Journal: Stem Cells Translational Medicine

Article Title: Wnt Signaling Orchestration with a Small Molecule DYRK Inhibitor Provides Long-Term Xeno-Free Human Pluripotent Cell Expansion

doi: 10.5966/sctm.2011-0033

Figure Lengend Snippet: A combination of ID-8 and Wnt ligand enhances human embryonic stem cell (hESC) survival and proliferation. (A): Screening of small molecule Wnt signaling modulators in replating assays with HES2 cells in mouse embryonic fibroblast conditioned medium (MEF-CM). Colony numbers per seeded cell numbers indicate replating efficiency. Statistical significance (p value) was calculated by Student's t test. Shown are cultured plates stained with ALP (B) and morphologies of typical colonies at day 7 (C). (D): ALP staining and OCT4 immunostaining of HES2 cells cultured in MEF-CM with 0.5 μM ID-8 and 100 ng/ml Wnt3a. (E): Replating efficiency of HES2, HES3, and H9 cells cultured in MEF-CM with or without ID-8 and Wnt3a. Statistical significance (p value) was calculated by Student's t test. Graphs show quantitative polymerase chain reaction analysis of pluripotent markers (F) and differentiation markers (G, H) in HES2 cells cultured in MEF-CM with or without ID-8 and Wnt3a. Scale bars = 200 μm; error bars indicate SD. Abbreviation: ALP, alkaline phosphatase.

Article Snippet: For feeder-free culture, the MEF feeder layer was removed via separation from embryonic stem cell clumps by sedimentation under gravity during passage, and cells were cultured in MEF-conditioned medium (MEF-CM) on 30-fold diluted Matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com )-coated culture dishes.

Techniques: Cell Culture, Staining, Immunostaining, Real-time Polymerase Chain Reaction

Journal: Stem Cells Translational Medicine

Article Title: Wnt Signaling Orchestration with a Small Molecule DYRK Inhibitor Provides Long-Term Xeno-Free Human Pluripotent Cell Expansion

doi: 10.5966/sctm.2011-0033

Figure Lengend Snippet: Characterization of human pluripotent cells maintained in xeno-free and feeder-free culture system. (A): Typical colony morphologies at passage 7. (B): Immunostaining of undifferentiated cell markers in HES3 cells at passage 24. Similar results were observed in H9 and HES2 cells (data not shown). (C): Flow cytometry analysis of undifferentiated cell markers in H9 cells at passage 26. White histograms indicate cells stained with secondary antibody; black histograms indicate cells stained with primary and secondary antibodies. Percentages of positive cells are listed on the bars. Similar results were observed in HES2 and HES3 cells (data not shown). (D): Quantitative polymerase chain reaction analysis of pluripotent stem cell markers in HES2 cells at passage 24. Error bars indicate SD. Similar results were observed in H9 and HES3 cells (data not shown). (E): G-banding karyotype at passage 22. Abbreviations: iPSC, induced pluripotent stem cell; MEF-CM, mouse embryonic fibroblast conditioned medium.

Article Snippet: For feeder-free culture, the MEF feeder layer was removed via separation from embryonic stem cell clumps by sedimentation under gravity during passage, and cells were cultured in MEF-conditioned medium (MEF-CM) on 30-fold diluted Matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com )-coated culture dishes.

Techniques: Immunostaining, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction