Date of Award

Spring 5-4-2020

Track

Biochemistry

Degree Type

Thesis

Degree Name

Master of Science in Chemical Sciences (MSCB)

Department

Chemistry

Committee Chair

Jonathan L. McMurry

Committee Member

Martin L. Hudson

Committee Member

Carol Chrestensen

Abstract

Somatic stem cells have been used as a regenerative medicine tool to treat diseases like leukemia for decades, but they are limited in their ability to self-renew and differentiate. A better alternative, induced pluripotent stem cells (iPSCs), has the ability to generate cells from all three germ layers and avoid tissue rejection as well as bypass the ethical concerns related to embryonic stem cells. In 2006, Takahashi and Yamanaka discovered that only four genes were required to induce pluripotency: Oct4, Sox2, Klf4, and c-Myc (OSKM). Since then, pluripotency has been induced with OSKM most effectively by transfection or transduction. However, both are difficult to control and can have undesirable off-target effects, e.g. random mutations in the genome. A safer approach to inducing pluripotency is exogenous delivery of OSKM proteins using cell-penetrating peptides (CPPs). Classically, CPP-mediated delivery has been inefficient because of poor penetration and entrapment in endosomes. Our lab’s novel CPP-adaptor system overcomes entrapment by relying on the cell’s natural calcium flux. A widely used CPP, TAT, is covalently linked to a calmodulin protein, creating TAT-CaM. The protein of interest that acts as a cargo, is engineered to have a calmodulin binding site (CBS). This allows TAT-CaM to bind cargo proteins outside the cell in the presence of Ca2+, but once they are endocytosed by the cell, the cargo can dissociate from TAT-CaM since intracellular Ca2+ concentration is low. The goals of the project were: (1) to induce pluripotency by delivering CBS-Oct4 with the TAT-CaM system; (2) to optimize reprogramming by addition of Sox2, and Klf4 to Oct4 and TAT-CaM; and (3) to combine OSK delivery with small molecule inhibitors to improve efficiency. Pluripotency was checked by using alkaline phosphatase staining and the iPSC colonies were cultured and expanded over multiple passages. Finally, stem cell state was further assayed by performing flow cytometry and immunostaining for Nanog, Oct4, and Sox2. Successful trials of this method could potentially lead to major changes in approaches to regenerative medicine.

Available for download on Friday, May 02, 2025

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