Abstract
Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative medicine applications. However, directing lineage-restricted differentiation of hESCs and obtaining a homogenous differentiated cell population is still a challenge. We previously described a micromass culture system as a model system to study chondrogenic commitment of the hESCs. Using this system, various growth factors including BMP2 and TGFβ1 direct chondrogenic differentiation and modulate cartilage-specific matrix gene expression in a distinctive manner. Furthermore, a high percentage of differentiated cells exhibit typical morphological characteristics of chondrocytes and express cartilage matrix proteins such as type II collagen and proteoglycans. Chondrogenic cells can be further isolated and cultured to form functional cartilage tissue in vitro. Here, we describe in detail our established protocols to analyze chondrogenic differentiation of hESCs, and possible isolation of chondrogenic cells to form functional cartilaginous tissue.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147.
Heng BC, Cao T, Lee EH. (2004) Directing stem cell differentiation into the chondrogenic lineage in vitro. Stem Cells 22, 1152–1167.
Toh WS, Yang Z, Liu H, Heng BC, Lee EH, Cao T. (2007) Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells 25, 950–960.
Toh WS, Yang Z, Heng BC, Cao T. (2008) Differentiation of human embryonic stem cells towards the chondrogenic lineage. Stem Cell Assays-Methods in Molecular Biology 407, 333–349.
Martin I, Jakob M, Schafer D, Dick W, Spagnoli G, Heberer M. (2001) Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints. Osteoarthritis Cartilage 9, 112–118.
Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.
Wiles MV, Johansson BM. (1999) Embryonic stem cell development in a chemically defined medium. Experimental Cell Research 247, 241–248.
Ahrens PB, Solursh M, Reiter RS. (1977) Stage-related capacity for limb chondrogenesis in cell culture. Developmental Biology 60, 69–82.
Mello MA, Tuan RS. (1999) High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development. In Vitro Cellular & Developmental Biology. Animal 35, 262–269.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC 2006
About this protocol
Cite this protocol
Toh, W.S., Lee, E.H., Richards, M., Cao, T. (2009). In Vitro Derivation of Chondrogenic Cells from Human Embryonic Stem Cells. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 584. Humana Press. https://doi.org/10.1007/978-1-60761-369-5_17
Download citation
DOI: https://doi.org/10.1007/978-1-60761-369-5_17
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-368-8
Online ISBN: 978-1-60761-369-5
eBook Packages: Springer Protocols