PLoS ONEplosplosonePLoS ONE1932-6203Public Library of ScienceSan Francisco, USAPONE-D-11-0176210.1371/journal.pone.0019386Research ArticleBiologyDevelopmental biologyEmbryologyFertilizationStem cellsProteomicsDevelopmental BiologyBiochemistryProteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface
Proteins on Human Embryonic Stem Cells and SpermsCommon Surface Protein Pattern of hESs and
hSpermsGuBin1ZhangJiarong1WuYing2ZhangXinzong2TanZhou1¤LinYuanji1HuangXiao3ChenLiangbiao4YaoKangshou2*ZhangMing1*The Institute of Genetics, College of Life
Sciences, Zhejiang University, Hangzhou, ChinaZhejiang Institute of Planned Parenthood
Research and Zhejiang Human Sperm Bank, Hangzhou, ChinaThe Institute of Cell and Developmental
Biology, College of Life Sciences, Zhejiang University, Hangzhou,
ChinaThe Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Beijing, ChinaBrivanlouAli HemmatiEditorRockefeller University, United States of Amercia* E-mail: zhangming_ls@zju.edu.cn (MZ); ksyao@163.net (KY)
Conceived and designed the experiments: BG LC KY MZ. Performed the
experiments: BG JZ YW XZ ZT YL. Analyzed the data: BG JZ XH LC KY MZ.
Contributed reagents/materials/analysis tools: JZ YW XZ ZT KY. Wrote the
paper: BG LC MZ.
Current address: Institute of Developmental and Regenerative Biology, College
of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou,
China
The authors have declared that no competing interests exist.
201135201165e19386201201128320112011Gu et alThis is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and
source are credited.Background
It has long been proposed that early embryos and reproductive organs exhibit
similar gene expression profiles. However, whether this similarity is
propagated to the protein level remains largely unknown. We have previously
characterised the promiscuous expression pattern of cell surface proteins on
mouse embryonic stem (mES) cells. As cell surface proteins also play
critical functions in human embryonic stem (hES) cells and germ cells, it is
important to reveal whether a promiscuous pattern of cell surface proteins
also exists for these cells.
Methods and Principal Findings
Surface proteins of hES cells and human mature sperms (hSperms) were purified
by biotin labelling and subjected to proteomic analyses. More than 1000
transmembrane or secreted cell surface proteins were identified on the two
cell types, respectively. Proteins from both cell types covered a large
variety of functional categories including signal transduction, adhesion and
transporting. Moreover, both cell types promiscuously expressed a wide
variety of tissue specific surface proteins, and some surface proteins were
heterogeneously expressed.
Conclusions/Significance
Our findings indicate that the promiscuous expression of functional and
tissue specific cell surface proteins may be a common pattern in embryonic
stem cells and germ cells. The conservation of gene expression patterns
between early embryonic cells and reproductive cells is propagated to the
protein level. These results have deep implications for the cell surface
signature characterisation of pluripotent stem cells and germ cells and may
lead the way to a new area of study, i.e., the functional significance of
promiscuous gene expression in pluripotent and germ cells.
This work was supported by grants from the Ministry of Science and Technology of
the People's Republic of China and the National Key Scientific Research
Program of China (2007947804). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.Introduction
At the beginning of life, terminally differentiated germ cells fuse to generate a
totipotent stem cell, the fertilised egg. After a series of cleavages, the last stem
cell type that can form any cell type, pluripotent stem cells, forms at the
blastocyst stage [1], [2]. A small group of pluripotent stem cells, the germline
stem cells, are set aside at this stage and will ultimately derive the germ cells of
the next generation and sustain the life of the species[3], [4]. Therefore, the terminally
differentiated germ cells and highly plastic pluripotent stem cells are two critical
points in the circle of life. The relationship between these two cell types,
distinct from the point of view of differentiation potential, is a basic question of
life science.
It has been postulated that pluripotent stem cells have similar gene expression
profiles compared to germ cells [5]. For example, many transcription factors that are critical
for pluripotency maintenance like OCT4 and DPPA3 are also expressed through
primordial germ cells to mature gametes [6]. A distinctive characteristic of
gene expression profiles is that the promiscuous expression of functional and tissue
specific genes is not supposed to exist in pluripotent and reproductive cells [7], [8]. However, this
characteristic has largely been demonstrated at the mRNA level [5], [7], [9], [10]. As pluripotent stem cells and
germline stem cells have loose chromatin structures and/or express transcription
factors that promote promiscuous gene expression, such as Aire, promiscuous gene
expression may be leaky expression and never lead to the translation of functional
proteins [11],
[12], [13], [14], [15], [16]. Determining
whether pluripotent stem cells and germ cells have similar promiscuous expression at
the protein level is important for the establishment of a functional relationship
between pluripotent stem cells and germ cells.
Cell surface proteins exercise critical functions in both pluripotent stem cells and
germ cells [17],
[18]. Our previous
study showed that mES cells, pluripotent stem cells derived from mouse blastocyst
inner cells mass, promiscuously express a large variety of functional and tissue
specific cell surface proteins through proteomic methods [19]. We also demonstrated that hES
cells, pluripotent stem cells derived from human blastocyst inner cell masses,
express some tissue specific surface proteins [19]. Whether the cell surface proteome
of hES cells have a similar promiscuous characteristic compared to mES cells and
whether this similarity extends to human germ cells are important questions.
In this study, we used an earlier described biotin-labelling coupled streptavidin
affinity purification method and purified cell surface proteins from hES cells and
normal mature human sperm. More than 1000 surface proteins were identified from both
cell types by LC-MS/MS analysis. A bioinformatic analysis showed that hES and hSperm
both promiscuously expressed diverse functional and tissue specific cell surface
proteins. Comparative analyses indicated that mES, hES and hSperm cells show a
similar surface proteomic pattern. Our results indicate that promiscuous gene
expression might be a conserved property of pluripotent stem cells and germ cells
and its functional significance deserve further study.
ResultsProteomic analyses of cell surface proteins on hES cells and hSperm
To explore the expression patterns of hES and hSperm surface proteins, we
purified cell surface proteins from these cell types by biotin labelling and
identified the proteins by LC-MS/MS. Before labelling, the quality of hES cells
and hSperm was evaluated. As shown in Fig. 1A, B and C, the hES cells used in this
study grew with a typical flattened colony morphology and homogeneously
expressed alkaline phosphatase (ALP), NANOG and SSEA3 [20]. Moreover, we mechanically
isolated hES cells with an undifferentiated morphology for proteomic study.
Therefore, most hES cells used in this study were undifferentiated. hES cell
surface proteins were labelled with membrane-impermeable biotin reagents.
Labelling efficiency was monitored by streptavidin-FITC staining. As shown in
Fig. 1D, most cells were
labelled with biotin on the cell surface, although some intracellular labelling
was observed, which can be explained by the staining of apoptotic cells that is
common in hES populations. As shown in Fig. 2A, the sperm cells displayed a normal
morphology, and the swim-up technique efficiently enriched our sample for motile
sperm cells (Fig. 2B). The
surface proteins of the hSperm were then labelled with membrane-impermeable
biotin reagents, and the labelling efficiency was monitored by streptavidin-FITC
staining. As shown in Fig.
2C, the majority of the biotin signal was located on the cell
surface.
10.1371/journal.pone.0019386.g001
Labelling of hES cell surface proteins.
A. hES cells exhibit a typical flattened colony morphology. B. hES cells
express alkaline phosphatase (ALP). C. ICC staining shows that hES cells
express the pluripotency markers NANOG and SSEA-3. D. Biotin labelling
of hES cell surface proteins. Streptavidin-FITC staining shows that the
biotin was labelled on the cell surface proteins.
10.1371/journal.pone.0019386.g002
Labelling of hSperm cell surface proteins.
A. Sperm showed a normal morphology after a modified Papanicolaou stain.
B. Sperm quantifications used in this study. As shown here, swim-up
efficiently enriched motile sperm to more than 85%. C. Biotin
labelling of sperm cell surface proteins. Streptavidin-FITC staining
showed that most of the biotin was labelled on the cell surface
protein.
The biotin-labelled proteins were resolved by SDS-PAGE and analysed by LC-MS/MS.
On hES cells, 5405 proteins were identified, and 3468 proteins were identified
on hSperm. The transmembrane structure and signal peptides were predicted using
SOSUI software [21]. Proteins annotated as ‘membrane’ in
UniProt Database or those predicted to contain transmembrane domains or signal
peptides were annotated as general membrane proteins. As shown in Figs. 3A and 3B, about
50% of the proteins identified on both cell types are general membrane
proteins, which is consistent with other reports that used these same
methods[22].
Transmembrane proteins and secreted proteins were annotated as cell surface
proteins for further analysis. To this end, 1560 and 1019 cell surface proteins
were identified on hES and hSperm cells, respectively (Tables S1,
S2).
We first evaluated the expression of 400 randomly selected surface proteins by
RT-PCR on hES cells, and 328 of them were confirmed to be expressed. Therefore,
our results should be at least 82% accurate when considering hES cells.
As we performed protein purification and identification under the same
experimental conditions to characterise hSperm, the accuracy should be similar.
A direct comparison of protein identifiers yielded 487 identical surface
proteins between the two cell types (Fig. 3C). It indicated that from the point of
view of the exact protein identity about half of the hSperm surface proteins
were identical to the hES cells.
10.1371/journal.pone.0019386.g003
Proteomic identification of hES and hSperm proteins.
A. Subcellular distribution of hES proteins. B. Subcellular distribution
of hSperm proteins. C. Cross comparison of hES and hSperm cell surface
proteins.
Thereafter, we performed gene ontology analyses according to the Molecular
Function annotations using DAVID software [23], [24]. As shown in Figs. 4A and 4B, the cell
surface proteins of hES and hSperm cells performed wide varieties of molecular
functions, and each functional category included many functional surface
proteins. The three functional categories that included the largest fraction of
cell surface proteins in hES and hSperm were both ‘transmembrane
transporter activity’, ‘signal transduction activity’ and
‘ion binding’, and the general distribution of cell surface protein
functions was similar. These data indicate that the cell surface proteins of hES
cells and hSperm possess a common functional pattern.
10.1371/journal.pone.0019386.g004
Functional categorisation of hES and hSperm cell surface
proteins.
A. Functional categorisation of hES cell surface proteins. B. Functional
categorisation of hSperm cell surface proteins.
ES cells and sperm both express diverse signal molecules
Signal ligands and receptors play critical roles in the self-renewal and
differentiation of ES cells, and they also play critical roles in sperm function
[25], [26], [27].
Consistently, surface proteins annotated to be ‘signal transducers’
are significantly enriched in both hES cells and hSperm (By Molecule Function
enrichment study by DAVID, Data not shown). As we have previously shown that mES
cells express signal receptors and ligands from 48 different signalling
pathways, we surveyed hES and hSperm cell data for signalling receptors and
ligands from these pathways and compared the results with mES[19]. As shown in
Table 1, except for
the AXL signal pathway and the vomeronasal receptors, receptors and ligands from
all these signal pathways were present on the cell surfaces of hES cells and
hSperm. As no obvious ortholog of the vomeronasal organ is present in humans, it
is reasonable that no vomeronasal receptors are present on human cells. Among
these signal pathways, some including the Wnt, FGF, TGF/Activin, Notch,
natriuretic peptide and EGF pathways have been characterised as functional in
hES cells and sperm [25], [26], [27], [28], [29], [30].
However, functions of most other signal pathways like olfactory receptor
pathways, semaphorin pathways, the Slit signal pathway and the TRP channel
pathway on hES and hSperm cells remain to be characterised. These data indicate
that mES cells, hES cells and hSperm cells also possess much more versatile
signal transforming abilities than ever thought.
10.1371/journal.pone.0019386.t001
Comparison of signal pathways on mES, hES and hSperm cells.
signal pathway
mES
hES
hSperm
Acetylcholine
+
+
+
angiopoietin
+
+
+
AXL
+
-
-
BMP
+
+
+
cannabinoid
+
+
+
chemokine
+
+
+
cholecystokinin
+
+
+
Cytokine
+
+
+
EGF
+
+
+
Eph
+
+
+
FGF
+
+
+
Flt
+
+
+
GABA
+
+
+
GDF
+
+
+
Glutamate
+
+
+
Glycine
+
+
+
Orphan GPCR
+
+
+
growth hormone
+
+
+
hedgehog
+
+
+
HGF
+
+
+
hormone
+
+
+
IGF
+
+
+
Insulin
+
+
+
interferon
+
+
+
interleukin
+
+
+
LIF
+
+
+
LPA
+
+
+
natriuretic peptide
+
+
+
netrin
+
+
+
neuropeptide
+
+
+
Neurotrophic factor
+
+
+
Nogo
+
+
+
Notch
+
+
+
olfactory
+
+
+
PCP
+
+
+
progestin
+
+
+
prolactin
+
+
+
prostaglandin
+
+
+
PTPR
+
+
+
relaxin
+
+
+
semaphorin
+
+
+
Sphingosine
+
+
+
Slit
+
+
+
Taste
+
+
+
TGF/Activin
+
+
+
TNF
+
+
+
Toll like receptor
+
+
+
TRP Channels
+
+
+
vomeronasal
+
+
+
Wnt
+
+
+
Besides proteomic characterisations, we also examined the expression of some
signalling molecules in situ by immunocytochemistry (ICC) and
flow cytometry. As hES cells are vulnerable during single cell separation, we
reasoned that flow cytometry analysis might introduce some artefacts considering
the expression pattern of signal molecules[31]. In order to show that
undifferentiated hES cells in the highly compacted colonies expressed the
signalling molecules, we costained the signalling molecules with OCT4 and
examined the staining samples under high magnificent microscope(1000X). As shown
in Fig. 5A, hES cells
expressed BMP2, EGFR and GM-CSFRa at the protein level. Co-staining of the
signal molecules with the pluripotent marker OCT4 demonstrated that the
signalling molecules were expressed on undifferentiated hES cells. ICC staining
also showed that the staining strength of the signalling molecules varied among
OCT4 positive cells, which indicates that hES cells heterogeneously express cell
surface signalling molecules. Moreover, it is also shown that the signalling
molecules were not homogeneously expressed on the cell surface of hES cells, but
formed foci like structures, which might indicate the existence of subcellular
functional complexes. For hSperm cells, we examined the expression of signalling
molecules by flow cytometry. As shown in Fig. 5B, hSperm expressed EGFR, GM-CSFRa and
c-Kit receptors. However, only a subset of hSperm strongly expressed these
receptors. These results indicate that hSperm heterogeneously express these cell
surface signalling molecules. As we have previously described that mES cells
globally express signal molecules, the global expression of signal molecules
might be a common characteristic of ES cells and sperms.
10.1371/journal.pone.0019386.g005
Signal molecules on hES and hSperm cells.
A. Immunocytochemistry staining showed that hES cells expressed BMP2,
EGFR and GM-CSFRα. First panel from the left, DAPI staining. Second
panel, ICC staining of cell surface proteins on hES cells. Third panel,
ICC staining of OCT4 on hES cells. Fourth panel, merge of surface
proteins and OCT4 staining, bars indicate 50 µm. B. Flow cytometry
analysis showed that hSperms heterogeneously expressed BMPR2, EGFR and
GM-CSFRα.
hES cells and hSperm express diverse tissue specific cell surface
proteins
It has been reported that hES cells promiscuously express tissue specific genes
at the mRNA level [9]. It has also been shown that hES cells and mouse
spermatogonial cells express the core regulator of promiscuous expression of
tissue specific genes in medullary thymic epithelial cells, the Aire gene [8], [15]. Therefore, it
is interesting to examine whether hES cells and hSperm promiscuously express
tissue specific cell surface proteins. To this end, we analysed the tissue
specificity of cell surface proteins from hES and hSperm cells according to
UniProt tissue specificity annotations using DAVID software. To our surprise, of
the 1560 hES cell surface proteins, 1441 were annotated as tissue specific. Of
the 1019 hSperm cell surface proteins, 958 were annotated as tissue specific. As
shown in Figs. 6A and B,
both hES cells and hSperm express a large variety of tissue specific cell
surface proteins. Brain specific surface proteins predominated the cell surface
proteins of both hES and hSperm cells, which may indicate a common gene
expression pattern between immunoprivileged entities like the brain and early
embryo. Both hES and hSperm cells also express a large variety of liver specific
genes. As many of these proteins are involved in de novo
synthesis processes, this may indicate some extension of the self-sustenance of
ES cells and germ cells. A significant difference between hES cells and hSperms
is that hES cells express diverse placenta-specific cell surface proteins while
hSperms do not. This might be a consequence of hES cells having the potential to
derive extraembryonic tissue including placenta while hSperms do not. Besides
these predominate tissues, both hES and hSperm cells also expressed tissue
specific proteins of several other tissues and distribution is fairly even. We
also compared data from hES and hSperm cells to data from previously obtained
mouse ES cell surface proteins. The results indicate that brain and liver
specific cell surface proteins predominated all three cell types and that all
three cell types expressed tissue specific cell surface proteins from many
tissues. These results further indicate an interspecies conservation of the
expression of tissue specific cell surface proteins in embryonic stem cells and
germ cells.
10.1371/journal.pone.0019386.g006
Tissue specificity of hES and hSperm cell surface proteins.
A. hES cells expressed tissue specific cell surface proteins of a wide
variety of tissue types. B. hSperm cells expressed tissue specific cell
surface proteins of a wide variety of tissue types.
Besides proteomic analyses, we also examined the expression of tissue specific
cell surface proteins in hES cells and sperm in situ by ICC and
FC. As shown in Fig. 7A, hES
cells express hematopoietic tissue specific surface protein CD34, liver specific
surface protein PAI3 and endothelium specific surface protein TIE1. Co-staining
with the pluripotent marker OCT4 demonstrated that tissue specific cell surface
proteins were expressed on undifferentiated hES cells. The results also showed
that the staining strength of tissue specific surface proteins among OCT4
positive hES cells varied, which indicates that the hES cells heterogeneously
express tissue specific cell surface proteins like mES cells. Then, we analysed
the expression of tissue specific cell surface proteins on hSperm by flow
cytometry. As shown in Fig.
7B, hSperm heterogeneously express T-cell specific surface protein
CD4, melanocyte specific surface protein CD146 and endothelium specific protein
TIE1. These results indicate that the global expression of tissue specific cell
surface proteins might be a common characteristic of ES cells and sperms.
10.1371/journal.pone.0019386.g007
Tissue specific cell surface proteins expressed on hES and
hSperm.
A. Immunocytochemistry staining showed that hES cells expressed CD34,
PAI3 and TIE1. First panel from the left, DAPI staining. Second panel,
ICC staining of cell surface proteins on hES cells. Third panel, ICC
staining of OCT4 on hES cells. Fourth panel, merge of surface proteins
and OCT4 staining, bars indicate 50 µm B. Flow cytometry analysis
showed that hSperms heterogeneously expressed CD4, CD146 and TIE1.
DiscussionA common pattern of promiscuous expression of cell surface proteins on ES
cells and germ cells
It is known that pluripotent stem cells from different species employ a similar
core transcriptional circuit that consists of Oct4, Sox2 and Nanog to sustain
pluripotent identity [17], [32], [33]. It is also known that germline cells from different
developmental stages express some pluripotent specific transcription factors
including OCT4 and DPPA3[6], [34], [35], [36], [37]. It has recently been proposed that pluripotent
embryonic and pluripotent germline stem cells possess an open chromatin
structure, and many functional and tissue specific genes in the genome are
poised for expression [14]. Since we previously demonstrated that mES cells
promiscuously express a large variety of functional and tissue specific cell
surface proteins at the protein level [19], it is interesting to ask
whether this promiscuous pattern is conserved between pluripotent stem cells and
germ cells from different species. Some previous studies using whole cell
proteomics have indicated that mouse multipotent germline stem cells have
similar proteomic patterns compared to pluripotent stem cells [38], [39]. However,
whether this similarity also exists in humans, whether it is propagated to
differentiated gametes and whether it exists for cell surface proteins are
important questions. Here, we demonstrate that like mES cells, hES cells and
hSperm promiscuously express functional and tissue specific cell surface
proteins in a heterogeneous manner. These results indicate that the similarity
of the transcription regulating network and the epigenetic characteristics
between pluripotent stem cells and germ cells are translated to a similar
surface protein pattern.
Complex signal network controls the behaviour of pluripotent stem cells and
germ cells
Some signal pathways have been demonstrated to play critical functions in
pluripotent stem cells and germ cells [26], [40], [41], [42]. However, our results
indicate that both pluripotent stem cells and germ cells express a large variety
of signal receptors and ligands of different signal pathways heterogeneously at
the protein level. Many of these have never been reported to function in these
cells types. These results indicate that the behaviour of pluripotent stem cells
and germ cells might be regulated by much more complex signalling networks than
previously thought, and the interaction between different subpopulations of
pluripotent stem cells and germ cells might be important. The heterogeneous
expression of cell surface proteins on hSperm cells might especially contribute
to the competition of sperm for fertilisation.
Implications into the differentiation potency determination of stem
cells
What determines the differentiation potency of different stem cell types is a
basic question in the biological science [43]. Previously, scientists
preferred a model that defined transcription circuits consisting of a small
number of stem cell type specific transcription factors that determined and
maintained differentiation potency [17], [32], [43]. However, recent studies have indicated that some
stem cell types express genes thought to be specific to their putative
differentiation descendants. Two examples are embryonic stem cells and
hematopoietic stem cells. It has been shown that both human and mouse
pluripotent stem cells promiscuously express many tissue specific genes at low
levels [7],
[9], [10]. It has also
been shown that many genes specific to differentiated hematopoietic lineages are
expressed in hematopoietic stem cells [44]. Therefore, it is
hypothesised that the extent of gene expression plasticity may contribute to the
differentiation potency determination and maintenance of stem cells [14], [44]. Our
results that both embryonic stem cells and sperm promiscuously express
functional and tissue specific cell surface proteins add several important lines
of evidence to this hypothesis. First, as sperm are generally transcriptionally
inert, it is reasonable to infer that sperm may inherit their promiscuous
expression of cell surface proteins from their progeny with a plastic
differentiation potential[45]. Therefore it's rational to imply that besides
pluripotent embryonic stem cells, pluripotent germline stem cells may also
promiscuously express cell surface proteins. This indicates that promiscuous
expression may be a characteristic not restricted to pluripotent embryonic stem
cells but also present in other pluripotent stem cells like germline stem cells.
Second, as cell surface proteins are the major mediator of extracellular stimuli
that affect cells, the versatile expression of cell surface proteins may endow
stem cells the ability to differentiate in response to diverse stimuli during
developmental or regeneration processes.
Implications to the cell surface signature of pluripotent stem cells and germ
cells
Cell surface markers and signatures are important for the identity
characterisation of pluripotent stem cells and germ cells [46]. There have been many
efforts to identify specific markers for pluripotent stem cells and germ cells.
For example, the SSEA antigens, Tra antigens and some other cell surface
proteins like Podocalyxin-like have been thought to be specific markers for
pluripotent stem cells and germline cells [47], [48], [49]. However, most of these
markers have been demonstrated to not be strictly specific for pluripotent stem
cells and germ cells [50], [51], [52]. Our results indicate that a conserved promiscuous
cell surface protein signature, rather than the expression of any specific
markers, may mark the identity of pluripotent stem cells and germ cells.
Therefore, a global view may be more important to identify pluripotent stem
cells and germ cells than some specific markers.
Materials and MethodsEthnical Statements
All the semen specimen donors signed a written Informed Consent Form approved by
the Ministry of Health (P.R. China) for the donation of semen for scientific
research use. The experiments involving semen donors and semen samples in this
article have been conducted according to the principles expressed in the
Declaration of Helsinki and have been approved by the review board of the
Zhejiang Institute of Planned Parenthood Research & Zhejiang Human Sperm
Bank (Hangzhou, China).
Cell lines and cell culture
Gamma irradiation inactivated mouse embryonic fibroblast (MEF) feeder cells
isolated from the embryos of ICR mice at gestational day 13.5 were purchased
from Invitrogen (Carlsbad, CA). MEFs were thawed in DMEM supplemented with
10% foetal bovine serum (Invitrogen) at 37°C and plated at a density
of 4×104 cells/cm2 for ES culture.
Human embryonic stem cells HUES3 were provided by Harvard University (Cambridge,
MA) and cultured on gamma irradiation inactivated MEFs in Knockout DMEM
supplemented with 20% KOSR (Invitrogen) and 1000 ng/ml bFGF (Millipore)
at 37°C in a 5% CO2 atmosphere [46]. The pluripotency of the
hES cells was routinely analysed using ALP staining (Sigma), SSEA-3 staining and
teratoma formation. In addition, the karyotype was routinely checked.
Semen sample collection and processing
Semen specimens were obtained from five donors 22–32 years old with normal
sperm quality. Sperm from each sample were stained by modified Papanicolaou
stain and evaluated manually for normal morphology. After liquefaction, semen
samples were subjected to swim-up as previously described[53]. Briefly, semen samples were
mixed with Quinn's 1023 culture medium at a ratio of 1∶3 and then
centrifuged at 200 g for 10 min. Then, the supernatant was
discarded and 0.75 ml Quinn's 1023 culture medium supplemented with
10% human serum was gently added. The samples were then incubated for 30
min in a 5% CO2 incubator at 37°C to allow motile sperm to
swim-up. The supernatants were collected and pooled together for proteomic
analysis. Sperm motility was analysed using a Hamilton CASA IVOS Integrated
Visual Optical System.
Cell surface protein labelling and affinity purification:
For biotin labelling, hES cells cultured on 50 10 cm tissue culture dishes
pre-seeded with MEF feeders were incubated with 1 mg/ml Sulfo-NHS-SS-Biotin
(Pierce, USA) in PBS for 30 min. Excess biotin was quenched using 10 mM glycine.
Colonies showing undifferentiated morphologies were then mechanically separated
from the culture under a phase contrast microscope. Next, the separated colonies
were lysed by homogenisation in ice cold lysis buffer (50 mM Tris-HCl (pH 7.4),
1% NP-40 substitute (Sigma), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF) using a
dounce homogeniser. The homogenate was placed on ice for 1 h with gentle
vortexing to extract membrane proteins. Then, the homogenate was centrifuged at
12,000 g to remove nuclei, unbroken cells and cell fragments.
The supernatant was mixed with streptavidin-coupled LATEX (300 nm diameter)
beads and vortexed at 4°C for 1 h. Contaminant proteins were excluded by
harsh washing as previously described [22], and purified proteins were
eluted with 100 mM DTT. About 200 µg of membrane proteins could be
purified from a preparation. Labelling efficiency was monitored using
FITC-streptavidin staining.
For biotin labelling of hSperm, 5×107 motile sperm were
incubated with 1 mg/ml Sulfo-NHS-SS-Biotin (Pierce, USA) in PBS for 30 min. Then
the cell surface proteins were purified as hES cells. About 50 µg of
membrane protein could be purified from 5×107 cells. Labelling
efficiency was monitored using FITC-streptavidin staining.
SDS-PAGE
Purified proteins were separated by 12.5% SDS-PAGE. Following
electrophoresis, gels were stained with Coomassie Blue. Gels were then dissected
and subjected to LC-MS/MS analysis.
Enzyme digestion, LC-MS/MS analysis and database searching
Enzyme digestion was performed as previously described [54]. Peptides from each
band were separated on a Paradigm MS4N Nano/Capillary HS MDLC (Michrom
Bioresources, Inc., USA) using a 100 µm ×150 mm C-18 reversed phase
column. LC separation was conducted on a linear gradient of 5–35%
buffer B for 50 min, followed by 35–90% buffer B for 10 min and
90% buffer B for 10 min (buffer A: 0.1% formic acid in a 2%
acetonitrile solution, buffer B: 0.1% formic acid in a 98%
acetonitrile solution) at a flow rate of 500 nl/min. Separated peptides were
then analysed on an LTQ-MS (Thermol, USA) coupled to a Michrome Advanced
nanospray apparatus (Microm). Peak list files were generated using Bioworks
software (Applied Biosystems) using the default parameters. They were searched
against databases for protein identification using the Sequest software. Search
parameters were: for bi or tri valent ions, Xcorr ≥ 2; for monovalent ion,
Xcorr ≥ 1.5; Deltacn ≥ 0.1. Two non-redundant peptides were identified in
each unique protein.
For double staining, hES cells cultured on coverslips pre-seeded with feeder
cells were fixed using 4% paraformaldehyde according to a standard
protocol, blocked with blocking/permeating buffer (PBS with 10% goat
serum and 0.3% Triton X-100) and then incubated with rat anti-human OCT4
monoclonal antibody overnight at 4°C. After washing, cells were incubated
with an Alexa 488-conjugated goat anti-rat antibody for 1 h at 37°C. After
washing, cells were incubated with rabbit polyclonal antibodies against cell
surface molecules for 1 h at 37°C. After washing, cells were incubated with
an Alexa 555-conjugated goat anti-rabbit antibody for 1 h at room temperature
and then observed under a Fluorescent Microscope (Olympus, Japan). For single
staining, cells were fixed using 4% paraformaldehyde according to a
standard protocol, blocked with blocking/permeating buffer (PBS with 10%
goat serum and 0.3% Triton X-100) and then incubated with primary
antibodies for 1 h at 37°C. After washing, cells were incubated with Alexa
488-conjugated secondary antibodies for 1 h at 37°C and then observed under
a fluorescent microscope (Olympus).
Biotin-labelled hES cells and hSperms were fixed with 4% paraformaldehyde
overnight at 4°C and then stained with FITC-conjugated streptavidin (Sigma)
for 30 min to monitor surface labelling.
Flow cytometry
Human sperms were washed with PBS containing 3% FBS. Cells were then
incubated with a primary antibody for 1 h on ice. After thorough washing, cells
were incubated with fluorescent secondary antibodies for 30 min on ice. Cells
were then washed with PBS and analysed by flow cytometry (BDLSR).
RT-PCR
RT-PCR was performed as previously described [15]. Total RNA was extracted using
the Trizol Reagent (Takara, Japan), retro-transcribed and then PCR-amplified.
Primers were designed using the PRIMER PREMIER 5 software.
ALP staining
ALP staining was performed with an ALP assay kit (Sigma).
Bioinformatic analyses
The subcellular localisations of the proteins were annotated according to
Swiss-Prot annotation, SOSUI prediction software and the literature. Proteins
containing transmembrane domains, secreted proteins and proteins annotated as
cell surface proteins by either Swiss-Prot or the literature were all considered
cell surface proteins. A gene ontology (GO) analysis was performed using the
DAVID software and database [23], [24]. Tissue specificity of the surface proteins was
annotated according to UniProt annotations.
Supporting Information
A list of cell surface proteins on hES cells identified in this study.
(DOC)
A list of cell surface proteins on hSperm cells identified in this study.
(DOC)
We thank Dr. Kerry Nugent and Mr. Yixin Zhu from Microm Inc. for their assistance in
mass spectrometry analyses.
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