Strategic Application of Epigenetic Regulators for Efficient Neuronal Reprogramming of Human Fibroblasts.

Background and Objectives: Cellular reprogramming in regenerative medicine holds great promise for treating patients with neurological disorders. In this regard, small molecule-mediated cellular conversion has attracted special attention because of its ease of reproducibility, applicability, and fewer safety concerns. However, currently available protocols for the direct conversion of somatic cells to neurons are limited in clinical application due of their complex nature, lengthy process, and low conversion efficiency. Methods and Results: Here, we report a new protocol involving chemical-based direct conversion of human fibroblasts (HF) to matured neuron-like cells with a short duration and high conversion efficiency using temporal and strategic dual epigenetic regulation. In this protocol, epigenetic modulation by inhibition of histone deacetylase and bromodomain enabled to overcome "recalcitrant" nature of adult fibroblasts and shorten the duration of neuronal reprogramming. We further observed that an extended epigenetic regulation is necessary to maintain the induced neuronal program to generate a homogenous population of neuron-like cells. Conclusions: Therefore, our study provides a new protocol to produce neurons-like cells and highlights the need of proper epigenetic resetting to establish and maintain neuronal program in HF.

Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes.

Astrocyte-to-neuron reprogramming is a promising therapeutic approach for treatment of neurodegenerative diseases. The use of small molecules as an alternative to the virus-mediated ectopic expression of lineage-specific transcription factors negates the tumorigenic risk associated with viral genetic manipulation and uncontrolled differentiation of stem cells. However, because previously developed methods for small-molecule reprogramming of astrocytes to neurons are multistep, complex, and lengthy, their applications in biomedicine, including clinical treatment, are limited. Therefore, our objective in this study was to develop a novel chemical-based approach to the cellular reprogramming of astrocytes into neurons with high efficiency and low complexity. To accomplish that, we used C8-D1a, a mouse astrocyte cell line, to assess the role of small molecules in reprogramming protocols that otherwise suffer from inconsistencies caused by variations in donor of the primary cell. We developed a new protocol by which a chemical mixture formulated with Y26732, DAPT, RepSox, CHIR99021, ruxolitinib, and SAG rapidly and efficiently induced the neural reprogramming of astrocytes in four days, with a conversion efficiency of 82 ± 6%. Upon exposure to the maturation medium, those reprogrammed cells acquired a glutaminergic phenotype over the next eleven days. We also demonstrated the neuronal functionality of the induced cells by confirming KCL-induced calcium flux.