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Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells


Hepatic stellate cells (HSCs) are nonparenchymal liver cells responsible for extracellular matrix homeostasis and are the main cells involved in the development of liver fibrosis following injury. The lack of reliable sources of HSCs has hence limited the development of complex in vitro systems to model liver diseases and toxicity. Here we describe a protocol to differentiate human induced pluripotent stem cells (iPSCs) into hepatic stellate cells (iPSC-HSCs). The protocol is based on the addition of several growth factors important for liver development sequentially over 12 d. iPSC-HSCs present phenotypic and functional characteristics of primary HSCs and can be expanded or frozen and used to perform high-throughput in vitro studies. We also describe how to coculture iPSC-HSCs with hepatocytes, which self-assemble into three-dimensional (3D) hepatic spheroids. This protocol enables the generation of HSC-like cells for in vitro modeling and drug screening studies.

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Fig. 1: Time course of the 12-d differentiation protocol.
Fig. 2: Characterization of iPSCs differentiation to HSC-like cells.
Fig. 3: Scheme of the downstream assays with iPSC-HSCs.
Fig. 4: Passage and thawed iPSC-HSCs characterization and stimulation.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper. Source data are provided with this paper.


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This work was performed in Center Esther Koplowitz and Vrije Universiteit Brussel. We thank the Advanced Optical Microscopy Unit from the Scientific and Technological Centers of the University of Barcelona for their support with the confocal image technique. We are indebted to the Cytomics Unit of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for flow cytometry analysis. Figures were created using P.S.-B. is supported by the Fondo de Investigación Sanitaria Carlos III, cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, ‘Una manera de hacer Europa’ (FIS PI20/00765, PI17/00673), DTS18/00088, COST Action H2020 PRO-EURO-DILI-NET CA17112 and Miguel Servet (CPII16/00041) to P.S.-B., PFIS (FI18/00215) to R.M. and APIF to J.V. MC FIS (PI18/00862). I.M. is supported by an FWO–V post-doctoral fellowship (12N5419 N) and S.V. is supported by an FWO–V postdoctoral fellowship (1243121N). L.A.v.G is supported by FWO projects G042719N, FWO-SBO-S001121 ‘iPSC-LiMiC’ and a ‘Wetenschappelijk Fonds Willy Gepts’ grant from the Vrije Universiteit Brussel. C.M.V. is supported by FWO-G0D9917N; IWT-140045, HILIM-3D; FWO-SBO-S001121, iPSC-LiMiC and the European Union’s Horizon 2020 research and innovation program under grant agreement 681002 (EU-ToxRisk).

Author information




J.V. and R.A.M. equally contributed to the optimization of expansion and freezing processes, characterization of the cells and drafting the manuscript. I.M. and A.S. performed the 3D spheroid experiments, and S.V. and. A.S. carried out parallel iPSC differentiations using a different iPSC line (not included in the manuscript). M.C. participated in the differentiation protocol design and reviewed the manuscript. S.A., T.R.-T., B.A.-B., C.M.-S. and D.B., participated in the differentiation protocol process. C.M.V. contributed to the design of the IPSCs differentiation protocol and reviewed the manuscript. L.A.v.G. guided the iPSC and spheroid studies in Brussels and critically reviewed the manuscript. P.S.-B. designed and supervised the study and critically reviewed the manuscript.

Corresponding authors

Correspondence to Catherine M. Verfaillie or Leo A. van Grunsven or Pau Sancho-Bru.

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Competing interests

M.C., C.M.V. and P.S.-B. have a patent application PCT/EP2016/079464, and L.A.v.G. has a patent application PCT/EP2015/062551.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key reference using this protocol

Coll, M. et al. Cell Stem Cell 23, 101–113.e7 (2018):

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Vallverdú, J., Martínez García de la Torre, R.A., Mannaerts, I. et al. Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells. Nat Protoc 16, 2542–2563 (2021).

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