Protocols were approved by the Stanford University Human Subjects Research Institutional Review Board. With informed written consent, two 2 mm skin punch biopsies were taken from each volunteer, diced with a scalpel, digested with 1 mg/mL collagenase IV (Life Technologies) for 2 h at 37 °C. Fibroblasts were then grown in DMEM with GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (FBS, US origin, Life Technologies) on 6-well plates (Greiner) coated with a 1:200 dilution of growth-factor reduced Matrigel (9 µg/cm2, Corning). Medium was changed every other day. When confluent, fibroblasts were passaged with TrypLE Express (Life Technologies) onto Matrigel-coated T225 flasks (Nunc).
For Sendai reprogramming (Supplemental Fig. 1a ), early passage (p2-p3) fibroblasts were seeded at 40,000 cells per well on Synthemax II-SC18 (625 ng/cm2, Corning)-coated 6-well plates. After 24 h, medium was changed to E841 (link). The E8 formula was modified to replace human-derived transferrin with an Oryza sativa-derived recombinant version, to make the formula completely chemically defined. The successful application of Synthemax II-SC at the low concentration of 625 ng/cm2 is in line with reports42 (link) that the minimal surface density of vitronectin protein is very low at 250 ng/cm2. E8 medium consisting of DMEM/F12 (10-092-CM, Corning), 20 µg/mL E. coli-derived recombinant human insulin (Dance Pharmaceuticals/CS Bio), 64 µg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 10.7 µg/mL Oryza sativa-derived recombinant human transferrin (Optiferrin, Invitria/Sigma-Aldrich), 14 ng/mL sodium selenite (Sigma-Aldrich), 100 ng/mL recombinant human FGF2 (154 amino acid, E. coli-derived, Peprotech), and 2 ng/mL recombinant human TGFβ1 (112 amino acid, HEK293-derived, Peprotech). To the medium was added four OSKM CytoTune-iPS Sendai Reprogramming Kit viral particle factors (Life Technologies)43 diluted ~1/5 based on manufacturer’s recommendations (3 × 105 cell infectious units (CIU) of each particle per well, multiplicity of infection (MOI) = 7.5). Medium was changed after 24 h and, thereafter once every day. For the first 7 days, cultures were maintained in E8 supplemented with 100 nM hydrocortisone (Sigma-Aldrich) and 200 µM sodium butyrate (Sigma-Aldrich)44 (link). At day 7 medium was swapped to E7N (E8 minus TGFβ1; supplemented with 200 µM sodium butyrate). Medium switched to E8 at day 20.
For plasmid-based reprogramming45 (link), pCXLE-hSK (27078), pCXLE-hUL (27080), and pCXLE-hOCT4-shp53 (27077) plasmids were obtained from Addgene. The OSKM codon optimized mini-intronic plasmid (CoMiP) was generated by S. Diecke. Plasmid-containing E. coli were grown in Miller’s LB (Life Technologies), and purified using Plasmid Maxi Kit (QIAGEN) following manufacturer’s instructions, and quantified using a NanoDrop 2000 (Thermo Scientific). 1 × 106 cells were electroporated with 6 µg total DNA (2 µg of each for 3 plasmid-based systems, 6 µg for CoMiP) using a Neon Transfection System (Life Technologies), with the settings: 1650 V, 3 pulses, 10 ms, and 100 µL tips, and Buffers R and E2. Cells were plated on Synthemax II-SC-coated 6-well plates.
For peripheral blood mononuclear cell (PBMC) reprogramming, 20 mL of blood was collected in EDTA-containing Vacutainer tubes (BD Biosciences). PBMCs were isolated using a Ficoll-Paque PLUS (1.077 g/mL) gradient (GE Healthcare) and plated at 1 million cells per mL in 2 mL of a humanized version of blood medium46 (link) comprised of 50:50 IMDM:F12 (both Life Technologies), 2 mg/mL recombinant human albumin, 1% v/v chemically defined lipid concentrate (Life Technologies), 10 µg/mL recombinant human insulin, 100 µg/mL recombinant human transferrin, 15 ng/mL sodium selenite, 64 µg/mL L-ascorbic acid 2-phosphate, 450 µM 1-thioglycerol (Sigma-Aldrich), 50 ng/mL SCF (Peprotech), 10 ng/mL IL3 (Peprotech), 2 U/mL EPO (EMD Millipore), 40 ng/mL IGF1 (Peprotech), and 1 µM dexamethasone (Sigma-Aldrich). Cells were cultured for 9 days with 50% medium changes every other day. After 9 days, 1 × 106 were plated in blood medium with Sendai virus, as above. Medium was changed every other day and were transferred to E7N in a Synthemax II-SC-coated 6-well plate at d3.
For all reprogramming methods, individual colonies with hESC morphology were picked into 1 well of a 12-well plate (1 colony per well) at d17-d25 in E8 with 2 µM thiazovivin for 24 h after picking. Subsequently, cells were expanded into 6-well plates by passaging 1:1, 1:4, 1:6, 1:8, and finally 1:12, using 0.5 mM EDTA (Life Technologies) in D-PBS without CaCl2 or MgCl2 (Life Technologies) for 7 min at RT.
For Sendai reprogramming (
For plasmid-based reprogramming45 (link), pCXLE-hSK (27078), pCXLE-hUL (27080), and pCXLE-hOCT4-shp53 (27077) plasmids were obtained from Addgene. The OSKM codon optimized mini-intronic plasmid (CoMiP) was generated by S. Diecke. Plasmid-containing E. coli were grown in Miller’s LB (Life Technologies), and purified using Plasmid Maxi Kit (QIAGEN) following manufacturer’s instructions, and quantified using a NanoDrop 2000 (Thermo Scientific). 1 × 106 cells were electroporated with 6 µg total DNA (2 µg of each for 3 plasmid-based systems, 6 µg for CoMiP) using a Neon Transfection System (Life Technologies), with the settings: 1650 V, 3 pulses, 10 ms, and 100 µL tips, and Buffers R and E2. Cells were plated on Synthemax II-SC-coated 6-well plates.
For peripheral blood mononuclear cell (PBMC) reprogramming, 20 mL of blood was collected in EDTA-containing Vacutainer tubes (BD Biosciences). PBMCs were isolated using a Ficoll-Paque PLUS (1.077 g/mL) gradient (GE Healthcare) and plated at 1 million cells per mL in 2 mL of a humanized version of blood medium46 (link) comprised of 50:50 IMDM:F12 (both Life Technologies), 2 mg/mL recombinant human albumin, 1% v/v chemically defined lipid concentrate (Life Technologies), 10 µg/mL recombinant human insulin, 100 µg/mL recombinant human transferrin, 15 ng/mL sodium selenite, 64 µg/mL L-ascorbic acid 2-phosphate, 450 µM 1-thioglycerol (Sigma-Aldrich), 50 ng/mL SCF (Peprotech), 10 ng/mL IL3 (Peprotech), 2 U/mL EPO (EMD Millipore), 40 ng/mL IGF1 (Peprotech), and 1 µM dexamethasone (Sigma-Aldrich). Cells were cultured for 9 days with 50% medium changes every other day. After 9 days, 1 × 106 were plated in blood medium with Sendai virus, as above. Medium was changed every other day and were transferred to E7N in a Synthemax II-SC-coated 6-well plate at d3.
For all reprogramming methods, individual colonies with hESC morphology were picked into 1 well of a 12-well plate (1 colony per well) at d17-d25 in E8 with 2 µM thiazovivin for 24 h after picking. Subsequently, cells were expanded into 6-well plates by passaging 1:1, 1:4, 1:6, 1:8, and finally 1:12, using 0.5 mM EDTA (Life Technologies) in D-PBS without CaCl2 or MgCl2 (Life Technologies) for 7 min at RT.
