Regulation of Human Thymic Epithelial Cell Development from ESC

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01629
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
The function of the immune system throughout life is essential for protection from infections and cancer. T lymphocytes are white blood cells that choreograph the multiple responses that the body uses to control infection. T lymphocytes are produced in the thymus, a specialized organ located in the chest in front of the heart. The production of new T lymphocytes (“thymopoiesis”) is abnormal in some children with genetic defects in the development of the thymus (DiGeorge syndrome [DGS]), but even in healthy people, thymic function declines with age. Thymic insufficiency, the decreased ability of the thymus to make new T lymphocytes, is a serious health problem. For example, if the T lymphocytes that have been previously made were to be destroyed, for example by HIV infection, chemotherapy or radiation therapy, or hematopoietic stem cell transplantation, the restoration of immune function requires the production of new T lymphocytes to replace those that were lost. For this reason, adults with such conditions have poorer recovery of immune function than children and the elderly have increasing risk of severe infection with age. For example, 10-40% of the elderly do not respond to annual influenza vaccination and as many as 50-100,000 may die of influenza annually. Thymic insufficiency is due to injury or death of skin-like cells called thymic epithelial cells (TEC), which produce a number of proteins such as interleukin-7 (IL-7) needed by developing T lymphocytes in the thymus (“thymocytes”). Like skin cells, TEC become more fragile and easily injured with age. Also like skin cells, TEC are destroyed by chemotherapy and radiation therapy. Clinical efforts to restore thymopoiesis in patients with HIV infection by transplantation of thymic tissue from unrelated donors have not been successful because of rejection of the transplanted tissue. Experimental efforts to correct the problem of decreased thymopoiesis have included attempts to replace TEC functions by injections of IL-7 or other cells that make IL-7; or to regenerate TEC by the injection of keratinocyte growth factor (KGF), a protein that stimulates the growth of TEC. Human embryonic stem cells (hESC) are a potential source of replacement TEC that could be used to regenerate the immune system in people whose pool of T lymphocytes has been decreased, e.g., the elderly, or those with HIV or cancer. In order to implement such a strategy, research on how to control the development of TEC from hESC are necessary. The proposed studies will test how the Tbx1 gene, which is abnormal in DGS, controls the development of TEC from hESC. In addition, the studies will develop model systems in mice for testing the ability of TEC to be transplanted, a necessary scientific tool for the assessment of future therapies that will use TEC progenitors to restore immune function.
Statement of Benefit to California: 
The research is aimed at understanding the generation of TEC in an effort to ultimately develop clinical strategies for thymic regeneration to treat thymic insufficiency. Thymic insufficiency occurs as both primary defects of TEC development and more commonly as acquired defects in TEC maintenance. Thymic insufficiency was first recognized in children with the rare DiGeorge syndrome (DGS), in which thymic hypoplasia occurs. More recent studies have shown that age-related thymic insufficiency is a common problem that progresses, and influences the outcome of many diseases. If an individual has a condition that results in destruction or increased turnover of mature T lymphocytes, their health will ultimately depend on the ability of the thymus to produce new T lymphocytes. An example is HIV infection, in which immunological recovery depends not just on the efficacy of anti-retroviral therapy to decrease the viral burden and T lymphocyte destruction, but also on the ability of the thymus to produce new T lymphocytes to replace those that were previously destroyed. The ability to do so is inversely related to age. Similar age-related thymic insufficiency occurs in recipients of high dose chemotherapy for cancer and in recipients of hematopoietic stem cell transplants (HSCT). Probably the largest group of individuals who are affected by thymic insufficiency are the elderly. There is evidence that the declining immune responsiveness of the elderly is a serious problem, particularly as it relates to common respiratory virus infections, such as influenza and respiratory syncytial virus, which together kill >50,000 Americans each year. In discussing the relevance of the studies to California, it must be recognized that this CIRM Basic Biology Grant is aimed at a set of basic questions that will not immediately translate into health benefits. Nevertheless, it is possible to make estimates of how many individuals have conditions that this work is directly related to. For example, DGS is thought to affect 5% of all children with congenital heart disease and 20-25% of those with severe CHD, especially those with conotruncal abnormalities. Using the estimated 500-600,000 births per year (http://cgi.rand.org) and an incidence of 0.4% of severe CHD, there are about 500-600 births of children with DGS in California per year. Based on CDC serosurveillance data, tens of thousands of Californians are HIV infected and tens of thousands others receive either intensive chemotherapy or HSCT annually. Finally, the 2000 census showed approximately 3.5 million Californians over the age of 65 (http://www.census.gov/census2000/states/ca.html). Thus, the research proposed in this grant is likely to be directly related to the health of millions of individuals in California as well as having large impact on health economics.
Progress Report: 
  • The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Work completed in the first reporting period of this project focused on a regulator named TCF-3. The role of this factor is known in mouse embryonic stem cell cultures to be a key regulator that helps define the pluripotent state in a very important way: loss of TCF-3 protein results in a refractory state of pluripotency and inability to differentiate. The molecular and cellular characteristics of TCF-3 and its function in human ESCs and iPSCs is not known. Therefore, it is not known whether human TCF-3 performs functions in hESC and hiPSC that are similar to its functions in mouse ESC. The work performed in the first funding period focused on defining the basic properties of human TCF-3 mRNA and protein expression under different culture conditions – preparatory experiments that will enable us to define its functions. Multiple conditions that enable maintenance of an embryonic stem cell state, or conditions that promote differentiation into different cell fate lineages were established. Biological tools were developed to study TCF-3 mRNA and protein under those conditions, including tools to re-introduce mutant and protein-modified TCF-3 and reagents to remove TCF-3 mRNA and protein from hESC cells. The following results were obtained: i) TCF 3 is expressed and active as a negative regulator, ii) TCF-3 protein is modified by protein cleavage and covalent modifications, iii) TCF-3 is an important regulator of differentiation genes. These data suggest that TCF-3 is an important modulator of stem cells and differentiation. While this might imply that TCF-3 performs similar functions in mouse and human ESC, we observe differences in the genes regulated and in hESC colony phenotypes when TCF-3 levels are modified. These observations suggest there are important differences in TCF-3 function that distinguish it from its counterpart in mouse ESC. Experiments have been designed to test this hypothesis and to define how the apparent differences of human TCF-3 are established at the molecular and cell signaling level.
  • The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Specifically, this project focused on the regulation and actions of TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs and iPSCs is not known, whether its functions are similar to its functions in mouse ESC. The work performed in the second funding period focused on mapping key protein modifications of TCF-3 including phosphorylation. Phosphorylation is a frequently used method to modify the actions of proteins – to either inhibit protein activity, stimulate activity, or change the ability to interact with other proteins. Human embryonic stem cell lines were modified to express a tagged version of TCF-3 for rapid isolation and purification from cell extracts. Purification was performed rapidly and under specialized conditions to prevent loss of any protein modifications. Mass spectrometry identified multiple phosphorylation sites and other forms of protein modification.
  • Work during this period also focused on developing strategies to identify key target genes regulated by TCF-3. Methods to remove TCF-3 from hESCs were developed and validated on levels to ensure bona fide removal without artefactual effects on hESCs. Removal of TCF-3, or “knockdown” will be used to investigate how hESCs cope in the absence of TCF-3. Preliminary results suggest that TCF-3 exerts control over a set of gene regulators as well as cellular signals. For these experiments, only early, immediate events that occur upon TCF-3 removal were assessed. A focus on early events is a purposeful effort to identify immediate-reacting, key regulatory steps. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.
  • The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Specifically, this project focused on the regulation and actions of TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs is not known. It is not known whether TCF-3 functions are similar to its functions in mouse ESC. The work performed in the third funding period focused on defining the genes that are modulated by TCF-3 expression. Genome-wide microarray analysis was performed in the presence and absence of TCF-3 and in the presence and absence of differentiation conditions.
  • Removal of TCF-3, or “knockdown” revealed a specific, small set of genes that are dynamically dependent on TCF-3 for expression. Signals for differentiation that mimic the environment in the developing embryo were used to understand how TCF-3 functions in this setting. Preliminary data suggest that TCF-3 exerts a strong, noticeable function within the new signaling context. Additional microarray analyses, validation and biochemical confirmation are being used to complement the microarray studies and better define the precise role of TCF-3 action. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.
  • The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs). Specifically, this project focused on the regulation and actions of the gene expression regulator TCF-3. The role of this factor in mouse embryonic stem cell cultures is known to protect the pluripotent state and cellular responsiveness in a very important way: loss of TCF-3 protein results in a permanent state of pluripotency and inability to differentiate. However, the molecular and cellular characteristics of TCF-3 and its function in hESCs is not known. It is not known whether TCF-3 functions are similar to its functions in mouse ESC. The work performed in the third funding period focused on defining the genes that are modulated by TCF-3 expression. Genome-wide microarray analysis was performed in the presence and absence of TCF-3 and in the presence and absence of differentiation conditions.
  • Removal of TCF-3, or “knockdown” revealed a specific, small set of genes that are dynamically dependent on TCF-3 for expression. Signals for differentiation that mimic the environment in the developing embryo were used to understand how TCF-3 functions in this setting. Our data suggests that TCF-3 exerts a strong, repressive function within the new signaling context. Microarray analyses, validation and biochemical confirmation have been used to better define the precise role of TCF-3 action. This past year utilized ChIP-seq approached to precisely define the pattern of genome occupancy that TCF-3 has in hESC cultures prior to differentiation. The results suggest that TCF-3 expression is dynamic, constitutively required, and that much of its function is to provide repressive actions. While this has general similarity to the role ascribed to TCF-3 in mouse embryonic stem cells, it appears that the types of genes and processes repressed by TCF-3 are distinct in hESC.

© 2013 California Institute for Regenerative Medicine