Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.
How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.
Institute of Reproduction and Stem Cell Engineering, Central South University, 8 Luyun Road, Changsha, 410000, Hunan, China.
Di
D
Zhou
Ge
G
Lin
Si-Cong
SC
Zeng
Bo
B
Xiong
Ping-Yuan
PY
Xie
De-Hua
DH
Cheng
Qing
Q
Zheng
Qi
Q
Ouyang
Xiao-Ying
XY
Zhou
Wei-Ling
WL
Tang
Yi
Y
Sun
Guang-Ying
GY
Lu
Guang-Xiu
GX
Lu
D017209:Apoptosis / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000473:pathology; D042822:Genomic Instability; D006801:Humans; D007621:Karyotyping; D016685:Mitomycin / Q000032:analysis / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
D016685:Mitomycin