 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.
The DNA methylation states of CpG dinucleotides in the OCT3/4 and NANOG promoter regions were evaluated by bisulfite genomic sequencing analysis. All The parental cells were highly (OCT3/4) or partially (NANOG) methylated, whereas all of the iPS cells were highly unmethylated (Fig. 2B). These data suggested that these promoters were active in these iPS cells.
All of the iPS cells expressed undifferentiated ES cell maker genes, such as OCT3/4, SOX2, NANOG, reduced expression 1 (REX1), undifferentiated embryonic cell transcription factor 1 (UTF1), growth and differentiation factor 3 (GDF3), developmental pluripotency-associated 2 (DPPA2), DPPA4, and DPPA5. However, the cells did not express retroviral transgenes (Tgs), such as Tg-OCT3/4, Tg-SOX2, and Tg-KLF4, indicating retroviral silencing. The cells before retroviral transduction (parental cell) expressed KLF4 and MYC but did not express other markers (Fig. 2C).
We also examined telomerase activity, which is known to be highly activated in cancer cells and ES cells. Each iPS cell showed high telomerase activity, whereas parental cells as well as heat-treated cells did not (Fig. 2D).
). Immunocytochemistry demonstrated the appearance of βIII-tubulin (red)- and tyrosine hydroxylase (green)-positive neural cells (Fig. 3F). Therefore, iPS cells had successfully differentiated into different types of cells.
FIGURE 3In 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).
RT-PCR analysis confirmed that these EB-derived various types of cells expressed AFP, FOXA2 (markers for endoderm), brachyury, TnTc (mesoderm), MAP2, and PAX6 (endoderm) (Fig. 3G). These data indicate that iPS cells derived from clonally expanded MSCs could differentiate into cell types of three embryonic germ layers.
To test for pluripotency, we injected iPS cells into the testis of severe combined immunodeficient mice. Nine weeks after injection, all showed tumor formation. Histological examination of the tumor demonstrated representative tissues of three embryonic germ layers, such as gut-like epithelium (endoderm), cartilage (mesoderm), and neuroepithelial rosettes (ectoderm) (Fig. 4, A–I). These findings indicated that the tumor was teratoma. Thus, like ES cells, iPS cells derived from clonally expanded MSCs have in vivo capability of differentiation into the three embryonic germ layers.
FIGURE 4Teratoma 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.
We also performed karyotype investigations by chromosomal G-band analysis (supplemental Fig. S3) and multicolor FISH analysis (data not shown). Two lines of iPS cells from 10F-101 showed an additional change in chromosome 16. However, the corresponding parental cell also showed the addition at the same locus, suggesting that the chromosomal aberration was already present in the parental cell (data not shown). Other iPS cells (10F-15 and HDF) showed normal karyotypes (supplemental Fig. S3).
). Microarray analysis showed that the mRNA levels of reprogramming factors, including OCT3/4, SOX2, NANOG, LIN28, and UTF1, were highly expressed in iPS cells when compared with those of the parental cells (Fig. 5A). The expression levels of MYC and p53 were similar in each parental cell and iPS cells. However, KLF4 expression was significantly lower (0.1-fold) in 10F-15 parental cells when compared with that of other parental cells and iPS cells. In addition, KLF5 expression was slightly higher (3-fold) when compared with that of the other cells (Fig. 5A).
FIGURE 5Global 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.
Scatter plot analysis showed that ES cell marker genes, such as OCT3/4, NANOG, LIN28, sal-like 4 (SALL4), and teratocarcinoma-derived growth factor 1 (TDGF1), were highly expressed in iPS cells (Fig. 5B). Heat map analysis showed that gene expression profiles of each iPS cell were similar but different from the corresponding parental cell. These profiles of iPS cells were also similar to those of BG01 and H9 human ES cells (Fig. 5C).
). Although expressions of KLF4, KLF2, KLF5, MYC, TP53, TBX3, and CHD1 were detected in each parental cell, it is hard to show statistical differences of these gene expressions between the high and low reprogramming cells (supplemental Table S2). Expressions of other reprogramming factors (OCT3/4, SOX family, KLF1, MYC family, NANOG, LIN28, UTF1, ESRRB, SALL4, and NR5A2) were not detected in each parental cell (supplemental Table S2). Although significant difference was not found, the KLF4 gene expressions of high reprogramming cells (10F-15, 10F-5, and 10F-8) were lower than cells of low reprogramming cells (10F-101, 10F-107, and HDF). Furthermore, real-time PCR analysis showed the same tendency of the expressions of the KLF4 gene (lower expressions in high reprogramming cells). Real-time PCR analysis also demonstrated the high expression of TP53 in HDF cells but almost the same expression levels in other cells (supplemental Fig. S4).
We then sought unknown factor(s) that facilitate the reprogramming in high reprogramming cells. From comparison of microarray data that was normalized by z-score transformation, we picked out the genes that demonstrated statistically significant difference between the high reprogramming cells (10F-15, 10F-5, and 10F-8) and the low reprogramming cells (10F-101, 10F-107, and HDF). Up-regulated and down-regulated genes are listed in supplemental Table S3.
In our preliminary experiments, we examined whether or not MSCs from the human third molars could be reprogrammed by transduction of three transcription factors, OCT3/4, SOX2, and KLF4, using commercially available lentivirus. We generated iPS cells using the lentivirus; however, the efficiency was extremely low (data not shown). Therefore, we used pMXs retrovirus in this study. We transduced these three transcription factors using the retrovirus into culture-expanded but not clonally expanded MSCs from third molars of 10-, 13-, and 16-year-old donors. We detected the different