Palatal periosteum-derived Mesenchymal stem cells in osteogenesis
Mesenchymal stem cells sourced from the palatal periosteum (PDCs) could be an alternative to using bone marrow aspiration procedures known to be associated with donor site morbidity. For safe use in human therapy, MSCs should be expanded in culture media that is free from animal or human-derived serum. Essential 8™ (E8™) medium is a well-defined serum-free and xeno-free DMEM/F12 based medium replacing FBS with only 7 essential components manufactured for the culture of induced pluoripotent cell lines, that has never been tested on MSCs. This study localised, quantified and characterised PDCs cultured in E8 medium. A portion of the periosteal tissues from 3 patients was dual-immunostained with MSC specific markers (CD105, CD90, CD73). The remaining portion was culture expanded and analysed using flow cytometry and by tri-lineage differentiation. Hepatocyte growth factor and its only known ligand MET (HGF/MET) signalling could play a crucial role in osteogenesis, therefore HGF and MET proteins were immunohistochemically localized. Production of HGF by PDCs cultured in E8™ medium, as well as HGF produced during osteogenesis with or without the blockage of HGF receptor MET by Capmatinib (INCB28060) was determined. The relative expression of MET, HGF, and other osteogenesis-related key genes of interest (SOX9, SP7, ATF4, RUNX2, FOSL1, ALPL, BGLAP, COL1A1, SP3, NELL1) by undifferentiated MSCs in E8 medium was compared with osteogenic differentiated cells as well as with the cells cultured in osteogenic medium supplemented with Capmatinib (INCB28060), in order to assess how blocking the activity of MET affects different stages of osteogenesis (day 7, 14 & 21). MSC were detected within the PP tissue as single cells or small colonies of cells in close proximity to blood vessels. Flow cytometry analysis revealed that 73±6.7% (mean±SD) of cultured cells were positive for MSC markers with only 0.5±0.3% (mean±SD) positive for the HSC markers. Tri-lineage differentiation analysis confirmed the PP cells could become adipoblasts, chondroblasts and osteoblasts. MET and HGF proteins were also localized within the periosteum in close association to the location of PDCs. A significant increase in the amount of HGF produced during osteogenesis was observed, suggesting a role for HGF in osteogenesis. Increased HGF during osteogenesis did not cause cell scattering. At the RNA level, cultured unstimulated PDCs in E8™ medium expressed all the osteogenic genes critical for the different stages of osteogenesis except NELL1. The results were comparable to those of the osteogenic differentiated cells at all different timepoints except for MET, ATF4, RUNX2, COL1A1, which were significantly downregulated during the final matrix mineralisation and maturation stage on day 21. No significant difference in gene expression was discovered between the PDCs cultured in the osteogenesis medium versus MET-blocked osteogenesis medium. An increasing level of NELL1 on day 14 with its peak level on day 21 was detected in only one of the cell lines, cultured in MET-blocked medium. In summary, this study validated PDCs cultured in E8™ medium as a viable, safe alternative source of MSCs for possible future clinical use to enhance bone repair and regeneration. A possible role for HGF/MET signalling during osteogenesis was explored and discussed.
Advisor: Duncan, Warwick; Coates, Dawn; De Silva, Rohana
Degree Name: Doctor of Philosophy
Degree Discipline: Dentistry, Sir John Walsh Research Institute
Publisher: University of Otago
Keywords: Hepatocyte growth factor; MET; Mesenchymal stem cells; Periosteum; Essential 8 medium; Serum free medium; Xeno free medium; HGF/c-MET signalling; Osteogenesis; Periosteum derived cells
Research Type: Thesis