ASCL1- and DLX2-induced GABAergic neurons from hiPSC-derived NPCs

Barretto, Natalie; Zhang, Hanwen; Powell, Samuel K.; Fernando, Michael B.; Zhang, Siwei; Flaherty, Erin; Ho, Seok‐Man; Slesinger, Paul A.; Duan, Jubao; Brennand, Kristen J.

doi:10.1016/j.jneumeth.2019.108548

Cited by 33 publications

(41 citation statements)

References 37 publications

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“…3A). GABAergic neuronal induction via overexpression of DLX2 and ASCL1 was efficient from both hiPSCs 59 and NPCs 54 (Fig. 3A).…”

Section: Demonstration Of Neuronal and Glial Generationmentioning

confidence: 95%

“…To ensure that the hiPSCs can robustly generate the neural subtypes most commonly linked to SZ (glutamatergic neurons 51 , GABAergic neurons 51 , dopaminergic neurons 52 , and astrocytes 53 ), each laboratory independently evaluated neural differentiation and induction using different methodologies already well-established at each site 23,30,[54][55][56] .…”

Section: Demonstration Of Neuronal and Glial Generationmentioning

confidence: 99%

“…3 A); the variability between hiPSC lines and induction batches was high, so we recommend that dopaminergic neuron differentiation be individually optimized for the remaining hiPSC lines. We queried expression of the 67 non-MHC SZ risk genes associated by prediXcan 63 with the SZ-PGC2 loci 2,64 ; finding that 62.6%, 79.1% and 76.1% are expressed at >0.5 log2RPKM, respectively, in our existing NGN2glutamatergic, ASCl1/DLX2-GABAergic and ASCL1, NURR1 and LMX1Adopaminergic neuron RNAseq datasets 54 .…”

Section: Demonstration Of Neuronal and Glial Generationmentioning

confidence: 99%

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Publicly available hiPSC lines with extreme polygenic risk scores for modeling schizophrenia

Rehbach

Zhang

Das

et al. 2020

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ABSTRACTSchizophrenia (SZ) is a common and debilitating psychiatric disorder with limited effective treatment options. Although highly heritable, risk for this polygenic disorder depends on the complex interplay of hundreds of common and rare variants. Translating the growing list of genetic loci significantly associated with disease into medically actionable information remains an important challenge. Thus, establishing platforms with which to validate the impact of risk variants in cell-type-specific and donor-dependent contexts is critical. Towards this, we selected and characterize a collection of twelve human induced pluripotent stem cell (hiPSC) lines derived from control donors with extremely low and high SZ polygenic risk scores (PRS). These hiPSC lines are publicly available at the California Institute for Regenerative Medicine (CIRM). The suitability of these extreme PRS hiPSCs for CRISPR-based isogenic comparisons of neurons and glia was evaluated across three independent laboratories, identifying 9 out of 12 meeting our criteria. We report a standardized resource of publicly available hiPSCs, with which we collectively commit to conducting future CRISPR-engineering, in order to facilitate comparison and integration of functional validation studies across the field of psychiatric genetics.

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“…3A). GABAergic neuronal induction via overexpression of DLX2 and ASCL1 was efficient from both hiPSCs 59 and NPCs 54 (Fig. 3A).…”

Section: Demonstration Of Neuronal and Glial Generationmentioning

confidence: 95%

Section: Demonstration Of Neuronal and Glial Generationmentioning

confidence: 99%

Section: Demonstration Of Neuronal and Glial Generationmentioning

confidence: 99%

See 1 more Smart Citation

Publicly available hiPSC lines with extreme polygenic risk scores for modeling schizophrenia

Rehbach

Zhang

Das

et al. 2020

Preprint

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“…Induced GABAergic neurons were produced from NPCs by overexpression of Ascl1 and Dlx2 37,38 . NPCs were transduced by spinfection with lentivirus encoding CMV-rtTA (Addgene 19780), TetO-Ascl1 -T2A-Puro (Addgene ID: 97329), TetO-Dlx2 -IRES-Hygro (Addgene ID: 97330) and the indicated RiboTag construct.…”

Section: Methodsmentioning

confidence: 99%

Cell-type specificin vitrogene expression profiling of stem-cell derived neural models

Gregory

Hoelzli

Abdelaal

et al. 2020

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Human induced pluripotent stem cells (hiPSCs) allow the production of defined, disease relevant cell types for transcriptomic and functional studies. A major challenge for hiPSCs disease modeling is to more closely mimic heterocellular networks observed in vivo. As hiPSC cultures become more complex, measuring cell-type specific transcriptomics will become more challenging. Here we report an extension of the RiboTag system, which uses cell-type restricted expression of epitope-tagged RPL22 in mouse tissue, applied to immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neural cells. We constructed lentiviral vectors expressing epitope-tagged RPL22 using constitutive and inducible promoters fused to HA, Flag, or V5 affinity tags in combination with fluorescent reporters; differential expression of uniquely tagged RPL22 in specific cell types allows mRNA from each population to be biochemically purified from mixed cultures. RiboTag expression enables efficient depletion of off-target RNA in mixed species co-cultures of immortalized cell lines using HA and V5 RiboTags. In monocultures of hiPSC-derived neural progenitor cells, motor neurons, and GABAergic neurons expressing RiboTags, we observed an enrichment of protein coding genes and concomitant decrease in mitochondrial and non-coding genes in immunoprecipitated RNA. Depletion efficiency varied across independent replicates of mixed-species co-cultures of hiPSC-derived motor neurons and primary mouse astrocytes. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed. In vitro differentiation of human induced pluripotent stem cells (hiPSC) into defined cell types is a powerful approach to study human neural cells 1 . A major goal for hiPSC disease modeling is to build increasingly complex in vitro cultures that retain the cellular diversity of neural tissue. Organoids, produced from directed or undirected differentiation of hiPSC aggregates, are increasingly being used to model cell-cell interactions in development and disease 2,3 . However, interrogating gene expression of specific cell types in increasingly complex heterocellular cultures, particularly those of low abundance, is difficult using conventional RNA-seq approaches. Complementary physical, biochemical, and computational approaches have been developed to identify cell-type specific transcriptomes.Physical methods for isolating cell-type specific RNA include fluorescence activated cell sorting (FACS)-based enrichment 4,5 , single cell RNA sequencing (scRNA-seq) 6,7 , and laser capture microdissection (LCM) 8-10 . Single-cell suspensions required for FACS and scRNA-seq can be challenging to prepare for highly arborized neural cells. scRNAseq provides cellular resolution data at low read depth and, while improvements have been made 11,12 , it remains prohibitively expensive for large scale studies. Low read depth and other technical considerations can bias scRNA-seq studies, with certain cell types or transcripts over-represented or missing in the ...

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“…Moreover, specific classes of GABAergic neurons, such as SST + and PV + neurons have been found to have a particularly strong influence on the E/I balance [30][31][32] . Although recent advances in differentiating human induced pluripotent stem cells (hiPSCs) into GABAergic neurons [33][34][35][36][37][38][39][40][41][42] , protocols that enable the generation of SST + and PV + human neurons are still challenging due to the long functional maturation of these cells 43 . Investigating E/I balance in human in vitro models for brain disorders ideally requires a model system that consists of a) neuronal networks In this study, we investigated the role of CDH13 in maintaining E/I balance in a human neuronal model.…”

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confidence: 99%

Cadherin-13 is a critical regulator of GABAergic modulation in human stem cell derived neuronal networks

Mossink

Rhijn

Wang

et al. 2020

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Activity in the healthy brain relies on concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders, however, obtaining mechanistic insight into these disruptions, with translational value for the human patient, has typically been hampered by methodological limitations.Cadherin-13 (CDH13) has strongly been associated to attention-deficit/hyperactivity disorder and comorbid disorders such as autism and schizophrenia. CDH13 localises at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and PV expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-β1 and Integrin-β3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type specific contribution of disease genes to the E/I balance. IntroductionNeuronal network activity is controlled by a tightly regulated interplay between excitation (E) and inhibition (I). In the healthy brain, this interplay maintains a certain E/I ratio via balanced synaptic communication between glutamatergic and GABAergic neurons 1, 2 , resulting in the so called 'E/I balance'. A growing number of studies imply that the E/I balance is disrupted in many neurodevelopmental disorders (NDDs) 3, 4 , including monogenic disorders, where the causative mutations are typically related to altered neuronal excitability and/or synaptic communication 5-7 , as well as polygenic disorders, such as autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and schizophrenia 4,8 . Mutations in Cadherin-13 (CDH13, also known as T-Cadherin or H-Cadherin) 9 have been associated with ADHD [10][11][12] and comorbid disorders such as ASD, schizophrenia, alcohol dependence and violent behaviour [13][14][15][16] . CDH13 is a special member of the cadherin superfamily since it lacks a transmembrane-and intracellular domain, and in contrast to other Cadherins, is anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor 17,18 . Because of this relatively weak connection to the outer membrane 19 , ...

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ASCL1- and DLX2-induced GABAergic neurons from hiPSC-derived NPCs

Cited by 33 publications

References 37 publications

Publicly available hiPSC lines with extreme polygenic risk scores for modeling schizophrenia

Publicly available hiPSC lines with extreme polygenic risk scores for modeling schizophrenia

Cell-type specificin vitrogene expression profiling of stem-cell derived neural models

Cadherin-13 is a critical regulator of GABAergic modulation in human stem cell derived neuronal networks

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