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Human induced pluripotent stem cells (iPSCs) are a powerful tool for studying development and disease. However, different iPSC lines show considerable phenotypic variation. The lack of common well-characterized cell lines that are used widely frustrates efforts to integrate data across research groups or replicate key findings. Inspired by model organism communities who addressed this issue by establishing a limited number of widely accepted strains, we characterised candidate iPSC lines in unprecedented detail to select a well-performing line to underpin collaborative studies. Specifically, we characterised the morphology, growth rates, and single-cell transcriptomes of iPSC lines in the pluripotent state and assessed their genomic integrity using karyotyping, DNA microarrays, whole genome sequencing, and functional assays for p53 activity. We further tested their ability to be edited by CRISPR/Cas9 and used single-cell RNA sequencing to compare the efficiency with which they could be differentiated into multiple lineages. We found that there was significant variability in the performance of lines across the tested assays that enabled the rational selection of a lead line, KOLF2.1J, which is a gene-corrected derivative of a publicly available line from the Human Induced Pluripotent Stem Cells Initiative (HipSci) resource. We are now using this line in an initiative from the NIH Center for Alzheimer’s and Related Dementias to derive hundreds of gene-edited and functionalized sub-clones to be distributed widely throughout the research community along with associated datasets, with the aim of promoting the standardisation required for large-scale collaborative science in the stem cell field.SummaryThe authors of this collaborative science study describe a deep characterization of widely available induced pluripotent stem cell (iPSC) lines to rationally select a line that performs well in multiple experimental approaches. Analysis of transcriptional patterns in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and differentiation efficiency towards multiple lineages identified KOLF2.1J as a well-performing cell line. The widespread distribution and use of this line makes it an attractive cell line for comparative and collaborative efforts in the stem cell field.HighlightsDeep genotyping and phenotyping reveals KOLF2.1J as well-performing cell line that is readily distributed and could serve as common reference lineDespite rare copy-neutral loss of heterozygosity (CN-LOH) events, iPSC lines retain genomic fidelity after CRISPR/Cas9-based gene editingOur multifactorial pipeline serves as a blueprint for future efforts to identify other lead iPSC linesGraphical abstract

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Tomosyns are soluble SNARE proteins proposed to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario using a novel mouse model, energy barrier recordings, and single-molecule force measurements. Tomosyn-1/2 deficiency drastically enhanced the probability that synaptic vesicles fuse at synapses, resulting in stronger synapses with faster depression and slower recovery. While wildtype tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Force measurements revealed that tomosyns SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that regions outside the SNARE motif contribute to tomosyns inhibitory function. These results reveal that tomosyns regulate synaptic transmission by preventing SNAP-25 binding to template complexes, increasing the energy barrier for synaptic vesicle fusion, and limiting synaptic depression.