Generation of Insulin-Producing Cells from Canine Adipose Tissue-Derived Mesenchymal Stem Cells

Abstract

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.

A novel hybrid polymer of PCL/fish gelatin nanofibrous scaffold improves proliferation and differentiation of Wharton's jelly‐derived mesenchymal cells into islet‐like cells

“…So as to, facilitate the differentiation of WJ‐MSCs into IPCs, After the Fish gelatin/PCL nanofiber scaffold was characterized and fabricated by electrospinning technique, Wharton's jelly‐derived MSCs were cultured on it as a 3D matrix and also cells were cultured on a 2D medium. To prove that cells culture on the 3D scaffold is more functional in comparison to cells cultured on 2D medium several analyses were done including Real‐time PCR, immunocytochemistry, ELISA, western blot, and microscopic images with regard to Nemattollahi et al and Takahiro Teshima et al studies 34,39 . We have planned a two‐step protocol for 18 days.…”

Immunomodulatory Effects of Canine Adipose Tissue Mesenchymal Stem Cell-Derived Extracellular Vesicles on Stimulated CD4+ T Cells Isolated from Peripheral Blood Mononuclear Cells

“…At 80-90% confluence, the cells were detached with trypsin-EDTA solution (Sigma-Aldrich) and passaged repeatedly. The expression of several markers, such as CD14-FITC, CD29-PE, CD34-PE, CD44-PE, CD45-FITC, and CD90-PE, on these cells was determined by flow cytometry using a CytoFLEX (BECKMAN COULTER, Tokyo, Japan) [ 24 ]. cADSCs at passage 3 were seeded in 150-mm dishes (4 × 10 6 cells/dish) and cultured in high glucose DMEM with 10% exosome-free FBS (Thermo Fisher Scientific) and a 1% antibiotic-antimycotic solution in a humidified atmosphere with 5% CO 2 at 37°C.…”

In vitro generation of transplantable insulin-producing cells from canine adipose-derived mesenchymal stem cells

“…The capacity of human MSCs (hMSCs) differentiated into IPCs as well as their clinical accomplishment has been shown in many previous studies 17 – 20 . Although a minority of research on IPCs has originated from canine MSCs (cMSCs) 21 – 23 , these cMSC-derived IPCs are still functionally inadequate and morphologically circumscribed. To fabricate the effective cMSC-derived IPCs, it is essential to advance the current differentiation protocols.…”

“…To fabricate the effective cMSC-derived IPCs, it is essential to advance the current differentiation protocols. In types of cMSCs, canine adipose-derived MSCs (cAD-MSCs) are an accessible candidate and possess the potency for IPC differentiation 22 , 23 . Therefore, this study focused on establishing a protocol for cAD-MSCs induction toward mature IPCs in vitro .…”