NeuroD and reaggregation induce beta-cell specific gene expression in cultured hepatocytes.
BACKGROUND
Our goal was to convert adult mouse hepatocytes to pancreatic beta-cells.


RESULTS
To facilitate conversion, cultured primary hepatocytes were dedifferentiated by the removal of dexamethasone (Dex) from the culture media. Removal of Dex caused detachment of hepatocytes from the culture dish, but the addition of betacellulin prevented this from happening. With the combination of lack of Dex and addition of betacellulin, albumin mRNA levels decreased. Cultured hepatocytes had a faint expression of insulin 2 mRNA, Nkx 6.1 and Pax 6 mRNA. Dedifferentiated hepatocytes were transduced with adenoviruses expressing NeuroD1, Ngn 3, or Pax 4. NeuroD1 transduction increased the insulin 2 mRNA but caused detachment of cells. However, when hepatocytes were allowed to reaggregate for 4 and 6 days in hydrophobic plates after transduction with NeuroD1, further increases of insulin 2 mRNA were found along with induction of PDX-1, IAPP, NeuroD1, Ngn3, Pax 4, Isl-1, PC1, PC2 and islet glucokinase. Additionally, glucagon, pancreatic polypeptide and somatostatin expression were induced, but neither elastase 1 nor insulin 1 mRNA could be detected. Ngn 3 and Pax 4 had effects similar to NeuroD1, but did not increase insulin 2 mRNA as much as NeuroD1.


CONCLUSIONS
We conclude that the combination of NeuroD1 and reaggregation promotes cultured dedifferentiated hepatocytes to differentiate towards a pancreatic beta-cell phenotype.
Yatoh
Shigeru
S
Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA.
Akashi
Tomoyuki
T
Chan
Phillip P
PP
Kaneto
Hideaki
H
Sharma
Arun
A
Bonner-Weir
Susan
S
Weir
Gordon C
GC
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D066259:Betacellulin; D002449:Cell Aggregation / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D020869:Gene Expression Profiling; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology*; D018398:Homeodomain Proteins / Q000096:biosynthesis; D007328:Insulin / Q000235:genetics; D050417:Insulin-Secreting Cells / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D051761:Paired Box Transcription Factors / Q000096:biosynthesis; D012709:Serum Albumin / Q000096:biosynthesis; D015534:Trans-Activators / Q000096:biosynthesis; D014161:Transduction, Genetic; D000509:alpha-Fetoproteins / Q000096:biosynthesis
D051792:Basic Helix-Loop-Helix Proteins; D066259:Betacellulin; D003907:Dexamethasone; D017228:Hepatocyte Growth Factor; D018398:Homeodomain Proteins; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D051761:Paired Box Transcription Factors; D012709:Serum Albumin; D015534:Trans-Activators; D000509:alpha-Fetoproteins; C587851:BTC protein, human; C587852:Btc protein, mouse; C498695:NEUROG3 protein, human; C491709:PAX4 protein, human; C084010:pancreatic and duodenal homeobox 1 protein; C093564:Neurogenic differentiation factor 1