A protocol for in vitro cell rejuvenation by cyclyc partial reprogramming (Poster)

CANATELLI-MALLAT M; CARRI, NG; LEHMANN M; CHIAVELLINI P; GOYA RG

Madrid

Conferencia; International Longevity and Cryopreservation Summit; 2017

The achievement of animal cloning and subsequent development of cell reprogrammingtechnology are having a profound impact on our view of the mechanisms of aging incomplex organisms. The experimental evidence showing that an adult somatic nucleusimplanted into an enucleated oocyte can give rise to a whole new individual stronglysuggests that the integrity of the genome of an adult nucleus is fully preserved. Here, wepropose a protocol for rejuvenation of skin fibroblasts from old mice by cyclic partialreprogramming. To this end we will use a high capacity adenovector (HD-AdSTEMCCA) harboring the four Yamanaka genes (STEMCCA expression cassette)under the control of a Tet-Off regulatable bidirectional promoter which also controls theexpression of the reporter gene for green fluorescent protein (GFP). The adenovectoralso harbors a constitutive cassette expressing the regulatory protein tTA (tetracyclynetransactivator). Primary cultures of tail tip fibroblasts from 2 years old mice will beestablished and transduced with our HD-Ad STEMCCA vector complexed withmagnetic nanoparticles and submitted for 30 min to the action of a magnetic field(magnetofection technique) in order maximize transduction efficiency. Transduced cellswill express green fluorescence. After 5 days, the antibiotic doxycycline (DOX) will beadded to the culture medium in order to repress the Yamanaka genes and the GFPreporter. Cells will become nonfluorescent. This will be the first cycle of partial cellreprogramming and the resulting fibroblasts should be slightly rejuvenated. After 7days, DOX will be removed from the medium and a new reprogramming cycle willbegin. Several cycles of partial reprogramming will be performed and at the end o ofthe series the signs of aging of the cells will be assessed. Specifically, expression of betagalactosidase (a marker of aging) will be measured in control and treated cells.Telomere length and proliferative capacity as well as the level of chromatin compaction(heterochromatin) will be also assessed in treated and control cells. These measurementswill be performed after varying a number of reprogramming cycles. Finally, the durationof the rejuvenation effects after or stopping a cyclic partial reprogramming series willbe assessed. The proposed studies should shed light on the process of cell rejuvenation by partiaL REPROGRAMMING.