Stem Cell Rev and Rep (2014) 10:429–446 DOI 10.1007/s12015-014-9501-8

Cryopreservation Effects on Wharton’s Jelly Stem Cells Proteome F. Di Giuseppe & L. Pierdomenico & E. Eleuterio & M. Sulpizio & P. Lanuti & A. Riviello & G. Bologna & M. Gesi & C. Di Ilio & S. Miscia & M. Marchisio & S. Angelucci

Published online: 12 March 2014 # Springer Science+Business Media New York 2014

Abstract Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tissue engineering. However, the freeze-thaw process strongly contributes to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation and altered physical cellular properties. Our previous proteomics investigation was carried out on Wharton’s Jelly Stem Cells (WJSCs) having similar properties to adult mesenchymal stem cells and thus representing a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJSCs proteome in different experimental conditions: fresh primary cell culture and frozen cell. To analyze changes in protein expression of WJSCs undergoing different culturing procedures, we performed a comparative proteomic analysis (2DE followed by MALDI-TOF MS/MS nanoESI-Q-TOF MS coupled with nanoLC) between WJSCs from fresh and

frozen cell culturing, respectively. Frozen WJSCs showed qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defence mechanism and metabolism, that could ensure freeze-thaw survival. The results of this study could play a key role in elucidating possible mechanisms related to maintaining active proliferation and maximal cellular plasticity and thus making the use of WJSCs in cell therapy safe following bio-banking. Keywords Wharton’s Jelly stem cell . Cryopreservation . Proteomic analysis . Cell therapy

Introduction Adult mesenchymal stem cells (MSCs) are generally considered the main tool box for cell-based therapies. Compared to

M. Marchisio and S. Angelucci as senior authors. Electronic supplementary material The online version of this article (doi:10.1007/s12015-014-9501-8) contains supplementary material, which is available to authorized users. F. Di Giuseppe : L. Pierdomenico : E. Eleuterio : M. Sulpizio : P. Lanuti : A. Riviello : C. Di Ilio : S. Miscia : M. Marchisio : S. Angelucci Aging Research Center, Ce.S.I., “Università G. d’Annunzio” Foundation, Via dei Vestini 31, 66013 Chieti, Italy F. Di Giuseppe : E. Eleuterio : M. Sulpizio : A. Riviello : C. Di Ilio : S. Angelucci Department of Experimental and Clinical Science, University “G. d’Annunzio” Chieti-Pescara, via dei Vestini 31, 66013 Chieti, Italy L. Pierdomenico : P. Lanuti : G. Bologna : S. Miscia : M. Marchisio Department of Medicine and Aging Science, School of Medicine and Health Sciences, University “G. d’Annunzio” Chieti-Pescara, via dei Vestini 31, 66013 Chieti, Italy

F. Di Giuseppe : L. Pierdomenico : E. Eleuterio : M. Sulpizio : P. Lanuti : C. Di Ilio : S. Miscia : M. Marchisio : S. Angelucci StemTeCh Group, Via Polacchi 13, 66013 Chieti, Italy M. Gesi Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via Risorgimento 36, 56126 Pisa, Italy S. Angelucci (*) Biochemistry, Department of Experimental and Clinical Science, School of Medicine and Health Sciences, University “G. d’Annunzio” Chieti-Pescara, via dei Vestini 31, 66013 Chieti, Italy e-mail: [email protected]

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embryonic stem cells (ESCs), adult MSCs exhibit the following advantages: accessibility with fewer ethical controversies [1], no reports of teratoma formation and versatile therapeutic applications [2–7]. In literature, the best suited markers currently used to characterize adult MSCs from different sources comprise CD44, CD73, CD90 and CD105 [8], together with the lack of expression of endothelial/hematopoietic markers (CD144, CD34, CD45). The umbilical cord (UC) is an extraembryonic structure essential for feeding the fetus during intrauterine development. It has been shown that Wharton’s jelly (WJ), representing the UC matrix around the umbilical vessels, contains a great number of stem cell mesenchymal like, which have been characterized as expressing aforementioned markers in both human and animal models (Fig. 1) [9–12]. Wharton’s jelly stem cells (WJSCs) are also able to differentiate towards a great number of mature cytotypes (adipocytes, bone, cartilage, skeletal muscle cells, cardiomyocytelike cells, and neural cells), show a long expansion capability in vitro and can be expanded up to 15 passages [13]. The abundant amount of WJ could represent an attractive source of MSCs for cell-based therapies [13, 14]. Our recent study, carried out on WJSCs having similar properties to MSCs, highlighted the changes in protein expression profiling during in vitro expansion which are probably related to the gradual reduction of their staminal plasticity and to the biological cellular mechanisms occurring in cellular aging [15]. Proteome characterization of WJSCs during their growth in vitro from 2nd to 12th culture passages allowed us to assert that more than 30 % of them belong to cytoskeleton compartment [15]. We also found several proteins as putative molecular targets involved in the proliferative potency and proteins synthetized at the end of cellular culturing, probably causing the impairment of cellular survival during replication and differentiation. We also confirmed that WJSCs are able to retain their stemness after long term expansion [15]. Recently, an interesting review report by Bongso and Fong summarized several biological properties like extensive

plasticity, stemness, multipotency, low immunogenicity and an attractive anti-cancer activity, suggesting possible challenges in their therapeutic use in clinical fields of chronic diseases and cancer [16]. In addition, WJSCs as conditioned medium or stromal support contribute to hMSC expansion ex vivo [17–19]. Gauthaman et al. looking into stress-related events associated with cell cryopreservation assert that WJSCs increase their thaw-survival, proliferation, stemness and differentiation potential after the thawing process [20]. Fong et al., by using a controlled-rate freezer to cryopreserve human umbilical cord stroma mesenchimal stem cells (hUCS-MSCs), described a 85– 90 % cell viability after thawing [21]; also dimethyl-sulphoxide (DMSO) toxicity tests on post-thaw samples confirmed a high relatively resistance of hUCS-MSCs to chemical injury [22]. With the development of cell- and tissue- based therapies, cryopreservation and bio-banking have become progressively important. In fact, a variety of preclinical and clinical investigations have shown the promising value of various stem and progenitor cell transplantation protocols [23]. Cryopreservation, now applied regularly to oocytes and embryos of many mammalian species, is to be considered the only procedure for long-term storage of viable cells and tissues in the use of cellular therapy, stem cell transplantation and/or tissue engineering even though physical and biological stress like osmotic and cold shock can induce irreversible damage to cell [22, 23]. It is well known that freeze-thaw processes strongly contribute to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation (IIF) and altered physical cellular properties [24, 25]. During freezing damage the production of free radicals is considered the possible trigger of cellular viability loss. An increase of reactive oxygen specie (ROS) levels induces oxidative damage (lipid peroxidation, protein oxidation and DNA degradation). Cellular response includes antioxidant molecules such as enzymatic activation (superoxide dismutase, catalase and peroxiredoxin) [26].

Fig. 1 Schematic representation of cord localization and developmental potency of WJSCs. A. Schematic representation of human umbilical cord. Wharton’s jelly is the intervascular connective tissue B. Model for the developmental position and potency of the fetal stem cells from several

fetal sources emerging during gestation. Fetal stem cells from these sources actually represent a new class developmentally and operationally located between the state of embryonic stem cells and adult stem cells, sharing and exhibiting multipotency features

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Apoptosis in response to low temperature exposure is well documented [27], and the involvement of apoptotic cell death in cryopreservation injury has been reported in a wide variety of cell types including haematopoietic stem cells [28]. Heng et al. demonstrated the involvement of apoptosis in early cell death in hESCs cryopreserved through slow cooling [29]. He observed that post-thaw immediate cell viability was initially very high but showed a gradual reduction in vitro culture at 37°C [30]. The role of cryopreservation in activating both the extrinsic and intrinsic pathways of apoptosis was recently investigated by Xu et al. [31]. They examined the effect of DMSO exposure and slow cooling on the production of ROS, p53 levels, the initiator levels of the apoptotic cascade (caspases 8 and 9) and cytoskeletal F-actin (which can contribute to the induction of apoptosis in response to environmental changes). Based on the biochemical aspects of cell biology, Olson et al. have demonstrated the crucial role of Rho kinases in an array of cellular processes such as adhesion, proliferation, differentiation and apoptosis strictly dependent on cell type [32]. Cryopreservation has been successfully used for many cell types, including well differentiated cell and mESCs and hMSCs from different species [33, 34]. In laboratory procedures, cryopreservation is normally composed of four steps: cryoprotectant agent (CPA) adding, freezing and keeping in liquid nitrogen to thawing and CPA removal. Nowadays, only slow freezing and vitrification can be used for both cell and tissue storage, though the slow freezing practice is more widely used in the cryopreservation of cell in suspension [35]. To date, research has focused on the biological aspects of freeze thaw survival of frozen MSCs evaluating post-thaw cell viability and proliferative rate, confirming possible cellular resistance to deleterious effects from cryopreservation [36]. The aim of the present work therefore was to analyze molecular changes that occur in WJSCs proteome in different culture conditions (fresh and frozen cell preparations) during each culture passage of in vitro growth so as to provide insight on the mechanisms related to cell recovery from cryopreservation.

Materials & Methods Apparatus and Reagents IPG strip gels were run on IPGphor III (GE Healthcare, Uppsala, Sweden). Casting and running second-dimensional SDS-PAGE (Dodeca Cell) were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Silver-stained gels were scanned using a Lab Scan (GE Healthcare, Uppsala, Sweden) and image analysis was carried out using Image Master 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden).

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C18ZipTip was purchased from Millipore (Bedford, MA, USA). Bruker-Daltonics AutoFlex Speed (MALDI TOF-TOF/ LIFT) and Maxis 4G (nano ESI-Q-TOF coupled on-line with Thermo Fisher Scientific Proxeon Easy-nLC) are the mass spectrometer used for the protein identification. Immobiline DryStrip (4–7) 24 cm, pharmalite 4–7, protease inhibitor mix, Drystrip cover fluid, IPG buffer, DeStreak reagent were purchased from GE Healthcare (Uppsala, Sweden). Sequencing grade, modified porcine trypsin were obtained from Promega (Madison, WI, USA). All other chemicals were of analytical reagent grade and purchased from Sigma Chemical (St. Louis, MO, USA). All buffers were prepared with Milli-Q water system (Millipore Bedford, MA, USA). Isolation and Cell Culture of WJSCs 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 h from partum to obtain the WJSCs as previously described [15]. After the removal of blood vessels, the extracellular matrix of WJ was scraped off, treated with collagenase IV (2 mg/ml) (Sigma) for 16 h at 37 °C, treated with 2.5 % trypsin for 30 min at 37 °C under agitation. The cells were then seeded in Human Mesenchymal Stem Cells Growth Medium (HMSCGM; Lonza) and were cultured in 5 % CO2 in a 37 °C incubator. The adherent cells were detached with 0.05 % trypsin-EDTA, counted with Trypan Blue exclusion, and reseeded at 3000 cells/cm2 to reach the 90 % of confluence after 3–4 population doublings. Cryopreservation and Thawing of WJSCs Fresh primary cell culture of WJSCs were frozen using 10 % dimethyl sulphoxide (Sigma) as a cryoprotectant, 40 % complete growth medium and 50 % FBS. Samples were frozen using passive cooling method. The cell suspension was transferred into cryotubes (Nalgene Labware) and were gradually frozen to −80 °C, with a 1 °C/min rate, overnight in a Mr. Frosty Freezing container (Nalgene Labware) filled with fresh isopropanol and subsequently placed in the liquid phase of liquid nitrogen at least four months. During thawing, the cryovials were removed from liquid nitrogen and placed in a preheated water bath (37 °C). Thawing lasted for 1–2 min. The content of the vials was removed and placed in falcon tubes. Ten milliliter of complete growth medium (D-MEM+10 % FBS) was added. The cell suspension was centrifuged at 200×g, at RT for 10 min. The supernatant was removed and the cell pellet was resuspended in 10 mL of complete growth medium. The cells were

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transferred to tissue culture dish (Φ100 mm) and cultured in 5 % CO2 in a 37 °C incubator. Immunophenotype by Flow Cytometry of WJSCs Washing buffer (0.1 % sodium azide and 0.5 % bovine serum albumine in PBS) was used for all washing steps. Samples were stained for surface or intracellular antigens, as previously described [37]. For surface staining, samples were resuspended in 100 μl washing buffer containing the appropriate amount of surface antibodies (S1; Supplemental Table 1); samples were incubated for 30 min at 4 °C in the dark. Cells were washed (3 ml of washing buffer), centrifuged (4 °C, 400×g, 8 min), resuspended with 1 ml PBS 0.5 % paraformaldehyde, incubated for 5 min at RT, washed, centrifuged (4 °C, 400×g, 8 min) and stored at 4 °C in the dark until their acquisition. For intracellular staining, cells were resuspended in 1 ml of FACS Lysing solution (BD), vortexed and incubated at room temperature (RT) in the dark for 10 min. Samples were centrifuged (4 °C, 400×g, 8 min); 1 ml of Perm 2 (BD) was added to each tube and cells were incubated at RT in the dark for 10 min. Samples were washed and centrifuged (4 °C, 400×g, 8 min). Cells were resuspended in 100 μl of washing buffer containing the appropriate amount of intracellular antibodies (S1) and incubated for 30 min at 4 °C in the dark. Cells were centrifuged (4 °C, 400×g, 8 min), resuspended with 1 ml PBS 0.5 % paraformaldehyde for 5 min at RT, washed, centrifuged (4 °C, 400×g, 8 min) and stored at 4 °C in the dark until the acquisition. Cells were analysed on a FACSCalibur flow cytometer (BD), using CellQuest™ software (BD). Flow Cytometry Measurement Quality control included regular check-ups with Rainbow Calibration Particles (BD Biosciences). Debris was excluded from the analysis by gating on morphological parameters; 20,000 non-debris events in the morphological gate were recorded for each sample. To assess nonspecific fluorescence we used isotype controls. All antibodies were titrated under assay conditions and optimal photomultiplier voltages (PMT) were established for each channel [38]. Data were analysed using FlowJo™ software (TreeStar, Ashland, OR). Mean Fluorescence Intensity Ratio (MFI Ratio) was calculated dividing the MFI of positive events by the MFI of negative events [39].

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incorporation, cells were washed with PBS and treated with 1 ml of a solution containing 2 N HCl and 0.5 % Triton X-100 (Sigma) for 30 min at room temperature. 1 ml per sample of 0.1 M Na2B4O7 (pH 8.57) was added to stop the HCl reaction. Cells were then washed with 1 ml PBS 0.5 % Triton X-100 1 % BSA, followed by an incubation with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (BD; dilution: 1:5 in PBS 0.5 % Triton X-100) for 30 min at room temperature in the dark. Cells were washed and resuspended in PBS containing 5 μg/ml Propidium Iodide (PI, Sigma) and 200 μg/ml RNase (Sigma). After 30 min of incubation biparametric BrdU/DNA data were acquired on a FACSCalibur flow cytometer (two-lasers, four-color configuration) with CellQuest 3.2.1.f1 (BD) software; data were analysed using FlowJo™ software (TreeStar, Ashland, OR). 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 [41]. Telomere Length Assay of WJSCs 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 using the Telo TAGGG kit (Roche) according to the manufacturer’s instructions. Appropriate controls by extracting DNA from cells with long or short telomere regions, were also provided with the kit [15]. Determination of Cell Senescence of WJSCs The amount of senescent cells was evaluated in different conditions by using the Senescence β-Galactosidase Staining Kit (Abcam, Cambridge, UK) in accordance to the manufacturer’s instructions, as previously described [15]. Adipogenic Differentiation of WJSCs To induce adipocyte differentiation, 10×103 cells/cm2 were cultured in DMEM high glucose (HG) (Sigma) supplemented with 10 % FBS, 0.5 mM isobutyl-methylxantine (Sigma), 200 μM indomethacin (Sigma), 1 μM dexamethasone (Sigma) and 10 μg/ml insulin (Sigma). Cells were cultured by replacing the medium every 2–3 days. After 2–3 weeks of culture, cells presented an increase of neutral lipids in fat vacuoles; they were fixed in 10 % formalin and stained with fresh oil red-O solution (Sigma) [15].

Cell Cycle Analysis of WJSCs Osteogenic Differentiation of WJSCs Exponentially growing cells were exposed to 10 μM bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) for 1 h, then fixed in 70 % ethanol and kept at 4 °C before labeling as previously described [40]. To detect BrdU

To induce osteogenic differentiation, 3×103 cells/cm2 were cultured in α-MEM (Sigma) supplemented with 10 % FBS, 10 mM β-glycerophosphate (Sigma), 0.2 mM ascorbic acid

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(Sigma), and 10 nM dexamethasone (Sigma), and cultured for 3–4 weeks by replacing the medium every 2–3 days. To demonstrate osteogenic differentiation, cultures were fixed and induced to the alkaline phosphatase reaction [15].

Proteome Analysis of WJSCs To generate 2D reference map of WJSCs and analyze protein changes of WJSCs during in vitro culture expansion between frozen and fresh cellular preparations we considered three passages during cellular growth 2nd, 8th and 12th respectively as described in previous proteomic analysis [15]. For each culture passage we used two independent donors and ran 3 gels (technical replicate) for all biological replicates. 16×106 stem cells from each population were treated with lysis buffer (40 mM Tris pH 7.4, 8 M urea, 4 % CHAPS) supplemented with a protease inhibitor mixture and 2 mM TBP as reducing agent. Protein concentration was determined by Bradford assay [15]. The resulting samples were loaded onto commercial 4–7 NL IPG strip and the second dimension was performed on a 9–16 % polyacrilamide gel. Silver staining and mass spectrometry compatibility staining were performed as previously described [15]. 2D gels once scanned were analyzed with Image Master 2D Platinum software version 6.0 (GE Healthcare). The intensity of the spots were normalized based on the total volumes on the gel. Protein spots with significant changes in expression level (paired t-test, P

Cryopreservation effects on Wharton's Jelly Stem Cells proteome.

Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tiss...
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