Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status
- Jason P Awe1,
- Patrick C Lee1,
- Cyril Ramathal2,
- Agustin Vega-Crespo1,
- Jens Durruthy-Durruthy2,
- Aaron Cooper3,
- Saravanan Karumbayaram4,
- William E Lowry4,
- Amander T Clark4,
- Jerome A Zack4,
- Vittorio Sebastiano2,
- Donald B Kohn4,
- April D Pyle4,
- Martin G Martin5,
- Gerald S Lipshutz6,
- Patricia E Phelps7,
- Renee A Reijo Pera2 and
- James A Byrne1, 4Email author
© Awe et al.; licensee BioMed Central Ltd. 2013
Received: 10 April 2013
Accepted: 17 July 2013
Published: 26 July 2013
The reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.
In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.
We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.
For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.
Human induced pluripotent stem cell reprogramming efficiencies from human dermal fibroblasts
Advantages of the STEMCCA reprogramming approach include the following: lentiviruses can transduce both dividing and nondividing cells; the STEMCCA polycistronic cassette was engineered for efficient production of multiple protein products from a single lentivirus and allows a characteristic stoichiometry of protein expression that reproducibly promotes consistent reprogramming success [15, 19]; the STEMCCA approach involves only a single transduction event, making it less labor intensive than more involved reprogramming methods such as synthetic mRNAs; the STEMCCA cassette is excisable, eliminating residual transgene expression that reportedly compromises differentiation potential ; and iPSCs can be generated to contain only one integration event and accurately mapped in the genome [16, 20, 21]. To date, a variety of cell types have been reprogrammed through polycistronic lentivirus-mediated reprogramming, including human keratinocytes, bone marrow cells, skin fibroblasts , and T cells from peripheral blood  and also from patients with diseases such as Huntington’s disease , heart failure , immunodeficiency disorders , lung disease , and neurodevelopmental disorders . Nevertheless, the majority (approximately 70%) of lentiviral integrations occur in actively transcribed genes [28, 29]. Because current safe-harbor criteria discard iPSC lines that result from a viral integration occurring in a gene , this greatly reduces the feasibility of STEMCCA-iPSC-based therapeutics. We and others have previously relied solely on microarray transcriptional analysis to assess the expression of genes following insertion of STEMCCA into the introns of genes [30, 31].
In this study, we use both microarray and sensitive real-time RT-PCR to investigate gene expression changes following both intron-based integration and excision of the STEMCCA cassette during the generation of human induced pluripotent stem cells (hiPSCs). We also fully characterized the post-excised iPSCs, differentiated them into four therapeutically useful cell types, and converted them into putative clinical-grade conditions.
Materials and methods
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.
Induced pluripotent stem cell generation and characterization
Nonrestrictive linear amplification PCR genomic mapping of integration into PRPF39 and pre-excised induced pluripotent stem cell characterization
Third-generation lentiviruses are capable of integrating into the host genome of primitive human repopulating cells multiple times, initially seeming to limit the practicality of using these viruses for reprogramming (for personalized cellular therapeutics) and warranting the need for new reprogramming methodologies that yield transductions with fewer copies per cell . Optimization of the multiplicity of infection to between 0.1 and 10, however, recently demonstrated that over 94% of iPSC colonies had a single stable integration . Extensive and site-specific genomic mapping to identify potential insertional mutagenesis and elucidate adverse gene expression effects is needed to establish therapeutically relevant and factor-free iPSC lines. To verify a single integrated STEMCCA line and specifically sequence and map the vector integration, nrLAM-PCR was used to analyze the vector-human genome location. Two lines (C3 and C8) demonstrated single intron-based integrations, and the third line (C11) demonstrated multiple integrations. The C3 iPSC line displayed one integration located in intron 5 of the lysosomal enzyme alpha-N-acetylgalactosaminidase (NAGA), and the C8 (pre-excised) iPSC line displayed one integration located in intron 4 of pre-mRNA-processing factor 39 (PRPF39), a protein known to interact with the spliceosome and play a role in pre-RNA processing . Mutations in NAGA have been associated with Schindler disease , whereas mutations in PRPF39 have not been correlated with any specific disease. We therefore focused our characterization and transcriptional analysis on the C8 line.
We next sought to generate a factor-free line void of any exogenous transgenic factors by expression of a nonintegrating adenovirus expressing both Cre-recombinase and puromycin resistance for selection of post-excised iPSC colonies. C3 and C8 iPSC cells were transduced for 24 hours with the Adeno-Cre-PuroR adenovirus and exposed to puromycin for 5 days, and then colonies were picked to establish three subclones from each colony (C3 subclones 1.1, 1.2, and 1.3 and C8 subclones 2.1, 2.2, and 2.3) after 2 weeks of recovery growth. We determined that successful Adeno-Cre-mediated excision of hSTEMCCA-loxP reprogramming construct occurred in only subclone 2.3, now called the post-excised iPSC subcloned line, as determined through PCR of genomic DNA with primers against endo-Myc-s and A-WPRE (Figure 1B). Expanded post-excised iPSCs were re-exposed to puromycin for 5 days, resulting in 100% cell death of all subcloned colonies and demonstrating that the Adeno-Cre-PuroR did not integrate into the genome following excision. Following Cre-mediated excision, post-excised iPSCs displayed a stable, uniform human ESC-like morphology for over 10 passages on MEFs and maintained pluripotent markers (alkaline phosphatase, NANOG, OCT4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) at a level comparable with that of the pre-excised iPSC line and control human ESCs (Figure 2A). Importantly, to avoid any MEF mRNA contamination issues in later applications, both the pre-excised and post-excised iPSC lines were transitioned into feeder-free conditions on Matrigel with mTeSR1 media. Post-excised cells also were able to maintain their pluripotency, as shown through their successful contribution to all three germ layers in teratoma formation (Figure 2B). Also, the post-excised iPSC line was able to maintain genomic stability for over 37 passages during the transition from pre-excised to post-excised hiPSCs as demonstrated by the normal karyotype maintained (Figure 2C). The completely factor-free post-excised iPSCs were therefore able to maintain pluripotency markers and a normal karyotype and to retain the ability to differentiate to representatives of all three germ layers in the teratoma assay.
PRPF39 gene expression and splicing analysis
Differentiation into clinically relevant cell types
Additional file 1: Is a video file showing spontaneous differentiation of transgene-free iPSCs into functional beating cardiomyocytes.(M4V 10 MB)
Transition from research-grade to putative clinical-grade induced pluripotent stem cells
In this study, we show successful derivation of hiPSCs from human adult somatic dermal fibroblasts that contain a single hSTEMCCA-loxP lentiviral integration. We used nrLAM-PCR technology to analyze both the number of integrations in each line and the site in the genome where the lentiviral provirus integrated. One pre-excised line was derived with a single integration found to map into intron 4 of PRPF39 (a gene not associated with any disease). Following Adeno-Cre-PuroR-mediated excision, a factor-free line, termed post-excised iPSCs, was derived and propagated.
Because previous studies using the polycistronic human STEMCCA lentivirus did not analyze the expression and splicing patterns of an integrated and subsequently excised hSTEMCCA construct in detail, we sought to characterize the expression and splicing patterns of our post-excised iPSC line. Small inactive viral LTRs left in the genome are thought to cause a small risk of insertional mutagenesis . A recent paper, however, argues that only transcriptionally active LTRs, and not transcriptionally inactive LTRs, are capable of forming myeloid tumors, even when multiple LTR copies are present . Previous studies also showed that HIV-based vectors have a clear correlation between increased gene activity hotspots and integration site preference , although not specifically into transcriptional start sites as seen with retroviruses . Therefore, despite the fact that oncogenic risk from an inactive LTR is low, the possibility of integration into a transcriptionally active location and gene is high, and therefore target gene expression and splicing data on the integration site are critical. Although we show abnormally increased gene expression in the pre-excised iPSC line, the gene expression levels were reduced to basal levels upon excision, and the post-excised line maintained a normal pluripotent stem cell phenotype. Fortunately, the post-excised iPSC line had proper splicing of PRPF39 mRNA, although this is probably due to the wild-type nonintegrated allele properly expressing PRPF39. It is important to show that the lentiviral integration does not cause dominant negative interactions with the wild-type allele, allowing normal expression. PRPF39 gene expression was therefore increased, probably due to an enhancer element like the woodchuck post-transcriptional regulatory element coded by the lentivirus causing a post-transcriptional increase in gene expression . If current safe-harbor criteria are expanded to include intron-based reprogrammed cells that have been characterized to demonstrate a normal post-excision integrated gene expression profile, such as the cells described in this study, this will increase the proportion of generated iPSC lines being considered for therapeutic applications and thereby increase the feasibility of iPSC-based therapeutics. We also demonstrated that the post-excised iPSC line was able to differentiate into multiple therapeutically important cell types, such as hepatocytes, cardiomyocytes, and oligodendrocyte progenitor cells .
Finally, we sought a regulatory path to convert these research-grade transgene-free hiPSCs into cells that could be used in future clinical therapeutics (clinical grade). This transition from research grade to clinical grade was previously performed for human ESCs initially derived in the presence of nonhuman serum, proteins, and cells . Geron converted their research-grade ESCs to clinical-grade ESCs by extended cultivation of their cells in defined xeno-free conditions free from nonhuman serum, proteins, and cells under current GMP manufacturing facilities. These facilities involve clean-room suites that are inspected and licensed by the state of California and use of qualified defined reagents and a standardized protocol, followed by standardized quality-control testing. In this study, we used the same approach and converted our research-grade transgene-free iPSCs into putative clinical-grade iPSCs. We discovered that while a small percentage (1%) of the research-grade cells still demonstrated detectible nonhuman sialic acid (which may induce an immunogenic response if these cells had been used for autologous cellular therapeutics ), the post-converted cells no longer demonstrated any detectible sialic acid, suggesting that these cells were now clean and could be used without risking an immunogenic response. However, several caveats must be kept in mind. First, the previous US Food and Drug Administration-approved conversion of research-grade human pluripotent stem cells to clinical-grade cells involved ESCs, not iPSCs, and it is not guaranteed that the same conversion criteria will apply for iPSCs. Second, the US Food and Drug Administration approval technically applied to one specific derivative (oligodendrocyte precursor cells) derived from the converted clinical-grade ESCs, and suggests that a separate US Food and Drug Administration approval would be required for each iPSC-derived, differentiated therapeutic product. How the US Food and Drug Administration will ultimately judge the clinical applicability of these iPSCs, their derivatives, and other future iPSC-based therapeutics initially derived under research-grade xeno-containing conditions remains to be determined.
In summary, we have demonstrated the derivation of a factor-free hiPSC line using a polycistronic human STEMCCA reprogramming virus. nrLAM-PCR-based genomic mapping showed that the line had a single integration into a relatively safe location in intron 4 of the PRPF39 gene. We then demonstrated proper expression levels following excision of the viral construct, correct splicing patterns, differentiation of the post-excised iPSCs into therapeutically relevant cell lineages, and transition into putative clinical-grade conditions.
Dulbecco’s modified Eagle’s medium
Dorsal root ganglion
Embryonic stem cell
Fetal bovine serum
Good manufacturing practice
- H & E:
Hematoxylin and eosin
Human induced pluripotent stem cell
Induced pluripotent stem cell
Mouse embryonic fibroblast
Nonrestrictive linear amplification
Polymerase chain reaction
Pluripotent stem cell
Stem cell cassette.
The authors would like to thank Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector and Katie Ingraham for assisting us with the Oligodendrocyte Precursor Cells co-cultures. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally-Obtained Derivatives Initiative to generate safe patient-specific cell-based therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc. and the UCLA CTSI Scholar’s Award to JAB, P01GM081621 (NIH) to ADP, 1R01HD058047 (NIH) to ATC, 1U01HL100397 (NHLBI) and TR3-05569 (CIRM) to RAR, P01 GM081621-01A1 (NIH) to JAZ, 5K08HD057555-05 and 1R01NS071076-03A1 (NIH) to GSL, and #RT2-01985 (CIRM) to MGM. All authors were funded by the aforementioned funding bodies or performed work/analysis that did not require extramural funding. None of the aforementioned funding bodies had any role in the design, collection, analysis or interpretation of data contained within this manuscript.
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