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Large numbers of hormone-releasing cells, approximately 10 9 endocrine cells, are required to treat type I diabetes patients by cell transplantation. The SOX9-positive pancreatic epithelium proliferates extensively during the early stages of pancreatic development. SOX9-positive pancreatic epithelium is thought to be an expandable cell source of β cells for transplantation therapy. In this study, we attempted to expand pancreatic progenitors (PPs: PDX1 + /SOX9 + ) derived from four human iPSC lines in three-dimensional (3D) culture using a chemically defined medium and examined the potential of the derived PPs to differentiate into β-like cells. PPs from four human iPSC lines were maintained and effectively proliferated in a chemically defined medium containing epidermal growth factor and R-spondin-1, CHIR99021, fibroblast growth factor-7, and SB431542. PPs derived from one iPSC line can be expanded by more than 10 4 -fold in chemically defined medium containing two of the fives, epidermal growth factor and R-spondin-1. The expanded PPs were also stable following cryopreservation. After freezing and thawing, the PPs proliferated without a decrease in the rate. PPs obtained after 50 days of culture successfully differentiated into insulin-positive β-like cells, glucagon-positive α-like cells, and somatostatin-positive δ-like cells. The differentiation efficiency of expanded PPs was similar to that of PPs without expansion culture.

Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10 5 –10 6 islet-like clusters per patient. Although thousands of uniform cell clusters can be produced using a conventional microwell plate, numerous obstacles need to be overcome for its clinical use. In this study, we aimed to develop a novel bag culture method for the production of uniform cell clusters on a large scale (10 5 –10 6 clusters). We prepared small-scale culture bags (< 10 5 clusters) with microwells at the bottom and optimized the conditions for producing uniform-sized clusters in the bag using undifferentiated induced pluripotent stem cells (iPSCs). Subsequently, we verified the suitability of the bag culture method using iPSC-derived pancreatic islet cells (iPICs) and successfully demonstrate the production of 6.5 × 10 5 uniform iPIC clusters using a large-scale bag. In addition, we simplified the pre- and post-process of the culture—a degassing process before cell seeding and a cluster harvesting process. In conclusion, compared with conventional methods, the cluster production method using bags exhibits improved scalability, sterility, and operability for both clinical and research use.

In the development of cell therapy products, immunocompromised animal models closer in size to humans are valuable for enhancing the translatability of in vivo findings to clinical trials. In the present study, we generated immunocompromised type 1 diabetic Göttingen mini-pig models and demonstrated the engraftment of human-induced pluripotent stem cell–derived pancreatic islet cells (iPICs). We induced hyperglycemia with a concomitant reduction in endogenous C-peptide levels in pigs that underwent thymectomy and splenectomy. After estimating the effective in vivo dose of immunosuppressants (ISs) via in vitro testing, we conducted exploratory implantation of iPICs using various implantation methods under IS treatments in one pig. Five weeks after implantation, histological analysis of the implanted iPICs embedded in fibrin gel revealed numerous islet-like structures with insulin-positive cells. Moreover, the area of the insulin-positive cells in the pre-peritoneally implanted grafts was greater than in the subcutaneously implanted grafts. Immunohistochemical analyses further revealed that these iPIC grafts contained cells positive for glucagon, somatostatin, and pancreatic polypeptides, similar to naturally occurring islets. The engraftment of iPICs was successfully reproduced. These data support the observation that the iPICs engrafted well, particularly in the pre-peritoneal space of the newly generated immunocompromised diabetic mini-pigs, forming islet-like endocrine clusters. Future evaluation of human cells in this immunocompromised pig model could accelerate and development of cell therapy products. Graphical Abstract

The differentiation of pancreatic endocrine cells from human pluripotent stem cells has been thoroughly investigated for their application in cell therapy against diabetes. Although non-endocrine cells are inevitable contaminating by-products of the differentiation process, a comprehensive profile of such cells is lacking. Therefore, we characterized non-endocrine cells in iPSC-derived pancreatic islet cells (iPIC) using single-cell transcriptomic analysis. We found that non-endocrine cells consist of (1) heterogeneous proliferating cells, and (2) cells with not only pancreatic traits but also liver or intestinal traits marked by FGB or AGR2 . Non-endocrine cells specifically expressed FGFR2 , PLK1 , and LDHB . We demonstrated that inhibition of pathways involving these genes selectively reduced the number of non-endocrine cells in the differentiation process. These findings provide useful insights into cell purification approaches and contribute to the improvement of the mass production of endocrine cells for stem cell-derived cell therapy for diabetes.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.