Pluripotent human stem cells for creation of disease-specific and genotypically diverse 3-D liver tissues for drug discovery and patient therapies
New Cell Lines
The goal of this proposal is to use adult cells that would normally be discarded after routine surgical procedures, or blood cells, to generate stem cells for regenerative medicine applications. Stem cells will develop into any of the tissues of the body under the proper conditions and will eventually be used to develop laboratory models of human disease and for drug discovery or for medical implantation. Stem cells are important because they are a renewable cell source and will eventually overcome the sourcing issues caused by the shortage of donated organs. We propose to induce these stem cells to become liver cells (hepatocytes) using a unique laboratory model of human liver tissue. In this model, hepatocytes are grown together with the other cells found in the liver (microenvironmental cells) in three dimensions (as in the body), forming a liver tissue. The microenvironmental cells support the hepatocytes and allow them to function for a long time in the laboratory. Because of this, certain measurements that could not be performed on hepatocytes alone (which survive for only a short time in the laboratory) are possible using these 3D liver tissue cultures (3DLC), for example, the assessment of the effects of chronic drug exposure and the replication of hepatitis viruses C and D for testing prospective antiviral drugs. Since certain “feeder” cells can influence stem cells to form specific types of tissue cells, we will use microenvironmental cells from adult or fetal liver to support the development of stem cells into hepatocytes. We will also do genetic profiling of stem cells and fetal and adult liver cells to identify genes that are important for hepatocytes to develop from stem cells. These genes can then be inserted into the stem cells to direct them to become hepatocytes. At present, drug discovery is studied using hepatocytes isolated from livers that are donated for transplant but found to be unsuitable for clinical use. These livers come from a pool of donors of diverse ages, genders, health status and ethnic backgrounds and their overall quality can also be highly variable. Regardless of overall quality, it is unusual for hepatocytes to survive in a highly functional state for more than a week in the laboratory and this severely limits the evaluation of new drugs and, in the case of hepatitis C, the development of antiviral therapies. Stem cell-derived hepatocytes would provide a renewable source of genetically-defined, reproducible, disease- and patient-specific hepatocytes for drug discovery and disease modeling using 3DLC. In addition, a database could be developed that, with time, could be used to identify such patient-specific outcomes as differential drug efficacy, idiosyncratic toxicity and adverse drug reactions. We have already produced 3DLC using mouse stem cell-derived hepatocytes and mouse liver microenvironmental cells indicating that this approach is feasible.
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 directly treat human conditions such as organ failure. For example, one prospective use of this model system is to advance new HCV antiviral therapies to the clinic. The incidence of hepatitis C (HCV) in the United States has been estimated at 1.8% , and more recently at 2.5% from a population-based sample of young women living in poorer neighborhoods in California , the state with the greatest number of HCV+ people. Computer modeling projects 165,900 deaths from chronic liver disease, 27,200 deaths from hepatocellular carcinoma, and $10.7 billion in direct medical expenditures for HCV from the year 2010 through 2019 . During this period, HCV may lead to 720,700 years of de-compensated cirrhosis and hepatocellular carcinoma and to the loss of 1.83 million years of life in those younger than 65 at a societal cost of $21.3 and $54.2 billion, respectively . HCV causes an estimated 8,000 to 10,000 deaths annually in the U.S. and accounts for 60 - 70 percent of chronic hepatitis cases, and 30 percent of cirrhosis, end-stage liver disease, and liver cancer cases. At least 75 percent of patients with acute hepatitis C ultimately develop chronic infection, and most of them have accompanying chronic liver disease. Liver failure from hepatitis C is the most common reason for a liver transplant. Currently, it is estimated there are about 180 million people worldwide who are infected with HCV, 4 million of those are in the United States. 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 create a collaborative team spanning major liver research and clinical centers throughout California, facilitating patient access to treatments and the teams access to normal and diseased, and 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.
Executive Summary The goal of the proposed research is to generate stem cell lines from adult somatic tissues and direct these cells to differentiate into hepatocytes for drug testing and regenerative medicine applications. Induced pluripotent stem (iPS) cells will be derived from adult adipose and blood cells. Hepatic lineage-inducing factors will be identified by comparative gene expression analysis of embryonic and differentiated tissue. Various procedures and conditions will be evaluated for the differentiation of stem cells into hepatocytes with particular focus on the application of 3D tissue culture containing cells representing the liver microenvironment. This proposal is focused on a significant goal. The ready availability of human hepatocytes would provide a valuable reagent for toxicity and drug testing, and could be employed in the development of cellular therapies for liver disease. The feasibility of the project was judged as low. The aims lack coherency or a consistent scientific objective. Many of the aims are not supported by any preliminary data and thus appear unfeasible. Important details about the different experimental approaches are lacking and alternative strategies are not discussed. There is no evidence that the PI or other project investigators are experienced in the required techniques, have sufficient background knowledge to complete the proposed experiments, or have established appropriate collaborations to augment needed expertise. Evidence of research productivity relevant to the proposed investigation is lacking. Reviewers had no confidence that research objectives could be accomplished or that any meaningful data would be obtained. Reviewers felt that the proposal was not responsive to the RFA. The applicant has emphasized hepatic differentiation rather than production of cell lines, and has not addressed the central goals of the RFA. Prospects for the generation of new pluripotent human cell lines appear poor. Reviewer One Comments Significance: The goal of this proposal is to generate stem cell lines from adipose tissue or blood and direct their differentiation into hepatocytes. A renewable source of human hepatocytes would provide a platform for toxicity testing, drug testing, and could even lead to insights into liver disease. Feasibility: This proposal outlines eight specific aims that lack a consistent and coherent experimental objective. Those aims (1, 2, and 4) which are feasible have very little to do with the stated goal or the RFA. The aims (3, 5, 6, 7, and 8) which appear to directly address the goal of the proposal or would create new cell lines are disconnected, unsupported by previous experiments or preliminary data, and are altogether unfeasible. Responsiveness to RFA: This proposal is not responsive to the RFA in that they will create cell lines of limited developmental potency and utility. Reviewer Two Comments Significance: Significance and Innovation: Low The author proposes to generate a pluripotent stem cell source from adult tissues (MSC) or iPSC, induce differentiation of the cells to hepatocytes and incorporate the neo-hepatocyes into a 3-D culture system they have been using for a number of years. The authors propose to use techniques established in other laboratories to isolate and culture MSC. Feasibility: Eight specific aims were presented. The author proposes to, 1) gene expression profile fetal and adult liver to identify candidate genes for hepatic lineage programming, 2) gene expression profile adipose derived MSC before and after hepatic induction, 3) evaluate the influence of fetal and adult liver derived ME in MSC-derived hepatocytes. Do similar experiments with MSC derived from peripheral blood and cord blood, and evaluate the effects of fetal and adult ME on the differentiation of these cells. They then suggest that they will use iPS technology to reprogram adipocytes, cord or peripheral blood derived MSC to generate pluripotent cell lines and finally characterize the hepatic functions of the cell types generated. The authors have virtually no experience with any of the techniques, cell types or experiments proposed in the application. With the exception of doing simple co-culture of hepatocytes with nonparenchymal cells, which are established hepatocyte culture techniques in many laboratories for over 20 years, all of the remaining techniques proposed in this application will require significant development in the laboratory just to get them started. The authors appear to have taken headline concepts and mixed them in good portion into an application. The lack of experience or a basic understanding of any of the techniques is apparent from the multitude of approaches proposed. There are no recent publications from this group to suggest any facility with the technology being proposed. Even the microenvironment (ME) technology they refer to was commonplace in many laboratories for over 20 years and the reference they cite is 18 years old. There is no evidence that the 3DME technology they propose will have any significant effect on hepatic differentiation of stem cells. Preliminary data included in the application shows endodermal differentiation (Albumin) of MSC, but no mature hepatic gene expression. There is nothing in the text or preliminary data presented in the application that suggests that the experiments proposed could be completed or that any meaningful data would be gained. PI and Personnel: Brian Naughton, Ph.D., 1978, Experimental Hematology, New York University, lists his experience mainly in the biotechnology industry with companies such as Advanced Tissue Sciences and now RegeneMed. He does have experience getting FDA approval for products in his prior position but that does not seem to be a significant part of the present application. There is research support listed on the CV, mainly through SBIR applications on projects not directly related to the present application. The other key person listed on the application was Dawn Applegate, Ph.D from MIT, 1992 in chemical engineering. Her experience is also mainly in biotech with Advanced Tissue Sciences and now she holds the position of President and CEO of RegeneMed. There are no recent peer-reviewed publications listed for either of these individuals in their CV’s. Other investigators listed in the application do not contribute stem cell background or experience to the application. Facilities and Environment: Good. Responsiveness to RFA: Poor. The emphasis of the application is on hepatic differentiation rather than line production as a goal. There is a plan for sharing cell lines stated. Reviewer Three Comments Significance: The studies encompassed by this proposal are directed at the generation of human hepatocyte cells from a variety of stem cell sources including various types of adult stem cell (MSC; HSC) and from reprogrammed somatic cells with pluripotent differentiation potential generated by means of the iPS approach first described by Yamanaka and colleagues. These studies, if successful, have the potential to be applied to drug discovery for currently incurable liver diseases and may also lead to the development of cellular therapies for liver disease. Feasibility: The PI Dr Brian Naughton is CSO of RegeneMed (San Diego), has current funding for the use of engineered 3-D liver tissue in oncology and hepatitis research. He has enlisted a group of researchers from RegeneMed including CEO Dr Applegate, senior scientists Dr New and Naughton and has established a collaboration with Dr Jeffrey Glenn (Stanford University) who, in addition to supplying fetal and adult human liver tissue and human adipose tissue, will provide access to the Stanford ESC facilities and cell line resources as well as stem cell propagation and characterization assays. The experimental strategy is clear and, for the most part, well conceived. The recognition of the potentially key role of the liver microenvironment (ME) in promoting the generation of hepatocytes is an important facet of the proposed studies. Under Aim 1, microarray comparison of human fetal vs. liver is proposed. The objective is to identify genes involved in specification of the hepatic lineage and those contributing to the liver ME. Details of the bioinformatic strategies to be employed are lacking. This seems a very open-ended approach and it is not clear this will achieve the stated aim. Further to this, the phenotypic evaluation of the expression of cell surface markers on fetal liver cell suspensions as a means to characterize the ME is not complemented by functional assays of the various cellular constituents (alone or in combination) identified by this means. Experience in the methodologies used to generate iPS cells appears to be lacking amongst the collective of investigators participating in this project and such expertise is not being sought through collaboration. No preliminary data attest to the capacity of the applicant to derive iPS cells. Where will the vectors be obtained or does the applicant propose to construct them in house? Too little attention is paid to possible problems that may be encountered during the research or to the development of alternative strategies that may circumvent such problems. It is not clear that in the absence of a collaborator with the appropriate level of training and experience, the applicant will be in a position to make well informed choices as to alternative approaches. This concern speaks to the feasibility of the iPS work proposed under Aims 6 and 7. Responsiveness to RFA: While part of this project seeks to derive pluripotent human cell lines using iPS technology, I am not convinced that such lines will be developed for the reasons stated above.