anti-tra-1-81 Search Results


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  • 93
    Abcam mouse anti tra 1 81
    Generation and Identification of Induced Pluripotent Stem Cells from the TSC Patient (A) Experimental scheme for the generation of iPSCs from peripheral blood mononuclear cells. (B) Morphology of iPSC lines in feeder-free medium. Scale bar, 100 μm. (C) Immunofluorescence for the pluripotency markers OCT4, SOX2, SSEA4, and <t>TRA-1-81</t> in iPSC lines. Scale bar, 100 μm. (D) Immunofluorescence for the endoderm (α-fetoprotein [AFP]), mesoderm (smooth muscle actin [SMA]), and ectoderm (NESTIN) markers in iPSC lines. Scale bar, 50 μm. (E) Teratoma formation for the TSC-patient-derived iPSC line shows tissues from the three germ layers. Scale bar, 100 μm. (F) Karyotype analysis of iPSC lines. See also Figures S1–S3 .
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    Millipore anti tra 1 81
    Generation of iPSCs from an AD patient harboring a PSEN1 (E120K) mutation, and an eldely normal subject. (A) Established iPSC lines from both control and PS1-E120K patient showing the expression of pluripotent stem cell markers, such as OCT4 (red), SOX2 (green), SSEA4 (red) and <t>TRA-1-81</t> (red). (B) Reverse transcription PCR (RT-PCR) showing the expression of pluripotency markers (OCT4, SOX2, NANOG, SSEA4 AND TRA-1-81) in both iPSC lines. (C) Genomic DNA sequences showing the presence of the heterozygous E120K mutation (GAA to AAA) in the PSEN1 gene of the PS1-E120K-iPSC line. (D) Immunofluorescence analysis showing the potential of iPSC lines to form three germ layers, including ectoderm (type III β-tubulin [TUJ1], green), mesoderm (smooth muscle actin [SMA], green), and endoderm (α-fetoprotein [AFP], red). Scale bar: 100 µm. (E) Karyotype analysis of the control and PS1-E120K iPSC lines. (F) Reverse-transcription PCR analysis showing the absence of integration of the Sendai virus vectors. (G) PCR analysis showing no contamination by mycoplasma.
    Anti Tra 1 81, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti tra 1 81
    WA09 hESCs subcultured for over 25 passages using hypertonic citrate retain their stem cell characteristics. (A) Immunodetection of Oct4, Sox2 and Nanog antigens (green); SSEA-4, Tra-1-60 or <t>Tra-1-81</t> antigens (red). Individual cell nuclei are visualized using DAPI (blue). Scale bar: 200 µM. (B) Flow cytometric analysis performed on cells cultured in either StemPro® or mTeSR™1 using antibodies that detect Oct4, SSEA-4, Tra-1-60, and Tra-1-81 antigens. Cells expressing each pluripotent antigen, detected using a specific antibody are illustrated in red. The isotype control used to detect non-specific binding is shown in gray. (C) Immunohistochemistry performed on embryoid bodies differentiated for 7 days in suspension culture and 7 days on gelatin-coated plates. Antibodies detecting Beta-III-Tubulin (TUJ1), Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown (green). Nuclear staining is shown using DAPI (blue). Scale bar: 200 µM. (D) Tissue sections of teratomas produced from undifferentiated hESCs contain cells from the indicated germ layers. Sections are shown counterstained with Hematoxylin and Eosin. Scale bar: 200 µm.
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    Santa Cruz Biotechnology anti tra 1 81
    hESCs maintain their morphology and stem cell markers expression upon Plasmocin TM prophylactic treatment. WA09 (H9) and H5 cells were treated with Plasmocin TM 5 µg/ml during 5 consecutive passages (one passage per week) (Prophylactic treatment) and then: (A) photographed using an inverted microscope in order to compare colony morphology; and grown on Matrigel TM coated plates until confluence and stained with primary antibodies that recognize stem cell markers. Control: untreated cells. Figure shows representative bright field and fluorescent images of hESCs immunostained or not with antibodies against SSEA-4, TRA-1-60, <t>TRA-1-81,</t> Nanog and Oct-4. Scale bars = 100 µm; (B) mRNA levels of oct-4 and nanog were analyzed by RT-Real Time PCR on each passage of the 5 consecutive passages of the prophylactic treatment. Control: untreated cells. Rpl7 expression was used as normalizer. Graph shows mRNA fold induction relative to human fibroblasts (HF). The mean ± S.E. from three independent experiments are shown. *=p
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    Stemgent anti tra 1 81
    In vitro characterization of the pluripotent state of EiPS cells. ( a ). Nanog, SSEA1, SSEA4, TRA-1-60 and <t>TRA-1-81</t> detected by fluorescent immunohistochemistry. Yellow scale bar 10 μm, white scalebar 20 μm. ( b ). Detection of gene expression of key pluripotency markers using RT-PCR primers specific for equine endogenous genes
    Anti Tra 1 81, supplied by Stemgent, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tra 1 81 millipore
    Real-time simultaneous monitoring of vector-encoded RF expression and pluripotency marker induction. ( A ) Vector expression in <t>Tra-1–81+</t> versus HLA-ABC high cells on day 12 ( Left ) and 20 ( Right ) after transduction. ( B ) Vector silencing in Tra-1–81+
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    Becton Dickinson mouse anti tra 1 81
    Real-time simultaneous monitoring of vector-encoded RF expression and pluripotency marker induction. ( A ) Vector expression in <t>Tra-1–81+</t> versus HLA-ABC high cells on day 12 ( Left ) and 20 ( Right ) after transduction. ( B ) Vector silencing in Tra-1–81+
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    Millipore mouse anti tra 1 81
    LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) <t>TRA-1-81</t> in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p
    Mouse Anti Tra 1 81, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson tra 1 81 alexa647
    LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) <t>TRA-1-81</t> in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p
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    Stemgent tra 1 81 09 0011
    LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) <t>TRA-1-81</t> in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p
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    Merck KGaA anti tra 1 81 fitc
    LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) <t>TRA-1-81</t> in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p
    Anti Tra 1 81 Fitc, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stemgent mouse anti tra 1 81
    LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) <t>TRA-1-81</t> in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p
    Mouse Anti Tra 1 81, supplied by Stemgent, used in various techniques. Bioz Stars score: 89/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec anti tra 1 81 pe
    Propagation and characterisation of urine-derived renal progenitors. (A) Representative pictures of the “rice grain”-like appearance of the cells from the initial attachment to an elongated MSC-like morphology. (B) Growth curve analysis of selected urine-derived renal progenitors carried out using the Resazurin metabolic assay. Data are presented as means ± SEMs. (C) Immune-phenotyping for SSEA4, <t>TRA-1-81</t> and TRA-1-60; and (D) immunofluorescence-based detection of the expression of pluripotency-associated stem cell- proteins SSEA4 (red), C-KIT (green), CD133 (red) and the mesenchymal-associated protein Vimentin (green); cell nuclei were stained using Hoechst/DAPI (scale bars: 100 µm and 50 µm). (E) Bisulfite sequencing of CpG island methylation patterns within the 5′- regulatory region of the OCT4 gene in UM51. Filled circles stand for methylated CpG dinucleotides. White circles stand for unmethylated CpGs. Arrows indicate the transcription start site. (F) In vitro Osteoblast, Chondrocyte and Adipocyte differentiation potential of urine-derived renal progenitors. (G) Cytokines secreted by urine-derived renal progenitors in culture media. Lists of significant GOs and KEGG pathways associated with the genes encoding the secreted cytokines are shown in Supplemental Fig. S1G .
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    Santa Cruz Biotechnology antibody mouse anti tra 1 81
    Propagation and characterisation of urine-derived renal progenitors. (A) Representative pictures of the “rice grain”-like appearance of the cells from the initial attachment to an elongated MSC-like morphology. (B) Growth curve analysis of selected urine-derived renal progenitors carried out using the Resazurin metabolic assay. Data are presented as means ± SEMs. (C) Immune-phenotyping for SSEA4, <t>TRA-1-81</t> and TRA-1-60; and (D) immunofluorescence-based detection of the expression of pluripotency-associated stem cell- proteins SSEA4 (red), C-KIT (green), CD133 (red) and the mesenchymal-associated protein Vimentin (green); cell nuclei were stained using Hoechst/DAPI (scale bars: 100 µm and 50 µm). (E) Bisulfite sequencing of CpG island methylation patterns within the 5′- regulatory region of the OCT4 gene in UM51. Filled circles stand for methylated CpG dinucleotides. White circles stand for unmethylated CpGs. Arrows indicate the transcription start site. (F) In vitro Osteoblast, Chondrocyte and Adipocyte differentiation potential of urine-derived renal progenitors. (G) Cytokines secreted by urine-derived renal progenitors in culture media. Lists of significant GOs and KEGG pathways associated with the genes encoding the secreted cytokines are shown in Supplemental Fig. S1G .
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    Cell Signaling Technology Inc anti tra 1 81
    Characterization of the two types of piPSCs. (A)The colonies of mpiPSCs were round and three-dimensional (bottom). The colonies of hpiPSCs were large and flat (top). Bar scale = 500 μm. The hpiPSCs and mpiPSCs were both positive for alkaline phosphatase (AP). Bar scale = 200 μm. (B) Immunocytochemical staining of Oct4, Sox2, SSEA-1 and SSEA-4 in hpiPSCs and mpiPSCs. Bar scale = 200 μm. (C) The surface marker <t>Tra-1-81</t> and Tra-1-60 were positive in hpiPSCs but not detected in mpiPSCs. Bar scale = 200 μm. (D) X chromosome activation state of hpiPSCs and mpiPSCs after immunostaining for H3K27me3. Positive signals of histone H3K27 me3 spots were observed in hpiPSCs but not in mpiPSCs. Bar scale = 200 μm. The white arrowheads indicate the H3K27me3 positive spot in the cell. (E) Immunocytochemical assay of 3-germ-layer cells in EBs derived from both types of piPSCs, the markers include β Ⅲ-Tubulin (ectoderm), α-SMA (mesoderm) and Sox17 (endoderm). Scale bar = 200 μm. (F) Hematoxylin and eosin staining of teratoma sections of piPSCs. Left: blood vessel of endothelium (ectoderm); middle: muscle (mesoderm); right: gut-like epithelium (endoderm). Scale bars = 200 μm. (G) Karyotype analysis of piPSCs.
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    Becton Dickinson tra 1 81 alexa fluor 647
    Characterization of the two types of piPSCs. (A)The colonies of mpiPSCs were round and three-dimensional (bottom). The colonies of hpiPSCs were large and flat (top). Bar scale = 500 μm. The hpiPSCs and mpiPSCs were both positive for alkaline phosphatase (AP). Bar scale = 200 μm. (B) Immunocytochemical staining of Oct4, Sox2, SSEA-1 and SSEA-4 in hpiPSCs and mpiPSCs. Bar scale = 200 μm. (C) The surface marker <t>Tra-1-81</t> and Tra-1-60 were positive in hpiPSCs but not detected in mpiPSCs. Bar scale = 200 μm. (D) X chromosome activation state of hpiPSCs and mpiPSCs after immunostaining for H3K27me3. Positive signals of histone H3K27 me3 spots were observed in hpiPSCs but not in mpiPSCs. Bar scale = 200 μm. The white arrowheads indicate the H3K27me3 positive spot in the cell. (E) Immunocytochemical assay of 3-germ-layer cells in EBs derived from both types of piPSCs, the markers include β Ⅲ-Tubulin (ectoderm), α-SMA (mesoderm) and Sox17 (endoderm). Scale bar = 200 μm. (F) Hematoxylin and eosin staining of teratoma sections of piPSCs. Left: blood vessel of endothelium (ectoderm); middle: muscle (mesoderm); right: gut-like epithelium (endoderm). Scale bars = 200 μm. (G) Karyotype analysis of piPSCs.
    Tra 1 81 Alexa Fluor 647, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA anti tra 1 81
    DP31 cells can be reprogrammed to iPSCs using OCT3/4 and SOX2. (a) We obtained a few ES-cell-like colonies from 5 × 10 5 DP31 cells transduced with OCT3/4 and SOX2 (OS), but we could not obtain any ES-cell-like colonies from DP75 cells. Human ES-cell-like colonies were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). (b) Genomic PCR using transgene-specific primers, with DP31 cells as a negative control, confirmed the insertion of only two transgenes in iPSCs derived from DP31 cells transduced with OS (iPS-DP31-OS) by PCR. The numbers denote different iPSC lines. We cropped the gels and blots for clarifying our presentation. The gels have been run under the same experimental conditions. (c) iPS-DP31-OS cells expressed pluripotency markers including SSEA3, TRA-1-60, <t>TRA-1-81,</t> and NANOG. Scale bar = 100 μm. (d) Pluripotency of iPS-DP31-OS cells was confirmed by EB-mediated differentiation and teratoma formation assay. Immunofluorescence staining showed that EB structures derived from iPS-DP31-OS cells expressed markers characteristic of the three germ layers including βIII-tubulin (ectoderm), α-smooth-muscle actin (mesoderm), and α-fetoprotein (endoderm). Nuclei were stained with Hoechst 33342. Scale bar = 100 μm. Hematoxylin- and eosin-stained sections of teratomas generated from iPS-DP31-OS cells are shown in the lower panels. The teratomas contained various tissues of all three germ layers, such as neural-tube-like structures (ectoderm), cartilage (mesoderm), and gut-like epithelial tissue (endoderm). Abbreviations: AFP, alpha-fetoprotein; α-SMA, alpha smooth muscle actin. Scale bar = 100 μm.
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    Millipore alexafluor 488 conjugated anti tra 1 81
    DP31 cells can be reprogrammed to iPSCs using OCT3/4 and SOX2. (a) We obtained a few ES-cell-like colonies from 5 × 10 5 DP31 cells transduced with OCT3/4 and SOX2 (OS), but we could not obtain any ES-cell-like colonies from DP75 cells. Human ES-cell-like colonies were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). (b) Genomic PCR using transgene-specific primers, with DP31 cells as a negative control, confirmed the insertion of only two transgenes in iPSCs derived from DP31 cells transduced with OS (iPS-DP31-OS) by PCR. The numbers denote different iPSC lines. We cropped the gels and blots for clarifying our presentation. The gels have been run under the same experimental conditions. (c) iPS-DP31-OS cells expressed pluripotency markers including SSEA3, TRA-1-60, <t>TRA-1-81,</t> and NANOG. Scale bar = 100 μm. (d) Pluripotency of iPS-DP31-OS cells was confirmed by EB-mediated differentiation and teratoma formation assay. Immunofluorescence staining showed that EB structures derived from iPS-DP31-OS cells expressed markers characteristic of the three germ layers including βIII-tubulin (ectoderm), α-smooth-muscle actin (mesoderm), and α-fetoprotein (endoderm). Nuclei were stained with Hoechst 33342. Scale bar = 100 μm. Hematoxylin- and eosin-stained sections of teratomas generated from iPS-DP31-OS cells are shown in the lower panels. The teratomas contained various tissues of all three germ layers, such as neural-tube-like structures (ectoderm), cartilage (mesoderm), and gut-like epithelial tissue (endoderm). Abbreviations: AFP, alpha-fetoprotein; α-SMA, alpha smooth muscle actin. Scale bar = 100 μm.
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    Merck KGaA mouse igm anti tra 1 81
    DP31 cells can be reprogrammed to iPSCs using OCT3/4 and SOX2. (a) We obtained a few ES-cell-like colonies from 5 × 10 5 DP31 cells transduced with OCT3/4 and SOX2 (OS), but we could not obtain any ES-cell-like colonies from DP75 cells. Human ES-cell-like colonies were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). (b) Genomic PCR using transgene-specific primers, with DP31 cells as a negative control, confirmed the insertion of only two transgenes in iPSCs derived from DP31 cells transduced with OS (iPS-DP31-OS) by PCR. The numbers denote different iPSC lines. We cropped the gels and blots for clarifying our presentation. The gels have been run under the same experimental conditions. (c) iPS-DP31-OS cells expressed pluripotency markers including SSEA3, TRA-1-60, <t>TRA-1-81,</t> and NANOG. Scale bar = 100 μm. (d) Pluripotency of iPS-DP31-OS cells was confirmed by EB-mediated differentiation and teratoma formation assay. Immunofluorescence staining showed that EB structures derived from iPS-DP31-OS cells expressed markers characteristic of the three germ layers including βIII-tubulin (ectoderm), α-smooth-muscle actin (mesoderm), and α-fetoprotein (endoderm). Nuclei were stained with Hoechst 33342. Scale bar = 100 μm. Hematoxylin- and eosin-stained sections of teratomas generated from iPS-DP31-OS cells are shown in the lower panels. The teratomas contained various tissues of all three germ layers, such as neural-tube-like structures (ectoderm), cartilage (mesoderm), and gut-like epithelial tissue (endoderm). Abbreviations: AFP, alpha-fetoprotein; α-SMA, alpha smooth muscle actin. Scale bar = 100 μm.
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    Image Search Results


    Generation and Identification of Induced Pluripotent Stem Cells from the TSC Patient (A) Experimental scheme for the generation of iPSCs from peripheral blood mononuclear cells. (B) Morphology of iPSC lines in feeder-free medium. Scale bar, 100 μm. (C) Immunofluorescence for the pluripotency markers OCT4, SOX2, SSEA4, and TRA-1-81 in iPSC lines. Scale bar, 100 μm. (D) Immunofluorescence for the endoderm (α-fetoprotein [AFP]), mesoderm (smooth muscle actin [SMA]), and ectoderm (NESTIN) markers in iPSC lines. Scale bar, 50 μm. (E) Teratoma formation for the TSC-patient-derived iPSC line shows tissues from the three germ layers. Scale bar, 100 μm. (F) Karyotype analysis of iPSC lines. See also Figures S1–S3 .

    Journal: Stem Cell Reports

    Article Title: Abnormal Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Partially Mimicked Development of TSC2 Neurological Abnormalities

    doi: 10.1016/j.stemcr.2017.02.020

    Figure Lengend Snippet: Generation and Identification of Induced Pluripotent Stem Cells from the TSC Patient (A) Experimental scheme for the generation of iPSCs from peripheral blood mononuclear cells. (B) Morphology of iPSC lines in feeder-free medium. Scale bar, 100 μm. (C) Immunofluorescence for the pluripotency markers OCT4, SOX2, SSEA4, and TRA-1-81 in iPSC lines. Scale bar, 100 μm. (D) Immunofluorescence for the endoderm (α-fetoprotein [AFP]), mesoderm (smooth muscle actin [SMA]), and ectoderm (NESTIN) markers in iPSC lines. Scale bar, 50 μm. (E) Teratoma formation for the TSC-patient-derived iPSC line shows tissues from the three germ layers. Scale bar, 100 μm. (F) Karyotype analysis of iPSC lines. See also Figures S1–S3 .

    Article Snippet: The primary antibodies used were as follows: rabbit anti-OCT4 (Abcam, catalog no. ab19857), rabbit anti-SOX2 (Abcam, catalog no. ab97959), mouse anti-SSEA4 (Abcam, catalog no. ab16287), mouse anti-TRA-1-81 (Abcam, catalog no. ab16289), mouse anti-AFP (Abcam, catalog no. ab3980), rabbit anti-SMA (Abcam, catalog no. ab5694), mouse anti-NESTIN (Life Technologies, catalog no. A24353), goat anti-SOX1 (Life Technologies, catalog no. A24354), rabbit anti-SOX2 (Life Technologies, catalog no. A24354), rabbit anti-PAX6 (Life Technologies, catalog no. A24354), rabbit anti-β-III-TUBULIN (TUJ1; Abcam, catalog no. ab18207), and rabbit anti-GFAP (Abcam, catalog no. ab7260).

    Techniques: Immunofluorescence, Derivative Assay

    Culture of hESCs on feeder cell-derived ECM. A , phase-contrast images of ECMs isolated from CD1 MEFs at P4 and P9 and from ihPSFs by alkali/detergent extraction. Scale bars , 100 μm. B , phase-contrast images showing morphology of HUES1 cells cultured for five consecutive passages on ECM derived from CD1 P4 and P9, ihPSF, and hPSF. Scale bars , 100 μm. C , HUES1 cells cultured on CD1 P4 and P9 ECM stained positively for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81 after multiple (up to four) passages. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm. D , HUES1 cells cultured on ihPSF-derived ECM stained positively for pluripotency-associated markers Nanog, Oct4, and Sox2 after multiple (up to four) passages, whereas HUES1 cells cultured on hPSF-derived ECM lost the expression of pluripotency-associated markers Nanog, Oct4, and Sox2. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance *

    doi: 10.1074/jbc.M113.463372

    Figure Lengend Snippet: Culture of hESCs on feeder cell-derived ECM. A , phase-contrast images of ECMs isolated from CD1 MEFs at P4 and P9 and from ihPSFs by alkali/detergent extraction. Scale bars , 100 μm. B , phase-contrast images showing morphology of HUES1 cells cultured for five consecutive passages on ECM derived from CD1 P4 and P9, ihPSF, and hPSF. Scale bars , 100 μm. C , HUES1 cells cultured on CD1 P4 and P9 ECM stained positively for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81 after multiple (up to four) passages. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm. D , HUES1 cells cultured on ihPSF-derived ECM stained positively for pluripotency-associated markers Nanog, Oct4, and Sox2 after multiple (up to four) passages, whereas HUES1 cells cultured on hPSF-derived ECM lost the expression of pluripotency-associated markers Nanog, Oct4, and Sox2. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 50 μm.

    Article Snippet: Primary antibodies and concentrations used were anti-Nanog (2 μg/ml; R & D Systems); anti-Oct4 (2.5 μg/ml; BD Biosciences); anti-TRA-1-81 (1 μg/ml; Abcam); anti-Sox2, anti-GATA4, anti-α-smooth muscle actin, anti-βIII-tubulin, anti-Sox17, anti-brachyury, anti-vimentin, and anti-α-fetoprotein (all 5 μg/ml; all R & D Systems); anti-fibronectin (1.25 μg/ml; Sigma-Aldrich); anti-tenascin C (10 μg/ml; Millipore); anti-collagen VI (10 μg/ml; Abcam); anti-human collagen XII and anti-mouse collagen XII (1:3000 and 1:1000, respectively; kind gift of M. Koch, University of Cologne); pan-specific anti-laminin (1:100; kind gift of D. R. Garrod, University of Manchester); anti-fibrillin-1 N-19 (N-terminal region) and PRO (proline-rich region) (1:50 and 1:200, respectively; kind gift of C. M. Kielty); and anti-fibulin-2 (2.5 μg/ml; kind gift of T. Sasaki).

    Techniques: Derivative Assay, Isolation, Cell Culture, Staining, Marker, Expressing

    Culture of hESCs on single ECM substrates or on substrates in combination with 5 μg/ml fibronectin. A , HUES1 cells were successfully cultured over three passages on fibrillin-1 coated at two different concentrations (10 and 20 μg/ml), similar to feeder-free culture on fibronectin (50 μg/ml). B , fibulin-2 and perlecan supported HUES1 cell culture only in combination with 5 μg/ml fibronectin; on these substrates, hESCs were maintained over three passages. C, HUES1 and HUES7 cells were successfully cultured over five passages on fibrillin-1 (10 μg/ml) and were positive for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance *

    doi: 10.1074/jbc.M113.463372

    Figure Lengend Snippet: Culture of hESCs on single ECM substrates or on substrates in combination with 5 μg/ml fibronectin. A , HUES1 cells were successfully cultured over three passages on fibrillin-1 coated at two different concentrations (10 and 20 μg/ml), similar to feeder-free culture on fibronectin (50 μg/ml). B , fibulin-2 and perlecan supported HUES1 cell culture only in combination with 5 μg/ml fibronectin; on these substrates, hESCs were maintained over three passages. C, HUES1 and HUES7 cells were successfully cultured over five passages on fibrillin-1 (10 μg/ml) and were positive for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm.

    Article Snippet: Primary antibodies and concentrations used were anti-Nanog (2 μg/ml; R & D Systems); anti-Oct4 (2.5 μg/ml; BD Biosciences); anti-TRA-1-81 (1 μg/ml; Abcam); anti-Sox2, anti-GATA4, anti-α-smooth muscle actin, anti-βIII-tubulin, anti-Sox17, anti-brachyury, anti-vimentin, and anti-α-fetoprotein (all 5 μg/ml; all R & D Systems); anti-fibronectin (1.25 μg/ml; Sigma-Aldrich); anti-tenascin C (10 μg/ml; Millipore); anti-collagen VI (10 μg/ml; Abcam); anti-human collagen XII and anti-mouse collagen XII (1:3000 and 1:1000, respectively; kind gift of M. Koch, University of Cologne); pan-specific anti-laminin (1:100; kind gift of D. R. Garrod, University of Manchester); anti-fibrillin-1 N-19 (N-terminal region) and PRO (proline-rich region) (1:50 and 1:200, respectively; kind gift of C. M. Kielty); and anti-fibulin-2 (2.5 μg/ml; kind gift of T. Sasaki).

    Techniques: Cell Culture, Marker, Staining

    Generation and characterization of ACL‐iPSC‐SW163A (from a second donor using protocol#2). ACL‐iPSC‐SW163 derived using the scoring technique were grown to passage 20 (six colonies with pluripotent morphology were observed from 50,000 somatic cells giving an estimated efficiency of 0.012%). (A) ACL‐iPSC‐SW163A stained positive for antibodies to pluripotency markers NANOG, OCT‐4, TRA‐1‐60, TRA‐1‐81, SSEA‐3, SSEA‐4, with lack of early differentiation marker SSEA‐1. (B) ACL‐iPSC‐SW163 displayed a normal karyotype. (C) ACL‐iPSC can form embryoid bodies (EBs), (Ci) morphology of EBs at day 10, (Cii‐Civ) phase images displaying a range of morphologies of following EB outgrowth for 5 days. (D) Following outgrowth, EBs produced cells positive for antibodies to markers of mesoderm (α‐SMA), endoderm (GATA6), and ectoderm (Neurofilament). [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: Journal of Orthopaedic Research

    Article Title: Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament

    doi: 10.1002/jor.24493

    Figure Lengend Snippet: Generation and characterization of ACL‐iPSC‐SW163A (from a second donor using protocol#2). ACL‐iPSC‐SW163 derived using the scoring technique were grown to passage 20 (six colonies with pluripotent morphology were observed from 50,000 somatic cells giving an estimated efficiency of 0.012%). (A) ACL‐iPSC‐SW163A stained positive for antibodies to pluripotency markers NANOG, OCT‐4, TRA‐1‐60, TRA‐1‐81, SSEA‐3, SSEA‐4, with lack of early differentiation marker SSEA‐1. (B) ACL‐iPSC‐SW163 displayed a normal karyotype. (C) ACL‐iPSC can form embryoid bodies (EBs), (Ci) morphology of EBs at day 10, (Cii‐Civ) phase images displaying a range of morphologies of following EB outgrowth for 5 days. (D) Following outgrowth, EBs produced cells positive for antibodies to markers of mesoderm (α‐SMA), endoderm (GATA6), and ectoderm (Neurofilament). [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R & D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R & D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R & D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R & D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific).

    Techniques: Derivative Assay, Staining, Marker, Produced

    Generation of ACL‐iPSC‐SW157A from anterior cruciate ligament (ACL) by scoring the culture on day 12 (protocol#2). (A) Isolated ACL cells were subject to the reprogramming protocol#2. (B) Successful reprogramming of ACL to ACL‐iPSC‐SW157A. (Bi–Bii) scoring with a pulled glass pipette (filled arrow) prevented the entire well from rolling up and allows putative induced pluripotent stem cell (iPSC) colonies to grow in the space provided (open arrow), (Biii) phase contrast image of isolated ACL‐iPSC‐SW157A. (C) Positive antibody staining of established passage 22 ACL‐iPSC‐SW157A for the pluripotency‐associated markers OCT‐4, SOX2, NANOG, SSEA‐4, TRA‐1‐60, TRA‐1‐81, SSEA‐3, and lack of early differentiation marker SSEA‐1. 4′,6‐diamidino‐2‐phenylindole (DAPI) nuclear staining shown in the top right quadrant for each. Eight pluripotent colonies were identified (four grown into established lines) from reprogramming of 50,000 cells, representing the efficiency of 0.020%. Scale bars represent 200 μm. [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: Journal of Orthopaedic Research

    Article Title: Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament

    doi: 10.1002/jor.24493

    Figure Lengend Snippet: Generation of ACL‐iPSC‐SW157A from anterior cruciate ligament (ACL) by scoring the culture on day 12 (protocol#2). (A) Isolated ACL cells were subject to the reprogramming protocol#2. (B) Successful reprogramming of ACL to ACL‐iPSC‐SW157A. (Bi–Bii) scoring with a pulled glass pipette (filled arrow) prevented the entire well from rolling up and allows putative induced pluripotent stem cell (iPSC) colonies to grow in the space provided (open arrow), (Biii) phase contrast image of isolated ACL‐iPSC‐SW157A. (C) Positive antibody staining of established passage 22 ACL‐iPSC‐SW157A for the pluripotency‐associated markers OCT‐4, SOX2, NANOG, SSEA‐4, TRA‐1‐60, TRA‐1‐81, SSEA‐3, and lack of early differentiation marker SSEA‐1. 4′,6‐diamidino‐2‐phenylindole (DAPI) nuclear staining shown in the top right quadrant for each. Eight pluripotent colonies were identified (four grown into established lines) from reprogramming of 50,000 cells, representing the efficiency of 0.020%. Scale bars represent 200 μm. [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R & D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R & D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R & D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R & D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific).

    Techniques: Isolation, Transferring, Staining, Marker

    Anterior cruciate ligament cells can be partially reprogrammed using a protocol optimized for dermal fibroblasts (protocol#1). (A) Isolated donor‐matched dermal fibroblast (DF) and anterior cruciate ligament (ACL) were subject to reprogramming protocol#1. (B) Successful reprogramming of DF to DF‐iPSC‐SW156A. (Bi) Isolated DF growing in monolayer in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal calf serum (FCS), (Bii) TRA‐1‐81 positive (StainAliveTM) induced pluripotent stem cell (iPSC) colony emerging between DF. (Biii) Phase‐contrast image of isolated DF‐iPSC‐SW156A. (C) Positive antibody staining of established passage 22 DF‐iPSC‐SW156A for the pluripotency‐associated markers, OCT‐4, SOX2, NANOG, SSEA‐4, TRA‐1‐60, SSEA‐3, and lack of early differentiation marker SSEA‐1. 4′,6‐diamidino‐2‐phenylindole (DAPI) nuclear staining shown in the top right quadrant for each. For DF reprogramming 33 pluripotent colonies were identified from reprogramming of 50,000 cells representing the efficiency of 0.066%, six colonies were grown into established lines. (D) Unsuccessful reprogramming of ACL. (Di) Isolated ACL cells, (Dii) TRA‐1‐81 positive (StainAlive™) cells emerging on top of ACL cells on day 12, (Diii) ACL spontaneously contracting into ligament‐like structure, TRA‐1‐81 positive cell became trapped and unable to form a colony, therefore were impossible to isolate. Scale bars represent 200 μm. [Color figure can be viewed at wileyonlinelibrary.com]

    Journal: Journal of Orthopaedic Research

    Article Title: Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament

    doi: 10.1002/jor.24493

    Figure Lengend Snippet: Anterior cruciate ligament cells can be partially reprogrammed using a protocol optimized for dermal fibroblasts (protocol#1). (A) Isolated donor‐matched dermal fibroblast (DF) and anterior cruciate ligament (ACL) were subject to reprogramming protocol#1. (B) Successful reprogramming of DF to DF‐iPSC‐SW156A. (Bi) Isolated DF growing in monolayer in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal calf serum (FCS), (Bii) TRA‐1‐81 positive (StainAliveTM) induced pluripotent stem cell (iPSC) colony emerging between DF. (Biii) Phase‐contrast image of isolated DF‐iPSC‐SW156A. (C) Positive antibody staining of established passage 22 DF‐iPSC‐SW156A for the pluripotency‐associated markers, OCT‐4, SOX2, NANOG, SSEA‐4, TRA‐1‐60, SSEA‐3, and lack of early differentiation marker SSEA‐1. 4′,6‐diamidino‐2‐phenylindole (DAPI) nuclear staining shown in the top right quadrant for each. For DF reprogramming 33 pluripotent colonies were identified from reprogramming of 50,000 cells representing the efficiency of 0.066%, six colonies were grown into established lines. (D) Unsuccessful reprogramming of ACL. (Di) Isolated ACL cells, (Dii) TRA‐1‐81 positive (StainAlive™) cells emerging on top of ACL cells on day 12, (Diii) ACL spontaneously contracting into ligament‐like structure, TRA‐1‐81 positive cell became trapped and unable to form a colony, therefore were impossible to isolate. Scale bars represent 200 μm. [Color figure can be viewed at wileyonlinelibrary.com]

    Article Snippet: Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R & D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R & D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R & D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R & D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific).

    Techniques: Isolation, Modification, Staining, Marker

    Generation of iPSCs from an AD patient harboring a PSEN1 (E120K) mutation, and an eldely normal subject. (A) Established iPSC lines from both control and PS1-E120K patient showing the expression of pluripotent stem cell markers, such as OCT4 (red), SOX2 (green), SSEA4 (red) and TRA-1-81 (red). (B) Reverse transcription PCR (RT-PCR) showing the expression of pluripotency markers (OCT4, SOX2, NANOG, SSEA4 AND TRA-1-81) in both iPSC lines. (C) Genomic DNA sequences showing the presence of the heterozygous E120K mutation (GAA to AAA) in the PSEN1 gene of the PS1-E120K-iPSC line. (D) Immunofluorescence analysis showing the potential of iPSC lines to form three germ layers, including ectoderm (type III β-tubulin [TUJ1], green), mesoderm (smooth muscle actin [SMA], green), and endoderm (α-fetoprotein [AFP], red). Scale bar: 100 µm. (E) Karyotype analysis of the control and PS1-E120K iPSC lines. (F) Reverse-transcription PCR analysis showing the absence of integration of the Sendai virus vectors. (G) PCR analysis showing no contamination by mycoplasma.

    Journal: Experimental Neurobiology

    Article Title: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia

    doi: 10.5607/en.2018.27.5.350

    Figure Lengend Snippet: Generation of iPSCs from an AD patient harboring a PSEN1 (E120K) mutation, and an eldely normal subject. (A) Established iPSC lines from both control and PS1-E120K patient showing the expression of pluripotent stem cell markers, such as OCT4 (red), SOX2 (green), SSEA4 (red) and TRA-1-81 (red). (B) Reverse transcription PCR (RT-PCR) showing the expression of pluripotency markers (OCT4, SOX2, NANOG, SSEA4 AND TRA-1-81) in both iPSC lines. (C) Genomic DNA sequences showing the presence of the heterozygous E120K mutation (GAA to AAA) in the PSEN1 gene of the PS1-E120K-iPSC line. (D) Immunofluorescence analysis showing the potential of iPSC lines to form three germ layers, including ectoderm (type III β-tubulin [TUJ1], green), mesoderm (smooth muscle actin [SMA], green), and endoderm (α-fetoprotein [AFP], red). Scale bar: 100 µm. (E) Karyotype analysis of the control and PS1-E120K iPSC lines. (F) Reverse-transcription PCR analysis showing the absence of integration of the Sendai virus vectors. (G) PCR analysis showing no contamination by mycoplasma.

    Article Snippet: The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz), anti-SOX2 (1:200, Millipore), anti-NANOG (1:200, R & D Systems), anti-SSEA-4 (1:100, Developmental Studies Hybridoma Bank)), anti-TRA-1-81 (1:100, CHEMICON), Tuj1 anti-tubulin beta III isoform (1:200, Millipore), anti-SMA (1:100, DAKO); anti-AFP (1:100, DAKO), anti-Nestin (1:200, R & D Systems), anti-Musashi (1:200, Millipore), anti-Map2 (1:200, Millipore), anti-TBR1 (1:100, Abcam), anti-CTIP2 (1:100, Abcam), 6E10 anti-Amyloid β (1:500, Covance), AT8 anti-p-tau (1:1000, ThermoFisher), anti-ChAT (1:200, Millipore) and anti-LC3B (1:500, Cell Signaling).

    Techniques: Mutagenesis, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

    WA09 hESCs subcultured for over 25 passages using hypertonic citrate retain their stem cell characteristics. (A) Immunodetection of Oct4, Sox2 and Nanog antigens (green); SSEA-4, Tra-1-60 or Tra-1-81 antigens (red). Individual cell nuclei are visualized using DAPI (blue). Scale bar: 200 µM. (B) Flow cytometric analysis performed on cells cultured in either StemPro® or mTeSR™1 using antibodies that detect Oct4, SSEA-4, Tra-1-60, and Tra-1-81 antigens. Cells expressing each pluripotent antigen, detected using a specific antibody are illustrated in red. The isotype control used to detect non-specific binding is shown in gray. (C) Immunohistochemistry performed on embryoid bodies differentiated for 7 days in suspension culture and 7 days on gelatin-coated plates. Antibodies detecting Beta-III-Tubulin (TUJ1), Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown (green). Nuclear staining is shown using DAPI (blue). Scale bar: 200 µM. (D) Tissue sections of teratomas produced from undifferentiated hESCs contain cells from the indicated germ layers. Sections are shown counterstained with Hematoxylin and Eosin. Scale bar: 200 µm.

    Journal: PLoS ONE

    Article Title: Scalable Passaging of Adherent Human Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0088012

    Figure Lengend Snippet: WA09 hESCs subcultured for over 25 passages using hypertonic citrate retain their stem cell characteristics. (A) Immunodetection of Oct4, Sox2 and Nanog antigens (green); SSEA-4, Tra-1-60 or Tra-1-81 antigens (red). Individual cell nuclei are visualized using DAPI (blue). Scale bar: 200 µM. (B) Flow cytometric analysis performed on cells cultured in either StemPro® or mTeSR™1 using antibodies that detect Oct4, SSEA-4, Tra-1-60, and Tra-1-81 antigens. Cells expressing each pluripotent antigen, detected using a specific antibody are illustrated in red. The isotype control used to detect non-specific binding is shown in gray. (C) Immunohistochemistry performed on embryoid bodies differentiated for 7 days in suspension culture and 7 days on gelatin-coated plates. Antibodies detecting Beta-III-Tubulin (TUJ1), Smooth Muscle Actin (SMA) and Alpha-Feto Protein (AFP) antigens are shown (green). Nuclear staining is shown using DAPI (blue). Scale bar: 200 µM. (D) Tissue sections of teratomas produced from undifferentiated hESCs contain cells from the indicated germ layers. Sections are shown counterstained with Hematoxylin and Eosin. Scale bar: 200 µm.

    Article Snippet: Extracellular antigens were detected on unfixed cells stained with PE-conjugated antigen-specific antibodies and respective isotypes: anti-TRA-1-60 (Becton Dickinson, 560193; 1∶50,000), anti-TRA-1-81 (Becton Dickinson, 560161; 1∶50,000), anti-IgG3 isotype (Becton Dickinson, 559926; 1∶200,000); anti-SSEA4 (Becton Dickinson, 560128; 1∶50,000) and anti-IgM isotype (Becton Dickinson, 555584; 1∶50,000).

    Techniques: Immunodetection, Flow Cytometry, Cell Culture, Expressing, Binding Assay, Immunohistochemistry, Staining, Produced

    hESCs maintain their morphology and stem cell markers expression upon Plasmocin TM prophylactic treatment. WA09 (H9) and H5 cells were treated with Plasmocin TM 5 µg/ml during 5 consecutive passages (one passage per week) (Prophylactic treatment) and then: (A) photographed using an inverted microscope in order to compare colony morphology; and grown on Matrigel TM coated plates until confluence and stained with primary antibodies that recognize stem cell markers. Control: untreated cells. Figure shows representative bright field and fluorescent images of hESCs immunostained or not with antibodies against SSEA-4, TRA-1-60, TRA-1-81, Nanog and Oct-4. Scale bars = 100 µm; (B) mRNA levels of oct-4 and nanog were analyzed by RT-Real Time PCR on each passage of the 5 consecutive passages of the prophylactic treatment. Control: untreated cells. Rpl7 expression was used as normalizer. Graph shows mRNA fold induction relative to human fibroblasts (HF). The mean ± S.E. from three independent experiments are shown. *=p

    Journal: PLoS ONE

    Article Title: Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status, Pluripotency, Cell Viability and Growth

    doi: 10.1371/journal.pone.0070267

    Figure Lengend Snippet: hESCs maintain their morphology and stem cell markers expression upon Plasmocin TM prophylactic treatment. WA09 (H9) and H5 cells were treated with Plasmocin TM 5 µg/ml during 5 consecutive passages (one passage per week) (Prophylactic treatment) and then: (A) photographed using an inverted microscope in order to compare colony morphology; and grown on Matrigel TM coated plates until confluence and stained with primary antibodies that recognize stem cell markers. Control: untreated cells. Figure shows representative bright field and fluorescent images of hESCs immunostained or not with antibodies against SSEA-4, TRA-1-60, TRA-1-81, Nanog and Oct-4. Scale bars = 100 µm; (B) mRNA levels of oct-4 and nanog were analyzed by RT-Real Time PCR on each passage of the 5 consecutive passages of the prophylactic treatment. Control: untreated cells. Rpl7 expression was used as normalizer. Graph shows mRNA fold induction relative to human fibroblasts (HF). The mean ± S.E. from three independent experiments are shown. *=p

    Article Snippet: Briefly, cells were rinsed with PBS and fixed in PBSA (PBS with 0.1% bovine serum albumin) with 4% formaldehyde for 45 min. After two washes cells were permeabilized with 0.1% Triton X-100 in PBSA with 10% normal goat serum for 30 min, washed twice and stained with the corresponding primary antibodies: murine monoclonal antibodies anti-SSEA4 (813-70) (sc-21704), anti-Tra-1-60 (sc-21705), anti-Tra-1-81 (sc-21706), anti-Oct-3/4 (C-10) (sc-5279), anti-Troponin T-C (CT3) (sc-20025), anti-AFP (F8) (sc-166325) from Santa Cruz Biotechnology (CA, USA); rabbit monoclonal antibody anti-Nanog (D73G4) XP (R) from Cell Signaling Technology Inc. (MA, USA) and rabbit polyclonal antibody anti-Nestin (AB5922) from Millipore (MA, USA).

    Techniques: Expressing, Inverted Microscopy, Staining, Real-time Polymerase Chain Reaction

    Infected hESCs cured with Plasmocin TM maintain their morphology, stem cell markers expression and karyotype. Mycoplasma sp. infected HUES-5 884 (H5 884) cells were treated with Plasmocin 25 µg/ml during 14 days (Curative treatment) and: (A) Mycoplasma sp. infection was followed by genomic DNA PCR analysis at day 0, 7 and 14 after antibiotic addition (lanes 4, 2 and 3), in non infected parental H5 line (lane 1) and in cured cells after 6 month post-treatment (lane 7) Control +: Mycoplasma orale genomic DNA (lanes 5 and 9). Control -: H 2 O (lanes 6 and 8); (B) Cured H5 884 cells were photographed using an inverted microscope in order to compare to HUES-5 (H5) colony morphology. Figure shows representative bright field photomicrographs. Scale bars = 100 µm; (C) H5 and cured H5 884 cells were grown on Matrigel TM coated plates until confluence and stained with primary antibodies that recognize stem cell markers. Figure shows representative fluorescent photomicrographs of hESCs immunostained with SSEA-4, TRA-1-60, TRA-1-81, Nanog and Oct-4. Scale bars = 100 µm; (D) H5 884 mRNA levels of oct-4 and nanog were analyzed by RT-Real Time PCR after 14 days of curative treatment and compared to H5 levels. Rpl7 expression was used as normalizer. Graph shows mRNA fold induction relative to human fibroblasts (HF). The mean ± S.E. from three independent experiments are shown. * = p

    Journal: PLoS ONE

    Article Title: Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status, Pluripotency, Cell Viability and Growth

    doi: 10.1371/journal.pone.0070267

    Figure Lengend Snippet: Infected hESCs cured with Plasmocin TM maintain their morphology, stem cell markers expression and karyotype. Mycoplasma sp. infected HUES-5 884 (H5 884) cells were treated with Plasmocin 25 µg/ml during 14 days (Curative treatment) and: (A) Mycoplasma sp. infection was followed by genomic DNA PCR analysis at day 0, 7 and 14 after antibiotic addition (lanes 4, 2 and 3), in non infected parental H5 line (lane 1) and in cured cells after 6 month post-treatment (lane 7) Control +: Mycoplasma orale genomic DNA (lanes 5 and 9). Control -: H 2 O (lanes 6 and 8); (B) Cured H5 884 cells were photographed using an inverted microscope in order to compare to HUES-5 (H5) colony morphology. Figure shows representative bright field photomicrographs. Scale bars = 100 µm; (C) H5 and cured H5 884 cells were grown on Matrigel TM coated plates until confluence and stained with primary antibodies that recognize stem cell markers. Figure shows representative fluorescent photomicrographs of hESCs immunostained with SSEA-4, TRA-1-60, TRA-1-81, Nanog and Oct-4. Scale bars = 100 µm; (D) H5 884 mRNA levels of oct-4 and nanog were analyzed by RT-Real Time PCR after 14 days of curative treatment and compared to H5 levels. Rpl7 expression was used as normalizer. Graph shows mRNA fold induction relative to human fibroblasts (HF). The mean ± S.E. from three independent experiments are shown. * = p

    Article Snippet: Briefly, cells were rinsed with PBS and fixed in PBSA (PBS with 0.1% bovine serum albumin) with 4% formaldehyde for 45 min. After two washes cells were permeabilized with 0.1% Triton X-100 in PBSA with 10% normal goat serum for 30 min, washed twice and stained with the corresponding primary antibodies: murine monoclonal antibodies anti-SSEA4 (813-70) (sc-21704), anti-Tra-1-60 (sc-21705), anti-Tra-1-81 (sc-21706), anti-Oct-3/4 (C-10) (sc-5279), anti-Troponin T-C (CT3) (sc-20025), anti-AFP (F8) (sc-166325) from Santa Cruz Biotechnology (CA, USA); rabbit monoclonal antibody anti-Nanog (D73G4) XP (R) from Cell Signaling Technology Inc. (MA, USA) and rabbit polyclonal antibody anti-Nestin (AB5922) from Millipore (MA, USA).

    Techniques: Infection, Expressing, Polymerase Chain Reaction, Inverted Microscopy, Staining, Real-time Polymerase Chain Reaction

    In vitro characterization of the pluripotent state of EiPS cells. ( a ). Nanog, SSEA1, SSEA4, TRA-1-60 and TRA-1-81 detected by fluorescent immunohistochemistry. Yellow scale bar 10 μm, white scalebar 20 μm. ( b ). Detection of gene expression of key pluripotency markers using RT-PCR primers specific for equine endogenous genes

    Journal: Stem Cell Reviews

    Article Title: Induced Pluripotent Stem Cell Lines Derived from Equine Fibroblasts

    doi: 10.1007/s12015-011-9239-5

    Figure Lengend Snippet: In vitro characterization of the pluripotent state of EiPS cells. ( a ). Nanog, SSEA1, SSEA4, TRA-1-60 and TRA-1-81 detected by fluorescent immunohistochemistry. Yellow scale bar 10 μm, white scalebar 20 μm. ( b ). Detection of gene expression of key pluripotency markers using RT-PCR primers specific for equine endogenous genes

    Article Snippet: The cells were then incubated overnight at 4°C with the following primary anti-mouse or anti-human antibodies; (i ) anti-Nanog (Reprocell #RCAB0002P-F), (ii ) anti-Oct4 (Santa Cruz #sc-5279), (iii ) anti-SSEA1 (Stemgent #09-0005), (vi) anti-SSEA4 (Stemgent #09-0006), (v ) anti-TRA-1-60 (Stemgent #09-0009), (vi ) anti-TRA-1-81 (Stemgent #09-0011).

    Techniques: In Vitro, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction

    Real-time simultaneous monitoring of vector-encoded RF expression and pluripotency marker induction. ( A ) Vector expression in Tra-1–81+ versus HLA-ABC high cells on day 12 ( Left ) and 20 ( Right ) after transduction. ( B ) Vector silencing in Tra-1–81+

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

    doi: 10.1073/pnas.0904825106

    Figure Lengend Snippet: Real-time simultaneous monitoring of vector-encoded RF expression and pluripotency marker induction. ( A ) Vector expression in Tra-1–81+ versus HLA-ABC high cells on day 12 ( Left ) and 20 ( Right ) after transduction. ( B ) Vector silencing in Tra-1–81+

    Article Snippet: For Tra-1–81 immunostaining, duplicate plates were fixed with 4% paraformaldehyde and incubated with an anti- Tra-1–81 antibody (Chemicon), followed by incubation with a horseradish peroxidase-linked anti-mouse IgM secondary antibody (Invitrogen).

    Techniques: Plasmid Preparation, Expressing, Marker, Transduction

    Silencing of ectopic factor expression in hiPSC lines. MFI of each co-expressed fluorescent protein, in 38 reprogrammed (Tra-1–81+, red circles) and 30 non-reprogrammed (Tra-1–81-, blue squares) clones. Error bars, SEM.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

    doi: 10.1073/pnas.0904825106

    Figure Lengend Snippet: Silencing of ectopic factor expression in hiPSC lines. MFI of each co-expressed fluorescent protein, in 38 reprogrammed (Tra-1–81+, red circles) and 30 non-reprogrammed (Tra-1–81-, blue squares) clones. Error bars, SEM.

    Article Snippet: For Tra-1–81 immunostaining, duplicate plates were fixed with 4% paraformaldehyde and incubated with an anti- Tra-1–81 antibody (Chemicon), followed by incubation with a horseradish peroxidase-linked anti-mouse IgM secondary antibody (Invitrogen).

    Techniques: Expressing, Clone Assay

    LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) TRA-1-81 in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p

    Journal: PLoS ONE

    Article Title: Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

    doi: 10.1371/journal.pone.0051866

    Figure Lengend Snippet: LAB-incorporated cell clusters express pluripotency markers. Immunocytochemistry for (A) NANOG, (B) OCT3/4, (C) SOX2, (D) SSEA-4, (E) SSEA-3, and (F) TRA-1-81 in LAB-incorporated cell clusters. (E–F) DAPI. (G) RT-PCR analysis on HDFs, LAB-incorporated cell clusters after 12 days of incorporation, and iPS cells. (H) The relative mRNA expression levels of NANOG , OCT4 , and SOX2 were quantified with LAB-incorporated cells and iPS cells by quantitative RT-PCR and normalized relative to the expression of endogenous human GAPDH . Error bars indicate the standard deviation. * p

    Article Snippet: The following primary antibodies were used for immunocytochemistry: rabbit anti-NANOG (ReproCELL), mouse anti-Oct-3/4 (Santa Cruz Biotechnology), rabbit anti-SOX2 (Millipore), rat anti-SSEA-3 (Millipore), mouse anti-SSEA-4 (Millipore), mouse anti-TRA-1-81 (Millipore), mouse anti-vimentin (Santa Cruz), rabbit anti-desmin (Thermo), mouse anti-Tuj1 (DSHB), and mouse anti-neurofilament (ZYMED).

    Techniques: Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Standard Deviation

    Propagation and characterisation of urine-derived renal progenitors. (A) Representative pictures of the “rice grain”-like appearance of the cells from the initial attachment to an elongated MSC-like morphology. (B) Growth curve analysis of selected urine-derived renal progenitors carried out using the Resazurin metabolic assay. Data are presented as means ± SEMs. (C) Immune-phenotyping for SSEA4, TRA-1-81 and TRA-1-60; and (D) immunofluorescence-based detection of the expression of pluripotency-associated stem cell- proteins SSEA4 (red), C-KIT (green), CD133 (red) and the mesenchymal-associated protein Vimentin (green); cell nuclei were stained using Hoechst/DAPI (scale bars: 100 µm and 50 µm). (E) Bisulfite sequencing of CpG island methylation patterns within the 5′- regulatory region of the OCT4 gene in UM51. Filled circles stand for methylated CpG dinucleotides. White circles stand for unmethylated CpGs. Arrows indicate the transcription start site. (F) In vitro Osteoblast, Chondrocyte and Adipocyte differentiation potential of urine-derived renal progenitors. (G) Cytokines secreted by urine-derived renal progenitors in culture media. Lists of significant GOs and KEGG pathways associated with the genes encoding the secreted cytokines are shown in Supplemental Fig. S1G .

    Journal: Scientific Reports

    Article Title: The FGF, TGFβ and WNT axis Modulate Self-renewal of Human SIX2+ Urine Derived Renal Progenitor Cells

    doi: 10.1038/s41598-020-57723-2

    Figure Lengend Snippet: Propagation and characterisation of urine-derived renal progenitors. (A) Representative pictures of the “rice grain”-like appearance of the cells from the initial attachment to an elongated MSC-like morphology. (B) Growth curve analysis of selected urine-derived renal progenitors carried out using the Resazurin metabolic assay. Data are presented as means ± SEMs. (C) Immune-phenotyping for SSEA4, TRA-1-81 and TRA-1-60; and (D) immunofluorescence-based detection of the expression of pluripotency-associated stem cell- proteins SSEA4 (red), C-KIT (green), CD133 (red) and the mesenchymal-associated protein Vimentin (green); cell nuclei were stained using Hoechst/DAPI (scale bars: 100 µm and 50 µm). (E) Bisulfite sequencing of CpG island methylation patterns within the 5′- regulatory region of the OCT4 gene in UM51. Filled circles stand for methylated CpG dinucleotides. White circles stand for unmethylated CpGs. Arrows indicate the transcription start site. (F) In vitro Osteoblast, Chondrocyte and Adipocyte differentiation potential of urine-derived renal progenitors. (G) Cytokines secreted by urine-derived renal progenitors in culture media. Lists of significant GOs and KEGG pathways associated with the genes encoding the secreted cytokines are shown in Supplemental Fig. S1G .

    Article Snippet: For the pluripotency-associated markers, TRA-1-60, TRA-1-81, and SSEA4 dye-coupled antibodies were used (anti-TRA-1-60-PE, human (clone: REA157), number 130-100-347; anti-TRA-1-81-PE, human (clone: REA246), number 130-101-410, and anti-SSEA-4-PE, human (clone: REA101), number 130-098-369; Miltenyi Biotec GmbH, Germany).

    Techniques: Derivative Assay, Metabolic Assay, Immunofluorescence, Expressing, Staining, Methylation Sequencing, Methylation, In Vitro

    Characterization of the two types of piPSCs. (A)The colonies of mpiPSCs were round and three-dimensional (bottom). The colonies of hpiPSCs were large and flat (top). Bar scale = 500 μm. The hpiPSCs and mpiPSCs were both positive for alkaline phosphatase (AP). Bar scale = 200 μm. (B) Immunocytochemical staining of Oct4, Sox2, SSEA-1 and SSEA-4 in hpiPSCs and mpiPSCs. Bar scale = 200 μm. (C) The surface marker Tra-1-81 and Tra-1-60 were positive in hpiPSCs but not detected in mpiPSCs. Bar scale = 200 μm. (D) X chromosome activation state of hpiPSCs and mpiPSCs after immunostaining for H3K27me3. Positive signals of histone H3K27 me3 spots were observed in hpiPSCs but not in mpiPSCs. Bar scale = 200 μm. The white arrowheads indicate the H3K27me3 positive spot in the cell. (E) Immunocytochemical assay of 3-germ-layer cells in EBs derived from both types of piPSCs, the markers include β Ⅲ-Tubulin (ectoderm), α-SMA (mesoderm) and Sox17 (endoderm). Scale bar = 200 μm. (F) Hematoxylin and eosin staining of teratoma sections of piPSCs. Left: blood vessel of endothelium (ectoderm); middle: muscle (mesoderm); right: gut-like epithelium (endoderm). Scale bars = 200 μm. (G) Karyotype analysis of piPSCs.

    Journal: PLoS ONE

    Article Title: Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions

    doi: 10.1371/journal.pone.0124562

    Figure Lengend Snippet: Characterization of the two types of piPSCs. (A)The colonies of mpiPSCs were round and three-dimensional (bottom). The colonies of hpiPSCs were large and flat (top). Bar scale = 500 μm. The hpiPSCs and mpiPSCs were both positive for alkaline phosphatase (AP). Bar scale = 200 μm. (B) Immunocytochemical staining of Oct4, Sox2, SSEA-1 and SSEA-4 in hpiPSCs and mpiPSCs. Bar scale = 200 μm. (C) The surface marker Tra-1-81 and Tra-1-60 were positive in hpiPSCs but not detected in mpiPSCs. Bar scale = 200 μm. (D) X chromosome activation state of hpiPSCs and mpiPSCs after immunostaining for H3K27me3. Positive signals of histone H3K27 me3 spots were observed in hpiPSCs but not in mpiPSCs. Bar scale = 200 μm. The white arrowheads indicate the H3K27me3 positive spot in the cell. (E) Immunocytochemical assay of 3-germ-layer cells in EBs derived from both types of piPSCs, the markers include β Ⅲ-Tubulin (ectoderm), α-SMA (mesoderm) and Sox17 (endoderm). Scale bar = 200 μm. (F) Hematoxylin and eosin staining of teratoma sections of piPSCs. Left: blood vessel of endothelium (ectoderm); middle: muscle (mesoderm); right: gut-like epithelium (endoderm). Scale bars = 200 μm. (G) Karyotype analysis of piPSCs.

    Article Snippet: The following primary antibodies were used: anti-Oct4 (Santa cruz, sc-5297), anti-Sox2 (Abcam, ab97959), anti-SSEA-1 (Cell Signaling Technology, 4744S), anti-SSEA-4 (Cell Signaling Technology, 4755P), anti-Tra-1-81 (Cell Signaling Technology, 4745P), anti-Tra-1-60 (Cell Signaling Technology, 4746P) and anti-H3K27me3 (Abcam, ab6002).

    Techniques: Staining, Marker, Activation Assay, Immunostaining, Derivative Assay

    DP31 cells can be reprogrammed to iPSCs using OCT3/4 and SOX2. (a) We obtained a few ES-cell-like colonies from 5 × 10 5 DP31 cells transduced with OCT3/4 and SOX2 (OS), but we could not obtain any ES-cell-like colonies from DP75 cells. Human ES-cell-like colonies were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). (b) Genomic PCR using transgene-specific primers, with DP31 cells as a negative control, confirmed the insertion of only two transgenes in iPSCs derived from DP31 cells transduced with OS (iPS-DP31-OS) by PCR. The numbers denote different iPSC lines. We cropped the gels and blots for clarifying our presentation. The gels have been run under the same experimental conditions. (c) iPS-DP31-OS cells expressed pluripotency markers including SSEA3, TRA-1-60, TRA-1-81, and NANOG. Scale bar = 100 μm. (d) Pluripotency of iPS-DP31-OS cells was confirmed by EB-mediated differentiation and teratoma formation assay. Immunofluorescence staining showed that EB structures derived from iPS-DP31-OS cells expressed markers characteristic of the three germ layers including βIII-tubulin (ectoderm), α-smooth-muscle actin (mesoderm), and α-fetoprotein (endoderm). Nuclei were stained with Hoechst 33342. Scale bar = 100 μm. Hematoxylin- and eosin-stained sections of teratomas generated from iPS-DP31-OS cells are shown in the lower panels. The teratomas contained various tissues of all three germ layers, such as neural-tube-like structures (ectoderm), cartilage (mesoderm), and gut-like epithelial tissue (endoderm). Abbreviations: AFP, alpha-fetoprotein; α-SMA, alpha smooth muscle actin. Scale bar = 100 μm.

    Journal: Scientific Reports

    Article Title: The homeobox gene DLX4 promotes generation of human induced pluripotent stem cells

    doi: 10.1038/srep07283

    Figure Lengend Snippet: DP31 cells can be reprogrammed to iPSCs using OCT3/4 and SOX2. (a) We obtained a few ES-cell-like colonies from 5 × 10 5 DP31 cells transduced with OCT3/4 and SOX2 (OS), but we could not obtain any ES-cell-like colonies from DP75 cells. Human ES-cell-like colonies were counted at 30 days post infection. Error bars indicate ± S.D. (n = 3). (b) Genomic PCR using transgene-specific primers, with DP31 cells as a negative control, confirmed the insertion of only two transgenes in iPSCs derived from DP31 cells transduced with OS (iPS-DP31-OS) by PCR. The numbers denote different iPSC lines. We cropped the gels and blots for clarifying our presentation. The gels have been run under the same experimental conditions. (c) iPS-DP31-OS cells expressed pluripotency markers including SSEA3, TRA-1-60, TRA-1-81, and NANOG. Scale bar = 100 μm. (d) Pluripotency of iPS-DP31-OS cells was confirmed by EB-mediated differentiation and teratoma formation assay. Immunofluorescence staining showed that EB structures derived from iPS-DP31-OS cells expressed markers characteristic of the three germ layers including βIII-tubulin (ectoderm), α-smooth-muscle actin (mesoderm), and α-fetoprotein (endoderm). Nuclei were stained with Hoechst 33342. Scale bar = 100 μm. Hematoxylin- and eosin-stained sections of teratomas generated from iPS-DP31-OS cells are shown in the lower panels. The teratomas contained various tissues of all three germ layers, such as neural-tube-like structures (ectoderm), cartilage (mesoderm), and gut-like epithelial tissue (endoderm). Abbreviations: AFP, alpha-fetoprotein; α-SMA, alpha smooth muscle actin. Scale bar = 100 μm.

    Article Snippet: The following antibodies were used: SSEA3 (1:10), TRA-1-81 (1:50), TRA-1-60 (1:50) (these antibodies were used at Kyoto University and were kind gifts from Dr. Peter W. Andrews), anti-SSEA4, anti-TRA-1-81, anti-TRA-1-60 (1:500, all contained in the ES Cell Characterization Kit from Merck Millipore; these antibodies were used at Gifu University), anti-NANOG (1:20, R & D Systems), anti-OCT3/4 (1:1000, Santa Cruz Biotechnology), anti-βIII-tubulin (1:200, Cell Signal Technology), anti-βIII-tubulin (1:2000, Covance), anti-α-SMA (1:500, DAKO), and anti-AFP (1:100, R & D).

    Techniques: Transduction, Infection, Polymerase Chain Reaction, Negative Control, Derivative Assay, Teratoma Formation Assay, Immunofluorescence, Staining, Generated