Abstract
Direct reprogramming is an appealing strategy to generate neurons from a somatic cell by forced expression of transcription factors. The generated neurons can be used for both cell replacement strategies and disease modelling. Using this technique, previous studies have shown that γ-aminobutyric acid (GABA) expressing interneurons can be generated from different cell sources, such as glia cells or fetal fibroblasts. Nevertheless, the generation of neurons from adult human fibroblasts, an easily accessible cell source to obtain patient-derived neurons, has proved to be challenging due to the intrinsic blockade of neuronal commitment. In this paper, we used an optimized protocol for adult skin fibroblast reprogramming based on RE1 Silencing Transcription Factor (REST) inhibition together with a combination of GABAergic fate determinants to convert human adult skin fibroblasts into GABAergic neurons. Our results show a successful conversion in 25 days with upregulation of neuronal gene and protein expression levels. Moreover, we identified specific gene combinations that converted fibroblasts into neurons of a GABAergic interneuronal fate. Despite the well-known difficulty in converting adult fibroblasts into functional neurons in vitro, we could detect functional maturation in the induced neurons. GABAergic interneurons have relevance for cognitive impairments and brain disorders, such as Alzheimer’s and Parkinson’s diseases, epilepsy, schizophrenia and autism spectrum disorders.
Highlights
Direct cell reprogramming offers unique access to human neurons from defined groups of patients for disease-modelling in vitro
We have previously shown that RE1 Silencing Transcription Factor (REST) inhibition is critical for a neuronal conversion of adult skin fibroblast [9], and REST inhibition was included in all reprogramming groups
To further improve the overall conversion efficiency, the reprogramming cocktail was tested with short hairpin RNAs against the REST complex under a U6 promotor (Figure 1A), as this has previously been shown to remove the reprogramming barrier associated with adult cells [9]
Summary
Direct cell reprogramming offers unique access to human neurons from defined groups of patients for disease-modelling in vitro. Direct reprogramming offers new approaches for disease modelling, as it is possible to reprogram skin cells from a human patient group into subtype-specific neurons and thereby generate human neurons with genetic backgrounds that are known to result in a particular disease. With such a human cell-based model, it is possible to study idiopathic forms of disease [2]. Given that these neurons retain the markers of aging, they are ideal candidates for modelling neuronal pathology in late-onset diseases, such as Parkinson’s and Alzheimer’s disease [3].
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