Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling.
A novel role for IGF-I in MSC chondrogenesis was determined. IGF-I effects were evaluated in the presence or absence of TGF-beta signaling by conditionally inactivating the TGF-beta type II receptor. We found that IGF-I had potent chondroinductive actions on MSCs. IGF-I effects were independent from and additive to TGF-beta.


BACKGROUND
Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow (BM), expanded, and differentiated into several cell types, including chondrocytes. The role of IGF-I in the chondrogenic potential of MSCs is poorly understood. TGF-beta induces MSC chondrogenic differentiation, although its actions are not well defined. 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-beta and in the presence or absence of TGF-beta signaling.


METHODS
Mononuclear adherent stem cells were isolated from mouse BM. 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-beta. We measured thymidine incorporation and stained 2-day-old pellets with TUNEL, cleaved caspase-3, peanut-agglutinin, and N-cadherin. 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 obtained MSCs from mice in which green fluorescent protein (GFP) was under the Collagen2 promoter and determined GFP expression by confocal microscopy. We conditionally inactivated the TGF-beta type II receptor (TbetaRII) in MSCs using a cre-lox system, generating TbetaRII knockout MSCs (RIIKO-MSCs).


CONCLUSIONS
IGF-I modulated MSC chondrogenesis by stimulating proliferation, regulating cell apoptosis, and inducing expression of chondrocyte markers. IGF-I chondroinductive actions were equally potent to TGF-beta1, and the two growth factors had additive effects. Using RIIKO-MSCs, we showed that IGF-I chondrogenic actions are independent from the TGF-beta signaling. We found that the extracellular signal-related kinase 1/2 mitogen-activated protein kinase (Erk1/2 MAPK) pathway mediated the TGF-beta1 mitogenic response and in part the IGF-I proliferative action. Our data, by showing the role of IGF-I and TGF-beta1 in the critical steps of MSC chondrogenesis, provide critical information to optimize the therapeutic use of MSCs in cartilage disorders.
Longobardi
Lara
L
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2579, USA.
O'Rear
Lynda
L
Aakula
Srikanth
S
Johnstone
Brian
B
Shimer
Kimberly
K
Chytil
Anna
A
Horton
William A
WA
Moses
Harold L
HL
Spagnoli
Anna
A
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects*; D051379:Mice; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D016212:Transforming Growth Factor beta; D007334:Insulin-Like Growth Factor I