Liver and Hepatocyte Transplantation: What Can Pigs Contribute?

About one-fifth of the population suffers from liver diseases in China, meaning that liver disorders are prominent causative factors relating to the Chinese mortality rate. For patients with end-stage liver diseases such as hepatocellular carcinoma or acute liver diseases with life-threatening liver dysfunction, allogeneic liver transplantation is the only life-saving treatment. Hepatocyte transplantation is a promising alternative for patients with acute liver failure or those considered high risk for major surgery, particularly for the bridge-to-transplant period. However, the lack of donors has become a serious global problem. The clinical application of porcine xenogeneic livers and hepatocytes remains a potential solution to alleviate the donor shortage. Pig grafts of xenotransplantation play roles in providing liver support in recipients, together with the occurrence of rejection, thrombocytopenia, and blood coagulation dysfunction. In this review, we present an overview of the development, potential therapeutic impact, and remaining barriers in the clinical application of pig liver and hepatocyte xenotransplantation to humans and non-human primates. Donor pigs with optimized genetic modification combinations and highly effective immunosuppressive regimens should be further explored to improve the outcomes of xenogeneic liver and hepatocyte transplantation.

Supportive Hepatocyte Transplantation after Partial Hepatectomy Enhances Liver Regeneration in a Preclinical Pig Model

<b><i>Background:</i></b> Hepatocyte transplantation (HTx) is regarded as a potential treatment modality for various liver diseases including acute liver failure. We developed a preclinical pig model to evaluate if HTx could safely support recovery from liver function impairment after partial hepatectomy. <b><i>Methods:</i></b> Pigs underwent partial hepatectomy with reduction of the liver volume by 50% to induce a transient but significant impairment of liver function. Thereafter, 2 protocols for HTx were evaluated and compared to a control group receiving liver resection only (group 1, <i>n</i> = 5). Portal pressure-controlled HTx was performed either immediately after surgery (group 2, <i>n</i> = 6) or 3 days postoperatively (group 3, <i>n</i> = 5). In all cases, liver regeneration was monitored by conventional laboratory tests and the novel noninvasive maximum liver function capacity (LiMAx) test with a follow-up of 4 weeks. <b><i>Results:</i></b> Partial hepatectomy significantly impaired liver function according to conventional liver function tests as well as LiMAx in all groups. A mean of 4.10 ± 1.1 × 10⁸ and 3.82 ± 0.7 × 10⁸ hepatocytes were transplanted in groups 2 and 3, respectively. All animals remained stable with respect to vital parameters during and after HTx. The animals in group 2 showed enhanced liver regeneration as observed by mean postoperative LiMAx values (621.5 vs. 331.3 μg/kg/h on postoperative day 7; <i>p</i> &#x3c; 0.001) whereas HTx in group 3 led to a significant increase in mean liver-specific coagulation factor VII (112.2 vs. 54.0% on postoperative day 7; <i>p</i> = 0.003) compared to controls (group 1), respectively. In both experimental groups, thrombotic material was observed in the portal veins and pulmonary arteries on histology, despite the absence of clinical symptoms. <b><i>Conclusion:</i></b> HTx can be performed safely and effectively immediately after a partial (50%) hepatectomy as well as 3 days postoperatively, with comparable results regarding the enhancement of liver function and regeneration.

Preferable Transplant Site for Hepatocyte Transplantation in a Rat Model

Intraportal injection is regarded as the current standard procedure of hepatocyte transplantation (HTx). In islet transplantation, which shares many aspects with HTx, recent studies have clarified that instant blood-mediated inflammatory reaction (IBMIR), characterized by strong innate immune responses, can cause poor engraftment, so other transplant sites to avoid such a reaction have been established. Although IBMIR was reported to occur in HTx, few reports have evaluated alternative transplant sites for HTx. In this study, we sought to determine the optimum transplant site for HTx. Rat hepatocytes (1.0 × 107) were transplanted at the 9 transplant sites (intraportal (IPO), intrasplenic (IS), liver parenchyma, subcutaneous, intraperitoneal, renal subcapsular, muscle, inguinal subcutaneous white adipose tissue, and omentum) of analbuminemic rats. The serum albumin levels, immunohistochemical staining (albumin, TUNEL, and BrdU), and in vivo imaging of the grafts were evaluated. The serum albumin levels of the IPO group were significantly higher than those of the other groups ( p < .0001). The BrdU-positive hepatocyte ratio of liver in the IS group (0.9% ± 0.2%) was comparable to that of the IPO group (0.9% ± 0.3%) and tended to be higher than that of the spleen in the IS group (0.5% ± 0.1%, p = .16). Considering the in vivo imaging evaluation and the influence of splenectomy, the graft function in the IS group may be almost entirely achieved by hepatocytes that have migrated to the liver. The present study clearly showed that the intraportal injection procedure is more efficient than other procedures for performing HTx

The Efficacy of the Hepatocyte Spheroids for Hepatocyte Transplantation

The safety and short-term efficacy of hepatocyte transplantation (HCTx) have been widely proven. However, issues such as reduced viability and/or function of hepatocytes, insufficient engraftment, and lack of a long-term effect have to be overcome for widespread application of HCTx. In this study, we evaluated hepatocyte spheroids (HSs), formed by self-aggregation of hepatocytes, as an alternative to hepatocytes in single-cell suspension. Hepatocytes were isolated from C57BL/6 J mice liver using a three-step collagenase perfusion technique and HSs were formed by the hanging drop method. After the spheroids formation, the HSs showed significantly higher mRNA expression of albumin, ornithine transcarbamylase, glucose-6-phosphate, alpha-1-antitrypsin, low density lipoprotein receptor, coagulation factors, and apolipoprotein E (ApoE) than 2 dimensional (2D)-cultured hepatocytes ( p < 0.05). Albumin production by HSs was significantly higher than that by 2D-cultured hepatocytes (9.5 ± 2.5 vs 3.5 ± 1.8 μg/dL, p < 0.05). The HSs, but not single hepatocytes, maintained viability and albumin mRNA expression in suspension (92.0 ± 2.8% and 1.03 ± 0.09 at 6 h). HSs (3.6 × 106 cells) or isolated hepatocytes (fSH, 3.6 × 106 cells) were transplanted into the liver of ApoE knockout (KO-/-) mice via the portal vein. Following transplantation, serum ApoE concentration (ng/mL) of HS-transplanted mice (1w: 63.1 ± 56.7, 4w: 17.0 ± 10.9) was higher than that of fSH-transplanted mice (1 w: 33.4 ± 13.0, 4w: 13.7 ± 9.6). In both groups, the mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, MCP-1, and MIP-1β) were upregulated in the liver following transplantation; however, no significant differences were observed. Pathologically, transplanted HSs were observed as flat cell clusters in contact with the portal vein wall on day 7. Additionally, ApoE positive cells were observed in the liver parenchyma distant from the portal vein on day 28. Our results indicate that HS is a promising alternative to single hepatocytes and can be applied for HCTx.

High Dose Versus Low Dose Syngeneic Hepatocyte Transplantation in Pex1-G844D NMRI Mouse Model is Safe but Does Not Achieve Long Term Engraftment

Genetic alterations in PEX genes lead to peroxisome biogenesis disorder. In humans, they are associated with Zellweger spectrum disorders (ZSD). No validated treatment has been shown to modify the dismal natural history of ZSD. Liver transplantation (LT) improved clinical and biochemical outcomes in mild ZSD patients. Hepatocyte transplantation (HT), developed to overcome LT limitations, was performed in a mild ZSD 4-year-old child with encouraging short-term results. Here, we evaluated low dose (12.5 million hepatocytes/kg) and high dose (50 million hepatocytes/kg) syngeneic male HT via intrasplenic infusion in the Pex1-G844D NMRI mouse model which recapitulates a mild ZSD phenotype. HT was feasible and safe in growth retarded ZSD mice. Clinical (weight and food intake) and biochemical parameters (very long-chain fatty acids, abnormal bile acids, etc.) were in accordance with ZSD phenotype but they were not robustly modified by HT. As expected, one third of the infused cells were detected in the liver 24 h post-HT. No liver nor spleen microchimerism was detected after 7, 14 and 30 days. Future optimizations are required to improve hepatocyte engraftment in Pex1-G844D NMRI mouse liver. The mouse model exhibited the robustness required for ZSD liver-targeted therapies evaluation.