Tgf β3

EBs (n=20) were transferred onto 6-well plates coated with Matrigel™ (Corning Incorporated). After 24 h, the medium was replaced with a chondrogenic medium (ChM) (day 0) composed of DMEM/F12 (Sigma-Aldrich; Merck KGaA), 2% KnockOut™ SR (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1 mM sodium pyruvate (Biowest, Nuaillé, France), 10⁻⁷ M dexamethasone, 50 µM ascorbic acid, 50 µM L-proline, 1% penicillin-streptomycin (all provided by Sigma-Aldrich; Merck KGaA), 1% ITS+ premix (Corning Incorporated), and 10 ng/ml TGF-β3 (ImmunoTools, Friesoythe, Germany). The EBs were cultured for 21 days and the medium was changed every second day.

The mature EBs were transferred onto 6-well plates (10 EBs per well) previously coated with 0.1% gelatin (Merck Millipore) and allowed to adhere for the next 24 h, following which the medium was replaced with a chondrogenic medium. This was either supplemented with TGF-β3 (10 ng/ml; ImmunoTools GmbH, Friesoythe, Germany), as a growth factor with the most chondrogenic potential, or conditioned on the HC-402-05a cell line as above. The positive influence of standard chondrogenic medium with the addition of exogenous TGF-β3 (10 ng/ml) on pluripotent SCs was previously tested and confirmed (18 (link)). The chondrogenic medium was changed every 48 h. The culture period lasted 21 days. In order to confirm that chondrocyte-like cells had been obtained, immunofluorescence analysis was performed on passage 0 (p0). Next, to evaluate the expression profile of chondrogenic markers (p3), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed (Fig. 1). In all analyses, the stable adult human articular chondrocyte cell line (HC-402-05a) served as a positive control, as the European Collection of Authenticated Cell Cultures recommended it for the evaluation of the differentiation process in in vitro model systems.

The mature EBs were transferred onto 6-well plates (10 EBs per well) previously coated with 0.1% gelatin (Merck Millipore) and allowed to adhere for the next 24 h, following which the medium was replaced with a chondrogenic medium This was either supplemented with TGF-β3 (10 ng/ml; ImmunoTools GmbH, Friesoythe, Germany), as a growth factor with the most chondrogenic potential, or conditioned with the HC-402-05a cell line as above. The positive influence of standard chondrogenic medium with the addition of exogenous TGF-β3 (10 ng/ml) on pluripotent stem cells was previously tested and confirmed by our group (16 (link)). The chondrogenic medium was changed every 48 h. The culture period lasted 21 days. In order to confirm that chondrocyte-like cells had been obtained, immunofluorescence analysis was performed on passage 0 (p0). Subsequently, to evaluate the expression profile of chondrogenic markers (p3), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed (14 (link)). In all analyses, the stable adult human articular chondrocyte cell line (HC-402-05a) served as a positive control, as the European Collection of Authenticated Cell Cultures recommended it for the evaluation of the differentiation process in in vitro model systems.