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

Shogo Ohta,Yu Saito,Mitsuo Shimada,Yuji Morine,Tetsuya Ikemoto,Satoru Imura,Yuma Wada,Shinichiro Yamada

doi:10.1038/s41598-019-55172-0

Shogo Ohta, Yu Saito + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-55172-0

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Journal: Scientific Reports	Publication Date: Dec 1, 2019
Citations: 17	License type: open-access

Affiliation: Tokushima University

Abstract

The generation of insulin-producing cells (IPCs) from pluripotent stem cells could be a breakthrough treatment for type 1 diabetes. However, development of new techniques is needed to exclude immature cells for clinical application. Dithizone staining is used to evaluate IPCs by detecting zinc. We hypothesised that zinc ion (Zn2+) dynamics reflect the IPC maturation status. Human adipose-derived stem cells were differentiated into IPCs by our two-step protocol using two-dimensional (2D) or 3D culture. The stimulation indexes of 2D -and 3D-cultured IPCs on day 21 were 1.21 and 3.64 (P < 0.05), respectively. The 3D-cultured IPCs were stained with dithizone during culture, and its intensity calculated by ImageJ reached the peak on day 17 (P < 0.05). Blood glucose levels of streptozotocin-induced diabetic nude mice were normalised (4/4,100%) after transplantation of 96 3D-cultured IPCs. Zn2+ concentration changes in the medium of 3D cultures had a negative value in the early period and a large positive value in the latter period. This study suggests that Zn2+ dynamics based on our observations and staining of zinc transporters have critical roles in the differentiation of IPCs, and that their measurement might be useful to evaluate IPC maturation as a non-destructive method.

Highlights

The generation of insulin-producing cells (IPCs) from pluripotent stem cells could be a breakthrough treatment for type 1 diabetes
Adipose-derived stem cells (ADSCs) can be procured less invasively and without ethical problems compared with some types of PSCs12
IPCs generated in 3D culture exhibited sphere-like formations until 21 days, whereas cell clusters had formed at around day 21 in conventional 2D culture (Fig. 1B)

Summary

Introduction

The generation of insulin-producing cells (IPCs) from pluripotent stem cells could be a breakthrough treatment for type 1 diabetes. Human adipose-derived stem cells were differentiated into IPCs by our two-step protocol using two-dimensional (2D) or 3D culture. The generation of insulin producing cells (IPCs) derived from pluripotent stem cells (PSCs) has the possibility to resolve the issue of donor shortages for islet transplantation in some countries[1]. 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. IPC maturation has been induced in vivo at a few months after transplantation[3,18]. 0.2 μm) in the cytoplasm of 3D-cultured IPCs on day 21, resembling secretory granules observed in human naïve β-cells[25] (right).

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