Decellularized Autologous Mesenchymal Stem Cell (MSC) Extracellular Matrix Enhances MSC Migration and Differentiation Potential

1Lin, H; 1Taboas, JM; 1Tan, J; +1Tuan, RS +1 Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery,

University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219, USA. rst13@pitt.edu

INTRODUCTION: Mesenchymal stem cell (MSC) derived extracellular matrix (ECM) is known to promote MSC proliferation and phenotype maintenance during culture expansion (i.e., the ability to differentiate down several mesenchymal lineages after multiple expansion) (1). Acellular 3-dimensional (3-D) scaffolds created with MSC ECM enhance MSC chondrogenesis with BMP-2 in vitro (2). We are interested in the application of MSC-derived ECM to potentiate regeneration of skeletal tissues, for example by using it as a coating for biomaterial scaffolds with optimal stiffness and porosity. We hypothesize that the components of the MSC-derived ECM will exhibit bioactivities favorable for tissue regeneration. We report here the development of a method to extract and solubilize bioactive ECM components secreted by MSCs, which are then used to coat tissue culture plastic, and the investigation of their effects on MSC proliferation, differentiation and migration (Fig 1).

METHODS: Cell culture Human bone marrow MSCs were obtained from total joint arthroplasty patient with IRB approval and used at passage 4 (P4). After 15 days of culture at 100% confluence on tissue culture plastic to produce ECM, cells were lyzed and removed with 0.1% Triton X-100 solution. ECM was collected by scrapping and treated with DNase I to remove DNA contaminiation. ECM Extraction Two solubilized ECM preparations were produced. ECM was extracted with 2M cold urea for 2 days (2.5 ml for each 150 cm2 flask), cleared by centrifugation, and dialyzed against PBS (referred to as U-MECM). The pellet after centrifugation was treated with 0.1% pepsin(w/v) in 0.01M HCl solution at 37C until pellet was dissolved(4 ml per pellet from 150 cm2 flask). The resultant solution was neutralized and dialyzed against PBS (HP-MECM). Proteins concentrations were determined via BCA assay. Proliferation assay Cell culture plates were coated with undiluted U-MECM (120 g/ml) or HP-MECM (270 g/ml) overnight at 4C. Plates, uncoated and coated with 1 mg/ml collagen type I, served as

controls. P4 human MSCs were cultured on coated surface for 3 days, and total DNA determined by Picogreen. Differentiation assay P4 human MSCs were seeded on the different surface for 4 hours, and then the medium switched to osteogenic and adipogenic differentiation media. After 14 days induction, cells were stained with

Alizarin Red or Oil red O to estimate differentiation. Migration Assay MSCs were plated inside a hollow stopper (10 cm2 area) placed in the center of a tissue culture well, with the surrounding coated with different ECM (see Fig 2). The stopper position was marked on the bottom of culture well. After 4 h, the ECM solution was drained, and the stopper was removed and growth medium added to the entire well. Cell migration was recorded under 40× magnification over time. Statistical Analyses Student’s paired t-tests were used. A value of p < 0.05(*) was considered significant (** , p<0.01). RESULTS: ECM extraction Typically, the yields were 0.3-0.4 mg U-MECM and 0.9-1.0 mg HP-MECM from each 150cm2 flask culture. Proliferation assay After 3 days, U-MECM coated plates displayed

significantly greater MSC proliferation. Total DNA in U-MECM group was 1.5 folder higher compared to uncoated group (Fig. 3). Dose response results showed that even the lowest 1.25 g/ml U-MECM coating promoted faster cell growth than the uncoated control group (Fig 4), and that 7.5 g/ml U-MECM was equivalent to 1 mg/ml collagen coating in promoting MSC proliferation. Differentiation assay After 14 days culture in adipogenesis medium, U-MECM group showed greater Oil red O staining (Fig 5). Normalizing staining density to total DNA showed that U-MECM significantly induced higher adipogenesis compared to other groups (e. g., 40% more than collagen), as well as enhanced osteogenic differentiation (data not shown). In summary, U-MECM coating enhanced MSC differentiation over plastic, and collagen and HP-MECM coating.

Migration Assay Co-culture with U-MECM promoted faster MSC proliferation and migration across non-coated surface over all other coatings (Fig 6). After 8 days, MSC populations on U-MECM group had crossed the 2 mm uncoated distances. In addition, MSCs also migrated faster on the U-MECM coated surface (data not shown).

DISCUSSION/CONCLUSIONS: Urea-extracted MSC-derived ECM (U-MECM) promotes MSC proliferation, migration and differentiation (with induction medium) in vitro to a greater extent than HP-MECM and collagen type I. U-MECM is strongly bioactive, efficiently enhancing MSC proliferation even at low concentrations. Faster MSC migration across the uncoated region and onto the U-MECM coating suggest that the release of soluble chemotactic factors. U-MECM also enhances MSC sensitivity to induced differentiation. In comparison, HP-MECM coating was similar to collagen coating for MSC proliferation, differentiation and migration. SDS-PAGE results showed that collagen was a major component of HP-MECM, whereas U-MECM contained several additional proteins (data not shown). Thus, non-collagenous components of U-MECM, e.g., biglycan or decorin, are likely responsible for its effects (1). Understanding the composition of U-MECM is critical to its use for tissue regeneration and the regulation of MSC fate determination. REFERENCES: (1) Lai, YL; et al. 2010. Stem cells Dev. Epub ahead of print. (2) Audo, W; et al. 2008. Tissue Eng Part A. 14(12):2041-9 ACKNOWLEDGEMENTS: Supported in part by a grant from the Commonwealth of Pennsylvania Department of Health

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Poster No. 1771 • ORS 2011 Annual Meeting