STEM CELLS AND DEVELOPMENT Volume 23, Number 10, 2014  Mary Ann Liebert, Inc. DOI: 10.1089/scd.2013.0367

A Mouse Bone Marrow Stromal Cell Line with Skeletal Stem Cell Characteristics to Study Osteogenesis In Vitro and In Vivo Sebastian Raeth,1 Benedetto Sacchetti,2 Georg Siegel,3 Ulrike A. Mau-Holzmann,4 Jan Hansmann,5 Gabriele Vacun,5 Thomas G. Hauk,1 Klaus Pfizenmaier,1 and Angelika Hausser1

Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, which are able to differentiate in vitro into osteocytes, adipocytes, and chondrocytes. Mouse BMSCs (mBMSCs) are a versatile model system to investigate factors involved in BMSC differentiation in vitro and in vivo as a variety of transgenic mouse models are available. In this study, mBMSCs were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy.

Introduction

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tem cells have the capacity to self-renew and differentiate into cells of various lineages. The bone marrow (BM) stroma contains progenitor cells of all tissues found in the skeleton, such as bone, cartilage, fibrous tissue, and fat [1]. In addition, these BM-derived progenitor cells give rise to cells that constitute the hematopoietic microenvironment (HME) [2] and are thus called BM stromal stem cells or skeletal stem cells [3]. Different laboratories have identified, under partly different isolation or culture conditions (eg, oxygen concentration), multipotent stromal cells [also called ‘‘mesenchymal stromal cell’’ (MSC)] from a panel of tissues, such as BM, adipose tissue, and umbilical cord. The differences in tissue origin, isolation method, and culture conditions likely contribute to diverse phenotypes and functions ascribed to MSCs [4]. Thus, for better characterization of MSCs, the International Society of Cellular Therapy defined MSCs by the following three criteria. First, MSCs must be adherent to plastic under standard tissue culture conditions.

Second, MSCs must express certain cell surface markers, such as CD73, CD90, and CD105, and lack expression of other markers, including CD45, CD34, CD14, or CD11b, CD79alpha or CD19, and HLA-DR surface molecules. Finally, MSCs must have the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts under in vitro conditions [5]. Still, these criteria are based on the phenotype of culture-expanded MSCs and reliable markers specific for both native and cultured MSCs remain to be identified (reviewed in Ref. [6]). The understanding of skeletal progenitor cells was extended by a study that demonstrates that in vivo transplantation of progenitor cells is the standard assay for confirming their multipotency. The authors postulated that only bone marrow stromal cells (BMSCs) are capable to differentiate into skeletal tissue and support the hematopoietic environment in vivo and are thus bona fide skeletal stem cells [7]. The definition and origin of MSCs and specifically the scope of potential clinical indications for MSC-based therapy is currently under heavy debate [8–12]. In particular,

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Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany. Department of Molecular Medicine, Sapienza University, Rome, Italy. 3 Institute of Clinical and Experimental Transfusion Medicine (IKET), University Hospital Tu¨bingen, Tu¨bingen, Germany. 4 Department of Medical Genetics, University of Tu¨bingen, Tu¨bingen, Germany. 5 Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany. 2

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despite a few promising reports of clinical benefits of MSC therapy, controlled clinical studies so far trail behind the expectations [8,13]. Regardless of this ongoing controversial debate, the efficacy of BMSCs to regenerate bone in vivo is widely accepted (reviewed in Refs. [14–16]). Transplantation of culture-expanded BMSCs deposited on natural or synthetic scaffolds has been successfully applied in animal models of large bone defects [17–19] and initial pilot studies in humans showed promising results [20,21]. The translation of these results into clinical practice, however, remains a challenge for many reasons, including safety concerns and technical aspects, such as expansion conditions of BMSCs. For repairing large bone defects, huge quantities of cells are required. However, transplants from late-passage cells form only bone and lack the ability to establish an HME in contrast to those derived from early passage cells [14]. This might be due to the fact that human MSCs get senescent and thus lose their multipotency during in vitro culture (reviewed in Ref. [22]). Thus, a significant understanding of skeletal stem cell biology in vitro and, more importantly, in vivo will be important to allow their application in routine clinical practice. Because of the ability to manipulate their genome, mice would be an excellent tool of choice to study the molecular mechanisms of MSC-related pathologies. However, the progenitor cell frequency in mouse BM is extremely low and has been estimated to be only 0.001%–0.0001%. Additionally, differences with respect to yield and growth kinetics among different mouse strains were reported [23,24] making the isolation of BMSCs still challenging. A particular problem is that isolates of BMSCs are often contaminated with hematopoietic cells of the myeloid/macrophage lineage, which adhere on top of the stromal cells [25]. Because of these difficulties, efforts to take full advantage of existing mouse models to study fundamental aspects of MSC biology are substantially hindered. We here show the successful establishment of a stable, multipotent BMderived stromal cell line from mouse. Notably, this line possesses skeletal stem cell characteristics in vivo, evident from bone tissue formation, and induction of host-derived vascularization as well as hematopoiesis when transplanted subcutaneously into mice.

Materials and Methods Isolation, culture, and maintenance of bone-marrow-derived stromal cells Isolation of BMSCs was performed as previously described [26,27]. Briefly, 8-week-old C57BL6 mice were sacrificed using cervical dislocation and rinsed with 70% ethanol. We have chosen animals of this young age since age-related effects on BMSCs are reported [28,29]. Additionally, cells of C57Bl6 origin are compatible with most transgenic mouse models available. The two tibias and femurs of each animal were cleaned of skin and transferred into alpha-MEM (Genaxxon) supplemented with 1% Pen/ Strep. After removing the connective tissue in a sterile bench, the ends of each bone were clipped off and the BM was flushed out with a 26G needle containing culture medium. The samples were stored on ice. Cells were separated with a 70-mm filter mesh followed by centrifugation (1,000

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g; 5 min) and resuspended in 1 mL of BMSC culture medium consisting of a-MEM (Genaxxon) supplemented with fetal calf serum (FCS) (15%; Hyclone). About 4 · 106 cells/ cm2 were plated (Greiner) and incubated at 37% and 5% CO2 in a humidified incubator. The culture medium was replaced after 3, 8, and 16 h. Seventy-two hours after isolation, the cells were washed with phosphate-buffered saline (PBS). Afterward, the medium was changed every 2–3 days until the cells were 70% confluent. Cells were split using Versene plus 0.5% trypsin (Invitrogen) and cultured in a 25cm2 flask or a 6-cm culture dish (Greiner). Cells were maintained in culture medium for further studies and passaged at a confluency of 6,700 cells/cm2 every 2–3 days if not mentioned otherwise. The number of population doublings (PDs) was calculated using the formula PD = (ln nch - ln ncs)/ln 2, where nch is number of cells harvested and ncs is number of cells seeded [30].

Immunofluorescence and microscopy BMSCs were grown on collagen-coated coverslips, washed with PBS, fixed in 4% paraformaldehyde at room temperature for 15 min, washed, permeabilized with 0.1% Triton-X 100 (5 min, room temperature), and blocked with blocking buffer (5% FCS in PBS) for 30 min. The cells were incubated with DAPI (1 mg/mL) for 15 min, washed, incubated with Alexa488 labeled phalloidin diluted in blocking buffer for 1 h, washed, mounted in Fluormount G (Southern Biotechnology), and analyzed using the Cellobserver (Zeiss). Cells were imaged with a Plan-Apochromat 20 · /0.8 DIC objective lens.

Flow cytometry Flow cytometry analysis of MSC populations was performed at passage 8, passage 23, and passage 43 employing an FACScan instrument (BD Biosciences) and BD CellQuest Pro software. Cells were incubated with (secondary) phycoerythrinlabeled mouse-specific antibodies [anti-CD4, -CD8a, -CD9, -CD11b, -CD11c, -CD14, -CD29, -CD31, -CD43, -CD44, -CD45R, -CD71, -CD73, -CD80, -CD86, -CD90, -CD105, -CD106, -CD117, -CD135, -CD140a, -CD144, -CD184, -CD195, -H-2K/H2D, -IA/IE, -SCA1, and -VEGFR2 (BD Biosciences); -CD34 (Abd Serotec); -CD39 (R&D); -CD140b (Epitomics); -CD146 (Bio Legend); and -CD271 (STEMCELL Technologies)] for 60 min on ice. Unspecific antibody binding was prevented by using the Mouse BD Fc Block (BD Biosciences) according to the manufacturer’s instructions. PE-conjugated or nonlabeled IgG, IgG2ak, IgG1k, IgG1l1, IgG2ck, IgG2bk, IgG2bk, and IgG1 antibodies (BD Biosciences) were used as isotype matched controls. As secondary antibodies, mouse anti-rat Ig, donkey anti-goat IgG, goat antirat Ig, rat anti-mouse IgG2a + b (BD Biosciences), or goat antirabbit Ig (Biotrend) was used. Dead cells were excluded by uptake of 7-Aminoactinomycin D. Analysis of percentage of antigen-positive cells and fluorescence intensity was performed using FlowJo software. The respective isotype control was subtracted from all samples to compensate unspecific antibody binding.

Osteogenic differentiation BMSCs were grown to 90%–100% confluence in 24-well plates and the culture medium was then replaced with

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osteogenic medium (a-MEM supplemented with 15% FCS plus 1% Penicillin/Streptomycin, 100 nM dexamethasone, 50 mg/mL ascorbate-2-phosphate, and 10 mM beta-glycerol phosphate). The medium was changed every 2–3 days. Osteogenic differentiation was assessed by Alizarin Red staining 21 days after initial osteogenic induction. In brief, cells were washed with PBS and allowed to dry for 5– 10 min. Afterward, cells were fixed with 50% ethanol for 20 min. The fixed cells were then stained with 1% Alizarin Red (Roth) at pH 6.4 for 30 min under continuous shaking. Subsequently, cells were rinsed three times with H2O, and transmitted light pictures were taken. As a negative control cells grown in culture medium for 21 days were used.

Adipogenic differentiation BMSCs were grown to confluence on Permanox 4-well chamber slides (Thermo Scientific). Adipogenic differentiation medium (a-MEM supplemented with 15% FCS plus 1% Penicillin/Streptomycin, 1 mM dexamethasone, 500 mM IBMX, 10 mg/mL human insulin, and 100 mM indomethacin) was added and renewed every 2–3 days. Twelve days after initial adipogenic induction, cells were washed with PBS and fixed for 10 min in 4% Histofix (Roth). Then, cells were rinsed once with H2O and incubated in 60% isopropanol for 5 min. Subsequently, the cells were incubated for 10 min with Oil Red O. Afterward, the cells were washed once with 60% isopropanol followed by H2O. Nuclei were counterstained with hemalaun. As a negative control cells grown in culture medium for 12 days were used.

Chondrogenic differentiation About 2.5 · 105 BMSCs were centrifuged (1,000 g; 5 min) in a 15 mL Falcon tube, the supernatant was discarded, and 1 mL of chondrogenic differentiation medium (a-MEM plus 1% Penicillin/Streptomycin, 50 mg/mL l-ascorbic acid-2phosphate, 100 nM dexamethasone, 100 mg/mL pyruvate, 40 mg/mL l-proline, and 1% insulin-transferrin-sodium selenite media supplement) was added to the cell pellet. TGFb3 (10 ng/mL) was added with every medium change. The negative controls were cultured in chondrogenic differentiation medium without TGF-b3. Medium was changed twice a week for 27 days. After fixing the pellets for 2 h in 4% Histofix (Roth), they were embedded in paraffin in cassettes using the automated system Shandon Citadel 1000 (Thermo Scientific). The cassettes with the pellets were transferred to a heating chamber at 60C after the program of the automated system was finished. The melted paraffin was removed from the cassettes and the cell pellets were transferred into a metal form filled with liquid paraffin. The paraffin solidified on a cooling plate. The obtained solid paraffin block containing the pellet was then sliced with a rotary microtome RM 2145 (Leica). The slices were deparaffinized using a descending sequence of xylol-ethanol. After 3 min in 0.1 N HCl, the slices were stained with 1% Alcian Blue (Roth) dissolved in 0.1 N HCl (pH 1.0) for 30 min. Two washing steps of 0.1 N HCl followed by H2O were performed to remove the unspecific bound dye. The slices were counterstained with nuclear fast red for 4 min. To dehydrate the pellet slice, ascending concentrations of ethanol were used. Isomount (LABOnord) was used to fix the slices on the object slides.

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Colony-forming unit fibroblast assay The proliferative induction capability of the M2 cell line was evaluated by colony-forming unit fibroblast (CFU-F) analysis. M2 BMSCs were seeded into 100 cm2 cell culture dishes at an initial density of three cells per cm2 in 12 mL medium. Cells were incubated for 7 days in a humidified 5% CO2 incubator at 37C. On day 7, cultures were simultaneously fixed and stained with 0.1% crystal violet in 20% methanol by incubating them at room temperature for 30 min and then washed twice with water. Crystal-violet-stained colonies with a minimum surface area of 1 mm2 were counted and then photographed. CFU-F frequency was calculated by dividing the number of colonies by the number of cells seeded. The experiment was performed in triplicates.

Single-clone analysis BMSCs (P42) were seeded with a concentration of 0.5 cells/well in a 96-well plate in preconditioned sterile-filtered a-MEM supplemented with 15% FCS. The wells were analyzed microscopically to record the single-cell seeding events 1 day after plating. Single-clone colonies were passaged after 17 days. Twenty-four clones were analyzed for osteogenic and adipogenic differentiation.

Nucleofection BMSCs (P20–P24) were nucleofected with Amaxa nucleofection kit for MSCs (Lonza) according to the manufacturer’s instructions. Briefly, 5 · 105 cells were resuspended in 100 mL of nucleofector solution, 1 mM of siRNA [ON-Targetplus SMART pool Mouse Runx2 (12393; Dharmacon) or the nontarget plus smartpool control] was added, and the cell suspension was transferred to a Nucleofector cuvette. Cells were nucleofected using program U23 of the Amaxxa Nucleofector (Lonza). Subsequently, the cell suspension was transferred into culture medium and incubated at 37C and 5% CO2. Successful knockdown was verified by qPCR 2 days postnucleofection.

Quantitative real-time PCR RNA was extracted using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions and concentration was measured using a NanoDrop (Peqlab). The 260/280 and 260/230 optical density ratios were measured to assess the purity of RNA samples. One-step real-time quantitative polymerase chain reaction (qPCR) was applied to quantitatively analyze mRNA levels. In brief, the reaction was performed on the CFX96 Touch qPCR System (BioRad) platform using QuantiTect SYBR Green RT-PCR Kit (Qiagen). The PCR mix contained 10 mL QuantiTect SYBR Green PCR Mix, 0.2 mL reverse transcriptase, 2 mL of the respective QuantiTect Primer Assay consisting of a mixture of predesigned forward and reverse primers for the gene of interest, and 100 ng RNA in 7.8 mL RNAse-free H2O. PCR was performed under the following conditions: 50C for 30 min, 95C for 15 min, followed by 40 cycles of 95C for 30 s, 55C for 30 s, and 72C for 30 s. A melt-curve analysis step was added with a melting profile of 65.0–95.0C (increment 0.5C). Quanti-Tect Primer Assays (Qiagen) were used for osteocalcin (Mm_Bglap_1_SG), osteopontin

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(Mm_Spp1_1_SG), integrin-binding-sialo-protein (Mm_ Ibsp_1_SG), Runx2 (Mm_Runx2_1_SG), GAPDH (Mm_ Gapdh_3_SG), PParg (Mm_Pparg_1_SG), Fabp (Mm_ Fabp4_1_SG), and DLK1 (Mm_Dlk1_1_SG). The relative expression was calculated by normalization to GAPDH using the DCq or the DDCq method.

Histology

Western blot analysis

Statistical analysis

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About 2.5 · 10 M2 cells were harvested via the hot lysis method. Briefly, supernatants were aspirated and adherent cells were lysed by addition of 300 mL of hot (95C) laemmli buffer [125 mM tris (pH 6.8), 10% glycerol, 4% SDS, 0.02% bromophenol blue, and 10% beta-mercaptoethanol] followed by incubation at 95C for further 10 min and sonication. Freshly prepared BM cells were lysed by resuspension in 300 mL of hot laemmli buffer. After centrifugation, supernatants were collected and 15 mL of each protein sample subjected to a 4%–12% gradient Bis-Tris SDS-PAGE (Life Technologies) was blotted onto nitrocellulose membranes and incubated with a p53-specific antibody (1C12; Cell Signaling). Equal loading was monitored by reprobing membranes with an antibody against actin (SigmaAldrich). HRP-conjugated secondary antibodies were from Jackson Immunoresearch Laboratories. Bands were visualized applying chemiluminescence SuperSignaldetection system (Pierce).

Anchorage-independent growth One thousand BMSCs were seeded in 50 mL of a-MEM supplemented with 15% FCS in the lid of a 100-mm culture dish. The lid was then inverted to create hanging drops. Images were captured with a 45 times magnification at an sz61 binocular (Olympus). The diameter of the spheres was monitored at days 3, 6, 8, 12, and 18.

In vivo transplantation To assess the osteogenic potential of the M2 cell line in vivo, mouse BMSC (mBMSC) M2 cell strains in early (P8–10) or late (P40–44) passages were seeded onto osteoconductive material [hydroxyapatite/tricalcium phosphate particles (HA/TCP)] as reported previously [31–33]. All animal procedures were approved by the relevant institutional committees. Briefly, 2 · 106 cells were loaded onto HA/TCP (40 mg, 100–200 mm; Zimmer) and embedded in a fibrin gel to generate carrier–cell constructs. The constructs and the cell-free carrier, as control, were subcutaneously transplanted in the backs of 6–15-week-old female SCID/beige mice (CB17.Cg-Prkdcscid Lystbg/Crl; Charles River Laboratories International, Inc.). In brief, operations were performed under sterile conditions under anesthesia achieved by intramuscular injection of a mixture of Zoletil 20 (Virbac; 5 mL/g of body weight) together with Rompun (Bayer; 1 mL/Zoletil 20 bottles). The mouse back was disinfected with betadine and midlongitudinal skin incisions of about 1 cm in length were made on the dorsal surface of each mouse. Subcutaneous pockets were formed by blunt dissection. A single transplant was placed into each pocket with up to four transplants per animal. The incisions were closed with surgical staples.

Heterotopic transplants were harvested after 8 weeks, fixed in 4% formaldehyde, decalcified in 10% ethylenediaminetetraacetic acid at pH 7.2, and embedded in paraffin. Subsequently, the deparaffinized and rehydrated sections were stained with hematoxylin and eosin.

Data are expressed as mean – standard error of the mean (SEM). A one-way ANOVA test followed by a Bonferroni post-test was used in statistical analysis for comparison, and P < 0.05 was used as the criterion for statistical significance.

Results Characterization of mBMSCs We aimed to establish a mouse MSC line as a tool to investigate osteogenesis in vitro and in vivo at a molecular level. Recent results indicate that only BMSCs have osteogenic potential in vivo [7]. In-vitro-cultured BMSCs isolated according to Nadri et al. [26] gave rise to six independent cell cultures: M2, M3, M4, M5, M6, and M7. All these cultures stopped proliferating after the initial passaging, entering a state of quiescence (Fig. 1A). However, the cells started to proliferate after several weeks in culture (50–150 days after initial passage). To investigate whether the stromal cell cultures contained multipotent BMSCs, early passage cells (P7–9) were cultured under various conditions as described in the ‘‘Materials and Methods’’ section to assess their capacity to differentiate into committed mesodermal lineages. In vitro multipotency toward the osteogenic, adipogenic, and chondrogenic lineages could be demonstrated for the cell cultures M2, M6, and M7 (Fig. 1B). M3, M4, and M5 only showed mineralization and hence osteogenic differentiation, but they failed to differentiate toward the adipogenic and chondrogenic lineages. This characterizes M3, M4, and M5 as committed osteogenic progenitors. The three multipotent cultures M2, M6, and M7 were further analyzed for surface marker expression profile using flow cytometry (Supplementary Fig. S1A–C; Supplementary Data are available online at www.liebertpub.com/scd). All stromal cell populations were positive for the classical mesenchymal markers CD9, CD29, CD44, CD71, CD105, and CD106 as well as the mouse stem cell marker Sca-1 and MCAM/CD146, whereas expression of hematopoietic and lymphoid markers CD34, CD45, CD11b, CD14, CD31, CD80, CD117, and CD135 was negative. Cells were also positive for H-2Kb (MHC class Ia) and negative for the MHC class IIa (data not shown). Of note, expression of CD90 was differentially regulated, with M2 cells lacking this marker completely whereas M6 and M7 cell lines contained a subpopulation of cells that were positive for CD90 (Supplementary Fig. S1A–C). The surface marker profile of the multipotent cell cultures is, thus, in accordance with a typical mBMSC phenotype. Finally, we also determined the proliferation capacity of the multipotent cell cultures over time. As Fig. 1C shows, cumulative PD increased steadily over time with increasing passage number. Our results thus show that the three cell cultures M2, M6, and M7 are potential BM-derived stromal cell lines.

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FIG. 1. Characterization of mouse bone marrow stromal cells (BMSCs). (A) Proliferation of BMSCs. Six independent cell cultures (M2–M7) were isolated from bone marrow (BM). Cell numbers were recorded from P1 until P5. (B) Differentiation of BMSCs. BMSCs were cultured in vitro in adipogenic, osteogenic, or chondrogenic media (indicated by + ) to assess multilineage differentiation capability. The cells were fixed and stained with Alizarin Red (osteogenesis), Oil Red O (adipogenesis), or Alcian Blue (chondrogenesis) for osteoblast, adipocyte, or chondrocyte differentiation. Cells cultivated in standard growth medium ( - ) served as a control. (C) Relationship between cell passages and cumulative population doubling (PD). BMSC cultures M2, M6, and M7 were continuously passaged in a-MEM supplemented with 15% fetal calf serum (FCS). Cell numbers were recorded and cumulative PD was calculated as described. Scale bars = 100 mm (chondrogenesis), 50 mm (adipogenesis), and 200 mm (osteogenesis).

The M2 cell line is a BM-derived stromal cell line Phenotypic and functional stability of M2 cells during in vitro culture. To investigate whether the established mouse cell

lines maintain stromal cell properties over time in vitro, we chose M2 cells for our further experiments. We analyzed the properties of these cells in early (P8–10), mid (P20–26), and late (P40–44) passages. First, we stained the cells with phalloidin to visualize F-actin. Microscopic analysis revealed a spindle-shaped fibroblast-like phenotype of the cells in passage 8 (Supplementary Fig. S1, left). This phenotype did not change during in vitro culture up to passage 40 (Supplementary Fig. S2, right, and data not shown). We then analyzed the density-independent growth of M2 cells using the CFU-F assay. Early passage cells formed discrete colonies indicative of density-independent growth with a CFU-F frequency of 12%. Colony number even increased in mid-passage and late-passage cells, resulting in CFU-F frequencies of 25% and 29%, respectively (Fig. 2A). This indicates that M2 cells are highly proliferative BMSCs with

a clonogenic potential. Phinney and colleagues demonstrated that long-term normoxic exposure of BMSC-derived cells selects for clones defective in p53 [34,35]. Indeed, western blot analysis of cell extracts revealed that M2 cells in early, mid, and late passages expressed high levels of p53 protein, whereas p53 protein expression was absent in freshly isolated BM cells (Fig. 2B). The M2 cells thus underwent spontaneous immortalization most likely by mutation of p53, liberating the growth arrest. Karyotyping was performed on early and latepassage cells and showed the presence of aneuploid cells (data not shown), which is typical for established mouse cell lines. However, anchorage-independent growth, a hallmark of several cancer cells, could not be observed in a hanging-drop assay (Supplementary Fig. S3). In line with this, the cell size and granularity did not change over time of cultivation as can be seen in flow cytometry dot-blots of early, mid-, and latepassage cells (Supplementary Fig. S4). Finally, subcutaneous transplantation of late-passage M2 cells in nude mice did not result in tumor formation (data not shown), proving that a malignant transformation has not occurred.

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FIG. 2. The M2 cell line is a BMSC line. (A) Left: CFU-F assays of M2 cells in passage P8–10, P24–26, or P41–44. Shown is the mean – standard error of the mean (SEM) of three independent experiments. **P < 0.01; ***P < 0.001. Right: Representative images of crystal-violet-stained CFU-F colonies formed from M2 cells. (B) Western blot analysis of early (P9), mid- (P23), and late- (P42) passage M2 cells and freshly prepared BM. Cells were lysed, proteins were separated by SDS-PAGE, and p53 was detected by western blot analysis using a specific antibody. Detection of actin served as a loading control. (C) Flow cytometry analysis of M2 cells in passages 8, 23, and 43. Cells were stained with indicated antibodies and binding was analyzed by flow cytometry (green). Unstained cells were used as a negative control (red). (D) Differentiation of M2 cells. M2 cells in passage 8, 20, or 40 were cultured in vitro in adipogenic, osteogenic, or chondrogenic media (indicated by + ) to assess multilineage differentiation capability. The cells were fixed and stained with Oil Red O (adipogenesis), Alizarin Red (osteogenesis), or Alcian Blue (chondrogenesis) for adipocyte, osteoblast, or chondrocyte differentiation. Cells cultivated in standard growth medium ( - ) served as a control. (E) Clones obtained by serial dilution were cultured in vitro in adipogenic or osteogenic media (indicated by + ) to assess multilineage differentiation capability. The cells were fixed and stained with Oil Red O (adipogenesis) or Alizarin Red (osteogenesis) for adipocyte or osteoblast differentiation. Cells cultured in standard growth medium ( - ) served as a control. A representative result from one clone is shown. Scale bars = 100 mm (chondrogenesis), 50 mm (adipogenesis), and 200 mm (osteogenesis). CFU-F, colonyforming unit fibroblast.

Cells were further analyzed at early (P8), mid (P23), and late (P43) passage numbers for the expression of CD9, CD11b, CD34, CD44, CD45, CD71, CD105, and Sca-1 (Fig. 2C and Supplementary Fig. S3A). For each culture, passage M2 cells were negative for CD11b, CD34, and

CD45. Comparison of common stromal cell surface markers in M2 cultures revealed a cell-passage-independent expression of CD9, CD71, and CD105, and Sca-1. Of note, late-passage cells even displayed a higher expression of Sca1 compared with early and mid-passage cells. Early, mid-,

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and late-passage cells were also positive for MCAM/CD146 and negative for CD90 (Supplementary Fig. S3A). FACS profiles thus demonstrate stability in marker expression during in vitro culture. Taken together, these data demonstrate phenotypic and functional stability of M2 cells during in vitro culture. Multipotency of M2 cells is retained during in vitro culture. Next, we investigated whether multipotency of M2

cells was retained during long-term in vitro culture. Indeed, osteogenic, chondrogenic, and adipogenic differentiation could be successfully induced in M2 cells of early (P8), mid (P20), and late (P40) passages (Fig. 2D). We also observed a spontaneous adipogenic differentiation in late-passage cells, however, only when cultured at high cell density (data not shown). Because the M2 cell line was developed from bulk culture and could be of polyclonal origin, we aimed to analyze whether the observed multipotency can be tracked to individual cell clones. We thus performed serial dilutions of late-passage cells (P42) and verified microscopically singlecell state immediately after plating. In total, progeny from 24 individual clones was analyzed with respect to their osteogenic and adipogenic differentiation potential. All clones were able to differentiate toward the adipogenic and osteogenic lineages in vitro (Fig. 2E and data not shown). The chondrogenic differentiation potential of these clones was not analyzed. M2 cells express osteogenic and adipogenic markers in vitro. The expression of genes specific for adipogenic or

osteogenic differentiation was analyzed by qPCR. Adipogenic and osteogenic differentiation was induced in midpassage (P21–24) M2 cells by culture in the respective differentiation media and gene expression was assessed after 12 and 21 days, respectively. As a control, we used cells incubated for 12 or 21 days in growth medium. To show adipogenic differentiation, we measured the mRNA levels of Fabp4 and PParg1, whose expression is upregulated in adipocytes [36–38]. Additionally, we analyzed DLK1 (Pref1), which has been reported to inhibit adipogenesis and is thus downregulated in adipocytes [39]. Indeed, Fabp4 and PParg1 expression was strongly upregulated in adipogenic differentiated M2 cells (Fig. 3A). By contrast, DLK1 was only weakly expressed by undifferentiated M2 cells and expression was completely lost upon adipogenesis (Fig. 3A). To verify osteogenic differentiation, we analyzed osteocalcin, osteopontin, and lbsp (bone sialoprotein), which were reported to be late, osteoblast-specific matrix proteins [40] (Fig. 3B). Indeed, we observed a strong upregulation of mRNA levels of these matrix proteins upon incubation of mid-passage M2 cells in osteogenic medium compared with the control (Fig. 3B). These data indicate that M2 cells are able to differentiate into mature adipocytes and osteoblasts in vitro. Together, these data indicate that M2 is a BMderived stromal cell line, which maintains in vitro multipotency over many in vitro passages.

In vitro osteogenic differentiation of stromal cell line M2 is Runx2 dependent The transcription factor Runx2 is the critical key regulator in osteogenic differentiation and regulates the expression of bone matrix proteins in terminally differentiated osteoblasts [41]. To analyze whether the Runx2-controlled

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osteogenic differentiation cascade is present in M2 cells, we transfected the mid-passage (P21–24) M2 cells with a Runx2-specific siRNA to deplete mRNA and Runx2 protein. Quantitative real-time PCR of cells transfected with the Runx2 siRNA 2 days post-transfection confirmed a reduction of Runx2 mRNA to about 40% of control siRNA (siNT) transfected, undifferentiated M2 cells (Fig. 3C). Subsequently, osteogenic differentiation was induced 2 days posttransfection by addition of osteogenic medium. After 21 days the expression of osteoblast-specific matrix proteins osteocalcin, osteopontin, and lbsp in siRunx2-transfected and control-transfected M2 cells was monitored by qPCR. Indeed, Runx2 depletion resulted in a 75% reduced expression of osteocalcin and Ibsp, compared with control cells. By contrast, osteopontin expression was unaffected by the initial Runx2 knockdown (Fig 3C). This was surprising as osteopontin expression has been reported to be dependent on Runx2 [42]. However, the incomplete knockdown might result in a residual Runx2 protein level sufficient to induce osteopontin expression.

The mouse stromal cell line M2 can generate heterotopic ossicles in vivo BMSCs generate heterotopic ossicles and are capable of establishing the hematopoietic environment in vivo (Friedenstein) [7]. We thus evaluated the capacity of M2 cells to establish bone and HME in mice. To assess the osteogenic potential, scaffolds of osteoconductive material (HA/TCP) loaded with M2 cells in early (P8–10) or late (P40–44) passages were generated [7,31,32]. When transplanted subcutaneously into immunocompromised mice, early and late-passage-derived M2 cell strains were able to generate complete heterotopic ossicles establishing full bone, adipocytes, vessels, and host-derived, heterotopic hematopoietic tissue clusters (Fig. 4), proving that the M2 cell line possesses full skeletal stem cell characteristics in vivo.

Discussion BM is the primary source of progenitor stromal cells, also called skeletal stem cells, required for in vivo bone formation. Here, we describe the isolation and establishment of a stable, MSC line from adult mouse BM harboring skeletal stem cell characteristics. Unlike human BMSCs, which typically loose both proliferative capacity and differentiation potential after several in vitro passages, the murine BM stromal cell line described here is a stable source for detailed studies on mechanisms of osteogenesis at a molecular level. The cell isolation was based on the protocol established by Nadri et al. [26,27], and already used with success by others [43]. This method employs frequent medium changes during the initial phase of culture to separate hematopoietic from stromal cells. Indeed, we could not detect expression of the hematopoietic markers CD45 and CD11b in the obtained cell population or the established cell lines, assuring that the isolation and maintenance of stromal cells was successful. Subsequently to first passaging, cells stopped growing and entered a phase of quiescence. After passing through this dormant state, the surviving cells entered a rapid growth phase. This phenomenon of spontaneous

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FIG. 3. Osteogenic differentiation of M2 cells is Runx2 dependent. Mid-passage M2 cells (P21–24) were cultured in vitro in adipogenic (A) or osteogenic (B) media. RNA was extracted and levels of adipogenic marker proteins Fabp4, DLK1, and PParg1 (A) and osteogenic marker proteins osteocalcin, osteopontin, and lbsp were measured by quantitative polymerase chain reaction (qPCR) analysis. As negative controls, cells cultured in a-MEM supplemented with 15% FCS were used. In all cases, the relative expression was calculated by normalization to GAPDH using the DCq method (n = 3, error bars represent SEM). (C) Mid-passage M2 cells (P21–24) were nucleofected with a control siRNA (siNT) or with an siRNA specific for mouse Runx2. Three days post-transfection, RNA was extracted to measure the level of Runx2 (left). Levels of osteocalcin, osteopontin, and lbsp were assessed from siNT- and siRunx2-nucleofected cells cultured for 21 days in osteogenic media. The fold expression was calculated by normalization to GAPDH using the DDCq method (right). Shown is the mean of four independent experiments; error bars represent SEM.

immortalization has been reported before and is known for rodent cells [44]. Parrinello et al. could show that atmospheric oxygen induces oxidative stress in primary mouse fibroblasts resulting in senescence [45]. Primary murine embryonic fibroblast (MEF) can bypass the proliferation block by a spontaneous acquisition of a p53 mutation. This allows outgrowth of the cell that lost function of the tumor suppressor and expansion of the subpopulation into an immortalized cell line [46,47]. Indeed, immortal MEF lines typically harbor either a p53 mutation or loss of p19 [48]. Accordingly, Phinney and colleagues could show that, under atmospheric oxygen, the poor growth and growth arrest of primary BMSCs is due to oxidative stress and is p53 depen-

dent [34]. Indeed, we could also detect high levels of p53 in early, mid-, and late-passage M2 cells, indicating that accumulation of p53 is most likely due to a loss-of-function mutation. Of note, the loss of p53 is necessary for spontaneous cellular immortalization but not sufficient to induce malignant transformation. In line with this, late-passage M2 cells did not form tumors when subcutaneously injected in nude mice (data not shown) nor showed anchorage-independent growth indicating that malignant transformation did not occur. To date, there is no agreement on a unique marker panel that characterizes cultured mBMSCs. The variances detected in surface antigen expression are likely due to different methods of isolation, cultivation, as well as the

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FIG. 4. Development of heterotopic bone and BM in transplants of M2 cells. (A–H) Eight weeks post-transplantation, both M2 cell strains in early (P8–10) and in late (P40–44) passages have formed abundant quantities of new full bone along with mesenchymal fibroblastic tissue (ft; in C, H). Bone contains differentiated osteoblasts (ob), osteocytes (oc), and adipocytes (ad; in D, H). Large caliber, branching sinusoids have formed within the fibroblastic tissue (in C, H). Hematopoietic cell clusters (hem; in B, F and H) are detectable around sinusoids (sin). hac, hydroxyapatite carrier.

different genetic backgrounds of the mouse strains used. Additionally, recent studies demonstrate the existence of different subsets of stromal cells in mouse BM in vivo [49]. However, several markers have been recently proposed to identify adult MSCs. For example, a subset of Sca1/ PDGFRa-positive mBMSCs were able to differentiate in the adipogenic, chondrogenic, and osteogenic lineages. When transplanted into mice, these cells differentiated into osteoblasts and adipocytes [50]. Further, it was demonstrated that nestin expression defines a pool of cells that display BMSC characteristics in vitro and in vivo and support the hematopoietic niche in vivo [51]. The transcription factor Mx1 was described as a marker of a subpopulation of BMSCs, which are tripotent in vitro but restricted to the osteogenic lineage in vivo [52]. Most of the surface markers expressed on the stromal cell line M2 have been described by others using mouse MSCs (CD29 + CD44 + CD105 +

CD106 + Sca1 + ) (reviewed in Ref. [6]). Additionally, we could show that the stromal cell line M2 expresses Runx2, the master regulator of osteogenesis [41], and that the loss of this transcription factor impedes osteogenesis in vitro. This is in line with findings that BMSC cultures have a stable expression of Runx2 [53]. Noteworthy, osteogenic markers, such as osteopontin and lbsp, are expressed in the stromal cell line M2. This is especially interesting because expression of lbsp has been proposed to be a predictor of osteogenic capacity in vivo [53]. In vivo transplantation of stromal cells verifies their osteogenic capacity by the formation of bone [32,54]. In the past, several BM-derived MSC lines of mouse origin have been established [53,55–62]; however, only some studies [53,56,59,62] demonstrated the generation of bone upon transplantation of cells in mice. BMC9 cells are conditionally immortalized and, upon subcutaneous implantation on a

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carrier of porous calcium phosphate ceramics, they formed bone in nude mice [56]. D1 cells, isolated from a Balb/C mouse, spontaneously differentiated into osteoblasts in vitro and formed vascularized bone without the presence of additional scaffold material [59]. Satomura et al. established five clonal stromal cell lines and three of these clones formed bone in vivo [53]. It has been reported that transplantation of BM stroma generates bone and establishes the hematopoietic environment at these sites [63]. Other studies revealed that the ability to generate bone tissue in vivo does not necessarily correlate with an establishment of hematopoiesis [7]. To our knowledge, the formation of hematopoiesis-accommodating bone upon transplantation of a stable mouse stromal cell line described here was not demonstrated so far. A unique feature of the M2 cell line described here, namely CD146 expression, is in accordance with its hematopoiesis-inducing activity and resembles the phenotype of human skeletal stem cells. Thus, high expression of CD146 distinguishes human BM stromal stem cells (skeletal stem cells) from other osteogenic progenitors and is a prerequisite for these cells to form bone and support hematopoiesis in vivo [7]. In line with this, CD146-positive cells could be identified in perivascular adventitial positions close to venous sinusoids but also as scattered fibroblasts in the marrow cavity in sections of human trabecular bone [64]. Mouse-BM-derived CD146 + CD105 + cells have been recently shown to possess osteogenic potential in vivo, supporting the specific role of CD146 + cells for murine osteogenesis, too [65]. Other studies using mouse BM tissue provide further evidence that a subset of CD105 + CD90 skeletal progenitors initiate formation of ectopic bones with marrow cavities containing hematopoietic stem cells [49,66]. Of relevance, the multipotent M2 cell line expresses CD146 and CD105 at high levels in early, mid, and late passages but lacks the expression of CD90. The expression profile of the early and late-passage M2 cells is thus in line with their capacity to form heterotopic ossicles and BM in nude mice. However, whether expression of CD146 and/or CD105 is of functional relevance and a prerequisite for the formation of the hematopoietic tissue clusters in vivo presently remains an open question. This can now be addressed with the M2 cell line by experimental silencing of the respective genes. In vivo models for bone regeneration have been applied to small and large animals [17,67]. For example, engineered constructs have been successfully used in small-animal, nonunion critical-sized femur defect models [18,19]. Cellbased approaches to bone regeneration are in clinical use and results of pilot trials are promising (reviewed in Refs. [16,68]); however, the repair of large bone defects requires huge quantities of BM-derived stromal progenitor cells expanded in vitro. Alternatively, the application of growth factors and antagonists of CXCR4 inducing in vivo stem cell mobilization is discussed as a promising approach for bone healing [69]. In both cases, a substantial understanding of skeletal stem cell biology is prerequisite for routine clinical use. The cell line described here now provides a model to experimentally approach these issues. In conclusion, the multipotent M2 cell line established here is a murine stromal cell line harboring skeletal stem cell properties in vitro and in vivo, evident from heterotopic bone formation and induction of hematopoiesis. Long-term prolif-

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eration potential under retention of a stable phenotype and multipotent differentiation capacity indicate that this cell line is a versatile tool to study tissue-specific differentiation pathways in vitro and verify/falsify these findings in vivo, thus complementing the scientific questions that can be addressed with human MSCs. As shown here, M2 cells are a useful model to study in detail osteogenesis in vivo, thereby aiding to improve current therapeutic strategies in bone regeneration.

Acknowledgments This work was supported by the German Federal Ministry of Education and Research (BMBF-SysTec, project no. 0315506A) and the Ministry of Science, Research and the Arts Baden-Wu¨rttemberg (project Biomimetic matrices). Conference abstract published in Journal of Stem Cells and Regenerative Medicine, Vol. VIII, Issue: 2 (Special Issue): ( JSRM Code: 008020700069).

Author Disclosure Statement No competing financial interests exist.

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Address correspondence to: Dr. Angelika Hausser Institute of Cell Biology and Immunology University of Stuttgart Allmandring 31 70569 Stuttgart Germany E-mail: [email protected] Received for publication August 5, 2013 Accepted after revision January 9, 2014 Prepublished on Liebert Instant Online January 9, 2014