title: Differentiation of Human-Induced Pluripotent Stem Cells Into Insulin-Producing Clusters by MicroRNA-7.
abstract: OBJECTIVE
  Diabetes results from inadequate insulin production from pancreatic β-cells. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all diabetic patients. Thus, finding a new source with effective maturation of β-cells is the major goal of many studies. MicroRNAs are a class of small noncoding ribonucleic acid that regulate gene expression through posttranscriptional mechanisms. MicroRNA-7 has high expression level during pancreatic islet development in humans, thereby playing a critical role in pancreatic β-cell function. We study aimed to develop a protocol to differentiate human-induced pluripotent stem cells efficiently into isletlike cell clusters in vitro by using microRNA-7.
  
  
  METHODS
  Human-induced pluripotent stem cell colonies were transfected with hsa-microRNA-7 by using siPORT NeoFX transfection agent. Total ribonucleic acid was extracted 24 and 48 hours after transfection. The expression of transcription factors which were important during pancreases development was also performed. On the third day, the potency of the clusters was assessed in response to high glucose levels. Diphenylthiocarbazone was used to identify the existence of the β-cells. The presence of insulin and Neurogenin-3 proteins was investigated by immunocytochemistry.
  
  
  RESULTS
  Morphologic changes were observed on the first day after chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The isletlike cell clusters were positive for insulin and Neurogenin-3 proteins in immunocytochemistry. The clusters were stained with Diphenylthiocarbazone and secreted insulin in a glucose challenge test.
  
  
  CONCLUSIONS
  MicroRNA-7 transcription factor network is important in pancreatic endocrine differentiation. Chemical transfection with microRNA-7 can differentiate human induced pluripotent stem cells into functional isletlike cell clusters in a short time.
authors.affiliation: From the Department of Genetics, Zarghan Branch, Islamic Azad University, Fars, Iran; and Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Anahita
authors.initials: A
authors.lastname: Shaer
authors.forename: Negar
authors.initials: N
authors.lastname: Azarpira
authors.forename: Mohammad Hosein
authors.initials: MH
authors.lastname: Karimi
authors.forename: Masood
authors.initials: M
authors.lastname: Soleimani
authors.forename: Sadrollah
authors.initials: S
authors.lastname: Dehghan
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D048430:Cell Shape; D005786:Gene Expression Regulation; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010641:Phenotype; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D007328:Insulin; D035683:MicroRNAs; D009419:Nerve Tissue Proteins; C549242:MIRN7-1 microRNA, human; C498695:NEUROG3 protein, human