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A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery. This requires well‐defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage‐related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells. Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.

The monoclonal antibody mAb 84, which binds to podocalyxin-like protein-1 (PODXL) on human embryonic stem cells (hESCs), was previously reported to bind and kill undifferentiated cells in in vitro and in vivo assays. In this study, we investigate the mechanism responsible for mAb 84-induced hESCs cytotoxicity. Apoptosis was likely not the cause of mAb 84-mediated cell death because no elevation of caspase activities or increased DNA fragmentation was observed in hESCs following incubation with mAb 84. Instead, it was preceded by cell aggregation and damage to cell membranes, resulting in the uptake of propidium iodide, and the leakage of intracellular sodium ions. Furthermore, examination of the cell surface by scanning electron microscopy revealed the presence of pores on the cell surface of mAb 84-treated cells, which was absent from the isotype control. This mechanism of cell death resembles that described for oncosis, a form of cell death resulting from membrane damage. Additional data suggest that the binding of mAb 84 to hESCs initiates a sequence of events prior to membrane damage, consistent with oncosis. Degradation of actin-associated proteins, namely, a-actinin, paxillin, and talin, was observed. The perturbation of these actin-associated proteins consequently permits the aggregation of PODXL, thus leading to the formation of pores. To our knowledge, this is the first report of oncotic cell death with hESCs as a model.

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 TGFbeta1 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.

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