Human dermal fibroblasts

For the scratch wound migration assay, human dermal fibroblasts (passage 5–6) (ATCC, Manassas, VA, USA) at a density of 6 × 10⁴ cells per well were seeded onto tissue-culture-treated polystyrene 48-well plates and cultured in DMEM complete medium containing 10% FBS) at 37 °C with 5% CO₂ and 95% humidity for 2 days. Scratch wounds were made on a confluent monolayer using a sterile 1 mL pipette tip. After removal of the medium from each well and rinsing with 0.4 mL/well of 1 × PBS, 0.2 mL/well of conditioned medium (collected as described above) was added to the wounds. Images of the wound areas were captured at 0 h, with two areas marked and monitored for each well. The plates were incubated at 37 °C for 24 h, and the exact same wound areas (with marker references) were imaged again at 24 h. Wound areas were measured using NIH Image J (Fiji for Mac OS X) software in arbitrary units (square pixels, px²). The migrated area was calculated as follows: $$\mathit{M}\mathit{i}\mathit{g}\mathit{r}\mathit{a}\mathit{t}\mathit{e}\mathit{d} \mathit{a}\mathit{r}\mathit{e}\mathit{a}=\mathit{A}\mathit{r}\mathit{e}{\mathit{a}}_{\mathbf{0}\mathit{h}}-\mathit{A}\mathit{r}\mathit{e}{\mathit{a}}_{\mathbf{24}\mathit{h}}$$

Primary human umbilical cord-derived mesenchymal stem cells (MSCs) from ATCC (Manassas, VA, USA) were maintained with low serum mesenchymal stem cell growth kit (ATCC). For reprogramming, on day −1, MSCs were plated onto six-well tissue culture plates at a density of 5 × 10⁵ cells/plate. On day 0, retrovirus carrying OSKM and other reprogramming factors were added with 10 μg/ml polybrene. The infected cells on day 4 were passaged onto mitomycin C treated mouse embryonic fibroblast (MEF) feeders in the presence of 10 μM Y-27632 (Selleckchem) ROCK inhibitor. On day 5, the medium was changed to a 1:1 mix of MSC medium and human ESC medium. Starting from day 7, the cells were maintained in complete human ESC medium, which contains 20% knockout serum replacement (KSR) in DMEM/F12, supplemented with 1 × NEAA, 1 × Glutamax, 0.5 × penicillin and streptomycin, 4 ng/ml human FGF2 (all from Thermo Fisher Scientific, Waltham, MA, USA), and 1 × β-mercaptoethanol (Merck Millipore, Billierica, MA, USA). iDOT1L and IWR1 were added in reprogramming as specified in the main text and maintained thereafter. The reprogramming of human dermal fibroblasts (ATCC) follows similar method and timeline except that fibroblasts were grown in medium containing DMEM plus 10% fetal bovine serum for the first 7 days of reprogramming before switching to human ESC medium without small chemicals.