RESUMO
All-trans-retinoic acid (ATRA) and its associated analogues are important mediators of cell differentiation and function during the development of the nervous system. It is well known that ATRA can induce the differentiation of neural tissues from human pluripotent stem cells. However, it is not always appreciated that ATRA is highly susceptible to isomerisation when in solution, which can influence the effective concentration of ATRA and subsequently its biological activity. To address this source of variability, synthetic retinoid analogues have been designed and synthesised that retain stability during use and maintain biological function in comparison to ATRA. It is also shown that subtle modifications to the structure of the synthetic retinoid compound impacts significantly on biological activity, as when exposed to cultured human pluripotent stem cells, synthetic retinoid 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid, 4a (para-isomer), induces neural differentiation similarly to ATRA. In contrast, stem cells exposed to synthetic retinoid 3-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid, 4b (meta-isomer), produce very few neurons and large numbers of epithelial-like cells. This type of structure-activity-relationship information for such synthetic retinoid compounds will further the ability to design more targeted systems capable of mediating robust and reproducible tissue differentiation.
Assuntos
Retinoides/síntese química , Retinoides/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Desenvolvimento Embrionário , Humanos , Luz , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Retinoides/química , Tretinoína/químicaRESUMO
Twofold sila-substitution (C/Si exchange) of the clinically used RXR-selective retinoid agonist bexarotene leads to disila-bexarotene, which displays pharmacological potency similar to that of the parent carbon compound, as shown in a HeLa-cell-based RXR assay. Formal exchange of the SiCH2CH2 Si group in disila-bexarotene with a SiCH2Si or SiOSi moiety leads to the disila-bexarotene analogues 8 and 9. The silicon compounds 8 and 9 were synthesized in multistep syntheses, starting from HC≡C(CH3)2SiCH2Si(CH3)2C≡CH and HC≡C(CH3)2SiOSi(CH3)2C≡CH, respectively. The key step in the syntheses of 8 and 9 is a cobalt-catalyzed [2+2+2] cycloaddition reaction that affords the 1,3-disilaindane and 2-oxa-1,3-disilaindane skeletons. Disila-bexarotene and its analogues 8 and 9 were studied for their biological effects relative to all-trans retinoic acid in cultured human pluripotent stem cells. The parent carbon compound bexarotene was included in some of these biological studies. Although the silicon-containing bexarotene analogues disila-bexarotene, 8, and 9 appear not to regulate the differentiation of TERA2.cl.SP12 stem cells, preliminary evidence indicates that these compounds may possess enhanced functions over the parent compound bexarotene, such as induction and regulation of cell death and cell numbers. The biological data obtained indicate that bexarotene, contrary to the silicon-containing analogues disila-bexarotene, 8, and 9, may partially act to induce cell differentiation.
Assuntos
Receptores X de Retinoides/agonistas , Silício/química , Células-Tronco/efeitos dos fármacos , Tetra-Hidronaftalenos/química , Bexaroteno , Diferenciação Celular , Proliferação de Células , Cristalografia por Raios X , Humanos , Conformação Molecular , Receptores X de Retinoides/metabolismo , Células-Tronco/citologia , Tetra-Hidronaftalenos/síntese química , Tetra-Hidronaftalenos/farmacologiaRESUMO
Retinoids are important molecules involved in the development and homeostasis of the nervous system. As such, various retinoid derivatives are often found in culture media and supplement formulations to support the growth and maintenance of neural cells. However, all-trans-retinoic acid (ATRA) and its associated derivatives are light sensitive and are highly susceptible to isomerisation. This can lead to variability in retinoid concentrations and the nature of the retinoid species present in culture solutions which in turn can influence biological activity and introduce inconsistency. We have previously described the development of the synthetic retinoid derivative, EC23, as a chemically and light stable alternative that does not degrade and has biological activity similar to ATRA. In this study we demonstrate that the addition of exogenous retinoid can significantly enhance neuronal differentiation of both human neuroprogenitor and human embryonic stem cells. In the former, both ATRA and EC23 induced increased maturation and stabilisation of the axonal cytoskeleton. However, EC23 was particularly potent at lower nanomolar concentrations resulting in significantly greater neurogenesis than ATRA. In ES cells enhanced motor neuron marker expression was also detected in response to both retinoids when incorporated into an established protocol for neuronal differentiation. We propose that synthetic retinoid EC23 represents a valuable addition to the formulation of new and existing culture supplements to enhance neuronal differentiation whilst enabling improved consistency.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Retinoides/farmacologia , Benzoatos/farmacologia , Células Cultivadas , Técnicas de Cocultura/métodos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Tetra-Hidronaftalenos/farmacologia , Tretinoína/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
The natural retinoid, all-trans retinoic acid (ATRA), is widely used to direct the in vitro differentiation of stem cells. However, substantial degradation and isomerisation of ATRA in response to UV-vis light has serious implications with regard to experimental reproducibility and standardisation. We present the novel application of proteomic biomarker profiling technology to stem cell lysates to rapidly compare the differentiation effects of ATRA with those of two stable synthetic retinoid analogues, EC19 and EC23, which have both been shown to induce differentiation in the embryonal carcinoma cell line TERA2.cl.SP12. MALDI-TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) protein profiles support previous findings into the functional relationships between these compounds in the TERA2.cl.SP12 line. Subsequent analysis of protein peak data enabled the semi-quantitative comparison of individual retinoid-responsive proteins. We have used ion exchange chromatographic protein separation to enrich for retinoid-inducible proteins, thereby facilitating their identification from SDS-PAGE gels. The cellular retinoid-responsive proteins CRABP-I, CRABP-II, and CRBP-I were up-regulated in response to ATRA and EC23, indicating a bona fide retinoid pathway response to the synthetic compound. In addition, the actin filament regulatory protein profilin-1 and the microtubule regulator stathmin were also elevated following treatment with both ATRA and EC23. The up-regulation of profilin-1 and stathmin associated with retinoid-induced neural differentiation correlates with their known roles in cytoskeletal reorganisation during axonal development. Immunological analysis via western blotting confirmed the identification of CRABP-I, profilin-1 and stathmin, and supported their observed regulation in response to the retinoid treatments.