(516a) PDGF and TGF-?1 Crosstalk Is Essential for Mesenchymal Stem Cell Stiffening

Mesenchymal stem cells (MSCs) are multipotent progenitor cells which can be isolated from bone marrow (BM), expanded rapidly and exhibit multi-lineage differentiation potential [1]. MSCs spontaneously home to tumor and wound healing sites, which secrete pro-inflammatory molecules that mediate MSC migration. In tissue microenvironments, MSCs secrete cytokines and chemokines for recruitment of other cell types (e.g. fibroblasts and endothelial cells), deposit and remodel local extracellular matrix, and differentiate into stromal cells. The role of MSCs in the tissues is dependent on niche-specific mechanical and chemical stimuli. For example, the tumor stroma is often more rigid than surrounding tissues with 13-fold higher rigidities of high-grade invasive ductal carcinoma compared to normal fibroglandular tissue [2]. Cells can feel this differential local tissue rigidity and respond by differentiating into specific cell types (e.g. myofibroblasts). In vivo microenvironments also include a multitude of growth factors that stimulate cell survival, migration, proliferation and differentiation using both autocrine and paracrine signaling mechanisms. Transforming growth factor-β1 (TGF-β1) [3, 4] and platelet derived growth factor-BB (PDGF-BB) [4-5] are two such widely studied growth factors that have been recognized to play key roles in tumors and regenerating tissues [7, 8].We systematically characterized the microscopic mechanical properties of MSCs, including their cytoskeletal rheology, morphology, cellular adhesion, and cell elongation, in response to TGF-β1 and PDGF-BB (referred as PDGF), individually and in combination. Small-molecule signaling inhibitors SB-505124 (1µM) and JNJ-10198409 (50nM) were used to block TGF-βRI and PDGFR-β dependent signaling, respectively. Affymetrix 430.2 mouse genome arrays were used to analyze genome wide expression of treated and untreated MSCs.

Although PDGF alone did not alter the morphology of MSCs, TGF-β1 individually and in combination with PDGF resulted in drastic elongation of MSCs and condensed both actin and microtubule organization. The viscoelasticity of MSCs, measured with multiple particle tracking microrheology (MPTM), was also severely affected by TGF-β1, alone and in combination with PDGF. The elastic modulus (G’) of the cytosol was increased 9-fold in TGF-β1 treated cells; whereas, the combination of PDGF and TGF-β1 treatment resulted in 100-fold change in the G’ compared to control cells after 24 hours. Blocking either TGF-β1 or PDGF signaling with inhibitors reversed the soluble factor induced cellular stiffening to control levels. Flow Cytometry results indicated significant increase in cell surface expression of β1 (CD29) and α_v(CD51) integrins for these conditions. Finally, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of genes involving cytoskeletal actin-binding proteins (ABPs). In conclusion, MSCs undergo dramatic elongation and stiffening under the influence of TGF-β1 (individually and combination with PDGF). However, our studies reveal that PDGF signaling is essential in activating ABPs required for TGF-β1dependent stiffening.

Acknowledgements:  Funding provided by the Georgia Tech and Emory Center for Regenerative Medicine and NSF.

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