Basic Biology III
Liver disease is one of the leading causes of death in the US. Liver transplantation is the only treatment for patients with acute or end-stage liver failure. However, donor shortage places a substantial number of patients on the waiting list for liver transplantation (around17,000 in the US and 3500 in California by December, 2010) and only a small proportion of patients actually received liver transplantation (6320 in the US and 551 in California in 2010). Many patients die from their disease unnecessarily while waiting for liver transplant. Recent advances in derivation of hepatocytes from human embryonic cells (hESCs) make cell therapy for liver patients a promising possibility in the future. However, one of the major obstacles for clinical applications of these hESC derived hepatocytes is that these hepatocytes are not fully matured to fulfill all functions of adult liver cells and still express the fetal hepatocyte protein, AFP, which is absent in the normal adult liver, but present in malignant liver cells. Therefore, these immature hepatocytes may impose a potential danger for tumorigenesis in patients. Another major obstacle for hepatocyte transplantation is the lack of an ideal transplantable scaffold for cell attachment, survival and function. Very few studies have addressed the role of cell microenvironment on hepatic lineage specification from hESCs. Even less is known about the mechanism regulating hepatocyte differentiation from hESCs by cell microenvironment. We propose to use natural liver extracellular microenvironment for further maturation of ESC-derived hepatocytes and maintenance of hepatic function. We will seek to provide fundamental understanding of the possible mechanism responsible for the expected enhanced maturation of ESC-derived hepatocytes induced by the liver extracellular microenvironment with focusing on integrin and growth factor signaling pathways. With the novel approach we propose here, we expect that successful completion of the proposed research will not only significantly advance our understanding of hepatocyte maturation but also provide a platform for future organ engineering, ultimately leading to human embryonic stem cell-based clinical application to treat patients suffering from liver disease.
Statement of Benefit to California:
Patients with acute or end-stage liver failure need liver transplantation to save their lives. However, donor shortage is a serious problem the US and even more serious in California. Only 16% of California patients (3500 in the year 2010) with liver diseases actually received liver transplantation in contrast to 37% of patients national wide (17,000 in the year 2010) received liver transplantation. Human embryonic cells provide an alterative promising cell source for treatment of liver patients. However, hESC-derived hepatocytes are immature, and therefore may not fulfill all functions of adult liver cells and impose a potential danger for tumorigenesis in patients. We will use a novel approach for further maturation of ESC-derived hepatocytes and maintenance of hepatic function using natural liver extracellular matrix. We will identify fundamental molecular mechanisms responsible for the expected enhanced maturation of ESC-derived hepatocytes. We expect that successful completion of the proposed research will ultimately lead to human embryonic stem cell-based clinical application to treat patients suffering from liver disease. Since the immaturity is a general problem in ESC differentiation toward targeted cell types in vitro, our approach can be applied to cell types other than hepatocytes and therefore also benefits patients with diseases other than liver failure in the long run. The benefits will first come to the California patients and their families. We also expect to obtain intellectual property for the state of California and continuously maintain the leading role in California in embryonic stem cell research toward clinical applications.
Project Synopsis: The goal of the proposed research is to determine if decellularized liver extracellular matrix (DLM), in concert with growth factors, can provide the natural liver microenvironment to facilitate maturation of human embryonic stem cell (hESC)-derived hepatocytes (hEHs) through integrin-mediated signaling pathways. To explore these hypotheses, a series of three aims has been proposed. First, the applicant will characterize the effects of DLM on hepatocyte differentiation from hESCs in vitro. Next, the mechanisms responsible for enhanced maturation will be explored through investigation of specific integrin and growth factor signaling pathways. In the third and final Aim, the applicant will employ an in vivo transplantation model to assess and validate the roles of DLM, integrin and signaling pathways in hEH maturation. Significance and Innovation: - This application addresses a major unsolved problem in stem cell biology, the inability to derive fully mature and functional hepatocytes from hESC. If successful, the proposed studies could generate new and important data towards understanding hepatocyte differentiation and the utility of DLM for studying hepatocyte function. - The proposal addresses a significant clinical problem. Liver disease is one of the leading causes of death in the United States, a fact that is exacerbated by shortages of donor organs. hESCs are envisioned as an alternative and potentially unlimited source of human hepatocytes, and thus insights towards improved derivation of these cells will be important for advancing the field. - The proposed studies are unlikely to have major impact. While somewhat innovative in the context of liver, extracellular matrix (ECM)-based approaches are being used with other tissue types, as the ECM and its embedded growth factors are important for differentiation in a variety of lineages. Furthermore, many aspects of the proposed work with commercially available ECM, transplantation experiments and integrin signaling have been previously described or published for hEH. - The studies are based on sound scientific rationale and focus largely on basic cellular and molecular mechanisms. The emphasis on integrins is logical but may be limiting. Feasibility and Experimental Design: - The feasibility of this project hinges on the success of Aim 1A. If DLM cannot facilitate the maturation of hESC-derived hepatocyte progenitors, the rationale for remaining portions will collapse. This considerable risk would have been alleviated by inclusion of more preliminary data with hEHs and DLM. - The proposed studies are well designed and represent logical extensions of the applicant’s previous work. Specific aims are well organized and achievable within the proposed timetable. Potential problems and alternative approaches have been considered. - The reliance on albumin secretion and urea processing as markers of hepatocyte maturity is not ideal. Reviewers suggested that cytochrome functionality would likely prove a better measure. Moreover, they recommended that the panel used for verifying maturity should be extended to include markers such as tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp2B6 and functions of glycogen storage, indocyanine green and low-density lipoprotein uptake, and inducible cytochrome P450 activity. Principal Investigator (PI) and Research Team: - The principal investigator (PI) is a junior investigator with numerous publications in well-regarded journals. He/she has the necessary expertise to successfully complete the project. The proposed 35% commitment is appropriate. - Collaborating investigators provide outstanding, extensive and complementary expertise in the areas of hESC-derived hepatocytes, liver repair and transplantation models. These collaborations are integral and critical to the success of the study. - The facilities and research environment are appropriate for conducting the proposed research. - The budget is reasonable, although some reviewers felt the annual supplies allocations were somewhat excessive. Responsiveness to the RFA: - The proposed research utilizes hESC and directly addresses molecular mechanisms driving the specification of adult hepatocyte identity. This project adequately and appropriately addresses the goals and objectives of the RFA.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was proposed, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.