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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279104 | GEO | 2025/09/10

REPOSITORIES: GEO

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Project description:Introduction: Preclinical models that are currently used for fibrosis research only partially mimic human disease processes. Particularly, traditional transforming growth factor beta 1 (TGFβ1) induced stellate cell models are lacking in activation of hypoxia-induced pathways, which is a relevant process in patients with liver disease. Here we investigated the synergic effect of two hypoxia-mimicking compounds (DMOG and IOX2) and TGFβ1, on fibrotic phenotype and in vivo disease recapitulation in primary human hepatic stellate cells (HSCs). Methods: Human primary HSCs were cultured, stimulated (TGFβ1 and hypoxia-mimicking compounds combinations) and harvested to identify optimal stimulation conditions for fibrotic phenotype, cell viability and toxicity levels. To assess the fibrotic phenotype, protein levels was assayed for fibrosis markers (collagen, TIMP-1, Fibronectin) were evaluated. RNA was isolated and sequenced through next generation sequencing (NGS). Bioinformatic tools using differential expression analysis (Deseq2) and Ingenuity Pathway Analysis (IPA) were used to identify differentially expressed genes (DEGs) and map these to biological functionality, respectively. Results: Our HSC model showed that stimulation with IOX2 (0.3 and 1 µM) in the presence of TGFβ1 significantly increased fibrotic markers levels (total collagen, TIMP-1, and fibronectin). DMOG showed no effect using low, non-toxic concentrations. Biological functionality from DEGs highlighted various fibrosis-related pathways, hypoxia-related genes and relevant crosslinking markers when IOX2 was added in a concentration-dependent manner. Furthermore, comparative analysis with human DEGs (Fibrotic vs non-fibrotic) showed improved disease representation in our HSC model with IOX2 addition. Conclusion: These findings suggest that IOX2 may aggravate fibrotic disease and improve in vivo disease recapitulation in vitro. Hypoxia-mimicking compounds like IOX2 hold promise for enhancing fibrosis in in vitro models, providing valuable insights into fibrosis pathogenesis, hypoxia-related genes and potential therapeutic strategies.

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

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