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Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Large cohorts of human induced pluripotent stem cells (iPSCs) from healthy donors are a potentially powerful tool for investigating the relationship between genetic variants and cellular behavior. Here, we integrate high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions. We identify genes that correlate in expression with intrinsic and extrinsic PEER factors and associate outlier cell behavior with genes containing rare deleterious non-synonymous SNVs. Our study, thus, establishes a strategy for examining the genetic basis of inter-individual variability in cell behavior., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)