|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 470-477
HEMATOPOIESIS
All-trans retinoic acid enhances the long-term repopulating
activity of cultured hematopoietic stem cells
Louise E. Purton,
Irwin D. Bernstein, and
Steven J. Collins
From the Clinical Research and Human Biology Divisions, Fred
Hutchinson Cancer Research Center, and The Department of Pediatrics,
The University of Washington, Seattle, WA.
 |
Abstract |
The retinoic acid receptor (RAR) agonist, 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. We previously reported that
pharmacologic levels (1 µmol) of ATRA enhanced the generation of
colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in
liquid suspension cultures of lin c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the
effects of ATRA as well as an RAR antagonist, AGN 193109, on the
generation of transplantable cells, including pre-CFU-S, short-term
repopulating stem cells (STRCs), and long-term repopulating stem cells
(LTRCs). ATRA enhanced the ex vivo maintenance and production of
competitive repopulating STRCs and LTRCs from lin c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these
primitive stem cells into more mature pre-CFU-S during the 14 days of
culture. In marked contrast, lin c-kit+ Sca-1+ cells
cultured with AGN 193109 for 7 days had virtually no short- or
long-term repopulating ability, but displayed an approximately 6-fold
increase in the pre-CFU-S population. The data suggest that the RAR
agonist ATRA enhances the maintenance and self-renewal of short- and
long-term repopulating stem cells. In contrast, the RAR antagonist AGN
193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an
important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation.
(Blood. 2000;95:470-477)
© 2000 by The American Society of Hematology.
| |
Introduction |
Retinoic acid (RA) and RA receptors (RARs)
play an important role in regulating the growth and differentiation of
a variety of different cells types.1 In hematopoiesis, the
RAR agonist, all-trans retinoic acid (ATRA) is predominantly known for
its differentiating effects, being a potent inducer of terminal
differentiation of malignant promyelocytes.2 In a recent
study, however, we showed that ATRA has different effects on cultured
hematopoietic cells depending on their maturational state.3
Specifically, the addition of pharmacologic levels (1 µmol) of ATRA
to liquid suspension cultures of lineage-negative,
c-kit-positive, Sca-1-positive (lin c-kit+
Sca-1+) hematopoietic precursors markedly enhanced the generation of
cells with colony-forming cell (CFC) and colony-forming unit-spleen
(CFU-S) potential. This effect of ATRA was restricted to a defined
population of these precursors, as ATRA did not enhance the generation
of cells with CFC potential from cultures of more mature, lin
c-kit+ Sca-1- progenitors. Furthermore, when the addition of
ATRA to cultures of primitive hematopoietic precursors was delayed
until the cultures had accumulated a significant number of committed
granulocyte/monocyte progenitors, ATRA accelerated the terminal
granulocytic maturation of these progenitors.3
The pleiotropic effects of ATRA are also evident in other developmental
systems. For example, during embryonic limb development in the mouse,
the application of pharmacological levels of ATRA had a
self-renewal-like effect, inducing the formation of supernumerary limbs.4,5 When ATRA was applied between 1030 and 1200 hours on 5.5 days postcoitum (dpc), limb duplications occurred, whereas no
duplications resulted when ATRA was administered after 1300 hours on
5.5 dpc.5 Moreover, when ATRA was given to the embryo at
later stages of development, between 10 and 12.5 dpc, it induced the
opposite effect of stunted limb development.6,7 Hence, ATRA
induces different effects on embryonic limb development in the mouse,
dependent upon the stage of embryonic development.
In a previous study, we observed that the addition of pharmacological
levels of ATRA to serum-containing liquid suspension cultures of
primitive hematopoietic precursors markedly enhanced the generation of
CFCs and CFU-S.3 Such cultures already contain endogenous
levels of ATRA, as ATRA is present both in serum-containing media and
in serum-free media, bound to the albumin present in these media.
Hence, in this study we were interested in further determining the
effects of both pharmacological and endogenous levels of ATRA on
cultured primitive hematopoietic precursors. To examine the effects of
endogenous ATRA, we used an RAR pan-antagonist, AGN 193109. This
antagonist binds to RARs with high affinity but does not activate
transcription, and thus acts as a competitive inhibitor of RAR
activation by endogenous levels of retinoic acid present in the
cultures.8,9 Our results indicate that ATRA enhanced the
maintenance and/or generation of short- and long-term repopulating stem
cells from lin c-kit+ Sca-1+ hematopoietic precursors
cultured in liquid suspension for 14 days. ATRA also prevented the
differentiation of these primitive stem cells into the more mature
pre-CFU-S population during the 7 to 14 days of culture. In marked
contrast, hematopoietic precursors lost their short- and long-term
competitive repopulating ability after 7 days of culture with AGN
193109, accompanied by an increase in the more mature pre-CFU-S. These
data suggest an unexpected role for ATRA in enhancing the in vitro
maintenance and/or inducing self-renewal of in vivo repopulating
hematopoietic stem cells.
| |
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
The lineage-negative, c-kit-positive, Sca-1-positive
(lin c-kit+ Sca-1+) hematopoietic precursor cells were
FACS enriched as previously described.3
Liquid suspension cultures of hematopoietic cells
Two thousand lin c-kit+ Sca-1+ precursor cells were
deposited into a single well of 24-well plates (Corning, Corning, NY) containing Iscove's modified essential medium (IMDM) supplemented with
20% fetal bovine serum (FBS) and cytokines (murine stem cell factor
[SCF], human Flt-3-ligand, human interleukin-6 [IL-6]; each at 100 ng/mL and human IL-11 [10 ng/mL]; PeproTech, Inc, Rocky Hill, NJ).
All-trans retinoic acid (Sigma) or the RAR pan-antagonist, AGN 193109 (Allergan Pharmaceuticals, Irvine, CA) were added to a portion of the
wells of cells in each experiment, to a final concentration of 1 µmol. Cultures were placed in incubation at 37°C with 5%
CO2 in air atmosphere. Cultures were replenished with fresh
media at periodic 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 or AGN 193109, where applicable. Different lots of FBS used in cultures were screened for their ability
to support colony growth of lin c-kit+ Sca-1+ cells in semisolid media. Initial studies showed that adding ATRA or AGN 193109 to the cultures more often than once weekly did not have any effect on
the experimental outcome.
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 as previously
described.3
CFU-S assay
The spleen colony assay of Till and McCulloch10 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 or 14 days of culture, all cells that grew
from 500 or 1000 Ly5.2 lin c-kit+ Sca-1+ cells,
respectively, were injected into lethally irradiated female Ly5.1 mice.
Transplanted mice were euthanized 8 or 12 days later, their spleens
dissected, fixed in Bouin's fixative for 5 minutes, then transferred
to 10% neutral buffered formalin (Sigma). Colony-forming unit-spleen
(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.
Pre-CFU-S assay
Pre-CFU-S was assayed as described by Hodgson et al.11
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 or 14 days of culture, all cells that grew
from 500 or 1000 Ly5.2 lin c-kit+ Sca-1+ cells,
respectively, were injected into lethally irradiated female Ly5.1 mice.
Thirteen days after the transplant, the primary recipients were
euthanized, and bone marrow from the femurs of 3 primary recipients
were pooled. Fractions of the pooled marrow were then injected into new
lethally irradiated recipients. Twelve days after transplant, the
secondary recipients were euthanized, their spleens dissected and fixed
for CFU-S evaluation as described previously. A total of 12 secondary
recipients were used per treatment group. The values given are for
pre-CFU-S generated from 1 femur and have been extrapolated from
colonies arising from 0.3 femurs injected per secondary recipient
(n = 4).
Short- and long-term competitive repopulation assay
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. The primitive hematopoietic precursors (lin
c-kit+ Sca-1+) were deposited into 24-well plates at an initial
density of 2000 cells per well in 1 mL of the culture medium described
previously, supplemented with or without 1 µmol ATRA or 1 µmol AGN
193109. One thousand freshly sorted female Ly5.2 lin
c-kit+ Sca-1+ were injected into the tail vein of irradiated
female Ly5.1 recipients, together with 1 × 105
normal male Ly5.1 bone marrow. After 7 and 14 days of culture, all
cells that grew in culture from 1000 Ly5.2 lin c-kit+
Sca-1+ cells under each culture condition were injected into irradiated female Ly5.1 mice, together with 1 × 105 normal
male Ly5.1 bone marrow.
Analysis of transplant recipients
Peripheral blood from each recipient was obtained from the
retro-orbital sinus at monthly intervals. The red blood cells were lysed with ammonium chloride lysis buffer, and the remaining cells were
washed in PBS/FBS, preincubated with FcRII block for 10 minutes at
4°C, and distributed into 12 × 75 mm polypropylene tubes
(Fisher Scientific) for immunofluorescent staining. Nucleated cells
were stained with biotinylated monoclonal antibodies specific for Ly5.2
(clone 104) and Ly5.1 (clone A20) (the kind gifts of Dr G. Spangrude)
or biotinylated mouse IgG2a (Pharmingen) for 30 minutes at 4°C. The
cells were then washed with PBS/FBS and stained with
streptavidin-phycoerythrin. At 3, 6, 9, and 12 months afer transplant,
donor cells in T-lymphocyte, B-lymphocyte, granulocyte, and
monocyte/macrophage lineages were analyzed by staining for donor
(Ly5.2) positive cells, along with FITC-conjugated monoclonal antibodies: anti-Thy1.2, anti-B220, anti-Gr-1, anti-CD11b, or FITC-conjugated isotype-matched control antibodies (Pharmingen). Host
(Ly5.1)-positive cells were also investigated in the B-lymphocyte lineage at these time points, to confirm accuracy of the donor cell
(Ly5.2) staining. The stained cells were washed with PBS/FBS and
resuspended in PBS/FBS containing 1 µg/mL PI and analyzed on a FACSCAN.
Statistical analysis
Data comparing the effects of ATRA versus No ATRA or AGN 193109 versus No AGN 193 109 in the short- and long-term competitive repopulation assays were analyzed with the Wilcoxon rank-sum test. The
transplant data of the 7-day cultured cells treated with or without
ATRA were further analyzed by fitting a regression model on the ranks
of the data, with treatment group being the explanatory variable of
interest and transplant number included as an additional explanatory variable.
| |
Results |
The RAR pan-antagonist, AGN 193109, enhances the production of
CFU-S day 8 but not CFU-S day 12 in cultures of lin
c-kit+ Sca-1+ hematopoietic precursors
The current studies were designed to investigate the effects of
retinoids on transplantable hematopoietic cells. In a previous study,
we observed that ATRA (1 µmol) markedly enhanced CFU-S production in
liquid suspension cultures of primitive hematopoietic precursors
(lin c-kit+ Sca-1+).3 We therefore further
explored the role of retinoids in the production of CFU-S by
determining the effects of the RAR pan-antagonist, AGN 193109, on the
generation of CFU-S from cultured hematopoietic precursors.
Lethally irradiated recipients were injected with 500 freshly sorted
lin c-kit+ Sca-1+ hematopoietic precursors or all cells that grew from 500 or 1000 hematopoietic precursors after 7 or 14 days
of culture, respectively, with or without 1 µmol AGN 193109. Mice
were euthanized at day 8 and day 12 after transplant, their spleens
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 spleen colonies were visible in these mice
(data not shown).
The results of these experiments are shown in Table
1. Hematopoietic precursors cultured for 7 days without AGN 193109 produced approximately 4- to 5-fold more CFU-S
D8 than the freshly sorted, noncultured lin c-kit+
Sca-1+ hematopoietic precursors (Table 1, experiment 2). This was
accompanied, however, by a 4-fold decrease in CFU-S D12 compared with
the noncultured precursors. The addition of 1 µmol AGN 193109 to the
cultures of hematopoietic precursors resulted in a 1.5- to 2-fold
increase in CFU-S D8 at 7 days of culture compared with the cells
cultured without AGN 193109, but there was no change in CFU-S D12
production. Again, this represented an increase in CFU-S D8 but a
decrease in CFU-S D12 when compared with freshly sorted lin
c-kit+ Sca-1+ precursors. After 14 days of culture, there were
few CFU-S D8 or D12 produced from either treatment groups (Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1.
The effect of AGN 193109 on CFU-S production from
cultured lineage-negative, c-kit-positive, Sca-1-positive
hematopoietic precursors
|
|
Effect of RAR agonist, ATRA, and RAR antagonist,
AGN 193109, on pre-CFU-S activity of cultured
hematopoietic precursors
To further delineate the effects of both the RAR agonist,
ATRA, and the RAR antagonist, AGN 193109, on cultured transplantable hematopoietic precursors, we determined their effects on cells within
the lin c-kit+ Sca-1+ population that are more primitive than CFU-S, the pre-CFU-S.12,13
The results of this experiment are shown in Table
2. Hematopoietic precursors cultured
without ATRA or AGN 193109 for 7 days had almost 2-fold the number of
pre-CFU-S compared with the freshly isolated, noncultured lin
c-kit+ Sca-1+ cells (Table 2). The addition of 1 µmol ATRA to
the cultures for 7 days resulted in fewer pre-CFU-S, at levels
slightly lower than the noncultured hematopoietic precursors. By day 14 of liquid suspension culture, however, hematopoietic precursors
cultured without ATRA could not rescue primary recipients to 13 days
after transplant, hence no values could be obtained for pre-CFU-S for
this time point. In contrast, hematopoietic precursors cultured with 1 µmol ATRA for 14 days maintained pre-CFU-S to levels similar to that
present at 7 days of culture in ATRA (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 2.
The effect of AGN 193109 and ATRA on pre-CFU-S
production from cultured lineage-negative, c-kit-positive,
Sca-1-positive hematopoietic precursors
|
|
The addition of the RAR pan-antagonist, AGN 193109, to cultures of
hematopoietic precursors for 7 days had a profound effect on the
generation of pre-CFU-S, increasing this compartment by approximately
3-fold compared with that of hematopoietic precursors cultured without
AGN 193109. This also represented approximately 6-fold and 14-fold
increases in pre-CFU-S when compared with pre-CFU-S potential of the
noncultured precursors and hematopoietic precursors cultured for 7 days
with 1 µmol ATRA, respectively (Table 2). By day 14 of liquid
suspension culture, however, hematopoietic precursors cultured with AGN
193109 could not rescue primary recipients to 13 days after
transplant, hence no values could be obtained for pre-CFU-S at this
time point.
ATRA enhances the ex vivo maintenance of in vivo long-term
repopulating stem cells
Our previous observations that ATRA enhances the generation of CFC
and CFU-S from cultured hematopoietic precursors,3 together with our current observation that pre-CFU-S levels are maintained, albeit at slightly lower levels, in such cultures (Table 2) suggest that ATRA may influence the production of even less mature
hematopoietic precursors. We therefore wished to determine whether
exogenous ATRA might enhance the production or maintenance of short-
and long-term repopulating stem cells.
The FACS-enriched primitive hematopoietic precursors were deposited
into 24-well plates at an initial density of 2000 cells per well in 1 mL of the culture medium described previously, with or without 1 µmol
ATRA. At day 0, 1000 freshly sorted Ly5.2+ lin c-kit+
Sca-1+ cells, together with 1 × 105 normal Ly5.1+
bone marrow cells, were injected into lethally irradiated Ly5.1+
recipients. Then, at days 7 and 14, all cells that grew in culture from
1000 of the initial lin c-kit+ Sca-1+ cells, together
with 1 × 105 normal Ly5.1+ bone marrow cells were
injected into lethally irradiated Ly5.1+ recipients. FACS analysis,
using antibodies specific for the Ly5.1 (host) and Ly5.2 (donor)
epitopes, allowed us to quantitate the percentage of residual host and
donor Ly5.1 (untreated) versus Ly5.2 donor (treated) cells in
peripheral blood samples serially harvested from these animals after
transplant. In all experiments, background staining of Ly5.2 in
nontransplanted Ly5.1 mice was < 3.0%.
These in vivo repopulation studies showed that the addition of ATRA to
cultures of hematopoietic precursors resulted in greater short- and
long-term repopulating activity compared with cultures without ATRA
(Figure 1). The number of cultured cells
injected per mouse for each of 2 experiments are shown in Table
3. In each experiment, mice transplanted
with noncultured precursors showed short-term ( 4 months) and
long-term ( 6 months) donor cell reconstitution (Figure 1A and D).
Mice receiving all cells that grew from 1000 initial hematopoietic
precursors cultured for 7 days with or without ATRA displayed similar
levels of long-term donor cell reconstitution (Figure 1B and E).
However, there were marked differences in the cell number injected into
mice in the 2 treatment groups, with significantly fewer cells present
in the ATRA-treated cultures at 7 days (Table 3). Therefore, we also
calculated the donor cell reconstitution per 105 cells
injected into the mice (Table 4). The
results of 3 separate experiments were used in this analysis, where
experiment 1 and experiment 2 are data from the respective experiments
in Figure 1, and experiment 3 data are from an additional transplant of 7-day cultured cells analyzed at 6 months. Analysis of these data showed a statistically significant difference in 2 of 3 experiments (P .02). In addition, there was an overall statistically
significant difference (P < .001) between the 2 groups,
with ATRA-treated cells having significantly higher donor cell
reconstitution per 105 cells injected into the mice. In
these studies, this long-term reconstitution in both treatment groups
was multilineage, as donor cells were detected in both myeloid
(Gr-1 and CD11b) and lymphoid (B220 and Thy1.2) populations (Table
5).
View larger version (25K):
[in this window]
[in a new window]
| Fig 1.
The effect of ATRA on liquid suspension cultures of
short- and long-term repopulating hematopoietic stem cells.
2000 FACS-enriched hematopoietic precursors (lin c-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 ATRA. Irradiated Ly5.1 recipients (10 mice per group) were
transplanted with 1 × 105 normal Ly5.1 bone marrow
cells together with 1000 noncultured ( ) Ly5.2 lin
c-kit+ Sca-1+ cells or with all cells that grew from 1000 of
these precursors after 7 or 14 days in liquid suspension culture. Data
are expressed as the mean ± SEM donor cell reconstitution in the
peripheral blood of transplanted recipients analyzed between 5 weeks
(1.25 mo) and 12 months after transplant. Results of noncultured cells
(A, D), 7-day cultured cells (B, E) and 14-day cultured cells (C, F)
are shown from 2 separate experiments (Exp. 1 and 2).
|
|
After 14 days of culture, there was a significant difference between
the repopulating ability of cells from the ATRA-treated and nontreated
cultures (Figure 1C and F). Of note, the number of cultured cells
injected into the mice in both treatment groups was similar at this
time point (Table 3). However, none of the 10 mice receiving cells
cultured for 14 days without ATRA showed any donor cell reconstitution,
whereas mice receiving hematopoietic precursors cultured with ATRA for
14 days showed significant short- and long-term donor cell
reconstitution (Figure 1C and F). Again, these reconstituting donor
cells gave rise to multilineage progeny (Table 5). This
difference in reconstituting ability of the ATRA-treated versus
untreated day 14 cultured cells was statistically significant at all
time points (P < .001, Wilcoxon rank-sum test).
The RAR antagonist, AGN 193109, abrogates the short- and long-term
repopulating activity of cultured stem cells
The marked increase in pre-CFU-S activity of hematopoietic
precursors cultured for 7 days with the RAR antagonist AGN 193109 (Table 2) may have resulted from a direct effect of the antagonist on
the pre-CFU-S population. Alternatively, it may be a reflection of an
effect of the antagonist on more primitive repopulating stem cells,
such as enhancing the differentiation of these cells into the
pre-CFU-S compartment. Given that the RAR agonist, ATRA, enhances the
production and/or maintenance, and likely blocks the
commitment/differentiation of short- and long-term repopulating stem
cells in ex vivo liquid suspension cultures, the latter seemed to be
the likely explanation. Therefore, we assessed the effects of the RAR
pan-antagonist on short- and long-term repopulating stem cells in the
liquid suspension cultures.
The short- and long-term competitive repopulation assays were performed
as described for the ATRA-treated cultures, except that cells were
cultured with or without 1 µmol AGN 193109 in liquid suspension culture.
The number of cells and CFC produced from 1000 initial hematopoietic
precursors after 7 days of culture with or without AGN 193109 was
similar in each of 2 experiments (Table 6).
In contrast, the AGN 193109-treated cells had significant differences
in their repopulating activity compared with precursors cultured
without the RAR pan-antagonist (Figure 2).
In each of 2 experiments, mice receiving precursors cultured for 7 days
without AGN 193109 showed short- and long-term donor cell
reconstitution (Figure 2). In marked contrast, the addition of the RAR
antagonist, AGN 193109, to cultures of hematopoietic precursors for 7 days significantly abrogated both short- and long-term repopulating
ability of these cells, with donor cell levels below the
background levels of 3% Ly5.2 positive cells in untransplanted Ly5.1
mice (P .002, Wilcoxon rank-sum test). Hematopoietic
precursors cultured for 14 days with or without the RAR pan-antagonist
did not contribute to short- or long-term reconstitution of the
recipients (data not shown).
View larger version (14K):
[in this window]
[in a new window]
| Fig 2.
The effect of the RAR antagonist, AGN 193 109, on liquid
suspension cultures of short- and long-term repopulating hematopoietic
stem cells.
2000 FACS-enriched hematopoietic precursors (lin c-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 AGN 193109. Irradiated Ly5.1 recipients (5 mice per group) were
transplanted with 1 × 105 normal Ly5.1 bone marrow
cells together with all cells that grew from 1000 lin
c-kit+ Sca-1+ precursors after 7 days in liquid suspension
culture. Data are expressed as the mean ± SEM donor cell
reconstitution in the peripheral blood of transplanted recipients
analyzed between 5 weeks (1.25 mo) and 6 months after transplant.
Results of 7-day cultured cells (A, B) are shown from 2 separate
experiments.
|
|
| |
Discussion |
In recent years, an increasing number of investigators have been
interested in ex vivo culture of hematopoietic precursor cells for
purposes such as stem cell expansion and retroviral-mediated gene
transduction. Invariably, the culture of these cells leads to a rapid
decline in stem cell activity, resulting in markedly impaired
transplantability of the cultured cell populations. The need to improve
on such methods is obvious, and, for gene therapy purposes using
retroviral vectors, it is essential for a hematopoietic stem cell to
divide but be prevented from differentiating during the culture period
in order to enhance the possibility of correcting a genetic deficiency
in hematopoietic stem cells.
The experiments described here involved attempts to determine whether
ATRA might alter the differentiation of lin c-kit+ Sca-1+ hematopoietic precursors cultured in liquid suspension. In a
previous study, we demonstrated that hematopoietic precursors cultured
in liquid suspension in pharmacologic levels (1 µmol) of ATRA were
maintained in a less differentiated state than those cultured without
ATRA as assessed by cell surface phenotyping and enhanced CFC and CFU-S
activity.3 The increase in CFC numbers was likely to be a
result of enhanced generation of a cell population more primitive than
CFC, as we also reported that ATRA did not enhance CFC production from
the more mature population of lin c-kit+
Sca-1-progenitors. The ATRA-induced increase in CFU-S and CFC
production in these cultures may therefore have resulted from either a
direct effect of ATRA on the self-renewal of CFU-S or from ATRA-induced
effects on a hematopoietic precursor more primitive than CFU-S. To
distinguish these possibilities, we determined the effects of ATRA on
the generation of pre-CFU-S, short-term repopulating stem cells
(STRCs) and long-term repopulating stem cells (LTRCs) in liquid
suspension cultures of lin c-kit+ Sca-1+ hematopoietic precursors.
The most striking observation involved the effect of the RAR agonist
and antagonist on the generation of STRCs and LTRCs. In the absence of
exogenous ATRA, the repopulating ability of the cultured hematopoietic
precursors gradually declined and, by 14 days, such ex vivo cultures no
longer harbored any stem cells as measured in an in vivo competitive
repopulating assay. The addition of 1 µmol ATRA to cultures of these
hematopoietic precursors, however, prolonged the maintenance of stem
cells so that after 14 days of culture both short- and long-term
competitive repopulating stem cells could still be detected in these
cultures. In marked contrast, the addition of the RAR antagonist to
these cultures had the opposite effect. Indeed, this compound
completely abrogated the production of both STRCs and LTRCs after 7 days of culture, a time at which considerable competitive repopulating stem cell activity was readily detected in the ATRA-treated and nontreated culture systems. This RAR antagonist-induced loss of functional primitive hematopoietic stem cells does not appear to merely
be the result of defective homing of these cultured cells,
because the AGN 193 109-treated hematopoietic precursors were
capable of homing to both marrow and spleen as demonstrated by their
marrow repopulating ability (pre-CFU-S) and CFU-S activity.
Interestingly, the pre-CFU-S have been previously reported to
resemble hematopoietic stem cells in that they share a pattern of low
rhodamine-123 fluorescence staining intensity.12,13 However, although the pre-CFU-S are relatively primitive hematopoietic precursors, our observed association of an increase in pre-CFU-S production in the RAR antagonist-treated cultures with a concomitant decrease in short- and long-term repopulating stem cell production indicates that the pre-CFU-S and competitive repopulating
hematopoietic stem cells represent discrete hematopoietic precursor compartments.
Our observation that the RAR antagonist abrogates STRC and LTRC
production in liquid suspension cultures strongly indicates that
endogenous levels of RA that are normally present in serum, or bound to
proteins present in serum-free media, can influence the generation and
maintenance of primitive hematopoietic stem cells in liquid suspension
culture, and underscores the importance of functioning RARs to the
integrity of hematopoietic stem cells. Previous studies involving mice
with "knockouts" of single and double RAR isoforms ( ,  ,
) have indicated numerous abnormalities in various organs,
particularly in double "knockouts," including malformations of
the head, vertebrae, limbs, neck, trunk, and abdominal
regions.14-19 However, only mice null for both the and
isoforms have hematopoietic defects,19 with the RAR
and RAR1 double null mutants displaying impaired
granulocytic differentiation in vitro.19
Interestingly, these mice have no observable stem cell defects, and
hematopoiesis, including granulopoiesis, is normal in vivo, suggesting
compensatory mechanisms in these mice. To our knowledge, however,
mutant mice null for all 3 isoforms have not been created, and, given
that double null mutants are often embryonically lethal or die shortly
after birth, the feasibility of creating such mutants is questionable.
Our transplant results using the RAR antagonist, AGN 193109, which is a
pan-(, , ) antagonist and effectively "knocks out" all
RAR activity,8 would suggest, however, that such triple
null mutant mice would have severe hematologic defects arising at the
primitive stem cell level.
Our observations on hematopoietic precursor and progenitor production
in RAR agonist and antagonist-treated cultures of lin c-kit+ Sca-1+ hematopoietic precursors noted both in the
current study and in our previous experimental efforts3 are
summarized in Figure 3. It is clear that
the RAR agonist and antagonist exert complex, pleiotropic effects in
these cultures that are likely heavily dependent on the maturational
state of the hematopoietic cell type being affected. ATRA appears to
maintain both STRCs and LTRCs in these cultures, whereas the RAR
antagonist depletes such stem cells most probably by encouraging their
differentiation to a more committed precursor, the pre-CFU-S. In
addition, although the LTRC, STRC and pre-CFU-S compartments are
maintained in ATRA-treated cultures, the CFU-S compartment is
dramatically increased, suggesting that ATRA most likely has a direct
effect in enhancing the self-renewal of CFU-S in these cultures.
The enhanced CFC production that we previously observed in the
ATRA-treated cultures is also likely secondary to enhanced production
and maintenance of more primitive CFU-S and marrow reconstituting stem
cells rather than a direct effect of ATRA on the CFC progenitors.
View larger version (17K):
[in this window]
[in a new window]
| Fig 3.
Schematic depiction of the effects of RAR agonists and
antagonists on cultured hematopoietic progenitor cells.
The effects of all-trans retinoic acid (ATRA) on various hematopoietic
progenitor cell compartments during culture of lin
c-kit+ Sca-1+ hematopoietic precursors for 7 and 14 days with 1 µmol RAR agonist, ATRA, or 1 µmol RAR antagonist, AGN 193109, compared with the magnitude of the progenitor compartments of the
noncultured hematopoietic precursors are shown. The magnitude of the
effect is indicated by the number of arrows, with 1 arrow representing
the smallest effect and 4 arrows representing the largest effect.
Arrows indicate as follows:  , maintenance in potential;  ,
increase in potential;  , decrease in potential. *denotes effects
seen only at 7 days of culture. Abbreviations: LTRC, long-term
repopulating stem cell; STRC, short-term repopulating stem cell;
pre-CFU-S, pre-colony-forming unit-spleen; CFU-S, colony-forming
unit-spleen; D8, day 8; D12, day 12; CFC, colony-forming cell.
|
|
We do not know whether our observed effect of ATRA in enhancing the
maintenance or production of transplantable hematopoietic stem cells in
liquid suspension culture results from a direct effect of ATRA on the
stem cells or an indirect effect through "accessory" cells that
may regulate stem cell behavior in these cultures. The ATRA-treated
cultures at 7 days exhibited reduced cell density compared with the
untreated cultures (Table 3), perhaps leading to changes in cellular
cross talk at this time point that might account for differences in
stem cell production/maintenance. However, cultures of the
hematopoietic precursors treated with the RAR antagonist, which
markedly abrogated transplantable stem cell activity at 7 days,
displayed comparable cell densities to cultures without the antagonist
(Table 6), suggesting that the differences in stem cell activity were a
direct effect of the antagonist.
The molecular mechanisms involved in the observed effects of ATRA
on the hematopoietic precursors have not yet been determined. ATRA
regulates its biologic activities by triggering the activation of
RAR-RXR heterodimers that serve as transcription factors to regulate
the expression of specific target genes. Key genes regulated by ATRA in
the mouse and chick during embryonic development include members of the
homeobox (HOX) superfamily.5,20,21 Hox genes are expressed
in hematopoietic cells and have been implicated in the regulation of
various aspects of hematopoiesis.22-28 Interestingly, there
are many parallels between our observations and those observed when the
HOX gene, HOX B4 was overexpressed in mouse bone
marrow.25 These observations included a maintenance of
pre-CFU-S, an increase in CFU-S D12 and a marked increase in CFC.
Our studies indicate that pharmacological levels, as well as endogenous
culture media-containing levels of ATRA influence the primitive
hematopoietic stem cells, as shown by the enhanced ex vivo maintenance
of repopulating stem cells in liquid suspension culture. Regardless of
whether ATRA enhances self-renewal of primitive hematopoietic stem
cells or slows their differentiation in liquid suspension cultures,
these results suggest that ATRA may be a useful tool for aiding
retroviral- or lentiviral-mediated gene transduction into primitive
hematopoietic stem cells. We are currently investigating this possibility.
| |
Acknowledgements |
We thank Cynthia Nourigat for her excellent technical assistance and Dr
Ted Gooley for statistical analysis.
| |
Footnotes |
Submitted June 10, 1999; accepted September 16, 1999.
Supported by National Institutes of Health (NIH) grants nos. HL54881
and CA58292. L.E.P. is a Leukemia Society of America Special Fellow.
I.D.B. is supported as a Clinical Research Professor by the American
Cancer Society.
Reprints: Louise E. Purton, Fred Hutchinson Cancer Research
Center, 1100 Fairview Ave N, C1-169, PO Box 19024, Seattle, WA
98109-1024.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Evans R.
The steroid and thyroid hormone receptor superfamily.
Science.
1988;240:889-895[Abstract/Free Full Text].
2.
Breitman T, Selonick S, Collins S.
Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid.
Proc Natl Acad Sci U S A.
1980;77:2936-2940[Abstract/Free Full Text].
3.
Purton LE, Bernstein ID, Collins SJ.
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.
Blood.
1999;94:483-495[Abstract/Free Full Text].
4.
Rutledge JC, Shourbaji AG, Hughes LA, et al.
Limb and lower-body duplications induced by retinoic acid in mice.
Proc Natl Acad Sci U S A.
1994;91:5436-5440[Abstract/Free Full Text].
5.
Niederreither K, Ward SJ, Dollé P, Chambon P.
Morphological and molecular characterization of retinoic acid-induced limb duplications in mice.
Dev Biol.
1996;176:185-198[Medline]
[Order article via Infotrieve].
6.
Kochhar DM.
Limb development in mouse embryos. I. Analysis of teratogenic effects of retinoic acid.
Teratology.
1973;7:289-298.
7.
Kochhar DM.
In vitro testing of teratogenic agents using mammalian embryos.
Teratog Carcinog Mutagen.
1980;1:63-74[Medline]
[Order article via Infotrieve].
8.
Johnson AT, Klein ES, Gillett SJ, et al.
Synthesis and characterization of a highly potent and effective antagonist of retinoic acid receptors.
J Med Chem.
1995;38:4764-4767[Medline]
[Order article via Infotrieve].
9.
Standeven AM, Johnson AT, Escobar M, Chandraratna RA.
Specific antagonist of retinoid toxicity in mice.
Toxicol Appl Pharmacol.
1996;138:169-175[Medline]
[Order article via Infotrieve].
10.
Till JE, McCulloch EA.
A direct measurement of the radiation sensitivity of normal mouse bone marrow cells.
Radiat Res.
1961;14:213[Medline]
[Order article via Infotrieve].
11.
Hodgson GS, Bradley TR, Radley JM.
The organization of hemopoietic tissue as inferred from the effects of 5-fluorouracil.
Exp Hematol.
1982;10:26-35[Medline]
[Order article via Infotrieve].
12.
Ploemacher RE, Brons RHC.
Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: evidence for a pre-CFU-S cell.
Exp Hematol.
1989;17:263-266[Medline]
[Order article via Infotrieve].
13.
Ploemacher RE, Brons NHC.
Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention.
J Cell Physiol.
1988;136:531-536[Medline]
[Order article via Infotrieve].
14.
Li E, Sucov HM, Lee K-F, Evans RM, Jaenisch R.
Normal development and growth of mice carrying a targeted disruption of the 1 retinoic acid receptor gene.
Proc Natl Acad Sci U S A.
1993;90:1590-1594[Abstract/Free Full Text].
15.
Lohnes D, Kastner P, Dierich A, Mark M, LeMeur M, Chambon P.
Function of retinoic acid receptor in the mouse.
Cell.
1993;73:643-658[Medline]
[Order article via Infotrieve].
16.
Lohnes D, Mark M, Mendelsohn C, et al.
Function of the retinoic acid receptors (RARs) during development. I. Craniofacial and skeletal abnormalities in RAR double mutants.
Development.
1994;120:2723-2748[Abstract].
17.
Mendelsohn C, Lohnes D, Dierich A, et al.
Function of the retinoic acid receptors (RARs) during development. II. Multiple abnormalities at various stages of organogenesis in RAR double mutants.
Development.
1994;120:2749-2771[Abstract].
18.
Kastner P, Mark M, Ghyselinck N, et al.
Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development.
Development.
1997;124:313-326[Abstract].
19.
Labrecque J, Allan D, Chambon P, Iscove NN, Lohnes D, Hoang T.
Impaired granulocytic differentiation in vitro in hematopoietic cells lacking retinoic acid receptors 1 and .
Blood.
1998;92:607-615[Abstract/Free Full Text].
20.
Helms J, Thaller C, Eichele G.
Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud.
Development.
1994;120:3267-3274[Abstract].
21.
Lu H-C, Revelli J-P, Goering L, Thaller C, Eichele G.
Retinoid signaling is required for the establishment of a ZPA and for the expression of Hoxb-8, a mediator of ZPA formation.
Development.
1997;124:1643-1651[Abstract].
22.
Sauvageau G, Lansdorp PM, Eaves CJ, et al.
Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells.
Proc Natl Acad Sci U S A.
1994;91:12,223-12,227[Abstract/Free Full Text].
23.
Perkins AC, Cory S.
Conditional immortalization of mouse myelomonocytic, megakaryocytic and mast cell progenitors by the Hox-2.4 homeobox gene.
EMBO J.
1993;12:3835-3846[Medline]
[Order article via Infotrieve].
24.
Lill MC, Fuller JF, Herzig R, Crooks GM, Gasson JC.
The role of the homeobox gene, HOX B7, in human myelomonocytic differentiation.
Blood.
1995;85:692-697[Abstract/Free Full Text].
25.
Sauvageau G, Thorsteinsdottir U, Eaves CJ, et al.
Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.
Genes Dev.
1995;9:1753-1765[Abstract/Free Full Text].
26.
Helgason CD, Sauvageau G, Lawrence HJ, Largman C, Humphries RK.
Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro.
Blood.
1996;87:2740-2749[Abstract/Free Full Text].
27.
Krishnaraju K, Hoffman B, Liebermann DA.
Lineage-specific regulation of hematopoiesis by HOX-B8 (HOX-2.4): inhibition of granulocytic differentiation and potentiation of monocytic differentiation.
Blood.
1997;90:1840-1849[Abstract/Free Full Text].
28.
Lansdorp PM.
Self-renewal of stem cells.
Biol Blood Marrow Transpl.
1997;3:171[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
R. Safi, G. G. Muramoto, A. B. Salter, S. Meadows, H. Himburg, L. Russell, P. Daher, P. Doan, M. D. Leibowitz, N. J. Chao, et al.
Pharmacological Manipulation of the RAR/RXR Signaling Pathway Maintains the Repopulating Capacity of Hematopoietic Stem Cells in Culture
Mol. Endocrinol.,
February 1, 2009;
23(2):
188 - 201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Chen, B. L. Esplin, K. P. Garrett, R. S. Welner, C. F. Webb, and P. W. Kincade
Retinoids Accelerate B Lineage Lymphoid Differentiation
J. Immunol.,
January 1, 2008;
180(1):
138 - 145.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Taschner, C. Koesters, B. Platzer, A. Jorgl, W. Ellmeier, T. Benesch, and H. Strobl
Down-regulation of RXR{alpha} expression is essential for neutrophil development from granulocyte/monocyte progenitors
Blood,
February 1, 2007;
109(3):
971 - 979.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Chute, G. G. Muramoto, J. Whitesides, M. Colvin, R. Safi, N. J. Chao, and D. P. McDonnell
Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells
PNAS,
August 1, 2006;
103(31):
11707 - 11712.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Walkley and S. H. Orkin
Rb is dispensable for self-renewal and multilineage differentiation of adult hematopoietic stem cells
PNAS,
June 13, 2006;
103(24):
9057 - 9062.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. E. Purton, S. Dworkin, G. H. Olsen, C. R. Walkley, S. A. Fabb, S. J. Collins, and P. Chambon
RAR{gamma} is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation
J. Exp. Med.,
May 15, 2006;
203(5):
1283 - 1293.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. D. Nadauld, D. N. Shelton, S. Chidester, H. J. Yost, and D. A. Jones
The Zebrafish Retinol Dehydrogenase, rdh1l, Is Essential for Intestinal Development and Is Regulated by the Tumor Suppressor Adenomatous Polyposis Coli
J. Biol. Chem.,
August 26, 2005;
280(34):
30490 - 30495.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Bug, H. Gul, K. Schwarz, H. Pfeifer, M. Kampfmann, X. Zheng, T. Beissert, S. Boehrer, D. Hoelzer, O. G. Ottmann, et al.
Valproic Acid Stimulates Proliferation and Self-renewal of Hematopoietic Stem Cells
Cancer Res.,
April 1, 2005;
65(7):
2537 - 2541.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Tsuzuki, K. Kitajima, T. Nakano, A. Glasow, A. Zelent, and T. Enver
Cross Talk between Retinoic Acid Signaling and Transcription Factor GATA-2
Mol. Cell. Biol.,
August 1, 2004;
24(15):
6824 - 6836.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Brunner, H. Laumen, P. J. Nielsen, N. Kraut, and T. Wirth
Expression of the Aldehyde Dehydrogenase 2-like Gene Is Controlled by BOB.1/OBF.1 in B Lymphocytes
J. Biol. Chem.,
November 14, 2003;
278(46):
45231 - 45239.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Perrotta, B. Nobili, F. Rossi, M. Criscuolo, A. Iolascon, D. Di Pinto, I. Passaro, L. Cennamo, A. Oliva, and F. Della Ragione
Infant hypervitaminosis A causes severe anemia and thrombocytopenia: evidence of a retinol-dependent bone marrow cell growth inhibition
Blood,
March 15, 2002;
99(6):
2017 - 2022.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Kastner, H. J. Lawrence, C. Waltzinger, N. B. Ghyselinck, P. Chambon, and S. Chan
Positive and negative regulation of granulopoiesis by endogenous RAR{alpha}
Blood,
March 1, 2001;
97(5):
1314 - 1320.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Lehmann, C. Paul, and H. Törmä
Retinoid Receptor Expression and Its Correlation to Retinoid Sensitivity in Non-M3 Acute Myeloid Leukemia Blast Cells
Clin. Cancer Res.,
February 1, 2001;
7(2):
367 - 373.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction