Generating human surrogate hepatocytes by integration- and oncogene-free lineage reprogramming

• Applicant: Dr. Milad Rezvani
• Subject Area: Gastroenterology
• Term: from 2015 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 272982029

The focus of this research proposal is on generating therapeutically effective human hepatocytes that can be expanded without creating a risk for malignant growth. For patients with chronic liver diseases suffering from a loss of hepatocyte function liver transplantation is the only established treatment. Much of the disease burden caused by waiting for scarce donor livers could be alleviated by cell-based therapies, but primary hepatocytes cannot fulfill all of the demand since they are isolated from donor livers and therefore subject to the same organ shortage. This leads to the investigation of alternative cell sources that replicate functions and regenerative abilities of primary adult hepatocytes. My previous work established the feasibility of turning human skin fibroblasts into fully functional hepatocytes that expand in vivo to repopulate chronically injured livers in the absence of tumorous growth. Part of the success was to use bioactive small molecules promoting proliferation without compromising function. In contrast, recent works by others demonstrating reprogramming of fibroblasts into induced hepatocytes (hiHeps) integrate potential oncogenes such as SV40 Large T into the host genome to facilitate expansion. Prompted by this unacceptable risk for translation, the central aim for this new research project is to use pro-proliferative small molecules instead of potentially cancer-causing transgenes in hiHep-generation to overcome the severe safety-concern of current protocols. Originally, the concept of lineage reprogramming by overexpression of small cohorts of fate-determining transcription factors has been designed to bypass cell states that could degenerate into malignancy. To meet this original requirement I conducted preliminary studies, obtaining first evidence that a combination of small molecules can replace potential oncogenes in generating hiHeps. Encouraged by this, in Aim 1, I propose to now refine my search for hiHep-expanding compounds by screening candidate molecules, and then broadening my study to an unbiased screen of chemical libraries on an established fluorescent imaging platform. In Aim 2, I will transplant expandable hiHeps into a mouse model that allows me to determine whether hiHeps can behave as functional hepatocytes, and proliferate in chronically injured livers without forming tumors. I plan to carry out the proposed research in the Willenbring lab at the University of California San Francisco that has extensive experience with a non-viral, non-integrating system for cell reprogramming, which is considered safe for clinical application. If, as expected, liver repopulation can be achieved without incurring a risk for hiHep-derived tumors, this project will establish safely generated hiHeps as hepatocytes for autologous cell therapy of chronic liver diseases. Moreover, this project prepares the long-term continuation of my research in Germany on the development of clinical-grade surrogate hepatocytes.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Holger Willenbring