Induced Pluripotent Stem Cells From Subjects With Primary Sclerosing Cholangitis Develop a Senescence Phenotype Following Biliary Differentiation

Abstract

Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient-specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three-dimensional matrigel-based culture to induce formation of cholangioids. RNA sequencing (RNA-seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA-seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three-dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC-derived cholangioids. Congruent with this, PSC-derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin-6, and chemokine (C-C motif ) ligand 2. Conditioned media (CM) from PSC-derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease-specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids. (Hepatology Communications 2021;0:1-16).

Biliary Epithelial Senescence in Liver Disease: There Will Be SASP

“…Further, NHC-sen cholangoids, as well as PSC cholangoids enhance macrophage recruitment compared to control (Guicciardi et al, 2018). Interestingly, isolated cholangiocytes from PSC patients have increased expression of genes associated with cell cycle arrest and senescence, as indicated by RNA-seq, and senescent cholangiocytes formed cholangoids of a smaller size that also lacked a lumen when compared to normal cholangiocytes (Jalan-Sakrikar et al, 2021). This work is supported by other findings showing that NHC-sen cells promote the proliferation of healthy cholangiocytes and monocyte migration, which was also found with plasma EVs from multidrug resistant cassette 2 knock out (Mdr2 −/− ) mice, a genetic murine model of PSC (Al Suraih et al, 2020).…”

Organoids and regenerative hepatology

“…Considered collectively, iPSC‐derived cholangiocyte organoids provide a unique platform for generating biliary epithelial cells from virtually any patient and from a broad variety of starting material (e.g., skin, blood, urine), using minimally invasive procedures. This approach does not require access to primary tissue; the resulting cells recapitulate key cholangiocyte functions and can be used successfully for disease modeling and drug screening 56,67–69,71. Despite the presence of many features of mature cholangiocytes, they still are limited by incomplete maturation, retention of some fetal characteristics, and the genetic instability that can be associated with iPSC 48…”

“…This approach does not require access to primary tissue; the resulting cells recapitulate key cholangiocyte functions and can be used successfully for disease modeling and drug screening. [56,[67][68][69]71] Despite the presence of many features of mature cholangiocytes, they still are limited by incomplete maturation, retention of some fetal characteristics, and the genetic instability that can be associated with iPSC. [48] Primary tissue-based systems…”

“…[70] However, the paracrine signals between cell types in multicellular systems remain difficult to disentangle. On the contrary, monocellular systems are chemically defined as they are solely based on key biliary factors such as Notch, Wnt, TGFβ, and FGF [56,67,69,71] reducing culture variability making monocellular systems more amenable for regenerative medicine applications. These cells express biliary markers such as CK7, CK19, CFTR, and SOX9, present typical morphological features, including primary cilia, and respond to secretory stimuli (secretin and somatostatin).…”

Bioengineering Liver Organoids for Diseases Modelling and Transplantation

“…Induced pluripotent stem cells (iPSC) are capable of differentiating into many different cell types, and iPSC-derived liver organoids can well replace the conventional liver tissue-derived organoid systems [ 50 ]. Protocols have been established to induce iPSC to differentiate into liver organoids of cholangiocytes and hepatocytes [ 51 , 52 , 53 , 54 , 55 ]. Typically, iPSCs were cultured in a medium containing bone morphogenetic protein 4 (BMP4), fibroblast growth factor-4 (FGF4), and B-27 supplements to induce differentiation of iPSC into hepatoblast-like cells, and subsequently cultured with epidermal growth factor (EGF) in matrigel to encourage cholangiocyte differentiation [ 56 ].…”