In vivo studies on non-viral transdifferentiation of liver cells towards pancreatic β cells.
Transdifferentiation in vivo is an attractive option for autologous replacement of pancreatic β cells in patients with type 1 diabetes. It has been achieved by adenoviral delivery of genes for transcription factors in the liver and pancreas of hyperglycaemic mice. However, these viral approaches are not clinically applicable. We used the hydrodynamic approach to deliver genes Pdx1, Ngn3 (Neurog3) and MafA singly and in combination to livers of normoglycaemic rats. Five expression plasmids were evaluated. Livers were removed 1, 3, 7, 14 and 28 days after gene delivery and assayed by quantitative PCR, semi-quantitative PCR and immunohistology. Functional studies on hyperglycaemic rats were performed. The highest and most sustained expression was from a CpG-depleted plasmid (pCpG) and a plasmid with an in-frame scaffold/matrix attachment region ((pEPI(CMV)). When Pdx1, Ngn3 and MafA were delivered together to normoglycaemic rats with these plasmids, insulin mRNA was detected at all time points and was ~50-fold higher with pCpG. Insulin mRNA content of livers at days 3 and 7 was equivalent to that of a pancreas, with scattered insulin-positive cells detected by immunohistology, but levels declined thereafter. Prohormone convertase 1/3 was elevated at days 3 and 7. In hyperglycaemic rats, fasting blood glucose was lower at days 1, 3 and 7 but not thereafter, and body weight was maintained to day 28. We conclude that hydrodynamic gene delivery of multiple transcription factors to rat liver can initiate transdifferentiation to pancreatic β cells, but the process is reversible and probably requires more sustained transcription factor expression.
Department of Hepatology and Transplantation, King's College London School of Medicine, James Black Centre, London SE5 9NU, UK.
Abdullah
A
Cim
Greta J
GJ
Sawyer
Xiaohong
X
Zhang
Haibin
H
Su
Louise
L
Collins
Peter
P
Jones
Michael
M
Antoniou
Jean-Paul
JP
Reynes
Hans-Joachim
HJ
Lipps
John W
JW
Fabre
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental / Q000628:therapy; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics; D006943:Hyperglycemia / Q000628:therapy; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000502:physiology; D008099:Liver / Q000166:cytology* / Q000502:physiology; D051473:Maf Transcription Factors, Large / Q000235:genetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics; D010179:Pancreas / Q000166:cytology / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D051473:Maf Transcription Factors, Large; D009419:Nerve Tissue Proteins; D015534:Trans-Activators; C485160:Mafa protein, mouse; C407737:Neurog3 protein, mouse; C084010:pancreatic and duodenal homeobox 1 protein