Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells

Burton, P. et al. (2010) Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells. Biochemical Journal, 432(3), pp. 575-584. (doi:10.1042/BJ20100726)

Burton, P. et al. (2010) Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells. Biochemical Journal, 432(3), pp. 575-584. (doi:10.1042/BJ20100726)

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Publisher's URL: http://dx.doi.org/10.1042/BJ20100726

Abstract

Human embryonic stem cells (hESCs) have enormous potential for use in pharmaceutical development and therapeutics, however to realise this potential there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an established inhibitor of adenosine deaminase (ADA) and cyclic nucleotide phosphodiesterase- 2 (PDE2). hESCs maintained in feeder-free conditions with EHNA for more than 10 passages showed no reduction in hESC associated markers including NANOG, POU5F1 and SSEA 4 compared to cells maintained in feeder-free conditions containing basic fibroblast growth factor (bFGF). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but upon removing EHNA hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure activity relationship analysis found the differentiation blocking properties of EHNA to reside in a pharmacophore comprised of a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large scale or cGMP compliant processes.

Item Type:Articles
Keywords:Adenosine deaminase (ADA), cell culture, differentiation, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), fibroblast growth factor, human embryonic stem cell
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Houslay, Professor Miles and Baillie, Professor George and Baker, Professor Andrew and Burton, Dr Peter and Gilmour, Dr Jane and Kane, Dr Nicole and Mountford, Dr Joanne and McCahill, Dr Angela and Kaupisch, Dr Alexandra and McAbney, Mr John and Milligan, Professor Graeme
Authors: Burton, P., Adams, D. R., Abraham, A., Allcock, R. W., Jiang, Z., McCahill, A., Gilmour, J., McAbney, J., Kaupisch, A., Kane, N. M., Baillie, G. S., Baker, A. H., Milligan, G., Houslay, M. D., and Mountford, J. C.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Journal Name:Biochemical Journal
Journal Abbr.:Biochem. J.
Publisher:Portland Press
ISSN:0264-6021
ISSN (Online):1470-8728
Published Online:05 October 2010
Copyright Holders:Copyright © 2010 Portland Press
First Published:First published in Biochemical Journal 432(3):575-584
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
438301Phosphodiesterase-4 isoforms - intracellular targeting, regulation and potential therapeutic targetsMiles HouslayMedical Research Council (MRC)G0600765Institute of Neuroscience and Psychology