PMC

Teratoma formation after in vivo implantation of iPS cells from clonally expanded MSCs and HDF. A–I, hematoxylin and eosin staining of harvested tumors. Each tumor (teratoma) contained three embryonic germ layer tissues including gut-like epithelium (A–C: endoderm), cartilage (D–F: mesoderm), and neuroepithelial rosettes (G–I: ectoderm). cl.58, colony derived from 10F-15 clonal cells; cl.8, colony derived from 10F-101 clonal cells; cl.1, colony derived from HDF.

Global gene expression analysis of iPS cells from clonally expanded MSCs and their parental cells. A, expression levels of reprogramming genes in parental cells and iPS cells. cl.39, colony derived from 10F-15 clonal cells; cl.58, colony derived from 10F-15 clonal cells; cl.5, colony derived from 10F-101 clonal cells; cl.8, colony derived from 10F-101 clonal cells; cl.1, colony derived from HDF. B, global gene expression comparison between parental cell and iPS cell. Red circles indicate the expression levels of OCT3/4, NANOG, SALL4, LIN28, and TDGF1. Red lines indicate the equivalent and 3-fold differences between the two samples. C, comparison of gene expression profiles of parental cells, iPS cells, and human ES (hES) cells. cl.8, colony derived from HDF.

Characterization of iPS cells from clonally expanded MSCs and HDF. A, immunocytochemistry of SSEA-3, SSEA-4, TRA-1–60, TRA-1–81, OCT3/4, and NANOG for iPS cells. Scale bars = 100 μm. B, DNA methylation states of the OCT3/4 and NANOG promoter regions. Open circles indicate unmethylated CpGs, and closed circles indicate methylated CpGs. C, RT-PCR analysis of ES cell marker genes and retroviral Tgs for iPS cells from different colonies (cl.58/cl.15 derived from 10F-15 clonal cells, cl.5/cl.8 derived from 10F-101 clonal cells, and cl.1/cl.8 derived from HDF). D, telomerase activity detected by TRAP assay. +, heat-treated; −, non-treated samples; P, parental cell. IC, internal control.

In vitro differentiation of iPS cells from clonally expanded MSCs and HDF. A, EB formation of iPS cells at day 8. B–E, immunocytochemistry for SOX17 (B), AFP (green) (C), desmin (red) (D), and α-smooth muscle actin βIII-tubulin (E). F, immunocytochemistry of differentiated iPS cells cultured on PA6 feeder cells at day 18. Tyrosine hydroxylase- and βIII-tubulin-positive cells are indicated as green and red cells, respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bars = 100 μm. G, RT-PCR analysis of three germ layer marker genes. P, parental cell; U, undifferentiated iPS cells; D, differentiated iPS cells (EB).

Generation of iPS cells from MSCs. A, reprogramming efficiency of non-cloned parental MSCs from the third molars of 10-, 16-, and 13-year-old donors. The bars represent the S.D. (n = 10). B, proliferation rate of clonally expanded MSCs from 10-year-old donor and HDF at day 5. The bars indicate S.D. (n = 3). C, Reprogramming efficiency of clonally expanded MSCs and HDF cells. The closed circles represent average of two independent experiments, and the bars indicate S.D. (n = 8). D, ALP staining of three factors transduced cells at day 30. E, viral transduction efficiency of the pMXs retroviral vector. The percentages indicate the ratio of DsRed-Express-positive cells. F, parental cells of clonally expanded MSC and HDF cells (upper panel), iPS cells from the parental cells (middle panel), and ALP staining of each iPS cell (lower panel). Scale bars = 100 μm.