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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4056-4064
By
From Aastrom Biosciences, Inc, Ann Arbor, MI.
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
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.
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
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.
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.
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
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).
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.
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
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.
Submitted January 14, 1997;
accepted December 31, 1997.
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
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 < 10
Abstract
20 µg/mL 5-FU for 24 hours at 37°C was highly toxic
to all cell populations (Fig 
-thalassemia and sickle cell anemia patients: Implications for evaluating human gene therapy protocols.
Hum Mol Genet
4:163,
1995