Osteogenic differentiation of human mesenchymal stem cells by type I BMP receptor, ALK-2

Abstract

[한글]

[영문]Human mesenchymal stem cells (hMSCs) render a potential interest to investigators in the fields of tissue engineering, gene therapy and cellular transplantation because hMSCs can differentiate to multifunctional mesenchyme-origin cells. Specially, with respect bone regeneration, many scientists have researched the osteogenic functions of bone morphogenetic proteins (BMPs) with hMSCs. The overexpression of bone morphogenetic protein-2 (BMP-2) gene can stimulate osteogenic differentiation of hMSCs and also induce bone formation in animal model have been reported. As well as, osteogenesis can be induced by the overexpression of type I BMP receptor gene in chondrocytes and myoblasts as similar as BMP-2 gene has been reported. In the present study, we investigated the osteogenic differentiation of hMSCs by the overexpression of activin receptor-like kinase-2 (ALK-2) gene as one of type I BMP receptors by in vitro experiments and verified bone formation in immunodeficiency mice by the implantation of genetic modified hMSCs.hMSCs were isolated from bone marrow of patients with spinal stenosis. Adenoviral vector containing with ALK-2 gene (Ad/ALK-2) was produced from 293A cell. After hMSCs were transduced with Ad/ALK-2, osteogenic staining and RT-PCR to osteogenic marker genes were performed to observe osteogenic differentiation of hMSCs. To verify the overexpression of ALK-2 gene in hMSCs trigger osteogenic cell signaling, westem blot to Smad1/5/8 and MAPK was performed. For 3-dimensional (3D) culture of Ad/ALK-2-transduced hMSCs, cells were seeded to bovine demineralized bone matrix (bDBM) as scaffold and the cell morphology was observed by scanning electron microscopy (SEM). Also, for analyzing newly synthesized surface material of ALK-2-overexpressed hMSCs, SEM-energy-dispersive X-ray spectroscopy (SEM-EDX) was performed. Finally, Ad/ALK-2-transduced hMSCs-adhered bDBMs were implanted to immunodeficiency mice. At 2 and 4 weeks, each group was sacrificed and the implants were harvested. For histological analysis, hematoxylin and eosin (H&E) staining and immunofluorescent (IF) staining were performed. We observed Ad/ALK-2-transduced hMSCs were stained by osteogenic staining and mRNA expression of osteocalcin in hMSCs was increased with depending on virus titer of Ad/ALK-2. Also, mRNA expression of osteogenic transcription factors such as runx2, osterix, dlx5 was activated by ALK-2. Western blot analysis of osteogenic cell signaling protein showed that the overexpression of ALK-2 activated Smad1/5/8 signaling pathway and increased phosphorylation of p38. On 3D culture, a deposit of calcium phosphate at surface on these cells was observed. On the in vivo experiment, the overexpression of ALK-2 could not stimulated enough to bone regeneration compared to the overexpression of BMP-2 as positive control at 2 weeks, but the ALK-2-overexpressed groups stimulated osteogenesis at 4 weeks were observed by histological staining. And ALK-2 protein in Ad/ALK-2-transduced hMSCs-adhered implants was overexpressed for 4 weeks was verified by immunofluorescent (IF) staining. Overexpression of type I BMP receptor, ALK-2 induced osteogenic phenotype in hMSC was verified by in vitro and in vivo studies. Therefore, ALK-2 is a potential therapeutic candidate for osteogenic gene therapy was confirmed by this study.