In vitro reversion of activated primary human hepatic stellate cells

Abstract

BackgroundLiver fibrosis is characterized by the excessive formation and accumulation of matrix proteins as a result of wound healing in the liver. A main event during fibrogenesis is the activation of the liver resident quiescent hepatic stellate cell (qHSC). Recent studies suggest that reversion of the activated HSC (aHSC) phenotype into a quiescent-like phenotype could be a major cellular mechanism underlying fibrosis regression in the liver, thereby offering new therapeutic perspectives for the treatment of liver fibrosis. Whether human HSCs have the ability to undergo a similar reversion in phenotype is currently unknown. The aim of the present study is to identify experimental conditions that can revert the in vitro activated phenotype of primary human HSCs and consequently to map the molecular events associated with this reversion process by gene expression profiling.ResultsWe find that epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) synergistically downregulate the expression of ACTA2 and LOX in primary human aHSCs. Their combination with oleic acid, palmitic acid, and retinol further potentiates a more quiescent-like phenotype as demonstrated by the abundant presence of retinyl ester-positive intra-cytoplasmic lipid droplets, low expression levels of activation markers, and a reduced basal as well as cytokine-stimulated proliferation and matrix metalloproteinase activity. Gene expression profiling experiments reveal that these in vitro reverted primary human HSCs (rHSCs) display an intermediary phenotype that is distinct from qHSCs and aHSCs. Interestingly, this intermediary phenotype is characterized by the increased expression of several previously identified signature genes of in vivo inactivated mouse HSCs such as CXCL1, CXCL2, and CTSS, suggesting also a potential role for these genes in promoting a quiescent-like phenotype in human HSCs.ConclusionsWe provide evidence for the ability of human primary aHSCs to revert in vitro to a transitional state through synergistic action of EGF, FGF2, dietary fatty acids and retinol, and provide a first phenotypic and genomic characterization of human in vitro rHSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-015-0031-z) contains supplementary material, which is available to authorized users.

Editor’s Highlight: Modeling Compound-Induced FibrogenesisIn VitroUsing Three-Dimensional Bioprinted Human Liver Tissues

“…This phenomenon is consistent with the outcome of elegant fate-mapping studies in mice that demonstrated a subset of HSCs are able to revert to a quiescent-like phenotype (Kisseleva et al, 2012) during the resolution of fibrotic injury. HSCs that have been previously activated exhibit a primed phenotype with rapid and robust patterns of reactivation in response to subsequent injury (Kisseleva et al, 2012;Taghdouini et al, 2015). This observation could explain the accelerated fibrogenic features observed in the current study, compared to the clinical setting in which fibrosis can take months or even years to develop.…”

Palmitic acid elicits hepatic stellate cell activation through inflammasomes and hedgehog signaling

“…As one of saturated fatty acids, PA was found to stimulate inflammasome NLRP3 activation in cultured Kupffer cells [32], but the results in HSCs are contradictory when different HSC lines and various concentrations were used. One study reported that PA at 75 μM led to growth rest and decrease in HSC activation [33], and another study claimed that PA at 100 μM in combination with EGF at 40 ng/mL, FGF at 220 ng/mL, oleic acid at 100 μM, and retinol at 5 μM, had a net effect of reverting HSCs to a quiescent status [34]. Whereas, in the present study, we confirmed that PA at 200 μM elicited HSC activation through the activated inflammasome NLRP3 molecule.…”

Human hepatic stellate cell isolation and characterization

“…As well, in isolated human HSCs, the results are not consistent. Positive immunostaining for desmin has been observed in primary isolated human HSCs in some studies [23,31,38], while negative immunoreactivity for desmin was reported in others [61,71].…”

“…The cultured hHSCs produced monocyte chemotactic protein-1 (MCP-1) [45], and responded to TGFb1 with increased PDGFRb [48]. It has been demonstrated that density gradient isolated human HSCs can be reverted to a quiescent-like phenotypes through synergistic action of epidermal growth factor (EGF), fibroblasts growth factor 2 (FGF2), dietary fatty acids (oleic acid, palmitic acid) and retinol, as demonstrated by the abundant presence of retinyl ester-positive intra-cytoplasmic lipid droplets, and low expression levels of activation markers [31].…”

“…Especially, mouse models utilizing HSC specific markers has greatly advanced our understanding of the function of HSCs [24][25][26][27]. Meanwhile, significant advancement has been made in human liver pathology studies as well as in vitro studies using isolated primary human HSCs, for example, to study their reverting capacity and function in retinoid metabolism [28][29][30][31][32][33]. There is increasing interest in translating research from mouse models and mouse HSCs to human HSCs and human disease.…”