Original Article
Expression Pattern of the Human ABC
Transporters in Pluripotent Embryonic Stem
Cells and in Their Derivatives
Zsuzsa Erdei,1
Reka L}orincz,2
Kornelia Szebenyi,1
Adrienn Pentek,1
Nora Varga,1
Istvan Liko,3
Gy€orgy Varady,1,2
Gergely Szakacs,4
Tamas I. Orban,1,5
Balazs Sarkadi,1,2
and Agota Apati1,2
*
1
Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of
Sciences, Budapest, Hungary
2
Molecular Biophysics Research Group of the Hungarian Academy of Sciences, Semmelweis University and
National Blood Service, Budapest, Hungary
3
Pharmacology and Drug Safety Research, Gedeon Richter Plc, Budapest, Hungary
4
Institute of Enzimology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest,
Hungary
5
Chemical Technology Transfer Ltd., Budapest, Hungary
Background: ATP-binding cassette (ABC) transporters have key roles in various physiological functions
as well as providing chemical defense and stress tolerance in human tissues. In this study, we have
examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs)
and in their differentiated progenies. We paid special attention to the cellular expression and localization
of multidrug transporter ABC proteins.
Methods: Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized
cell types were separated mechanically. Cellular features regarding pluripotency and tissue
identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy, and
qPCR-based low-density arrays.
Results: Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells, and mesenchymal
stem cells) were distinguished by morphology, immunostaining markers, and selected
mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins
also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug
and lipid transporter ABC protein expression was examined by using well characterized specific
antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded
well to the mRNA expression results. Moreover, the cellular localization of these important human
ABC transporter proteins could be established in the pluripotent and differentiated hESC derived
samples.
Conclusions: These studies provide valuable information regarding ABC protein expression in human
stem cells and their differentiated offspring. The results may also help to obtain further information concerning
the specialized cellular functions of selected ABC transporters. VC 2014 International Clinical Cytometry
Society
Key words: human pluripotent stem cell; mesenchymal cell; cardiomyocyte; neural cells; ABC transporters;
pluripotency; stem cells; flow cytometry; microscopy
Additional Supporting Information may be found in the online version
of this article.
*Correspondence to: Agota Apati, Institute of Molecular Pharmacology,
Research Centre for Natural Sciences, Hungarian Academy of Sciences,
and National Blood Service, Dioszegi 64, Budapest, 1113,
Hungary. E-mail: apati@biomembrane.hu
Grant sponsor: OTKA; Grant numbers: NK83533, KTIA_AIK_12-1-
2012-0025; KMR_12-1-2012-0112.
Received 2 September 2013; Revised 27 January 2014; Accepted
10 February 2014
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.
com).
DOI: 10.1002/cyto.b.21168
Cytometry Part B (Clinical Cytometry) 00B:00–00 (2014)
VC 2014 International Clinical Cytometry Society
How to cite this article: Erdei Z, L}orincz R, Szebenyi K, Pentek A, Varga N, Liko I, Varady G, Szakacs G, Orban
TI, Sarkadi B, Apati A. Expression Pattern of the Human ABC Transporters in Pluripotent Embryonic Stem Cells
and in Their Derivatives. Cytometry Part B: Clinical Cytometry 2014;00B:000–000.
INTRODUCTION
The ATP-binding cassette (ABC) protein family is present
from bacteria to humans, and the 48 human ABC
proteins include several transporters, channels, as well
as nonmembrane proteins (1). During the past decades
a large amount of information became available about
the expression and function of these human proteins,
while there are still numerous unresolved questions in
this field. We have shortly summarized the recent
knowledge about the human ABC proteins in Supporting
Information Table 1 [see references (9,35-38) cited
therein, and for more information see http://humanabc.
4t.com/main.htm].
Certain ABC transporters form a special network of
chemo-defense system, as these ATP-dependent active
transporters extrude a wide variety of substrates from
the cells, including endo- and xenobiotics. While these
ABC transporter (with a key role of ABCB1/Pgp, ABCC1/
MRP1, and ABCG2/BCRP) proteins play an important
role in protecting our body, they are also involved in
causing multidrug resistance in cancer cells (2,3). One
member of these multidrug transporters, ABCG2, has
been shown to play a major role in the protection of
stem cells against toxic compounds. In addition, the
ABCB6 protein has been indicated to play a major role in
the defense against toxic heme derivatives, while the
ABCA1 protein has a key role in the extrusion of excess
cellular lipid derivatives, especially cholesterol (see
Refs. 4–9).
Since the human embryonic stem cells (hESC) require
special protection during development, and some of the
differentiated tissues are also well protected against toxic
agents, an important task is to follow the changes in the
expression and localization of the ABC multidrug transporters
during these early developmental processes.
In our previous studies we have shown that the
HUES9 embryonic stem cells express the ABCG2 protein
at the cell surface (10), and this transporter has an
important role in defending the HUES9 cells during
stress conditions (5). In the current work we have
examined the full pattern of 48 human ABC protein
mRNA expression levels by qPCR-based microarray, in
order to provide information about changes in their tissue
expression patterns in undifferentiated and selectively
differentiated hES cells. We have also followed the
expression of selected ABC transporter proteins by flow
cytometry and immunostaining during early human cell
differentiation. In addition to provide basic information
about ABC protein expression, these studies also
allowed to examine the cellular localization of these
transporter proteins, which was not exactly known for
embryonic stem cells and their derivates.
MATERIALS AND METHODS
Cell Culture and Differentiation
hES cell lines HUES9, HUES3, and HUES4 (originally
provided by Dr. Douglas Melton, Harvard University)
were maintained on mitotically inactivated mouse
embryonic fibroblasts (MEF) and spontaneous differentiation
were performed via embryoid body (EB) formation
as described previously (11). After 6 days EB were
placed onto gelatin coated 24 well plates, where they
attached to the surface and underwent spontaneous differentiation.
The desired cell types were separated as
follows:
Some of the EB-outgrowths start spontaneously beating
within a few days postplating. These rhythmically
contracting areas were isolated mechanically and hES
cell-derived cardiomyocytes were replated on gelatin
coated 8 well confocal chambers or harvested to RNA
isolation. Neuronal progenitor cells with rosette-like
morphology were mechanically isolated for generation
of hES cell-derived neural cells, between days 8 and 10.
These rosettes were able to reattach onto gelatin coated
surfaces and continue their further differentiation to
mature neural cell types. The RNA isolation and immunostaining
were performed at day 24.
The mesenchymal stem cell like (MSCl) cells were
generated and maintained as described previously
(12). For more details see the Supporting Information
Methods.
Flow Cytometry
Single cell suspensions were prepared by gentle trypsinization,
and the cells were labeled in PBS containing
0.5% bovine serum albumin with appropriate antibodies.
In all hESC samples an anti mouse Sca-1 (Ly-6A/E) (FITC
or PE, BD Pharmingen) antibody was employed for gating
out the positively labeled mouse feeder cells.
We used the following directly labeled anti-human
antibodies: SSEA4-APC and PODXL-PE (R&D Systems) for
investigation of pluripotency. For indirect staining of
ABC transporters the following monoclonal antibodies
were applied: 5D3 (BD Pharmingen) for ABCG2, MRK16
(Alexis Biochemicals) for ABCB1, anti-Lan (OSK43 (generated
and kindly provided by Hideo Takahashi Japanese
Red Cross Osaka Blood Center, Japan) (13) for ABCB6,
R1 (Abcam) for ABCC1 and AB H10 (Abcam) for ABCA1
labeling. The 5D3 labeling was performed in the presence
of Ko143, a specific inhibitor of ABCG2, which
maximizes 5D3 binding (14). Control staining with
appropriate isotype-matched control mAbs was
included. For fixation and permeabilization of the cells
the Fix&Perm (Invitrogen) solution was used, according
to the manufacturer’s instruction. Samples were
2 ERDEI ET AL.
Cytometry Part B: Clinical Cytometry
analyzed by a BD FACSCantoII flow cytometer (Becton
Dickinson Immunocytometry Systems [BDIS]). To ascertain
the specificity of the antibodies we have performed
various studies, including Western blotting in several
cell lines (see Refs. 15–18 and data not shown). For
more details see the Supporting Information Methods.
Immuno-Cytochemical Staining
Immunostaining of all cell types was performed as
described previously (10), except the cell surface
labeling of ABCG2, which was carried out as
described by Ref. 5. For labeling of pluripotent and
differentiation markers the following primary antibodies
were applied: Oct4 (SantaCruz), Nanog, SSEA4,
and PODXL (RnD Systems) cardiac Troponin-I (Sigma),
Nestin (Abcam), b-III Tubulin (RnD Systems), and CD-
44-FITC (BD Pharmigen). The ABC transporters were
studied by the same antibodies used for flow cytometry.
Hoechst33342 (Invitrogen) was used for nuclear
staining. The stained samples were examined by an
Olympus FV500-IX confocal laser scanning microscope.
For more details see the Supporting Information
Methods.
Gene Expression Analysis
Total RNA was isolated from the cells using the Trizol
reagent (Life Technologies). RNA integrity was checked
by standard gel electrophoresis, RNA concentration was
determined by spectrophotometry using a NanoDrop
2000 Spectrophotometer (Thermo Scientific). Gene
expression profiles were determined by analyzing
TaqManVR Low Density Arrays (TLDA cards, Life Technologies)
designed for measuring pluripotency marker
genes (TaqManVR Array Human Stem Cell Pluripotency
Panel, cat. #: 4385344) or ABC transporters (TaqManVR
Array Human ABC Transporter Panel, cat. #: 4378700).
Briefly, 500 ng of total RNA was used to prepare cDNA
samples using the Reverse Transcription System Kit
(Promega), according to the manufacturer’s instruction.
cDNA samples corresponding to 100 ng of total RNA
were combined with 23 TaqManVR Gene Expression
Master Mix and loaded on one channel of the appropriate
TLDA card; real-time PCR reactions were run on a
7900HT System according to the manufacturer’s protocol
(Life Technologies). For data analysis, the DataAs-
sistTM
Software v3.0 (Life Technologies) was used. Gene
expression values were determined by the DDCt method
using multiple endogenous control genes on the TLDA
cards (ACTB and GAPDH genes for the pluripotency
array data and RPLP0 and PPIA genes for the ABC transporter
array data). During further analysis we have
selected and excluded several genes with very low
expression levels (e.g., genes with Ct > 38, such as HBB,
INS, and PAX4 on the pluripotency panel and ABCA6,
ABCB5, ABCC11, and ABCC12 on the ABC transporter
panel). The average linkage clustering method was
applied to analyze the expression data and to calculate
Pearson’s correlation values to define distances of our
samples sets.
RESULTS
Differentiation of hES Cells—Characterization by
Differentiation Markers
For studying the expression of human ABC proteins
in pluripotent cells and in their differentiated derivatives,
we have generated cardiac cells, mesenchymal-like
stem cells [MSCls (12)] and neural cells from the HUES9
cells, by using a method of spontaneous differentiation,
via EB formation (Supporting Information Fig. 1). With
this method the desired cell types could be generated
from hESC without the addition of special chemicals,
and the selection of the differentiated progenies was
performed by enzymatic digestion and/or mechanical
selection, without applying any drug selection or cell
sorting. All the differentiated cell types were cultured in
the same media and under similar conditions (see Materials
and Methods section). These uniform conditions
were used because the expression levels and localization
of several ABC proteins have been shown to be influenced
by chemical inducers, selection drugs or antibiotics
(see Refs. 19,20).
When selecting various tissue types, we performed a
detailed phenotypic characterization of the parental and
differentiated cell types, in order to assure that any further
analysis should be performed by using properly
selected populations. This characterization included the
investigation of several cell type specific markers by
flow-cytometry and immunocytochemistry.
As shown in Figure 1A, by flow cytometry studies we
found that the pluripotent hES cells (HUES9) highly
expressed the SSEA-4 and PODXL pluripotency markers
on the cell surface. Using immunostaining and confocal
microscopy, we could clearly show the presence of
the nuclear pluripotency markers, Oct4, and Nanog
(Fig. 1B).
During differentiation studies, cell surface markers for
hESC-s and hESC derived MSCls were analyzed (Supporting
Information Fig. 2) as described in detail in our previous
work (see Ref. 12). In all cases, immunostaining
revealed proper separation of cells showing positive
staining for representative markers of cardiac (cardiac
troponin-cTNI), neural (neuron specific b-III tubulin and
Nestin), and mesenchymal (CD44) cell types (Fig. 1C).
After this phenotypic selection and verification we
have analyzed the mRNA expression patterns of the
selected cell types derived from HUES9 cells by using a
TLDA pluripotency array. Besides the differentiated cell
types, the common progenies of the differentiated cells
(mesenchymal, cardiac, and neural) from 6 days old EBs
were included in the further investigation. The array
included 36 pluripotency, 19 mesoderm, 13 ectoderm,
17 endoderm, and 4 trophectoderm markers, along with
several housekeeping genes. An overview of these data
(summarized in Supporting Information Table 2) is demonstrated
in a heat-map (Fig. 2A), showing relative gene
expression levels. The cluster analysis (see Methods section),
documented in Figure 2B, shows close correlation
of the mRNA expression pattern with the differentiation
ABC TRANSPORTERS IN PLURIPOTENT HESC 3
Cytometry Part B: Clinical Cytometry
status of the cell samples. As shown, the biological parallels
(for hESC and MSCl) clustered together, while the
undifferentiated hESC samples are clearly separated
from the partially differentiated sample (6 days EB). The
mRNA clusters of the differentiated cell types are noticeably
different from that of the hESCs and from one
another and the biological replicates of the most differentiated
cell types (the MSCl cells, after 80 days of differentiation)
are located away from the undifferentiated
cells with the longest distance in the dendrogram. As
shown by the highest expression levels found in the pluripotency
array data, the hES cells showed high levels of
the pluripotency mRNA markers, while the derivative
cells showed upregulated levels of the proper lineagespecific
markers (Fig. 2C).
ABC Transporter Expression in Pluripotent and
Differentiated hES Cells—Flow Cytometry Studies
In these experiments we investigated the ABC transporter
expression in the pluripotent and differentiated
FIG. 1. Characterization of HUES9 cells and their derivates by following the expression of selected proteins. (A) Investigation of the pluripotent
state of HUES9 cells by flow cytometry. More than 90% of the cells show cell-surface SSEA4 and PODXL expression. M1: cell population with
marker positivity was gated based on the relevant isotype-matched control mAbs. (B) Investigation of the pluripotent state of HUES 9 cells by immunomicroscopy.
HUES 9 cells were grown on MEF feeder cells for two days in eight-well chambers for confocal microscopy. Coculture of HUES9 and
feeder cells were fixed, permeabilized, and stained with the antibodies recognizing the Nanog (red) and Oct4 (green), markers. Nuclei were counterstained
with Hoechst33342 (blue). Nanog and Oct4 transcription factors showed nuclear localization. (C) Investigation of the differentiated forms of
HUES9 cells by immunomicroscopy. HUES9-derived cell types were differentiated as described in Materials and Methods section and were transferred
mechanically into eight-well chambers for confocal microscopy. The samples were fixed, permeabilized, and stained with antibodies recognizing
specific proteins for each cell type; cTNI (green) for cardiac, b-III tubulin (green), and Nestin (red) for neural and CD44 (green) for mesenchymal
cell type. Nuclei were counterstained with Hoechst33342 (blue).
4 ERDEI ET AL.
Cytometry Part B: Clinical Cytometry
cells at the protein level. The application of specific and
properly reactive antibodies is a crucial question in
these experiments. Especially, in the case of the flow
cytometry studies, we used antibodies against extracellular
epitopes to examine the expression and cell surface
localization of ABCG2 (mAb 5D3), ABCB1 (mAb
MRK16), and ABCB6 (mAb OSK43). However, most of
the antibodies recognizing human ABC transporters
have been generated against intracellular epitopes, and
in several experiments we have used such antibodies
(see Methods section). In each case, the specific protein
recognition was assured by examining various cell types
overexpressing these ABC transporters (Supporting
Information Fig. 3).
The two major cell types in which ABC transporter
expression could be studied without major cellular damage
were the undifferentiated hES cells and the MSCs. In
these cell types we focused on the expression of ABC
multidrug or lipid transporters with key roles in cellular
chemodefense and stress response. As shown in Figure 3,
in the case of ABCB1, ABCB6, and ABCG2 we used
the respective antibodies recognizing extracellular epitopes,
while in the case of ABCC1 and ABCA1 we
employed antibodies generated against intracellular protein
domains.
Figure 3A shows flow cytometry detection of five
ABC transporters (ABCB1, ABCB6, ABCG2, ABCC1, and
ABCA1) in HUES9 cells under nonpermeabilized conditions
using antibodies recognizing external epitopes,
while panel B of Figure 3 shows similar measurements
after permeabilization of hESCs. As documented, in all
cases we found a very low level expression in the
FIG. 2. TLDA analysis of differentiation status of HUES9 cells and their differentiated offspring. (A) Heat map representation of human pluripotency
marker gene mRNA expressions in 7 samples of 4 cell types (hESCs a and b, cardiac, neural, and MSCs a and b) and the 6 days old EB culture
(the common progenitors of differentiated cell types). The pluripotency genes are shown on the y-axis, the 7 samples are ordered on the x-axis
with biological replicates (“a” and “b”). The color code ranges from high (dCt 5 22, light red) through medium (black), to low (dCt 5 16, light green)
levels of gene expression relative to selected housekeeping genes. (B) Hierarchical clustering of 7 samples allowing a separation based on differentiation
status of the samples. (C) Genes with the highest expressions values for each cell type show the proper characteristics for a given differentiation
status. The table shows dCt data relative to the geometric mean of housekeeping control genes (ACTB and GAPDH). [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
ABC TRANSPORTERS IN PLURIPOTENT HESC 5
Cytometry Part B: Clinical Cytometry
nonpermeabilized cells except ABCG2, which showed
elevated level of cell surface expression, while a slight
increase in the apparent expression of all these transporters,
especially ABCA1 could be found in the permeabilized
hESCs. Only ABCG2 expression level showed an
apparent reduction under these conditions, showing
that the 5D3 antibody binding was reduced by the
applied fixation-permeabilization. Similar results were
obtained when analyzing HUES3 and HUES4 cells as
documented in Supporting Information Figures 4 and 5.
Figure 4 documents similar studies carried out with
hES-derived MSCls by flow cytometry. In this case the
expression levels of all studied transporters were negligible
without permeabilization (Fig. 4A), but were well
detectable in the permeabilized cells in the case of
ABCB6, ABCC1, and especially of ABCA1. It is important
to note that ABCG2 expression was not detectable in
these cells, with or without permeabilization.
In the case of ABCB1 and ABCB6 (antibodies reacting
with external membrane epitopes) the potential expression
of these proteins in intracellular membrane compartments
may be suggested by these data. Since the
ABCC1 and ABCA1 antibodies recognize intracellular
epitopes, the increased protein levels found in the permeabilized
cells most probably correspond to increased
general expression levels, and in this case the plasma
membrane or intracellular membrane expression levels
cannot be distinguished. Similar results were obtained
when analyzing HUES3 and HUES4 cells as documented
in Supporting Material Figures 6 and 7.
ABC Transporter Expression in Pluripotent and
Differentiated hES Cells—Immunomicroscopy Studies
Flow cytometry examinations could not be performed
with vulnerable cell types such as cardiomyocytes and
neural cells, without injuring the cells. Comparative
examination of the hESC and hESC derived cell types by
immunohistochemistry is thus an additional method to
characterize protein expression and yield more informative
cellular localization data than flow cytometry.
In Figure 5 we summarize the results of the immunostaining
experiments. This figure compiles representative
data for each cell type and for the above described
antibodies recognizing selected ABC transporters. As
shown in Supporting Information Figure 8, we have
examined several selected cell types, overexpressing
these ABC transporters, as positive controls (see also
Materials and Methods section).
As documented in Figure 5, in the case of ABCA1 all
hESC-derived cell types showed well measurable expression,
and in the undifferentiated hESCs and the MSCls,
the expression was homogenously high showing both
plasma membrane and cytosolic localization. In contrast,
only a small number of cardiac and neural cells showed
ABCA1 expression, and this expression was mostly localized
in the cytosol. In the case of ABCB1, we found
measurable expression only in the differentiated cardiac
and neural cell types, which was in agreement with the
flow cytometry data indicating no ABCB1 expression in
the undifferentiated hESCs and the MSCls. The ABCB6
FIG. 3. Flow cytometry analysis of ABC transporter expression in hES cells. Single cell suspensions from HUES9 cells were obtained as described
in Materials and Methods section. Nonviable cells were gated out by 7AAD staining. Monoclonal antibodies specific for ABCB1, ABCB6, ABCG2,
ABCC1, and ABCA1 were used to detect transporter expression (A) in intact cells (without fixation and permeabilization) and (B) in fixed and permeabilized
cells (for details see Materials and Methods section). R1: cell population with marker positivity was gated based on the relevant isotypematched
control mAbs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
6 ERDEI ET AL.
Cytometry Part B: Clinical Cytometry
protein was expressed ubiquitously in all of the examined
cell types, and the expression was found in intracellular
compartments. These data are again in line with
the flow cytometry results, which also indicated intracellular
expression of this protein. For ABCC1, a well measurable
expression could be observed for most of the cell
types, except for the cardiac cells, showing a much
lower protein level. In all cases, ABCC1 expression was
detected in the plasma membrane compartment.
As shown earlier in Figure 3 and Supporting Information
Figures 4 and 5, in the undifferentiated hES cells high level
of ABCG2 protein expression was detected on the cell surface
by flow cytometry and the immunostaining experiments
shown in Figure 5 verified this phenomenon. In
contrast, we could not detect a significant ABCG2 expression
in the differentiated MSCl cell types and some expression
could be detected on cardiac and neural cultures
however this expression was confined to the border of tissues.
In Figure 5, we have also included immunostaining
data for the ABCC6 protein, as this transporter has been
indicated to play an important role in cardiac tissues
(21,22). However, we could not detect ABCC6 expression
in any of the examined hESC-derived cell types.
ABC Protein mRNA Expression in Pluripotent and
Differentiated hES Cells
Because of the lack of reliable antibodies and staining
protocols for numerous human ABC proteins, a systematic
analysis can only be performed for the respective
mRNA expression levels. For this purpose we used a
quantitative RT-PCR technology and ABC protein specific
microarray, and examined the same mRNA samples
as used in the pluripotency array analysis (see Fig. 2),
obtained from pluripotent hESCs and the differentiated
cell types. A visual overview of these gene expression
data is demonstrated in a heat-map (Fig. 6A), while the
dCt data are presented in the Supporting Information
Table 3.
Cluster analysis based on ABC protein expression patterns
is shown in Figure 6B. Remarkably, clustering
based on ABC transporter profiles was found to be similar
to the hierarchy defined by pluripotency markers
(Fig. 2), indicating that all the examined cell types have
characteristic expression patterns (fingerprints) for the
ABC proteins, depending on the cell maturation status.
As shown in Figure 6C, some of the ABC proteins
showed similar (medium) level of expression in all cell
types (Fig. 6C, left panel), although most of these ABC
proteins are not well described transporters, and some
are not even membrane proteins (Supporting Information
Table 1). These data suggest a role of these
ABC proteins in the general maintenance of cellular
homeostasis.
In the following analysis, we focused on the recognized
ABC membrane transporters, and excluded those
proteins, which showed high Ct values, indicating
extremely low or no expression (Fig. 6C, right panel).
FIG. 4. Flow cytometry analysis of ABC transporter expression on MSCl cells. Single cell suspensions from MSCl cells were obtained by gentle trypsinization.
Nonviable cells were gated out by 7AAD staining. Monoclonal antibodies specific for ABCB1, ABCB6, ABCG2, ABCC1, and ABCA1 were
used to detect transporter expression (A) on intact cells (without fixation and permeabilization) and (B) in fixed and permeabilized cells (for details
see Materials and Methods section). R1: cell population with marker positivity was gated based on the relevant isotype-matched control mAbs. [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ABC TRANSPORTERS IN PLURIPOTENT HESC 7
Cytometry Part B: Clinical Cytometry
We compared the relative expression of ABC transporter
mRNAs in differentiated cells to those in undifferentiated
hES cells (Supporting Information Table 4).
As shown in Figure 7 and presented in Supporting
Information Table 4, we found that most of the ABC
transporters were expressed at significantly higher level
(although in different magnitudes, see Fig. 7A and B) in
the differentiated cell types than in the pluripotent
hESCs. When analyzing the cell-type dependent expression,
we found that several ABC transporter mRNA levels
showed large increase in differentiated progenies. These
included the expression of ABCA8, ABCC3, ABCC9, and
ABCG1. In contrast, there were several transporter
mRNA expression levels characteristic for certain cell
types: changes of ABCB1 and ABCC6 expressions were
higher in cardiac cells and ABCA4 was higher in neural
cells as compared to hESC (Fig. 7A). Note, that the only
ABC transporter, which showed a decreased expression
level in all differentiated cell types was ABCG2 (Fig. 7B).
This cell type-specific expression indicates a possible
role of these proteins in the differentiated tissues and
allows a further characterization of their physiological
roles.
DISCUSSION
ABC transporters play an important role in various
physiological functions, maintain cellular homeostasis,
and provide defense mechanism against toxic endo- and
xenobiotics. These ABC transporters work in a general
defense network system, having overlapping functions
and substrate specificity. On the basis of their promiscuous
recognition of toxic agents, they can functionally
substitute each other. While many of the ABC multidrug
transporters also have broad tissue distribution, their
expression levels may provide a tissue-specific
“fingerprint,” corresponding to special metabolic or
other cellular functions, as well as tolerance against
stress and drugs.
In this study, we have examined the protein level and
cellular localization of the major multidrug transporters,
as well as the mRNA expression pattern of all 48 ABC
proteins in pluripotent hESC and in their selected, differentiated
progenies. To investigate the human ABC transporter
expression “fingerprint” in differentiating cells,
we have used and characterized an experimental system
involving four human cell types of isogenic origin. We
have cultured human pluripotent hESCs, and in a
FIG. 5. Immuncytochemical detection of ABC transporters in HUES9 cells and their derivatives. HUES 9 cells were grown on MEF feeder cells for
two days in eight-well chambers and HUES9-derived cell types were differentiated as described in Materials and Methods section and were transferred
mechanically into eight-well chambers for confocal microscopy. The samples were stained with specific antibodies to visualize ABCA1,
ABCB1, ABCB6, ABCC1, ABCG2, and ABCC6 proteins (red) as described in the “Materials and Methods” section. Nuclei were counterstained with
Hoechst33342 (blue). Stained samples were examined by an Olympus FV500-IX confocal laser scanning microscope. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
8 ERDEI ET AL.
Cytometry Part B: Clinical Cytometry
spontaneous differentiation model generated well separable
differentiated cell types, including cardiac cells,
neural cells, and MSC-like cells. The major advantage of
this system is that the hES cells and the differentiated
progenies have the same genetic background and cell
differentiation did not involve major changes in the culturing
conditions.
In these experiments first we examined pluripotency
and tissue-specific marker protein expression in the
selected cell types by flow cytometry and
FIG. 6. TLDA analysis of ABC protein gene expression in HUES9 cells and their differentiated offspring. (A) Heat map representation of human
ABC protein gene expressions in 7 samples of 4 cell types (hESCs a and b, cardiac, neural, and MSCls a and b) and the 6 days old EB culture (the
common progenitors of differentiated cell types). The ABC genes are shown on the y-axis, the 7 samples are ordered on the x-axis with biological
replicates (“a” and “b”). The color code ranges from high (dCt 5 4, light red) through medium (black), to low (dCt 5 14, light green) gene expression
relative to selected housekeeping genes. (B) Hierarchical clustering of 7 samples allowing a separation based on differentiation status of samples.
(C) The listed highest ABC gene expressions (27 < Ct > 32, bold) and lowest gene expressions (Ct > 38, normal) were common for each cell type
showing universal expression pattern for certain ABC proteins. [Color figure can be viewed in the online issue, which is available at wileyonlineli-
brary.com.]
ABC TRANSPORTERS IN PLURIPOTENT HESC 9
Cytometry Part B: Clinical Cytometry
immunostaining. We have also carried out the investigation
of the mRNA expression pattern of a wide range of
pluripotency and differentiation markers by using RTPCR
based quantitative arrays. These studies established
a proper characterization of the respective cell types.
Surprisingly, fibronectin (FN1) showed the highest relative
expression level in all samples; however this is
likely caused by the similar cell culturing conditions.
When looking for ABC protein expression, we found
that the mRNA expression levels of all the 48 human
ABC proteins also clearly distinguished the pluripotent
and the respective differentiated cell types. According to
recent studies, distinct ABC protein patterns were
observed when hESCs (HES2 and HES3) were compared
to hematopoietic stem cells, unrestricted somatic stem
cells and mesenchymal stem cells (23). It has also been
shown that mRNA expression levels are different in
hESCs and hESC-derived hMSCs (24). However, none of
these studies have investigated the ABC transporter
expression at protein level. To perform a detailed characterization,
we have used three hES cell lines (HUES9,
HUES3, and HUES4) and selected several key ABC transporters
involved in multidrug resistance, providing
defense against stress conditions, and modulating cellular
lipid metabolism. These transporters were examined
by using well characterized specific antibodies both in
FIG. 7. Relative gene expressions of ABC transporters in HUES9 derived cell types as compared to undifferentiated cells. (A) The relative gene
expression level values (dCt) of each transporter in HUES9 are set arbitrarily at 1, and the fold differences in expression (ddCt) in other cell types
are presented on the graph. (B) The relative mRNA expression (dCt) of selected transporters in HUES9 cells. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
10 ERDEI ET AL.
Cytometry Part B: Clinical Cytometry
flow cytometry and confocal microscopy experiments.
This way the cellular expression pattern and also the
subcellular localization of these important human ABC
transporters could be established in the pluripotent and
differentiated hES cell derived samples. It is important
to note that the obtained ABC transporter protein
expression data closely corresponded to the respective
mRNA expression results (Fig. 7B). An exception was
ABCC6 expression, which was under the detection limit
of immunostaining, while the cardiac samples showed
relatively high mRNA levels.
During our investigations, we found that the most
prevalent multidrug transporter in the undifferentiated
hES cells, in accordance with previous data, was the
ABCG2/BCRP protein. Several other articles demonstrated
ABCG2 expression at mRNA level in various
human embryonic stem cell lines (23,25–29), while only
one study indicated that human ES cells do not express
ABCG2 (30). Recently, ABCG2 mRNA expression was
confirmed in 10 different embryonic stem cell lines by
Padmanabhan et al., while the ABCG2 protein was under
the detection limit in this work. These discrepancies may
be caused by the different hESC lines studied, the different
culturing conditions, or the protein detection techniques.
This latter can be significantly enhanced by using
the 5D3 monoclonal antibody together with a specific
inhibitor of ABCG2, as described earlier (10,14,31).
According to our data, the ABCG2 protein is expressed
on the cell surface and, although this expression is heterogeneous,
may significantly contribute to the defense
mechanisms in pluripotent stem cells (see Refs.
5,10,23,24,32,33).
Another multidrug transporter found at low levels in
the undifferentiated cells was ABCC1 (on the cell surface),
while we could not detect ABCB1/MDR1 in this
cell type. Two other ABC transporters found to be
expressed in the hES cells were ABCA1, an important
player in lipid/cholesterol extrusion, and ABCB6, a potential
player in protecting against toxic heme derivatives.
Interestingly, this transporter, noted to be also in the
plasma membranes of various cells (16,34), showed only
intracellular expression in all cell types examined here.
In the human ES-derived neural cells we observed high
level expression for ABCB6 and ABCC1, and in certain
cell regions for that of ABCA1 and ABCB1. In the cardiomyocytes
high level expression of ABCB6 and lower levels
for ABCB1 and ABCA1 were observed. Interestingly, certain
cells in the external regions of cardiac and neural tissue
samples also showed ABCG2 expression. In the MSCs
a predominant, but mostly intracellular expression was
observed for ABCA1, ABCB6, and ABCC1, while there
was no measurable expression for ABCB1 or ABCG2. As
of note, we did not find any significant ABCC6 expression
at protein level in any of the cell types examined here.
As a summary, these studies may provide important
information for a selective ABC protein expression pattern
in human stem cells and in their differentiated offsprings.
However, further studies are needed for the establishment
of specialized intertissue and interorgan distribution, as
well as for the cellular functions of the key human ABC
transporters during early human development.
ACKNOWLEDGMENTS
The authors are indebted to Beata Haraszti for excellent
technical assistance, Andras Varadi, and Laszlo
Homolya for providing positive control cell lines and
antibodies and Dr. Anna Brozik for useful suggestions.
The authors appreciate the gift of HUES9 cell line by Dr.
Douglas Melton, HHMI.
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