REST suppression mediates neural conversion of adult human fibroblasts via microRNA‐dependent and ‐independent pathways

Abstract

Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient‐ and disease‐specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient‐derived material for large‐scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST‐controlled neuron‐specific microRNAs miR‐9 and miR‐124, we show that the effect of REST inhibition is only partially mediated via microRNA up‐regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one‐step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single‐vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease.

Dual modulation of neuron‐specific microRNAs and the REST complex promotes functional maturation of human adult induced neurons

“…For biopsy sampling information, see Drouin-Ouellet et al [22]. For biopsy sampling information, see Drouin-Ouellet et al [22].…”

“…The single vector containing Ascl1, Brn2, and shREST was used at MOI of 20 [22]. The DNA plasmids used are expressed in the open reading frames (ORFs) for Ascl1, Brn2 with short hairpin RNA (shRNA) targeting REST or ORFs for Ascl1, Brn2 with miRNA loops for miR9/9* and miR124 in combination with shRNA targeting REST.…”

“…The following protocol, used for direct conversion, was previously described in Shrigley et al [25] and Drouin-Ouellet et al [22], with the omission of the replating step at day 12 of conversion for some of the experiments (see Results). Prior to plating, the wells were coated overnight with a combination of PFL: polyornithine (15 µgÁmL À1 ), fibronectin (0.5 ngÁµL À1 ), and laminin (5 µgÁmL À1 ).…”

“…Also, microRNAs [13][14][15][16] and small molecules [17][18][19][20] have been shown to play a critical role in the conversion process. Additionally, we have shown that while Ascl1 and Brn2 efficiently convert human fetal fibroblasts to neurons, human fibroblasts from adult donors require codelivery of miR9 and miR124 and/or knockdown of RE1-silencing TF (REST) [22]. The forced expression of miR124 and another neuron-specific microRNA, miR9/9*, is sufficient for the cells converted from adult fibroblasts to adopt neuronal features [14].…”

“…Overexpression of neuron-specific microRNA miR124 triggers a neuronal gene expression profile in a number of different cell types: HeLa cells, human embryonic carcinoma stem cells (NT2), mouse neuronal progenitor cells (N2A), human retinal epithelial cells (ARPE19), and primary mouse embryonic fibroblasts [21]. The effect of short hairpin-mediated knockdown of REST (RESTi) on neuronal conversion efficiency is mediated at least in part via the upregulation of miR9 and miR124 as an effect of RESTi, but it is becoming clear that RESTi also acts via microRNA-independent pathways [22]. Additionally, we have shown that while Ascl1 and Brn2 efficiently convert human fetal fibroblasts to neurons, human fibroblasts from adult donors require codelivery of miR9 and miR124 and/or knockdown of RE1-silencing TF (REST) [22].…”