Written approvals for human skin biopsy procedures and human fibroblast derivation, culture, and experimental use were obtained from the Stanford University Institutional Review Board (Stanford IRB protocol #10368) and the Stanford University Stem Cell Research Oversight Committee (Stanford SCRO protocol #40), and written informed consent was obtained from each individual participant. Cells used in this study were initially derived at Stanford University and transferred to UCLA through a material transfer agreement (UCLA MTA #2011-00000147). Written approvals for the experiments performed in this study were obtained from the UCLA Institute Biosafety Committee (UCLA IBC protocol #123.10.0-f), the Animal Research Committee (UCLA ARC protocol #2006-119-21) and the Stem Cell Research Oversight Committee (UCLA SCRO protocol #2010-010-02).
In vitro culture of primary human skin cells
The human skin-derived (HUF1) primary cell line used in this study was obtained from a 4-mm adult skin punch biopsy and was cultured as described . Two other fibroblast lines were also used in this study: an infant fibroblast line (MGM2) and a fibroblast line from Fibrocell Science, Inc. (Exton, PA, USA) (azficel-T (LAVIV) part #DR01/RMS-5519v00). All human biopsy-derived cells and fibroblast lines were cultured in complete DMEM/F-12 media consisting of DMEM nutrient mixture/F-12 supplemented with 10% fetal bovine serum (FBS), 1× minimum essential medium nonessential amino acid, 1× Glutamax, and 100 IU/ml penicillin–streptomycin (all from Invitrogen/Gibco, Grand Island, NY, USA) and maintained at 37°C in a 5% CO2 incubator. Culture media were changed every 2 days. Cells were allowed to expand to 80 to 90% confluency before passaging with 0.05% trypsin–ethylenediamine tetraacetic acid (Invitrogen) and replating at a 1:3 ratio. A large bank of early-passage HUF1 cells was cryopreserved in culture media supplemented with 10% dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO, USA). All research adhered to National Academy of Sciences guidelines.
In vitro culture of stem cell lines
Human-1, human-2, and human-9 embryonic stem cell (ESC) lines were provided by the UCLA Broad Stem Cell Research Center-Stem Cell Core. Multiple integration iPSCs were derived as previously published . The mRNA hiPSCs were derived using Stemgent’s mRNA reprogramming factor set (Stemgent, San Diego, CA, USA). The adult pre-excision line (termed C-8, or pre-excised iPSC) and the adult post-excision line (termed 2.3, or post-excised iPSC), derived as explained below, were all initially maintained on 0.2% gelatin-coated six-well plates covered with 35,000 cells/cm2 irradiated mouse embryonic fibroblasts (MEFs) (GlobalStem, Rockville, MD, USA) with standard ESC media consisting of DMEM/F-12 supplemented with 20% Knockout Serum Replacement, 1× Glutamax, 1× nonessential amino acid, 100 IU/ml penicillin–streptomycin (all from Invitrogen), 1× β-mercaptoethanol (Millipore, Billerica, MA, USA), and 10 ng/ml recombinant human basic fibroblast growth factor (Globalstem). All cells were transitioned into a feeder-free system and subsequently maintained on reduced growth factor Matrigel (BD Biosciences, San Jose, CA, USA) in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 10 ng/ml basic fibroblast growth factor (Globalstem) and 1× Primocin (InvivoGen, San Diego, CA, USA). Media were changed daily. Cells were passaged every 4 to 5 days, depending on colony density and size. Differentiation was removed daily from colonies using pulled glass pipettes. To passage the pluripotent stem cells, an 18-gauge needle was used to cross-hatch colonies in a grid format, with subsequent gentle agitation to remove the pieces with a P200 pipette. Usually, 4 to 8 colonies were passaged onto freshly coated Matrigel plates.
Lentivirus production and infection
For pre-excised and post-excised iPSC lines, lentiviral human STEMCCA vector was synthesized and packaged as published  and was concentrated to 100×. The day before infection, 100,000 cells/well were plated in a six-well plate grown in standard DMEM/F-12 media without antibiotics. On the day of transduction, 100× lentiviral supernatant was thawed, and 2 ml MEF conditioned media from each well of fibroblasts to be infected was taken out and mixed with 2× and 4× viral supernatant concentrations, respectively, with 8 μg/ml polybrene (Millipore). This virus-containing mixture was quickly added to the cells to avoid drying, shaken gently, and placed at 37°C in a 5% CO2 incubator overnight. From day 2 through day 6, media were changed every day with DMEM/F-12 medium with antibiotics. Irradiated xCF1 fibroblasts harvested from day 8 mouse embryos were plated on day 6, and 50,000 and 100,000 cells from one well in a six-well plate were plated on day 7 onto an MEF-plated 10-cm plate and left to sit at 37°C in a 5% CO2 incubator overnight. The next day, MEF media were replaced with human ESC medium for the duration of the reprogramming and changed daily. Colonies were picked on the parental plate when colonies reached the size of 60 to 70% of 5× field view or became three-dimensional/differentiated into cell aggregates. Each parental colony was cut into two or three pieces and seeded onto a 24-well plate preseeded with xCF1 mouse feeders, one clone per well. Colonies were grown and further subcloned out according to optimal growth and colony morphology (flattened, very little differentiation, and high nucleus-to-cytoplasm ratio) and when colonies reached 60 to 70% of 5× field. Subcloning into a 12-well plate required 8 to 10 pieces from each clone per well from a 24-well plate be placed into an xCF1 MEF precoated 12-well plate. The pieces were then eventually subcloned out to a six-well plate for further characterization.
Vector integration site analysis by nonrestrictive linear amplification PCR
DNA was isolated from iPSCs using the PureLink Genomic DNA Mini Kit (Invitrogen). Approximately 100 ng genomic DNA was used to perform nonrestrictive linear amplification (nrLAM) PCR . Briefly, 100 cycles of linear amplification were performed with primer HIV3linear (Biotin-agtagtgtgtgcccgtctgt). Linear reactions were purified using 1.5 volumes of AMPure XP beads (Beckman Genomics, Indianapolis, IN, USA) and captured onto 100 μg of M-280 Streptavidin Dynabeads (Invitrogen Dynal), prepared in accordance with the instructions of the manufacturer. Captured ssDNA was ligated to read 2 linker (Phos-agatcggaagagcacacgtctgaactccagtcac-3C Spacer) using CircLigase II (Epicentre, Madison, WI, USA) in a 10 μl reaction at 65° for 2 hours. PCR was performed on these beads using primer HIV3right (aatgatacggcgaccaccgagatctacactgatccctcagacccttttagtc) and an appropriate indexed reverse primer (caagcagaagacggcatacgagat-index-gtgactggagttcagacgtgt). PCR products were mixed and quantified by probe-based quantitative PCR, and appropriate amounts were used to load Illumina v3 flow cells (Illumina, San Diego, CA, USA). Paired-end 50-base-pair sequencing was performed on an Illumina HiSeq 2000 instrument using a custom read 1 primer (ccctcagacccttttagtcagtgtggaaaatctctagca). Reads were aligned to the hg19 build of the human genome with Bowtie , and alignments were condensed and annotated using custom Perl and Python scripts to locate vector integrations.
Infection of induced pluripotent stem cells with adeno-Cre
Excision of STEMCCA was performed by transient transduction of a defective adenoviral vector expressing Cre-recombinase-puromycin (Adeno-Cre-puroR), which was generated by Vector BioLabs (Philadelphia, PA, USA) to express Cre recombinase and puromycin resistance, into the parental pre-excised iPSC line. We used 45 and 5 μl concentrated Adeno-Cre-puroR virus with 8 μg/ml polybrene (Millipore) in standard ESC media for 24 hours. After 24 hours (on day 1), the mixed viral supernatant was removed, and the cells were washed twice with ESC media and then cultured in fresh ESC media containing 2 μg/ml puromycin (Invitrogen) for a period of 5 days. Individual colonies still growing after 5 days were subcloned into 12-well plates and expanded as described above.
Genomic and RT-PCR analysis
Genomic DNA was isolated from pluripotent stem cells (PSCs) grown in feeder-free conditions with the PureLink Genomic DNA Mini Kit (Invitrogen) in accordance with the instructions of the manufacturer. PCR was performed using the KAPA HiFi Hotstart ReadyMix PCR kit (KAPA, Woburn, MA, USA) with a five-step PCR protocol as follows: initial denaturation at 95°C for 3 minutes; 35 cycles of each of the following: denaturation at 98°C for 20 seconds, primer annealing at 62°C for 15 seconds, and extension at 72°C for 15 seconds; followed by a single cycle final extension at 72°C for 3 minutes. Ten nanograms of template DNA were used. Primers specific for exogenous integrations of the STEMCCA lentivirus are listed as follows: gDNA-hendo-MycS-forward, 5′-acgagcacaagctcacctct-3′; gDNA-hWPRE-reverse, 5′-tcagcaaacacagtgcacacc-3′. gDNA PCR was normalized to beta-actin: gDNA-hACTB-forward, 5′-ggagaatggcccagtcctc-3′; and gDNA-hACTB-reverse, 5′-ggtctcaagtcagtgtacagg-3′ . Total RNA was isolated using PSCs grown only on feeder-free conditions to prevent MEF mRNA contamination issues with Roche’s High Pure RNA Isolation Kit in accordance with the instructions of the manufacturer (Roche, Indianapolis, IN, USA). Then 700 ng PSCs and 300 ng all fibroblast lines’ RNA were reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit, using anchored-oligo(dT)18 and random hexamer primers (Roche). PCR was performed using the KAPA HiFi Hotstart ReadyMix PCR kit (KAPA) with a five-step PCR protocol: initial denaturation at 95°C for 5 minutes; 28 cycles of each of the following: denaturation at 98°C for 20 seconds, primer annealing at 64°C for 15 seconds, and extension at 72°C for 15 seconds; followed by a single cycle final extension at 72°C for 5 minutes. In total, 75 ng RNA was used per reaction, and 12 μl with 3 μl loading dye was loaded into a 3% agarose gel in accordance with the recommendations of the manufacturer. Primers specific to exon 4/5 splice junction analysis were: RT-hexon4/5-forward, 5′-tgagcatgctgttctagctgcagga-3′; and RT-hexon4/5-reverse, 5′-accaggaggaccatcatcaccac-3′. RT-PCR gene expression was normalized to beta-actin: RT-hACTB-forward, 5′-ggagaatggcccagtcctc-3′; and RT-hACTB-reverse, 5′-ggtctcaagtcagtgtacagg-3′.
Global transcriptional meta-analysis
Pre-excised and post-excised iPSCs were grown in standard feeder-free culture conditions as stated above and harvested for total mRNA using a High Pure RNA Isolation Kit in accordance with the instructions of the manufacturer (Roche). Microarray analysis was carried out as published . Affymetrix data adhered to the standards proposed by the Functional Genomics Data Society and were deposited in a MIAME-compliant format into the Gene Expression Omnibus  [GEO:GSE48830]. Each CEL file was uploaded to GeneSifter (VisX Labs, Seattle, WA, USA) using the Advanced Upload Method and normalized using the Affymetrix Microarray Analysis Suite (MAS) 5.0 (Santa Clara, CA, USA) algorithm. GeneSifter pairwise analysis between samples was performed using all mean normalization and t-test statistical analysis (P <0.05). For each pairwise analysis, two replicates from each cell line were compared. Probe sets were considered significantly different when P <0.05 and fold change ≥2.
Quantitative reverse transcription-polymerase chain reaction
Total RNA was isolated using PSCs grown only on feeder-free conditions to prevent MEF mRNA contamination issues as stated above. Primers and probes were designed and ordered from Roche’s Universal ProbeLibrary. Quantitative PCR relative expression experiments used a LightCycler 480 Real-Time PCR System (Roche), and data were further analyzed with LightCycler 480 Software release 1.5.0. Primers for the genes are listed as follows – primers specific for pre-loxP site analysis: QRT-hPRPF39-forward, 5′-caggattttacaggctgggta-3′ and QRT-hPRPF39-reverse, 5′-tcctggcagccatcaagt-3′, probe #2; QRT-hPOU5F1-forward, 5′-gaagttaggtgggcagcttg-3′ and QRT-hPOU5F1-reverse, 5′-tgtggccccaaggaatagt-3′, probe #13; QRT-hSOX2-forward, 5′-gggggaatggaccttgtatag-3′ and QRT-hSOX2-reverse, 5′-gcaaagctcctaccgtacca-3′, probe #65; QRT-hNANOG-forward, 5′-cagtctggacactggctgaa-3′ and QRT-hNANOG-reverse, 5′-cacgtggtttccaaacaaga-3′, probe #55; and gene expression was normalized using HPRT1 and GAPDH primers: QRT-hHPRT1-forward, 5′-tgaccttgatttattttgcatacc-3′ and QRT-hHPRT1-reverse, 5′-cgagcaagacgttcagtcct-3′, probe #73; and QRT-GAPDH-forward, 5′-gctctctgctcctcctgttc-3′ and QRT-GAPDH-reverse, 5′-acgaccaaatccgttgactc-3′, probe #60. Five nanograms per sample were used in a 20 μl reaction that consisted of 10 μM UPL probe, 2× LightCycler 480 Probes Master, and 20 μM forward and reverse primers. Triplicate experimental samples were paired using the all-to-mean pairing rule with two housekeeping genes run in duplicate for advanced relative quantification.
Total RNA was extracted as stated above and amplified with the hexon 4/5 primers and purified with a PCR purification kit (Qiagen, Valencia, CA, USA). Samples were sent for full-service sequencing at UCLA’s Genotyping and Sequencing Core (Los Angeles, CA, USA) using Invitrogen/Applied Biosystems 3730 Capillary DNA Analyzers, and sequence results were analyzed on ApE by (M. Wayne Davis; ).
Cultured cells were fixed in 4% paraformaldehyde/1× PBS for 15 minutes, washed twice with 1× PBS supplemented with 100 mM glycine for 5 minutes, and then incubated, when needed, with permeabilization buffer consisting of 0.1% Triton X-100 (Sigma-Aldrich) in 1× PBS for 30 minutes at room temperature. Blocking was performed with 4% goat serum in Blocker Casein in PBS (Thermo Scientific, Rockford, IL, USA) for 60 minutes at room temperature. The cells were then incubated for 2.5 hours with primary antibody at room temperature. Cells were washed with PBS after primary antibody staining and following each subsequent step. Following primary antibody incubation, the coverslips/wells were incubated with Alexa Fluor secondary antibodies (Invitrogen) at room temperature for 1 hour and mounted in Prolong Gold with 4′,6-diamidino-2-phenylindole (Invitrogen). Cultures were visualized with an AxioCam MR Monocolor Camera and AxioVision Digital Image Processing Software (Axio Observer Inverted Microscope; Carl Zeiss, Jena, Germany).
The primary antibodies used for PSC characterization are mouse anti-Oct-3/4 (C-10) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat anti-SSEA-3 (1:200; Millipore), mouse anti-SSEA-4 (1:200; Millipore), mouse anti-TRA-1-60 (1:200; Millipore), mouse anti-TRA-1-81 (1:200; Millipore), and rabbit anti-NANOG (1:100; Abcam, Cambridge, MA, USA) . For oligodendrocyte progenitor and oligodendrocyte cells, the following primary antibodies were used: mouse anti-NG2 (1:25; eBioscience, San Diego, CA, USA), rabbit anti-PDGFRα (1:20; Abcam), rabbit anti-SOX10 (1:20; Abcam), mouse anti-OLIG1 (1:200; Millipore), mouse anti-A2B5 (1:50; Millipore), mouse anti-O4 (1:40; R&D Systems, Minneapolis, MN, USA), mouse anti-O1 (1:40; R&D Systems), and rat anti-Myelin Basic Protein (1:40; Abcam). To analyze oligodendrocyte and neuronal co-culture, and to ensure oligodendrocyte human origin, rabbit anti-TUJ-1 (1:2500; Covance, Inc., Emeryville, CA, USA) and mouse anti-human mitochondria (1:40; Millipore) antibodies were used, respectively. For hepatocyte cells, the following primary antibodies were used: mouse anti-CK18 (1:50; Dako, Carpinteria, CA, USA), mouse anti-serum albumin (1:50; R&D Systems), and mouse anti-alpha-fetoprotein (1:100; Invitrogen). For cardiomyocytes, the following primary antibodies were used: mouse anti-Troponin I (1:50; Millipore) and mouse anti-alpha-actinin (Sarcomeric) (1:100; Sigma-Aldrich). For fibroblast differentiation, the following primary antibody was used: mouse anti-COL3A1 (1:40; Santa Cruz Biotechnology).
Induced pluripotent stem cell-directed differentiation
For oligodendrocyte progenitor and mature oligodendrocyte differentiation, embryoid bodies (EBs) were made on day 1 by 1 mg/ml collagenase treatment for 10 minutes, followed by gentle scraping with a 5-ml serological pipette. Detached colonies were collected and transferred to low-adhesion plates (Sigma-Aldrich) in a 50:50 combination of mTeSR1 and Glial Restrictive Media and differentiated as published . For co-culture experiments, rat dorsal root ganglion (DRG) neurons were dissected and cultured as previously described, except for the substitution of rat DRG neurons . DRG neurons were cultured on Matrigel (BD Biosciences) for a period of 7 days before post-excised derived oligodendrocyte progenitor cells were plated on top of the DRG neurons at a density of 15,000 cells/well in a 24-well plate. All cells were cultured in Glial Restrictive Media. Co-cultured cells were cultured for a period of 7 days before fixation and immunostaining.
For EB-directed beating cardiomyocyte differentiation, post-excised iPSCs were incubated with 1 mg/ml collagenase for 10 minutes and then quenched with standard differentiation media consisting of standard DMEM as listed above but with 20% FBS and also with inclusion of 50 μg/ml ascorbic acid (Sigma-Aldrich), followed by making strips of iPSCs with a 5-ml serological pipette and subsequent placement into low-adhesion plates (Sigma-Aldrich). Media were changed every day with fresh media until day 5, when EBs were plated onto 0.2% gelatin-coated plates. The FBS concentration was reduced to 5% on day 10, and media were changed every 4 to 5 days with fresh ascorbic acid .
For non-EB-directed cardiomyocyte differentiation, post-excised iPSCs cultured on Matrigel were changed to DMEM/F-12 (Invitrogen) supplemented with 1× N2, 2 mM l-glutamine, 1 mM nonessential amino acid, 1× B27 supplement (all from Invitrogen), 0.5 mg/ml bovine serum albumin (Fraction V; Sigma-Aldrich), and 0.11 mM 2-mercaptoethanol (Millipore) (N2/B27-CDM) supplemented with 50 ng/ml recombinant human BMP-4 and 50 ng/ml recombinant human activin A (both from PeproTech, Rocky Hill, NJ, USA) for 3 or 4 days and cultured in N2/B27-CDM without additional factors for an additional 8 to 10 days. The medium was changed daily .
For hepatocyte differentiation, post-excised iPSCs were grown on Matrigel as stated above until reaching a 60 to 70% confluence upon which endoderm induction was initiated by replacing the post-excised iPSCs for 24 hours with RPMI 1640 medium (Invitrogen/Gibco, Rockville, MD, USA), supplemented with 0.5 mg/ml albumin fraction V (Sigma-Aldrich), and 100 ng/ml Activin A (PeproTech). On the following 2 days, 0.1 and 1% insulin–transferrin–selenium (Invitrogen/Gibco) were added to the medium, respectively. Post-excised iPSCs were then cultured in hepatocyte culture medium (Lonza, Walkersville, MD, USA) containing 30 ng/ml fibroblast growth factor-4 and 20 ng/ml BMP2 (PeproTech) for 4 days. The now-differentiated cells were then incubated in hepatocyte culture medium containing 20 ng/ml hematopoietic growth factor and 20 ng/ml keratinocyte growth factor (PeproTech) for 6 days, in hepatocyte culture medium containing 10 ng/ml oncostatin-M (R&D Systems) plus 0.1 μM dexamethasone (Sigma-Aldrich) for 5 days, and in DMEM containing N2, B27, 1× Glutamax, 1× nonessential amino acid, and 1× β-mercaptoethanol (all from Invitrogen/Gibco) for 3 more days. Media were changed daily during differentiation .
For fibroblast differentiation, EBs were cultured in adherent conditions on 0.2% gelatin using standard fibroblast media with 10% FBS and were passaged until typical fibroblast morphology was seen .
Post-excised iPSCs were passaged onto a 25-cm2 flask to 60 to 70% confluency and sent out for G-band karyotyping analysis (Cell Line Genetics, Madison, WI, USA).
Teratomas for the pre-excised and post-excised iPSC lines were generated by injecting 8 × 106 cells resuspended in Hanks’ balanced salt solution (Invitrogen) into the two testes in a severe combined immunodeficient adult male beige mouse. All tumors were dissected 6 to 8 weeks after injection and fixed in 4% formaldehyde, and sections were paraffin-embedded and then stained with H & E for further analysis at the UCLA Translational Pathology Laboratory. All animal experiments were performed in accordance with the UCLA Animal Research Committee and the UCLA Division of Laboratory Animal Medicine.
Good manufacturing practice conversion and analysis
Post-excised iPSCs were slowly transitioned from mTeSR1 media conditions to a 1:1 ratio of mTeSR1 and NutriStem (Stemgent) and finally to a 1:1 ratio of TeSR2/NutriStem (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 1× Primocin (InvivoGen) and 1× basic fibroblast growth factor (GlobalStem), which are both defined xeno-free media (containing no animal proteins). This conversion used 0:100, 20:80, 50:50, 80:20, and 100:0 mTeSR1/NutriStem:TeSR2/NutriStem ratios, with each condition lasting for 3 days. Regular passaging was maintained every 4 or 5 days based on cell morphology and density. Once cells were converted to the 1:1 TeSR2/NutriStem, the cells were mechanically passaged with an 18-gauge needle in the presence of 1× ROCK inhibitor (Stemgent), preconditioned in the media for 1 hour, and then transferred to a xeno-free substrate (Synthemax; Sigma-Aldrich). Cells were initially fibroblastic in nature, and continual differentiation of the iPSCs had to be taken out with a hand-pulled glass pipette. Specific selection of proper iPSC colonies over a period of 2 or 3 weeks generated morphologically homogeneous and standard-looking iPSCs. Cells that were converted to xeno-free conditions were then transferred to the UCLA good manufacturing practice (GMP)-compatible facility and underwent extended cultivation (for over 3 months) under xeno-free conditions. The cells were then subjected to standardized quality-control testing to ensure viability, sterility, and appropriate cellular composition, which included immunocytochemical analysis of stem cell markers, confirmation that the cells were free from nonhuman contaminants, including bacteria, fungi, mycoplasma or sialic acid (Neu5Gc) contamination, and confirmation they possessed a normal karyotype, and were cryobanked for potential future clinical applications as previously described . To further show the broad applicability of our slow transition methodology across media and synthetic matrices, we also converted the post-excised cells to a fully defined, synthetic matrix called CELLstart (Invitrogen) and cultured in NutriStem media alone.
Flow cytometry-based detection of sialic acid contamination
Flow cytometry was performed on the BD LSRII flow cytometer and all data were analyzed with BD FACSDiva Version 6.1.3 Software (BD Biosciences). The cell surface expression of nonhuman sialic acid Neu5Gc (N-glycolylneuraminic acid) was detected utilizing the chicken anti-Neu5Gc IgG (1:200) (Sialix anti-Neu5Gc Basic Pack Kit; Sialix San Diego, CA, USA) and labeled with FITC-conjugated donkey anti-chicken IgG (H + L) (1:200; Jackson ImmunoResearch, West Grove, PA, USA). 4’,6-Diamidino-2-phenylindole (Invitrogen) was included as previously published . Standard conditions and experimental controls were performed as per manufacturer recommendations (Sialix). hiPSCs that were derived and maintained under xeno-free clinical grade conditions and mouse embryonic fibroblasts (Globalstem) served as negative and positive controls, respectively. Additionally, post-excised iPSCs in mTeSR1 plated on Matrigel and post-excised iPSCs in xeno-free NutriStem plated on CELLstart were utilized for this assay.
Results are presented as means ± standard deviations. The statistical significance of differences for PRPF39 gene expression was evaluated using SPSS 20 (IBM Corporation, Chicago, IL, USA). Analysis of variance, a t test for independent samples, and Kruskal–Wallis nonparametrical one-way analysis of variance tests were considered statistically significant with P <0.05.