Volume 132, Issue 5, Pages 1937-1946 (May 2007) Gene Expression Profiles During Hepatic Stellate Cell Activation in Culture and In Vivo  Samuele De Minicis, Ekihiro Seki, Hiroshi Uchinami, Johannes Kluwe, Yonghui Zhang, David A. Brenner, Robert F. Schwabe  Gastroenterology  Volume 132, Issue 5, Pages 1937-1946 (May 2007) DOI: 10.1053/j.gastro.2007.02.033 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Combination of collagenase-pronase based density centrifugation and MACS achieves high purity of quiescent and activated hepatic stellate cells. (A and B) Hepatic stellate cells were isolated from normal mice and mice that underwent BDL for 2 weeks and were either depleted of CD11b- and F4/80-expressing cells or left untreated. Expression of CD11b and F4/80 was analyzed by flow cytometric analysis (A). Expression of CD68 in quiescent HSCs (“Q”), culture-activated HSCs (“A”), BDL-activated HSCs (“B”), and CCl4-activated HSCs (“C”) was measured by quantitative real-time PCR (B). Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison to Kupffer cells (“KC” = 100%). (C and D) Contamination of Kupffer cell-depleted HSCs with hepatocytes (C) and endothelial cells (D) was measured by quantitative real-time PCR for albumin and CD31, respectively. Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison with primary hepatocytes (“Hep” = 100%) and primary hepatic endothelial cells (“EC” = 100%). (E) To demonstrate purity and activation status, HSCs were isolated from transgenic mice expressing GFP under the collagen α1(I) promoter. GFP and retinoid autofluorescence were checked 1 day after isolation in quiescent HSC and HSC isolated from bile duct-ligated (“BDL”) and CCl4-treated mice and after 5 days in culture-activated HSCs. Gastroenterology 2007 132, 1937-1946DOI: (10.1053/j.gastro.2007.02.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Combination of collagenase-pronase based density centrifugation and MACS achieves high purity of quiescent and activated hepatic stellate cells. (A and B) Hepatic stellate cells were isolated from normal mice and mice that underwent BDL for 2 weeks and were either depleted of CD11b- and F4/80-expressing cells or left untreated. Expression of CD11b and F4/80 was analyzed by flow cytometric analysis (A). Expression of CD68 in quiescent HSCs (“Q”), culture-activated HSCs (“A”), BDL-activated HSCs (“B”), and CCl4-activated HSCs (“C”) was measured by quantitative real-time PCR (B). Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison to Kupffer cells (“KC” = 100%). (C and D) Contamination of Kupffer cell-depleted HSCs with hepatocytes (C) and endothelial cells (D) was measured by quantitative real-time PCR for albumin and CD31, respectively. Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison with primary hepatocytes (“Hep” = 100%) and primary hepatic endothelial cells (“EC” = 100%). (E) To demonstrate purity and activation status, HSCs were isolated from transgenic mice expressing GFP under the collagen α1(I) promoter. GFP and retinoid autofluorescence were checked 1 day after isolation in quiescent HSC and HSC isolated from bile duct-ligated (“BDL”) and CCl4-treated mice and after 5 days in culture-activated HSCs. Gastroenterology 2007 132, 1937-1946DOI: (10.1053/j.gastro.2007.02.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 The gene expression pattern of culture-activated HSCs is different from that of BDL- and CCl4-activated HSCs. Hepatic stellate cells were isolated from normal mice and mice that underwent BDL for 2 weeks or 4 CCl4 injections. HSCs were considered culture activated after 5 days of culture in 10% fetal bovine serum on uncoated cell culture dishes. After isolation of mRNA and generation of cRNA, microarray was performed. (A) Shown is a representative graph of 3 separate HSC isolations per group (“1,” “2,” and “3”) containing all genes that were more than 2-fold up-regulated (shown in red) or down-regulated (shown in blue) in at least 1 of the 3 groups of activated HSCs in comparison with quiescent HSCs (t test, P < .05 followed by Benjamini–Hochberg correction). (B) The relative expression of all 45,000 probe sets of the array was correlated among culture-activated HSCs, BDL-activated HSCs, and CCl4-activated HSCs using Spearman rank correlation. (C) The Venn diagram shows overlapping patterns of probe sets that were significantly (P < .05) and at least 2-fold up-regulated and down-regulated in 1 of the 3 groups of activated HSCs. Probe sets that were 2-fold up-regulated or down-regulated in one group were considered down-regulated or up-regulated in the other groups if they showed at least 1.5-fold up-regulation or down-regulation. Gastroenterology 2007 132, 1937-1946DOI: (10.1053/j.gastro.2007.02.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Identification of previously known and novel differentially regulated genes during HSC activation in vivo and in culture. (A) Expression of differentially regulated genes was confirmed in quiescent HSCs (“Q”), culture-activated HSCs (“A”), BDL-activated HSCs (“B”), and CCl4-activated HSCs (“C”) by qPCR. Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison with quiescent HSCs. Each group of HSC samples consisted of at least 3 different isolations. (B) Expression of selected genes was confirmed by Western blot analysis. Each Western blot is based on HSCs isolated from 3 normal, BDL-, or CCl4-treated mice and is representative of at least 2 independent experiments. Gastroenterology 2007 132, 1937-1946DOI: (10.1053/j.gastro.2007.02.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Exposure of HSCs to Kupffer cells or LPS during culture shifts the gene expression toward the pattern of in vivo-activated HSCs. (A and B) Genes that showed a different expression pattern during culture and in vivo activation were examined after 5 days of culture activation in the presence or absence of Kupffer cells or LPS (100 ng/mL) by quantitative real-time PCR as described in the Materials and Methods section (“Q,” quiescent; “A,” culture activated without additional stimuli; “K,” Kupffer cell treated; “L,” LPS treated). Shown is the mean of PCRs from 3 independent HSC isolations. (C) The influence of Kupffer cells and LPS on the expression of selected genes was confirmed by Western blot analysis. For this purpose, HSCs were cultured in the presence or absence of Kupffer cells in a noncontact-dependent manner for 5 days or in the presence of LPS (100 ng/mL). This experiment is representative of at least 2 independent HSC isolations. Gastroenterology 2007 132, 1937-1946DOI: (10.1053/j.gastro.2007.02.033) Copyright © 2007 AGA Institute Terms and Conditions