Effects of DLX3 on the osteogenic differentiation of induced pluripotent stem cell‑derived mesenchymal stem cells.

Osteoporosis is a disease characterized by the degeneration of bone structure and decreased bone mass. Induced pluripotent stem cell‑derived mesenchymal stem cells (iPSC‑MSCs) have multiple advantages that make them ideal seed cells for bone regeneration, including high‑level proliferation, multi‑differentiation potential and favorable immune compatibility. Distal‑less homeobox (DLX)3, an important member of the DLX family, serves a crucial role in osteogenic differentiation and bone formation. The present study aimed to evaluate the effects of DLX3 on the proliferation and osteogenic differentiation of human iPSC‑MSCs. iPSC‑MSCs were induced from iPSCs, and identified via flow cytometry. Alkaline phosphatase (ALP), Von Kossa, Oil Red O and Alcian blue staining methods were used to evaluate the osteogenic, adipogenic and chondrogenic differentiation of iPSC‑MSCs. DLX3 overexpression plasmids were constructed and transfected into iPSC‑MSCs to generate iPSC‑MSC‑DLX3. iPSC‑MSC‑GFP was used as the control. Reverse transcription‑quantitative PCR (RT‑qPCR) and western blotting were performed to measure the expression of DLX3 2 days after transfection. Subsequently, cell proliferation was assessed using a Cell Counting Kit‑8 assay on days 1, 3, 5 and 7. RT‑qPCR and western blotting were used to analyze osteogenic‑related gene and protein expression levels on day 7. ALP activity and mineralized nodules were assessed via ALP staining on day 14. Statistical analysis was performed using an unpaired Student's t‑test. Flow cytometry results demonstrated that iPSC‑MSCs were positive for CD73, CD90 and CD105, but negative for CD34 and CD45. iPSC‑MSC‑DLX3 had significantly lower proliferation compared with iPSC‑MSC‑GFP on days 5 and 7 (P<0.05). mRNA expression levels of osteogenic markers, such as ALP, osteopenia (OPN), osteocalcin (OCN) and Collagen Type I (COL‑1), were significantly increased in iPSC‑MSC‑DLX3 compared with iPSC‑MSC‑GFP on day 7 (P<0.05). Similarly, the protein expression levels of ALP, OCN, OPN and COL‑1 were significantly increased in iPSC‑MSC‑DLX3 compared with iPSC‑MSC‑GFP on day 7 (P<0.05). The number of mineralized nodules in iPSC‑MSC‑DLX3 was increased compared with that in iPSC‑MSC‑GFP on day 14 (P<0.05). Thus, the present study demonstrated that DLX3 serves a negative role in proliferation, but a positive role in the osteogenic differentiation of iPSC‑MSCs. This may provide novel insight for treating osteoporosis.