Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

The small intestine and liver play important role in determining oral drug’s fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, Cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid (ARA), enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

DOI： 10.1093/pnasnexus/pgae070

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Language：English   Publishing type：Research paper (scientific journal)  

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC-MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.

DOI： 10.1016/j.heliyon.2024.e24590

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Type 1 diabetes mellitus (T1DM) affects 8.4 million people worldwide, with patients primarily relying on exogenous insulin injections to maintain blood glucose levels. Islet transplantation via the portal vein has allowed for the direct internal release of insulin by glucose-sensitive islets. However, this method might not be desirable for future cell therapy transplanting pluripotent stem cell-derived β cells, facing challenges including difficulties in cell retrieval and graft loss due to the instant blood-mediated inflammatory reaction (IBMIR). Here, we established a subcutaneous transplantation protocol using an atelocollagen sponge as a scaffold. While the subcutaneous site has many advantages, the lack of a vascular bed limits its application. To address this issue, we performed angiogenesis stimulation at the transplantation site using bFGF absorbed in a gelatin sponge (Spongel), significantly improving the microvascular area. Our in vivo experiments also revealed angiogenesis stimulation is crucial for reversing hyperglycemia in streptozotocin (STZ)-induced diabetic mice. In addition to the angiogenic treatment, an atelocollagen sponge is used to carry the islets and helps avoid graft leakage. With 800 mouse islets delivered by the atelocollagen sponge, the STZ-induced diabetic mice showed a reversal of hyperglycemia and normalized glucose intolerance. Their normoglycemia was maintained until the graft was removed. Analysis of the harvested islet grafts exhibited a high vascularization and preserved morphologies, suggesting that using an atelocollagen sponge as a scaffold helps maintain the viability of the islet grafts.

DOI： 10.1177/09636897241277980

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2023.08.064

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Language：English   Publishing type：Research paper (scientific journal)  

Primary Human Hepatocyte (PHH) remains undefeated as the gold standard in hepatic studies. Despite its valuable properties, partial attachment loss due to the extraction process and cryopreservation remained the main hurdle in its application. We hypothesized that we could overcome the loss of PHH cell attachment through thawing protocol adjustment and medium composition. We reported a novel use of a medium designed for iPSC-derived hepatocytes, increasing PHH attachment on the collagen matrix. Delving further into the medium composition, we discovered that removing BSA and exposure to cAMP activators such as IBMX and Forskolin benefit PHH attachment. We found that activating EPAC2, the cAMP downstream effector, by S-220 significantly increased PHH attachment. We also found that EPAC2 activation induced bile canaliculi formation in iPS-derived hepatocytes. Combining these factors in studies involving PHH or iPS-hepatocyte culture provides promising means to improve cell attachment and maintenance of hepatic function.

DOI： 10.1038/s41598-023-39712-3

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Language：English   Publishing type：Research paper (scientific journal)  

Wolfram Syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1 proteins by studying the stability of full-length wild-type WFS1, a missense mutant P724L, and two C-terminally truncated mutants, W837X and Y652X. We compared their stability by overexpressing them in MIN6 and HEK293T cells. The C-terminally truncated mutants W837X and Y652X are degraded more rapidly than the missense P724L mutant or wild-type WFS1 in MIN6 cells. In contrast, Y652X is more stable than WT or other mutant WFS1 proteins in HEK293T. In conclusion, we found that C-terminally truncated WFS1 mutants are selectively degraded in a cell type-specific manner.

DOI： 10.1002/2211-5463.13674

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.xpro.2023.102183

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cytochrome P450 3A4 (CYP3A4) is involved in first-pass metabolism in the small intestine and is heavily implicated in oral drug bioavailability and pharmacokinetics. We previously reported that Vitamin D3 (VD3), a known CYP enzyme inducer, induces functional maturation of iPSC-derived enterocyte-like cells (iPSC-ent). Here, we identified a Notch activator and CYP modulator Valproic Acid (VPA), as a promotor for the maturation of iPSC-ent. We performed bulk RNA sequencing to investigate the changes in gene expression during the differentiation and maturation periods of these cells. VPA potentiated gene expression of key enterocyte markers ALPI, FABP2, and transporters such as SULT1B1. RNA sequencing analysis further elucidated several function-related pathways involved in fatty acid metabolism, significantly upregulated by VPA when combined with VD3. Particularly, VPA treatment in tandem with VD3 significantly upregulated key regulators of enterohepatic circulation, such as FGF19, apical bile acid transporter SLCO1A2 and basolateral bile acid transporters SLC51A and SLC51B. To sum up, we could ascertain the genetic profile of our iPSC-ent cells to be specialized towards fatty acid absorption and metabolism instead of transporting other nutrients, such as amino acids, with the addition of VD3 and VPA in tandem. Together, these results suggest the possible application of VPA-treated iPSC-ent for modelling enterohepatic circulation.

DOI： 10.1093/stmcls/sxad042

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/stmcls/sxac082

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Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

ABSTRACT

Traditional cell culture media do not accurately represent the availability of the nutrients in plasma. They usually contain a supraphysiological concentration of nutrients such as glucose, amino acids, etc. These high nutrients can alter the metabolism of cultured cells and induce metabolic phenotypes that do not reflect in vivo conditions. We demonstrate that the supraphysiological levels of nutrients interfere with endodermal differentiation. Refinement of media formulations has a potential application in maturity modulation of stem cell-derived β-cells (SC-β) generation in vitro. To address these issues, we established a defined culture system to derive SC-β-cells using a blood amino acids-like medium (BALM). Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into the definitive endoderm, pancreatic progenitors, endocrine progenitors, and SC-β in BALM-based med. The differentiated cells secreted C-peptide in vitro in response to high glucose levels and expressed several pancreatic β-cell markers. In conclusion, amino acids at the physiological levels are sufficient for deriving functional SC-β cells.

DOI： 10.1242/bio.059581

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Other Link： https://journals.logists.com/bio/bio/article-pdf/doi/10.1242/bio.059581/2647580/bio059581.pdf

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Pluripotent stem cells (PSCs) exhibit a unique feature that requires S-adenosylmethionine (SAM) for the maintenance of their pluripotency. Methionine deprivation in the medium causes a reduction in intracellular SAM, thus rendering PSCs in a state potentiated for differentiation. In this study, we find that methionine deprivation triggers a reduction in intracellular protein-bound Zn content and upregulation of Zn exporter SLC30A1 in PSCs. Culturing PSCs in Zn-deprived medium results in decreased intracellular protein-bound Zn content, reduced cell growth, and potentiated differentiation, which partially mimics methionine deprivation. PSCs cultured under Zn deprivation exhibit an altered methionine metabolism-related metabolite profile. We conclude that methionine deprivation potentiates differentiation partly by lowering cellular Zn content. We establish a protocol to generate functional pancreatic β cells by applying methionine and Zn deprivation. Our results reveal a link between Zn signaling and methionine metabolism in the regulation of cell fate in PSCs.

DOI： 10.1016/j.celrep.2022.111120

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Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

DOI： 10.1038/s41598-021-84861-y

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We aimed to establish an in vitro differentiation procedure to generate matured small intestinal cells mimicking human small intestine from human-induced pluripotent stem cells (iPSCs). We previously reported the efficient generation of CDX2-expressing intestinal progenitor cells from embryonic stem cells (ESCs) using 6-bromoindirubin-3'-oxime (BIO) and (3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT) to treat definitive endodermal cells. Here, we demonstrate the generation of enterocyte-like cells by culturing human iPSC-derived intestinal progenitor cells on a collagen vitrigel membrane (CVM) and treating cells with a simple maturation medium containing BIO, DMSO, dexamethasone, and activated vitamin D3. Functional tests further confirmed that these iPSC-derived enterocyte-like cells exhibit P-gp- and BCRP-mediated efflux and cytochrome P450 3A4 (CYP3A4)-mediated metabolism. We concluded that hiPS cell-derived enterocyte-like cells can be used as a model for the evaluation of drug transport and metabolism studies in the human small intestine.

DOI： 10.1016/j.stemcr.2020.12.017

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Human pluripotent stem cells (PSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising cell sources in regenerating pancreatic islets through in vitro directed differentiation. Recent progress in this research field has made it possible to generate glucose-responsive pancreatic islet cells from PSCs. Single-cell RNA sequencing techniques have been applied to analyze PSC-derived endocrine beta-cells, which are then compared with human islets. This has led to the identification of novel signaling pathways and molecules involved in lineage commitment during pancreatic differentiation and maturation processes. Single-cell transcriptomics are also used to construct a detailed map of in vivo endocrine differentiation of developing mouse embryos to study pancreatic islet development. Mimicking those occurring in vivo, it was reported that differentiating PSCs can generate similar islet cell structures, while metabolomics analysis highlighted key components involved in PSC-derived pancreatic islet cell function, providing information for the improvement of in vitro pancreatic maturation procedures. In addition, cell transplantation into diabetic animal models, together with the cell delivery system, is studied to ensure the therapeutic potentials of PSC-derived pancreatic islet cells. Combined with gene-editing technology, the engineered mutation-corrected PSC lines originated from diabetes patients could be differentiated into functional pancreatic islet cells, suggesting possible autologous cell therapy in the future. These PSC-derived pancreatic islet cells are a potential tool for studies of disease modeling and drug testing. Herein, we outlined the directed differentiation procedures of PSC-derived pancreatic islet cells, novel findings through transcriptome and metabolome studies, and recent progress in disease modeling.

DOI： 10.1186/s41232-020-00152-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Diabetes Association  

Vesicular monoamine transporter 2 (VMAT2) uptakes cytoplasmic monoamines into vesicles for storage. VMAT2 plays a role in modulating insulin release by regulating dopamine levels in the pancreas, although the exact mechanism remains elusive. We found that VMAT2 expression in β-cells specifically increases under high blood glucose conditions. The islets isolated from β-cell-specific Vmat2 knockout (βVmat2KO) mice show elevated insulin secretion levels in response to glucose stimulation. Under prolonged high-fat diet feedings, the βVmat2KO mice exhibit impaired glucose and insulin tolerance and progressive β-cell dysfunction. Here we demonstrate VMAT2 uptake of dopamine to protect dopamine from degradation by monoamine oxidase, thereby safeguarding β-cells from excess reactive oxygen species (ROS) exposure. In the context of high demand for insulin secretion, the absence of VMAT2 leads to elevated ROS in β-cells, which accelerates β-cell dedifferentiation and β-cell loss. Therefore, VMAT2 controls the amount of dopamine in β-cells, thereby protecting pancreatic β-cells from excessive oxidative stress.

DOI： 10.2337/db20-0207

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Other Link： https://syndication.highwire.org/content/doi/10.2337/db20-0207

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Pancreatic cells derived from pluripotent stem cells using the stem cell-derived-β (SC-β) differentiation protocol are composed of four main cell types, namely β-like cells (SC-β-cells), α-like cells, enterochromaffin-like cells and non-endocrine cells. Single-cell dissociation and reaggregation depleted non-endocrine cells and improved β-cell function. Veres et al. succeeded in efficiently purifying SC-β by magnetic cell sorting using CD49a antibody and reaggregation, so that the final SC-β-cell ratio increased to 80%.

DOI： 10.1111/jdi.13140

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/jdi.13140

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We recently reported that a treatment with tauroursodeoxycholic acid (TUDCA), a secondary bile acid, improved developmentally-deteriorated hepatic steatosis in an undernourishment (UN, 40% caloric restriction) in utero mouse model after a postnatal high-fat diet (HFD). We performed a microarray analysis and focused on two genes (Cidea and Cidec) because they are enhancers of lipid droplet (LD) sizes in hepatocytes and showed the greatest up-regulation in expression by UN that were completely recovered by TUDCA, concomitant with parallel changes in LD sizes. TUDCA remodeled developmentally-induced histone modifications (dimethylation of H3K4, H3K27, or H3K36), but not DNA methylation, around the Cidea and Cidec genes in UN pups only. Changes in these histone modifications may contribute to the markedly down-regulated expression of Cidea and Cidec genes in UN pups, which was observed in the alleviation of hepatic fat deposition, even under HFD. These results provide an insight into the future of precision medicine for developmentally-programmed hepatic steatosis by targeting histone modifications.

DOI： 10.1038/s41598-019-52943-7

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We previously demonstrated that androgen signaling expands pancreatic β-cell mass in the sexual maturation period (Am J Physiol Endocrinol Metab 314: E274-E286, 2018). The aim of this study was to elucidate whether fetal androgen signaling plays important roles in β-cell mass development and β-cell function in adulthood, defects of which are associated with type 2 diabetes mellitus. In the pancreas of male fetuses, androgen receptor (AR) was strongly expressed in the cytoplasm and at the cell membrane of Nkx6.1-positive β-cell precursor cells but was markedly reduced in insulin-positive β-cells. Administration of the anti-androgen flutamide to pregnant dams during late gestation reduced β-cell mass and Ki67-positive proliferating β-cells at birth in a male-specific manner without affecting body weight. The decrease of β-cell mass in flutamide-exposed male rats was not recovered when rats were fed a standard diet, whereas it was fully recovered when rats were fed a high-fat diet (HFD), at 6 and 12 wk of age. Flutamide exposure in utero led to the development of glucose intolerance in male rats due to a decrease in insulin secretion when fed HFD but not standard diet. Insulin sensitivity did not differ between the two groups irrespective of diet. These results indicated that the action of fetal androgen contributed to β-cell mass expansion in a sex-specific manner at birth and to the development of glucose intolerance by decreasing the secretion of insulin in HFD-fed male rats. Our data demonstrated the involvement of fetal androgen signaling in hypothesized sex differences in the developmental origins of health and disease by affecting pancreatic β-cell function.

DOI： 10.1152/ajpendo.00173.2019

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BACKGROUND: Management of vascular access (VA) is essential in hemodialysis (HD) patients. However, VA often fails and percutaneous transluminal angioplasty (PTA) is required. Conventional hemostasis at the puncture site is associated with complications. This study aimed to analyze the efficacy and safety of a hemostatic wound dressing made of calcium alginate at the puncture site of VA after PTA and evaluate other factors affecting hemostasis. METHODS: After PTA for VA, 200 HD patients were randomized to a calcium alginate sheet (CA) group (n = 100) or a no drug-eluting sheet (control) group (n = 100). We recorded time to hemostasis at the puncture site every 5 min, noting any complications. RESULTS: In the CA group, rates of hemostatic achievement at 5, 10, 15 and >15 min were 57, 25, 8 and 10%, respectively. In the control group, the rates were 39, 28, 14 and 19%, respectively. Rates of hemostatic achievement at 5 min were significantly higher in the CA group (P = 0.01). In logistic regression analysis, factors affecting hemostasis within 5 min were use of the CA sheet [odds ratio (OR) 2.33; 95% confidence interval (CI) 1.26-4.37], platelet count ≤100 000/μL (OR 0.19; 95% CI 0.04-0.69), number of antithrombotic tablets used per day ≥1 tablet (OR 0.50; 95% CI 0.26-0.94) and upper arm VA (OR 0.16; 95% CI 0.03-0.55). CONCLUSIONS: A CA sheet can safely reduce time to hemostasis at the puncture site after PTA, and should be considered for treating patients with a bleeding tendency.

DOI： 10.1093/ndt/gfy143

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

Differentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of AFP decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as ALB, ASGR1 Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.

DOI： 10.1242/bio.042192

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Language：Japanese   Publisher：日本組織培養学会  

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Insulin-producing and -secreting cells derived from mouse pluripotent stem cells (PSCs) are useful for pancreatic development research and evaluating drugs that may induce insulin secretion. Previously, we have established a differentiation protocol to derive insulin-secreting cells from mouse embryonic stem cells (ESCs) using a combination of growth factors, recombinant proteins, and a culture substratum with net-like fibers. However, it has not been tested which materials and diameters of these fibers are more effective for the differentiation. Therefore, the present study aimed to produce net-like culture substratum formed from polyamide (PA) and polyacrylonitrile (PAN) fibers. Substrata were delineated into PA100, 300, 600, PAN100, 300, and 600 groups based on fiber diameters. The differentiation efficiencies of mouse ESCs cultured on the substrata were then examined by insulin 1 (Ins1) expression. Expression was found to be highest in PA300 differentiated cells, indicating the potential to produce high levels of insulin. To understand any differences in substratum properties, the adsorption capacities of laminin were measured, revealing that PA300 had the highest for it. We next examined the stage of differentiation affected by incubation with PA300. This showed that Sox17- and Pdx1-GFP-positive cells increased during the first step of differentiation. To show the production of insulin without absorption from the medium, we confirmed the expression of insulin C-peptide after differentiation. Finally, we tested the effects of PA300 on the differentiation of human-induced PSC, and found more Sox17-positive cells with the PA300 substratum at the definitive endoderm stage. Furthermore, these cells expressed insulin C-peptide and had glucose-responsive C-peptide secretion. In summary, our study identified and validated a novel substratum which is suitable for pancreatic differentiation of mouse and human PSCs.

DOI： 10.1088/1748-605X/ab261c

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Pluripotent stem cells, including induced pluripotent stem (iPS) cells, are promising cell sources for regenerative medicine to replace injured tissues, and tissue engineering technologies enable engraftment of functional iPS cell-derived cells in vivo for prolonged periods. However, the risk of tumor formation is a concern for the use of iPS cells. Bioengineered tissues provide a suitable environment for cell survival, which requires vigorous efforts to eliminate remaining iPS cells and prevent tumor formation. We recently reported three iPS cell elimination strategies, including methionine-free medium, TRPV1 activation through 42°C cultivation, and dinaciclib, a cyclin-dependent kinase 1/9 inhibitor. However, it remains unclear how many iPS cells in bioengineered tissues can be eliminated using these strategies alone or in combination, as well as the mode of subsequent tumor prevention. In the present study, we found that 2 days of cultivation at 42°C sufficiently eliminated 1 × 102 iPS cells in fibroblast sheets and prevented tumor formation. After screening for suitable combinations of these strategies based on Lin28 expression in co-cultures of fibroblasts and 1 × 104 iPS cells, we found that 1 day of cultivation at 42°C in methionine-free culture medium with or without dinaciclib remarkably decreased Lin28 expression and prevented tumor formation. Furthermore, these culture strategies did not affect spontaneous beating or the cell number of human iPS cell-derived cardiomyocytes. These quantitative findings may contribute to decreasing tumor formation risk and development of regenerative medicine using iPS cells.

DOI： 10.1089/ten.TEC.2018.0228

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A deficient pancreatic β-cell mass increases the risk of type 2 diabetes mellitus. Here, we investigated the effects of testosterone on the development of pancreatic β-cell mass in male rats. The β-cell mass of male rats castrated at 6 wk of age was reduced to ~30% of that of control rats at 16 wk of age, and castration caused glucose intolerance. Loss of β-cell mass occurred because of decreases in islet density per pancreas and β-cell cluster size. Castration was negatively associated with the number of Ki-67-positive β-cells and positively associated with the number of TUNEL-positive β-cells. These β-cell changes could be prevented by testosterone treatment. In contrast, castration did not affect β-cell mass in male mice. Androgen receptor (AR) localized differently in mouse and rat β-cells. Testosterone enhanced the viability of INS-1 and INS-1 #6, which expresses high levels of AR, in rat β-cell lines. siRNA-mediated AR knockdown or AR antagonism with hydroxyflutamide attenuated this enhancement. Moreover, testosterone did not stimulate INS-1 β-cell viability under high d-glucose conditions. In INS-1 β-cells, d-glucose dose dependently (5.5-22.2 mM) downregulated AR protein levels both in the presence and absence of testosterone. The intracellular calcium chelator (BAPTA-AM) could prevent this decrease in AR expression. AR levels were also reduced by a calcium ionophore (A23187), but not by insulin, in the absence of the proteasome inhibitor MG132. Our results indicate that testosterone regulates β-cell mass, at least in part, by AR activation in the β-cells of male rats and that the β-cell AR is degraded under hyperglycemic conditions.

DOI： 10.1152/ajpendo.00211.2017

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DOI： 10.1152/ajpcell.00071.2016

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Other Link： http://orcid.org/0000-0001-6708-9976

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DOI： 10.1186/s12896-017-0331-z

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DOI： 10.1016/j.stemcr.2016.05.015

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DOI： 10.1186/s12861-016-0120-2

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Umeda K, Shiraki N, Kume S, Methods in molecular biology (Clifton, N.J.), 2016, vol. 1357, pp. 375-381

DOI： 10.1007/7651_2014_146

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Yamazoe T, Shiraki N, Kume S, Methods in molecular biology (Clifton, N.J.), 2016, vol. 1357, pp. 475-483

DOI： 10.1007/7651_2014_138

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Publishing type：Research paper (scientific journal)   Publisher：OMICS Publishing Group  

DOI： 10.4172/2155-9872.1000295

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Human embryonic stem cells (ESCs) show a characteristic feature in that they are highly dependent on methionine metabolism. Undifferentiated human ESCs cannot survive under condition that methionine is deprived from culture medium. We describe here a procedure for definitive endoderm differentiation from human ESCs, in which human ESCs are subject to 10 days' (d) differentiation combined with methionine deprivation between differentiation days (d) 8 to (d) 10. Methionine deprivation results in elimination of undifferentiated cells from the culture with no significant loss of definitive endoderm cells, as compared to those cultured under complete condition throughout the whole culture period.

DOI： 10.1007/7651_2015_272

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Sakano D, Shiraki N, Kume S, Methods in molecular biology (Clifton, N.J.), 2016, vol. 1341, pp. 417-423

DOI： 10.1007/7651_2015_217

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DOI： 10.1111/gtc.12308

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Shiraki N, Kume S, Nihon rinsho. Japanese journal of clinical medicine, 2015, vol. 73, no. 5, pp. 765-772

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DOI： 10.1093/jmcb/mju029

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DOI： 10.1016/j.cmet.2014.03.017

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DOI： 10.1074/jbc.M113.524363

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DOI： 10.1371/journal.pone.0095451

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DOI： 10.1016/j.scr.2014.01.004

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DOI： 10.1111/j.1365-2443.2008.01201.x

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DOI： 10.1634/stemeells.2007-0608

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Kume S, Shiraki N, Rinsho byori. The Japanese journal of clinical pathology, 2006, vol. 54, no. 4, pp. 386-392

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DOI： 10.1111/j.1365-2443.2005.00854.x

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DOI： 10.1248/bpb.23.1528

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DOI： 10.14952/SEIKAGAKU.2017.890903

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Human embryonic stem cells (ESCs) show a characteristic feature in that they are highly dependent on methionine metabolism. Undifferentiated human ESCs cannot survive under the condition that methionine is deprived from culture medium. We describe here a procedure for definitive endoderm differentiation from human ESCs, in which human ESCs are subject to 10 days (d) differentiation combined with methionine deprivation between differentiation day (d) 8 to d10. Methionine deprivation results in elimination of undifferentiated cells from the culture with no significant loss of definitive endoderm cells, as compared to those cultured under complete condition throughout the whole culture period.

DOI： 10.1007/7651_2015_224

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DOI： 10.1002/stem.1344

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DOI： 10.14894/faruawpsj.48.9_852

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DOI： 10.11213/tonyobyo.54.268

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Other Link： http://search.jamas.or.jp/link/ui/2010265250

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Language：Japanese   Publisher：The Japanese Society of Internal Medicine  

DOI： 10.2169/naika.98.3148

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DOI： 10.11477/mf.1542102092

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Language：Japanese   Publisher：Japanese Society of Pharmaceutical Health Care and Sciences  

We investigated the cytotoxicity of erythromycin stearate, roxithromycin, clarithromycin and minocycline using lactate dehydrogenase (LDH) in cultured human intestinal epithelial cells Caco- 2, in vitro, and rat intestinal enzymes LDH and alkaline phosphatase (ALP) as end points of toxicity, in viro. The cytotoxicity after the addition of roxithromycin and clarithromycin to the Caco- 2 cells tended to be larger than that after the addition of erythromycin and minocycline. The intestinal toxicity induced by repeated 5 -day orally administered macrolide antibiotics was examined. The reduction in intestinal ALP after the administration of erythromycin and clarithromycin to rats tended to be larger than that after the addition of roxithromycin and minocycline. However, there were no significant changes in the intestinal LDH when macrolide antibiotics were orally administered. In addition, there were no significant changes or differences in either the body weight gain or the histological findings of the rat intestine. These results do not suggest any substantial risk for intestinal disorders related to the use of erythromycin, clarithromycin and roxithromycin.

DOI： 10.5649/jjphcs.27.351

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Applicant：国立大学法人 熊本大学, 国立大学法人東京工業大学

Application no：JP2015064380  Date applied：2015.5

Announcement no：WO2015-178397  Date announced：2015.11

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Applicant：国立大学法人 熊本大学, 国立大学法人京都大学

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Announcement no：WO2012-150707  Date announced：2012.11

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Applicant：国立大学法人 熊本大学, 国立大学法人京都大学

Application no：特願2013-513086  Date applied：2012.5

Patent/Registration no：特許第6051378号  Date issued：2016.12

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Applicant：国立大学法人 熊本大学, LSIPファンド運営合同会社

Application no：特願2012-541844  Date applied：2011.10

Patent/Registration no：特許第5875007号  Date issued：2016.1

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Applicant：国立大学法人 熊本大学, つちやゴム株式会社

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Applicant：国立大学法人 熊本大学, 持立 克身

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Applicant：国立大学法人 熊本大学

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Patent/Registration no：特許第5589216号  Date issued：2014.8

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Applicant：国立大学法人 熊本大学

Application no：特願2007-517854  Date applied：2006.5

Patent/Registration no：特許第5092124号  Date issued：2012.9

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