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

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.

Syngeneically transplanted insulin producing cells differentiated from adipose derived stem cells undergo delayed damage by autoimmune responses in NOD mice

“…1 d). The staining intensity of these NOD-IPCs was very similar to the reported intensity of isolated fresh human islets (the intensity; 244) 8 . The mRNA expression of Insulin and MafA in the generated NOD-IPCs on day 28 was significantly higher than that in ADSCs (both P < 0.05, Mann–Whitney U test, Fig.…”

“…The SIs of the generated NOD-IPCs were comparable to those of previously reported human IPCs 5 – 10 . We previously reported that electron microscopy revealed that the nuclei of the IPCs contained dense cystic microstructures and organelles such as rough endoplasmic reticulum and mitochondria, and these dense cystic microstructures in the generated IPCs morphologically resembled insulin secretion granules observed in normal islets from naïve rat pancreas as a characteristic analysis 6 , 8 . Moreover, we described various positive markers, such as dithizone, C-peptide, insulin and PDX-1 morphological markers, and INS , MAFA , SLC39A4 , and SLC30A8 mRNA expression.…”

“…The mean gray saturation values at three random points in a spheroid area were measured in filtered images as the staining intensity. Dithizone staining intensity of highly functional IPCs was defined as > 180, as previously reported 8 . For the immunohistochemical analysis, sample images were filtered using a hue threshold of 20 to 28 after dividing images into three channels.…”

The Fragility of Cryopreserved Insulin-producing Cells Differentiated from Adipose-tissue-derived Stem Cells

“…This will be important when considering the transplantation of cryopreserved IPCs. We previously reported that IPCs with poor cell quality did not secrete sufficient amounts of insulin 5 , 32 and that the transplantation of such IPCs into streptozotocin-induced immunodeficient mice did not improve blood glucose levels 8 . We have already investigated the mechanism of this phenomenon in detail (in press).…”

Effective <i>in vitro</i> differentiation of adipose-derived stem cells into Schwann-like cells with folic acid supplementation

“…Adipose-derived stem cells (ADSCs) have been focused on because of their multipotency and ease of harvest. We have investigated ADSC multipotency and reported scientific evidence of insulin producing cells (IPCs) for clinical applications (7)(8)(9)(10)(11). Thus, we hypothesized that ADSCs can also self-renew and differentiate into ectodermal lineage cells, including Schwann-like cells (SLCs).…”