High-Throughput Screening for Modulators of Mesenchymal Stem Cell Chondrogenesis

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine and the study of skeletal development. Despite considerable interest in MSC chondrogenesis, the signal transduction and molecular mechanisms underlying this process remain largely undefined. To explore the signaling topology regulating chondrogenic differentiation, as well as to discover novel modulators, we developed and validated a high-throughput screening (HTS) assay for MSC chondrogenesis. Adapting standard assay procedures to enable HTS, we successfully minimized cell number, handling, and culture duration. Using our optimized methodology with automation, we evaluated a comprehensive screen using four growth factors, TGF-β3, BMP-2, IGF-1, and FGF-2, to demonstrate the feasibility of large combinatorial screens. We examined the chondrogenic effects of these growth factors in different combinations and doses (81 combinations total with 16 replicates per group) and found variable effects on GAG content with different combinations. In general, TGF-β3 had a pro-chondrogenic effect while FGF-2 had a proliferative effect. BMP-2 was both proliferative and pro-chondrogenic while the effect of IGF-1 in our system was variable. We also carried out an HTS campaign of the National Institute of Neurological Disorders and Stroke (NINDS) chemical library of small molecules (1040 compounds) and identified 5 potential inducers and 24 potential inhibitors of chondrogenesis. Of these compounds, several were identified from the hypnotic, anti-neoplastic, or anti-protein synthesis classes of molecules. These studies demonstrate our ability to carry out high-throughput screening assays for modulators of chondrogenesis.

[1]  Manas Kumar Majumdar,et al.  BMP‐2 and BMP‐9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL‐1 , 2001, Journal of cellular physiology.

[2]  G. Verbruggen.  Chondroprotective drugs in degenerative joint diseases. , 2006, Rheumatology.

[3]  V. Goldberg,et al.  FGF‐2 enhances the mitotic and chondrogenic potentials of human adult bone marrow‐derived mesenchymal stem cells , 2005, Journal of cellular physiology.

[4]  M E Bolander,et al.  Ciprofloxacin Inhibition of Experimental Fracture-Healing* , 2000, The Journal of bone and joint surgery. American volume.

[5]  J. Parvizi,et al.  Antibiotics for Local Delivery Systems Cause Skeletal Cell Toxicity In Vitro , 2007, Clinical orthopaedics and related research.

[6]  R Kujat,et al.  Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge. , 1999, Tissue engineering.

[7]  R. Tuan,et al.  Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[9]  P. Prendergast,et al.  A comparison of the involvement of p38, ERK1/2 and PI3K in growth factor-induced chondrogenic differentiation of mesenchymal stem cells. , 2008, Biochemical and biophysical research communications.

[10]  F. Beier,et al.  Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis* , 2007, Journal of Biological Chemistry.

[11]  R. Tuan,et al.  p38 MAP Kinase Regulation of AP‐2 Binding in TGF‐β1‐Stimulated Chondrogenesis of Human Trabecular Bone‐Derived Cells , 2002, Annals of the New York Academy of Sciences.

[12]  W. Patrick Walters,et al.  A guide to drug discovery: Designing screens: how to make your hits a hit , 2003, Nature Reviews Drug Discovery.

[13]  P. Benya,et al.  Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression , 1988, The Journal of cell biology.

[14]  Rocky S Tuan,et al.  Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. , 2008, Arthritis and rheumatism.

[15]  B. A. Byers,et al.  Regulation of Cartilaginous ECM Gene Transcription by Chondrocytes and MSCs in 3D Culture in Response to Dynamic Loading , 2007, Biomechanics and modeling in mechanobiology.

[16]  Kenneth Dixon,et al.  Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. , 2002, Arthritis and rheumatism.

[17]  F. Guilak,et al.  Effects of Transforming Growth Factor β1 and Dexamethasone on the Growth and Chondrogenic Differentiation of Adipose-Derived Stromal Cells , 2003 .

[18]  Peter G Schultz,et al.  A small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells. , 2002, Journal of the American Chemical Society.

[19]  K. Gibson,et al.  Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine. , 1976, Biochimica et biophysica acta.

[20]  Charles C Hong,et al.  Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. , 2008, Nature chemical biology.

[21]  G. Sukhikh,et al.  Mesenchymal Stem Cells , 2002, Bulletin of Experimental Biology and Medicine.

[22]  F. Beier,et al.  RhoA/ROCK Signaling Regulates Sox9 Expression and Actin Organization during Chondrogenesis* , 2005, Journal of Biological Chemistry.

[23]  C. Kaps,et al.  BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. , 2003, Differentiation; research in biological diversity.

[24]  Eric Lejeune,et al.  Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial , 2001, The Lancet.

[25]  Yubo Sun,et al.  Effects of Cyclic Compressive Loading on Chondrogenesis of Rabbit Bone‐Marrow Derived Mesenchymal Stem Cells , 2004, Stem cells.

[26]  Boon Chin Heng,et al.  Combined effects of TGFβ1 and BMP2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation , 2005, Growth factors.

[27]  R. Tuan,et al.  Glucosamine promotes chondrogenic phenotype in both chondrocytes and mesenchymal stem cells and inhibits MMP-13 expression and matrix degradation. , 2007, Osteoarthritis and cartilage.

[28]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[29]  C. Anast,et al.  Amino acid, potassium, and sulfate transport and incorporation by embryonic chick cartilage: the mechanism of the stimulatory effects of serum. , 1966, Biochimica et biophysica acta.

[30]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[31]  Andre Steinert,et al.  Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[32]  A H Huang,et al.  Tensile properties of engineered cartilage formed from chondrocyte- and MSC-laden hydrogels. , 2008, Osteoarthritis and cartilage.

[33]  A. Hollander,et al.  Pharmacological Regulation of Adult Stem Cells: Chondrogenesis Can Be Induced Using a Synthetic Inhibitor of the Retinoic Acid Receptor , 2007 .

[34]  G. Im,et al.  Chondrogenic differentiation of mesenchymal stem cells isolated from patients in late adulthood: the optimal conditions of growth factors. , 2006, Tissue engineering.

[35]  H. Pondenis,et al.  Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. , 2007, American journal of veterinary research.

[36]  J. Welter,et al.  Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay. , 2007, BioTechniques.

[37]  F. Guilak,et al.  Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. , 2006, Arthritis and rheumatism.

[38]  Junzo Tanaka,et al.  Growth factor combination for chondrogenic induction from human mesenchymal stem cell. , 2004, Biochemical and biophysical research communications.

[39]  Sheng Ding,et al.  A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells , 2007, Proceedings of the National Academy of Sciences.

[40]  F. Guilak,et al.  Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells. , 2003, Tissue engineering.

[41]  W. Hozack,et al.  Transforming Growth Factor-β-mediated Chondrogenesis of Human Mesenchymal Progenitor Cells Involves N-cadherin and Mitogen-activated Protein Kinase and Wnt Signaling Cross-talk* , 2003, Journal of Biological Chemistry.

[42]  Jean F Welter,et al.  High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells. , 2005, BioTechniques.

[43]  F. Beier,et al.  RhoA/ROCK Signaling Regulates Chondrogenesis in a Context-dependent Manner* , 2006, Journal of Biological Chemistry.

[44]  F. Barry,et al.  Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. , 2001, Experimental cell research.

[45]  J. Elisseeff,et al.  Reorganization of actin filaments enhances chondrogenic differentiation of cells derived from murine embryonic stem cells. , 2006, Biochemical and biophysical research communications.

[46]  D. Prockop.  Marrow Stromal Cells as Stem Cells for Nonhematopoietic Tissues , 1997, Science.

[47]  R. Tuan,et al.  Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. , 2006, Osteoarthritis and cartilage.

[48]  H. Moses,et al.  Effect of IGF‐I in the Chondrogenesis of Bone Marrow Mesenchymal Stem Cells in the Presence or Absence of TGF‐β Signaling , 2005, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[49]  Richard Tuli,et al.  Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. , 2005, Biomaterials.