Proteome analysis of human Wharton's jelly cells during in vitro expansion

Background The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine. Results To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time. Conclusions Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.


Background
Since the first identification of non-hematopoietic stem cells in the bone marrow as colony-forming unit-fibroblasts (CFU-Fs) and the detailed characterization and description of the tri-lineage potential of the mesenchymal stem cells (MSCs), our knowledge of the molecular properties of these cells has made great progress [1,2]. MSCs have a great appeal for tissue engineering and therapeutic applications because of their high in vitro expansion potential, self renewal capacity and multipotentiality [3][4][5]. However, considering the invasive procedure related to their availability, there is an increasing interest in investigating the presence of MSCs in adult and fetal sources and especially their presence in fetal membranes such as umbilical cord matrix [6][7][8][9].
In the umbilical cord two arteries and one vein, surrounded by mucoid connective tissue, called Wharton's jelly are present (figure 1). It contains fibroblast-like cells (WJCs), which show properties similar to the MSCs and may represent a rich source of primitive cells [10,11]. Like bone marrow stromal cells, WJCs are plastic adherent, positively stained for markers of the mesenchymal cells and negatively stained for markers of the hematopoietic lineage [10][11][12][13][14].
WJCs can be expanded longer than MSCs before its multeplicity is compromised [11]. For this reason they could be induced to form different cellular lineages i.e. adipose tissue, bone, cartilage, skeletal muscle cells, cardiomyocyte-like cells and neural cells enabling them to be used in biomedical engineering applications [15][16][17].
The aim of the present work was to build a 2D reference map as a proteomic dataset, useful to define the molecular characteristics of WJCs and also to discover a number of candidate biomarkers specifically expressed in these extra-embryonic stem cells. In addition we also studied the molecular change that occurs during the WJCs in vitro growth in order to find proteins possibly related to their long expansion ability and fast growth in vitro.

Isolation and cell culture
For cell culture institutional review board approval was obtained for all procedures. With the consent of the parents, fresh human umbilical cords were obtained from full-term births, aseptically stored in sterile saline and processed within 6 hours from partum to obtain the umbilical cord matrix mesenchymal stem cells. After removal of blood vessels, the abundant extracellular matrix of Wharton's jelly was scraped off with a scalpel, finely cut and centrifuged at 250 × g for 5 minutes at room temperature and the pellet was washed with serumfree Dulbecco's modified Eagle's medium (DMEM). Next, the cells were treated with collagenase (2 mg/ml) (Sigma) for 16 hours at 37°C, washed in PBS(1X), and treated with 2.5% trypsin for 30 minutes at 37°C under agitation (Wang, Sarugaser). Finally, the cells were washed in PBS(1X) and seeded in complete growth medium (HMSCGM) with growth supplements, all from Cambrex Bio Science (now Lonza, Walkersville, Inc., Walkersville, MD), in 5% CO 2 in a 37°C incubator (ref Sarugaser, Weiss, Mitchell). The cells were cultured with the medium exchange every 3-4 days until reaching the confluence. The adherents cells were detached with 0,05% trypsin-EDTA, counted with Trypan Blue exclusion, and reseeded at 3000 cells/cm 2 to reach the 90% of confluence after 3-4 population doublings.

Cell cycle
5 × 10 5 cells/sample for each passage were fixed by adding 500 μl of 70% cold ethanol and then stored at 4°C. After 24 h cells were washed and stained by adding 500 μl of a solution containing 50 μg/ml propidium iodide (PI) and 200 μg/ml RNase. Cells were incubated for 3 h at 4°C in the dark and acquired on a FACSCalibur flow cytometer using the CellQuest™ software (both from Becton Dickinson, USA). Debris was excluded from the analysis by gating a forward scatter versus side scatter plot. Cell aggregates were excluded by gating FL2 area versus FL2 width. The low flow rate mode (about 400-500 events/ second) was used to record 10,000 non-debris events for each sample. PI fluorescence data were collected using linear amplification. DNA content was assessed, placing the G1 peak around the channel 400. Finally, data were analysed using ModFit LT™ software (Verity Software House, Toshan, ME, USA).

Telomere length assay
DNA extraction was performed using Wizard Genomic DNA Purification Kit (Promega) following the manufacturer's instructions. The length of telomere regions of genomic DNA was assessed on DNA from cells at different passages using the Telo TAGGG kit (Roche) according to the manufacturer's instructions. Appropriate controls, DNA extracted from cells with long or short telomere regions, were also provided with the kit.

Determination of cell senescence
The amount of senescent cells was evaluated at different conditions by the use of the Senescence β-Galactosidase Staining Kit (Abcam, Cambridge, UK) in accordance to the manufacturer's instructions. Thus, the cells at different experimental conditions (2 nd , 4 th , 8 th and 12 th passage) were plated a density of 10,000 cells/cm 2 for 24 h before senescence-associated β-galactosidase staining. After completion of the staining procedures, 5 representative images were taken from diverse areas of each cell culture, using phase-contrast microscopy to asses the number of positive cells.

Proteome characterization
To generate 2D map of WJCs and analyze their protein changes during culture condition we chose four passages (2 nd , 4 th , 8 th and 12 th ) in vitro expansion that were processed for protein extraction. For all culture passages we considered three independent donors and we ran 3 gels for each biological replicate. 16 × 10 6 stem cells of each population were treated with lysis buffer (40 mM Tris pH 7.4, 8 M urea, 4% CHAPS) supplemented with protease inhibitor mixture and 2 mM TBP as reducing agent. Protein concentration was determined by using the Bradford assay with bovine serum albumin (BSA) as standard [18].
The lysated samples were loaded onto commercial 4-7 IPG strip and the second dimension was performed on a 9-16% acrylamide gel. Analytical gels were stained with ammoniacal silver nitrate, while gels used for MALDI-TOF MS protein identification were silver-stained without glutaraldehyde, according to the mass compatible method described by Shina et al. [19,20]. After staining, gels were scanned with a MagicScan scanner (GE Healthcare, formerly Amersham Biotech, Uppsala, Sweden) in transparency mode at 800 dpi and the images were stored as TIFF images. Once digitized, the gel images were analyzed with Image Master 2D platinum software, 6.0 ver-sions (GE Healthcare, formerly Amersham Biotech, Uppsala, Sweden). A positional gel calibration was carried out by using a 2D calibration method included in the analysis package that calculates the position of protein spots in terms of their isoelectric point (pI) and Molecular Weight (MW) values. Several spots, Thioredoxin (pI 5.13-47.09 kDa), Endoplasmin (pI 4.60-90.13 Da), spectrin alpha chain (pI 5.6-118.17 kDa), 75 kDa glucose regulated protein (pI 6.2-74.02 kDa), Glycerol-3-phosphate dehydrogenase (pI6.1 -38.65 kDa), distributed throughout the reference gel and identified by mass spectrometry analysis were chosen as marker proteins. Then a protein list including these marker proteins was created and pI and MW were calibrated in all maps simultaneously by interpolating between the known pI and MW values of the markers. In order to construct a synthetic gel from all conditions included in the present study three different gel runs for each sample were performed and then subjected to image analysis with Image Master 2D evolution software (Ge Healthcare, formerly Amersham Biotech, Uppsala, Sweden). A spot was considered only if it was detected in three out of all experimental replicates for each sample and defined "common spot" (i.e. always present and in the same position on all gels). The quantity of each spot was normalized with respect to the total spot volume detected in the gel. Relative spot volumes were determined by modelling the optical density in individual spot segments using a two-dimensional Gaussian analysis.
Following gel analysis, protein spots on preparative WJCs 2-DE gels were excised and analyzed by the peptide mass finger printing (PMF) approach on a MALDI-TOF MS. Protein spots, once excised from the gel, were washed with 100% ethanol and 100 mM ammonium bicarbonate (NH4HCO3). The pieces of gel corresponding to a single protein were incubated for 60 min at 56°C in a volume of 50 mM NH4HCO3 supplemented with 10 mM DTT and then for 30 min in the dark in 55 mM iodoacetamide in 100 mM NH4HCO3 at room temperature. Finally, the gel was reswollen in 50 mM NH4HCO3 containing trypsin and incubated at 37°C overnight [21]. Peptide extract was applied to a C18ZipTip (Millipore, Bedford, MA, USA), rinsed with a 0.1% TFA and eluted directly on the MALDI target with 0.5 μl of a saturated αcyano-4-hydroxycinnamic acid (1:1 = ACN: 0.1% TFA) solution.
For all MS analysis, PMF was obtained using REFLEX-IV MALDI-TOF (Bruker Daltonics, Germany) in the reflectron operation mode at a potential of 20 kV and a delayed extraction of 400 ns. The instrument was calibrated with external standards such as bradykinin (fragment 1-7) 757,39 m/z, angiotensin II 1046,54 m/z, ACTH (fragment 18-39) 2465,19 m/z, Glu Fibrinopeptide B 1571,57 m/z, and renin substrate tetradecapeptide por-cine 1760,02 m/z. Each spectrum was produced by accumulating data from 100 laser shots over the m/z range of 700-3000 Da. All mass spectra were calibrated internally by trypsin (Promega Bioscience, CA) autolysis products at 842, 50 Da, 1.046,56 Da, 2.212,11 Da and 2.284,19 Da. Trypsin and keratin contamination peaks were excluded from the peak list used in the database searching.
The results from the PMF were employed to search the human NCBIn protein database by Mascot search engine, which compare the experimentally determined tryptic peptide masses with theoretical peptide masses calculated for protein from these databases. Search parameters are as follows: type of search, peptide mass fingerprint; enzyme, trypsin; fixed modification, carbamidomethylation (Cys); variable modifications, oxidation (Met); mass values, monoisotopic; peptide charge state, 1+; maximum missed cleavages, 1; and peptide mass tolerance, 100 ppm.

Cellular isolation, culturing and characterization
As can be seen in figure 2A, the cells isolated from the human Wharton's jelly and used in the present work, display a consistent spindle-shaped elongated morphology similar to fibroblastoid cells. This feature appears to be maintained up to the end of culturing. A representative immunophenotype of the cells used in our experiment is reported in figure 2B. A positive response for CD105 (endoglin), CD73, CD90, CD29, CD166, CD13, CD44 (hyaluronan receptor) markers and a negative reactivity for CD34, CD45 and CD14 hematopoietic markers were detected at each time of culture passages. This high homogeneous expression of the above mentioned markers suggests that the samples used in the present study were not significantly contaminated by no-stem cell population (figure 2B). Figure 2C illustrates the ability of WJCs to differentiate in osteogenic, adipogenic and chondrogenic lineages further evidencing their pluripotency.
As reported in figure 2D cells, after expansion, showed a homogeneous diploid content in the G1 cell-cycle phase. Furthermore, in figure 2D it can be also seen that the G1 and the G2 cell-cycle checkpoints appeared intact. This finding is consistent with cells actively cycling. The long telomeric end of DNA extracted from cells at all passages examined also confirms that these cells preserve their capability to undergo a high number of cellular divisions up to the 12 th passage ( figure 2E). Accordingly, a low frequency of β-galactosidase positive staining cells was found in all different passages (2 nd , 4 th , 8 th and 12 th ) (Additional file 1). Overall, the data reported in figure 2 strongly indicate that the cells derived from Warthon's jelly belong to the mesenchimal stem-cell population.
In order to characterize the proteome profile of WJCs, the cells prepared at the 2 nd , 4 th , 8 th and 12 th culture passages were analyzed. Longer term in vitro culture passage led to an impairment of the cell expansion ability as accounted by the cell number count (figure 2F) and by the analysis of the doubling time ( figure 2G). For these reasons they were not included in the present investigation.

2DE analysis and MS protein identification
The master gels of the 2 nd , 4 th , 8 th and 12 th passage constructed for each step are reported in figure 3, by using cells prepared from three different human donors. On average 2450 ± 76, 2330 ± 91, 2200 ± 103 and 1750 ± 74 spots were detected from the 2 nd to the 12 th in vitro expansion passages respectively (Additional file 2).
In order to evaluate the change of protein pattern expression occurring during in vitro cell expansion, the master gels, resulting from each culture passages, were compared with the synthetic gel constructed by using the gels resolved from all culture passages (figure 3).
Image analysis of the synthetic gel (figure 4A) revealed the presence of 2150 spots. Since these spots are common to all passages they can therefore be considered house- 4B), i.e. cytoskeleton and motility, metabolism, protein biosynthesis folding and degradation, nucleotide biosynthesis and cell signaling. Overall 60% of total proteome have been identified as proteins belonging to the cytoskeleton compartment and involved in protein biosynthesis, folding and degradation. The great number of proteins of these two groups may probably give to WJCs the proliferative capacity for the quickly change of their phenotype in response to adequate external stimuli.
The large number of proteins associated with the energetic metabolism may confer to WJCs the necessary power for their intense proliferation. Antioxidant proteins such as thioredoxin, peroredoxin and gluthatione transferases, are also present in these cells. They may play a key role in preserving WJCs from oxidative injury during in vitro expansion.
Proteins involved in nucleotide biosynthesis and signal transduction seem to be poorly represented in WJCs. It has to be noted that myocyte's characterizing proteins like caldesmon, α-actinin, tropomyosin, and vinculin are expressed up to the last culture passages [22]. Furthermore cardiomyogenic and hepatic putative markers such as tropomyosin and pyruvate kinase were also detected in these cells [23][24][25][26].
The presence of vimentin (VIME), which is the most ubiquituos intermediate filament protein and the first protein to be expressed during cell differentiation is common to other MSCs such as bone marrow and cord blood derived non-hematopoietic (mesenchymal, stromal) progenitor cells, fibroblasts and endothelial cells [27,28]. VIME is one of the most prominent phosphoprotein present in MSCs and its phosphorylation is significantly enhanced during cell division [29,30]. Six different isoforms of phosphorilated VIME were identified in WJCs. However, one of them i.e. Vimentin 4.9/47.4 (table 1; figure 5F) was no longer expressed after the 2 nd passage, whereas another one i.e. Vimentin 5.1/45.7 (table 2; figure  5F) appeared only at the end of the last culture passage.
It is interesting to note that proteins reported to be markers of MSCs such as Annexin 1, Annexin 2, or heat shock protein 27 b (HSP27 b) and proteins considered markers for hESCs such as elongation factor Tu (TUFM), isocitrate dehydrogenase (IDH1), peroxiredoxins 1, 2, and 6 (PRDX1, PRDX2, PRDX6) are simultaneously expressed  in WJCs [31]. Furthermore, WJCs contain nuclesphosmin, a nucleolus protein, which is highly expressed in proliferating cell as well as in undifferentiated hESCs [32][33][34][35]. Two signal transduction proteins, i.e. 14-3-3 protein zeta/delta and Twinfillin-like protein 2, not yet found in any of the stem cell lines so far investigated, are present in WJCs. The possibility that they may be WJCs specific proteins needs to be further investigated. The gel matching between the master gels and the synthetic gel has allowed to detect 47 proteins that were no longer expressed after the initial passages (figure 5A).
8 out of 47 were assigned by PMF ( figure 5B) and six of them were validated by Western blotting experiments (table 1 and figure 5E, F). These proteins are involved in important cellular physiological functions including protein biosynthesis, cellular division, internal cellular motility and neuronal differentiation.
Shootin 1 (SHOT1) and two Adenylate Kinase 5 isoenzymes (KAD5) have been identified as brain specific proteins that may play a key role in neurogenesis [36][37][38]. The eukaryotic translation initiation factor 3 subunit 1 (EIF3I) has an essential role in the rate-limiting initiation phase of translation and is required for several steps in the initiation of protein synthesis [39]. Although the physiological role of Plasminogen activator inhibitor 2 (PAI2) has not yet been well defined, several intracellular functions, including capacity to alter genes expression, ability to influence the cellular proliferation and differentiation or inhibition of apoptosis, have been attributed to this protein [40,41].
The inability of WJCs to express, after the initial stage of replication, specific proteins involved in neuronal differentiation, may indicate that WJCs are progressively reducing their differentiation capacity during in vitro expansion.
Gel matching analysis has also allowed to identify 27 new proteins that appear only at the end of culturing (figure 5C). 5 out of 27 (table 2) were assigned by PMF ( figure 5D) including two isoforms of Prolyl-4-hydroxylase i.e. the subunit alpha-1(4PHA1) and the subunit alpha-2 (4PHA2). Western blotting analysis was carried out on them and on ERO1-like protein alpha; the data obtained are shown in figure 5F. 4PHAs promote the post-translational formation of 4hydroxyproline in hypoxia-inducible factor (HIF) alpha proteins which physiologically regulates a broad range of relevant cellular functions including apoptosis, cellular survival and energy metabolism [42]. 4PHAs are considered cellular oxygen sensors able to modulate the cellular response towards hypoxia [43][44][45].
In addition, these enzymes are involved in the intracellular collagen maturation by catalyzing the hydroxylation of proline residues of the procollagen. Their presence A) Entry code number from SWISS PROT database. B) Score is -10*Log(P), where P is the probability that the observed match is a random event, it is based on Swiss-Prot database using the MASCOT searching program as MALDI-TOF data. C)Sequence coverage means the ratio of portion sequence covered by matched peptide to the full length of the protein sequence.
may probably be a consequence of the impairment of the cellular expansion ability that occurs to WJCs at the end of cellular in vitro expansion (figure 2F, G). Accordingly, Miyaishi et al. have found that human fibroblasts at low population doubling rate increase the level of 4PHA1 and 4PHA2 expression [46].
At the end of culturing we have also found ERO1-like protein alpha (ERO1A). This enzyme is responsible for ROS cellular production during oxidative stress [47][48][49]. It is well known that ROS are considered the major contributors to cellular aging [50]. This protein, induced by hypoxia and regulated via HIF-pathway may play a key role in the maintenance of cellular redox homeostasis.
An interesting protein we have found to be expressed only at the end of culture expansion is the Aspartyl-tRNA synthetase. This enzyme interacting with differentiationrelated gene 1 protein has a growth inhibitory role on cellular proliferation [51].
Taking into account the functional roles attributed to these proteins, it cannot be excluded that they are related to the molecular mechanism of WJCs in vitro selfrenewal and multipotency.

Conclusion
In this report we describe an extensive study of human WJCs proteome profiling. Several important proteins, including shootin 1, adenylate kinases 5 isoenzyme and plasminogen activator inhibitor 2, are no longer expressed after the early stage of cellular in vitro replica-tion. This is probably correlated with the gradual reduction of their staminal multipotency. In addition, at the end of the cellular expansion, new proteins probably involved in the impairment of cellular survival during replication and differentiation time were identified. These proteins could be related to the biological cellular mechanism occurring in the cellular WJCs in vitro senescence.
Further investigation will be needed to elucidate biological mechanisms involved in maintaining active proliferation and maximal cellular plasticity in order to optimize in vitro culturing procedure.
WJCs obtained from the Wharton jelly umbilical cord therefore appear to be an inexpensive biological source for the isolation of stem cells thus circumventing the ethical constraint that arises from the use of the embryonic tissue.
In this context, this study carried out to increase the knowledge about the molecular features of WJCs, may give a great contribution to the field of regenerative medicine.  A) Entry code number from SWISS PROT database. B) Score is -10*Log(P), where P is the probability that the observed match is a random event, it is based on Swiss-Prot database using the MASCOT searching program as MALDI-TOF data. C) Sequence coverage means the ratio of portion sequence covered by matched peptide to the full length of the protein sequence.