All animal procedures were approved by the Animal Care and Use Committee of the Zhejiang Sci-Tech University (Hangzhou, China). One guinea pig (Dunkin-Hartley; female; age, 1 year; 40 days pregnant; weight, 1,200 g) and 8 mice (BALB/c-nu; female; age, 6–8 weeks; weight, 18–25 g) were provided by the Laboratory Animal Center of Ningxia Medical University (Yinchuan, China) and the Laboratory Animal Center of Zhejiang University (Hangzhou, China). Animals were maintained at room temperature with normal humidity, and were given free access to food and water under a 12-h dark/light diurnal cycle.

Guinea pig iPS (giPS) were cultured on feeder layers of mitomycin C-treated guinea pig fibroblast cells. All reagents were purchased from Sigma Aldrich; Merck Millipore (Darmstadt, Germany) unless otherwise indicated. giPS were passaged every 3 days. giPS were cultured in KnockOut^™ Dulbecco's modified Eagle's medium (DMEM) containing 0.5% glutamine, 1.0% nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 15% ESC-qualified fetal bovine serum (FBS; SH30406.02E; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin, 100 mg/ml streptomycin (15070–063; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1,000 U/ml mouse leukemia inhibitory factor (LIF; ESG1106; EMD Millipore, Billerica, MA, USA). To determine whether the giPS colonies could grow under feeder-free culture conditions, giPS were alternatively cultured in feeder-free culture conditions on gelatin-coated dishes using ESGRO Complete Plus Clonal Grade medium (SF001-500P; EMD Millipore) containing 15% FBS. giPS were cryopreserved in ESC-qualified fetal bovine serum containing 10% dimethyl sulfoxide (cell culture grade; A3672; AppliChem GmbH, Darmstadt, Germany). The 293FT packaging cells (Invitrogen; Thermo Fisher Scientific, Inc.) (19,20), which were used to produce lentiviruses, were cultured in DMEM containing 10% FBS, 50 U/ml penicillin and 50 mg/ml streptomycin.

To isolate guinea pig fetal fibroblasts, a 40-day-pregnant guinea pig was washed with phosphate-buffered saline (PBS) followed by 75% alcohol. The animal was then sacrificed and a fetus was collected, and their heads and visceral tissues were removed. The remaining fetal tissues were washed in fresh PBS, transferred into a 0.1 mM trypsin/1 mM EDTA solution (3 ml mixture/embryo) and incubated at 37°C for 20 min. Following incubation, 3 ml 0.1 mM trypsin/1 mM EDTA solution was added to each embryo, and embryos were incubated at 37°C for 20 min. Following trypsinization, 6 ml DMEM containing 10% FBS was added to each embryo, pipetting up and down a few times to dissociate cells. Samples were incubated at room temperature for 5 min and transferred into a new tube. Cells were collected by room temperature centrifugation for 5 min at 1,000 × g, and then resuspended in fresh medium. For the first passage, 1×10⁶ cells were cultured on 100-mm dishes at 37°C with 5% CO₂. The present study used guinea pig fibroblasts at passages 3–5 to avoid replicative senescence.

To obtain high-quality fetal fibroblasts, guinea pig fibroblasts were isolated and morphologically characterized in vitro. The lentiviral plasmid, FUW-OSKM (Addgene plasmid no. 20328; www.addgene.org/20328/), which is a single polycistronic virus encoding octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), Kruppel-like factor 4 (Klf4) and c-Myc, was donated by Dr. Rudolf Jaenisch (Massachusetts Institute of Technology, Cambridge, MA, USA) (15). giPS were generated from fibroblasts and used at passage3-5, as described by Carey et al (15). Briefly, 293FT cells were plated at a density of 2×10⁶ cells per 60-mm dish. The following day, cells were transfected with 12 µg/ml FUW-OSKM as well as 9 µg/ml PsPAX2 and 3.6 µg/ml PMD.2G (PMD.2G encodes the viral protein V-G, and PsPAX2 is a packaging vector. These two plasmids were donated by Dr. Peter Hornsby (University of Texas Health Science Center at San Antonio) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following transfection for 24 h, the supernatant of the transfectant was collected and filtered using a 0.45-mm pore-size Whatman^® cellulose acetate filter (Sigma-Aldrich; Merck Millipore) and concentrated by centrifugation at 10,000 × g for 3 h at 4°C. Guinea pig fibroblasts were seeded at 4×10⁵ cells per 35-mm dish and the following day the medium was replaced with virus-containing supernatant supplemented with 8 µg/ml polybrene (Nacalai Tesque, Inc., Kyoto, Japan) prior to incubation for 24 h; this process was repeated three times. A total of 12 h following the last infection, the medium was replaced with fibroblast culture medium. Fibroblasts were passaged using trypsin and plated at densities between 5×10⁴ and 5×10⁵ cells/10-cm on gelatin-coated dishes of guinea pig fibroblast feeder layers, following infection for 5 days. For reprogramming, the culture medium was replaced 24 h later by giPS medium in the presence of 1 mM valproic acid and 10 µg/ml vitamin C. The resultant giPS colonies were picked mechanically based on morphology and maintained according to previously used mouse iPS protocols (1). Colonies that were compact with clear edges were manually selected and expanded on guinea pig fibroblast feeder layers. One of the picked cell lines was selected for further study and passaged >30 times.

Cells were prepared and lysed using Qproteome Mammalian Protein Prep kit (Qiagen, Inc., Valencia, CA, USA), and protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of total protein (60 µg/lane) were boiled in denaturing loading buffer (200 mM Tris-HCl at pH 6.8, 50% glycerol, 8% SDS, 400 mM DTT, 0.4% bromophenol blue), separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked in 5% non-fat milk powder in TBS containing 0.1% Tween-20 (TBST) at pH 7.6 following the protocol of the antibody manufacturer. PVDF membranes were incubated with the following primary antibodies following a brief wash in TBST: Rabbit anti-Nanog (1:1,000; 14295-1-AP), rabbit polyclonal β-actin (1:1,000; 20536-1-AP), rabbit anti-Sox2; 1:2,000; 11064-1-AP) and rabbit anti-Oct4; 1:500; 11263-1-AP), all purchased from ProteinTech Group, Inc., Chicago, IL, USA. Antibodies were diluted in 2% non-fat milk powder in TBST, and incubated overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (IgG) antibodies (1:2,000; SA00001-2; ProteinTech Group, Inc.) for 60 min at room temperature, followed by detection with an Enhanced Chemiluminescence reagent (GE Healthcare Life Sciences, Chalfont, UK). β-actin served as a loading control.

To assess alkaline phosphatase activity and expression of pluripotency markers in giPS, cells were washed three times in PBS, fixed in 4% paraformaldehyde at room temperature for 30 min and washed a further three times with PBS. For alkaline phosphatase analysis, cells were subsequently incubated with SIGMAFAST5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma-Aldrich; Merck Millipore), according to the manufacturer's protocol. For immunofluorescence, cells were incubated with the following primary antibodies: Rabbit anti-Oct4 (1:200), mouse anti-stage-specific embryonic antigen 1 (SSEA1; 1:100; ab16285; Abcam, Cambridge, UK), and rabbit anti-Nanog (1:200) and incubated at 4°C overnight. The following day, the cells were incubated at room temperature for 60 min with the following secondary antibodies: Goat anti-rabbit IgG (H+L)-fluorescein isothiocyanate conjugated or donkey anti-mouse IgG(H+L)-Alexa Fluor 555-labeled (A0562 and A0460; both from Beyotime Institute of Biotechnology, Haimen, China). Cells were counterstained with DAPI (C1005; Beyotime Institute of Biotechnology). Images were captured using a fluorescence microscope.

Total RNA was isolated using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA was prepared from guinea pig fibroblasts and mouse induced pluripotent stem cells (donated by Dr. Duanqing Pei; Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences) to serve as the control. A total of 500 ng RNA was treated using the DNaseI, RNase-free kit (Takara Biotechnology Co., Ltd., Dalian, China) to remove any potential genomic DNA contamination. The RevertAid First Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd.) was used to synthesize cDNA using a random hexamer primer, according to the manufacturer's protocol. qPCR reactions were performed in duplicate using the Power SYBR-Green Master mix (Takara Biotechnology Co., Ltd.), and the Bio-Rad iCycler iQ and iQ5 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: Initial cycle at 95°C for 30 sec, followed by 40 cycles of 95°C denaturation for 5 sec, 60°C annealing for 30 sec and 72°C extension for 15 sec. Expression values were normalized to those of the β-actin housekeeping gene using the ΔΔCq method (21). Primer sequences are presented in Table I.

Karyotyping was performed using conventional techniques as described by Wu et al (22). Briefly, chromosomes were analyzed from actively proliferating cultures of giPS. Cells were treated with colchicine at 37°C for 6 h, trypsinized, centrifuged for 5 min at 1,000 × g at room temperature and incubated with 0.04 M KCI at 37°C for 20 min. The cells were subsequently fixed for 20 min using ice-cold 1:3 (v/v) acetic acid/methanol and collected. The resultant dispersed giPS suspension was smeared onto a cold slide, dried, stained with 10% Giemsa for 20 min at room temperature and observed microscopically.

To assess the differentiation potential of giPS, giPS were implanted into severe combined immunodeficient mice. A total of 2×10⁶ cells were suspended in 100 µl culture medium containing 50% Matrigel (23,24) and injected subcutaneously into the mice. Mice injected with guinea pig fibroblast cells served as controls. A total of eight weeks later, mice were sacrificed. Tumors were collected, fixed in 4% paraformaldehyde and examined using conventional histological procedures (7). The H&E method was used to stain the sections (6 µm).

Data were presented as the mean ± standard deviation of three experiments with n=3 repeats. All statistical analyses were performed using SPSS version 13.0 (SPSS, Inc., Chicago, IL). One-way analysis of variance followed by Scheffe's post hoc test was used for analysis of multiple groups. An independent samples Student's t-test was used for comparison of two groups. P<0.01 was considered to indicate a statistically significant difference.

Following the infection of guinea pig fibroblasts with a single polycistronic virus encoding Oct4, Sox2, Klf4 and c-Myc, cultures were maintained in ESC conditions in the presence of valproic acid (25) and vitamin C. Following 10 to 14 days, a number of small colonies with altered morphology appeared in the confluent fibroblast cultures. The cells expressed a high nuclear/cytoplasmic ratio and prominent nucleoli as presented in Fig. 1. Colonies that were compact with clear edges were manually selected and expanded on guinea pig fibroblast feeder layers. One of the picked cell lines was selected for further study and passaged >30 times. giPS at passage 30 with mouse ESC-like morphology are presented in Fig. 2. When giPS were cultured in media not containing LIF, their typical ESC morphology and differentiation was altered. This indicated that giPS were similar to mouse ESC. When such colonies were fixed and stained for alkaline phosphatase activity analysis, they were positive for alkaline phosphatase (Fig. 2). To determine whether colonies could grow under feeder-free culture conditions, cells were alternatively plated on gelatin-coated dishes in ESGRO Complete Plus Clonal Grade Medium containing 15% FBS. As presented in Fig. 3A, cells continued to grow rapidly. In addition, alkaline phosphatase staining analysis demonstrated that the dense patches of small rapidly dividing cells were alkaline phosphatase positive (Fig. 3B). Clones used for detailed studies in the present research have been grown continuously in culture at normal growth rates for >30 passages.

In karyotypic analysis performed at passage 30, giPS exhibited a normal guinea pig karyotype of 64 XY, as presented in Fig. 4.

Immunostaining analyses demonstrated that pluripotency markers of giPS were expressed at the protein level (Fig. 5). giPS stained positive for Nanog (Fig. 5A and B) and Oct4 (Fig. 5C and D). The cells were additionally positive for the cell-surface marker SSEA1 (Fig. 5E and F), further verification that giPS exhibited pluripotency.

RT-qPCR was performed to assess the expression of pluripotency genes in giPS, using guinea pig fibroblasts as a control. Relative gene expression levels for pluripotency genes Oct4, Sox2, T-box 3 (Tbx3) and Nanog are presented in Fig. 6. The mRNA expression levels of Oct4, Sox2, Tbx3 and Nanog increased significantly (P<0.01) in giPS compared with guinea pig fibroblasts, and were similar to those of mouse iPS.

To further assess protein expression of pluripotency factors in giPS, western blot analyses were performed. The results revealed that Oct4, Sox2 and Nanog were expressed in giPS, but not in guinea pig fibroblasts, which further confirmed the pluripotency of giPS (Fig. 7).

To assess whether giPS have the ability to differentiate into all three germ layer cell types in vivo, giPS were transplanted into immunodeficient mice by subcutaneous injection. Teratomas developed at the subcutaneous sites. H&E staining demonstrated that the teratomas were comprised of a number of different tissue types, which represented all three germ layers, including the ectoderm (neural rosettes), mesoderm (cartilage and muscle) and endoderm (gland like cells), as presented in Fig. 8.