TISSUE-SPECIFIC STEM CELLS
Brief Report: Aldehyde Dehydrogenase Activity Is a Biomarker of
Primitive Normal Human Mammary Luminal Cells
PETER EIREW,aNAGARAJAN KANNAN,aDAVID J.H.F. KNAPP,aFRANC ¸OIS VAILLANT,b,cJOANNE T. EMERMAN,d
GEOFFREY J. LINDEMAN,b,eJANE E. VISVADER,b,cCONNIE J. EAVESa,f
aTerry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;bBreast Cancer
Laboratory, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia;cDepartment
of Medical Biology, University of Melbourne, Parkville, Victoria, Australia;dDepartment of Cellular and
Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada;eDepartment of
Medicine, University of Melbourne, Parkville, Victoria, Australia andfDepartment of Medical Genetics,
University of British Columbia, Vancouver, British Columbia, Canada
Key Words. Tissue-specific stem cells•Clonal assays•Progenitor cells•Breast cancer
Elevated aldehyde dehydrogenase (ALDH) expression/
activity has been identified as an important biomarker of
primitive cells in various normal and malignant human
tissues. Here we examined the level and type of ALDH
expression and activity in different subsets of phenotypi-
cally and functionally defined normal human mammary
cells. We find that the most primitive human mammary
stem and progenitor cell types with bilineage differentia-
tion potential show low ALDH activity but undergo a
marked, selective, and transient upregulation of ALDH
activity at the point of commitment to the luminal lineage.
This mirrors a corresponding change in transcripts and
protein levels of ALDH1A3, an enzyme involved in reti-
noic acid synthesis and the most highly expressed ALDH
gene in normal human mammary tissue. In contrast,
ALDH1A1 is expressed at low levels in all mammary epi-
thelial cells. These findings raise interesting questions
about the reported association of ALDH activity with
breast cancer stem cells and breast cancer prognosis.
STEM CELLS 2012;30:344–348
Disclosure of potential conflicts of interest is found at the end of this article.
Much attention is now being focused on elucidating the molec-
ular mechanisms that regulate the different early stages of nor-
mal mammary cell differentiation, with the goal of identifying
those relevant to the cells that maintain human breast cancers
or that may serve as useful markers of these cells. The aldehyde
dehydrogenases (ALDHs), which are encoded by a large gene
family (http://www.aldh.org), are of interest in this regard ,
although the results of analyses of their expression in normal
and neoplastichuman breast
(reviewed in ). Here we analyzed the ALDH activity and iso-
form expression in different phenotypically and functionally
defined subsets of normal human mammary cells. We show that
ALDH activity in the normal human mammary gland is largely
due to ALDH1A3 and is low or absent in normal human mam-
mary stem cells but is then elevated when these transition into
progenitor cells committed to the luminal lineage.
MATERIALS AND METHODS
These are detailed in Supporting Information.
We first analyzed the ALDH activity present in each of the
five biologically and phenotypically distinct populations that
are found in dissociated normal human mammary tissue and
can be separately isolated based on their differential expres-
sion of CD49f, epithelial cell adhesion molecule (EpCAM,
also known as CD326), CD31, and CD45 (Fig. 1A) [3–5].
Three of these populations are subsets of mammary epithelial
cells: (a) a basal (CD49fþEpCAM?/lowCD31?CD45?) fraction
that contains all of the mammary stem cells, all of the biline-
age- and myoepithelial-restricted progenitors, and all of the
Author contributions: P.E.: conception and design, collection of data, data analysis and interpretation, and manuscript writing; N.K. and
F.V.: collection of data and data analysis and interpretation; D.J.H.F.K.: data analysis and interpretation; G.J.L. and J.E.V.: conception
and design and manuscript writing; J.T.E.: provision of study material; C.J.E.: conception and design, data analysis and interpretation,
manuscript writing, and final approval of manuscript.
Correspondence: Connie J. Eaves, Ph.D., Terry Fox Laboratory, 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3,
Canada. Telephone: 604-675-8122; Fax: 604-877-0712; e-mail: email@example.com
November 11, 2011; first published online in STEM CELLS EXPRESS November 30, 2011. V
STEM CELLS 2012;30:344–348 www.StemCells.com
Received August 16, 2011; accepted for publication
C AlphaMed Press 1066-5099/2012/$30.00/0
mature myoepithelial cells; (b) a primitive luminal (or ‘‘lumi-
fraction that contains all of the luminal-restricted progenitors
and some of the more differentiated luminal cells, and (c) a
mature luminal (CD49f?EpCAMþCD31?CD45?) fraction that
consists of fully differentiated luminal cells. Because mam-
mary stem cells cannot be identified directly, they are referred
to as ‘‘mammary repopulating units’’ (MRUs) based on their
ability to individually regenerate a full mammary gland struc-
ture within 4–6 weeks when transplanted into an appropriate
environment in immunodeficient mice [3, 4]. Mammary pro-
genitors are likewise referred to operationally as mammary
‘‘colony-forming cells’’ (CFCs), based on their ability to pro-
duce colonies within 8–10 days in vitro that contain differen-
tiated adherent cells of one or both mammary lineages.
CD49f/EpCAM/CD31/CD45 staining also yields another two
populations of cells that are present in high numbers in nor-
mal breast tissue but are not epithelial, that is, a stromal
(CD49f?EpCAM?CD31?CD45?) fraction and a fraction that
contains a mixture of hematopoietic (CD45þ) and endothelial
We incubated normal human mammary cells with Alde-
fluor (a fluorescent reagent that is activated by ALDH to pro-
duce an intracellularly retained fluorescent derivative, bodipy
aminoacetate [BAA]) and then with fluorescently conjugated
antibodies. Comparison of the resulting fluorescence profiles
obtained in the presence and absence of diethylaminobenzal-
dehyde (DEAB), a specific inhibitor of ALDH enzyme activ-
ity, showed that the cells in all fractions had DEAB-sensitive
ALDH activity, as indicated by their decreased fluorescence
when DEAB was present (Fig. 1B, Supporting Information
Fig. 1). However, in all tissue samples examined (four fresh and
seven previously cryopreserved), we found the median fluores-
cence intensity (MFI) to be much higher (on average, 18-fold,
p < .001) in the primitive luminal fraction when compared with
the basal fraction (Fig. 1C) and this difference was sustained
when CD10 was used as an additional marker to discriminate
the luminal progenitor-enriched and basal fractions [6, 7] (with
a 17-fold higher MFI in CD49fþEpCAMþCD10?population on
average when compared with the CD49fþEpCAM?/lowCD10þ
cells; Supporting Information Fig. 2). The mature luminal cells
exhibited intermediate activity (on average, fourfold higher MFI
than the basal cells, p < .001, Fig. 1C). In the same cell suspen-
sions, the MFI of the stromal cell fraction was also much higher
(on average, 13-fold, p < .001) than the MFI of the basal fraction.
Thus, the elevated ALDH activity within bulk populations of
human mammary cells is determined predominantly by the preva-
lence of primitive luminal cells and stromal cells, both typically
present in high numbers. As a result, even after fractionation,
minor contamination of the more primitive basal fraction with
either of these subpopulations would give false-positive readings.
Examination of two published global gene expression
datasets [5, 8] for these subpopulations of cells that used
microarray or LongSAGE
ALDH1A3 is the most prominently expressed ALDH of all
the members of this gene family in the normal human breast
and, in both datasets, ALDH1A3 expression is highest in the
CD49fþEpCAMþ(or CD24þ) primitive luminal populations
(Supporting Information Fig. 3A). We also compared these
datasets with another gene expression microarray dataset
activated cell sorting gating approach used to analyze fractions of the different cell types present in dissociated normal human breast tissue, based
on differential expression of EpCAM, CD49f, CD31, and CD45, as previously described [4, 5]. (B): Representative BAA fluorescence profiles of
the fractions identified in Panel (A) after incubation with Aldefluor in the presence (solid gray) or absence (open profiles) of DEAB and then
with antibodies. (C): MFI values for the BAA fluorescence of cells in each fraction in Panel (B) expressed relative to the MFI of the correspond-
ing basal fraction. Bars show the mean 6 SEM for mammoplasty samples that were freshly processed (green bars, n ¼ 4) or previously cryopre-
served (yellow bars, n ¼ 7). Abbreviations: BAA, bodipy aminoacetate; DEAB, diethylaminobenzaldehyde; EpCAM, epithelial cell adhesion
molecule; FSC, forward scatter; MFI, median fluorescence intensity.
Elevated aldehyde dehydrogenase activity is specific to the primitive luminal epithelial and stromal cell fractions. (A): Fluorescence-
Eirew, Kannan, Knapp et al.
generated on similarly fractionated cells obtained from 3-day
cultures of dissociated primary mammoplasty tissue cells .
The results were largely confirmatory, ALDH1A3 remaining as
one of the most highly expressed ALDH family members in
these cultured cells, with highest ALDH1A3 expression again in
enriched fraction (Supporting Information Fig. 3B). However,
some notable differences were seen (e.g., marked upregulation of
ALDH1B1, 3B1, 3B2, 5A1, and 16A1 expression in the cultured
vs. freshly isolated mammary cells), highlighting the likelihood
that mammary cells maintained in culture may not retain all of
the phenotypic properties they possess in vivo.
To obtain a more accurate assessment of the changes in
expression of ALDH1A3 during the differentiation of human
mammary stem cells in vivo, we performed quantitative reverse
trancriptase polymerase chain reaction and Western blotting
analyses for ALDH1A3 transcripts and protein on cells isolated
from three normal breast tissue samples. The results (Fig. 2A,
2B) consistently paralleled the ALDH activity profiles deter-
mined by Aldefluor staining (Fig. 1B, 1C). In contrast, the
ALDH1A1 transcript and protein levels were consistently low
in all epithelial subsets (Fig. 2A, 2B). Interestingly, we found
that the stromal cells present in mammary tissue express high
levels of ALDH1A1 transcripts and protein compared with any
of the subsets of mammary epithelial cells and low levels of
ALDH1A3 compared with the luminal subsets (Fig. 2A, 2B).
To determine directly the ALDH activity of CFCs and
MRUs, we subjected cells dissociated from another eight nor-
mal breast tissue samples to the same surface marker- and
Aldefluor-based fractionation strategy (Fig. 3A, 3B, left and
middle panels) and then further subdivided the cells as fol-
lows. In the first set of experiments, all of the three mammary
epithelial subsets plus the stromal cells were separated as a
single group into an ALDHþand ALDH?fraction using a
single stringent gate to discriminate between DEAB-sensitive
and DEAB-insensitive BAA fluorescence. In the second set of
experiments, we subdivided the individually defined primitive
luminal and basal subsets (only) into low, medium, and highly
BAA-fluorescent fractions. The cells from each of the eight
fractions from both designs (2 þ 6) were then isolated by fluo-
rescence-activated cell sorting (FACS) and assayed for CFC ac-
tivity in vitro and MRU activity in vivo (the latter using a sub-
renal capsule xenotransplantation assay that facilitates sensitive
quantitative measurements of human MRU frequencies [4, 9]).
The results we obtained from the first experiments showed that
more than 90% of the MRUs as well as the bilineage and
myoepithelial CFCs are in the ALDH?(BAAlow-med) fraction,
whereas most (76 6 10%) of the luminal CFCs are in the
ALDHþ(BAAhigh) fraction (right panel of Fig. 3A; Supporting
Information Table 1). The results of the second experiments
confirmed the segregation of luminal versus basal CFCs and
MRUs in the EpCAMþand EpCAM?subsets of CD49fþcells,
respectively (right panel of Fig. 3B; Supporting Information
Table 2). In addition, they showed the luminal CFCs to be
split between the three ALDH activity fractions in a ratio of
5:62:33 (BAAlow:BAAmed:BAAhigh), with a reverse distribution
(75:24:1 and 81:18:1) for the myoepithelial plus bilineage
CFCs and the MRUs, respectively (right panel of Fig. 3B; Sup-
porting Information Table 2).
These results establish that the most primitive normal human
mammary epithelial cell types defined by their bilineage
developmental potential are characterized by low ALDH
activity, and that this activity is first and selectively increased
by more than an order of magnitude at the time of commitment
to the luminal lineage before further proliferative ability is lost.
A similar pattern occurs in bovine mammary tissue where the
stem cells are detected using the same in vivo transplant assay
as used here for human MRUs . The present findings are
also in agreement with those found in the mouse mammary
gland where ALDH1A3 expression has been found to be upreg-
ulated at the point of luminal progenitor generation .
ALDH1A3 transcripts are also detected at high levels in luminal
progenitor-enriched fractions obtained from short-term adherent
cultures of freshly isolated normal human mammary cells ;
however, a more detailed study of different in vitro systems is
required to investigate the stability of this phenotype.
ALDH1A3 plays a catalytic role in the biosynthesis of
retinoic acid in mammary cells  and approximately two-
thirds of human genes with binding sites for Gata-3 (a tran-
scription factor that regulates the luminal differentiation pro-
gram ) contain retinoic acid regulatory elements .
Activation of retinoic acid signaling (using all-trans retinoic
acid) in breast cancer cell lines was reported to result in a
reduced frequency of cells with mammosphere-forming activ-
ity, and blocking of ALDH activity (using DEAB) had the
reverse effect . These findings encourage future investiga-
tion of the possibility that ALDH activity plays a role in acti-
vating the luminal cell differentiation program in the mam-
mary gland through retinoid signaling pathways.
the primitive luminal cell fraction. (A): ALDH1A3 and ALDH1A1 tran-
script levels in the different fluorescence-activated cell sorted fractions
of cells are determined by quantitative reverse transcription polymerase
chain reaction and normalized relative to GAPDH. Mean 6 SEM val-
ues for three independently studied mammoplasty samples are shown.
(B): Representative Western blots showing different ALDH1A3 and
ALDH1A1 protein levels in the different fractions of cells. Histone H3
is shown as the loading control. Abbreviation: GAPDH, glyceralde-
ALDH1A3 transcript and protein levels are elevated in
ALDH Is a Luminal Progenitor Biomarker
The high ALDH activity reported for cells with breast
cancer-initiating properties in xenograft assays [16–18] stands
in direct contrast with the results for normal mammary stem
cells and correlates better with normal luminal progenitors.
This adds further weight to the possibility that cells with
breast cancer stem cell activity in clinical samples may be
more closely related to luminal progenitor cells. A case for
this model has recently been made in BRCA1-associated
breast cancer, in which aberrant luminal progenitor activity
has been described in human breast tissue that is still pheno-
typically normal . It is noteworthy that more ALDHþcells
have been reported in breast tissue from BRCA1 mutation car-
riers , where they appear to have a luminal rather than a
basal location. Alternatively, it may be that in certain breast
cancers, the oncogenic process itself perturbs the control of
ALDH expression and/or activity. Examples of the latter have
been demonstrated in leukemia [20–23], suggesting that the
picture may be equally heterogeneous in breast cancer.
Our study demonstrates that elevated ALDH activity charac-
terizes luminal progenitor cells in the normal human mam-
kinson, G. Edin, and the staff of the Flow Cytometry Facilities of
the Terry FoxLaboratory andthe Walter andEliza Hall Institute.
Mammoplasty tissue, generously donated by patients, was
obtained with the assistance of Drs. J. Sproul, P. Lennox, N. Van
Laeken, and R. Warren (Canada) and the Victorian Cancer Bio-
bank (Australia). This work was supported by the Canadian
Breast Cancer Research Alliance (Grant CBCRA 019343), the
U.S. Department of Defense Breast Cancer Research Program
(Predoctoral Fellowship number W81XWH-06-1-0702), the Ca-
nadian Breast Cancer Foundation BC/Yukon (Fellowship to N.
Kannan), the National Health and Medical Research Council
ment through the Victorian Cancer Agency/Victorian Breast
Cancer Research Consortium and an Operational Infrastructure
Supportgrant, andthe AustralianCancer Research Foundation.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
activity. Both left and middle panels of (A) and (B) show the two gating approaches used to isolate the fractions to be subsequently assessed func-
tionally for their CFC and MRU content as described in Supporting Information Methods. The proportions of total cells in each of these fractions
(mean 6 SEM of non-diethylaminobenzaldehyde [DEAB]-treated cells) are indicated in the middle panels. The right panels show the distributions
of luminal CFCs (red diamonds), combined myoepithelial plus bipotent CFCs (blue diamonds), and MRUs (blue circles) in each set of fractions
assayed. Each symbol shows the result from an individual experiment, and the bars show the mean of the values measured (n ¼ 5 samples in (A),
n ¼ 3 samples in (B)). Abbreviations: BAA, bodipy aminoacetate; CFC, colony-forming cell; EpCAM, epithelial cell adhesion molecule; MRU,
mammary repopulating unit.
Human MRUs and bipotent CFCs have low aldehyde dehydrogenase (ALDH) activity whereas luminal CFCs exhibit high ALDH
Eirew, Kannan, Knapp et al.
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ALDH Is a Luminal Progenitor Biomarker