ESTABLISHMENT OF AN IMMORTALIZED HUMAN PANCREATIC BETA CELL LINE

• DOI: 10.1097/00007890-200407271-01669
• Publication Date: 2004-09-03T09:01:14Z
• Authors: M Narushima; N Kobayashi; K A Westerman; T Fukazawa; M Sakaguchi; Y Tanaka; Jonathan RT. Lakey; Philippe Leboulch; N Tanaka
• Keywords: Transduction (biophysics); Multiplicity of infection; PDX1

P1174 Aims: It is of extreme importance to establish human pancreatic islet cell lines, especially beta lines develop diabetes-targeted therapies. In the present study, we have established an immortalized line using a retroviral transduction simian virus 40 large T antigen (SV40T) and telomerase reverse transcriptase (hTERT). Methods: Human islets were isolated defined protocols enzymatic dissociation purification discontinuous gradients Ficoll refrigerated Cobe 2991 machine. The resulting shipped Japan then subjected experiments. following vectors utilized: 1) SSR#69 encoding LoxP-flanked SV40T hygromycin resistance cDNAs 2) SSR#197 expressing hTERT green fluorescent protein (GFP) cDNAs. After 3 times transduction, cells selected with (100 μg/ml) subsequently sorted by flow cytometer MoFlo recover GFP-positive populations. vitro clones characterized biochemical studies, including northern-blot, western-blot, electrophoretic mobility sift assay. vivo transplantation was conducted into sub-renal capsule 220 mg/kg streptozotocin (STZ)-induced diabetic SCID mice. Results: One 8 clones, NAKT-13, grew well in tissue culture flask without obvious crises. NAKT-13 positive for beta-specific transcriptional factors Pdx1, Isl1, Nkx6.1, Pax6. transplant, blood glucose level mice decreased normal range; contrast control maintained hyperglycemia (> 400 mg/dl). Histoimmunochemical examination revealed insulin-positivity transplanted Conclusions: Using SSR#197, has been successfully established. Transplantation ameliorated STZ-induced This study presented novel strategy facilitate