Direct Reprogramming of Human Cell Lines in vitro Towards a β-Cell Phenotype Ryan Connor Nicholson Submitted under the supervision of Dr. James Dutton to the University Honors Program at the University of Minnesota-Twin Cities in partial fulfillment of the requirements for the degree of Bachelor Science, summa cum laude in Biochemistry. April 2018 Acknowledgements I would like to thank Dr. James Dutton for the time and effort he has dedicated to advising me in conducting work for the thesis and other projects in the past several years. I would also like to thank Caitlin Marie Hill for her time instructing and guiding me through the scientific process for this and other work. i Abstract Type 1 diabetes results from the loss of insulin producing β-cells of the pancreas, causing dependence on exogenous insulin for those affected. β-cell transplantation has been a promising treatment method, but lack of donor cells and requirement for repeated treatments currently limit this approach. Alternative treatments for type 1 diabetes are investigating reprogramming cell types toward a β-cell-like phenotype to generate a reliable source of insulin-secreting cells for transplant therapies. The pancreatic transcription factors Pdx1, Ngn3, and MafA have been combined in an adenoviral vector (Ad-PNMa) and used to reprogram rodent cells to generate insulin-secreting cells both in vivo and in vitro. However, studies examining similar reprogramming of human cell lines have met with little success to date. Here, we attempt to reprogram a human hepatocarcinoma cell line with the Ad-PNMa vector, and human pancreatic epithelial ductal (HPDE) cell lines with the Ad-PNMa vector and new Ad-PN and Ad-PNMb vectors expressing human Pdx1, Ngn3, and MafB. It was found that qRT-PCR detected weak insulin gene expression in Ad-PNMa infected HepG2 cells. Infection of HPDE cells with Ad- PNMb vector caused approximately 64% of cells to express the three vector cargo genes when treated at a concentration of 1.7x1010 ifu/ml. No insulin production was detected in HDPE cells following vector treatments, indicating that expression of these genes is not sufficient to induce insulin expression in HPDE cells. It may be possible to reprogram the HPDE cells to a β-cell phenotype with the expression of additional genes. ii Table of Contents Acknowledgements .......................................................................................................................... i Abstract ........................................................................................................................................... ii Table of Contents ........................................................................................................................... iii List of Tables ...................................................................................................................................v List of Figures ................................................................................................................................ vi Introduction ......................................................................................................................................1 Scope and Definition of Diabetes ........................................................................................1 Type 2 Diabetes ...................................................................................................................1 Type 1 Diabetes ...................................................................................................................2 Gestational Diabetes ............................................................................................................4 Surgical Diabetes .................................................................................................................4 Pancreatic Development ......................................................................................................5 Reprogramming Techniques ................................................................................................7 Reprogramming Cells to a β-Cell Phenotype in Mouse Models .........................................8 Reprogramming Human Cells to a β-Cell Phenotype..........................................................9 Past Studies of the Dutton Lab...........................................................................................10 Goal of the Current Study ..................................................................................................11 Methods..........................................................................................................................................12 Cell Culture ........................................................................................................................12 iii Testing Ad-PNMa, Ad-PN, and Ad-PNMb Vectors .........................................................12 Immunohistochemistry and Cell Counting ........................................................................13 RNA Extraction from HepG2 Cells ...................................................................................14 cDNA Synthesis .................................................................................................................14 qRT-PCR for Gene Expression ..........................................................................................14 Calculation of Fold Insulin Gene Expression Change .......................................................15 Results ............................................................................................................................................15 HepG2 A12 Ad-PNMa Assay ............................................................................................15 qRT-PCR of A12 Ad-PNMa Infected HepG2 Cells ..........................................................16 HPDE C13 Ad-PNMa Assay .............................................................................................17 HPDE Ad-PN Assay ..........................................................................................................17 HPDE Ad-PNMb Assay ....................................................................................................18 Discussion ......................................................................................................................................18 References ......................................................................................................................................23 Tables .............................................................................................................................................28 Figures............................................................................................................................................30 iv List of Tables Table 1. Titers for the Ad-PNMa, Ad-PN, and Ad-PNMb vectors ...............................................28 Table 2. Primary antibodies and dilutions .....................................................................................28 Table 3. Secondary antibodies and dilutions .................................................................................28 Table 4. PrimeTime primers for human Gapdh and insulin genes used for qRT-PCR .................29 v List of Figures Figure 1. Transcription factors involved in pancreatic tissue specification ...................................30 Figure 2. Differential expression over time of MafA and MafB transcription factors in the adult β-cells of humans and mice ...........................................................................................................31 Figure 3. Format of the Ad-PNMa vector ......................................................................................31 Figure 4. Antibody detection of Pdx1 and insulin in HepG2 cells treated with dilutions (ifu/ml) of A12 Ad-PNM ............................................................................................................................32 Figure 5. Percent expression of Pdx1 protein in HepG2 cells infected with dilutions of A12 Ad- PNM ...............................................................................................................................................33 Figure 6. Fold expression change of insulin gene expression in HepG2 cells infected with dilutions of A12 Ad-PNMa ............................................................................................................34 Figure 7. Antibody detection of Pdx1 and insulin in HPDE cells treated with dilutions (ifu/ml) C13 Ad-PNM .................................................................................................................................35 Figure 8. Percent expression of Pdx1 protein in HPDE cells transfected with dilutions of C13 Ad-PNMa .......................................................................................................................................36 Figure 9. Antibody detection of MafB in HPDE cells treated with a dilution (ifu/ml) of Ad- PNMb .............................................................................................................................................37 Figure 10. Antibody detection of Pdx1 and insulin in HPDE cells treated with dilutions (ifu/ml) Ad-PN ............................................................................................................................................38 vi Figure 11. Percent expression of Pdx1 protein