ReProgramming of Fibroblast using mRNA

The isolation of plasmids was performed using the QIAprep Spin Miniprep Kit (Qiagen). Linearized DNA templates were generated using the FastDigest MluI restriction enzyme (Thermo Fisher Scientific). Therefore, 36 μg plasmid was incubated for 3 h at 37°C with 5 U enzyme, 20 μl 1x reaction buffer, and nuclease-free water in a total volume of 200 μl. Afterwards, linearized DNA was purified using the ISOLATE II PCR and Gel Kit (Bioline) and the complete linearization, purity, and specific length were analyzed by 1% agarose gel electrophoresis. Next, IVT was performed using the RiboMAX Large-Scale Production System T7 Kit (Promega, Madison, USA) according to the manufacturer’s instructions. The IVT reaction was prepared with 10 μg template DNA and contained 40 U of the RNAse Inhibitor (Thermo Fisher Scientific) to prevent the degradation of srRNA. Afterwards, 5-end capping (Cap1) was performed using the ScriptCap Cap1 Capping System followed by 3-end polyadenylation with the A-Plus Poly(A) Polymerase Tailing Kit (both from CELLSCRIPT, Madis

Cultivation of Fibroblasts Neonatal human foreskin fibroblasts (NuFFs, untreated, passage 9, Amsbio, Milton Park, UK) were cultivated in DMEM high glucose supplemented with 10% FBS, 1x GlutaMAX, 10 mM HEPES, and 50 μg/ml gentamicin B. These cell culture reagents were obtained from Thermo Fisher Scientific. Cells were cultivated at 37°C with 5% CO2 (normoxia), and medium was changed every 2-3 days. Cells were detached at about 70% confluency using 0.04% trypsin/0.03% EDTA, and then trypsin neutralizing solution (TNS, 0.05% trypsin inhibitor in 0.1% BSA, PromoCell, Heidelberg, Germany) was added. Afterwards, cells were centrifuged for 5 min at 300 x g, resuspended in culture medium, and seeded at the desired cell density for reprogramming.

To generate inactivated feeder cells, NuFFs and mouse embryonic fibroblasts (MEFs, CF-1, untreated, passage 3, Amsbio) were treated with 10 mg/ml mitomycin C (Merck, Darmstadt, Germany) and frozen in 10% DMSO containing cell culture medium. The wells of 6-well plates were coated with 0.1% gelatin (Sigma-Aldrich Chemie GmbH) in fibroblast culture medium for 4 h at 37°C. For the mRNA-based reprogramming inactivated NuFFs or for the cultivation of primarily picked mRNA-iPSCs inactivated MEFs were seeded per well of a 0.1% gelatin coated 6-well plate and cultivated overnight.

2.4. Reprogramming of Cells Using Synthetic mRNA To perform synthetic mRNA-mediated reprogramming, fibroblasts were seeded per well of a 6-well plate, which was preseeded with inactivated NuFF feeder cells in cultivation medium. The next day, Pluriton reprogramming medium (Stemgent, Cambridge, USA) was equilibrated at hypoxia (5% O2 and 5% CO2, at 37°C) for 2 h and supplemented with 200 ng/ml B18R interferon inhibitor protein (Thermo Fisher Scientific) to repress synthetic mRNA-mediated immune activation. Then, the mRNA transfection cocktail was prepared with a molar ratio of 3 : 1 : 1 : 1 : 1 : 1 for Oct4, Klf4, cMyc, Sox2, Lin28, and eGFP mRNA, respectively. For each transfection, 1.2 μg (100 ng/μl) mRNA cocktail and 4 μl Lipofectamine 2000 (Thermo Fisher Scientific) were incubated in 120 μl Opti-MEM™ I Reduced Serum Medium (Opti-MEM, Thermo Fisher Scientific) for 15 min at room temperature (RT) to form lipoplexes. The transfection complexes were then added dropwise to the cells and incubated for 4 h at hypoxia. Afterwards, the complexes were aspirated and 2 ml B18R containing Pluriton medium was added to the transfected fibroblasts and incubated for 24 h at hypoxia. The transfection of cells was performed daily for 20 days. At day 6, the medium was changed to NuFF-conditioned Pluriton medium. NuFF-conditioned medium was obtained by seeding inactivated NuFFs in T75 cell culture flasks and incubating cells with 25 ml of Pluriton medium supplemented with 4 ng/ml bFGF (PeproTech, Hamburg, Germany). The medium was collected 6x after overnight incubation, pooled, and sterile-filtered using a 0.2 μm filter.

Cultivation of iPSCs Primary iPSC colonies were stained with mouse anti-human StainAlive™ SSEA-4 DyLight™ 550 antibody (Stemgent). iPSCs obtained by the synthetic mRNA delivery were picked manually and transferred into one well of a 12-well plate preseeded with iMEF feeder cells. The cultivation was performed in a standard stem cell medium containing DMEM/F12 high glucose supplemented with 20% KnockOut™ Serum Replacement, 1x GlutaMAX, 1x minimum essential medium (MEM) nonessential amino acids (NEAA), 20 ng/ml bFGF (PeproTech), 100 μM 2-mercaptoethanol, and 1x penicillin/streptomycin. After 2-3 passages, iPSCs were adapted to feeder-free conditions by cultivation on surfaces coated with 0.5 mg/cm2 truncated recombinant human vitronectin (VTN-N) in Essential 8 (E8) medium. Unless otherwise indicated, all cell culture reagents were obtained from Thermo Fisher Scientific. iPSCs obtained by the srRNA delivery were also picked manually and cultivated on 0.5 mg/cm2 VTN-N coated 12-well plates in E8 medium. Passaging of iPSCs was performed every 5-7 days at a split ratio of 1 : 10 with 0.5 mM EDTA in Dulbecco’s phosphate-buffered saline (DPBS) for 5-10 min at RT. After the aspiration of ETDA solution, colonies were detached using the E8 medium supplemented with a 10 μM Y-27632 ROCK inhibitor (Enzo Life Science, Lörrach, Germany) to increase single cell survival.