In vitro and in vivo effects of insulin-producing cells generated by xeno-antigen free 3D culture with RCP piece

Abstract

To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from adipose-derived stem cells (ADSCs). We established a 3D culture using a human recombinant peptide (RCP) petaloid μ-piece with xeno-antigen free reagents. Briefly, we employed our two-step protocol to differentiate ADSCs in 96-well dishes and cultured cells in xeno-antigen free reagents with 0.1 mg/mL RCP μ-piece for 7 days (step 1), followed by addition of histone deacetylase inhibitor for 14 days (step 2). Generated IPCs were strongly stained with dithizone, anti-insulin antibody at day 21, and microstructures resembling insulin secretory granules were detected by electron microscopy. Glucose stimulation index (maximum value, 4.9) and MAFA mRNA expression were significantly higher in 3D cultured cells compared with conventionally cultured cells ( P < 0.01 and P < 0.05, respectively). The hyperglycaemic state of streptozotocin-induced diabetic nude mice converted to normoglycaemic state around 14 days after transplantation of 96 IPCs under kidney capsule or intra-mesentery. Histological evaluation revealed that insulin and C-peptide positive structures existed at day 120. Our established xeno-antigen free and RCP petaloid μ-piece 3D culture method for generating IPCs may be suitable for clinical application, due to the proven effectiveness in vitro and in vivo .

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

“…The study demonstrated that intraperitoneal transplantation of 1.5 × 10 6 BM-MSCs that differentiated into IPCs into diabetic rats led to the improvement of the hyperglycemic states. However, in a recent report on the transplantation of IPCs generated from human AT-MSCs with μ -pieces into diabetic mice, 4.8 × 10 7 cells were transplanted under the kidney capsule or intramesentery region and led to normoglycemic states [ 44 , 45 ]. One of these studies also calculated the cell number of generated IPCs and found that 1.8 × 10 4 IPCs were obtained from 5 × 10 5 AT-MSCs, indicating that 3.6% of the initial AT-MSCs differentiated into IPCs; thus, approximately 1.7 × 10 6 IPCs are needed per mouse to gain the normoglycemic states in STZ-induced diabetic mice [ 45 ].…”

The Differences in the Characteristics of Insulin-producing Cells Using Human Adipose-tissue Derived Mesenchymal Stem Cells from Subcutaneous and Visceral Tissues

“…Moreover, when considering the proper transplantation site, it is proposed that the intra-mesentery transplantation identified in a previous study is one reasonable and less-invasive transplantation site 12 . As professional hepato-biliary-pancreatic and transplant surgeons, the authors routinely undergo high-risk laparoscopic surgeries 35–37 .…”

“…Generated cells and extirpated organs were prepared and stained by methods already reported 12 . Briefly, generated IPCs were fixed using a cell fixing kit (Funakoshi), recipient mice were sacrificed and IPCs bearing kidneys were extirpated.…”

“…Thus, the focus of this study is on the ADSCs as a new cell source of MSCs 6–8 and the establishment of a new 2-step protocol for the differentiation of IPCs from ADSCs with a short culture duration and functional efficiency 9 . Moreover, this 2-step protocol was modified into a xeno-antigen free and three-dimensional (3D) culture protocol 10,11 which improved cell quality and function in vitro / in vivo and resulted in the normalization of diabetic nude mice blood glucose over 120 days 12 . In other words, this modified protocol is reaching the pre-clinical level of proof of a concept.…”

A change in the zinc ion concentration reflects the maturation of insulin-producing cells generated from adipose-derived mesenchymal stem cells

“…Thus, we focused on ADSCs as a new cell source for IPCs 12,14,15 . We have previously reported a new two-step differentiation protocol 16 and its modification to a more effective xeno-free and three-dimensional culture protocol 17 to generate functional IPCs from ADSCs. For clinical application, it is important to exclude immature IPCs.…”