measured at 2 × 104 cells per well was 63.8 per
105 MNCs, a 15-fold difference.
Accessory cells increased nonlinearity in the 2° CFC assay.
The effect of assay plating density on the measured 2° CFC
frequency within BM MNC samples may have been due to the significant number of accessory cells that are present in each culture in addition
to the primitive cells. To examine this hypothesis, assays were
performed on CD34-enriched cells and MNCs from the same donors in
parallel. CD34-enriched BM cells were plated at 1,000 and 2,500 CD34+lin cells per well. As control,
MNCs from the same donors were plated in parallel at densities to give
1,000 and 2,500 CD34+lin cells per well,
as determined by flow cytometry. Representative results from four of
eight experiments are shown (Fig 2). Over the eight experiments, CD34-enriched cell assays inoculated at 1,000 CD34+lin cells per well gave a 1.1-fold
higher 2° CFC frequency readout than those inoculated at 2,500 CD34+lin cells per well (1,787 v
1,632 per 105, P = .30). In contrast, the paired
MNC samples inoculated at 1,000 CD34+lin
cells per well gave a 1.7-fold higher 2° CFC frequency readout than
those inoculated at 2,500 CD34+lin cells
per well (75 v 43 per 105, P = .02).
Although CD34-enriched and MNC samples were inoculated to contain the
same number of CD34+lin cells per well,
presumably containing the same number of LTC-IC, the assay readout from
CD34-enriched samples was less influenced by plating density.
Therefore, the presence of accessory cells in MNC samples increased
assay nonlinearity.
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| Fig 2.
Measurement of 2° CFC frequencies (per
105 cells) from a series of paired BM MNCs and
CD34-enriched cell samples. Both MNCs and CD34-enriched cells were
plated at densities to give 1,000 and 2,500 CD34+lin cells per well. Each point
represents the mean 2° CFC frequency (±SEM) from three to six
replicate cultures, and lines connect the points performed with the
same cells. Paired CD34-enriched cells and MNCs obtained from the same
BM sample are indicated by use of the same plot symbol.
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Bulk 2° CFC assay of cells after an ex vivo expansion procedure.
The above results demonstrated nonlinearity in the bulk 2° CFC
assay, which was in part attributed to the presence of accessory cells.
Because ex vivo cell expansion procedures result in cell populations
that have considerable accessory cell content, the assay of cells after
expansion culture was assessed next. BM MNCs expanded in 12-day
perfusion cultures with PIXY321, KL, and Epo, as previously
described,28 were assayed for 5-week 2° CFC content at
several plating densities. These MNC samples displayed nonlinearity in
the assay both before and after the expansion procedure
(Fig 3). The consequences of this
nonlinearity for the determination of expansion ratios were
significant. In a representative experiment, using the various
combinations of fresh and expanded cell assay plating densities, the
2° CFC expansion ratio was calculated to be anywhere from 0.4- to
4.2-fold (Fig 4).
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| Fig 3.
Measurement of 2° CFC frequencies (per
105 cells) from a series of paired fresh and expanded BM
MNC samples. Each point represents the mean 2° CFC frequency from
three to six replicate cultures, and lines connect the points performed
with the same cells. Paired fresh and expanded cells obtained from the
same BM sample are indicated by use of the same plot symbol.
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| Fig 4.
Calculated 2° CFC expansion ratios as a function of
assay plating densities. Fresh and expanded BM MNCs were assayed at
different plating densities (the data set represented by triangles
in Fig 3) and the 2° CFC frequencies at each density were used
to calculate an expansion ratio. Calculated 2° CFC expansion varied
significantly as the different points of Fig 3 for fresh and expanded
2° CFC frequencies were used for the calculation.
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Although fresh CD34-enriched cells were found to be less affected by
assay nonlinearity than MNC (Fig 2), it is known that expansion culture
of CD34-enriched cells results in a population that contains many
accessory cells.35 Therefore, CD34-enriched cells and MNCs
from three donors were expanded and assayed in parallel. Each
experiment showed that expanded cells, whether from a CD34-enriched or
MNC inoculum, resulted in a population that gave nonlinear 2° CFC
assay readout (Fig 5). Importantly, expanded MNCs consistently contained a higher frequency of 2° CFC
than expanded CD34-enriched cells, even though the 2° CFC frequency
was considerably higher in the CD34-enriched fraction before the
expansion procedure. Although these data are consistent with previous
reports on the increased LTC-IC output from MNC cultures as compared
with CD34-enriched cell cultures,29,36 the nonlinearity of
the assay makes absolute quantitation of the differences difficult. The
magnitude of the difference varied with assay plating density, but the
trend of greater 2° CFC frequency in expanded MNCs was maintained
throughout the range examined.
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| Fig 5.
Measurement of 2° CFC frequencies (per
105 cells) from a series of paired expanded BM MNCs and
CD34-enriched cell samples assayed over a range of plating densities.
Each point represents the mean 2° CFC frequency from three to six
replicate cultures, and lines connect the points performed with the
same cells. Paired MNC and CD34-enriched cells obtained from the same
BM sample are indicated by use of the same plot symbol.
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LDA.
The high 5-week 2° CFC frequencies obtained from bulk assays at low
plating densities (Table 1) began to approach values that might be
expected for CFU-GM assays performed on fresh BM (typically 200 to 500 per 105 MNCs). However, the 5-week 2° CFC assay
presumably measures a primitive cell that is significantly more rare
than CFU-GM. This discrepancy can only be explained if the number of
2° CFC obtained per LTC-IC is significantly greater than the value
of four that has been reported.9 Also, plating densities in
the bulk 2° CFC assay could not be reduced below 2 × 104 MNCs per well to search for a linear range, because the
number of 2° CFCs scored per well became very low and statistically
insignificant given the number of replicates performed in the bulk
assay.
These two issues were addressed by performing LTC-IC LDA assays on a
series of BM MNC samples using 96-well plates inoculated at various
densities. Even under these limiting dilution conditions, the measured
2° CFC frequencies continued to increase as the plating density was
decreased (Table 2). At the lowest plating
density of 2,500 MNCs per well, the measured 2° CFC frequency was
80.0 per 105 MNCs. Because 24-well plates have fivefold
more surface area than 96-well plates (1.8 v 0.35 cm2), the data of Table 2 are consistent with the data of
Table 1. In fact, when the average 2° CFC frequencies from the
different experimental series are plotted versus the assay plating
density per square centimeter (instead of per well), the consistency of the long-term culture assay data is quite remarkable
(Fig 6), even though these 137 BM samples
were assayed over a period of 3 years using the different assay methods
and different irradiated stromal layer sources. Therefore, the ability
to generate 2° CFC in a 5-week culture, on a per inoculum cell
basis, is related to the surface density of cells plated in the assay
in addition to the absolute number of stem cells plated.
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| Fig 6.
Plot of average 2° CFC frequencies (per
105 MNCs) versus the assay plating density (in cells per
square centimeter). Consistency of the long-term culture assay data and
dependence on assay plating density is demonstrated with the data from
137 BM samples (Tables 1 and 2).
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LDA of the data was also performed using an iterative maximum
likelihood solution method.30,31 Several groups of plating densities were used to perform the assay. The mean LTC-IC frequency ranged from 8.2 per 105 MNCs at the highest group of
plating densities to 22.4 per 105 MNCs at the lowest group
of plating densities (Table 2). Because of the large number of samples
analyzed with each group of plating densities, these differences were
highly significant. Therefore, using the higher plating densities for
LDA, LTC-ICs were found to be present at a frequency of 1 per 12,195 MNCs, which is comparable to previously reported values.29
However, when the assay was performed at lower plating densities, the
LTC-IC frequency was found to be 1 per 4,464 MNCs, which is
significantly less rare (P < 104). A
frequency histogram of measured LTC-IC densities (using the lowest
assay plating densities of Table 2) showed a log-normal distribution
within the donor population that covered a 175-fold range (0.6 to 105 LTC-ICs per 105 MNCs; Fig 7A).
Because the LTC-IC frequency calculated by LDA decreased with
increasing plating density in a fashion similar to the decrease in
2° CFC frequencies, the number of 2° CFC generated per LTC-IC
was not dependent on the assay plating density. Instead, the average
number of 2° CFCs per LTC-IC varied from donor-to-donor approximately within the range of one to four, with an apparent bimodal
distribution (Fig 7B).
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| Fig 7.
Frequency histogram of LTC-IC densities (expressed as
number per 105 MNCs) in 53 BM samples measured by LDA
using 2.5 × 103, 5 × 103,
104, and 2 × 104 MNCs per well. (A) BM MNC
LTC-IC densities in the donor population were log-normally distributed.
(B) The number of 2° CFCs generated per LTC-IC varied from
donor-to-donor, with an apparent bimodal distribution.
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LDA assay of MNCs after exposure to 5-FU.
The results given above demonstrated that 2° CFC and LTC-IC cannot
be definitively quantitated in the presence of accessory cells, but
that this can be overcome by CD34-enrichment of the cell population to
be assayed. However, this is not a practical approach for use in a
typical laboratory setting with a large number of experiments.
Therefore, the use of the chemical purging agent, 5-FU, was explored as
a potential means of reducing accessory cell nonlinearity. A series of
10 experiments was performed to determine the time and dose of exposure
that would deplete mature cells (eg, CFU-GM and burstforming
unit-erythroid [BFU-E]) and accessory cells (eg,
CFU-fibroblast [CFU-F]) while sparing the majority of LTC-IC.
Exposure to  20 µg/mL 5-FU for 24 hours at 37°C was highly toxic
to all cell populations (Fig 8). An
exposure time of 0.5 to 2 hours at a dose of 250 µg/mL gave a
consistent high kill (>85%) of CFU-F and a moderate kill (60% to
85%) of CFU-GM, whereas most LTC-ICs were spared. Control untreated BM MNCs again gave nonlinear results, with the measured 2° CFC
frequency being 2.2-fold greater when assayed at 2.5 × 103 per well as compared with 2 × 104 per
well (P < .05, Table 3). In
contrast, MNCs exposed to 250 µg/mL 5-FU for 1 to 2 hours gave a
linear 2° CFC measurement over the assay plating density range
tested (P = .47). The frequency of 5-FU-resistant
LTC-ICs was 7.2 per 105 MNCs, 49% of the total LTC-IC
compartment under these conditions (P < .05). Cells obtained
after ex vivo expansion of BM MNCs (using the perfusion method
described above) were also assessed, and 42% of these LTC-IC were 5-FU
resistant (n = 3), which is similar to the fresh MNC result. However,
the response of expanded cells to 5-FU will probably vary greatly
depending on the method of expansion chosen.
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| Fig 8.
Effect of time and dose of 5-FU exposure on various BM
MNC populations. BM MNCs from 10 donor samples were exposed to various doses of 5-FU for 0.5, 2, and 24 hours at 37 °C. The mean (±SEM) percentages of (A) CFU-GM, (B) CFU-F, and (C) LTC-IC killed by the
various exposures are shown.
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DISCUSSION |
The assay of primitive human hematopoietic cells is of great clinical
and scientific interest. In vitro assays for these cells have focused
on the ability of a cell population to generate progenitor cells over
an extended period of time in culture. A number of studies have shown
that cells enriched to contain long-term in vivo repopulating ability
are also enriched in long-term in vitro repopulating
ability.11-13 On this basis, the development of the LTC-IC
assay as a quantitative stem cell assay was undertaken.9,11 To be considered quantitative, an assay must deliver a predictable output in response to varying input cell numbers. Although it was
originally thought that the LTC-IC assay fulfills this criterion in a
linear fashion,9,26 this hypothesis has not been borne out
during the accumulated use of this assay with hundreds of BM samples.
In fact, the large majority of samples assayed displayed declining
2° CFC and LTC-IC frequencies as the assay plating density was
increased. This nonlinearity compromises the ability of the assay to
quantitatively measure LTC-IC within BM populations that contain
accessory cells.
LTC-IC assay results, whether performed in bulk culture using 2°
CFC readout or under LDA conditions, were found to be nonlinear with
respect to the input number of MNCs over the entire range tested.
Because the number of 2° CFCs per LTC-IC did not vary with assay
plating density, results obtained from bulk 2° CFC measurements
were comparable to results obtained by LDA, consistent with previous
reports.9 However, if only one assay density is used for
bulk 2° CFC cultures, there is a significant risk of obtaining too
few (<10) raw colonies per plate (resulting in statistically weak
data) or too many (>70) raw colonies per plate (resulting in
inaccurate counts from crowded plates). Because the measured 2° CFC
and LTC-IC frequencies in human BM samples was found to cover a greater
than 100-fold range, no single plating density could accurately cover
this range. In contrast, LDA typically use multiple plating densities,
thereby reducing the probability of this pitfall.
The assay readout was less influenced by plating density when
CD34-enriched cells were used, suggesting that the observed nonlinearity was, at least in part, due to the presence of
CD34 accessory cells. However, assays of
CD34-enriched cells also became nonlinear at high density, so a general
cell crowding/inhibition effect appears to be at work. This nonspecific
cell crowding/inhibition effect is considerably more pronounced with
nonpurified cells, because so many nonpurified cells must be plated to
obtain a measurable signal from the rare LTC-IC population. Because
most cell populations subjected to LTC-IC assay contain accessory
cells, the LTC-IC assay generally cannot be considered quantitative.
For example, the measurement of LTC-IC expansion requires different
cell populations to be assayed, often containing very different
accessory cell populations that would be expected to influence LTC-IC
assay readout in an unpredictable manner. In fact, the net result of
differing accessory cell content of pre-expansion and post-expansion
populations resulted in ambiguous data (eg, Fig 4).
Although LTC-IC assay of nonpurified cell mixtures was not definitively
quantitative, the assay displayed a semiquantitative quality. For
instance, the comparative ranking of different BM samples with respect
to LTC-IC content was generally conserved when the assay was performed
at different densities, even though the absolute number of LTC-ICs
varied with density. Therefore, assay of nonpurified cells can give a
semiquantitative indication of LTC-IC when comparing one population
with another. This type of semiquantitative analysis would be most
accurate when the different cell populations are plated at densities
that yield similar raw colony counts. For example, a population
containing 10 2° CFC per 105 cells should be plated at
a density that is 10-fold greater than a population containing 100 2° CFC per 105, such that the number of colonies
counted from both sets of plates will be similar, within the
operational range of 10 to 70 colonies per plate and therefore within
the same range of the nonlinear assay response curve.
Although CD34-enriched cells gave linear assay output, the use of cell
purification for every LTC-IC assay is impractical in most laboratory
settings. Therefore, if quantitative LTC-IC assays are to be performed,
an alternative method for depleting accessory cells while sparing stem
cells would be useful. The chemical purging agent, 5-FU, has been shown
to kill certain mature cell populations while sparing primitive cells.
In fact, this purging strategy has been used for the development of the
pre-CFU-GM delta assay37 and for the
functional isolation of primitive hematopoietic cells.38
The current study showed that a relatively short exposure of BM MNCs to
5-FU preferentially depleted accessory cells, particularly CFU-F, to a
greater extent than LTC-ICs. After 5-FU exposure, 2° CFC
measurement became linear, thereby providing a method that is a simple
and attractive alternative to CD34-enrichment. As an additional benefit
of this procedure, 5-FU resistance selects noncycling LTC-ICs that are
more primitive38 and perhaps more representative of
quiescent cells, which are reported to have greater in vivo
repopulating activity.39,40
In conclusion, primitive cell measurement using the bulk 2° CFC or
LDA LTC-IC methods is semiquantitative unless care is taken to
eliminate assay nonlinearity due to accessory cells. Linear assay
response can be achieved from BM cells after CD34-enrichment or after a
relatively simple exposure to 5-FU. Similar study of assay linearity is
warranted for cord blood cells, which are known to behave in a
nonlinear manner (not shown), and mobilized peripheral blood cells.
Although the bulk and LDA methods yield similar data, multiple plating
densities are required in both methods due to the greater than 100-fold
range in primitive cell frequencies present in the normal human donor
population.
| |
FOOTNOTES |
Submitted January 14, 1997;
accepted December 31, 1997.
Address reprint requests to Alan K. Smith, PhD, Aastrom Biosciences,
Inc, PO Box 376, Ann Arbor, MI 48106.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Robert J. Maher, Maritza Oxender, Mahshid Palsson,
and Jason Williams for excellent technical assistance, and Drs Randy
Broun (St Louis University, St Louis, MO), Albert Deisseroth (M.D.
Anderson, Houston, TX), Melissa Fenner (University of Michigan, Ann
Arbor, MI), Voravit Ratanatharathorn (Harper Hospital, Detroit, MI),
Lyle Sensenbrenner (Harper Hospital), and Joseph Uberti (Harper
Hospital) for BM specimens. We also thank Dr Kristin
Goltry for critical reading of the manuscript.
| |
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Abstract
Introduction
Abstract