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

Abstract

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 (DL X)3, an important member of the DL X family, serves a crucial role in osteogenic differentiation and bone formation. The present study aimed to evaluate the effects of DL X3 on the proliferation and osteogenic differentiation of human iPSC-MSCs. iPSC-MSCs were induced from iPSCs, and identified via flow cytometry. Alkaline phosphatase (AL P), Von Kossa, Oil Red O and Alcian blue staining methods were used to evaluate the osteogenic, adipogenic and chondrogenic differentiation of iPSC-MSCs. DL X3 overexpression plasmids were constructed and transfected into iPSC-MSCs to generate iPSC-MSC-DL X3. iPSC-MSC-GFP was used as the control. Reverse transcription-quantitative PCR (RT-qPCR ) and western blotting were performed to measure the expression of DL X3 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. AL P activity and mineralized nodules were assessed via AL P 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 CD 73, CD 90 and CD 105, but negative for CD 34 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 AL P, 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 AL P, 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-DL X3 was increased compared with that in iPSC-MSC-GFP on day 14 (P<0.05). Thus, the present study demonstrated that DL X3 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.