Pinacidil enhances survival of cryopreserved human embryonic stem cells

Cryobiology 63 (2011) 298–305

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Pinacidil enhances survival of cryopreserved human embryonic stem cells q Ivana Barbaric a,⇑, Mark Jones a, Kristina Buchner a, Duncan Baker b, Peter W. Andrews a, Harry D. Moore a a b

Centre for Stem Cell Biology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK Sheffield Diagnostic Genetic Services, Sheffield Children’s Hospital, Sheffield S10 2TH, UK

a r t i c l e

i n f o

Article history: Received 11 March 2011 Accepted 6 October 2011 Available online 17 October 2011 Keywords: Human embryonic stem cell Cryopreservation Pinacidil

a b s t r a c t Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. Ó 2011 Elsevier Inc. All rights reserved.

Introduction Pluripotent stem cells derived either from human embryonic stem cells (hESCs), or by reprogramming somatic cells (induced pluripotent stem cells, iPSCs) have the capacity of unlimited proliferative capacity and differentiate into many cell types of the body [26,24,25]. There is great interest in using these cells in regenerative medicine to treat degenerative diseases [22]. However, the efficient cryopreservation methods are of utmost importance for any future clinical applications. The overall efficiency of hESC or iPSC recovery after cryopreservation is determined by several factors. The ultimate endpoint for a cryopreserved aliquot of pluripotent stem cells is a proliferating colony of undifferentiated cells for expansion and subsequent differentiation to the cell of choice. Therefore, aside from the immediate effects of protection from cryo-injury, the rates of cell apoptosis and differentiation after thawing can have a major impact on recovery of the cells and their amplification. Pluripotent stem cells are maintained in a niche created in culture as the colony develops, either in the presence of feeder cells (usually mitotically inactivated mouse or human embryonic fibroblasts), or more recently with extracellular matrix components (e.g. Matrigel) and q Statement of funding: This research was funded by MRC and the ESTOOLS consortium under the Sixth Research Framework Programme of the European Union contract LSHG-CT-2006-018739. ⇑ Corresponding author. E-mail address: i.barbaric@sheffield.ac.uk (I. Barbaric).

0011-2240/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2011.10.002

defined culture medium [18]. They rapidly undergo apoptosis or differentiation when dispersed to single cells or very small colonies when recovered from monolayer adherent culture [13], prior to cryopreservation in suspension, and following thawing and culture. In this regard, the Rho-associated kinase (ROCK) inhibitor (Y-27632) has been shown to prevent the apoptosis of single pluripotent stem cells and improve their clonogenic potential after passage [27]. Moreover the supplementation of culture and/or cryopreservation medium with Y-27632 substantially increased the efficiency of post-thaw cell recovery [20,21]. Recently, in a high-throughput screening assay we identified a drug with similar properties to Y-27632 in protecting dissociated pluripotent stem cells from excessive death by promoting their attachment to matrix and decreasing apoptosis [9]. This compound is pinacidil (N-cyano-N0 -4-pyridinyl-N00 -(1,2,2-trimethylpropyl) guanidine monohydrate), an FDA approved vasodilator drug for the treatment of hypertension [7,6]. We reasoned that pinacidil might also enhance survival of cryopreserved hESCs and therefore compared the supplementation of ES culture and cryopreservation medium with pinacidil versus the ROCK inhibitor Y-27632.

Materials and methods Human embryonic stem cell culture hESC lines used in this study were Shef4, Shef5, Shef7 [3] and H7 line [26]. All cultures of hESC lines had a normal karyotype as

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assessed by G-banding of metaphase chromosome spreads. Stock cultures of hESC lines were maintained as colonies on mitoticallyinactivated mouse embryonic fibroblasts (MEFs) in hESC medium consisting of KnockOut DMEM (KO-DMEM) (Invitrogen, Paisley, UK) supplemented with 20% KnockOut Serum Replacement (Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM b-mercaptoethanol (Sigma–Aldrich, Poole, Dorset, UK), 1 non-essential amino acids (Invitrogen) and 4 ng/ml basic FGF (Invitrogen). Cells were grown at 37 °C in 5% CO2 in air. For routine maintenance, cells were passaged by treating with collagenase type IV (1 mg/ml) (Invitrogen) for 7 min followed by scraping off the flask surface. Chemicals

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conditions, the final concentration of pinacidil was 100 lM and final concentration of Y-27632 was 10 lM. Control cells were similarly prepared and plated but without addition of either drug. All conditions were tested in triplicate wells. After 48 h in culture, the cells were washed once in PBS and fixed with 4% PFA for 15 min at room temperature. Nuclei were stained with 10 lg/ml Hoechst 33342 (Invitrogen). The cells were imaged using the InCell Analyzer 1000 with a 4 objective. Developer Toolbox software (GE Healthcare) was used for automated cell counting. For RNA extraction, karyotype analysis and flow cytometry, 1–2  106 thawed cells were plated in 25 cm2 flasks on MEFs in hESC medium with or without 100 lM pinacidil. After 48 h, pinacidil was removed and cells were further grown and passaged in hESC medium only.

Pinacidil and Y-27632 were purchased from Sigma–Aldrich. Assessment of post-thaw cell viability Antibodies The following primary monoclonal antibodies were prepared in house as pre-titred supernatants from hybridomas: MC631 (antiSSEA-3) [23], MC813-70 (anti-SSEA-4) [15], TRA-1-60 [5] and P3X63Ag8 [16]. Rabbit polyclonal antibody to OCT4, mouse monoclonal antibody to Nestin, mouse monoclonal antibody to alphafetoprotein, and mouse monoclonal antibody to alpha-smooth muscle actin (SMA) were purchased from Abcam (Cambridge, UK). Secondary antibodies used were Dylight-594-conjugated anti-goat l-chain specific antibody (Jackson ImmunoResearch), fluorescein-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) or Dylight-488-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), as appropriate to the isotype of the primary antibody. Colony forming assay Colony forming assays were performed in 6-well plates coated in Matrigel (BD Biosciences, Oxford, UK) (1:25 dilution in KO-DMEM). Shef4 cells were dissociated to single cells using trypsin for 3 min at 37 °C. After washing once in media the cells were resuspended in mTESR media (STEMCELL Technologies, Vancouver, Canada) and 5000 cells were plated per well. After 7 days, cells were fixed with 4% paraformaldehyde (PFA) and stained for OCT4 and SSEA3. Plates were imaged using the InCell Analyzer 1000 (GE Healthcare, Little Chalfont, UK) automated microscopy system. Colony number, cell number and number of OCT4 and SSEA3-positive cells were counted using the Developer Toolbox (GE Healthcare) image analysis software. Cryopreservation of hESCs HESCs were dissociated to single cells by treatment with trypsin (Sigma–Aldrich) for 3 min at 37 °C. After washing with medium, 0.5–2  106 cells were frozen in 1 ml of freezing medium (10% ME2SO (Sigma–Aldrich), 90% foetal calf serum (Hyclone, ThermoScientific, Epsom, UK)) per cryovial (Sarstedt, Leicester, UK). To test the effect of pinacidil during cryopreservation of hESCs, 100 lM pinacidil was included in the freezing medium. The cryovials were placed into a Mr. Frosty slow-rate cooling freezing container (Nalgene, Roskilde, Denmark) at 80 °C overnight. The following day, the cryovials were placed into liquid nitrogen and kept there for at least 48 h prior to thawing. Cell thawing and quantification of cell survival Cryopreserved cells were thawed quickly in a waterbath at 37 °C, washed once in hESC media and counted. Cells (105) were plated per well of a 12 well plate on a layer of MEFs. For test

Immediately after thawing the cells, 0.1 ml of cell suspension was mixed with 0.1 ml of 0.4% Trypan Blue (Sigma), and cells were counted using a hematocytometer. Post-thaw viability was defined as a ratio of viable to total cell numbers. Assessment of post-thaw cell attachment Immediately after thawing the cells, 10,000 cells were plated per well of a 96-well lclearÒ flat-bottom plate (Greiner Bio-one, Stonehouse, UK) coated in Matrigel (BD Biosciences) (1:25 dilution in KO-DMEM), in mTESR medium (control), mTESR medium supplemented with increasing concentrations of pinacidil (1, 10 and 100 lM) or mTESR medium supplemented with 10 lM Y-27632. Four hours after plating, cells were fixed with 4% PFA for 15 min at room temperature and permeabilised with 0.1% Triton-X in PBS for 30 min. Fixed cells were then incubated with Fluorescein Isothiocyanate-Labeled Phalloidin (Sigma) to visualise F-actin. Nuclei were counterstained with 10 lg/ml Hoechst 33342 (Invitrogen). The cells were imaged using the InCell Analyzer 1000 with a 10 objective. Developer Toolbox software (GE Healthcare) was used for automated cell counting. Embryoid body (EB) culture HESCs grown on MEFs were treated with 1% collagenase type IV (Invitrogen) for 20 min at 37 °C. The detached clumps of cells were washed once in hESC medium and transferred into non-adhesive Petri dishes (Sterilin, Caerphilly, UK) and incubated at 37 °C in 5% CO2. After 7 days, RNA extraction was performed using the RNeasy Mini Kit (QIAGEN, Crawley, UK). Some of the EBs were plated out in 48-well plates (Nunc, Langenselbold, Germany) pre-coated with 0.1% gelatin for 20 min, and grown for a further 8 days in DMEM (Invitrogen) supplemented with 10% FCS (Hyclone). Cells were then fixed with 4% PFA for 15 min and stained for lineage-specific markers. RNA extraction, reverse transcription reaction and PCR Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 lg RNA using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR was performed using gene-specific primers and Taq polymerase (Invitrogen) in 25 ll reactions. Each reaction contained 0.2 mM dNTPs, 0.5 units Taq polymerase (Invitrogen), 1 manufacturer’s buffer, 5 mM MgCl2, 5 pmol of each of the forward and reverse primers, and 1 ll of cDNA template. Samples were denatured at 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 52–62 °C for 45 s, and 72 °C for 45 s. The final extension step was at 72 °C

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for 7 min. Primer sequences were obtained either from published studies or selected from target sequences using the Primer3 program (http://frodo.wi.mit.edu/primer3). Sequences are listed in Supplementary Table S1. Immunocytochemistry Cells were fixed with 4% PFA for 15 min at room temperature, blocked in PBS containing 10% foetal calf serum (blocking buffer) for 1 h and then permeabilized using 0.1% Triton-X. Cells were then incubated with the primary antibody (anti-SSEA3, OCT4, Nestin, alpha-fetoprotein or alpha-smooth muscle actin) in blocking buffer for 1 h. After three washes with PBS, the cells were incubated with the secondary antibody in the blocking buffer for 1 h. Nuclei were counter-stained with 10 lg/ml Hoechst 33342 (Invitrogen). The cells were imaged using the InCell Analyzer 1000 (GE Healthcare).

Flow cytometry Cells were harvested using trypsin (Sigma–Aldrich) and resuspended in PBS supplemented with 10% foetal calf serum. Cells (5  105) were incubated for 1 h with a primary antibody to SSEA3, SSEA4 and TRA-1-60. After washing three times in PBS, cells were labelled with FITC-conjugated secondary antibody for 1 h. This was followed by washing the cells three times with wash buffer and analysing cell fluorescence on a CyAnADP flow cytometer with O2 optics (Beckman Coulter, Brea, USA). The gate for FITC-positive cells was set using control cells that were incubated with a negative control antibody obtained from the parent myeloma cell line P3X63Ag8 [16]. Karyotyping The karyotype analysis was assessed using the G-banding technique. Cells from a 25 cm2 flask were treated with 0.1 lg/ml

Fig. 1. Pinacidil increases the colony forming efficiency of hESCs. (a) Single cells were plated in 6-well plates on Matrigel in mTESR media supplemented with 0.1% ME2SO (control, upper panels) or mTESR supplemented with 100 lM pinacidil (lower panels). After 7 days, the cells were fixed and stained for stem cell markers SSEA3 (red, middle panels) and OCT4 (green, right panels). Nuclei were counterstained with Hoechst 33342 (blue, left panels). Higher colony numbers, and increased colony sizes, were observed in pinacidil-treated wells. (b) Colony forming efficiencies expressed as a ratio of the number of colonies after 7 days post-plating versus the number of single cells plated. Values shown are mean ± SD of triplicate wells. ⁄⁄⁄P < 0.001, Student’s t test. (c) Quantification of the percentage of cells expressing SSEA3 and OCT4 in the colony formation assay. Colonies in the pinacidil-treated wells retain the expression of SSEA3 and OCT4. Values shown are mean ± SD of triplicate wells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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colcemid (Invitrogen) for up to 4 h. After dissociation with 0.25% trypsin/versene (Gibco, Invitrogen), the cells were re-suspended in pre-warmed 0.0375 M KCl hypotonic solution and incubated for 10 min at room temperature. Cells were then pelleted and resuspended in fixative (3:1 methanol:acetic acid). Metaphase spreads were prepared on glass microscope slides and G-banded by brief exposure to trypsin and stained with 4:1 Gurr’s/Leishmann’s stain (Sigma). A minimum of 10 metaphase spreads were analysed and a further 20 counted. Results Pinacidil increases clonogenic efficiency of hESCs The impetus for this study came from our earlier observation that the FDA-approved drug pinacidil improves survival of dissociated hESCs [9]. As a more stringent test of the ability of pinacidil to promote hESC survival, we examined the clonogenic efficiency of cells in the presence of pinacidil. After dissociation to single cells and plating of cells at low density, more colonies emerged in the wells where pinacidil was present, with the clonogenic efficiency increasing from 1% for control cells to 10% for pinacidil-treated

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cells (Fig. 1a and b). Pinacidil-treated cells remained positive for SSEA3 and OCT4 [1], indicating that the cells were in an undifferentiated state (Fig. 1a and c). Given this ability of pinacidil to increase clonogenic efficiency of hESCs, we reasoned that it might also improve survival of cryopreserved hESCs. Effect of pinacidil on survival of cryopreserved cells We initially examined the rate of hESC survival upon adding pinacidil to the post-thaw media. Dissociated human ES cells were cryopreserved by slow freezing and recovered by rapid thawing, and then plated in the presence or absence of pinacidil. As a positive control, we included Y-27632 ROCK inhibitor, as this small molecule was previously reported to improve cell recovery after cryopreservation [20]. By 48 h after plating, the pinacidil-treated wells had a strikingly higher number of cells (Fig. 2a). Pinacidil improved the cell survival by more than 65% in all 3 hESC lines tested, similar to the effect of the Y-27632 ROCK inhibitor (Fig. 2b–d). Adding pinacidil to the cryoprotectant only (i.e. with no addition post-thaw) had no effect on the cell numbers after thawing. Adding pinacidil to the cryoprotectant and the post-thaw culture medium did not further enhance cell numbers compared to the effects

Fig. 2. Pinacidil improves survival of cryopreserved hESCs. (a) Cryopreserved H7 cells were thawed and plated on feeder cells in hESC media (control, left panel), hESC media supplemented with 100 lM pinacidil (middle panel) or 10 lM Y-27632 (right panel). Forty-eight hours after plating, the cells were fixed and stained with Hoechst 33342 to visualize the nuclei. Counting of the nuclei 48 h after plating hESC lines (b) Shef7, (c) H7 and (d) Shef4 with or without pinacidil or Y-27632. Values shown are mean ± SD of triplicate wells. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, Student’s t test.

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Fig. 3. Effect of adding pinacidil during the freezing and thawing processes. (a) H7, Shef5 and Shef4 cells were frozen and thawed in medium with or without pinacidil. Fortyeight hours after thawing, nuclei were counterstained with Hoechst 33342 and counted. Values shown are mean ± SD of triplicate wells. (b) Assessment of post-thaw cell viability in the medium with or without pinacidil or Y-27632, using Trypan Blue exclusion method. Values shown are mean ± SD of triplicate samples. (c) Pinacidil promotes cell attachment. Cryopreserved H7 cells were thawed and plated on Matrigel in mTESR media with increasing concentrations of pinacidil or in 10 lM Y-27632. The number of attached cells was counted 4 h after plating. Values shown are mean ± SD of triplicate wells. (d) Appearance of cells stained with Phalloidin-FITC (green) and Hoechst 33342 (blue), 4 h after thawing and plating on Matrigel in mTESR media (control, left panel), mTESR supplemented with 100 lM pinacidil (middle panel) or mTESR supplemented with Y-27632 (right panel). Apart from a higher number of cells, pinacidil and Y-27632-treated cells show reduced membrane blebbing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

observed with pinacidil in post-thaw culture medium only (Fig. 3a). Immediate post-thaw viability of cells assessed by Trypan Blue exclusion method was relatively high in our control conditions (85 ± 4%) (Fig. 3b), similar to what was observed by Mollamohammadi et al. when using 90% FCS/10% DMSO as freezing media [21]. Neither pinacidil nor Y-27632 further improved immediate

post-thaw viability of cells. No difference in the immediate postthaw viability of cells with or without pinacidil, versus a significant difference in cell numbers at 48 h after plating in pinacidil-containing culture medium, indicated that pinacidil might be exerting its effects on cell survival by promoting cell adhesion of cryopreserved cells. Indeed, at 4 h after plating, the number of attached cells was

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Fig. 4. Cryopreserved cells thawed in pinacidil media retain an undifferentiated phenotype and the ability to differentiate. (a) H7 cells 7 days after thawing in pinacidilcontaining medium show normal colony morphology and express the OCT4 transcription factor (right panel). Nuclei are counterstained with Hoechst 33342 (left panel). (b) Expression of pluripotency-associated antigens SSEA-3, SSEA-4 and TRA-1-60 on cells after thawing in normal hESC medium with or without pinacidil. (c) RT-PCR analysis of pluripotency-associated and lineage-specific markers for cells thawed in pinacidil media (left lane) and after 7-day differentiation of these cells in embryoid bodies (middle lane). Markers are: pluripotency – OCT4 and NANOG; ectoderm – PAX6; endoderm – AFP, SOX7; mesoderm – MSX1 and GATA2. (d) Immunocytochemistry of embryoid bodies derived from cells thawed in pinacidil medium for the endodermal marker a-FETOPROTEIN (left panel), the ectodermal marker NESTIN (middle panel) and the mesodermal marker SMA (right panel) (all labelled in green). Nuclei are counterstained with Hoechst 33342 (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

increased by about 70% in the medium supplemented with 100 lM pinacidil and this effect was dose-dependent (Fig. 3c). In addition to the change in cell numbers, the cells in pinacidil-containing medium also showed less membrane blebbing compared to the control cells (Fig. 3d). Overall, we concluded that pinacidil exerts positive effects on hESC survival at the time of plating after cell thawing and should, therefore, be included in the post-thaw culture medium.

Characterisation of cryopreserved cells after thawing Following the thawing of cells in culture medium supplemented with pinacidil, we carried out tests to determine the properties and behaviour of the cells. Cells formed colonies with the characteristic hESC morphology and expressed the OCT4 marker of pluripotency (Fig. 4a). Pinacidil appeared to improve retention of pluripotency

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pinacidil does not cause gross genomic alterations or select for cells containing such alterations (Fig. 5).

Discussion

Fig. 5. Cryopreserved cells thawed in pinacidil-containing medium retain a normal karyotype. Representative images of G-banded metaphase spreads for (a) Shef4 and (b) H7 hESCs.

markers as a higher proportion of cells expressed SSEA-3, SSEA-4 and TRA-1-60 markers [1] than in the control sample (without pinacidil) as assessed by flow cytometry (Fig. 4b). To assess whether cells thawed in pinacidil media truly maintained a pluripotent phenotype, we assessed their ability to differentiate into all three embryonic germ layers upon formation of embryoid bodies (EBs). Cells were passaged four times in normal hESC culture medium and then differentiated by induction of EBs in culture. RT-PCR analysis of stem cell and lineage-specific markers was carried out for undifferentiated cells and EBs (Fig. 4c). Undifferentiated cells expressed the pluripotency-associated markers OCT4 and NANOG, which were down-regulated upon EB differentiation [1]. Lineage choices that a pluripotent stem cell can make include ectodermal, mesodermal and endodermal fate. Thus, we selected a repertoire of markers to assess the ability of cells to contribute to these lineages. We detected markers of all three embryonic germ layers upon EB differentiation by RT-PCR (Fig. 4c). Representative markers of endoderm (a-FETOPROTEIN), ectoderm (NESTIN) and mesoderm (SMOOTH MUSCLE ACTIN, SMA) were also detected by immunocytochemistry (Fig. 4d). Finally, although we have previously shown that maintenance of ES cells in pinacidil does not alter their karyotype [9], we extended this analysis by analyzing the karyotype of cryopreserved Shef4 and H7 hESCs early after thawing in pinacidil-containing medium. No karyotypic abnormalities were detected in cells two passages after thawing, indicating that

The efficiency of cryopreservation of pluripotent stem cells is of major practical importance in both research and clinical settings. However, it is paramount for seed and working cell banks used for the clinic. Poor cryopreservation may contribute to genetic mutation in the cell line that, if not detected, could potentially result in serious long-term consequences for the patient. Stresses imposed on the cell during a cryopreservation and thawing cycle are considerable and a genetic (or epigenetic) change giving a selective advantage to a cell(s) to cope with this process (adaptation) may be favoured and rapidly spread through the culture [12,8] thereby altering the final cell phenotype. Hence, it is imperative to minimise this risk with cryogenic procedures by reducing any selection pressure on the cell line. Previously it was shown that the ROCK inhibitor (Y-27632) could improve the post-thaw survival and proliferation of hESCs and iPSCs by preventing apoptosis and increasing clonogenic capacity [20,21]. However, additional compounds that could replace Y-27632 would be beneficial, given the high price of the compound as well as patent issues related to use in commercial applications. We report here that pinacidil is an alternative to Y27632 for increasing survival of cryopreserved hESCs. Only transient exposure to the compound is required during the immediate post-thaw period. We have previously assessed the effect of pinacidil on survival of dissociated hESCs in a range of concentrations from 1 to 100 lM and found 100 lM pinacidil to be the most effective [9]. Thus, we reasoned that this concentration of pinacidil should also be effective in enhancing post-thaw survival of hESCs. Indeed, cell attachment shortly after plating the thawed cells was significantly increased in the presence of 100 lM pinacidil in the post-thaw media compared to the control, and this effect of pinacidil was dose-dependent. The concentration of the vehicle solvent (ME2SO) at 100 lM pinacidil is 0.1% (v/v). Although treatment of cells with ME2SO can cause cytotoxicity and induce differentiation [14,2], a 7-day treatment of cells with 0.1% ME2SO in our clonogenic assay caused no decrease in POU5F1 (OCT4) and SSEA-3 expression. It is possible that high levels of bFGF (100 ng/ml) present in the mTESR media used in the clonogenic assay prevented cell differentiation. Under the post-thaw conditions (hESC culture medium that contained 4 ng/ml of bFGF), we did observe some differentiation of cells in 0.1% ME2SO, based on the expression of SSEA-3, SSEA-4 and TRA-1-60 markers. Interestingly, pinacidil-treated cells showed less differentiation than 0.1% ME2SO control. Martin-Ibanez et al. noticed a similar effect of Y-27632 on decreasing spontaneous differentiation of cells after cryopreservation [20]. These observations highlight the possibilities of using pinacidil for reducing differentiation during routine hESC maintenance. Our previous studies addressed longer exposure of cells to pinacidil as well as long-term effects of passaging cells in pinacidil and revealed no adverse effect of the compound on differentiation ability or karyotypic stability [9]. Further studies need to address whether epigenetic changes occur during this exposure and whether they may subsequently affect the cell fates. Moreover, detailed genetic analysis is warranted to detect a possible presence of mutations that cannot be detected by karyotyping. Despite the initially high post-thaw viability of cells in the control conditions, a significant proportion of cells never survive re-plating. Low cell survival indicates the potential for intense selection in culture. If by chance, a mutation arises that confers competitive advantage on a cell, the cell with the advantageous

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mutation is likely to outcompete other cells and overtake the culture. Indeed, such ‘culture-adapted’ cells that harbour genetic mutations are frequently observed in hESC cultures [12,8]. The occurrence of culture-adapted cells is particularly worrying if the hESCs are to be used in clinical applications [10]. Addition of pinacidil to the post-thaw culture medium significantly improves attachment and thereby survival of hESC population and this is likely to lessen the selection pressure, which might otherwise favour the selection of mutant cells. We showed recently that pinacidil strongly inhibits ROCK activity in vitro [9]. This suggests that pinacidil and Y-27632 may act via similar pathways to promote hESC survival. ROCK plays a key role in a complex network of signalling mechanisms that regulate the cytoskeleton. In particular, ROCK promotes the stabilization of filamentous actin and triggers a signalling cascade that leads to coupling of actin–myosin filaments to the plasma membrane, resulting in actin–myosin contractility and membrane blebbing [17,4,19]. ROCK inhibition by siRNA or chemical inhibitors prevents excessive actin–myosin contractile force in dissociated cells, allowing the cells to re-attach and survive [11]. Indeed, we have shown that pinacidil reduces membrane blebbing and promotes cell attachment of cryopreserved hESCs, leading to a better cell survival. In addition to ROCK, pinacidil inhibits a number of other kinases, many of which are also targets of Y-27632 [9]. Notably, both compounds reduce PRK2 activity by over 90%. Other overlapping targets include MNK1, RSK1 and AMPK. However, the role of these kinases in cell survival remains unclear but warrant further investigation given they are implicated in the mechanism of action of both pinacidil and Y-27632. In conclusion, our results show that pinacidil effectively enhances post-thaw survival of hESCs. Efficient survival of hESCs thawed after cryopreservation is important to reduce selective pressures on cells in culture that could otherwise lead to genetic instability. Pinacidil provides a cheaper alternative to Y-27632 inhibitor and has been used clinically in the past. Acknowledgments We are grateful for technical support from Qiushi Huang. This research was funded by MRC and the ESTOOLS consortium under the Sixth Research Framework Programme of the European Union contract LSHG-CT-2006-018739. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cryobiol.2011.10.002. References [1] O. Adewumi, B. Aflatoonian, L. Ahrlund-Richter, M. Amit, P.W. Andrews, G. Beighton, P.A. Bello, N. Benvenisty, L.S. Berry, S. Bevan, B. Blum, J. Brooking, K.G. Chen, A.B. Choo, G.A. Churchill, M. Corbel, I. Damjanov, J.S. Draper, P. Dvorak, K. Emanuelsson, R.A. Fleck, A. Ford, K. Gertow, M. Gertsenstein, P.J. Gokhale, R.S. Hamilton, A. Hampl, L.E. Healy, O. Hovatta, J. Hyllner, M.P. Imreh, J. ItskovitzEldor, J. Jackson, J.L. Johnson, M. Jones, K. Kee, B.L. King, B.B. Knowles, M. Lako, F. Lebrin, B.S. Mallon, D. Manning, Y. Mayshar, R.D. McKay, A.E. Michalska, M. Mikkola, M. Mileikovsky, S.L. Minger, H.D. Moore, C.L. Mummery, A. Nagy, N. Nakatsuji, C.M. O’Brien, S.K. Oh, C. Olsson, T. Otonkoski, K.Y. Park, R. Passier, H. Patel, M. Patel, R. Pedersen, M.F. Pera, M.S. Piekarczyk, R.A. Pera, B.E. Reubinoff, A.J. Robins, J. Rossant, P. Rugg-Gunn, T.C. Schulz, H. Semb, E.S. Sherrer, H. Siemen, G.N. Stacey, M. Stojkovic, H. Suemori, J. Szatkiewicz, T. Turetsky, T. Tuuri, S. van den Brink, K. Vintersten, S. Vuoristo, D. Ward, T.A. Weaver, L.A. Young, W. Zhang, Characterization of human embryonic stem cell lines by the international stem cell initiative, Nat. Biotechnol. 25 (2007) 803–816.

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