Pathology response in primary human Hepatic Stellate cells determined by donor’s phenotype

Introduction: Liver disease has increased worldwide in both developing and developed world. Preclinical research has intensively delved into pathology mechanisms and target identification using hepatic stellate cells. However, there is a lack of knowledge in interindividual variability, fibrosis progression differences and connection of preclinical fibrosis in vitro models with patient subpopulations. Here, we aimed to investigate individual response of human primary hepatic stellate cells (HSC) from distinct (fibrotic/non-fibrotic) donors. Methods: Human primary HSCs from fibrotic (Fibrosis Fscore 3, 4) and non-fibrotic (Fibrosis Fscore 0) donors were cultured and stimulated with TGFβ1 to investigate disease recapitulation and interindividual variability on relevant fibrotic markers (Collagen, Fibronectin, TIMP1) as well as their biological functionality through NGS data. To assess fibrotic phenotype, fibrotic parameters were assayed in the cell/matrix fraction or in conditioned culture media. RNA was isolated and sequenced through next generation sequencing (NGS) to elucidate biological functionality and identify differentially expressed genes (DEGs) using Deseq2 and Ingenuity Pathway Analysis (IPA), respectively. Results: First, all primary HSCs donors (except one) showed induction after fibrosis stimulation with TGFβ1 (4 ng/ml) on the various fibrosis markers (total collagen, TIMP-1 and fibronectin). Of note, on the group level, fibrotic donors showed higher disease memory compared to non-fibrotic evidenced by less DEGs (4919 vs 7631) in the stimulated condition (TGFβ1). In addition, when each donor individually (control and stimulated, separately) was compared against healthy pooled controls (D#5-D#8), three out of four fibrotic donors (D#1, D#2, D#4) showed high upregulation in fibrosis pathways in control condition. Conclusion: Here we show that HSCs cultured in vitro from distinct human donors seem to respond equally to fibrosis induction. However, it is relevant to consider that the in vivo metabolic phenotype is retained in unstimulated conditions, thereby showing metabolic memory when cultured in vitro.