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A mesenchymal stromal cell line resistant to paclitaxel that spontaneously differentiates into osteoblast-like cells

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The mesenchymal stromal cell line SR-4987 has been established in our laboratory from the bone marrow of BDF/1 mice. Recent information on mesenchymal stem cells biology and the need to deal with well-characterized cell lines suggest to critically
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  A mesenchymal stromal cell line resistant to paclitaxelthat spontaneously differentiates into osteoblast-like cells Augusto Pessina  &  Francesca Sisto  &  Valentina Coccè  &  Loredana Cavicchini  & Emilio Ciusani  &  Laura Gribaldo  &  Arianna Bonomi Received: 14 August 2010 /Accepted: 9 December 2010 /Published online: 29 December 2010 # Springer Science+Business Media B.V. 2010 Abstract  The mesenchymal stromal cell line SR-4987 has been established in our laboratory from the bone marrow of BDF/1 mice. Recent information onmesenchymal stem cells biology and the need to dealwith well-characterized cell lines suggest to criticallyconsider the existent data on this cell line by updatingthem with new investigations on growth parameters,in vitro plasticity, and drug sensitivity to anti-cancer,anti-inflammatory, and a histone deacetylase inhibitor.SR-4987 cells show a population doubling time of 24.5±5.4 h, a plating efficiency of 2.87±1.19%, andunder stimulation maintain only in part their multi- potency by differentiating towards chondro-osteogeniclineages but not into adipogenic. Surprisingly, thesemesenchymal stromal cells differentiate spontaneouslyinto osteoblast-like cells and this is significantlystimulated by valproic acid. SR-4987 cells show adramatic resistance to paclitaxel (PTX) with aresistance index of 39.6 times (evaluated versusMOLT-4 leukemia) and of 68.2 (versus HT-29colorectal carcinoma). SR-4987 resistance is reversed by verapamil and correlates with high expression of P-glycoprotein that is down-modulated by PTX. Takentogether, our results indicated that SR-4987 line is avery interesting cell model useful to investigate bothdrug sensitivity resistance and physiopathologicalaspects related to mesenchymal cell function. Keywords  Drugsensitivity.Mesenchymalstemcells.Osteogenesis.Paclitaxel resistance.SR-4987 Introduction The presence of fibroblast-like cells, termed colony-forming unit   —  fibroblast (CFU-F) in the bone mar-row, described since many years by Friedenstein et al.(1966), appeared as cells capable of limited self-renewing but able to differentiate into variousconnective tissue lineages. When these cells have been better studied and characterized, they resultedconstituted by a homogeneous cell population capableof extensive self-renewal and with a wide range of differentiation potential (e.g., bone, cartilage, tendon,adipocytes, muscle, hepatocytes) if stimulated withappropriate factors and were named mesenchymalstromal cells (MSCs) (Haynesworth et al. 1992). Therecent progress in using MSCs in regenerative Cell Biol Toxicol (2011) 27:169  –  180DOI 10.1007/s10565-010-9179-xA. Pessina ( * ) :  F. Sisto : V. Coccè :  L. Cavicchini : A. BonomiDepartment of Public Health  –  Microbiology  –  Virology,University of Milan,Via Pascal 36,20133 Milan, Italye-mail: augusto.pessina@unimi.it E. CiusaniFondazione IRCCS Istituto Neurologico Besta,Milan, ItalyL. GribaldoIHCP, Joint Research Centre,Ispra, VA, Italy  medicine confirms the high plasticity degree of thesecells, arising also questions on their biological activitythat need to be more investigated also by using invitro experimental models. A murine MSC line (SR-4987) originated and established in our laboratoryfrom adherent cells of a long-term bone marrowculture (Pessina et al. 1992) has a fibroblast like-morphology, is very susceptible to the clonogeniceffect of FGFs, and, very singularly, together withtypical characters of stromal cells (as the productionof M-CSF), expresses also B cell markers. Moreover,it is tumorigenic and able to produce sarcomas insyngeneic mice (Pessina et al. 1997). This line has been used in porous carriers to construct a three-dimensional hematopoietic culture system (Takagiet al. 1999; Takagi 2005) and for gene transfection studies (Opavsky et al. 2001).The recent knowledge acquired on the mesenchy-mal stem cell biology, together with the need to dealwith well-characterized cell lines, suggested our laboratory to critically consider all the existent dataon SR-4987 and update them by new investigationsconcerning growth parameters as plating efficiency(PE) and population doubling time (PDT), its in vitro plasticity in comparison to other mesenchymal stemcells, and its drug sensitivity in comparison to tumor cell lines. As SR-4987 cells srcinated from mesen-chymal stem cells, we studied if the main differenti-ation multipotency described for MSCs (Dennis et al.1999) was maintained and found that under appropri-ate stimulation this mesenchymal cell line maintainsonly in part its multipotency (being able to differen-tiate toward chondro-osteogenic lineages but not intoadipogenic). A very interesting characteristic of SR-4987 cells is their ability to differentiate spontane-ously into osteoblast-like cells, and their resistance to paclitaxel (PTX) correlated to high P-glycoprotein(P-gp) expression. Materials and methods DrugsAspirin (ASA) was purchased from Cayman (Tallin,Estonia); indomethacin (IMC), 5-fluorouracil (5-FU),and doxorubicin hydrochloride (DOX) from EnzoLife Sciences (Lausen, Switzerland); valproic acid(VPA) and camptothecin (CPT) from Sigma-Aldrich(St Louis, MO, USA); PTX from Serva (Mannheim,Germany); and verapamil (VP) from Abbott. All thestock solutions, with the exception of VPA and VP,were prepared in DMSO at a concentration of 100 mg/ml (ASA), 400 mg/ml (IMC), 0.2 mg/ml (5-FU), 5 mg/ml (DOX and PTX), and 10 mg/ml (CPT);VPA stock was prepared in PBS (50 mg/ml). VP was purchased as solution for i.v. injection (Isoptin) at aconcentration of 2.5 mg/ml. All the stock solutionswere stored at   − 20°C except the VP that was storedat +4°C. Working solutions were prepared freshaccording to the experimental design by serialdilutions in culture medium.Cell linesSR-4987 is a bone marrow mesenchymal cell lineestablished in our laboratory from a long-term bonemarrow cell culture of BDF/1 mice (Pessina et al.1992, 1997) and currently available by ATCC (CRL- 2028). The cell line, cultured in RPMI 1640 mediumsupplemented with 5% fetal bovine serum and 2 mM L -glutamine (EuroClone, UK), was weekly trypsi-nized by 0.05% trypsin/0.02% EDTA (EuroClone,UK) and splitted 1:5 to 1:10 into 25-cm 2 flasks(Corning, USA). For our differentiation experiments,cells from passage 120 to 160 were used.Human cell lines MOLT-4 (acute lymphoblasticleukemia) (Minowada et al. 1972) and HT-29 (colo-rectal adenocarcinomas) (Fogh and Trempe 1975)have been kindly supplied by Dr. Maura Ferrari(IZSLER, Brescia). Cells were maintained in IMDMsupplemented with FBS 10% (MOLT-4) or FBS 5%(HT-29) by weekly 1:3 passages.Determination of PDT and PEPDT has been evaluated as suggested by McAteer andDevis (1994). SR-4987 cells were cultured in 24multi-well plates at three different starting densities(1,000  –  5,000 and 10,000 cells/well), in quadruplicate.PDT value was calculated measuring the cell growthafter 72, 96, 120, and 144 h from the seeding, and thecells were counted by Trypan blue in a Burker chamber.Plating efficiency has been evaluated by culturingcells in 35-mm Petri dishes (Nunc, Germany) at threedifferent densities: 100  –  200 and 1,000 cells/dish (intriplicate). After 72 h at 37°C, 5% CO 2 , by an 170 Cell Biol Toxicol (2011) 27:169  –  180  inverted microscope (20  –  25× magnification) wescored the number of colonies adherent to the Petrisurface.Mesenchymal stromal cells from human bone marrowMononuclear cells from human bone marrow (BM-MSC) were purchased frozen in liquid nitrogenfrom Lonza (Switzerland) and stored at   − 120°Cuntil use. Then the cells were thawed according toa procedure detailed for umbilical cord blood(Pessina et al. 2004) previously used. Briefly, cellswere quickly thawed at 37°C and then transferred toa conical tube by adding IMDM+10% FBS (Euro-Clone) and DNase I 10 U/ml (Serva, Germany).After centrifugation (15 min at 200×  g  ), most of thesupernatant was removed and cell pellet resuspendedin 1  –  2 ml of the remaining medium. Fresh mediumwas added to the cell suspension and then centri-fuged as above. Cell pellet was resuspended in non-hematopoietic expansion medium (Miltenyi Biotec,Germany) and, after evaluation of cell number andviability, cells were plated in 25-cm 2 flasks (Corn-ing) at 2×10 6 cells/ml. After 24 h at 37°C, 5% CO 2 ,floating cells were discarded and fresh medium wasadded to adherent cells. Medium was changedweekly until cells reached 80% confluency. Then,cells were trypsinized and plated in differentiationmedia according to the conditions described for their differentiation.Osteo-, chondro-, and adipo-differentiationSR-4987 cells were tested for their capacity todifferentiate into osteocytes, chondrocytes, and adi- pocytes according to the methods suggested byPittenger et al. (1999) with some modifications. As positive control, we used human bone marrowmesenchymal cells (hMSC) prepared as describedabove. Briefly, cells were plated into 35-mm Petridishes (Nunc) at 50 cells/cm 2 density in 1 ml/Petri of Stemline Mesenchymal Stem Cell Expansion Medium(Sigma-Aldrich) supplemented with 3% FBS and4 mM  L -glutamine; after 72 h of incubation at 37°C,5% CO 2 , culture medium was replaced with specificdifferentiation media described below.According to the method suggested by Tropel et al.(2004), to induce osteogenic differentiation, SR-4987cells were cultured for 14 days in Stemline supple-mented with 10 nM dexamethasone, 10 mM glycerol-2-phospate, and 300 nM  L -ascorbic acid. Differenti-ation medium was replaced every 3  –  4 days. Culturemonolayers were then fixed for 5 min in cooledmethanol at   − 20°C and then processed for alkaline phosphatase staining, using SIGMA FAST BCIP/  NTB (Sigma-Aldrich). The presence of more maturestage of osteoblasts was checked by alizarin stainingthat identifies calcium in cultured cells.Spontaneous osteogenesis was evaluated by main-taining SR-4987 in growth medium without supple-ments. Both spontaneous and induced osteogenesiswere also evaluated in the presence of different drugs:two anti-inflammatory, ASA (100 and 300  μ  g/ml) andIMC (50 and 150  μ  g/ml), one anti-tumor, PTX (10,30, and 50 ng/ml), and an anti-convulsant and mood-stabilizing drug, VPA (50 and 150  μ  g/ml).To induce chondrocytic differentiation, cells werecultured in hMSC chondrogenesis induction medium(Provitro, Germany) according to the micro-massmethods (Johnstone et al. 1998; Tallheden et al.2003) with some modifications. Cultures wererefeeded every 4 to 5 days, and after 21 days the cellmass was spread and smeared on slides and cellsfixed for 30 min at room temperature with formalinsolution, neutral buffered 10% (Sigma-Aldrich), andthen processed both by toluidine staining and byimmunocytochemistry technique for aggrecan. Brief-ly, for toluidine staining, fixed cells were stained bytoluidine blue 0.1% (dissolved in ethanol 70%) for 3 min and then washed with distilled water. For immunocytochemistry, cells were permeabilized by0.3% Triton X-100 for 45 min, treated 10 min withH 2 O 2 , incubated overnight at 4°C with a mouse anti-human aggrecan monoclonal antibody (1:100; Milli- pore, USA) and then 1 h at room temperature withgoat anti-mouse HRP-conjugated antibody (1:50;Santa Cruz Biotechnology, CA, USA). Cells werefinally developed with 3,3 ′ -diaminobenzidine solution(MP Biomedicals, Irvine, Canada), 0.5 mg/ml in bidistilled water, for 10 min.To induce adipocytic differentiation, cells werecultured in Stemline supplemented with indomethacin200  μ  M (Alexis Biochemicals, USA), isobutylme-thylxanthine 0.5 mM (Applichem, Germany), dexa-methasone 1  μ  M, hydrocortisone 1  μ  M, and insulin10  μ  g/ml (all from Sigma-Aldrich). After 10 days of culture, cells were fixed for 30 min at roomtemperature with formalin solution, neutral buffered Cell Biol Toxicol (2011) 27:169  –  180 171  10% (Sigma-Aldrich), and then processed for Oil redO staining and Sudan Black (Sigma-Aldrich).Effect of drugs on cell proliferationThe effect of the four anti-cancer drugs (PTX, DOX,CPT, and 5-FU), two anti-inflammatory drugs (ASA,IMC), and an anti-convulsant (VPA) on cell prolifer-ation of SR-4987 has been studied in 96 multi-well plates (Sarstedt, Germany) in comparison to the effect exerted on other cell lines HT-29 and MOLT-4.Briefly, 1:2 serial dilutions of 2× concentrations of the drugs were prepared in 50  μ  l of culture medium/ well according to different ranges as determined in preliminary experiments not reported here. Accordingto the experimental design, to each well were added50  μ  l containing 0.5×10 3 (for SR-4987) or 10 3 cells(for HT-29 and MOLT-4). After 7 days of culture at 37°C, 5% CO 2 , cell viability was evaluated by MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetra-zolium) assay as previously described (Mossman1983; Pessina et al. 1994). For each cell line and drug, the inhibitory concentrations (IC) 50 and 90were determined according to the Reed and Muench(1938) formula.Study of cell cycle and P-gp expression by FACSanalysisDNA content for cell cycle phase detection was performed as follows: Cells at T 0  and after 24 h of treatment with 2  μ  g/ml PTX were trypsinized,washed, suspended in saline GM solution (glucose6 mM, NaCl 150 mM, KCl 5 mM, Na 2 HPO 4  1 mM,KH 2 PO 4  1 mM, EDTA 0.7 mM), and fixed with 96%(v/v) ethanol for 1 h at 4°C. After PBS wash, cellswere suspended in propidium iodide 50  μ  g/ml inPBS. Cells were incubated overnight at 4°C andanalyzed by flow cytometry (FacsVantageSE; Becton-Dickinson, USA). For evaluation of P-gp expression,cells were first incubated with a monoclonal antibodymouse anti-human P-gp (clone C1; Ylem, Italy) for 30 min a 4°C, washed with PBS, and incubated with aFITC-conjugated goat anti-mouse immunoglobulin(Becton-Dickinson) for further 30 min at 4°C. Cellswere then washed once and analyzed by flowcytometry using a specific software (CellQuest Pro;Becton-Dickinson). Data reported refer to at least three independent experiments expressed as the ratio between intensity detected with the specific antibodyand the basal fluorescence with isotypic antibody. Table 1  Main biological characteristics of mesenchymal stromal cells SR-4987ReferencesTissue of origin Long-term bone marrow from a female BDF/1 mouse Pessina et al. 1992MorphologykaryotypeFibroblast-likeTetraploidClonogenicitytumsrcenicityPoor clonogenicity in agar (0.6%)Induction of sarcoma in syngeneic miceMarkers Positivity for: CD5, CD44, CD45R (B220), (CD73), vimentin,GM3Pessina et al. 1997 Negativity for: ras, fms, GM1a ganglioside Negative for reverse transcriptaseCytokines productionSecreting M-CSF Pessina et al. 1992, 1997 Expressing mRNA for bFGF, IL-7, GM-CSF, and SCFCytokinessensitivity bFGF and aFGF dramatically enhance agar clonogenicity Pessina et al. 1995, 1998 M-CSF, G-CSF, GM-CSF, IL-3, IL-7, TNF α  , PDGF, and EGFdo not modulate clonogenicityCholera toxin produces a dramatic increase in intracellular cAMPwithout inhibiting cell growthApplications Metabolic conversion of doxorubicin Gribaldo et al. 1999; Takagi et al. 1999, 2005; Opavsky et al. 2001Bioassays of bFGF Construction of three-dimensional hematopoietic culture systemGene transfection studies172 Cell Biol Toxicol (2011) 27:169  –  180  Results Growth charactersThe main characters of SR-4987 cells are summarizedin Table 1. This line expresses both features of stromal cell and others that are more specific of hemolymphopoietic cell line. SR-4987 cells arenegative for reverse transcriptase (RT) and for theexpression of p-21 ras, although they are tumsrcenicin vivo (Pessina et al. 1997). The new investigationson the cell growth kinetics show that SR-4987 cellshave a mean PDT value of 24.5±5.4 h with aninoculum of 10,000 cells/cm 2 . As expected, the PDTvalue was found to be dependent on the cell density at the startingseeding (number ofcells/cm 2 ) and increasessignificantly over the density of 10,000 cells/cm 2 .Plating efficiency, expressed as the percentage of cellsable to adhere on the substrate to give colonies at 72 h,was 2.87±1.19%.Differentiation capacityThe reference differentiation pattern of BM-MSC isreported in Fig. 1, whereas Fig. 2 shows the differen- tiating capacity of SR-4987 cells. As human MSCsfrom bone marrow, SR-4987 cells also have thecapacity to differentiate into osteoblasts and chondro-cytes (Fig. 2b  –  e), but are unable to differentiate intomature adipocytes (Fig. 2h, i). Of interest is that SR-4987 cells are capable of differentiating spontaneouslyinto osteoblasts because in the absence of specificstimulation with osteogenic medium the cell monolay-er evidenced many focal plaques of spontaneousosteogenesis (one of it is documented in the Fig. 2f, g).Drug sensitivity of SR-4987 proliferationto anti-cancer The sensitivity of SR-4987 to four anti-cancer drugswas evaluated by MTT assay in comparison to two ALP stainingNegative controlChondrogenesisBlue ToluidineAggrecanAlizarin redOsteogenesis Oil Red OSudan Black BAdipogenesis acbdefg Fig. 1  Plasticity of human bone marrow mesenchymal stem cells(hu-MSCs).  a  Negative control (hu-MSC in culture mediumwithout stimulation and without staining).  b ,  c  Toluidine stainingand immunocytochemistry for aggrecan to evaluate chondro-differentiation (micropellet slide spread of cells cultured 20 daysin chondro medium).  d ,  e  Alkaline phosphatase (ALP) andalizarin staining in methanol-fixed cells to evaluated osteo-differentiation (14-day cultures in osteo medium).  f  ,  g  Oil red andSudan Black staining on formalin-fixed cells to evaluate adipo-differentiation (10-day culture in adipo medium)Cell Biol Toxicol (2011) 27:169  –  180 173
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