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.
