Plating human iPSC lines on micropatterned substrates reveals role for ITGB1 nsSNV in endoderm formation.

Alice Vickers,Franca Fraternali,Anna Laddach,Fiona M Watt,Martina Poletti,Mukul Tewary,Vasiliki Salameti,Davide Danovi

doi:10.1016/j.stemcr.2021.09.017

Abstract

SummaryQuantitative 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.

Highlights

Human induced pluripotent stem cells provide an accessible resource for the in vitro study of human development and disease mechanisms and have demonstrated their potential to provide patient-specific cells for regenerative medicine (Fatehullah et al, 2016; Liu et al, 2018; Mandai et al, 2017; Park et al, 2008; Takahashi et al, 2007; Yamanaka, 2020)
Using the Human Induced Pluripotent Stem Cell Initiative (HipSci) resource, we have previously combined cell-based assays, high-content imaging, and genome sequencing datasets to identify rare, deleterious, non-synonymous single nucleotide variants in genes related to cell adhesion that are associated with outlier induced pluripotent stem cell (iPSC) phenotypes in the pluripotent state (Vigilante et al, 2019)
Building upon our previous study (Vigilante et al, 2019), we selected iPSC lines from the HipSci cell bank that harbor rare and deleterious non-synonymous single nucleotide variants (nsSNVs) in genes related to cell adhesion and/or germ layer differentiation

Summary

Introduction

Human induced pluripotent stem cells (iPSCs) provide an accessible resource for the in vitro study of human development and disease mechanisms and have demonstrated their potential to provide patient-specific cells for regenerative medicine (Fatehullah et al, 2016; Liu et al, 2018; Mandai et al, 2017; Park et al, 2008; Takahashi et al, 2007; Yamanaka, 2020). The Human Induced Pluripotent Stem Cell Initiative (HipSci) was established to create a large, high-quality reference panel of iPSCs with accompanying genetic, proteomic, and phenotypic data. Diverse, large-scale collections of iPSCs such as HipSci have enabled the identification of genetic factors that influence gene expression and cellular phenotypes in both pluripotent and differentiated cells (Bonder et al, 2021; Carcamo-Orive et al, 2017; Kilpinen et al, 2017; Panopoulos et al, 2017; Schwartzentruber et al, 2018; Warren et al, 2017). Using the HipSci resource, we have previously combined cell-based assays, high-content imaging, and genome sequencing datasets to identify rare, deleterious, non-synonymous single nucleotide variants (nsSNVs) in genes related to cell adhesion that are associated with outlier iPSC phenotypes in the pluripotent state (Vigilante et al, 2019)

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