Cytotherapy, 2013; 15: 805e819

Effect of calcium on the proliferation kinetics of synovium-derivedmesenchymal stromal cells

HELEN DRY1, KRISTEN JORGENSON1, WATARU ANDO2, DAVID A. HART2,CYRIL B. FRANK2 & ARINDOM SEN1,2

1Pharmaceutical Production Research Facility, Schulich School of Engineering, and 2McCaig Institute for Bone andJoint Health, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

AbstractBackground aims. Synovium-derived mesenchymal stromal cells (S-MSCs) have potential utility in clinical joint repairapplications. However, their scarcity in tissues means S-MSCs cannot be isolated in large quantities and need to be expandedin culture. Because synovial tissues in vivo are exposed to higher calcium (Ca2þ) levels than typically found in culture media,this study examined the impact of Ca2þ supplementation on the rate of S-MSC proliferation in culture. Methods. S-MSCswere serially cultured with or without Ca2þ supplementation. The effect of inhibiting Ca2þ uptake was assessed using Ca2þ

channel blockers. After extended exposure to elevated Ca2þ concentrations, S-MSCs were characterized by evaluatingsurface marker profiles, performing reverse transcriptase quantitative polymerase chain reaction and carrying out tri-lineagedifferentiation assays. Results. Elevated Ca2þ concentrations resulted in enhanced S-MSC proliferation. Peak growthoccurred at 5.0 mmol/L Ca2þ, with an average fold increase of 4.52 � 0.65 per passage over 8 passages compared with2.03 � 0.46 in un-supplemented medium. Proliferation was inhibited by Ca2þ channel blockers. Ca2þ-supplemented cellsshowed enhanced capacity toward osteogenesis (17.82 � 4.21 mg Ca2þ deposited/sample vs. 12.70 � 2.11 mg Ca2þ

deposited/sample) and adipogenesis (0.47 � 0.04 mg oil red O/sample vs. 0.352 � 0.005 mg oil red O/sample) and retainedtheir capacity to undergo chondrogenesis (1.37 � 0.07 mg glycosaminoglycan/pellet vs. 1.33 � 0.17 mg glycosaminoglycan/pellet). S-MSCs cultured in elevated Ca2þ 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 Ca2þ has not beenreported previously. These findings indicate that S-MSC population expansion rates may be up-regulated by Ca2þ

supplementation without compromising defining cell characteristics. This study exemplifies the need to consider mediumcomposition 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 aredefined in part by their ability to adhere to a plasticsurface and replicate while maintaining the potentialto differentiate along osteogenic, adipogenic andchondrogenic lineages (1). Cell populations exhibitingthese traits have been isolated from several species andexpanded in vitro for extended periods via traditionalserial sub-culturing techniques (2e7). Because oftheir multi-lineage capacity, these cells have attractedgreat research interest in the areas of tissue engi-neering and cell therapy. MSCs are most commonlyisolated from bone marrow, but cells exhibiting MSCcharacteristics can be found in many other tissues,including adipose (8), skin (9), umbilical cord blood

Correspondence: Arindom Sen, PhD, PEng, Schulich School of Engineering, UnT2N 1N4. E-mail: asen@ucalgary.ca

(Received 23 May 2012; accepted 18 January 2013)

ISSN 1465-3249 Copyright � 2013, International Society for Cellular Therapy. Phttp://dx.doi.org/10.1016/j.jcyt.2013.01.011

(10) and synovium (4). Synovium-derived mesen-chymal stromal cells (S-MSCs) have been reported toexhibit comparable growth characteristics to bonemarrow-derived MSCs but may have a greaterpropensity to differentiate toward a chondrogenicphenotype (6,11). In addition, it has been reportedthat these cells could be induced to form three-dimensional constructs, which were able to repairdamaged cartilage when transplanted into a chondraldefect site in pigs (12e14). The new cartilage exhibi-ted mechanical and viscoelastic properties similar tonative articular cartilage. Based on these traits as wellas their relative ease of isolation during standardarthroscopic surgery, S-MSCs represent an attractivecell type for use in cell-based therapies for bone

iversity of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada

ublished by Elsevier Inc. All rights reserved.

806 H. Dry et al.

and joint disorders. However, because of the limitedamount of synovial tissue that can be procuredsurgically, and the relative scarcity of stem cellswithin adult tissues, S-MSCs can be isolated inonly very limited quantities, hindering the develop-ment of new therapies. In vitro culture methodshave to be employed to generate large numbersof S-MSCs without compromising their definingcharacteristics.

MSCs are typically cultured in static culturevessels (e.g., tissue culture flasks) using basal media,such as Dulbecco’s modified Eagle’s medium(DMEM), supplemented with 10% fetal bovineserum (FBS). Under these conditions, doublingtimes have been reported to range from 1e4 days(7,15e17). After several passages in culture, the cellshave been shown to retain tri-lineage differentiationcapacity. As reported in the literature, currentprotocols used to expand S-MSCs are based on bonemarrow-derived MSC expansion methods. However,the culture protocols presently being used may notbe optimized for S-MSCs; this includes the standardculture medium (10% FBS DMEM) that is used bymany research groups for the culture of differentmammalian cell types, including adult mesenchymalstem and progenitor cells.

Standard culture medium typically has a calcium(Ca2þ) concentration of approximately 1.8e2.2mmol/L, depending on the FBS lot used for supple-mentation. The Ca2þ concentration in synovial fluidhas been reported to be �4.0 mmol/L (18). Duringbone remodeling, which involves bone precursor cellsand can occur in joints, the Ca2þ concentration hasbeen reported to be 40 mmol/L at bone resorptionsites (19). Ca2þ also plays an important role in carti-lage, electrostatically binding to proteoglycans andfacilitating the maintenance of tissue structure andfunction (20). Cells in vivo near a joint may beaccustomed to a higher level of Ca2þ than is presentin standard culture medium. In co-culture, synovialcells and chondrocytes were shown to communicatevia Ca2þ signaling (21). Osteoclast-precursor cellsresponded to higher levels of Ca2þ by differentiatingto osteoclasts and rapidly proliferating (22). In addi-tion, elevated levels of basic calcium phosphate crys-tals in synovial fluid have been reported to accompanyincreased synovial cell proliferation (23). S-MSCs,which are naturally present in joints, may be respon-sive to extracellular Ca2þ levels greater than levelstypically found in 10% FBS DMEM.

In addition to affecting cells present in joints,Ca2þ has been shown to affect the proliferation ofmany other mammalian cell types. Elevated Ca2þ

levels resulted in enhanced proliferation of dentalstem cells (24), osteoblasts and osteoblast progenitorcells (19,25,26), human lens epithelial cells (27),

Chinese hamster lung fibroblasts (28), mousemesangial cells (29) and human foreskin fibroblasts(30). Although the mechanisms governing Ca2þ-related pathways that affect proliferation are not fullyunderstood, normal mammalian cell division doesappear to be affected by intracellular and extracel-lular Ca2þ levels (30e32). Ca2þ release from internalstores, mediated by inositol triphosphate, was foundto mark the start of the G1 phase of the cell cycle(27). In addition, a spike in intracellular calciumconcentration ([Ca2þ]i) has been observed duringnuclear breakdown at the G1/S and metaphase/anaphase transitions (17,33). Extracellular Ca2þ mayalso play a role in regulating [Ca2þ]i and cell cycleevents and proliferation. Martinez and Santibañez(34) showed inhibited growth of two mammalian celllines using Ca2þ channel blockers, Ca2þ-specificchelators and Ca2þ-depleted medium, suggestingthat extracellular Ca2þ concentration can affectproliferation. Other researchers have reported similarfindings using Ca2þ channel blockers, showingdiminished proliferation for human lens epithelialcells (27) and human MSCs (17,35). Dental stemcells implanted using a biomaterial scaffold consist-ing of calcium hydroxide (Ca(OH)2) resulted inimproved migration, proliferation and differentiationof the stem cells compared with a scaffold withoutCa(OH)2 (24).

To our knowledge, no studies to date have re-ported the effect of Ca2þ on S-MSCs in culture.Because S-MSCs may be exposed to higher Ca2þ

concentrations in vivo and considering that tissueengineering applications such as bone tissue engi-neering often use MSCs combined with biomaterialscaffolds that contain Ca2þ, we hypothesized thatelevated Ca2þ concentrations in the culture mediummay have an impact on the in vitro behavior ofS-MSCs. We report here that supplementing theculture medium with Ca2þ significantly increased therate of cell proliferation and that this effect could bemaintained during extended serial sub-culture with-out compromising the cells’ capacity to differentiatetoward osteogenic, adipogenic and chondrogenicphenotypes when induced using conventional dif-ferentiation protocols.

Methods

Cell isolation and culture

The current study was carried out with cells fromthree porcine donors. Pigs are an accepted largeanimal model that have been used to evaluate potentialnew therapies for bone, cartilage, diabetes, athero-sclerosis and cardiovascular disorders (13,36,37).Published studies indicate that porcine MSCs have

Calcium effects on synovium-derived mesenchymal stromal cells 807

characteristics similar to human MSCs, suggestingthat information gained using porcine cells may bedirectly applicable to human cells (2,13,38).Moreover,porcineMSCs can be used to carry out autologous andallogeneic studies in pigs, thereby contributing to thedevelopment of therapies for bone and joint disordersfor humans. For these reasons, porcine S-MSCs werechosen for the current study.

Synovial tissue was surgically procured from theknee joints of 4-month-old juvenile female Yorkshirepigs as described previously (13) using protocolsapproved by the Animal Care Committee at theUniversity of Calgary. Briefly, tissue from eachdonor was rinsed with 1� phosphate-buffered saline(PBS), minced using a scalpel in sterile 100-cm2

Petri dishes and digested with 6 mL of 0.1% colla-genase IV (Sigma-Aldrich, St Louis, MO, USA) ina 15-mL conical tube for 1 h at 37�C. DMEM(6 mL) was added to the conical tube to dilute thecollagenase and limit further digestion, and the celland tissue suspension was centrifuged (600g for5 min). The cell/tissue sample was washed twicewith DMEM. After the second wash, the cells wereisolated from the tissue debris by filtration, and thecells were centrifuged (300g for 5 min) and washedtwice with PBS. Finally, the cells were re-suspendedin 10% FBS DMEM and inoculated into two25-cm2 Nunc tissue culture flasks (T-25 flasks;Sigma Aldrich, St. Louis, MO, USA) each con-taining 5 mL of culture medium. A completemedium change was performed after 24 h to removenon-adherent cells, and the remaining cells werecultured until almost fully confluent. The cells wereharvested using 0.05% trypsin-EDTA (Cat. No.25300-120; Invitrogen, Carlsbad, CA, USA) for4 min (2 mL per T-25 flask) after two washes in 1�PBS. After blocking trypsin activity with an equalvolume of culture medium (10% FBS DMEM), thecells were collected by centrifugation (600g for5 min) and counted using trypan blue exclusion.Fresh cultures were inoculated at 5000 cells/cm2 inT-25 Nunc tissue culture flasks containing 10% FBSDMEM. The cells were maintained in a humidifiedincubator at 37�C with 5% carbon dioxide (CO2)and were sub-cultured approximately every 5 daysthereafter. The FBS used in this study was fromInvitrogen (Cat. No. 12483-020, Lot No. 439145;Grand Island, NY, USA), and the DMEM was fromMediaTech Inc (Cat. No. 90-113-PB, Lot No.90113011; Manassas, VA, USA). All stock solutionsand media used in this study were made using cellculture grade water and sterilized through a 0.2-mmfilter. For volumes >50 mL, bottle-top filters wereused (Cat. No. 290-4520; Nalgene, Sigma-Aldrich).Volumes <50 mL were filtered using syringe-topfilters (Cat. No. 83.1826.001; VWR, Radnor, PA,

USA), with an appropriate-sized syringe (30 mL or10 mL, Cat No. 309650 or 309604; BD, FranklinLakes, NJ, USA).

Ca2þ supplementation

A calcium chloride (CaCl2) stock solution wasprepared by adding 0.5 g calcium chloride dihydrate(147 g/mol; Cat. No. C3881; Sigma-Aldrich) to10 mL of water to a final concentration of 50 g/Lor 0.34 mol/L. 10% FBS DMEM (which normallyhas a Ca2þ concentration of 1.9 mmol/L), was sup-plemented with the CaCl2 stock solution to finalconcentrations of 3.4 mmol/L, 5.0 mmol/L or6.9 mmol/L.

Ca2þ channel blockers: Verapamil and Nifedipine

The Ca2þ channel-blocking drugs Verapamil (Cat.No. V4629; Sigma-Aldrich) and Nifedipine (Cat.No. N7634; Sigma-Aldrich) were used to test theeffect of blocking Ca2þ channels on the growth ofS-MSCs. Both drugs were prepared according to themanufacturer’s specifications to stock concentra-tions of 12.5 mmol/L. Verapamil (molecular weight491.1 g/mol) stock solution was prepared by adding0.0085 g to 1.38 mL double-distilled water. Nifedi-pine (molecular weight 346.3 g/mol) stock solutionwas prepared by adding 0.0065 g to 1.5 mL ethanol.The final concentration of the drugs in the culturemedium was 25 mmol/L. For Nifedipine, the finalconcentration of ethanol in the culture medium was0.2%; a control case with 0.2% ethanol in culturemedium was included in this particular experiment.These drugs are both light-sensitive, and the stocksolutions were prepared on the day of the experimentand stored in a foil-covered container that restrictedlight exposure before use.

Surface marker analysis: flow cytometry

The presence (or absence) of surface markers wasdetermined using a FACSCalibur flow cytometer(BD Biosciences, San Jose, CA, USA) with the dataacquisition and analysis software CellQuest (BDBiosciences). The surface marker panel was assessedusing monoclonal antibodies against CD14 (Cat.No. MCA1568GA; AbD Serotec, Raleigh, NC,USA), CD29 (Cat. No. 552369; BD BioSciences),CD45 (Cat. No. ab23918; ABcam, Cambridge, MA,USA), CD90 (Cat. No. 555593; BD BioSciences)and CD105 (Cat. No. ab53318; ABcam). A fluo-rescein isothiocyanate-conjugated secondary anti-body was used (Cat No. ab6785; ABcam), and theFACSCaliber was calibrated using CaliBRITE beads(Cat. No. 340486; BD Biosciences).

808 H. Dry et al.

Differentiation

Differentiation toward osteogenic, adipogenic andchondrogenic lineages was evaluated using alizarinred (osteogenic), oil red O (adipogenic) and alcianblue (chondrogenic). Cells were cultured formultiple passages in either 10% FBS DMEM(control) or 10% FBS DMEM with an elevated Ca2þ

level of 5.0 mmol/L. The cells were removedfrom these conditions and subjected to traditionalosteogenic, adipogenic or chondrogenic inductionconditions (6,11e13,39) to determine if prior expo-sure to elevated Ca2þ levels negatively affected theirability to subsequently differentiate down thesethree specialized cell lineages. Elevated Ca2þ levelswere not used during the differentiation processesthemselves.

Osteogenic differentiation was induced in six-wellplates. Each well was inoculated with 3.0 � 104 cells(3.1 � 103 cells/cm2) in 2.5 mL 10% FBS DMEM.Cells were allowed to attach to the tissue culturesurface for 24 h at 37�C with 5% CO2. The culturemedium was replaced with osteogenic inductionmedium, and samples were cultured at 37�C with5% CO2 for approximately 35 days. Osteogenicinduction medium consisted of 10% FBS DMEMsupplemented with 10 mmol/L b-glycerophosphate(Cat. No. 35675; Calbiochem-EMD Millipore,Darmstadt, Germany), 0.1 mmol/L dexamethasone(Cat. No. D2915; Sigma-Aldrich) and 0.05 mmol/Lascorbate-2-phosphate (Cat. No. A4544; Sigma-Aldrich). Control samples were cultured in 10%FBS DMEM for the duration of the differentiationperiod and were not exposed to osteogenic inductionmedium. A complete medium change was performedtwice weekly. Ca2þ deposition was detected usingalizarin red (Cat. No. A5533; Sigma-Aldrich). Inaddition, an Arsenazo III CalciumAssay Kit (Cat. No.140-20; Diagnostic Chemicals Limited, Charlotte-town, PE, Canada) was used to quantify Ca2þ depo-sition. Samples were washed with 2 mL PBS, lysedwith 1.5 mL 10% formic acid (Cat No. 251364, LotNo. 07012EE; Sigma-Aldrich) for 15min and scrapedusing a cell scraper. The lysed cells and supernatantwere collected and stored in 1.5 mL microcentrifugetubes at�80�C until analysis could be performed. Foranalysis, 10 mL of sample and 200 mL of the ArsenazoIII solution were added per well of a 96-well plate. Thewell plate was incubated at 37�C for 30 min andanalyzed using a plate reader at 650 nm.

Adipogenesis was induced in six-well plates. Eachwell was inoculated with 2.0 � 105 cells (2.1 � 104

cells/cm2) in 2.5 mL 10% FBS DMEM. The cellswere allowed to reach confluence (3e5 days), at whichtime the medium was replaced with adipogenicinduction medium and cultured for an additional

28 days at 37�C with 5% CO2. Adipogenic inductionmedium consisted of 10% FBS DMEM supple-mented with 0.5 mmol/L isobutylmethylxanthine(Cat. No. I5879; Sigma-Aldrich), 0.5 mmol/L dexa-methasone (Cat. No. D2915; Sigma-Aldrich) and0.1 mmol/L indomethacin (Cat. No. I7373; Sigma-Aldrich). Control samples were cultured in 10%FBS DMEM for the duration of the differentiationperiod. A complete medium change was performedtwice weekly. Intracellular lipid production wasdetected using oil red O (Cat. No. O0625; Sigma-Aldrich) in isopropanol. Intracellular oil wasquantified by eluting the oil red O taken up by theintracellular lipids and measuring the absorbance ofthe eluted sample using a spectrophotometer at510 nm.

Chondrogenesis was induced using a standardpellet culture system, with each pellet initially con-sisting of 2.5 � 105 viable cells. The differentiationwas carried out using a chondrogenic differentiationkit (Cat. No. PT-3003; Lonza, Basel, Switzerland)and transforming growth factor (TGF)-b3 (Cat. No.PT-4124; Lonza). After harvesting cells from statictissue culture flasks, the desired number of cells wastransferred into 15-mL conical tubes. The cells werecentrifuged for 5 min at 300g, and each cell pelletwas re-suspended in incomplete chondrogenicinduction medium (iCIM; induction case) or 10%FBS DMEM (control case), to a final concentrationof 7.5 � 105 cells/mL. The samples were centrifugeda second time and re-suspended in complete chon-drogenic induction medium (cCIM; induction case)or 10% FBS DMEM (control case) to a concentra-tion of 5.0 � 105 cells/mL. A volume of 0.5 mL of thecell suspension (2.5 � 105 cells) was aliquoted intonew 15-mL conical tubes and centrifuged once more(300g for 5 min) to form the pellets for differentia-tion. Pellets were cultured in cCIM or controlmedium (10% FBS DMEM) for 35 days at 37�Cwith 5% CO2. A complete medium change wasperformed twice weekly, at which time each tubewas gently flicked to ensure that the pellet wasfree-floating. cCIM consisted of chondrogenic dif-ferentiation basal medium supplemented withL-glutamine, gentamicin sulfate/amphotericin B,dexamethasone, ascorbate, proline, sodium pyru-vate, insulin-transferrin-selenium þ supplement andTGF-b3. iCIM did not contain TGF-b3. At the endof the differentiation period, each pellet was fixed in10% neutral buffered formalin, embedded in paraffinand sectioned (3 mm thick) on a Leica 2125 RotaryMicrotome (Leica Biosystems, Concord, Ontario,Canada). The sections were mounted on StarfrostAdhesive Plus slides (Mercedes Medical, Sarasota,FL, USA) and stained with alcian blue (Cat. No.

Calcium effects on synovium-derived mesenchymal stromal cells 809

A3157, Lot No. 116K1055; Sigma-Aldrich) at a pHof 1.0 to detect glycosaminoglycans (GAGs).

The extent of chondrogenesis was quantified usingmethods previously described (13). Briefly, pelletswere digested in 50 mL of papain solution for 4 h at65�C. The papain solution was prepared by adding12.5 mg of papain (Cat. No. P4762; Sigma-Aldrich)and 16.32 mg of N-acetyl-L-cysteine (Cat. No.A9165; Sigma-Aldrich) to 50 mL of 50 mmol/Lphosphate buffer. Chondroitin sulfate (Cat. No.C9819; Sigma-Aldrich) in 50 mmol/L phosphatebuffer was used to make up the standard solutions.GAGs were detected using the 1,9-dimethyl-methy-lene blue (DMB) binding assay. The DMB solutionwas made by adding 8mg of DMB (Cat. No. 341088;Sigma-Aldrich) to 2.5 mL of ethanol and mixingthis with 500 mL double-distilled water containing1.0 g sodium formate and 1.0 mL formic acid. Foranalysis, 10 mL of sample and 200 mL of the DMBsolution were added per well of a 96-well plate. Thewell plate was incubated at 37�C for 30 min andanalyzed using a plate reader at 510 nm.

Reverse transcriptase quantitative polymerasechain reaction

Total RNA was isolated from S-MSCs after beingsub-cultured for five passages in either controlmedium or Ca2þ-supplemented culture medium.The cells were lysed in TRIzol reagent (InvitrogenLife Technologies Inc, Grand Island, NY, USA)using the trypsin method described by Reno andcolleagues (40). Quantification of messenger RNA(mRNA) levels for 18S, SOX-9, TGF-b1, collagen I,peroxisome proliferator activated receptor gamma(PPAR-g), a2P, osteocalcin, osterix and cbfa-1 wasperformed using real-time primer sets validated foruse with porcine molecules and polymerase chainreaction (PCR) with an iCycler (Bio-Rad, Hercules,CA, USA). Levels of gene expression for moleculesof interest were normalized to levels for 18S(housekeeping gene).

Statistics

Data were statistically analyzed using one-way anal-ysis of variance and the Tukey-Kramer method. AP value of 5% was considered significant (P < 0.05).

Results

Supplementation of 10% FBS DMEM with Ca2þ

enhances S-MSC proliferation

The effect of elevated Ca2þ levels on the prolifera-tion of S-MSCs was evaluated by serial sub-culture

in 10% FBS DMEM supplemented with Ca2þ.S-MSCs were inoculated at a concentration of 5000cells/cm2 into un-supplemented 10% FBS DMEM(control medium; 1.9 mmol/L Ca2þ) or 10% FBSDMEM supplemented with CaCl2 to a final Ca2þ

concentration of 3.4 mmol/L, 5.0 mmol/L or6.9 mmol/L. The osmolality of all conditions wasmeasured and found to be <330 mOsm/kg.Previous studies in our laboratory found thatmedium osmolalities above this value may havea negative impact on cell growth kinetics (unpub-lished data). As shown in Figure 1, enhanced cellgrowth was observed for all Ca2þ-supplementedconditions compared with the control cultures. Aconcentration of 5.0 mmol/L Ca2þ resulted in thegreatest increase in growth after 5 days comparedwith lower and higher Ca2þ concentrations. Inaddition, the cells appeared smaller in size andnarrower in morphology when cultured in Ca2þ-supplemented conditions compared with the controlmedium. These results suggested that S-MSC ex-pansion could be improved by increasing the Ca2þ

concentration in the medium but that an upper limitexists beyond which additional Ca2þ supplementa-tion no longer promotes proliferation. To study theeffect of Ca2þ further, subsequent experiments werecarried out in 10% FBS DMEM supplemented to5.0 mmol/L Ca2þ.

Ca2þ channel blockers reverse enhanced proliferationobserved under elevated Ca2þ conditions

To determine if the elevated extracellular Ca2þ

concentration was responsible for the observedincrease in proliferation, the Ca2þ channel-blockingdrugs Verapamil and Nifedipine were added to cellscultured in both 10% FBS DMEM and Ca2þ-supplemented 10% FBS DMEM (final concentra-tion of 5.0 mmol/L Ca2þ). These drugs are known toblock voltage-dependent Ca2þ channels, restrictingthe influx of Ca2þ from the culture medium. Bothdrugs have been reported to inhibit cell growth ina dose-dependent manner (34). In the current study,these drugs were used at a concentration of 25 mmol/L, which has been reported to significantly inhibit theproliferation of other mammalian cell types (34). Thecontrol case for each condition was not exposed toeither drug. Because Nifedipine was prepared inethanol (final concentration of 0.2%), an additionalcontrol case with 0.2% ethanol in 10% FBS DMEMwas included. As shown in Figure 2, the additionof the Ca2þ channel-blocking drugs resulted in a30e40% inhibition of cell growth compared with therespective control cases for both conditions. It isevident that ethanol (0.2%) alone did not have aneffect on cell growth, indicating that any diminished

Figure 1. Effect of Ca2þ concentration on the fold increase of S-MSCs. (A) S-MSCs were cultured in 10% FBS DMEM (control; 1.9 mmol/L Ca2þ) or 10% FBS DMEM supplemented with CaCl2 to a final Ca2þ concentration of 3.4 mmol/L, 5.0 mmol/L or 6.9 mmol/L. Thecultures were maintained in a humidified 37�C incubator that contained 5% CO2. Inoculation density was 5000 cells/cm2. Data werecollected in duplicate. Error bars represent the range of data values collected for each data point. *P< 0.05, #P< 0.01 compared with controlcondition or with other indicated test conditions. (B) Photomicrographs show the progression of cell expansion over the course of 5 days inun-supplemented 10% FBS DMEM (1.9 mmol/L Ca2þ) or supplemented 10% FBS DMEM (5.0 mmol/L Ca2þ). (Scale bars ¼ 200 mm.)

810 H. Dry et al.

growth was a result of the Ca2þ channel-blockingdrugs. Because Verapamil and Nifedipine specificallyblock the uptake of extracellular Ca2þ, these resultssuggested that the enhanced proliferation observedin the Ca2þ-supplemented medium (as shown inFigure 1) was likely related to the additional Ca2þ inthe culture medium.

Elevated Ca2þ levels are able to support enhancedlong-term proliferation

The effect of elevated Ca2þ concentration on cellgrowth was monitored over eight serial passages.S-MSCs from the same stock were inoculated intoeither 10% FBS DMEM (1.9 mmol/L Ca2þ; controlmedium) or 10% FBS DMEM supplemented with

Ca2þ (final Ca2þ concentration of 5.0 mmol/L). Asshown in Figure 3, the elevated Ca2þ concentrationconsistently sustained enhanced growth of S-MSCsover multiple passages compared with the un-supplemented medium. Over the course of eightserial sub-cultures, the average cell fold expansion perpassage in Ca2þ-supplemented medium was 4.52 �0.65 (mean � standard deviation) compared witha 2.03 � 0.46 fold increase in un-supplementedmedium. The cumulative fold increase over the entire8 passages was approximately 1.6 � 105 in theCa2þ-supplemented medium but only 650 in thecontrol medium. To determine if the effect of Ca2þ

was transient or permanent, a third condition wasstudied. In this condition, cells that had been grownin Ca2þ-supplemented medium for 3 passages were

Figure 2. Effect of adding Ca2þ channel blockers Verapamil andNifedipine on the cell fold increase of S-MSCs. S-MSCs werecultured in 10% FBS DMEM or 10% FBS DMEM supplementedwith Ca2þ to a final concentration of 5.0 mmol/L. The effect ofadding 25 mmol/L Verapamil or Nifedipine was evaluated in bothconditions. The control case was not exposed to either drug.Because Nifedipine was prepared in 0.2% ethanol (EtOH), anadditional control consisting of 10% FBS DMEM supplementedwith 0.2% EtOH was also included. Inoculation density for allcases was 5000 cells/cm2. The cells were cultured in a humidifiedincubator for 5 days at 37�C with 5% CO2. All data were collectedin duplicate. Error bars represent the range of data values collectedfor each data point. *P < 0.05 compared with control case.

Calcium effects on synovium-derived mesenchymal stromal cells 811

cultured in un-supplemented medium for an addi-tional 5 passages. This experimental condition wastermed “post-Ca2þ.” In the first passage post-Ca2þ,cell growth was comparable to that of cells in

Figure 3. Effect of elevated Ca2þ (5 mmol/L) on the long-termgrowth of S-MSCs. S-MSCswere serially sub-cultured for 8 passagesin 10% FBS DMEM (1.9 mmol/L Ca2þ) or Ca2þ-supplemented10%FBSDMEM (5.0 mmol/L Ca2þ). “Post-Ca2þ” is the conditionin which cells expanded for 3 passages in Ca2þ-supplementedmedium were subsequently cultured in un-supplemented mediumfor 5 additional passages. Inoculation density for all cases was 5000cells/cm2. The cells were cultured in a humidified incubator for5 days per passage at 37�C with 5% CO2. All data were collected induplicate. Error bars represent the range of data collected. *P< 0.05compared with control condition at each passage.

Ca2þ-supplemented medium. Subsequently, how-ever, proliferation in the post-Ca2þ condition exhi-bited growth characteristics similar to cells in controlmedium. The average cell fold expansion per passagefor post-Ca2þ cells was 2.15 � 0.39 over the final 4passages of the study (i.e., passage numbers 5e8 inFigure 3), which is comparable to the average foldexpansion measured in the control condition. Over-all, these results suggest that the enhanced growth ofS-MSCs was dependent on the presence of elevatedextracellular Ca2þ levels and that the proliferationbehavior of S-MSCs exposed to elevated Ca2þ

concentrations was not altered permanently by thatexposure.

Cells generated under elevated Ca2þ levels maintainMSC characteristics

When expanding MSCs in culture, it is important tocharacterize the resulting cell population to ensurethat the defining characteristics of these cells havebeen maintained. MSC populations are defined inpart by their specific surface marker profile and theircapacity to differentiate along osteogenic, adipogenicand chondrogenic lineages.

The surface marker profile of the S-MSCs wasanalyzed after multiple passages in 10% FBSDMEM (control medium) or Ca2þ-supplementedmedium. In contrast to human MSCs, there is nostandard panel of surface markers for porcine MSCs.Based on the scarce literature for porcine cells(10,41,42) and cross-referencing with the acceptedstandard human MSC surface markers (1), thefollowing panel was used in this study: CD14, CD29,CD45, CD90 and CD105. As shown in Figure 4, itwas found that the surface marker profile of porcineS-MSCs was generally not affected by the levelof Ca2þ in the culture medium. The cell popula-tions were strongly positive (>95% expression) forCD29 and CD90 and were strongly negative (<5%expression) for CD14 and CD45. The expressionof CD105 was negative for S-MSCs in controlmedium but appeared to increase slightly in Ca2þ-supplemented cultures.

Multipotentiality was assessed by tri-lineagedifferentiation toward osteogenic, adipogenic andchondrogenic fates. Ca2þ levels were not elevated(i.e., there was no Ca2þ supplementation) during anydifferentiation process. Rather, standard differentia-tion protocols were employed to evaluate if cell pop-ulations expanded under elevated Ca2þ levels (i.e.,cells cultured in Ca2þ-supplemented medium beforedifferentiation) could still exhibit multipotentiality. Itwas determined that S-MSCs exposed to elevatedCa2þ levels before differentiation maintained theirability to differentiate along all three lineages.

Figure 4. Surface marker analysis of porcine S-MSCs using flow cytometry. S-MSCs were cultured in 10% FBS DMEM containing either1.9 mmol/L Ca2þ (control) or 5.0 mmol/L Ca2þ for multiple passages before this analysis. Cells in the control medium had never beenexposed to elevated Ca2þ. (A) Bar chart shows a synopsis of the percentage of cells in tested populations that were positive for a particularmarker. (B) FACSCalibur histogram plots for surface markers associated with MSCs. Plots show specific antibody staining (solid) versusisotype control staining (line).

812 H. Dry et al.

Osteogenic differentiation was detected by posi-tive alizarin red staining. Cells previously grown incontrol medium and under elevated Ca2þ conditionsboth stained positive for Ca2þ deposition, as shownin Figure 5A and B. Qualitatively, no significant

differences could be visually observed. However,quantitative analysis revealed greater average miner-alization (Ca2þ deposition) for S-MSCs culturedin elevated Ca2þ compared with control medium(Figure 5C); osteogenically-induced S-MSCs from

Figure 5. Osteogenic differentiation of S-MSCs. S-MSCs were exposed to osteogenic induction medium for 35 days, at 37�C with 5% CO2,following 5 passages in either 10% FBS DMEM or Ca2þ-supplemented medium. Ca2þ deposition is an indication of osteogenesis and wasdetected using alizarin red. (A) Osteogenic induction of S-MSCs cultured beforehand in un-supplemented 10% FBS DMEM (control).(B) Osteogenic induction of S-MSCs cultured in Ca2þ-supplemented medium for 5 passages before differentiation. (C) Extent of osteogenicdifferentiation was quantified using an Arsenazo III assay for Ca2þ mineralization. Three samples were measured per condition. Error barsrepresent the standard deviation. *P < 0.05. (A and B, Magnification �5, scale bar ¼ 200 mm.)

Calcium effects on synovium-derived mesenchymal stromal cells 813

Ca2þ-supplemented medium had 17.82 � 4.21 mgCa2þ deposition per well compared with 12.70 �2.11 mg Ca2þ deposition per well for S-MSCsgenerated in the 10% FBS DMEM control.

Adipogenic differentiation of cells cultured inCa2þ-supplemented or control conditions bothresulted in the generation of intracellular oil dropletsthat stained positive for oil redO (Figure 6).However,a greater number of cells containing oil droplets couldbe seen in samples cultured under elevated Ca2þ

conditions prior to differentiation. The quantitativeanalysis confirmed that the adipogenic potential ofS-MSCs was enhanced when the cells were expandedin additional Ca2þ. For adipogenically-inducedS-MSCs expanded in Ca2þ-supplemented medium,0.47 � 0.04 mg oil red O were absorbed per wellcompared with 0.352 � 0.005 mg oil red O absorbedper well for cells from control cultures (P < 0.05).

Chondrogenic differentiation was also inducedfor both Ca2þ-supplemented cells and control cells,with both conditions staining positive for alcian blue(Figure 7). No significant differences were observedbetween pellets from the different culture conditions.Chondrogenically-induced S-MSC pellets using cellsexpanded in Ca2þ-supplemented medium produced1.37 � 0.07 mg GAGs/pellet compared with 1.33 �

0.17 mg GAGs/pellet for cells from control cultures.These results indicate that the enhanced proliferationcaused by elevated Ca2þ did not compromise thechondrogenic ability of the cells. The chondrogeniccell pellets were also stained with alizarin red todetermine if the exposure to elevated Ca2þ promotedmineralization of the cartilage after differentiation.However, no such staining was observed (data notshown). Overall, S-MSCs retained their tri-lineagedifferentiation potential after serial exposure toelevated Ca2þ levels.

Reverse transcriptase quantitative PCR analysis ofmRNA levels in S-MSCs cultured in the presence orabsence of supplemental Ca2þ

In addition to the above-described characterizationstudies, reverse transcriptase quantitative PCRanalysis was performed to see if expression of specificgenes was altered by culturing the cells in mediumsupplemented with Ca2þ. The genes chosen foranalysis were related to osteogenesis, adipogenesisand chondrogenesis. The cells examined here weresimply sub-cultured in the two different media andwere not intentionally induced to differentiate downany of these lineages. Levels of gene expression were

Figure 6. Adipogenic differentiation of S-MSCs. S-MSCs were exposed to adipogenic induction medium for 28 days, at 37�C with 5%CO2,following 5 passages in either 10% FBS DMEM or Ca2þ-supplemented medium. Intracellular lipid production is an indication of adipo-genesis and was detected using oil red O. (A) Adipogenic induction of S-MSCs cultured beforehand in un-supplemented 10% FBS DMEM(control). (B) Adipogenic induction of S-MSCs cultured in Ca2þ-supplemented medium for 5 passages before differentiation. (C) Extent ofadipogenic differentiation was quantified by measuring oil red O absorbed by the samples, indicating amount of intracellular oil dropletformation. Three samples were measured per condition. Error bars represent the standard deviation. *P < 0.05. (A and B,Magnification �20, scale bars ¼ 50 mm.)

814 H. Dry et al.

normalized to levels for 18S (housekeeping gene),which we determined was not influenced bysupplemental Ca2þ (data not shown). As shown inFigure 8, S-MSCs cultured in elevated Ca2þ

exhibited significantly enhanced mRNA levels forSOX-9 (chondrogenic lineage) and PPAR-g(adipogenic lineage) compared with S-MSCs fromcontrol culture conditions (un-supplemented 10%FBS DMEM). Elevated Ca2þ conditions lead tosignificantly depressed mRNA levels for collagen I(which is difficult to assign to a specific lineage) anda trend toward decreased TGF-b1 expression.mRNA levels for a2P, osteocalcin, CBfa1 and osterixalso were not significantly affected.

Discussion

Effect of Ca2þ on S-MSC growth kinetics

In this study, the in vitro proliferation of S-MSCs wasfound to be enhanced by increasing the concentra-tion of Ca2þ in the culture medium. Other cell typeshave also responded positively to Ca2þ; elevatedCa2þ levels (3e10 mmol/L) have been linked toimproved proliferation of mouse osteoblasts (19) and

Chinese hamster lung fibroblasts (at 1e10 mmol/LCa2þ) (28). More recently, enhanced recruitmentand proliferation of dental stem cells was achievedusing a Ca(OH)2-containing biomaterial scaffold ata concentration of 10 mg/mL (24). However, incontrast to the current study, rabbit bone marrow-derived MSCs were unaffected by Ca2þ concentra-tions >1.8 mmol/L (5), and low Ca2þ concentrationswere reported to maintain proliferation of murinemesencephalic neural precursor cells, with concen-trations >0.6 mmol/L having no further effect (43).Porcine osteoblasts exhibited no change in prolifer-ation at Ca2þ concentrations up to 5 mmol/L in long-term culture but showed inhibited growth at higherconcentrations, as measured by DNA concentration(25). Contrary to the current study, human adiposetissue-derived MSCs exhibited enhanced growthin Ca2þ-reduced (0.09 mmol/L) medium (44).Although it is clear that extracellular Ca2þ concen-tration affects the growth kinetics of many mamma-lian cells, the response to Ca2þ is not uniform andappears to be cell type or cell source dependent.

A Ca2þ concentration of 5.0 mmol/L appearedto have a more significant effect on S-MSC pro-liferation compared with the higher concentration

Figure 7. Chondrogenic differentiation of S-MSCs, using a standard pellet culture method. S-MSCs were exposed to chondrogenicinduction medium for 35 days, at 37�C with 5% CO2, following 5 passages in either 10% FBS DMEM or Ca2þ-supplementedmedium. GAG production is an indication of chondrogenesis and was detected using alcian blue. (A) Chondrogenic induction ofS-MSCs cultured in un-supplemented 10% FBS DMEM (control). (B) Chondrogenic induction of S-MSCs cultured in Ca2þ-supple-mented medium for 5 passages before differentiation. (C) Extent of chondrogenic differentiation was quantified by measuring the GAGcontent per pellet. Three samples were measured per condition. Each tissue section was 3 mm thick. (A and B, Magnification �10, scalebars ¼ 100 mm.)

Calcium effects on synovium-derived mesenchymal stromal cells 815

tested (6.9 mmol/L), indicating that an optimalin vitro concentration may exist. Similarly, mouseosteoblasts exhibited enhanced growth at elevatedCa2þ concentrations, reaching a peak at 5 mmol/L,with no further effect up to 20 mmol/L (19), andporcine osteoblasts exhibited diminished prolifera-tion at concentrations >5 mmol/L (25), which isconsistent with the present findings. For bone-related and joint-related mesenchymal cells such asosteoblasts and S-MSCs, the optimal level of Ca2þ

in the culture medium may be in the range of5 mmol/L. Supplementing the medium with Ca2þ

also had an impact on average cell size; S-MSCs wereobserved to be smaller on average when cultured inCa2þ-supplemented medium. Smaller cell sizes havebeen associated with higher growth rates for bonemarrow-derived MSCs (7,45), and considering thesimilarities between bone marrow-derived MSCs

and synovium-derived MSCs, the same may be truefor the cells used in this study.

Long-term culture in Ca2þ-supplementedmedium showed enhanced growth compared withthe control condition over 8 serial passages. Toassess the impact of transient exposure to elevatedCa2þ, some of the S-MSCs were removed fromCa2þ-supplemented medium after 3 passages andsubsequently cultured in control medium for5 additional passages. During the first passage afterremoval from Ca2þ-supplemented medium, thecells (termed “post-Ca2þ”) exhibited comparableproliferation to the Ca2þ-supplemented condition.Although the cells were no longer exposed to ele-vated extracellular Ca2þ at this time, they may haveretained elevated intracellular concentrations fromthe previous passage. Intracellular Ca2þ has beenshown to play a role in cell cycle activity (32,33), and

Figure 8. Reverse transcriptase quantitative PCR analysis ofmRNA levels in S-MSCs cultured under normal conditions or inthe presence of elevated Ca2þ levels. (A) mRNA levels for Sox9,TBG-b1 and ap2. (B) mRNA levels for cbfa1, osterix and osteo-calcin. Three samples were evaluated per analysis. Error barsrepresent standard deviation. *P ¼ 0.01, zP ¼ 0.026, xP ¼ 0.032.

816 H. Dry et al.

an elevated intracellular concentration may haveresulted in the enhanced growth compared with thecontrol condition. Beyond the first passage post-Ca2þ, S-MSC populations exhibited expansioncomparable to control cells, suggesting that transientexposure to elevated Ca2þ did not have a long-termimpact on cell proliferation and that the observedincrease in proliferation was a result of the elevatedextracellular Ca2þ concentration. It may be possibleto exert some control on the in vitro proliferation rateof S-MSCs by simply manipulating the extracellularCa2þ concentration.

Extracellular Ca2þ concentration affects cell proliferation

Several studies have been published that may help toexplain the mechanisms by which S-MSC prolifera-tion is affected by elevated Ca2þ levels. Oneproposed mechanism is that extracellular Ca2þ mayactivate the calcium-sensing receptor (CaSR). CaSRis a cell surface receptor that is expressed in manytissues and organs, such as the parathyroid gland,intestine, kidney and bone growth plate. It is thoughtto play an important role in Ca2þ homeostasis bydirectly sensing the extracellular Ca2þ concentra-tion and indirectly inducing the release of Ca2þ

from internal stores (28,29,46). More recently, cells

isolated from the human parathyroid gland werecharacterized as being similar to MSCs in termsof morphology, phenotype and tri-lineage differenti-ation potential and were shown to express CaSR(47). The effect of Ca2þ on the proliferation ofmesenchymal-like cell populations, such as theS-MSCs in this study, may be related to the activa-tion of the CaSR.

Another mechanism by which extracellular Ca2þ

may influence intracellular concentrations is viavoltage-gated Ca2þ channels, which allow transportof Ca2þ into the cell in response to membranedepolarization. Verapamil and Nifedipine are Ca2þ

channel-blocking drugs that are known to preventCa2þ influx into the cell by blocking voltage-gatedchannels (34). In the current study, use of these drugsresulted in growth inhibition for both the control andthe Ca2þ-supplemented conditions to nearly the sameextent (approximately 30e40% inhibition). Levels ofproliferation in the presence of elevated Ca2þ togetherwith Ca2þ channel-blocking drugs were still near, orin excess of, that observed in standard medium (10%FBSDMEM)without the drugs (Figure 2). There arelikely multiple mechanisms by which elevated levelsof Ca2þ enhance growth other than via these chan-nels. More recently, ion channels have been identifiedin bone marrow-derivedMSCs frommurine (48) andhuman (17,35) sources. Lepski et al. (35) reportedthat MSCs contain at least a small proportion of Ca2þ

voltage-gated channels, as determined by the inter-ruption of a Ca2þ current into the cell by Nifedipine.Cell cycle Ca2þ oscillation events were significantlyreduced in MSCs exposed to the Ca2þ channel-blocking drug, resulting in diminished proliferation(17). Similarly, growth of normal murine bonemarrow cells and human lens epithelial cells has beenshown to be inhibited in the presence of these drugs(27,34). In the current study, Ca2þ voltage-gatedchannel blocking drugs resulted in inhibition ofS-MSC growth, suggesting that similar to bonemarrow-derived MSCs and other mammalian cells,the influx of Ca2þ into S-MSCs via these channelsmay affect the intracellular Ca2þ concentration,which affects cell proliferation.

Effect of Ca2þ on differentiation ability

In addition to enhanced proliferation, the differen-tiation ability of S-MSCs was studied to assesswhether exposure to elevated Ca2þ levels hadaffected the multipotency of the cells. Differentiationalong osteogenic, adipogenic and chondrogeniclineages was successfully shown for S-MSCscultured in Ca2þ-supplemented medium and in un-supplemented control medium. This finding indi-cates that culturing these cell populations in medium

Calcium effects on synovium-derived mesenchymal stromal cells 817

containing elevated Ca2þ levels did not compromisetheir subsequent differentiation ability. Increases inaverage osteogenic and adipogenic potential wereobserved for cells cultured in elevated Ca2þ com-pared with the un-supplemented control condition,although Ca2þ mineralization was not detected dur-ing any of the chondrogenic differentiation assays.However, the chondrogenic capacity of the cellpopulation generated in elevated Ca2þ conditionswas shown to be similar to cell populations generatedin unsupplemented control medium. Cells generatedin elevated Ca2þ conditions did not express higherlevels of GAGs compared with populations culturedin the control medium. There was an increase inthe CD105þ sub-population under elevated Ca2þ

conditions. It has been reported that CD105-expressing cells are present in articular cartilage (49)and that the CD105þ cell fraction sorted fromsynovial tissue expresses Sox-9 and exhibits thecapacity to undergo chondrogenic differentiation(50). It is possible that increasing the Ca2þ level tothat more typically found in synovial joints selectivelypromoted the expansion of a CD105þ cell sub-pop-ulation in culture.

The above-described functional assessments areinteresting in light of the reverse transcriptasequantitative PCR results, which indicated thatculturing S-MSCs in supplemental Ca2þ led tosignificantly elevated mRNA levels for SOX-9and PPAR-g in the undifferentiated state (Figure 8).The elevations in mRNA levels of a chondrogenic(SOX-9) and adipogenic (PPAR-g) marker did nottranslate to enhanced lineage-specific differentiation.Either a basal threshold level of these molecules is allthat is required for subsequent lineage differentia-tion, or a unique subset of S-MSCs expresses SOX-9and PPAR-g and increased levels do not mean morecells committed to those lineages. Relevant to thispoint are more recent studies in our laboratory aimedat cloning S-MSCs from porcine synovium (data notshown). Those results indicate that some clonalpopulations appear to have a unique chondrogenic-adipogenic phenotype, consistent with the previousspeculation. Similarly, the decreases in collagen ImRNA levels in the presence of supplemental Ca2þ

did not overtly affect differentiation to any of thelineages assessed, including osteogenesis wherecollagen I is likely involved. Another interestingresult was that elevated Ca2þ in the culture mediumdid not lead to up-regulated TGF-b1, Cbfa1,osteocalcin or osterix, which are considered osteo-genic lineage markers. Growth of MSCs on Ca2þ-containing scaffolds has been reported to up-regulatedifferentiation toward bone. In the present study, theosteogenesis achieved by cells that had been culturedin Ca2þ-supplemented medium was not significantly

different from cells grown in control medium afterinducing functional differentiation. The influence ofexogenous Ca2þ on undifferentiated MSCs duringcell expansion did not translate into overt effects onsubsequent differentiation of the cells in suitableinduction media. However, these studies involvedpopulations of cells (i.e., multiple clones), and it wasimpossible to determine whether the changes inmRNA levels were reflective of all cells in the pop-ulation or a unique subset. The use of single MSCclones in future studies could help resolve this issue.

Many tissue engineering applications for boneregeneration use biomaterial scaffolds consisting ofCa2þ that also appear to enhance differentiation.Rabbit MSCs exhibited greater mineralization atelevated Ca2þ concentrations (5), and dental pulpstem cells implanted in a Ca(OH)2 scaffold showedenhanced differentiation and mineralization (24). Ithas also reported that for osteogenically-inducedMSCs, Ca2þ influx into the cells activates the Ca2þ

mediated RK/calmodulin/protein kinase II pathway,resulting in increased mineralization (51). Ca2þ hasalso been shown to induce differentiation of othercell types, including keratinocytes (22) and epithelialcells (52). Further studies are needed to determinethe precise mechanisms governing Ca2þ-stimulateddifferentiation of S-MSCs.

In conclusion, the work presented here suggeststhat when expanding cell populations, such asMSCs, in culture, it is important to pay attention tothe type of medium being used and to make efforts tooptimize the concentration of individual mediumcomponents for the cell type being cultured. Basedon the rationale that in vivo, S-MSCs typically residein an environment with elevated Ca2þ concentra-tions, we have shown here that increasing the extra-cellular Ca2þ concentration from 1.9 mmol/L to5.0 mmol/L in a commonly used culture mediumcan have a significant and positive impact on theproliferation kinetics of S-MSCs. The multi-lineagedifferentiation capacity of the cells was retained, asshown by functional differentiation assays. Giventhat in vitro expansion is necessary to generate largenumbers of stem cells such as S-MSCs, the role ofcommonly used medium components such as Ca2þ

should be investigated further.

Acknowledgments

The authors acknowledge the Natural Sciences andEngineering Research Council of Canada (NSERC),the Canadian Institutes of Health Research (CIHR),Alberta InnovateseTechnology Futures (NewFaculty Grant) and Alberta Innovates Health Solu-tions (Osteoarthritis Interdisciplinary Team Grant)for financial support.

818 H. Dry et al.

Disclosure of interest: The authors have nocommercial, proprietary, or financial interest in theproducts or companies described in this article.

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