Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy / 生物工程学报
Chinese Journal of Biotechnology
; (12): 4098-4107, 2023.
Article
em Zh
| WPRIM
| ID: wpr-1008014
Biblioteca responsável:
WPRO
ABSTRACT
Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.
Palavras-chave
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Puromicina
/
Diferenciação Celular
/
Sirolimo
/
Células-Tronco Pluripotentes
/
Caspase 9
/
Células-Tronco Pluripotentes Induzidas
/
RNA Guia de Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2023
Tipo de documento:
Article