Stem cells are the hope for many people suffering from some form of organ or tissue dysfunction. The renowned capacity of stem cells and their ability to give rise to multiple types of adult cells (pluripotent) makes them so appealing for cell therapies. Stem cells originally derived from early embryos, often referred to us, as embryonic stem (ES) cells. They have extensive growth potential, self-replication capacity, can mature into adult cells, and hence can reconstitute damaged tissue when are injected to the organism. When isolated from early embryos, ES cells can be maintained in a dish under specific conditions without loosing their ability to expand indefinitely. A critical requirement for maintenance of ES cells in cultures is a layer of murine cells that supply unidentified signals vital to the ES cells. This requirement, obviously involves a risk for clinical therapies. Therefore, there is considerable effort ongoing by many investigators to define these factors. When needed, ES cells can be removed from the murine cell layer and spontaneously and without the ability to control it, can form all adult tissues. This event occurs in a very disorganized way resulting in “monster” tumors. Intensive investigation controlling the fate of the cells still is undergoing. In this project, we are suggesting to identify and characterize the factors that are differentiating ES cells from their murine layer cells, and from their matured cells. The main goal is to control the factors that are responsible to their pluripotentacy, and enriched the factors that are participating in the maturation of the cells. In order to get matured functional cells, particularly liver cells. Controlling maturation of ES cells into active matured liver cells is proposed as ideal technique for end-stage liver diseases, because their ability to expand extensively, differentiate into all mature liver cells, and reconstitute liver tissue when transplanted. In this project, we are proposing to analyze and identify factors that are differentiating cells in their embryonic stem state from cells that were already committed to liver cells. In a three well designed specific aims, we are suggesting to examine changes in factors within ES cells before and after they have committed to differentiate into liver cells as a function of time and culture conditions. With state of the art technologies and an interdisciplinary research teams this project goals are to be compliance with the highest medical and ethical standards.
Statement of Benefit to California:
This proposed project is directly relevant to the mission of proposition 71 and specifically will benefit the State of California and its citizens in the following ways: A. The knowledge and data from this project will directly improve the California health care system and reduce the long-term health care cost burden on California through the development of therapies that treat disease and injuries with the ultimate goal to cure them. B. The knowledge and data from the proposed project will specifically provide opportunity for the state of California to benefit from royalties, patents and licensing fees that result from this project. C. This project will benefit the state of California economy by creating jobs and projects to its citizens, will generate millions of dollars in new tax revenues to the state. D. The knowledge and data from this project potentially will advance biotech industry in the state of California E. This project will benefit the scientists, researchers and medical doctors working on this specific project by bringing them to a world-class recognition and leadership in the stem cell research.
SYNOPSIS: The general goal of this proposal is to gain new insights into what might be necessary to control the differentiation of human ES cells into mature hepatocytes. The specific approach will be to develop new molecular markers of the undifferentiated state, as well as new markers for differentiation along the hepatocyte lineage. The will be accomplished by a combination of metabolic fingerprinting and metabolic profiling. In Aim 1 the applicants will use LC-MS-TOF to describe the metabolic products present in the culture supernatants of murine feeder cells, feeder cells with hES cells, and hepatocytes. This will complement previous work, from others, that used similar technology to describe the proteins present in the supernatants of cultured human ES cells. In Aim 2 the applicants will compare similar cells, using tandem mass spec to focus on eiconosoids, which are regulatory lipids in the arachidonic acid cascade. SIGNIFICANCE AND INNOVATION: Metabolic fingerprinting and profiling of hESCs and hESC-derived hepatocytes has not been previously reported. This approach has the potential to provide new insights into biological pathways that are important to maintain the pluripotent state, and important for the transition to the hepatocyte lineage. If successful, this work will bring us a step forward toward controlled differentiation of hESC to hepatocyte lineages for toxicology research and screening and for therapeutics. STRENGTHS: This proposal brings together two PIs with expertise in stem cell biology and metabolic profiling. It is an intriguing blend, and the reviewer supported the idea of CIRM's encouraging the entry of new scientists with special expertise into the field of stem cell biology. The roles of small molecules, such as lipid metabolites, in ES cell biology is potentially important, and likely to be overlooked by many investigators who do not have the expertise or background to go in that direction. Strengths of this proposal include the innovation and significance stated previously. Additionally, the assembled team is experienced in liver biology research and in metabolomic and proteomic technologies. Strong support of this project and team is evident in support by the collaborators. WEAKNESSES: Undoubtedly an enormous amount of information will emerge from the proposed studies, as is always the case in these kinds of globally descriptive approaches. However, the application contains no discussion of how this information will be used to gain new insights into ES cell biology. How will the applicants use their observations to understand hepatocye differentiation? There are certainly going to be huge differences in the metabolic signatures of ES cells and hepatocytes. How will the applicants sort through this information to focus on differences that are biologically significant? The application is also weak in its description of the cells to be analyzed. A key step here is the purification of hepatocyte-like cells that derive from human ES cells. The application provides no information on how efficiently these cells can be obtained, and especially whether they can be purified in sufficient numbers for the proposed experiments on culture supernatants. The focus in Aim 2 on the eiconosoid pathway is not justified by any prior data showing that this pathway is especially significant in ES cells or in hepatocyte differentiation. What biological experiments might be inspired by results obtained in Aim 2? No clear hypothesis was stated. There are many part-time members of the research team. There are 6 team members with the most committed allocating 47% of her time. The PI and co-PI both are only 10% committed. This signals a lack of leadership and a lack of coherence in the work. Dr. Reid from North Carolina is an integral member of the team. She serves as a paid collaborator. It is not clear what her exact role is, nor what experiments will be carried out in her laboratory. The budget includes an annual payment of $20,000 to this collaborator. The proposed analysis is not completely defined in the research design section. For example, in the Anticipated Difficulties paragraph the PI states that "quantitative methods will be developed to deliver instrument independ data...." It is not clear what these methods will be, how they will be developed, or what exactly they will address. The metabolomic fingerprinting as proposed will be performed on cells grown in undefined conditions - e.g. in medium containing serum or undefined serum replacer. If is not clear how exact results can be obtained from this type of system. There is a lack of hESC experience on the research team. The yields of hESC-derived hepatocytes may be extremely low. The PI proposes to isolate these cells by laser microdisection. A discussion of the number of cells obtainable vs the number of cells needed for the analyses is warranted. DISCUSSION: There was no further discussion following the reviewers' comments.