McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15203, USA.
Liver Transpl. 2012 Feb;18(2):226-37. doi: 10.1002/lt.22322.
Although hepatic cell transplantation (CT) holds the promise of bridging patients with end-stage chronic liver failure to whole liver transplantation, suitable cell populations are under debate. In addition to hepatic cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being considered as alternative cell sources for initial clinical cell work. Fetal liver (FL) tissue contains potential progenitors for all these cell lineages. Based on the collagenase incubation of tissue fragments, traditional isolation techniques yield only a fraction of the number of available cells. We report a 5-step method in which a portal vein in situ perfusion technique is used for tissue from the late second trimester. This method results in the high viabilities known for adult liver vascular perfusion, addresses the low cell yields of conventional digestion methods, and reduces the exposure of the tissue to collagenase 4-fold. We used donated tissue from gestational weeks 18 to 22, which yielded 1.8 ± 0.7 × 10(9) cells with an average viability of 78%. Because HSC transplantation and MSC transplantation are of interest for the treatment of hepatic failure, we phenotypically confirmed that in addition to hepatic progenitors, the resulting cell preparation contained cells expressing typical MSC and HSC markers. The percentage of FL cells expressing proliferation markers was 45 times greater than the percentage of adult hepatocytes expressing these markers and was comparable to the percentage of immortalized HepG2 liver hepatocellular carcinoma cells; this indicated the strong proliferative capacity of fetal cells. We report a case of human FL CT with the described liver cell population for clinical end-stage chronic liver failure. The patient's Model for End-Stage Liver Disease (MELD) score improved from 15 to 10 within the first 18 months of observation. In conclusion, this human FL cell isolation protocol may be of interest for further clinical translation work on the development of liver cell-based therapies.
虽然肝细胞移植 (CT) 有望为终末期慢性肝功能衰竭患者提供桥接治疗，直至进行全肝移植，但合适的细胞群体仍存在争议。除了肝细胞之外，间充质干细胞 (MSCs) 和造血干细胞 (HSCs) 也被认为是替代细胞来源，可用于初步的临床细胞工作。胎肝 (FL) 组织中包含所有这些细胞谱系的潜在祖细胞。基于组织碎片的胶原酶孵育，传统的分离技术只能获得可用细胞数量的一小部分。我们报告了一种 5 步方法，其中门静脉原位灌注技术用于妊娠晚期的组织。这种方法产生了类似于成人肝脏血管灌注的高细胞活力，解决了传统消化方法中细胞产量低的问题，并将组织暴露于胶原酶的时间减少了 4 倍。我们使用捐赠的妊娠 18 至 22 周的组织，得到了 1.8 ± 0.7×10(9)个细胞，平均活力为 78%。由于 HSC 移植和 MSC 移植对肝功能衰竭的治疗具有重要意义，我们通过表型证实，除了肝祖细胞外，所得细胞制剂还包含表达典型 MSC 和 HSC 标志物的细胞。表达增殖标志物的 FL 细胞百分比是表达这些标志物的成人肝细胞的 45 倍，与永生化 HepG2 肝癌细胞的百分比相当；这表明胎儿细胞具有强大的增殖能力。我们报告了一例使用所描述的肝细胞群体进行的人类胎肝 CT，用于治疗临床终末期慢性肝功能衰竭。患者的终末期肝病模型 (MELD) 评分从观察的前 18 个月内从 15 分提高到 10 分。总之，这种人类 FL 细胞分离方案可能对进一步的临床转化工作，即开发基于肝细胞的治疗方法具有重要意义。
