Funding opportunities

Detection of Cell Lineages Among Stem Cell Progeny by microRNA Profiling

Funding Type: 
SEED Grant
Grant Number: 
RS1-00241
Funds requested: 
$529 006
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Many currently untreatable degenerative diseases are caused by the loss or dysfunction of cells in the body. Human embryonic stem (hES) cells are the first type of human cell cultured in the laboratory that have the potential to become any of the several hundred cell types in the developing human. The goal, therefore, of regenerative medicine is to develop the means of transforming hES cells into populations of cells that can be used to restore function in tissues lost as a result of disease. A critical milestone in the path from the lab bench to the hospital bedside is the development of techniques for manufacturing protocols to reliably obtain pure populations of desired cell types (in some cases relatively rare cell types). Some of the central regulators of cell fate (that is, whether hES cells become lung, heart, or brain) are known such as the “homeodomain” proteins. It is well-established that the homeodomain proteins are central regulators in the construction of the body plan, but no one has described an efficient means of influencing these regulatory proteins to makes cells of interest from hES cells. The recent discovery of a new class of RNA molecules called microRNAs (miRNAs) is stimulating a great deal of interest in the scientific community because these molecules appear to play a master regulatory role over many of the genes in the cell including some of the homeodomain proteins. However, because of profound differences between species, data being obtained in mice is not predictive of how these RNAs govern human cell fate decisions. Therefore medical research would benefit from a method to define how these molecules control hES cell differentiation. The aim of this grant is to accomplish a comprehensive profling of these miRNAs during hES cell differentiation in vitro. We have developed a new method of sorting out the many kinds of cells that originate from hES cells. We will utilize this bank of 100+ diverse hES-derived clonal differentiated cell lines each of which has a complete gene expression profile. We will obtain a large-scale miRNA profile for representative members of these purified cell types and then, using mathematical methods, we will identify the potential master regulatory genes that these miRNAs target. Lastly, we anticipate that our experimental system will allow us to then test these miRNAs to determine whether our hypothesis is actually correct and whether we can use these master regulatory molecules to steer hES cells into the lineages most needed in medicine, including those useful in the discovery of new drugs, and for human therapy to restore function in a broad array of diseases where the the loss or dysfunction of cells is the cause of the disease.
Statement of Benefit to California: 
California, like much of the United States, is facing a staggering challenge to its health care system. The large investments made in recent decades by the National Institutes of Health (NIH) have largely ignored the problems of age-related degenerative disease. As a result, increasingly physicians are treating the chronic, debilitating, and therefore expensive diseases associated with aging. This is made all the worse by the demographic wave caused by the entry of the Baby Boomers into retirement. It is estimated that by the year 2010, the Baby Boomers will be 25 percent of the population of California. By 2020 they will be approaching 64 years of age. As a result, the percentage of the elderly in California is expected to grow from 14 percent in 1990 to 22 percent in 2030. (Source: California Department of Finance, Population Projections 1993). Many of the chronic devastating diseases of an aging population are the degenerative diseases. Generally speaking, degenerative diseases are those diseases caused by the loss or dysfunction of cells. Examples include osteoarthritis (loss of cartilage cells that protect the ends of the bones), Parkinson’s disease (the loss of dopminergic neurons), osteoporosis (dysfunction of osteoblasts), macular degeneration (dysfunction of retinal pigment cells) and so on. More significantly, the loss or dysfunction of cells in the heart (or the vessels that supply the heart with blood) results in heart disease, the most frequent cause of death in California. In 2001 (the most recent year data is available) heart disease caused 68,234 deaths (29% of all the deaths in the state). Stroke is also a vascular disease and the third leading cause of death in California. In 2001, stroke caused 18,088 deaths (8% of all of the deaths in the state). Regenerative medicine represents the effort of cell biologists to invent a new approach to the problem of degenerative disease. Human embryonic stem (hES) cells have the potential to become all of the cells in the human body, and their unique properties give researchers the hope that from these primitive cells new therapies can result that may be available in time for the looming health care crisis. It is estimated that are over 200 cell-types in the adult human and hES cells are capable of making all of these. However, to turn this new technology into actual therapies that can alleviate human suffering, researchers need new tools to generate large numbers of purified cell types. This proposal describes a project to identify molecules that could be used to generate large numbers of important cell types from hES cells. The results from the successful completion of this proposed research are anticipated to be useful in numerous therapeutic applications of regenerative medicine.
Review Summary: 
SYNOPSIS: In this proposal the investigators plan to define the complement of miRNAs expressed in ESCs and ESCs allowed to commit to different lineages while remaining proliferative; these cell lines were generated from the proprietary MAO3 cell line by ACT. The underlying hypothesis is that specific complements of miRNAs will regulate Hox gene expression, which regulates differentiation from ESCs. Additional cell lines will be generated and evaluated to further extend the complement of cells with specific Hox gene expression; miRNAs within these cells will be characterized and then the influence of HOX gene miRNAs will be tested for their effect on differentiation. INNOVATION AND SIGNIFICANCE: The in vivo function of many if not most miRNA genes is unknown. There are >450 miRNAs in vertebrates (including humans) and it has been estimated that there might be as many as 2,000. It will be important to determine the role of the miRNAs, as they likely play important roles in governing key steps of self renewal and differentiation during normal development and in disease. The goal of this proposal is to identify the complement of miRNAs active in ESCs that govern expression levels of HOX genes, key orchestrators of differentiation and lineage specification. This could yield important insights into the mechanisms underlying ESC differentiation and lineage specification. The use of a large number of clonally differentiated population of hES cell lines for a miRNA array analysis is innovative. STRENGTHS: A strength is the availability of the clonal ESC derived cell lines committed to specific lineages and expressing specific complements of Hox genes and the expectation that a large amount of data will be obtained regarding the expression of genes (miRNAs and mRNAs) in these lines. This data would be of interest to the many labs that work on hES cells. Another strength is the expertise of the investigators; the proposal is well within the experimental expertise of the lab and they have generated a large amount of preliminary data. The experiments proposed are well thought out and should generate a lot of data. However, there are reservations regarding how useful these data will be in answering the scientific question proposed (see below). WEAKNESSES: Although it is well accepted that miRNAs play important roles in regulation of cell differentiation and specification, it is not clear that large scale screens for expression patterns of miRNAs followed by target identification in clonally derived hESC derivatives specified to certain lineages will further our insights in these roles. It is not clear how well the clonally derived ESC cell lines with a given specification recreate normal human development, as no such data was provided by the investigators. There is no obvious reason why these experiments need to be performed in human ESC. It might be better to perform these studies using human purified progenitors from given tissues, that like these clonal cell lines express specific complements of Hox genes, as the exact function of such cells and spcific role of such cells in organs / tissues would be better characterized than that of the clonal cell lines. It is likely that a more accurate picture of the potential roles of miRNAs wcould be derived from such studies. Although it is likely that many new collections of miRNAs will be identified, a problem with the proposal is that there is no explanation of how a these collections of miRNAs from various cell lines will aid researchers in determining the function of these genes. Various computer programs have generated a huge number of “predicted” targets for each miRNA, however, the vast majority of these putative targets have not been validated in vivo or in vitro. Although some information may come from misexpression, inhibition or overexpression studies proposed, to do this large scale may be premature as the readouts proposed may not yield very informative data about the functions or targets of these genes. At this stage it might be more opportune to identify specific targets in a given Hox gene, identify miRNAs for these targets, and then specifically test whether modification of miRNAs will interfere with this Hox gene expression and lead to altered differentiation. The author makes a strong point that miRNAs regulate Hox genes, in particular the miR196 and miR10 family. In mouse, it is clear that miR196 binding to HoxB8 results in a decrease in HoxB8 mRNA. In addition, in vivo Hoxb8 and miR196 are expressed in a complementary expression pattern, suggesting that miR196 is responsible for the downregulation of Hoxb8 mRNA. A reviewer agrees with the author that miRNAs probably play key roles in regulating Hox genes, but disagrees that a huge screen in hES cells is the best way to investigate this question. If the author wants to determine in humans if miR196 regulates Hixb8 wouldn’t it be easier to take a cell line expressing HoxB8 and introduce miR196? The role miRNAs play in regulating gene expression can better be answered using known human cell lines. If the author is interested in the regulation of specific homeodomain genes then miRNAs target sites should first be identified in these genes and then cell lines expressing or lacking expressing of the corresponding miRNAs could be identified and characterized. There is no need to perform an in depth and expensive global screen for miRNAs in hES cells to determine the role miRNAs play in regulating gene expression in humans. DISCUSSION: There was no further discussion following the reviewers' comments.
Conflicts: 

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