Defining the epigenetic blockage that limits in vitro human oligodentrocyte terminal differentiation

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02165
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
At its most basic, epigenetics is the study of changes in gene expression and cellular phenotype that do not involve alterations to the genetic code, which is DNA sequence. Even though all of the cells in our body share the exact same DNA, they exhibit dramatic differences in morphology and function. Epigenetic regulation is the reason for this. In other words, epigenetic mechanisms govern the gene regulation and cell differentiation, including oligodendrocyte differentiation. Oligodendrocyte is one important cell type in the nervous system. It provides the myelin sheaths around the axons of neurons to keep their integrity and normal function. Oligodendrocyte demyelination causes Multiple sclerosis (MS). It also contributes to clinical deficits followed by stroke, inflammatory attack, spinal cord injury or trauma. The recent advances in our understanding of stem cell biology has launched stem cell based therapy as one of the most exciting and difficult challenges in today’s biology world. However, for oligodendrocyte differentiation, while mouse oligodendrocyte progenitor cells (OPC) derived from mouse embryonic stem cells (mESCs) are committed and readily differentiate into myelinating mature oligodendrocytes. Yet, human OPCs derived from hESCs, even with the current lengthy in vitro differentiation protocol, fail to enter this terminal myelination-competent stage. In this study, we will differentiate mESCs and hESCs in parallel and perform genetic and epigenetic profiling analysis at different differentiation stages. After identifying the epigenetic regulatory elements via strategic bioinformatic data mining, we will manipulate the epigenetic regulation system and alleviate the blockage that prohibits the in vitro hOPC terminal differentiation. The findings of this proposal will provide valuable information for us to dissect out the crucial mechanisms that promote the full development of human oligodendrocytes. This information will be helpful in the future to effectively differentiate ESCs or iPS cells derived from patients for cell replacement therapy as well as to understand the causes of oligodendrocyte related diseases.
Statement of Benefit to California: 
Oligodendrocytes are the myelin-forming cells in the CNS and are essential for the integrity and proper functioning of neural circuits. Oligodendrocyte demyelination causes Multiple Sclerosis (MS), It also plays a part in clinical deficits followed by diseases, such as stroke, spinal cord injury, inflammatory attack, or trauma. Recent studies have shown that age-related myelin breakdown leads to cognitive decline and Alzheimer's disease; meanwhile Schizophrenia and bipolar brains show downregulation of key oligodendrocyte and myelination genes, including transcription factors that regulate these genes, when compared to normal brains. As we all know, stroke is the third leading cause of death in the US and the leading cause of permanent disability, which costs us over $50 billion dollars annually. Spinal cord injury, trauma and Alzheimer’s disease can be equally tragic to the patients they affect. For multiple sclerosis (MS) patients, who are diagnosed in their 20s-40s, they must live the rest of their life with neurologic disabilities, which creates a huge emotional and financial burden for their families, and our society as well. Results from small clinical studies have demonstrated that transplantation of autologous hematopoietic stem cells can bring some positive effects on severe forms of MS by blocking uncontrolled inflammation. However, the real solution to fix the chronically abnormal neural system will rely on restoring mature oligodendrocytes into the system, which are capable of remyelinating. Therefore, to find and remove the epigenetic blockage that limits in vitro human oligodentrocyte terminal differentiation is a critical step for the translational study to develop stem cell based therapies for so many oligodendrocyte demyelination related diseases that creates major burdens on the citizens of California.
Progress Report: 
  • 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.
  • 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.
  • 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 20 times more endothelial cells depending upon the time of administration than in cells without this added factor. In live animal studies by other groups, it was shown that expression of this gene after the time of normal expression has little effect on the animal . In human embryonic stem cells, we found that the same effect is seen when the gene is administered at the peak of expression or up to 7 days after the peak of expression.
  • All of our methods thus far have used a technique that alters the genetic makeup of the cells we are testing. We are now exploring methods that do not alter the genes of the cells that could be used to generate cells that could be administered to patients.

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