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Increasing Efficiency of Inducing Pluripotency with a Novel Cell Penetrating Peptide‐Adaptor System
Author(s) -
Oja LauraMaria,
Clearman Kelsey R.,
Hudson Martin L.,
McMurry Jonathan L.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.636.10
Subject(s) - signal transducing adaptor protein , microbiology and biotechnology , sox2 , induced pluripotent stem cell , endocytosis , endosome , stem cell , embryonic stem cell , transfection , chemistry , cell , biology , signal transduction , biochemistry , gene , intracellular
Somatic stem cells have been used to treat a number of disorders for decades but are limited in their ability to differentiate and self‐renew. Induced pluripotent stem cells (iPSCs) not only have the ability to generate cells of all three germ layers, but bypass the ethical concerns related to embryonic stem cells. They also hold the prospect of utilizing a patient's own cells, thus avoiding host‐vs‐graft issues. In 2006, Takahashi and Yamanaka discovered that expression of only four genes were required to induce pluripotency: Oct4, Sox2, Klf4 and c‐Myc (OSKM). Since then, pluripotency has been induced most effectively by transfection or transduction of these genes. However, efficiencies are low, expression is difficult to control, and off‐target effects are a concern. Inducing pluripotency by exogenous delivery of OSKM proteins using cell‐penetrating peptides (CPPs), while promising, is also inefficient due to of poor penetration and entrapment in endosomes. Our novel CPP‐adaptor system overcomes entrapment by relying on the calcium flux during end o somal trafficking. The adaptor, TAT‐CaM, is a recombinant protein consisting of commonly used CPP, TAT, fused to calmodulin. The protein of interest (the ‘cargo’) has an engineered calmodulin binding site (CBS). TAT‐CaM binds purified CBS‐cargo outside the cell where the Ca 2+ concentration is relatively high, but after CPP‐mediated endocytosis, cargo dissociates from TAT‐CaM inside endosomes as calcium concentrations drop, leaving the CPP‐adaptor trapped but cargo free to escape to the cytoplasm and beyond. We have used our adaptor system to deliver submicromolar doses of CBS‐Oct4 protein, inducing pluripotency as measured by expression of stem state markers and alkaline phosphatase staining. CBS‐Sox2 and CBS‐Klf4 have also been delivered and while insufficient to induce pluripotency, play an efficiency enhancing role. While not directly comparable to published results, CPP‐mediated efficiency as measured expression of stem‐state markers is higher than current transfection‐ and transduction‐mediated methods. Parameters such as concentration and timing of doses and molar ratios of OSK to each other and TAT‐CaM affect efficiency. iPSCs created via our CPP‐adaptor system can be cultured, expanded over multiple passages and differentiated into other cell types such as cardiomyocytes. Success in this work will lead to a virus‐ and nucleic acid‐free, nontoxic, tunable method for inducing pluripotency. Support or Funding Information This work was supported by NIH grant R15GM120691 and R15NS100632 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .