January 2012

Document Type


Degree Name



Dept. of Molecular and Medical Genetics


Oregon Health & Science University


Liver disease and type 1 diabetes combined affect over 28 million people in the USA alone, with the incidence of both diseases increasing significantly. Medically refractory liver failure is treated by orthotopic liver transplantation. Similarly whole pancreas transplantation is the only definitive intervention for type 1 diabetes. An alternative to whole organ transplantation is a cell therapy approach to restore the cellular deficit caused by hepatocyte loss or beta cell destruction in the respective disease. Importantly, initial hepatocyte and islet transplantations in humans have demonstrated that these procedures can be performed safely and effectively, but their widespread use has been limited by a number of factors, with one of the major restrictions being the paucity of transplantable material. Therefore, new methods to generate transplantable, functional cells are needed urgently. My thesis research focused on two different approaches to address this problem. Firstly, we created fumarylacetoacetate hydrolase (Fah)­null heterozygote pigs by gene targeting and somatic cell nuclear transfer (SCNT) in order to eventually generate Fah-null homozygote pigs to expand human hepatocytes in. In a novel approach we used the chimeric adeno­associated virus DJ serotype (AAV­DJ) and homologous recombination to target and disrupt the porcine Fah gene. The AAV­DJ vector was used to deliver the Fah knockout construct to fetal pig fibroblasts with an average knockout targeting frequency of 5.4%. Targeted Fah-null heterozygote fibroblasts were used as nuclear donors for SCNT to porcine oocytes, and multiple viable Fah-null heterozygote pigs were generated. Fah-null heterozygotes were phenotypically normal, but had decreased Fah transcriptional and enzymatic activity compared to wild-type animals. The second focus of my research was to generate pancreatic beta cells from mouse gall bladder cells (GBCs) using a direct reprogramming approach that could eventually be used to treat patients with type 1 diabetes. Murine GBCs were robustly expanded in vitro, allowing the generation of billions of cells from a single gall bladder. We determined that expression of Neurog3, Pdx1 and MafA to be the minimal required transcription factors for reprogramming to the beta cell fate. Reprogramming towards the beta-cell fate occurred rapidly within 48 hours, and was augmented by retinoic acid and by inhibition of notch signaling. Using flow cytometry to isolate reprogrammed cells, we confirmed reprogrammed GBCs were differentiating towards a beta-like cell fate by both gene and protein analyses, including whole genome RNA-Sequencing analysis. In order to determine if these cells could be used to reverse hyperglycemia in vivo, we transplanted these cells into diabetic mice. Although the transplanted cells were unable to consistently reverse the hyperglycemia, the transplanted cells were able to engraft long term in these mice and were insulin-positive for at least 12 weeks post-transplantation. In summary, both these studies explore different approaches to generate new cells for the purpose of cell therapy. There is an urgent need to generate transplantable cells for treating myriad liver diseases and type 1 diabetes. Chapter one of this thesis outlines the background to the problems of liver disease and type 1 diabetes, and the different approaches currently being attempted to treat these disorders by cell therapy. In chapters two and three, I describe in detail my efforts in generating Fah-null heterozygote pigs and mouse pancreatic beta cells, respectively. Finally, in chapter four I summarize the significance of my research, the current obstacles with both approaches and the future directions I see this research going, as well as speculating on the general challenges associated with all cell therapies currently focused on treating liver disease and type 1 diabetes.




School of Medicine



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