The characteristics of human iChon cell lines.

<p>(<b>A</b>) Phase images of human iChon cell lines and HDFs. The photos were taken when the cell numbers had expanded and reached 10⁷. Bar: 100 µm. (<b>B</b>) The mRNA levels of <i>cMYC</i>, <i>KLF4</i> and <i>SOX9</i> in iChon cells. The relative expression levels in comparison to human HDFs or HFCs are shown. The mRNA levels were determined by a real-time RT-PCR analysis using primers specific for endogenous transcripts (white columns) and those common for both transgenic and endogenous transcripts (black columns). The error bars indicate ± SD (<i>n</i> = 3). (<b>C</b>) The growth curves of human iChon cells and parental HDFs. (<b>D</b>) The karyotype of human iChon cells. iChon cell line #87-18 was examined at passages 15. A total of 20 cells for each cell line were examined. HDF, human dermal fibroblasts; HFC, redifferentiated human fetal chondrocytes.</p

The generation and selection of induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture.

<p>(<b>A</b>) Top, a schematic diagram of the gene transduction. Middle left, HDFs. Middle right, polygonal-shaped cells generated by transduction of c-MYC, KLF4, and SOX9. Bottom left, nodules formed by polygonal-shaped cells. Bottom right, nodules were intensely stained with alcian blue, suggesting the production of glycosaminoglycan. Bars in top panels, 100 µm; Bars in bottom panels, 500 µm. (<b>B</b>) Top, a schematic diagram of the gene transduction. Left middle and bottom panels, phase and GFP images of cell nodules formed in HDF culture at 18 days after transduction with c-MYC, KLF4 and SOX9. Right middle and bottom panels, magnification of the boxed region in the left panels. Cells were cultured in the absence of puromycin Bars in left panels, 500 µm; Bars in right panels, 100 µm. (<b>C</b>) HDF cultures which had been transduced with lentiviral <i>COL11A2-</i> reporter vectors and nucleofected with <i>Slc7a1</i> were transduced with retroviral c-MYC, KLF4 and SOX9, or DsRed fluorescent protein, or SOX5, SOX6 and SOX9. Cells were cultured in the absence of puromycin. Dishes (10 cm in diameter) were stained with alcian blue 21 days after retroviral transduction. The numbers of nodules with positive alcian blue staining were counted. (<b>D</b>) HDF cultures which had been transduced with lentiviral <i>COL11A2-</i> reporter vectors and nucleofected with <i>Slc7a1</i> were transduced with retroviral c-MYC, KLF4 and SOX9, or DsRed fluorescent protein, or SOX5, SOX6 and SOX9. Puromycin was added to the medium 7 days after retroviral transduction. Dishes (10 cm in diameter) were stained with crystal violet and alcian blue 21 days after retroviral transduction. The numbers of all colonies stained with crystal violet (white bars) and the numbers of colonies with positive alcian staining (black bars) were counted. In (C and D), after nucleofection of <i>Slc7a1</i>, HDFs were replated at a density 5×10⁵ cells per 10 cm dish for retroviral transduction. Cells were split 1∶5 onto 10 cm dishes immediately after completion of the retroviral transductions. The numbers of nodules in five 10 cm dishes which were derived from one identical dish were added together. Error bars indicate ± SD (<i>n</i> = 3 dishes).</p

The origins of iChon cells in HDF culture.

<p>HDFs were transduced with the lentiviral <i>COL11A2</i>-reporter vector, nucleofected with Slc7a1, and transduced with retroviral c-MYC, KLF4 and SOX9 vectors. Cells were replated onto a well of a 6 well plate immediately after completion of the retroviral transduction. The well was cultured in the absence of puromycin and subjected to time-lapse GFP observation using the Biostation CT (Nikon). (<b>A</b>) The entire wells were each scanned using a total of 64 images (8 rows×8 columns), and a tiled image was reconstituted. The time-lapse GFP fluorescence of the tiled images at 3, 8 and 14 days after transduction of retroviral c-MYC, KLF4 and SOX9 vectors (3 left panels), and a phase contrast image 14 days after transduction (right panel), spanning an entire well of a 6 well plate are shown. GFP fluorescence was not observed at 3 days after retroviral transduction. Some cell clusters expressed <i>COL11A2-GFP</i> fluorescence at 8 days after transduction (arrowheads), and gradually formed nodules, increasing the level of GFP fluorescence. At 14 days after transduction, some nodules (arrowheads) specifically expressed <i>COL11A2-GFP</i> and others did not. Bar, 10 mm. (<b>B</b>) The magnification of the boxed regions in (A). At 1 day after transduction, no cells expressed <i>COL11A2-GFP</i>, suggesting that they were not chondrogenic cells. A cluster of cells with polygonal morphology started to express <i>COL11A2-GFP</i> weakly (arrow) at 5 days after transduction. The cells in the cluster increased in number and expressed <i>COL11A2-GFP</i> (half-arrow) at 7 days after transduction. A cell cluster formed multiple layers, forming a nodule which expressed <i>COL11A2-GFP</i> strongly at 14 days after transduction. These results suggest that iChon cells are derived from non-chondrogenic cells which did not express <i>COL11A2-GFP</i>. Bar, 100 µm. (<b>C</b>) Magnification of the boxed regions in (B). GFP images were enhanced to detect weak fluorescent signals. Only the polygonal cell clusters expressed <i>COL11A2-GFP</i>, but the surrounding fibroblast cells did not express <i>COL11A2-GFP</i>. Bar, 50 µm.</p

<i>In vivo</i> cartilage formation by human iChon cells in the subcutaneous spaces of nude mice (A and B) and articular cartilage defects created in SCID rat (C).

<p>The iChon cells at passage 7 were used. Mice were sacrificed at 4 weeks after subcutaneous injection of iChon cells, and nodules at the injected sites were collected. Rats were sacrificed 4 weeks after implantation. (<b>A</b>) The histological features of nodules formed by injected iChon cells into subcutaneous spaces of nude mice. Safranin O-fast green-iron hematoxylin staining. Cartilaginous matrix was specifically stained with Safranin O as an orange color. Bars in top panels, 500 µm; Bars in bottom panels, 100 µm. (<b>B</b>) The expression of differentiation-related proteins in nodules formed by injected iChon cells into subcutaneous spaces of nude mice. Semiserial sections of nodules derived from injected 87-18 human iChon cell were stained with Safranin O-fast green-iron hematoxylin and immunostained with anti-type I collagen, anti-type II collagen and anti-human vimentin antibodies. The bars in top panels, 500 µm; in bottom panels, 100 µm. (<b>C</b>) Human iChon cells (line #117-3) were implanted into defects created in the articular cartilage of the distal femurs of SCID rats. Four weeks after implantation the rats were sacrificed. Semiserial sections were stained with hematoxylin-eosin and immunostained with anti-type II collagen, anti-type X collagen, and anti-human vimentin antibodies. Brackets indicate regions of defects created in the articular cartilage. The bars, 100 µm.</p

Marker gene expression of human iChon cell lines.

<p>RNA samples were extracted from iChon cells at passage 7. (<b>A</b>) The quantitative expression analyses of chondrocyte and fibroblast marker genes in human iChon cell lines, HDFs and HFC. Error bars indicate the ± SD (<i>n</i> = 3). *P<0.01 compared with iChon cell lines by Student’s t-test. (<b>B</b>) The quantitative expression analyses of chondrocyte hypertrophy and terminal differentiation marker genes in human iChon cell lines, HDFs and CD-hBMSCs. Error bars indicate the ± SD (<i>n</i> = 3). The relative expression levels of <i>COL10A1</i> and <i>MMP13</i> mRNAs were zero in iChon cell lines. (<b>C</b>) Methylation of the regulartory region of the <i>COL1A1</i> gene. Bisulfite genomic sequencing of the regulartory regions of <i>COL1A1</i> was performed using DNA derived from iChon cell lines and HDFs. Each horizontal row of circles represents an individual sequencing result from one amplicon. Open circles indicate unmethylated CpG dinucleotides, while closed circles indicate methylated CpGs. The nucleotide numbers for <i>COL1A1</i> are indicated at the bottom. The ATG translation initiation codon is set as +1 (GenBank accession number NC 000017, nt 48261457). HDFs, human dermal fibroblasts; HFC, redifferentiated human fetal chondrocytes; CD-hBMSCs, chondrogenically differentiated human bone marrow stem cells.</p

The response of human iChon cells to osteogenic conditions.

<p>(<b>A</b>) Human iChon cells (line #117-37) were cultured in the osteogenic medium (α-MEM supplemented with 10% FBS, 10 mM β-glycerophosphate, 50 µg/ml ascorbic acid, and 10⁻⁷ M dexamethasone with the absence or presence of various concentrations of BMP2 as indicated at the bottom of the graphs) for 21 days. RNAs were extracted and subjected to a real-time RT-PCR expression analysis. Error bars indicate the ± SD (<i>n</i> = 3). (<b>B</b>) Human iChon cells were implanted into defects created in the calvaria of SCID mice. Three weeks after implantation, the mice were sacrificed. Semiserial sections were stained with hematoxylin-eosin and safranin O, and immunostained with anti-human vimentin antibodies. The magnification of boxed regions in the left panel is shown in the right three panels, respectively. The bars in the left panels, 250 µm; in the other panels, 50 µm.</p

Investiční záměr pro zástavbu lokality Libouchec-Knínice(okres Ústí n.L)

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta stavební. Katedra (222) městského stavitelstv

Additional file 2: Figure S2. of Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

SYO-1 and HS-SY-II (PDGFRα-dependent) SS cells were treated with 0.001–10 μM of TAS-115 or control (0.1% DMSO) for 3 h, followed by an additional treatment with 10-ng/ml rhPDGF-BB for the last 15 min. (PPTX 178 kb