Etsrp/ER71 mediated stem cell differentiation into vascular lineage

Etsrp/ER71 mediated stem cell differentiation into vascular lineage

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02165
Award Value: 
$1,382,400
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
Human embryonic stem cells (hESC) have the potential to differentiate into all of the cell types that make up the body. Therefore, hESCs are promising tools for the treatment of degenerative diseases and for use in regenerative medicine. One highly desirable use of hESCs is to treat cardiovascular disease. Cardiovascular disease is a leading cause of mortality and morbidity in the state and country. Cardiovascular disease is caused by damage to blood vessels and the ability to repair this damage will improve disease outcomes. However, the ability to efficiently differentiate hESCs down cardiovascular lineages to generate large numbers of cells on a therapeutically relevant scale is lacking. The goal of this project is to develop a protocol for the differentiation of hESCs into vascular endothelial cells for the treatment of cardiovascular disease. Initially we will study the expression of vascular precursor cell genes during embryoid body formation from hESCs. Then, using gene transfer technology and regulatable gene expression of transcription factors that induce the vascular cell lineage, we will “tune” treated hESCs to optimize derivation of endothelial cells. We will then use these cells in a pre-clinical mouse model of ischemic heart damage to test their ability to integrate into the damaged tissue and restore circulatory function. Additionally, we will use these cells to study the molecular mechanisms of endothelial cell differentiation. These studies will increase our knowledge of hESC biology, endothelial cell development, and suggest methods for therapeutic use of hESCs.
Statement of Benefit to California: 
Cardiovascular disease, including heart disease, heart failure, and stroke, is the number one cause of death in the state of California. Additionally, patients suffering with these conditions have decreased quality of life and represent a significant financial and emotional burden to both their families and the state in general. Novel treatments to block or reverse the progression of cardiovascular disease will benefit the patient physically, the caregiver emotionally, and relieve the financial burden to everybody. Recently, the use of human stem cells has been proposed as a therapeutic treatment for cardiovascular disease. Addition of these cells to injured tissue has the potential to prevent the loss of more tissue and regenerate lost tissue. However, before such treatments become available, the basic biology of these cells must be understood to maximize treatment efficiency and prevent unwanted side effects. We have identified a critical gene in the development of blood vessel precursor cells and propose to use it to optimize the generation of blood vessel cells from human embryonic stem cells. In addition we will carefully examine the molecular events underlying the transition from stem cell to blood vessel precursor. The potential knowledge gained from these studies has implications for controlling blood vessel overgrowth in diseases such as cancer and diabetic retinopathy as well as blood vessel dysfunction in diseases such as cardiovascular disease and peripheral artery disease. These findings will benefit the field of stem cell research and basic developmental biology with future research directions based on these results. In addition to the clinical and scientific applications of these studies, the young scientists being trained in the laboratory will provide the foundation for future research in academia or industry in California. The success of this project will enhance the already stellar reputation of the state of California as a leader in the stem cell field.
Progress Report: 

Year 1

Cardiovascular disease is a major concern for medicine and is caused by damage to blood vessels. We have begun a project to generate endothelial cells, the cells which line the blood vessels, from human embryonic stem cells (hESCs) using gene transfer technology and regulated gene expression. Little is known about the early stages of blood vessel endothelial differentiation of the human embryo. It is imperative that we understand normal development in order to mimic it in the laboratory. We have used hESCs to model embryonic development and determine the pattern of gene expression in the early stages of differentiation. Using what is known about mouse embryonic development as a model, we have determined that gene expression in differentiating human cells closely follows that of differentiating mouse cells. In particular, we have determined the timing of the expression pattern of a gene that is required for the generation of endothelial cells. This knowledge will allow us to induce expression of this gene at the proper time during differentiation in the cells in the laboratory to increase the number of blood vessel cells we can generate. Timing of gene expression during development is extremely important and improper timing can result in cells being unable to respond to the signal generated by the gene or unable to progress further in development. The factor required for blood vessel development is only required for a short window of time and then must be removed from the system for the cells to progress to mature blood vessels. Using a viral vector to introduce the modified genes to cells, we are taking advantage of a system that allows us to regulate the expression of an endothelial gene by the addition of a common drug to the cells. Once the drug is removed from the system, gene expression is ended. This allows us to mimic the pattern of the factor seen in normal development of blood vessel cells. We have established a method in the laboratory to reliably generate endothelial cells from unmodified hESCs based on methods from previously published studies. These laboratory generated cells mimic human endothelial cells in many tests including gene expression and surface protein expression. In addition, we have shown that expression of the transcription factor required for endothelial cell development in hESCs induces the cells to express other genes associated with blood vessel endothelium. We are in the process of introducing the viral system to the hESCs so that we can temporally induce the endothelial gene and increase the numbers of endothelial cells that we generate using our differentiation method. To test the ability of the cells that we have generated in the laboratory to aid in human condition, we have been testing models of cardiovascular blockage in a mouse. We have thus far tested models that mimic a complete coronary artery blockage in the heart, a complete blockage in a leg artery and a model which tests how well the introduced cells are able to integrate into the mouse circulatory system. All of these models will be further tested to determine which is most effective for the endothelial cells we have generated.

Year 2

Cardiovascular disease is a major concern for medicine and is caused by damage to blood vessels. We have begun a project to generate endothelial cells, the cells which line the blood vessels, from human embryonic stem cells (hESCs) using gene transfer technology and regulated gene expression. Little is known about the early stages of blood vessel endothelial differentiation of the human embryo. It is imperative that we understand normal development in order to mimic it in the laboratory. We have used hESCs to model embryonic development and determine the pattern of gene expression in the early stages of differentiation. Using what is known about mouse embryonic development as a model, we have determined that gene expression in differentiating human cells closely follows that of differentiating mouse cells. In particular, we have determined the timing of the expression pattern of a gene that is required for the generation of endothelial cells. This knowledge will allow us to induce expression of this gene at the proper time during differentiation in the cells in the laboratory to increase the number of blood vessel cells we can generate. We have established a method in the laboratory to reliably generate endothelial cells from unmodified hESCs. Timing of gene expression during development is extremely important and improper timing can result in cells being unable to respond to the signal generated by the gene or unable to progress further in development. We have found that introduction of a single factor into the differentiating hESCs results in either as little as two or as much as six times more endothelial cells depending upon the time of administration than in cells without this added factor. These cells behave similarly to cells generated without the addition of the factor in all tests that we have performed on the cells. To test the ability of the cells that we have generated in the laboratory to aid in human condition, we have been testing mouse models of retinopathy of prematurity (ROP). Premature infants are often placed in a very high oxygen environment to help with their underdeveloped lungs. While this aids their survival, the high levels of can disrupt the vessels in the retina and result in blindness. We are using this model to test the ability of administered hESC derived endothelial cells to aid in the recovery of retinal vessels from exposure to a high oxygen environment. So far we have found that endothelial cells derived from hESCs with and without the addition of the single factor mentioned above result in an improved vessel network in the eyes of tested mice.

© 2013 California Institute for Regenerative Medicine