Mathematic Optimization of Multiple Signals for Stem Cell Differentiation into Hepatocytes

Funding Type: 
Basic Biology I
Grant Number: 
ICOC Funds Committed: 
Public Abstract: 
Hepatitis C virus (HCV) is a major human health concern with an estimated 170 million people infected with HCV worldwide. In comparison, 40 million people are infected with HIV in the world. HCV causes liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Approximately 5 million people in the United State are infected with HCV. Persons with HCV often show no signs or symptoms of the disease until they have reached end-stage liver disease, 20, 30 even 50 years after the initial infection. Many people do not realize that they are infected. Treatment for HCV is only efficient in a portion of patients. Once a patient has developed end-stage liver disease, their only treatment option is often a liver transplant, which only extends their life for a short period of time. Liver failure due to HCV infection presents an excellent case calling for improvement of stem cell technology as a potential therapeutic approach. Liver tissue is one of the tissues that can be most easily regenerated, offering a favorable opportunity to demonstrate the promise of stem cell therapy. There are a few rate limiting steps in stem cell therapy. One is the low efficiency of differentiation into specific cell types. The differentiation process involves multiple signals. Defining the optimal combination of multiple signals is a challenge to traditional biology. We will use a mathematic approach to overcome this problem. The mathematic modeling and search algorithms will allow us to understand the interactions among multiple signals that control the cell differentiation and thereby optimize the process. Another rate limiting step in cell therapy is how to replace the existing cells with the newly derived cells. Here the cell killing due to HCV infection offers the opportunity. We can modify the stem cells to confer resistance to HCV infection, which will provide selection advantage over the original liver cells that are more susceptible to HCV infection. The selection pressure imposed by HCV can be used to efficiently replace the original liver cells with modified liver cells derived from stem cells. Thus, the proposed study can be a good test case. This proposal addresses fundamental issues in stem cell biology while providing potential application. It combines the expertise of cell biology and virology with mathematics to define and optimize stem cell differentiation process. The utilization of a tractable mouse model that directly reflects human cell differentiation, viral infection, and tissue regeneration process, establishes a solid foundation for future clinical application.
Statement of Benefit to California: 
An estimated 600,000 Californians are infected with HCV. Many of them are unaware of the fact that they are infected. California’s public and private healthcare systems have experienced increased demand for Hepatitis C services, including patient and provider education and case management. California’s public and private healthcare systems are therefore under great economic pressure to treat Hepatitis C, a life threatening liver disease. Hepatitis C impacts the families, friends, employees and communities of those with HCV. California’s economic loss due to HCV infection is several billion dollars annually. The treatment for HCV is only partially effective. Once a patient has advanced to end-stage liver disease, current drug treatments may not be well tolerated and could worsen the health status of the patient. For these patients the only available treatment option is liver transplantation. Liver transplantation however can only extend the life and cannot eliminate viral infection, which will recur soon. Although several HCV-drugs are in pipelines, the potential emerging of drug-resistant HCV is well expected for fast mutating RNA viruses. Up to 10 percent of HCV+ individuals are also HIV+. Roughly 6 percent of individuals co-infected with both viruses will not develop antibodies to HCV, making detection of the virus and the treatment more complicated. Thus a new treatment approach is urgently needed. Stem cell therapy is one of the promising approaches to treat HCV infected patients. California Hepatitis C Task Force is one of the organizations that strongly support the legislation that resulted in the establishment of the California Institute for Regenerative Medicine. One of the bottle necks of applying stem cell treatment is the low efficiency of differentiating progeny cells into liver cells. Here we are taking a novel approach by combining mathematics with updated stem cells technology to generate liver cells. Furthermore, we will modify the cells to make them resistant to HCV infection or slow down their replication. Such HCV-resistant cells will be injected into the patients’ liver and will gradually replace the original susceptible liver cells. Thus this liver regeneration approach will cure or prevent liver failure with a relatively simple procedure in comparison with liver transplantation. Although this proposal focuses on liver cell regeneration and the potential therapeutic application for HCV infected patients, the methods being developed in this proposal are generally applicable to stem cell biology as well as the applications. Therefore, this research can significantly accelerate the advancement of CIRM’s mission.
Progress Report: 
  • Induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine: these cells are similar to embryonic stem cells (ESCs) but can be derived upon “reprogramming” of any mature cell type isolated from a patient. Thus, tissue-specific stem cells derived from iPSCs and re-injected into the same patient may not trigger immune rejection. However, before the full potential of iPSCs is achieved, we need to learn how to better generate these cells, control their maturation into tissue-specific stem cells and progenitors, and harness their tumorigenic potential. Interestingly, ESCs and iPSCs share many characteristics of cancer cells, including their unlimited proliferation potential, and emerging evidence suggests that the mechanisms underlying the infinite proliferation of cancer cells and ESCs are intimately intertwined. Similarly, the progressions stages of tumorigenesis and cellular reprogramming to iPSCs share several characteristics, including changes in the packaging of the chromosomes.
  • Based on these observations, we proposed to directly study the function of a major cancer pathway, the RB pathway, in cellular reprogramming and iPSCs. RB is a key tumor suppressor in humans. RB acts as a cellular brake that restricts cell division but has several other cellular functions, including in the control of cellular maturation. When RB is mutated, cells divide faster and become more immature, two features of cancer cells, but also of cells undergoing reprogramming. We hypothesized that RB is an important regulator of cellular reprogramming and will test this idea using mouse and human cell types in culture. In the last year, we have performed experiments that largely support this hypothesis. We have found that, similar to its role in normal cell cycle, RB acts as a brake to normally restrict the reprogramming of cells into iPSCs. We have also found that RB is regulated in cells by enzymes that normally control the coating structure of chromosomes; these enzymes are thought to play a role in reprogramming, suggesting that RB may be a critical regulator of reprogramming by controlling the ability of reprogramming factors to modify the structure of the DNA. These experiments now provide a powerful system to analyze the molecular mechanisms underlying cellular reprogramming.
  • Our general goal is to better understand the differences and similarities between cancer cells and embryonic stem cells, to prevent tumor formation following stem cell transplantation but also to gain novel insights into the mechanisms of tumorigenesis and into the biology of embryonic stem cells. To this end, we have been studying how a tumor suppressor named Rb controls the dedifferentiation (or "reprogramming") of cells into induced pluripotent stem cells (iPS cells), which are similar to embryonic stem cells. (ES cells).
  • We have found that, similar to other tumor suppressors such as p53, Rb normally restricts the reprogramming process, both in human and mouse cells. We have also found that loss of RB does not change the proliferation rate of cells during reprogramming, suggesting that the enhanced efficiency of reprogramming observed in the absence of Rb is not due to a simple increase cell number. We are currently investigating the mechanisms by which Rb normally restricts the reprogramming process.
  • Our overarching goal is to understand the mechanisms controlling the balance between stem cell pluripotency, self-renewal, and tumorigenesis, to harness the full therapeutic promise of human embryonic stem cells (hESCs). To this end, we study the function of the RB gene family in stem cells. Our initial hypothesis was that RB family genes may control the reprogramming of somatic cells into iPSCs by interacting with chromatin remodeling factors to induce specific changes in the chromatin structure and control the expression of a specific program of genes. We found that loss of RB, but not of its family members p107 and p130, results in enhanced reprogramming of fibroblasts to iPS cells. In the past year, we have investigated this unique function of RB. In particular, we have performed high throughput RNA-seq and ChIP-seq experiments for RB early in the differentiation process to explore the mechanisms by which loss of RB may enhance reprogramming. We have also performed ChIP-Seq experiments with various chromatin marks to explore the relationship between RB loss and change sof the chromatin structure of cells early in reprogramming.
  • During the reporting period, we have pursued our work on the role of the retinoblastoma tumor suppressor during the reprogramming of mouse and human cells into induced pluriptoent stem cells (iPS cells). We have performed and analyzed genome-wide RNA-seq and ChIP-seq experiments to investigate how loss of RB promotes reprogramming. We have also tested candidate downstream mediators of RB in reprogramming using mouse genetics in vivo.

© 2013 California Institute for Regenerative Medicine