Project description:We generated intrahepatic cholangiocyte organoids and fetal hepatocyte organoids to compare their transcriptomic profile with liver tissue, primary human hepatocytes, and other hepatocyte model systems.
Project description:The myriad of available hepatocyte in vitro models provides researchers the possibility to select hepatocyte-like cells (HLCs) for specific research goals. However, direct comparison of hepatocyte models is currently challenging. We systematically searched the literature and compared different HLCs, but reported functions were limited to a small subset of hepatic functions. To enable a more comprehensive comparison, we developed an algorithm to compare transcriptomic data across studies that tested HLCs derived from hepatocytes, biliary cells, fibroblasts, and pluripotent stem cells, alongside primary human hepatocytes (PHHs). This revealed that no HLC covered the complete hepatic transcriptome, highlighting the importance of HLC selection. HLCs derived from hepatocytes had the highest transcriptional resemblance to PHHs regardless of the protocol, whereas the quality of fibroblasts and PSC derived HLCs varied depending on the protocol used. Finally, we developed and validated a web application (HLCompR) enabling comparison for specific pathways and addition of new HLCs. In conclusion, our comprehensive transcriptomic comparison of HLCs allows selection of HLCs for specific research questions and can guide improvements in culturing conditions.
Project description:Mass spectrometry based secretomics approaches preferentially utilize serum-free culture conditions to circumvent serum induced interference and enable sufficient analytical depth. However, this negatively affects a wide range of cellular functions and thus reduces the biological relevance. Moreover, the experimental time range is limited by the viability of cells under serum free culture conditions which results in potentially missing out a large fraction of transcriptionally regulated secretion events and feedback-loops. We present an “interval-based” secretomics workflow, that addresses the shortcomings of the serum-free secretomics approach, by probing protein secretion rates in short serum-free time windows. We applied this approach to unravel the time-resolved secretion in the hepatocyte model systems HepG2 and HepaRG after stimulation of the acute-phase response (APR), a cellular reaction to inflammatory stimuli that typically manifests over days. The integration of relative quantification using tandem mass tags (TMT) enabled precise monitoring of time dependent changes in the secretome over days. Cell line and cytokine specific changes were observed: IL1b directed the APR towards pathogen defense over three distinct chronological phases. In contrast, IL6 directed the APR towards regeneration. Cell surface shedding was pronounced upon IL1b stimulation and small molecule inhibition of ADAM and matrix metalloproteases identified induced as well as constitutive shedding events. Inhibition of ADAM proteases by TAPI-0 not only led to reduced shedding of the sorting receptor SORT1, but also an attenuated cytokine response suggesting a direct link between cell surface shedding and cytokine secretion rates.
Project description:Pancreatic cancer is an aggressive disease with a poor prognosis for which current standard chemotherapeutic treatment options offer little survival benefit. In recent years, receptor tyrosine kinases (RTK)s have garnered interest as therapeutic targets to augment or replace standard chemotherapeutic therapies because of their high expression levels in various cancers and their ability to promote cell growth, migration, and survival. Met and Ron, which are homologous RTKs activated by the ligands hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), respectively, are over-activated in many of the least treatable cancers. In pancreatic adenocarcinoma, Met expression is linked to poor patient survival and Ron expression is generally higher in tumor samples relative to normal tissue, although its prognostic significance in pancreatic cancer remains unclear. Despite the structural homology between Met and Ron, studies that have directly compared the functional outcomes of these systems in any context are limited. To address this, we sought to determine if the HGF/Met and MSP/Ron systems produce overlapping or divergent contributions towards a malignant phenotype by performing a characterization of MSP and HGF driven signaling, behavioral, and transcriptomic responses in pancreatic cancer cells in vitro. We found HGF and MSP both encouraged cell migration and activated the MAPK/Erk pathway both at the transcript and protein level. HGF uniquely increased proliferation in addition to regulating a wider variety of transcripts compared to MSP. Although HGF and MSP produced a differing breadth of responses, overlapping pro-cancer signaling, behavioral, and transcriptional effects suggest dual inhibition of the MSP/Ron and HGF/Met systems in pancreatic cancer may provide a more complete anti-cancer effect compared to individually targeting either system.