Biology subjects

Spee, B.

Publications and source records attributed to Spee, B..

4 recordsLinked to original sources

bioRxiv2024

Novel hepatocyte-like liver organoids recapitulate crucial mature hepatic functions

Accurate liver disease modeling and drug toxicity testing still remain challenging as liver cells in vitro poorly resemble adult hepatocytes, as we previously demonstrated using whole transcriptome and cell identity analysis. To address this, we used our insights into hepatic modeling to develop hepatocyte-like liver organoids (HeLLOs), a novel human organoid model with mature hepatocyte functions superior to existing models. HeLLOs are easily established from (small) healthy or diseased liver tissues and rapidly expanded for an extended period in optimized culture conditions. Transcriptomic and functional analyses revealed that differentiated HeLLOs closely resemble fresh primary human hepatocytes (PHHs) and perform key hepatic functions such as gluconeogenesis, drug metabolism, and bile acid synthesis. We developed a HeLLO-based toxicity assay with higher sensitivity in predicting liver toxicity of known liver-toxic drugs compared to the gold-standard PHHs. By modeling disease-related mechanisms, such as bile acid transport, HeLLOs uncover transport-inhibition toxicity mechanisms of known liver toxic drugs. Single cell sequencing analysis of HeLLOs identified a heterogeneous cluster of cells with cholangiocyte-like and hepatocyte-like cells, overall resembling liver regenerative cells. As such, HeLLOs hold great promise for advancing liver disease modeling and drug testing. To our knowledge, HeLLOs are the best expandable liver model for predicting adverse drug reactions as well as modeling various liver disease mechanisms.

cell biology↗

bioRxiv2024

A hollow fiber membrane-based liver organoid-on-a-chip model for examining drug metabolism and transport

Liver-on-a-chip models predictive for both metabolism as well as canalicular and blood transport of drug candidates in humans are lacking. Here, we established an advanced, bioengineered and animal component-free hepatocyte-like millifluidic system based on 3D hollow fiber membranes (HFMs), recombinant human laminin 332 coating and adult human stem cell-derived organoids. Organoid fragments formed polarized and tight monolayers on HFMs with improved hepatocyte-like maturation, as compared to standard 3D organoid cultures in Matrigel from matched donors. Gene expression profiling and immunofluorescence revealed that hepatocyte-like monolayers expressed a broad panel of phase I (e.g., CYP3A4, CYP2D6) and II (UGTs, SULTs) drug-metabolizing enzymes and drug transporters (e.g., OATP1B3, MDR1 and MRP3). Moreover, statically cultured monolayers displayed phase I and II metabolism of a cocktail of six relevant compounds, including midazolam and 7-hydroxycoumarin. We also demonstrated the disposition of midazolam in the basal/blood-like circulation and apical/canalicular-like compartment of the millifluidic chip. Finally, we connected the system to the other two PK/ADME-most relevant organ systems, i.e. small intestine- and kidney proximal tubule-like to study the bioavailability of midazolam and coumarin, and excretion of metformin. In conclusion, we generated a proof-of-concept liver organoid-on-a-chip model for examining metabolism and transport of drugs, which can be further developed to predict PK/ADME profiles in humans.

pharmacology and toxicology↗

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↗

bioRxiv2021

Locational memory of macrovessel vascular cells is transcriptionally imprinted

The locational predisposition of vascular pathologies illustrates the need for a better insight into vascular heterogeneity. To investigate the transcriptomic basis of angiodiversity, we isolated and analyzed transcriptomes from endothelial cells and vascular smooth muscle cells from nine different adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We identified both reported and novel expression patterns defining specialized adult blood vessels. Our findings also show that adult vascular cells in culture express a remarkably high number of transcription factors crucial to organ development in the embryo. The persistent expression of these genes in culture indicates that these genes are not regulated by the flow or surrounding cell types but are rather fixed in the molecular memory. Therefore, our findings prompt the re-thinking of the extrapolation of results from single-origin endothelial cell systems.

cell biology↗