Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential
Figure 4
Pluripotency and stability of stem cells after laser-mediated passage. (a) Colony morphology (brightfield, BF) and immunocytochemical analysis of Oct4, Sox2, Nanog, alkaline phosphatase (AP), SSEA4, TRA1-60, and TRA1-81 expression in human ESCs (H9) after 24 consecutive laser-mediated passages. Hoechst was used as a nuclear counterstain. Scale bar, 250 μm. (b) Whole well brightfield images of human iPSC (BIMR 6) cultures after 10 consecutive laser-mediated passages or collagenase passages. Scale bar, 5 mm. (c) QRT-PCR analysis of stem-cell-associated gene expression in iPSCs (BIMR 6) after 10 consecutive laser-mediated passages or collagenase passages. The asterisks (*) indicate values that are statistically significant compared with the starting population of iPSCs. The data are presented as mean ± s.d. (). Statistical analysis was performed using -test, with a value ≤ 0.05 considered to be significant. (d) Normal karyotype of H9 human ESCs after 24 laser-mediated passages (6 months). (e) Schematic depicting genomic abnormalities of H9 ESCs at P35 (starting population) and at P59, after 24 laser-mediated passages (6 months), as determined by aCGH. No new subkaryotypic abnormalities were detected after 24 passages. Red bars indicate a deletion. Green bars indicate an amplification. See Table 1 for complete list of aberrations found in these cells.
