Promotion of the efficient metabolic maturation of human pluripotent stem cell-derived hepatocytes by correcting specification defects (original) (raw)

Promoting the functional maturation of the desired cell types derived from human pluripotent stem cells (hPSCs) remains a major challenge, especially for hepatocytes, as routine access to metabolically functional hepatocytes would enable their use in drug toxicity screening. Although previous attempts to induce hepatic specification from hPSCs yielded cells possessing some hepatic features1,2,3, most of these cells showed poor metabolic activities, and the responsible mechanisms are unknown. In this study, we explored the intrinsic defects in hPSC-derived immature hepatocytes and tested whether correcting these defects would promote the metabolic maturation of the differentiated cells.

One possible explanation for the inability to derive mature hepatic cells is incomplete specification of the differentiated cells4 caused by the lack of some key transcription factors. To test this hypothesis, immunofluorescence staining was carried out to assess the co-expression of a panel of crucial transcription factors, including FOXA2, GATA4, HNF4A, GATA6, PROX1, HNF6 and TBX3, along with the mature hepatocyte markers ALB and CYP3A4 in hepatocyte-like cells differentiated from human embryonic stem cells (hESCs), according to a previously published protocol5. Although ALB was efficiently expressed and co-localized with HNF4A in hepatocyte-like cells differentiated from hESCs, CYP3A4 was rarely observed in the differentiated cultures (Supplementary information, Data S1 and Figure S1A–S1B). Surprisingly, the limited expression of CYP3A4 correlated well with the expression of PROX1 and HNF6, which also were rarely observed in the cultures. When cells at the hepatoblast stage were characterized, similar defects were already present (Supplementary information, Figure S1C–S1E). Although FOXA2, GATA4, HNF4A and GATA6 were expressed ubiquitously (> 95%) in hESC-derived AFP-positive cells, PROX1 and HNF6 were barely detectable (< 0.1%). Interestingly, in mouse embryos, a deficiency in PROX1 or HNF6 in hepatoblasts does not disturb the emergence of Alb+ cells from the endoderm6,7. Therefore, these results suggest that two populations can be distinguished during hepatic differentiation from hESCs based on the expression of PROX1 and HNF6. The cells lacking PROX1 and HNF6 expression at an early hepatoblast stage, although retaining ALB expression, are incompletely programmed and fail to complete late metabolic maturation.

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Acknowledgements

We thank Iain C Bruce for critical reading of the manuscript, Yizhe Zhang and Liying Du for technical support on real-time PCR and FACS analysis, respectively. This work was supported by grants from the National Basic Research Program of China (973 program; 2012CD966401 and 2009CB522501), Bill & Melinda Gates Foundation Grant (1023963 and 37871), the Ministry of Education of China (111 project), Beijing Science and Technology Plan (Z121100005212001) and National Science and Technology Major Project (2012ZX10004-503) to HD.

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Author notes

  1. Dongxin Zhao and Song Chen: These two authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, 100871, China
    Dongxin Zhao, Song Chen, Shuguang Duo, Chengang Xiang, Jun Jia, Mina Guo & Hongkui Deng
  2. Center for Life Sciences, Peking University, Beijing, 100871, China
    Dongxin Zhao, Song Chen & Hongkui Deng
  3. Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking University, Shenzhen, Guangdong, China
    Chengang Xiang, Jun Jia & Hongkui Deng
  4. Beijing You-An Hospital, Capital Medical University, Beijing, 100069, China
    Wei Lai & Shichun Lu

Authors

  1. Dongxin Zhao
  2. Song Chen
  3. Shuguang Duo
  4. Chengang Xiang
  5. Jun Jia
  6. Mina Guo
  7. Wei Lai
  8. Shichun Lu
  9. Hongkui Deng

Corresponding author

Correspondence toHongkui Deng.

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( Supplementary information is linked to the online version of the paper on the Cell Research website.)

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Zhao, D., Chen, S., Duo, S. et al. Promotion of the efficient metabolic maturation of human pluripotent stem cell-derived hepatocytes by correcting specification defects.Cell Res 23, 157–161 (2013). https://doi.org/10.1038/cr.2012.144

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