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Significance Thyroid hormone deficiencies are the most common preventable causes of intellectual disability. We report that mutations in the thyroid hormone receptor α1 gene (THRA) that result in intellectual disability also reduce brain size. Using human THRA mutation stem cell models, we studied the impact of THRA mutations on human brain development by combining quantitative lineage analysis, gene expression analyses, and novel assays of neuroepithelium formation. We found that THRA regulates the balance between progenitor self-renewal and neurogenesis, and thus overall brain size. Importantly, these in vitro results are consistent with in vivo evidence from magnetic resonance imaging of people with these mutations, advancing our understanding of thyroid hormone action in human brain development., Mutations in the thyroid hormone receptor α 1 gene (THRA) have recently been identified as a cause of intellectual deficit in humans. Patients present with structural abnormalities including microencephaly, reduced cerebellar volume and decreased axonal density. Here, we show that directed differentiation of THRA mutant patient-derived induced pluripotent stem cells to forebrain neural progenitors is markedly reduced, but mutant progenitor cells can generate deep and upper cortical layer neurons and form functional neuronal networks. Quantitative lineage tracing shows that THRA mutation-containing progenitor cells exit the cell cycle prematurely, resulting in reduced clonal output. Using a micropatterned chip assay, we find that spatial self-organization of mutation-containing progenitor cells in vitro is impaired, consistent with down-regulated expression of cell–cell adhesion genes. These results reveal that thyroid hormone receptor α1 is required for normal neural progenitor cell proliferation in human cerebral cortical development. They also exemplify quantitative approaches for studying neurodevelopmental disorders using patient-derived cells in vitro.

Urbančič et al. developed an open-source platform called Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia at their tips and bases concurrently with their morphological and dynamic properties. This customizable tool therefore enables researchers to determine the relationship between protein localization and filopodium behavior., Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.

SummaryVariation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output.

Summary The early stages of Alzheimer’s disease are associated with synaptic dysfunction prior to overt loss of neurons. To identify extracellular molecules that impair synaptic plasticity in the brain, we studied the secretomes of human iPSC-derived neuronal models of Alzheimer’s disease. When introduced into the rat brain, secretomes from human neurons with either a presenilin-1 mutation, amyloid precursor protein duplication, or trisomy of chromosome 21 all strongly inhibit hippocampal long-term potentiation. Synaptic dysfunction caused by presenilin-1 mutant and amyloid precusor protein duplication secretomes is mediated by Aβ peptides, whereas trisomy of chromosome 21 (trisomy 21) neuronal secretomes induce dysfunction through extracellular tau. In all cases, synaptotoxicity is relieved by antibody blockade of cellular prion protein. These data indicate that human models of Alzheimer’s disease generate distinct proteins that converge at the level of cellular prion protein to induce synaptic dysfunction in vivo., Graphical Abstract, Highlights • Secretomes of human iPSC-derived models of AD inhibit long-term potentiation in vivo • Familial AD neurons release forms of Aβ that inhibit hippocampal LTP • Neurons with trisomy of chromosome 21 release a form of tau that blocks LTP • Blockade of cellular prion protein prevents LTP inhibition by all secretomes, Hu et al. find that human iPSC-derived neurons with autosomal dominant Alzheimer’s disease mutations or trisomy of chromosome 21 release Aβ peptides and tau derivatives that each inhibit a form of long-term synaptic plasticity, LTP, in vivo. This disruption occurs via a common pathway that requires cellular prion protein.

INTRODUCTION Scientists are building detailed maps of the cellular composition in the human brain to learn about its development. In the human cortex, the largest area of the mammalian brain, neural circuits are formed through anatomical refinement, including axon and synaptic pruning, and the emergence of complex patterns of network activity during early fetal development. Cellular analyses in the human brain are restricted to postmortem material, which cannot reveal the process of development. Model organisms are, therefore, commonly used for studies of brain physiology, development, and pathogenesis, but the results from model organisms do not always translate to humans. RATIONALE Systems to model human neuron dynamics and their dysfunction in vivo are needed. While biopsy specimens and the generation of neurons from induced pluripotent stem cells (iPSCs) could provide the necessary human genetic background, two- and three-dimensional cultures lack factors that normally support neuronal development, including blood vessels, immune cells, and interaction with innervating neurons from other brain areas. On the basis of previous stem cell transplantation studies in mice, we reasoned that the physiological microenvironment of the adult mouse brain could support the growth of human cortical tissue grafts that had been generated from iPSC-derived neuronal progenitors. With human neurons implanted into the mouse brain, high-resolution, real-time in vivo monitoring of human neuron dynamics for periods of time spanning the range from subseconds to several months becomes feasible. RESULTS We found that transplanted human iPSC–derived neuronal progenitors consistently assembled into vascularized territories with complex cytoarchitecture, mimicking key features of the human fetal cortex, such as its large size and cell diversification. Single-cell-resolution intravital microscopy showed that human neuronal arbors were refined via branch-specific retraction, rather than degeneration. Human synaptic networks restructured over the course of 4 months, while maintaining balanced rates of synapse formation and elimination. Human functional neurons rapidly and consistently acquired oscillatory population activity, which persisted over the 5-month observation period. Lastly, we used cortical tissue grafts derived from the fibroblasts of two individuals with Down syndrome, caused by supernumerary chromosome 21. We found that neuronal synapses in cells derived from these individuals were overly stable and that oscillatory neural activity was reduced in these grafts, revealing in vivo cellular phenotypes not otherwise apparent. CONCLUSION By combining live imaging in a multistructured tissue environment in mice with a human-specific genetic background, we provide insights into the earliest stages of human axon, synaptic, and network activity development and uncover cellular phenotypes in Down syndrome. Our work provides an alternative experimental system that can be used to study other disorders affecting the developing human cortex.

Summary The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2), which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations., Highlights • Microglia can be efficiently differentiated from human iPSCs • iPSC microglia resemble human primary microglia transcriptionally and functionally • Disease-causing mutations in TREM2 affect receptor processing and expression • TREM2 mutant microglia differentiate, respond to pathogenic stimuli, and phagocytose, Brownjohn and colleagues report methods to generate microglia from induced pluripotent human stem cells, which they demonstrate are highly similar to cultured primary human microglia. Microglia differentiated from patient-derived stem cells carrying neurological disease-causing mutations in the TREM2 receptor differentiate normally and respond appropriately to pathogenic stimuli, despite the absence of functional TREM2 receptor on the plasma membrane.

A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology., Summary: Human PSC-derived cerebral cortex neurons form large-scale functional networks that change over time and mimic those found in the developing cerebral cortex in vivo.

Summary In Alzheimer’s disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon., Graphical Abstract, Highlights • Extracellular tau protein enters human neurons by endocytosis and micropinocytosis • Aggregated tau enters human neurons primarily by endocytosis • Tau antibodies reduce uptake and are carried into neurons by tau • Findings suggest that tau uptake is dependent on carrier proteins or receptors, In contrast with predictions that transfer of the microtubule-associated protein tau between neurons is a toxic gain-of-function process in dementia, Evans et al. show that healthy human neurons efficiently take up both normal and aggregated tau, by distinct but overlapping uptake mechanisms.

Summary Oocytes have a remarkable ability to reactivate silenced genes in somatic cells. However, it is not clear how the chromatin architecture of somatic cells affects this transcriptional reprogramming. Here, we investigated the relationship between the chromatin opening and transcriptional activation. We reveal changes in chromatin accessibility and their relevance to transcriptional reprogramming after transplantation of somatic nuclei into Xenopus oocytes. Genes that are silenced, but have pre-existing open transcription start sites in donor cells, are prone to be activated after nuclear transfer, suggesting that the chromatin signature of somatic nuclei influences transcriptional reprogramming. There are also activated genes associated with new open chromatin sites, and transcription factors in oocytes play an important role in transcriptional reprogramming from such genes. Finally, we show that genes resistant to reprogramming are associated with closed chromatin configurations. We conclude that chromatin accessibility is a central factor for successful transcriptional reprogramming in oocytes., Graphical Abstract, Highlights • ATAC-seq reveals chromatin accessibility changes during reprogramming in oocytes • Genes with open promoters are preferentially activated during reprogramming • Transcription factors play a role in transcriptional reprogramming in oocytes • Closed chromatin is associated with reprogramming-resistant genes, Miyamoto et al. show genome-wide changes in chromatin accessibility during transcriptional reprogramming in oocytes using the frog nuclear transfer system. They demonstrate that donor cell chromatin states affect transcriptional reprogramming and changes in open chromatin during reprogramming are associated with specific transcription factors.

Summary Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP) processing and the accumulation of APP-derived amyloid β (Aβ) peptides are key processes in Alzheimer's disease (AD). We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation., Highlights • Phenotypic drug screening of a human stem cell model of Alzheimer's disease • Avermectins identified as modifiers of APP processing in health and disease • Avermectins increase short Aβ peptides at the expense of longer, toxic forms • Effect is independent of known avermectin targets and the core γ-secretase complex, In this article, Livesey and colleagues perform a phenotypic drug screen in a human stem cell model of Alzheimer's disease. The anthelminthic avermectins are identified as a family of compounds that increase the production of short Aβ peptides over longer more toxic Aβ forms. The effect is analogous to existing γ-secretase modulators, but is independent of the core γ-secretase complex.

Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an organ-like configuration. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.

BACKGROUND: Chromatin-modifying complexes have key roles in regulating various aspects of neural stem cell biology, including self-renewal and neurogenesis. The methyl binding domain 3/nucleosome remodelling and deacetylation (MBD3/NuRD) co-repressor complex facilitates lineage commitment of pluripotent cells in early mouse embryos and is important for stem cell homeostasis in blood and skin, but its function in neurogenesis had not been described. Here, we show for the first time that MBD3/NuRD function is essential for normal neurogenesis in mice. RESULTS: Deletion of MBD3, a structural component of the NuRD complex, in the developing mouse central nervous system resulted in reduced cortical thickness, defects in the proper specification of cortical projection neuron subtypes and neonatal lethality. These phenotypes are due to alterations in PAX6+ apical progenitor cell outputs, as well as aberrant terminal neuronal differentiation programmes of cortical plate neurons. Normal numbers of PAX6+ apical neural progenitor cells were generated in the MBD3/NuRD-mutant cortex; however, the PAX6+ apical progenitor cells generate EOMES+ basal progenitor cells in reduced numbers. Cortical progenitor cells lacking MBD3/NuRD activity generate neurons that express both deep- and upper-layer markers. Using laser capture microdissection, gene expression profiling and chromatin immunoprecipitation, we provide evidence that MBD3/NuRD functions to control gene expression patterns during neural development. CONCLUSIONS: Our data suggest that although MBD3/NuRD is not required for neural stem cell lineage commitment, it is required to repress inappropriate transcription in both progenitor cells and neurons to facilitate appropriate cell lineage choice and differentiation programmes.

Background Phenotypically identical cells demonstrate predictable, robust behaviours. However, there is uncertainty as to whether phenotypically identical cells are equally similar at the underlying transcriptional level or if cellular systems are inherently noisy. To answer this question, it is essential to distinguish between technical noise and true variation in transcript levels. A critical issue is the contribution of sampling effects, introduced by the requirement to globally amplify the single cell mRNA population, to observed measurements of relative transcript abundance. Results We used single cell microarray data to develop simple mathematical models, ran Monte Carlo simulations of the impact of technical and sampling effects on single cell expression data, and compared these with experimental microarray data generated from single embryonic neural stem cells in vivo. We show that the actual distribution of measured gene expression ratios for pairs of neural stem cells is much broader than that predicted from our sampling effect model. Conclusion Our results confirm that significant differences in gene expression levels exist between phenotypically identical cells in vivo, and that these differences exceed any noise contribution from global mRNA amplification.

Comparison of the performance of three methods for amplifying single-cell amounts of RNA for use in expression profiling shows that PCT amplification is more reliable than linear amplification., Background Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Several methods for RNA amplification are in general use, but little consideration has been given to the comparative analysis of those methods in terms of the overall validity of the data generated when amplifying from single-cell amounts of RNA, rather than their empirical performance in single studies. Results We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings. Conclusion Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios.

Summary Accumulation of Aβ peptide fragments of the APP protein and neurofibrillary tangles of the microtubule-associated protein tau are the cellular hallmarks of Alzheimer’s disease (AD). To investigate the relationship between APP metabolism and tau protein levels and phosphorylation, we studied human-stem-cell-derived forebrain neurons with genetic forms of AD, all of which increase the release of pathogenic Aβ peptides. We identified marked increases in intracellular tau in genetic forms of AD that either mutated APP or increased its dosage, suggesting that APP metabolism is coupled to changes in tau proteostasis. Manipulating APP metabolism by β-secretase and γ-secretase inhibition, as well as γ-secretase modulation, results in specific increases and decreases in tau protein levels. These data demonstrate that APP metabolism regulates tau proteostasis and suggest that the relationship between APP processing and tau is not mediated solely through extracellular Aβ signaling to neurons., Graphical Abstract, Highlights • Neurons from different genetic forms of Alzheimer’s disease differ in APP processing • APP mutations increase total and phosphorylated tau; PSEN1 mutations do not • Pharmacological manipulation of APP processing changes tau protein levels • APP regulation of tau proteostasis is not solely mediated through extracellular Aβ, Moore et al. use neurons made from familial Alzheimer’s disease stem cells to reveal how three proteins involved in the disease are linked in a pathway that controls disease progression. They show that drugs that target this pathway change levels of a protein involved in neurodegeneration, microtubule-associated protein tau, opening up a potential therapeutic pathway.

INTRODUCTION: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation. METHODS: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction. RESULTS: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components. CONCLUSION: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.