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.
Subject(s)
Humans , Induced Pluripotent Stem Cells , Sirolimus/metabolism , Caspase 9/metabolism , RNA, Guide, CRISPR-Cas Systems , Pluripotent Stem Cells/metabolism , Cell Differentiation , Puromycin/metabolismABSTRACT
El trasplante de órganos, uno de los avances más sorprendentes de la medicina junto con el desarrollo de agentes inmunosupresores cada vez más eficaces, modificó el curso de enfermedades terminales devastadoras. Sin embargo, esta práctica médica nos enfrenta a una población cada vez más longeva que padece las complicaciones secundarias de la inmunosupresión crónica, necesaria para asegurar la supervivencia del injerto. La elevada incidencia de neoplasias interna y, en particular, cutáneas plantea la necesidad de un manejo interdisciplinario. En este sentido, el médico dermatólogo desempeña un papel protagónico en la prevención, el diagnóstico y el tratamiento de estos procesos malignos.