Pinacidil enhances survival of cryopreserved human embryonic stem cells.
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 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.
Centre for Stem Cell Biology, University of Sheffield, Western Bank, Sheffield, UK. i.barbaric@sheffield.ac.uk
Ivana
I
Barbaric
Mark
M
Jones
Kristina
K
Buchner
Duncan
D
Baker
Peter W
PW
Andrews
Harry D
HD
Moore
D000577:Amides / Q000494:pharmacology; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000235:genetics / Q000378:metabolism; D000959:Antihypertensive Agents / Q000494:pharmacology*; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D058732:Embryoid Bodies / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005615:Freezing; D015870:Gene Expression; D006801:Humans; D007621:Karyotyping; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020110:Pinacidil / Q000494:pharmacology*; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D055746:Stage-Specific Embryonic Antigens / Q000235:genetics / Q000378:metabolism
D000577:Amides; D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; D000959:Antihypertensive Agents; D015415:Biomarkers; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011509:Proteoglycans; D011725:Pyridines; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C052924:stage-specific embryonic antigen-3; C066584:stage-specific embryonic antigen-4; C108830:Y 27632; D020110:Pinacidil