Molecular analyses of human induced pluripotent stem cells and embryonic stem cells

Abstract

Recent work from our group and others has argued that human induced pluripotent stem cells (hiPSCs) generated by the introduction of four viruses bearing reprogramming factors differ from human embryonic stem cells (hESCs) at the level of gene expression (Chin et al., 2009). Many of the differences seen were common across independent labs and, at least to some extent, are thought to be a result of residual expression of donor cell-specific genes (Chin et al., 2009; Ghosh et al., 2010; Marchetto et al., 2009). Two new reports reanalyze similar expression data sets as those used in Chin et al. (2009) and come to different conclusions (Newman and Cooper, 2010; Guenther et al., 2010). We compare various approaches to perform gene expression meta-analysis that all support our original conclusions and present new data to demonstrate that polycistronic delivery of the reprogramming factors and extended culture brings hiPSCs transcriptionally closer to hESCs.

Figure 1. Overlap of differentially expressed genes between hiPSCs and hESCs from different labs, determined using the Baysian T-test with FDR correction and 1.5 fold cutoff

Differentially expressed genes between hiPSCs and hESCs were determined for each of the three indicated reprogramming experiments (Chin et al., 2009; Lowry et al., 2008; Maherali et al., 2008; Maherali and Hochedlinger, 2008; Yu et al., 2009) using a Bayesian T-test with a FDR < 0.05 along with a greater than 1.5 fold change requirement. In (A), overlap among the differentially expressed gene lists was only considered if they were differentially expressed in the same direction between hESCs and hiPSCs. In (B), directionality was not taken into consideration for the overlap.

Figure 2. Variation in gene expression between hiPSCs and hESCs generated in our laboratories using different technical strategies

Hierarchical clustering of expression data obtained for hESCs (blue highlight), various hiPSCs at different passage (p) (yellow for single pMX-retroviral hiPSCs, pink for polycistronic pMIP retroviral hiPSCs, and purple for polycistronic STEMCCA lentiviral hiPSCs), and fibroblasts (F, gray). The Supplemental Methods section provides further identification for each cell line.

Figure 3. Extended passaging brings the gene expression signature of hiPSCs closer to that of hESCs

(A) Correlation table comparing normalized and filtered microarray data for hESC lines to hiPSCs generated with 4 separate pMX retroviruses, which were profiled at different passages (p) as indicated in the yellow highlight. Values present the Pearson’s correlation coefficient between two cell lines. (B) Correlation table comparing hESC lines to hiPSCs generated with the polycistronic STEMCCA lentivirus at different passages. (C) Correlation table comparing hESCs to hiPSCs at early passage derived from the three unique reprogramming experiments: 4-factor pMX-hiPSCs (yellow), polycistronic STEMCCA lentivirus (purple), and polycistronic pMIP retrovirus (pink).