Differentiation of human embryonic stem cells into hepatocytes and their in vivo application for hepatitis C viral production

  • Author / Creator
    Alsagheir, Ali I
  • Abstract Introduction: Chronic hepatitis C virus (HCV) infection is a global problem. The World Health Organization estimates that about 170 million individuals around the world are infected with HCV. Chronic HCV has a high rate of morbidity and mortality due to cirrhosis and hepatocellular carcinoma. It is a major indication for liver transplantation. The current treatment is interferon α and ribavirin of which only 50% of cases show sustained virological responses and clinical signs of improvement, indicating the need for further exploration of novel anti HCV drugs 1. Several small animal models capable of supporting HCV infection in vivo have been achieved by the transplantation and expansion of primary human hepatocytes into the livers of mice2. The major limitations of these models are the generation of a supply of hepatocytes, which must come from human donors, and the technical difficulties associated with their isolation. Human embryonic stem cells (hESC) are pluripotent cells derived from the inner cell mass of blastocytes during early embryonic life 3. These cells are capable of self-regeneration and differentiation into any adult cell type in the human body. In the last few years, multiple centers around the world have successfully generated mature hepatocytes from human embryonic stem cells. Therefore, it is possible that hESCs can be used as a substitute for primary human hepatocytes in a small animal mouse model. Our primary objective was to explore the possibility of differentiating hESCs into hepatocyte-like cells that could be used as substitutes for primary human hepatocytes in an SCID/UPA mouse model. As such, these studies are expected to increase the accessibility and utility of the SCID/UPA mouse model for a variety of applications, including the testing of the efficacy of antiviral strategies targeting the HCV lifecycle. Methods: According to a published procedure4, human embryonic stem cells underwent a multi-stage differentiation protocol to render them hepatocyte-like. The successful transition of hESCs to hepatocytes was monitored by indirect immunofluorescence detection of various protein markers at each stage of the differentiation process. The ability of the differentiated human hepatocytes to engraft and support productive HCV infection was evaluated in vivo subsequent to their transplantation to the livers of SCID/UPA mice using procedures previously established in our lab. Results: Successful differentiation of hESCs into hepatocyte-like cells was demonstrated with indirect immunofluorescence. Thirty-five SCID/uPA mice were transplanted with undifferentiated hESCs (n=7), primary human hepatocytes (PHHs) (n=9), or differentiated human hepatocytes (DHHs) (n=19). After transplantation, serum analysis of mice from the DHH group showed undetectable levels of human alpha-1 antitrypsin (hAAT) and HCV viral production. By contrast, mice transplanted with PHH secreted hAAT values ranging from 229–1515 ng/ml, and 3 out of the 9 mice showed detectable HCV RNA levels. At the end point of the study, the mice with the transplanted liver cells were collected and examined for the presence of Alu repeat sequences using PCR and in situ hybridization techniques. DHH group showed weak signals with PCR and no evidence of histologically intergrated human cells with in situ hybridization; while histologically integrated human cells were readily detected in mice transplanted with PHH or undifferentiated hESCs. Conclusion: Our in vivo studies showed no evidence of engraftment or HCV viral production in SCID/UPA mice transplanted with DHHs. Unfortunately; our experiments provided unsatisfactory or negative data to support the use of DHHs as a substitute for PHHs in SCID/uPA mice.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Surgery
  • Specialization
    • Experimental Surgery
  • Supervisor / co-supervisor and their department(s)
    • Dr. Kneteman, Norman ( Department of Surgery)
    • Dr. Korbutt, Gregory (Department of Surgery)
    • Dr. Douglas, Donna (Department of Surgery)
  • Examining committee members and their departments
    • Dr. Churchill, Thomas (Department of Surgery)
    • Dr. Korbutt, Gregory (Department of Surgery)
    • Dr. Adesida, Adetola (Department of Surgery)
    • Dr. Douglas, Donna (Department of Surgery)
    • Dr. Kneteman, Norman ( Department of Surgery)