(270g) Engineering Nanog Protein for Effective Protein Transduction: A Possible Alternative to Reverse the Effects of Organismal Aging On Mesenchymal Stem Cells

Previously, our group demonstrated that overexpression of a single transcription factor, Nanog, can reverse the effects of organismal aging on mesenchymal stem cells (MSCs) proliferation and myogenic differentiation [1]. We also found that short-term Nanog overexpression by using doxycycline-control system is sufficient to reverse the effects of aging, thereby suggesting the permanent genetic modification may not be necessary. These results prompted us to hypothesize that delivery of Nanog proteins into MSCs may overcome the effects of cellular senescence. However, it has been proven difficult to deliver proteins into cells, especially into MSCs. To address this problem, we first engineered Nanog proteins, which contain a cell penetrating peptide (nona-arginine) for efficient protein delivery. To increase the stability of Nanog protein in the cells, ubiquitination target sequence (PEST) was removed. These engineered Nanog proteins were expressed in E. coli (BL21) and purified by denaturation/renaturation process followed by preparative chromatography. Chloroquine was used to enhance endosomal escape of internalized Nanog proteins. Human hair follicle MSCs were used in this study. Our results have demonstrated that fusion of the CPP can efficiently increase the binding of Nanog proteins on the cells and help internalization. Also, removal of PEST sequence did not have detrimental effects on Nanog transcription factor activity and increased the stability, as demonstrated by its prolonged half-life in the cells. Chloroquine effectively promoted endosomal escape of Nanog proteins that might increase the chance of nuclear localization. Furthermore, we introduced the SV40 virus nuclear localization signal (NLS) to Nanog proteins and its effects on nuclear delivery after endocytosis are under investigation. This study provides a possible alternative to overcome the difficulty of Nanog protein transduction by rationally engineering Nanog protein. Our results imply that this strategy may be employed to reverse the effects of organismal aging or culture senescence on MSC without viral transduction and permanent genome modification.

[1] Han J, Mistriotis P, Lei P, Wang D, Liu S, Andreadis ST. Nanog reverses the effects of organismal aging on mesenchymal stem cell proliferation and myogenic differentiation potential. Stem Cells. 2012;30:2746-59.