FMR1 loss in a human stem cell model reveals early changes to intrinsic membrane excitability.

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. While cortical hyperexcitability is a hallmark of FXS, the reported phenotypes and underlying mechanisms, including alterations in synaptic transmission and ion channel properties, are heterogeneous and at times contradictory. Here, we report the generation of new isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9 to facilitate the study of how complete FMRP loss, independent of genetic background, drives molecular and cellular alterations relevant for FXS. After differentiating these stem cell tools into excitatory neurons, we systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over time. Using whole-cell patch clamp analyses, we found that FMR1y/- neurons overall showed an increased intrinsic membrane excitability compared to age-matched FMR1y/+ controls, with no discernable alternations in synaptic transmission. Surprisingly, longitudinal analyses of cell intrinsic defects revealed that a majority of significant changes emerged early following in vitro differentiation and some were not stable over time. Collectively, this study provides a new isogenic hPSC model which can be further leveraged by the scientific community to investigate basic mechanisms of FMR1 gene function relevant for FXS. Moreover, our results suggest that precocious changes in the intrinsic membrane properties during early developmental could be a critical cellular pathology ultimately contributing to cortical hyperexcitability in FXS.

Genomic, molecular, and cellular divergence of the human brain.

While many core biological processes are conserved across species, the human brain has evolved with unique capacities. Current understanding of the neurobiological mechanisms that endow human traits as well as associated vulnerabilities remains limited. However, emerging data have illuminated species divergence in DNA elements and genome organization, in molecular, morphological, and functional features of conserved neural cell types, as well as temporal differences in brain development. Here, we summarize recent data on unique features of the human brain and their complex implications for the study and treatment of brain diseases. We also consider key outstanding questions in the field and discuss the technologies and foundational knowledge that will be required to accelerate understanding of human neurobiology.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells.

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly.

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

Why both sides of the gender equation matter.

Despite efforts to increase gender diversity in science, technology, engineering, mathematics and medicine (STEMM), men continue to hold most tenured and leadership positions. Moreover, the specific population shifts and timelines which may be required to achieve gender parity have not been well delineated. It is obvious that if women are statistically underrepresented in a field, then men must be statistically overrepresented: however, male overrepresentation and related gender-based advantages are rarely mentioned in conversations about gender equality. It is important that actions to address both overrepresentation and underrepresentation are elements of any strategy that seeks to move STEMM fields closer to gender parity.

Molecular convergence between Down syndrome and fragile X syndrome identified using human pluripotent stem cell models.

Down syndrome (DS), driven by an extra copy of chromosome 21 (HSA21), and fragile X syndrome (FXS), driven by loss of the RNA-binding protein FMRP, are two common genetic causes of intellectual disability and autism. Based upon the number of DS-implicated transcripts bound by FMRP, we hypothesize that DS and FXS may share underlying mechanisms. Comparing DS and FXS human pluripotent stem cell (hPSC) and glutamatergic neuron models, we identify increased protein expression of select targets and overlapping transcriptional perturbations. Moreover, acute upregulation of endogenous FMRP in DS patient cells using CRISPRa is sufficient to significantly reduce expression levels of candidate proteins and reverse 40% of global transcriptional perturbations. These results pinpoint specific molecular perturbations shared between DS and FXS that can be leveraged as a strategy for target prioritization; they also provide evidence for the functional relevance of previous associations between FMRP targets and disease-implicated genes.

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia.

It is unclear how the 22q11.2 deletion predisposes to psychiatric disease. To study this, we generated induced pluripotent stem cells from deletion carriers and controls and utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Here, we show that upon differentiation into neural progenitor cells, the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including autism. In excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common and rare variants. To understand how the deletion contributed to these changes, we defined the minimal protein-protein interaction network that best explains gene expression alterations. We found that many genes in 22q11.2 interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways. Our findings suggest that the 22q11.2 deletion impacts genes that may converge with psychiatric risk loci to influence disease manifestation in each deletion carrier.

De novo DNA methyltransferases DNMT3A and DNMT3B are essential for XIST silencing for erosion of dosage compensation in pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell-based therapies. However, the long non-coding RNA XIST, which is essential for the establishment and maintenance of X chromosome inactivation, is repressed during culture, thereby causing erosion of dosage compensation in female hPSCs. Here, we report that the de novo DNA methyltransferases DNMT3A/3B are necessary for XIST repression in female hPSCs. We found that the deletion of both genes, but not the individual genes, inhibited XIST silencing, maintained the heterochromatin mark of H3K27me3, and did not cause global overdosage in X-linked genes. Meanwhile, DNMT3A/3B deletion after XIST repression failed to restore X chromosome inactivation. Our findings revealed that de novo DNA methyltransferases are primary factors responsible for initiating erosion of dosage compensation in female hPSCs, and XIST silencing is stably maintained in a de novo DNA-methylation-independent manner.

Using human pluripotent stem cell models to study autism in the era of big data.

Advances in human pluripotent stem cell (hPSC) biology coupled with protocols to generate diverse brain cell types in vitro have provided neuroscientists with opportunities to dissect basic and disease mechanisms in increasingly relevant cellular substrates. At the same time, large data collections and analyses have facilitated unprecedented insights into autism genetics, normal human genetic variation, and the molecular landscape of the developing human brain. While such insights have enabled the investigation of key mechanistic questions in autism, they also highlight important limitations associated with the use of existing hPSC models. In this review, we discuss four such issues which influence the efficacy of hPSC models for studying autism, including (i) sources of variance, (ii) scale and format of study design, (iii) divergence from the human brain in vivo, and (iv) regulatory policies and compliance governing the use of hPSCs. Moreover, we advocate for a set of immediate and long-term priorities to address these issues and to accelerate the generation and reproducibility of data in order to facilitate future fundamental as well as therapeutic discoveries.

A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells.

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. Collectively, our results provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

Fly model sheds light on brain disease.

Experiments on flies suggest that a gain-of-function mechanism in a protein called CSPɑ contributes to the progressive brain disease CLN4.

Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission.

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.

A Scaled Framework for CRISPR Editing of Human Pluripotent Stem Cells to Study Psychiatric Disease.

Scaling of CRISPR-Cas9 technology in human pluripotent stem cells (hPSCs) represents an important step for modeling complex disease and developing drug screens in human cells. However, variables affecting the scaling efficiency of gene editing in hPSCs remain poorly understood. Here, we report a standardized CRISPR-Cas9 approach, with robust benchmarking at each step, to successfully target and genotype a set of psychiatric disease-implicated genes in hPSCs and provide a resource of edited hPSC lines for six of these genes. We found that transcriptional state and nucleosome positioning around targeted loci was not correlated with editing efficiency. However, editing frequencies varied between different hPSC lines and correlated with genomic stability, underscoring the need for careful cell line selection and unbiased assessments of genomic integrity. Together, our step-by-step quantification and in-depth analyses provide an experimental roadmap for scaling Cas9-mediated editing in hPSCs to study psychiatric disease, with broader applicability for other polygenic diseases.

Generation of a TLE1 homozygous knockout human embryonic stem cell line using CRISPR-Cas9.

Here, we generated a biallelic mutation in the TLE1 (Transducin Like Enhancer of Split 1) gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC) line WA01. The homozygous knockout cell line, TLE1-464-G04, displays loss of TLE1 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.

Generation of a TLE3 heterozygous knockout human embryonic stem cell line using CRISPR-Cas9.

Here, we generated a monoallelic mutation in the TLE3 (Transducin Like Enhancer of Split 3) gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC) line WA01. The heterozygous knockout cell line, TLE3-447-D08-A01, displays partial loss of TLE3 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.

Self-renewal does not predict tumor growth potential in mouse models of high-grade glioma.

Within high-grade gliomas, the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis, we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity, while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly, Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further, eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival, while knockdown of Olig2 within Id1(low) cells has a significant survival benefit, underscoring the importance of non-self-renewing lineages in disease progression.

Differential modulation by nicotine of substantia nigra versus ventral tegmental area dopamine neurons.

Midbrain dopamine (DA) neurons are found in two nuclei, the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). The SNc dopaminergic projections to the dorsal striatum are involved in voluntary movement and habit learning, whereas the VTA projections to the ventral striatum contribute to reward and motivation. Nicotine induces profound DA release from VTA dopamine neurons but substantially less from the SNc. Nicotinic acetylcholine receptor (nAChR) expression differs between these nuclei, but it is unknown whether there are differences in nAChR expression on the afferent projections to these nuclei. Here we have compared the nicotinic modulation of excitatory and inhibitory synaptic inputs to VTA and SNc dopamine neurons. Although nicotine enhances both the excitatory and inhibitory drive to SNc DA cells with response magnitudes similar to those seen in the VTA, the prevalence of these responses in SNc is much lower. We also found that a mixture of nAChR subtypes underlies the synaptic modulation in SNc, further distinguishing this nucleus from the VTA, where alpha7 nAChRs enhance glutamate inputs and non-alpha7 receptors enhance GABA inputs. Finally, we compared the nicotine sensitivity of DA neurons in these two nuclei and found larger response magnitudes in VTA relative to SNc. Thus the observed differences in nicotine-induced DA release from VTA and SNc are likely due to differences in nAChR expression on the afferent inputs as well as on the DA neurons themselves. This may explain why nicotine has a greater effect on behaviors associated with the VTA than the SNc.

Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor.

Multiple nuclear transcription factors including E-26-like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.