Developmental Regulation of Human Embryonic Stem Cells by microRNAs

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Stem cells are remarkable cells which have two unique properties: self-renewal (self replication) and pluripotency (the ability to regenate a wide range of tissue-specific cells). These properties and the associated potential to use these cells to cure a wide range of degenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease, and heart disease, have made stem cells a subject of intense scientific and medical interest. The recent discovery of cancer stem cells implies that stem cell research will also have important implications for cancer therapy, since cancer stem cells must be targeted by cancer drugs to prevent relapse of tumors. The mechanisms by which stem cells self-renew and differentiate are poorly understood. Several genes have been isolated and are thought to be essential to mammalian stem cell renewal and differentiation. Recent evidence has identified a new and potentially important molecular mechanism for regulating these genes in ESCs. These molecules are small bits of RNA, of approximately 22 nucleotides (nt) in length, and often termed microRNAs (miRNAs). miRNAs appear to play an important part in regulating gene activity. These small RNAs “turn off” genes by directly binding to them and preventing the production of proteins based on the gene’s genetic code. Several recent studies indicate that the types of miRNAs present in stem cells (miRNA “expression profiles”) are different from other cells and tissues. We propose to identify candidate miRNAs that are playing key roles in hESCs and to characterize the effects of their actions on self-renewal and differentiation. Our preliminary data also indicates that miRNA “expression profiles” differ depending on whether a stem cell is differentiating, self-renewing, or quiescent. This project will employ a wide range of techniques in molecular biology, including microarrays, bioinformatics, and bioluminescent reporter gene assays, to determine which specific miRNAs show differential expression between stem cell states and we will identify the genes they target. Having identified candidate miRNAs that play an important role in determining stem cell fate, we will manipulate their expression levels using biotechnology techniques known as RNA oligos and plasmid or viral expression vectors. We will then determine if these manipulations change the cell’s decision-making process in regard to differentiation or self-renewal. This will be done using molecular biology and biochemical assays on undifferentiated and differentiated cells. Ultimately we will investigate the underlying regulatory mechanisms in hESCs that control miRNA expression. We anticipate that our results will contribute to understanding the transitions between stem cells and differentiated cells, as well as normal and cancer cells. This type of information can be invaluable in designing new therapeutic approaches for stem cell replacement or cancer treatment.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) hold the potential to revolutionize human medicine by making cell replacement therapies, drug delivery, or in vivo modifications of cell populations a reality. In degenerative conditions we may be able to replace dead cells with functional new ones derived from hESCs. This approach could be used to treat Parkinson’s Disease, Alzheimer’s Disease, heart disease, diabetes, or paralytic spinal cord injuries. There is also potential for new cancer treatments, as cancer stem cells share many properties of hESCs. This type of medical technology would benefit the citizens of California in several general ways. It could offer hope to Californians suffering from these diseases. It could help relieve the pain and suffering for affected individuals and their families and loved ones. Finally, the economic impact of hESC-based therapies is likely to be significant. Chronic diseases will no longer incapacitate patients and they can return to productive work lives. State government and private expenditures on health care will actually decrease. Companies will organize to commercialize these hESC-based therapies and this will stimulate the California economy by providing new jobs and tax revenue. Most medical economists believe that significant revenues from patents, royalties, and licenses will flow from scientific discoveries pioneered in California based stem cell research centers. This project will also enhance California’s competitive position in biomedical research and push the State to the forefront of research not only in America, but also the world. It will be a reflection of the enormous ongoing investment in science on the part of the state and private institutions, fueled in recent decades by the high-tech and biotechnology industries. All of the research will be done in California. The project focuses on the role of microRNA (miRNA) molecules in controlling the fate of hESCs. miRNAs are recently discovered molecules that play an important part in gene regulation. The expression profiles of miRNAs in stem cells are different from other tissues, and miRNAs may play an essential role in stem cell self-renewal and differentiation. Many potential target genes for miRNAs are essential players in stem cell renewal and differentiation. Some of the most exciting and innovative work on miRNAs has taken place in California and this project will help confirm the State’s leading role in miRNA research and California’s role as a place where miRNA researchers’ specific application to stem cell biology is being studied.
Progress Report: 
  • Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss, culminating in extensive disability through defects in neurologic function. The demyelination that defines MS pathology is progressive over time; however, studies indicate that myelin repair can occur during the course of disease in patients with MS and in animal models designed to mimic the immunopathogenesis of MS. While it is generally thought that endogenous oligodendrocyte precursor cells (OPCs) are largely responsible for spontaneous remyelination, it is unclear why these cells are only able to transiently induce myelin repair in the presence of ongoing disease. Along these lines, two therapies for demyelinating diseases look promising; implanting OPCs into sites of neuroinflammation that are directly capable of inducing remyelination of the damaged axons and/or modifying the local environment to stimulate and support remyelination by endogenous OPCs. Indeed, we have shown that human embryonic stem cell (hESC)-derived oligodendrocytes surgically implanted into the spinal cords of mice with virally induced demyelination promoted focal remyelination and axonal sparing. We are currently investigating how the implanted OPCs positionally migrate to areas of on-going demyelination and the role these cells play in repairing the damaged CNS. The purpose of this research is to identify the underlying mechanism(s) responsible for hESC-induced remyelination.
  • Oligodendrocyte progenitor cells (OPCs) are important in mediating remyelination in response to demyelinating lesions. As such, OPCs represent an attractive cell population for use in cell replacement therapies to promote remyelination for treatment of human demyelinating diseases. High-purity OPCs have been generated from hESC and have been shown to initiate remyelination associated with improved motor skills in animal models of demyelination. We have previously determined that engraftment of hESC-derived OPCs into mice with established demyelination does not significantly improve clinical recovery nor reduce the severity of demyelination. Importantly, remyelination is limited following OPC transplantation. These findings highlight that the microenvironment is critical with regards to the remyelination potential of engrafted cells. In addition, we have determined that human OPCs are capable of migrating in response to proinflammatory molecules often associated with human neuroinflammatory diseases such as multiple sclerosis. This is an important observation in that it will likely be necessary for engrafted OPCs to be able to positionally navigate within tissue in order to move from the site of surgical transplantation to areas of damage to initiate repair and tissue remodeling. Finally, we have also made a novel discovery of a unique signaling pathway that protects OPCs from damage/death in response to treatment with proinflammatory cytokines. We believe this is an important and translationally relevant observation as OPCs are critical in contributing to remyelination and remyelination failure is an important clinical feature for many human demyelinating diseases inclusing spinal cord injury and MS. We have identified a putative protective ligand/receptor interaction affords protection from cytokine-induced apoptosis. These findings may reveal novel avenues for therapeutic intervention to prevent damage/death of OPCs and enhance remyelination.

© 2013 California Institute for Regenerative Medicine