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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 96-106
Single Adult Human
CD34+/Lin /CD38 Progenitors
Give Rise to Natural Killer Cells, B-Lineage Cells,
Dendritic Cells, and Myeloid Cells
By
Jeffrey S. Miller,
Valarie McCullar,
Michael Punzel,
Ihor R. Lemischka, and
Kateri A. Moore
From the Department of Medicine, University of Minnesota Cancer
Center, Minneapolis, MN; and the Department of Molecular Biology,
Princeton University, Princeton, NJ.
 |
ABSTRACT |
Marrow stromal cultures support adult
CD34+/Lin /HLA-DR or
CD34+/Lin/CD38 cell
differentiation into natural killer (NK) or myeloid cells, but unlike
committed lymphoid progenitors
(CD34+/Lin/CD45RA+/CD10+),
no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2,
and AFT024, a murine fetal liver line, which supports culture of
transplantable murine stem cells. NK cells,
CD10+/CD19+ B-lineage cells and dendritic
cells (DC) developed from the same starting population and IL-7, FL,
and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin/CD38 cells onto
AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time
addition of IL-3 at culture initiation was essential for multilineage
differentiation from single cells. Single and double lineage progeny
were frequently detected, but more importantly, 2% of single cells
could give rise to at least three lineages (NK cells, B-lineage cells,
and DC or myeloid cells) providing direct evidence that NK and
B-lineage differentiation derive from a common lymphomyeloid
hematopoietic progenitor under the same conditions. This study provides
new insights into the role of the microenvironment niche, which governs
the earliest events in lymphoid development.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THERE HAS BEEN MUCH interest in
mechanisms by which the marrow microenvironment governs lymphoid
differentiation through its supportive extracellular matrix, cell
surface ligands, and production of soluble cytokines and proteoglycans.
We have shown that culture of
CD34+/Lin/DR cells
from adult marrow will induce differentiation into phenotypic and
functional natural killer (NK) cells if progenitors are grown in direct
contact with normal allogeneic stroma and interleukin-2 (IL-2).1 In mice, the ability of stroma to induce
differentiation is, at least in part, regulated by the transcription
factor interferon-regulatory factor-1 (IRF-1).2 IRF-1
knockout mice exhibit a severe NK deficiency, which is mediated by
failure of transcriptional regulation of IL-15. In human studies, IL-15
made by stroma and monocytes plays a role in NK development and
survival by interaction with components of the IL-2
receptor.3,4 The production of IL-15 by monocytes may
explain why NK differentiation can occur in the absence of exogenous
IL-15 or stroma.5 However, stroma still provides other
factors to induce the most primitive adult marrow progenitors to
develop along the NK lineage. The requirement for direct contact with
intact stroma distinguishes this population from more committed
progenitors (CD34+/CD7+), which do not require
direct contact with stroma for differentiation.6
Culture of human primitive progenitors with IL-2 and stroma results in
terminal differentiation of NK cells. Progeny of long-term NK cultures
cannot initiate secondary long-term NK cultures or support other
lymphoid or myeloid lineages. The role of other defined cytokines in NK
cell commitment and differentiation was assessed by studies using
CD34+/CD33 progenitors after 14-day
culture in IL-3 and macrophage inflammatory protein
(MIP)-1 , which are highly clonogenic for myeloid
long-term culture-initiating cells (LTC-IC).7,8
CD34+/CD33 cells from these cultures do
not differentiate into NK cells when cultured with IL-2 and stroma
until IL-7 and c-kit ligand (KL) were added.9 We have also
been exploring the molecular events, which may occur using in vitro
culture. Fresh double sorted CD34+/Lin/DR cells do
not express transcripts for RAG-2, CD3 , CD3 , or CD3 . However,
culture with stromal-conditioned media, IL-7, FL, KL, IL-2, and IL-3
not only induced CD3, CD3, or CD3, but also RAG-2 suggesting
possible early T-or B-cell development.10 The role of FL in
lymphopoiesis is further highlighted by defects identified in
flt3-deficient mice,11 and recently, the overlapping and
distinct roles of FL and KL in hematopoiesis have been
reviewed.12
There are many similarities between NK cell and B-cell development. The
importance of stroma for B-lymphoid progenitor differentiation has been
described for several lymphoid culture systems and is the basis for the
Whitlock-Witte culture.13 The control of B lymphopoiesis
requires survival, proliferation, and differentiation signals from the
bone marrow microenvironment.13-15 In the absence of
stroma, immature B-cell progenitors rapidly die by
apoptosis.16 Normal B-cell progenitors adhere to stroma
through 4 1 integrin.17 Disruption of this adhesive
interaction results in decreased B-cell differentiation. Normal human
B-cell progenitors cultured while physically separated from stroma by a
microporous membrane lead to decreased proliferation of
CD34+/CD10+ pro-B cells suggesting the
importance of direct stromal contact.18 Similar experiments
with B-cell acute lymphocytic leukemia demonstrated decreased cell
survival by an apoptotic mechanism when stroma contact was
prohibited.19 Moreover, maximal proliferation of murine
pre-B cells in a M2-10B4-dependent culture occurs through RGD-dependent binding of B-cell precursors to fibronectin,
which is lost when differentiation occurs.20
In this report, we questioned whether an alternate microenvironment
could induce human adult marrow progenitors to differentiate along the
B-cell lineage, which is not found when culturing
CD34+/Lin/DR
progenitors on primary adult stromal layers using current conditions and multiple cytokines. Given the heterogeneity of human stroma containing fibroblasts, adipocytes, macrophages, and endothelial cells,
we focused on murine stromal cell lines known to support murine
primitive cells. M2-10B4 has been characterized for the ability to
support human myeloid LTC-IC.21 More recently, Moore et
al22 have developed a cell line, called AFT024, which is derived from day 14 gestational fetal liver adherent cells immortalized by introduction of a retrovirus containing a temperature-sensitive SV40
T antigen. Culture of as few as 100 highly purified murine marrow or
fetal liver stem cells on AFT024 for 4 to 7 weeks resulted in
multilineage reconstitution after transplantation into irradiated mice.
In vitro, AFT024 induced proliferation of primitive progenitors and
expansion of pro-B-cell progenitors suggesting a novel role for AFT024
in expansion and differentiation of murine hematopoietic progenitors.
Therefore, we use the murine AFT024 cell line and defined cytokines to
study human hematopoiesis in vitro.
| |
MATERIALS AND METHODS |
Normal bone marrow.
Bone marrow was obtained from normal donors after informed consent
using guidelines approved by the Committee on the Use of Human Subjects
in Research at the University of Minnesota. Bone marrow mononuclear
cells were obtained by Ficoll-Hypaque (specific gravity, 1.077) (Sigma
Diagnostics, St Louis, MO) density gradient centrifugation.
Purification of primitive progenitors.
Bone marrow mononuclear cells were enriched for CD34+ cells
using an avidin-biotin column as recommended by the manufacturer (Cellpro, Bothel, WA). Resultant cells were stained with CD34-biotin (Cellpro) for multicolor sorting, as previously
described.10,23 Fluorescein isothiocyanate
(FITC)-conjugated antibodies against CD2, CD3, CD4, CD5, CD7, CD8,
CD10, and CD19 were used for the lineage (Lin) cocktail (Becton
Dickinson [BD], San Jose, CA). Phycoerythrin (PE)-conjugated
anti-HLA-DR (BD) or anti-CD38 (BD) was used and streptavidin SA670
(GIBCO-BRL, Grand Island, NY) as the third fluorescent color. Single
CD34+/Lin/DR or
CD34+/Lin/CD38 were
sorted directly into 96-well plates using the Automatic Cell Deposition
Unit of the FACS Star Plus (BD). For single cell deposition
experiments, the Automatic Cell Deposition Unit was set up in a low
event "through-put" (200 events/second) and single droplet
sorting was used instead of packet sorting to insure deposition of
single cells.
Stromal cell lines.
Murine stromal cell lines were grown to confluency in 96-well plates
and then irradiated (2,000 rad) before use. The M2-10B4 cell line was
cultured as described.21,24 The AFT024 was cloned from
murine fetal liver using described techniques25 and
supports the ex vivo culture of murine transplantable stem
cells.22 AFT024 was maintained at 33°C in Dulbecco's
Modified Eagle's Medium (DMEM) (GIBCO Laboratories, Grand Island, NY)
supplemented with 20% fetal calf serum (HyClone Laboratories, Logan,
UT) and 50 µmol/L 2-mercaptoethanol (Bio-Rad, Hercules, CA) and
subcultured in 96-well plates precoated with 0.1% gelatin (Specialty
Media, Lavalette, NJ).
Culture of hematopoietic progenitors.
CD34+/Lin/DR or
CD34+/Lin/CD38 cells
were plated in a 2:1 (vol/vol) mix of DMEM/Ham's F12-based medium
without stroma or in direct contact with stromal cell lines as
indicated. The DMEM/F12-based medium (DMEM and Ham's F12 were obtained
from GIBCO Laboratories), developed to maximize NK cell
growth,26 was supplemented with 24 µmol/L
2-mercaptoethanol, 50 µmol/L ethanolamine, 20 mg/L L-ascorbic acid, 5 µg/L sodium selenite (Na2SeO3), 100 U/mL
penicillin, 100 U/mL streptomycin (GIBCO), 20% heat inactivated human
AB serum (North American Biologicals, Miami, FL) at culture initiation reduced to 10% for subsequent media changes. Progenitors were plated
in limiting dilution assays (22 replicates at four dilutions: 1,000 to
1,200, 300 to 400, 100 to 130, and 33 to 45 cells/well) or by single
cell deposition in 96-well plates. The cloning frequency of NK, B, and
dendritic cell progenitors was determined by immunophenotyping and was
calculated as the reciprocal of the concentration of cells that
resulted in 37% negative wells using Poisson statistics and the
weighted mean method.27,28 In limiting dilution
experiments, multiple wells were analyzed and reported irrespective of
cell growth. In single cell deposition experiments, only those wells which exhibited visual cell growth (>100 cells/well) were analyzed further with three-color immunophenotyping, the remaining wells were
considered as having no growth. Cultures were maintained in a
humidified atmosphere at 37°C and 5% CO2 and medium
was half changed weekly with the indicated cytokines. Cytokines were
supplemented as indicated with 1,000 U/mL IL-2 (a gift from Amgen,
Thousand Oaks, CA), 10 ng/mL flt3 ligand (FL, a gift from Immunex,
Seattle, WA), 20 ng/mL c-kit ligand (KL or stem cell factor, a gift
from Amgen), 20 ng/mL IL-7 (R&D Systems, Minneapolis, MN), and 5 ng/mL IL-3 (R&D Systems). Secondary dendritic cell conditions contained 10 ng/mL tumor necrosis factor (TNF) (R&D Systems), 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immunex, Seattle, WA), and 5 ng/mL IL-4 (R&D Systems). Cytokines were added weekly or only once at the time of culture initiation as indicated.
Phenotype, cell quantitation, cytotoxicity, and allogeneic mixed
lymphocyte reaction.
FITC- and PE-coupled control immunoglobulins or specific antibodies
directed at CD2, CD3, CD4, CD7, CD8, CD10, CD11c, CD34, CD19, HLA-DR,
kappa light chain, lambda light chain, IgM (all from BD), and mu heavy
chain (clone ATTC HB57 from T. LeBien, University of
Minnesota Cancer Center, Minneapolis, MN) were used to evaluate progeny
of long-term cultures. Three-color phenotype analysis was used to
determine multilineage progeny of starting progenitors using CD56-PE,
CD19-PerCP, and either CD1a-FITC (PharMingen, San Diego, CA), CD14-FITC
(BD), or CD15-FITC (BD). Absolute cell numbers were determined by
addition of 3 × 105 polystyrene microspheres
(Polysciences, Warrington, PA) to the total progeny of a culture well
and after gating out debris, absolute cell numbers were calculated
using the method described by Pribyl et al.29,30 The
absolute number of cells/well was calculated as: [(total number of
beads added/well)/(number of beads collected) × (number of cells
in the phenotype gate of interest)]. The relationship between
polystyrene microspheres and absolute cell numbers using this technique
was linear between 2.1 × 102 cells/well and 3.3 × 105 cells/well. All analyses were performed with a
FACSCalibur (BD) and CELLQuest software (BD). Cytotoxicity assays
were performed from progeny of single cells in triplicate using the
K562 (American Type Culture Collection, Rockville, MD)
cell lines in a 4-hour Cr51 release assay.31
Allogeneic mixed lymphocyte reactions were performed as described with
modifications.32 Briefly, 105 allogeneic
monocyte-depleted mononuclear cells were incubated in round bottom
96-well tissue culture plates with graded doses of stimulators (30,000 to 3,300) irradiated at 3,000 rads. After 5 days, cultures were pulsed
with 1 µCi/well of 3H-thymidine (New England Nuclear,
Boston, MA) for 18 hours before harvesting and counting.
Terminal deoxynucleotidyl transferase (TdT)
determination by in situ staining.
Cytospin preparations of sorted CD19+ or CD56+
cells were fixed for 30 minutes in absolute methanol at 4°C
immediately before staining. Rabbit anti-TdT (Supertechs, Bethesda, MD)
is diluted 1:10 in phosphate-buffered saline (PBS) buffer and 20 µL
is applied to each slide for 30 minutes. After washing, an equal amount
of a 1:10 dilution of secondary FITC-goat antirabbit (Supertechs) is
applied. TdT positivity is determined within 24 hours of staining using
a fluorescent microscope.
TdT determination by PCR.
After determining the phenotypic presence of CD19 positive cells by
flow cytometry, the remaining progeny of single cell cultures were used
for determination of TdT by reverse transcriptase-polymerase chain
reaction (RT-PCR). Total mRNA was extracted using RNeasy spin columns
according to the manufacturer's recommendations (Qiagen, Santa
Clarita, CA). Reverse transcription was performed as previously described.10 Briefly, samples were subjected to 40 cycles
of denaturation at 95°C for 20 seconds, annealing at 55°C for
15 seconds, and extension at 72°C for 1 minute in a Perkin Elmer 480 thermal cycler (Applied Biosystems, Foster City, CA).
Oligonucleotide primer sequences were: TdT 5 primer:
5-ACACGAATGCAGAAAGCAGGA-3; TdT 3 primer:
5-AGGCAACCTGAGCTTTTCAAA-3 (provided by Dr T. LeBien);
-actin 5 primer: 5-TACCTCATGAAGATCCTCA-3;
-actin 3 primer: 5-TTCGTGGATGCCACAGGAC-3.
Amplified products were size separated on 1.5% agarose gels and
transferred to Hybond N+ nucleic acid transfer membranes
(Amersham, Arlington Heights, IL). Probes were labeled with
32P-deoxyadenosine triphosphate (dATP) using a
TdT 3-end labeling kit (Boehringer Mannheim, Indianapolis, IN)
using probe sequences: TdT 5-ACACGAATGCAGAAAGCAGGA-3
(provided by Dr T. LeBien); -actin 5-CCATCTCTT-GCTCGAAGTC-3.
Statistics.
Results of experimental points obtained from multiple experiments were
reported as mean ± 1 standard error of the mean (SEM). Significance
levels were determined by two sided Student's t-test analysis.
| |
RESULTS |
Although primitive
CD34+/Lin/DR
progenitors can be induced to differentiate along the NK cell lineage
when cultured with IL-2 in contact with primary adult marrow allogeneic
stroma, these cultures result in terminal differentiation and do not
support B-lineage cells or other lineages. Therefore, we designed
experiments to assess stromal feeder cell lines, which may support
differentiation along the NK and B-cell lineage to establish a link
between these two lymphoid lineages where differentiation is known to
occur in the marrow microenvironment.
CD34+/Lin/DR cells
were isolated from adult marrow and plated with multiple cytokines at
culture initiation (IL-7, FL, KL, and IL-2) in the absence of stroma or
with two murine feeder cell lines, M2-10B4 and AFT024 (n = 3). Analysis
of progenitors cultured without stroma showed little growth and
complete absence of NK cell or B-cell progeny. M2-10B4, which supports
proliferation of mature NK cells and primitive myeloid
cells,21,33 inefficiently gave rise to NK cell progeny
(36% of wells positive at 1,200 cells/well) and no B-lineage cells
could be identified. In contrast, coculture of
CD34+/Lin/DR cells on
AFT024 in the presence of IL-7, FL, KL only at culture initiation and
IL-2 throughout culture resulted in both NK and B-cell progeny from the
same wells (Table 1). Culture of a
population of lymphoid-committed cells already expressing one of the
lymphoid antigens (CD34+/Lin+) also gave rise
to both NK and B-lineage cell progeny (data not shown). In contrast to
the committed CD34+/Lin+ population where
lymphoid progeny were identified by 14 days, differentiation of the
more primitive
CD34+/Lin/DR
population required longer (>28 days) culture intervals.
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Table 1.
AFT024 Supports Differentiation of
CD34+/Lin/DR Cells Into
Both NK Cell and B-Lineage Cell Progeny When Cultured With IL-7,
FL, KL, and IL-2
|
|
B-lineage cells from the AFT024 cultures were CD19 and CD10 positive
and were found to have very low forward and side scatter, significantly
smaller than NK cells within the same cultured population (Fig 1). Phenotype analysis showed that the
B-lineage cells were negative for CD34 (n = 20), IgM (n = 8), mu (n = 30), kappa light chains (n = 20), and lambda light chains (n = 20),
irrespective of the cytokine combination used. Small numbers of
CD19+ B-lineage cells were positive for CD20 (18% ± 5.5%, n = 6; P =.005) and CD21 (14% ± 2.9%, n = 13;
P = .009) compared with isotype controls (4.3% ± 1.5%).
Given this immature phenotype, cells were analyzed further for TdT in
situ fluorescent staining. CD56+ NK cells were essentially
negative (<2%) for characteristic nuclear TdT staining by
fluorescence microscopy (n = 4). In contrast, CD19+
B-lineage cells from the same cultured population were strongly positive in 23% to 55% of cells (n = 4).
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| Fig 1.
Progeny of AFT024 cultures give rise to NK cells and
CD10+/CD19+ B-lineage cells.
CD34+/Lin/DR cells were
cultured in limiting dilutions on AFT024 with IL-7, FL, KL, and IL-2
added at culture initiation. Media was half changed weekly with fresh
media supplemented with IL-2 alone. Individual wells were harvested and
analyzed by flow cytometry after three-color staining (CD56-PE,
CD10-FITC, CD19-PerCP) using appropriate isotype controls. The
representative example shown is from a culture well initiated with 130 cells. (A) Shows the two-color analysis of CD56 and CD19. CD19 positive
cells were then backgated onto the forward and side scatter plot (B).
The CD19+ B-lineage cells (black dots) were significantly
smaller than NK cells (medium gray) found within the lymphoid window of
the same culture. Backgating to find B-lineage cells in this very small
lymphocyte gate (R2) was used as an absolute criteria for all cultures
determined positive for B-lineage cells. (C) Shows a representative
example of the CD10+/CD19+ B-lineage cells
based on the R2 gate.
|
|
The precise role of the individual cytokines was assessed by sequential
deletion of single cytokines from the four-cytokine cocktail (IL-7, FL,
KL, IL-2) using sorted
CD34+/Lin/DR cells.
Comparative cell growth was estimated by flow cytometry as the number
of events (per minute) from progeny of each well. Omitting IL-2 from
the four-cytokine combination significantly decreased total cell
proliferation from 14,660 ± 3,197 to 3,561 ± 312 events per minute (n = 22 wells initiated with 330 cells/well from two
donors; P = < .001). However, the number of wells positive for NK cells and B-lineage cells was not significantly different, suggesting that IL-2 is not critical for either NK or B-lineage cell
differentiation in the presence of IL-7, FL, and KL. IL-7, FL, and KL
were then each omitted from the four-cytokine combination. Deletion of
IL-7 slightly decreased relative proliferation (P = .082), did
not effect the number of wells with NK cell growth, but significantly
decreased the number of wells with B-lineage cell progeny
(Fig 2). Although elimination of KL
significantly affected relative proliferation, there was no difference
in the percent of wells positive for either NK or B-lineage cells. In contrast, omitting FL significantly decreased relative proliferation and the frequency of wells with NK and B-lineage cells (Fig 2), suggesting critical roles for FL and IL-7 in lymphoid differentiation on AFT024. Weekly addition of cytokines lead to increased NK and B-lineage cell proliferation (data not shown), therefore weekly cytokines were used in all subsequent experiments.
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| Fig 2.
Proliferation and differentiation in AFT024 cultures
depends on addition of defined cytokines.
CD34+/Lin/DR cells (1,000 cells/well) were cultured on AFT024 in 96-well plates with the cytokine
combinations indicated. Cytokines were added only once at culture
initiation and weekly half media changes contained fresh IL-2 alone.
Abbreviations for cytokines in all figures are as follows: 7, IL-7; F,
FL (Flt3 ligand); K, KL (c-kit ligand); 2, IL-2; 3, IL-3. After 28 to
35 days in culture, 42 wells per condition from cells derived from
three donors were analyzed for proliferation and presence or absence of
NK and B-lineage cells. (A) Relative proliferation was determined by
flow cytometry as the number of events analyzed per minute. Data are
the mean ± SEM for replicate wells analyzed in parallel with the
total contents of a harvested well analyzed in a constant volume of
approximately 180 µL. The P values shown are comparisons
between adjacent cytokine combinations. (B) The percentage of positive
wells (of 42 wells analyzed per condition) is shown for NK cells (black
bars) and B-lineage cells (hatched bars) for each cytokine
combination.
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Having identified a role for IL-7 and FL, experiments were then
performed to determine cloning frequency and lineage-specific cell
proliferation by sequential addition of IL-7, FL, KL, and IL-2. IL-3
was also added to this analysis to determine if this primitive acting
cytokine would increase cloning frequency and proliferation or decrease
lymphoid capacity, as has been suggested by others.34 A
known number of polystyrene microspheres was added to progeny of each
well,30 allowing precise quantitation of the absolute
number of cells by flow cytometry. For these experiments, cytokines
were added weekly except for IL-3, which was only added once at culture
initiation, because weekly addition of IL-3 lead to cell death from
myeloid overgrowth (data not shown). Total cell number (all lineages)
was determined for cultures inoculated with 130 CD34+/Lin/DR cells on
AFT024 with or without sequential addition of cytokines (n = 20 to 38 wells per condition from cells derived from four normal donors). After
35 to 42 days, proliferation was poor with either no cytokines (653 ± 29 cells/well) or IL-7 alone (512 ± 21 cells/well). However,
addition of IL-7 + FL (2,717 ± 407 cells/well), IL-7 + FL + KL
(21,454 ± 2,390 cells/well), IL-7 + FL + KL + IL-2 (59,595 ± 10,612 cells/well), and IL-7 + FL + KL + IL-2 + IL-3 (108,380 ± 11,712 cells/well) lead to a significant increase in total cell
proliferation with each additional cytokine (P = < .03 for
each added cytokine).
Based on phenotype studies, a proportion of cells derived from AFT024
cultures were neither NK or B-lineage cells, suggesting the presence of
other lineages. Wright-Giemsa staining showed the presence of myeloid
cells (from promyelocyte to neutrophils), monocyte/macrophage cells,
and cells with abundant cytoplasmic projections distinct from other
cells. Sorting of CD15+ cells from cultures and subsequent
cytospin staining showed that all cells were myeloperoxidase positive,
further verifying their myeloid origin. Sorting of CD14+
cells from a 4-week culture showed mixed morphologic monocytes, macrophages, and some myeloid cells. CD14+ cells from
cultures greater than 8 weeks old showed only macrophages that were
myeloperoxidase negative. Sorting CD1a+ cells identified
cells with cytoplasmic projections morphologically consistent with
dendritic cells (DC). These cells were CD15 and
CD14/dim+. Transfer of progeny from 5 week AFT024
culture into media containing GM-CSF, TNF, and IL-4 further enriched
for a population of cells, which were CD1a+,
CD11c+, HLA-DR+, CD4+,
CD15, and CD14 consistent with
the phenotype of cultured DC.32,35 Cultured cells exhibited
characteristic DC function, as primary progeny of AFT024 cultures or
progeny of secondary cultures supplemented with GM-CSF, TNF, and IL-4
were capable of stimulating allogeneic T cells in mixed lymphocyte
reaction (Fig 3). We could not detect any
CD3+ T cells or CD4+/CD8+ T cells
in cultured progeny of AFT024 cultures with any of the cytokine
combinations tested. Despite the identification of NK, B-lineage,
myeloid, and DC, there was always a population of AFT024 cultured
progeny, which was negative for any of the antigens tested, raising the
possibility that other lineages may be present as well.
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| Fig 3.
Progeny of AFT024 cultures function to stimulate
allogeneic mixed lymphocyte reactions. Monocyte-depleted peripheral
blood mononuclear cells (105) from the same donor were
mixed with irradiated (3,000 rads) autologous CD14+ fresh
monocytes ( ), 6-week progeny of AFT024 cultures ( ), and 5-week
progeny of AFT024 cultures transferred for an additional week to media
containing TNF, GM-CSF, and IL-4 ( ). Proliferation (counts per
minute [cpm]) was assessed after 5 days by pulsing for 18 hours with
3H-thymidine and data are presented as the mean ± SEM cpm
of triplicate wells from a representative experiment.
|
|
The role of cytokines (added weekly except for IL-3) was then evaluated
in AFT024 cultures by plating
CD34+/Lin/DR in
limiting dilutions to determine the cloning frequency for NK cells,
B-lineage cells, and DC. From these same cultures, the absolute number
of cells derived from each positive well could also be calculated. The
NK cell cloning frequency of
CD34+/Lin/DR cells
grown without cytokines or with IL-7 alone was less than 0.2% and only
slightly increased to 0.5% when IL-7 and FL were combined. However,
addition of KL to IL-7 and FL significantly increased (P = .028) the NK cloning frequency to over 3%
(Fig 4A). Further addition of IL-2 and IL-3
to IL-7, FL, and KL did not change cloning frequency. The role of
cytokines on proliferation differed from the role of cytokines on
cloning frequency. Proliferation was determined from wells started with
130 CD34+/Lin/DR
cells cultured for 35 to 42 days (Fig 4B). Even though IL-7, FL, and KL
had the greatest impact on cloning frequency, the number of absolute NK
cells per well was low (507 ± 100, n = 36). In contrast, addition
of IL-2 and further addition of IL-3 at culture initiation
significantly increased NK proliferation. The absolute number of NK
cells derived from 130 cells when all cytokines were used was 69,354 ± 10,108 NK cells/well (n = 38).
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| Fig 4.
NK cell cloning frequency and absolute NK cell
proliferation in AFT024 cultures is dependent on the addition of
exogenous cytokines.
CD34+/Lin/DR cells were
plated in limiting dilutions (replicates of 1,200 cells/well, 400 cells/well, 130 cells/well, and 45 cells/well) on AFT024 in 96-well
plates with the cytokine combinations indicated. Cultures were
maintained with weekly half media changes and fresh cytokines were
added weekly except for IL-3, which was only added once at culture
initiation. (A) After 35 to 42 days of culture, wells were analyzed
using three-color flow cytometry for the presence of
CD56+ NK cells to calculate the cloning frequency of
initially plated
CD34+/Lin/DR cells. Cells
were gated on viable cells and any well containing greater than 20 absolute CD56+ cells was counted as positive. Each bar
represents the mean ± SEM cloning frequency from four donors. (B) The
absolute number of NK cells per positive well initiated with 130 CD34+/Lin/DR cells is shown
for each cytokine combination. Absolute cell counts per harvested well
was determined by addition of a known number of polystyrene
microspheres to each sample before analysis by flow cytometry as
described in Materials and Methods. Each condition represents the mean ± SEM of 20 to 38 individual wells initiated with
CD34+/Lin/DR cells derived
from four donors. P values listed are for comparisons between
adjacent conditions.
|
|
The cloning frequency of CD1a positive DC was similar to that of NK
cells when a minimum of IL-7, FL, and KL were added to cultures
(Fig 5A). This did not change when IL-2 or
IL-3 were added. Although the addition of all cytokines consistently
resulted in the highest number of absolute DC (2,546 ± 492, n = 37)
derived from 130 CD34+/Lin/DR cells,
individual cytokine combinations had less influence on proliferation
until only IL-7 and FL were added, which resulted in 278 ± 122 DC
per positive well (n = 20). B-lineage cell cloning frequency and
proliferation was significantly less than either NK cells or DC.
Culture of
CD34+/Lin/DR cells in
limiting dilutions on AFT024 with IL-7, FL, and KL (n = 8) resulted in
a significantly higher B-lineage cell cloning frequency than any other
cytokine combination tested (Fig 5B). Although this same combination
induced the highest number of B-lineage cells per well (89 ± 19),
given the low overall cloning frequency, the B-lineage cell
proliferation did not change significantly with the different cytokine
combinations.
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| Fig 5.
DC and B-lineage cell cloning frequency in AFT024
cultures is dependent on the addition of exogenous cytokines.
CD34+/Lin/DR cells were
plated in limiting dilutions on AFT024 in 96-well plates with the
cytokine combinations indicated for 35 to 42 days. (A) Multiple
replicates were analyzed using three-color flow cytometry for the
presence of CD1a+ cells to calculate the cloning
frequency of initially plated
CD34+/Lin/DR cells. Cells
were gated on viable cells and any well containing greater than 20 absolute CD1a+ cells was counted as positive. Each bar
represents the mean ± SEM cloning frequency from four donors. (B)
Replicates were analyzed for the presence of CD19+ cells
to calculate the B-lineage cell cloning frequency. Cells were gated on
a very low forward and side scatter (Fig 1B designated R2) and any well
containing greater than 10 absolute CD19+ cells was
counted as positive. Each bar represents the mean and SEM cloning
frequency from four donors except for the condition using IL-7, FL, and
KL (7/F/K) where eight donors were used. All P values shown are
compared with the frequency using IL-7, FL, and KL.
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Bulk cultures identified conditions optimal for inducing NK cell,
B-lineage cell, and DC differentiation and proliferation from adult
marrow primitive progenitors. However, we still could not conclude that
all of these cell lineages were derived from a single cell. We also
questioned whether the role of certain cytokines may be more important
at the single cell level rather than in bulk cultures. To answer these
questions, experiments were performed by single cell deposition of
primitive progenitors from four normal donors onto AFT024 stromal
layers using flow cytometry. To maximize outgrowth of primitive
progenitors, single cells were sorted
CD34+/Lin/CD38 based
on findings that this population may contain a higher frequency of the
primitive human progenitors than the
CD34+/Lin/DR
population. Sorting windows were chosen so that the
CD34+/Lin/CD38
population accounted for 2.4% ± 1.2% of total CD34+
cells.
A total of 3,872 single
CD34+/Lin/CD38 cells
were sorted onto AFT024 with IL-7, FL, and KL with or without the
addition of IL-2, IL-3, or the combination
(Table 2).
CD34+/Lin/CD38 cells
plated without IL-3 exhibited significantly less single cell growth
with 6.3% positive wells versus 27% when IL-3 was included. There was
only one positive well of 1,584 plated without IL-3 that gave rise to
B-lineage cells compared with 46 of 2,288 wells positive for B-lineage
cells when IL-3 was included only at culture initiation. More than 10%
of wells with IL-3 at culture initiation gave rise to NK cells and
either DC or myeloid cells. Most importantly, 1.5% to 2.0% of single
CD34+/Lin/CD38 cells
plated under optimal conditions gave rise to three lineages (NK cells,
B-lineage cells, and either CD1a+ DC, CD15+
myeloid cells, or CD14+ monocytes) demonstrating origin
from the same cell (Fig 6).
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Table 2.
NK Cell, B-Lineage Cell, Dendritic Cell, and Myeloid
Progeny From Single Sorted
CD34+/Lin/CD38 Cells
Cultured on AFT024 With IL-7, FL, KL, ±IL-3 ± IL-2
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| Fig 6.
Multiple lineages are present from the cultured progeny
of single cells. Single
CD34+/Lin/CD38 cells were
cultured on AFT024 for 42 days with IL-7, FL, KL, IL-2, and IL-3 (A and
B are from progeny of the same single cell) or with IL-7, FL, KL, and
IL-3 (C and D are from progeny of the same single cell). Data are
examples of the three-color phenotypes summarized in Table 2 showing
the multilineage differentiation, which resulted from single cell
cultures gated on all viable cells (panel A and C) or a smaller
lymphocyte gate (panel B and D).
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Single-cell proliferation was analyzed using data from Table 2.
Exogenous cytokines played a significant role in cell proliferation from single cells (Fig 7A). Although the
frequency of single cells giving rise to any progeny was most
influenced by initial addition of IL-3, analysis for the absolute
number of cells derived from single cells showed contributions to NK
cell and DC proliferation from both IL-2 and IL-3 (Fig 7B and C). The
absolute number of B-lineage cells in positive wells cultured with
IL-7, FL, KL, IL-3, and IL-2 was 190 ± 83.
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| Fig 7.
Proliferation of single sorted
CD34+/Lin/CD38 cells is
optimal with IL-7, FL, KL, IL-2, and IL-3. The absolute number of total
cells (A), NK cells (B), and DC (C) were analyzed from progeny of 2,640 single sorted
CD34+/Lin/CD38 cells
summarized in Table 2. Results in (A) are from all wells evaluated per
cytokine condition. Results in (B) and (C) are from wells determined
positive for NK cell or DC progeny, respectively. The P values
listed are comparisons between adjacent bars where significant.
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To further evaluate the B-lineage cells from AFT024 cultures, progeny
of single cells were evaluated for TdT mRNA by RT-PCR. Progeny of
single CD34+/Lin/CD38
cells were analyzed by three-color flow cytometry for the presence of
NK cells, B-lineage cells, and other lineages. The remaining cells were
used for mRNA extraction after identifying the phenotypic presence or
absence of CD19+ cells. All of the single cell progeny (n = 9), which were phenotypically positive for B-lineage cells were
positive for TdT transcripts. In contrast, in wells where B-lineage
cells could not be phenotypically identified, seven of 17 were still
positive for TdT. The finding of TdT in 40% of phenotypic B-lineage
cell negative populations suggests that the number of wells giving rise
to B-lineage cells may be underscored by phenotypic identification
alone. Finally, after 42 days of culture with IL-7, FL, KL, IL-3, and
IL-2, progeny of single
CD34+/Lin/CD38 cells
cocultured with AFT024 were selected based on growth for expansion in
24-well plates with IL-2 alone to obtain enough NK cells for functional
and NK cell subset analysis. The 16 populations derived from single
CD34+/Lin/CD38 cells
gave rise to an average of 8.3 × 105 cells (range, 3 to 22 × 105) of which 94% ± 1.0% were
CD56+/CD3, 11% ± 2.0% were
CD56+/CD2+, and 21% ± 5% were
CD56+/CD7+. These NK cells exhibited
characteristic function against K562 targets in cytotoxicity assays
(Fig 8).
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| Fig 8.
NK cells derived from single cells exhibit cytotoxic
activity against K562 targets. Single
CD34+/Lin/CD38 progenitors
were cultured on AFT024 with IL-7, FL, KL, IL-2, and IL-3 (added only
at culture initiation) for 42 days and wells with the highest
proliferation were transferred to 24-well plates in media containing
IL-2 alone for an additional 14 days. Cells were then counted and
tested for cytotoxicity against chromium-labeled K562 targets. Results
are from 13 NK cell populations derived from progeny of 13 single cells
from two normal donors (data represent the mean ± SEM of the average
of triplicate wells from each population).
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DISCUSSION |
We have developed a novel long-term culture assay using the murine
fetal stromal cell line, AFT024, and human cytokines (IL-7, FL, KL,
IL-2, and IL-3) to induce multilineage lymphoid and myeloid differentiation from adult human marrow progenitors. The multilineage potential of this murine stromal-based system using one culture condition was striking. In addition to inducing NK differentiation more
efficiently than found previously with adult human allogeneic stroma,6 CD10+/CD19+ B-lineage
cells were also generated from the same starting cells. Limiting
dilution assays using three-color immunophenotype analysis and absolute
quantitation of various cell types, determined the role of cytokines on
NK cell, B-lineage cell, and DC cloning frequency and proliferation.
Single cell sorting of individual
CD34+/Lin/CD38 cells
verified that not only NK cells and B-lineage cells derive from the
same cell, but DC and mixed myeloid cells (granulocytic/monocytic) as
well. Finding that most single cells that resulted in B-lineage cell
differentiation also resulted in NK cell and DC or myeloid differentiation (at least three lineages from one cell) suggests origin
from a very primitive progenitor.
In experiments eliminating single cytokines, the B-lineage cell
differentiating capacity of this culture was dependent on FL, IL-7, and
stromal ligands in agreement with previous reports in human and murine
lymphopoiesis showing a role for one or more of these
factors.36-41 Although the B-lineage cell development from
CD34+/Lin/DR cells
occurred without KL, its addition optimizes growth for readout
detection. IL-7, FL, and KL were required to induce development along
the NK cell lineage. This is in agreement with Silva et al,5 who demonstrate that NK differentiation is independent of IL-2 as long as IL-7 is present. The finding that KL potentiates outgrowth fits with the finding of c-kit receptor on lymphoid progenitors and more primitive CD56+bright blood NK
cells.42,43 The ability of AFT024 to induce NK cell, B-lineage cell, and other myeloid lineages from the same cell depends
on properties of the murine AFT024 fetal cell line. M2-10B4 produces
soluble factors and provides contact-mediated growth promoting ligands
to mature NK cells.24,33 Although M2-10B4 can support
primitive myelopoiesis,21 it poorly supports NK cell
differentiation and does not support B-cell development from primitive
human progenitors. The capacity of the AFT024 cell line to support
lymphoid differentiation may be, in part, related to its developmental
fetal origin. Pribyl and LeBien44 have shown that human
fetal stroma can differentiate fetal
CD34++/Lin cells into mu/kappa or
mu/lambda expressing B cells and differentiation was IL-7-independent.
Alternatively, there may be unique properties of murine stromal cell
lines to support B-cell differentiation such as the S17 cell line
reported by Rawlings et al45,46 or by the MS-5 cell line
described by Berardi et al.47 However, the
cytokine-independent differentiation observed with S17 and MS-5 is in
contrast to results shown here with the AFT024 cell line. In addition
to different murine stroma cell lines, results may be explained in part
by our use of adult marrow progenitors contrasted to progenitors from
cord blood, which may contain more immature stem cells with different
cytokine requirements than adult bone marrow.48
The B-lineage cells, which developed from adult marrow progenitors,
were developmentally blocked at the pro-B cell stage by the absence of
surface heavy and light chains and the presence of TdT. The detection
of CD20 and CD21 on a small proportion of CD19+ cells may
suggest early progression to B-cell maturation with D-J gene
rearrangement but the absence of surface IgM shows they are not able to
develop into mature B cells.49,50 The phenotype of
B-lineage cells resulting from AFT024 cultures is similar to that shown
by Rawlings et al45 with the exception that less than 3%
of their S17 cultured B-lineage cells were TdT positive, while more
AFT024 B-lineage cell progeny were positive for TdT by both
in situ staining and RT-PCR.
Single-cell experiments differed from bulk cultures in the absolute
requirement of a one time addition of 5 ng/mL of IL-3 at culture
initiation. Whether this is required for mere survival or proliferation
induction is uncertain. The role of early acting cytokines can alter
the self-renewal, viability, or proliferation of single
CD34+CD38 cells.51 For
example, in studies of single CD34+/CD38
progenitors, 60 ng/mL IL-3 in addition to 300 ng/mL of FL and KL were
necessary to obtain optimal amplification of myeloid LTC-IC and
colony-forming cells (CFC).52 However, when
10 ng/mL of FL and KL were used, the same concentration of IL-3
decreased LTC-IC expansion and increased CFC expansion, suggesting that the net effect of multiple cytokines is determined not only by the
cytokines themselves, but also their relative concentrations. The
requirement for IL-3 early in the culture period differs from several
murine studies by Ogawa's group demonstrating that IL-3 is inhibitory
to B cell, T-cell, and NK cell development.34,42,53,54 However, the concentration of IL-3 in our cultures was low and distant
(4 to 6 weeks) from the readout of lymphoid progeny. In addition,
IL-3-induced lymphoid suppression may not be absolute, as the addition
of IL-4 in combination with IL-11 or IL-6 reversed the IL-3-induced
inhibition on early B-cell development.55 The sequential,
low concentration, one time addition of IL-3 clearly increased NK
proliferation, possibly by upregulation of IL-2 receptors by
simultaneous exposure to KL, as proposed by Shibuya et
al.56
The cloning frequency from the single cell experiments was consistently
higher than from the limiting dilution assays. This may be explained by
competitive interactions within the AFT024 cultures. In addition to the
interaction with AFT024, the contribution of human progeny cell
interactions induced by AFT024 may be critical to the resultant
multilineage lymphoid and myeloid differentiation. Differentiation of
DC progeny expressing costimulatory molecules or monocyte progeny
secreting IL-15 may interact with B-lineage cells and NK cells,
respectively. Similarly, transforming growth factor- (TGF-), a
potent costimulator of FL-induced DC growth,57 may be
present in culture by activation of latent TGF- in serum by the
extracellular matrix component thrombospondin or by production of
TGF- from developing NK cells.33 The complexities of
these interactions will need further study.
In summary, using the murine fetal liver cell line, AFT024, and defined
cytokines, long-term in vitro culture can differentiate single adult
human CD34+/Lin/CD38
cells into NK cells, B-lineage cells, DC, and myeloid lineages using a
single culture condition. Our results are consistent with the NK cell,
B-cell and DC differentiation pattern derived from the more committed
CD34+/Lin/CD45RA+/CD10 |
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Abstract
Introduction