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Direct Neural Conversion of Adult Human Fibroblasts – Generation of Dopaminergic Neurons for Cell-Based Replacement Therapy

Authors: Shelby Shrigley; Maria Pereira; Deirdre B Hoban; Janelle Drouin-Ouellet; Malin Parmar;

Direct Neural Conversion of Adult Human Fibroblasts – Generation of Dopaminergic Neurons for Cell-Based Replacement Therapy

Abstract

Cell-based replacement therapy has great potential to improve the clinical outcome of Parkinson’s disease (PD), a neurodegenerative disease resulting from the progressive loss of dopamine within the nigrostriatal system. Induced neurons (iNs) provide a unique resource for cell replacement therapies that negate the ethical concerns often associated with embryonic stem (ES) derived cells. We have previously shown that the dopaminergic (DA) neuronal fate could be generated from human fetal fibroblasts using forced expression of Ascl1 and specific DA fate determinants including: Lmx1a, Lmx1b, FoxA2 and Otx2. However, this early iN conversion protocol has shown low conversion efficiencies when converting adult fibroblasts. Here, we build on our newly developed reprogramming method to convert adult fibroblasts in a very efficient manner to generate dopaminergic iNs by transducing adult fibroblasts with a new combination of transcription factors combined with a REST knock-down. High content screening analysis showed that 21% of converted cells express tyrosine hydroxylase (TH) using our new reprogramming cocktail. Furthermore, quantitative PCR analysis revealed that those induced DA neurons strongly express other DA markers, including PITX3, DAT and AADC. We are now investigating in vivo the potential of these DA iNs to survive, convert and integrate in the host circuitry of the rat striatum. This study provides a more robust and efficient protocol for the generation of DA iNs from human adult fibroblasts. Results from this first transplantation study will help answer important questions related to the optimal conversion stage for DA iNs to be grafted in order to ensure survival in the host circuitry, and will pave the way for studies assessing their potential for brain repair.

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This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
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This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
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