The present study was approved by the Henan Provincial People’s Hospital (Zhengzhou, Henan, China), Tianjin Central Hospital of Obstetrics and Gynecology (Tianjin, China), and Affiliated Hospital of Medical College, Qingdao University (Qingdao, Shandong, China), and no financial benefits were involved in the donation process. Written informed consent was obtained from the patients. It was guaranteed to the donors that biopsied tissue and the resulting fibroblast cells were only for use in basic scientific research. One piece of skin tissue was obtained by biopsy from each of six patients with heart disease. The tissue was minced with scissors following rinsing in phosphate-buffered saline (PBS), and dissociated into single cells using 0.5% trypsin. The cells were then transferred to culture medium containing 85% Dulbecco’s Modified Eagle’s medium supplemented with 15% fetal bovine serum, 2 mM L-glutamine, 1% minimum essential medium nonessential amino acids and 1% penicillin-streptomycin. These primary-passage fibroblast cells were divided following seven days of incubation, prior to being passaged every three to four days. Cells at passage four were prepared for iPSC generation.

A lentivirus with octamer-binding transcription factor 4 (Oct-4), SRY-related HMG-box gene 2 (Sox-2), Kruppel-like factor 4 (Klf-4) and cellular myelocytomatosis oncogene (c-Myc) was provided as a commercial kit (SCR544; Millipore, Billerica, MA, USA), and the procedure for generating iPSCs was performed according to the product specifications. Fibroblast cells were prepared and used as they reached 30% confluence. On the first day, 1 μl lentivirus solution (10⁹ TU/ml) was added to the cell culture medium together with 8 μg/ml polybrene. The culture medium was changed on the second day, and a further 1 μl lentivirus solution was added with 8 μg/ml polybrene. The culture medium with lentivirus was discarded on the third day and new culture medium was added. The fibroblast culture medium was changed to a stem cell culture medium on the sixth day, and the cells were digested with 0.25% trypsin. The iPS cell culture medium consisted of 80% KO-DMEM, 20% KO-serum, 1% L-glutamine, 0.5% penicillin/streptomycin, 1% non-essential amino acids, 1 mM β-mercaptoethanol and 10 μg/ml bFGF The transfected cells were then plated onto mitomycin C-treated mouse embryo fibroblast cells, and cultured for 12 days until small colonies appeared. After 20 days, the colonies were removed using a glass needle and cut into pieces. One part was cultured in Matrigel-coated tissue culture dishes, and the other part was cultured in mouse feeder cell-coated tissue culture dishes. The iPSCs were passaged every four to seven days.

Karyotyping analysis was performed as previously described (15). iPSCs were treated with colchicine and blocked at mitotic metaphase. The nuclear membrane was then broken by potassium chloride treatment. Following fixation in methanol:glacial acetic acid, the cells were transferred onto glass slides, the chromosome spreads were stained with Giemsa and their images were captured. The chromosomes were visualized using standard G-band staining. For each sample, ≥50 metaphase cells were examined.

Stem cells were characterized using specific stem cell markers. iPSCs were fixed using 4% paraformaldehyde following rinsing with PBS, and subsequently incubated with the primary antibodies to tumor rejection antigen-1-60 (TRA-1-60; MAB4381, Millipore), Oct-4 (ab27985) and Sox2 (ab75485, Abcam, Cambridge, UK) at 1:100 dilution overnight at 4°C. The cells were then washed with PBS and the secondary antibody (1:200) was added immediately. The secondary antibody was fluorescein isothiocyanate (FITC)-conjugated antibody (ab97224) and Texas Red-conjugated antibody (ab6757) (Abcam). Cell nuclei were stained with Hoechst 33342 (Sigma-Aldrich). Cells were examined using a confocal laser scanning system (LSM 510 META; Carl Zeiss, Jena, Germany).

Total RNA was extracted using a TRIzol^® RNA isolation kit (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cytoplasmic RNA from human iPSCs and their derived embryoid bodies (EB) were reverse-transcribed to single-stranded cDNA. This cDNA was subjected to PCR amplification using primers for genes representative of the three germ layers, including α-fetoprotein (AFP, endoderm), NEUOD-1 (ectoderm) and HBZ (mesoderm).

The differentiation ability of iPSCs was analyzed in vitro by EB formation, as previously described (15). Clumps of iPSCs were suspended in culture medium without basic fibroblast growth factor, following digestion with 1 mg/ml type IV collagenase. EB formation was observed five to seven days later, and the three germ layers were analyzed regarding their representative gene expression.

iPSC colonies were digested using 1 mg/ml type IV collagenase, and 2–5×10⁵ cells were collected and injected into the back leg of severe combined immunodeficiency (SCID) mice. The SCID mice were purchased from the animal center in the Second Affiliated Hospital, Harbin Medical University. The production and sale licenses were obtained from the ZPSTD, Inspection and Quarantine Bureau (Harbin, China) and Price Bureau (Harbin, China). These mice were fed in accordance with national legislation for animal care. Teratomas, which developed after three months, were biopsied and identified using immunohistochemistry, and representative images for the three germ layers were observed using an inverted microscope (80i, Nikon, Inc., Tokyo, Japan).

Six iPSC lines were derived successfully using fibroblast cells from patients with MI (Fig. 1A), including three cell lines using feeder-layer cells (HTH-iPS-1-3) and three cell lines generated under feeder-free conditions (HTH-iPS-4-6). Small colonies were able to be observed on day 12 after transfection with four transcription factors, and the primary colonies were passaged on day 20 (Fig. 1B). The cells had normal stem cell morphology and exhibited the features of human ESCs, including a high nucleus/cytoplasm ratio and tightly packed colonies when observed under an inverted microscope. There were no differences in the morphology of iPSCs grown under different culture conditions (Fig. 1C and D). The iPSCs showed high levels of alkaline phosphatase activity, and had normal karyotypes following long-term propagation (Fig. 2A–C).

To identify the characteristics of human heart disease-specific iPSCs, cell surface markers were detected by immunofluorescence and pluripotent gene expression was detected by RT-PCR. All iPSCs expressed the pluripotent markers Oct-4 and Nanog, and the cell surface marker TRA-1-60 (Fig. 3A–C).

To evaluate the differentiation abilities of the iPSC lines, the ability of the iPSCs to differentiate into EBs in vitro when cultured in suspension was assessed. Following spontaneous differentiation for 7–10 days, the clumps of cells formed EBs, which were then subjected to RT-PCR analysis (Fig. 4A). Genes uniquely expressed in all three embryonic germ layers were detected, including AFP (endoderm), NEUOD-1 (ectoderm) and HBZ (mesoderm) (Fig. 4B).

Furthermore, the in vivo differentiation ability of the iPSCs was evaluated by teratoma formation. All the human iPSCs formed teratomas following injection into the back leg of SCID mice. Histopathological examination of the resulting teratomas revealed the presence of various tissue types, including thyroid (endoderm), cartilage (mesoderm) and sebaceous gland (ectoderm) (Fig. 4C). These results demonstrate that human iPSCs are capable of differentiating into derivatives of all three embryonic germ layers in vivo.