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Transcriptome Analysis of the Normal Human Mammary Cell Commitment and Differentiation Process

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Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.
Changes in Gene Expression Seen during the Differentiation of CFCs In Vitro Are Similar to Those Evident in 3-Day Cultured Cells and Evidence of a Role for Notch in Normal Human Mammary Epithelial Cell Commitment to the Luminal Lineage (A) Transcript levels of six genes in 8-day colonies produced by the luminal-restricted CFCs were compared to the levels of the same transcripts in 8-day colonies produced by the bipotent CFCs (in vitro ratio). These ratios are compared to the transcript levels in the mature luminal [EPCAM + CD49f ? (MUC1/CD133) + CD10 ? THY1 ? ] or myoepithelial [EPCAM + CD49f ? MUC1 ? CD133 ? (CD10/THY1) + ] cells isolated from 3-day cultured mammoplasty samples (n = 3). (B) Bipotent and luminal-restricted CFC-enriched fractions were purified from six different 3-day precultured mammoplasty samples and then assayed for CFC with (10 mM) or without (+ DMSO) DAPT. Colony counts were performed 8 days later. (C) Representative FACS profiles of the EPCAM + cells present in the 8-day colonies generated in (B). Staining with antibodies against EPCAM allowed human mammary cells to be discriminated from contaminating mouse fibroblast feeder cells. Mean fluorescence intensities (MFI) were calculated using FlowJo software (Ashland, OR). The arrow indicates the decreased expression of the luminal cell markers (MUC1 and/or CD133) when bipotent CFCs were cultured in the presence of DAPT as compared to the DMSO control. (D) A representative FACS profile of the cells present in 8-day colonies generated from bipotent CFCs infected with a lenti-dnMAML-GFP or a control lenti-GFP virus (from one of two experiments) after staining the harvested cells as in (C). The mean fluorescence (MFI) values show the decreased expression of MUC1 and/or CD133 in the GFP + cells. (E) Representative FACS profile of the cells present in 8-day colonies generated from bipotent CFCs or luminal-restricted CFCs infected with either LentishN3 or control Lenti-NS virus (from one of two experiments) after staining the harvested cells as in (C). The mean fluorescence (MFI) values show the decreased expression of MUC1 and/or CD133 in the GFP + cells. The error bars represent the standard error of the mean.
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Cell Stem Cell
Resource
Transcriptome Analysis of the Normal Human Mammary
Cell Commitment and Differentiation Process
Afshin Raouf,
1
Yun Zhao,
1
Karen To,
1
John Stingl,
1,7
Allen Delaney,
2
Mary Barbara,
4
Norman Iscove,
4
Steven Jones,
2
Steven McKinney,
3
Joanne Emerman,
5
Samuel Aparicio,
3
Marco Marra,
2,6
and Connie Eaves
1,6,
*
1
Terry Fox Laboratory
2
Genome Sciences Centre
3
Molecular Oncology and Breast Cancer Program
British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada
4
Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, ON M5G 2M9, Canada
5
Departments of Pathology and Laboratory Medicine, Anatomy and Cell Biology
6
Department of Medical Genetics
University of British Columbia, Vancouver, BC V6T 1Z4, Canada
7
Present address: Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
*Correspondence: ceaves@bccrc.ca
DOI 10.1016/j.stem.2008.05.018
SUMMARY
Mature mammary epithelial cells are generated from
undifferentiated precursors through a hierarchical pro-
cess, but the molecular mechanisms involved, partic-
ularly in the human mammary gland, are poorly under-
stood. To address this issue, we isolated highly
purified subpopulations of primitive bipotent and
committed luminal progenitor cells as well as mature
luminal and myoepithelial cells from normal human
mammary tissue and compared their transcriptomes
obtained using three different methods. Elements
unique to each subset of mammary cells were identi-
fied, and changes that accompany their differentiation
in vivo were shown to be recapitulated in vitro. These
include a stage-specific change in NOTCH pathway
gene expression during the commitment of bipotent
progenitors to the luminal lineage. Functional studies
further showed NOTCH3 signaling to be critical for
this differentiation event to occur in vitro. Taken to-
gether, these findings provide an initial foundation for
future delineation of mechanisms that perturb primi-
tive human mammary cell growth and differentiation.
INTRODUCTION
The normal female mammary gland grows rapidly at puberty to
produce an elaborate bilayered tree-like structure composed
of an inner layer of luminal cells surrounded by an outer layer
of myoepithelial cells. Later cycles of expansion and involution
occur during each estrous cycle and even more dramatically
with each pregnancy (Howlin et al., 2006; Russo and Russo,
2004). This dynamic activity suggests the lifelong maintenance
within the normal mammary gland of a population of self-renew-
ing undifferentiated mammary stem cells. Support for this con-
cept was first provided by vector integration site analysis of
serially transplantable outgrowths generated in precleared
mammary fat pads of mice transplanted with retrovirally marked
tissue fragments (Kordon and Smith, 1998). More recently,
a quantitative in vivo assay for the cell of origin of these clonal
outgrowths has been identified and used to enable their partial
characterization and phenotypic distinction from most other
mammary epithelial cells in mice, including those capable of col-
ony formation in vitro (Asselin-Labat et al., 2006; Shackleton
et al., 2006; Stingl et al., 2006a).
Several lines of evidence suggest that the mammary gland of
normal adult women also contains a population of mammary
stem cells. These include in situ studies of X chromosome inac-
tivation patterns in normal human mammary tissue indicating
a common origin of cells in adjacent lobules and ducts (Tsai
et al., 1996) and in vitro experiments identifying a unique and
rare subset of human mammary progenitor cells that generate
mixed colonies containing both luminal and myoepithelial cells,
as well as other subsets of clonogenic cells that produce only
one or the other mature cell type (Stingl et al., 1998, 2001). The
properties exploited most effectively for the prospective isolation
of these different types of colony-forming cells (CFCs) include
their shared expression of an epithelial marker known as epithe-
lial cell adhesion molecule (EPCAM) (Stingl et al., 1998, 2001)
and a
6
-integrin (CD49f), a frequent marker of epithelial progeni-
tors (Stingl et al., 2001). The undifferentiated (bipotent) CFCs
have also been further distinguished by their expression of the
common acute lymphoblastic leukemia antigen (CALLA, also
called CD10), a marker of the more basally situated mature my-
oepithelial cells (Stingl et al., 1998), whereas the luminal-re-
stricted CFCs selectively express Mucin-1 (MUC1), a specific
marker of mature luminal cells in the mammary gland.
We now describe a procedure that utilizes additional markers
to subdivide normal human mammary epithelial cells into four
fractions, two of which are more highly and exclusively enriched
in bipotent CFCs and luminal-restricted CFCs than previously
achievable, the other two fractions representing the mature my-
oepithelial and luminal cells. Long serial analysis of gene expres-
sion (LongSAGE) and single-channel expression microarray (Af-
fymetrix) technologies were used to generate global transcript
profiles for each of these four subpopulations, and some of the
interesting differences were then further examined by quantita-
tive real-time PCR (Q-RT-PCR) measurements. The results
Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc. 109
identify many gene expression changes that accompany the dif-
ferentiation of primitive normal human mammary cells both in
vivo and in vitro. They also provide new evidence for a previously
unrecognized and nonredundant role of NOTCH3 signaling in the
commitment of bipotent mammary cells to the luminal lineage.
RESULTS
Isolation of Highly Purified Populations of Primitive and
Mature Subsets of Human Mammary Epithelial Cells
Human mammary CFCs constitute approximately 1% of freshly
dissociated, fibroblast-depleted suspensions of normal reduc-
tion mammoplasty samples, and this value can be increased
several-fold by culturing the cells in bulk for 3 days (Stingl
et al., 2001). Using such ‘‘precultured’’ cells and improved assay
conditions, we first showed that these allow CFC progenitor fre-
Figure 1. Detection and Purification of
Distinct, Functionally Defined Subsets of
Normal Human Mammary Epithelial Cells
(A) Linearity of colony formation in assays of un-
separated EPCAM
+
cells from 3-day cultured
mammoplasty cells (green, red, and blue lines
show data for three different samples, r R0.95
in each case). Values shown are the mean ±
SEM of three to six replicates in each experiment.
(B) Linearity of colony formation in assays of frac-
tions enriched in bipotent (dashed lines) or lumi-
nal-restricted (solid lines) CFCs isolated as shown
in (C) from the three samples shown in (A) (and
similarly color-coded, r R0.95 in each case).
(C) Strategy used to isolate subpopulations of nor-
mal human mammary EPCAM
+
viable (PI
) epithe-
lial cells selectively enriched in their content of bi-
potent CFCs, luminal-restricted CFCs, mature
myoepithelial cells, or mature luminal cells. A rep-
resentative FACS plot is shown. Typical mixed and
luminal colonies generated in assays of 50 cells
from the purified subsets indicated are shown
(magnified 323).
(D) Distribution of bipotent (circles) and luminal-
restricted (triangles) CFCs in the subsets indicated
(n = 16).
(E) To calculate the yield of bipotent and luminal-
restricted CFCs in each fraction, the values for
each progenitor type in the EPCAM
+
CD49f
+
frac-
tion were set = 100%.
The error bars represent the standard error of the
mean.
quencies to be measured independent of
the number of cells plated over a wide
range (Figures 1A and 1B). Additional ex-
periments showed that highly purified
and distinct populations of bipotent and
luminal-restricted CFCs were consis-
tently obtained by initial immunomag-
netic selection of the EPCAM
+
cells pres-
ent in 3-day cultures of dissociated
mammoplasty samples followed by sub-
dividing both the CD49f
+
and CD49f
compartments according to their com-
bined expression of either MUC1 and CD133, or CD10 and
THY1 (CD90) (Figure 1C). Using this protocol, we found that
the CD49f
fraction contained very few CFCs (2 ± 1 CFCs per
100 starting CD49f
cells, n = 4) in contrast to the CD49f
+
cells,
of which approximately 50% were CFCs and thus contained
>95% of the EPCAM
+
CFCs. Accordingly, the yield of CFCs
present in subsets of CD49f
+
cells analyzed according to their
expression of MUC1 and CD133, or CD10 and THY1, was
used to calculate progenitor yields from different starting popu-
lations. The MUC1
CD133
(CD10/THY1)
+
subset of the CD49f
+
fraction contained the majority (57%) of the bipotent CFCs
at a purity of 45% ± 3% with very few luminal-restricted CFCs
present (only 3% of this subset). The remaining (MUC1/
CD133)
+
CD10
THY1
CD49f
+
cells contained most (96%) of
the luminal-restricted CFCs, which were present in this fraction
at a purity of 32% ± 3% and were contaminated by the presence
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
110 Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc.
of very few bipotent CFCs (only 5% of this subset) (Figures 1D
and 1E).
We next asked whether cells coexpressing cytokeratin 14 and
18 would be found in either of the CD49f
+
subsets and, if so,
whether their numbers would correlate with the number of CFC
present, since coexpression of these two cytokeratins has been
associated with a primitive state of mammary epithelial cells
(Smalley et al., 1999; Welm et al., 2002). Immunostaining of sorted
MUC1
CD133
(CD10/THY1)
+
and (Muc1/CD133)
+
CD10
THY1
cells from three different reduction mammoplasty samples re-
vealed that both of these fractions consistently contained detect-
able, albeit variable, frequencies of dually cytokeratin 14
+
and 18
+
cells. However,relatively high CFC frequencieswere seen in some
samples where dual cytokeratin 14/18
+
cells were rare and vice
versa (see Table S1 available online), thus indicating nonidentity
of this phenotype with any type of CFC.
Transcriptional Profiling of Different Purified Subsets
of Human Mammary Epithelial Cells
cDNA preparations were generated from each of the four sub-
sets of normal human mammary cells using PCR-amplification
methods previously shown to preserve transcript representation
in both LongSAGE libraries (Zhao et al., 2007) and array analyses
(Iscove et al., 2002). LongSAGE libraries were made from all four
fractions obtained from one mammoplasty sample (Table 1),
and each library was then sequenced to a depth of 200,000
tags (Gene Expression Omnibus [GEO] accession number
GSE11395). We found that each of these libraries contained
approximately 34,000 unique tags when a 99.9% sequence
quality cut-off was used. Twelve Affymetrix array hybridizations
were also performed, one for each of the four fractions obtained
from the same sample used for the LongSAGE libraries and the
other eight from the four fractions isolated from two additional
mammoplasty samples (Table 1 and GEO accession number
GSE11395). An unsupervised hierarchical clustering of the data
obtained by Pearson correlation analysis of the LongSAGE
library data demonstrated that the population of cells that was
most enriched in bipotent CFCs was most closely related to
the differentiated myoepithelial cells. Conversely, the population
most enriched in luminal-restricted CFCs was most closely re-
lated to the differentiated luminal cells (Figure 2A). Similar results
were obtained from the larger Affymetrix dataset (Figure 2B).
We then used DiscoverySpace software (Robertson et al.,
2007) to identify all tags in the LongSAGE libraries that mapped
to a RefSeq transcript at position 3 or lower and were signifi-
cantly differentially expressed (at a confidence level of 95%) in
pairwise comparisons of the four LongSAGE libraries (Tables
S2–S5). In terms of cell surface markers, both CD29 and PROCR
(endothelial protein C receptor/EPCR) transcripts were found to
be upregulated in the bipotent CFC-enriched fraction. Expres-
sion of the CD29 gene is also upregulated in mouse mammary
stem cells (Shackleton et al., 2006), and increased expression
of PROCR/EPCR is a feature of hematopoietic and hair-follicle
stem cells (Balazs et al., 2006; Blanpain et al., 2004).
From the Affymetrix data, we identified differentially expressed
transcripts using a 1.5-fold cut-off and a p value %0.05 (Tables
S6–S9). To determine whether and how these differences might
correlate with functionally distinct cell types, Volcano plots were
used to identify the most differentially expressed transcripts from
each of the six possible pairwise comparisons, and a K-mean
clustering algorithm was then performed using centered Pear-
son correlation analysis. From this analysis we obtained four
gene expression clusters (Figure 2C). One of these clusters
was exclusive for the luminal-restricted CFCs and included tran-
scripts for both CD24 and ALDH5A1. CD24 is of interest because
it was initially found to be expressed on certain breast cancer
stem cells (Al-Hajj et al., 2003) and subsequently was found to
be highly expressed on purified human luminal epithelial cells
(Jones et al., 2004), on luminal-restricted CFCs in the mouse (As-
selin-Labat et al., 2007; Stingl et al., 2006a), and at lower level on
mouse mammary stem cells (Asselin-Labat et al., 2007; Shackle-
ton et al., 2006; Stingl et al., 2006a). Consistent with these
Table 1. Transcriptome Profiling of Highly Purified Normal Human Mammary Epithelial Progenitor Populations
Transcriptome Profiling
Colony Phenotype LongSAGE Library Affymetrix Analysis
Sample Purified Fraction Luminal Mixed Myoepithelial
Amount of
RNA Used Total Tags
Total Tag
Types
Amount of
RNA Used
1 Bipotent CFC-enriched 0.5 51 0 10 ng 205,221 40,640 50 pg
Luminal-restricted CFC-enriched 28 6.5 0 10 ng 203,246 35,236 50 pg
Mature myoepithelial ND ND ND 10 ng 210,834 38,336 50 pg
Mature luminal ND ND ND 10 ng 201,037 32,632 50 pg
2 Bipotent CFC-enriched 3.3 46 1 50 pg
Luminal-restricted CFC-enriched 40.5 0.5 0 50 pg
Mature myoepithelial ND ND ND 50 pg
Mature luminal ND ND ND 50 pg
3 Bipotent CFC-enriched 5 37 2 50 pg
Luminal-restricted CFC-enriched 33 5 0 50 pg
Mature myoepithelial ND ND ND 50 pg
Mature luminal ND ND ND 50 pg
Bipotent and luminal-restricted progenitor-enriched fractions were isolated from three different reduction mammoplasty samples. The progenitor
frequency in each subfraction was calculated as described in the Experimental Procedures. ND, not determined.
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc. 111
findings, we determined from FACS analyses that virtually all of
the CD24
+
EPCAM
+
cells coexpressed MUC1 and/or CD133
and accounted for approximately 82%–96% of the (MUC1/
CD133)
+
subset (Figure S1). High ALDH5A1 expression in the lu-
minal CFC-enriched fraction is of interest in view of the recent re-
port that primitive normal and malignant human mammary cells
exhibit uniquely elevated ALDH activity (Ginestier et al., 2007;
Liu et al., 2008). In contrast to the luminal-restricted CFCs, a dis-
tinct cluster for the bipotent CFCs was not resolved. Instead,
a cluster that included both the bipotent CFCs and the differenti-
ated myoepithelial cells was identified. This cluster contained
Figure 2. Relatedness of Undifferentiated,
Lineage-Restricted, and Mature Normal
Human Mammary Epithelial Subpopula-
tions Revealed by Global Transcriptome
Comparisons
(A) Relatedness of the LongSAGE libraries deter-
mined by Pearson correlation analysis.
(B) Relatedness of the transcriptomes identified by
Affymetrix analyses of all four subsets of mam-
mary cells (three samples each) derived from an
unsupervised hierarchical clustering analysis.
(C) Cluster analysis performed by applying a
K-mean clustering algorithm of the most highly dif-
ferentially expressed transcripts identified in the
Affymetrix data comparisons of gene expression
in the four subsets of human mammary cells stud-
ied here.
(D) Distribution according to the Biological Pro-
cess (D) and Molecular Function (E) GO categories
at level 5 of transcripts found to be overexpressed
in the bipotent as compared to the luminal-re-
stricted CFC-enriched subpopulations (70 genes,
red bars) and in the luminal-restricted CFC-
enriched cells as compared to the bipotent CFC-
enriched subpopulations (108 genes, blue bars)
from both the LongSAGE and Affymetrix data
comparisons based on their assignment.
transcripts for THY1/CD90, which was
used to isolate these populations, and
also for both JAGGED1 (JAG1) and the
NOTCH4 receptor (Figure 2C).
A total of 178 genes were identified
as differentially expressed in both the
LongSAGE and Affymetrix comparisons
of the fractions enriched in bipotent ver-
sus luminal-restricted CFCs, 70 of which
were more highly expressed in the bipo-
tent CFC fraction and the other 108 of
which were more highly expressed in
the luminal CFC fraction (Table S10). Fur-
ther categorization of these differentially
expressed genes according to their
Gene Ontology Biological Process and
Molecular Function groups revealed that
the bipotent CFC molecular signature
contained a preponderance of tran-
scripts for proteins involved in cell migra-
tion, shape control, and morphogenesis,
and in calcium, integrin, and insulin signaling (Figures 2D and
2E). In contrast, the luminal-restricted CFC molecular signature
was found to contain a preponderance of transcripts for proteins
involved in transcription, RNA metabolism, phosphotransferase
activity, and retinoic acid synthesis (ALDH1A3) and signaling
(CRAPB2 and RBP7). Shared differentially expressed transcripts
for other comparisons are listed in Tables S11–S13, and their
numbers are shown diagrammatically in Figure S2. Together,
these data suggest at least 332 genes that undergo changes in
expression during normal human mammary epithelial cell differ-
entiation. Interestingly, CD44, a cell surface adhesion protein
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
112 Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc.
whose expression has been reported to allow selective enrich-
ment of breast tumor-initiating cells (Al-Hajj et al., 2003), was
not found to be differentially expressed in either CFC-enriched
fraction by either transcriptome profiling method. This failure to
find CD44 expression to be a discriminating feature of any of
the subsets of EPCAM
+
cells studied here was further supported
by FACS analysis of antibody-stained preparations (Figure S1).
These showed 30%–50% of all EPCAM
+
cells are CD44
+
and
that among the CD49f
+
cells (which includes both the luminal
and bipotent CFCs subtypes, Figure 1), approximately 90% of
the cells coexpress CD44.
We then selected 32 genes for more extensive analysis of their
expression levels as determined by Q-RT-PCR. These genes in-
cluded 18 identified as differentially expressed in different mam-
mary subsets either in the LongSAGE libraries or the Affymetrix
data sets as well as 14 genes implicated in differentiation and/or
breastcancer. Transcriptlevels for each test genewere normalized
to GAPDH transcript levels in the same extract, which gave similar
results to those obtained when an alternative highly expressed
gene (RPL36) was used for the normalization (data not shown).
The results of the Q-RT-PCR assays demonstrated consistent dif-
ferentialexpression by 15 of the18 genes identified from the global
gene expression studies and 19 of all 32 surveyed (Table S14).
Included among these were the estrogen and progesterone re-
ceptor genes. These showed high progesterone receptor and low
estrogen receptor transcript levels in the bipotent CFC-enriched
fraction, which changed to low levels of progesterone receptor
transcripts and high levels of estrogen receptor transcripts in
Figure 3. Comparison of Q-RT-PCR Ex-
pression Data for LIF and NOTCH Pathway
Genes Expressed in Different Subsets of
Human Mammary Cells
(A–D) The level of expression of each transcript
type was assessed by normalizing its level to the
level of GAPDH transcripts in the same extract
and then to the levels of the same test transcript
in the bipotent CFC-enriched fraction (set = 1, dot-
ted lines). The error bars represent the standard
error of the mean.
the luminal-restricted CFC-enriched frac-
tion (Table S14). Q-RT-PCR also con-
firmed the LongSAGE data indicating LIF
expression to be upregulated (9-fold) in
the fraction enriched in luminal-restricted
CFCs as compared to the fraction en-
riched in bipotent CFCs and then similarly
reduced (9-fold) in the differentiated lu-
minal cells but remaining unchanged in
the mature myoepithelial cell fraction (Fig-
ure 3). Q-RT-PCR data further demon-
strated transcripts for both components
of the LIF receptor (LIFR and GP130) to
be present in all four cell fractions exam-
ined, albeit at constant levels.
Another regulatory gene that showed
a marked but progressive change in ex-
pression during luminal cell differentiation
(30-fold overall decrease from bipotent progenitors to mature lu-
minal cells) was the Iroquois-class homeobox gene IRX4 (Table
S14). IRX4 has been associated with the development of the
lungs and heart (Bruneau et al., 2001; van Tuyl et al., 2006). Inter-
estingly, transcripts for IRX2, another Iroquois-class homeobox
gene, were previously found to be preferentially expressed by lu-
minal and not basal epithelial cells in the mouse mammary gland
(Lewis et al., 1999).
All profiling methods also showed that transcripts for the
NOTCH ligand JAGGED2 were highest in the bipotent CFC-en-
riched fraction as compared to the luminal-restricted CFC-en-
riched fraction, with the opposite scenario for the NOTCH signal-
ing receptor, NOTCH3 (Table S10 and Figure 3). Expression of
NOTCH1 and NOTCH2 was also found to increase during the
process of luminal differentiation, although the changes in tran-
script levels for these genes were more modest. NOTCH4 ex-
pression showed the opposite pattern; transcripts levels were
relatively high in the bipotent CFC-enriched fraction and then de-
creased more than 50-fold during luminal differentiation and ap-
proximately 2-fold during myoepithelial cell differentiation. The
Q-RT-PCR data also showed higher (10-fold) expression of the
NOTCH receptor ligand genes DLL1,JAGGED1, and JAGGED 2
in the bipotent CFC-enriched fraction as compared to the lumi-
nal-restricted CFC-enriched fraction (Figure 3 and Table S14). In-
terestingly, transcripts for HES1,HES6, and HEY1, known target
genes of NOTCH, were found to be upregulated in parallel with
the upregulated expression of the NOTCH1,-2, and -3 receptors
in the luminal-restricted CFC-enriched fraction (Figure 3).
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc. 113
The CFC Assay Recapitulates Human Mammary
Cell Differentiation
As a first use of these gene expression data we asked the extent
to which the differentiation programs obtained in the in vitro CFC
assay replicate those executed largely in vivo. Accordingly,
we analyzed the levels of expression of human LIF,GAS6,
JAGGED2, and NOTCH3 in the predominantly myoepithelial
and luminal cells present after 8 days in cultures initiated with
EPCAM
+
cells enriched in either bipotent or luminal-restricted
CFCs, respectively, and compared these with the directly iso-
lated mature EPCAM
+
myoepithelial and luminal cells (that had
been in culture for a maximum of 3 days). The four genes se-
lected were chosen because they had shown large differences
between the CFC-enriched fractions and the two populations
of mature mammary cells (Table S14). To facilitate comparison
of lineage-specific differences in the expression of these four
genes in differently derived cells from multiple samples, we ex-
amined the ratio of the relative transcript levels in the two mature
cell types isolated from each source (Figure 4A). As can be seen
the relative levels of expression of the four selected genes in the
two sets of culture-derived ‘‘mature’’ cells show a pattern of
change that is similar to that characteristic of the in vivo genera-
tion of their mature counterparts.
NOTCH Signaling Regulates the Restriction of Bipotent
Mammary Cells to the Luminal Pathway
Given the gene profiling data suggesting that bipotent CFCs un-
dergo marked changes in NOTCH receptor expression when
they generate luminal-restricted CFCs, we hypothesized that
NOTCH expression may have an important functional role in
this lineage restriction process. To investigate this possibility,
we cultured purified bipotent and luminal CFCs in the presence
Figure 4. Changes in Gene Expression Seen
during the Differentiation of CFCs In Vitro
Are Similar to Those Evident in 3-Day Cul-
tured Cells and Evidence of a Role for Notch
in Normal Human Mammary Epithelial Cell
Commitment to the Luminal Lineage
(A) Transcript levels of six genes in 8-day colonies
produced by the luminal-restricted CFCs were
compared to the levels of the same transcripts
in 8-day colonies produced by the bipotent
CFCs (in vitro ratio). These ratios are compared
to the transcript levels in the mature luminal
[EPCAM
+
CD49f
(MUC1/CD133)
+
CD10
THY1
]or
myoepithelial [EPCAM
+
CD49f
MUC1
CD133
(CD10/THY1)
+
] cells isolated from 3-day cultured
mammoplasty samples (n = 3).
(B) Bipotent and luminal-restricted CFC-enriched
fractions were purified from six different 3-day
precultured mammoplasty samples and then as-
sayed for CFC with (10 mM) or without (+ DMSO)
DAPT. Colony counts were performed 8 days later.
(C) Representative FACS profiles of the EPCAM
+
cells present in the 8-day colonies generated in
(B). Staining with antibodies against EPCAM al-
lowed human mammary cells to be discriminated
from contaminating mouse fibroblast feeder cells.
Mean fluorescence intensities (MFI) were calcu-
lated using FlowJo software (Ashland, OR). The
arrow indicates the decreased expression of the
luminal cell markers (MUC1 and/or CD133) when
bipotent CFCs were cultured in the presence of
DAPT as compared to the DMSO control.
(D) A representative FACS profile of the cells pres-
ent in 8-day colonies generated from bipotent
CFCs infected with a lenti-dnMAML-GFP or a con-
trol lenti-GFPvirus (from one of two experiments) af-
ter staining the harvested cells as in (C). The mean
fluorescence (MFI) values show the decreased ex-
pression of MUC1 and/or CD133 in the GFP
+
cells.
(E) RepresentativeFACS profile of the cells present
in 8-day colonies generated from bipotent CFCs or
luminal-restricted CFCs infected with either Lenti-
shN3 or control Lenti-NS virus (from one of two ex-
periments) after staining the harvested cells as in
(C). The mean fluorescence (MFI) values show the
decreased expression of MUC1 and/or CD133 in
the GFP
+
cells.
The error bars represent the standard error of the
mean.
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
114 Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc.
or absence of a g-secretase inhibitor, N-[N-(3, 5-difluorophen-
acetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 10 mM)
and then measured the effect on the numbers and types of col-
onies obtained. To ensure that any effects on initial cell attach-
ment were avoided, the DAPT (or vehicle control) was not added
until after the first 16 hr of culture and then every other day there-
after. We found no effect of this treatment on the size or total
number of colonies obtained in assays of either the bipotent or
luminal CFC-enriched fractions (Figure 4B and data not shown).
However, DAPT consistently and significantly (p = 0.0006,
Student’s t test) reduced the number of colonies that contained
a detectable luminal component in the assays of the bipotent
CFC-enriched fraction (Figure 4B). At the same time, the number
of apparently ‘‘pure’’ myoepithelial colonies was proportionately
increased (p = 0.005, Student’s t test). In contrast, DAPT had no
effect on the ability of the luminal-restricted CFCs to form colo-
nies of differentiated luminal cells (Figure 4B), in spite of an
equivalent extent of DAPT-mediated suppression of NOTCH
signaling as shown by a reduction in HEY1 transcripts in cultures
initiated with either purified bipotent or luminal-restricted CFCs
(2- and 6-fold, and 2- and 2-fold, respectively, in two experi-
ments). The differential effects of DAPT on bipotent and lumi-
nal-restricted CFCs were confirmed by flow cytometric analysis
of the immunophenotype of the cells present in 8-day colonies
derived from purified progenitors. These analyses showed a spe-
cific loss of (MUC1/AC133)
+
cells exclusively among the progeny
produced by the DAPT-treated bipotent CFCs and no change in
output of (CD10/THY1)
+
cells (Figure 4C).
As a second approach to investigating the requirement of bi-
potent cells for NOTCH signaling to undergo restriction to the
luminal pathway, we infected suspensions of purified bipotent
CFCs with a lentivirus expressing a dominant-negative (dn)
form of the human mastermind-like-1 gene (MAML) fused to
GFP (Lenti-dnMAML) or a Lenti-GFP control virus and then as-
sessed the phenotype of the GFP
+
cells harvested from bulk
CFC assays 8 days later (Figure 4D). MAML is a coactivator of
NOTCH signaling, and the dn cDNA used here was previously
shown to suppress NOTCH signaling (Maillard et al., 2004;
Weng et al., 2003). As shown in Figure 4D, forced expression
of the dnMAML also decreased the output of (MUC1/AC133)
+
cells from the bipotent progenitors.
We next asked whether this restriction step was directed by
specific activation of the NOTCH3 receptor using a similar strat-
egy in which purified bipotent CFCs were first transduced with
a GFP lentivirus also expressing a short hairpin (sh) NOTCH3
RNA or a nonsilencing control RNA. As shown in Figure 4E,
knockdown of the expression of NOTCH3 receptor in the bipotent
CFCs (90% and 85% in the two experiments undertaken; data not
shown) reduced their ability to generate (MUC1/AC133)
+
luminal
cells. Thus, activation of NOTCH3 appears to be critical for the
restriction of most bipotent progenitors to the luminal pathway,
and other NOTCH receptors cannot substitute for this activity.
DISCUSSION
The development of specific assays for cells at distinct stages
of differentiation in parallel with strategies for their selective
isolation at high purities constitute critical steps in delineating
the molecular mechanisms that regulate normal cell populations
and are likely targets for malignant transformation. In applying
this approach to the normal human mammary gland, we demon-
strate here the robustness of the 2D colony assay conditions
now available to distinguish undifferentiated (bipotent) and line-
age-restricted mammary progenitors of human origin. Second,
we describe a cell separation method that allows each of these
progenitor types to be isolated routinely at purities of more
than 30% and with less than 5% contamination with each other.
Attainment of such purities depends, however, on the use of
a starting population that has been cultured for an initial period
of 3 days to selectively enrich for CFCs while also allowing cells
damaged by the initial enzymatic dispersion process to be re-
moved. The CFC purification scheme we describe also takes ad-
vantage of the fact that the majority of bipotent progenitors share
a number of features with mature myoepithelial cells and lack
features of mature luminal cells, whereas the converse is true
for luminal-restricted progenitors (Russo and Russo, 2004; Stingl
et al., 1998; Tsai et al., 1996), in addition to the previous obser-
vation that primitive mammary cells with significant proliferative
potential express high levels of the a
6
-integrin (CD49f) in contrast
to the majority of mature luminal and myoepithelial mammary
cells (Stingl et al., 2001, 2006b).
Our first application of this technology enabled us to refute the
notion that dual expression of cytokeratins 14 and 18 is a feature
of undifferentiated or even primitive luminal-restricted progeni-
tors in the normal human mammary gland. A second application
was to generate molecular signatures for each of the four differ-
ent cell populations from their global gene expression profiles.
These allowed us to demonstrate that the differentiated cells
produced in the colony assays resemble their counterparts iso-
lated directly after only 3 days in vitro. However, the fact that
all of the gene expression data were from cells that had been
in culture for 3 days would predict that there would be some dif-
ferences with the corresponding cell types present in vivo. The
importance of culture conditions in replicating in vivo transfor-
mation events in human mammary cells has recently been docu-
mented in an elegant study by Ince et al. (2007).
The gene expression data presented here have also offered
some new insights into the molecular characteristics of normal
undifferentiated human mammary cells and their immediate lu-
minal-restricted progeny. For example, we found that bipotent
CFCs contain transcripts for the progesterone receptor but not
the estrogen receptor, with an opposite picture for the luminal-
restricted CFCs. Both of these patterns thus differ from the dou-
ble-negative profile described for normal mouse mammary stem
cells (Asselin-Labat et al., 2006; Shackleton et al., 2006) or any of
the four categories of human breast cancer defined by expres-
sion profiling methods (Hu et al., 2006). We also examined the
expression of transcripts for LIF and its receptor elements.
This cytokine has been previously implicated in controlling apo-
ptosis in the mammary gland (Kritikou et al., 2003) and is down-
regulated in some breast cancers (Hu et al., 2004). In addition,
Dontu et al. (2003) noted that LIF expression was elevated in cul-
tured human mammospheres, which represent mixed popula-
tions but contain elevated frequencies of primitive mammary
cells. The purity of the discrete populations obtained here now
demonstrates that the expression of LIF is markedly upregulated
specifically at the time of progenitor commitment to the luminal
differentiation pathway, at which time transcripts for the receptor
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc. 115
are also present. It is thereforeinviting to speculate that these cells
may be the most important physiological targets for paracrine or
autocrine effects of LIF in the normal mammary epithelium.
A similar stage-specific change in gene expression emerged
from a survey of transcripts for genes associated with the evo-
lutionary conserved NOTCH pathway. This pathway is widely
involved in cell-fate decisions (Fuchs and Raghavan, 2002;
Osborne and Minter, 2007) and has been shown to have a role
in mammary gland development and transformation (Callahan
and Egan, 2004; Farnie et al., 2007; Leong et al., 2007; Pece
et al., 2004; Shi and Harris, 2006). Upon binding of their ligands,
the NOTCH receptors (NOTCH1–4) undergo proteolytic cleav-
age, thereby releasing the NOTCH intracellular domain (NICD),
which then moves into the nucleus and activates genes such
as HES1 and HES6 (Callahan and Egan, 2004; Osborne and
Minter, 2007). Notch4 was first identified as an oncogene (Int3)
in mice by MMTV insertional activation (Raafat et al., 2004).
More recently, higher levels of NOTCH4 transcripts were found
in extracts of whole human mammospheres than in mature hu-
man mammary cells, and addition of soluble ligand to these cul-
tures increased the generation of CFCs in the mammospheres
(Dontu et al., 2004). We now show that NOTCH4 gene expres-
sion is highest in undifferentiated human clonogenic mammary
progenitors and is then markedly downregulated when these
cells become committed to the luminal lineage but before they
have lost their proliferative activity. We also found an opposite
pattern to hold for NOTCH3 and HES6, and to a lesser extent
for NOTCH1 and NOTCH2. This is interesting because the
NOTCH4 receptor possesses a shorter EGF-like repeat se-
quence in its extracellular domain than NOTCH3 and also lacks
cytokine response sequences in its intracellular domain (Allens-
pach et al., 2002; Bigas et al., 1998). Importantly, we also found
that two different methods of nonspecifically blocking NOTCH
signaling in bipotent progenitors (by exposing the cells to a
g-secretase inhibitor or by forced expression in them of a dn
form of MAML) selectively prevented them from generating
mature luminal progeny without affecting their ability to prolifer-
ate and generate mature myoepithelial cells. Nevertheless, the
same treatment had no effect on the production of differentiated
luminal cells from already committed luminal progenitors. Inter-
estingly, knockdown of NOTCH3 expression was sufficient to
produce this same result, suggesting that NOTCH1 and -2,
which appear to be coexpressed at similar levels in these cells,
do not share this unique commitment-requiring function of
NOTCH3 (Figure 5). The recent demonstration of an increased
production during alveogenesis of basal cells in Rbp-j or Pofut1
null mice (Buono et al., 2006) is consistent with this model, as is
the original observation that WAP-Int3 (=Notch4) transgenic
mice display a block in mammary cell differentiation.
The present analysis thusillustrates a number of key changes in
signaling molecules and points to a specificand previously unrec-
ognized role of NOTCH in the process of restriction of bipotent
progenitors to the luminal lineage. They also provide a compre-
hensive descriptionof the gene expression profiles of distinct sub-
sets of normal human mammary epithelial cells that establish an
important new framework for further interrogation of pathways in-
volved in the regulation of normal human mammary cell behavior.
EXPERIMENTAL PROCEDURES
Preparation and Isolation of Mammary Cell Subsets
Discarded reduction mammoplasty tissue was obtained with appropria te con-
sent; the cells were processed, frozen, and thawed; and single-cell suspen-
sions were prepared as described (Stingl et al., 2005). The cells were then cul-
tured for 3 days on top of irradiated 3T3 mouse fibroblasts (8 310
3
per cm
2
)in
EpiCult-B media supplemented with 5% fetal calf serum (FCS) (both from
StemCell Technologies Inc.), and the adherent cells were resuspended using
trypsin. EPCAM
+
cells (92.3% ± 4.7% EPCAM
+
, n = 7) were isolated immuno-
magnetically using the Human EPCAM positive selection kit (StemCell Tech-
nologies). Subsequently, cells were stained with anti-MUC1 (1:100 dilution,
StemCell Technologies), anti-CD133 (AC133, 1:100 dilution, Miltenyi Biotech),
anti-a6 integrin (CD49f) conjugated to fluorescein isothiocyanate (FITC) (1:40
dilution, Becton Dickenson PharMingen), anti-CD10 (CALLA, 1:10 dilution,
StemCell Technologies) conjugated to R-phycoerythrin (PE), and anti-CD90
(THY-1 = 5E10, 1:125 dilution, from Dr. P. Lansdorp, Terry Fox Laboratory,
Vancouver, BC, Canada). A goat anti-mouse antibody conjugated to allophy-
cocyanin (APC, 1:500 dilution, PharMingen) was used to detect cells express-
ing either MUC1 and/or CD133. IgG antibodies directly conjugated to FITC or
PE were used as isotype controls. To distinguish between live and dead cells,
propidium iodide (PI, Sigma) was added at 1 mg/ml to each sample (82% ± 4%
PI
cells in 12 experiments). Cells were sorted on a FACS Vantage SE using
gates that excluded 99.9% of events present in negatively stained control sam-
ples. Events with very high forward and side light scatter profiles were also ex-
cluded to improve sort efficiency. This was estimated to be 80%–98% as mea-
sured by sorting 10
5
MUC1
+
cells into a tube, and viable cell count was obtained
by a hemocytometer. To obtain RNA extracts for gene expression studies, al-
iquots of cells were sorted directly into Trizol reagent (Invitrogen, http://www.
invitrogen.com/) and DNase treated, and the quality of the RNA samples was
examined with the Agilent 2100 Bioanalyzer (Agilent Technologies).
Lentiviral Transduction of Isolated Progenitors
Human dnMAML-GFP(Maillardet al., 2004) was obtainedfrom Dr. A. Weng (Terry
Fox Laboratory, Vancouver, BC, Canada) and cloned into the KA391 lentiviral
vectorand virus-containingsupernatantsgenerated as previously described(Im-
ren et al., 2004). Bipotent and luminal-restricted CFC-enriched populationswere
infectedin 100 ml of EpiCult-Bgrowth media containing5% FCS, 5 310
5
lentiviral
particles(eitherLenti-dnMAML-GFP or Lenti-GFP,orLenti-shN3-GFP [Open Bio-
systems] or the Lenti-shNS-GFP [Open Biosystems]), and 5 mg/ml of protamine
sulfate for 4 hr at 37C. Subsequently, cells were washed three times in 2%
FCS in Hank’s solution and platedat clonal densities in CFC assays (see below).
CFC Assays
CFC assays were performed as described (Stingl et al., 1998, 2001) with the
modification that the plates were first precoated with type I collagen by adding
a solution of 70 mg/ml (Vitrogen 100, Collagen Biomaterials) for 1 hr at 37C
Figure 5. Model of the Proposed Role of NOTCH in Regulating the
Differentiation of Normal Human Mammary Progenitors
The initial commitment process is uniquely dependent on NOTCH3 signaling,
whereas subsequent execution of the luminal differentiation program pro-
ceeds independently.
Cell Stem Cell
Transcript Analysis of Human Mammary Cell Subsets
116 Cell Stem Cell 3, 109–118, July 2008 ª2008 Elsevier Inc.
followed by washing with PBS to remove unpolymerized collagen. After 8 days
the cultures were fixed with methanol acetone (1:1 ratio) and stained with
Wright-Giemsa (Sigma), and colonies containing R50 cells were scored and
typed using a microscope.
Construction and Analysis of LongSAGE Libraries
RNA from each isolated subpopulation was converted to cDNA and then am-
plified as described (Zhao et al., 2007). LongSAGE libraries were constructed
and sequenced using standard protocols (Saha et al., 2002). The relatedness
of the libraries was determined by calculating Pearson correlation values, and
the results were displayed as a tree diagram using Phylip software (http://
evolution.genetics.washington.edu/phylip.html). The complete data set has
been uploaded onto the GEO website (accession number GSE11395).
Microarray Analyses
RNA from each sample was exponentially amplified as described (Iscove et al.,
2002) except that the total number of PCR cycles was restricted to 42 and ex-
ecuted in a single reaction. From each sample, 50 pg aliquots were amplified in
each of ten replicate 20 ml reactions, each yielding 2–3 mg of amplified product .
The amplified cDNA preparations were then purified using the High Pure PCR
Product purification kit (Roche Applied Science), end-labeled with Biotin-N6
ddATP (StemCore Laboratories, OHRI, Ottawa, Ontario, Canada), and hybrid-
ized to Affymetrix human X3P GeneChip arrays according to the manufac-
turer’s protocol (Tietjen et al., 2003). The complete data set has been uploaded
onto the GEO website (accession number GSE11395).
Q-RT-PCR Analyses
cDNA was prepared from 1 ng of RNA using the SuperScript III reverse tran-
scriptase enzyme (Invitrogen) according to the manufacturer’s protocol and
subjected to real-time PCR (7500 Sequence Detection System, Applied Bio-
systems) using gene-specific primers for each transcript analyzed. To quantify
the relative expression of transcripts from the mature luminal and myoepithelial
cells generated in vitro, RNA was extracted from the pooled 8-day progeny of
cells from purified bipotent and luminal-restricted CFC-enriched fractions cul-
tured at 50 cells per plate and pooled for extraction. Relative levels of expres-
sion of each test transcript were calculated by normalizing to the level of
GAPDH transcripts in the same extract, and to compare results in particular
subsets isolated from different samples, values were normalized to those mea-
sured in the bipotent CFC-enriched fractions.
Statistical Analysis
Differentially expressed transcripts from different LongSAGE libraries were iden-
tified using a 95% statistical cut-off as determined by DiscoverySpace software
(Robertson et al., 2007). Transcripts represented by tags expressed only once
in any library (singletons) were excluded from further analysis. Differentially
expressed transcripts from the Affymetrix hybridizations were analyzed using
ArrayAssist software (http://Stratagene.com/software solutions) and R/Bio-
Conductor (Gentleman et al.,2004). The implemented GC-RMA algorithm, which
accountsfor probecomposition,was used to subtractthe backgroundand deter-
minethe correctedprobe intensityfor each probeset cluster.Transcriptsshowing
>1.5-folddifferences with p values%0.05 (after Bonferroni-Holmescorrectionfor
multiple comparisons) in a Student’s t test across replicates were deemed to be
significant. Student’s t test was used for pairwise comparisons to determinesta-
tistical significance. Volcano plots were generated using ArrayAssist software to
identifythe most differentially expressed transcripts. K-mean clustering was car-
riedout using Cluster3.01software(Eisen et al.,1998) using centered correlations.
ACCESSION NUMBERS
All raw data are available online under Gene Expression Omnibus (GEO)
accession number GSE11395.
SUPPLEMENTAL DATA
The Supplemental Data include two figures and 14 tables and can be found
with this article online at http://www.cellstemcell.com/cgi/content/full/3/1/
109/DC1/.
ACKNOWLEDGMENTS
The authors acknowledge excellent technical contributions from Darcy Wilkin-
son and Margaret Hale and the staff of the Flow Cytometry Facility of the Terry
Fox Laboratory, which is funded in part by the Michael Smith Foundation for
Health Research. Mammoplasty tissue was obtained with the assistance of
Drs. Jane Sproul, Peter Lennox, Nancy Van Laeken, and Richard Warren.
This project was funded by grants from Genome BC/Genome Canada, the
Stem Cell Network, The BC/Yukon Division of the Canadian Breast Cancer
Foundation (CBCF), and the Canadian Institute of Health Research (CIHR), in-
cluding the CIHR Centre for Molecular Pathology at the BC Cancer Agency.
A.R. was funded by CBCF and CIHR Fellowships. Y.Z. was funded by a Leuke-
mia Research Foundation of Canada Fellowship, and J.S. was funded by
a CBCF Fellowship and an Industrial Fellowship cofunded by the Natural Sci-
ences and Engineering Research Council of Canada and StemCell Technolo-
gies, Inc.
Received: November 5, 2007
Revised: April 9, 2008
Accepted: May 15, 2008
Published: July 2, 2008
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... The distribution of the Notch signaling pathway components in human breast epithelium was investigated by Raouf et al. [34]. Based on gene expression, human breast epithelial cells were divided into four distinct subpopulations: bipotent colony-forming cells (CFCs), luminal-restricted CFCs, mature luminal cells, and mature myoepithelial cells. ...
... Similarly, transcription of the target genes HES1, HES6, and HEY1 was upregulated in luminal-restricted CFCs. In contrast, NOTCH4 expression showed the opposite profile, with relatively high transcription levels in bipotent CFCs [34]. In situ hybridization also showed that NOTCH3 was located to the luminal epithelium, while JAG1-positive cells were located within the basal layer [16]. ...
... Further functional study indicated that inhibition of Notch activity could induce myoepithelial colony formation ability and reduce the number of luminal colonies. Knockdown of NOTCH3 in the bipotent CFCs could reduce their ability to generate luminal cells, showing that NOTCH3 signaling is critical for breast progenitor cell differentiation in vitro [34]. Raouf et al. used immunohistochemistry to detect the location of Notch receptors and ligands proteins. ...
Article
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Mammary gland development primarily occurs postnatally, and this unique process is complex and regulated by systemic hormones and local growth factors. The mammary gland is also a highly dynamic organ that undergoes profound changes at puberty and during the reproductive cycle. These changes are driven by mammary stem cells (MaSCs). Breast cancer is one of the most common causes of cancer-related death in women. Cancer stem cells (CSCs) play prominent roles in tumor initiation, drug resistance, tumor recurrence, and metastasis. The highly conserved Notch signaling pathway functions as a key regulator of the niche mediating mammary organogenesis and breast neoplasia. In this review, we discuss mechanisms by which Notch contributes to breast carcinoma pathology and suggest potentials for therapeutic targeting of Notch in breast cancer. In summary, we provide a comprehensive overview of Notch functions in regulating MaSCs, mammary development, and breast cancer.
... However, by late embryogenesis, the gland contains two unipotent progenitor cells that form and maintain the luminal and myoepithelial cell layers of the ductal structures within the gland during puberty and adult life and a quiescent multipotent adult MaSC that is only reactivated upon injury (Woodward et al., 2005;Watson and Khaled, 2020). Notch signalling promotes the differentiation of the foetal MaSCs into the unipotent luminal progenitor cell and prevents this cell differentiating into mature luminal epithelial cells to maintain the population (Dontu et al., 2004;Buono et al., 2006;Bouras et al., 2008;Raouf et al., 2008;Lafkas et al., 2013;Šale et al., 2013;Rodilla et al., 2015;Lilja et al., 2018). Upon ablation of the luminal epithelial cells in the adult mammary gland, Notch signalling can also promote the conversion of unipotent myoepithelial progenitor cells into unipotent luminal progenitors to repopulate the luminal lineage (Centonze et al., 2020). ...
... On the other hand, there is abundant evidence that Notch signalling plays a significant role in driving MaSCs toward the unipotent luminal progenitor fate. Notch1-3 are more highly expressed in luminal cells, whilst the Notch pathway inhibitors Numb and Numb-like are found in myoepithelial cells (Bouras et al., 2008;Raouf et al., 2008;Raafat et al., 2011;. Functional studies using knockout and transgenic mouse models and primary human cells have confirmed that Notch signalling controls the luminal vs. myoepithelial lineage balance (Smith et al., 1995;Dontu et al., 2004;Kiaris et al., 2004;Buono et al., 2006;Hu et al., 2006;Bouras et al., 2008;Raouf et al., 2008;Yalcin-Ozuysal et al., 2010;Santoro et al., 2016;Onoyama et al., 2020). ...
... Notch1-3 are more highly expressed in luminal cells, whilst the Notch pathway inhibitors Numb and Numb-like are found in myoepithelial cells (Bouras et al., 2008;Raouf et al., 2008;Raafat et al., 2011;. Functional studies using knockout and transgenic mouse models and primary human cells have confirmed that Notch signalling controls the luminal vs. myoepithelial lineage balance (Smith et al., 1995;Dontu et al., 2004;Kiaris et al., 2004;Buono et al., 2006;Hu et al., 2006;Bouras et al., 2008;Raouf et al., 2008;Yalcin-Ozuysal et al., 2010;Santoro et al., 2016;Onoyama et al., 2020). In the absence of Notch signalling, there is an accumulation of myoepithelial cells, whilst increased Notch signalling leads to an expansion of the of the luminal lineage (Smith et al., 1995;Kiaris et al., 2004;Buono et al., 2006;Hu et al., 2006;Bouras et al., 2008;Yalcin-Ozuysal et al., 2010;Onoyama et al., 2020). ...
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The Notch signalling pathway is a highly conserved developmental signalling pathway, with vital roles in determining cell fate during embryonic development and tissue homeostasis. Aberrant Notch signalling has been implicated in many disease pathologies, including cancer. In this review, we will outline the mechanism and regulation of the Notch signalling pathway. We will also outline the role Notch signalling plays in normal mammary gland development and how Notch signalling is implicated in breast cancer tumorigenesis and progression. We will cover how Notch signalling controls several different hallmarks of cancer within epithelial cells with sections focussed on its roles in proliferation, apoptosis, invasion, and metastasis. We will provide evidence for Notch signalling in the breast cancer stem cell phenotype, which also has implications for therapy resistance and disease relapse in breast cancer patients. Finally, we will summarise the developments in therapeutic targeting of Notch signalling, and the pros and cons of this approach for the treatment of breast cancer.
... Sustained Notch activation also severely impairs alveologenesis in Elf5−/− mice, which have MGs containing a surplus of stem/progenitor cells (Chakrabarti et al., 2012;Choi et al., 2009). All four Notch receptors are expressed in temporally and spatially restricted patterns in subpopulations of basal and luminal mammary epithelial cells (Bach et al., 2017;Bouras et al., 2008;Raafat et al., 2011;Raouf et al., 2008), with lineage-tracing studies suggesting distinct roles for these receptors in directing stem/progenitor cell activity Rodilla et al., 2015;Sale et al., 2013). By comparison, expression of Notch ligands (JAG1, JAG2 and DLL1) is largely confined to the basal compartment (Bach et al., 2017;Bouras et al., 2008;Raafat et al., 2011;Raouf et al., 2008;Xu et al., 2012). ...
... All four Notch receptors are expressed in temporally and spatially restricted patterns in subpopulations of basal and luminal mammary epithelial cells (Bach et al., 2017;Bouras et al., 2008;Raafat et al., 2011;Raouf et al., 2008), with lineage-tracing studies suggesting distinct roles for these receptors in directing stem/progenitor cell activity Rodilla et al., 2015;Sale et al., 2013). By comparison, expression of Notch ligands (JAG1, JAG2 and DLL1) is largely confined to the basal compartment (Bach et al., 2017;Bouras et al., 2008;Raafat et al., 2011;Raouf et al., 2008;Xu et al., 2012). Thus, while there is an indisputably important role for Notch signaling in governing alveolar development, how Notch receptors are regulated in subsets of mammary progenitor cells by different Notch ligands remains unclear. ...
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In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating β-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy.
... To profile the cell-types present within the normal and TNBC organoids, we used a combination of flow cytometry and scRNA-seq. We assayed for mammary epithelial lineages using EPCAM and CD49f as luminal and basal cell markers, respectively (21,22,53,67). As previously shown (22), the normal-derived organoids recapitulate the EPCAM þ luminal lineage, EPCAM þ /CD49f þ LP cells, and EPCAM À /CD49f þ basal cell lineages (NM07NL in Fig. 5A). ...
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... From the statistical result, 1.5 fold-change of genes expression (corresponds to 0.58 in logarithmic scale) in a Student's t-test across replicas helps select the significantly variant genes 18 . Note that positive fold-change refers to up-regulation of genes and negative value refers to down-regulation. ...
Preprint
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Stroke is one of the leading causes of death in humans. Even if patients survive from stroke, they may suffer sequelae such as disability. Treatment for strokes remains unsatisfactory due to an incomplete understanding of its mechanisms. This study investigates Ischemic Stroke (IS), a primary subtype of stroke, through analyses based on microarray data. Limma (in R)derives differentially expressed genes, and the protein-protein interaction (PPI) network is mapped from the database. Gene co-expression patterns are obtained for clustering gene modules by the Weighted Correlation Network Analysis (WGCNA), and genes with high connectivity in the significantly co-expressed modules are selected as key regulators. Common hubs are identified as Cdkn1a, Nes and Anxa2. Based on our analyses, we hypothesize that these hubs might play a key role in the onset and progression of IS. Result suggests the potential of identifying unexplored key regulators by the systemic method used in this work. Further analyses aim at expanding candidate genes for screening biomarkers for IS, and experimental validation is required on identified potential hubs.
... A fold change cutoff of 1.5 and adjusted p-value cutoff of < 0.05 were applied [7,8]. To determine DEGs, a moderated t-test was implemented with a Westfall Young Permutative (WYP) multiple testing correction, which provides a good balance between Type I and Type II error [2]. ...
Presentation
Integrated omics technologies have been instrumental in a host of applications including disease discovery. In ESR 12, the main objectives include the identification of the molecular signatures (using multi-omics analysis of transcriptomic and metabolic data) of co-exposure to neurotoxicants in the human biosamples. Multi-omics are a powerful tool in disease discovery that have undergone large advancements in recent years; available technologies allow for the generation of datasets with tens of thousands of outputs. This requires large computational power and advanced statistical analysis for datasets. While the in-house methods for metabolomics analysis are well-established, there is a need for a statistical pipeline for transcriptomic data analysis. Unfortunately, there is debate within the scientific community over the ideal methods to process raw data, analyze and identify differentially expressed genes (DEGs), and generate robust results while limiting Type I and Type II errors. We developed two statistical method pipelines using Linear Models for Microarray Data (LIMMA; R software) and moderated t-test (Agilent GeneSpring™ software) for DEG discovery and functionality comparisons. R is an open-source software that allows extensive control of statistical pipelines and parameters and the ability to handle complex statistical designs, although it requires basic knowledge of programming languages. GeneSpring™ is a commercially available, user-friendly analysis software that is efficient for basic statistical analyses with the benefit of freely available technical support. However, Genespring™ is geared towards parametric data analysis and tends to be rigid in its pipeline capabilities. We analyzed Agilent™ microarray data generated from three real datasets from experiments from within the lab group (One-Color, SurePrint Zebrafish Gene Expression v3 4 x 44k Microarray, design ID: 026437) that aim to detect the molecular mechanisms involved in metabolic disorders associated with environmental contaminants. 3-day post fertilization (dpf) zebrafish larvae (n = ~17 per replicate, 4 replicates) were exposed for 48 hours (sampled at 5-dpf) to two concentrations of the plasticizer bis(2-ethylhexyl) phthalate (DEHP; 25 nM and10 µM), positive control (amiodarone; 1 µM), and a carrier control [dimethyl sulfoxide (DMSO); 0.1%]. Samples were processed following manufacturer protocols and features were extracted using Agilent Feature Extraction™ software. Raw data was exported and analyzed using the above methods. Preliminary results suggest that LIMMA is more conservative than the moderated t-test and generates fewer DEGs. Overall, this work is instrumental in future efforts to generate statistically robust and reliable computational models and systems biology for the prediction of a host of metabolic diseases.
... From the statistical result, 1.5 fold-change of genes expression (corresponds to 0.58 in logarithmic scale) in a Student's t-test across replicas helps select the significantly variant genes [33]. Note that positive fold-change refers to up-regulation of genes and negative value refers to downregulation. ...
Preprint
Full-text available
Stroke is one of the leading causes of death in humans. Even if patients survive from stroke, they may suffer sequelae such as disability. Treatment for strokes remains unsatisfactory due to an incomplete understanding of its mechanisms. This study investigates Ischemic Stroke (IS), a primary subtype of stroke, through analyses based on microarray data. Limma (in R)derives differentially expressed genes, and the protein-protein interaction (PPI) network is mapped from the database. Gene co-expression patterns are obtained for clustering gene modules by the Weighted Correlation Network Analysis (WGCNA), and genes with high connectivity in the significantly co-expressed modules are selected as key regulators. Common hubs are identified as Cdkn1a, Nes and Anxa2. Based on our analyses, we hypothesize that these hubs might play a key role in the onset and progression of IS. Result suggests the potential of identifying unexplored key regulators by the systemic method used in this work. Further analyses aim at expanding candidate genes for screening biomarkers for IS, and experimental validation is required on identified potential hubs.
... In order to profile the cell-types present within the normal and TNBC organoids, we used a combination of flow cytometry and single cell RNA-seq (scRNAseq). We assayed for mammary epithelial lineages using EPCAM and CD49f as luminal and basal cell markers respectively (Dekkers et al., 2020;Lim et al., 2009;Raouf et al., 2008;Rosenbluth et al., 2020). As previously shown (Rosenbluth et al., 2020), the normal derived organoids recapitulate the EPCAM+ luminal lineage, EPCAM+/CD49f+ luminal progenitor cells and EPCAM-/CD49f+ basal cell lineages (NM07NL in Fig 6A). ...
Preprint
Full-text available
Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with poor patient outcomes, and an unmet clinical need for targeted therapies and better model systems. Here, we developed and comprehensively characterized a diverse biobank of normal and breast cancer patient-derived organoids (PDOs) with a focus on TNBCs. PDOs recapitulated patient tumor intrinsic properties and a subset of PDOs can be propagated for long-term culture (LT-TNBCs). Single cell profiling of PDOs identified cell types and gene candidates affiliated with different aspects of cancer progression. The LT-TNBC organoids exhibit signatures of aggressive MYC-driven basal-like breast cancers and are largely comprised of luminal progenitor (LP)-like cells. The TNBC LP-like cells are distinct from normal LPs and exhibit hyperactivation of NOTCH and MYC signaling. Overall, our study validates TNBC PDOs as robust models for understanding breast cancer biology and progression, paving the way for personalized medicine and better treatment options.
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Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions1–4. How these metabolic features are established remains unclear. Here we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of new organelles is proteomically immature and they respire less. After cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells that inherit old mitochondria. Cells that inherit newly synthesized mitochondria with low levels of Rieske iron–sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria. Döhla et al. show that selectively and asymmetrically inherited mitochondria impose a metabolic bias on progeny in mammary stem-like cells that alters the balance between stem cell self-renewal and differentiation.
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BRCA1 germline mutation carriers are predisposed to breast cancers. Epigenomic regulations have been known to strongly interact with genetic variations and potentially mediate biochemical cascades involved in tumorigenesis. Due to the cell-type specificity of epigenomic features, profiling of individual cell types is critical for understanding the molecular events in various cellular compartments within complex breast tissue. Here, we produced cell-type-specific profiles of genome-wide histone modifications including H3K27ac and H3K4me3 in basal, luminal progenitor, mature luminal and stromal cells extracted from a small pilot cohort of pre-cancer BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+), using a low-input ChIP-seq technology that we developed. We discovered that basal and stromal cells present the most extensive epigenomic differences between mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+), while luminal progenitor and mature luminal cells are relatively unchanged with the mutation. Furthermore, the epigenomic changes in basal cells due to BRCA1 mutation appear to facilitate their transformation into luminal progenitor cells. Taken together, epigenomic regulation plays an important role in the case of BRCA1 mutation for shaping the molecular landscape that facilitates tumorigenesis.
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Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and differentiation of progenitor cells along a particular lineage. Moreover, depending on the cellular and developmental context, the Notch pathway acts as a regulator of cell survival and cell proliferation. Abnormal expression of Notch receptors has been found in different types of epithelial metaplastic lesions and neoplastic lesions, suggesting that Notch may act as a proto-oncogene. The vertebrate Notch1 and Notch4 homologs are involved in normal development of the mammary gland, and mutated forms of these genes are associated with development of mouse mammary tumors. In order to determine the role of Notch signaling in mammary cell-fate determination, we have utilized a newly described in vitro system in which mammary stem/progenitor cells can be cultured in suspension as nonadherent 'mammospheres'. Notch signaling was activated using exogenous ligands, or was inhibited using previously characterized Notch signaling antagonists. Utilizing this system, we demonstrate that Notch signaling can act on mammary stem cells to promote self-renewal and on early progenitor cells to promote their proliferation, as demonstrated by a 10-fold increase in secondary mammosphere formation upon addition of a Notch-activating DSL peptide. In addition to acting on stem cells, Notch signaling is also able to act on multipotent progenitor cells, facilitating myoepithelial lineage-specific commitment and proliferation. Stimulation of this pathway also promotes branching morphogenesis in three-dimensional Matrigel cultures. These effects are completely inhibited by a Notch4 blocking antibody or a gamma secretase inhibitor that blocks Notch processing. In contrast to the effects of Notch signaling on mammary stem/progenitor cells, modulation of this pathway has no discernable effect on fully committed, differentiated, mammary epithelial cells. These studies suggest that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells, affecting self-renewal and lineage-specific differentiation. Based on these findings we propose that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells.
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We have compared the ability of two mammalian Notch homologs, mouse Notch1 and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.
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