Cell loss during pseudoislet formation Hampers profound improvements in islet lentiviral transduction efficacy for transplantation purposes
Cell loss during pseudoislet formation Hampers profound improvements in islet lentiviral transduction efficacy for transplantation purposes
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DOI：
10.3727/000000007783464948
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发表时间：
2007-01-01
影响因子：
3.3
通讯作者：
Mathieu, C.
中科院分区：
文献类型：
--
作者：
Callewaert, H.;Gysemans, C.;Mathieu, C.
胰岛移植是1型糖尿病的一种有希望的治疗方法，但需要长期免疫抑制是广泛应用的主要障碍。通过慢病毒载体离体引入试剂--提高β细胞对免疫攻击的抵抗力--是一条有吸引力的途径。本研究的目的是研究在病毒感染前将胰岛分离成单细胞和在移植前重新聚集是否会提高病毒转导效率而无细胞毒性。该程序将LV-pWPT-CMV-EGFP构建体的转导效率从MOI 50时在整个胰岛中的11.2 +/-4.1%提高到MOI 5时的80.0 +/-2.8%。活力（通过Hoechst/PI测量）和功能（通过葡萄糖激发测量）保持较高水平。移植后，转染的假胰岛聚集体保持EGFP阳性超过90天，并且EGFP的表达主要与胰岛素阳性β细胞共定位。在体外或体内未观察到对免疫攻击的脆弱性增加。这些数据表明，在慢病毒转染之前分散胰岛和在移植之前再聚集是将感兴趣的基因引入胰岛中用于体外和体内移植目的的高效方式，但是当使用分散的细胞聚集体时，胰岛正常化所需的β细胞的量比未操作的胰岛高8倍以上。在胰岛细胞中达到稳定和强的转基因表达所付出的高昂代价当然是重要的细胞损失。
Islet transplantation is a promising treatment in type 1 diabetes, but the need for chronic immunosuppression is a major hurdle to broad applicability. Ex vivo introduction of agents by lentiviral vectors-improving beta-cell resistance against immune attack-is an attractive path to pursue. The aim of this study was to investigate whether dissociation of islets to single cells prior to viral infection and reaggregation before transplantation would improve viral transduction efficacy without cytotoxicity. This procedure improved transduction efficacy with a LV-pWPT-CMV-EGFP construct from 11.2 +/- 4.1% at MOI 50 in whole islets to 80.0 +/- 2.8% at MOI 5. Viability (as measured by Hoechst/PI) and functionality (as measured by glucose challenge) remained high. After transplantation, the transfected pseudoislet aggregates remained EGFP positive for more than 90 days and the expression of EGFP colocalized primarily with the insulin-positive beta-cells. No increased vulnerability to immune attack was observed in vitro or in vivo. These data demonstrate that dispersion of islets prior to lentiviral transfection and reaggregation prior to transplantation is a highly efficient way to introduce genes of interest into islets for transplantation purposes in vitro and in vivo, but the amount of beta-cells needed for normalization of glycemia was more than eightfold higher when using dispersed cell aggregates versus unmanipulated islets. The high price to pay to reach stable and strong transgene expression in islet cells is certainly an important cell loss.