Effect of calcium on the proliferation kinetics of synovium-derived mesenchymal stromal cells HELEN DRY 1 , KRISTEN JORGENSON 1 , WATARU ANDO 2 , DAVID A. HART 2 , CYRIL B. FRANK 2 & ARINDOM SEN 1,2 1 Pharmaceutical Production Research Facility, Schulich School of Engineering, and 2 McCaig Institute for Bone and Joint Health, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Abstract Background aims. Synovium-derived mesenchymal stromal cells (S-MSCs) have potential utility in clinical joint repair applications. However, their scarcity in tissues means S-MSCs cannot be isolated in large quantities and need to be expanded in culture. Because synovial tissues in vivo are exposed to higher calcium (Ca 2þ ) levels than typically found in culture media, this study examined the impact of Ca 2þ supplementation on the rate of S-MSC proliferation in culture. Methods. S-MSCs were serially cultured with or without Ca 2þ supplementation. The effect of inhibiting Ca 2þ uptake was assessed using Ca 2þ channel blockers. After extended exposure to elevated Ca 2þ concentrations, S-MSCs were characterized by evaluating surface marker profiles, performing reverse transcriptase quantitative polymerase chain reaction and carrying out tri-lineage differentiation assays. Results. Elevated Ca 2þ concentrations resulted in enhanced S-MSC proliferation. Peak growth occurred at 5.0 mmol/L Ca 2þ , with an average fold increase of 4.52  0.65 per passage over 8 passages compared with 2.03  0.46 in un-supplemented medium. Proliferation was inhibited by Ca 2þ channel blockers. Ca 2þ -supplemented cells showed enhanced capacity toward osteogenesis (17.82  4.21 mg Ca 2þ deposited/sample vs. 12.70  2.11 mg Ca 2þ deposited/sample) and adipogenesis (0.47  0.04 mg oil red O/sample vs. 0.352  0.005 mg oil red O/sample) and retained their capacity to undergo chondrogenesis (1.37  0.07 mg glycosaminoglycan/pellet vs. 1.33  0.17 mg glycosaminoglycan/ pellet). S-MSCs cultured in elevated Ca 2þ expressed enhanced messenger RNA levels for SOX-9 and peroxisome pro- liferator activated receptor gamma and depressed levels for collagen I. Conclusions. S-MSC sensitivity to Ca 2þ has not been reported previously. These findings indicate that S-MSC population expansion rates may be up-regulated by Ca 2þ supplementation without compromising defining cell characteristics. This study exemplifies the need to consider medium composition when culturing stem cells. Key Words: calcium, cell culture medium, culture conditions, differentiation, mesenchymal stromal cells, mesenchymal stromal cells, proliferation, synovium Introduction Mesenchymal stromal cell (MSC) populations are defined in part by their ability to adhere to a plastic surface and replicate while maintaining the potential to differentiate along osteogenic, adipogenic and chondrogenic lineages (1). Cell populations exhibiting these traits have been isolated from several species and expanded in vitro for extended periods via traditional serial sub-culturing techniques (2e7). Because of their multi-lineage capacity, these cells have attracted great research interest in the areas of tissue engi- neering and cell therapy. MSCs are most commonly isolated from bone marrow, but cells exhibiting MSC characteristics can be found in many other tissues, including adipose (8), skin (9), umbilical cord blood (10) and synovium (4). Synovium-derived mesen- chymal stromal cells (S-MSCs) have been reported to exhibit comparable growth characteristics to bone marrow-derived MSCs but may have a greater propensity to differentiate toward a chondrogenic phenotype (6,11). In addition, it has been reported that these cells could be induced to form three- dimensional constructs, which were able to repair damaged cartilage when transplanted into a chondral defect site in pigs (12e14). The new cartilage exhibi- ted mechanical and viscoelastic properties similar to native articular cartilage. Based on these traits as well as their relative ease of isolation during standard arthroscopic surgery, S-MSCs represent an attractive cell type for use in cell-based therapies for bone Correspondence: Arindom Sen, PhD, PEng, Schulich School of Engineering, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4. E-mail: asen@ucalgary.ca Cytotherapy, 2013; 15: 805e819 (Received 23 May 2012; accepted 18 January 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.01.011