Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

Abstract

Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance. O ur general aim is to identify genes and pathways of early neural differentiation that are relevant for the development and function of the human nervous system. In vitro differentiation of human embryonic stem cells (hESCs) yielding developmentally competent neuronal progenitors is an attractive model for these studies and several approaches for this have been developed, including those by our group (1).Large-scale loss-of-function analysis by RNA interference (RNAi) using small hairpin (shRNA) or small interfering RNA (siRNA) -mediated knockdown of genes is a powerful screening approach in mammalian cells (2-7). ShRNAs provide the opportunity for long-term silencing by stable infection of cells maintained under selection pressure. Furthermore, pooled libraries of shRNAs can be efficiently used instead of arrayed individual clones. Screening experiments can be designed for detection of shRNAs that produce a desired effect in a cell (positive screens) or for the depletion of shRNA species that silence a necessary gene (dropout screens). The abundance of shRNAs has been measured by using barcodes and array hybridization (8, 9), but this procedure is subject to cross-hybridization and nonlinear responses. Most positive screens have been limited to studies of cellular proliferation or survival because it is easier to analyze the recovered enriched shRNAs (10). For dropout screens it is critical to accurately measure the abundance of shRNAs that are retained in cells at the end of the experiment to determine which shRNAs were depleted (8). In part because of this challenge, ...

Aberrant expression of miR‐218 and miR‐204 in human mesial temporal lobe epilepsy and hippocampal sclerosis—Convergence on axonal guidance

“…The results from this initial screening should be interpreted with caution, as there are only two control samples; thus a second patient cohort with TLE and HS was included for further validation of miRNA expression. As neuronal loss and neurodegeneration have common denominators with neurodevelopment, we investigated the expression patterns of these 30 miRNAs in the hippocampus of domestic pigs during embryonic development (E42–E115) and 14 miRNAs showed >twofold up‐regulation or down‐regulation (Fig. ).…”

EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy

“…A general role of autophagy in neuronal embryogenesis is supported by the finding of severe neural tube defects in the Ambra1 knockout mouse but remains to be further elucidated ( Cecconi et al , 2007 ; Fimia et al , 2007 ). Moreover, certain stem cells involved in neurodevelopment may also have a role in neurodegeneration, suggesting that defects in the pathways leading to aberrant development may also result in later neurodegeneration, due to a pleiotropic effect of some neurodevelopmental genes on the nervous system ( Zhang et al , 2013 ). These observations place EPG5 -related Vici syndrome within an emerging group of early-onset neurodevelopmental and neurodegenerative disorders associated with defective autophagy (reviewed in Ebrahimi-Fakhari et al , 2014 , 2015 ) such as static encephalopathy in childhood with neurodegeneration in adulthood (SENDA) due to recessive mutations in WDR45 ( Haack et al , 2012 ), and a recently recognized syndromic form of early-onset ataxia due to recessive mutations in SNX14 ( Thomas et al , 2014 ; Akizu et al , 2015 ).…”

Genome-Scale CRISPR Screening Identifies Novel Human Pluripotent Gene Networks

“…Despite the huge potential, genetic screening using hPSCs has been limited by their expensive and tedious cell culture requirements (Chen et al, 2011), and reduced genetic manipulation efficiencies (Ihry et al, 2018). Only a few shRNA screens have been conducted in hPSCs (Chia et al, 2010; Zhang et al, 2013), however shRNAs have a high level of off targets and do not cause a complete loss of function, which is difficult to interpret (DasGupta et al, 2005; Echeverri et al, 2006; Kampmann et al, 2015; McDonald et al, 2017). Currently, the CRISPR/Cas9 system is the genetic screening tool of choice because it can efficiently cause loss-of-function alleles (Cong et al, 2013; Jinek et al, 2012; Mali et al, 2013).…”