Development of cell therapies using reversibly immortalized human pancreatic beta cell lines

用UW溶液解冻冻存的胰岛后，胰岛细胞显示出有效的单层形成，并促进有效的慢病毒介导的转导。小鼠胰岛的单层形成有效地实现了使用的细胞外基质来源于804 G细胞。我们成功地建立了猪糖尿病模型，进行全胰腺切除术。这种糖尿病猪在手术后10天内死于酮症酸中毒。该模型将有助于评估糖尿病靶向细胞疗法的疗效。尚未获得功能上等同于原代β细胞的人胰腺β细胞系。我们建立了一个可逆的永生化的人β细胞克隆（NAKT-15），通过转染原代人β细胞与逆转录病毒载体含有猿猴病毒40大T抗原（SV 40 T）和人端粒酶逆转录酶（hTERT）的cDNA两侧的配对重组靶loxP，这允许删除SV 40 T和hTERT基因的Cre重组酶。恢复的NAKT-15细胞表达β细胞转录因子（Isl-1、Pax 6、Nkx 6.1、Pdx-1）、激素原转化酶1/3和2以及分泌颗粒蛋白，并且响应于葡萄糖而分泌胰岛素，类似于正常人胰岛。将NAKT-15细胞移植到链脲佐菌素诱导的糖尿病SCID小鼠中，在2周内血糖得到了完美的控制;小鼠保持正常血糖超过30周。β细胞系的建立是通过移植潜在治愈糖尿病的一步。

After thawing the cryopreserved islets with UW solution, the islet cells showed an efficient monolayer formation and facilitated effective lentivirus-mediated transduction. Monolayer formation of mouse islets was efficiently achieved by the use of extracellular matrices derived from 804G cells. We successfully made a diabetes model in pigs to perform total pancreatectomy. Such diabetic pigs died of ketoacidosis within 10 days after surgery. The model would be useful to assess the efficacy of diabetes-targeted cell therapies. A human pancreatic beta cell line that is functionally equivalent to primary beta cells has not been available. We established a reversibly immortalized human beta cell clone (NAKT-15) by transfection of primary human beta cells with a retroviral vector containing simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired recombination target loxPs, which allows deletion of SV40T and hTERT genes by Cre recombinase. Reverted NAKT-15 cells expressed beta cell transcription factors (Isl-1,Pax 6,Nkx 6.1,Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic SCID mice resulted in perfect control of blood glucose within 2 weeks ; mice remained normoglycemic for longer than 30 weeks. The establishment of a beta cell line is one step toward the potential cure of diabetes by transplantation.