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.
Highlights
Islet transplantation (ICTx) is one of the therapeutic options for patients with type 1 diabetes mellitus and can remove the need for insulin injections and associated complications[1,2]
We focused on these cells because adipose-derived stem cells (ADSCs) can be obtained from the patient’s own fat tissue under local anaesthesia, and auto-ADSC transplantation has fewer ethical problems compared with the use of induced pluripotent stem or embryonic stem cells
We focused on ADSCs among mesenchymal stem cells, as these cells can be used for procurement with less invasiveness methods and ADSCs were reported to have more multipotency compared with other cell sources[26,27]
Summary
Islet transplantation (ICTx) is one of the therapeutic options for patients with type 1 diabetes mellitus and can remove the need for insulin injections and associated complications[1,2]. ICTx still faces several obstacles, such as the requirement of a large islet yield[2], the fragility of the isolated islets[3] and severe donor shortages in some counties, such as Japan[4], which need to be addressed before widespread clinical use To solve these issues, we have focused on stem cells for generating insulin-producing cells (IPCs). We investigated adipose-derived stem cells (ADSCs) as a new cell source[5,6], for their trophic effects and for their multi-potency We focused on these cells because ADSCs can be obtained from the patient’s own fat tissue under local anaesthesia, and auto-ADSC transplantation has fewer ethical problems compared with the use of induced pluripotent stem or embryonic stem cells. CellSaic is xeno-antigen free and clinical grade has been achieved[23]
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