Induction of mesenchymal stem cell chondrogenesis through sequential administration of growth factors within specific temporal windows.

Human mesenchymal stem cells (hMSCs) are capable of differentiating into chondrocyte-like cells but fail to produce the quality or quantity of cartilage matrix compared to articular chondrocytes using current differentiation protocols. In this study, we aim to improve the chondrogenic differentiation of hMSCs through the sequential administration of multiple growth factors (GFs). We began by looking at differentiating hMSCs' cell surface GF receptor expression every 3 days throughout differentiation using flow cytometry and found that not only was receptor expression dynamic throughout differentiation, but ligand sensitivity was positively correlated with receptor expression, suggesting that differentiating hMSCs may have varying GF requirements depending on their stage of differentiation. We then constructed GF sequences by administering several prochondrogenic GFs singly every 3 days throughout differentiation and assaying the expression of a variety of cartilage-related genes using qPCR. The resulting chondrocytic phenotype of sequentially induced hMSCs was then compared to that of hMSCs induced under standard culture conditions using qPCR, dimethylmethylene blue assay, and histology. We found that while the initial GF sequence was unable to improve hMSC chondrogenesis, withdrawal of GF treatment at Day 9 of differentiation in pellet culture vastly improved the success of differentiation beyond that induced by TGFβ1 alone. Additional modifications allowed us to further improve chondrogenesis to levels comparable to that obtained by co-administration of TGFβ1 and BMP7 throughout differentiation. Taken together, we demonstrated the ability to improve the chondrocytic phenotype of differentiated hMSCs through the sequential administration of multiple GFs.