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Illumina Human Polycystic Liver Disease and Normal Biliary Stem Cell RNAseq

ABSTRACT: Background & aims: Polycystic liver disease (PLD) is an autosomal dominantly inherited disorder caused by mutations in genes such as PRKCSH and SEC63. It has been thought that cysts develop from biliary progenitor cells due to loss-of-heterozygosity (LOH), leading to aberrant proliferation or defects in differentiation. Cyst expansion can be suppressed by somatostatin analogues such as lanreotide. There is no human in vitro model available that truly recapitulates polycystic liver disease. We hypothesize that PLD progenitors can form bipotent liver organoids that carry key features of cyst development. To find gene expression differences between Human Polycystic Liver Disease and Normal Biliary Stem Cells. Methods: Cells from normal biliary duct (n=6), cyst biliary epithelium (n=60) and cyst fluid (n=31) were isolated and placed under conditions suitable for expansion of human adult liver stem cells. We analyzed genetic LOH, gene expression, differentiation capacity, response to lanreotide and cilium formation of these organoids. Results: Cholangiocytes from cyst biliary epithelium (47/60) and cyst fluid (9/31) proved capable of expanding as bipotent liver organoids. Multiple cyst organoids displayed LOH surrounding PRKCSH or SEC63 regions. Organoids formed cilia when proliferation was inhibited. Neither hepatocyte nor biliary differentiation of PLD organoids was impaired. RNAseq revealed no significantly dysregulated pathway in PLD organoids. Lanreotide significantly decreased expansion of liver organoids in comparison to negative control (197% ± 46% versus 547% ± 28%; p: 0.038). Conclusion & discussion: Biliary progenitor cells from patient cyst epithelium and fluid can expand into liver organoids. They recapitulate key characteristics of PLD, and are a promising human in vitro model for research, diagnostics and treatment of polycystic liver diseases and cholangiociliopathies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE73579 | GEO | 2015/09/30

SECONDARY ACCESSION(S): PRJNA297355

REPOSITORIES: GEO

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Project description:Polycystic liver disease (PLD) is a group of genetic disorders characterized by bile duct dilatation and/or progressive development of multiple intrahepatic fluid-filled biliary cysts (>10), which are the main cause of morbidity. Current surgical and/or pharmacological therapies exert short-term and modest benefits, and thus liver transplantation represents the only curative option. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. Here, we hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. In this regard, ER stress was assessed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy; TEM) and functional (20S proteasome activity) levels in different models of PLD (i.e., human and rat). Moreover, PCK rat (animal model of PLD) was employed to determine the effects of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or inducers [tunicamycin (TM)] in hepatic cytogenesis and, consequently, in the progression of the disease. Likewise, the impact of the aforementioned ER stress modulators was also analyzed on the expression of unfolded protein response (UPR) factors, the 20S proteasome activity, the mechanisms involved in the maintenance of ER proteostasis and the cell fate decisions (i.e., survival/apoptosis). Interestingly, the expression levels of the UPR factors were markedly upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Additionally, cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (i.e., synthesis, folding, trafficking and degradation of nascent proteins), marked enlargement of the ER lumen (by TEM), and hyperactivation of the 20S proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline or TM administration compared to control groups. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized, at least in part, proteomic profiles related to protein synthesis, folding, trafficking and degradation, and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. Conclusion: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel and promising therapeutic strategy.

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Illumina Human Polycystic Liver Disease and Normal Biliary Stem Cell RNAseq

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