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 Centre for Stem Cell Biology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK b Sheffield Diagnostic Genetic Services, Sheffield Children’s Hospital, Sheffield S10 2TH, UK article info Article history: Received 11 March 2011 Accepted 6 October 2011 Available online 17 October 2011 Keywords: Human embryonic stem cell Cryopreservation Pinacidil abstract Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to dif- ferentiate 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 recov- ering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y- 27632. 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 prolif- erative capacity and differentiate into many cell types of the body [26,24,25]. There is great interest in using these cells in regenera- tive 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 cryopreser- vation 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 dif- ferentiation to the cell of choice. Therefore, aside from the imme- diate effects of protection from cryo-injury, the rates of cell apoptosis and differentiation after thawing can have a major im- pact on recovery of the cells and their amplification. Pluripotent stem cells are maintained in a niche created in culture as the col- ony develops, either in the presence of feeder cells (usually mitot- ically inactivated mouse or human embryonic fibroblasts), or more recently with extracellular matrix components (e.g. Matrigel) and 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 cul- ture. In this regard, the Rho-associated kinase (ROCK) inhibitor (Y-27632) has been shown to prevent the apoptosis of single plu- ripotent 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-N 0 -4-pyridinyl-N 00 -(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 0011-2240/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2011.10.002 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). Cryobiology 63 (2011) 298–305 Contents lists available at SciVerse ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo