title: A new system to evaluate the influence of immunosuppressive drugs on pancreatic islets using epigenetic analysis in a 3-dimensional culture.
abstract: OBJECTIVE
  The present study aimed to establish a new method to evaluate the influence of immunosuppressive drugs on pancreatic islets in short-term in vitro cultures using epigenetic analysis in a 3-dimensional culture.
  
  
  METHODS
  For this purpose, we selected (a) a 3-dimensional culture system utilizing thermoreversible gelation polymer, (b) pancreatic duodenal homeobox-1 (pdx-1)-Venus transgenic pigs expressing the green fluorescent protein, (c) FK506 as an immunosuppressive drug of the evaluation, and (d) the bisulfite sequencing technique to evaluate the methylation levels of pdx-1 and insulin genes. Each isolated pancreatic islet was cultured with several doses of FK506. The viability of the each islet was evaluated by analyzing the emission of Venus in real time and by propidium iodide staining. Epigenetic analysis was performed at several time points.
  
  
  RESULTS
  Each single pancreatic islet was stably cultured for 30 days in this system. At day 30 in culture, we observed that insulin DNA methylation levels in the group that received a high dose of FK506 dramatically increased, although there was no change in pdx-1 DNA methylation level and configuration of the islets.
  
  
  CONCLUSIONS
  Our system may be useful to determine immunosuppressive drugs that are specifically suitable for islet transplantation.
authors.affiliation: From the *Meiji University International Institute for Bio-Resource Research; †Department of Immunology, St Marianna University School of Medicine; and ‡Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagaya
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Arai
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsunari
authors.forename: Michiyo
authors.initials: M
authors.lastname: Honda
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nakano
authors.forename: Miki
authors.initials: M
authors.lastname: Maehara
authors.forename: Naomi
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Mirina
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Rieko
authors.initials: R
authors.lastname: Sakai
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Asano
authors.forename: Masahito
authors.initials: M
authors.lastname: Watanabe
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Umeyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019175:DNA Methylation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D013552:Swine; D016559:Tacrolimus / Q000494:pharmacology*; D013997:Time Factors; D046509:Tissue Culture Techniques; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; D007166:Immunosuppressive Agents; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins; D016559:Tacrolimus