Generation of two gene edited iPSC-lines carrying a DOX-inducible NGN2 expression cassette with and without GFP in the AAVS1 locus

Schmid, Benjamin; Holst, Bjørn; Poulsen, Ulla; Jørring, Ida; Clausen, Christian; Rasmussen, Mette; Mau-Holzmann, Ulrike A.; Steeg, Rachel; Nuthall, Hugh; Ebneth, Andreas; Cabrera-Socorro, Alfredo

doi:10.1016/j.scr.2021.102240

Cited by 16 publications

(9 citation statements)

References 5 publications

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“…Another in-house bulk RNA-seq dataset was generated to assess batch-to-batch variability of iPSC-derived NGN2 neurons similar to those described by Schmid et al 24 . The CellMap output indicated low batch-to-batch variability and the cells reaching fully differentiated states by day 21 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Characterizing the composition of iPSC derived cells from bulk transcriptomics data with CellMap

Ouyang¹,

Bourgeois-Tchir

Lyashenko

et al. 2022

Sci Rep

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Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

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Section: Resultsmentioning

confidence: 99%

Characterizing the composition of iPSC derived cells from bulk transcriptomics data with CellMap

Ouyang¹,

Bourgeois-Tchir

Lyashenko

et al. 2022

Sci Rep

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“…iPSCs were maintained on Matrigel-coated plates (VWR) in Essential 8 (E8) medium supplemented with 0.1% Penicillin/Streptomycin (P/S, Fisher Scientific). BIONi010-C-13 iPSCs contain doxycycline-inducible neurogenin-2 (NGN2) within the AAVS1 safe-harbor locus [ 56 ]. Therefore, it was not necessary to infect the cells with a construct of the NGN2 gene for neuronal induction.…”

Section: Methodsmentioning

confidence: 99%

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F

Öttl,

Toonen,

Verhage

2024

Human Molecular Genetics

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MUNC18-1 is an essential protein of the regulated secretion machinery. De novo, heterozygous mutations in STXBP1, the human gene encoding this protein, lead to a severe neurodevelopmental disorder. Here, we describe the electrophysiological characteristics of a unique case of STXBP1-related disorder caused by a homozygous mutation (L446F). We engineered this mutation in induced pluripotent stem cells from a healthy donor (STXBP1LF/LF) to establish isogenic cell models. We performed morphological and electrophysiological analyses on single neurons grown on glial micro-islands. Human STXBP1LF/LF neurons displayed normal morphology and normal basal synaptic transmission but increased paired-pulse ratios and charge released, and reduced synaptic depression compared to control neurons. Immunostainings revealed normal expression levels but impaired recognition by a mutation-specific MUNC18-1 antibody. The electrophysiological gain-of-function phenotype is in line with earlier overexpression studies in Stxbp1 null mouse neurons, with some potentially human-specific features. Therefore, the present study highlights important differences between mouse and human neurons critical for the translatability of pre-clinical studies.

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“…The BIONi010-C-13 line (donor biosample SAMEA103988285) with doxycycline (DOX)-inducible neurogenin 2 ( NGN2 ) transgene was obtained and is available from the European Bank for Induced pluripotent Stem Cells (EBiSC, www.ebisc.org ) [ 14 ]. The line is derived from a dermal fibroblast sample donated by an 18 year-old healthy male of African-American descent.…”

Section: Methodsmentioning

confidence: 99%

Functional characterization of a single nucleotide polymorphism associated with Alzheimer’s disease in a hiPSC-based neuron model

Stolzenburg,

Esmaeeli,

Kulkarni

et al. 2023

PLoS ONE

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Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer’s disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the FosB proto-oncogene (FOSB) and ERCC excision repair 1 (ERCC1) genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants.

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Generation of two gene edited iPSC-lines carrying a DOX-inducible NGN2 expression cassette with and without GFP in the AAVS1 locus

Cited by 16 publications

References 5 publications

Characterizing the composition of iPSC derived cells from bulk transcriptomics data with CellMap

Characterizing the composition of iPSC derived cells from bulk transcriptomics data with CellMap

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F

Functional characterization of a single nucleotide polymorphism associated with Alzheimer’s disease in a hiPSC-based neuron model

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