Two MSCs: Marrow stromal cells and mesenchymal stem cells

Two MSCs: Marrow stromal cells and mesenchymal stem cells

Marrow stromal cells are able to generate a series of terminally differentiated cells in vitro. However, most of these experiments are performed with heterogeneous stromal cells, which had been obtained by adherence to plastic culture dishes. Since it is demonstrated that bone marrow-derived stromal cells are purified to a homogeneous population that met the criteria for nonhematopoietic stem cells, these cells have been termed mesenchymal stem cells because they generate an array of cells, defined as mesenchymal cells. In contrast, mesenchymal stem cells are multipotent cells capable of differentiating into cells of mesoderm-origin regardless of cell sources. Mesenchymal stem cells can be recovered from a variety of other adult tissues such as fat, muscle, menstrual blood, endometrium, placenta, umbilical cord, cord blood, skin, and eye. The terms mesenchymal stem cell and stromal cell, both of which are most plausible cells for regenerative medicine, have been used interchangeably in emerging literature; we here re-organize our understanding on two MSC biology and further clinical trail in this review.

Introduction
http://blogs.yahoo.co.jp/akihiroumezawa/19883767.html

Shortening of human cell life span by platelet-derived growth factor via induction of p16ink4a

Shortening of human cell life span by platelet-derived growth factor via induction of p16ink4a

Cells vary in their requirements in a cell type-specific manner, and cells can therefore be categorized or defined by their requirements. In this study, we attempted to prolong life span of a marrow-derived mesenchymal stem cell using a combination of growth factors and hormones. Epidermal growth factor, platelet-derived growth factor-BB, acidic fibroblast growth factor (FGF), basic FGF, and leukemia inhibitory factor were found to be key factors for the mesenchymal stem cell proliferation. The combination of these growth factors showed extremely strong mitogenic activity, and simultaneously induced the expression of cyclin-dependent kinase inhibitor p16ink4a protein and premature senescence more rapidly than serum-supported culture conditions. The induction of p16ink4a by growth factors was mediated through the mitogen-activated protein kinase (MAPK) cascade. Excess growth stimulation by growth factors was thus one of the culture stress signals and a trigger of premature senescence at least in human cells.

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.

Can a human adult stem cell spontaneously transform?

ヒト細胞に関する、培養過程での癌化の可能性について言及しました。下の文章には、多少間違い（染色体異常の出現率）がありますが、基本的な考え方は問題ありません。




Can a human adult stem cell spontaneously transform?

Correspondence re: Rubio, D., et al., Spontaneous human adult cell transformation. Cancer Res., 65: 3035-3039, 2005.


In vitro "Human adult stem cell transformation" is a major concern for those working on regenerative medicine or cell-based therapy. An article by Rubio et al. provides a caution that human mesenchymal stem cells undergo spontaneous transformation following long-term in vitro culture, i.e., 4 to 5 months. We agree that this was the first report of in vitro spontaneous transformation of human adult stem cells. Theoretically, normal human cells including mesenchymal stem cells have been shown to undergo a limited number of divisions in culture and then enter a non-dividing state referred to as "senescence" and do not spontaneously transform during culture period.

We have performed a similar experiment to determine if such chromosomal abnormality is detected after long-term in vitro culture of human mesenchymal stem cells or mesenchymal cells derived from bone marrow, umbilical cord, planceta, endometrium, and menstrual blood. Karyotypic analysis (G-banding and SKY) revealed that no genomic abnormalities except were found in the mesenchymal cells employed in this study. These abnormalities included translocations, a deletion, other rearrangements, and polyploidy. Our study raised some different points concerning chromosomal abnormality after long-term culture except for clonally inherited minor abnormalities. In addition, no telomerase activity was detected by the TRAP assay in the mesenchymal cells at all of the PDs tested, unlike in the case of multipotent adult progenitor cells (Verfaillie, et al.). Likewise, the telomere length of the mesenchymal cells decreased with the number of PDs.

We also monitored for in vivo malignant transformation of the mesenchymal cells for 3 months after inoculation and then sacrificed by cervical location. The cells did not undergo malignant transformation. They stopped dividing after reaching confluence, and they did not form any foci after confluence in vitro. Nor did the cells grafted into the subcutaneous and muscle tissue of nude mice or immunodeficient NOD/SCID/IL-2 receptor gamma-/- mice produce tumors, at least during the monitoring period (more than 100 days). Immunohistochemical analysis using an antibody against human specific vimentin revealed that injected mesenchymal cells survived but did not proliferate at the injection sites.

In the light that biosafety of mesenchymal stem cells as a source of cell-based therapy is very important, we, off course, should keep in mind that even a unlikely possiblity of human cell transformation during ex vivo expansion cannot be neglected and the human cell transformation may depend on culture condition employed in each laboratory or cell processing center. However, it should not escaped from our idea, i.e. almost zero expectation of spontaneous transformation of human adult stem cells after long-term ex vivo cultivation, obtained from our own results using cells derived from bone marrow, umbilical cord, planceta, endometrium, and menstrual blood.

We would not say the zero risk of spontaneous transformation during cultivation of mesenchymal stem cells and even one litterature (ref. 1) often win the title in this type of discussion to the controversy. However, we here emphasize that possiblity of spontaneous transformation in cultured human mesenchymal stem cell without treatment of storng mutagen or transfection of oncogenes is extremely low or considered to be nearly zero from the scientific viewpoint and tons of literature.