Evidence exists that cells of mesenchymal origin show a differentiation plasticity that depends on their differentiation state. We used in vitro differentiation of embryonic stem cells through embryoid bodies as a model to analyze chondrogenic and osteogenic differentiation because embryonic stem cells recapitulate early embryonic developmental phases during in vitro differentiation. Here, we show that embryonic stem cells differentiate into chondrocytes, which progressively develop into hypertrophic and calcifying cells. At a terminal differentiation stage, cells expressing an osteoblast-like phenotype appeared either by transdifferentiation from hypertrophic chondrocytes or directly from osteoblast precursor cells. Chondrocytes isolated from embryoid bodies initially dedifferentiated in culture but later re-expressed characteristics of mature chondrocytes. The process of redifferentiation was completely inhibited by transforming growth factor β3. In clonal cultures of chondrocytes isolated from embryoid bodies, additional mesenchymal cell types expressing adipogenic properties were observed, which suggests that the subcultured chondrocytes indeed exhibit a certain differentiation plasticity. The clonal analysis confirmed that the chondrogenic cells change their developmental fate at least into the adipogenic lineage. In conclusion, we show that chondrocytic cells are able to transdifferentiate into other mesenchymal cells such as osteogenic and adipogenic cell types. These findings further strengthen the view that standardized selection strategies will be necessary to obtain defined cell populations for therapeutic applications.

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Cell-based approaches using mesenchymal stromal precursor cells (MSCs) for the regeneration of intervertebral discs are attracting increased interest, even though the intervertebral disc is a very demanding environment. Implanted cells eventually face acidic pH, hypoxia, and a lack of nutrients. While the regenerative potential of MSCs for skeletal tissues has been well described, it is still questionable whether human MSCs can be prepared for prolonged survival and proper functioning and whether they can differentiate under the adverse conditions encountered in the disc. Here we examined the influence of hypoxia during expansion and differentiation on the chondrogenesis of MSCs. Chondrogenic differentiation was performed in in situ solidifying gelatin hydrogels, which represent a suitable matrix for delivering and anchoring cells within the disc tissue. To consider limitations in nutrition in the intervertebral disc, differentiation was performed at low cell concentrations in the gelatin hydrogels. Standard high-density micromass cultures served as reference controls. To determine the quality of chondrogenesis we analyzed typical marker molecules such as collagen types I, II, X, Sox-9, MIA, and aggrecan mRNA using RT-qPCR and determined protein deposition by histological stainings and biochemical methods. We could demonstrate that in gelatin-based hydrogels chondrogenic differentiation of human MSCs is possible at low cell concentrations. The quality of chondrogenic differentiation could be improved by hypoxia. Best results were obtained when the entire in vitro process, including MSC expansion and subsequent differentiation, was done under hypoxic conditions. MSCs that were expanded under reduced oxygen tension were primed for a chondrogenic differentiation.