pWPT lentiviral vectors (plasmid cat. no. 12255; Addgene, Inc., Cambridge, MA, USA) were constructed using the coding regions of mouse GATA4, MEF2C, TBX5 and HAND2 genes, which were amplified via reverse transcription-polymerase chain reaction (RT-PCR, RR014A; Takara Biotechnology Co., Ltd., Dalian, China). The primers sequences used are listed in Table I, and denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 2 min, and 35 cycles were used. The sequences were inserted into the BamHI/SalI or MluI/SalI site of the pWPT plasmid. A Kozak sequence, GCCACC, was inserted prior to the ATG codon; amplification of the target cDNA fragment was achieved using a 5′-primer containing a Kozak sequence and an ATG codon, and a 3′-primer with unique restriction sites. Primer sequences were introduced into the multiple cloning site of the pWPT lentiviral vector; a stop codon at the 3′-end of the target sequence was omitted.

The tail-tip fibroblasts (TTFs) used in the present study were isolated from 10 male ICR mice (5 day-old). Briefly, mice were anesthetized with 2% diethyl ether for 15 sec, and tails were removed and then rinsed in ethanol followed by PBS. The tissue was minced, transferred into a solution constituting 0.25% trypsin, 0.2% EDTA and 0.1% collagenase (Roche Diagnostics, Basel, Switzerland), and incubated at 37°C for 1 h. Following centrifugation at 500 × g at room temperature for 5 min, isolated cells were resuspended in Dulbecco's modified Eagle's medium [DMEM; containing 10% fetal bovine serum (FBS); Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA] and seeded onto 100 mm dishes. TTFs of passages 2 to 3 were employed for cardiomyocyte induction. 293T cells (CRL-3216; American Type Culture Collection, Manassas, VA, USA), which were used to produce lentiviruses, were maintained in DMEM (containing 10% FBS; Gibco; Thermo Fisher Scientific, Inc.). All procedures with experimental animals were approved by the Institutional Animal Care and Use Committee of Chinese PLA General Hospital (Beijing, China) and carried out according to the guidelines of China Council on Animal Care and Use (permit no. 2014-X9-09).

Prior to transduction, 293T cells were seeded at 8×10⁶ cells/100 mm dish. On the subsequent day, pWPT lentiviral vectors were transduced into 293T cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) transfection reagent according to the manufacturer's instructions. A total of 35 µl transfection reagent, 8 µg lentiviral expression plasmid, 4 µg psPAX2 and 2 µg pMD2.G (all from Addgene, Inc.) were diluted in 3 ml Opti-MEM^® I medium (Thermo Fisher Scientific, Inc.), and then added dropwise onto 293T cells of ~85% confluence; cells were maintained in 10 ml fresh DMEM on 100 mm dishes. pWPT-GFP (Addgene, Inc.) was used as a negative control. At 48 and 72 h following transduction, the viral supernatants were collected, filtered using a 0.45 µm pore size filter (EMD Millipore, Billerica, MA, USA) and concentrated 30-fold with PEG-it^™ Virus Precipitation Solution (System Biosciences, Palo Alto, CA, USA). They were centrifuged at 1,500 × g at 4°C for 30 min, and subsequently resuspended in PBS.

Mouse TTFs were seeded onto gelatin-coated 100 mm dishes at 1.5×10⁴ cells/cm². Following initial cell culture, the culture medium was replaced with 10 ml fresh DMEM containing 10% FBS and 10 µg/ml polybrene (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)-this was classed as day 0. Prior to overnight incubation at 37°C, the same volume (500 µl) of supernatant containing each of the GMTH lentiviruses were mixed and transferred to the dishes. In the control group, TTFs were untransduced or transduced with green fluorescent protein (GFP) lentivirus (Addgene, Inc.) On day 1, the medium of certain plates of GMTH-transduced TTFs was replaced with 10 ml cardiac inductive medium (CIM) constituting DMEM/medium199 (4:1), 10% FBS, 10% conditioned medium obtained from neonatal mouse cardiomyocyte culture, 1% non-essential amino acids, vitamin mixture, 5% horse serum, B-27, insulin-selenium-transferrin and sodium pyruvate (Invitrogen; Thermo Fisher Scientific, Inc.). GMTH-transduced cells were not treated with CIM as control. The medium was replaced every 2 days. Conditioned medium was filtered usinga 0.22 µm pore size filter (EMD Millipore).

TTFs (1.5×10⁶) treated with CIM following GMTH transduction were dissociated, resuspended and loaded onto a discontinuous Percoll gradient on day 9. Percoll (GE Healthcare, Chicago, IL, USA) was diluted in a buffer solution containing 150 mM NaCl and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid. The gradient consisted of a 40.5% Percoll layer above a layer of 58.5% Percoll; cell layers were observed following centrifugation for 30 min at 1,500 × g at room temperature. Cells of different layers were collected, washed with PBS and resuspended in DMEM medium. They were then plated for immunostaining, or collected for reverse transcription (RT)-qPCR analysis. The fractionated cells were seeded onto chamber slides, cultured for an additional few days and immunostained prior to immunocytochemical analysis.

Total RNA was isolated from cultured cells (TTFs, TTFs + GMT and TTFs + GMTH) using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For RT-sqPCR, cDNA was synthesized with RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. PCR analyses were performed with 2X Taq PCR MasterMix (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. Thermocycling condition were: Denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec, for 30 cycles. The products were run in 1% agarose gel with ethidium bromide, and images were captured with Gel Doc™ XR+Gel Documentation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). qPCR was performed with the Rotor-Gene^® 2000 system using the QuantiTect SYBR Green one-step RT-PCR kit and QuantiTect Primer Assay (both from Qiagen Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The thermocycling conditions were as follows: 95°C for 15 sec, 60°C for 1 min, for 40 cycles. The results were analyzed using the 2^−ΔΔCq method (20) with GAPDH as a reference gene and GFP-transduced TTF cells as a calibrator. Primer sequences are presented in Table II.

Immunostaining was performed as previously described (21). Briefly, transduced and untransduced TTF cells were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were washed with PBS three times and subsequently treated with 0.5% Triton X-100 for 30 min at room temperature. Unspecific binding sites were blocked by 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. The primary antibodies against GATA4 (1:100, cat. no. sc-25310), MEF2C (1:100, cat. no. sc-13268), TBX5 (1:100, cat. no. sc-376952), HAND2 (1:100, cat. no. sc-9409), α-myosin heavy chain (MHC, 1:100, cat. no. sc-20641) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), α-sarcomeric actinin (1:100, cat. no. BM0003; Boster Biological Technology, Wuhan, China) and cardiac troponin T (cTnT, 1:200, cat. no. MAB-0374; Fuzhou Maixin Biotechnology, Fuzhou, China) were applied without washing and incubated at 4°C overnight. Following washing with PBS, cells were incubated with the secondary antibodies, fluorescein isothioyanate (FITC)-labeled or phalloidin-tetramethyl rhodamine-labeled goat anti-mouse immunoglobulin G (IgG, 1:50; OriGene Technologies, Inc., Rockville, MD, USA) in the dark at room temperature for 2 h. Nuclei were stained with 0.1 µg/ml DAPI (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature. Images were captured by an inverted phase contrast fluorescence microscopy (Olympus Corporation, Tokyo, Japan) and confocal laser scanning microscopy (Zeiss GmbH, Jena, Germany).

Cells were washed with PBS, dissociated with 0.25% trypsin and 0.04% EDTA, and then filtered through a 35 µm cell-strainer to remove cell clumps. The resulting single cell suspensions were centrifuged at 300 × g for 5 min at room temperature, resuspended in 1 ml PBS, pipetted into 1.5 ml centrifuge tubes, further centrifuged at 400 × g for 5 min at room temperature, and resuspended in 1 ml 4% paraformaldehyde at room temperature for 10 min. Cells were collected by centrifugation at 400 × g for 5 min at room temperature and rinsed with PBS three times, and were then resuspended in 0.5% Triton X-100 for 30 min at room temperature. Subsequently, TTFs were stained with Anti-Myosin Heavy Chain Alexa Fluor^® 488 (1:100, cat. no. 53-6503-82; eBioscience; Thermo Fisher Scientific, Inc.) for 30 min at 37°C, followed by three washes with PBS and centrifugation at 400 × g for 5 min at room temperature. The isotype control antibody was FITC-conjugated mouse IgG1; TTF pellets were resuspended in 500 µl PBS to be detected with flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) for flow cytometric analysis (CellQuest Pro software version 5.1; BD Biosciences).

Results were repeated three times and expressed as the mean ± standard deviation. Differences between groups were examined by Student's t-test for statistical significance using SPSS software version 4.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The generated individual lentiviruses efficiently expressed each of the four cardiac-specific transcription factors GATA4, MEF2C, TBX5 and HAND2 within mouse TTFs. Transduced or untransduced TTFs were examined on day 2 post-transduction, and the majority of transduced cells expressed cardiac transcription factors. Immunofluorescence staining for GMTH proteins demonstrated their nuclear localization (Fig. 1), and viral transduction efficiency was detected to be >96%.

The effects of GMT were compared with those of GMTH in the present study. RT-PCR revealed that cardiac-specific genes were not expressed within the control TTFs compared with the GMT- and GMTH-transduced TTFs. The expression of some cardiac-specific genes within the GMT-transduced TTFs were detected; however, α-MHC expression was not detected 14 days post-transduction. Conversely, GMTH-transduced TTFs demonstrated increased expression levels of cardiomyocyte markers, including α-MHC, β-MHC, atrial natriuretic factor, NK2 homeobox 5 (Nkx2.5) and cTnT compared with GMT transduction (Fig. 2A). Flow cytometry analysis confirmed that ~3.6% of GMTH-transduced TTFs were positive for α-MHC expression; however, α-MHC⁺ TTFs were not detected two weeks following GMT-transduction (Fig. 2B). Additionally, immunofluorescence staining indicated that some GMTH-transduced TTFs expressed α-MHC, compared with in the control and GMT-transduced groups, in which no positive cells were detected (Fig. 2C).

To enhance the productivity of α-MHC⁺ cells, CIM was employed. CIM treatment stimulated the expression of α-MHC within GMTH-transduced TTFs by 3-fold, when compared with in untreated GMTH-transduced TTFs (Fig. 3A); no effect was detected in control TTFs. The duration of cardiomyocyte induction from TTFs was analyzed by flow cytometry. α-MHC⁺ cells were detected as early as 3 days following GMTH induction via CIM treatment, the number of α-MHC⁺ cells slowly increased to ≤18% at day 9 (Fig. 3B). The percentage of α-MHC⁺ cells peaked at day 9 post-transduction, and declined thereafter due to overgrowth of non-reprogrammed fibroblasts (Fig. 3C).

CIM-treated TTFs at day 9 post-GMTH transduction were collected and transferred to a discontinuous Percoll gradient (40.5 over 58.5%). Two layers of cells were generated following centrifugation (Fig. 4A). One layer was observed above the layer of Percoll (fraction I), and the second layer observed was between the two layers of Percoll (fraction III). The input cells, and cells within the 40.5% Percoll layer (fraction II) and the 58.5% Percoll layer (fraction IV) were collected for α-MHC immunocytochemical staining (Fig. 4B). Quantitative detection of triplicate samples revealed that fraction III contained 27.2±4.1% α-MHC⁺ cells and fraction IV contained 72.4±5.5% α-MHC⁺ cells, while fraction I and II contained only 0.1 to 1.5% α-MHC⁺ cells. When compared with the starting input that constituted 14.7±1.1% α-MHC⁺ cells, fraction IV revealed a 3.6-fold enrichment of α-MHC⁺ cells (Fig. 4C). These results demonstrated a marked enrichment of cardiomyocytes using the method of discontinuous Percoll gradient centrifugation.

The GMTH-transduced cells were collected from fractions III and IV following Percoll separation, and the cardiac gene expressions were compared among TTFs, transduced and unenriched cells, transduced and enriched cells, and neonatal mouse cardiomyocytes. qPCR analysis of gene expression patterns revealed that several cardiac-specific genes were upregulated; however, the fibroblast marker gene, collagen type 1 a2 (COL1A2), was inhibited in Percoll-enriched, GMTH transduced cells (Fig. 5F). Higher expressions of cardiac genes myosin heavy chain 6 (MYH6), troponin 2 (TNNT2), NKX2-5, natriuretic peptide A (NPPA), and gap junction α1 (GJA1) were observed in the enriched cells when compared with transduced and unenriched cells; however, they were not detected in TTFs by qPCR (Fig. 5A-E). These data indicated that iCMs could be enriched from GMTH-transduced TTFs. Enriched iCMs had an expression pattern similar to neonatal ventricular cardiomyocytes, and full maturation may be a slow course that occurred over a few of weeks.

To characterize the Percoll-enriched cell populations, immunocytochemistry was performed using antibodies against cTnT and α-actinin. Confocal laser scanning microscopy revealed that Percoll-enriched cells had strong immunofluorescence for the sarcomeric proteins cTnT and α-actinin, and it confirmed the extent of differentiation. The enriched cells exhibited typical cardiomyocyte morphology and possessed distinct myofilaments. The well-developed sarcomeric structures were observed by cTnT and α-actinin immunocytochemistry (Fig. 6). Noncardiac cells expressed SMA or vimentin, which was indicative of the existence of fibroblasts or smooth muscle cells in the Percoll-enriched populations (data not shown).