Defining the epigenetic blockage that limits in vitro human oligodentrocyte terminal differentiation
Basic Biology III
$1 382 400
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.
Project Synopsis: The proposed research aims to identify epigenetic mechanisms that underlie the terminal in vitro differentiation of human oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. To achieve this goal, the applicant proposes three specific aims. First, they will perform transcriptome profiling of human and mouse cells at different stages during an oligodendroglial differentiation process. Second, isolated RNA and chromatin samples will be collected at the intermediate and final stages of differentiation of mouse oligodendrocytes, and these will be used for various transcriptome and epigenetic dynamic analyses such as assessment of DNA methylation and histone modifications. Finally, the applicant will apply bioinformatic analysis to the expression and epigenetic profiling data to identify elusive core oligodendroglial transcription factors. Significance and Innovation: - Reviewers questioned the significance of the project’s focus and were not convinced that studying the terminal differentiation of oligodendrocytes in vitro would substantially contribute to approaches for generating oligodendrocytes for transplantation. - The project does not address a major unsolved problem in stem cell biology or regenerative medicine. - The level of innovation of the proposal is moderate and primarily involves the translation of successes already obtained in the mouse system to the human system. Feasibility and Experimental Design: - Reviewers seriously doubted the feasibility of the proposed study. - Much of the proposed work is descriptive in nature, and it is unclear how the data that is obtained in Specific Aims 1 and 2 will contribute to the rest of the project. - Reviewers questioned whether comparisons between the mouse and human cells would provide meaningful data, as different properties of the cells such as the different rates of differentiation between mouse and human oligodendrocytes were not addressed in the proposal. - Much of the project is not hypothesis driven but rather appears to be motivated by available technology. - The proposal provided little information about alternative approaches. - Description of the transplantation validation experiments were lacking in appropriate detail and explanation. - The research facilities available to the team appeared adequate to support the proposed research. Principal Investigator (PI) and Research Team: - The PI has only a limited publication record and little evident experience leading a research program. Responsiveness to the RFA: - The proposal is responsive to the RFA as it is focused on fate determinations during human cell differentiation and regulatory mechanisms underlying the developmental potential of human stem cells.