The first advantage is to be able to explore genes and pathways expressed at physiological levels without the need to introduce exogenous gene expression systems. This was exemplified in studies that screened compounds for genetic forms of ASD induced by the loss of function of FRMP
[1][2][3][104,105,106] and duplication of a segment of chromosome 7
[4][107]. FXS was the first neurodevelopmental disorder to be modeled with this aim using iPSCs. FXS is a neurodevelopmental disorder characterized by mild-to-severe intellectual disability and abnormal behaviors, such as attention deficit, anxiety and depression
[5][108]. FXS is also the most common known monogenic cause of autism, with 43–67% of male patients meeting the criteria of autism spectrum disorders. At the genetic level, FXS is linked to mutations (triplet repeats) in the 5′-untranslated region of the fragile X mental retardation 1 (FMR1) gene, which results in the absence of the FMRP protein. FMRP is a brain-specific RNA-binding protein that regulates the transport and translation of many mRNAs that play an important role in learning and memory
[6][109]. Consequently, screenings were conducted in neural progenitors to identify compounds that could increase FRMP levels. Kaufmann and collaborators used FXS-patient iPSCs to develop an image-based HTS assay measuring the levels of FRMP in neural stem cells using immunofluorescence
[1][104]. In all, 50,000 compounds were screened, including epigenetic regulators with known mode of action (7%), molecules covering a broad chemical space and biological diversity (46%) and a set of randomly selected compounds from an internal archive (47%). Four hits were identified and further confirmed for efficacy and absence of toxicity in dose-response experiments but were not further investigated for their mode of action or evaluated in an animal model. At the same time, Kumari and collaborators described the screening of 5000 known tool compounds and approved drugs in neural stem cells differentiated from an FXS patient-derived iPSC line using time-resolved fluorescence resonance energy transfer assay for FMRP detection
[2][105]. Interestingly, the primary screening was performed in a 1536-well plate format, a format rarely used for cell-based assays and six compounds were identified that modestly increased FMR1 gene expression in FXS patient cells. Although none of these studies resulted in clinically relevant compounds, they provide strong proof of principle of the assays performed on patient-derived neural stem cells in a very high-throughput format to identify new lead compounds for FXS drug development. More recently, Li and collaborator used the newly described CRISPR/Cas9 system to create a reporter line for detecting FMR1 gene reactivation in human neural cells and used it to screen 1262 bioactive compounds
[3][106]. This revealed two epigenetic regulators, 5-aza-dC and 5-aza-C, that significantly restored FRMP levels in disease cells. This study demonstrated that CRISPR/Cas9 can successfully be combined with iPSC-derived neural cells to design customized screening lines by knocking the luciferase reporter into endogenous target genes in order to obtain reporter lines and to reduce screening costs while increasing screening performance. This was possible only because iPSC-derived neural stem cells physiologically express endogenous levels of FRMP and proved the value of screening in human cells differentiated from PSCs. Together, these three studies demonstrate the feasibility and relevance of HTS in the neural progeny of PSCs for neurodevelopmental disorders.