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
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