Efficient transformation of small hepatocytes into insulin-expressing cells by forced expression of Pdx1.
OBJECTIVE
The expression of ectopic pancreatic and duodenal homeobox factor 1 (Pdx1) can transform hepatocytes into pancreatic endocrine cells. Small hepatocytes (SHs) have a high possibility to be a cellular source for islet cell transplantation. However, the efficacy of the transformation of SHs into pancreatic endocrine cells is not fully understood. The focus of our study was to compare the efficacy of the transformation into pancreatic endocrine cells of SHs and mature hepatocytes (MHs).


METHODS
MHs and SHs were cultured for 3 and 10 days, respectively, before Adeno-Pdx1 gene transduction. Western blot analysis was performed for pancreatic transcription factors, and reverse-transcription polymerase chain reaction (RT-PCR) was performed for the gene expression of pancreatic hormones. Confocal laser microscanning analysis was used to observe insulin and glucagon expression.


RESULTS
Although the pancreatic transcription factors Pdx1, Ngn3, NeuroD, and Pax6 were induced in both SHs and MHs after Adeno-Pdx1 gene expression, the pancreatic transcription factors Nkx2.2 and Nkx6.1 were induced in SHs more than in MHs. Glucagon mRNA expression was seen in both SHs and MHs, whereas insulin mRNA expression was higher in SHs than in MHs. Confocal laser microscanning analysis showed that SHs expressed both insulin and glucagon, whereas MHs predominantly expressed glucagon.


CONCLUSIONS
SHs were transformed into both insulin-and glucagon-expressing cells, and the efficacy of the transformation into insulin-expressing cells of SHs was higher than that for MHs. Thus, SHs could be a more suitable source of future cell therapy than MHs.
Kawasaki
Hiroyuki
H
Department of Surgery I, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-Ku, Sapporo, Hokkaido 060-8543, Japan.
Mizuguchi
Toru
T
Oshima
Hideki
H
Nobuoka
Takayuki
T
Shibata
Toshihito
T
Kaji
Shinsuke
S
Kokai
Yasuo
Y
Katsuramaki
Tadashi
T
Mitaka
Toshihiro
T
Hirata
Koichi
K
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D005934:Glucagon / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism*
D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; C000715628:Nkx2-2 protein, rat; C491327:Nkx6-1 protein, rat; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005934:Glucagon