Development of genetic manipulation approach for in vitro production of islet-like cell cluster (ILCCs) or insulin-producing cells (IPCs) from canine bone marrow-derived mesenchymal stem cells (cBM-MSCs)

Abstract

In this study, trend of stem cell-based treatment for diabetes type 1 in veterinary practice has been preliminarily investigated. Production of pancreatic lineages by canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) using genetic manipulation approach has been studied. Transfection efficiency of second-generation lentiviral vector on cBM-MSCs employing “pLenti CMV GFP Puro (658-5)” (Addgene plasmid #17448) was investigated and the results suggested the susceptibility of the cell to such transfection. Further study was performed to evaluate the efficiency of PDX1 transfection on cBM-MSCs, focusing on cell fate after transfection. The lentiviral vector containing “pWPT-PDX1 plasmid” (Addgene plasmid # 12256) was used. The multiplicity of infection (MOI) 20, 30, and 50 were employed. The results illustrated that PDX-1 transfection could enhance dose- and time-dependent cell morphological change toward colony-like structure. Some of pancreatic gene markers were also upregulated. Upon the induction by modified three-dimension (3D) micro-environmental manipulating protocol, “hanging drop-culture technique” and “hanging-drop culture technique with Matrigel-formed dome culture technique” could effectively enhance pancreatic differentiation by cBM-MSCs. Further study on integrating hanging drop-cell culture technique with genetic manipulation illustrated that cBM-MSCs could be differentiated toward pancreatic lineage with dramatic expression of pancreatic mRNA markers. Thus, this study demonstrated that cBM-MSCs could be used as the source of pancreatic lineage derivation by using integration of genetic and microenvironmental manipulating protocol in vitro