Supporting materials

Isolation and cell culture of human mesenchymal stem cells.
After obtaining signed informed consent, mesenchymal stem cells and mesenchymal cells were harvested from human donors with the approval of the Ethics Committee of Keio University School of Medicine (approval number: 13-1 and 12-1) and the Ethics Committee of National Research Institute for Child Health and Development, Tokyo (approval number: 25, 26, 27, 49, 55, 88, 89, 90, 91, 146, and 156). Cells were resuspended in growth medium (the M061101 medium, MED SHIROTORI Co., Ltd., Tokyo; MSCGM, PT-3238 and PT-4105, Cambrex Bio Science Walkersville, inc. Walkersville, MD) and cultured as previously described (1-3). Cells were deposited to the RIKEN CELL BANK (http://www.brc.riken.go.jp/lab/cell/english/guide.shtml) and the Health Science Research Resources Bank (http://www.jhsf.or.jp/English/index_gc.html).

G-banding karyotypic analysis and spectral karyotyping (SKY) analysis.
We performed a karyotypic analysis (G-banding and SKY) of both the mesenchymal stem cells and mesenchymal cells (Fig. 1, 2. A: Yub10F; B: Yub623; C: Yub625; D: Yub627; E: UCB302; F: UCB408; G: UCB432; H: PL502; I: PL503; J: PL504; K: PL505; L: PL506; M, N: PL507). Metaphase spreads were prepared from at least 50 cells treated with Colcemid (Karyo Max, Gibco Co. BRL, 100 ng/ml for 6 hours). We performed a standard G-banding karyotypic analysis on metaphase spreads for each population. SKY permits a detailed analysis of all complex markers and provides insights into their involvement in subsequent rearrangements (4). No genomic abnormalities were found in the cells analysed as shown by G-banding (Fig. 1) and SKY analysis (Fig. 2).

Cell transplantation and in vivo tumor formation assay
Freshly collected confluent cells (106 cells) were subcutaneously and intramuscularly injected into Balb/c nu/nu mice (Sankyo Laboratory, Hamamatsu, Japan) and NOD/SCID/IL-2 receptor common gamma knockout (NOG) mice. Animals were monitored for malignant transformation of the injected cells after inoculation and then sacrificed by cervical location. Nor did the cells grafted into the subcutaneous and muscle tissue of nude mice and NOG mice produce tumors. Furthermore, the cells did not undergo malignant transformation in vitro: They stopped dividing after reaching confluence, and they did not form any foci after confluence in vitro.

Telomerase activity and telomere length in human mesenchymal stem cells.
Telomerase activity was determined with a TRAP assay kit “Telo TAGGG telomerase PCR ELISA plus” (Roche, Indianapolis, IN) according to the manufacturer’s instructions. No telomerase activity was detected by the TRAP assay in the mesenchymal stem cells and mesenchymal cells. For the telomere length assay, genomic DNA was extracted from cultured cells. Restriction enzyme digestion of genomic DNA was carried out with Hinf I and Rsa l. The fragments obtained were resolved on 0.7% agarose gels, transferred to a Hybond N membrane (Amersham, UK), and hybridized with digoxigenin (DIG)-labeled (TTAGGG)3 probe. The membrane was then incubated with anti-DIG alkaline phosphatase and detection was performed with a chemiluminescence solution. The size range and intensity were determined with X-ray film. The telomere length of the mesenchymal stem cells and mesenchymal cells decreased with the number of PDs.

Reference
1. Mori, T., T. Kiyono, H. Imabayashi, Y. Takeda, K. Tsuchiya, S. Miyoshi, H. Makino, K. Matsumoto, H. Saito, S. Ogawa, M. Sakamoto, J.-i. Hata, and A. Umezawa, Combination of hTERT and Bmi-1, E6 or E7 induce prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential. Mol Cell Biol, 25: 5183–5195, 2005.
2. Imabayashi, H., T. Mori, S. Gojo, T. Kiyono, T. Sugiyama, R. Irie, T. Isogai, J. Hata, Y. Toyama, and A. Umezawa, Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis. Exp Cell Res, 288: 35-50, 2003.
3. Terai, M., T. Uyama, T. Sugiki, X.K. Li, A. Umezawa, and T. Kiyono, Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16INK4a/RB Braking Pathway. Mol Biol Cell, 16: 1491-1499, 2005.
4. Schrock, E., T. Veldman, H. Padilla-Nash, Y. Ning, J. Spurbeck, S. Jalal, L.G. Shaffer, P. Papenhausen, C. Kozma, M.C. Phelan, E. Kjeldsen, S.A. Schonberg, P. O'Brien, L. Biesecker, S. du Manoir, and T. Ried, Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities. Hum Genet, 101: 255-62, 1997.