Fig. 13

1 The similarity in appearance between induced pluripotent stem (iPS) clones and human embryonic stem cells (HESC). In panel A', the figure shows colonies of NHDF1 (normal human dermal fibroblast) cells infected with different viruses. These viruses include an empty virus, a GFP-containing virus, or a combination of six viruses, each carrying one of five specific transcription factors or GFP. The colonies are observed under phase contrast microscopy, revealing their diverse morphologies. Panels B-B” provide phase-contrast images of specific colonies from the cultures transduced with the combination of five transcription factors and GFP. These images are merged with live TRA-1–81 staining (shown in red) and GFP fluorescence (shown in green) derived from the pMX-GFP virus. The upper images show the merged view, while the lower images display only the TRA-1–81 channel. It is noteworthy that only a small fraction of colonies exhibit TRA-1–81 positivity, as indicated in panels B and B'. Importantly, the TRA-1–81 staining in these positive colonies closely resembles that of HESC. Panels C–C” display phase-contrast images of iPS clones at different passages, highlighting their morphological characteristics. Finally, panels D-D”' present “live” TRA-1–81 staining merged with the phase-contrast appearance of specific iPS clones at passage 5. 2 The induced pluripotent stem (iPS) clones exhibit important markers found in human embryonic stem cells (HESC). In panel A and A', polymerase chain reaction (PCR) was performed on genomic DNA obtained from various sources: iPS clones, “early” OCT4/C-MYC clones, NHDF1 (normal human dermal fibroblast) cells infected with control or defined factor viruses, and HSF1 or H9 HESC. The PCR targeted specific regions of integrated viruses, with a loading control PCR for a genomic region on the X chromosome within the XIST locus. Additionally, iPS clones 24 and 29 were included in panel A' as a positive control for the PCR conditions. In panel B, reverse transcription PCR (RT-PCR) was conducted to analyze pMX retroviral transcription and the expression of endogenous counterparts of the defined factors, as well as other genes specific to HESC (TDGF1 through REX1), in iPS clones, NHDF1 cells, HSF1 HESC, and OCT4/CMYC clones. It is worth noting that iPS clones 24 and 29, as well as the OCT4/CMYC clones, displayed limited suppression of expression from the viruses they received. 3 The comparison of the transcriptome (gene expression profile) between induced pluripotent stem (iPS) clones and human embryonic stem cells (HESC). In panel A, the expression values of various cell types are presented on a scatter plot using genome-wide microarray expression data. The cell types include fibroblasts infected with control viruses or viruses carrying specific factors, iPS clones 2 and 5, and the HSF1 HESC line. It is observed that iPS clones 2 and 5 exhibit a high similarity to the HSF1 HESC, while iPS lines 1 and 7 show slightly less similarity. Panel B represents the global Pearson correlation analysis of the entire expression data between the different cell types, indicating the degree of similarity in gene expression. Panel C displays the hierarchical clustering of gene-expression data using the indicated cell types. The analysis involved normalization and expression analysis with DNA-chip analyzer (dChip), filtering genes based on a 20% presence call, and removing redundant probe sets. In panel D, the 2,000 most up- and down-regulated genes in HSF1 versus NHDF (normal human dermal fibroblast) were identified from genome-wide expression datasets. The expression of these genes was further analyzed to determine if they were up-regulated, down-regulated, or showed no change in expression between iPS clones (or infected fibroblast pools) and NHDF. The terms “MI” and “MD” represent statistically marginal increase and decrease, respectively. 4 The formation of embryoid bodies (EBs) by induced pluripotent stem (iPS) cells, which is comparable to human embryonic stem cells (HESCs). Panel A displays phase-contrast images of EBs created from iPS clones 2 and 5. Panel B demonstrates the growth of iPS-derived EBs when placed on adherent tissue culture dishes using three distinct media conditions. One of the media conditions includes the presence of bone morphogenetic protein 4 (BMP). 5 The pluripotency of induced pluripotent stem (iPS) cells and the increased expression of markers associated with ectoderm, endoderm, and mesoderm. In panel A, a real-time RT-PCR analysis compares the expression of pluripotency genes in iPS cells and control human embryonic stem cells (HESC) after inducing differentiation through embryoid body (EB) formation and subsequent plating under specific conditions (BMP4, FBS, and retinoic acid). The analysis measures the fold change in gene expression relative to the reference gene GAPDH. Notably, the down-regulation of pluripotency markers like OCT4 and NANOG is observed during EB differentiation. In panel B, a similar analysis is conducted, but this time the expression of marker genes associated with different germ layers is examined. Each marker is specific to a particular germ layer, as indicated. The y-axis represents the fold induction of gene expression compared to undifferentiated cells. While the extent of induction of lineage markers may vary between HESC and iPS clones, the overall pattern remains consistent. Reprinted from [728]with permission from the PNAS