Epigenetic gene regulation during the differentiation of human embryonic stem cells: Impact on neural repair

Epigenetic gene regulation during the differentiation of human embryonic stem cells: Impact on neural repair

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00111
Approved funds: 
$2,412,995
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
Human embryonic stem cells (hESCs) have the potential to become all sorts of cells in human body including nerve cells. Moreover, hESCs can be expanded in culture plates into a large quantity, thus serving as an ideal source for cell transplantation in clinical use. However, the existing hESC lines are not fully characterized in terms of their potential to become specific cell types such as nerve cells. It is also unclear if the nerve cells that are derived from hESCs are totally normal when tested in cell transplantation experiments. One of the goals for our proposal is to compare the quality and the potential of eight lines of hESCs in their capacity to become nerve cells. To measure if the nerve cells that are derived from hESCs are normal when compared to the nerve cells in normal human beings, we will examine the levels of gene expression and the mechanisms that control gene expression in hESC-derived nerve cells. Specifically, we will examine the pattern of DNA modification, namely DNA methylation, in the DNA of nerve cells. This DNA modification is involved in the inhibition of gene expression. It is known that if DNA methylation pattern is abnormal, it can lead to human diseases including cancer and mental retardation disorders. We will use a DNA microarray technology to identify DNA methylation pattern in the critical regions where gene expression is controlled. Our recent results suggest that increased DNA methylation is observed in hESC-derived nerve cells. In this proposal, we will also test if we can balance the level of DNA methylation through pharmacological treatment of enzymes that are responsible for DNA methylation. Finally, we will test if hESC-derived nerve cells can repair the brain after injury . A mouse stroke model will be used for testing the mechanisms stem cell-mediated repair and recovery in the injured brain and for selecting the best nerve cells for cell transplantation. Our study will pave the way for the future use of hESC-derived nerve cells in clinical treatment of nerve injury and neurodegenerative diseases such as stroke and Parkinson’s disease.
Statement of Benefit to California: 
Neurodegenerative diseases such as stroke are the leading cause of adult disability. Stroke produces an area of damage in the brain which frequently causes the loss of crucial brain functions such as sensory and movement control, language skills, and cognition capability. Stem cell transplantation has emerged as a method that may improve recovery in these brain areas. Studies of stem cell transplantation after stroke have been limited because many of the transplanted cells do not survive, the appropriate regions for transplantation have not been identified, and the mechanisms by which transplanted stem cells improve recovery have not been determined. Also, there have been no studies of human embryonic stem cell transplantation after stroke. For the use of stem cell therapy in stroke patients, human embryonic stem cell lines have to be grown and tested for their efficacy in repairing the brain after stroke. We have recently found that the process of growing human embryonic stem cells in culture introduces genetic modifications in some of these cell lines that may decrease survival of the cells in the brain and impair their ability to repair the injured brain. The experiments in this grant will determine which human embryonic stem cell lines do not undergo this negative genetic modification. The optimum human embryonic stem cell lines will then be systematically tested for the location in the stroke brain that produces survival and integration, and the mechanisms of repair that these cells mediate in the brain after stroke. These studies will specifically test the role of human embryonic stem cells in improving sensory and movement functions after stroke. In summary, these studies will establish protocols for the proper growth of human embryonic stem cell lines, the lines that are most effective for repairing the brain after stroke, and the principles behind how human embryonic stem cells repair the brain. These results are applicable to other kinds of neurodegenerative conditions, such as Parkinsons, Alzheimer’s and Huntington’s diseases, and to the growth and culture of human embryonic stem cells in general for repair of disease of other human tissues.
Progress Report: 

Year 1

Summary of Research Progress: Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair. In the past year, we have made progress in guiding several lines of human stem cells into nerve cells. We are now ready to compare the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also begin to produce and characterize a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We also test the pattern of DNA methylation in different lines of human stem cells. By engineering stem cells carrying different levels of methylation, we aim to find the optimal levels of DNA methylation for efficient nerve cell differentiation. Finally, we also made excellent progress on the procedure of cell transplantation. We have found a suitable substrate that can be used to enhance neuronal survival after cell transplantation and we expect to publish a research paper in this new method of cell transplantation.

Year 2

Summary of Research Progress: Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair. In the past year, we have made great progress in converting several lines of human stem cells into nerve cells. We have compared the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also begin to produce and characterize a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We also test the pattern of DNA methylation in different lines of human stem cells. By engineering stem cells carrying different levels of methylation, we aim to find the optimal levels of DNA methylation for efficient nerve cell differentiation. Finally, we also made excellent progress on the procedure of cell transplantation. We have found a suitable substrate that can be used to enhance neuronal survival after cell transplantation and we expect to publish a research paper in this new method of cell transplantation.

Year 3

Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair. In the past year, we have made great progress in converting several lines of human stem cells into nerve cells. We have compared the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also succeeded in making a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We have tested the pattern of DNA methylation in different lines of human stem cells, including mutant cell lines from patients who exhibit defects in DNA methylaiton. Finally, we also made excellent progress on the procedure of cell transplantation and we characterized gene expression and epigenetic changes in transplanted nerve cells from human embryonic stem cells. Our studies allow us to optimize methods of neural cell differentiation and transplantation. We plan to publish additional two research papers in the near future.

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