Fig. 3

1 The process of generating piPSC colonies from PFF (pluripotent stem cells derived from preimplantation embryos). (A) The first image is a phase contrast image of PFF. (B) The second image shows granulated piPSC colonies similar to mouse and human iPSC that begin to appear approximately three weeks after viral infection. (C) The third image represents a representative piPSC colony after multiple passages, resembling hESC (human embryonic stem cells), shown at a lower magnification. (D) The fourth image is a higher magnification of the same piPSC colony shown in (C). (E) The piPSC colonies express alkaline phosphatase (AP), as depicted in the image. (F) The image shows nuclear localization of OCT4 (green) and surface SSEA1 (red) in the piPSC colonies. (G) Some piPSC colonies have a tendency to undergo spontaneous differentiation, as indicated by the area (arrow) on the right side of the colony. The differentiated cells exhibit cobblestone morphology with a relatively low nucleus to cytoplasm ratio. 2 The results of gene expression analysis conducted on piPSC (porcine-induced pluripotent stem cells) compared to PFF (porcine fetal fibroblast) and H9 hESC (human embryonic stem cells). The analysis involved different techniques, as described in the following paragraphs. In panel A, the researchers used RT-PCR (Reverse Transcription Polymerase Chain Reaction) to examine the expression of specific pluripotency genes in piPSC, PFF, and H9 hESC. The primers used were designed to target porcine genes rather than their human counterparts. However, it was observed that the primers for pc-MYC and pKLF4 also showed some level of cross-reactivity. Panel B displays the results of hierarchical clustering analysis performed on microarray data from three piPSC lines (IC1, ID4, and ID6) and two PFF cells (1 and 2). The clustering was based on Pearson-centered single-linkage rule, and it aimed to identify patterns of gene expression similarity or dissimilarity among the samples. The analysis included all genes (totaling 8,015) that exhibited a fold-change of at least 1.3 in their normalized expression between piPSC and PFF, with a significance level (P value) of 0.05 or lower. The values indicated next to the branches represent Pearson distances, which indicate the degree of dissimilarity between the gene expression profiles. In panel C, the fold differences (Log2) in gene expression between piPSC and PFF are presented. The black bars on the right-hand side of the axis represent genes that were up-regulated (showed increased expression) in piPSC compared to PFF, while the gray bars on the left side represent down-regulated (showed decreased expression) genes. The significance of the differences was assessed using P values, with '*' indicating a significance level of 0.05 or lower and '**' indicating a significance level of 0.01 or lower. 3 The results of immunofluorescence staining carried out on piPSC colonies cultured on MEF, focusing on pluripotent markers. The upper panels (A, B, and C) depict the immunofluorescence staining of OCT4, NANOG, and SOX2 respectively. The lower panels (A–C) confirm the specific localization of these markers to the nuclei, as indicated by the blue staining with DAPI. 4 The measurement of telomerase activity in different types of cells. The telomerase activities of several piPSC lines (IC1 passage 10, ID4 passage 10, ID6 passage 10, IIIB2 passage 3, and IB3 passage 8) are compared to their parental cells, including EGFP-PFF passage 10, MEF passage 4, and H9 hESC passage 41. The assay was conducted using triplicate samples, each containing 0.2 μg of total cell protein, and the TRAPESE-RT Telomerase Detection Kit (Chemicon) was utilized. The telomerase activity is represented by the value in amole, which indicates the number of extended primers containing telomeric repeats. 5 The process of differentiating piPSC (pluripotent induced pluripotent stem cells) into embryoid bodies (EB). In Part A, Day 0 shows piPSC cells plated on MEF (mouse embryonic fibroblasts). Day 1 shows an image of the resulting EB obtained on the next day, while Day 5 displays an image after 5 days of differentiation. Finally, Day 9 exhibits cells treated with 5% FBS (fetal bovine serum) for a duration of 9 days. Part B presents the results of real-time RT-PCR analysis, which measures the relative concentrations of transcript molecules of pluripotent and lineage-specific genes in various cell lines. These cell lines include piPSC lines (IC1, ID4, and ID6), PFF (pluripotent fetal fibroblasts), and piPSC that were differentiated into EB using BMP4, FBS, or retinoic acid (RA) as differentiation agents. The y-axis represents the fold change relative to the expression of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), which is a reference gene commonly used in gene expression studies. 6 A microscopic image of a tumor taken from the peritoneum of a hairless mouse. The tumor, which was surgically removed, was formed by injecting cells from the piPSC line ID6 under the skin of the mouse. The tumor exhibited a high level of differentiation and consisted of various types of tissues. These tissues included neural epithelium (ectoderm) on the left side, striated muscle (mesoderm) in the middle, and epithelium with a brush border (endoderm) on the right side. The magnification used for all three tissues is the same. An inset on the right side provides a closer view of the brush border, indicated by a red arrow, and the scale bar in the image corresponds to 5 μm. Reprinted from [83] with permission from the PNAS