Liver Disease

Coding Dimension ID: 
301
Coding Dimension path name: 
Liver Disease

Molecular dissection of adult liver regeneration to guide the generation of hepatocytes from pluripotent stem cells

Funding Type: 
New Faculty II
Grant Number: 
RN2-00950
ICOC Funds Committed: 
$3 032 510
Disease Focus: 
Liver Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
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.

Gene therapy-corrected autologous hepatocyte-like cells from induced pluripotent stem cells for the treatment of pediatric single enzyme disorders

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06831
ICOC Funds Committed: 
$1 801 629
Disease Focus: 
Liver Disease
Genetic Disorder
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Liver transplantation (LT) has been used to treat a variety of liver diseases. Within hours after birth, neonates can present with disorders of the urea cycle (UCDs), the critical metabolic liver pathway needed to detoxify waste nitrogen from the diet and cellular turnover. The overall incidence of UCDs is estimated to be 1 in 8200 births. An increase in ammonia concentrations is particularly toxic to the central nervous system (CNS), causing brain edema, with multiple episodes affecting survival and often resulting in mental retardation and cerebral palsy. While LT is recommended in neonatal-onset patients with these single-enzyme defects, organ availability is a major limitation and transplantation requires lifelong immunosuppression. In addition, transplant morbidity and mortality are not inconsequential: 1-year survival is about 91.9%, and 5-year survival in children transplanted at less than 5 kg is 74%. Transplantation of genetically corrected embryonic stem cell-derived hepatocytes from the affected patients themselves is a potential way to replace LT for the treatment of metabolic liver disease and will likely not require immunosuppression; importantly, the supply will be limitless, allowing early transplantation before CNS injury. We propose to explore this approach by using a new and robust liver repopulation UCD mouse to advance this therapy: treatment of single-enzyme liver defects with patient-derived and genetically corrected stem cell-induced liver cells.
Statement of Benefit to California: 
Unfortunately there is a substantial wait for people in California who need a liver transplant, resulting in many who develop significant disability or die while waiting. While many are adults with chronic disease, some are children with metabolic disorders including urea cycle disorders (UCDs). UCDs are caused by mutations resulting in enzyme deficiencies responsible for removing waste nitrogen, which as ammonia can cause irreversible brain damage, coma and/or death. Newborns can become catastrophically ill within 36-48 hours after birth. These and other inborn errors represent a substantial cause of brain damage and death among newborns and infants and because many cases remain undiagnosed, or infants with the disorders die without a definitive diagnosis, the exact incidence is unknown and likely underestimated. Present treatment is dietary for most which is onerous & incomplete; definitive therapy is liver transplantation which is challenging in infants who have greater rates of complications and morbidity. In this proposal we will develop genetically-corrected hepatocyte-like cells from induced pluripotent stem cells from patients with arginase deficiency, a UCD. These will be tested for the ability to correct the disorder in a unique UCD liver repopulation mouse model. The advantage of the proposed methodology over current therapy is that genetically-corrected cells will be limitless and will require no major surgery or immunosuppression and its short- & long-term risks.

Development of a cell and gene based therapy for hemophilia

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06809
ICOC Funds Committed: 
$2 322 440
Disease Focus: 
Blood Disorders
Liver Disease
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma and affects 1/30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient and plagued by increased risk for blood-borne infections (HCV, HIV), high costs and limited availability. This has fueled a search for gene/cell therapy based alternatives. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance and easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers and chronic immunosuppression; making iPSC-based cell therapy an attractive prospect. As part of this project, we plan to generate iPSCs from hemophilic patients that will then be genetically corrected by inserting DNA capable of making FIX. After validation for correction, we will then differentiate these iPSCs into liver cells that can be transplanted into our mouse model of hemophilia that is capable of accepting human hepatocytes and allowing their proliferation. These mice exhibit disease symptoms similar to human patients and we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical and physiological assays. If successful, this will provide a long-term cure for hemophilia and other liver diseases.
Statement of Benefit to California: 
Generation of iPSCs from adult cells unlocked the potential of tissue engineering, replacement and cell transplant therapies to cure a host of debilitating diseases without the ethical concerns of working with embryos or the practical problems of immune-rejection. We aim to develop a POC for a novel cell- and gene-therapy based approach towards the treatment of hemophilia B. In addition to the obvious and direct benefit to the affected patients and families by providing a potential long-term cure; the successful development of our proposal will serve as a POC for moving other iPSC-based therapies to the clinic. Our proposal also has the potential to treat a host of other hepatic diseases like alpha-1-antitrypsin deficiency, Wilson’s disease, hereditary hypercholesterolemia, etc. These diseases have devastating effects on the patients in addition to the huge financial drain on the State in terms of the healthcare costs. There is a pressing need to find effective solutions to such chronic health problems in the current socio-economic climate. The work proposed here seeks to redress this by developing cures for diseases that, if left untreated, require substantial, prolonged medical expenditures and cause increased suffering to patients. Being global leaders in these technologies, we are ideally suited to this task, which will establish the state of California at the forefront of medical breakthroughs and strengthen its biomedical/biotechnology industries.

iPSC-derived Hepatocytes as Platforms for Research in Viral Hepatitis and Non-alcoholic Steatohepatitis

Funding Type: 
Tissue Collection for Disease Modeling
Grant Number: 
IT1-06563
ICOC Funds Committed: 
$865 370
Disease Focus: 
Liver Disease
Infectious Disease
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Hepatitis C and fatty liver disease are the two most common liver diseases in California. Individuals from different backgrounds are susceptible to these liver diseases, but they have unique genetic profiles that may influence the severity of disease and the response to specific therapies. Technology now makes it possible to generate stem cells from a person’s own skin. These cells can subsequently be used to generate liver cells identical to those from the original donor. Using this approach, scientists can perform research directly on an individual’s own liver cells to identify features that make the cells susceptible or resistant to disease and drug therapy. In this project, the research team will collect blood and skin tissue from people with liver disease and from healthy control subjects. The donated tissues will be placed in a "bank" for the production of stem cells. The overall goal is for the donated cells to be made available to scientists who will convert them to liver cells, and then carefully study them to learn more about liver disease. Research such as this is extremely valuable because it allows patients and volunteers to make a very personal contribution to the understanding of liver disease. The materials donated to this tissue "bank" will be a resource to the scientific community for many years.
Statement of Benefit to California: 
Hepatitis C and fatty liver disease are the two most common liver diseases affecting the citizens of California. Together they afflict one in every 12 people in the state and kill roughly 4,000 state residents each year. Researchers in California are actively seeking new information about the causes of and treatments for liver disease; their progress will be greatly accelerated by the opportunity to directly study the biology of diseased patients. The goal of this project is to build a "bank" of stem cells from local patients with liver disease. Patient donors will come from many different backgrounds, reflecting the great diversity of California. The bank, once established, will be a tremendous resource for medical research because the banked cells will be renewable and made available to the entire research community. Banked stem cells will enable researchers to study the genetics and biology of liver disease and to test new therapies. Importantly, they will give researchers an opportunity to study liver disease in its most important context - the affected patient. The research made possible through this effort will greatly enhance our understanding of liver disease; this will in turn reduce the negative impact of liver disease on the health and well-being of California residents.

Generation of safe and therapeutically effective human induced hepatocyte-like cells

Funding Type: 
Early Translational III
Grant Number: 
TR3-05542
ICOC Funds Committed: 
$1 544 170
Disease Focus: 
Liver Disease
Stem Cell Use: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
Although the liver can regenerate itself, chronic or overwhelming damage can cause life-threatening liver failure. Currently, the only therapy for liver failure is liver transplantation. Because the supply of cadaveric livers or liver tissue from living donors far exceeds the demand, physicians and researchers seek to develop new therapies to save the lives of patients with liver failure. One promising strategy is transplantation of hepatocytes, the cells of the liver that provide most of its functions and that are defective in liver failure. To make hepatocyte transplantation available to all patients who could benefit, a cell source other than scarce donor livers has to be established. In contrast to hepatocytes, skin cells can be readily obtained and expanded in culture. Therefore, the recent discovery that skin cells can be converted into hepatocytes by transfer of a few genes suggests a promising new source of hepatocytes. To develop transplantation of such cells as a therapy for liver failure, we aim to identify which readily available human cell type—skin, blood or fat cells—can be most efficiently converted into hepatocytes using methods of temporary gene transfer. Importantly, the therapeutic efficacy and safety of these induced hepatocytes will be rigorously tested in animal models of human liver failure. If successful, our project will establish the feasibility of therapy of liver failure with cells derived from a patient’s own readily available non-liver cells.
Statement of Benefit to California: 
Like in most states in the US, the number of Californians in need of a liver transplant significantly exceeds the number of available donor organs. Most of these patients have liver cirrhosis due to hepatitis C infection, alcoholic liver disease or cholestatic diseases. Other indications for liver transplantation include acute liver failure, hepatitis B virus infection, metabolic liver diseases and cancer. While the incidence of these liver diseases has been relatively stable, non-alcoholic steatohepatitis (NASH), which was first described only 10 years ago, is rapidly emerging and predicted to become the leading indication for liver transplantation in the future. Because Hispanics have an increased risk of developing NASH, California, the state with the largest Hispanic population in the US, will be particularly impacted by this epidemic. Thus, developing an abundant source of cells for liver cell therapy, as proposed in this project, will not only benefit the Californians currently awaiting liver transplantation, but may also help the state’s medical system to respond to this future challenge.
Progress Report: 
  • The overall goal of this project is to generate safe and therapeutically effective human induced hepatocyte-like cells from readily accessible cell sources like skin, fat or blood cells. In the first year of this project we have identified genetic factors effective in inducing hepatocyte differentiation of skin and fat cells. We have also identified genetic factors that allow the expansion of these induced hepatocyte-like cells. In addition, we have identified chemical factors that promote the function of human induced hepatocyte-like cells.

Generation of hepatic cell from placental stem cell for congenital metabolic disorders

Funding Type: 
Early Translational III
Grant Number: 
TR3-05488
ICOC Funds Committed: 
$1 750 375
Disease Focus: 
Liver Disease
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Approximately 1 in 1,500 children has a congenital metabolic disorder. These inborn errors of metabolism are caused by deficiencies of different enzymes and result in accumulation of various substances inside cells. These substances affect the function of vital organs, and in many cases are lethal. Transplantation of cells that possess the particular deficient enzyme carries the potential to cure these diseases. Currently, a shortage of human liver cells for transplantation prohibits clinical use of this therapy. The human placenta contains cells that may acquire hepatic function. Following delivery of a baby, these cells can be collected from the placenta which is in most cases is treated as medical waste and discarded. The therapeutic potential of this cell type has been shown in animal models. We propose to first develop a method to separate these cells from non liver like cells, and secondly use these cells to treat multiple mouse models of human inborn errors of metabolism. We will also establish a clinically applicable small-scale preparatory Bio-banking system to provide immunotype-matched cells to patients affected by these diseases. These immunotype-matched cells can replace the missing enzyme function in patients who suffer from congenital liver metabolic disorders, and potentially will be cure the condition. Although this proposal focuses on the congenital liver metabolic disorders, success may lead to the use of these cells in other liver diseases.
Statement of Benefit to California: 
We propose to develop a technology to isolate and derive functional hepatic cells from discarded human placentae. The therapeutic cells will be utilized to treat congenital metabolic disorders. Current therapy for congenital metabolic disorders requires life-long treatment. It is easy to imagine how the economical burden afflicts the patients' families and society. If successful, immuotype matched hAEC-derived cell replacement therapy may completely cure some of the congenital metabolic disorders. The benefit of this new regenerative medicine will be tremendous not only for the patients' quality of life but also for our society. Although this proposal focuses on the congenital liver metabolic disorders, the target disease can potentially be extended to other liver diseases. This cell therapy would be the first cell therapy for liver disease and could benefit thousands of patients in California who suffer various liver diseases. Furthermore, once this therapeutic potential is demonstrated, a placenta collection system, placental stem cell banking system, and a stem cell-derived hepatic cell distribution system might be a novel industry or industries that could provide job opportunities to the citizens of California.
Progress Report: 
  • We took human amniotic epithelial cells (hAECs) from placentae and isolated the cells with the enzyme activities that are lacking in three inherited metabolic disorders: mucopolysaccharidosis type I (MSP I, or Hurler syndrome), maple syrup urine disease and ornithine transcarbamylase deficiency (OTC). By transplanting these enzymatically-active cells into mice, we demonstrated an effective treatment for these disorders.
  • Our group and others have demonstrated that hAECs possess unique stem cell-like qualities, such as the ability to differentiate. More importantly, hAECs are genetically stable and therefore don’t form tumors upon transplantation in mice and humans.
  • During the first year of the project, we identified markers on the surface of hAECs that indicate the presence of the genes that code for the desired enzymes. We successfully established colonies of mice with each of the three metabolic disorders and defined the protocols for the radiological and biochemical tests, or assays. We also performed several hAEC transplantations to mice with MSP I.
  • The first case of hAEC transplantation demonstrated a very promising result: the pathologic protein concentration in the urine of the treated MSP I mouse was dramatically decreased. We will confirm this result by investigating it further in more mice.
  • As proposed, we have also started building a small-scale bio-bank of hAECs from 24 placentae. These hAECs will be used to determine whether hAECs retain their therapeutic potential after cryopreservation, or freezing.

Antibody tools to deplete or isolate teratogenic, cardiac, and blood stem cells from hESCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
ICOC Funds Committed: 
$1 869 487
Disease Focus: 
Blood Disorders
Heart Disease
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells. We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile. Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine. Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.
Statement of Benefit to California: 
Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state. hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.
Progress Report: 
  • Our program is focused on improving methods that can be used to purify stem cells so that they can be used safely and effectively for therapy. A significant limitation in translating laboratory discoveries into clinical practice remains our inability to separate specific stem cells that generate one type of desired tissue from a mixture of ‘pluripotent’ stem cells, which generate various types of tissue. An ideal transplant would then consist of only tissue-specific stem cells that retain a robust regenerative potential. Pluripotent cells, on the other hand, pose the risk, when transplanted, of generating normal tissue in the wrong location, abnormal tissue, or cancer. Thus, we have concentrated our efforts to devise strategies to either make pluripotent cells develop into desired tissue-specific stem cells or to separate these desired cells from a mixture of many types of cells.
  • Our approach to separating tissue-specific stem cells from mixed cultures is based on the theory that every type of cell has a very specific set of molecules on its surface that can act as a signature. Once this signature is known, antibodies (molecules that specifically bind to these surface markers) can be used to tag all the cells of a desired type and remove them from a mixed population. To improve stem cell therapy, our aim is to identify the signature markers on: (1) the stem cells that are pluripotent or especially likely to generate tumors; and (2) the tissue-specific stem cells. By then developing antibodies to the pluripotent or tumor-causing cells, we can exclude them from a group of cells to be transplanted. By developing antibodies to the tissue-specific stem cells, we can remove them from a mixture to select them for transplantation. For the second approach, we are particularly interested in targeting stem cells that develop into heart (cardiac) tissue and cells that develop into mature blood cells. As we develop ways to isolate the desired cells, we test them by transplanting them into animals and observing how they grow.
  • Thus, the first goal of our program is to develop tools to isolate pluripotent stem cells, especially those that can generate tumors in transplant recipients. To this end, we tested an antibody aimed at a pluripotent cell marker (stage-specific embryonic antigen-5 [SSEA-5]) that we previously identified. We transplanted into animals a population of stem cells that either had the SSEA-5-expressing cells removed or did not have them removed. The animals that received the transplants lacking the SSEA-5-expressing cells developed smaller and fewer teratomas (tumors consisting of an abnormal mixture of many tissues). Approaching the problem from another angle, we analyzed teratomas in animals that had received stem cell transplants. We found SSEA-5 on a small group of cells we believe to be responsible for generating the entire tumor.
  • The second goal of the program is to develop methods to selectively culture cardiac stem cells or isolate them from mixed cultures. Thus, in the last year we tested procedures for culturing pluripotent stem cells under conditions that cause them to develop into cardiac stem cells. We also tested a combination of four markers that we hypothesized would tag cardiac stem cells for separation. When these cells were grown under the proper conditions, they began to ‘beat’ and had electrical activity similar to that seen in normal heart cells. When we transplanted the cells with the four markers into mice with normal or damaged hearts, they seemed to develop into mature heart cells. However, these (human) cells did not integrate with the native (mouse) heart cells, perhaps because of the species difference. So we varied the approach and transplanted the human heart stem cells into human heart tissue that had been previously implanted in mice. In this case, we found some evidence that the transplanted cells differentiated into mature heart cells and integrated with the surrounding human cells.
  • The third goal of our project is to culture stem cells that give rise only to blood cells and test them for transplantation. In the past year, we developed a new procedure for treating cultures of pluripotent stem cells so that they differentiate into specific stem cells that generate blood cells and blood vessels. We are now working to refine our understanding and methods so that we end up with a culture of differentiated stem cells that generates only blood cells and not vessels.
  • In summary, we have discovered markers and tested combinations of antibodies for these markers that may select unwanted cells for removal or wanted cells for inclusion in stem cell transplants. We have also begun to develop techniques for taking a group of stem cells that can generate many tissue types, and growing them under conditions that cause them to develop into tissue-specific stem cells that can be used safely for transplantation.
  • Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
  • In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
  • In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
  • In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
  • In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.
  • The main focus of our program is to improve methods to generate pure populations of tissue-specific stem cells that form only heart tissue or blood. Such tissue-specific stem cells are necessary for developing safe and effective therapies. If injected into patients with heart damage, heart-forming stem cells might be used to regenerate healthy heart tissue. Blood-forming stem cells are capable of regenerating the blood-forming system after cancer therapy and replacing a defective blood forming-system. We showed that blood-forming stem cells from a given donor induce in the recipient permanent transplant tolerance of all organs, tissues, or other tissue stem cells from the same donor. Therefore, having a single biological source of each of the different types of stem cells (e.g., blood and heart) would revolutionize regenerative medicine.
  • Our projects involve generating tissue-specific stem cells from pluripotent stem cells (PSCs), the latter of which are stem cells that can form all tissues of the body. PSCs (which include embryonic stem cells and induced pluripotent stem cells) can turn into all types of more specialized cells in a process known as “differentiation.” Because PSCs can be grown to very large numbers, differentiating PSCs into tissue-specific stem cells could lead to banks of defined tissue stem cells for transplantation into patients—the ultimate reason to conduct PSC research.
  • However, current methods to differentiate PSCs often generate mixtures of various cell types that are unsafe for injection into patients. Therefore, generating a pure population of a desired cell type from PSC is pivotal for regenerative medicine—purity is a key concern for cell therapy as it is with medications.
  • We have invented technologies to purify desired types of cells from complex cell populations, allowing us to precisely isolate a pure population of tissue-specific stem cells from differentiating PSCs for cell therapy. For instance, in our work on heart-forming cells, we developed labels for cells that progressively give rise to heart cells. We used these labeled cells to clarify the natural, stepwise, differentiation process that leads from PSCs to heart-forming stem cells, and finally to different cells within the heart. Exploiting these technologies to isolate desired cell types, we have completed the first step of turning human PSCs into heart-forming stem cells. In the laboratory, when we transplanted these heart-forming stem cells into a human heart, they integrated with the surrounding tissue and participated in correctly timed beating. In the future we hope to deliver heart-forming stem cells into the damaged heart to regenerate healthy tissue.
  • We have also attempted to turn PSCs into blood-forming stem cells by understanding the complex process of blood formation in the early embryo. As mentioned above, if blood-forming stem cells are transplanted into patients, they create in the recipient an immune system that will tolerate (i.e., not reject) other tissues and types of tissue stem cells (e.g., for skin or heart) from the same donor. Thus, turning PSCs into blood-forming stem cells will provide the basis for all regenerative medicine, whereby the blood-forming stem cells and the needed other tissue stem cells can be generated from the same pluripotent cell line and be transplanted together.
  • In parallel studies to those above, we have turned PSCs into liver-forming stem cells. In the embryo, the liver emerges from a cell type known as endoderm, whereas the blood and heart emerge from a different cell type known as mesoderm. We learned that PSCs could only be steered to form endoderm (and subsequently, liver) by diverting them away from the path that leads to mesoderm. Through this approach, we could turn human PSCs into endoderm cells (at >99% purity) and then into liver-forming stem cells that, when injected into the mouse liver, gave rise to human liver cells. This could be of therapeutic importance for human patients with liver damage.
  • Finally, we have developed methods to deplete PSCs from mixtures of cells differentiated from PSCs, because residual PSCs in these mixtures can form tumors (known as teratomas). These methods should increase the safety of PSC-derived tissue stem cell therapy.
  • In summary, we have developed methods to turn PSCs to tissue-specific stem cells that exclusively develop into only heart, blood cells, or liver cells. This work has involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.

Liver Cell Transplantation

Funding Type: 
Early Translational II
Grant Number: 
TR2-01857
ICOC Funds Committed: 
$5 199 767
Disease Focus: 
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
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) would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics. The objective of this proposal is to focus a team effort to determine which differentiated hESC will be the most effective liver-like cells in cell culture and in animal studies, and to then use the best cells in clinical trials of cell transplantation in patients with advanced liver disease. In the proposed studies, the team will differentiate hESC 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 result in tumors, the cells will be assessed in clinically-relevant models using techniques that can then be adapted to future human clinical trials. One of the ways cells can be evaluated is to label the cells which will provide a means to monitor them with various imaging systems. The intent in these studies is to determine which will be the most effective cells to use in human clinical trials. Once this is determined, the best cells can then be employed in human patients. If the studies are successfully undertaken, we will have established 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 whole liver transplantation. Moreover, all people who have 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, the cells will be assessed in clinically-relevant models using techniques that can then be adapted to future human clinical trials. In our studies, we will compare human embryonic stem cells with other stem cells to determine which will be the most effective cells to transplant into people. In the future, we will employ the best cells in clinical trials in humans. If the studies are successfully undertaken, we will have established 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 whole liver transplantation. Moreover, all people who have liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
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) would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics. The objective of this proposal is to focus a team effort to determine which differentiated hESC will be the most effective liver-like cells in cell culture and in animal studies, and to then use the best cells in clinical trials of cell transplantation in patients with advanced liver disease.
  • In the past year, we have been able to differentiate hESC so that they act like liver cells in culture. The cells are acting like liver cells by producing normal human liver proteins, and they metabolize drugs in a manner similar to liver cells, and they do not cause tumors when transplanted. Now we are ready to assess the cells in clinically-relevant models using techniques that can then be adapted to future human clinical trials. The intent in these studies is to determine which will be the most effective cells to use in human clinical trials. Once this is determined, the best cells can then be employed in human patients.
  • If the studies are successfully undertaken, we will have established 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 whole liver transplantation. Moreover, all people who have liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
  • Some patients with life-threatening liver disease can be effectively treated with liver transplantation. However, this requires a major surgical procedure that is associated with considerable morbidity and mortality. More importantly, the long-standing shortage of donor livers has rendered this treatment unavailable to most patients. Consequently, thousands of patients with end-stage liver disease die each year while on a waiting list for liver transplantation, and tens of thousands are never put on this list. Since human embryonic stem cells replicate virtually indefinitely in culture, these cells, if differentiated into liver cells (hepatocytes), represent an infinite source of cells that can be made available to treat patients with liver failure.
  • The objective of this award than is to develop a reproducible and efficient differentiation method to produce metabolically active human embryonic stem cell-derived hepatocytes. The initial requirement is that at least 90-95% of the cells must have liver-specific gene expression and possess metabolic function comparable to freshly isolated human hepatocytes. In addition, the yield from differentiation should be adequate to support preclinical studies. We believe that we made excellent progress during the grant award period towards meeting the success criteria for the quality of the cells in in vitro analysis. We firmly believe that our cells represent an exceedingly good level of hepatocyte function in culture, and that the remaining tasks are to scale-up their production and to determine a strategy to enhance their in vivo function.
  • The objective of this Early Translational II Research Award is to develop a reproducible and efficient differentiation method to produce metabolically active human embryonic stem cell-derived hepatocytes (hEDH)(also called liver cells). We have determined that at least 90-95% of the cells have liver-specific gene expression and possess biotransformation ability comparable to freshly isolated human hepatocytes. This means that our cells that we have differentiated from embryonic stem cells are acting like human liver cells. In addition, the yield from differentiation should be adequate to support preclinical studies. We believe that we made excellent progress during the grant award period towards meeting the success criteria for the quality of the cells and that further work may well yield cells that can be transplanted into people.

Development of an hES Cell-Based Assay System for Hepatocyte Differentiation Studies and Predictive Toxicology Drug Screening

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01012
ICOC Funds Committed: 
$971 558
Disease Focus: 
Liver Disease
Toxicity
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
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.

An in vitro and in vivo comparison among three different human hepatic stem cell populations.

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00359
ICOC Funds Committed: 
$2 504 614
Disease Focus: 
Liver Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
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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