Tesr2 medium

In this work, the hiPSC cell line iPS-DF6-9-9 T.B, purchased from WiCell Bank, was used. This cell line is vector free and was derived from foreskin fibroblasts with a karyotype 46, XY. Maintenance of hiPSC culture was performed using mTeSR1 medium (STEMCELL Technologies) in 6-well tissue culture plates coated with Matrigel (BD Biosciences) diluted 1 : 30 in DMEM/F12. Prior to differentiation, hiPSCs were adapted in the same xeno-free culture system used for differentiation by using TeSR2 medium (STEMCELL Technologies) or Essential 8 medium (Thermo Fisher Scientific). Plates were coated with Synthemax II-SC (Corning), a synthetic vitronectin-based peptide, at a concentration of 5 μg/cm², Vitronectin XF (STEMCELL Technologies), a defined human recombinant protein, at a concentration of 1.25 μg/cm² or laminin from murine origin (Sigma) at a concentration of 2.5 μg/cm². Enzyme-free passaging was performed using EDTA (Thermo Fisher Scientific) solution diluted in PBS at a concentration of 0.5 mM. Cells were incubated for 5 min with EDTA at room temperature and flushed with culture medium. For cell counting, a sample of 100 μL was incubated in 400 μL of Accutase for 7 min at room temperature and samples were diluted in trypan blue. Phase contrast images were obtained using a Leica DMI 3000B microscope and a digital camera Nikon DXM 1200.

The nonintegrating vector used for reprogramming of fibroblasts to iPSCs was the modified, noninfectious, self-replicating Venezuelan Equine Encephalitis (VEE) virus RNA replicon RNA system from EMD Millipore (catalog number SCR550). This synthetic polycistronic RNA replicon has all four reprogramming factors on a single RNA strand, thereby eliminating the need to transfect multiple individual mRNAs, and increasing the reprogramming efficiency over DNA-based and protein-based reprogramming methods. Briefly, fibroblasts were transfected with the vector, and selected with puromycin for 9–11 days in the presence of B18R protein (GF156; EMD Millipore). Removal of the B18R protein mediates the elimination of the RNA replicon system from the cultures. Selected cells were passaged onto Matrigel (354277; BD) and allowed to grow for 3–4 weeks, during which time iPSC colonies began to form. These colonies were picked and passaged to establish individual iPSC lines, which were subsequently maintained in culture under feeder-free conditions, using a 1:1 formulation of TeSR2 medium (05860; Stem Cell Technologies) and NutriStem® medium (01-0005; Stemgent). A bioinformatics assay for pluripotency, PluriTest, was performed by Cedars-Sinai. Karyotyping was performed by Cell Line Genetics.