Application of Hanging Drop Culture for Retinal Precursor-Like Cells Differentiation of Human Adipose-Derived Stem Cells Using Small Molecules

Abstract

Retinal degenerative diseases lead to blindness due to poorly regenerative potential of the retina. Recently, cell therapy is more considered for degenerative diseases. Autologous mesenchymal stem cells derived from adipose tissue are a suitable source for this purpose. Therefore, we conducted a stepwise efficient method to differentiate human adipose-derived stem cells (hADSCs) into retinal precursor-like cells in vitro. We compared two differentiation protocols, monolayer and hanging drop cultures. Through the defined medium and 3D hanging drop culture method, we could achieve up to 75 retinal precursor gene expression profile (PAX6, RAX, CHX10, and CRX) from hADSCs. By imitation of in vivo development, for direct conversion of stem cells into retinal cells, the suppression of the BMP, Nodal, and Wnt signaling pathways was carried out by using three small molecules. The hADSCs were primarily differentiated into anterior neuroectodermal cells by expression of OTX2, SIX3, and Beta-TUB III and then the differentiated cells were propelled into the retinal cells. According to our data from real-time PCR, RT-PCR, immunocytochemistry, and functional assay, it seems that the hanging drop method improved retinal precursor differentiation yield which these precursor-like cells respond to glutamate neurotransmitter. Regarding the easy accessibility and immunosuppressive properties of hADSCs and more efficient hanging drop method, this study may be useful for future autologous cell therapy of retinal degenerative disorders.