Functional characterization of a bioengineered liver after heterotopic implantation in pigs

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

The beta-catenin-target Fascin-1, altering hepatocyte differentiation, is a new marker of immature cells in hepatoblastomas

OBJECTIVE: Beta-catenin is a well-known effector of the Wnt pathway and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of beta-catenin are highly frequent in pediatric liver primary tumors. Those mutations are mostly heterozygous allowing the co-expression of wild-type (WT) and mutated beta-catenins in tumor cells. We investigated the interplay between WT and mutated beta-catenins in liver tumor cells, and searched for new actors of the beta-catenin pathway. DESIGN: Using an RNAi strategy in beta-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of beta-catenin, carried mainly by, respectively, WT and mutated proteins. Their impact was characterized using transcriptomic and functional analyses. We studied mice that develop liver tumors upon activation of beta-catenin in hepatocytes (APCKO and beta-catenin∆exon3 mice). We used transcriptomic data from mouse and human HB specimens and analyzed samples by immunohistochemistry. RESULTS: We highlighted an antagonist role of WT and mutated beta-catenins on hepatocyte differentiation as attested by alteration of hepatocyte markers expression and bile canaliculi formation. We characterized Fascin-1 as a target of beta-catenin involved in hepatocyte differentiation. Using mouse models, we found that Fascin-1 is highly expressed in undifferentiated tumors. Finally, we found that Fascin-1 is a specific marker of the embryonal component in human HBs. CONCLUSION: In mice and human, Fascin-1 expression is linked to loss of differentiation and polarity of hepatocytes. Thus, we highlighted Fascin-1 as a new player in the modulation of hepatocyte differentiation associated to beta-catenin pathway alteration in the liver.

The Effect of miR122 and miR185 on Hepatic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells

Background: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been considered as a promising cell source in liver diseases. miRNAs have been shown to play an important role in hepatic differentiation of hUC-MSCs. The study seeks to explore whether miR122 and miR185 could affect induction of hUC-MSCs into hepatic differentiation. Methods: miR122 and miR185 stable overexpression by hUC-MSCs were firstly created, then hUC-MSCs were cultured by hepatic differentiation conditional medium. After 28 days culture in hepatic inducing conditional medium, hepatic markers expressed by these cells were detected by qRT-PCR and western-blot. The cell functions were also evaluated by PAS staining and ICG phagocytosis. Results: Our results demonstrated that at the end of 28 days, hUC-MSCs overexpressing miR122 had increasing expression of hepatocyte markers including AFP, ALB, CK18, CK19 and HNF4α in both mRNA level and protein ecpression, while in the miR185 overexpression group, hUC-MSCs showed decreasing expression of hepatocyte markers. Moreover, there was also improvement of glycogen deposits as well as ICG phagocytosis ability in the hepatic inducing miR122 overexpression cells, while in the hepatic inducing miR185 overexpression group, hUC-MSCs showed decreasing glycogen deposits and ICG phagocytosis ability. Conclusions: We thus speculate that it is possible to promote hepatic differentiation of hUC-MSCs by overexpression of miR122 and this effect may be inhibited by miR185 overexpression. It seems miR122 and miR185 have an antagonistic effect on hUC-MSCs hepatic differentiation. Overexpression of certain kind of miRNA in cells by transfection or other gene modification skills could be an effective way to modulate stem cell fate.

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRβ), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFβ1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

Clinicopathological features of neoplasms with neuroendocrine differentiation occurring in the liver

Background/aimsWe investigated the clinicopathological features of hepatic neuroendocrine tumours (NET) and neuroendocrine carcinoma (NEC), which remain largely unknown.Material and methodsWe examined 1235 tumours from 1048 patients who had undergone curative hepatectomy for liver neoplasms at Kurume University Hospital. Pathological diagnoses were based on the 2010 WHO Classification of Tumours of the Digestive System. We performed immunostaining for hepatocyte markers (eg, hepatocyte paraffin (HepPar)-1), neuroendocrine markers (eg, chromogranin A (CGA)) and the proliferation marker (Ki-67).ResultsThere were four cases of NET G2 (0.38%) and five of hepatic malignant tumours with an NEC component (HNEC) (0.48%). HNEC cases were classified into three types, that is, transitional, intermediate and separate types, according to their histological and immunohistochemical features. In the former two types, the NEC component intermingled with the moderately to poorly differentiated hepatocellular carcinoma (HCC) component or intermediate component consisting of tumour cells showing the colocalisation of CGA and HepPar-1. In the separate type, the NEC and poorly differentiated HCC components were present separately, whereas the sarcomatous HCC component was detected in the vicinity of the NEC component. Ki-67 labelling indices of the NET G2, HCC and NEC components of HNEC were 6.8%, 14.9% and 58.9%, respectively.ConclusionsPrimary hepatic NET and NEC are very rare tumours. The NEC component in HNEC showed high proliferative activity and influenced patient prognoses.

An Efficient Protocol for Deriving Liver Stem Cells from Neonatal Mice: Validating Its Differentiation Potential

The success of liver regeneration depends on the availability of suitable cell types and their potential to differentiate into functional hepatocytes. To identify the stem cells which have the ability to differentiate into hepatocytes, we used neonatal liver as source. However, the current protocol for isolating stem cells from liver involves enzymes like collagenase, hyaluronidase exposed for longer duration which limits the success. This results in the keen interest to develop an easy single step enzyme digestion protocol for isolating stem cells from liver for tissue engineering approaches. Thus, the unlimited availability of cell type favors setting up the functional recovery of the damaged liver, ensuring ahead success towards treating liver diseases. We attempted to isolate liver stem derived cells (LDSCs) from mouse neonatal liver using single step minimal exposure to enzyme followed byin vitroculturing. The cells isolated were characterized for stem cell markers and subjected to lineage differentiation. Further, LDSCs were induced to hepatocyte differentiation and validated with hepatocyte markers. Finally, we developed a reproducible, efficient protocol for isolation of LDSCs with functional hepatocytes differentiation potential, which further can be used asin vitromodel system for assessing drug toxicity assays in various preclinical trials.