Date on Master's Thesis/Doctoral Dissertation

12-2015

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Degree Program

Physiology and Biophysics, PhD

Committee Chair

Boyd, Nolan

Committee Co-Chair (if applicable)

Arteel, Gavin

Committee Member

Arteel, Gavin

Committee Member

Harris, Patrick

Committee Member

Hoying, James

Committee Member

Joshua, Irving

Committee Member

Williams, Stuart

Author's Keywords

liver; induced pluripotent stem cell; stromal vascular fraction; iPSC; SVF; tissue mimic

Abstract

This dissertation describes the incorporation of several technologies (stem cells, gene therapy, tissue engineering and regenerative medicine) into a single project that aims to produce a liver-tissue mimic for therapeutic applications. The liver is arguably one of the most complex organs in the body. In addition to its remarkable capacity to regenerate, it performs a host of vital functions. As a result, its impairment has widespread systemic consequences. The work described herein focused on the liver in the context of cardiovascular disease and used the heritable disorder Familial Hypercholesterolemia (FH) as a clinical disease model. As (a) the only definitive cure for FH is currently liver transplant and (b) the availability of quality liver organs for transplant is critically low, these studies seek to develop a liver-tissue mimic comprised of two parts: functional hepatocyte-like cells (derived from induced pluripotent stem cells, or iPSC) and vascular support (provided by adipose-derived stromal vascular fraction (SVF)). The dissertation is divided into six sections. Chapter I provides an introductory overview and lists the aims and hypotheses for the dissertation. Chapter II provides a four-part background discussing cholesterol metabolism, the liver organ, stem cells, and the vasculature. Chapter III describes our efforts to generate a proof-of-concept liver-tissue mimic, using HepG2 as a hepatocyte model cell source and SVF cells as the vascular support system. As vascular support is critical for parenchymal survival and function, Chapter IV examines the mechanisms of spontaneous SVF vascular self-assembly. Chapter V discusses development of a patient-specific, therapeutic cell system. FH-patient dermal fibroblasts were programmed into iPSC using modRNA technology, and eventually subjected the iPSC to directed differentiation into hepatocyte-like cells. Yet, as the iPSC were derived from an FH patient, the cells required functional restoration of their LDL-R in order to impart any therapeutic benefit. To accomplish this, a novel episomal LDL-R plasmid containing (a) upstream regulatory control sequences that confer physiological feedback control of LDL-R expression and (b) Epstein-Barr sequences for episomal retention and replication was used. To mitigate any potential concerns associated with viral vectors, the iPSC were derived and corrected using non-viral modalities. These cells were combined with an SVF derived vascular support system to assess iPSC-HLC survival characteristics in vivo. Chapter VI provides a comprehensive discussion regarding our experimental efforts. These experiments demonstrate the development of a vascularized, iPSC-derived hepatocyte liver-tissue mimic that could potentially be used for therapeutic applications, such as for the treatment of FH. Efforts to create this tissue engineered liver construct were guided by three aims: (1) Assess the role of adipose SVF in providing vascular support to implanted parenchymal cells, (2) Evaluate and define the mechanism(s) of SVF vascular self-assembly, and (3) Restore the functionality of monogenic-deficient FH cells. These studies provide several proofs-of-principle towards the development of effective cell-based treatments, not only for FH, but also for other diseases classically requiring whole-organ transplantation.

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