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

Abstract

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.

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

“…Our maturation step using the cocktail FYLDSp generated TUJ1 + /MAP2 + /NeuN + /vGLUT1 + neuron-like cells, indicating that the challenges posed by JQ-1(+) during neuronal maturation can be addressed. To this end, we investigated whether these reprogrammed cells attained neuronal functionality by analyzing the calcium influx/outflow, which is one of the functional properties of neurons ( 28 , 29 ). Incubating the cells with Rhod 2-AM, a high affinity Ca 2+ indicator, showed spontaneous calcium influx followed by a gradual outflow of calcium over period of 50 sec.…”

Reprogramming stem cells in regenerative medicine

“…At the same time, previous small-molecule reprogramming often has the disadvantage of being multistep, complex, and lengthy, and thus its application can be limited. Fernandes et al 49 developed a new chemical-based approach in which a chemical mixture formulated with RepSox, DAPT, Y26732, CHIR99021, ruxolitinib, and SAG was used on the mouse astrocyte cell line C8-D1a to rapidly, simply, and efficiently induce the reprogramming of astrocytes to neurons within 4 days with 82 � 6% conversion efficiency. These reprogrammed cells gained a glutamatergic phenotype for the next 11 days and the neuronal function of the induced cells was demonstrated by identifying the KCL-induced calcium flux.…”

Cell-Type-Dependent Role for nsP3 Macrodomain ADP-Ribose Binding and Hydrolase Activity during Chikungunya Virus Infection

“…To directly compare the replication kinetics of CHIKV 181/25 (WT) in astrocytic and neuronal cell lines in vitro, well-characterized mouse C8-DIA astrocytic cells [60,64] and NSC-34 motor neuronal cells [53,61,65] were infected at an MOI of 5, and the production of infectious virus was quantified by plaque formation on Vero cells (Figure 1A). Virus was produced more rapidly and to higher titer throughout 24 h by C8-D1A cells compared to previously studied NSC-34 cells.…”