The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism

Vivian Lehmann, Imre F Schene, Arif I Ardisasmita, Nalan Liv, Tineke Veenendaal, Judith Klumperman, Hubert P J van der Doef, Henkjan J Verkade, Monique M A Verstegen, Luc J W van der Laan, Judith J M Jans, Nanda M Verhoeven-Duif, Peter M van Hasselt, Edward E S Nieuwenhuis, Bart Spee, Sabine A Fuchs*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.

Original languageEnglish
Pages (from-to)353-365
Number of pages13
JournalJournal of Inherited Metabolic Disease
Volume45
Issue number2
DOIs
Publication statusPublished - Mar 2022

Bibliographical note

Funding Information:
The authors are grateful for the collaborative “United for Metabolic Diseases (UMD)” efforts to improve care for patients with (genetic) metabolic diseases. Moreover, the authors thank prof. Dr. Jeffrey Beekman for kindly providing relevant medications and inhibitors for CF related experiments. This work was supported by Metakids funding (to Sabine A. Fuchs), a Clinical Fellows grant (40-00703-97-13537 to Sabine A. Fuchs) and a grant from the research program Applied and Engineering Sciences (15498 to Bart Spee), both from The Netherlands Organization for Health Research and Development. Mechanistic studies and treatment development for rare diseases are limited by the small number and geographic distribution of patients. Recently developed organoid models promise exciting possibilities for patient-specific preclinical studies. However, it is currently unknown for which specific metabolic functions and diseases ICOs can be effectively used. To address this, we present our experience with ICOs and evaluate the potential and limitations of patient-derived ICOs to study metabolic functions. We show that ICOs can be used to study basic metabolism, more specific hepatic functions, and transport functions, exemplified respectively by pathways of BCAA metabolism, copper metabolism, and chloride transport. We noticed that IEM gene expression in ICOs and fibroblasts varied within each metabolic category. Cell model choice should be done case by case with focus on the IEM gene/pathway of interest. ICO transcriptome variance was supported by TEM analysis which revealed that ICOs are composed of intermediate cell types with progenitor cell, hepatic, and biliary characteristics. Likely, the cells' ductal origin as well as the environmental stimuli offered to ICOs promote this intermediary cell type. Indeed, it is well known that environmental stimuli affect cell fate.34–38 Adjustments in DM and hydrogel composition could favor expression of some as of yet absent metabolic functions. We and other research groups are currently exploring different approaches to achieve improved separate hepatic or cholangiocyte differentiation of ICOs.35 We anticipate new insights to arise and be adopted widely in the coming decade. Until then, this paper addresses the recurring queries on ICO use for current clinical and research questions. Several cell models are available for IEM research, including, ICOs, fibroblasts, cell lines, primary hepatocytes, and induced pluripotent stem cells (Table 1). Most of these cell models are suitable for personalized medicine approaches and biobanking. Cell model choice will depend on the specific study goal, availability of patient cells through skin and/or liver biopsy, representation of metabolic function, costs, and expertise. Although ICOs require some expertise and investment, they score high for other categories. ICOs not only express a variety of IEM genes, but time to first assay and ease of handling are additional advantages. Moreover, ICO culture is versatile; both long-term 3D and 2D transwell cultures are possible, as previously reported for gut organoids.39 Abbreviations: iPSCs, induced pluripotent stem cells; PHH, primary human hepatocytes; +, applicable; ++, very applicable; −, not applicable; (+)+/−(−), applicable with limitations, while ++ and − − indicate better or worse; 2D, two-dimensional; 3D, three-dimensional. Moreover, ICO generation and differentiation does not require genetic reprogramming or immortalization.40,41 Previous in vitro copper metabolism studies were all performed in genetically induced fibroblasts or embryonic stem cells.42–44 We show this can also be done in ICOs which retain the original patient (epi)genome.6,41,45 Further studies are needed to determine applicability of Wilson disease ICOs for personalized drug testing.46,47 To study basic metabolism, a hepatic phenotype is not required. Although transcriptome analysis favors ICOs to study MMA (Figures 3 and S2B,C), several studies have reported successful phenotyping of MMA in patient fibroblasts and immortalized kidney tubule cells, derived from patient urine.48–50 This illustrates that lower expression of genes does not necessarily result in absence of a disease phenotype. In ICOs derived from MMA patients, we discerned significantly increased concentrations of the clinical biomarker propionylcarnitine, which represents a first step toward studying MMA treatment response in a personalized setting in ICOs. Importantly, ICO differentiation capacity varies between donors. It is currently unclear whether this relates to a specific biopsy, isolation or a donor's genetic background or age. It has been shown that extrahepatic cholangiocyte progenitors cannot differentiate to hepatocytes, indicating that the location of cholangiocyte progenitors is crucial for their differentiation potential.51,52 Yet, this interdonor variability does not hamper studying intradonor differences after treatment. When patient material is scarce, patient mutations may be introduced in cells.42 Current CRISPR-based technologies are also applicable to ICOs.22,53,54 Moreover, mechanistic insight can be achieved by editing different genes in a pathway. Evidently, artificial IEM ICO models may be helpful in investigating the gene in isolation, but not for personalized strategies. Complete absence of a key pathway gene is likely to hamper studies thereof. For example, we expect oxalate metabolism studies in ICOs to be impeded by absence of expression of the peroxisomal AGXT gene. Peroxisomal assembly genes such as the PEX, PPAR, and ABCD families were well expressed, suggesting availability of peroxisome machinery in ICOs. In contrast, electron microscopy analysis revealed a reduced peroxisome size. Peroxisome biogenesis is highly plastic and dependent on nutrient availability and culture confluency.55–59 This has been shown for HepG2 peroxisomes which transiently become tubular rather than spherical during periods of rapid growth.59 The smaller peroxisomes in ICOs might represent this transient morphology in peroxisome biogenesis. Provision of relevant substrates in culture media as well as improved differentiation conditions might promote peroxisome maturation and expression of yet absent genes. Initially ICOs were described as a liver model to study specific hepatic functions. Concurrently, ICOs were shown to eliminate urea, metabolize drugs, and secrete albumin.6,14,15 ICOs are derived from the oval cell, or bi-potent liver progenitor, but current methods do not suffice to generate a pure population of mature hepatocytes. With current methods, ICOs are suitable for studying a selection of basic, hepatic, and cholangiocyte metabolic functions. Prior to using ICOs for a specific research question, expression of the corresponding pathway and/or function should be considered. If the full spectrum of mature hepatic functions is required, a different cell model is more suitable. Recently, hepatic liver organoids (HLOs) were established from foetal hepatocytes.60 These showed an improved hepatic phenotype compared to ICOs. We anticipate that generation of HLOs from pediatric and adult tissue will provide an improved hepatic patient-specific in vitro model and will fill some gaps in patient-related IEM research. To conclude, we provide an overview of metabolic functions and IEMs, which can be studied with ICOs. Presence of mitochondria, lysosomes and the ER combined with good gene expression in energy, amino acid, and lipid metabolism suggest that ICOs are suitable to study related functions and diseases. Furthermore, the 3D nature of ICOs renders the model highly suitable for transepithelial transport studies. We present several functional assays with which to study drug responses preclinically. This is especially relevant for IEMs where global patient numbers and geographic distribution do not allow for standard clinical drug testing. Our transcriptome data may be of help to decide whether ICOs are a suitable model for a specific research question. For diseases that can currently not be studied with ICOs, we anticipate that improved culturing conditions and/or adult HLOs will be available in the near future. The authors are grateful for the collaborative “United for Metabolic Diseases (UMD)” efforts to improve care for patients with (genetic) metabolic diseases. Moreover, the authors thank prof. Dr. Jeffrey Beekman for kindly providing relevant medications and inhibitors for CF related experiments. This work was supported by Metakids funding (to Sabine A. Fuchs), a Clinical Fellows grant (40-00703-97-13537 to Sabine A. Fuchs) and a grant from the research program Applied and Engineering Sciences (15498 to Bart Spee), both from The Netherlands Organization for Health Research and Development.

Funding Information:
The authors are grateful for the collaborative “United for Metabolic Diseases (UMD)” efforts to improve care for patients with (genetic) metabolic diseases. Moreover, the authors thank prof. Dr. Jeffrey Beekman for kindly providing relevant medications and inhibitors for CF related experiments. This work was supported by Metakids funding (to Sabine A. Fuchs), a Clinical Fellows grant (40‐00703‐97‐13537 to Sabine A. Fuchs) and a grant from the research program Applied and Engineering Sciences (15498 to Bart Spee), both from The Netherlands Organization for Health Research and Development.

Publisher Copyright:
© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.

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