Open AccessIdentification of Insulin‐Producing Cells Derived from Embryonic Stem Cells by Zinc‐Chelating Dithizone
Author(s) -
Shiroi Akira,
Yoshikawa Masahide,
Yokota Hiroshi,
Fukui Hiroshi,
Ishizaka Shigeaki,
Tatsumi Kouko,
Takahashi Yoshiko
Publication year - 2002
Publication title -
stem cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.159
H-Index - 229
eISSN - 1549-4918
pISSN - 1066-5099
DOI - 10.1634/stemcells.20-4-284
Subject(s) - dithizone , biology , embryonic stem cell , stem cell , zinc , chelation , microbiology and biotechnology , induced pluripotent stem cell , staining , cell culture , in vitro , cell , regenerative medicine , silver stain , biochemistry , genetics , chemistry , organic chemistry , gene , chromatography
Background and Aims . Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc‐chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ‐stained cellular clusters originating from ES cells. Methods . Embryoid bodies (EBs), formed by a 5‐day hanging drop culture of ES cells, were allowed to form outgrowths in the culture. The outgrowths were incubated in DTZ solution (final concentration, 100 μg/ml) for 15 minutes before being examined microscopically. The gene expression of endocrine pancreatic markers was also analyzed by reverse transcriptase‐polymerase chain reaction. In addition, insulin production was examined immunohistochemically, and its secretion was examined using enzyme‐linked immunosorbent assay. Results . DTZ‐stained cellular clusters appeared after approximately 16 days in the EB culture and became more apparent by day 23. They were found to be immunoreactive to insulin and expressed pancreatic‐duodenal homeobox 1 (PDX1), proinsulin 1, proinsulin 2, glucagon, pancreatic polypeptide, glucose transporter‐2 (GLUT2), and islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP) mRNA. They were also able to secrete detectable amounts of insulin. Conclusions . ES cell‐derived DTZ‐positive cellular clusters possess characteristics of the endocrine pancreas, including insulin secretion. Further, DTZ staining is a useful method for the identification of differentiated pancreatic islets developed from EBs in vitro.