Fig 1.
A. Description of the four culture conditions used for the expansion of hESC WA09. B. Culture expansion and sample collection strategy.
Fig 2.
Graphs illustrating the number (A) and total length (B) of genetic aberrations.
Aberrations were identified in the samples using CNV analysis of high resolution SNP genotyping data from the WA09 hESC line. Duplications and deletions; regions of loss of homozygosity (LOH) were categorized as deletions and complex CNVs were categorized as duplications. The x-axis indicates the number of passages for the current study. All cultures started from a source culture that had been passaged for 37 passages. Genetic aberrations present in this source culture were not counted, and therefore at the start of the study, the number of duplications and deletions were set at 0. Of note, each culture condition was assigned a unique color that is consistent throughout the manuscript.
Fig 3.
Overview of genetic aberrations.
Results are shown for the WA09 hESC line (A) and the HDF51IPS1, HDF51IPS7, and HDF51IPS11 hiPSC lines (B). Fig. 3A shows regions of duplication and deletion in the 4culture conditions for the WA09 hESC line. Duplications (3 or 4 copies) (identified by CNVPartition 3.2.0) are shown below the chromosome in a red scale. Deletions (0 or 1 copy) (identified by CNVPartition 3.2.0 and replicate error analysis) are shown below the regions of duplication in a blue scale. Red and blue scales are used to represent the passage when the event (duplication or deletion) first occurred, and they range from light colors (early passages) to darker colors (late passages). White and black hatches represent partial mutations (duplications or deletions that are present in only a subpopulation of cells) and complex CNVs (CNVs that appear to be composed of both duplicated and deleted regions, for which the precise copy numbers cannot confidently be called based on the SNP genotyping data), respectively. Asterisks represent mutations that were present only in earlier passages and were not detectable in later passages. For each CNV in each condition, the replicate(s) in which the event was detected is indicated by a letter to the right of each bar. Fig. 3B shows regions of duplication, deletion and LOH in the 2 culture conditions for the hiPSC lines. Duplications are shown in blue, deletions are shown in red and regions of LOH are shown in green. The chromosome ideograms are from http://genomics.energy.gov.
Fig 4.
Chromosome 12 detail in MefEnz.
Detailed view of the duplications in chromosome 12 for MefEnz. A. The green areas indicate the regions of duplication that were identified by CNVPartition 3.2.0. The areas that were detected manually rather than by the software are indicated in a scale from grey to black. Estimates of the percentage of the population carrying the duplication were performed using the BAF distance for heterozygous SNPs as described [3]. B. LogR Ratio (LRR) and BAF plots of replicate A of MefEnz showed changes in genetic aberrations over time. C. The CNV data were further validated via TaqMan Copy Number Assays. Probe 1 corresponds to Hs03295596_cn, Probe 2 to Hs04404518_cn, Probe 3 to Hs03812366_cn, Probe 4 to Hs03841181_cn, Probe 5 to Hs06947059_cn, and Probe 6 to Hs06363301_cn. D. Karyotyping results confirmed the CNV data.
Fig 5.
Detail of recurrent deletions in chromosome 17.
A. The short arm of the chromosome is enlarged and the regions that showed deletions in the MefEnz, EcmMech, and EcmEnz conditions are indicated by red bars. Blue lines enclose the common area among all the conditions and the genes in the common area are indicated in the lower part of the figure. Genes for which the level of expression correlated with copy number are highlighted by red squares. B. Graphs representing the expression levels of TP53, SENP3, and SOX15, as measured by gene expression microarray. The ideogram of the chromosome was from the U.S. Department of Energy Genome Programs.
Fig 6.
Horizontal brackets indicate significant differences between the specified conditions, as calculated using ANOVA (p-value<0.05). A. The MTT assay measures cell proliferation, with absorbance being correlated with cell growth. B. Doubling time was calculated from cell counts taken over 3 days (average of 3 replicates per condition). C. The EdU incorporation was determined via FACS, and reveals the cell cycle distribution. The graph shows the percent of cells in S phase. D. Relative telomerase activity is shown. E. Average number of telomere repeat fragments (TRF, average of 3 replicates) for each condition. F. Percent of apoptotic cells, determined using TUNEL-FACS. G. Example of an OCT4-positive focus in a teratoma section. H. Bar graph showing, for each culture condition, the percent of teratoma sections containing at least one OCT4-positive focus, and the average number of foci per section. The error bar indicates the standard deviation of counts from 6 (EcmEnz), 7 (MefMech), or 8 (MefEnz and EcmMech) sections. Raw counts can be found in S4 Table).
Fig 7.
Differences in gene expression with time in culture, passaging method, and media type.
Heatmaps showing differentially expressed genes for early vs. late passage (A), mechanical vs. enzymatic passage (B), and Mef vs. Ecm substrate (C). In heatmap (A), the yellow boxes indicate genes for which the expression levels in the late passage MefMech (P103) samples was similar to those in the early passage samples. Probes were selected by multivariate regression. Enrichments in KEGG pathways identified by functional enrichment analysis are shown. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. D. Diagram of the TP53 signaling pathway, showing genes downregulated in the late vs. early passage samples, and the Ecm vs. Mef substrate comparisons.
Fig 8.
A. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (S6A Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. B. DNA methylation at imprinted loci. Methylation levels for imprinted probes (S6B Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. C, D, E. Heatmaps showing differential DNA methylation genes for early vs. late passage (C), mechanical vs. enzymatic passage (D), and Mef vs. Ecm substrate (E). In heatmap (C), the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [13]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).