Development of Serum-Free,Chemically Defined Conditions for Human Embryonic Stem Cell–Derived Fibrochondrogenesis

Abstract

This study established serum-free, chemically defined conditions to generate fibrocartilage with human embryonic stem cells (hESCs). Three sequential experimental phases were performed to eliminate serum because of its variability and antigenic potential and characterize the performance of hESCs in serum-free and serum-based conditions. Each phase used a two-stage modular experiment: chondrogenic differentiation followed by scaffold-less tissue engineering, called self-assembly. Phase I studied serum effects, and showed that a 1% serum chondrogenic medium (CM) during differentiation resulted in uniform constructs, whereas a 20% serum CM did not. Furthermore, a no-serum CM during self-assembly led to a collagen content 50% to 200% greater than a 1% serum CM. Thus, a “serum standard” of 1% serum during differentiation and no serum during self-assembly was carried forward. Phase II compared this with serum-free formulations, using 5% knock-out serum replacer or 1-ng/mL transforming growth factor beta 1 (TGF-β1). The TGF-β1 group was chosen as a “serum-free standard” because it performed similarly to the serum standard in terms of morphological, biochemical, and biomechanical properties. In Phase III, the serum-free standard had significantly more collagen (100%) and greater tensile (∼150%) and compressive properties (∼80%) than the serum standard with TGF-β1 treatment during self-assembly. These advances are important to the understanding of mechanisms of chondrogenesis and creating clinically relevant stem cell therapies.