Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Volpato V., Smith J., Sandor C., Ried JS., Baud A., Handel A., Newey SE., Wessely F., Attar M., Whiteley E., Chintawar S., Verheyen A., Barta T., Lako M., Armstrong L., Muschet C., Artati A., Cusulin C., Christensen K., Patsch C., Sharma E., Nicod J., Brownjohn P., Stubbs V., Heywood WE., Gissen P., De Filippis R., Janssen K., Reinhardt P., Adamski J., Royaux I., Peeters PJ., Terstappen GC., Graf M., Livesey FJ., Akerman CJ., Mills K., Bowden R., Nicholson G., Webber C., Cader MZ., Lakics V.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

DOI

10.1016/j.stemcr.2018.08.013

Type

Journal article

Journal

Stem Cell Reports

Publication Date

09/10/2018

Volume

11

Pages

897 - 911

Keywords

cortical neurons, cross-site experimental variation, gene expression profile, induced pluripotent stem cell, molecular profiling, proteomic profiles, public-private partnership, reproducibility, single-cell sequencing, stembancc, Cell Differentiation, Cell Line, Factor Analysis, Statistical, Gene Expression Regulation, Genotype, Humans, Induced Pluripotent Stem Cells, Neurons, Phenotype, Proteomics, Reproducibility of Results, Transcriptome

Permalink Original publication