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.
Highlights
New advances in somatic cell reprogramming offer unique access to human neurons from defined patient groups for modeling neurological disorders in vitro
When expressed in human fetal fibroblasts, the three constructs resulted in different levels of expression of the conversion genes (Fig 1B and C), and we found that the pB.pA construct, yielding the highest ASCL1 to BRN2 protein expression ratio, resulted in the highest level of neural conversion (Fig 1D)
This type of conversion makes it possible to study otherwise hard to access patient- and disease-specific neurons and holds great promise for creating age-relevant models of neurological disorders. induced neurons (iNs), that are obtained via direct conversion, present a faster route by which to generate neurons compared to conventional reprogramming approaches using induced pluripotent stem cells followed by directed differentiation
Summary
New advances in somatic cell reprogramming offer unique access to human neurons from defined patient groups for modeling neurological disorders in vitro. The most common route to patient- and disease-specific neurons to date is through reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), followed by directed neural differentiation (Nityanandam & Baldwin, 2015) This approach has led to important insights into neurodevelopmental disorders and mechanisms underlying neural pathologies (Ebert et al, 2009; Lee et al, 2009; Lafaille et al, 2012), a number of studies show that reprogramming into pluripotency resets the age of the cells such that the resulting neurons are very young (Maherali et al, 2007; Meissner et al, 2008; Lapasset et al, 2011; Miller et al, 2013; Mertens et al, 2015).
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