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High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers

Abstract

Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes 4 h to complete.

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Figure 1: Isolated HSCs reflect gene expression found in fibrotic livers.
Figure 2: Retinoid-based FACS sorting improves the purity of HSC isolates without impairing the expression of HSC activation markers.
Figure 3: Overview of the HSC isolation procedure.

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Acknowledgements

R.F.S. was supported by grants 5R01AA020211, 1U01AA021912 and 5R01DK076920. D.H.D. was supported by grant F31DK091980. I.M. was supported by grant from the German Research Foundation (ME 3723/1-1) and the American Liver Foundation. S.A. was supported by a grant from the Spanish Association for the Study of the Liver (AEEH).

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Authors and Affiliations

Authors

Contributions

I.M. performed primary cell isolations and data analysis, recorded and cut the video files and drafted the manuscript. D.H.D. performed in vivo injury models, primary cell isolations, data acquisition including RNA isolation and qPCRs, data analysis and drafted the manuscript. S.A. performed primary cell isolations and data acquisition. H.U. optimized the isolation procedure. R.F.S. designed and oversaw the study, performed data analysis and drafted the manuscript.

Corresponding author

Correspondence to Robert F Schwabe.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Increased fibrogenic gene expression is a product of HSC expansion in the injured liver.

Mice were treated with four injections of CCl4 (0.25 μl/g for the first dose and 0.5 μl/g i.p. for subsequent doses), dissolved in corn oil at a ratio of 1:3, injected every 3 days (n=5 for untreated mice, n=10 for CCl4-treated). Expression of genes that change in response to HSC activation was determined in livers and isolated HSCs (from the same mice). Gene expression was normalized to Pdgfrb. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University.

Supplementary Figure 2 HSCs express characteristic retinoid fluorescence.

a. Vitamin A fluorescence detected by using 405–407 nm laser for excitation and a 450/50 nm bandpass filter for retinoid detection shows typical retinoid fluorescence which can be abrogated by bleaching of HSCs with UV-light. b. For FACS analysis, cells were initially gated by FSC-A/SSC-A, followed by FSC-A/FSC-H.

Supplementary Figure 3 Retinoid-based FACS isolation does not significantly alter expression of typical HSC genes and proliferation markers.

Expression of HSC markers Lhx2, Lrat, Hand2, Vim and Pdgfrb and of proliferation markers Ccnb1 and Ccnb2 was determined by qPCR (n=5 untreated mice, n=10 CCl4-treated mice). Analysis of liver, unsorted, mock-sorted and FACS-sorted HSCs was performed using the same mice. Data are presented as fold induction in comparison to untreated controls. All data are shown as means ± s.e.m. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University. n.s., non-significant.

Supplementary Figure 4 Retinoid-low cell populations do not express high levels of HSC activation markers or characteristic HSC genes, but have high expression of hepatocyte, LSEC, macrophage and cholangiocyte markers.

Gene expression was compared between FACS-sorted retinoid-high HSCs and the remaining FACS-sorted retinoid-low population in untreated mice (n=4-5) and CCl4-treated mice (n=10). a. Expression of HSC activation markers Acta2, Col1a1 and Lox was determined. Data are displayed as fold induction relative to untreated. b. Expression of non-HSC cell contamination markers (Alb for hepatocytes, vWF for endothelial cells, Emr1 for macrophages, and Krt19 for cholangiocytes/liver progenitor cells) in FACS-sorted low-retinoid population and high-retinoid HSCs. Data are displayed as percent of pure populations (set as 100%) of cholangiocytes, macrophages, LSEC and of liver (as a surrogate for hepatocytes) c. Expression of HSC characteristic genes Lhx2, Lrat, Pdgfrb and Hand2 was determined by PCR. Data of these HSC-enriched genes are displayed as fold induction versus normal liver. All data are shown as means ± s.e.m. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University. * P<0.05, ** P<0.01, *** P<0.001

Supplementary Figure 5 FACS-based HSC ultrapurification in DDC diet-induced biliary fibrosis.

a. FACS-based ultrapurification of gradient-purified HSCs from DDC diet-treated mice. b. HSC activation was determined by qPCR for Col1a1, Acta2, Lox and Hhip in mock-sorted and FACS-ultrapurified HSCs. Data are presented as fold induction in comparison to HSCs from control liver. c-d. Gene expression of non-HSC cell contamination markers (Alb for hepatocytes [“Hep“], Emr1 for liver macrophages [“KC“], Krt19 for cholangiocytes [“Chol“] and vWF for liver sinusoid endothelial cells [“LSEC“], was compared between high retinoid FACS-sorted and mock-sorted HSCs (c) and high retinoid FACS-sorted HSCs and low-retinoid populations (d) from DDC-treated mice. Gene expression is expressed as percentage compared to pure isolates of hepatocytes, cholangiocytes, LSEC and liver macrophages (each set as 100% value). e-f. Expression of characteristic HSC genes Lhx2, Lrat, Pdgfrb and Hand2 was compared between high retinoid FACS-sorted and mock-sorted HSCs (e) and high retinoid FACS-sorted HSCs and low retinoid populations (f) from DDC-treated mice. Data of these HSC-enriched genes are displayed as fold induction versus normal liver. All data are shown as means ± s.e.m. n=5 control mice and n=5 DDC-diet-treated mice. All animal procedures were approved by the Institutional Animal Care and Use Committee at Columbia University. **P<0.01 and ***P<0.001; n.s., non-significant.

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Mederacke, I., Dapito, D., Affò, S. et al. High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc 10, 305–315 (2015). https://doi.org/10.1038/nprot.2015.017

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