Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

Abstract

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.

Figure 1

Characterization of optimized and large-scale expanded hiHeps. (A) Optimized hiHeps show high percentage of albumin and AAT double-positive cells as determined by flow cytometry. (B) q-PCR analyses of hepatic gene expression in optimized hiHeps, including albumin (ALB), ATP-binding cassette subfamily B member 11 (BSEP), transthyretin (TTR), cadherin 1 (CDH1), transferrin, asialoglycoprotein receptor 1 (ASGPR1), AAT, tyrosine aminotransferase (TAT). (C) Testosterone elimination of optimized hiHeps was determined by LC-MS/MS. (D) The ability of eliminating ammonia was measured in optimized hiHeps by enzymatic colorimetric assay. (E) Schematic outline of the large-scale expansion of hiHeps. hiHeps were generated and enriched in 6 cm petri dish and then expanded to 2.4 × 10⁸ in 10 cm dishes. hiHeps were finally expanded in Hyperflasks. (F) Large-scale expansion of hiHep cultures from 5 × 10⁶ to ∼3 × 10⁹ cells in 20 days. (G-I) Large-scale expanded hiHeps maintained hepatic gene expression levels (G), efficient testosterone clearance (H) and ammonia elimination (I). HFF, human fetal fibroblast. PHH, primary human hepatocyte. *P < 0.05, t-test.

Figure 2

hiHep-BAL rescues ALF pigs. (A) Schematic diagram depicts the configuration of the hiHep-BAL support system. Approximately 3 × 10⁹ hiHeps were implanted into the bioreactor. (B) The outline of the hiHep-BAL treatment of ALF Bama miniature pigs. (C) Bama miniature pigs treated by Empty-BAL and hiHep-BAL. Note that ALF pigs after hiHep-BAL treatment were apparently active on day 5. (D) Serum biochemical parameters of ALF pigs in No-BAL, Empty-BAL and hiHep-BAL groups. Serum levels of ALT, AST, ammonia, TBIL, albumin and prothrombin time (PT) were measured. Because most animals of No-BAL and Empty-BAL groups were dead or extremely sick from day 3, blood samples were not collected in these two groups after day 3. (E) Kaplan-Meier survival curve of No-BAL, Empty-BAL- and hiHep-BAL-treated ALF pigs (n = 6 for No-BAL and Empty-BAL, n = 8 for hiHep-BAL group). *P < 0.01, log-rank test.

Figure 3

Therapeutic effects of hiHep-BAL on ALF pigs. (A) Human albumin and ATT were measured in the pig serum after Empty-BAL and hiHep-BAL treatment. Human protein-specific ELISA kit was used in the assay. (B) Hematoxylin and Eosin (HE) staining of pig livers of No-BAL, Empty-BAL and hiHep-BAL groups. Livers of No-BAL and Empty-BAL groups were collected on day 2 or 3 after D-gal induction. Livers of hiHep-BAL group were collected on day 7. Red arrowheads indicate liver damage, including karyorrhexis, karyolysis and hemorrhage. Black arrowheads indicate local infiltration of inflammatory cells. Necro-inflammation was scored according to the Scheuer system. Scale bar, 100 μm. (C) mRNA expression of inflammatory cytokine genes was determined by q-PCR in pig livers of No-BAL, Empty-BAL and hiHep-BAL groups, including IL1β, IL2, IL6, IL8, TNFα and IFNγ. (D) Serum levels of the indicated cytokines were determined by ELISA with antibodies specific for pig proteins. (E) Histological analyses of proliferating hepatocytes in hiHep-BAL-treated pigs on day 7. Red arrowheads indicate hepatocytes at division phase. High magnification images of division phase are inserted.