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bFGF and Activin A function to promote survival and proliferation of single iPS cells in conditioned half-exchange mTeSR1 medium

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Abstract

Human induced pluripotent stem (iPS) cells can be well maintained by clonal growth. The pluripotent growth of single iPS cells is limited by low survival. To facilitate robust single iPS cells cultured in vitro, half-exchange mTeSR1 medium (HM), whole-exchange medium (WM) and iPS cell-derived conditioned medium (iPS-CM) culture were used. The effects of bFGF and Activin A on the growth of single iPS cells were explored. The dissociation and propagation of single iPS cells also included Accutase enzymatic isolation, Rho-associated protein kinase (ROCK) inhibitor Y27632 protection and high-density single-cell seeding (1 × 106 cells/well). CCK-8 assays demonstrated that the viability of clonal iPS cells in mTeSR1 medium and single iPS cells in HM, iPS-CM or WM supplemented with 100 ng/ml bFGF and 10 ng/ml Activin A was significantly higher than that in WM. Annexin v and propidium iodide (PI) assay, Calcein AM and EthD-III double staining also confirmed the similar results. ELISA assays showed that the levels of bFGF and Activin A of single iPS cells in HM and iPS-CM were higher than single iPS cells in WM. Meanwhile, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), quantitative Polymerase Chain Reaction (qPCR), Western Blotting (WB), Immunofluorescence (IF) and karyotype analysis revealed that HM culture was able to maintain undifferentiated markers of Nanog, Klf4, Sox2, Oct4, and did not affect the karyotype of iPS cells. Undifferentiated single iPS cells in HM displayed homogenized growth. These findings demonstrate that bFGF and Activin A are important for the survival and growth of single iPS cells. HM culture system combined Accutase, Y27632 and high-density single-cell seeding can facilitate short-term growth of single iPS cells in vitro.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81371689), collaborate grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060) and the Natural Science Foundation of Guangdong Province (S2013010013391).

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Authors and Affiliations

  1. Key Laboratory for Regenerative Medicine, Ministry of Education, **an University, Guangzhou, China

    **aoling Guo, Yonglong Guo & Jiansu Chen

  2. Eye Institute, Medical College of **an University, Guangzhou, China

    Jiansu Chen

  3. Department of Ophthalmology, the First Clinical Medical College of **an University, Guangzhou, China

    Ruiling Lian, Qing Liu & Jiansu Chen

  4. Department of Ophthalmology, Affiliated Anhui Provincial Hospital of Anhui Medical University, Hefei, China

    Qingshan Ji

  5. Institute of Ophthalmology, Medical College, **an University, Guangzhou, 510632, China

    Jiansu Chen

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  1. **aoling Guo
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Correspondence to Jiansu Chen.

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Guo, X., Lian, R., Guo, Y. et al. bFGF and Activin A function to promote survival and proliferation of single iPS cells in conditioned half-exchange mTeSR1 medium. Human Cell 28, 122–132 (2015). https://doi.org/10.1007/s13577-015-0113-7

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