Pmxs hsox2

Retrovirus was made by transfecting 293FT cells with pUMVC and pCMV-VSV-g plasmids, and individual plasmids: pMXs-hKLF4, pMXs-hSOX2, pMXs-hOCT4 or pMXs-hcMYC (Addgene, Cambridge, MA, USA) at a 1:1:1 ratio using Lipofectamine 2000 (Invitrogen, 11668-019). Virus supernatant was collected and combined, filtered through a 0.45-μm filter (EMD Millipore, SCHVU01RE, Billerica, MA, USA) and concentrated 80–100-fold in a centrifugal filter with a 1 00 000 MWCO (EMD Millipore, UFC910008). The concentrate containing all four factors was then used immediately to transduce fibroblasts at a final concentration between 3- and 12-fold.

On the day before transfection, GP2-293 packaging cells (Clontech, Germany) were seeded at a density of 4 × 10⁶ cells per 100 mm dish; pMXs-hOCT4, pMXs-hSOX2, pMXs-hKLF4, and pMXs-hc-MYC (Addgene, USA) were transfected using retroviral packaging vector VSV-G (Thermo Scientific) and lipofectamine 2000 reagent (Thermo Scientific). The collected medium was centrifuged at 1,300 rpm for 3 min to remove debris, and the supernatant was filtered using a 0.45 μm syringe filter. Then, the filtered supernatant was loaded on an Amicon® Ultra-15 10 kDa Centrifugal Filter (Merck, USA) and centrifuged at 4,000×g and 4 °C for 20 min. The resulting solution was resuspended in fresh growth medium containing 8 μg mL⁻¹ polybrene (Sigma Aldrich). Each OG-MEFs and ASCs were pre-seeded at a density of 1 × 10⁶ and 2 × 10⁵ cells, respectively, in 100 mm dishes before transduction. About 10 mL of the growth medium containing the retrovirus and polybrene was added to each dish, and the medium was replaced with a new growth medium after 24 h. After 48 h for complete expression, the transduction efficiencies of the OG-MEFs (>90 %) and ASCs (>50 %) were confirmed, and the detached cells were suspended in each hydrogel solution at a density of 2 × 10⁶ cells mL⁻¹. The transduced cells encapsulated hydrogel was replaced with the iPSC medium every day.