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

用UW溶液解冻冷冻保存的胰岛细胞后，胰岛细胞显示出有效的单层形成，并促进了慢病毒介导的有效转导。利用来源于804G细胞的细胞外基质有效地形成了小鼠胰岛的单层。我们成功地制作了糖尿病猪模型来进行全胰腺切除术。这些糖尿病猪在术后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治疗的疗效。人类胰腺β细胞系在功能上与原代β细胞相当，目前还没有发现。我们用含有类人猿病毒40大T抗原（SV40T）和人端粒酶逆转录酶（hTERT） cdna的逆转录病毒载体转染原代人β细胞，建立了一个可逆永生化的人β细胞克隆（NAKT-15），该逆转录病毒载体的两侧是配对重组靶loxPs，允许Cre重组酶删除SV40T和hTERT基因。恢复的NAKT-15细胞表达β细胞转录因子（isl1, pax6, nkx6.1,Pdx-1），激素原转化酶1/3和2，分泌颗粒蛋白，并分泌胰岛素以响应葡萄糖，类似于正常的人胰岛。将NAKT-15细胞移植到链脲佐菌素诱导的糖尿病SCID小鼠体内，2周内血糖得到完全控制；小鼠保持血糖正常的时间超过30周。β细胞系的建立是通过移植治疗糖尿病的潜在一步。

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.