All-trans-retinoic acid (ATRA) and its stereoisomer, 9-cis-retinoic acid (9-cis-RA), are two active metabolites of vitamin A which are known to regulate a broad range of biological processes, including vertebrate development, growth, proliferation and cell differentiation. Retinoids are understood to induce cell differentiation by binding to distinct families of ligand-activated nuclear receptors (RARs) and retinoid X receptors (RXRs).
Naturally occurring retinoids, including ATRA, 9-cis-RA (9CRA) and 13-cis-retinoic acid (13CRA), can be described as chromophores since they selectively absorb light. Such molecules are essentially composed of three structurally distinct regions: a hydrophobic end, a polyene linker and an acidic group. The polyene linker in naturally occurring retinoids is highly conjugated and it is this region that gives it the ability to absorb light (at a frequency of 300-400 nm depending on the solvent). It is due to this feature that these molecules are particularly susceptible to photoisomerisation and can degrade into a mixture of different retinoic acid isomers. The resulting concentration of retinoid levels has also been shown to decrease markedly over time in culture and this could be a consequence of both their degradation and metabolism. Moreover, retinoids such as ATRA are temperature sensitive and are known to oxidise readily.
Isomerisation of ATRA is understood to be an important part of its metabolic pathway because the resulting isomers have different mechanisms of action. This is an important point that is all too often over-looked by users of ATRA in the cell culture laboratory. It has been reported that the isomers of ATRA differentially affect the ability of mammalian stem cells to differentiate along alternative lineages and stated that extreme care should be taken to protect retinoic acids from isomerisation in such experiments (Murayama et al J. Nutr. Sci. Vitaminol 43(167) 1977. This is particularly relevant when the cellular response is determined by the concentration(s) of the isomer(s) present in solution. For example, the induction of the differentiation of pluripotent stem cells using retinoids is quite often variable, resulting in the formation of heterogeneous cultures of cells composed of different proportions of alternative cell types. To reduce such variability in differentiation response and improve reproducibility, it is essential that whatever is used to induce cell differentiation is in the same form and concentration every time it is used. Currently, this cannot be guaranteed when using reagents such as ATRA and its stereoisomers, all of which are light and heat sensitive and are prone to undergo isomerisation under sample preparation conditions, under storage of stock solution conditions and in culture conditions.
It is because the different isomers have diverse effects on cells that some attempts have been made to control ATRA's sensitivity and tendency to isomerise. For example, a number of additives preventing cis-trans interconversions or oxidation of retinoic acids have been evaluated, including bovine serum albumin (BSA), fibrogen, lysozyme, phosphatidylcholine N-ethylmaleimide and vitamin C (Chen et al. J. Am. Chem. Soc. 126 (410) 1995; Wang et al. J Chromatogr 796, 283, 2003). However, the addition of such molecules to cell culture media is not desirable; for example, the use of BSA would not be possible in serum free culture media.
There is a need for improved alternatives to ATRA that do not suffer from the aforementioned stability problems and which can readily be used in cell culture applications.