Adipose-derived mesenchymal stem cells (ADSCs) have been suggested their benefits in regenerative medicine for various diseases. Lipomas, benign neoplasms in adipose tissue, have been reported as a potential source of stem cells. These lipoma-derived mesenchymal stem cells (LDSCs) may be useful for regenerative medicine. However, the detailed characteristics of LDSCs have not been fully elucidated. This study investigated the cellular proteomics and secretomes of canine LDSCs in addition to morphology and proliferation and differentiation capacities. Some LDSCs isolated from canine subcutaneous lipomas were morphologically different from ADSCs and showed a rounded shape instead of fibroblast-like morphology. The phenotype of cell surface markers in LDSCs was similar to those in ADSCs, but CD29 and CD90 stem cell markers were more highly expressed compared with those of ADSCs. LDSCs had noticeably high proliferation ability, but no significant differences were observed compared with ADSCs. In regard to differentiation capacity compared to ADSCs, LDSCs showed higher adipogenesis, but no differences were observed with osteogenesis. Cellular proteomic analysis using two-dimensional gel electrophoresis revealed that over 95% of protein spots showed similar expression levels between LDSCs and ADSCs. Secretome analysis was performed using iTRAQ and quantitative cytokine arrays. Over 1900 proteins were detected in conditioned medium (CM) of LDSCs and ADSCs, and 94.0% of detected proteins showed similar expression levels between CM of both cell types. Results from cytokine arrays including 20 cytokines showed no significant differences between CM of LDSCs and that of ADSCs. Our results indicate that canine LDSCs had variability in characteristics among individuals in contrast with those of ADSCs. Cellular proteomics and secretomes were similar in both LDSCs and ADSCs. These findings suggest that LDSCs may be suitable for application in regenerative medicine.

The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide μ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.