Characterization and reduction of non-endocrine cells accompanying islet-like endocrine cells differentiated from human iPSC.

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.

Microwell bag culture for large-scale production of homogeneous islet-like clusters.

Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 105-106 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 (105-106 clusters). We prepared small-scale culture bags (< 105 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 × 105 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.

Fabrication of cultured oral gingiva by tissue engineering techniques without materials of animal origin.

BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.

Involvement of cholinergic neurons in the regulation of the ghrelin secretory response to feeding in sheep.

We previously demonstrated that a transient surge in plasma levels of ghrelin occurs just prior to a scheduled meal and that this surge is modified by the feeding regimen. This suggests that the ghrelin secretion is regulated by the autonomic nervous system, especially the cholinergic projections to the stomach. To test this hypothesis, we investigated changes in plasma ghrelin levels at feeding time in rams by administering cholinergic blockers (atropine and hexamethonium) and a cholinergic accelerator (metoclopramide). The average food intake in each group infused with atropine, hexamethonium, metoclopramide, and saline was 150+/-28, 137+/-46, 153+/-50, and 1075+/-25g, respectively. Plasma ghrelin concentrations increased (P<0.05) after i.v. infusion of hexamethonium and gradually decreased (P<0.05) after i.v. infusion of metoclopramide. Plasma ghrelin levels in hexamethonium-treated animals were greater (P<0.05) than those of atropine-treated animals. Plasma ghrelin levels were significantly (P<0.05) higher in sheep given i.v. infusions of atropine or hexamethonium than the levels in normal- or pair-fed sheep infused with saline. Plasma ghrelin levels were similar in metoclopramide-treated, pair-fed, and control animals. These results support the possibility that ghrelin secretion is regulated by cholinergic neurons of the vagus and that cholinergic activity suppresses ghrelin secretion in sheep.

A transient surge of ghrelin secretion before feeding is modified by different feeding regimens in sheep.

Ghrelin is a recently identified orexigenic hormone secreted by the stomach and has been implicated in meal-time hunger. Several experiments demonstrate a transient surge in ghrelin secretion shortly before a scheduled meal, suggesting from the involvement of cephalic mechanisms. If ghrelin secretion is stimulated by hunger in sheep, plasma levels of ghrelin should be modified by different feeding regimens that affect hunger drive. To test this hypothesis, we investigated changes in plasma ghrelin concentrations in fed Suffolk rams ad libitum and in rams either twice or four times daily. Plasma ghrelin levels increased (P<0.05) abruptly just before every feeding period in sheep fed twice and four times daily and then fell shortly after feeding. Peak levels of the pre-prandial ghrelin surge were higher (P<0.01) in animals fed twice daily than in animals fed four times daily, leading to greater (P<0.05) areas under response curves over 12h. In contrast, the plasma ghrelin levels remained relatively low and constant in sheep fed ad libitum, with no evidence of surges in plasma ghrelin levels. These results confirm that the transient surge in plasma ghrelin levels occurs just before feeding and demonstrate that this can be modified by the feeding regimen in sheep.