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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 483-495
All-Trans Retinoic Acid Delays the Differentiation of Primitive
Hematopoietic Precursors
(lin c-kit+Sca-1+)
While Enhancing the Terminal Maturation of Committed
Granulocyte/Monocyte Progenitors
By
Louise E. Purton,
Irwin D. Bernstein, and
Steven J. Collins
From the Clinical Research and Molecular Medicine Divisions, Fred
Hutchinson Cancer Research Center, Seattle, WA; and The Department of
Pediatrics, The University of Washington, Seattle, WA.
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ABSTRACT |
All-trans retinoic acid (ATRA) is a potent inducer of terminal
differentiation of malignant promyelocytes, but its effects on more
primitive hematopoietic progenitors and stem cells are less clear. In
this study, we investigated the effect of ATRA on highly enriched
murine hematopoietic precursor cells
(lin c-kit+Sca-1+)
grown in liquid suspension culture for 28 days. ATRA initially slowed
the growth of these hematopoietic precursors but prolonged and markedly
enhanced their colony-forming cell production compared with the
hematopoietic precursors cultured in its absence. At 7 and 14 days of
culture, a substantially greater percentage of cells cultured with ATRA
did not express lineage-associated antigens (55.4% at day 7 and 68.6%
at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9%
at day 14) compared with cells grown in its absence (lin
cells: 31.5% at day 7 and 4% at day 14; Sca-1+: 10.4%
at day 7 and 0.7% at day 14). Moreover, a marked inhibition of
granulocyte production was observed in cultures continuously incubated
with ATRA. Significantly, ATRA markedly prolonged and enhanced the
production of transplantable colony-forming unit-spleen (CFU-S) during
14 days of liquid suspension culture. In contrast with its effects on
primitive
linc-kit+Sca-1+
hematopoietic precursors, ATRA did not exert the same effects on the
more committed
linc-kit+Sca-1
progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has
different effects on hematopoietic cells depending on their
maturational state, preventing and/or delaying the differentiation of
primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RETINOIDS AND in particular retinoic acid
(RA) regulate the growth and differentiation of a wide variety of cell
types.1 The biologic effects of RA are mediated through
nuclear receptors that are members of the steroid/thyroid hormone
superfamily of transcription factors.1 The RA receptor  (RAR) appears to be of particular interest in hematopoiesis, because
hematopoietic cells preferentially express this particular RA receptor
isoform.2,3
RA has well-documented effects on hematopoietic cell proliferation and
differentiation, particularly in cells of the granulocytic lineage. As
a single agent, all-trans RA (ATRA) induces granulocytic differentiation of primary leukemia cells from patients with acute promyelocytic leukemia (APL) both in vitro and in vivo, and APL cells
harbor an aberrant PML-RAR fusion transcript.4-8 In
addition, RA is known to induce granulocytic differentiation of HL-60
myeloid leukemia cells, and this is mediated directly through
RAR.7
In contrast, the roles that RA might play in regulating aspects of
normal hematopoiesis have not been clearly defined. Studies of the
actions of RA on hematopoietic progenitor cells have reached variable
conclusions, with some reports showing that RA enhances the growth of
progenitor cells, including erythroid and myeloid precursors,9-11 and others demonstrating an inhibitory role
of RA on the proliferation and differentiation of hematopoietic
progenitor cells.12-15 Moreover, studies on isolated
primitive hematopoietic populations, such as human lineage-negative
(lin), CD34+ cells and murine
lineage-negative, Sca-1-positive (Sca-1+) cells, have
indicated that RA has complex effects on these cells, with some studies
suggesting that RA can stimulate the growth of these
cells16 and others indicating that RA has inhibitory effects.17,18 In this study, we examined the effects of
exogenous ATRA on the growth and differentiation of highly enriched
linc-kit+Sca-1+
murine hematopoietic precursors and
linc-kit+Sca-1
progenitor cells in liquid suspension culture. We measured the colony-forming potential of these cells in secondary culture systems after ATRA was removed from the cells. Our results indicate that ATRA
enhances the generation of colony-forming cells, including both
granulocyte/macrophage as well as the more primitive macroscopic, multilineage colony-forming cells. In contrast, the delayed addition of
ATRA to cultures of hematopoietic precursors led to decreased granulocyte/macrophage colony-forming activity, presumably by inducing
the terminal maturation of committed granulocyte/monocyte progenitors
present in these cultures. Furthermore, in vivo colony-forming unit-spleen (CFU-S) studies demonstrated that the
linc-kit+Sca-1+
hematopoietic precursors cultured in ATRA-containing media for 7 and 14 days had markedly greater CFU-S activity than the same hematopoietic
precursors cultured without ATRA. These data suggest that the effect of
ATRA on hematopoietic development is dependent on the maturational
state of the target cell and that ATRA enhances the in vitro production
and/or self-renewal of primitive hematopoietic precursors.
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MATERIALS AND METHODS |
Mice.
C57BL/6J (Ly5.2) female mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). Congenic C57BL/6.SJL-Ly5.1-Pep3b (Ly5.1)
mice were bred at the Fred Hutchinson Cancer Research Center (Seattle,
WA). All animals were housed in specific pathogen free conditions and
maintained on acidified drinking water and autoclaved chow ad libitum.
Mice were used at 8 to 12 weeks of age.
Enrichment of hematopoietic precursor cells.
For enrichment of hematopoietic precursor cells, single-cell
suspensions of Ly5.2 bone marrow cells were obtained by flushing femurs
and passaging the marrow through a 23-gauge needle into phosphate-buffered saline (PBS) containing 2% heat-inactivated fetal
bovine serum (PBS/FBS). Low-density cells were enriched by equilibrium
centrifugation over a cushion of Ficoll-Hypaque at a density of 1.077 g/mL. Retrieved cells were washed and then resuspended at a density of
5 × 107 cells/mL in PBS/FBS containing a
predetermined saturating solution of monoclonal antibodies specific for
murine T lymphocytes (CD2, CD3, CD5, and CD8), B lymphocytes (B220),
macrophages (Mac-1; CD11b), granulocytes (Gr-1), and erythrocytes
(TER-119; the kind gifts of Dr G. Spangrude, University of Utah, Salt
Lake City, UT).19,20
After 30 minutes of incubation on ice, the cells were washed,
resuspended in PBS/FBS at a concentration of 108 cells/mL
and transferred to a 50-mL conical tube. Twice-washed immunomagnetic
particles (Dynabeads M-450, sheep antirat IgG specificity; Dynal Inc,
Great Neck, NY) were slowly added to the cells to a final ratio of 4 beads/cell. The cell/bead suspension was allowed to stand for 5 minutes
at room temperature and was then centrifuged at 400 rpm
for 3 minutes. The cells and bead mixture were resuspended in 1 mL of
PBS/FBS. The magnetic particle-free fraction was retrieved by exposure
to a magnetic field, and the magnetic depletion procedure was repeated
on this fraction.
The bead-free cells were then preincubated with Fc RII block (clone
2.4G2; Pharmingen, San Diego, CA) for 10 minutes on ice and then
stained with biotinylated anti-Ly6A/E (Sca-1, rat IgG2a, clone
E31-161.7) and fluorescein isothiocyanate (FITC)-conjugated anti-CD117
(c-kit, rat IgG2b, clone 2B8) monoclonal antibodies (Pharmingen) for 30 minutes on ice. Aliquots of the bead-free cells
were also stained with isotype-matched controls. The cells were then
washed with 10 mL PBS/FBS, stained with streptavidin-phycoerythrin (Pharmingen) for 30 minutes on ice, washed, and resuspended in PBS/FBS
containing 1 µg/mL propidium iodide (PI; Sigma Chemical Co, St Louis,
MO). The sample was filtered through a 70-µm pore size nylon screen.
Fluorescence-activated cell sorting was performed on a FACS VANTAGE
cell sorter (Becton Dickinson, San Jose, CA) using the CloneCyt direct
cloning application (Becton Dickinson). Cells were sequentially
selected for sorting as being PI,
c-kit+, Sca-1+, with intermediate
forward- and right-angle scatters.
Liquid suspension cultures of hematopoietic cells.
For prolonged liquid culture studies, 50 linc-kit+Sca-1+
precursor cells were deposited into a single well of round-bottom
96-well plates (Corning, Corning, NY) containing Iscove's modified
essential medium (IMDM) supplemented with 20% FBS and cytokines
(murine stem cell factor [SCF], human Flt-3L, human
interleukin-6 [IL-6], each at 100 ng/mL, and human IL-11 [10
ng/mL]; PeproTech, Inc, Rocky Hill, NJ). ATRA (Sigma) was added to a
portion of the wells of cells in each experiment to a final
concentration of 1 µmol/L. Each group consisted of five wells of
cells for each time point. For experiments assessing the effect of ATRA
on
linc-kit+Sca-1
progenitor cells, the final sort window was changed so that 100 cells
lacking expression of Sca-1 were deposited into each well. Cultures
were placed in incubation at 37°C with 5% CO2 in air atmosphere. Cultures were replenished with fresh media at weekly intervals by removing half the media in each well and replacing it with
an equal volume of media containing 2× concentration of cytokines
and ATRA where applicable. Different lots of FBS used in cultures were
screened for their ability to support colony growth of
linc-kit+Sca-1+
cells in semisolid media. Initial studies showed that adding ATRA to
the cultures more often than once weekly did not have any effect on the
experimental outcome.
For experiments investigating the effect of ATRA on single primitive
hematopoietic precursor cells, a single
linc-kit+Sca-1+ cell
was deposited into a single well of Nunclon 72 microwell plates (Nalge
Nunc International, Naperville, IL) in 15 µL total volume/well, in
media described above. The outer wells of each plate were filled with
sterile distilled water (15 µL/well) to prevent media evaporating
from the cell cultures. For each experiment, at least 120 wells of each
group were established. After sorting, each well was individually
viewed for the presence of a single cell, and wells in which no cell or
more than 1 cell were initially observed were not included in the
experiment. Wells were monitored at 24-hour intervals, and the number
of cells in each well of each plate was recorded daily.
In vitro colony assay.
The cultured cells were analyzed for colony formation in 35-mm culture
dishes (Nalge Nunc International) containing methylcellulose-based semisolid medium. At each time point, aliquots of cells were plated at
a predetermined concentration so that the average number of colonies
per plate did not exceed 50. The final concentration of methylcellulose
(Fisher Scientific, Norcross, GA) was 1.2% in -modified essential
medium containing 30% FBS, 1% deionized bovine serum albumin (BSA;
Sigma), 0.1 mmol/L 2-mercaptoethanol (Sigma), 3 U/mL Epo (Amgen,
Thousand Oaks, CA), 100 ng/mL each of SCF and IL-6, 50 ng/mL
thrombopoietin (Tpo; PeproTech), and 5% WEHI conditioned
medium (a source of IL-3).21 Cells were plated in 1 mL of
this methylcellulose mix and scored at 8 days for erythroid growth and
12 days for mixed and granulocyte/ macrophage (CFU-GM)
colonies. Erythroid colony types were routinely confirmed by staining
selected plates at day 8 with benzidine (Sigma). Mixed colonies
(HPP-mix) were larger than 1.5 mm in diameter and
consistently contained erythrocytes, macrophages, and granulocytes. In
addition, May-Grünwald-Giemsa-stained cytospin preparations of
these colonies often showed the presence of megakaryocytes.
Cell surface phenotyping.
Cultured cells were preincubated with FcRII block for 10 minutes at
4°C and then stained at 4°C for 30 minutes with predetermined concentrations of phycoerythrin (PE)-conjugated surface markers: Gr-1,
CD11b, TER-119, Thy1.2, B220, Sca-1 (Pharmingen), and F4/80 (Caltag,
South San Francisco; CA). Isotype-matched control antibodies were used
to determine background staining. After staining, the cell preparations
were washed, resuspended in PBS/FBS containing 1 µg/mL PI, and
analyzed on a FACSCAN (Becton Dickinson). Cells with a surface antigen
fluorescence intensity greater than 95% of control staining were
considered positive, whereas cells with a surface antigen fluorescence
intensity less than the top 5% of control staining were considered
negative. The total percentage of positive cells for each marker was
obtained by subtracting background staining of the isotype control from
the surface marker-stained sample.
CFU-S assay.
The spleen colony assay of Till and McCulloch22 was
applied. Ly5.1 female recipients 8 to 12 weeks of age were exposed to a
single dose of 10.0 Gy of radiation from dual opposed
60Co sources at an exposure rate of 20 cGy/min on the day
of transplantation. After 7 days of culture, all cells that grew in
culture from 500 Ly5.2
linc-kit+Sca-1+
cells were injected into lethally irradiated female Ly5.1 mice. After
14 days of culture, all cells that grew in culture from 1,000 Ly5.2
linc-kit+Sca-1+
cells were injected into irradiated female Ly5.1 mice. Transplanted mice were euthanized 8 or 12 days later, and their spleens were dissected, fixed in Bouin's fixative for 5 minutes, and then
transferred to 10% neutral buffered formalin (Sigma). CFU-S were
counted under a dissecting microscope. The number of colonies in
recipient spleens has been directly stated without correction for
seeding (f) factor.
Statistical analysis.
Data comparing the effects of ATRA versus no ATRA on the cell
proliferation and colony-forming output of cultured hematopoietic precursors were analyzed by two-way analysis of variance (ANOVA). For
experiments in which ATRA was added at different time intervals, the
Kruskal-Wallis test was used to compare the median values of
CFU-GM and mixed colonies between the groups in which
RA was added at 0 to 6 days or not added at all with the median values of the group in which RA was added at day 7 after culture initiation. Dunn's procedure was used to adjust for multiple comparisions, with
adjustments considered separately for the analysis of CFU-GM and
HPP-mix.
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RESULTS |
ATRA alters the growth and colony-forming cell (CFC)
production from cultured
linc-kit+Sca-1+
hematopoietic precursors.
To determine the effects of ATRA on relatively primitive hematopoietic
cells, we chose to use a combination of cytokines similar to one
previously reported23 that enhances the survival of
long-term reconstituting stem cells in liquid suspension for at least 7 days.
Linc-kit+Sca-1+ bone
marrow precursor cells were directly deposited by FACS into 96-well
plates at initial densities of 50 cells/well. Cells were cultured in
liquid suspension in serum-containing medium supplemented with 100 ng/mL each of SCF, IL-6, and Flt-3L and 10 ng/mL IL-11, with half of
the wells of cells containing 1 µmol/L ATRA. At various times during
the culture, the contents of 5 separate wells of each group were
counted, harvested, and plated for colony formation in the absence of
ATRA, and cells were also analyzed for cell surface phenotype after 7 and 14 days of liquid culture.
During the first 7 days of culture, the growth of the
linc-kit+Sca-1+
precursor cells was significantly slower in the ATRA-treated cultures
compared with cells cultured in the absence of ATRA
(Fig 1A and B), whereas viability was
comparable in both culture conditions (data not shown). In contrast,
beyond 12 days of culture, there was an increased number of cells in
cultures containing ATRA (Fig 1A and B). The maximum cell number for
the ATRA-negative cultures was observed between days 7 and 12, whereas
peak production of cells in the ATRA-treated cultures was noted at day
21. This difference in the average cell counts between the two groups
across the time period examined was statistically significant (two-way
ANOVA, P < .001 for both experiments).
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| Fig 1.
The effect of ATRA on cell growth and colony formation
from linc-kit+Sca-1+
hematopoietic precursor cells. Fifty FACS-enriched hematopoietic
precursors
(linc-kit+Sca-1+)
were added to wells containing media and cytokines (SCF, IL-6,
IL-11, and Flt-3-ligand) and cultured without ( ) or with ( ) 1 µmol/L ATRA. Cells were removed from liquid suspension at periodic
intervals, washed, counted, and replated in semisolid medium without
ATRA. The cell counts (A and B) and colony outputs of CFU-GM (C and D)
and HPP-mix (E and F) are given as the total number per 50 starting
hematopoietic precursors. Results are shown from two separate
experiments and are expressed as the mean ± SEM from 5 separate wells
for each time point. Two additional experiments were evaluated
periodically over 21 days, each with similar results, with ATRA-treated
cells exhibiting an initial slow growth followed by an accelerated cell
growth associated with enhanced CFU-GM and HPP-mix production.
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Of particular interest was our observation that ATRA prolonged and
enhanced the colony-forming cell output of hematopoietic precursor
cells (Fig 1C through F), including both mature CFU-GM and the more
immature mixed progenitors (HPP-mix). Cells grown in the absence of
ATRA exhibited colony-forming activity during the first 14 days of
liquid culture, after which production of CFU-GM (Fig 1C and D) and
HPP-mix (Fig 1E and F) was minimal. In contrast, cultures of
hematopoietic precursors stimulated with ATRA formed both CFU-GM and
HPP-mix during 21 days of liquid culture, with some colonies detectable
as late as day 28 (Fig 1C through F). The CFU-GM production was
enhanced in ATRA-treated cells by fourfold at day 7 and by more than
30-fold at day 14 in experiment no. 1, and by 160-fold at day 12 and
70-fold at day 14 in experiment no. 2. In addition, HPP-mix was
increased in ATRA-treated cells by more than 20-fold at and after 14 days in experiment no. 1, and by threefold at day 8 and more than
70-fold at days 12 and 14 in experiment no. 2. In both experiments,
there were significant differences in both CFU-GM and HPP-mix output
between the two groups (two-way ANOVA, P < .001).
In contrast to the CFU-GM and HPP-mix production, there was no effect
of ATRA on the number or size of erythroid colonies generated from
hematopoietic precursor cells cultured in liquid suspension (data not shown).
To further assess the ATRA-induced growth inhibition of
linc-kit+Sca-1+
precursor cells, single cells were deposited into each well of 72 microwell plates and the number of cells in each well was monitored daily. Immediately after sorting, each well was individually viewed for
the presence of a single cell, and wells in which no cell or more than
1 cell were observed were not included in the experiment. The addition
of ATRA to the cultures did not alter the time of the first cell
division, with the vast majority of cells cultured in either condition
dividing by day 3 (Fig 2A). In contrast,
ATRA slowed the subsequent cell growth, and by day 4 the majority of wells containing ATRA harbored between 2 and 50 cells, whereas in the
absence of ATRA there was a marked increase in the percentage of wells
containing more than 50 cells (Fig 2B).
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| Fig 2.
The effect of ATRA on the growth of single
linc-kit+Sca-1+
hematopoietic precursor cells. Single FACS-enriched hematopoietic
precursors
(linc-kit+Sca-1+)
were added to wells containing media and cytokines (SCF, IL-6, IL-11,
and Flt-3-ligand) and cultured without () or with () 1 µmol/L
ATRA. After sorting, each well was individually viewed for the presence
of a single cell, and wells in which no cell or more than one cell were
initially observed were not included in the experiment. The number of
cells in each well were counted at daily intervals after culture
initiation. The cumulative percentage of wells containing at least one
cell division (A) and the percentage of wells with 1 to 16, 17 to 50, and greater than 50 cells per well at daily intervals (B) are shown. In
(B), () indicates no ATRA and ( ) indicates ATRA. Results are
shown from one experiment but are indicative of three separate
experiments, each initiated with 120 single cells per treatment.
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Primitive
linc-kit+Sca-1+
hematopoietic precursors cultured in the presence of ATRA exhibit a
more immature surface phenotype than those grown in its absence.
The expression of differentiation antigens by cultured
linc-kit+Sca-1+
precursor cells was analyzed by flow cytometry after 7 and 14 days of
liquid suspension culture. To have enough cells for analysis of the
multiple cell markers at these time points, cultures were established
in 24-well plates at an initial density of 1,000 cells/well in 1 mL of
the culture medium described previously. To determine that this
different initial cell density did not markedly change the experimental
outcome, the production of CFC from these cultures was also analyzed
and the results were comparable to those from the 96-well plate
cultures described above (data not shown).
We observed that ATRA enhanced the expression of the monocytic marker,
F4/80, by the cultured cells at both 7 and 14 days of culture
(Table 1). There was negligible expression
(<3%) of this marker by cells grown in the absence of ATRA (Table
1). In contrast, the granulocyte marker, Gr-1, was predominantly
expressed by cells grown in the absence of ATRA (Table 1).
Significantly fewer cells cultured with ATRA expressed this marker
(Table 1).
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Table 1.
Flow Cytometric Analysis of
linc-kit+Sca-1+
Cells Cultured With or Without 1 µmol/L ATRA for 7 and 14 Days in
Liquid Suspension Culture
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There was little or no expression of the erythroid marker, TER-119, or
the T-lymphoid (Thy1.2) and B-lymphoid (B220) lineage markers after
either 7 or 14 days of culture (data not shown). The total percentage
of lin+ cells in these experiments was therefore considered
to be the summation of F4/80-positive and Gr-1-positive populations.
For cells grown in the absence of ATRA, at 7 days the total percentage of lin+ cells was 68.5%, increasing to 96.0% of the
population at day 14. In contrast, 44.6% of cells grown in the
presence of ATRA expressed F4/80 or Gr-1 at 7 days of incubation, and
31.4% of the population was positive for these lineage markers at 14 days of incubation.
Evaluation of the expression of Sca-1 showed that a large proportion of
the cells expressed Sca-1 when ATRA was included in the culture,
especially at day 14 of culture (Table 1). In contrast, the percentage
of Sca-1+ cells in cultures grown without ATRA decreased
over 14 days (Table 1).
Effect of 1,25 dihydroxyvitamin D3
[1,25(OH)2D3] on cultured
linc-kit+Sca-1+
primitive hematopoietic precursors.
To determine whether other ligands for the nuclear hormone receptor
superfamily might have a similar effect on the growth and
colony-forming capacity of the cultured murine hematopoietic precursors, similar experiments were performed using
1,25(OH)2D3. This ligand was chosen because
it also has well-documented activity in the regulation of certain
aspects of hematopoiesis.24-26 Hematopoietic precursor
cells
(linc-kit+Sca-1+)
were incubated with or without 1 µmol/L ATRA, 1 µmol/L
1,25(OH)2D3, or 10 nmol/L
1,25(OH)2D3 in the liquid suspension
cultures. At 1 week of culture, total viable cells and colony-forming
cell output was assessed.
Similar to the cells incubated with ATRA, precursor cells cultured with
1,25(OH)2D3 exhibited a marked reduction in
cell number compared with cells cultured in the absence of either
ligand (Fig 3A). Vitamin D3 was
more inhibitory than ATRA, with little cell growth occurring in 1 µmol/L ( 100 cells/well), and even at 10 nmol/L, there were fewer
cells in wells incubated with 1,25(OH)2D3 than in those cultured with 1 µmol/L ATRA. In contrast with
ATRA-treated cells, the colony output was markedly reduced from
precursor cells cultured with 1,25 dihydroxyvitamin D3,
with no colony growth observed from cells grown in 1 µmol/L
1,25(OH)2D3, and markedly fewer colonies
produced from cells cultured in 10 nmol/L
1,25(OH)2D3 compared with those incubated in
ATRA or in the absence of any ligand (Fig 3B). No HPP-mix were produced
from 10 nmol/L 1,25 dihydroxyvitamin D3-treated cells,
and the colonies grown from cells cultured in liquid suspension in the
presence of this ligand were predominantly mature macrophages (data not
shown).
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| Fig 3.
The effect of 1 ,25 dihydroxyvitamin D3 on
cell growth and colony formation from
linc-kit+Sca-1+
hematopoietic precursor cells. Fifty enriched hematopoietic precursors
(linc-kit+Sca-1+)
were added to wells containing media and cytokines (SCF, IL-6, IL-11,
and Flt-3-ligand) and cultured with or without 1 µmol/L ATRA, 1 µmol/L 1,25 dihydroxyvitamin D3 (D3), or 10 nmol/L D3
in the media. Cells were removed from liquid suspension after 7 days,
washed, counted, and replated in semisolid medium without ATRA or D3.
The cell counts (A) and total colonies (B) are given as the total
number per 50 starting hematopoietic precursors. Results are shown from
one experiment but are indicative of two separate experiments and are
expressed as the mean ± SEM from 5 separate wells for each time
point.
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The enhanced CFC production from cells cultured with ATRA is observed
only with highly enriched
linc-kit+Sca-1+
primitive hematopoietic precursors.
The enhanced and prolonged detection of both CFU-GM and HPP-mix in
ATRA-containing liquid suspension cultures of
linc-kit+Sca-1+
precursor cells (Fig 1) suggested that ATRA affects a relatively primitive hematopoietic precursor. We therefore tested if ATRA could
affect more mature progenitors
(linc-kit+Sca-1).
These cells were deposited into 96-well plates at a density of 100 cells/well, and wells were assayed for cell number and colony-forming
production (calculated as the total number of CFU-GM and HPP-mix per
well) at days 4, 7, and 14 of culture.
Similar to its effects on primitive
linc-kit+Sca-1+
hematopoietic precursors, ATRA slowed the proliferation of these more
committed linc-kit+Sca-1
progenitor cells (Fig 4A and B). However,
in contrast, the cell output from this ATRA-treated population never
exceeded the cell production from
linc-kit+Sca-1
progenitor cells grown in the absence of ATRA (Fig 4A and B). Moreover,
ATRA did not enhance or prolong the production of colonies from these
progenitors; in fact, the total colony output was lower in the
ATRA-treated cultures at and after 7 days of primary liquid suspension
culture (Fig 4C and D).
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| Fig 4.
The effect of ATRA on cell growth and colony formation
from linc-kit+Sca-1
hematopoietic progenitor cells. One hundred FACS-enriched
hematopoietic progenitors
(linc-kit+Sca-1)
were added to wells containing media and cytokines (SCF, IL-6, IL-11,
and Flt-3-ligand) and cultured without () or with () 1 µmol/L
ATRA. Cells were removed from liquid suspension at periodic intervals,
washed, counted, and replated in semisolid medium without ATRA. The
cell counts (A and B) and total colony outputs (C and D) are given as
the total number per 100 starting hematopoietic progenitors. Results
are shown from two separate experiments and are expressed as the mean ± SEM from 5 separate wells for each time point.
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The enhanced colony-forming cell production from the cultured
linc-kit+Sca-1+
precursor cells requires the early addition of ATRA after initiation of
cultures.
The enhanced CFC production observed in the ATRA-treated cultures of
primitive
linc-kit+Sca-1+
hematopoietic precursors was observed when ATRA was continuously present in the culture medium from the time of culture initiation (Fig
1). To determine whether there was a specific time interval during
which the addition of ATRA resulted in this enhanced generation of CFC,
we cultured
linc-kit+Sca-1+
cells in 24-well plates at an initial density of 1,000 cells per well
and then added ATRA (1 µmol/L final concentration) at daily intervals
from 0 to 7 days after culture initiation to a separate group of wells
for each time point. Cells were then harvested at day 8 and counted,
and colony assays were performed.
In each of two experiments, the addition of ATRA at each time point
caused a reduction in cell growth, with the lowest cell counts obtained
when ATRA was continuously present from day 0 of culture
(Fig 5A and B). There was then a gradual
increase in the cell number corresponding with each daily addition of
ATRA, with the maximum cell output obtained from wells of cells that were cultured in the absence of ATRA for the entire 8-day period.
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| Fig 5.
The effect of the delayed addition of ATRA on cell growth
and colony formation from cultured
linc-kit+Sca-1+
hematopoietic precursors. FACS-enriched hematopoietic precursors
(linc-kit+Sca-1+)
were added to wells containing media and cytokines (SCF, IL-6, IL-11,
and Flt-3-ligand). At daily intervals from 0 to 7 days after culture
initiation, ATRA (1 µmol/L final concentration) was added to a
separate group of wells for each time point. Cells were harvested at
day 8, washed, counted, and assayed for colony-forming cell activity.
The results from two separate experiments are shown. Experiment no. 1 was initiated at 50 cells/well in 96-well plates (200 µL media/well),
and experiment no. 2 was initiated at 1,000 cells/well in 24-well
plates (1 mL media/well). Cell counts (A and B) are shown as total cell
number per well for each experiment. Colony output, CFU-GM (C and D)
represent the total number of CFU-GM colonies per 50 starting
hematopoietic precursors for both experiments. Colonies are shown as
the mean ± SEM of triplicate plates per well.
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The delayed addition (days 1, 2, and 3) of ATRA did not affect the
production of CFU-GM in comparison with cultures that were initiated
with ATRA (Fig 5C and D). In contrast, the late addition of ATRA (day
7) resulted in a significant decrease in CFU-GM production (P = .008, Kruskal-Wallis test, adjusted by Dunn's procedure) compared with
cultures in which ATRA was added during the first 3 days (Fig 5C and D).
The late addition of ATRA to cultured
linc-kit+Sca-1+
precursor cells enhances granulocytic differentiation.
The marked decrease in CFU-GM production observed in the cultured
linc-kit+Sca-1+
cells when ATRA was added relatively late (day 7; Fig 5C and D)
suggested that ATRA influences the proliferative and differentiative potential of granulocyte/monocyte progenitors that have accumulated in
the cultures over the initial 7 days. Because ATRA is a well-known inducer of the differentiation of leukemic promyelocytes to
granulocytes,4 we determined whether the late addition of
ATRA might enhance terminal granulocytic differentiation of cells
generated by the cultured hematopoietic precursors.
Surface phenotype analysis by flow cytometry was performed on the
cultured cells in which ATRA was successively added at days 0 to 7 of
culture, with analysis on day 8 after culture initiation. Results of
four separate experiments reproducibly showed an increase in the
percentage and absolute number of Gr-1-expressing cells (Fig 6A and B) in the cultures in which
ATRA was added on and after day 3, with a decrease in F4/80-expressing
cells (Fig 6C and D) in these cultures.
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| Fig 6.
Expression of granulocyte and monocyte/macrophage markers
after the delayed addition of ATRA during liquid suspension culture.
One thousand FACS-enriched
linc-kit+Sca-1+
hematopoietic precursors were added to wells containing media and
cytokines (SCF, IL-6, IL-11, and Flt-3 ligand). At daily intervals from
0 to 7 days after culture initiation, ATRA (1 µmol/L final
concentration) was added to a separate group of wells for each time
point. Cells were harvested at day 8 and washed, and the surface
phenotypes of granulocytes (Gr-1) and monocyte/macrophages (F4/80) were
analyzed by flow cytometry. Results are expressed as the percentage and
absolute number of Gr-1-expressing (A and B) or F4/80-expressing (C
and D) cells per well for each day of addition. Results are shown from
one experiment, but multiple repeat experiments yielded similar
results.
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The percentage and absolute number of Gr-1-expressing cells present in
cultures grown without ATRA and to which ATRA was added on day 7 were
similar. However, the expression of Gr-1 did not discriminate between
immature and mature granulocytic cells in these populations. To examine
the effect of ATRA on the maturation of granulocytic cells, we
therefore prepared cytospins of these cultures and analyzed them for
morphological differences. We observed that the addition of ATRA on day
7 resulted in an increased number of mature granulocytes in the
cultures as assessed 24 hours later using
May-Grünwald-Giemsa-stained cytospins
(Table 2). These observations, taken
together, suggest that ATRA enhances the terminal granulocytic
differentiation of the CFU-GM that are generated during the 7-day
culture of the
linc-kit+Sca-1+
precursors initially grown in the absence of ATRA.
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Table 2.
The Effect of the Late (Day 7) Addition of ATRA on the
Granulocytic Differentiation of Cultured Hematopoietic Precursors
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ATRA enhances the production of CFU-S from cultured
linc-kit+Sca-1+
hematopoietic precursors.
Given its different effects on hematopoietic cells depending on their
maturational state, we wished to determine whether ATRA enhanced the in
vitro generation of hematopoietic cells that are more primitive than
colony-forming cells. We therefore investigated the effect of ATRA on
the production of CFU-S from cultured
linc-kit+Sca-1+ precursors.
The primitive hematopoietic precursors were deposited into 24-well
plates at an initial density of 2,000 cells/well in 1 mL of the culture
medium described previously. At days 7 and 14 of culture, the wells of
cells in each group were pooled, washed, and resuspended in PBS
containing 2% FBS for injection into lethally irradiated recipients.
An initial titration curve was performed using cells cultured without
ATRA to determine the optimal cell number to inject into mice after 7 and 14 days of culture (data not shown). In two studies, at day 7 of
culture, all cells that grew in culture from 500 linc-kit+Sca-1+
cells were injected into lethally irradiated female recipients. At day
14, all cells that grew in culture from 1,000 linc-kit+Sca-1+
cells were injected into lethally irradiated female recipients. Mice
were euthanized at day 8 or day 12 posttransplant, and their spleens
were removed, fixed, and counted for CFU-S. Endogenous CFU-S were also
measured in mice that were lethally irradiated but injected with
PBS/2% FBS only, and no CFU-S were visible in these mice (data not shown).
The results of these experiments are shown in
Table 3. At both days 7 and 14 of culture,
cells that were cultured with ATRA had markedly greater CFU-S activity
than cells cultured without ATRA. In fact, 500 or 1,000 hematopoietic
precursors cultured with ATRA for 7 or 14 days, respectively, produced
so many CFU-S that the spleen colonies were grossly confluent at both
days 8 and 12. The weights of the spleens of these mice were
approximately twice that of the spleens of mice injected with cells
grown without ATRA (Table 3). Therefore, to better quantitate CFU-S
number, the ATRA-treated cells were further titrated in experiment no. 2, and these results were used to derive the data shown in Table 3.
Specifically, the spleen colonies of mice injected with all cells that
grew from 20 ATRA-treated
linc-kit+Sca-1+
cells at both days 7 and 14 were quantifiable and thus were used to
extrapolate values for CFU-S potential of cells that grew from 500 or
1,000 hematopoietic precursors after culture in ATRA-containing medium
for 7 or 14 days, respectively. In this experiment, we determined that,
after 7 days of culture, the ATRA-treated cells had approximately
12-fold more CFU-S on day 8 (D8) and 31-fold more CFU-S on
day 12 (D12) than cells cultured without ATRA. After 14 days of culture, the ATRA-treated cells had 625-fold more CFU-S D8 than
hematopoietic precursors cultured without ATRA. At day 12, no mice
injected with cells cultured without ATRA had survived, so the CFU-S
D12 fold-increase could not be measured.
There was also a marked difference in the CFU-S numbers from both
cultured populations when compared with the CFU-S numbers of uncultured
linc-kit+Sca-1+
cells (experiment no. 2; Table 3). Freshly sorted
linc-kit+Sca-1+
cells gave rise to approximately 4 CFU-S D8 and 30 CFU-S D12 per 500 cells, similar to values previously reported by other investigators.27 Cells cultured without ATRA for 7 days had approximately fivefold more CFU-S D8 than the uncultured hematopoietic precursors. However, this was accompanied by a fourfold decrease in
CFU-S D12 potential. After 14 days of culture, the hematopoietic cells
cultured without ATRA had approximately fourfold less the number of
CFU-S D8 than 1,000 uncultured
linc-kit+Sca-1+
cells, and no measurable CFU-S D12. In marked contrast, hematopoietic precursors cultured with ATRA for 7 days had a 61-fold increase in
CFU-S D8 and a sevenfold increase in CFU-S D12 potential over the
uncultured
linc-kit+Sca-1+
hematopoietic precursors. In addition, 1,000 hematopoietic precursors cultured for 14 days with ATRA had approximately 157-fold more CFU-S D8
and fivefold more CFU-S D12 than the same number of uncultured linc-kit+Sca-1+
hematopoietic precursors.
The radioprotective ability of the cells cultured with or without ATRA
was also markedly different. Only 1 of 5 mice injected with cells that
grew from 1,000 hematopoietic precursors cultured without ATRA for 14 days survived by day 12 in experiment no. 1, and none of 3 similarly
injected mice survived in experiment no. 2. In contrast, all 5 mice
receiving cells that grew from 1,000 hematopoietic precursors cultured
in ATRA in experiment no. 1 and all 3 mice in experiment no. 2 had
survived and looked healthy when euthanized at day 12. In addition, in
experiment no. 2, all 3 mice receiving cells that grew from 100 hematopoietic precursors cultured with ATRA survived by day 12 (and had
confluent spleens) and 1 of 3 mice receiving cells that grew from 20 of these cultured hematopoietic precursors survived by day 12.
Taken together, these observations indicate that ATRA markedly enhances
the production and/or self-renewal of both CFU-S D8 and CFU-S D12 as
well as hematopoietic cells exhibiting radioprotective ability in
liquid suspension cultures of primitive hematopoietic precursors.
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DISCUSSION |
When highly enriched hematopoietic precursors are cultured in liquid
suspension in the presence of specific hematopoietic growth factors,
they undergo extensive proliferation accompanied by progressive lineage
commitment and terminal differentiation that, in some respects, may
mimic the proliferation and differentiation that normal hematopoietic
stem cells undergo in vivo.28,29 In this study, we provide
evidence that ATRA, a potent inducer of differentiation of
promyelocytic leukemia cells,4 can markedly alter the
growth and colony-forming capacity of highly enriched (linc-kit+Sca-1+)
hematopoietic precursors cultured in liquid suspension.
ATRA initially slowed the proliferation of these primitive
hematopoietic precursors, although this delayed cell production was
temporary and was followed by relatively rapid proliferation in the
ATRA-treated cultures. Surprisingly, ATRA enhanced and prolonged CFC
generation, including the immature, multipotent HPP-mix and the more
lineage-committed CFU-GM. Cells cultured in ATRA also exhibited fewer
lineage-specific markers (Gr-1 and F4/80) and displayed enhanced
expression of the marker associated with immature cells, Sca-1.
A striking effect of ATRA on the cultured hematopoietic precursors was
an inhibition of granulocytic production, as measured by expression of
the marker Gr-1. In the bone marrow, this antigen has previously been
shown to also be expressed transiently on the monocyte
lineage30; however, in the periphery it specifically recognizes neutrophils.31 The specificity of this marker
for cultured cell populations is unknown. However, given that there is
negligible expression of the monocyte-specific marker, F4/80, by cells
cultured without ATRA, which largely express Gr-1, we believe that Gr-1
is likely to be a granulocyte-specific marker in our cultured cell populations.
The significance of the Sca-1 expression, which is enhanced in cells
from ATRA-treated cultures, is less clear. This marker is expressed not
only on multipotent hematopoietic stem
cells,32 but also on subpopulations of bone marrow B cells
and myeloid cells33 as well as on certain populations of
resting and activated T cells.34,35 The lack of expression
of B-lymphocyte and T-lymphocyte markers by cells cultured with or
without ATRA indicates that the expression of Sca-1 is not by these
cell types. Although it is possible that Sca-1 is expressed by myeloid
cells in the cultured cell populations, the percentage of myeloid cells
(Gr-1-positive and F4/80-positive) in the ATRA-treated cultures does
not account for the high percentage of Sca-1-expressing cells,
especially at day 14 of culture. Likewise, the low percentage of
Sca-1-expressing cells in the untreated cultures does not correspond
with the high percentage of myeloid cells in these cultures. Although
it is unlikely that all the Sca-1-positive cells in the ATRA-treated cultures represent primitive hematopoietic stem cells, nevertheless, their presence correlates with the enhanced production of CFU-S noted
in these cultures.
The addition of ATRA to liquid suspension cultures of
linc-kit+Sca-1+
hematopoietic precursors strikingly enhanced the production of both
day-8 CFU-S and day-12 CFU-S compared with both uncultured linc-kit+Sca-1+
cells and the hematopoietic precursors cultured for 7 and 14 days
without ATRA. In contrast, hematopoietic precursors cultured without
ATRA showed a progressive loss in CFU-S activity during the 14 days of
liquid suspension culture compared with the CFU-S potential in
uncultured
linc-kit+Sca-1+
cells. Whereas there was a fivefold increase in CFU-S D8 in the 7-day
cultured cells over the uncultured hematopoietic precursors, this was
accompanied by a fourfold decrease in CFU-S D12 at this time point. The
CFU-S populations are more primitive than colony-forming cells, with
CFU-S D8 reflecting a more mature cell population than CFU-S
D12.36 The shift caused by a loss of CFU-S D12 in conjunction with an increase in CFU-S D8 in the cultures at 7 days
suggests that the primitive hematopoietic precursors cultured without
ATRA were progressively differentiating to more mature precursors. This
progressive differentiation appeared to continue by day 14 in the cells
cultured without ATRA, where we could detect very few CFU-S D8 and no
CFU-S D12. However, in marked contrast, the prolonged and enhanced
production of CFU-S D8 and CFU-S D12 in the ATRA-treated cultures
suggests that ATRA may be enhancing the self-renewal of hematopoietic
cells with CFU-S activity in these cultures. Alternatively, ATRA may be
influencing the self-renewal of hematopoietic cells that are even more
primitive than CFU-S,37 which could also result in enhanced
CFU-S production. To distinguish these possibilities, we are currently
determining whether ATRA enhances the production of short- and
long-term repopulating stem cells in liquid suspension cultures of
primitive hematopoietic precursors.
In other studies assessing the effect of ATRA on murine hematopoietic
precursor cells, Jacobsen et al18 cultured
linSca-1+ cells in liquid suspension in
the presence or absence of ATRA but did not investigate the
colony-forming cell or CFU-S potential of these cells after this
initial liquid suspension culture. However, they did morphologically
characterize cytospin preparations of the
linSca-1+ cells and noticed a threefold
to fourfold increase in the relative number of immature blasts that
formed after 12 to 14 days of incubation of
linSca-1+ cells in the presence of
either SCF and IL-3 or SCF and granulocyte colony-stimulating factor
(G-CSF), consistent with our own observation that ATRA maintains the
cultured hematopoietic precursors in a more undifferentiated state.
In addition, Jacobsen et al18 noted a marked decrease in
HPP-CFC formation when linSca-1+
hematopoietic precurs |
TOP
ABSTRACT
INTRODUCTION