Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Measuring intravascular migration of mouse Ly6C(low) monocytes in vivo using intravital microscopy.

This unit describes methods for intravital imaging of monocytes in the vasculature of the dermis and the mesentery in vivo using fluorescent reporter mice, fluorescent dyes, and antibodies. Cx3cr1(gfp/gfp (or +)), Rag2(-/-), Il2rg(-/-) mice expressing eGFP at the locus of the Cx3cr1 gene, on the Rag2(-/-) Il2rg(-/-) C57Bl/6 background, are used. Although aimed at specifically tracking Ly6C(low) monocytes, these protocols could readily be adapted to investigate the interaction of other blood leukocytes with the vascular endothelium by use of other fluorescent reporter mice and fluorescently labeled antibodies.

Time-lapse FRET microscopy using fluorescence anisotropy.

We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein-protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.

Intercellular transfer and supramolecular organization of human leukocyte antigen C at inhibitory natural killer cell immune synapses.

After accumulation of target cell human leukocyte antigen (HLA)-C at inhibitory natural killer (NK) cell immune synapses, some HLA-C transfers from target cells to NK cell plasma membranes and cytoplasm. This unexpected intercellular transfer of HLA-C is dependent on NK receptor recognition, since HLA-Cw6 or -Cw4 but not -Cw3 transfer to an NK transfectant expressing killer Ig-like receptor (KIR)2DL1. Strikingly, live-cell time-lapse laser scanning confocal microscopy shows vesicles containing target cell green fluorescent protein-tagged HLA-C migrating away from immune synapses into NK cells. Unlike clustering of HLA-C at the immune synapse, intercellular transfer of HLA-C is dependent on NK cell ATP, but not target cell ATP. However, the intercellular transfer of HLA-C is not dependent on active polymerization of the actin cytoskeleton. In addition, different arrangements of HLA-C are seen at inhibitory NK immune synapses, and these alter as NK synapses mature, but in a fashion distinct from that seen upon T cell activation.