Thymidine solution

iPS cells were plated on Matrigel-coated T25 flasks and cultivated for 2–3 days in mTeSR1 medium. Chromosome analysis was carried out according to established protocols. In brief, cells were synchronized using thymidine solution (Sigma-Aldrich) and subsequently treated with colcemid (Roche) for 10 min at 37°C. After detachment with trypsin—EDTA, cells were centrifuged, and the cell pellet was re-suspended and maintained in hypotonic solution (75 mM KCl) for 12 min at 37°C. Cells were then fixed in methanol and acetic acid. Metaphase spreads were prepared on cover slips, dried overnight and Giemsa stained (Sigma-Aldrich) after trypsin pre-treatment.

For numerical and structural chromosome analyses of dermal fibroblasts and hiPSCs, high-resolution karyotyping was performed. Fibroblasts were grown in T25 culture flasks in fibroblast growth medium. Human iPSCs were cultivated in T25 flasks coated with hESC-qualified Matrigel in mTeSR™1 maintenance medium. Cells were synchronised using thymidine solution (Sigma-Aldrich, Munich, Germany) and subsequently treated with colcemid (Roche, Mannheim, Germany) for 10 min at 37 °C. After detachment with trypsin–EDTA, cells were centrifuged, and the cell pellet was re-suspended and maintained in hypotonic solution (75 mM KCl) for 12 min at 37 °C. Cells were then fixed in methanol and acetic acid. Metaphase spreads were prepared on cover slips, dried overnight and Giemsa stained (Sigma-Aldrich, Munich, Germany) after trypsin pre-treatment.

Cells were blocked for 18 h with 2 mM thymidine solution (Sigma-Aldrich, T1895). Cells were then released by washing with PBS followed by addition of complete medium and incubation for 9 h. Then, 2 mM thymidine solution was added again for 15 h. Cells were next collected (t = 0 h), then washed with PBS and complete medium added. Cells were collected every 2 h until 12 h. For the M phase control, unsynchronized cells were treated with 330 nM of nocodazole (Sigma-Aldrich, M1404) for 18 h.