| Project/Area Number |
18390414
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Orthopaedic surgery
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
TOGUCHIDA Junya Kyoto University, Institute for Frontier Medical Sciences, Professor (40273502)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Takashi Kyoto University, Graduate School of Medicine, Professor (10201675)
AOYAMA Tomoki Kyoto University, Institute for Frontier Medical Sciences, Assistant Professor (90378886)
|
|---|
| Project Period (FY) |
2006 – 2007
|
| Project Status |
Completed (Fiscal Year 2007)
|
| Budget Amount *help |
¥16,360,000 (Direct Cost: ¥14,800,000、Indirect Cost: ¥1,560,000)
Fiscal Year 2007: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000)
Fiscal Year 2006: ¥9,600,000 (Direct Cost: ¥9,600,000)
|
|---|
| Keywords | Mesenchymal stem cell / Transformation / Mutation / Methylation / p16 / 発現誘導システム |
|---|
| Research Abstract |
Clinical applications of mesenchymal stem cells (MSC), one of tissue stem cells with multidirectional differentiation potential, have been conducted in a various fields of regeneration medicine. Transformation of transplanted MSC-derived cells is the most serious complication, for which no definite guideline has been established to monitor the process. In this research project, we aimed to establish the proper monitoring system to detect cells with genomic or epigenomic alterations at the earliest stage. At first, we have analyzed the growth profile of 29 cases of bone-marrow derived human MSCs. Median value of the culture period and the final population doubling (PD) was 151 days and 27 PD, respectively. The average length of telomere at the initial passage and the age of donor correlated with the number of final PD. Among cell cycle regulators, the expression level of p16 was closely associated with the number of PD and the morphological sign of cellular senescence. The inhibition of p16 expression successfully enabled cells to regain the growth potential and escape from senescence. Among 29 cases, the silencing of p16 expression took place during in vitro expansion in four cases, which was caused by the methylation of the core promoter region. One of these four cases, which continued to proliferate over 300 days, contained a chromosomal aberration. These results indicated the methylation of the p16 gene should be monitored as an early epigenetic alteration in ex vivo culture of MSC. Then we have established the rapid and sensitive method to detect the methylation of p16 gene by the combination of methylation specific PCR and Real time PCR detector. The entire process will be finished within 12hours and the sensitivity was 0.01%, meaning one cell with methylated p16 gene could be detected among 10,000 cells with unmethylated p16 gene.
|
