Detection of Cell Lineages Among Stem Cell Progeny by microRNA Profiling
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.
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.