Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.
OBJECTIVE
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 microg/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.
Division of Developmental Biology, Department of Parasitology, Nara Medical University, Kashihara, Nara, Japan.
Akira
A
Shiroi
Masahide
M
Yoshikawa
Hiroshi
H
Yokota
Hiroshi
H
Fukui
Shigeaki
S
Ishizaka
Kouko
K
Tatsumi
Yoshiko
Y
Takahashi
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods* / Q000639:trends; D002478:Cells, Cultured; D002614:Chelating Agents; D003920:Diabetes Mellitus / Q000628:therapy*; D004230:Dithizone; D005260:Female; D051273:Glucose Transporter Type 2; D005952:Glucose-6-Phosphatase; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D011384:Proinsulin / Q000235:genetics; D011506:Proteins / Q000235:genetics; D033581:Stem Cell Transplantation / Q000379:methods* / Q000639:trends; D039901:Totipotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D015032:Zinc / Q000378:metabolism
D002614:Chelating Agents; D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D011506:Proteins; D004230:Dithizone; D011384:Proinsulin; D005952:Glucose-6-Phosphatase; C117983:G6pc2 protein, mouse; D015032:Zinc