Abstract
Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogenous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.
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Acknowledgements
M.H.S. was supported by a Canadian Institutes of Health Research Canada Graduate Scholarship doctoral award. This work was supported by Canadian Institutes of Health Research, Ontario Ministry of Research Innovation and a Canada Research Chair award to M.B. We thank T. Schuesler and C. von Kalle for providing and troubleshooting the LAM-PCR methods and applications to hESC lines.
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M.H.S. designed, performed, and analyzed all experiments, and wrote and revised the manuscript. S.C.B. contributed intellectual input to design and analysis of experiments and results. M.L.-M. performed all FACS. M.B. provided intellectual support toward design and interpretation of the results, and wrote and revised the manuscript.
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Stewart, M., Bendall, S., Levadoux-Martin, M. et al. Clonal tracking of hESCs reveals differential contribution to functional assays. Nat Methods 7, 917–922 (2010). https://doi.org/10.1038/nmeth.1519
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DOI: https://doi.org/10.1038/nmeth.1519
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