Combinatorial induction with valproic acid and fibroblast growth factor-9 enhances neural differentiation and cell fate commitment in adipose tissue-derived mesenchymal stem cells

Abstract

Recent advances in neural differentiation have unveiled new possibilities that could potentially be applied to the development of human-relevant non-animal models, for use in fields such as biomedical research and drug screening. Thus, the directed differentiation of tissue stem cells toward neural progenitor cells or neural stem cells, by small molecules and growth factors without the need for genetic manipulation, has attracted great attention. The in vitro generation of neural progenitor cells, and their proliferation and lineage commitment are regulated by signaling pathways activated by small molecules and growth factor families, including various fibroblast growth factors (FGFs). FGF-9 regulates the differentiation of neural stem cells, not only during embryonic development, but also contributes to adult neurogenesis and the protection of degenerating neurons. Here, we investigate an improved protocol for neural stem cell proliferation, differentiation and lineage commitment of adipose tissue-derived mesenchymal stem cells (AD-MSCs). We evaluated a cocktail of valproic acid (VPA), CHIR99021 and FGF-9, both alone and in combination, for the potential to induce cell differentiation. The AD-MSCs were isolated from human omentum fat and characterised immunologically by the presence of specific mesenchymal markers and multi-lineage differentiation potential. To assess the potential toxic effect of each cocktail, cell proliferation and viability were determined, followed by confirmation of neural differentiation via quantitative reverse transcriptase real-time PCR (qRT-PCR) and immunocytochemistry. The combined administration of VPA and FGF-9 promoted neurogenesis and neuronal fate commitment of AD-MSCs cultures, leading to significantly increased expression of the neural stem cell markers Nestin and Sox-2, as well as the neuronal cell marker MAP-2. This study contributes to our understanding of the role of FGF-9 in neural differentiation from human AD-MSCs. Our simplified protocol uses VPA and FGF-9 to efficiently generate neural progenitor cells, avoiding complex and prolonged induction methods, making it ideal for use in, for example, in vitro human-relevant drug screening for neurotoxicity.