Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of ...cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.
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•Coupling NGN2 expression and SMAD/WNT inhibition yields human patterned induced neurons (hpiNs)•Single-cell analysis indicates excitatory, neuronal identity, with variable maturity•A CAMK2A::GFP reporter gene isolates more differentiated neurons from progenitors•CAMK2A+ hpiNs display AMPAR- and NMDAR-mediated synaptic transmission
Nehme et al. combine two strong neuralizing factors (transcription factor programming and small molecule patterning) to generate human excitatory neurons from stem cells. They further undertake single-cell and reporter gene approaches to select highly differentiated neurons with increased functionality, augmenting their utility in the modeling of nervous system disorders.
While there are a growing number of human pluripotent stem cell repositories, genetic diversity remains limited in most collections and studies. Here, we discuss the importance of incorporating ...diverse ancestries in these models to improve equity and accelerate biological discovery.
DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of ...replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.
Sensory information may be represented in the brain by stereotyped mapping of axonal inputs or by patterning that varies between individuals. In olfaction, a stereotyped map is evident in the first ...sensory processing centre, the olfactory bulb (OB), where different odours elicit activity in unique combinatorial patterns of spatially invariant glomeruli. Activation of each glomerulus is relayed to higher cortical processing centres by a set of ∼20-50 'homotypic' mitral and tufted (MT) neurons. In the cortex, target neurons integrate information from multiple glomeruli to detect distinct features of chemically diverse odours. How this is accomplished remains unclear, perhaps because the cortical mapping of glomerular information by individual MT neurons has not been described. Here we use new viral tracing and three-dimensional brain reconstruction methods to compare the cortical projections of defined sets of MT neurons. We show that the gross-scale organization of the OB is preserved in the patterns of axonal projections to one processing centre yet reordered in another, suggesting that distinct coding strategies may operate in different targets. However, at the level of individual neurons neither glomerular order nor stereotypy is preserved in either region. Rather, homotypic MT neurons from the same glomerulus innervate broad regions that differ between individuals. Strikingly, even in the same animal, MT neurons exhibit extensive diversity in wiring; axons of homotypic MT pairs diverge from each other, emit primary branches at distinct locations and 70-90% of branches of homotypic and heterotypic pairs are non-overlapping. This pronounced reorganization of sensory maps in the cortex offers an anatomic substrate for expanded combinatorial integration of information from spatially distinct glomeruli and predicts an unanticipated role for diversification of otherwise similar output neurons.
Genomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that ...act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these “replication timing quantitative trait loci” (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences.
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•Replication timing, a driver of locus-specific mutation rates, varies among humans•Whole genome sequence data can be used to study DNA replication activity•Replication timing associates with common polymorphisms near replication origins•Replication timing QTLs have megabase-scale effects on replication and transcription
Replication timing varies among humans, and genetic variants associate with replication timing. These replication timing quantitative trait loci are cis-acting modifiers of replication timing, gene expression levels, and local mutation rates and are an unexpected source of potential wide-reaching variation between individuals.
To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare ...protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.
We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs ...were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.
Abnormalities of brain connectivity and signal transduction are consistently observed in individuals with schizophrenias (SZ). Underlying these anomalies, convergent in vivo, post mortem, and genomic ...evidence suggest abnormal oligodendrocyte (OL) development and function and lower myelination in SZ. Our primary hypothesis was that there would be abnormalities in the number of induced pluripotent stem (iPS) cell-derived OLs from subjects with SZ. Our secondary hypothesis was that these in vitro abnormalities would correlate with measures of white matter (WM) integrity and myelination in the same subjects in vivo, estimated from magnetic resonance imaging. Six healthy control (HC) and six SZ iPS cell lines, derived from skin fibroblasts from well-characterized subjects, were differentiated into OLs. FACS analysis of the oligodendrocyte-specific surface, glycoprotein O4, was performed at three time points of development (days 65, 75, and 85) to quantify the number of late oligodendrocyte progenitor cells (OPCs) and OLs in each line. Significantly fewer O4-positive cells developed from SZ versus HC lines (95% CI 1.0: 8.6, F
= 8.06, p = 0.02). The difference was greater when corrected for age (95% CI 5.4:10.4, F
= 53.6, p < 0.001). A correlation between myelin content in WM in vivo, estimated by magnetization transfer ratio (MTR) and number of O4-positive cells in vitro was also observed across all time points (F
= 4.3, p = 0.07), reaching significance for mature OLs at day 85 in culture (r = 0.70, p < 0.02). Low production of OPCs may be a contributing mechanism underlying WM reduction in SZ.
The effect of the substitutional and vacancy type defects on the H
2
adsorption energy over a monolayer hexagonal boron nitride (h-BN) substrate has been studied by using the van der Waals density ...functional theory calculations. Carbon doping at the boron site or formation of boron vacancy can be an effective way to increase the adsorption energy value of a pristine h-BN substrate. The repulsive lateral interaction present in between the two H
2
molecules plays a vital role in case of multiple H
2
molecule adsorption over the substrate. Also, the carbon cluster formation during doping can have a favorable effect in the overall storage capacity of the h-BN substrate.
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