The experiment protocol of the present study was approved by the Ethical Review Committee of Jinan University (approval no. 2015-045). Human iPSC line was obtained from the South China Institute for Stem Cell Biology and Regenerative Medicine Group of the Chinese Academy of Sciences. iPSCs were cultured in mTeSR1 medium (Stemcell Technologies, Inc.) on Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco; Thermo Fisher Scientific, Inc.) coated dishes for 5 days. Then, the medium was replaced with DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine, 1% penicillin/streptomycin and 0.1 mM non-essential amino acids (Gibco; Thermo Fisher Scientific, Inc.). When these cells reached 80% confluence, they were passaged. When most of the cells presented spindle-like morphology, they were collected and applied for subsequent experiments.

Flow cytometry was used to evaluate surface markers of iPSC-MSCs. Briefly, the cells were harvested, and then 3% BSA (Gibco; Thermo Fisher Scientific, Inc.) was used to block non-specific antigens on the cell surface at 37°C for 30 min. Subsequently, the cells were incubated with monoclonal antibodies (all purchased from BioLegend, Inc.) against CD73 (cat. no. 344005), CD90 (cat. no. 328113), CD105 (cat. no. 323207), CD34 (cat. no. 343607) and CD45 (cat. no. 368511). Then, the cells were washed three times with BSA to remove non-specific antibodies. A Guava^® easyCyte™ flow cytometer and Guava^® Suite Software 3.4 (both EMD Millipore) were used to analyze surface antigens.

The osteogenic, adipogenic and chondrogenic differentiation capabilities of iPSC-MSCs were analyzed. To study osteogenic differentiation, iPSC-MSCs at 1×10⁴/well density were seeded on 6-well plates (Corning, Inc.) and incubated with 2 ml StemPro™ Osteogenesis Differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 3 weeks. The Osteogenesis Differentiation medium was replaced every 3 days. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature. The ALP activity of iPSC-MSCs was assayed using an ALP staining kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions, and the calcified matrix deposition was detected using a Von Kossa staining kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions and observed under a light microscope (Zeiss Axio Observer.Z1; Carl Zeiss AG).

In order to study adipogenic differentiation, iPSC-MSCs at 1×10⁴/well density were seeded on 6-well plates and incubated with 2 ml StemPro™ Adipogenesis Differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 3 weeks. Adipogenesis Differentiation medium was replaced every 3 days, and subsequently the cells were assayed with an Oil Red O kit (Nanjing Jiancheng Bioengineering Institute) for 15 min at room temperature.

To study chondrogenic differentiation, iPSC-MSCs at 1×10⁴/well density were seeded on 6-well plates and incubated with 2 ml StemPro™ Chondrogenesis Differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 3 weeks. The Chondrogenesis Differentiation medium was replaced every 3 days, and subsequently the cells were assayed with an Alcian blue kit (Nanjing Jiancheng Bioengineering Institute) for 30 min at room temperature.

Human DLX3 gene primers (PrimerBank ID:38327640c1, Table I) were designed according to PrimerBank online (The Massachusetts General Hospital) (22). Subsequently, PCR amplification was performed according to our previous study (23). The amplified DLX3 primers were treated with EcoRI and BamHI, and then combined into the lentivirus vector pCDH-CMV-MCS-EF1-copGFP (pCDH; System Biosciences, LLC) to construct the recombinant plasmid pCDH-DLX3. The target gene was transduced into 4×10⁵ 293FT cells per well in 6 well plate using a combination of enveloping plasmid, packaging plasmid and recombinant lentiviral plasmid (3rd generation system; Cyagen Biosciences, Inc.) at 37°C. Then, 48 h later, 293FT cells were centrifuged at 10,000 × g at 4°C for 4 h and the supernatant was collected to infect the 3rd passage of iPSC-MSCs (iPSC-MSC-DLX3) at 37°C. The MOI was 50, and concentration of Polybrene was 5 µg/ml. Cells were cultured at 37°C in DMEM containing 10% FBS with 1 µg/ml puromycin for 3 days in order to select puromycin-resistant cells and 0.25 µg/ml puromycin was used for maintenance. Similarly, iPSC-MSCs transfected with blank lentivirus vector (iPSC-MSC-GFP) were generated and used as the control. After 2 days, reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis were performed to evaluate the expression of DLX3 in the two groups.

Proliferation of iPSC-MSC-GFP and iPSC-MSC-DLX3 was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.). On the first day, cells were seeded on 96-well plates at 2×10³ cells/well, and then cultured with DMEM plus 10% FBS. Cell viability was evaluated on day 1, 3, 5 and 7 post-transfection. Optical density (OD) at 450 nm was recorded using an enzyme immunoassay reader (Bio-Rad Laboratories, Inc.).

Total RNA was isolated from iPSC-MSC-GFP and iPSC-MSC-DLX3 using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) on day 7. NanoDrop™ 2000 system (Thermo Fisher Scientific, Inc.) was used to test RNA concentrations. The extracted RNA was reverse-transcribed into cDNA using iScript™ gDNA Clear cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. qPCR analysis was operated using PowerUp SYBR-Green Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) and measured by spectrofluorimetric iQ5 Thermal iCycler (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. The primer sequences of DLX3, ALP, osteopontin (OPN), osteocalcin (OCN), Collagen Type I (COL-1) and GAPDH are presented in Table I. The relative expression of target genes was determined using the 2^−ΔΔCq method (24) and normalized to GAPDH. Each sample was tested three times.

A total of 7 days after transfection, iPSC-MSC-GFP and iPSC-MSC-DLX3 were rinsed with PBS and lysed in 0.1 ml RIPA buffer containing 10 mg/ml proteinase inhibitor PMSF (Invitrogen; Thermo Fisher Scientific, Inc.) on ice for 30 min. The lysed cells were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was collected. During electrophoresis, 20 µg target total protein/lane was separated via SDS-PAGE on a 10% gel (Beyotime Institute of Biotechnology), which were subsequently transferred to PVDF membranes (Thermo Fisher Scientific, Inc.) at 200 mA for 2 h. PVDF membranes were blocked using 5% non-fat milk with TBS with 0.1% Tween-20 at room temperature for 2 h, and then incubated with the primary antibodies at 4°C overnight. The primary antibodies used were as follows: DLX3 (1:500; cat. no. ab64953; Abcam), ALP (1:500; cat. no. ab83259; Abcam), OPN (1:500; cat. no. ab8448; Abcam), OCN (1:500; cat. no. ab93876; Abcam), COL-1 (1:500; cat. no. ab34710; Abcam) and GAPDH (1:2,500; cat. no. ab9485; Abcam). Then, PVDF membranes were incubated with a secondary antibody (1:2,500; cat. no. ab97051; Abcam) at 37°C for 2 h. GAPDH was used as the control. The blotting results were visualized with chemiluminescent western blotting detection reagents (Pierce; Thermo Fisher Scientific, Inc.) and measured by Image-Pro Plus version 6.0 (Media Cybernetics, Inc.).

The transfected cells were seeded on 6-well plates at an initial density of 3×10⁴ cells/well and cultured for 14 days to 80% confluence in DMEM containing 10% FBS. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature. The ALP Staining kit (Nanjing Jiancheng Bioengineering Institute) was used to stain cells for 30 min at room temperature on day 14 after transfection. In total, three randomized observation views were selected, and the number of mineralized nodules was counted under a microscope (Zeiss Axio Observer.Z1; Carl Zeiss AG).

SPSS 20.0 software (IBM Corp.) was used to analyze the data, which are presented as the mean ± SD. Statistical significance was calculated using an unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

As illustrated in Fig. 1A, human iPSCs presented as packed clones with clear margins. After culturing in MSC medium for 14 days, iPSCs were gradually induced into MSCs. Under the microscope, borders of the colonies were eliminated (Fig. 1B), and cells exhibited homogeneous spindle-like morphology (Fig. 1C). The iPSC-derived cells were positive for CD73 (99%), CD90 (71%) and CD105 (95%), and negative for CD34 and CD45, as determined by flow cytometric analysis (Fig. 1D).

After osteogenic induction, iPSC-derived cells displayed positive ALP staining (Fig. 1E) and Von Kossa staining (Fig. 1F). Additionally, iPSC-derived cells had positive Oil Red O and Alcian blue staining after adipogenic (Fig. 1G) and chondrogenic induction (Fig. 1H). Thus, iPSCs were successfully induced into iPSC-MSCs.

Both iPSC-MSC-GFP and iPSC-MSC-DLX3 groups had a GFP expression percentage of ~100% (Fig. 2A). RT-qPCR results indicated that the relative mRNA expression of DLX3 in iPSC-MSC-GFP and iPSC-MSC-DLX3 was 1.00±0.05 and 5.84±0.89, respectively (Fig. 2B). Moreover, the western blotting results demonstrated that the relative DLX3 expression in iPSC-MSC-GFP and iPSC-MSC-DLX3 was 1.00±0.10 and 4.05±0.81, respectively (Fig. 2C and D). Based on these results, it was suggested that the DLX3 gene was successfully transfected into iPSC-MSCs.

The OD values of iPSC-MSC-GFP on day 1, 3, 5 and 7 were 0.21±0.02, 0.25±0.03, 0.33±0.03 and 0.50±0.05, while the OD values of iPSC-MSC-DLX3 on day 1, 3, 5 and 7 were 0.19±0.01, 0.22±0.03, 0.26±0.02 and 0.37±0.03, respectively (Fig. 3). There was no significant difference in cell numbers between the two groups on days 1 and 3 (P>0.05). However, iPSC-MSC-DLX3 demonstrated significantly lower proliferative activity compared with the iPSC-MSC-GFP group on days 5 and 7 (P<0.05).

After 7 days of transfection of iPSC-MSC-GFP and iPSC-MSC-DLX3, the relative expression levels of ALP were 1.00±0.33 and 11.99±0.24, those of OPN were 1.00±0.49 and 5.80±0.07, those of OCN were 1.00±0.06 and 8.64±0.11, those of COL-1 were 1.00±0.53 and 5.98±0.02. The results indicated that the mRNA expression levels of osteogenic markers in iPSC-MSC-DLX3 were significantly higher compared with those in iPSC-MSC-GFP (P<0.05; Fig. 4A).

Similarly, the relative expression levels of ALP were 1.00±0.13 and 7.77±0.05, those of OPN were 1.00±0.04 and 2.05±0.04, those of OCN were 1.00±0.05 and 3.79±0.17, and those of COL-1 were 1.00±0.11 and 3.38±0.22. The expression levels of the osteogenic proteins in iPSC-MSC-DLX3 were significantly increased compared with those in iPSC-MSC-GFP (P<0.05; Fig. 4B and C).

As presented in Fig. 5A, ALP was stained as a golden color. ALP staining in the iPSC-MSC-DLX3 group was increased and brighter compared with that in the iPSC-MSC-GFP group. Mineralized nodules were stained as black/brown in color. The number of mineralized nodules in iPSC-MSC-DLX3 was significantly higher compared with that in iPSC-MSC-GFP (P<0.05; Fig. 5B).