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Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension

Nature Biotechnology volume 28, pages 361–364 (2010)Cite this article

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Abstract

Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies1,2,3,4,5,6,7. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies8. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.

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Figure 1: Human ESCs remain pluripotent after 10 weeks propagation in suspension.
Figure 2: Derivation of hESCs in suspension.
Figure 3: Controlled conversion of the hESC clusters in suspension into neural precursor spheres.

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Acknowledgements

We are grateful to the following members of the Hadassah Human Embryonic Stem Cell Research Center: M. Gropp, M. Aharonowiz and O. Singer for technical assistance; S. Tennenbaum for editing the manuscript. We thank K.M. Yamada (National Institute of Dental and Craniofacial Research, National Institutes of Health) for providing anti-fibronectin antibody, N. Benvenisty for the QPCR primers and WiCell Research Institute for providing H7 hESCs. This research was supported by a gift from Judy and Sidney Swartz, the Sidney Swartz Chair in Human Embryonic Stem Cell Research and Legacy Heritage Fund.

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Authors and Affiliations

  1. The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy,

    Debora Steiner, Hanita Khaner, Malkiel Cohen, Sharona Even-Ram, Yaniv Gil, Pavel Itsykson, Tikva Turetsky, Maria Idelson, Yael Berman-Zaken & Benjamin Reubinoff

  2. Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

    Einat Aizenman

  3. Department of Genetics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

    Rita Ram

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  1. Debora Steiner
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Contributions

D.S. designed and performed the experiments, analyzed the data and wrote the manuscript; H.K. and M.C. performed the neural differentiation study; S.E.-R. conducted immunostainings and confocal analysis; Y.G. performed the teratoma studies; P.I. contributed to developing the concept of suspension culture; T.T. contributed to the experiments; M.I. performed PCR analysis; E.A. contributed to embryo recruitment, culture and isolation of inner cell masses; R.R. and Y.B.-Z. conducted karyotype analysis. B.R. conceived the study and wrote the paper.

Corresponding author

Correspondence to Benjamin Reubinoff.

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Competing interests

B.R. is the CSO and holds shares in CellCure Neurosciences Ltd. However, the project was not funded by CellCure Neurosciences Ltd. and the company has no rights in its results.

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Steiner, D., Khaner, H., Cohen, M. et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 28, 361–364 (2010). https://doi.org/10.1038/nbt.1616

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