Abstract
Liver disease is a very serious problem that kills tens of thousands of people each year. In 2010, 50,000 people in the United States died due to some type of liver disease according to the Centers for Disease Control and Prevention. The American Cancer Society estimates that in 2013, more than 22,000 men and about 8,000 women in the United States will be diagnosed with primary liver cancer and intrahepatic bile duct cancer. Additionally, the incidence of primary hepatocellular carcinoma (liver cancer) is on the rise in the United States. A promising avenue of research for treating liver disease is the use of cellular therapy mediated approaches to support or replace damaged liver cells. It is possible to inject in vitro cultured human hepatocytes (hHCs) into liver injury sites in patients with diseased livers to take over or supplement the role of a patient's natural hHCs. Through our work, we are furthering our understanding of the differentiation of hHCs from human embryonic stem cells (ESCs) and endodermal progenitor cells (EPCs) so that hHCs can be cultured more effectively; bringing potentially lifesaving therapies closer to clinical application. Our specific aims were to optimize EPC culture protocols, characterize the hHC like phenotype of EPC derived hHCs, experiment with xenobiotic free or fully characterized EPC/EPH culture conditions, and identify upregulated demethylases in the ESC to hHC differentiation process. We cultured EPCs for up to 12 passages, optimized growth and differentiation protocols and tested their growth on different matrices, including vitronectin and human feeders . Our analysis ofEPC growth on human feeder cells, and EPH growth on feeder free and defined matrices/media conditions served as a pre-translational step towards the goal of bringing these cells to a clinical stage. We also assessed the effects of MEF concentration on EPC growth and phenotype, and found that using greater than previously recommended MEF concentrations did not have deleterious effects on EPC phenotype. We also did some preliminary work with xenobiotic free culture of EPCs that met with limited success. Human foreskin fibroblasts were used in lieu of Swiss-Webster MEFs. The human fibroblasts successfully formed a network, but the EPCs failed to grow and expand. Through qPCR we found meaningful increases over the differentiation period of genes encoding the secreted proteins albumin (ALB), and alpha-fetoprotein (AFP), functional proteins glucose transporter 2 (GLUT2), and metabolic/enzymatic proteins: histone acetyl transferase (HAT), fumarylacetoacetate hydrolase (FAH), alcohol dehydrogenase IA (ADRIA), cytochrome P450 4Al l (CYP4Al 1), and cytochrome P450 2D6 (CYP2D6). ELISA analysis confirmed the presence of albumin and its increased expression as EPHs matured. Our next step was to perform flow cytometry to confirm the presence of hepatic proteins. Through flow cytometry we saw an increase in positive expression of ALB, alpha-1-antitrypsin (AIAT), and AFP. The majority ofEPHs tested high for AIAT and AFP early on, with only slight increases in positive expression as the cells progressed through the differentiation process. However, cells showed increases in mean signal intensity for all markers over the course of differentiation. These signs were positive indicators of an increasing hHC like phenotype among the EPHs. As for studies on epigenetic modifications, we analyzed ESC derived definitive endoderm for 28 different demethylases via qPCR and identified 7 genes (HR, JMID3, JMJD6, JHDM LD, UTX, JMJD IA, and JMJD2A) that showed clear trends of dynamic expression changes over the course of the differentiation period. Five of the seven demethylases (JMJD3, JMJD6, UTX, JMJDJA, and JMJD2A) showed a trend of increasing transcription over the differentiation period. Western blots were also performed on DE protein extracts to verify the qPCR results. The westerns confirmed the presence of JMID3 in both day O and day 8 DE. In summary we found that EPCs were efficient to maintain in culture for extensive passages and expanded readily. In addition, MEF quality was found to play an important role in the expansion capability of EPC cells. In our experiments, human feeders were an inadequate substitute for MEFs and were unable to maintain EPC growth. EPC derived EPHs showed increasing expression of key hepatic markers as they progressed through the differentiation process. Lastly, the differentiation of DE from ESCs was shown to correspond with increasing expression of several histone demethylases as shown through both qPCR and western blotting.