Synthesis of mRNAs
Plasmids for generating KLF4, cMYC, OCT4 and SOX2 (abbreviated as KMOS) mRNAs in vitro were obtained from Addgene: pcDNA3.3_KLF4 (catalog # 26815); pcDNA3.3_OCT4 (catalog # 26816); pcDNA3.3_SOX2 (catalog # 26817); pcDNA3.3_c-MYC (catalog # 26818), and prepared as previously described [45]. RNAs were synthesized and purified as previously described [10]. KMOS mRNA stocks were mixed in 1:1:3:1 ratio to prepare 100 ng/μl (total) combined mRNA reprogramming cocktails.
RNA-based reprogramming
Human dermal fibroblasts (HDFs) and human hair follicle cells (HHFCs) [46] were isolated from skin biopsies and hair follicles obtained with informed consent and with an approval from The Medical Ethics Review Committees of Hangzhou Normal University Affiliated Hospital. One day before transfection, primary HDFs were plated onto Geltrex-coated six-well plates (Coring) at 50,000 cells per well in fibroblast medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 × non-essential amino acids solution (NEAA), 1 × GlutaMAX supplement, 1 × Penicillin-Streptomycin (P/S), all from Gibco). The following day the medium was changed to KOSR medium ((DMEM/F12 with GlutaMAX Supplement, 20% KOSR, 1 × MEM NEAA, 55 μM of 2-mercaptoethanol (β-ME), 1 × P/S, all from Gibco) supplemented with 100 ng/ml bFGF (Peprotech), 200 ng/ml B18R (eBioscience), 1 mM VPA (Sigma) and 10 mM Y27632 ROCK inhibitor (BioMol) equilibrated at 5% CO2 for 2 hours. The first transfection was performed 2 h after changing the medium. RNA and RNAiMAX Reagent (Invitogen) were first diluted in Opti-MEM (Gibco), the RNA dose was 1,000 ng per well (6-well plate) and was diluted 5 ×, and 5μl of RNAiMAX Reagent per microgram of RNA was diluted 10 ×. After dilution, these components were mixed together and incubated for 20 minutes at room temperature (RT). The transfection mixtures were then dispensed to each well containing cells. Four hours later, the culture supernatant was replaced with KOSR medium supplemented with fresh 100 ng/ml bFGF, 200 ng/ml B18R, 1 mM VPA and 10 mM Y27632. The transfections were performed every 48 h. After three transfections (day 6), cells were digested with TrypLE Select recombinant protease (Invitrogen), and passaged onto gamma-irradiated human fibroblast feeders with a split ratio of 1:6 followed by the other four mRNA transfections. B18R supplementation was discontinued the day after the final transfection and the cells were grown up to day 21 when the iPSC-like colonies were mechanically picked and transferred to MEF-coated 24-well plates with KOSR medium containing 10 ng/ml bFGF and 5 mM Y27632.
Preparation of protein extracts
For expressing reprogramming proteins, human transcription factors (KLF4, cMYC, OCT4 and SOX2) were fused with 9R and the myc tag [11]. To establish clones stably expressing all 4 factors, 293T cells were transfected with pCMV hOct4-9R-myc, pCMV hSox2-9R-myc, pCMV hKlf4-9R-myc, and pCMV hc-Myc-9R-myc vectors, respectively and were grown in the presence of 500 μg/ml neomycin (G418). After 12 days of screening, G418 resistant clones were picked up and tranferred into 24-well plates for further amplification. Stable 293T clones with high expression of K,M,O or S protein were identified by western blotting analysis. For preparation of cell extracts, cells were washed in 1 × PBS and pelleted by centrifugation at 400 × g for 5 min at 4°C followed by suspension in 1 volume of cold cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and a cocktail of protease inhibitors (Roche)), and incubated for additional 40 min on ice. Cells were sonicated on ice, followed by centrifugation at 15,000g for 15 min at 4°C to remove the insoluble components. After filtration though a 0.2 μm membrane, the protein extracts from four transfected 293T cell lines were combined at a 1:1:1:1 ratio, diluted to the final concentration of 500 ng/μl, and frozen rapidly.
Establishment of vigorous HDF clones
Primary HDFs were plated into gelatin-coated 6-well plate. On the following day, cells were treated with combined 293T cell extracts at 100 μg per well for 16 hr. After washing with 1 × PBS, cells were incubated in fibroblast medium for another 6 days with medium being changed every other day. Cells were then digested with TrypLE Select recombinant protease and passaged onto gelatin-coated culture dishes with a split ratio of 1:6. After 4 cycles of protein treatment and subculturing, cells were digested and plated into gelatin-coated dishes at clonal density via fibroblast medium, change the medium every two days. The vigorous HDF clones were mechanically picked and transferred to 24-well plates for amplification.
Karyotyping
For karyotyping, Colcemid (Gibco) was added to each well, mixed gently and incubated at 37 °C, 5% CO2 for 2 h. After washing with 1 × PBS, cells were digested with trypsin. After centrifugation, the supernatants were discarded and cell pellets were re-suspended in 5 ml of 37 °C hypotonic solution (0.075 M KCl), incubated at 37 °C for further 10 min followed by centrifugation. Cell pellets were gently re-suspended in 5 ml cold fixative, and placed on ice for 20 min, and then centrifuged and re-suspended for three times. After the final centrifugation, the cells were suspended in a few drops of cold fixative, 1-2 drops were placed onto wet and clean slides and were left dry at 37 °C for 3 days. After trypsin treatment, the slides were stained with Giemsa solution for 10 minutes, followed by proper washing and drying. After sealing, first used a low-power lens for a comprehensive inspection, then switched to a high-power lens, observed and took pictures. At least 30 genomes are selected from each sample for analysis. Karyotyping was performed according to ISCN (2016).
RT-PCR
Total RNA was purified with Trizol reagent (Invitrogen). One microgram of total RNA was used for reverse transcription reaction with Reverse Transcription System (Promega) and Oligo (dT15) Primer, according to the manufacturer’s instructions. Then PCR was performed with ExTaq (Takara, Japan). Primer sequences are shown in Supplemental Table3.
Teratoma formation
HiPSCs were suspended in KOSR medium containing 10 ng/ml bFGF, SCID mice were anesthetized with diethyl ether and the cell suspension (1 × 106 cells) were injected subcutaneously into the flank of 6-week-old SCID mice. Tumors harvested at 6-10 weeks were fixed in 4% PFA, and embedded in paraffin. Sections were stained with H&E.
Immunostaining
Immunochemical analysis was carried out as previously described [47]. Antibodies used include anti-SSEA-1 (1:200, FCMAB117P, Merck), anti-SSEA-4 (1:200, MAB4304, Merck), anti-TRA 1-60 (1:200, MAB4360C3, Merck), anti-TRA-1-81 (1:200, MAB4381C3, Merck), anti-NANOG (1:500, MABD24C3, Merck), anti-OCT-4 (1:200, AB3209MI, Millipore), anti-Vimentin (1:500, ab45939, Abcam). The Alexa-488 or Alexa-594 conjugated second ary antibodies were obtained from Molecular Probes (Thermo fisher). The nucleic acid dye 40,6-diamidino-2-phenylindole (DAPI) was obtained from Roche.
Western immunoblotting
Western blotting was carried out as previously described [48]. Briefly, cells were lysed in sample buffer plus a cocktail of protease inhibitors (Roche). For each sample, 20 mg of protein was used for electrophoresis in SDS-PAGE gel. Primary antibodies were used as follows: anti-rabbit γH2AX (1:200, ab229914, Abcam), anti-DNA Ligase IV (1:800, ab193353, Abcam), anti-Rad51 (1:2000, ab133534, Abcam), and anti-Rad52 (1:1000, ab124971, Abcam). Horseradish peroxidase (HRP)-conjugated secondary antibody (Promaga) was used at 1:2500. Chemiluminescent signals were detected by autoradiography using the ECL System (Amersham, Piscataway, NJ, USA).
Statistical Analysis
All quantitative data are presented as Means ± SD. Statistical significance of the difference was evaluated by Student’s t-test. P-value < 0.05 was considered statistically significant.