Common genetic variants can have profound effects on cellular function, but studying these effects in primary human tissue samples and during development is challenging. Human induced pluripotent stem cell (iPSC) technology holds great promise for assessing these effects across a range of differentiation contexts. Here, we use an efficient multiplexing strategy to differentiate 215 iPS cell lines towards a midbrain neural fate, including dopaminergic neurons, and profile over 1 million cells sampled across three differentiation timepoints using single cell RNA sequencing. We find that the proportion of neuronal cells produced by each cell line is highly reproducible over different experimental batches, and identify robust molecular markers in pluripotent cells that predict line-to-line differences in cell fate. We identify expression quantitative trait loci (eQTL) that manifest at different stages of neuronal development, and in response to rotenone-induced oxidative stress. We find 1,284 eQTL that colocalise with a known risk locus for a neurological trait, 46% of which are not found in the GTEx catalogue. Our study illustrates how coupling single cell transcriptomics with long-term iPSC differentiation can profile mechanistic effects of human trait-associated genetic variants in otherwise inaccessible cell states.
Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We performed saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identified 3,432 functionally abnormal variants, in three distinct classes. We trained a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants could account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation.
Common genetic variants can have profound effects on cellular function, but studying these effects in primary human tissue samples and during development is challenging. Human induced pluripotent stem cell (iPSC) technology holds great promise for assessing these effects across different differentiation contexts. Here, we use an efficient pooling strategy to differentiate 215 iPS cell lines towards a midbrain neural fate, including dopaminergic neurons, and profile over 1 million cells sampled across three differentiation timepoints using single cell RNA sequencing. We find that the proportion of neuronal cells produced by each cell line is highly reproducible over different experimental batches, and identify robust molecular markers in pluripotent cells that predict line-to-line differences in cell fate. We identify expression quantitative trait loci (eQTL) that manifest at different stages of neuronal development, and in response to oxidative stress, by exposing cells to rotenone. We find over one thousand eQTL that colocalise with a known risk locus for a neurological trait, nearly half of which are not found in GTEx. Our study illustrates how coupling single cell transcriptomics with long-term iPSC differentiation can profile mechanistic effects of human trait-associated genetic variants in otherwise inaccessible cell states.
KPTN-related disorder (KRD) is an autosomal recessive disorder associated with germline variants in KPTN (kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KRD, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn-/- mice display many of the key KRD phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. Assessment of affected individuals has identified concordant selectivity of cognitive deficits, postnatal onset of brain overgrowth, and a previously unrecognised KPTN dosage-sensitivity, resulting in increased head circumference in their heterozygous parents. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape, and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated iPSC models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. Increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KRD in the broader group of mTORC1 related disorders affecting brain structure, cognitive function, and network integrity.
KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn-/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n=6) and postnatal onset of brain overgrowth (n=19). By analysing head size data from their parents (n=24), we have identified a previously unrecognised KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape, and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated iPSC models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we find that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1 related disorders affecting brain structure, cognitive function, and network integrity.
Efficient and effective methods for converting human induced pluripotent stem cells (iPSC) into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells (NSCs). We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of ICC Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.
This protocol outlines the steps of differentiating human induced pluripotent stem cells (iPSCs) into cortical linage neural stem cells (NSCs) using a dual-SMAD and Wnt-inhibition method. The cells are cultured in 6 well plates over the course of 14 days, generating approximately 10 million NSCs per well. An immunocytochemistry quality control assay is used to assess the expression of NSC markers including PAX6, FOXG1 and NESTIN.
Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.
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