Figure 1
Figure 1. Induction of MPS IH somatic cells into MPS-iPS cells. (A) Live culture stained with TRA-1-60 antibody 4 weeks after transduction. (B) Phase contrast (PC) image of the same human ES cell-like colony. To confirm their ability to express ES cell proteins, human KCs derived from P1 were stained with alkaline phosphatase (C) and immunostained with TRA-1-81 (D), SSEA-3 (E), SSEA-4 (G), OCT4 (H), TRA-1-60 (J), and Nanog (K). Corresponding images stained with 4,6-diamidino-2-phenylindole (DAPI) show nuclei of individual cells in the colonies (F,I,L). Images in panels A-C were obtained with a Leica DMIL scope, magnification 10×/0.22. Images were acquired with an Optronics camera and Optronics MagnaFire software. Fluorescein isothiocyanate color block was used. Images in panels D-L were obtained with an Olympus BX61 FV500 Confocal Microscope, magnification 10×/.40. Argon, green HeNe, and blue diode lasers were used to acquire the images in Olympus FluoView software Version 4.3. All images were taken at room temperature. (M) Quantitative reverse transcription PCR analysis of OCT4, SOX2, NANOG, KLF4, c-MYC, LIN28, ABCG2, DNMT3b, and REX1 expression levels in KC-derived iPS cells from P1 (red bars), KC-derived iPS cells from P2 (green bars), and MSC-derived iPS cells from P2 (blue bars), respectively. All values were normalized against endogenous GAPDH expression. Because MPS-KCs and MPS-MSCs expressed statistically indistinguishable levels of these ES cell marker genes, their values were plotted against expression levels of parental MPS-KCs from P1 (black bars). (N) Bisulfite sequencing of the OCT4 and NANOG promoters in parental MPS-KCs (from P1), parental MPS-MSCs (from P2), and MPS-iPS cells derived from KCs in P1. Open circles denote unmethylated CpGs, and filled circles represent methylated CpGs. CpG position relative to the downstream transcriptional start site is shown above each column. Sequencing reactions of specific amplicons are represented by each row of circles. KC indicates keratinocytes; MSC, mesenchymal stromal cells; iPS cells, induced pluripotent cells; P, patient; MPS IH, mucopolysaccharidosis type I, Hurler syndrome.

Induction of MPS IH somatic cells into MPS-iPS cells. (A) Live culture stained with TRA-1-60 antibody 4 weeks after transduction. (B) Phase contrast (PC) image of the same human ES cell-like colony. To confirm their ability to express ES cell proteins, human KCs derived from P1 were stained with alkaline phosphatase (C) and immunostained with TRA-1-81 (D), SSEA-3 (E), SSEA-4 (G), OCT4 (H), TRA-1-60 (J), and Nanog (K). Corresponding images stained with 4,6-diamidino-2-phenylindole (DAPI) show nuclei of individual cells in the colonies (F,I,L). Images in panels A-C were obtained with a Leica DMIL scope, magnification 10×/0.22. Images were acquired with an Optronics camera and Optronics MagnaFire software. Fluorescein isothiocyanate color block was used. Images in panels D-L were obtained with an Olympus BX61 FV500 Confocal Microscope, magnification 10×/.40. Argon, green HeNe, and blue diode lasers were used to acquire the images in Olympus FluoView software Version 4.3. All images were taken at room temperature. (M) Quantitative reverse transcription PCR analysis of OCT4, SOX2, NANOG, KLF4, c-MYC, LIN28, ABCG2, DNMT3b, and REX1 expression levels in KC-derived iPS cells from P1 (red bars), KC-derived iPS cells from P2 (green bars), and MSC-derived iPS cells from P2 (blue bars), respectively. All values were normalized against endogenous GAPDH expression. Because MPS-KCs and MPS-MSCs expressed statistically indistinguishable levels of these ES cell marker genes, their values were plotted against expression levels of parental MPS-KCs from P1 (black bars). (N) Bisulfite sequencing of the OCT4 and NANOG promoters in parental MPS-KCs (from P1), parental MPS-MSCs (from P2), and MPS-iPS cells derived from KCs in P1. Open circles denote unmethylated CpGs, and filled circles represent methylated CpGs. CpG position relative to the downstream transcriptional start site is shown above each column. Sequencing reactions of specific amplicons are represented by each row of circles. KC indicates keratinocytes; MSC, mesenchymal stromal cells; iPS cells, induced pluripotent cells; P, patient; MPS IH, mucopolysaccharidosis type I, Hurler syndrome.

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