Isolation of murine mesenchymal progenitor cells
All animals used for progenitor cell preparation were treated according to institutional guidelines. Preparation of murine progenitor cells was performed after cervical dislocation of NMRI mice (sex: female, age: 10 months; Charles River Laboratories Inc., Wilmington, DE, USA). Progenitor cells derived from PAT were obtained after laparotomy and surgical preparation of the retroperitoneal and perirenal tissue. Murine MST specimens were obtained after separation of the costo-sternal joints, luxation of the breast bone and surgical preparation of the anterior mediastinum. Murine PAT and MST samples were removed and separated from adjacent connective tissue. Tissue samples were digested with basal media supplemented with 1 mg/ml collagenase A (Sigma, München, Germany) for 45 min at 37°C, sieved, centrifuged, and washed with basal media. The isolation of BM derived stem/progenitor cells was performed by preparation of the lower extremities and exposition of the bone marrow cavity. Marrow cells were flushed out of the femur as well as the tibia using a 27-gauge needle with 10 ml of heparinised (5000 IE) basal media. The number of cells was determined and the cell suspension was plated onto 25 cm2 tissue culture flasks (TPP, Trasadingen, Switzerland). The approximate number of cells obtained directly after isolation was 4 × 105 cells (PAT), 2.5 × 105 cells (MST) and 1.5 × 105 cells (BM). Non adherent cells were removed by the first medium change after two days. Single colonies of adherent fibroblast-like cells were first visible after 72 hours of cultivation. All cultivations were performed at 37°C and 5% CO2.
Cultivation of murine mesenchymal progenitor cells
Progenitor cells were cultured in basal medium consisting of Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Paisley, U.K.) supplemented with 1% sodium pyruvate (PAA, Pasching, Austria), 1% L-glutamine (PAA, Pasching, Austria), 1% MEM non-essential amino acids (Invitrogen, Paisley, U.K.), 1% penicillin/streptomycin (PAA, Pasching, Austria) and 10% fetal bovine serum (PAA, Pasching, Austria). Once adherent cells reached approximately 80-90% confluence, they were washed with phosphate buffered saline (PBS), trypsinized, and centrifuged for 5 min at 250 g. The cells were plated at a density of 1 × 104/cm2 and passaged every 4 to 10 days up to passage 15. Karyotype analysis in early (passage p5) as well as later (BM: passage p15 as well as PAT and MST: p13; see Additional file 2) stages was performed using standard methods for G- and C-Banding . When reaching confluence, the cells were phenotyped using FACS analysis (passage p4) or replated for differentiation. Adipogenic, chondrogenic and osteogenic differentiation was analyzed in three independent samples (passages p5, p9 and p12) per experimental group in all three progenitor cell isolates. The daily doubling index was used to determine the proliferation and growth properties of the murine progenitor cells. Murine BM-, PAT- and MST-isolates were plated at a density of 1 × 104 cells per cm2 and the increase in cell number was determined every 24 hours to calculate the daily doubling index.
Differentiation of murine mesenchymal progenitor cells via classical "micro mass body" (MMB) and monolayer cultivation
Chondrogenic differentiation was performed using MMB cultivation. Cells were trypsinized, counted, and basal medium was replaced by chondrogenic induction medium. Aliquots of 2 × 105 cells in 0.5 ml chondrogenic induction medium were centrifuged at 65 g in 15 ml polypropylene conical tubes. Chondrogenic induction medium consisted of basal medium supplemented with 0.1 μM dexamethasone (Merk, Darmstadt, Germany), 300 μM ascorbic acid (Sigma, München, Germany), 1 mM L-proline (Sigma, München, Germany), 10 ng/ml transforming growth factor (TGF) β3, (R&D, Wiesbaden, Germany) and 1% ITS premix (Becton Dickinson, Heidelberg, Germany: 6.25 μg/ml insulin; 6.25 μg/ml transferrin; 6.25 μg/ml selenious acid; 1.25 mg/ml bovine serum albumin; 5.35 mg/ml linoleic acid). Samples of MMBs were taken for RNA-isolation (4 MMB per sample per day), hisotchemical or immunhistochemical analysis (1 MMB per sample per day) during the course of chondrogenic differentiation in three independent samples per experimental group (n = 3). MMBs prepared for histochemical and immunhistochemical staining were embedded in Tissue-Tek O.C.T. (Sakura Finetechnical, Tokyo, Japan), frozen at -80°C and cryosectioned (10 μm) for further analysis. To screen for proteoglycan deposits or marker protein expression within the chondrogenic MMBs, cryosections were fixed and stained with Alcian blue (AB)  or immunostained. Uninduced MMBs were stained as negative controls.
To analyse adipogenic and osteogenic differentiation, isolated progenitor cells were differentiated via monolayer protocols. Adipogenic and osteogenic induction of the progenitor cells was performed at 80-90% confluence. To induce osteogenic differentiation cells were treated with osteogenic medium for 25 days. Osteogenic medium consisted of basal medium supplemented with 0.1 μM dexamethasone (Merk, Darmstadt, Germany), 10 mM β-glycerolphosphate (Sigma, München, Germany) and 300 μM ascorbic acid (Sigma, München, Germany). To induce adipogenic differentiation cells were treated with adipogenic induction medium and adipogenic maintenance medium for 25 days. Induction medium consisted of basal medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX Sigma, München, Germany), 1 μM dexamethasone (Merk, Darmstadt, Germany), 200 μM indomethacin (Sigma, München, Germany) and 2 μM insulin (Sigma, München, Germany). Following a four-day induction period, the adipogenic induction medium was replaced with adipogenic maintenance medium consisting of basal medium supplemented with 2 μM insulin for three days. This cycle was repeated three times and ultimately followed by a four-day period of adipogenic maintenance culture.
Lipid accumulation during adipogenic differentiation was demonstrated by Sudan III staining. Cells were washed with PBS followed by staining with a 0.2% solution of Sudan III (Sigma, München, Germany) in 70% ethanol. Alkaline Phosphatase (AP) activity of progenitor cells differentiating along the osteogenic lineage was demonstrated using protocols for fixation (2.5 ml citrate solution (Sigma, München, Germany), 6.5 ml acetone (Roth, Karlsruhe, Germany), 0.8 ml formaldehyde 37% (Merck, Darmstadt, Germany)) and AP staining (125 μl FRV-alkaline solution (Sigma, München, Germany), 125 μl sodium nitrite solution (Sigma, München, Germany) 125 μl naphthol AS-BI alkaline solution (Sigma, München, Germany), 5.63 ml aqua dest.).
Differentiation of murine progenitor cells via three-dimensional "mesenchymal microsphere" (MMS) cultivation
To compare classical adipogenic and osteogenic monolayer and chondrogenic MMB differentiation with a cell-saving three-dimensional model of in vitro cell differentiation, isolated mesenchymal progenitor cells were differentiated via the MMS protocol (see Fig. 3A). For MMS cultivation 20 μl aliquots of a primary cell suspension at a density of 2.5 × 105 cells/ml were pipetted onto the bottom of a 100 mm bacteriological petri dish (GBO, Essen, Germany). By turning it upside down "hanging droplets" were obtained. To avoid evaporation, a lid of a 60 mm culture dish was filled with PBS and placed within the MMS cultivation chamber. The "hanging droplets" were cultured for six days until the formation of spheroids was visible. On the 6th day of MMS formation, cellular aggregates were flushed down with basal medium and collected using a 100 μl pipette. MMSs were plated onto 60 mm culture dishes (TPP, Trasadingen, Switzerland; 10 MMS per dish for total RNA isolation) and 2-well-chamber slides (B&D, Franklin Lakes, NJ, USA; 4 MMS per well for Sudan III, alkaline phosphatase and AB staining and for immunostaining, respectively). Four days after plating of the MMS, the spheroids firmly adhered and were differentiated using adipogenic, chondrogenic or osteogenic induction media as described above in three independent samples (passages p5, p9 and p12) per experimental group (n = 3). The time point of the application of induction media was denominated as 0 d. During the course of differentiation MMS remained adherent to the cell culture surface and a cellular outgrowth could be observed. The differentiation was analyzed at 0 d and at least at three different time points after induction medium was applied (9 d, 18 d and 25 d).
Quantitative analysis of histochemical staining
To compare monolayer and MMS differentiation of mesenchymal progenitor cells derived from BM, PAT and MST by AP or Sudan III staining, ten areas of 0.77 mm2 for osteogenic differentiation and ten areas of 0.235 mm2 for adipogenic differentiation were quantified per sample per day in three independent samples per experimental group (n = 3). The stained areas were measured in relation to the total analyzed area of cells using ImageJ software (NIH, Bethesda, MD, USA) and quantified in percent.
MMS cultured on chamber slides or MMB cryosections were rinsed three times with PBS, fixed for 5 min with pre-cooled (-20°C) methanol-acetone at 4°C, washed four times with PBS and incubated at room temperature for 30 min with 7.5% bovine serum albumin. Specimens were then incubated for 1 hour with a primary antibody in a humidified chamber at 37°C. Antibodies specific for the following proteins were used (designation, dilution ratio in PBS as well as reference are given in parentheses): collagen type II (II-II-6B3; 1:20; ), collagen type X (XAC9; 1:20; ), osteopontin (MPIIIB101; 1:20; ), bone sialoprotein I+II (WVID1(9C5); 1:20; ). The antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA). After rinsing four times with PBS, slides were incubated for 1 hour at 37°C with either fluorescein isothiocyanate (FITC, Dianova, Hamburg, Germany; 1:200) or cyanine3 (Cy3, Dianova, Hamburg, Germany; 1:600) labelled anti-mouse IgG as well as 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, Taufkirchen, Germany). Slides were washed four times in PBS and briefly washed in distilled water. The method to couple immunostaining with fluorescence in situ hybridization has been previously described . The probes used to detect Sox5 and Sox6 have been described elsewhere . After immunostaining the specimens were embedded in Vectashield mounting medium (Vector, Burlingame, CA, USA) and analyzed with the fluorescence microscope Axioskop (ZEISS, Oberkochen, Germany). Negative controls were performed using only the secondary antibody. In addition, negative controls without the application of induction media were performed showing no differentiation (see Additional file 3).
Mesenchymal progenitor cells differentiated via MMS or MMB were collected at different time points, washed twice with PBS, and total RNA was isolated using a standardized RNA Isolation Kit (Macherey&Nagel, Düren, Germany). The RNA concentrations were determined by measuring the absorbance at 260 and 280 nm. Samples of 500 ng RNA were reverse transcribed using oligo-dT primer and Superscript II reverse transcriptase following the manufacturer's recommendations (Invitrogen, Paisley, U.K.). Aliquots of 1 μl from the reverse transcriptase reactions were used for amplification of transcripts using primers specific for the analyzed genes and Taq polymerase according to the manufacturer's instructions (Fermentas, St.Leon, Germany). Reverse transcriptase reactions were denatured for 2 min at 95°C, followed by amplification for 30-40 cycles of 40 s denaturation at 95°C, 40 s annealing at the primer-specific temperature and 50 s elongation at 72°C. Primers specific for the following genes were used (sequence, annealing temperature, size as well as cycle numbers are given in parentheses): PPARγ (5'-GCC TAA GTT TGA GTT TGC TGT G-3', 5'-TGT CAT CTT CTG GAG CAC CTT-3', 58°C, 226 bp, 36), aP2 (5'-ATG CCT TTG TGG GAA CCT-3', 5'-GCT TGT CAC CAT CTC GTT TT-3', 58°C, 333 bp, 30), adipsin (5'-CTG ACA GCC TTG AGG ACG A-3', 5'-AGA GCC CCA CGT AAC CAC A-3', 58°C, 356 bp, 36), osteopontin (5'-TCA CTC CAA TCG TCC CTA CA-3' 5'-TGC TCA AGT CTG TGT GTT TCC-3', 58°C, 289 bp, 36), osteocalcin (5'-GCA GGA GGG CAA TAA GGT AG-3', 5'-CAG GGC AGA GAG AGA GGA CA-3', 58°C, 267 bp, 36), collagen type II (5'-ACG GTG GCT TCC ACT TCA-3', 5'-TAC ATC ATT GGA GCC CTG GA-3' 58°C, 383 bp, 35), Sox9 (5'-CTC TGG AGG CTG CTG AAC G-3', 5'-TTG TAA TCG GGG TGG TCT TTC TT-3', 60°C, 82 bp, 40), and GAPDH (5'-GGA AGG GCT CAT GAC CAC A-3', 5'-CCG TTC AGC TCT GGG ATG AC-3', 58°C, 164 bp, 30). Electrophoretic separation of PCR products was carried out on 2% agarose gels (2% (w/v) agarose (Roth, Karlsruhe, Germany), 0.7 ng/ml ethidium bromide (Roth, Karlsruhe, Germany)). The fragments were analyzed by computer-assisted densitometry in relation to GAPDH gene expression. The densitometric values of each marker were calculated in relation to GAPDH. From these values the highest of each marker was taken as 100%. Distilled water and no-RT reactions were always included as a negative control.
Statistical analysis was performed using SigmaPlot 2000 software (Systat, Erkrath, Germany) and calculated according to the student's t-test. Probes were analysed in three independent samples per experimental group (n = 3).
Fluorescence activated cell sorting (FACS) of murine mesenchymal progenitor cells
Tyrpsin/EDTA- (0.25%) treated cells were washed twice with FACS buffer (PBS, 1% BSA and 0.1% NaN3) and adjusted to approximately 5 × 105 cells/ml and subsequently stained. A 100 μl cell suspension was incubated with 10 μl phycoerythrin (PE) conjugated monoclonal antibodies (mAbs), 10 μl of FITC conjugated mAbs, 10 μl of allophycocyanin (APC) mAbs or alternatively 10 μl non-conjugated mAbs and a secondary rat anti-mouse IgG-FITC at 4°C for 30 min. To discriminate mesenchymal progenitor cells from cells of hematopoietic origin, isolates were stained for CD34 and CD45. In addition, the following antigens were included to the phenotyping profile: CD29, CD44, CD49d, CD54, CD73, CD81, CD105, CD106, CD140b, CD166 as well as Oct 1/3. Prior to the FACS analysis, all samples were filled up to a total volume of 500 μl with FACS buffer. Cells were analyzed on a Cytomics FC 500 flow cytometer using cytomics CXP software (Beckman Coulter, Krefeld, Germany). At least 10,000 events were acquired and analyzed using a three parametric protocol (FL1, FL2 and FL4). Cell debris and aggregates were excluded by gating (FSC/SSC dotplot). Non-specific isotype-matched controls (IgG1, IgG2a, IgG2b, and IgM) were used to determine background fluorescence. All antibodies used were purchased from Becton Dickinson (Heidelberg, Germany), except CD105, CD166, and Oct 3/4 (RD Systems, Abingdon, U.K.) as well as CD34 and CD140b (eBioscience, San Diego, CA, USA).