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The proteomic analysis of hepatic stellate cell differentiation from iPSCs identifies RORalpha as an antifibrogenic target
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International audience. Background and aims: Understanding the embryonic development of Hepatic stellate cells (HSCs) and their activation is fundamental to develop new therapeutic strategies. We developed a protocol to differentiate iPSCs to HSCs by sequential addition of growth factors. We hypothesis that the differentiation protocol may be an excellent tool for drug discovery. By performing a time course proteomic characterization, we envision to understand the protein dynamics across differentiation and identify transcription factors involved in the maintenance HSC phenotype and cell activation. Method: MS proteomics was performed in 4 independent differentiations of iPSC to HSCs and primary human HSCs. Seven staggerer mice (RORA−/−) and their wildtype littermates were treated with CCl4 during 4 weeks. Fourteen mice were treated with CCl4 and 7 were treated with the RORA agonist SR1078. qPCR, immunofluores-cence and immunohistochemistry assays were used as validation. RORA expression was evaluated in a cohort of 25 patients with alcohol and metabolic associated chronic liver d isease. Results: Proteomic results indicated that iPSC-HSC differentiation occurred in three stages: undifferentiated phase (Day 0 to 4), intermediate fetal stage (Day 6 to 8) and final maturation stage (Day 10 to 12). Developmental markers of fetal HSC, such as VIM, ALCAM, FBLN1 and DCN were sequentially expressed and followed by mature HSC markers, indicating the recapitulation of the embryonic development in vitro. Pathway analysis of iPSC-HSCs and primary HSCs revealed RORA as an important transcription factor in HSC phenotype and commitment. Experimentally, we first evaluated the effect of RORA on HSC development. iPSC-HSC across differentiation were treated with SR1078, which increased the expression of HSC phenotype and quiescence markers such as RELN, PCDH7, LRAT and LHX2. Next, we evaluated the role of RORA on HSC activation by treating iPSC-HSCs, which decreased the expression of ACTA2 and increased the expression of quiescent markers (LRAT and LHX2). In vivo studies with the staggerer mice, showed an increased fibrotic content by Sirius Red staining and an increased gene expression of fibrogenic markers such as ACTA2, collagens and MMPs in the mutant group. In addition, SR1078 treatment of CCl4 mice showed a reduction of collagen deposition and aSMA staining, together with a reduced expression of fibrogenic markers. In patients, expression of RORA correlated negatively with fibrogenic markers and MELD Score. These results suggest that RORA plays a role in the progression of liver fibrosis. Conclusion: The present study demonstrates that the differentiation protocol is a new and reliable tool for the study of HSC biology and the discovery of new antifibrogenic targets. Moreover, we identify RORA as a targetable transcription factor to mitigate liver fibrosis.
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