肝细胞球状体作为在小鼠和猪模型中进行离体基因治疗后移植的替代物，优于单细胞。
Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models.
机构信息
Department of Surgery, Mayo Clinic, Rochester, MN; Faculty of Medicine, University of Barcelona, Spain.
Department of Surgery, Mayo Clinic, Rochester, MN; Department of Molecular Medicine, Mayo Clinic, Rochester, MN.
出版信息
Surgery. 2018 Sep;164(3):473-481. doi: 10.1016/j.surg.2018.04.012. Epub 2018 Jun 6.
BACKGROUND
Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes.
METHODS
Pig and mouse hepatocytes were isolated, labeled with zirconium-89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography-computed tomography. Fumarylacetoacetate hydrolase-deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single-cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single-cell or spheroid hepatocytes were followed through histologic analysis and animals' ability to thrive off 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione.
RESULTS
Positron emission tomography-computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid- and single-cell-transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase-positive hepatocytes in the liver, correlating with similar weight stabilization curves.
CONCLUSION
Ex vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase-deficient pigs can be performed using hepatocyte spheroids or single-cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures.
背景
体外基因治疗后自体肝细胞移植是代谢性肝病肝移植的替代方法。在此，我们评估了体外基因治疗后，再移植单细胞或球体状肝细胞的效果。
方法
从猪和鼠肝脏中分离并标记锆-89 后，将其作为单细胞或球体状重新植入肝脏。通过正电子发射断层扫描-计算机断层扫描评估生物分布。从缺乏延胡索酰乙酰乙酸水解酶的猪肝细胞中分离出来，并通过含有 Fah 基因的慢病毒载体进行转导。在体外基因传递后，通过门静脉输注单细胞或球体状自体肝细胞。测量门静脉压力，并通过超声评估血栓形成。通过组织学分析和动物脱离 2-(2-硝-4-三氟甲基苯甲酰基）-1,3-环已二酮的能力，跟踪体外校正的单细胞或球体状肝细胞的植入和扩增差异。
结果
与单细胞相比，正电子发射断层扫描-计算机断层扫描成像显示球体状肝细胞的生物分布异质性增加，在肝脏中的分布更加均匀。接受球体状细胞的动物门静脉压力的平均变化高于接受单细胞的动物（P <.01）。此外，球体组的两只动物出现了门静脉血栓，需要全身抗凝。对接受球体状和单细胞移植的动物进行免疫组织化学分析显示，在肝脏中，具有延胡索酰乙酰乙酸水解酶活性的肝细胞的植入和扩增率相似，这与相似的体重稳定曲线相关。
结论
体外基因校正法可用于缺乏延胡索酰乙酰乙酸水解酶的猪自体肝细胞，可使用肝细胞球体或单细胞，其中球体状在肝脏中的分布更为不均匀，且具有更高的门静脉血栓形成和门静脉压力升高的风险。
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