Organoids and Spheroids as Models for Studying Cholestatic Liver Injury and Cholangiocarcinoma

Abstract

Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two-dimensional monolayer cell cultures do not resemble intercellular cell-to-cell interaction and communication; however, three-dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three-dimensional hepatic cell cultures as in vitro models of cholangiopathies.

In vitro cell culture models are key in liver research to supplement the lack of human samples or in vivo animal models.⁽¹⁾ Importantly, the human body consists of three-dimensional (3D) cellular interactions and structures with noncellular components, and classic two-dimensional (2D) monolayer culture models do not mimic this interaction between cells and components or matrices. In the past decade, techniques of 3D cell culture systems, such as organoids and spheroids, have been developed to mimic hepatic structure and cellular interaction in vitro.⁽²⁾ These 3D cell culture models have more accurate physiological environmental conditions resembling in vivo complex architecture, microenvironment, and cellular functions and may be more appropriate for biomedical studies than monolayer models.^(2,3)

The definitions of the terms “organoid” and “spheroid” can vary or overlap depending on previous studies. Organoids are generally recognized as an in vitro “miniorgan” or complex tissue-like structure with multiple cell types.⁽³⁾ Spheroids are often referred to as “simple cell aggregates” with a single cell type. Organoids are generated from (1) tissue-derived primary cells, (2) progenitors, (3) embryonic stem cells (ESCs), or (4) induced pluripotent stem cells (iPSCs), which can self-assemble and differentiate into organ-like cell clusters when cultured in medium supplemented with growth factors that resemble the tissue/organ development signals during the embryonic stage.⁽⁴⁾ Scaffold techniques, such as extracellular matrix (ECM)–based hydrogels, support organoid formation by cell-to-matrix interaction.⁽³⁾ Spheroids are often generated from immortalized or cancer cell lines, and both scaffold and scaffold-free techniques can be used for spheroid formation. Scaffold-free methods, such as the hanging drop method or cell culture plates with an ultralow attachment surface, allow cells to float freely in culture media and form spheroidal cell aggregates instead of forming monolayers on the plate surface. Although this review focuses on the terms “organoids” and “spheroids,” cancerous organoids generated from human primary tumor tissues may be referred to as “tumoroids,” and biliary organoids/spheroids generated from primary ductal cells or cholangiocyte cell lines may be referred to as “cholangioids.” Figure 1 summarizes the differences in methodologies between organoids and spheroids.

FIG. 1.

Generation of hepatic organoids and spheroids. Liver organoids are generally established from ESCs, iPSCs, HPCs, or primary cells isolated from liver or bile duct tissue samples. Cells are embedded with ECM components, such as Matrigel or collagen type I, to support organoid formation. Cells differentiate into hepatic cells depending on culture media. Mature organoids are functional and consist of multiple cell types, such as hepatocytes and cholangiocytes. Hepatocytes in organoids express HNF4α and albumin, as well as show tight junction, bile canaliculi, and CYP3A4 activity. Populating cholangiocytes can form cyst-like structures and have primary cilia and GGT activity expressing biliary markers, such as CK-7 and CK-19. Cell aggregates established from a single cell type, especially immortalized or cancer cells, are referred to as “spheroids.” Spheroid formation can be achieved using scaffold or scaffold-free techniques. Scaffold-free methods inhibit cells to adhere on the bottom surface by hanging drop culture media, constant rotation of flasks, or culture plates with an ultralow attachment surface. Although spheroids do not have multiple cell types, they are suitable for gene manipulation and drug screening tests.

Primary sclerosing cholangitis (PSC), primary biliary cholangitis, and biliary atresia (BA) are cholangiopathies that are characterized by biliary obstruction and damage as well as liver inflammation and fibrosis.⁽⁵⁾ Cholangiopathies are progressive disorders, and detailed mechanisms of the pathophysiology are still undefined. Curative treatments have not been established, and liver transplantation is required for patients in the advanced stages of cholangiopathies.⁽⁶⁾ In addition, patients with cholangiopathies, such as late-stage PSC, have a high risk for developing cholangiocarcinoma (CCA).⁽⁷⁾ Cholangiopathies are highly heterogeneous, but experimental models for developing personalized treatments are currently limited.⁽⁵⁾ Although classic monolayer or coculture systems have been standard in in vitro experiments, in vivo liver cells interact and communicate with each other and orchestrate the coordinated response to diseased conditions.⁽⁸⁾ Cholangiocytes secrete signals to induce autocrine responses in other cholangiocytes and paracrine events in hepatic stellate cells (HSCs), leading to biliary inflammation and liver fibrosis during injury.^(8,9) Classic 2D systems do not mimic 3D cell-to-cell interaction, architecture, communication, and microenvironment. In addition, protein expression patterns and cell functions differ between 2D and 3D culture systems.⁽¹⁰⁾ In CCA studies, 2D cultures for CCA cell lines and xenograft animal models are standard experimental models; however, these models fail to recapitulate critical features of a growing tumor in vivo, and 3D cultures are an advanced technology in which the cells can recapitulate the native physiology of cells in the healthy tissue of origin as well as the pathology of the cells in patient-derived tissues.⁽³⁾ Previous studies have developed liver organoids with various techniques and have demonstrated that 3D cell culture systems can contribute to cholangiopathy research as experimental models.⁽¹¹⁾ This review focuses on biliary disorders as well as biliary cells and summarizes current methodologies and techniques to generate biliary organoids/spheroids as models of cholangiopathies.

Current Methodologies and Sources to Generate Organoids With Biliary Phenotypes

PLURIPOTENT STEM CELLS

For organoid generation, the most common approach is to use pluripotent stem cells, such as ESCs or iPSCs, with Matrigel or collagen in order to provide ECM. These cells differentiate into hepatocytes and/or cholangiocytes, resulting in the formation of liver organoids. A previous study differentiated human iPSCs into definitive endoderm, foregut, and hepatic organoids on Matrigel.⁽¹²⁾ Immunofluorescence showed that some cells populated in the organoid were cytokeratin (CK)–19 positive biliary phenotypes and that some were hepatocyte nuclear factor 4α (HNF4α)–positive hepatocyte-like cells.⁽¹²⁾ Immunostaining also identified expression of zonula occludens-1 (ZO-1), the tight junction marker, in hepatic organoids and primary cilia in biliary cells, showing matured and differentiated hepatocytes and cholangiocytes in organoids.⁽¹²⁾ Abundant bile acids were detected in organoid supernatant, indicating that the hepatic organoid generated in this system is functional and able to synthesize bile acids in vitro.⁽¹²⁾ In another study, human iPSCs were differentiated into endoderm and mesoderm, and hepatic organoids were generated on Matrigel with cholesterol and other small molecules.⁽¹³⁾ Generated organoids consisted of HNF4α⁺ hepatocyte-like cells and epithelial cell adhesion molecule–positive (EpCAM⁺) CK-7⁺ cholangiocyte-like cells.⁽¹³⁾ Liver organoids secreted albumin and urea, as well as metabolized drugs, as evidenced by cytochrome P450 3A4 (CYP3A4) activity, showing functional “miniliver” features.⁽¹³⁾ Definitive endoderm and posterior foregut can be differentiated under 2D culture conditions from human ESCs or iPSCs and embedded in Matrigel to form hepatic organoids that consist of a mixed cell population with albumin+ hepatocytes and CK-7⁺ cholangiocytes.⁽¹⁴⁾ Liver organoids generated in this system were functional, showing albumin secretion and CYP3A4 activity.⁽¹⁴⁾ A fluorescent compound, 5-(and-6)-carboxy-2′7′-dichlorofluorescein (CDF), and its diacetate (CDFDA) were used to visualize the bile canaliculi network in liver organoids.⁽¹⁵⁾ Cell-permeable CDFDA diffuses into hepatocytes and is hydrolyzed into CDF. Detection of CDF revealed a bile canaliculi network in organoids, as well as CDF flow from the network into cholangiocyte cyst-like structures for CDF storage.⁽¹⁴⁾ Treatment of organoids with troglitazone, which causes intrahepatic cholestasis, inhibited CDF flow and induced apoptosis in organoids, indicating that liver organoids can mimic liver function and drug-induced cholestatic liver injury.⁽¹⁴⁾ Treatment of organoids with a cocktail of free fatty acids increased lipid deposition, triglyceride accumulation, and oxidative stress in organoids, indicating a potential for liver organoids to serve as a model of non-alcoholic fatty liver disease (NAFLD).⁽¹⁴⁾ A previous study generated the definitive endoderm from human ESCs and iPSCs seeded in Matrigel for organoid formation.⁽¹⁶⁾ Single-cell RNA sequencing for cells populated in liver organoids identified cell clusters expressing different hepatic cell markers, such as hepatocyte-like cells expressing HNF4α (HNF4A), cholangiocyte-like cells expressing CK-7 (CK7), and HSC-like cells expressing collagen type I.⁽¹⁶⁾ Liver organoids were incubated with oleic acid, and lipid and triglyceride deposition as well as proinflammatory cytokine production were up-regulated in organoids.⁽¹⁶⁾ Organoid stiffness was increased by oleic acid treatments due to fibrogenesis in organoids, indicating that liver organoids mimic pathophysiological cellular events in the liver and could be used as a model of NAFLD or nonalcoholic steatohepatitis (NASH).⁽¹⁶⁾ Another study generated organoids from human ESCs on Matrigel, and populating cells showed hepatic progenitor cell (HPC)–like gene expression, such as SRY-box transcription factor 9 (SOX9) and EpCAM.⁽¹⁷⁾ These cells could further differentiate into mature hepatocyte-like cells, and organoids showed functional characteristics including albumin and urea secretion and CYP3A4 activity.⁽¹⁷⁾ This study cocultured ESC-derived organoids with human fetal liver mesenchymal cells to form mixed organoids, and 7-day ethanol treatments induced fibrogenesis and collagen secretion in those organoids, indicating the potential of organoids as a meaningful in vitro model of alcohol-associated liver injury.⁽¹⁷⁾ Biliary spheroids can be generated from pluripotent stem cells, and a previous study described procedures for ductular spheroid formation.⁽¹⁸⁾ Human iPSCs seeded on Matrigel formed cell aggregates that highly express EpCAM, and these aggregates formed a spheroid-like structure or tubule-like ductal structure expressing biliary markers including CK-7 and CK-19.⁽¹⁸⁾

HEPATIC PROGENITOR CELLS

HPCs are liver stem cells located in the canal of Hering. HPCs can proliferate and differentiate into hepatocytes or cholangiocytes during liver injury.⁽¹⁹⁾ HPCs are identified by various markers, such as leucine-rich repeat-containing G protein–coupled receptor 5 (Lgr5),^(19,20) and liver organoids can be generated using HPCs instead of pluripotent stem cells. Broutier et al. have described detailed procedures to generate hepatic organoids from human or murine Lgr5⁺ HPCs isolated from liver tissues.⁽²¹⁾ Matured organoids generated by this system consisted of albumin⁺ or HNF4α⁺ hepatocytes and EpCAM⁺ cholangiocytes.⁽²¹⁾ The authors demonstrated that transfection and transduction using viral or expression vector had an effect on cells populating in organoids.⁽²¹⁾ A previous study generated murine liver organoids from isolated Lgr5⁺ HPCs on Matrigel and collagen type I.⁽²²⁾ Populating cells in organoids generated in this study had primary cilia as well as CK-7 and CK-19 expression but little albumin expression, indicating that most of the populating cells had biliary phenotypes.⁽²²⁾ The fluorescent compound rhodamine 123 was incubated with biliary organoids, and organoids could take up and transport fluorescence inside the organoid structure, showing their functional transporting activities.⁽²²⁾

LIVER AND BILE DUCT TISSUES

Primary hepatic cells from liver tissues can be a source of organoid generation because these cells contain HPCs. A previous study seeded human primary liver cells from liver biopsies on Matrigel and generated liver organoids.(23) Populating cells included EpCAM^– albumin⁺ hepatocytes and EpCAM⁺ CK-19⁺ cholangiocytes, and interestingly, cholangiocytes, but not hepatocytes, contributed to organoid formation in this system, showing the ability of biliary phenotypes to form organoids.⁽²³⁾ These tissue-derived liver organoids showed liver functions, such as albumin secretion, CYP3A4 activity, glycogen uptake, and LDL uptake.⁽²³⁾ The authors have developed the method for homology-directed repair–mediated knock-in of exogenous DNA in cells that populate in liver organoids using CRISPR–CRISPR-associated 9 (Cas9) technology, showing a potential for gene manipulation in organoids.⁽²⁴⁾ Primary cells from bile duct tissues can be used to generate biliary organoids, and a previous study isolated primary cholangiocytes from human extrahepatic bile ducts and seeded them on Matrigel to generate organoids.⁽²⁵⁾ Populating cells expressed biliary markers, such as CK-7, CK-19, and secretin receptor; and organoids showed functional features including rhodamine 123 transportation, alkaline phosphatase activity, and γ-glutamyl transferase (GGT) activity.⁽²⁵⁾ Previous studies reported detailed procedures for biliary organoid formation using primary cells from mouse or human extrahepatic bile ducts.^(26,27) Cells populating organoids generated from primary ductal cells showed CK-7, CK-19, and GGT expression as well as GGT activity but did not show albumin expression, indicating that those cells were cholangiocytes and did not differentiate into hepatocytes.⁽²⁷⁾ Bile ducts and cholangiocytes are heterogeneous, and their functions and responses against injury can differ depending on phenotypes.⁽²⁸⁾ Therefore, the functional features of biliary organoids may also differ depending on the cell source. A previous study generated biliary organoids that were derived from human intrahepatic or extrahepatic bile ducts or gallbladder.⁽²⁹⁾ Cells in organoids expressed higher levels of Lgr5 and CK-7 but low levels of albumin and HNF4α, indicating cholangiocyte-like and HPC-like, but not hepatocyte-like, phenotypes.⁽²⁹⁾ RNA sequencing revealed that transcriptomic profiles were unique and different between organoids derived from intrahepatic and extrahepatic bile ducts, indicating that characteristics of biliary organoids could vary with different locations of originated cells.⁽²⁹⁾ Liver organoids can be generated using a mixture of hepatic cells without ECM. Human primary hepatocytes, cholangiocytes, and liver sinusoidal endothelial cells (LSECs) were cocultured on plates with an ultralow attachment surface. Generated organoids consisted of albumin⁺ hepatocytes, CK-19⁺ cholangiocytes, and aquaporin 1–positive (AQP1⁺) LSECs (Fig. 2). CYP3A4 expression was also identified in organoids generated in this system, indicating that primary hepatic cells can form organoids without scaffold; and these organoids could help cholangiopathy studies as an in vitro model (Fig. 2).

FIG. 2.

Generation of liver organoids using isolated human hepatic cells and scaffold-free methods. (A) Left: Representative phase contrast microscopic image showing the isolated human intrahepatic cholangiocyte in culture. Right: Immunofluorescent staining showing CK-19 expression in isolated cholangiocytes. (B) Representative microscopic image showing the formation of liver organoids by coculturing of primary human hepatocytes, cholangiocytes, and LSECs for 14 days at low adhesive plates. (C) Immunofluorescent staining shows the expression of albumin and CYP3A4 in hepatocytes populating the generated organoid. (D) Immunofluorescence shows the expression of AQP1 in LSECs and CK-19 in cholangiocytes populating the organoid. Abbreviation: CHO, cholangiocyte.

IMMORTALIZED CHOLANGIOCYTES AND CCA CELL LINES

Cell aggregates generated from stem cells or primary hepatic cells are referred to as “organoids,” and those formed from a single cell type, especially immortalized cell lines, are recognized as “spheroids” or “cysts,” although clear definitions have not been established (Fig. 1). Immortalized normal rat cholangiocyte lines were generated, and biliary spheroids were established using these cell lines on Matrigel or polyethylene glycol hydrogel.⁽³⁰⁾ Over 70% of the populating cells were viable in spheroids, and accumulation of rhodamine 123 was identified in those biliary spheroids, showing transporter activity.⁽³⁰⁾ A previous study cultured the CCA cell lines SK-ChA-1 and Mz-ChA-1 on classic 2D culture plates or in a gyratory rotation incubator.⁽¹⁰⁾ This rotary cell culture system inhibited CCA cells adhering on the bottom surface and allowed 3D spheroid formation.⁽¹⁰⁾ Immunoblotting and 2D polyacrylamide gel electrophoresis analysis using cell lysates showed different protein expression levels and patterns between 2D-cultured and 3D-cultured CCA cells, indicating that functional characteristics of CCA cells may differ depending upon culture systems.⁽¹⁰⁾ CCA tumors are often accompanied with dense stroma, which is referred to as the “tumor microenvironment.”⁽³¹⁾ Activated HSCs or fibroblasts, which are recognized as cancer-associated fibroblasts (CAFs), secrete ECM components and contribute to fibrogenesis and development of the tumor microenvironment associated with CCA progression and metastases.(31) A previous study has generated rat cholangiocyte lines and alpha-smooth muscle actin + CAF lines from a rat model of CCA, and two cell lines were cocultured on collagen type I hydrogel to form organoids.⁽³²⁾ The authors have demonstrated that profibrogenic cytokine transforming growth factor beta 1 (TGF-β1) promotes proliferation and accumulation of CAFs in organoids as well as organoid expansion, indicating the pathological role of TGF-β1 in development of the CCA tumor microenvironment.⁽³²⁾

Organoids as Experimental Models of Cholangiopathies

PRIMARY SCLEROSING CHOLANGITIS

PSC is a cholestatic liver disease characterized by ductular reaction, biliary inflammation, and fibrosis.⁽⁶⁾ The cause of PSC is undefined, and there is no effective curative therapy.⁽⁶⁾ Patients with end-stage disorders often require liver transplantation, and PSC is one of the most common risk factors for CCA development.⁽⁷⁾ Cellular senescence in cholangiocytes is characteristic in PSC, and senescent cholangiocytes undertake a senescence-associated secretory phenotype (SASP) secreting elevated amounts of proinflammatory cytokines, such as interleukin (IL)-6 and C-C motif chemokine ligand 2 (CCL2) when compared to quiescent cholangiocytes.⁽³³⁾ Inhibition of cholangiocyte senescence may be a therapeutic approach for cholestatic liver injury to decrease ductular reaction and liver fibrosis.⁽⁹⁾ A previous study isolated and cultured cholangiocytes from patients with PSC and demonstrated that PSC cholangiocytes were senescent and secrete SASP markers including IL-1β, IL-6, IL-8, and CCL2.⁽³⁴⁾ The authors generated biliary spheroids using these cholangiocytes derived from patients with PSC with Matrigel on dishes precoated with poly(2-hydroxyethyl methacrylate) to prevent cell attachment on the surface.⁽³⁵⁾ Electron microscopy identified primary cilia and tight junctions between cholangiocytes populating biliary spheroids.⁽³⁵⁾ PSC-derived spheroids were more senescent than spheroids derived from normal cholangiocytes, and hydrogen peroxide treatments induced senescence in normal biliary spheroids.⁽³⁵⁾ Senescent spheroids secreted SASP makers, indicating that these biliary spheroids resembled 3D interaction and connection between cholangiocytes and demonstrated secretory functions in diseased conditions.⁽³⁵⁾ However, spheroids were established by cholangiocytes alone in these studies, and they lacked the support cells, such as HSCs, Kupffer cells, and LSECs, which do not mimic the in vivo environment and cell-to-cell interaction of cholangiocytes and other hepatic cells.

As mentioned previously, primary ductal cells isolated from bile ducts can form organoids with biliary phenotypes.⁽²⁹⁾ Soroka et al. have demonstrated organoid formation using cells isolated from bile samples of patients with PSC.⁽³⁶⁾ Bile samples ere filtered, and cells were isolated by centrifugation and seeded on Matrigel.⁽³⁶⁾ Populating cells were positive for biliary markers including EpCAM and CK-7, and organoids showed GGT expression and transporter activity detected by rhodamine 123.⁽³⁶⁾ RNA sequencing identified different gene expression profiles in organoid cells derived from the bile of patients with PSC compared to control organoid cells, indicating that bile samples are sufficient to generate biliary organoids that can be used for gene expression profiling to identify candidate therapeutic targets for PSC (Table 1).⁽³⁶⁾

TABLE 1.

Studies Using Organoids/Spheroids as a Model of Cholangiopathies

Source	Hydrogel	Populating Cells	Data Shown for Potential Models as
Human iPSCs(12)	Matrigel	Hepatocytes, cholangiocytes	Alagille syndrome
Human ESCs and iPSCs(14)	Matrigel	Hepatocytes, cholangiocytes	Drug-induced cholestasis, NAFLD
Human ESCs and iPSCs(16)	Matrigel	Hepatocytes, cholangiocytes, HSCs, HPCs, Kupffer cells	NAFLD, NASH, Wolman disease
Human ESCs(17)	Matrigel	Hepatocytes, cholangiocytes	Alcohol-associated liver disease
Isolated cholangiocytes from patients with PSC(35)	Matrigel on dishes coated with poly-HEMA	Cholangiocytes	PSC
Isolated cells from bile samples(36)	Matrigel	Cholangiocytes	PSC
Immortalized murine cholangiocyte line(38–40)	Matrigel and collagen type I	Cholangiocytes	BA
EpCAM+ cells from liver biopsies(41)	Matrigel	Hepatocytes and cholangiocytes for BA organoids	BA
Primary human cells from liver biopsy(45,46)	BME type 2	CCA tumor cells	CCA

Abbreviation: HEMA, hydroxyethylmethacrylate.

BILIARY ATRESIA

BA is a cholangiopathy affecting infants exclusively, characterized by biliary inflammation and liver fibrosis.⁽³⁷⁾ Viral infections or toxins are suggested to cause insufficient bile duct development leading to BA, although the detailed mechanisms are undefined.⁽³⁷⁾ Although injection of rhesus rotavirus into newborn mice is a common animal model, other experimental models are limited for BA.⁽⁵⁾ A previous study has identified an isoflavonoid, referred to as “biliatresone,” from plants, Dysphania genus, that causes morphological biliary destruction in zebrafish larvae.⁽³⁸⁾ This study generated biliary spheroids using an immortalized murine cholangiocyte line and demonstrated that biliatresone induced morphological disruption and lumen closure in biliary spheroids, indicating that this isoflavonoid causes BA-like symptoms.⁽³⁸⁾ Biliatresone also induced mislocalization of ZO-1 in biliary spheroids.⁽³⁹⁾ Detection of loaded rhodamine in spheroids demonstrated that biliatresone-treated spheroids were leaky compared to untreated spheroids, suggesting destroyed tight junctions and cell-to-cell architecture in spheroids.⁽³⁹⁾ The authors have demonstrated that biliatresone-induced cholangiocyte destruction is mediated by decreased levels of glutathione and SOX17.⁽³⁹⁾ Biliary organoids generated in this system were used to identify signaling pathways in biliatresone-induced cholangiocyte destruction.⁽⁴⁰⁾ Decreased glutathione levels in populating cholangiocytes induced up-regulation of Wnt and Notch signaling pathways, leading to down-regulation of SOX17 and cholangiocyte damage.⁽⁴⁰⁾ Babu et al. isolated EpCAM⁺ cells from liver biopsies of patients with BA and generated organoids on Matrigel.⁽⁴¹⁾ Organoids derived from patients with BA showed slow organoid expansion and aberrant morphology with scattered ZO-1 expression compared to control organoids.⁽⁴¹⁾ Rhesus rotavirus A–infected neonatal mice were also used for organoid generation, and infected mice-derived organoids showed similar BA-like morphological aberration compared to organoids derived from uninfected mice.⁽⁴¹⁾ Populating cells in human control organoids were CK-19⁺ and HNF4α^–, indicating biliary phenotypes; but cells in BA organoids were CK-19⁺ and HNF4α⁺, indicating transdifferentiation from cholangiocytes to hepatocytes with unknown reasons or mechanisms.⁽⁴¹⁾ The authors identified accumulation of beta-amyloid in cholangiocytes of liver samples from patients with BA and BA-derived organoids and demonstrated that beta-amyloid exposure induces cholangiocyte destruction in organoids.⁽⁴¹⁾ These studies support the potential of biliary organoids/spheroids as in vitro models to understand the pathophysiological mechanisms of BA.

CHOLANGIOCARCINOMA

CCA is a highly malignant biliary tract cancer. CCA heterogeneity on both the clinical and molecular levels and scarce knowledge of etiology have limited the discovery of biomarkers and impeded the development of tools for early diagnosis and effective treatments.⁽⁷⁾ Because early diagnosis of CCA is challenging and treatment options are limited, therapeutic approaches are necessary.⁽⁴²⁾ However, there are no gold-standard experimental models of CCA, and the most common animal models are xenograft models, which do not mimic CCA development and the tumor microenvironment.⁽⁴³⁾ Organoids generated from primary cells of tumor biopsies are also referred to as “tumoroids,” which means tumor-like organoids resembling 3D tumor complexity and the tumor microenvironment.⁽⁴⁴⁾ CCA-derived organoids/spheroids may provide experimental models for CCA studies. A previous study generated organoids from liver biopsies from patients with primary liver cancers, hepatocellular carcinoma (HCC) or CCA, with basement membrane extract (BME) type 2 as ECM components.⁽⁴⁵⁾ CCA organoids, but not HCC organoids, were EpCAM⁺.⁽⁴⁵⁾ HCC and CCA organoids retained gene expression profiles and genetic alterations from the original tumor tissues.⁽⁴⁵⁾ This study performed drug screening and identified a candidate therapeutic agent, extracellular signal–regulated kinase inhibitor SCH772984, indicating the promising potential use of organoids as CCA in vitro models.⁽⁴⁵⁾ Another study has demonstrated that cancer organoids can be generated from needle biopsies (~1 mm in diameter and 5–10 mm in length).⁽⁴⁶⁾ HCC and CCA organoids preserved histological characteristics and genetic alterations from original tumor tissues.⁽⁴⁶⁾ Patient-derived organoids were treated with sorafenib and showed that the drug response could differ depending on individuals, suggesting the potential of organoids for designing personalized chemotherapy regimens.⁽⁴⁶⁾ A total of 27 patient-derived cancer organoids were generated from patients with primary liver cancers including CCA, and drug screening was performed for 129 different drugs.⁽⁴⁷⁾ Results showed that drug response varied depending on individuals and identified candidate drugs with universal effects across all organoids.⁽⁴⁷⁾ Patient-derived organoids may contribute to the identification of pan-effective cancer drugs, as well as the development of personalized chemotherapy.

CCA tumors often have genetic aberrations, such as isocitrate dehydrogenase (IDH) mutations and fibroblast growth factor receptor fusions.⁽⁴⁸⁾ Mutant IDH proteins have malfunctions producing abnormal amounts of oncometabolite D-2-hydroxyglutarate (2-HG), leading to CCA development.⁽⁴⁹⁾ A previous study generated organoids using primary liver cells isolated from mouse liver tissues with Matrigel and performed genetic transduction with lentiviral vectors to express human mutant IDH1 in murine hepatic organoids.⁽⁵⁰⁾ Populating cells in organoids were CK-19⁺, and higher levels of 2-HG were detected in cell extracts of organoids expressing mutant IDH1 compared to those with wild-type IDH1.⁽⁵⁰⁾ Mutant IDH1 expression facilitated organoid formation as well as glucose metabolism in organoids, indicating the pathological roles of IDH1 mutations in CCA.⁽⁵⁰⁾ Breast cancer 1 associated protein 1 (BAP1) is a deubiquitinating enzyme that functions as a tumor suppressor, and absent expression of BAP1 is often observed in patients with intrahepatic CCA.⁽⁵¹⁾ Liver organoids were generated from human liver biopsies with BME matrix, and BAP1 was knocked out by CRISPR-Cas9 technology.⁽⁵²⁾ BAP1 depletion induced scattered ZO-1 expression and morphological destruction in organoids, as well as alterations of gene expression in junctional and cytoskeleton components, which are associated with tumorigenesis.⁽⁵²⁾ These studies suggest that cancer-derived organoids/tumoroids are suitable for transfection or transduction to study certain proteins or gene aberrations of the donor patient. Most recently, CCA organoids were formed using human CCA lines Mz-ChA-1 and SG231 together with human mast cells and/or HSCs on plates with an ultralow attachment surface for organoid formation.⁽⁵³⁾ This study showed that administration of histamine receptor inhibitors decreased expression of genes associated with tumor progression and metastases through stimulator of interferon genes in CCA organoids, indicating the roles of mast cells and histamine signaling in CCA.⁽⁵³⁾ Another study generated organoids with human CCA cell lines, normal primary cholangiocytes, and HSCs.⁽⁵⁴⁾ The authors demonstrated that CCA organoids enable study of the tumor microenvironment for expression of cancer/progenitor markers, apoptosis, collagen formation, and invasion/metastasis under chemotherapy.⁽⁵⁴⁾

Conclusions and Future Perspectives

Current studies have demonstrated that (1) hepatic organoids/spheroids can be generated from the differentiation of pluripotent stem cells or HPCs, primary cells isolated from whole liver or bile duct tissues, or cancer/immortalized cell lines; (2) organoids generated from stem cells or HPCs consist of multiple hepatic cells, such as hepatocyte-like cells and cholangiocyte-like cells; (3) organoids can have functions, such as transporter or enzyme activity; (4) patient-derived organoids can be generated to identify morphological and functional abnormalities as well as mutation profiles; (5) drug screening tests using patient-derived organoids may allow evaluation of effectiveness and sensitivity for the donor, to design personalized chemotherapy; and (6) genetic manipulation can be performed for organoids to study functional roles of the gene of interest in cell structure, morphology, cell-to-cell interaction, and functions through RNA sequencing, immunofluorescence, and enzyme-linked immunosorbent assay (Fig. 3). These features overcome common problems in classic 2D monolayer models, such as lack of 3D cellular architecture, organization, and microenvironment. Liver organoids can be cultured for long periods of time, which is a useful feature compared to 2D models.⁽²³⁾ Although cholangiopathies are bile duct disorders, not only cholangiocytes but also other hepatic cells are involved in the pathophysiological responses, such as inflammatory responses in hepatic macrophages and fibrogenic activities of HSCs.⁽⁸⁾ Because liver organoids can consist of various hepatic cells, 3D cell culture systems may be more useful than classic 2D systems to study intercellular communication between hepatic cells during injury.⁽¹⁶⁾ In addition, ductular reaction, which is proliferative and responsive to bile duct expansion, is identified not only in cholangiopathies but also in other liver diseases, such as NAFLD, indicating pathological involvement of cholangiocytes.⁽¹⁹⁾ Liver organoids containing biliary phenotypes may be more suitable for experimental models for various liver diseases than hepatocyte-only culture systems, to resemble pathophysiological events in liver tissues more appropriately.

FIG. 3.

Experimental potentials of hepatic organoids. Generated liver organoids/spheroids are suitable for drug screening and gene manipulation, such as transfection and transduction. Effects can be obtained by RNA sequencing for gene expression profiling, immunofluorescence for protein expression and localization, dose–response curve analysis for drug sensitivity tests, and enzyme-linked immunosorbent assay for protein secretion into culture media. Patient-derived organoids can be used to observe morphology as well as mutation screening to identify gene aberrations. Abbreviation: ELISA, enzyme-linked immunosorbent assay.

Both hepatocytes and cholangiocytes are plastic and can transform into the other hepatic cell type for liver regeneration and disease pathogenesis. Hepatocytes can transdifferentiate into cholangiocytes during biliary damage, and cholangiocytes can transdifferentiate into hepatocytes during severe hepatocyte loss, which indicates the importance of their crosstalk.⁽¹⁹⁾ Although previous studies have identified associated signaling pathways, such as Notch and Wnt signaling, data can be controversial depending on experimental conditions; and detailed mechanisms are still undefined.⁽³⁴⁾ Liver organoids may be a powerful tool for the study of fate and plasticity of hepatic cells. Feeding with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet induces biliary damage in rodents, and Aloia et al. isolated EpCAM⁺ biliary cells from liver tissues of DDC-fed mice and generated organoids with Matrigel.⁽⁵⁵⁾ The authors found that cells in organoids underwent genome-wide remodeling in transcriptional profiles due to DDC-induced biliary damage, which may be critical in transdifferentiation and liver regeneration.⁽⁵⁵⁾ Another study transfected cells in biliary organoids with adeno-associated virus for HNF4α overexpression.⁽⁵⁶⁾ Biliary cells became hepatocyte-like cells with HNF4α overexpression, expressing hepatocyte marker genes, such as albumin, transthyretin, and cytochrome P450 3A11.⁽⁵⁶⁾ Future studies may reveal detailed mechanisms of hepatic transdifferentiation and liver regeneration using liver organoids.

In conclusion, 3D organoid/spheroid culture systems derived from human cells are emerging as the next generation in vitro research models of cholangiopathies including CCA, which cannot be achieved with classic 2D cell culture systems and animal models.

Abbreviations:

AQP1

aquaporin 1

BA

biliary atresia

BAP1

breast cancer 1 associated protein 1

BME

basement membrane extract

CAF

cancer-associated fibroblast

CCA

cholangiocarcinoma

CDF

5-(and-6)-carboxy-2’7’-dichlorofluorescein

CK

cytokeratin

CYP3A4

cytochrome P450 3A4

2D

two-dimensional

3D

three-dimensional

DDC

3,5-diethoxycarbonyl-1,4-dihydrocollidine

ECM

extracellular matrix

EpCAM

epithelial cell adhesion molecule

ESCs

embryonic stem cells

GGT

γ-glutamyl transferase

HCC

hepatocellular carcinoma

HNF4α

hepatocyte nuclear factor 4α

HPCs

hepatic progenitor cells

HSCs

hepatic stellate cells

IDH

isocitrate dehydrogenase

IL

interleukin

iPSCs

induced pluripotent stem cells

Lgr5

leucine-rich repeat-containing G-protein coupled receptor 5

LSECs

liver sinusoidal endothelial cells

NAFLD

non-alcoholic fatty liver disease

PSC

primary sclerosing cholangitis

SASP

senescence-associated secretory phenotype

SOX

SRY-box transcription factor

ZO-1

zonula occludens-1