anti-ssea-1 Search Results


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  • 93
    Thermo Fisher ssea 1 pe
    Ssea 1 Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti ssea 1
    Insulin released in response to low glucose (5.6 mM) and high glucose (25 mM) stimulation in culture medium of insulinsecreting cells generated from bone marrow derived <t>SSEA-1+</t> SCs (day 24) was measured. Undifferentiated bone marrow derived SSEA-1 positive cells were used as a control. Each value represents mean ± SD of three experiments. Statistical significance was tested by a Student’s t test. *; P
    Anti Ssea 1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore ssea 1
    Clone-sorted myofiber-associated stem cells reprogram at high efficiency. ( A ) Experimental strategy for clone-sorting and reprogramming sorted cells. Myofiber-associated cells from transgenic mice carrying dox-inducible transgenes (Oct4, Sox2, c-Myc, Klf4) and labeled with constitutively-expressed Tdtomato [22] , were double-sorted for purity, and seeded into 96-well plates, at one cell per well, on irradiated MEFs. After three weeks in dox-containing media, emergent colonies were trypsinized and passaged in the absence of dox, and iPS lines were established which showed embryonic stem cell-like morphology, stained for the pluripotent marker <t>SSEA-1,</t> and showed alkaline phosphatase activity (data not shown).
    Ssea 1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc anti ssea 1
    Maintenance of self-renewal and pluripotency of J1 mES on methanol fixed fibroblasts. J1 mES cells were cultured on methanol fixed MEF (MT-MEF) and NIH3T3 (MT-3T3) cells, and Mitomycin C treated MEF (MMC-MEF). ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 mES. ( C ) qRT-PCR analysis of pluripotent genes in J1 mES. ( D , E ) Immunofluorescence ( D ) and flow cytometry analysis ( E ) of pluripotent markers OCT4 and <t>SSEA-1</t> in J1 mES. Nuclei were stained by Hoechst 33342 (Hoe). ( F ) Teratoma formation of J1 mES. Arrows indicate tissues from the three germ layers. Scale bar, 400 μm for A, 200 μm for F, and 100 μm for D.
    Anti Ssea 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology ssea 1
    Inhibition of EGFR by AG1478 impairs mESC self-renewal and pluripotency, and induces differentiation. (A) Enzymatic activity for AP was analyzed in control and AG1478 treated mESCs. (B–D) IF staining against <t>SSEA-1</t> (red), OCT-4 (green), and Nanog (red) in control and AG1478 treated mESCs. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole, scale bar: 200 μm. (E and F) Quantitative PCR analysis of mRNA levels of pluripotency factor s and differentiation related genes in control and AG1478 treated mESCs. The amounts of each mRNA were normalized to GAPDH mRNA and are shown relative to the amounts in control mESCs (set to 1). (G) Protein expression of OCT4 and GATA4 in control and AG1478 treated mESCs. β-actin served as a loading control. (H) Quantified relative band intensity ratio of OCT4 and GATA4. The data are presented as mean ± SD ( n = 3; ** P
    Ssea 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank ssea 1
    Excision of the reprogramming cassette and its effect on pluripotency markers in differentiating iPSCs. The iPSCs on differentiation displayed many areas consisting of small, rounded cells (A), which stained positive for OCT4 (B), <t>SSEA-1</t> (C) and alkaline phosphatase (AP) (D). Crystal violet staining of the differentiated iPSCs pre- (E, upper well) and post- (E, lower well) ganciclovir selection showed that the transgenes were not silenced on differentiation, thereby leading to massive cell death in the lower well. Therefore, to stop/decrease the expression of reprogramming transcription factors during differentiation we transfected the iPSC colonies (F) transiently with a CRE:GFP plasmid (G). Massive cellular death ensued after Ganciclovir selection (H)as visualized by the lack of visible colonies in the upper well of figure (I). However, after few days of culture the colonies again started to appear back (lower well of figure I). Genomic PCR confirmed transgene excision in two iPSC clones: pre- (clone #1 and clone #2) and post-excision (clone #1Ex and Clone #2Ex). Genomic Gapdh was used as a control (upper lane) and two areas of the transgene: i) HSV-tk to WPRE (middle lane) ii) Klf4 to 2A (lower lane), were used as the tests. Both the excised clones lacked the test bands. (J). AP staining showed maintenance of pluripotency in the emerging colonies (K). RT-PCR for Oct4 and Nanog in differentiating iPSCs showed a considerable decrease in the expression of both the markers post-excision in both the clones (1 and 2). No HSV-tk and WPRE expression was seen after excision (L). After excision the expression of Oct4, SSEA-1 and Alkaline phosphate were also reduced on ICC in the differentiating cells (M–P). Scale bar = 100 µm.
    Ssea 1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stemgent ssea 1
    Excision of the reprogramming cassette and its effect on pluripotency markers in differentiating iPSCs. The iPSCs on differentiation displayed many areas consisting of small, rounded cells (A), which stained positive for OCT4 (B), <t>SSEA-1</t> (C) and alkaline phosphatase (AP) (D). Crystal violet staining of the differentiated iPSCs pre- (E, upper well) and post- (E, lower well) ganciclovir selection showed that the transgenes were not silenced on differentiation, thereby leading to massive cell death in the lower well. Therefore, to stop/decrease the expression of reprogramming transcription factors during differentiation we transfected the iPSC colonies (F) transiently with a CRE:GFP plasmid (G). Massive cellular death ensued after Ganciclovir selection (H)as visualized by the lack of visible colonies in the upper well of figure (I). However, after few days of culture the colonies again started to appear back (lower well of figure I). Genomic PCR confirmed transgene excision in two iPSC clones: pre- (clone #1 and clone #2) and post-excision (clone #1Ex and Clone #2Ex). Genomic Gapdh was used as a control (upper lane) and two areas of the transgene: i) HSV-tk to WPRE (middle lane) ii) Klf4 to 2A (lower lane), were used as the tests. Both the excised clones lacked the test bands. (J). AP staining showed maintenance of pluripotency in the emerging colonies (K). RT-PCR for Oct4 and Nanog in differentiating iPSCs showed a considerable decrease in the expression of both the markers post-excision in both the clones (1 and 2). No HSV-tk and WPRE expression was seen after excision (L). After excision the expression of Oct4, SSEA-1 and Alkaline phosphate were also reduced on ICC in the differentiating cells (M–P). Scale bar = 100 µm.
    Ssea 1, supplied by Stemgent, used in various techniques. Bioz Stars score: 92/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam antibodies ssea 1
    Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker <t>SSEA-1</t> (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a , Nanog , and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. ∗ Compared to the expression level of cultured cESCs in passage 3, P
    Antibodies Ssea 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Merck KGaA ssea 1
    Characteristics of ntESCs and iPSCs from APCs (A) Morphology and alkaline phosphatase expression of ntESCs and iPSCs. Scale bars, 100 μm. (B) Immunofluorescence staining of pluripotent markers <t>SSEA-1</t> (red), Nanog (green), Sox2 (green), and Oct4 (red) in AN and AI lines. Nuclei were stained with DAPI. Scale bars, 50μm. (C) Quantitative PCR analysis of the pluripotent markers Sall4, Oct4 and Nanog in AN and AI lines. Relative mRNA expression is normalized to GAPDH and is represented relative to expression in fibroblasts (37MEF). The experiments were performed in triplicate (means±SD; n=3). The primers used are listed in Supplementary Table 2 . (D) H E staining of teratomas generated from AN1 that formed tissues from all three germ layers. 1: Ectoderm; 2: Mesoderm; 3: Endoderm. Scale bars, 100 μm. A detailed description of all of the cell lines is given in Supplementary Table 1 . (E and F) Quantitative PCR analysis showing the upregulation of markers for all three germ layers during the in vitro differentiation of AN and AI lines. Relative mRNA expression is normalized to GAPDH and represented relative to expression in undifferentiated AN and AI lines. The experiments were performed in triplicate (mean±SD; n=3). The primers used are listed in Supplementary Table 2 . (G) Germline transmission of an AN1 tetraploid mouse. Details on all of the cell lines are in Supplementary Table 1 .
    Ssea 1, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    STEMCELL Technologies Inc anti ssea 1
    The morphologies and characteristics of rat bone marrow mesenchymal stem cells (BMMSCs) and multilineage‐differentiating stress‐enduring (Muse cells) . (A) The morphology (a1) of BMMSCs lipoblasts (a2) and osteoblasts (a3) differentiated from BMMSCs were observed by microscopy. BMMSCs were positive for CD29 (a4), CD90 (a5), and RT1A (a6) and negative for CD34 (a4), CD45 (a5), RT1B (a6), SSEA‐3 (a7) and <t>SSEA‐1</t> (a8) detected by flow cytometry (FCM). (B) Muse cells formed the Muse‐cell‐clusters (M‐clusters) in suspension cultivation and long‐shuttle types in adherent cultivation (b1–b3). Rat Muse cells were positive for CD29 (b4), CD90 (b5), and RT1A (b6) and negative for CD34 (b4), CD45 (b5), RT1B (b6) similar to BMMSCs, but positively expressed SSEA‐3 (75.6 ± 2.8% vs. 2.3 ± 0.3%, b7) SSEA‐1 (74.8 ± 3.1% vs. 2.1 ± 0.2%). In addition, 77.62 ± 5.3% of SSEA‐1 (+) cells expressed SSEA‐3 (b9). Scale bars = 50 μm.
    Anti Ssea 1, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ssea 1  (Sony)
    91
    Sony ssea 1
    The morphologies and characteristics of rat bone marrow mesenchymal stem cells (BMMSCs) and multilineage‐differentiating stress‐enduring (Muse cells) . (A) The morphology (a1) of BMMSCs lipoblasts (a2) and osteoblasts (a3) differentiated from BMMSCs were observed by microscopy. BMMSCs were positive for CD29 (a4), CD90 (a5), and RT1A (a6) and negative for CD34 (a4), CD45 (a5), RT1B (a6), SSEA‐3 (a7) and <t>SSEA‐1</t> (a8) detected by flow cytometry (FCM). (B) Muse cells formed the Muse‐cell‐clusters (M‐clusters) in suspension cultivation and long‐shuttle types in adherent cultivation (b1–b3). Rat Muse cells were positive for CD29 (b4), CD90 (b5), and RT1A (b6) and negative for CD34 (b4), CD45 (b5), RT1B (b6) similar to BMMSCs, but positively expressed SSEA‐3 (75.6 ± 2.8% vs. 2.3 ± 0.3%, b7) SSEA‐1 (74.8 ± 3.1% vs. 2.1 ± 0.2%). In addition, 77.62 ± 5.3% of SSEA‐1 (+) cells expressed SSEA‐3 (b9). Scale bars = 50 μm.
    Ssea 1, supplied by Sony, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nordic BioSite pe anti ssea 1
    ), association data of upregulated gene interaction with <t>SSEA-1+</t> vs SSEA-1neg porcine embryonic fibroblasts (pEFS), which includes protein and genetic interaction pathways, co-expression, co-localization and protein domain similarity.
    Pe Anti Ssea 1, supplied by Nordic BioSite, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend anti ssea 1
    Epithelial derived LTβR controls mTECp development. ( a ) Thymic stromal cells were prepared from neonatal Ltbr fl/fl K14 Cre and Ltbr fl/+ K14 Cre control mice. mTECp is identified as Cld3,4 hi <t>SSEA-1</t> + within CD45 − EpCAM + TEC population. ( b ) Statistic data are shown as mean ± SD of more than 3 mice each group. One representative FACS plot and statistic result from more than 3 repeats are shown. An unpaired two-tailed Student’s t -test is used: *P
    Anti Ssea 1, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Developmental Studies Hybridoma Bank mouse anti ssea 1
    Immunofluorescence staining of FS101 and FS111 PC PGCs. (a) Confocal images of <t>SSEA-1-</t> (red) and CVH- (green) stained FS101 PGCs. (b) Higher magnification of FS101 cells (white square indicates the cells on (a)). (c) Confocal images of SSEA-1- (red) and CVH- (green) stained FS111 PGCs. (d) Higher magnification of the FS111 cells (white square indicates the cells on (c)). For nuclear staining (NS), we used TO-PRO-3 (blue). Scale bars: 25 μ m (a, c) and 5 μ m (b, d). PC PG cells: Partridge colour Hungarian chicken embryo-derived primordial germ cells.
    Mouse Anti Ssea 1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 89/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec anti ssea 1
    Generation and Characterization of iPSCs from WT and oc/oc Mice (A) Experimental outline for the generation of WT and oc/oc iPSC clones with excised reprogramming vector (VCN = 0) and carrying the correct chromosome complement (see text for details). (B) Expression of alkaline phosphatase on WT and oc/oc iPSC colonies. (C) Expression of Oct4, Sox2, Nanog, and <t>SSEA-1</t> on WT and oc/oc iPSCs as revealed by immunofluorescence. Nuclei are stained with DAPI. Scale bars, 100 μm. (D) Karyotype analysis of WT and oc/oc iPSC clones. (E) Generation of the three germ layers in vitro by differentiated WT and oc/oc iPSCs is revealed by immunofluorescence. Expression of Brachyury, AFP, and Nestin indicate formation of mesoderm, endoderm, and ectoderm, respectively. Scale bars, 50 μm. (F) H E staining of teratomas generated after subcutaneous injection of WT and oc/oc iPSC clones into NSG mice, demonstrating differentiation into the three germ layer derivatives (i, mesoderm-cartilage; ii, mesoderm-adipose tissue; iii, endoderm-glandular structure; iv, endoderm-glandular structures (intestinal paneth or pancreatic acinar cell-like); v, ectoderm-primitive and mature neuroepithelium; vi, ectoderm-primitive neuroepithelium). Scale bars, 50 μm. See also Figure S1 .
    Anti Ssea 1, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti ssea 1
    Characteristics of EB differentiation. a EB immunofluorescent staining of undifferentiated state marker <t>SSEA-1</t> and surface antigens, including cytokeratin ( CK ), CD73, and CD151. Cell nuclei were stained with DAPI. Bars = 100 μm. b Morphological appearance of pESC-derived and ESC-derived EBs and outgrowths of EBs. Bars = 100 μm. c Expression of Oct3/4 (stemness), nestin (ectoderm), Afp (entoderm), and mesoderm markers Snai1 , Hand1 , and Gata2 in pESC-derived and ESC-derived EB outgrowths. * p
    Mouse Anti Ssea 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stemgent mouse anti ssea 1
    Puma is not required to suppress DNA damage during reprogramming. A. Confocal analysis of day 14 <t>SSEA-1</t> positive colonies for γH2AX and DAPI. B. FACS analyses of day 14 to 18 OSK(M) cultures co-stained with γH2AX and SSEA-1. (Wild-type,
    Mouse Anti Ssea 1, supplied by Stemgent, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies anti ssea 1 cd15
    Puma is not required to suppress DNA damage during reprogramming. A. Confocal analysis of day 14 <t>SSEA-1</t> positive colonies for γH2AX and DAPI. B. FACS analyses of day 14 to 18 OSK(M) cultures co-stained with γH2AX and SSEA-1. (Wild-type,
    Anti Ssea 1 Cd15, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Insulin released in response to low glucose (5.6 mM) and high glucose (25 mM) stimulation in culture medium of insulinsecreting cells generated from bone marrow derived SSEA-1+ SCs (day 24) was measured. Undifferentiated bone marrow derived SSEA-1 positive cells were used as a control. Each value represents mean ± SD of three experiments. Statistical significance was tested by a Student’s t test. *; P

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Insulin released in response to low glucose (5.6 mM) and high glucose (25 mM) stimulation in culture medium of insulinsecreting cells generated from bone marrow derived SSEA-1+ SCs (day 24) was measured. Undifferentiated bone marrow derived SSEA-1 positive cells were used as a control. Each value represents mean ± SD of three experiments. Statistical significance was tested by a Student’s t test. *; P

    Article Snippet: Briefly the SSEA-1 positive cells were stained with antibodies to SSEA-1 (1:400, anti-SSEA-1, BD Pharmingen, USA), OCT-4 (1:500, anti-OCT-4; Abcam, USA).

    Techniques: Generated, Derivative Assay

    Immunostaining of pluripotency markers in bone marrow derived SSEA-1 positive cells. Expression of A. OCT-4, B. SSEA-1 in purified bone marrow derived SSEA-1 positive cells evaluated by immunofluorescence staining. Nuclei were counterstained with DAPI and C. Flow cytometric analysis of bone marrow derived SSEA-1 positive cells show that they expressed SSEA-1, CXCR4 and SCA-1 but expression of CD45 was much lower or undetectable. SSEA-1; Stage-specific embryonic antigen 1, OCT-4; Octamer-binding transcription factor 4, DAPI; 4´, 6-diamidino-2-phenylindole, CXCR4; C-X-C chemokine receptor type 4 and SCA-1; Stem cells antigen-1.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Immunostaining of pluripotency markers in bone marrow derived SSEA-1 positive cells. Expression of A. OCT-4, B. SSEA-1 in purified bone marrow derived SSEA-1 positive cells evaluated by immunofluorescence staining. Nuclei were counterstained with DAPI and C. Flow cytometric analysis of bone marrow derived SSEA-1 positive cells show that they expressed SSEA-1, CXCR4 and SCA-1 but expression of CD45 was much lower or undetectable. SSEA-1; Stage-specific embryonic antigen 1, OCT-4; Octamer-binding transcription factor 4, DAPI; 4´, 6-diamidino-2-phenylindole, CXCR4; C-X-C chemokine receptor type 4 and SCA-1; Stem cells antigen-1.

    Article Snippet: Briefly the SSEA-1 positive cells were stained with antibodies to SSEA-1 (1:400, anti-SSEA-1, BD Pharmingen, USA), OCT-4 (1:500, anti-OCT-4; Abcam, USA).

    Techniques: Immunostaining, Derivative Assay, Expressing, Purification, Immunofluorescence, Staining, Flow Cytometry, Binding Assay

    Identification of bone marrow derived SSEA-1 positive cells differentiated into insulin producing cells. A. Primary explant cultures of bone marrow derived SSEA-1 positive cells. Undifferentiated SSEA-1 positive cells were spherical in shape, B. Colony of purified SSEA-1 positive cells after culturing on MEF-coated dishes and C. Dithizone staining of insulin secreting cellsderived from bone marrow derived SSEA-1 positive cells. SSEA-1; Stage-specific embryonic antigen 1 and MEF; Mouse embryonic fibroblasts.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Identification of bone marrow derived SSEA-1 positive cells differentiated into insulin producing cells. A. Primary explant cultures of bone marrow derived SSEA-1 positive cells. Undifferentiated SSEA-1 positive cells were spherical in shape, B. Colony of purified SSEA-1 positive cells after culturing on MEF-coated dishes and C. Dithizone staining of insulin secreting cellsderived from bone marrow derived SSEA-1 positive cells. SSEA-1; Stage-specific embryonic antigen 1 and MEF; Mouse embryonic fibroblasts.

    Article Snippet: Briefly the SSEA-1 positive cells were stained with antibodies to SSEA-1 (1:400, anti-SSEA-1, BD Pharmingen, USA), OCT-4 (1:500, anti-OCT-4; Abcam, USA).

    Techniques: Derivative Assay, Purification, Staining

    Electropherograms of RT-PCR product of mRNA extracted from bone marrow derived SSEA-1 positive cells induced into insulin secreting cells (lane 2), undifferentiated bone marrow derived SSEA-1 positive cells (lane 3) and pancreatic β-cells as a positive control (lane 4). The leftmost lane represents the DNA ladder and distilled H2O (lane 1) was used as a negative control. β-actin amplification served as an internal control. A. Pdx1 , B. Ngn3 , C. glucagon , D. Amylase , E. Insulin1 , F. Insulin2 and G. β-actin. RT-PCR; Reverse transcription polymerase chain reaction and SSEA-1; Stage-specific embryonic antigen 1.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Electropherograms of RT-PCR product of mRNA extracted from bone marrow derived SSEA-1 positive cells induced into insulin secreting cells (lane 2), undifferentiated bone marrow derived SSEA-1 positive cells (lane 3) and pancreatic β-cells as a positive control (lane 4). The leftmost lane represents the DNA ladder and distilled H2O (lane 1) was used as a negative control. β-actin amplification served as an internal control. A. Pdx1 , B. Ngn3 , C. glucagon , D. Amylase , E. Insulin1 , F. Insulin2 and G. β-actin. RT-PCR; Reverse transcription polymerase chain reaction and SSEA-1; Stage-specific embryonic antigen 1.

    Article Snippet: Briefly the SSEA-1 positive cells were stained with antibodies to SSEA-1 (1:400, anti-SSEA-1, BD Pharmingen, USA), OCT-4 (1:500, anti-OCT-4; Abcam, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Negative Control, Amplification

    Immunofluorescence reveals bone marrow derived SSEA-1 positive cells differentiated into insulin secreting cells in vitro . The insulin secreting cells at days 24 were fixed and stained with antibodies against Pdx1 and Glut2 and visualized with secondary antibody (FITC). 4′, 6-diamidino-2-phenylindole (DAPI) was used to counter-stain DNA (blue) (scale bar; 50 μm). A. Anti Pdx1 immunofluorescence, B. Nuclear counterstain with DAPI, C. Merged image of A and B, D. AntiGlut2 immunofluorescence, E. Nuclear counterstain with DAPI and F. Merged image of D and E. SSEA-1; Stage-specific embryonic antigen 1.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Immunofluorescence reveals bone marrow derived SSEA-1 positive cells differentiated into insulin secreting cells in vitro . The insulin secreting cells at days 24 were fixed and stained with antibodies against Pdx1 and Glut2 and visualized with secondary antibody (FITC). 4′, 6-diamidino-2-phenylindole (DAPI) was used to counter-stain DNA (blue) (scale bar; 50 μm). A. Anti Pdx1 immunofluorescence, B. Nuclear counterstain with DAPI, C. Merged image of A and B, D. AntiGlut2 immunofluorescence, E. Nuclear counterstain with DAPI and F. Merged image of D and E. SSEA-1; Stage-specific embryonic antigen 1.

    Article Snippet: Briefly the SSEA-1 positive cells were stained with antibodies to SSEA-1 (1:400, anti-SSEA-1, BD Pharmingen, USA), OCT-4 (1:500, anti-OCT-4; Abcam, USA).

    Techniques: Immunofluorescence, Derivative Assay, In Vitro, Staining

    ZFX Over clones resist spontaneous differentiation. A. ZFX Over clones and controls were expanded in conditions promoting self-renewal before SSEA-3 and SSEA-1 FACS analysis. B. ZFX Over clones and controls were expanded in suboptimal conditions before SSEA-3 and SSEA-1 FACS analysis. The quantitation compares ZFX Over clones to controls in three independent experiments and error bars represent the S.E.M. See Figure S5 for all 3 independent experiments.

    Journal: PLoS ONE

    Article Title: ZFX Controls the Self-Renewal of Human Embryonic Stem Cells

    doi: 10.1371/journal.pone.0042302

    Figure Lengend Snippet: ZFX Over clones resist spontaneous differentiation. A. ZFX Over clones and controls were expanded in conditions promoting self-renewal before SSEA-3 and SSEA-1 FACS analysis. B. ZFX Over clones and controls were expanded in suboptimal conditions before SSEA-3 and SSEA-1 FACS analysis. The quantitation compares ZFX Over clones to controls in three independent experiments and error bars represent the S.E.M. See Figure S5 for all 3 independent experiments.

    Article Snippet: Aliquots of the cells were stained with anti-SSEA-3 (SSEA-3 Alexa Fluor® 647; unpublished, BD Pharmingen, La Jolla, CA) and anti-SSEA-1 antibodies (SSEA-1 Alexa Fluor® 647; BD Pharmingen #560120, La Jolla, CA) before quantitation on a FACSAria.

    Techniques: Clone Assay, FACS, Quantitation Assay

    Clone-sorted myofiber-associated stem cells reprogram at high efficiency. ( A ) Experimental strategy for clone-sorting and reprogramming sorted cells. Myofiber-associated cells from transgenic mice carrying dox-inducible transgenes (Oct4, Sox2, c-Myc, Klf4) and labeled with constitutively-expressed Tdtomato [22] , were double-sorted for purity, and seeded into 96-well plates, at one cell per well, on irradiated MEFs. After three weeks in dox-containing media, emergent colonies were trypsinized and passaged in the absence of dox, and iPS lines were established which showed embryonic stem cell-like morphology, stained for the pluripotent marker SSEA-1, and showed alkaline phosphatase activity (data not shown).

    Journal: PLoS ONE

    Article Title: Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

    doi: 10.1371/journal.pone.0026406

    Figure Lengend Snippet: Clone-sorted myofiber-associated stem cells reprogram at high efficiency. ( A ) Experimental strategy for clone-sorting and reprogramming sorted cells. Myofiber-associated cells from transgenic mice carrying dox-inducible transgenes (Oct4, Sox2, c-Myc, Klf4) and labeled with constitutively-expressed Tdtomato [22] , were double-sorted for purity, and seeded into 96-well plates, at one cell per well, on irradiated MEFs. After three weeks in dox-containing media, emergent colonies were trypsinized and passaged in the absence of dox, and iPS lines were established which showed embryonic stem cell-like morphology, stained for the pluripotent marker SSEA-1, and showed alkaline phosphatase activity (data not shown).

    Article Snippet: Immunostaining and alkaline phosphatase staining Immunostaining was performed with antibodies to Oct4 (Abcam ab19857) and SSEA-1 (Chemicon MAB4301).

    Techniques: Transgenic Assay, Mouse Assay, Labeling, Irradiation, Staining, Marker, Activity Assay

    SMPs and Sca-1+ mesenchymal progenitors reprogram into pluripotent iPS cells with high efficiency. ( A–F ) iPS cells generated from SMPs (“SMP iPS” cells) show embryonic stem cell-like morphology ( A ), and stain for the pluripotency markers Oct 4 ( B ) and SSEA-1 ( C ). Brightfield and corresponding fluorescence images (red) are shown for B and C. SMP iPS cells also show alkaline phosphatase activity ( D ). When SMP iPS cells were injected into C57Bl/6 blastocysts, the resulting chimeras had agouti coat color ( E ), indicating contribution of the SMP iPS cells to chimeric tissues. Offspring from matings between SMP iPS cell chimeric mice and C57Bl/6 mice have brown coat color ( F ), demonstrating the capacity of SMP iPS cells to provide germline transmission.

    Journal: PLoS ONE

    Article Title: Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

    doi: 10.1371/journal.pone.0026406

    Figure Lengend Snippet: SMPs and Sca-1+ mesenchymal progenitors reprogram into pluripotent iPS cells with high efficiency. ( A–F ) iPS cells generated from SMPs (“SMP iPS” cells) show embryonic stem cell-like morphology ( A ), and stain for the pluripotency markers Oct 4 ( B ) and SSEA-1 ( C ). Brightfield and corresponding fluorescence images (red) are shown for B and C. SMP iPS cells also show alkaline phosphatase activity ( D ). When SMP iPS cells were injected into C57Bl/6 blastocysts, the resulting chimeras had agouti coat color ( E ), indicating contribution of the SMP iPS cells to chimeric tissues. Offspring from matings between SMP iPS cell chimeric mice and C57Bl/6 mice have brown coat color ( F ), demonstrating the capacity of SMP iPS cells to provide germline transmission.

    Article Snippet: Immunostaining and alkaline phosphatase staining Immunostaining was performed with antibodies to Oct4 (Abcam ab19857) and SSEA-1 (Chemicon MAB4301).

    Techniques: Generated, Staining, Fluorescence, Activity Assay, Injection, Mouse Assay, Transmission Assay

    SSEA-1 + fibroblasts differentiate into non-fibroblastic cells during tumour growth ( a,b ) Flow cytometric analysis ( a ) and immunohistochemical detection (brown) ( b ) of SSEA-1 in primary tumours. Human H-2Kd − cells were gated and plotted. Pictures of four adjacent fields were combined. Scale bar: 150 μm. ( c ) Immunohistochemical detection (brown) of the indicated proteins in primary tumours resulting from injection of GFP-labeled SSEA-1 + fibroblasts mixed with unlabeled SSEA-1 − fibroblasts. Cells immunopositive for hCD34 and tubulin-β-III are indicated by arrows. Wide areas of myosin-positive cells were detected. Scale bars: 80 μm (upper and middle panels) and 50 μm (bottom panels). ( d ) Flow cytometric analysis of CD166 and SSEA-1 in transformed fibroblasts, a primary tumour, and fibroblasts isolated from the tumour. ( e ) Abundance of cells expressing high levels of the indicated antigens measured by flow cytometric analysis in transformed fibroblasts, three representative primary tumours and two representative tumour fibroblast populations. The gate was set based on the values measured in the transformed fibroblasts. ( f ) Quantitative RT-PCR measuring expression levels of the mesenchymal markers CDh2 and FGF2 in the indicated samples. Values are normalized to the housekeeping gene PPIA and represent means from two replicates. Statistical significance of the differences compared to the tumour cells (p

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: SSEA-1 + fibroblasts differentiate into non-fibroblastic cells during tumour growth ( a,b ) Flow cytometric analysis ( a ) and immunohistochemical detection (brown) ( b ) of SSEA-1 in primary tumours. Human H-2Kd − cells were gated and plotted. Pictures of four adjacent fields were combined. Scale bar: 150 μm. ( c ) Immunohistochemical detection (brown) of the indicated proteins in primary tumours resulting from injection of GFP-labeled SSEA-1 + fibroblasts mixed with unlabeled SSEA-1 − fibroblasts. Cells immunopositive for hCD34 and tubulin-β-III are indicated by arrows. Wide areas of myosin-positive cells were detected. Scale bars: 80 μm (upper and middle panels) and 50 μm (bottom panels). ( d ) Flow cytometric analysis of CD166 and SSEA-1 in transformed fibroblasts, a primary tumour, and fibroblasts isolated from the tumour. ( e ) Abundance of cells expressing high levels of the indicated antigens measured by flow cytometric analysis in transformed fibroblasts, three representative primary tumours and two representative tumour fibroblast populations. The gate was set based on the values measured in the transformed fibroblasts. ( f ) Quantitative RT-PCR measuring expression levels of the mesenchymal markers CDh2 and FGF2 in the indicated samples. Values are normalized to the housekeeping gene PPIA and represent means from two replicates. Statistical significance of the differences compared to the tumour cells (p

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: Flow Cytometry, Immunohistochemistry, Injection, Labeling, Transformation Assay, Isolation, Expressing, Quantitative RT-PCR

    Model for reprogramming of somatic cells to CSCs Inhibition of tumour suppressor cellular mechanisms and concomitant oncogenic activation convert somatic differentiated cells into pre-malignant SSEA-1 − cells. This cellular intermediate is not tumorigenic. Additional, stochastic modifications lead to full reprogramming of somatic cells to SSEA-1 + CSCs, conferring self-renewal and differentiation ability. These cellular properties allow reprogrammed somatic cells to initiate and maintain tumours. During tumour growth, SSEA-1 + CSCs differentiate into phenotypically diverse, non-tumorigenic cancer cell, thereby generating heterogeneous and hierarchically-organized tumours.

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: Model for reprogramming of somatic cells to CSCs Inhibition of tumour suppressor cellular mechanisms and concomitant oncogenic activation convert somatic differentiated cells into pre-malignant SSEA-1 − cells. This cellular intermediate is not tumorigenic. Additional, stochastic modifications lead to full reprogramming of somatic cells to SSEA-1 + CSCs, conferring self-renewal and differentiation ability. These cellular properties allow reprogrammed somatic cells to initiate and maintain tumours. During tumour growth, SSEA-1 + CSCs differentiate into phenotypically diverse, non-tumorigenic cancer cell, thereby generating heterogeneous and hierarchically-organized tumours.

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: Inhibition, Activation Assay

    Self-renewal ability of SSEA-1 + transformed fibroblasts ( a ) Serial transplantation of SSEA-1 + fibroblasts in BALB/cAnNCr- nu/nu mice. 3 × 10 6 cells were injected for all passages. ( b ) Percentages of SSEA-1 + cells in the indicated samples measured by flow cytometric analysis. Values represent mean ± s. d. from 2 to 6 tumours. Statistical significance of the differences is indicated. ( c ) Quantitative analysis of soft agar assay using unsorted cells from primary, secondary and tertiary tumours. The frequency of clonogenic cells is indicated. Values represent mean ± s. d. from three replicates. Statistical significance of the differences is indicated.

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: Self-renewal ability of SSEA-1 + transformed fibroblasts ( a ) Serial transplantation of SSEA-1 + fibroblasts in BALB/cAnNCr- nu/nu mice. 3 × 10 6 cells were injected for all passages. ( b ) Percentages of SSEA-1 + cells in the indicated samples measured by flow cytometric analysis. Values represent mean ± s. d. from 2 to 6 tumours. Statistical significance of the differences is indicated. ( c ) Quantitative analysis of soft agar assay using unsorted cells from primary, secondary and tertiary tumours. The frequency of clonogenic cells is indicated. Values represent mean ± s. d. from three replicates. Statistical significance of the differences is indicated.

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: Transformation Assay, Transplantation Assay, Mouse Assay, Injection, Flow Cytometry, Soft Agar Assay

    The presence of the stage-specific embryonic antigen SSEA-1 in populations of differentiated cells correlates with acquisition of tumorigenicity upon transformation ( a ) Relative abundance of cells immunopositive for the indicated antigens in two independent cell lines of in vitro -transformed fibroblasts compared to the corresponding hTERT-immortalized control cell lines. The average percentage of positive cells in the two transformed cell lines for each antigen measured by flow cytometric analysis is indicated. The red line indicates a relative abundance of 1 and values above the line indicate enrichment of the immunopositive cells in transformed fibroblasts compared to hTERT-immortalized cells. ( b,c ) Immunodetection of SSEA-1 in the Transformed-2 cell line and the corresponding hTERT-immortalized control cell line by flow cytometry ( b ) and fluorescence microscopy ( c ). The percentage of SSEA-1 + cells and the significance of the difference between the two populations determined by K-S test are indicated. The signal intensities and the percentage of SSEA-1 + cells in the hTERT-immortalized cell lines were not different (p > 0.05) from the background measured in unstained cells or cells stained with an isotype control antibody ( Supplementary Fig.S2a ). Scale bar: 40 μm. ( d ) Flow cytometric analysis of SSEA-1 in primary and in vitro -transformed mammary epithelial cells. The percentage of SSEA-1 + cells is indicated. ( e ) Immunohistochemical detection of SSEA-1 in clinical samples from one inflammatory myofibroblastoma (top left panel) and three malignant fibrohistiocytomas. Representative images of varying abundance and distribution of SSEA-1 + cells observed in the tissue microarray are shown. No SSEA-1 + positive cells were detected in normal control tissues (bottom panels). Scale bar: 150 μm. ( f ) Abundance of SSEA-1 + cells in a polyclonal population of primary fibroblasts transformed by serial introduction of transforming factors under conditions of high infection efficiency or in four clones obtained by simultaneous introduction of the transforming factors under conditions of low infection efficiency. Injection of the clones marked by an asterisk induced tumour formation in mice.

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: The presence of the stage-specific embryonic antigen SSEA-1 in populations of differentiated cells correlates with acquisition of tumorigenicity upon transformation ( a ) Relative abundance of cells immunopositive for the indicated antigens in two independent cell lines of in vitro -transformed fibroblasts compared to the corresponding hTERT-immortalized control cell lines. The average percentage of positive cells in the two transformed cell lines for each antigen measured by flow cytometric analysis is indicated. The red line indicates a relative abundance of 1 and values above the line indicate enrichment of the immunopositive cells in transformed fibroblasts compared to hTERT-immortalized cells. ( b,c ) Immunodetection of SSEA-1 in the Transformed-2 cell line and the corresponding hTERT-immortalized control cell line by flow cytometry ( b ) and fluorescence microscopy ( c ). The percentage of SSEA-1 + cells and the significance of the difference between the two populations determined by K-S test are indicated. The signal intensities and the percentage of SSEA-1 + cells in the hTERT-immortalized cell lines were not different (p > 0.05) from the background measured in unstained cells or cells stained with an isotype control antibody ( Supplementary Fig.S2a ). Scale bar: 40 μm. ( d ) Flow cytometric analysis of SSEA-1 in primary and in vitro -transformed mammary epithelial cells. The percentage of SSEA-1 + cells is indicated. ( e ) Immunohistochemical detection of SSEA-1 in clinical samples from one inflammatory myofibroblastoma (top left panel) and three malignant fibrohistiocytomas. Representative images of varying abundance and distribution of SSEA-1 + cells observed in the tissue microarray are shown. No SSEA-1 + positive cells were detected in normal control tissues (bottom panels). Scale bar: 150 μm. ( f ) Abundance of SSEA-1 + cells in a polyclonal population of primary fibroblasts transformed by serial introduction of transforming factors under conditions of high infection efficiency or in four clones obtained by simultaneous introduction of the transforming factors under conditions of low infection efficiency. Injection of the clones marked by an asterisk induced tumour formation in mice.

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: Transformation Assay, In Vitro, Flow Cytometry, Immunodetection, Cytometry, Fluorescence, Microscopy, Staining, Immunohistochemistry, Microarray, Infection, Clone Assay, Injection, Mouse Assay

    SSEA-1 identifies a biologically distinct subpopulation of transformedfibroblasts ( a ) Abundance of SSEA-1 + cells in populations of sorted SSEA-1 + (top) and SSEA-1 − fibroblasts (bottom) over time in culture in a representative time course experiment. About 1% of SSEA-1 + cells were present in the unsorted population. ( b,c ) Immunodetection of SSEA-1 in untreated cells ( b ) or cells treated with 25mM blebbistatin for 24h ( c ) by fluorescence microscopy. Pairs of daughter cells were identified as described in Supplementary Fig. S3 and Methods. Scale bar: 20 μm. The percentage of cells undergone each type of cell division is indicated. ( d ) Immunodetection of SSEA-1 in an isolated single SSEA-1 − -sorted fibroblast and in its daughter cells generated after 3 weeks of culture by fluorescence microscopy. Scale bar: 80 μm. ( e ) Flow cytometric analysis of SSEA-1 in three independent clonal populations resulting from single SSEA-1 − fibroblasts after three weeks of culture. Variable percentages of SSEA-1 + cells in each clone suggest that conversion to SSEA-1 + fibroblasts occurs in a stochastic manner. ( f ) Heatmap representing relative expression levels of genes showing a difference of at least 1.5-fold between SSEA-1 + and SSEA-1 − fibroblasts with a p value

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: SSEA-1 identifies a biologically distinct subpopulation of transformedfibroblasts ( a ) Abundance of SSEA-1 + cells in populations of sorted SSEA-1 + (top) and SSEA-1 − fibroblasts (bottom) over time in culture in a representative time course experiment. About 1% of SSEA-1 + cells were present in the unsorted population. ( b,c ) Immunodetection of SSEA-1 in untreated cells ( b ) or cells treated with 25mM blebbistatin for 24h ( c ) by fluorescence microscopy. Pairs of daughter cells were identified as described in Supplementary Fig. S3 and Methods. Scale bar: 20 μm. The percentage of cells undergone each type of cell division is indicated. ( d ) Immunodetection of SSEA-1 in an isolated single SSEA-1 − -sorted fibroblast and in its daughter cells generated after 3 weeks of culture by fluorescence microscopy. Scale bar: 80 μm. ( e ) Flow cytometric analysis of SSEA-1 in three independent clonal populations resulting from single SSEA-1 − fibroblasts after three weeks of culture. Variable percentages of SSEA-1 + cells in each clone suggest that conversion to SSEA-1 + fibroblasts occurs in a stochastic manner. ( f ) Heatmap representing relative expression levels of genes showing a difference of at least 1.5-fold between SSEA-1 + and SSEA-1 − fibroblasts with a p value

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: Immunodetection, Fluorescence, Microscopy, Isolation, Generated, Flow Cytometry, Expressing

    SSEA-1 + fibroblasts are responsible for tumour initiation ( a ) Experimental design to track cells responsible for tumour initiation among in vitro -transformed fibroblasts and possible outcomes. Higher percentage of GFP + cells in the tumours compared to that in the injected cells indicates greater tumorigenicity of labelled SSEA-1 + cells than competing unlabeled SSEA-1 − cells. ( b ) Percentages of GFP + cells in tumours induced by the indicated combinations of unlabeled and labelled cells measured by flow cytometric analysis. Dots represent individual tumours at 6 weeks after injection. Crosses are injections that failed to form tumours. Solid lines indicate median values of GFP + cells for each group. Statistical significance of the differences between the experimental values and the expected values under the hypothesis of equal tumorigenicity of the injected unlabeled and labelled cells (see Methods) is indicated for each group. ( c ) Immunohistochemical detection of GFP (brown) in tumours resulting from injection of the indicated cells. Pictures of four adjacent fields were combined for each tumour. Scale bar: 150 μm.

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: SSEA-1 + fibroblasts are responsible for tumour initiation ( a ) Experimental design to track cells responsible for tumour initiation among in vitro -transformed fibroblasts and possible outcomes. Higher percentage of GFP + cells in the tumours compared to that in the injected cells indicates greater tumorigenicity of labelled SSEA-1 + cells than competing unlabeled SSEA-1 − cells. ( b ) Percentages of GFP + cells in tumours induced by the indicated combinations of unlabeled and labelled cells measured by flow cytometric analysis. Dots represent individual tumours at 6 weeks after injection. Crosses are injections that failed to form tumours. Solid lines indicate median values of GFP + cells for each group. Statistical significance of the differences between the experimental values and the expected values under the hypothesis of equal tumorigenicity of the injected unlabeled and labelled cells (see Methods) is indicated for each group. ( c ) Immunohistochemical detection of GFP (brown) in tumours resulting from injection of the indicated cells. Pictures of four adjacent fields were combined for each tumour. Scale bar: 150 μm.

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: In Vitro, Transformation Assay, Injection, Flow Cytometry, Immunohistochemistry

    Hierarchical organization of tumours induced by in vitro -transformed fibroblasts ( a ) Soft agar assay using unsorted cells form a primary tumour and SSEA-1 + tumour fibroblasts. Scale bar: 400 mm. Similar results were obtained with cells from 5 other tumours. ( b , c ) Quantitative analysis of soft agar assay ( b ) and non-adherent sphere formation assay ( c ) using the indicated cells from a primary tumour resulting from injection of 3,000 transformed fibroblasts. The frequency of clonogenic cells is indicated. Values represent means ± s. d. from three replicates. Statistical significance of the differences compared to the unsorted population is indicated by one (p

    Journal: Nature cell biology

    Article Title: In vitro generation of human cells with cancer stem cell properties

    doi: 10.1038/ncb2308

    Figure Lengend Snippet: Hierarchical organization of tumours induced by in vitro -transformed fibroblasts ( a ) Soft agar assay using unsorted cells form a primary tumour and SSEA-1 + tumour fibroblasts. Scale bar: 400 mm. Similar results were obtained with cells from 5 other tumours. ( b , c ) Quantitative analysis of soft agar assay ( b ) and non-adherent sphere formation assay ( c ) using the indicated cells from a primary tumour resulting from injection of 3,000 transformed fibroblasts. The frequency of clonogenic cells is indicated. Values represent means ± s. d. from three replicates. Statistical significance of the differences compared to the unsorted population is indicated by one (p

    Article Snippet: Immunodetection was performed by incubating cells in medium containing an anti-SSEA-1 antibody (Millipore, 1:50) for 15 min at 37 °C, followed by addition of a secondary antibody and Hoechst 33342 at a final concentration of 20 μgml−1 and 1.5 μgml−1 , respectively, for additional 15 min at 37 °C.

    Techniques: In Vitro, Transformation Assay, Soft Agar Assay, Tube Formation Assay, Injection

    Pluripotency genes are partially repressed in embryonal carcinoma cells. UD: undifferentiated; D4, D8: Days post-induction of differentiation; ESCs: embryonic stem cells and F9 ECCs: F9 embryonal carcinoma cells, PpGs: pluripotency genes. (A, B, and E) Gene expression analysis by RT-qPCR of PpGs in (A) F9 ESCs, (B) ECCs (E) lineage specific genes in F9 ECCs. The C t values for each gene were normalized to Gapdh and expression is shown relative to that in undifferentiated cells (dotted line). In F9 ECCs, the lineage specific genes show a 5 to 60-fold induction of gene expression (E) whereas the expression of PpGs is on average reduced to about 50% post-differentiation (B). Average and SEM of two biological replicates are shown for each gene. (C) Alkaline phosphatase staining and (D) SSEA-1 immunofluorescence of ESCs and F9 ECCs pre- and post-differentiation. Positive signal indicates pluripotency that is lost post-differentiation in ESCs. The scale bar is a 100 µm.

    Journal: bioRxiv

    Article Title: Oct4-mediated inhibition of Lsd1 activity promotes the active and primed state of pluripotency enhancers

    doi: 10.1101/672451

    Figure Lengend Snippet: Pluripotency genes are partially repressed in embryonal carcinoma cells. UD: undifferentiated; D4, D8: Days post-induction of differentiation; ESCs: embryonic stem cells and F9 ECCs: F9 embryonal carcinoma cells, PpGs: pluripotency genes. (A, B, and E) Gene expression analysis by RT-qPCR of PpGs in (A) F9 ESCs, (B) ECCs (E) lineage specific genes in F9 ECCs. The C t values for each gene were normalized to Gapdh and expression is shown relative to that in undifferentiated cells (dotted line). In F9 ECCs, the lineage specific genes show a 5 to 60-fold induction of gene expression (E) whereas the expression of PpGs is on average reduced to about 50% post-differentiation (B). Average and SEM of two biological replicates are shown for each gene. (C) Alkaline phosphatase staining and (D) SSEA-1 immunofluorescence of ESCs and F9 ECCs pre- and post-differentiation. Positive signal indicates pluripotency that is lost post-differentiation in ESCs. The scale bar is a 100 µm.

    Article Snippet: SSEA-1 immunofluorescence was performed using the following antibodies: anti-SSEA-1 (Millipore, MAB430) and AlexaFluor 555 nm (Life Technologies, A21422).

    Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence

    Maintenance of self-renewal and pluripotency of J1 mES on methanol fixed fibroblasts. J1 mES cells were cultured on methanol fixed MEF (MT-MEF) and NIH3T3 (MT-3T3) cells, and Mitomycin C treated MEF (MMC-MEF). ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 mES. ( C ) qRT-PCR analysis of pluripotent genes in J1 mES. ( D , E ) Immunofluorescence ( D ) and flow cytometry analysis ( E ) of pluripotent markers OCT4 and SSEA-1 in J1 mES. Nuclei were stained by Hoechst 33342 (Hoe). ( F ) Teratoma formation of J1 mES. Arrows indicate tissues from the three germ layers. Scale bar, 400 μm for A, 200 μm for F, and 100 μm for D.

    Journal: Scientific Reports

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    doi: 10.1038/s41598-018-26238-2

    Figure Lengend Snippet: Maintenance of self-renewal and pluripotency of J1 mES on methanol fixed fibroblasts. J1 mES cells were cultured on methanol fixed MEF (MT-MEF) and NIH3T3 (MT-3T3) cells, and Mitomycin C treated MEF (MMC-MEF). ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 mES. ( C ) qRT-PCR analysis of pluripotent genes in J1 mES. ( D , E ) Immunofluorescence ( D ) and flow cytometry analysis ( E ) of pluripotent markers OCT4 and SSEA-1 in J1 mES. Nuclei were stained by Hoechst 33342 (Hoe). ( F ) Teratoma formation of J1 mES. Arrows indicate tissues from the three germ layers. Scale bar, 400 μm for A, 200 μm for F, and 100 μm for D.

    Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.

    Techniques: Cell Culture, Staining, Quantitative RT-PCR, Immunofluorescence, Flow Cytometry, Cytometry

    Application of methanol fixed fibroblasts for drug screening and repeated usage. ( A ) Diagram of drug screening. ( B ) The stably transfected cell lines derived from J1 cells transfected by miR70/EGFP and piPS cells transfected by METTL3-EGFP and miR370/EGFP, respectively, which were cultured on MT-MEF and MT-3T3. ( C , D ) Repeated usage of MT-MEF ( C ) and MT-3T3 ( D ) for four times (R0-R3). ( E , F ) Flow cytometry analysis of SSEA-1 expression in J1 mES cultured on MT-MEF ( E ) and MT-3T3 ( F ), which were used repeatedly for four times (R0-R3). Scale bar, 100 μm for B, 200 μm for C and D.

    Journal: Scientific Reports

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    doi: 10.1038/s41598-018-26238-2

    Figure Lengend Snippet: Application of methanol fixed fibroblasts for drug screening and repeated usage. ( A ) Diagram of drug screening. ( B ) The stably transfected cell lines derived from J1 cells transfected by miR70/EGFP and piPS cells transfected by METTL3-EGFP and miR370/EGFP, respectively, which were cultured on MT-MEF and MT-3T3. ( C , D ) Repeated usage of MT-MEF ( C ) and MT-3T3 ( D ) for four times (R0-R3). ( E , F ) Flow cytometry analysis of SSEA-1 expression in J1 mES cultured on MT-MEF ( E ) and MT-3T3 ( F ), which were used repeatedly for four times (R0-R3). Scale bar, 100 μm for B, 200 μm for C and D.

    Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.

    Techniques: Stable Transfection, Transfection, Derivative Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P

    Journal: Scientific Reports

    Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

    doi: 10.1038/s41598-018-26238-2

    Figure Lengend Snippet: Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P

    Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.

    Techniques: Cell Culture, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Microscopy

    Inhibition of EGFR by AG1478 impairs mESC self-renewal and pluripotency, and induces differentiation. (A) Enzymatic activity for AP was analyzed in control and AG1478 treated mESCs. (B–D) IF staining against SSEA-1 (red), OCT-4 (green), and Nanog (red) in control and AG1478 treated mESCs. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole, scale bar: 200 μm. (E and F) Quantitative PCR analysis of mRNA levels of pluripotency factor s and differentiation related genes in control and AG1478 treated mESCs. The amounts of each mRNA were normalized to GAPDH mRNA and are shown relative to the amounts in control mESCs (set to 1). (G) Protein expression of OCT4 and GATA4 in control and AG1478 treated mESCs. β-actin served as a loading control. (H) Quantified relative band intensity ratio of OCT4 and GATA4. The data are presented as mean ± SD ( n = 3; ** P

    Journal: PeerJ

    Article Title: EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells

    doi: 10.7717/peerj.6314

    Figure Lengend Snippet: Inhibition of EGFR by AG1478 impairs mESC self-renewal and pluripotency, and induces differentiation. (A) Enzymatic activity for AP was analyzed in control and AG1478 treated mESCs. (B–D) IF staining against SSEA-1 (red), OCT-4 (green), and Nanog (red) in control and AG1478 treated mESCs. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole, scale bar: 200 μm. (E and F) Quantitative PCR analysis of mRNA levels of pluripotency factor s and differentiation related genes in control and AG1478 treated mESCs. The amounts of each mRNA were normalized to GAPDH mRNA and are shown relative to the amounts in control mESCs (set to 1). (G) Protein expression of OCT4 and GATA4 in control and AG1478 treated mESCs. β-actin served as a loading control. (H) Quantified relative band intensity ratio of OCT4 and GATA4. The data are presented as mean ± SD ( n = 3; ** P

    Article Snippet: Cells were then blocked by 5% BSA in PBS at room temperature for 30 min, and were incubated with primary antibodies for EGFR (1:500; CST, Danvers, MA, USA), SSEA-1 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), OCT4 (1:500; CST), and Nanog (1:500; CST) overnight at 4 °C.

    Techniques: Inhibition, Activity Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

    DBA/2J ES cell derivation. ( A ) In each round of derivation, 11–60% of embryos gave rise to stable embryonic stem cell lines (see also Table 1 ). ( B ) Approximately ∼20% of lines were male with normal chromosome counts, regardless of culture conditions. (C) All of the ES cell lines, regardless of culture conditions, expressed the essential pluripotency markers NANOG, SSEA-1 and OCT-3/4 (POU5F1).

    Journal: PLoS ONE

    Article Title: Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J, a Model of Glaucoma and Other Complex Diseases

    doi: 10.1371/journal.pone.0050081

    Figure Lengend Snippet: DBA/2J ES cell derivation. ( A ) In each round of derivation, 11–60% of embryos gave rise to stable embryonic stem cell lines (see also Table 1 ). ( B ) Approximately ∼20% of lines were male with normal chromosome counts, regardless of culture conditions. (C) All of the ES cell lines, regardless of culture conditions, expressed the essential pluripotency markers NANOG, SSEA-1 and OCT-3/4 (POU5F1).

    Article Snippet: Primary antibodies were anti-OCT 3/4 (1∶250, Santa Cruz Biologicals, #sc-5279), anti-NANOG (1∶250, Abcam, #ab21603) and anti-SSEA-1 (1∶250, Santa Cruz Biologicals, #sc-21702).

    Techniques:

    Excision of the reprogramming cassette and its effect on pluripotency markers in differentiating iPSCs. The iPSCs on differentiation displayed many areas consisting of small, rounded cells (A), which stained positive for OCT4 (B), SSEA-1 (C) and alkaline phosphatase (AP) (D). Crystal violet staining of the differentiated iPSCs pre- (E, upper well) and post- (E, lower well) ganciclovir selection showed that the transgenes were not silenced on differentiation, thereby leading to massive cell death in the lower well. Therefore, to stop/decrease the expression of reprogramming transcription factors during differentiation we transfected the iPSC colonies (F) transiently with a CRE:GFP plasmid (G). Massive cellular death ensued after Ganciclovir selection (H)as visualized by the lack of visible colonies in the upper well of figure (I). However, after few days of culture the colonies again started to appear back (lower well of figure I). Genomic PCR confirmed transgene excision in two iPSC clones: pre- (clone #1 and clone #2) and post-excision (clone #1Ex and Clone #2Ex). Genomic Gapdh was used as a control (upper lane) and two areas of the transgene: i) HSV-tk to WPRE (middle lane) ii) Klf4 to 2A (lower lane), were used as the tests. Both the excised clones lacked the test bands. (J). AP staining showed maintenance of pluripotency in the emerging colonies (K). RT-PCR for Oct4 and Nanog in differentiating iPSCs showed a considerable decrease in the expression of both the markers post-excision in both the clones (1 and 2). No HSV-tk and WPRE expression was seen after excision (L). After excision the expression of Oct4, SSEA-1 and Alkaline phosphate were also reduced on ICC in the differentiating cells (M–P). Scale bar = 100 µm.

    Journal: PLoS ONE

    Article Title: A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0064342

    Figure Lengend Snippet: Excision of the reprogramming cassette and its effect on pluripotency markers in differentiating iPSCs. The iPSCs on differentiation displayed many areas consisting of small, rounded cells (A), which stained positive for OCT4 (B), SSEA-1 (C) and alkaline phosphatase (AP) (D). Crystal violet staining of the differentiated iPSCs pre- (E, upper well) and post- (E, lower well) ganciclovir selection showed that the transgenes were not silenced on differentiation, thereby leading to massive cell death in the lower well. Therefore, to stop/decrease the expression of reprogramming transcription factors during differentiation we transfected the iPSC colonies (F) transiently with a CRE:GFP plasmid (G). Massive cellular death ensued after Ganciclovir selection (H)as visualized by the lack of visible colonies in the upper well of figure (I). However, after few days of culture the colonies again started to appear back (lower well of figure I). Genomic PCR confirmed transgene excision in two iPSC clones: pre- (clone #1 and clone #2) and post-excision (clone #1Ex and Clone #2Ex). Genomic Gapdh was used as a control (upper lane) and two areas of the transgene: i) HSV-tk to WPRE (middle lane) ii) Klf4 to 2A (lower lane), were used as the tests. Both the excised clones lacked the test bands. (J). AP staining showed maintenance of pluripotency in the emerging colonies (K). RT-PCR for Oct4 and Nanog in differentiating iPSCs showed a considerable decrease in the expression of both the markers post-excision in both the clones (1 and 2). No HSV-tk and WPRE expression was seen after excision (L). After excision the expression of Oct4, SSEA-1 and Alkaline phosphate were also reduced on ICC in the differentiating cells (M–P). Scale bar = 100 µm.

    Article Snippet: IPSCs were then stained with primary antibodies against OCT4 (Rabbit polyclonal, ABCAM: ab19857), SSEA-1 (Mouse monoclonal, Developmental Studies Hybridoma Bank, MC-480) and NANOG (Rabbit polyclonal, ABCAM: ab80892) at a dilution of 1∶100 for 2 hours and subsequently were incubated with fluorophore-labeled secondary antibodies (Life technologies) at a final dilution of 1∶500.

    Techniques: Staining, Selection, Expressing, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry

    Immunocytochemistry and RT-PCR for stemness and pluripotency markers in transgene-excised iPSCs. The transgene-excised iPSCs were imaged for stemness markers. Phase contrast imaging revealed compact colony morphology (A) with small cells and a prominent nucleus on DAPI staining (B). The colonies stained positive for alkaline phosphatase (C), SSEA-1 (D), OCT4 (E) and NANOG (F). On RT-PCR both the transgene-excised iPSC lines (1Ex and 2Ex) and mouse ES cell line D3 (MESC) expressed stemness markers like Oct4, Sox2, Klf4, Nanog, Gdf3, and Rex1. Gapdh was used as the PCR control (G). Scale bar = 100 µm.

    Journal: PLoS ONE

    Article Title: A Robust Strategy for Negative Selection of Cre-LoxP Recombination-Based Excision of Transgenes in Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0064342

    Figure Lengend Snippet: Immunocytochemistry and RT-PCR for stemness and pluripotency markers in transgene-excised iPSCs. The transgene-excised iPSCs were imaged for stemness markers. Phase contrast imaging revealed compact colony morphology (A) with small cells and a prominent nucleus on DAPI staining (B). The colonies stained positive for alkaline phosphatase (C), SSEA-1 (D), OCT4 (E) and NANOG (F). On RT-PCR both the transgene-excised iPSC lines (1Ex and 2Ex) and mouse ES cell line D3 (MESC) expressed stemness markers like Oct4, Sox2, Klf4, Nanog, Gdf3, and Rex1. Gapdh was used as the PCR control (G). Scale bar = 100 µm.

    Article Snippet: IPSCs were then stained with primary antibodies against OCT4 (Rabbit polyclonal, ABCAM: ab19857), SSEA-1 (Mouse monoclonal, Developmental Studies Hybridoma Bank, MC-480) and NANOG (Rabbit polyclonal, ABCAM: ab80892) at a dilution of 1∶100 for 2 hours and subsequently were incubated with fluorophore-labeled secondary antibodies (Life technologies) at a final dilution of 1∶500.

    Techniques: Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Imaging, Staining, Polymerase Chain Reaction

    Cellular organization and guidance cue expression are similar in the ventral diencephalon of Dscam del17 WT and mutant littermates. ( A ) Immunostaining of the SSEA-1–positive neurons located posterior to the developing optic chiasm and RC2-positive

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

    Article Title: DSCAM promotes axon fasciculation and growth in the developing optic pathway

    doi: 10.1073/pnas.1618606114

    Figure Lengend Snippet: Cellular organization and guidance cue expression are similar in the ventral diencephalon of Dscam del17 WT and mutant littermates. ( A ) Immunostaining of the SSEA-1–positive neurons located posterior to the developing optic chiasm and RC2-positive

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti–neuron- specific β-tubulin (TUJ1; MMS-435P, 1:1,000; Cambridge Biosciences), anti–TAG-1 (4D7, 1:40; Developmental Studies Hybridoma Bank), anti–SSEA-1 (MC-480; 1:9; Developmental Studies Hybridoma Bank), RC2 (1:50; Developmental Studies Hybridoma Bank), anti-BRN3A (MAB1585, 1:300; Millipore,), anti-phosphohistone H3 (06–570, 1:50; Millipore), anti–cleaved caspase-3 (9664, 1:100; New England Biolabs), and anti-ZiC2 (a gift from Eloisa Herrera), followed by the appropriate secondary antibody: Alexa Fluor 488 goat anti-mouse IgG (1:200; Thermo Fisher Scientific), Cy3 goat anti-rabbit IgG, or Cy3 goat anti-mouse IgG (1:1,000; Jackson ImmunoResearch).

    Techniques: Expressing, Mutagenesis, Immunostaining

    Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker SSEA-1 (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a , Nanog , and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. ∗ Compared to the expression level of cultured cESCs in passage 3, P

    Journal: Stem Cells International

    Article Title: An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro

    doi: 10.1155/2018/2157451

    Figure Lengend Snippet: Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker SSEA-1 (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a , Nanog , and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. ∗ Compared to the expression level of cultured cESCs in passage 3, P

    Article Snippet: The primary antibodies SSEA-1 (Abcam, 1 : 100) and Nanog (Abcam, 1 : 150), both of which were diluted in the blocking buffer, were incubated along with the fixed cells overnight at 4°C.

    Techniques: Stable Transfection, In Vitro, Cell Culture, Marker, Staining, Expressing, High Content Screening

    Characteristics of ntESCs and iPSCs from APCs (A) Morphology and alkaline phosphatase expression of ntESCs and iPSCs. Scale bars, 100 μm. (B) Immunofluorescence staining of pluripotent markers SSEA-1 (red), Nanog (green), Sox2 (green), and Oct4 (red) in AN and AI lines. Nuclei were stained with DAPI. Scale bars, 50μm. (C) Quantitative PCR analysis of the pluripotent markers Sall4, Oct4 and Nanog in AN and AI lines. Relative mRNA expression is normalized to GAPDH and is represented relative to expression in fibroblasts (37MEF). The experiments were performed in triplicate (means±SD; n=3). The primers used are listed in Supplementary Table 2 . (D) H E staining of teratomas generated from AN1 that formed tissues from all three germ layers. 1: Ectoderm; 2: Mesoderm; 3: Endoderm. Scale bars, 100 μm. A detailed description of all of the cell lines is given in Supplementary Table 1 . (E and F) Quantitative PCR analysis showing the upregulation of markers for all three germ layers during the in vitro differentiation of AN and AI lines. Relative mRNA expression is normalized to GAPDH and represented relative to expression in undifferentiated AN and AI lines. The experiments were performed in triplicate (mean±SD; n=3). The primers used are listed in Supplementary Table 2 . (G) Germline transmission of an AN1 tetraploid mouse. Details on all of the cell lines are in Supplementary Table 1 .

    Journal: Oncotarget

    Article Title: High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells

    doi: 10.18632/oncotarget.17185

    Figure Lengend Snippet: Characteristics of ntESCs and iPSCs from APCs (A) Morphology and alkaline phosphatase expression of ntESCs and iPSCs. Scale bars, 100 μm. (B) Immunofluorescence staining of pluripotent markers SSEA-1 (red), Nanog (green), Sox2 (green), and Oct4 (red) in AN and AI lines. Nuclei were stained with DAPI. Scale bars, 50μm. (C) Quantitative PCR analysis of the pluripotent markers Sall4, Oct4 and Nanog in AN and AI lines. Relative mRNA expression is normalized to GAPDH and is represented relative to expression in fibroblasts (37MEF). The experiments were performed in triplicate (means±SD; n=3). The primers used are listed in Supplementary Table 2 . (D) H E staining of teratomas generated from AN1 that formed tissues from all three germ layers. 1: Ectoderm; 2: Mesoderm; 3: Endoderm. Scale bars, 100 μm. A detailed description of all of the cell lines is given in Supplementary Table 1 . (E and F) Quantitative PCR analysis showing the upregulation of markers for all three germ layers during the in vitro differentiation of AN and AI lines. Relative mRNA expression is normalized to GAPDH and represented relative to expression in undifferentiated AN and AI lines. The experiments were performed in triplicate (mean±SD; n=3). The primers used are listed in Supplementary Table 2 . (G) Germline transmission of an AN1 tetraploid mouse. Details on all of the cell lines are in Supplementary Table 1 .

    Article Snippet: After permeabilization with 0.5% Triton-X and blocking with 0.5% bovine serum albumin (BSA), the cells were incubated with primary antibodies against Oct4 (1:500, Santa Cruz, Dallas, TX, USA), Sox2 (1:500, Santa Cruz), Nanog (1:500, COSMO BioCo, Tokyo, Japan) and SSEA-1 (1:50, Merck, Millipore).

    Techniques: Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Generated, In Vitro, Transmission Assay

    The morphologies and characteristics of rat bone marrow mesenchymal stem cells (BMMSCs) and multilineage‐differentiating stress‐enduring (Muse cells) . (A) The morphology (a1) of BMMSCs lipoblasts (a2) and osteoblasts (a3) differentiated from BMMSCs were observed by microscopy. BMMSCs were positive for CD29 (a4), CD90 (a5), and RT1A (a6) and negative for CD34 (a4), CD45 (a5), RT1B (a6), SSEA‐3 (a7) and SSEA‐1 (a8) detected by flow cytometry (FCM). (B) Muse cells formed the Muse‐cell‐clusters (M‐clusters) in suspension cultivation and long‐shuttle types in adherent cultivation (b1–b3). Rat Muse cells were positive for CD29 (b4), CD90 (b5), and RT1A (b6) and negative for CD34 (b4), CD45 (b5), RT1B (b6) similar to BMMSCs, but positively expressed SSEA‐3 (75.6 ± 2.8% vs. 2.3 ± 0.3%, b7) SSEA‐1 (74.8 ± 3.1% vs. 2.1 ± 0.2%). In addition, 77.62 ± 5.3% of SSEA‐1 (+) cells expressed SSEA‐3 (b9). Scale bars = 50 μm.

    Journal: Cell Biology International

    Article Title: Study of the protective effect on damaged intestinal epithelial cells of rat multilineage‐differentiating stress‐enduring (Muse) cells

    doi: 10.1002/cbin.11255

    Figure Lengend Snippet: The morphologies and characteristics of rat bone marrow mesenchymal stem cells (BMMSCs) and multilineage‐differentiating stress‐enduring (Muse cells) . (A) The morphology (a1) of BMMSCs lipoblasts (a2) and osteoblasts (a3) differentiated from BMMSCs were observed by microscopy. BMMSCs were positive for CD29 (a4), CD90 (a5), and RT1A (a6) and negative for CD34 (a4), CD45 (a5), RT1B (a6), SSEA‐3 (a7) and SSEA‐1 (a8) detected by flow cytometry (FCM). (B) Muse cells formed the Muse‐cell‐clusters (M‐clusters) in suspension cultivation and long‐shuttle types in adherent cultivation (b1–b3). Rat Muse cells were positive for CD29 (b4), CD90 (b5), and RT1A (b6) and negative for CD34 (b4), CD45 (b5), RT1B (b6) similar to BMMSCs, but positively expressed SSEA‐3 (75.6 ± 2.8% vs. 2.3 ± 0.3%, b7) SSEA‐1 (74.8 ± 3.1% vs. 2.1 ± 0.2%). In addition, 77.62 ± 5.3% of SSEA‐1 (+) cells expressed SSEA‐3 (b9). Scale bars = 50 μm.

    Article Snippet: Flow cytometry (FCM) Rat BMMSCs and Muse cells were incubated with the desired antibodies (e.g., anti‐CD29, anti‐CD90, anti‐RT1A, anti‐CD34, anti‐CD45, anti‐RT1B, anti‐SSEA‐3 [BioLegend, San Diego, USA], and anti‐SSEA‐1, [STEMCELL Technologies, Vancouver, BC, Canada]).

    Techniques: Microscopy, Flow Cytometry

    ), association data of upregulated gene interaction with SSEA-1+ vs SSEA-1neg porcine embryonic fibroblasts (pEFS), which includes protein and genetic interaction pathways, co-expression, co-localization and protein domain similarity.

    Journal: Cell Cycle

    Article Title: Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    doi: 10.1080/15384101.2017.1315490

    Figure Lengend Snippet: ), association data of upregulated gene interaction with SSEA-1+ vs SSEA-1neg porcine embryonic fibroblasts (pEFS), which includes protein and genetic interaction pathways, co-expression, co-localization and protein domain similarity.

    Article Snippet: Cells were labeled with PE anti-SSEA-1 (Nordic BioSite, # 301906) according to manufacturer's instructions in PBS containing 2% BSA with fluorochrome-conjugated antibodies for 60 min at rt, followed by washing twice in PBS containing 2% BSA.

    Techniques: Expressing

    Reprogramming of SSEA-1 positive cells reveals enhanced reprogramming capabilites. (A) Schematic of reprogramming SSEA-1+, SSEA-1neg and unsorted D1 pEFs using lentiviral transfection. (B) Flow cytometry analysis of SSEA-1+ cell fraction in D1 pEFs. (C) Representative Alkaline Phosphatase positive (AP+) colony forming unit with ESC-like morphology (CFU) derived from D1 SSEA-1+ cells 21 d post transduction. (D) Number of AP+ CFUs derived from the 3 cell populations. (E) Schematic of magnetic cell sorting (MACS) targeting SSEA-1+ cell fraction and subsequent episomal plasmid-based reprogramming of SSEA-1+ and SSEA-1neg fractions. (F) Total mean number of AP+ CFUs derived from MACS sorted SSEA-1+ and SSEA-1neg fractions. (G) Representative image of AP+ CFUs from MACS sorted SSEA-1+ and SSEA-1neg fractions.

    Journal: Cell Cycle

    Article Title: Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    doi: 10.1080/15384101.2017.1315490

    Figure Lengend Snippet: Reprogramming of SSEA-1 positive cells reveals enhanced reprogramming capabilites. (A) Schematic of reprogramming SSEA-1+, SSEA-1neg and unsorted D1 pEFs using lentiviral transfection. (B) Flow cytometry analysis of SSEA-1+ cell fraction in D1 pEFs. (C) Representative Alkaline Phosphatase positive (AP+) colony forming unit with ESC-like morphology (CFU) derived from D1 SSEA-1+ cells 21 d post transduction. (D) Number of AP+ CFUs derived from the 3 cell populations. (E) Schematic of magnetic cell sorting (MACS) targeting SSEA-1+ cell fraction and subsequent episomal plasmid-based reprogramming of SSEA-1+ and SSEA-1neg fractions. (F) Total mean number of AP+ CFUs derived from MACS sorted SSEA-1+ and SSEA-1neg fractions. (G) Representative image of AP+ CFUs from MACS sorted SSEA-1+ and SSEA-1neg fractions.

    Article Snippet: Cells were labeled with PE anti-SSEA-1 (Nordic BioSite, # 301906) according to manufacturer's instructions in PBS containing 2% BSA with fluorochrome-conjugated antibodies for 60 min at rt, followed by washing twice in PBS containing 2% BSA.

    Techniques: Transfection, Flow Cytometry, Cytometry, Derivative Assay, Transduction, FACS, Magnetic Cell Separation, Plasmid Preparation

    This data are based on 5 biologic replicates (A) The significantly differentially expressed Genes from the Volcano Plot built by comparing “SSEA-1+ vs SSEA-1-neg,” using Multiple Testing Correction: Benjamini and Hochberg False Discovery Rate. Differentially expressed genes were defined by Fold Difference: 1.2 and a P-value Cutoff: 0.05. (B) A gene condition tree heat map clustering of all genes was built by comparing “SSEA-1+ vs SSEA-1-neg.” Similarity Measurement, Distance. The clustering algorithm was performed using average linkage. (C) Gene condition tree heat map clustering of all genes built by comparing “SSEA-1+ vs SSEA-1-neg.” Discarded genes with no gene symbol annotation from the starting conditions. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage.

    Journal: Cell Cycle

    Article Title: Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    doi: 10.1080/15384101.2017.1315490

    Figure Lengend Snippet: This data are based on 5 biologic replicates (A) The significantly differentially expressed Genes from the Volcano Plot built by comparing “SSEA-1+ vs SSEA-1-neg,” using Multiple Testing Correction: Benjamini and Hochberg False Discovery Rate. Differentially expressed genes were defined by Fold Difference: 1.2 and a P-value Cutoff: 0.05. (B) A gene condition tree heat map clustering of all genes was built by comparing “SSEA-1+ vs SSEA-1-neg.” Similarity Measurement, Distance. The clustering algorithm was performed using average linkage. (C) Gene condition tree heat map clustering of all genes built by comparing “SSEA-1+ vs SSEA-1-neg.” Discarded genes with no gene symbol annotation from the starting conditions. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage.

    Article Snippet: Cells were labeled with PE anti-SSEA-1 (Nordic BioSite, # 301906) according to manufacturer's instructions in PBS containing 2% BSA with fluorochrome-conjugated antibodies for 60 min at rt, followed by washing twice in PBS containing 2% BSA.

    Techniques:

    Epithelial derived LTβR controls mTECp development. ( a ) Thymic stromal cells were prepared from neonatal Ltbr fl/fl K14 Cre and Ltbr fl/+ K14 Cre control mice. mTECp is identified as Cld3,4 hi SSEA-1 + within CD45 − EpCAM + TEC population. ( b ) Statistic data are shown as mean ± SD of more than 3 mice each group. One representative FACS plot and statistic result from more than 3 repeats are shown. An unpaired two-tailed Student’s t -test is used: *P

    Journal: Scientific Reports

    Article Title: Epithelial LTβR signaling controls the population size of the progenitors of medullary thymic epithelial cells in neonatal mice

    doi: 10.1038/srep44481

    Figure Lengend Snippet: Epithelial derived LTβR controls mTECp development. ( a ) Thymic stromal cells were prepared from neonatal Ltbr fl/fl K14 Cre and Ltbr fl/+ K14 Cre control mice. mTECp is identified as Cld3,4 hi SSEA-1 + within CD45 − EpCAM + TEC population. ( b ) Statistic data are shown as mean ± SD of more than 3 mice each group. One representative FACS plot and statistic result from more than 3 repeats are shown. An unpaired two-tailed Student’s t -test is used: *P

    Article Snippet: The antibodies used included anti-CD45 (30-F11, eBioscience), anti-EpCAM (G8.8, Biolegend), anti-Ly51 (6C3, Biolegend), FITC labelled Ulex europaeus agglutinin-1 (UEA-1; Vector Laboratory), anti-IA/IE (M5/114.15.2, Biolegend), anti-SSEA-1 (MC-480, Biolegend), anti-LTβR (eBio3C8, eBioscience).

    Techniques: Derivative Assay, Mouse Assay, FACS, Two Tailed Test

    Immunofluorescence staining of FS101 and FS111 PC PGCs. (a) Confocal images of SSEA-1- (red) and CVH- (green) stained FS101 PGCs. (b) Higher magnification of FS101 cells (white square indicates the cells on (a)). (c) Confocal images of SSEA-1- (red) and CVH- (green) stained FS111 PGCs. (d) Higher magnification of the FS111 cells (white square indicates the cells on (c)). For nuclear staining (NS), we used TO-PRO-3 (blue). Scale bars: 25 μ m (a, c) and 5 μ m (b, d). PC PG cells: Partridge colour Hungarian chicken embryo-derived primordial germ cells.

    Journal: Stem Cells International

    Article Title: Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

    doi: 10.1155/2018/1780679

    Figure Lengend Snippet: Immunofluorescence staining of FS101 and FS111 PC PGCs. (a) Confocal images of SSEA-1- (red) and CVH- (green) stained FS101 PGCs. (b) Higher magnification of FS101 cells (white square indicates the cells on (a)). (c) Confocal images of SSEA-1- (red) and CVH- (green) stained FS111 PGCs. (d) Higher magnification of the FS111 cells (white square indicates the cells on (c)). For nuclear staining (NS), we used TO-PRO-3 (blue). Scale bars: 25 μ m (a, c) and 5 μ m (b, d). PC PG cells: Partridge colour Hungarian chicken embryo-derived primordial germ cells.

    Article Snippet: Then, cells were washed three times with PBS and were incubated with each of the primary antibodies including mouse anti-SSEA-1 (1 : 10, Developmental Studies Hybridoma Bank, US) and rabbit anti-VASA (1 : 1000; kindly provided by Bertrand Pain, Lyon, France).

    Techniques: Immunofluorescence, Staining, Derivative Assay

    Generation and Characterization of iPSCs from WT and oc/oc Mice (A) Experimental outline for the generation of WT and oc/oc iPSC clones with excised reprogramming vector (VCN = 0) and carrying the correct chromosome complement (see text for details). (B) Expression of alkaline phosphatase on WT and oc/oc iPSC colonies. (C) Expression of Oct4, Sox2, Nanog, and SSEA-1 on WT and oc/oc iPSCs as revealed by immunofluorescence. Nuclei are stained with DAPI. Scale bars, 100 μm. (D) Karyotype analysis of WT and oc/oc iPSC clones. (E) Generation of the three germ layers in vitro by differentiated WT and oc/oc iPSCs is revealed by immunofluorescence. Expression of Brachyury, AFP, and Nestin indicate formation of mesoderm, endoderm, and ectoderm, respectively. Scale bars, 50 μm. (F) H E staining of teratomas generated after subcutaneous injection of WT and oc/oc iPSC clones into NSG mice, demonstrating differentiation into the three germ layer derivatives (i, mesoderm-cartilage; ii, mesoderm-adipose tissue; iii, endoderm-glandular structure; iv, endoderm-glandular structures (intestinal paneth or pancreatic acinar cell-like); v, ectoderm-primitive and mature neuroepithelium; vi, ectoderm-primitive neuroepithelium). Scale bars, 50 μm. See also Figure S1 .

    Journal: Stem Cell Reports

    Article Title: Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts

    doi: 10.1016/j.stemcr.2015.08.005

    Figure Lengend Snippet: Generation and Characterization of iPSCs from WT and oc/oc Mice (A) Experimental outline for the generation of WT and oc/oc iPSC clones with excised reprogramming vector (VCN = 0) and carrying the correct chromosome complement (see text for details). (B) Expression of alkaline phosphatase on WT and oc/oc iPSC colonies. (C) Expression of Oct4, Sox2, Nanog, and SSEA-1 on WT and oc/oc iPSCs as revealed by immunofluorescence. Nuclei are stained with DAPI. Scale bars, 100 μm. (D) Karyotype analysis of WT and oc/oc iPSC clones. (E) Generation of the three germ layers in vitro by differentiated WT and oc/oc iPSCs is revealed by immunofluorescence. Expression of Brachyury, AFP, and Nestin indicate formation of mesoderm, endoderm, and ectoderm, respectively. Scale bars, 50 μm. (F) H E staining of teratomas generated after subcutaneous injection of WT and oc/oc iPSC clones into NSG mice, demonstrating differentiation into the three germ layer derivatives (i, mesoderm-cartilage; ii, mesoderm-adipose tissue; iii, endoderm-glandular structure; iv, endoderm-glandular structures (intestinal paneth or pancreatic acinar cell-like); v, ectoderm-primitive and mature neuroepithelium; vi, ectoderm-primitive neuroepithelium). Scale bars, 50 μm. See also Figure S1 .

    Article Snippet: In order to avoid possible MEFs contamination in PCR analysis, all clones were purified with anti-SSEA-1 (stage-specific embryonic antigen-1) magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions and passaged three times on gelatin-coated plates before collecting genomic DNA and RNA for analysis.

    Techniques: Mouse Assay, Clone Assay, Plasmid Preparation, Expressing, Immunofluorescence, Staining, In Vitro, Generated, Injection

    Insulin released in response to low glucose (5.6 mM) and high glucose (25 mM) stimulation in culture medium of insulinsecreting cells generated from bone marrow derived SSEA-1+ SCs (day 24) was measured. Undifferentiated bone marrow derived SSEA-1 positive cells were used as a control. Each value represents mean ± SD of three experiments. Statistical significance was tested by a Student’s t test. *; P

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Insulin released in response to low glucose (5.6 mM) and high glucose (25 mM) stimulation in culture medium of insulinsecreting cells generated from bone marrow derived SSEA-1+ SCs (day 24) was measured. Undifferentiated bone marrow derived SSEA-1 positive cells were used as a control. Each value represents mean ± SD of three experiments. Statistical significance was tested by a Student’s t test. *; P

    Article Snippet: BMMNCs were resuspended in cold MACS buffer (Miltenyi Biotec, Germany) and incubated with microbeadconjugated anti-SSEA-1 antibody (Miltenyi Biotec, Germany) for 45 minutes at 4˚C on a plateshaker.

    Techniques: Generated, Derivative Assay

    Immunostaining of pluripotency markers in bone marrow derived SSEA-1 positive cells. Expression of A. OCT-4, B. SSEA-1 in purified bone marrow derived SSEA-1 positive cells evaluated by immunofluorescence staining. Nuclei were counterstained with DAPI and C. Flow cytometric analysis of bone marrow derived SSEA-1 positive cells show that they expressed SSEA-1, CXCR4 and SCA-1 but expression of CD45 was much lower or undetectable. SSEA-1; Stage-specific embryonic antigen 1, OCT-4; Octamer-binding transcription factor 4, DAPI; 4´, 6-diamidino-2-phenylindole, CXCR4; C-X-C chemokine receptor type 4 and SCA-1; Stem cells antigen-1.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Immunostaining of pluripotency markers in bone marrow derived SSEA-1 positive cells. Expression of A. OCT-4, B. SSEA-1 in purified bone marrow derived SSEA-1 positive cells evaluated by immunofluorescence staining. Nuclei were counterstained with DAPI and C. Flow cytometric analysis of bone marrow derived SSEA-1 positive cells show that they expressed SSEA-1, CXCR4 and SCA-1 but expression of CD45 was much lower or undetectable. SSEA-1; Stage-specific embryonic antigen 1, OCT-4; Octamer-binding transcription factor 4, DAPI; 4´, 6-diamidino-2-phenylindole, CXCR4; C-X-C chemokine receptor type 4 and SCA-1; Stem cells antigen-1.

    Article Snippet: BMMNCs were resuspended in cold MACS buffer (Miltenyi Biotec, Germany) and incubated with microbeadconjugated anti-SSEA-1 antibody (Miltenyi Biotec, Germany) for 45 minutes at 4˚C on a plateshaker.

    Techniques: Immunostaining, Derivative Assay, Expressing, Purification, Immunofluorescence, Staining, Flow Cytometry, Binding Assay

    Identification of bone marrow derived SSEA-1 positive cells differentiated into insulin producing cells. A. Primary explant cultures of bone marrow derived SSEA-1 positive cells. Undifferentiated SSEA-1 positive cells were spherical in shape, B. Colony of purified SSEA-1 positive cells after culturing on MEF-coated dishes and C. Dithizone staining of insulin secreting cellsderived from bone marrow derived SSEA-1 positive cells. SSEA-1; Stage-specific embryonic antigen 1 and MEF; Mouse embryonic fibroblasts.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Identification of bone marrow derived SSEA-1 positive cells differentiated into insulin producing cells. A. Primary explant cultures of bone marrow derived SSEA-1 positive cells. Undifferentiated SSEA-1 positive cells were spherical in shape, B. Colony of purified SSEA-1 positive cells after culturing on MEF-coated dishes and C. Dithizone staining of insulin secreting cellsderived from bone marrow derived SSEA-1 positive cells. SSEA-1; Stage-specific embryonic antigen 1 and MEF; Mouse embryonic fibroblasts.

    Article Snippet: BMMNCs were resuspended in cold MACS buffer (Miltenyi Biotec, Germany) and incubated with microbeadconjugated anti-SSEA-1 antibody (Miltenyi Biotec, Germany) for 45 minutes at 4˚C on a plateshaker.

    Techniques: Derivative Assay, Purification, Staining

    Electropherograms of RT-PCR product of mRNA extracted from bone marrow derived SSEA-1 positive cells induced into insulin secreting cells (lane 2), undifferentiated bone marrow derived SSEA-1 positive cells (lane 3) and pancreatic β-cells as a positive control (lane 4). The leftmost lane represents the DNA ladder and distilled H2O (lane 1) was used as a negative control. β-actin amplification served as an internal control. A. Pdx1 , B. Ngn3 , C. glucagon , D. Amylase , E. Insulin1 , F. Insulin2 and G. β-actin. RT-PCR; Reverse transcription polymerase chain reaction and SSEA-1; Stage-specific embryonic antigen 1.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Electropherograms of RT-PCR product of mRNA extracted from bone marrow derived SSEA-1 positive cells induced into insulin secreting cells (lane 2), undifferentiated bone marrow derived SSEA-1 positive cells (lane 3) and pancreatic β-cells as a positive control (lane 4). The leftmost lane represents the DNA ladder and distilled H2O (lane 1) was used as a negative control. β-actin amplification served as an internal control. A. Pdx1 , B. Ngn3 , C. glucagon , D. Amylase , E. Insulin1 , F. Insulin2 and G. β-actin. RT-PCR; Reverse transcription polymerase chain reaction and SSEA-1; Stage-specific embryonic antigen 1.

    Article Snippet: BMMNCs were resuspended in cold MACS buffer (Miltenyi Biotec, Germany) and incubated with microbeadconjugated anti-SSEA-1 antibody (Miltenyi Biotec, Germany) for 45 minutes at 4˚C on a plateshaker.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Negative Control, Amplification

    Immunofluorescence reveals bone marrow derived SSEA-1 positive cells differentiated into insulin secreting cells in vitro . The insulin secreting cells at days 24 were fixed and stained with antibodies against Pdx1 and Glut2 and visualized with secondary antibody (FITC). 4′, 6-diamidino-2-phenylindole (DAPI) was used to counter-stain DNA (blue) (scale bar; 50 μm). A. Anti Pdx1 immunofluorescence, B. Nuclear counterstain with DAPI, C. Merged image of A and B, D. AntiGlut2 immunofluorescence, E. Nuclear counterstain with DAPI and F. Merged image of D and E. SSEA-1; Stage-specific embryonic antigen 1.

    Journal: Cell Journal (Yakhteh)

    Article Title: In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    doi:

    Figure Lengend Snippet: Immunofluorescence reveals bone marrow derived SSEA-1 positive cells differentiated into insulin secreting cells in vitro . The insulin secreting cells at days 24 were fixed and stained with antibodies against Pdx1 and Glut2 and visualized with secondary antibody (FITC). 4′, 6-diamidino-2-phenylindole (DAPI) was used to counter-stain DNA (blue) (scale bar; 50 μm). A. Anti Pdx1 immunofluorescence, B. Nuclear counterstain with DAPI, C. Merged image of A and B, D. AntiGlut2 immunofluorescence, E. Nuclear counterstain with DAPI and F. Merged image of D and E. SSEA-1; Stage-specific embryonic antigen 1.

    Article Snippet: BMMNCs were resuspended in cold MACS buffer (Miltenyi Biotec, Germany) and incubated with microbeadconjugated anti-SSEA-1 antibody (Miltenyi Biotec, Germany) for 45 minutes at 4˚C on a plateshaker.

    Techniques: Immunofluorescence, Derivative Assay, In Vitro, Staining

    Characteristics of EB differentiation. a EB immunofluorescent staining of undifferentiated state marker SSEA-1 and surface antigens, including cytokeratin ( CK ), CD73, and CD151. Cell nuclei were stained with DAPI. Bars = 100 μm. b Morphological appearance of pESC-derived and ESC-derived EBs and outgrowths of EBs. Bars = 100 μm. c Expression of Oct3/4 (stemness), nestin (ectoderm), Afp (entoderm), and mesoderm markers Snai1 , Hand1 , and Gata2 in pESC-derived and ESC-derived EB outgrowths. * p

    Journal: Stem Cell Research & Therapy

    Article Title: Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent

    doi: 10.1186/s13287-016-0407-z

    Figure Lengend Snippet: Characteristics of EB differentiation. a EB immunofluorescent staining of undifferentiated state marker SSEA-1 and surface antigens, including cytokeratin ( CK ), CD73, and CD151. Cell nuclei were stained with DAPI. Bars = 100 μm. b Morphological appearance of pESC-derived and ESC-derived EBs and outgrowths of EBs. Bars = 100 μm. c Expression of Oct3/4 (stemness), nestin (ectoderm), Afp (entoderm), and mesoderm markers Snai1 , Hand1 , and Gata2 in pESC-derived and ESC-derived EB outgrowths. * p

    Article Snippet: Cells were then blocked with 10 % bovine serum albumin (BSA; Sigma-Aldrich) for 45 min and incubated overnight at 4 °C with 1:200 diluted primary antibodies, including goat anti-OCT3/4, rabbit anti-NANOG; and mouse anti-SSEA-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Staining, Marker, Derivative Assay, Expressing

    Puma is not required to suppress DNA damage during reprogramming. A. Confocal analysis of day 14 SSEA-1 positive colonies for γH2AX and DAPI. B. FACS analyses of day 14 to 18 OSK(M) cultures co-stained with γH2AX and SSEA-1. (Wild-type,

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma

    doi: 10.1002/stem.1054

    Figure Lengend Snippet: Puma is not required to suppress DNA damage during reprogramming. A. Confocal analysis of day 14 SSEA-1 positive colonies for γH2AX and DAPI. B. FACS analyses of day 14 to 18 OSK(M) cultures co-stained with γH2AX and SSEA-1. (Wild-type,

    Article Snippet: Cells were washed with PBS and stained with Alexa fluor-555 secondary antibody (Invitrogen); Hoechst33342 (Invitrogen) and mouse anti-SSEA-1 (Stemgent) in PBS containing 1% BSA.

    Techniques: FACS, Staining

    Loss of Puma and p21 permits generation of stable mouse iPSCs. A. Representative iPSCs (passage 5-6) from each genotype stained for alkaline phosphatase (AP) or SSEA-1 and Oct3/4. B. Real time PCR analysis of pluripotency marker gene expression relative

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma

    doi: 10.1002/stem.1054

    Figure Lengend Snippet: Loss of Puma and p21 permits generation of stable mouse iPSCs. A. Representative iPSCs (passage 5-6) from each genotype stained for alkaline phosphatase (AP) or SSEA-1 and Oct3/4. B. Real time PCR analysis of pluripotency marker gene expression relative

    Article Snippet: Cells were washed with PBS and stained with Alexa fluor-555 secondary antibody (Invitrogen); Hoechst33342 (Invitrogen) and mouse anti-SSEA-1 (Stemgent) in PBS containing 1% BSA.

    Techniques: Staining, Real-time Polymerase Chain Reaction, Marker, Expressing