Example 3
Pluripotent stem cell-derived cells (2.5×105 cells), having been passaged at least five times, were collected in 15-ml conical tubes and centrifuged at 150 g for 5 min after which they were transferred to serum-free chondrogenic media (Lonza Basel Switzerland) in the presence or absence of TGFβ3 (10 ng/ml; Peprotech, Rocky Hill, N.J.). The media was changed twice weekly. At the end of 3 weeks, some cell pellets were fixed with Z-Fix (Anatech, Battle Creek, Mich.), paraffin-embedded, sectioned, and assessed for their chondrogenic differentiation status as detailed below for histochemical stains, immunocytochemical markers, and mRNA as described below.
Total RNA was extracted from cell pellets with RNeasy kit (Invitrogen, Carlsbad, Calif.) and was reverse transcribed to cDNA with SuperScript (Invitrogen, Carlsbad, Calif.). Real-time RT-PCR of collagen IIA1 and aggrecan was performed using Taqman-® Gene expression assays as per manufacturer's instructions (Applied Biosystems, Foster City, Calif.).
