Induction of Tendon-Specific Markers in Adipose-Derived Stem Cells in Serum-Free Culture Conditions

Differentiation of stem cells as a cell-based therapy for repairing, replacing, or restoring damaged tissues such as bone, cartilage, and tendon is becoming increasingly attractive within the field of musculoskeletal tissue engineering. Toward this end, there are numerous published and well-defined protocols to differentiate stem cells toward cartilage and bone tissues, but the protocols toward tendon tissue are still emerging and thus less developed. Recent studies focused on the induction of tendon-specific markers in cultured stem cells using different growth factors (GFs), including bone morphogenetic proteins (BMPs) and transforming growth factor (TGF) isoforms. However, the inclusion of serum in relatively high concentration across these studies is less favorable, since the components within serum may interfere with the induction of the markers. Alternatively, in vitro studies with low concentration or absence of serum would be ideal. In this study, we assessed the induction effect of BMP-12 and TGF-beta 1 on tendon-specific markers in adipose-derived stem cells (ADSCs), in serum-free conditions. Specifically, we investigated the temporal and dosing effects of both GFs on several markers. Our results demonstrate that BMP-12 induces late expression of the transcription factors Scleraxis (SCX) and Mohawk (MKX), whereas TGF-beta 1 induced their earlier expression. Moreover, BMP-12 induced Decorin (DCN), but was inhibited by TGF-beta 1. Other markers such as collagen I alpha 1 (COL1A1) likewise showed this pattern. Importantly, the protein analysis generally supported the gene expression data. Interestingly, differences were observed in the cellular localization of SCX between BMP-12 and TGF-beta 1 stimulations. Furthermore, the addition of ascorbic acid with either BMP-12 or TGF-beta 1 resulted in increased deposition of collagen I. Our results enhance the existing protocols for the differentiation of ADSCs toward the tenogenic lineage in serum-free conditions and contribute to the understanding and the development of tenogenic induction protocols. Impact Statement Herein, we describe the tenogenic effect of bone morphogenetic protein-12 and transforming growth factor-beta 1 in cultured adipose-derived stem cells (ADSCs) in serum-free conditions. This culture system provides an insight into serum-free culture conditions in stem cell differentiation protocols. A positive response of the ADSCs to the tenogenic induction was observed. In particular, the different growth factors used in this study displayed notable differences both on the gene and on the protein expression of the tendon-specific markers. The results underline the positive outcome of the serum removal in tenogenic differentiation protocols, contributing to the development of future cell-based therapies for tendon regeneration and repair.