Regarding the role of IGF-I in the chondrogenic potential of MSCs, it has been reported that MSCs express IGF-I, but most studies have shown that IGF-I treatment has no effect on MSCs or has effect only in combination with TGF-β. 7- 9 The biological action of IGFs is transduced by the type I tyrosine kinase receptor (IGFIR) that classically activates the mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase 1/2 (MEK1/2) extracellular signal-regulated kinase 1/2 (Erk1/2) mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase-Akt (PI3K-Akt) pathways. Insulin-like growth factors (IGFs) play a critical role in skeletal development, as indicated by the fact that Igf gene knockout in mice leads to severe growth failure. Research efforts focused on identifying growth factors (GFs) that regulate the chondrogenic potential of MSC are crucial to optimize the therapeutic use of MSCs in cartilage disorders. 2- 6 Despite growing information concerning MSCs, the mechanisms that govern their chondrogenic potential are poorly understood. Adult bone marrow (BM) contains nonhematopoietic mesenchymal stem cells (MSCs) capable of differentiating into cells of numerous tissue lineages including chondrocytes. This has led to efforts to develop alternative means to restore damaged cartilage. 1 Because cartilage lacks regenerative ability, treatment for cartilage diseases is primarily palliative. I n the developing skeleton, chondrogenesis is initiated by the migration of mesenchymal chondroprogenitors to the future long bone segment sites, where they undergo condensation, proliferation, and chondrocyte differentiation and hypertrophy. Our data, by showing the role of IGF-I and TGF-β1 in the critical steps of MSC chondrogenesis, provide critical information to optimize the therapeutic use of MSCs in cartilage disorders. We found that the extracellular signal-related kinase 1/2 mitogen-activated protein kinase (Erk1/2 MAPK) pathway mediated the TGF-β1 mitogenic response and in part the IGF-I proliferative action. Using RIIKO-MSCs, we showed that IGF-I chondrogenic actions are independent from the TGF-β signaling. IGF-I chondroinductive actions were equally potent to TGF-β1, and the two growth factors had additive effects. Results and Conclusions: IGF-I modulated MSC chondrogenesis by stimulating proliferation, regulating cell apoptosis, and inducing expression of chondrocyte markers. We conditionally inactivated the TGF-β type II receptor (TβRII) in MSCs using a cre-lox system, generating TβRII knockout MSCs (RIIKO-MSCs). We obtained MSCs from mice in which green fluorescent protein (GFP) was under the Collagen2 promoter and determined GFP expression by confocal microscopy. Seven-day-old pellets were measured in size, stained for proteoglycan synthesis, and analyzed for the expression of collagen II and Sox-9 by quantitative real time PCR. We measured thymidine incorporation and stained 2-day-old pellets with TUNEL, cleaved caspase-3, peanut-agglutinin, and N-cadherin. Chondrogenic differentiation was induced by culturing high-density MSC pellets in serum- and insulin-free defined medium up to 7 days, with or without IGF-I and/or TGF-β. Materials and Methods: Mononuclear adherent stem cells were isolated from mouse BM. The aim of our study was to define the biological role of IGF-I on proliferation, chondrogenic condensation, apoptosis, and differentiation of MSCs into chondrocytes, alone or in combination with TGF-β and in the presence or absence of TGF-β signaling. TGF-β induces MSC chondrogenic differentiation, although its actions are not well defined. The role of IGF-I in the chondrogenic potential of MSCs is poorly understood. Introduction: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow (BM), expanded, and differentiated into several cell types, including chondrocytes. IGF-I effects were independent from and additive to TGF-β. We found that IGF-I had potent chondroinductive actions on MSCs. IGF-I effects were evaluated in the presence or absence of TGF-β signaling by conditionally inactivating the TGF-β type II receptor. A novel role for IGF-I in MSC chondrogenesis was determined.
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