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Rotary Suspension Culture Enhances Mesendoderm Differentiation of Embryonic Stem Cells Through Modulation of Wnt/β-catenin Pathway

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Abstract

Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.

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Acknowledgments

We thank Miss Qiao Jingqiao (Institute of Zoology, CAS) for tissue section and staining with hematoxylin and eosin. We thank Dr. Zhao Tongbiao (Institute of Zoology, CAS) for the J1 cell line and Dr. Mark Mercola for the T/Brachyury-eGFP Rex Neo plasmid. This work was supported by grants from the National Basic Research Program of China (2011CB710905 to ED), Strategic Priority Research Program of the Chinese academy of Sciences (XDA 01010202 to ED), National Natural Science Foundation of China (31201099 to SL).

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The authors indicate no potential conflicts of interest.

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Authors and Affiliations

  1. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, China

    Xiaohua Lei, Zhili Deng, Huishan Zhang, Huashan Zhao, Shuang Liu, Qi Chen, Lina Ning, Yujing Cao, Xinyue Wang, Xudong Zhang & Enkui Duan

  2. University of Chinese Academy of Sciences, Beijing, 100190, China

    Zhili Deng, Huishan Zhang, Huashan Zhao, Xinyue Wang & Xudong Zhang

  3. State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China

    Jiaxi Zhou

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  1. Xiaohua Lei
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Correspondence to Enkui Duan.

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Xiaohua Lei and Zhili Deng contribute equally to this work.

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Lei, X., Deng, Z., Zhang, H. et al. Rotary Suspension Culture Enhances Mesendoderm Differentiation of Embryonic Stem Cells Through Modulation of Wnt/β-catenin Pathway. Stem Cell Rev and Rep 10, 526–538 (2014). https://doi.org/10.1007/s12015-014-9511-6

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