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Directed differentiation of human embryonic stem cells toward chondrocytes

Abstract

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95–97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

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Figure 1: Schematic of directed differentiation protocol in three stages.
Figure 2: Morphology of hESC cultures (HUES1) at different stages of the protocol.
Figure 3: Gene expression analysis of hESCs at different stages of the protocol.
Figure 4: Sulfated glycosaminoglycan accumulation during directed differentiation of hESCs (HUES1) to chondrocytes.
Figure 5: Immunofluorescence of SOX9 and collagen II.
Figure 6: Flow cytometry analyses of HUES1-derived cells at the end of stage 3 (day 14).

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Acknowledgements

This work was funded by the North West Regional Development Agency; North West Embryonic Stem Cell Centre is also supported by the UK Medical Research Council and the UK National Institute for Health Research Biomedical Research Funding Scheme. We thank N. Hanley (University of Manchester) for the gift of human fetal cDNA.

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R.A.O., D.R.B., T.E.H. and S.J.K. were responsible for study concept and design, analysis and interpretation of data and preparation of the manuscript. R.A.O., M.A.B., E.T.L., N.B., F.S. and L.M.G. were responsible for the acquisition of data.

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Correspondence to Susan J Kimber.

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Competing interests

R.A.O., D.R.B., T.E.H. and S.J.K. are named inventors on UK Intellectual Property Office patent applications GB 1012495.6 (filed 26 July 2010) and GB 1012559.9 (filed 27 July 2010).

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Oldershaw, R., Baxter, M., Lowe, E. et al. Directed differentiation of human embryonic stem cells toward chondrocytes. Nat Biotechnol 28, 1187–1194 (2010). https://doi.org/10.1038/nbt.1683

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