Neurogenic differentiation 1 directs differentiation of cytokeratin 19-positive human pancreatic nonendocrine cells into insulin-producing cells.
BACKGROUND
It has been reported that the human pancreatic nonendocrine fraction, which remains after islet isolation, can be differentiated toward beta cells. However, the optimal method to accomplish this goal has not been established. In this study, we introduced the human neurogenic differentiation 1 (NeuroD1) gene into human nonendocrine pancreatic epithelial cells (NEPECs) and promoted insulin-producing cells in vitro.


METHODS
The human pancreatic nonislet fractions were obtained from brain-dead donors and cultured in suspension for 2-3 days followed by culture with G418 for 4 days. These cells (NEPECs) were then plated on dishes. The NEPECs spread into a cell monolayer within 7 days and all of the cells were cytokeratin-19 (CK19) positive. Seven days after plating, plasmids encoding human NeuroD1 gene under human CK19 promoter were transfected 3 times every other day (termed NEPEC+ND). Seven days after starting induction, these cells were characterized.


RESULTS
Seven days after starting the induction of human NeuroD1, NEPEC+ND strongly expressed NeuroD1 and insulin mRNA. The ratio of NeuroD1-positive cells in NEPEC+ND was significantly higher than in NEPEC. Human insulin-positive cells in NEPEC+ND were also significantly greater than in NEPEC. Human insulin and C-peptide levels in culture medium in NEPEC+ND were significantly higher than in NEPEC.


CONCLUSIONS
These findings demonstrated that human NeuroD1 under control of the CK19 promoter can induce the differentiation of CK19-positive NEPECs into insulin-producing cells.
Shimoda
M
M
Division of Cardiology, Department of Internal Medicine, Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, Texas, USA.
Chen
S
S
Noguchi
H
H
Matsumoto
S
S
Grayburn
P A
PA
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000494:pharmacology*; D001926:Brain Death; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division; D017931:DNA Primers; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D053539:Keratin-19 / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000494:pharmacology*; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D007328:Insulin; D053539:Keratin-19; D009419:Nerve Tissue Proteins; C093564:Neurogenic differentiation factor 1