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Conversion of Fibroblasts to Hepatocytes In Vitro

  • Pengyu Huang
  • Lulu Sun
  • Ludi Zhang
  • Lijian Hui
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1905)

Abstract

Primary hepatocytes are widely used in regenerative medicine, drug metabolism analysis, and in vitro drug screens. To overcome the shortage of liver donors, several strategies, such as differentiation of pluripotent stem cells and transdifferentiation from somatic cells, were developed to generate hepatocytes from alternative sources. Here, we describe in detail lenti-virus-based procedure for direct conversion of human fibroblasts to hepatocytes (hiHep cells) in vitro. A detailed protocol for preparation of human fibroblasts from scar tissues is also provided. Based on this protocol, FOXA3, HNF1A, and HNF4A are introduced into SV40-large-T-antigen-expressing human scar fibroblasts by lenti-virus. It usually takes about 5–7 days to get epithelial hiHep colonies. SV40-large-T-antigen-expressing hiHep (hiHepLT) cells are proliferative and can be expanded to a large number for potential uses.

Key words

Hepatocytes HiHep cells Transdifferentiation Reprogramming 

References

  1. 1.
    Scarpelli DG, Rao MS (1981) Differentiation of regenerating pancreatic cells into hepatocyte-like cells. Proc Natl Acad Sci U S A 78(4):2577–2581CrossRefGoogle Scholar
  2. 2.
    Shen CN, Horb ME, Slack JM, Tosh D (2003) Transdifferentiation of pancreas to liver. Mech Dev 120(1):107–116CrossRefGoogle Scholar
  3. 3.
    Reddy JK, Rao MS, Qureshi SA, Reddy MK, Scarpelli DG, Lalwani ND (1984) Induction and origin of hepatocytes in rat pancreas. J Cell Biol 98(6):2082–2090CrossRefGoogle Scholar
  4. 4.
    Shen CN, Slack JM, Tosh D (2000) Molecular basis of transdifferentiation of pancreas to liver. Nat Cell Biol 2(12):879–887.  https://doi.org/10.1038/35046522CrossRefGoogle Scholar
  5. 5.
    Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui L (2011) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475(7356):386–389.  https://doi.org/10.1038/nature10116CrossRefGoogle Scholar
  6. 6.
    Huang P, Zhang L, Gao Y, He Z, Yao D, Wu Z, Cen J, Chen X, Liu C, Hu Y, Lai D, Hu Z, Chen L, Zhang Y, Cheng X, Ma X, Pan G, Wang X, Hui L (2014) Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 14(3):370–384.  https://doi.org/10.1016/j.stem.2014.01.003CrossRefGoogle Scholar
  7. 7.
    Shi XL, Gao YM, Yan YP, Ma HC, Sun LL, Huang PY, Ni X, Zhang LD, Zhao X, Ren HZ, Hu D, Zhou Y, Tian F, Ji Y, Cheng X, Pan GY, Ding YT, Hui LJ (2016) Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes. Cell Res 26(2):206–216.  https://doi.org/10.1038/cr.2016.6CrossRefGoogle Scholar
  8. 8.
    Zhou T, Benda C, Dunzinger S, Huang YH, Ho JC, Yang JY, Wang Y, Zhang Y, Zhuang Q, Li YH, Bao XC, Tse HF, Grillari J, Grillari-Voglauer R, Pei DQ, Esteban MA (2012) Generation of human induced pluripotent stem cells from urine samples. Nat Protoc 7(12):2080–2089.  https://doi.org/10.1038/nprot.2012.115CrossRefGoogle Scholar
  9. 9.
    Gao Y, Zhang X, Zhang L, Cen J, Ni X, Liao X, Yang C, Li Y, Chen X, Zhang Z, Shu Y, Cheng X, Hay DC, Lai D, Pan G, Wei G, Hui L (2017) Distinct gene expression and epigenetic signatures in hepatocyte-like cells produced by different strategies from the same donor. Stem Cell Reports 9(6):1813–1824.  https://doi.org/10.1016/j.stemcr.2017.10.019CrossRefGoogle Scholar
  10. 10.
    Du Y, Wang J, Jia J, Song N, Xiang C, Xu J, Hou Z, Su X, Liu B, Jiang T, Zhao D, Sun Y, Shu J, Guo Q, Yin M, Sun D, Lu S, Shi Y, Deng H (2014) Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell 14(3):394–403.  https://doi.org/10.1016/j.stem.2014.01.008CrossRefGoogle Scholar
  11. 11.
    Ji S, Zhu L, Gao Y, Zhang X, Yan Y, Cen J, Li R, Zeng R, Liao L, Hou C, Gao Y, Gao S, Wei G, Hui L (2017) Baf60b-mediated ATM-p53 activation blocks cell identity conversion by sensing chromatin opening. Cell Res 27(5):642–656.  https://doi.org/10.1038/cr.2017.36CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Pengyu Huang
    • 1
  • Lulu Sun
    • 2
  • Ludi Zhang
    • 2
  • Lijian Hui
    • 1
    • 2
  1. 1.School of Life Science and TechnologyShanghai Tech UniversityShanghaiChina
  2. 2.State Key Laboratory of Cell BiologyCAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of SciencesShanghaiChina

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