Characterization of undifferentiated rESC and riPSC under feeder-dependent and feeder-free culture conditions.
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(A) Rat PSCs form floating or loosely attached colonies on mitotically inactivated MEFs (MEF-2iLIF). Both cell types are positive for alkaline phosphatase activity and show SSEA-1 and Oct4 expression. Scale bars: 200 μm. (B) Flow cytometry analyses of both rPSC types for Oct4 expression. (C) Representative diploid karyograms of rPSCs originating from MEF-2iLIF conditions. P indicates the passage number under MEF-2iLIF conditions. rESCs in passage 14 showed a normal female rat karyotype (42, XX). riPSCs in passage 27 presented with an aberrant male karyotype characterized by a translocation between one homolog of chromosome 3 and the X chromosome (arrows) and two marker chromosomes (mar), presumably composed of material from chromosomes 17 and 20 which are missing. (D) No significant difference in population doubling time was detected between rESC and riPSC. Mean ± SEM, n = 20–21, unpaired t-test, P = 0.897. (E) Also in feeder-free monolayer culture (Geltrex-2iLIF), undifferentiated rPSCs express pluripotency markers. Phase contrast images reveal the typical high nucleus-to-cytoplasm ratio of pluripotent stem cells. In addition, cells show alkaline phosphatase activity, and are immuno-positive for SSEA-1 and Oct4. Scale bars: 200 μm. (F) Flow cytometry analysis revealed Oct4pos expression levels comparable to feeder-based cultures for both rPSC types. (G) Representative karyograms of rPSCs after several passages in Geltrex-2iLIF conditions. P indicates the passage number of cells originating from MEF-2iLIF conditions plus additional passages in Geltrex-2iLIF. After 16 passages, rESC still show a normal rat karyotype in the majority of metaphase plates. After 5 passages, riPSCs also showed no differences to the karyotype found under MEF-2iLIF conditions. However, after 18 passages in Geltrex-2IiLIF, the majority of cells showed a tetraploid karyotype (see S1 Fig). (H) Proliferation rates show a significant difference of rESCs versus riPSCs in Geltrex-2iLIF culture. Mean ± SEM, n = 21, unpaired t-test, *P < 0.001.