Liver Disease

Coding Dimension ID: 
301
Coding Dimension path name: 
Liver Disease
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01012
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$971 558
Disease Focus: 
Liver Disease
Toxicity
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Drug-induced liver toxicity, including that from FDA-approved drugs, is the leading cause of liver failure in the US. One of the biggest road blocks to testing drug-induced liver toxicity prior to clinical studies or release of the drug into the market is the absence of a good model of human drug metabolism in the liver. Development of a clinically predictive drug screening system would allow earlier detection of drug-induced liver toxicity, thus decreasing drug costs, decreasing the scale of pre-clinical animal testing, and increasing drug safety. Unfortunately, use of primary human liver cells for drug screening is hampered by their limited availability and poor viability in culture. Human embryonic stem (hES) cells, however, could provide a renewable, scalable, relevant source of liver cells since they can be induced to turn into these types of cells. Unfortunately, though, current hES protocols yield primarily immature liver cells, even though mature adult-like liver cells would be needed for drug screening. Here we propose development of a new hES cell line tool that attaches a fluorescent molecule to a protein found in mature liver cells. This would be a very powerful tool for two key avenues of study and development. First, it will facilitate testing of new methods to enhance the maturation of hES-derived liver cells, ultimately leading to better protocols for transplantation and regenerative medicine purposes. Second, it will also be instrumental in screening for drug-induced liver toxicity effects. While [REDACTED] interests lie more with the usage of this tool for drug screening purposes, we plan to openly share this tool with the scientific community under standard licensing agreements so that rapid progress can be made in both these areas.

Statement of Benefit to California: 

This proposal has been submitted by a California company whose mission is to develop and commercialize ES cell-based assays to aid in drug discovery and development. Using stem cells to model how the liver metabolizes drugs would lead to earlier detection of drug-induced liver toxicity, keeping many of the more dangerous clinical drugs from ever reaching the market. This would increase drug safety, decrease drug costs, and decrease the scale of pre-clinical animal testing that is currently used in drug development benefiting all Californians.

The work outlined in this proposal will also bring significant revenue into California, in various different forms. Whenever possible, we will continue to order supplies and/or use services through our standing relationships with California vendors, such as E&K Scientific (Santa Clara, CA), Invitrogen (Carlsbad, CA), and Stanford University Core Research facilities in order to support California businesses and universities. Partnering and licensing of the technology developed in this proposal, as well as commercialization of new and safer drugs, would bring revenue and additional jobs into California. In addition, patents arising from these technologies are also potentially significant for California, due to the licensing revenue fees that would go back to the state.

The ES cell line tool generated as a result of this proposal, along with the reagents used to make it, will be shared openly with the scientific community under transfer and/or licensing agreements that are standard to both academic and industrial scientific entities. This will facilitate rapid progress toward two key avenues of research and development: 1) screening drugs for potential toxic effects on the liver and 2) for better understanding liver development, ultimately leading to better protocols for transplanting ES-derived liver cells into patients with liver disease or drug-induced liver toxicity. This would also result in more jobs in California to carry out this work and would form the basis of additional academic and corporate collaborations that would increase California's leadership role in stem cell applications.

Progress Report: 
  • The leading cause of liver failure in the US is drug-induced liver toxicity. Currently there is an absence of a good model of human drug metabolism in the liver, which poses one of the biggest road blocks to testing drug-induced liver toxicity prior to clinical studies or release of the drug into the market. We are using human embryonic stem (hES) cells to develop a clinically predictive drug screening system that should allow earlier detection of drug-induced liver toxicity, thus decreasing drug costs, decreasing the scale of pre-clinical animal testing, and increasing drug safety. There are two arms to this work. The first is to engineer a new hES cell line that attaches a fluorescent molecule to a protein found in mature liver cells. To date we have completed the genetic molecules necessary for development of this cell line, and we are currently using these molecules to generate the engineered hES cell line. The second arm is to test new methods to enhance the maturation of hES-derived liver cells, since current hES protocols only yield immature liver cells. As part of this approach, we are testing a novel 3D culture system that has already been shown to improve maturation of other cell types, such as heart cells and fresh liver cells from humans. By combining our new hES cell line with improved protocols for generating mature hES-derived liver cells, we will have a powerful system not only for screening drugs for potential liver toxicity effects but also for improving protocols for transplantation and regenerative medicine purposes. We plan to openly share this new cell line with the scientific community under standard licensing agreements so that rapid progress can be made in both these areas.
  • We have developed and validated two human ES cell clones that have the BLA reporter correctly targeted into the CYP3A4 gene. In addition, we have made major improvements to the hepatocyte differentiation protocols, resulting in cultures with greater than 85% hepatocytes, which express significant levels of mature hepatocyte proteins and drug metabolizing enzymes, including albumin, CYP1A2 and CYP3A4. Furthermore, these cultures demonstrate CYP1A2-dependent metabolism of acetaminophen, which is approximately 20% of the activity, on a per cell basis, seen from primary human hepatocytes. This is significantly more than we have seen with any other protocols. We expect to use these cells in our drug development programs as screening assays for liver metabolism and toxicity studies.
  • We have successfully achieved the major aim of this grant, which was to develop a new human embryonic stem (hES) cell line in which a fluorescent molecule is attached to a protein found in mature liver cells. This protein is responsible for metabolizing the majority of drugs currently in the market, so it is critical to understand the effect that different drugs have on the function of this protein so that drug-induced liver toxicity, which is the leading cause of liver failure in the US, can be reduced. We have done extensive validation of this cell line tool, and we are currently in the process of developing it into an assay system for screening compounds for drug-induced liver toxicity effects. There is a great need for this type of assay since there is currently an absence of a robust model of human drug metabolism in the liver. As part of this development, we have also done extensive work optimizing the generation of liver cells from hES cells. We now can differentiate hES cells into nearly pure populations of cells that are precursors to liver cells and we are currently using our new cell line to facilitate testing new methods to enhance the maturation of these precursors into hES-derived liver cells. The combination of using our new cell line tool in an optimized protocol for generating hES-derived liver cells will hopefully result in a clinically predictive drug screening system that will allow earlier detection of drug-induced liver toxicity, decreased pre-clinical animal testing, and increased drug safety. We are committed to aggressively continuing this work even beyond the close of this grant funding to achieve the goal of a human cell-based, clinically predictive liver toxicity drug screening system.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00950
Investigator: 
ICOC Funds Committed: 
$3 032 510
Disease Focus: 
Liver Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

The liver is a promising target for cell therapy since it supports and functionally integrates transplanted cells. Human liver contains more than 50 billion cells and more than 10% replacement will be required for most liver diseases. Hence, embryonic stem cells (ESC), which have unlimited growth capacities, represent one of the few cell types with potential for liver cell therapy. However, to be functionally effective and safe, ESC have to be differentiated into hepatocytes, the cells of the liver that provide its typical functions, before transplantation. Unfortunately, current ESC differentiation protocols generate cells that are not fully differentiated or functional. To achieve levels of differentiation that would be therapeutic we propose to identify the mechanisms that establish hepatocyte function in progenitor cells in the adult liver. Adult liver progenitors are typically absent from the normal liver but become apparent in liver disease when hepatocytes are damaged. Remarkably, adult liver progenitors can differentiate into fully functional hepatocytes within a few days. We hope to identify the genes that enable this rapid maturation process in order to apply it to immature cells derived from ESC. If maturation can be induced and these hepatocyte-like cells function to correct a mouse model of a human liver disease we will have provided proof-of-principle for the potential of ESC for liver cell therapy.

Statement of Benefit to California: 

Liver transplantation is the only curative option for patients with severe liver diseases. As donor livers are rare, many Californians are currently waiting to receive a transplant. In fact, many patients on the waiting list die before a donor organ becomes available. If successful, the proposed project would help to alleviate the need for donor organs by establishing embryonic stem cells as a source of hepatocytes for transplantation. As hepatocyte transplantation would be less invasive and expensive than orthotopic liver transplantation, funds might become available that could be used to benefit the citizens of California in other areas.

Progress Report: 
  • The overall goal of this project is to establish new strategies for liver regeneration. For this purpose, we aim at identifying molecular mechanisms regulating liver regeneration that could be exploited for therapeutic purposes. Along these lines, we have identified small RNAs that either promote or inhibit the proliferation of hepatocytes, the cells in the liver that provide its manifold functions. We are now working on developing tools to modify the levels of these small RNAs in the liver with the goal to enhance liver regeneration. In addition, we aim at developing hepatocyte replacement strategies for liver diseases that cause irreparable hepatocyte damage. We focus on immune-compatible pluripotent stem cells because they can in principle generate the large numbers of hepatocytes required for therapeutically effective cell therapy and would not require life-long immune suppression. We have established proof-of-principle for the therapeutic efficacy of hepatocytes derived from mouse pluripotent stem cells, and are now working towards recapitulating these results in human cells.
  • In the past year we have made several discoveries that move us closer to our goal to improve the proliferation and function and thus therapeutic efficacy of hepatocytes derived from pluripotent stem cells.
  • Some of these discoveries have elucidated the role of microRNAs, a class of non-coding small RNAs, in liver regeneration and function. For example, we found that miR-21 acts as a promoter of hepatocyte proliferation during liver regeneration. In addition, we identified several other microRNAs that establish differentiated function in hepatocytes.
  • Other discoveries of ours have revealed which type of pluripotent stem cell is best for liver cell therapy that does not require chronic immune suppression. Our results show that induced pluripotent stem cells derived from fibroblasts are as effective in reversing liver failure as normal hepatocytes.
  • In the last year we have made significant progress towards our goal of "Molecular dissection of adult liver regeneration to guide the generation of hepatocytes from pluripotent stem cells". We have identified the mechanism of how microRNA-21 promotes liver regeneration. We are currently working on translating this understanding into a therapeutic strategy for liver failure. We have also gained in-depth insight into the molecular regulation of differentiation of liver progenitor cells into hepatocytes. We have begun to use this insight to direct the differentiation of pluripotent stem cells into hepatocytes that are effective in liver cell therapy.
  • Being able to generate hepatocytes from human pluripotent stem cells would advance many important research efforts, including studies of the pathobiology of liver diseases and the development of liver cell therapies. Unfortunately, realizing this potential has been hampered by shortcomings of human hepatocyte-like cells (HLCs) generated with current in vitro-differentiation protocols, not only as it pertains to replicating the function of primary human hepatocytes, but also their ability to proliferate in vivo. We have made significant progress toward our goal of identifying regulators of hepatocyte differentiation. In addition, we have established the feasibility of liver repopulation of immune-deficient mice with HLCs generated in vitro, thereby proving their ability to mature and proliferate after transplantation
  • We have made significant progress toward our goal of generating in the laboratory human liver cells that are therapeutically effective in mouse models of human liver failure. Because these surrogate human liver cells can be derived from readily accessible cell types like skin cells, they have potential for autologous liver cell therapies requiring nor or little immune suppression. Much of this progress was afforded by insight into mouse liver development and regeneration obtained from the investigations performed under this grant.
  • The overall goal of the research funded by this award was to establish the feasibility of generating therapeutically effective human hepatocytes in the laboratory. Another goal was to generate these hepatocytes so that they would not require immune suppression after transplantation. For this we investigated how hepatocyte differentiation and proliferation are regulated in mice and humans and applied this insight for the directed differentiation of human fibroblasts fully or partially reprogrammed to pluripotency. Our results showed that human fibroblasts can be converted into cells that replicate both function and proliferation of primary human hepatocytes, thereby establishing the feasibility of autologous liver cell therapy not requiring immune suppression.
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00359
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$2 504 614
Disease Focus: 
Liver Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Because there is still considerable morbidity and mortality associated with the process of transplantation, and because more than a thousand people die each year while on the liver transplantation list, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial assist device. Developing such a cell line from human embryonic stem cells (hESC) or from other human stem cell sources would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics.

In the proposed studies, we will differentiate human embryonic stem cells or fetal liver cells or bone-marrow derived cells so that they act like liver cells in culture. Once it has been established that the cells are acting like liver cells by producing normal human liver proteins, and that they do not act like cancer cells, the cells will be injected into the livers of immunoincompetent mice that do not rejects human cells. Then we will evaluate whether the cells grow and thrive in the mouse livers, whether they still produce high levels of human liver-specific proteins, whether they produce tumors in the mouse livers, and whether they can replace damaged mouse liver cells with human cells. One of the ways this will be done is to label the cells with a marker gene and to image the marker gene in the livers of the mice with special x-ray machines that can distinguish a few hundred human cells in the mouse liver. Finally, we will infuse the human liver stem cells into the liver of monkeys to determine if they will grow in the monkey livers, because the monkeys are more similar to man. Such studies should be done in nonhuman primates before clinical studies are undertaken to employ these cells to replace abnormal liver cells in man. Our intent is these studies is to compare and contrast three types of stem cells to determine which will be the most effective cells to use in human studies.

If the studies are successfully undertaken, we will establish a clinically useful and safe liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with liver transplantation; moreover, all people who had liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.

Statement of Benefit to California: 

In California, as in all parts of the US, there are not enough livers available for transplantation for all the people who need them. The result is that many more people die of liver failure than is necessary. One way to improve this situation is the transplantation of liver cells rather than whole organ transplantation. We are attempting to develop liver cell lines from stem cells that will act like normal liver cells. If the cells that we develop function well and do not act like cancer cells in culture, we will then transplant them into special mouse models of liver disease and see if the human cells can rescue the mouse from its liver disease. As a final test, we will see if the cells function in primate livers. In our studies, we will compare human embryonic stem cells with adult stem cells to determine which will be the most effective cells to transplant into people. If the studies are successfully undertaken, it means that we will have a stem cell line that can then be employed in human studies to determine their safety and effectiveness.

Progress Report: 
  • Because there is still considerable morbidity and mortality associated with the process of whole liver transplantation, and because more than a thousand people die each year while on the liver transplantation list, and tens of thousands more never get on the list because of the lack of available livers, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial liver. Developing such a cell line from human embryonic stem cells (hESC) or from other human stem cell sources would provide a valuable tool for cell-based therapeutics.
  • In the past year, we have improved on our ability to differentiate the hESC towards liver cells in culture. They are producing normal human liver proteins. They also are capable of metabolizing drugs and other substances in the same manner of normal liver cells in culture. This means that they have the most important attributes of normal liver cells. Also, we have employed these cells in clinically-relevant models using techniques that can then be adapted to future human clinical trials. Moreover, they do not produce tumors.
  • In addition, we are employing adult stem cells derived from the bone marrow in collaborative studies with colleagues in Egypt. These stem cells have been differentiated so that they act like liver cells, and they have been transplanted into patients with advanced liver disease. The patients that have received the cells have improvement in their blood tests, and they are living longer than would have been expected without the transplantation.
  • Thus we are making some progress in establishing a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with liver transplantation.
  • Because there is still considerable morbidity and mortality associated with the process of whole liver transplantation, and because more than a thousand people die each year while on the liver transplantation list, and tens of thousands more never get on the list because of the lack of available livers, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial liver. Developing such a cell line from human embryonic stem cells (hESC) or from other human stem cell sources would provide a valuable tool for cell-based therapeutics.
  • In the past year, we have improved on our ability to differentiate the hESC towards liver cells in culture. They are producing normal human liver proteins. They also are capable of metabolizing drugs and other substances in the same manner of normal liver cells in culture. This means that they have the most important attributes of normal liver cells. Also, we have employed these cells in clinically-relevant models using techniques that can then be adapted to future human clinical trials. Moreover, they do not produce tumors.
  • We have also worked to differentiate human induced pluripotent cells (hiPSC) to become liver-like cells in culture. The hiPSC behave very much like hESC, in that they are pluripotent. However, they are derived from adult somatic cells and thus do not have the ethical concerns associated with hESC. Our differentiation protocol has been successful in deriving cells that again have most of the important attributes of normal liver cells. Thus, we are hopeful that they also may be helpful for cell-based therapeutics in the future.
  • In addition, we are employing adult stem cells derived from the bone marrow in collaborative studies with colleagues in Egypt. These stem cells have been differentiated so that they act like liver cells, and they have been transplanted into patients with advanced liver disease. The patients that have received the cells have improvement in their blood tests, and they are living longer than would have been expected without the transplantation.
  • Thus we are making some progress in establishing a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with liver transplantation.
  • Because there is still considerable morbidity and mortality associated with the process of whole liver transplantation, and because more than a thousand people die each year while on the liver transplantation list, and tens of thousands more never get on the list because of the lack of available livers, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial liver. Developing such a cell line from human embryonic stem cells (hESC) or from other human stem cell sources would provide a valuable tool for cell-based therapeutics.
  • In the past year, we have improved on our ability to differentiate the hESC towards liver cells in culture. They are producing normal human liver proteins. They also are capable of metabolizing drugs and other substances in the same manner of normal liver cells in culture. This means that they have the most important attributes of normal liver cells. Also, we have employed these cells in clinically-relevant models using techniques that can then be adapted to future human clinical trials. Moreover, they do not produce tumors.
  • We have also worked to differentiate human induced pluripotent cells (hiPSC) to become liver-like cells in culture. The hiPSC behave very much like hESC, in that they are pluripotent. However, they are derived from adult somatic cells and thus do not have the ethical concerns associated with hESC. Our differentiation protocol has been successful in deriving cells that again have most of the important attributes of normal liver cells. Thus, we are hopeful that they also may be helpful for cell-based therapeutics in the future.
  • Thus we are making some progress in establishing a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with liver transplantation.

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