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

Change log
Volpato, Viola 
Sandor, Cynthia 
Ried, Janina S 
Baud, Anna 

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.

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
Journal Title
Stem Cell Reports
Conference Name
Journal ISSN
Volume Title
Elsevier BV
European Commission and European Federation of Pharmaceutical Industries and Associations (EFPIA) FP7 Innovative Medicines Initiative (IMI) (115439)
Wellcome Trust (101052/Z/13/Z)
Medical Research Council (MR/L023784/1)
Initiative Joint Undertaking under grant agreement no. 115439, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. A.H., S.C., and M.Z.C. were also funded by the NIHR (Oxford BRC). K.M. and A.B. were also supported by the NIHR GOSH BRC.