Preparation of MEF-ECM-coated wells
MEF was isolated from 13.5 dpc embryos of C57BL/6 × DBA/2 hybrid background and maintained as described . For preparation of MEF-ECM-coated wells, confluent passage one MEF cultures in 6 well plates were allowed to continue for another day. The cultured cells were treated with 20 mM NH3 and 0.5% Triton X-100 for 5 min at room temperature, the lysis buffer was removed, and the plates were placed without cover for 20 min to allow the ECM adhere firmly to the substrata of the wells. The substratum was washed five times with Hanks' balanced salt solution (HBSS; Invitrogen) and stored at 4°C for subsequent uses.
Isolation and expansion of mMSCs
Young B6D2F1 mice (3-5 weeks old) were sacrificed by cervical dislocation using protocol approved by the Institutional Animal Committee. Whole bone marrow was harvested by flushing femurs and tibiae as described . The marrow cells were plated into 6-well cell culture plate at a density of 2 × 105 cells/cm2 in 2.5 ml DMEM containing 20% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin antibiotics (Invitrogen). In some experiments, a combination of antioxidants (800 μM alpha -phenyl-t-butyl nitrone (PBN) and 5 mM N-acetylcysteine (NAC)) were added in the medium. In others, bone marrow cells were seeded directly onto MEF-ECM-coated wells. Cells were cultured at an atmosphere of 2% O2 (2% O2, 93% N2, 5% CO2) at 37°C. Non-adherent cells were removed after 24 hours by washing twice with phosphate-buffered saline (PBS), and fresh medium added. At day 5-6, adherent cells were treated with 0.25% trypsin/0.02% EDTA (Invitrogen) for 3 min at 37°C. The cells lifted within 3 min were collected and replated at 1000 cells/cm2 onto uncoated 6-well plates or plates coated with MEF-ECM. The medium was replaced every 2-3 days until cells reached 90% confluence. Subsequent passaging was carried out using the same conditions.
For initial experiments to optimize culture conditions under 20% O2, the marrow cells were plated at a higher density of 2 × 106 cells/cm2, and subcultures were performed at a split ratio of 1:3 when cells reached 90% confluence.
For immunodepletion experiments, retrieved primary mMSCs were stained with PE-conjugated anti-CD45 and anti-Ter119 antibodies (Miltenyi Biotec), and then stained with anti-PE magnetic microbeads (Miltenyi Biotec) and immunodepeleted according to the manufacturer's instruction. Cells negative for CD45 and Ter119 were seeded onto 6-well plates at 10, 000 cells/cm2.
In vitro Differentiation of mMSCs
To induce osteogenic differentiation, 70-80% confluent mMSCs were incubated in osteogenic medium (DMEM supplemented with 20% FBS, 20 mM β-glycerol phosphate, 1 nM dexamethasone and 0.5 μM ascorbate 2-phosphate) for one week with medium changed once, based on a method with slight modification . The cells were fixed with 3.7% paraformaldehyde and stained with Alizarin Red (pH 4.1).
To induce adipogenic differentiation, 70-80% confluent mMSCs were incubated in adipogenic medium (DMEM supplemented with 20% FBS, 0.5 μM hydrocortisone, 0.5 mM isobutylmethylxanthine (IBMX) and 60 μM indomethacin , for one week and the medium was changed once. The cells were fixed in 3.7% paraformaldehyde and stained with 0.5% Oil Red in propylene glycol.
Chondrogenic differentiation was performed in 70-80% confluent monolayer or in a pellet  formed by centrifugation of 2 × 105 mMSCs. The chondrogenic medium consisted of high-glucose DMEM supplemented with 500 ng/ml bone morphogenetic protein-2 (BMP-2; Peprotech), 10 ng/ml transforming growth factor β3 (TGF-β3; Peprotech), 10-7 M dexamethasone, 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate, 1 × insulin-transferrin-selenium and 1 × lenolenic acid-bovine serum albumin (LA-BSA) (Sigma). The monolayers or pellets were incubated in this medium for 1-2 weeks with medium change twice a week. The monolayers were fixed and stained with Alcian blue. The pellets were embedded in tissue-freezing medium (Leica), sectioned into 8-μm slices, fixed with cold acetone and stained with Alcian blue.
Immunostaining and FACS analysis
For flow cytometry analysis of mMSCs epitope, following PE conjugated antibodies were used: CD31, CD34, Flk-1, Sca-1, CD44 (eBioscience), CD45, CD11b (Miltenyi Biotec). Cells were divided into aliquots (5 × 104 each), stained with PE-conjugated antibodies at final concentration of 2 μg/ml at room temperature for 30 min, washed with PBS and analyzed by flow cytometry. For flow cytometry analysis of γ-H2AX, cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked in 3% normal goat serum (Sigma) in PBS. After incubation with 2 μg/ml anti-γ-H2AX antibody (Upstate, 05-636) at 4 °C overnight, the cells were washed and stained with Alexa 488-conjugated goat anti-mouse IgG (1:100 v/v, Molecular Probes) for 1 hour, and washed and analyzed as above. Immunostaining of pluipotency-associated markers was performed with following primary antibodies: Oct-4 (sc5279, Santa Cruz, CA), Nanog (Abcam, ab10626) and SSEA1 (DSHB, MC-480), and secondary antibodies: Texas red conjugated anti-mouse IgG (Vector, TI-2000, CA) or Alexa fluor 488 goat anti-mouse IgM (Molecular Probes). Immunofluorescence was observed and imaged using a Leica microscope equipped with epi-fluorescence and appropriate filters.
Intracellular reactive oxygen species (ROS) was determined by flow cytometry using 5-b-chloromethyl-2'7'-dichlorodihydrofluorescin diacetate (CM-H2DCFDA; Molecular Probes) [49, 57]. mMSCs were harvested by trypsin-EDTA and incubated in serum-free medium containing 10 μM CM-H2DCFDA for 30 min at 37°C. Then the cells were washed, resuspended, and analyzed by flow cytometry. The median fluorescence intensity was used to estimate intracellular ROS levels.
Micronucleus (MN) assay
Micronucleus assay was performed as described with slight modification . Cultured mMSCs at 50-80% confluence were incubated with 4.5 μg/ml cytochalasin-B (Sigma) for 24 hours and harvested. After a brief hypotonic treatment in 0.075 M KCl, cells were fixed in methanol/acetic acid (5:1), stained with 5 μg/ml Hoechst 33342 (Molecular Probes) in PBS and mounted for immunofluorescence microscopy. Frequency of micronucleus was assessed on 1000 binucleated cells for each culture in three independent experiments.
Karyotype analysis and telomere fluorescence in situ hybridization (FISH)
mMSCs at 50-80% confluence were incubated with 0.1 μg/ml colcemid (Sigma) for 5 h. The cells were incubated in hypotonic solution (0.075 M KCl), fixed in methanol/acetic acid (3:1) and spread onto slides. Cell spreads were stained with 5 μg/ml Hoechst 33342 (Molecular Probes) in PBS, examined by immunofluorescence microscope, and number of chromosomes were counted. FISH with FITC-labelled telomere-specific (CCCTAA)3 peptide nucleic acid (PNA) probe (Applied Biosystems, Framingham, MA, USA) was performed as described .
2 × 106 mMSCs were subcutaneously injected into 4 weeks old BALB/c nude mice. The animals were monitored for tumor formation for two months. Formed tumors were excised, fixed in formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin.
Generation of iPS cells
iPS cells were induced by transduction of four Yamanaka's factors according to the protocol of Okita et al.  with slight modifications. Briefly, pMXs-based retroviral vectors (pMXs-Sox2, Klf4, Oct4, c-Myc) were introduced into Plat-E cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. The viral supernatant were collected, filtered (0.45 μm pore size) and supplemented with 8 μg/ml polybrene before use. The day before infection, primary mMSCs and tail-tip fibroblasts (TTFs) were plated at 5 × 104 cells/well into 6-well plates coated with MEF-ECM or gelatin. The mMSCs and TTFs were infected with virus/polybrene-containing supernatant twice within forty eight hours. Three days after infection, the cells were replated in ES medium (knock-out DMEM supplemented with 20% FBS, 1000 U/ml LIF, 0.1 mM β-mercaptoethanol, 1 mM L-glutamine and 0.1 mM nonessential amino acids). The cells were passaged at day 5 onto MEF feeders and the medium was changed every day. ES-like colonies were picked by day 10 to 13, and passaged onto MEF feeders.
Alkaline phosphatase staining
Alkaline phosphatase assay was performed using the Vector Blue Aalkaline Phosphatase Substrate Kit (Vector Laboratories, DAKO, Carpinteria, CA) according to manufacturer's instruction.
The data were analyzed by Student's t-test. Results are expressed as mean ± SD. A value of p < 0.05 was considered significant by comparison. All experiments were performed at least three times.