Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells.
ABSTRACT Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.
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ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and is responsible for a quarter of a million deaths annually. The survival rate for HNSCC patients is poor, showing only minor improvement in the last three decades. Despite new surgical techniques and chemotherapy protocols, tumor resistance to chemotherapy remains a significant challenge for HNSCC patients. Numerous mechanisms underlie chemoresistance, including genetic and epigenetic alterations in cancer cells that may be acquired during treatment and activation of mitogenic signaling pathways, such as nuclear factor kappa-light-chain-enhancer-of activated B cell, that cause reduced apoptosis. In addition to dysfunctional molecular signaling, emerging evidence reveals involvement of cancer stem cells (CSCs) in tumor development and in tumor resistance to chemotherapy and radiotherapy. These observations have sparked interest in understanding the mechanisms involved in the control of CSC function and fate. Post-translational modifications of histones dynamically influence gene expression independent of alterations to the DNA sequence. Recent findings from our group have shown that pharmacological induction of post-translational modifications of tumor histones dynamically modulates CSC plasticity. These findings suggest that a better understanding of the biology of CSCs in response to epigenetic switches and pharmacological inhibitors of histone function may directly translate to the development of a mechanism-based strategy to disrupt CSCs. In this review, we present and discuss current knowledge on epigenetic modifications of HNSCC and CSC response to DNA methylation and histone modifications. In addition, we discuss chromatin modifications and their role in tumor resistance to therapy.World journal of stem cells. 11/2014; 6(5):511-525.
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ABSTRACT: Common cancer theories hold that tumor is an uncontrolled somatic cell proliferation caused by the progressive addition of random mutations in critical genes that control cell growth. Nevertheless, various contradictions related to the mutation theory have been reported previously. These events may be elucidated by the persistence of residual tumor cells, called Cancer Stem Cells (CSCs) responsible for tumorigenesis, tumor maintenance, tumor spread, and tumor relapse. Herein, we summarize the current understanding of CSCs, with a focus on the possibility to identify specific markers of CSCs, and discuss the clinical application of targeting CSCs for cancer treatment.Cancer Investigation 09/2014; · 2.06 Impact Factor
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ABSTRACT: Cancer stem cells (CSCs) have been reported to play an important role in chemoradiation resistance. Although the association of CSC markers with clinicopathological outcomes after neoadjuvant chemoradiotherapy (NACRT) has been reported in various types of cancers, there have been no such reports for pancreatic cancer. Here we examined the sequential changes in CSC marker expressions after NACRT in patients with pancreatic adenocarcinoma (PA) and the impact of these changes on the prognosis.BMC Cancer 09/2014; 14(1):687. · 3.32 Impact Factor
Inhibition of aldehyde dehydrogenase and retinoid
signaling induces the expansion of human
hematopoietic stem cells
John P. Chute*†, Garrett G. Muramoto*, John Whitesides‡, Michael Colvin§, Rachid Safi¶, Nelson J. Chao*,
and Donald P. McDonnell¶
*Division of Cellular Therapy,‡Human Vaccine Institute,§Division of Hematology?Oncology, and¶Department of Pharmacology and Cancer Biology,
Duke University Medical Center, Durham, NC 27710
Communicated by Bert W. O’Malley, Baylor College of Medicine, Houston, TX, May 12, 2006 (received for review January 27, 2006)
Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in
the liver and is required for the conversion of retinol (vitamin A) to
retinoic acids. ALDH is also highly enriched in hematopoietic stem
cells (HSCs) and is considered a selectable marker of human HSCs,
study, we demonstrate that ALDH is a key regulator of HSC
differentiation. Inhibition of ALDH with diethylaminobenzalde-
hyde (DEAB) delayed the differentiation of human HSCs that
otherwise occurred in response to cytokines. Moreover, short-term
culture with DEAB caused a 3.4-fold expansion in the most prim-
itive assayable human cells, the nonobese diabetic?severe com-
bined immunodeficiency mouse repopulating cells, compared with
day 0 CD34?CD38?lin?cells. The effects of DEAB on HSC differ-
entiation could be reversed by the coadministration of the retinoic
acid receptor agonist, all-trans-retinoic acid, suggesting that the
ability of ALDH to generate retinoic acids is important in deter-
mining HSC fate. DEAB treatment also caused a decrease in retinoic
acid receptor-mediated signaling within human HSCs, suggesting
directly that inhibition of ALDH promotes HSC self-renewal via
reduction of retinoic acid activity. Modulation of ALDH activity and
retinoid signaling is a previously unrecognized and effective strat-
egy to amplify human HSCs.
retinoic acid ? self-renewal ? diethylaminobenzaldehyde ? long-term
progeny throughout the lifetime of an individual (1, 2). Several
molecular pathways that regulate HSC self-renewal have now been
identified, including Notch (3), HOXB4 (4), Wnt (5), and bone
morphogenetic protein signaling pathways (6). The osteoblastic
niche for HSCs within the bone marrow (BM) has also been
characterized (7, 8). Despite these advances in understanding HSC
biology, clinical methods to amplify human HSCs have yet to be
realized, and characterization of the pathways that regulate HSC
self-renewal continues to evolve.
Two decades ago, Colvin et al. (9, 10) demonstrated that the
intracellular enzyme, aldehyde dehydrogenase (ALDH), protected
BM progenitors from the cytotoxic effects of cyclophosphamide by
deactivation of its metabolite, 4-hydroxycyclophosphamide (9, 10).
Several isoforms of ALDH have been identified, with ALDH1
being the primary isoform expressed within human hematopoietic
progenitors (11, 12). Recent studies have shown that human and
murine hematopoietic progenitors can be isolated by using a
fluorescently labeled dye specific for ALDH activity (13–16) and
cord blood (CB) ALDHbrlin?cells are enriched for nonobese
repopulating cells [SCID-repopulating cells (SRCs)] (15, 16). Al-
though these data demonstrate that ALDH is a selectable marker
for human stem?progenitor cells, the HSC-specific function of
ALDH remains unknown. In the liver, ALDH1 contributes pri-
marily to the metabolism of retinol (vitamin A) into retinoic acid
self-renew and give rise to all mature lymphohematopoietic
(17). Because ALDH1 is also highly concentrated in HSCs, it is
plausible that the primary function of ALDH1 in HSCs relates to
its production of retinoids.
The biological actions of retinoids are mediated by the retinoic
acid receptor (RAR) and retinoid X receptor (RXR), ligand-
dependent transcription factors that are expressed in the nuclei of
target cells (18–20). Through its actions on these receptors, all-
trans-retinoic acid (ATRA) induces cellular differentiation, tissue
patterning, and embryonic development in vertebrates (18–22).
ATRA is also used therapeutically to induce the differentiation of
on retinoid signaling and that inhibition of ALDH, which is
required for production of retinoic acids, could interfere with HSC
differentiation. In this study, we demonstrate that ALDH activity
is necessary for normal HSC differentiation to occur in response to
cytokines and that inhibition of ALDH, coupled with early acting
cytokines, is sufficient to induce the quantitative expansion of
human SRCs. Our findings indicate that inhibition of ALDH
activity and retinoid signaling can impart a robust expansion of
Inhibition of ALDH Delays the Differentiation of HSCs in Culture. We
first determined whether inhibition of ALDH activity in primary
BM and CB CD34?CD38?lin?cells affected the differentiation of
HSCs when cultured for 7 days in the presence of thrombopoietin
(20 ng?ml), stem cell factor (SCF; 100 ng?ml), and flt-3 ligand (50
proliferation of human HSCs in culture (24). FACS-sorted human
shown to be the most highly enriched population for human HSCs
(25). Treatment with 100 ?M diethylaminobenzaldehyde (DEAB)
? TSF significantly reduced ALDH activity in CD34?CD38?cells
of day 0 CB CD34?CD38?lin?cells demonstrated ALDH activity
(Fig. 1a, P ? 0.001 and P ? 0.001). The progeny of CB and BM
CD34?CD38?lin?cells after 7 days of culture with DEAB ? TSF
contained significantly higher percentages of primitive
(Fig. 1b and Fig. 5a, which is published as supporting information
Conflict of interest statement: No conflicts declared.
cord blood; HSC, hematopoietic stem cell; NOD?SCID, nonobese diabetic?severe combined
immunodeficiency; SRC, SCID-repopulating cell; CFC, colony-forming cell; DEAB, diethyl-
aminobenzaldehyde; ATRA, all-trans-retinoic acid.
†To whom correspondence should be addressed at: Division of Cellular Therapy, Depart-
ment of Medicine, Duke University Medical Center, 2400 Pratt Street, Box 3961, Durham,
NC 27710. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
August 1, 2006 ?
vol. 103 ?
no. 31 ?
on the PNAS web site; P ? 0.02 and P ? 0.01, respectively). DEAB
? TSF cultures supported a mean 4-fold total cell expansion and a
maintenance of absolute numbers of BM CD34?CD38?cells
compared with day 0. Conversely, TSF culture supported a mean
15-fold increase in total cells, but this increase was associated with
a 5-fold decrease in BM CD34?CD38?cell numbers compared
with input (P ? 0.01). Table 1 summarizes the effect of DEAB on
HSC expansion in vitro. As expected, HSC-enriched day 0
CD34?CD38?lin?cells demonstrated little colony-forming cell
cells with TSF alone caused a 5-fold increase in CFCs compared
with input, indicating HSC differentiation during culture. Con-
versely, the progeny of CD34?CD38?lin?cells cultured with
impeded HSC maturation during culture (P ? 0.002). As further
confirmation of the inhibitory effect of DEAB on HSC maturation
in vitro, extended 14-day cultures of CB CD34?CD38?lin?cells
with DEAB ? TSF continued to maintain a discrete population of
CD34?CD38?cells at day 14 (Fig. 5b; mean 17.5% ? 6.1).
revealed a predominance of cells with high nuclear:cytoplasmic
ratios and prominent nucleoli, whereas TSF-cultured progeny
contained primarily bands and myelocytes, suggesting that DEAB
treatment maintained more immature progenitors during culture
(data not shown). To determine the effect of DEAB alone on
human HSCs, CB CD34?CD38?lin?cells were also placed in
culture with 100 ?M DEAB in the absence of TSF and, at day 7,
did not induce the proliferation or self-renewal of human HSCs
in the absence of hematopoietic cytokines. Taken together, these
results indicated that inhibition of ALDH with DEAB impeded
HSC differentiation that otherwise occurred in response to
Culture. To determine whether inhibition of ALDH promoted the
self-renewal of HSCs in culture, we performed limiting dilution
repopulation assays as we and others have described (24–27) to
their progeny after culture with DEAB. Analysis of NOD?SCID
mice at 8 weeks posttransplant demonstrated that the progeny of
significantly increased SRC capacity than either day 0 CB
CD34?CD38?lin?cells or the progeny of cells cultured with TSF
alone (Fig. 2a). At a dose of 0.5 ? 103, no mice transplanted with
TSF alone or TSF ? DEAB demonstrated human hematopoietic
cell engraftment. At doses of 1–2.5 ? 103cells, only 3 of 15 mice
(20%) transplanted with day 0 CD34?CD38?lin?cells showed
huCD45?cell engraftment (Fig. 2b). Similarly, only 1 of 9 mice
transplanted with the progeny of this dose after culture with TSF
alone demonstrated human engraftment. In contrast, 8 of 16 mice
Table 1. Expansion of CD34?CD38?lin?cells after treatment with DEAB and ATRA
Day 0Day 7
Total (?103)CD34?(?103) CD34?CD38?(?103)
Cell count Fold changeCell countFold changeCell countFold change
TSF ? DEAB
TSF ? DEAB
TSF ? ATRA
71.4 ? 4
19.2 ? 2
950.0 ? 80
220.0 ? 60
290.0 ? 70
26.4 ? 1
14.4 ? 1
100.0 ? 10
83.0 ? 2
10.0 ? 1
1.1 ? 0.1
4.3 ? 0.3
80.0 ? 1.0
58.0 ? 2.0
Primary human BM or CB CD34?CD38?lin?cells were placed in culture with either TSF alone, TSF ? DEAB, or TSF ? ATRA. At day 7, the number of total cells,
CD34?cells, and CD34?CD38?cells in each culture condition was quantified and compared with input.
For each culture condition, 3–6 experiments were performed.
ferentiation of human HSCs. FACS-sorted
CB CD34?CD38?lin?cells were analyzed
the ALDH activity level in the CD34?CD38?
progeny of 7-day cultures with thrombo-
poietin, SCF, and flt3-ligand (TSF) alone
versus TSF ? DEAB (a). (b) The surface ex-
pression of CD34 and CD38 on day 0 CB
eny after culture with TSF alone (Middle)
versus TSF ? DEAB (Bottom) is shown. Cul-
ture with TSF alone caused a marked in-
crease in CFC content compared with day 0
CD34?CD38?lin?cells, whereas the prog-
eny of DEAB ? TSF cultures contained little
differentiation during culture (c).*indi-
cates a statistically significant difference
between the DEAB ? TSF treated group
versus TSF alone.
ALDH inhibition impedes the dif-
www.pnas.org?cgi?doi?10.1073?pnas.0603806103Chute et al.
(50%) transplanted with the progeny of CD34?CD38?lin?cells
(Fig. 2b). Mice engrafted with DEAB-cultured progeny (2.5 ? 103
dose) also demonstrated 1 log higher repopulation (mean 8.0%
huCD45?cells, range 1.5–21.6%) as compared with mice trans-
planted with day 0 CB CD34?CD38?lin?cells (mean 0.9%, range
0.02–3.5%, P ? 0.048) or their progeny after culture with TSF
(0.7%, range 0.4–1.3%), indicating that DEAB-cultured progeny
or TSF-cultured cells. Poisson statistical analysis (24–28) indicated
that the SRC frequency within day 0 CB CD34?CD38?lin?cells
was 1 in 7,500 cells (95% confidence interval of 1?2,900 to
1?30,000). The SRC frequency within the progeny of TSF-cultured
CB CD34?CD38?lin?cells was more than 2-fold reduced at 1 in
17,000 cells (confidence interval of 1?3,800 to 1?290,000). In
contrast, the SRC frequency within the progeny of DEAB ?
TSF-cultured CB CD34?CD38?lin?cells was 1 in 2,200 cells
(confidence interval of 1?1,100 to 1?4,900), which was 3.4-fold
higher than day 0 CB CD34?CD38?lin?cells and 7.7-fold higher
The human cell engraftment observed in all primary transplanted
NOD?SCID mice is summarized in Table 2, which is published as
supporting information on the PNAS web site. These data dem-
onstrate that inhibition of ALDH not only inhibited the differen-
in NOD?SCID mice is shown at week 8 in mice transplanted with 1 ? 103day 0 CB CD34?CD38?lin?cells (Top), their TSF-cultured progeny (Middle), or their progeny
increased frequency of human engraftment (?1.0%) and percent huCD45?cell repopulation compared with day 0 CB CD34?CD38?lin?cells or their progeny after
culture with TSF alone. The mean levels of huCD45?cells per culture condition are indicated by horizontal lines. (c) Multilineage engraftment of CD45?cells, CD34?
progenitor cells, CD19?B cells, and CD33?13?myeloid cells is shown in the BM of a representative NOD?SCID mouse transplanted with the progeny of 2.5 ? 103CB
staining (i), huCD45 versus muCD45 staining (ii), IgGFITC versus IgG-phycoerythrin control staining (iii), huCD34 versus huCD38 staining (iv), huCD19 versus huCD3
staining (v), and huCD13 versus huCD33 staining (vi). (d) PCR analysis for a 1,171-bp segment of the human chromosome 17-specific ?-satellite region demonstrates
identifies 100% human CB cell DNA; ‘‘Mo’’ identifies 100% mouse BM cell DNA; and ‘‘N’’ identifies the no template control (dH20).
Chute et al.
August 1, 2006 ?
vol. 103 ?
no. 31 ?
tiation of HSCs but also promoted the amplification of HSCs in
culture. Detailed flow cytometric analysis revealed extensive
CD34?progenitor cell, CD19?B lymphoid, and CD33?13?my-
eloid differentiation in mice transplanted with DEAB ? TSF-
cultured cells, demonstrating that a pluripotent repopulating cell
was sustained during culture with DEAB (Fig. 2c). Of note, the
ratio of human CD19?B lymphoid cells to CD33?CD13?myeloid
cells detectable at 8 weeks in mice transplanted with day 0
1.7% CD13?33?cells), whereas this ratio was 2.7:1 in mice trans-
planted with the progeny of CD34?CD38?lin?cells after culture
with DEAB ? TSF. These data suggest that inhibition of ALDH
HSCs expanded under this condition.
Although our primary limiting dilution NOD?SCID transplan-
(20–50%) of human CD45?cell chimerism to allow for secondary
transplantation studies, we did perform a limited number of
secondary transplants to assess for the presence of long-term
repopulating stem cells within the DEAB-cultured populations. At
from primary recipients of DEAB ? TSF-cultured cells (2.5 ? 103
chromosome 17-specific ?-satellite region (Fig. 2d) (29). These
results confirmed that inhibition of ALDH activity promoted the
differential maintenance of long-term repopulating stem cells in
DEAB Specifically Inhibits ALDH1 and Decreases Retinoic Acid Activity
in HSCs. To further test our hypothesis that ALDH contributes to
HSC differentiation through the production of retinoic acids, we
first evaluated the effects of ATRA on primary CB
which maintained a population of CD34?CD38?cells in culture at
day 7 (Fig. 3a–c), treatment of CB CD34?CD38?lin?cells with
TSF ? 1 ?M ATRA resulted in a marked decline in CD34?cells
in culture compared with TSF alone, and no CD34?CD38?cells
were detectable at day 7 (Fig. 3d). Moreover, when we cultured CB
CD34?CD38?lin?cells with 1 ?M ATRA ? 100 ?M DEAB ?
TSF, phenotypic differentiation of CD34?CD38?cells again oc-
the effects of DEAB-induced inhibition of ALDH (Fig. 3e). Taken
together, these results suggested that ALDH might contribute to
HSC differentiation through its production of intracellular retin-
oids. Analysis of CFC content within cultures treated with ATRA
? TSF demonstrated a ?10-fold reduction in CFCs within ATRA-
treated cultures compared with the progeny of TSF alone, indicat-
ing that ATRA either inhibited CFC formation or promoted
terminal progenitor cell differentiation, thereby reducing CFC
numbers (data not shown). As further support of the relationship
between ALDH activity and retinoid production in HSCs, we also
cultured human CB CD34?CD38?lin?cells with 100 nM of
propenyl) benzoic acid (TTNPB), an RAR-specific agonist, with
TSF and observed a marked differentiation of cells by day 7 of
culture. The effect of TTNPB also overcame the effect of DEAB
toward inhibiting HSC differentiation (Fig. 6 a and b, which is
published as supporting information on the PNAS web site). The
addition of 100 ?M of LGD101268, a synthetic agonist of the RXR
(courtesy of Ligand Pharmaceuticals, San Diego, CA), also com-
pletely overcame the inhibitory effect of DEAB on HSC differen-
of 1,25-dihydroxyvitamin D3(vitamin D), which has been shown to
induce the differentiation of human hematopoietic progenitor cells
(30), also overcame the effects of DEAB on HSCs in culture (Fig.
6 e and f). Interestingly, the addition of DEAB did appear to
increase the maintenance of CD34? progenitor cells in culture
when combined with either the RAR agonist, the RXR agonist, or
vitamin D, suggesting that inhibition of ALDH activity moderately
slowed the differentiation of HSCs that occurred in response to
these three ligands (Fig. 6 a–f). Nonetheless, these data demon-
strated that the addition of exogenous retinoids, rexinoids, or
vitamin D generally overcame the effect of DEAB toward inhib-
iting HSC differentiation, further supporting our hypothesis that
ALDH mediates its effects on HSC fate via its contribution to
FACS-sorted CB CD34?CD38?lin?cells (a) were cultured with TSF alone (b), and
their phenotype was compared with the progeny of cells cultured with TSF ?
? TSF (d), which induced a marked loss of CD34?cells and CD34?CD38?cells in
culture as compared with input or TSF culture, consistent with accelerated dif-
ferentiation during culture. The addition of ATRA to TSF also overcame the
retinoids can overcome the effect of ALDH inhibition on HSCs. (f) Real-time PCR
analysis demonstrated a 50% reduction in cEBP? expression in DEAB ? TSF-
for ALDH1, retinaldehyde, markedly increased cEBP? expression in HSCs, the
ALDH1 activity, thereby impeding retinoid signaling in HSCs.
DEAB inhibits ALDH1 activity and decreases retinoid signaling in HSCs.
www.pnas.org?cgi?doi?10.1073?pnas.0603806103Chute et al.
Because several isoforms of ALDH exist (31), we sought to
determine whether DEAB was specifically inhibiting ALDH1,
which is known to be highly expressed in HSCs (11, 14, 32) and is
the predominant regulator of retinoic acid synthesis in mammals
(31), versus other isoforms. First, we observed that treatment of
HSCs with DEAB for 7 days caused a 2-fold decrease in the
expression of cEBP? (Fig. 3f), which is under the control of RAR
signaling (33), confirming that inhibition of ALDH activity with
DEAB caused a decrease in retinoid activity in human HSCs.
Secondly, the addition of 1 ?M retinaldehyde, which is a specific
substrate for ALDH1, to HSC cultures caused an 8.4-fold increase
in cEBP? expression in HSCs, confirming the contribution of
ALDH1 to retinoic acid activity in these cells. However, when
DEAB was added to HSC cultures supplemented with retinalde-
hyde, the effect of retinaldehyde on cEBP? expression was signif-
icantly reduced, demonstrating that DEAB significantly inhibited
the activity of ALDH1 in HSCs, which consequently caused a
marked decrease in retinoid signaling in these primitive cells
Inhibition of ALDH Activity Up-Regulates HOXB4 Expression in Human
HSCs. Because HOXB4 and Notch have established roles in HSC
self-renewal (3, 4), we sought to determine whether ALDH inhi-
of either of these target genes. Interestingly, culture of primary CB
CD34?CD38?lin?cells with TSF alone caused a 5-fold decrease in
HOXB4 transcription compared with day 0 CB CD34?CD38?lin?
cells, whereas the addition of DEAB to TSF maintained HOXB4
expression at 70% of input levels (Fig. 4). Conversely, treatment
with DEAB did not alter Notch transcription compared with TSF
may also promote HSC self-renewal via discrete interactions with
other established pathways, such as HOXB4, although the organi-
zation of these signals is yet to be elucidated.
Improved characterization of the pathways that regulate HSC
self-renewal will facilitate the development of therapies to
we have characterized the novel contributions of the enzyme
ALDH and retinoid signaling to human HSC differentiation and
self-renewal. ALDHs are NAD(P)?-dependent enzymes that
oxidize a large number of aldehydes to their corresponding
carboxylic acids (17, 31). Several different ALDH isoforms (30)
have been identified that are responsible for the metabolism of
ethanol (34), catecholamines, (14) and the conversion of vitamin
A to its active metabolite, retinoic acid (17). ALDH is also a
selectable marker of human stem?progenitor cells (13, 15, 16).
However, the contribution of ALDH activity to HSC function
has remained unknown. In this study, we show that inhibition of
ALDH activity with DEAB delayed the phenotypic and func-
tional maturation of HSCs in response to thrombopoietin, SCF,
and flt-3 ligand. ALDH inhibition, coupled with TSF, also gave
rise to a 3.4-fold increase in SRCs in short-term culture, whereas
treatment with TSF alone was associated with a 2-fold reduction
in SRC content compared with input. Importantly, secondary
transplant studies confirmed that long-term repopulating stem
cells were maintained in cultures treated with DEAB. These
studies indicate that ALDH plays a critical role in human HSC
differentiation. Moreover, inhibition of ALDH, when combined
with early acting cytokines, is sufficient to induce the amplifi-
cation of human HSCs.
In light of the observed effects of ALDH inhibition on the
amplification of human HSCs in culture, we also sought to deter-
mine the mechanism through which this effect occurred. Because
ALDH1 is the predominant isoform within HSCs (11, 14, 32) and
is the dominant isoform in mammals that regulates the conversion
of retinaldehydes to retinoic acids (31), we tested whether DEAB
specifically inhibited ALDH1 activity in HSCs and the effect this
had on RAR-response genes. Our studies confirmed that DEAB
treatment blocked the capacity for HSCs to convert retinaldehyde
into retinoic acids by virtue of a marked decrease in expression of
cEBP?, which is an RAR-specific response gene. Because ALDH1
is the dominant isoform required for the conversion of retinalde-
hyde to retinoic acids, these results also confirmed that the effect
of DEAB on HSC differentiation was predominantly mediated
through inhibition of ALDH1. Taken together, these data provide
strong evidence that ALDH1 mediates the differentiation of HSCs
via production of intracellular retinoic acids and that targeted
inhibition of this enzyme promotes HSC self-renewal via inhibition
of retinoic acid signaling.
signaling can induce the expansion of human HSCs. There are
several implications of these observations. First, the functional role
of ALDH1 in HSC fate and the link between ALDH1 activity,
retinoid signaling, and HSC self-renewal has not been previously
described. Interestingly, Purton et al. (35, 36) reported that culture
of murine c-kit?sca-1?lin?cells with ATRA for 14 days enhanced
the maintenance of cells with in vivo repopulating capacity as
compared with culture with cytokines alone. These results appear
to contrast with our observations that inhibition of ALDH1 and
retinoid signaling induces the expansion of human HSCs. These
differences may be explained by differences in the contribution of
ALDH1 activity to HSC fate between mice and humans or differ-
ences in the repopulating assays being performed. We have initi-
ated additional studies to determine whether the function of
ALDH1 in hematopoiesis is conserved in both humans and mice.
Our data also suggest that cytokines, such as thrombopoietin, SCF,
and flt-3 ligand, induce HSC differentiation via induction of
retinoid signaling, perhaps mediated through increased ALDH1
activity. This hypothesis is supported by the recent observation that
another cytokine, IL-3, induces hematopoietic progenitor cell dif-
ferentiation via activation of Stat5 which, in turn, activates retinoid
signaling (37). Our results also have implications for the develop-
performed these studies on primary human HSCs, and our obser-
vations are therefore directly translatable to clinical protocols to
expand human HSCs. Moreover, in contrast to other reported
strategies to expand HSCs in vitro (5, 38),the approach we have
described does not depend on the genetic modification of HSCs or
coculture with surrogate stromal cell niches to achieve potency (39,
40). Finally, the observed in vivo multilineage differentiation of
DEAB-treated HSCs transplanted in NOD?SCID mice demon-
differentiation program of human HSCs. It will be important to
isolated from multiple replicates of FACS-sorted CB CD34?CD38?lin?cells at
day 0 and their progeny after culture with TSF alone or DEAB ? TSF. The RNA
was reverse-transcribed, and the expression of HOXB4 and Notch 1 was
analyzed by quantitative real-time PCR. (Left) The expression of HOXB4 in
whereas treatment with DEAB prevented the down-regulation of HOXB4
expression over time. (Right) The expression of Notch was also significantly
reduced after TSF culture and was not altered by treatment with DEAB.
Treatment with DEAB sustains HOXB4 expression in HSCs. RNA was
Chute et al.
August 1, 2006 ?
vol. 103 ?
no. 31 ?
further augment the expansion of HSCs described here via the
combination of ALDH inhibitors with other ligands capable of
inhibiting HSC differentiation programs. In addition, it will also be
HSCs. We have observed that a selective RXR modulator causes
highly comparable to ALDH inhibition (J.P.C., D.P.M., G.G.M.,
and R.S., unpublished data).
In summary, our data suggest that ALDH1 functions fundamen-
tally in HSCs to promote differentiation via the production of
retinoic acids. The notion that ALDH1 is both a selectable marker
of stem and progenitor cells (13, 14) and a critical regulator of stem
cell differentiation appears counterintuitive. However, because a
fundamental property of HSCs is the ongoing production of all
mature hematopoietic cells, it is not surprising that HSCs would
possess a differentiation program (e.g., ALDH1 activity, produc-
tion of retinoic acids) that can be activated early in their lifespan,
particularly in response to external stimuli (e.g., cytokines). Pro-
duction of large numbers of HSCs lacking such an early differen-
tiation capacity would also be potentially pathologic. Therefore, we
believe it is consistent with normal hematopoiesis that HSCs might
possess in vivo repopulating capacity while also carrying the critical
capacity for fairly rapid differentiation in response to external
signals. The results presented here demonstrate that inhibition of
ALDH and retinoid activity is sufficient to induce the expansion of
Isolation of Human BM and CB CD34?CD38?lin?Cells. We obtained
whole BM and CB units from the Duke University Stem Cell
Laboratory within 48 h of collection. CB was volume-reduced, and
lineage depletion was conducted by using the Human Progenitor
Enrichment Mixture (StemCell Technologies, Vancouver). For
details, see Supporting Methods, which is published as supporting
information on the PNAS web site.
Lin?CB or BM cells were thawed and washed once in Iscove’s
modified Dulbecco’s medium (Invitrogen) containing 10% FBS
and 1% penicillin?streptomycin. Immunofluorescent staining and
sterile cell sorting to isolate CD34?CD38?and CD34?CD38?
subsets was performed as described in ref. 24. For additional
details, see Supporting Methods.
Analysis of in Vitro Hematopoietic Activity of Human CD34?CD38?lin?
Cells After Culture with DEAB. Primary human BM and CB
CD34?CD38?lin?cells were placed in culture with 20 ng?ml
thrombopoietin, 100 ng?ml stem cell factor, and 50 ng?ml flt-3
ligand (TSF, R & D Systems), a cytokine combination which we
have previously found to induce human stem and progenitor cell
proliferation and differentiation in vitro (24). BM or CB
CD34?CD38?lin?cells at a dose of 0.5–1 ? 104were cultured for
7 days with and without 100 ?M DEAB (courtesy of M. Colvin,
Duke University). At day 7, cell counts and immunophenotypic
and compared with day 0 (input) staining. Fourteen-day methyl-
cellulose CFC assays were performed in triplicate as we have
previously described (24, 26) (for details, see Supporting Methods).
BM and CB CD34?CD38?lin?cells were also placed in culture
with TSF with and without ATRA (Sigma-Aldrich), TTNPB
ma-Aldrich) with or without DEAB to determine the relationship
between ALDH inhibition and retinoic acid signaling in human
HSCs (see Supporting Methods for details).
In Vivo Long-Term Repopulating Assays in NOD?SCID Mice. NOD?
SCID mice (41) were transplanted with either day 0 FACS-sorted
lin?cells cultured with TSF alone or TSF supplemented with 100
primary and secondary mice, and SCID-repopulating cell (SRC)
For details, see Supporting Methods.
Real-Time PCR Analysis of Gene Expression in HSCs. Total RNA
isolation from Day 0 CB CD34?CD38?lin?and the resultant day
7 progeny was conducted on 1 ? 104cells per sample by using the
RNAqueous-Micro kit (Ambion, Austin, TX) following the man-
ufacturer’s suggested protocol. See Supporting Methods for details.
Analysis of ALDH Activity. ALDH enzyme activity in day 0
CD34?CD38?lin?cells and their progeny was assayed by using the
ALDEFLUOR staining kit (StemCell Technologies) as described
in ref. 16. See Supporting Methods for details.
We thank Dr. David Venzon for his critical assistance with the statistical
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