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Human embryonic stem cells (hESCs) are an exciting cell source for fibrocartilage engineering. In this study, the effects of differentiation time and cell line, H9 versus BG01V, were examined. Embryoid bodies (EBs) were fibrochondrogenically differentiated for 1, 3, or 6 weeks and then used to engineer tissue constructs that were grown for an additional 4 weeks. Construct matrix was fibrocartilaginous, containing glycosaminoglycans (GAGs) and collagens I, II, and VI. A differentiation time of 3 or 6 weeks produced homogeneous constructs, with matrix composition varying greatly with cell line and differentiation time: from 2.6 to 17.4 μg of GAG per 10 6  cells and from 22.3 to 238.4 μg of collagen per 10 6  cells. Differentiation for 1 week resulted in small constructs with poor structural integrity that could not be mechanically tested. The compressive stiffness of the constructs obtained from EBs differentiated for 3 or 6 weeks did not vary significantly as a function of either differentiation time or cell line. In contrast, the tensile properties were markedly greater with the H9 cell line, 1,562–1,940 versus 32–80 kPa in the BG01V constructs. These results demonstrate the dramatic effects of hESC line and differentiation time on the biochemical and functional properties of tissue‐engineered constructs and show progress in fibrocartilage tissue engineering with an exciting new cell source.  Disclosure of potential conflicts of interest is found at the end of this article.

Fibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines