Ectopic expression of neurogenin 3 in neonatal pig pancreatic precursor cells induces (trans)differentiation to functional alpha cells.
OBJECTIVE
Neurogenin 3 (NEUROG3), a basic helix-loop-helix transcription factor that is needed for endocrine cell development in the embryonic pancreas, has been shown to induce transdifferentiation of duct cells from adult pancreas towards a neuro-endocrine phenotype. Our study explored the endocrine transdifferentiation potential of NEUROG3 in neonatal pancreatic precursor cells.


METHODS
A replication-deficient adenovirus expressing Neurog3 and green fluorescent protein (GFP) (Ad-NEUROG3) was used to infect neonatal pig pancreatic cell preparations enriched for endocrine islet and cytokeratin-positive precursor cells. GFP-positive cells were sorted using flow cytometry on days 3 and 8 after infection and characterised at the transcript and protein level. For in vivo experiments, the total population of Ad-NEUROG3-infected pancreatic cells was transplanted, then later removed for determination of graft hormone content and immunohistochemistry.


RESULTS
Among the GFP-positive cells, the fraction of precursor cells decreased by more than 85% at day 8 after infection, while the fraction of glucagon-positive cells increased 2.5-fold and the beta cell number remained the same. Transplantation of the Ad-NEUROG3-infected pancreatic cell preparation failed to reverse streptozotocin-induced hyperglycaemia, while non-infected cells and a control cell preparation infected with replication-deficient adenovirus expressing only GFP were able to do so. At day 109 after transplantation, kidneys grafted with Ad-NEUROG3-infected pancreatic cells contained significantly decreased insulin and increased glucagon levels. Abundant glucagon-immunopositive cells were seen in Ad-NEUROG3-infected grafts, which were virtually devoid of proliferating insulin-positive cells.


CONCLUSIONS
In summary, adenoviral delivery of NEUROG3 to pancreatic precursor cells from neonatal pig pancreas promotes alpha cell differentiation in vitro and in vivo.
Harb
G
G
Department of Surgery, Surgical-Medical Research Institute, 1074 Dentistry Pharmacy Center, University of Alberta, Edmonton, Alberta T6G 2N8, Canada.
Heremans
Y
Y
Heimberg
H
H
Korbutt
G S
GS
D000256:Adenoviridae; D000818:Animals; D030801:Animals, Genetically Modified; D000831:Animals, Newborn; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D002454:Cell Differentiation; D017931:DNA Primers; D003921:Diabetes Mellitus, Experimental / Q000601:surgery; D005934:Glucagon / Q000032:analysis; D050416:Glucagon-Secreting Cells / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007150:Immunohistochemistry; D007328:Insulin / Q000032:analysis; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D053552:Keratin-7; D007633:Keratins / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D016708:Synaptophysin / Q000032:analysis; D014183:Transplantation, Heterologous
D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D005934:Glucagon; D049452:Green Fluorescent Proteins; D007328:Insulin; D053552:Keratin-7; D007633:Keratins; D009419:Nerve Tissue Proteins; D016708:Synaptophysin; C507496:Krt7 protein, mouse; C407737:Neurog3 protein, mouse