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Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β

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Abstract

In a previous study, the Notch pathway inhibited with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (also called DAPT) was shown to promote the differentiation of fetal liver stem/progenitor cells (FLSPCs) into hepatocytes and to impair cholangiocyte differentiation. The precise mechanism for this, however, was not elucidated. Two mechanisms are possible: Notch inhibition might directly up-regulate hepatocyte differentiation via HGF (hepatocyte growth factor) and HNF (hepatocyte nuclear factor)-4α or might impair cholangiocyte differentiation thereby indirectly rendering hepatocyte differentiation as the dominant state. In this study, HGF and HNF expression was detected after the Notch pathway was inhibited. Although our initial investigation indicated that the inhibition of Notch induced hepatocyte differentiation with an efficiency similar to the induction via HGF, the results of this study demonstrate that Notch inhibition does not induce significant up-regulation of HGF or HNF-4α in FLSPCs. This suggests that Notch inhibition induces hepatocyte differentiation without the influence of HGF or HNF-4α. Moreover, significant down-regulation of HNF-1β was observed, presumably dependent on an impairment of cholangiocyte differentiation. To confirm this presumption, HNF-1β was blocked in FLSPCs and was followed by hepatocyte differentiation. The expression of markers of mature cholangiocyte was impaired and hepatocyte markers were elevated significantly. The data thus demonstrate that the inhibition of cholangiocyte differentiation spontaneously induces hepatocyte differentiation and further suggest that hepatocyte differentiation from FLSPCs occurs at the expense of the impairment of cholangiocyte differentiation, probably being enhanced partially via HNF-1β down-regulation or Notch inhibition.

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Abbreviations

CT:

Cycle threshold

CYP:

Cytochrome P450

CK:

Cytokeratin

DAPT:

N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester

DMSO:

Dimethylsulfoxide

ED:

Embryonic day

FLSPCs:

Fetal liver stem/progenitor cells

GAPDH:

D-Glyceraldehyde-3-phosphate dehydrogenase

G6P:

Glucose-6-phosphatase

GGT:

γ-Glutamyltransferase

HGF:

Hepatocyte growth factor

HNF:

Hepatocyte nuclear factor

ICG:

Indocyanine green

LDL:

Low-density lipoprotein

PAS:

Periodic-acid-Schiff’s staining

RNAi:

RNA interference

shRNA:

Short hairpin RNA

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Acknowledgments

We thank Dr. Peng Luo (Department of Neurosurgery, Xijing Hospital, The Fourth Military Medical University, China) and Fuqing Zhang, Nan You, and Xing Wang (Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, China) for substantial help with the experiments.

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Authors and Affiliations

  1. General Surgery Center, Chengdu Military General Hospital, Chengdu, Sichuan Province, 610083, China

    Tao Wang, Tao Chen, Hong-yin Liang, Hong-tao Yan, Ning Lin, Li-ye Liu, Hao Luo, Zhu Huang, Wei-hui Liu & Li-jun Tang

  2. Department of Vascular and Endocrine Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, 710032, China

    Nan-lin Li

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  1. Tao Wang
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  2. Tao Chen
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  3. Hong-yin Liang
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  4. Hong-tao Yan
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  5. Ning Lin
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  6. Li-ye Liu
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  7. Hao Luo
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  8. Zhu Huang
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  9. Nan-lin Li
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  10. Wei-hui Liu
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  11. Li-jun Tang
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Corresponding authors

Correspondence to Wei-hui Liu or Li-jun Tang.

Additional information

Tao Wang, Tao Chen, Hong-yin Liang, Hong-tao Yan and Ning Lin contributed equally to this work.

The authors declare that they have no competing interests.

This work was supported by the Chinese National Natural Science Foundation (grant nos. 81030010, 81001695, 81072179, 81170419 and 81302168), Science Plan Program of Sichuan Province (grant no. 2013JY0046) and Science Fund of Chengdu Military General Hospital (grant no. 424121 J2).

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Wang, T., Chen, T., Liang, Hy. et al. Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β. Cell Tissue Res 357, 173–184 (2014). https://doi.org/10.1007/s00441-014-1825-9

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