. 2022 Dec 1;29(12):1685-1702.e22.

doi: 10.1016/j.stem.2022.11.004.

A reference human induced pluripotent stem cell line for large-scale collaborative studies

Caroline B Pantazis¹, Andrian Yang², Erika Lara¹, Justin A McDonough³, Cornelis Blauwendraat⁴, Lirong Peng⁵, Hideyuki Oguro⁶, Jitendra Kanaujiya⁶, Jizhong Zou⁷, David Sebesta⁸, Gretchen Pratt⁸, Erin Cross⁸, Jeffrey Blockwick⁸, Philip Buxton⁸, Lauren Kinner-Bibeau⁸, Constance Medura⁸, Christopher Tompkins⁸, Stephen Hughes⁸, Marianita Santiana¹, Faraz Faghri⁹, Mike A Nalls⁹, Daniel Vitale⁹, Shannon Ballard⁹, Yue A Qi¹, Daniel M Ramos¹, Kailyn M Anderson¹, Julia Stadler¹, Priyanka Narayan¹⁰, Jason Papademetriou¹, Luke Reilly¹, Matthew P Nelson¹, Sanya Aggarwal¹¹, Leah U Rosen¹², Peter Kirwan¹¹, Venkat Pisupati¹³, Steven L Coon¹⁴, Sonja W Scholz¹⁵, Theresa Priebe¹⁶, Miriam Öttl¹⁶, Jian Dong¹⁶, Marieke Meijer¹⁶, Lara J M Janssen¹⁶, Vanessa S Lourenco¹⁶, Rik van der Kant¹⁷, Dennis Crusius¹⁸, Dominik Paquet¹⁹, Ana-Caroline Raulin²⁰, Guojun Bu²⁰, Aaron Held²¹, Brian J Wainger²², Rebecca M C Gabriele²³, Jackie M Casey²³, Selina Wray²³, Dad Abu-Bonsrah²⁴, Clare L Parish²⁵, Melinda S Beccari²⁶, Don W Cleveland²⁶, Emmy Li²⁷, Indigo V L Rose²⁷, Martin Kampmann²⁷, Carles Calatayud Aristoy²⁸, Patrik Verstreken²⁸, Laurin Heinrich²⁹, Max Y Chen²⁹, Birgitt Schüle²⁹, Dan Dou³⁰, Erika L F Holzbaur³⁰, Maria Clara Zanellati³¹, Richa Basundra³¹, Mohanish Deshmukh³¹, Sarah Cohen³¹, Richa Khanna³², Malavika Raman³², Zachary S Nevin³³, Madeline Matia³³, Jonas Van Lent³⁴, Vincent Timmerman³⁴, Bruce R Conklin³³, Katherine Johnson Chase²⁰, Ke Zhang²⁰, Salome Funes³⁵, Daryl A Bosco³⁵, Lena Erlebach³⁶, Marc Welzer³⁶, Deborah Kronenberg-Versteeg³⁶, Guochang Lyu³⁷, Ernest Arenas³⁷, Elena Coccia³⁸, Lily Sarrafha³⁸, Tim Ahfeldt³⁸, John C Marioni³⁹, William C Skarnes⁴⁰, Mark R Cookson⁴¹, Michael E Ward⁴², Florian T Merkle⁴³

Affiliations

¹ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
² European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK; Wellcome Trust - Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK; Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK.
³ The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
⁴ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA.
⁵ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International LLC, Washington, DC, USA; Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD, USA.
⁶ The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA; Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA.
⁷ iPS Cell Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
⁸ KromaTiD Inc., Longmont, CO, USA.
⁹ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International LLC, Washington, DC, USA.
¹⁰ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Genetics and Biochemistry Branch, NIDDK, NINDS, National Institutes of Health, Bethesda, MD 20814, USA.
¹¹ Wellcome Trust - Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK; Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK.
¹² European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
¹³ Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK; John van Geest Centre for Brain Repair, University of Cambridge, Cambridge CB2 0PY, UK.
¹⁴ Molecular Genomics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
¹⁵ Neurodegenerative Diseases Research Unit, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA; Department of Neurology, Johns Hopkins University, Baltimore, MD 21287, USA.
¹⁶ Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam de Boelelaan 1087, 1081 HV Amsterdam, the Netherlands.
¹⁷ Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam de Boelelaan 1087, 1081 HV Amsterdam, the Netherlands; Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Amsterdam UMC, Amsterdam, the Netherlands.
¹⁸ Institute for Stroke and Dementia Research, University Hospital, LMU Munich, 81377 Munich, Germany.
¹⁹ Institute for Stroke and Dementia Research, University Hospital, LMU Munich, 81377 Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany.
²⁰ Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
²¹ Department of Neurology, Sean M. Healey & AMG Center for ALS, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
²² Department of Neurology, Sean M. Healey & AMG Center for ALS, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Anesthesiology, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA; Broad Institute of Harvard University and MIT, Cambridge, MA, USA.
²³ Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK.
²⁴ The Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Pediatrics, University of Melbourne, Parkville, VIC 3052, Australia.
²⁵ The Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia.
²⁶ Department of Cellular and Molecular Medicine and Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA.
²⁷ Institute for Neurodegenerative Diseases and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA.
²⁸ VIB-KU Leuven Center for Brain & Disease Research, 3000 Leuven, Belgium; KU Leuven, Department of Neurosciences, Leuven Brain Institute, Mission Lucidity, Leuven, Belgium.
²⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
³⁰ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
³¹ Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
³² Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA.
³³ Gladstone Institutes, San Francisco, CA, USA.
³⁴ Peripheral Neuropathy Research Group, Department of Biomedical Sciences, University of Antwerp, Antwerp 2610, Belgium.
³⁵ Department of Neurology, UMass Chan Medical School, Worcester, MA, USA.
³⁶ Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
³⁷ Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
³⁸ Nash Family Department of Neuroscience; Departments of Neurology and Cell, Developmental and Regenerative Biology; Ronald M. Loeb Center for Alzheimer's Disease; Friedman Brain Institute; Black Family Stem Cell Institute at Mount Sinai, New York, NY, USA.
³⁹ European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK; Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.
⁴⁰ The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. Electronic address: bill.skarnes@jax.org.
⁴¹ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA. Electronic address: cookson@mail.nih.gov.
⁴² Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA. Electronic address: michael.ward4@nih.gov.
⁴³ Wellcome Trust - Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK; Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address: fm436@medschl.cam.ac.uk.

PMID: 36459969
PMCID: PMC9782786
DOI: 10.1016/j.stem.2022.11.004

A reference human induced pluripotent stem cell line for large-scale collaborative studies

Caroline B Pantazis et al. Cell Stem Cell. 2022.

. 2022 Dec 1;29(12):1685-1702.e22.

doi: 10.1016/j.stem.2022.11.004.

Authors

Affiliations

¹ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
² European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK; Wellcome Trust - Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK; Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK.
³ The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
⁴ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA.
⁵ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International LLC, Washington, DC, USA; Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD, USA.
⁶ The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA; Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA.
⁷ iPS Cell Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
⁸ KromaTiD Inc., Longmont, CO, USA.
⁹ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International LLC, Washington, DC, USA.
¹⁰ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Genetics and Biochemistry Branch, NIDDK, NINDS, National Institutes of Health, Bethesda, MD 20814, USA.
¹¹ Wellcome Trust - Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK; Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK.
¹² European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
¹³ Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK; John van Geest Centre for Brain Repair, University of Cambridge, Cambridge CB2 0PY, UK.
¹⁴ Molecular Genomics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
¹⁵ Neurodegenerative Diseases Research Unit, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA; Department of Neurology, Johns Hopkins University, Baltimore, MD 21287, USA.
¹⁶ Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam de Boelelaan 1087, 1081 HV Amsterdam, the Netherlands.
¹⁷ Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam de Boelelaan 1087, 1081 HV Amsterdam, the Netherlands; Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Amsterdam UMC, Amsterdam, the Netherlands.
¹⁸ Institute for Stroke and Dementia Research, University Hospital, LMU Munich, 81377 Munich, Germany.
¹⁹ Institute for Stroke and Dementia Research, University Hospital, LMU Munich, 81377 Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany.
²⁰ Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
²¹ Department of Neurology, Sean M. Healey & AMG Center for ALS, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
²² Department of Neurology, Sean M. Healey & AMG Center for ALS, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Anesthesiology, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA; Broad Institute of Harvard University and MIT, Cambridge, MA, USA.
²³ Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK.
²⁴ The Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Pediatrics, University of Melbourne, Parkville, VIC 3052, Australia.
²⁵ The Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia.
²⁶ Department of Cellular and Molecular Medicine and Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA.
²⁷ Institute for Neurodegenerative Diseases and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA.
²⁸ VIB-KU Leuven Center for Brain & Disease Research, 3000 Leuven, Belgium; KU Leuven, Department of Neurosciences, Leuven Brain Institute, Mission Lucidity, Leuven, Belgium.
²⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
³⁰ Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
³¹ Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
³² Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA.
³³ Gladstone Institutes, San Francisco, CA, USA.
³⁴ Peripheral Neuropathy Research Group, Department of Biomedical Sciences, University of Antwerp, Antwerp 2610, Belgium.
³⁵ Department of Neurology, UMass Chan Medical School, Worcester, MA, USA.
³⁶ Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
³⁷ Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
³⁸ Nash Family Department of Neuroscience; Departments of Neurology and Cell, Developmental and Regenerative Biology; Ronald M. Loeb Center for Alzheimer's Disease; Friedman Brain Institute; Black Family Stem Cell Institute at Mount Sinai, New York, NY, USA.
³⁹ European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK; Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.
⁴⁰ The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. Electronic address: bill.skarnes@jax.org.
⁴¹ Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA. Electronic address: cookson@mail.nih.gov.
⁴² Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA. Electronic address: michael.ward4@nih.gov.
⁴³ Wellcome Trust - Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK; Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address: fm436@medschl.cam.ac.uk.

PMID: 36459969
PMCID: PMC9782786
DOI: 10.1016/j.stem.2022.11.004

Abstract

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Keywords: CRISPR; differentiation; iPSC; karyotype; p53; pluripotent; reference; single-cell; stem cell; whole-genome.

Published by Elsevier Inc.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests S.W.S. is on the scientific advisory council of the Lewy Body Dementia Association and the MSA Coalition. S.W.S. is an editorial board member for the Journal of Parkinson Disease and JAMA Neurology. S.W.S. received research support from Cerevel Therapeutics. M.K. serves on the scientific advisory boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, and Alector and is a consultant to Modulo Bio and Recursion Therapeutics. Participation by researchers from Data Tecnica International, LLC in this project was part of a competitive contract awarded to Data Tecnica International, LLC by the National Institutes of Health to support open science research. M.A.N. also currently serves on the scientific advisory board for Clover Therapeutics and is an advisor to Neuron23 Inc. E.A. is founder, shareholder, and scientific advisor of Cholestenix, Ltd.

Figures

**Figure 1:. Growth, gene expression, and P53 pathway integrity of candidate cell sub-lines.**
(A) Representative phase contrast photomicrographs of colony morphology of the eight iPSC sub-lines on day 3 after replating. Scale bars indicate 100 μm. (B) Mean and SEM (n=4) of the total number of cells 48h after plating 30,000 cells/well. (C) Mean and SEM (n=6) of percent confluence at 0, 24 and 48 h after plating 30,000 cells/well. (D) UMAP projection of 2,270 iPSC cells color-coded by cell sub-line. There are two distinct groupings of cells, with the large group being composed of the 6 out of 7 sub-line and the small outlier group being composed of the LNGPI1 sub-line. (E) Beeswarm plots showing expression of selected genes associated with undifferentiated stem cells (top row) or poor neuronal differentiation potential (bottom row). (F) A mCherry PiggyBac reporter assay confirms the baseline activity (left plot) of the p53 pathway in KOLF2.1J (red) relative to *TP53* knockout cells (blue), which is further inducible in response to the DNA damaging agent doxorubicin (right plot).

**Figure 2.. Genetic analyses of eight candidate iPSC sub-lines.**
(A) The percentage of genetic variant types present in the 8 candidate iPSC sub-lines, grouped by their bioinformatically predicted consequences on coding sequences. (B) The number of rare (gnomAD AF<0.001) predicted deleterious (CADD phred >30) variants identified in the 8 iPSC sub-lines versus the total number of identified variants. (C) Polygenic risk scores (PRS) for Alzheimer’s disease (AD) and Parkinson’s disease (PD) are shown alongside the population-centered Z score distribution for PD PRS (y-axis) in 2995 PD cases (black) and 96,215 controls (grey) from the UK Biobank, and a similar PRS distribution for AD polygenic risk score (x-axis) in 2337 AD cases (grey) and the same controls.

**Figure 3.. Comparative gene editing efficiency.**
(A) Schematic of the gene editing experiment, showing how a CRISPR/Cas9-induced double strand break can lead to the formation of insertion/deletion mutations (indels) via the non-homologous end joining (NHEJ) pathway or be repaired with a single-stranded oligodeoxynucleotide via the homology-directed repair (HDR) pathway to introduce a single nucleotide variant (SNV) of interest and resulting in 6 potential alleles. Figure created with BioRender.com (B) The number of clones out of 24 expressing each possible genotype at the targeted SNV for each analyzed sub-line. (C) Number of genomic abnormalities observed among 20–24 analyzed clones across the eight analyzed sub-lines. (D) Ideogram of chromosome 22 with the *TIMP3* gene in 22q12.3 indicated by a red bar and black arrow, using the UCSC Genome Browser. NeuroArray genotyping revealed Chr22 CN-LOH from chr22q12.3-ter in clones derived from the KOLF2.1J sub-line. Upper plots show Log R ratio (LRR) where the mean LRR (red line) is 0 for the normal clone and >1 for the abnormal clones, and middle panels show the B allele frequency (BAF) for bi-allelic probes along the arrays with evidence of duplicated alleles across the chromosome.

**Figure 4.. Differentiation potential of candidate cell sub-lines.**
(A) Schematic of experimental design for four differentiation protocols evaluated in this study: hypothalamus and cortical differentiation (growth factor-based protocols), and hNGN2 and hNIL differentiation (transcription factor-based protocols). See STAR methods for detailed descriptions of differentiation protocols. Figure created with BioRender.com (B and C) UMAP plot for each differentiation colored by cell sub-line (B) and cell type (C). Cell type classification is derived from grouping each cluster into target neuron, neuron, neuroblast and progenitor/other categories based on the cluster annotation (Figure S4). (D) Bar plot showing proportion of cells assigned to each cell type per cell sub-line.

**Figure 5.. Whole genome sequencing of KOLF2.1J and the KOLF2-C1 parental line.**
(A) Flowchart of the discovery, filtering, annotations, and comparisons of SNP/indel variants in the *ARID2*-corrected KOLF2.1J and its parental line KOLF2-C1. Schematic created with BioRender.com (B) The consensus variant calls for KOLF2.1J from two Illumina short sequencing services (Psomagen and Jax) and one linked-read sequencing platform (10x Genomics). (C) The genetic compositions of the variant classes (left) and their effect on coding genes (right) for the 3.28 million high-confidence SNPs and indels in KOLF2.1J.

**Figure 6.. Performance of KOLF2.1J and established hPSC lines in head-to-head comparisons**
In all figure panels, the performance of KOLF2.1J is shown relative to a comparison line, with the providing laboratory and differentiation method indicated. Photomicrographs of immunostaining (or recording traces) are shown to the left of each panel, where scale bars represent 50 μm and nuclear markers are in blue. Quantification of phenotypic outcomes are given on the right, where N=3 differentiations were analyzed for each cell line and unpaired t-tests were used to calculate significance unless otherwise stated. *p<0.05, **p<0.01. (A) Multi-lineage differentiation to endoderm (SOX17), mesoderm (brachyury), or ectoderm (nestin) reveals no significant differences between KOLF2.1J and APOE3 ALSTEM. All data not shown. (B) NGN2-induced neuronal differentiation quantified by the expression of the neuronal marker TUJ1 (grey) and the cortical layer 2/3 marker BRN2 (green) show similar expression across KOLF2.1J and comparison lines ND50003, ND50004, and 11a. n=7 wells/line, N=1 differentiation. Kruskal-Wallis test for each marker. (C) Patch clamp analysis of NGN2-induced neurons derived from BioniC13 and KOLF2.1J lines show spontaneous miniature excitatory postsynaptic currents (mEPSCs, left) in similar frequencies between the lines (center), though KOLF2.1J-derived cells appeared to have a lower excitatory EPSC amplitude (right). (D) A greater proportion of KOLF2.1J-derived cortical neurons generated by directed differentiation expressed the deep-layer cortical marker CTIP2 and TUJ1 than the Thermo Fisher A18944 comparison line (D). (E) In another group, directed cortical neuron differentiation yielded similar expression of Tuj1 (grey) CTIP2 (red) as the comparison lines RBi001 and Ctrl1. Comparison lines averaged together, N=3–6 differentiations/line. (F) KOLF2.1J and H9 neurons had similar expression of BRN2 (green), CTIP2 (red), and TBR1 (grey) following directed differentiation. (G) Greater efficiency of Islet1 expression was observed following differentiation of KOLF2.1J into motor neurons, compared to the CVB cell line. (H) KOLF2.1J and WTC11 cell lines were differentiated into astrocytes and immunostained for S100β (green) and NFIA (red, left), revealing a higher intensity of S100β immunoreactivity in KOLF2.1J. N=8 differentiations. (I) Microglial differentiation of KOLF2.1J and A18944 revealed no significant differences in the expression of the IBA1, TREM2, or PU.1 marker genes. Mann-Whitney test for each marker. (J) Differentiation of KOLF2.1J and SFC065 towards ventral midbrain dopaminergic neurons showed similar percentages of cells expressing the marker genes EN1 (green) and FOXA2 (red). Mann-Whitney test for EN1 expression.

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A reference human induced pluripotent stem cell line for large-scale collaborative studies

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A reference human induced pluripotent stem cell line for large-scale collaborative studies

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