Derivation of hepatic lineage cells from embryonic stem cells following differentiation in 3D liver microenvironmental cell cultures for large-scale application to cell and tissue tools, devices, diagnostics and therapeutics
Tools and Technologies I
By virtue of their unlimited self-renewal potential, stem cells offer the promise of an inexhaustible source of genetically-defined cells that could dramatically change the study and treatment of human disease, including personalized therapies, regenerative medicine and circumvention of immune rejection for tissue replacements. The inability to direct stem cell differentiation into specified primary cell type(s) with high efficiency and at large scale has limited their widespread application. In the body, stem cell differentiation is guided by several developmental mechanisms including modifications in gene expression that is induced by cell-cell and cell-tissue component interactions and structure, and environmental factors including signalling factors and other proteins expressed by cells and communicated through the tissue. Researchers have attempted to improve the efficiency of directed stem cell differentiation in laboratory cultures through alteration of one or more of these factors. However, yields of specific differentiated cell types remain low, and the ability to select individual cell types from the differentiated population for use in specific patient therapies is limited by a lack selection markers for each cell type. The goal of the proposed work is to enable efficient differentiation of embryonic stem cells (ESC) into desired cell types by mimicking in laboratory systems, the conditions in the body that drive stem cell differentiation. To achieve this, ESC will be grown in tissue cultures that have been proven to function as tissue replacements, repairing host tissue after transplantation into the body. These tissue cultures are formed on three-dimensional scaffolds and contain all of the cells of the native tissue, which like bees make a bee hive, the cells express all of the native tissue components to recreate tissue structure and function in the lab. All native tissues of the body contain progenitor cells that replenish cell types of the tissue as they are damaged or die. We have already proven that addition of these progenitor cells to the tissue cultures that are devoid of given cell types results in rapid differentiation of the progenitors to replenish these cell types. Using ESC results in the same differentiation, but at lower efficiency. The aims of proposed work are to increase this efficiency of differentiation of ESC into tissue-specific cell lineages by growing the ESC in the tissue replacements. To assist in inducing differentiation of ESC in the tissues, genes that regulate ESC differentiation from embryos will be identified and inserted into the ESC to enhance differentiation down desired lineages. Because of its early development from embryos, liver will be chosen as the model tissue for these studies. The ability of liver tissue cultures and hepatic lineage genes identified by profiling liver versus other organs generated from embryos to drive ESC differentiation to hepatic lineage cells will be identified.
Statement of Benefit to California:
The goal of this proposal is to develop a stem cell-based human liver tissue model for use in the laboratory to study human disease, to facilitate the development of new drug therapies and to eventually directly treat human conditions such as organ failure. Lack of human predictive laboratory liver tissue models results in toxic and non-efficacious drugs reaching costly clinical trials. Liver toxicity alone is responsible for 2/3 of drug failures in clinical trials, 1/3 of drug withdrawals from the market, 1/2 of all black box warning labels on approved drugs, and 40% of liver failures are drug induced. Human predictive models in the lab would enable pharmaceutical companies to identify toxic drugs early in development when costs are low, enabling only those drugs that are safe to advance to clinical trials where costs are high, focusing time and money on effective drugs. The laboratory tissue models will also find utility in efficacy assessment of new drugs, in identification of biomarkers of disease, as antiviral screening platforms for liver-related diseases such as Hepatitis, and in studying the effects of other diseases with known impacts on the liver such as HIV, diabetes and obesity. They will also eventually serve as extracorporeal devices for treatment of liver failure, personalized and regenerative therapies and liver transplants. California has the greatest number of Hepatits C positive people of any state in the country. It also is among the top ten employers for the pharmaceutical and biotechnology industries. Hence, dramatic savings could be realized for the state upon commercialization of the human liver tissue products. In addition to this direct benefit to the citizens of California, indirect benefits include development of novel core facilities and shared equipment resources, experienced collaborative research teams that can attract millions of dollars of additional funding to the state, spinout companies from new technology development, and employment opportunities resulting from this new technology as well as increased tax revenues. The program would also build on a collaborative team spanning major liver research and clinical centers throughout California, facilitating researcher access to products, patient access to eventual treatments and the team's access to fetal and adult tissues and multiple clinical trial sites. The research teams involved have been successful largely because of the strong biotechnology community in California enabling partnership to expedite product commercialization. These partnerships have brought millions of dollars of government grants to California as well as the promise of a growing, profitable tissue engineering and stem cell industry that will deliver innovative cell and tissue technologies to revolutionize patient care. There are few cities in the US that have the biotechnology infrastructure and collaborative environment to foster multidisciplinary teams and expedite discovery.
This proposal focuses on the development of methods for hepatocyte differentiation of mouse embryonic stem (mESCs) and the generation of three-dimensional (3-D) liver models in culture. The Principal Investigator (PI) proposes to generate a new mESC line, identify key transcription factors (TFs) for hepatocyte and liver endoderm differentiation and then force expression of these TFs in mESCs. These cells would then be cultured in the presence of extra-parenchymal cells and matrix components from liver to generate 3-D cultures. The reviewers found this proposal convoluted and lacking experimental detail. They raised doubts about its feasibility, given the large number of aims and lack of clear milestones. The reviewers also questioned the necessity of certain aspects of the research plan, including generation of a new mESC line and a novel method for hepatocyte differentiation. The reviewers agreed that, in principle, this application addresses an important area of research: development of in vitro liver tissue models. These models would be extremely valuable for drug discovery and toxicity studies. However, reviewers commented that the potential impact of this project was greatly diminished by the use of mESCs. Translation of the work to human ES cells was given only vague mention in the proposal. Even so, one reviewer raised significant questions about the potential application of the proposed techniques to humans. Availability of the human tissue material required for such studies would be difficult. The reviewer commented that studies examining artificial three-dimensional substrates would seem to be a better long-term bet. The reviewers raised serious doubts about the feasibility of this proposal. They commented that it was overly ambitious with poorly defined goals and milestones. One reviewer noted that the project aims both to define and use pro-differentiation TFs to promote hepatic cell generation from mESCs and then also explore their function in 3-D cultures. The reviewer thought that either aim alone represents a significant project and would require two years. As hepatic differentiation from ESCs, both mouse and human, has been described by a number of groups, the reviewer suggested that the 3-D culture work might form a more useful focus and one where the applicant clearly has a scientific advantage in the field. In addition, reviewers raised several questions about the experimental design. One reviewer commented that the forced expression of TFs for hepatocyte differentiation might not be needed, as the process is well established in many labs. The problem instead is that the hepatocytes achieved to date are immature, and it’s unclear that the proposed methods will solve this. A reviewer noted that the investigators do not address the separation of definitive endoderm from endoderm for these studies. Finally and importantly, reviewers were unsure why a new mESC line needs to be generated for these studies. The PI and co-investigators are well suited for this project and have experience with both cell culture and extracellular matrices. One reviewer thought that stem cell experience in the team was weak. The budget was difficult to assess given the lack of milestones for year two. One reviewer commented that the consumables budget was excessive. Overall, this proposal was difficult to follow, overly ambitious and lacking in experimental detail. The application addresses an important area of research, but the research plan needs significant refinement.