Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and in vitro disease modeling

Abstract

Summary Hepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate in vitro model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein ( RFP ) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle ( ACTA2 ). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.

Generation of functionally competent hepatic stellate cells from human stem cells to model liver fibrosis in vitro

“…The pHSCs were already activated during the isolation and culture; some of them even activated in the tissue, which showed gene expression similar to that of aHSCs and fibroblasts. However, combined with other data, our iHSC-like cells more resemble qHSCs, indicating that this procedure promotes mature and quiescent iHSC differentiation ( Coll et al., 2018 ; Koui et al., 2021 ). In addition, the acquisition of a mature state by iHSCs in the differentiation medium was characterized by the wound-healing response after TGF-β treatment.…”

“…Compared with previous studies, the iHSCs differentiated by our new strategy are similar in terms of morphology, gene expression patterns, and functionality to qHSCs ( Coll et al., 2018 ). Importantly, iHSCs from our protocol are more quiescent than others, and showed almost no collagen I and α-SMA expression in the quiescent iHSCs, but nearly all cells showed high expression during activation ( Coll et al., 2018 ; Koui et al., 2021 ). These data suggest that iHSCs generated by our strategy are promising for large-scale applications and mechanism studies of HSC activation in vitro and may resolve the shortage of activation phenotype of pHSCs.…”

“…Interestingly, Koui et al. recently reported a similar strategy, in that BMP4, FGF2, and VEGF combined could induce iHSC differentiation, which supports that VEGF plays an important role in iHSC differentiation ( Koui et al., 2021 ). In addition, PPARγ was discovered as a new transcription factor for regulating the phenotype of HSCs to prevent activation of HSCs, which could help us to maintain the qHSC features ( Liu et al., 2020b ).…”

“…To further evaluate the overall characteristics of iHSCs, we did RNA sequencing to generate transcriptomic profiles of endoderm-derived iHSCs (iHSCs_end), mesoderm-derived iHSCs (iHSCs_M2), purchased pHSCs (purHSCs) from Lonza, fibroblasts from Lonza, and pHSCs cultured for 4 days from liver isolation. Gene expression data of human qHSCs ( Liu et al., 2020a ) ( GSE141100 ), hepatocytes ( GSE43984 ), cultured hepatocytes (GSES98710) ( Koui et al., 2017 ), and iPSC-derived qHSC-like cells (TkDN4_M_qHSCs and FF_1_qHSCs) ( Koui et al., 2021 ) ( GSE155017 ) were extracted from publicly available databases and processed for comparison. As shown in Figure 3 A, the hierarchical clustering analysis demonstrated that the expression profile of iHSCs_end was similar to those of human qHSCs, TkDN4_M_qHSCs, and FF_1_qHSCs.…”

Potential Use of Patient-Specific Induced Pluripotent Stem Cell for Liver Fibrosis Thalassemia Treatment Management

“…MSCs attenuate liver inflammation by regulating the immune cell function creating a hepato-protective environment through paracrine effect via cell-cell interaction. HSCs also have essential roles in treatment of liver fibrosis by changing their characteristic from quiescent cells to an activated state, producing ECMs [25], [26].…”