Biology subjects

Fuchs, S. A.

Publications and source records attributed to Fuchs, S. A..

5 recordsLinked to original sources

bioRxiv2026

Multiscale three-dimensional ultrastructural mapping of intestinal tissues and organoids

Understanding cell biology in native environments requires imaging of subcellular organization in three dimensions. In the intestinal epithelium, multiple cell types organize along the crypt-villus axis, where cell-cell interfaces and subcellular architecture control cell differentiation, tissue organization and epithelial function. Resolving these features volumetrically remains challenging: light microscopy offers molecular specificity but has limited resolution, whereas electron microscopy provides ultrastructural detail but is poorly suited to volumetric acquisition combined with specific protein labeling. Here, we show that expansion microscopy enables the multiscale volumetric study of epithelial ultrastructure in tissue sections and organoid models. Using an optimized workflow, we resolve epithelial tissue architecture, cell types and subcellular features within volumes across scales. Application to a microvillus inclusion disease (MVID) organoid model revealed disease-associated ultrastructural phenotypes that were only observed using electron microscopy. Our results establish expansion microscopy as key technology for studying three-dimensional cell biology within intestinal tissue and tissue mimics.

cell biology↗

bioRxiv2024

Mass Generation and Long-term Expansion of Hepatobiliary Organoids from Adult Primary Human Hepatocytes

Adult primary human hepatocytes (PHHs) are the gold standard in ex vivo toxicological studies and possess the clinical potential to treat patients with liver disease as advanced therapy medicinal products (ATMPs). However, the utility of this valuable cell type has been limited by short-term functionality and limited expansion potential in vitro. While notable advances have been made in the long-term maintenance of primary hepatocytes, there has been limited success in driving the efficient generation and expansion of adult PHH-derived organoids which recapitulate both liver tissue architecture and function, hampering in vitro studies and regenerative medicine applications. Here we describe the mass generation and long-term expansion of hepatobiliary organoids with functionally interconnected hepatic and biliary-like structures from adult primary human hepatocytes. Hepatobiliary organoids retain the expression of lineage and functional markers, closely resembling PHH, while also acquiring the expression of regeneration, fetal and biliary markers. Organoids perform key hepatocyte functions while proliferating and can be matured to enhance their functionality. As a proof-of-principle, we demonstrate that hepatobiliary organoids can recapitulate hallmarks of cholestasis and steatosis in vitro. Moreover, we show that hepatocytes can be transfected, transduced and gene edited in 3D prior to organoid generation, facilitating a wide range of applications. Our novel hepatobiliary organoid system bridges the gap between short-term functionality of primary human hepatocytes and the need for scalable, long-term organoid models of the adult liver, offering immense potential for drug testing, disease modeling, and advanced therapeutic applications.

cell biology↗

bioRxiv2023

Tracer-based lipidomics identifies novel disease-specific biomarkers in mitochondrial β-oxidation disorders

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and possible influence on cellular lipid homeostasis are, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore new disease markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two new candidate disease markers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second biomarker is an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. These are hypothesized to be degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two new disease markers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

molecular biology↗

bioRxiv2021

Mutation-specific reporter for the optimization and enrichment of prime editing

We present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) and any prime editor protein, including novel variants with flexible PAM recognition. Successful reporter editing enriches for genomic editing. FluoPEER can be employed for efficient correction of patient cells and to elucidate cellular mechanisms needed for successful prime editing.

molecular biology↗

bioRxiv2020

Prime editing for functional repair in patient-derived disease models

Prime editing is a novel genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. We develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding {beta}-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers diseasecausing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

genomics↗