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  • 90
    Abcam anti ssea 4
    Cells obtained from human amniotic membrane mesoderm displayed mesenchymal stromal cells characteristics. Phase-contrast micrograph of hAMSC adhered to a polystyrene cell culture plate at 3 rd passage showing fibroblast morphology; the photograph was taken at 40x of magnification, scale bar 100 μm ( A ). The cells were cultured for ten days and stained with crystal violet, and a direct light micrograph was performed in order to identify the UFC (arrows); the photograph was magnified at 35x in a stereoscopic microscope, scale bar 100 μm ( B ). Fluorescence micrographs of hAMSC stained with pluripotent embryonic markers OCT-4 (left panel), and <t>SSEA-4</t> (right panel). DAPI was used to identify their nuclei in both panels; scale bars represent 20 μm ( C ). hAMSC cells from 4 th passage were trypsinized and stained with antibodies against the indicated cell surface antigens and analyzed by flow cytometry. As shown, cells were positive to ( > 90%) CD105, CD73, CD90, CD44 and CD29; in contrast, they were negative to the expression of CD34 and CD45 hematopoietic-cells markers, inner numbers represent the mean ± SD ( D ). These are representative images from three different independent assays.
    Anti Ssea 4, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson anti ssea4
    Characterization of current good manufacturing practice-compliant human-induced pluripotent stem cell (hiPSC) lines (LiPSC 18R and LiPSC ER2.2). (A) LiPSC 18R positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, <t>SSEA4,</t> and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (B) LiPSC 18R were induced to spontaneously differentiate into three germ layers through embryoid bodies (EB) formation. Differentiated cells expressed the markers for endoderm [Alpha-Feto Protein (AFP)], mesoderm [Smooth Muscle Actin (SMA)], and early ectoderm (beta-III-Tubulin or TUJ1). (C) Flow-cytometry analysis showed that the LiPSC 18R expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). Light blue exhibits the isotype control. (D) LiPSC 18R demonstrated normal karyotype after eight passages in culture as shown by G-banding analysis. (E) LiPSC ER2.2 positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (F) LiPSC ER2.2 were induced to spontaneously differentiate into three germ layers through EB formation. Differentiated cells expressed the markers for endoderm (AFP), mesoderm (SMA), and early ectoderm (beta-III-Tubulin or TUJ1). (G) Flow-cytometry analysis showed that the LiPSC ER2.2 expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). (H) LiPSC 18R demonstrated normal karyotype after 15 passages in culture as shown by G-banding analysis.
    Anti Ssea4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend anti ssea4
    Overview of experimental Design. Primary mesenchymal cell populations were isolated from control (N = 3) and IPF (N = 3) human lung tissue and used between passages 2 and 6. FACS was used to isolate a single cell suspension of <t>SSEA4</t> hi cells (hereafter referred to as MPCs). Cells were subjected to live/dead assessment, microscopic evaluation to ensure single cells in each chamber and sequenced on the Fluidigm C1 platform according to the manufacturer’s protocol.
    Anti Ssea4, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti ssea4
    Expression of pluripotent markers and CDX2 in CDX2-KD and control bESCs. OCT4 ( A , A′ – C , C′ ), SOX2 ( D , D′ – F , F′ ), NANOG ( G , G′ – I , I′ ), E-CAD ( J , J′ – L , L′ ), SSEA1 ( M , M′ – O , O′ ), <t>SSEA4</t> ( P , P′ – R , R′ ) and CDX2 ( S , S′ – U , U′ ). GFP expression in CDX2-KD bESCs was shown in inserted picture. Nuclear was stained by DAPI. Bar = 100 μm.
    Anti Ssea4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore anti ssea4
    Reprogramming fibroblast cells in fully defined condition (a) Schematic of procedure to derive integration-free iPS cells from fibroblast cells in fully defined conditions. ( b ) The micrographs show a typical iPS colony 25 days after reprogramming (left, scale bar = 200 µm.) and prior to picking, and after first passage (right, scale bar = 25 µm.). This particular iPS clone was maintained in E8 (NODAL) on Matrigel. ( c ) FACS analysis of OCT4 and <t>SSEA4</t> of a typical iPS cell line derived from foreskin fibroblasts and maintained in E8 for 20 passages. Gree n peak: OCT4 staining; unshaded peak: mouse IgG control. ( d – g ) Human foreskin fibroblasts were reprogrammed in the indicated media (see Methods for details). The plots show reprogramming efficiency scored after 30 days. In (d), sodium butyrate (100 µM) was added to both conditions to improve efficiency. (In (d, f, g), *p
    Anti Ssea4, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    R&D Systems ssea4
    Fluorescence-activated cell sorter analysis of hAFSCs. Representative fluorescence-activated cell sorter analyses for the expression of surface markers CD44, CD45, CD73, CD90, CD 105, CD166, and <t>SSEA4</t> and the intracellular transcription factor Oct4. The gray line indicates the isotypic controls. All analyzed hAFSCs ( n = 12) showed similar results in passages 2 to 5. PE, phycoerythrin; FITC, fluorescein isothiocyanate.
    Ssea4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank ssea4
    Images of orangutan iPSCs and derivatives. Data pertaining to KB10973 iPSC colony 1 ( Panels a – k ) and KB10460 iPSC colony 1 ( panels l – v ) are provided, as described in “ Methods ” Section. Light microscope images of iPSCs are shown in panels ( a , l ). Alkaline phosphatase staining is depicted in panels ( b , m ). Images of iPSCs immunostained for TRA-1-60 ( panels c , n ), TRA-1-81 ( panels d , o ), <t>SSEA4</t> ( panels e , p ), OCT4 ( panels f , q ), SOX2 ( panels g , r ), and NANOG ( panels h , s ) are shown. DAPI nuclear counterstaining is not shown for the purpose of image clarity. Panels t – v provide the results of in vitro differentiation assays conducted on KB10973 iPSC colony 1 ( panels i – k ) and KB10460 iPSC colony 1. Cell populations derived from each of the three germ layers were detected by immunostaining for AFP (endoderm; panels i , t ), SMA (mesoderm; panels j , u ), and beta-III-tubulin (ectoderm; panels k , v ). Nuclei were counterstained with DAPI ( blue ) in panels t – v
    Ssea4, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ssea4
    Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, OCT4, SOX2, and <t>SSEA4</t> expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and
    Ssea4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stemgent ssea4
    Verification of genes identified in transcriptome analysis in normal- and patient-derived iPSCs and neural rosettes. (A) Morphology of the expanded human iPSCs (Scale bar, 100 μm). (B) Alkaline phosphatase staining of iPSCs (Scale bar, 100 μm). (C) The expression of OCT4 and <t>SSEA4</t> , which are human ESC-specific markers, was detected by immunocytochemistry. DAPI signals indicate the total cell presence in the image (scale bars, 100 μm). (D) The expression of OCT4 , SOX2 and NANOG , which are human ESC-specific markers, was detected by RT-PCR (lane1, ALS patient-derived iPSCs; lane 2, normal-derived iPSCs; and lane 3, human fibroblast as a negative control). (E) The expression of NESTIN , PAX6 , and FOXG1 which are the specific markers for the human neural rosette, was detected by RT-PCR (lane 1, ALS patient-derived neural rosettes; lane 2, normal-derived neural rosettes; and lane 3, human fibroblast as a negative control). (F) Quantitative comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in iPSCs. (G) Quantitative comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in neural rosettes. NR, neural rosette, * P
    Ssea4, supplied by Stemgent, used in various techniques. Bioz Stars score: 92/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA anti ssea4
    Flow cytometry analysis. ( A ) Expression of exosome markers (CD9 and CD63) and hiPSC markers [rBC2LCN, TRA-1-60, <t>SSEA4,</t> R-10G, podocalyxin (Pod)] on EVs and cells. ( B ) Correlation between marker expression of cells (y-axis) and EVs (x-axis).
    Anti Ssea4, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank anti ssea4
    Generation of CMT2F patient and dHMN2B patient-derived iPSCs. (a) Experimental timeline for iPSC generation. KOSR, KnockOut™ serum replacement; bFGF, basic fibroblast growth factor. (b) Morphology of fibroblasts from normal individual and patients (original magnification, 50x). Scale bars: 200 μ m. (c) iPSC colonies showed ESC-like morphology, such as a flat cobblestone-like appearance with individual cells having a high nucleus-to-cytoplasm ratio (original magnification, 50x). Scale bars: 200 μ m. (d) CMT2F-iPSCs and dHMN2B-iPSCs had preserved point mutation sites in the HSPB1 gene, verified by sequencing of RT-PCR products. (e) CMT2F-iPSCs and dHMN2B-iPSCs maintained normal karyotype. (f) Expression of total and endogenous Klf4, Oct3/4, Sox2 , and c-Myc in CMT2F-iPSCs and dHMN2B-iPSCs was verified by RT-PCR. Two clones from each of the patients-derived iPSCs were tested (clone 1 and clone 2). (g) ESCs and iPSCs expressed stem cell markers such as NANOG (in the nucleus; original magnification, 200x) and <t>SSEA4</t> (in the cytoplasm; original magnification, 100x). Scale bars: 200 μ m. (h) EB-mediated in vitro spontaneous differentiation of ESCs and iPSCs resulted in the expression of three-germ-layer markers such as AFP (endoderm), SMA (mesoderm), and nestin (ectoderm; original magnification, 200x). Scale bars: 100 μ m. (i) ESCs and iPSCs showed in vivo pluripotency by forming teratomas 8 weeks after subcutaneous injection into NOD/SCID mice. Teratomas consisted of various three-germ-layer tissues including columnar gland epithelial cells with secretions [Gl] for endodermal tissue, muscle [M] and cartilage with calcification [Ca] for mesodermal tissue, and neuroectodermal tissue [NE] and nerve axonal bundle [N] for ectodermal tissue (original magnification, 400x). Scale bars: 100 μ m.
    Anti Ssea4, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 91/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cells obtained from human amniotic membrane mesoderm displayed mesenchymal stromal cells characteristics. Phase-contrast micrograph of hAMSC adhered to a polystyrene cell culture plate at 3 rd passage showing fibroblast morphology; the photograph was taken at 40x of magnification, scale bar 100 μm ( A ). The cells were cultured for ten days and stained with crystal violet, and a direct light micrograph was performed in order to identify the UFC (arrows); the photograph was magnified at 35x in a stereoscopic microscope, scale bar 100 μm ( B ). Fluorescence micrographs of hAMSC stained with pluripotent embryonic markers OCT-4 (left panel), and SSEA-4 (right panel). DAPI was used to identify their nuclei in both panels; scale bars represent 20 μm ( C ). hAMSC cells from 4 th passage were trypsinized and stained with antibodies against the indicated cell surface antigens and analyzed by flow cytometry. As shown, cells were positive to ( > 90%) CD105, CD73, CD90, CD44 and CD29; in contrast, they were negative to the expression of CD34 and CD45 hematopoietic-cells markers, inner numbers represent the mean ± SD ( D ). These are representative images from three different independent assays.

    Journal: Scientific Reports

    Article Title: Human Amniotic Membrane Mesenchymal Stem Cells inhibit Neutrophil Extracellular Traps through TSG-6

    doi: 10.1038/s41598-017-10962-2

    Figure Lengend Snippet: Cells obtained from human amniotic membrane mesoderm displayed mesenchymal stromal cells characteristics. Phase-contrast micrograph of hAMSC adhered to a polystyrene cell culture plate at 3 rd passage showing fibroblast morphology; the photograph was taken at 40x of magnification, scale bar 100 μm ( A ). The cells were cultured for ten days and stained with crystal violet, and a direct light micrograph was performed in order to identify the UFC (arrows); the photograph was magnified at 35x in a stereoscopic microscope, scale bar 100 μm ( B ). Fluorescence micrographs of hAMSC stained with pluripotent embryonic markers OCT-4 (left panel), and SSEA-4 (right panel). DAPI was used to identify their nuclei in both panels; scale bars represent 20 μm ( C ). hAMSC cells from 4 th passage were trypsinized and stained with antibodies against the indicated cell surface antigens and analyzed by flow cytometry. As shown, cells were positive to ( > 90%) CD105, CD73, CD90, CD44 and CD29; in contrast, they were negative to the expression of CD34 and CD45 hematopoietic-cells markers, inner numbers represent the mean ± SD ( D ). These are representative images from three different independent assays.

    Article Snippet: Purified antibodies: anti-elastase, anti-albumin, anti-collagen II, anti-TSG-6, anti-SSEA-4 and anti-Oct-4 were purchased from Abcam (Cambridge, England).

    Techniques: Cell Culture, Staining, Microscopy, Fluorescence, Flow Cytometry, Cytometry, Expressing

    In vivo reprogramming reactive glia to embryonic stem-like cells. Tracing the fates of retrovirus infected cells in the injured cortex 2 weeks and 4 weeks after traumatic brain injury (TBI) with immunostaining. ( a ) Cells expressing EGFP (green) around the injury area 2 weeks after TBI. ( b ) Cell clusters (white arrows) expressing 4 human transcriptional factors (hOCT4, hKLF4, hSOX2 and hcMYC, hOKSM) and EGFP (green) around the injury area 2 weeks after TBI. ( c ) Cells expressing hOKSM-EGFP (green) also colabeled with Nanog (red). c1–4. Images of single focal section images to show colabeling of hOKSM/EGFP (green) with Nanog (red) in the cells within the white box from panel ( c) . ( d–g) hOKSM/EGFP (green) expressing cells form cluster and expressed SSEA4 4 weeks after TBI. ( h (x–z)). Images of three-dimensional reconstruction to confirm that a hOKSM/EGFP expressing cell in the white box of panel ( g ) also expressed SSEA4.

    Journal: Scientific Reports

    Article Title: In vivo reprogramming reactive glia into iPSCs to produce new neurons in the cortex following traumatic brain injury

    doi: 10.1038/srep22490

    Figure Lengend Snippet: In vivo reprogramming reactive glia to embryonic stem-like cells. Tracing the fates of retrovirus infected cells in the injured cortex 2 weeks and 4 weeks after traumatic brain injury (TBI) with immunostaining. ( a ) Cells expressing EGFP (green) around the injury area 2 weeks after TBI. ( b ) Cell clusters (white arrows) expressing 4 human transcriptional factors (hOCT4, hKLF4, hSOX2 and hcMYC, hOKSM) and EGFP (green) around the injury area 2 weeks after TBI. ( c ) Cells expressing hOKSM-EGFP (green) also colabeled with Nanog (red). c1–4. Images of single focal section images to show colabeling of hOKSM/EGFP (green) with Nanog (red) in the cells within the white box from panel ( c) . ( d–g) hOKSM/EGFP (green) expressing cells form cluster and expressed SSEA4 4 weeks after TBI. ( h (x–z)). Images of three-dimensional reconstruction to confirm that a hOKSM/EGFP expressing cell in the white box of panel ( g ) also expressed SSEA4.

    Article Snippet: The sections were put on the slides and mounted using Fluorescent Mount G. Primary antibodies and their final concentrations were as follows: anti-EGFP (1:1000, chicken, Abcam), anti-Nanog (1:200, rabbit, Abcam), anti-SSEA4 (1:100, mouse, Abcam), anti-nestin (1:1000; rabbit; Covance), anti-GFAP (1:1000, mouse, Sigma), anti-CD11b (1:200, rabbit, Millipore), anti-IbaI (1:200, goat, Abcam), anti-NG2 (1:200, rabbit, Millipore), anti-Dcx (1:1000, Guniea pig, Millipore), anti-NeuN (1:1000, mouse, Millipore), anti-Brachyury (1:100, rabbit, Abcam), anti-Gata4 (1:100, rabbit, Abcam).

    Techniques: In Vivo, Infection, Immunostaining, Expressing

    The expression of stem cell markers SSEA-4, Nanog and nucleostemin. a , d , g MMSCs expressed a high level of stem cell markers, SSEA-4, Nanog and nucleostemin, respectively. Insets show enlarged views of positive staining with three stem cell markers.

    Journal: Cytotechnology

    Article Title: A study to identify and characterize the stem/progenitor cell in rabbit meniscus

    doi: 10.1007/s10616-016-9949-2

    Figure Lengend Snippet: The expression of stem cell markers SSEA-4, Nanog and nucleostemin. a , d , g MMSCs expressed a high level of stem cell markers, SSEA-4, Nanog and nucleostemin, respectively. Insets show enlarged views of positive staining with three stem cell markers.

    Article Snippet: The stem cell markers were tested at 4 °C overnight incubation using mouse anti-SSEA-4 (1:1000; Abcam, Cat. #ab16287, Cambridge, MA, USA); mouse anti-CD105 (1:1000, Thermo Scientific, Cat. #MA5-17041, Rockford, IL, USA); mouse anti-CD90 (1:1000; ab92574, Cambridge, MA, USA); and mouse anti-CD73 (1:1000; Santa Cruz Biotechnology, Cat. #sc-32299, Dallas, TX, USA) antibodies, respectively.

    Techniques: Expressing, Staining

    Immunofluorescent staining of Nucleostemin (A, green), CD44 (B, green), SSEA4 (C, red) and CD90 (D, green) in P3 TSCs. All scale bars = 25 μm.

    Journal: PLoS ONE

    Article Title: Dual-modal photoacoustic and magnetic resonance tracking of tendon stem cells with PLGA/iron oxide microparticles in vitro

    doi: 10.1371/journal.pone.0193362

    Figure Lengend Snippet: Immunofluorescent staining of Nucleostemin (A, green), CD44 (B, green), SSEA4 (C, red) and CD90 (D, green) in P3 TSCs. All scale bars = 25 μm.

    Article Snippet: The cells were then reacted with primary anti-SSEA4 antibody (1:100; Abcam, ab16287), anti-Nucleostemin antibody (1:100; Abcam, ab70346) anti-CD44 antibody (1:50; Abcam, ab194987) and anti-CD90 antibody (1:100; Abcam, ab92574) for overnight at 4°C.

    Techniques: Staining

    Cells on VN+HSA+UV beads with neutral or positive surface charge cultured in stirred suspension are shown (A) at different culture times (FDA staining; bars: 200 μm) along (B) with their concentration (solid lines) and viability (dashed lines). (C) Concentration (solid line) and viability (dashed line) of hESCs cultured on VN+HSA+UV beads with neutral charge in stirred-suspension with E8 medium supplemented with pluronic F68. (D) Their expression of OCT4 and SSEA4 is also shown. Red curves: E8 supplemented with 0.02% F68, blue curves: E8 (no F68), Black curve: isotype control.

    Journal: ACS biomaterials science & engineering

    Article Title: Engineering Xeno-Free Microcarriers with Recombinant Vitronectin, Albumin and UV Irradiation for Human Pluripotent Stem Cell Bioprocessing

    doi: 10.1021/acsbiomaterials.6b00253

    Figure Lengend Snippet: Cells on VN+HSA+UV beads with neutral or positive surface charge cultured in stirred suspension are shown (A) at different culture times (FDA staining; bars: 200 μm) along (B) with their concentration (solid lines) and viability (dashed lines). (C) Concentration (solid line) and viability (dashed line) of hESCs cultured on VN+HSA+UV beads with neutral charge in stirred-suspension with E8 medium supplemented with pluronic F68. (D) Their expression of OCT4 and SSEA4 is also shown. Red curves: E8 supplemented with 0.02% F68, blue curves: E8 (no F68), Black curve: isotype control.

    Article Snippet: Cells were fixed with 4% paraformaldehyde (Sigma) in PBS, permeabilized/blocked in PBS with 0.1% Triton X-100 (Mallinckrodt Baker, Phillipsburg, NJ) and 1% bovine serum albumin (BSA; Sigma) for 30 min and incubated overnight at 4 °C with primary antibodies: Mouse anti-SSEA4 (AbCam, Cambridge, MA) and rabbit anti-OCT4 (SantaCruz Biotechnology Inc., Santa Cruz, CA), goat anti-SOX17 (R & D Systems, Minneapolis, MN), rabbit anti-FOXA2 (Cell Signaling Technology, Danvers, MA), rabbit anti-MOX1 (Novus Biologicals, Littleton, CO), mouse anti-KDR (PE-conjugated, R & D Systems), rabbit anti-β-Tubulin III (TUJ1, Sigma, St. Louis, MO), mouse anti-Nestin (R & D Systems).

    Techniques: Cell Culture, Staining, Concentration Assay, Expressing

    Human ESCs (H9) expanded on VN+HSA+UV microcarriers in stirred suspension for multiple successive passages. (A) Cell growth (solid line), viability (dashed line) and (B) LDH activity profiles are shown. (C) Expression of pluripotency genes from the first (P1) to the last passage (P5) determined by qPCR. (D) Flow cytometric analysis of the fractions of OCT4 + and SSEA4 + hESCs. (E) Results of the karyotypic analysis of hESCs after 5 passages in microcarrier cultures.

    Journal: ACS biomaterials science & engineering

    Article Title: Engineering Xeno-Free Microcarriers with Recombinant Vitronectin, Albumin and UV Irradiation for Human Pluripotent Stem Cell Bioprocessing

    doi: 10.1021/acsbiomaterials.6b00253

    Figure Lengend Snippet: Human ESCs (H9) expanded on VN+HSA+UV microcarriers in stirred suspension for multiple successive passages. (A) Cell growth (solid line), viability (dashed line) and (B) LDH activity profiles are shown. (C) Expression of pluripotency genes from the first (P1) to the last passage (P5) determined by qPCR. (D) Flow cytometric analysis of the fractions of OCT4 + and SSEA4 + hESCs. (E) Results of the karyotypic analysis of hESCs after 5 passages in microcarrier cultures.

    Article Snippet: Cells were fixed with 4% paraformaldehyde (Sigma) in PBS, permeabilized/blocked in PBS with 0.1% Triton X-100 (Mallinckrodt Baker, Phillipsburg, NJ) and 1% bovine serum albumin (BSA; Sigma) for 30 min and incubated overnight at 4 °C with primary antibodies: Mouse anti-SSEA4 (AbCam, Cambridge, MA) and rabbit anti-OCT4 (SantaCruz Biotechnology Inc., Santa Cruz, CA), goat anti-SOX17 (R & D Systems, Minneapolis, MN), rabbit anti-FOXA2 (Cell Signaling Technology, Danvers, MA), rabbit anti-MOX1 (Novus Biologicals, Littleton, CO), mouse anti-KDR (PE-conjugated, R & D Systems), rabbit anti-β-Tubulin III (TUJ1, Sigma, St. Louis, MO), mouse anti-Nestin (R & D Systems).

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

    Cultispher-S microcarriers support expansion of undifferentiated SHEF3 hESCs. 3 × 10 6 SHEF3 hESCs were seeded onto Cultispher-S microcarriers in KO-DMEM supplemented with 20% (v/v) KO-SR and 4 ng/mL FGF-2 in the presence (3D CM) or absence (3D Non-CM) of MEF conditioned medium (CM). Cells were cultured for 7 days, passaged and then cultured for a further 7 days. A: (i) Cell numbers plated and recovered at each passage for each condition are presented. (ii) The mean number of viable cells attached to microcarriers (±SD) are shown for each condition for the culture periods d0–d7 (upper panel) and d7–d14 (lower panel). B: Assessment of markers of pluripotency. (i) Expression of SSEA4, Tra-1–60 and Tra-1–85, were determined by flow cytometry. 2D indicates hESCs controls maintained in 2D culture on MEF plus FGF-2. RNA and protein were extracted from hESCs cultured in 2D or 3D conditions for the times indicated and expression of OCT-4 and NANOG determined by (ii) semi-quantitative RT-PCR; (iii) quantitative RT-PCR normalized to β-actin (±SD) or (iv) immunoblotting, normalized to SHP-2.

    Journal: Biotechnology and Bioengineering

    Article Title: Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells

    doi: 10.1002/bit.22850

    Figure Lengend Snippet: Cultispher-S microcarriers support expansion of undifferentiated SHEF3 hESCs. 3 × 10 6 SHEF3 hESCs were seeded onto Cultispher-S microcarriers in KO-DMEM supplemented with 20% (v/v) KO-SR and 4 ng/mL FGF-2 in the presence (3D CM) or absence (3D Non-CM) of MEF conditioned medium (CM). Cells were cultured for 7 days, passaged and then cultured for a further 7 days. A: (i) Cell numbers plated and recovered at each passage for each condition are presented. (ii) The mean number of viable cells attached to microcarriers (±SD) are shown for each condition for the culture periods d0–d7 (upper panel) and d7–d14 (lower panel). B: Assessment of markers of pluripotency. (i) Expression of SSEA4, Tra-1–60 and Tra-1–85, were determined by flow cytometry. 2D indicates hESCs controls maintained in 2D culture on MEF plus FGF-2. RNA and protein were extracted from hESCs cultured in 2D or 3D conditions for the times indicated and expression of OCT-4 and NANOG determined by (ii) semi-quantitative RT-PCR; (iii) quantitative RT-PCR normalized to β-actin (±SD) or (iv) immunoblotting, normalized to SHP-2.

    Article Snippet: Flow Cytometry and Immunofluorescence For flow cytometry, TrypLE™ (Invitrogen) dissociated hESC were washed, resuspended in wash buffer (PBS containing 5% (v/v) FCS, 0.1% (w/v) sodium azide) and stained, on ice, with the following antibodies: 1:100 SSEA4 (Abcam, ab16287); 1:100 Tra-1–60 (ab16288; Abcam), or 5:100 phycoerythrin (PE)-conjugated Tra-1–85 (FAB3195P; R & D systems, Abingdon, UK).

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

    Characterization of current good manufacturing practice-compliant human-induced pluripotent stem cell (hiPSC) lines (LiPSC 18R and LiPSC ER2.2). (A) LiPSC 18R positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (B) LiPSC 18R were induced to spontaneously differentiate into three germ layers through embryoid bodies (EB) formation. Differentiated cells expressed the markers for endoderm [Alpha-Feto Protein (AFP)], mesoderm [Smooth Muscle Actin (SMA)], and early ectoderm (beta-III-Tubulin or TUJ1). (C) Flow-cytometry analysis showed that the LiPSC 18R expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). Light blue exhibits the isotype control. (D) LiPSC 18R demonstrated normal karyotype after eight passages in culture as shown by G-banding analysis. (E) LiPSC ER2.2 positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (F) LiPSC ER2.2 were induced to spontaneously differentiate into three germ layers through EB formation. Differentiated cells expressed the markers for endoderm (AFP), mesoderm (SMA), and early ectoderm (beta-III-Tubulin or TUJ1). (G) Flow-cytometry analysis showed that the LiPSC ER2.2 expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). (H) LiPSC 18R demonstrated normal karyotype after 15 passages in culture as shown by G-banding analysis.

    Journal: Frontiers in Medicine

    Article Title: Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers

    doi: 10.3389/fmed.2018.00069

    Figure Lengend Snippet: Characterization of current good manufacturing practice-compliant human-induced pluripotent stem cell (hiPSC) lines (LiPSC 18R and LiPSC ER2.2). (A) LiPSC 18R positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (B) LiPSC 18R were induced to spontaneously differentiate into three germ layers through embryoid bodies (EB) formation. Differentiated cells expressed the markers for endoderm [Alpha-Feto Protein (AFP)], mesoderm [Smooth Muscle Actin (SMA)], and early ectoderm (beta-III-Tubulin or TUJ1). (C) Flow-cytometry analysis showed that the LiPSC 18R expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). Light blue exhibits the isotype control. (D) LiPSC 18R demonstrated normal karyotype after eight passages in culture as shown by G-banding analysis. (E) LiPSC ER2.2 positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (F) LiPSC ER2.2 were induced to spontaneously differentiate into three germ layers through EB formation. Differentiated cells expressed the markers for endoderm (AFP), mesoderm (SMA), and early ectoderm (beta-III-Tubulin or TUJ1). (G) Flow-cytometry analysis showed that the LiPSC ER2.2 expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). (H) LiPSC 18R demonstrated normal karyotype after 15 passages in culture as shown by G-banding analysis.

    Article Snippet: Extracellular antigens were detected on unfixed cells stained with PE-conjugated antigen-specific antibodies and respective isotypes using the manufacturer’s recommended concentration: anti-TRA-1-81 (Becton Dickinson, 560161), anti-TRA-1-60 (Becton Dickinson, 560193), anti-IgG3 isotype (Becton Dickinson, 556659), anti-SSEA4 (Becton Dickinson, 560128), and anti-IgM isotype (Becton Dickinson, 555584).

    Techniques: Staining, Flow Cytometry, Cytometry

    The expression of stemness genes and surface markers in primary endometrial cancer cells. (A) RT-PCR showed both two patients expressed stemness related genes including c-Myc , Sox-2 , Nanog , Oct4A , ABCG2 , BMI-1 , CK-18 , Nestin and β - actin , β - actin is the negative control. (B-1 and B-2) The expression levels of CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3, and SSEA4 by flow cytometry. (C) The mRNA expression of comparison and analysis between CD24, CD133, and CXCR4 positive and negative subpopulation in the two patients by RT-PCR. (D) The double CD133+CXCR4+ cells ration is 7.2% and 9.3%, respectively.

    Journal: Translational Oncology

    Article Title: Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4

    doi: 10.1016/j.tranon.2017.07.007

    Figure Lengend Snippet: The expression of stemness genes and surface markers in primary endometrial cancer cells. (A) RT-PCR showed both two patients expressed stemness related genes including c-Myc , Sox-2 , Nanog , Oct4A , ABCG2 , BMI-1 , CK-18 , Nestin and β - actin , β - actin is the negative control. (B-1 and B-2) The expression levels of CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3, and SSEA4 by flow cytometry. (C) The mRNA expression of comparison and analysis between CD24, CD133, and CXCR4 positive and negative subpopulation in the two patients by RT-PCR. (D) The double CD133+CXCR4+ cells ration is 7.2% and 9.3%, respectively.

    Article Snippet: Cells (1 × 106 ) were stained with antibodies against CD24 (anti CD24-FITC; BD Pharmingen, Franklin Lakes, NJ, USA), CD133 (anti CD133-FITC; Miltenyi Biotec, Germany), CD47 (anti CD47-FITC; Santa Cruz Biotechnology, USA), CD29 (anti CD29-PE; BD Pharmingen), CD44 (anti CD44-PE; BD Pharmingen), CXCR4 (anti CXCR4-PE; Beckman Coulter, USA), SSEA3 (anti SSEA3-FITC; BD Pharmingen), and SSEA4 (anti SSEA4-PE; BD Pharmingen), then washed twice with PBS and analyzed using the FACS Canto II system (BD Biosciences, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Flow Cytometry, Cytometry

    Overview of experimental Design. Primary mesenchymal cell populations were isolated from control (N = 3) and IPF (N = 3) human lung tissue and used between passages 2 and 6. FACS was used to isolate a single cell suspension of SSEA4 hi cells (hereafter referred to as MPCs). Cells were subjected to live/dead assessment, microscopic evaluation to ensure single cells in each chamber and sequenced on the Fluidigm C1 platform according to the manufacturer’s protocol.

    Journal: Scientific Reports

    Article Title: Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory

    doi: 10.1038/s41598-020-66630-5

    Figure Lengend Snippet: Overview of experimental Design. Primary mesenchymal cell populations were isolated from control (N = 3) and IPF (N = 3) human lung tissue and used between passages 2 and 6. FACS was used to isolate a single cell suspension of SSEA4 hi cells (hereafter referred to as MPCs). Cells were subjected to live/dead assessment, microscopic evaluation to ensure single cells in each chamber and sequenced on the Fluidigm C1 platform according to the manufacturer’s protocol.

    Article Snippet: Immunohistochemistry/ImmunofluorescenceFixed human lung IPF tissue samples (individual or combined in TMAs) underwent serial sectioning and were stained with Hematoxylin-Eosin, or probed with the following antibodies: anti-SSEA4 (Biolegend, #330401, 1:50), anti-CD44 (Abcam, ab101531, 1:500), anti-MARCKS (Novus, NB110-58875SS, 1:500), anti-pro-collagen I (Abcam, ab64409, 1:500).

    Techniques: Isolation, FACS

    Cells expressing CD44, SSEA-4 and MARCKS reside in the cellular perimeter region of the fibroblastic focus. An Idiopathic Pulmonary Fibrosis (IPF) specimen was serially sectioned at 4 μm and processed for histology, immunohistochemistry (IHC) and immunofluorescence (IF). IHC: Representative images for Hematoxylin and Eosin (H E) staining (scale bar represents 50 μm left and 20 μm right) with an asterisk labeling a fibroblastic focus; Immunostaining for anti-procollagen type I (brown, scale bar 20 μm); anti-CD44 (red, scale bar 20 μm, dashed outline box, scale bar 20 μm); anti-SSEA4 (brown, scale bar 20 μm, dashed outline box, scale bar 10 μm). Lower panel: Immunostaining anti-SSEA-4 (green), MARCKS (red), DAPI (blue, scale bar 20 μm). A small apoptotic body is noted adjacent to the cell on the right. Immunofluorescence images obtained at the perimeter of the fibroblastic focus.

    Journal: Scientific Reports

    Article Title: Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory

    doi: 10.1038/s41598-020-66630-5

    Figure Lengend Snippet: Cells expressing CD44, SSEA-4 and MARCKS reside in the cellular perimeter region of the fibroblastic focus. An Idiopathic Pulmonary Fibrosis (IPF) specimen was serially sectioned at 4 μm and processed for histology, immunohistochemistry (IHC) and immunofluorescence (IF). IHC: Representative images for Hematoxylin and Eosin (H E) staining (scale bar represents 50 μm left and 20 μm right) with an asterisk labeling a fibroblastic focus; Immunostaining for anti-procollagen type I (brown, scale bar 20 μm); anti-CD44 (red, scale bar 20 μm, dashed outline box, scale bar 20 μm); anti-SSEA4 (brown, scale bar 20 μm, dashed outline box, scale bar 10 μm). Lower panel: Immunostaining anti-SSEA-4 (green), MARCKS (red), DAPI (blue, scale bar 20 μm). A small apoptotic body is noted adjacent to the cell on the right. Immunofluorescence images obtained at the perimeter of the fibroblastic focus.

    Article Snippet: Immunohistochemistry/ImmunofluorescenceFixed human lung IPF tissue samples (individual or combined in TMAs) underwent serial sectioning and were stained with Hematoxylin-Eosin, or probed with the following antibodies: anti-SSEA4 (Biolegend, #330401, 1:50), anti-CD44 (Abcam, ab101531, 1:500), anti-MARCKS (Novus, NB110-58875SS, 1:500), anti-pro-collagen I (Abcam, ab64409, 1:500).

    Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Labeling, Immunostaining

    Expression of pluripotent markers and CDX2 in CDX2-KD and control bESCs. OCT4 ( A , A′ – C , C′ ), SOX2 ( D , D′ – F , F′ ), NANOG ( G , G′ – I , I′ ), E-CAD ( J , J′ – L , L′ ), SSEA1 ( M , M′ – O , O′ ), SSEA4 ( P , P′ – R , R′ ) and CDX2 ( S , S′ – U , U′ ). GFP expression in CDX2-KD bESCs was shown in inserted picture. Nuclear was stained by DAPI. Bar = 100 μm.

    Journal: Scientific Reports

    Article Title: Establishment of bovine embryonic stem cells after knockdown of CDX2

    doi: 10.1038/srep28343

    Figure Lengend Snippet: Expression of pluripotent markers and CDX2 in CDX2-KD and control bESCs. OCT4 ( A , A′ – C , C′ ), SOX2 ( D , D′ – F , F′ ), NANOG ( G , G′ – I , I′ ), E-CAD ( J , J′ – L , L′ ), SSEA1 ( M , M′ – O , O′ ), SSEA4 ( P , P′ – R , R′ ) and CDX2 ( S , S′ – U , U′ ). GFP expression in CDX2-KD bESCs was shown in inserted picture. Nuclear was stained by DAPI. Bar = 100 μm.

    Article Snippet: Cells were incubated in primary antibody overnight at 4 °C and secondary antibody at 37 °C for 1 h. Primary antibodies were used that were anti-OCT-3/4 (Santa Cruz), anti-NANOG (Abcam), anti-SOX2 (Cell signaling), anti-SSEA1 (Santa Cruz), anti-SSEA4 (Santa Cruz), anti-E-CADHERIN (BD Bioscience), anti-KLF4 (Stemgent), anti-CDX2 (Biogenex).

    Techniques: Expressing, Staining

    Maintenance and characterization of hNPC PSA-NCAM+ . (A–B) Neural markers (Sox1, Sox2, Nestin, and Musashi1) were extensively and consistently expressed throughout multiple passages. (C) RT-PCR analysis shows that the expression of Sox1 and Pax6 were sustained from the neural rosette stage to hNPC PSA-NCAM+ ; furthermore, hTERT was continuously expressed throughout the maintenance culture. (D–E) During multiple passages, about 70% of cultured cells were positive for Ki67, supporting their self-renewing potential. (F) At passage 9, SSEA4-positive cells were rarely detected. (G) hNPC PSA-NCAM+ exhibited a normal karyotype at passage 20. (H–J) Along with Zo-1 expression in the lumen-side of the rosette structure (H), co-expression of PLZF with Sox1 (I) as well as prominent expression of “neural rosette-specific” genes (J) indicate that hNPC PSA-NCAM+ exhibited the characteristics of typical neural rosette cells. (K–L) Although NPs did not express either PMP2 or HOP , they did express AQP4 and S100β to some extent, which were proposed to be specifically expressed in FGF/EGF-expanded neural cells (NPC FGF/EGF ). Scale bars: 50 µm.

    Journal: PLoS ONE

    Article Title: Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes

    doi: 10.1371/journal.pone.0039715

    Figure Lengend Snippet: Maintenance and characterization of hNPC PSA-NCAM+ . (A–B) Neural markers (Sox1, Sox2, Nestin, and Musashi1) were extensively and consistently expressed throughout multiple passages. (C) RT-PCR analysis shows that the expression of Sox1 and Pax6 were sustained from the neural rosette stage to hNPC PSA-NCAM+ ; furthermore, hTERT was continuously expressed throughout the maintenance culture. (D–E) During multiple passages, about 70% of cultured cells were positive for Ki67, supporting their self-renewing potential. (F) At passage 9, SSEA4-positive cells were rarely detected. (G) hNPC PSA-NCAM+ exhibited a normal karyotype at passage 20. (H–J) Along with Zo-1 expression in the lumen-side of the rosette structure (H), co-expression of PLZF with Sox1 (I) as well as prominent expression of “neural rosette-specific” genes (J) indicate that hNPC PSA-NCAM+ exhibited the characteristics of typical neural rosette cells. (K–L) Although NPs did not express either PMP2 or HOP , they did express AQP4 and S100β to some extent, which were proposed to be specifically expressed in FGF/EGF-expanded neural cells (NPC FGF/EGF ). Scale bars: 50 µm.

    Article Snippet: Primary antibodies used in our study were as follows: SSEA4 (1∶500, Santa Cruz Biotechnology), Oct4 (1∶200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Sox1 (1∶200, Millipore), Pax6 (1∶200, DSHB, Iowa, IA, USA), Nestin (1∶1000, Millipore), Sox2 (1∶200, Millipore), PSA-NCAM (1∶300, Millipore), AP2 (1∶100, DSHB), HNK1 (1∶500, Sigma), Tuj1 (1∶1000, Covance, Berkeley, CA, USA), NeuN (1∶200, Millipore), GFAP (1∶300, Millipore), O4 (1∶200, R & D Systems), TH (1∶300, Pel-freez Biologicals, Rogers, AR, USA; or1∶500, Millipore), Nurr1 (1∶300, Millipore), Pitx3 (1∶400, Millipore), HB9 (1∶200, Millipore), HoxB4 (1∶70, DSHB), Ki67 (1∶300, Vision Biosystems, Newcastle, UK), Zo-1 (1∶100, BD Biosciences), PLZF (1∶50, Calbiochem, San Diego, CA., USA), S100β (1∶1000, BD Biosciences).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

    Reprogramming fibroblast cells in fully defined condition (a) Schematic of procedure to derive integration-free iPS cells from fibroblast cells in fully defined conditions. ( b ) The micrographs show a typical iPS colony 25 days after reprogramming (left, scale bar = 200 µm.) and prior to picking, and after first passage (right, scale bar = 25 µm.). This particular iPS clone was maintained in E8 (NODAL) on Matrigel. ( c ) FACS analysis of OCT4 and SSEA4 of a typical iPS cell line derived from foreskin fibroblasts and maintained in E8 for 20 passages. Gree n peak: OCT4 staining; unshaded peak: mouse IgG control. ( d – g ) Human foreskin fibroblasts were reprogrammed in the indicated media (see Methods for details). The plots show reprogramming efficiency scored after 30 days. In (d), sodium butyrate (100 µM) was added to both conditions to improve efficiency. (In (d, f, g), *p

    Journal: Nature methods

    Article Title: Chemically defined conditions for human iPS cell derivation and culture

    doi: 10.1038/nmeth.1593

    Figure Lengend Snippet: Reprogramming fibroblast cells in fully defined condition (a) Schematic of procedure to derive integration-free iPS cells from fibroblast cells in fully defined conditions. ( b ) The micrographs show a typical iPS colony 25 days after reprogramming (left, scale bar = 200 µm.) and prior to picking, and after first passage (right, scale bar = 25 µm.). This particular iPS clone was maintained in E8 (NODAL) on Matrigel. ( c ) FACS analysis of OCT4 and SSEA4 of a typical iPS cell line derived from foreskin fibroblasts and maintained in E8 for 20 passages. Gree n peak: OCT4 staining; unshaded peak: mouse IgG control. ( d – g ) Human foreskin fibroblasts were reprogrammed in the indicated media (see Methods for details). The plots show reprogramming efficiency scored after 30 days. In (d), sodium butyrate (100 µM) was added to both conditions to improve efficiency. (In (d, f, g), *p

    Article Snippet: Antibodies: anti-OCT4 (Santa Cruz), anti-SSEA4 (Millipore) and Alexa-488 anti-mouse IgG (Invitrogen).

    Techniques: FACS, Derivative Assay, Staining

    Immunocytochemical analyses of hESCs cryopreserved in SCK or DAP for the stem cell markers octamer-binding transcription factor 3/4 (Oct3/4), stage-specific embryonic antigen-4 (SSEA4), and Tra-1-60. Images of hESCs in bright-field, immunofluorescence, and 4′,6-diamidino-2-phenylindole (DAPI) staining were obtained on a microscope at a magnification of 40x. Scale bars: 100 um. hESCs were fixed with 4% PFA, permeabilized, and reacted with primary antibodies against Oct3/4, SSEA4, or Tra-1-60 at 4° C overnight, and further reacted with fluorescently tagged secondary antibodies in the dark. Mounting medium containing DAPI was added, and cells were observed on a fluorescence microscope. (a-c) hESC/SCK/Oct3/4. (d-f) hESC/DAP/Oct3/4. (g-i) hESC/SCK/ SSEA4. (j-l) hESC/DAP/SSEA4. (m-o) hESC/SCK/Tra-1-60. (p-r) hESC/DAP/Tra-1-60.

    Journal: Cell Transplantation

    Article Title: StemCell Keep™ is Effective for Cryopreservation of Human Embryonic Stem Cells by Vitrification

    doi: 10.3727/096368916X692654

    Figure Lengend Snippet: Immunocytochemical analyses of hESCs cryopreserved in SCK or DAP for the stem cell markers octamer-binding transcription factor 3/4 (Oct3/4), stage-specific embryonic antigen-4 (SSEA4), and Tra-1-60. Images of hESCs in bright-field, immunofluorescence, and 4′,6-diamidino-2-phenylindole (DAPI) staining were obtained on a microscope at a magnification of 40x. Scale bars: 100 um. hESCs were fixed with 4% PFA, permeabilized, and reacted with primary antibodies against Oct3/4, SSEA4, or Tra-1-60 at 4° C overnight, and further reacted with fluorescently tagged secondary antibodies in the dark. Mounting medium containing DAPI was added, and cells were observed on a fluorescence microscope. (a-c) hESC/SCK/Oct3/4. (d-f) hESC/DAP/Oct3/4. (g-i) hESC/SCK/ SSEA4. (j-l) hESC/DAP/SSEA4. (m-o) hESC/SCK/Tra-1-60. (p-r) hESC/DAP/Tra-1-60.

    Article Snippet: The primary antibodies used were Oct3/4 (Santa Cruz Biotechnology), SSEA4 (Millipore, Billerica, MA, USA), Nanog (ReproCELL), and Tra-1-60 (Millipore).

    Techniques: Binding Assay, Immunofluorescence, Staining, Microscopy, Fluorescence

    Fluorescence-activated cell sorter analysis of hAFSCs. Representative fluorescence-activated cell sorter analyses for the expression of surface markers CD44, CD45, CD73, CD90, CD 105, CD166, and SSEA4 and the intracellular transcription factor Oct4. The gray line indicates the isotypic controls. All analyzed hAFSCs ( n = 12) showed similar results in passages 2 to 5. PE, phycoerythrin; FITC, fluorescein isothiocyanate.

    Journal: The American Journal of Pathology

    Article Title: Stem Cells Derived from Human Amniotic Fluid Contribute to Acute Kidney Injury Recovery

    doi: 10.2353/ajpath.2010.091245

    Figure Lengend Snippet: Fluorescence-activated cell sorter analysis of hAFSCs. Representative fluorescence-activated cell sorter analyses for the expression of surface markers CD44, CD45, CD73, CD90, CD 105, CD166, and SSEA4 and the intracellular transcription factor Oct4. The gray line indicates the isotypic controls. All analyzed hAFSCs ( n = 12) showed similar results in passages 2 to 5. PE, phycoerythrin; FITC, fluorescein isothiocyanate.

    Article Snippet: Fluorescein isothiocyanate- and phycoerythrin-conjugated antibodies with specificities against CD44, CD45, CD90, CD105 (AbD Serotec, Raleigh, NC), CD73, CD166 (BD Pharmingen, San Diego, CA), Oct3/4, and SSEA4 (R & D Systems, Minneapolis, MN) were used.

    Techniques: Fluorescence, Expressing

    Images of orangutan iPSCs and derivatives. Data pertaining to KB10973 iPSC colony 1 ( Panels a – k ) and KB10460 iPSC colony 1 ( panels l – v ) are provided, as described in “ Methods ” Section. Light microscope images of iPSCs are shown in panels ( a , l ). Alkaline phosphatase staining is depicted in panels ( b , m ). Images of iPSCs immunostained for TRA-1-60 ( panels c , n ), TRA-1-81 ( panels d , o ), SSEA4 ( panels e , p ), OCT4 ( panels f , q ), SOX2 ( panels g , r ), and NANOG ( panels h , s ) are shown. DAPI nuclear counterstaining is not shown for the purpose of image clarity. Panels t – v provide the results of in vitro differentiation assays conducted on KB10973 iPSC colony 1 ( panels i – k ) and KB10460 iPSC colony 1. Cell populations derived from each of the three germ layers were detected by immunostaining for AFP (endoderm; panels i , t ), SMA (mesoderm; panels j , u ), and beta-III-tubulin (ectoderm; panels k , v ). Nuclei were counterstained with DAPI ( blue ) in panels t – v

    Journal: BMC Research Notes

    Article Title: Derivation of induced pluripotent stem cells from orangutan skin fibroblasts

    doi: 10.1186/s13104-015-1567-0

    Figure Lengend Snippet: Images of orangutan iPSCs and derivatives. Data pertaining to KB10973 iPSC colony 1 ( Panels a – k ) and KB10460 iPSC colony 1 ( panels l – v ) are provided, as described in “ Methods ” Section. Light microscope images of iPSCs are shown in panels ( a , l ). Alkaline phosphatase staining is depicted in panels ( b , m ). Images of iPSCs immunostained for TRA-1-60 ( panels c , n ), TRA-1-81 ( panels d , o ), SSEA4 ( panels e , p ), OCT4 ( panels f , q ), SOX2 ( panels g , r ), and NANOG ( panels h , s ) are shown. DAPI nuclear counterstaining is not shown for the purpose of image clarity. Panels t – v provide the results of in vitro differentiation assays conducted on KB10973 iPSC colony 1 ( panels i – k ) and KB10460 iPSC colony 1. Cell populations derived from each of the three germ layers were detected by immunostaining for AFP (endoderm; panels i , t ), SMA (mesoderm; panels j , u ), and beta-III-tubulin (ectoderm; panels k , v ). Nuclei were counterstained with DAPI ( blue ) in panels t – v

    Article Snippet: The following primary antibodies were used: TRA-1-81 (mouse monoclonal against human, 1:100 dilution, EMD Millipore, Catalog #MAB4381), TRA-1-60 (mouse monoclonal against human, 1:100 dilution, EMD Millipore, Catalog #MAB4360), OCT4 (goat polyclonal against human, 1:100 dilution, R & D Systems, Catalog #AF1759), NANOG (goat polyclonal against human, 1:50 dilution, R & D systems, Catalog #1997), SOX2 (goat polyclonal against mouse, rat and human, 1:100 dilution, Santa Cruz Biotechnology, Catalog #sc17320), SSEA4 (mouse monoclonal against human, 1:100, Developmental Studies Hybridoma Bank, Catalog #MC-813-70), α-fetoprotein (AFP) (mouse monoclonal against human, 1:250 dilution, Sigma, Catalog #A8452), βIII-Tubulin (rabbit monoclonal against rat, 1:100 dilution, Covance Catalog #MRB-435P) and alpha smooth muscle actin (αSMA) (1:500 dilution, Abcam, Catalog #ab5694).

    Techniques: Light Microscopy, Staining, In Vitro, Derivative Assay, Immunostaining

    Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, OCT4, SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and

    Journal: Stem Cells Translational Medicine

    Article Title: Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells

    doi: 10.5966/sctm.2013-0179

    Figure Lengend Snippet: Characteristics of the established hiPSCs. (A): Immunofluorescence analysis of NANOG, OCT4, SOX2, and SSEA4 expression in transgene-residual (KeI6) and transgene-free (KeI6-R1) hiPSCs. (B): Quantitative RT-PCR analysis of ESC-specific genes (OCT3/4 and

    Article Snippet: The cells were incubated with antibodies against NANOG (Cell Signaling Technology, Beverly, MA, ; 1:200, rabbit polyclonal), OCT4 (Cell Signaling Technology; 1:200, rabbit polyclonal), SOX2 (Cell Signaling Technology, 1:200, rabbit polyclonal), SSEA4 (Cell Signaling Technology; 1:200, mouse IgG3 monoclonal), TRA-1-60 (Cell Signaling Technology; 1:200, mouse IgM monoclonal), TRA-1-81 (Cell Signaling Technology; 1:200, mouse IgM monoclonal), human keratin 5/14 (Covance, Princeton, NJ, ; 1:500, rabbit IgG polyclonal), and each negative control antibody (Dako) overnight at 4°C.

    Techniques: Immunofluorescence, Expressing, Quantitative RT-PCR

    Generation of hSOX10-2A-Nano-lantern reporter hiPS cells. (A) A schematic representation of targeting cassette for hSOX10 2A-NL-fNeo/+ or hSOX10 2A-NL/+ knockin allele. (B) Genotyping PCR with genome DNA extracted from hiPS 201B7 and hSOX10 2A-NL-fNeo/+ hiPS cells. Genomic recombined 5’fragment of targeting cassette was detectable by F1 and R1 primers, and also 3’ fragment by F2 and R2 primers. (C) DNA Copy number check by quantitative PCR with genome DNA extracted from hiPS 201B7, Pax3 EGFP-fNeo/+ , and hSOX10 2A-NL-fNeo/+ cells. All error bars indicate ±s.e.m. (n = 3) (D) The excision of loxP-Neo-loxP genomic fragment in hSOX10 2A-NL-fNeo/+ hiPS cells with Cre recombinase treatment to establish hSOX10 2A-NL/+ hiPS cells. (E) hSOX10 2A-NL-fNeo/+ cells were immunostained with indicated undifferentiated pluripotent cell markers, anti-OCT3/4 (red, upper panel) and anti-SSEA4 (green, upper panel), anti-NANOG (red, lower panel) and anti-TRA1-60 (green, lower panel) antibodies. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 100 μm. (F) Differentiation capacity into ectoderm ( PAX6 ), mesoderm ( T ), endoderm ( FOXA2 ) markers with embryonic bodies (EB day7) from hSOX10 2A-NL/+ cells.

    Journal: PLoS ONE

    Article Title: SOX10-Nano-Lantern Reporter Human iPS Cells; A Versatile Tool for Neural Crest Research

    doi: 10.1371/journal.pone.0170342

    Figure Lengend Snippet: Generation of hSOX10-2A-Nano-lantern reporter hiPS cells. (A) A schematic representation of targeting cassette for hSOX10 2A-NL-fNeo/+ or hSOX10 2A-NL/+ knockin allele. (B) Genotyping PCR with genome DNA extracted from hiPS 201B7 and hSOX10 2A-NL-fNeo/+ hiPS cells. Genomic recombined 5’fragment of targeting cassette was detectable by F1 and R1 primers, and also 3’ fragment by F2 and R2 primers. (C) DNA Copy number check by quantitative PCR with genome DNA extracted from hiPS 201B7, Pax3 EGFP-fNeo/+ , and hSOX10 2A-NL-fNeo/+ cells. All error bars indicate ±s.e.m. (n = 3) (D) The excision of loxP-Neo-loxP genomic fragment in hSOX10 2A-NL-fNeo/+ hiPS cells with Cre recombinase treatment to establish hSOX10 2A-NL/+ hiPS cells. (E) hSOX10 2A-NL-fNeo/+ cells were immunostained with indicated undifferentiated pluripotent cell markers, anti-OCT3/4 (red, upper panel) and anti-SSEA4 (green, upper panel), anti-NANOG (red, lower panel) and anti-TRA1-60 (green, lower panel) antibodies. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 100 μm. (F) Differentiation capacity into ectoderm ( PAX6 ), mesoderm ( T ), endoderm ( FOXA2 ) markers with embryonic bodies (EB day7) from hSOX10 2A-NL/+ cells.

    Article Snippet: Fixed samples were pre-treated with BlockingOne (Nacalai Tesque) for 30 min at RT, and incubated with anti-OCT3/4, anti-NANOG, anti-SSEA4, anti-TRA1-60 (Cell Signaling Technology; diluted 1/200), anti-GFP (Molecular Probes; diluted 1/500), anti-SOX10 (SantaCruz Biotechnology; diluted 1/100) antibodies in 5% of BlockingOne in PBST for overnight at 4°C.

    Techniques: Knock-In, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Staining

    Verification of genes identified in transcriptome analysis in normal- and patient-derived iPSCs and neural rosettes. (A) Morphology of the expanded human iPSCs (Scale bar, 100 μm). (B) Alkaline phosphatase staining of iPSCs (Scale bar, 100 μm). (C) The expression of OCT4 and SSEA4 , which are human ESC-specific markers, was detected by immunocytochemistry. DAPI signals indicate the total cell presence in the image (scale bars, 100 μm). (D) The expression of OCT4 , SOX2 and NANOG , which are human ESC-specific markers, was detected by RT-PCR (lane1, ALS patient-derived iPSCs; lane 2, normal-derived iPSCs; and lane 3, human fibroblast as a negative control). (E) The expression of NESTIN , PAX6 , and FOXG1 which are the specific markers for the human neural rosette, was detected by RT-PCR (lane 1, ALS patient-derived neural rosettes; lane 2, normal-derived neural rosettes; and lane 3, human fibroblast as a negative control). (F) Quantitative comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in iPSCs. (G) Quantitative comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in neural rosettes. NR, neural rosette, * P

    Journal: PLoS ONE

    Article Title: Elucidation of Relevant Neuroinflammation Mechanisms Using Gene Expression Profiling in Patients with Amyotrophic Lateral Sclerosis

    doi: 10.1371/journal.pone.0165290

    Figure Lengend Snippet: Verification of genes identified in transcriptome analysis in normal- and patient-derived iPSCs and neural rosettes. (A) Morphology of the expanded human iPSCs (Scale bar, 100 μm). (B) Alkaline phosphatase staining of iPSCs (Scale bar, 100 μm). (C) The expression of OCT4 and SSEA4 , which are human ESC-specific markers, was detected by immunocytochemistry. DAPI signals indicate the total cell presence in the image (scale bars, 100 μm). (D) The expression of OCT4 , SOX2 and NANOG , which are human ESC-specific markers, was detected by RT-PCR (lane1, ALS patient-derived iPSCs; lane 2, normal-derived iPSCs; and lane 3, human fibroblast as a negative control). (E) The expression of NESTIN , PAX6 , and FOXG1 which are the specific markers for the human neural rosette, was detected by RT-PCR (lane 1, ALS patient-derived neural rosettes; lane 2, normal-derived neural rosettes; and lane 3, human fibroblast as a negative control). (F) Quantitative comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in iPSCs. (G) Quantitative comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in neural rosettes. NR, neural rosette, * P

    Article Snippet: The iPSCs were incubated with primary antibodies such as OCT3/4 (Santa Cruz, Texas, USA) and SSEA4 (Stemgent, MA, USA).

    Techniques: Derivative Assay, Staining, Expressing, Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Negative Control, Quantitative RT-PCR

    Flow cytometry analysis. ( A ) Expression of exosome markers (CD9 and CD63) and hiPSC markers [rBC2LCN, TRA-1-60, SSEA4, R-10G, podocalyxin (Pod)] on EVs and cells. ( B ) Correlation between marker expression of cells (y-axis) and EVs (x-axis).

    Journal: Scientific Reports

    Article Title: Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray

    doi: 10.1038/s41598-018-22450-2

    Figure Lengend Snippet: Flow cytometry analysis. ( A ) Expression of exosome markers (CD9 and CD63) and hiPSC markers [rBC2LCN, TRA-1-60, SSEA4, R-10G, podocalyxin (Pod)] on EVs and cells. ( B ) Correlation between marker expression of cells (y-axis) and EVs (x-axis).

    Article Snippet: The beads were also incubated with a 100-fold dilution of anti-CD9 antibody (clone#: 12A12, SHI-EXO-M01, COSMO BIO Co. Ltd.), a 100-fold dilution of anti-CD63 (Clone#: 8A12, Cat#: SHI-EXO-M02, COSMO BIO Co. Ltd.), anti-podocalyxin goat pAb (Cat#: AF1658, R & D Systems, Inc., Minneapolis, MN), a 100-fold dilution of anti-SSEA4 (clone#: MC-813-70, Cat#: MAB4304, Merck KGaA), anti-TRA1-60 (clone#: TRA-1-60, Cat#: MAB4360, Merck KGaA), or R-10G (Clone#: R-10G, Cat#: 011-25811, Wako Pure Chemical Industries, Ltd.) followed by R-phycoerythrin (PE)-conjugated AffiniPure Donkey Anti-Goat IgG (H+L) (Cat#: 705-115-147, Jackson ImmunoResearch Inc.,) or PE-Goat Anti-Mouse IgG (Cat#: 550589, BD).

    Techniques: Flow Cytometry, Cytometry, Expressing, Marker

    Generation of CMT2F patient and dHMN2B patient-derived iPSCs. (a) Experimental timeline for iPSC generation. KOSR, KnockOut™ serum replacement; bFGF, basic fibroblast growth factor. (b) Morphology of fibroblasts from normal individual and patients (original magnification, 50x). Scale bars: 200 μ m. (c) iPSC colonies showed ESC-like morphology, such as a flat cobblestone-like appearance with individual cells having a high nucleus-to-cytoplasm ratio (original magnification, 50x). Scale bars: 200 μ m. (d) CMT2F-iPSCs and dHMN2B-iPSCs had preserved point mutation sites in the HSPB1 gene, verified by sequencing of RT-PCR products. (e) CMT2F-iPSCs and dHMN2B-iPSCs maintained normal karyotype. (f) Expression of total and endogenous Klf4, Oct3/4, Sox2 , and c-Myc in CMT2F-iPSCs and dHMN2B-iPSCs was verified by RT-PCR. Two clones from each of the patients-derived iPSCs were tested (clone 1 and clone 2). (g) ESCs and iPSCs expressed stem cell markers such as NANOG (in the nucleus; original magnification, 200x) and SSEA4 (in the cytoplasm; original magnification, 100x). Scale bars: 200 μ m. (h) EB-mediated in vitro spontaneous differentiation of ESCs and iPSCs resulted in the expression of three-germ-layer markers such as AFP (endoderm), SMA (mesoderm), and nestin (ectoderm; original magnification, 200x). Scale bars: 100 μ m. (i) ESCs and iPSCs showed in vivo pluripotency by forming teratomas 8 weeks after subcutaneous injection into NOD/SCID mice. Teratomas consisted of various three-germ-layer tissues including columnar gland epithelial cells with secretions [Gl] for endodermal tissue, muscle [M] and cartilage with calcification [Ca] for mesodermal tissue, and neuroectodermal tissue [NE] and nerve axonal bundle [N] for ectodermal tissue (original magnification, 400x). Scale bars: 100 μ m.

    Journal: Stem Cells International

    Article Title: HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

    doi: 10.1155/2016/9475981

    Figure Lengend Snippet: Generation of CMT2F patient and dHMN2B patient-derived iPSCs. (a) Experimental timeline for iPSC generation. KOSR, KnockOut™ serum replacement; bFGF, basic fibroblast growth factor. (b) Morphology of fibroblasts from normal individual and patients (original magnification, 50x). Scale bars: 200 μ m. (c) iPSC colonies showed ESC-like morphology, such as a flat cobblestone-like appearance with individual cells having a high nucleus-to-cytoplasm ratio (original magnification, 50x). Scale bars: 200 μ m. (d) CMT2F-iPSCs and dHMN2B-iPSCs had preserved point mutation sites in the HSPB1 gene, verified by sequencing of RT-PCR products. (e) CMT2F-iPSCs and dHMN2B-iPSCs maintained normal karyotype. (f) Expression of total and endogenous Klf4, Oct3/4, Sox2 , and c-Myc in CMT2F-iPSCs and dHMN2B-iPSCs was verified by RT-PCR. Two clones from each of the patients-derived iPSCs were tested (clone 1 and clone 2). (g) ESCs and iPSCs expressed stem cell markers such as NANOG (in the nucleus; original magnification, 200x) and SSEA4 (in the cytoplasm; original magnification, 100x). Scale bars: 200 μ m. (h) EB-mediated in vitro spontaneous differentiation of ESCs and iPSCs resulted in the expression of three-germ-layer markers such as AFP (endoderm), SMA (mesoderm), and nestin (ectoderm; original magnification, 200x). Scale bars: 100 μ m. (i) ESCs and iPSCs showed in vivo pluripotency by forming teratomas 8 weeks after subcutaneous injection into NOD/SCID mice. Teratomas consisted of various three-germ-layer tissues including columnar gland epithelial cells with secretions [Gl] for endodermal tissue, muscle [M] and cartilage with calcification [Ca] for mesodermal tissue, and neuroectodermal tissue [NE] and nerve axonal bundle [N] for ectodermal tissue (original magnification, 400x). Scale bars: 100 μ m.

    Article Snippet: Primary antibodies were anti-SSEA4 (1 : 100; mouse IgG3 , MC-813-70, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), anti-NANOG (1 : 500; mouse IgG1 , NNG-811, Abcam), anti-α -fetoprotein (AFP; 1 : 100; mouse IgG2b , 2A9, Abcam), anti-α -smooth muscle actin (SMA; 1 : 100; mouse IgG2a , 1A4, Abcam), anti-nestin (1 : 1000; mouse IgG1, 10C2, Abcam), anti-HB9 (1 : 100; mouse IgG1, 81.5C10, DSHB), anti-islet-1/2 (ISL1/2; 1 : 50; mouse IgG2b , 39.4DS, DSHB), anti-neurofilament H nonphosphorylated (SMI32; 1 : 500; mouse IgG1 , Covance, Princeton, NJ, USA), anti-neuron-specific beta III tubulin (Tuj1; 1 : 1000; rabbit polyclonal, Abcam), anti-microtubule-associated protein 2 (MAP2; 1 : 200; rabbit polyclonal, Millipore, Billerica, MA, USA), anti-choline acetyltransferase (ChAT; 1 : 1000; rabbit polyclonal, Abcam), anti-synapsin 1 (1 : 1000; rabbit polyclonal, Abcam), anti-α -tubulin (1 : 500; rabbit polyclonal, Abcam), and anti-acetylated α -tubulin (1 : 200; mouse IgG2b , 6-11B-1, Abcam) antibodies.

    Techniques: Derivative Assay, Knock-Out, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, In Vitro, In Vivo, Injection, Mouse Assay