Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods

Many reprogramming methods can generate human induced pluripotent stem cells (hiPSCs) that closely resemble human embryonic stem cells (hESCs). This has led to assessments of how similar

hiPSCs are to hESCs, by evaluating differences in gene expression, epigenetic marks and differentiation potential. However, all previous studies were performed using hiPSCs acquired from

different laboratories, passage numbers, culturing conditions, genetic backgrounds and reprogramming methods, all of which may contribute to the reported differences. Here, by using

high-throughput sequencing under standardized cell culturing conditions and passage number, we compare the epigenetic signatures (H3K4me3, H3K27me3 and HDAC2 ChIP-seq profiles) and

transcriptome differences (by RNA-seq) of hiPSCs generated from the same primary fibroblast population by using six different reprogramming methods. We found that the reprogramming method

impacts the resulting transcriptome and that all hiPSC lines could terminally differentiate, regardless of the reprogramming method. Moreover, by comparing the differences between the hiPSC

and hESC lines, we observed a significant proportion of differentially expressed genes that could be attributed to polycomb repressive complex targets.

This study was funded by the Canadian Institute of Health Research 201210MFE-289547 (J.M.C.), National Institutes of Health 1K99HL128906 (J.M.C.), PCBC_JS_2014/4_01 (J.M.C.), National

Research Foundation of Korea 2012R1A6A3A03039821 (J.L.), the Burroughs Wellcome Foundation, National Institutes of Health R01 HL123968, HL128170, R01 HL126527 (J.C.W.), and P01 GM099130

(M.P.S.). The authors would like to thank the Stanford Stem Cell Institute Genome Center for their sequencing knowledge, V. Sebastiano for hESC culturing, and B. Huber for his help with the

teratoma assay. We would also like to thank J. Brito and B. Wu for their help in editing the manuscript.

Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA

Jared M. Churko, Jaecheol Lee, Mohamed Ameen, Mingxia Gu, Sebastian Diecke, Karim Sallam, Joseph D. Gold & Joseph C. Wu

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, 94305, USA

Jared M. Churko, Jaecheol Lee, Mohamed Ameen, Mingxia Gu, Sebastian Diecke, Karim Sallam & Joseph C. Wu

Department of Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA

Jared M. Churko, Jaecheol Lee, Mohamed Ameen, Mingxia Gu, Sebastian Diecke, Karim Sallam & Joseph C. Wu

Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA

J.D.G., N.S., M.P.S. and J.C.W. supervised and planned the project. J.M.C. wrote the manuscript, performed data analysis, generated and cultured hiPSC lines, and performed RNA-seq. N.S. and

M.V. performed integration analysis. H.I. helped analyse RNA-seq. J.L. performed ChIP-seq experiments. M.A. and M.G. performed FACS analysis on differentiated cardiomyocytes. G.W. and K.S.

helped to culture hiPSC and hESC lines. S.D. generated minicircle hiPSC lines.

Reads per kilobase of transcript per million mapped reads of each Ensembl ID, calculated via AltAnalyze.

Gene-expression differences, calculated via a Bayes moderated t-test p-value (unpaired), assuming unequal variance, and p