Discovery of chemical means to reverse aging and restore cellular function
]. The assays developed in this study, combined with robotics and the increasing power of artificial intelligence, will facilitate increasingly larger screens for genes, biologics, and small molecules that safely reverse mammalian aging, and, given that aging is the single greatest contributor to human disease and suffering, these advances cannot come soon enough.
The human iPSC line AG27602 was used as a positive control for staining of iPSC cell markers and was cultured in mTeSR™ Plus according to manufacturer guidelines.To generate NCC-stable cells, we used FugeneHD for transfection of pLVX-EF1alpha-2xGFP:NES-IRES-2xRFP:NLS , psPAX2 , and pMD2.G into 293FT cells following the provided instructions. The 293FT cell medium was collected two and four days post-transfection and filtered through a 0.45-micron filter.
For cloning of the pLVX-UbC-rtTA-hOSK-Neo vector, the Ngn2:2A:EGFP and PuroR cassettes on pLVX-UbC-rtTA-Ngn2:2A:EGFP were swapped with the hOCT4:2A:hSOX2:2A:hKLF4 and NeoR cassettes, respectively. NCC fibroblasts were transduced with hOSK lentivirus using the same procedure as mentioned earlier to achieve stable hOSK expression. Two days post lentiviral transduction, the cells were cultured in DMEM supplemented with 15% Tet-free FBS, 1% P/S, 0.1 mM β-ME, and 200 μg/ml G418.
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