Sequential introduction and dosage balance of defined transcription factors affect reprogramming efficiency from pancreatic duct cells into insulin-producing cells.
While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-β cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes.
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Kazuyuki
K
Miyashita
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; Department of Medicine, Metabolism and Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan; Center for Molecular Diabetology, Juntendo University Graduate School of Medicine, Tokyo, Japan. Electronic address: miyatsuka-takeshi@umin.net.
Takeshi
T
Miyatsuka
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Taka-Aki
TA
Matsuoka
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Shugo
S
Sasaki
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Satomi
S
Takebe
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Tetsuyuki
T
Yasuda
Department of Medicine, Metabolism and Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan; Center for Molecular Diabetology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Hirotaka
H
Watada
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Hideaki
H
Kaneto
Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Iichiro
I
Shimomura
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D002460:Cell Line; D065150:Cellular Reprogramming; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D007328:Insulin / Q000235:genetics; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D051473:Maf Transcription Factors, Large / Q000235:genetics*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010183:Pancreatic Ducts / Q000166:cytology*; D015534:Trans-Activators / 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
Insulin-producing cells; Mafa; Neurog3; Pancreatic duct cells; Pdx1; Reprogramming