Yosef Buganim 1, Styliani Markoulaki 2, Niek van Wietmarschen 3, Heather Hoke 4, Tao Wu 5, Kibibi Ganz 2, Batool Akhtar-Zaidi 2, Yupeng He 6, Brian J Abraham 2, David Porubsky 3, Elisabeth Kulenkampff 2, Dina A Faddah 4, Linyu Shi 2, Qing Gao 2, Sovan Sarkar 2, Malkiel Cohen 2, Johanna Goldmann 2, Joseph R Nery 6, Matthew D Schultz 6, Joseph R Ecker 6, Andrew Xiao 5, Richard A Young 7, Peter M Lansdorp 8, Rudolf Jaenisch 9
Abstract
Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.
Figures
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Figure 1. Characterization of SNEL-iPSC Lines
(A) Schematic presentation of Bayesian network demonstrates the hierarchy of a subset of pluripotent genes that leads to a stable and transgene independent pluripotency state (Buganim et al., 2012). Sall4, Nanog, Esrrb, and Nanog (SNEL) are marked by a red circle. (B) Representative images of two stable dox-independent, GFP-positive colonies (Nanog-GFP SNEL#1 and Oct4-GFP SNEL#3) and immunostaining for Sall4, Sox2, Utf1, and Esrrb. (C) Heatmap demonstrating the relative expression levels of Dppa3, Dppa2, Zfp42 (Rex1), and Lin28 normalized to the Hprt housekeeping control gene in the indicated samples. (D) Hematoxylin and eosin staining of teratoma sections generated from Oct4-GFP SNEL#1 showing structures from all three layers. See also Figure S1.
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Figure 2. SNEL-iPSCs Produce “All-iPSC” Mice with High Success Rates as Compared to OSKM
(A) Percent of injected blastocysts surviving to birth are plotted for OSKM and SNEL lines, with the number of blastocysts noted on the x axis. Blue represents the number of pups that merely survived delivery, red the number of pups additionally foster-nursed. Percentages were compared by χ2 test to compute significance. (B) Representative images of 4n adult mice produced from Oct4-GFP SNEL#1 and Oct4-GFP SNEL#4 lines and their F1 generation. (C) Confirmation of origin of “all-iPSC” mice by PCR for strain-specific polymorphisms. Two different simple sequence polymorphism (SSLP) markers were tested using genomic DNA isolated from tissues of “all-iPSC” mice. Genomic DNA from the parental iPSCs (donor cells), a 129 Sv/Jae mouse (donor strain), and a B6D2F1 mouse (host blastocyst strain) served as controls. See also Figure S2.
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Figure 4. The Transcript Number of Key Master Regulators and the Establishment of ESC-Specific Superenhancers Are Comparable between Poor- and High-Quality iPSCs
(A) sm-mRNA-FISH experiments depict the transcript number of Oct4 versus Sox2 and Oct4 versus Esrrb in single cells from the indicated iPSC lines. n, represents the number of single cells analyzed. (B) ChIP-seq binding profiles for Oct4, Sox2, and Nanog (merged, OSN) in V6.5 mESCs and Med1 for the indicated cell lines at the Sox2 locus. Location of the superenhancer, as defined in V6.5 mESCs (Whyte et al., 2013), is indicated by the red bar. Rpm/bp, reads per million per base pair. (C) Hierarchical clustering of Med1 densities in superenhancers recapitulates phylogeny of cell types. ChIP-seq read densities for Med1 were calculated in mES superenhancers. Clustering these densities indicates that cell types of similar origin have similar signal of Med1 in superenhancers. All ChIP-seq was performed with a Bethyl Laboratories antibody (A300-793A, lot A300-783A), except for the farthest right V6.5 ChIP, which was performed with a Santa Cruz Biotechnology antibody (SC-5334X, lot A1112). See also Figure S4.