Establishment of new generation liver tissue engineering approach

利用肝细胞移植的肝组织工程已被提出作为全器官移植或肝定向基因治疗的替代方案，以纠正各种类型的肝功能不全。在同基因小鼠肾包膜下移植时，肝细胞不能维持。然而，当我们用从Engelbreth-Holm-Swarm细胞中提取的细胞外基质成分移植肝细胞时，肝细胞存活了至少140天，并形成了小的肝组织。血友病A小鼠的肝脏工程重建了正常凝血活性的5%至10%，足以减少出血时间并具有治疗效益。相反，使用Engelbreth Holm-Swarm凝胶基质的肝细胞不能在皮下空间持续存活。我们假设在移植部位建立局部血管网络可以减少移植物损失。为了验证这一想法，我们在肝细胞移植到皮下间隙之前提供了一种有效的血管生成剂。通过这种方法，在实验期间（120天）实现了持续生存。为了证实这些工程肝组织在体内也保留了其天然再生潜力，我们对未成熟肝脏诱导了两种不同的增殖刺激模式，并证实肝外组织内的肝细胞再生的活性与未成熟肝脏相似。总之，我们的研究表明，肝组织可以在肝外部位进行工程化和维持，在体内保持其再生能力，并成功地用于治疗遗传疾病。

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.