Pilz, F. | bioRxiv Science

bioRxiv2024

A perfusion-independent high-throughput method to isolate liver sinusoidal endothelial cells

Liver sinusoidal endothelial cells (LSECs) critically regulate homeostatic liver function and liver pathogenesis. However, the isolation of LSECs remains a major technological bottleneck in studying molecular mechanisms governing LSEC functions. Current techniques to isolate LSECs, relying on perfusion-dependent liver digestion, are cumbersome with limited throughput. We here describe a perfusion-independent high-throughput procedure to isolate LSECs with high purity. Indifferently from previous perfusion-independent approaches, chopped liver tissue was incubated in the digestion mix for 30 minutes with intermittent mixing with a serological pipette. This led to the safeguarding of LSEC integrity and yielded 10 {+/-} 1.0 million LSECs per adult mouse liver, which is far higher than previous perfusion-independent protocols and comparable yield to established perfusion-dependent protocols for isolating LSECs. Combining magnetic and fluorescence-activated cell sorting (FACS), LSECs from different zones of the hepatic sinusoid can now be isolated in high numbers in less than two hours for downstream applications including proteomics. Our protocol enabled the isolation of LSECs from fibrotic liver tissues from mice and healthy liver tissues from higher vertebrate species (pigs), where traditional perfusion-based digestion protocols have very limited application. In conclusion, these technical advancements reduce post-mortem changes in the LSEC state and aid in reliable investigation of LSEC functions.

physiology↗

bioRxiv2023

Single-cell time series analysis reveals the dynamics of in vivo HSPC responses to inflammation

Hematopoietic stem and progenitor cells (HSPCs) are known to respond to acute inflammation; however, little is understood about the dynamics and heterogeneity of these stress responses in HSPCs. Here, we performed single-cell sequencing of HSPCs during the sensing, response and recovery phases of the inflammatory response of HSPCs to treatment with the pro-inflammatory cytokine IFN to investigate the HSPCs dynamic changes during acute inflammation. For the analysis of the resulting datasets, we developed a computational pipeline for single-cell time series. Using a semi-supervised response-pseudotime inference approach, we discover a variety of different gene responses of the HSPCs to the treatment. Interestingly, we were able to associate reduced myeloid differentiation programs in HSPCs with reduced myeloid progenitor and differentiated cells following IFN treatment. Altogether, single-cell time series analysis have allowed us to unbiasedly study the heterogeneous and dynamic impact of IFN on the HSPCs.

cell biology↗