Identification and Analysis of Genome-wide Intra- and Inter-chromosomal Associations in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Vision Loss
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
We used to think of genes as discrete segments of DNA that code for proteins and that are regulated or controlled by nearby or adjacent DNA sequences. Recently, it has been shown that DNA sequences on the same chromosome that are very distant from a gene may regulate its function, determining if the gene will be turned on or turned off. We have now discovered that DNA sequences that are even on different chromosomes from the protein-coding gene may control this gene. These interactions are often totally unexpected. For example, the NF1 gene, which causes neurofibromatosis, a genetic disease that may lead to the development of brain tumors, lies on chromosome 11 in the mouse, but the production of its protein product is partially under the control of a DNA segment near a growth factor gene(IGF2) on mouse chromosome 7; the same relationship appears to hold true in humans. No one had ever suspected such an interaction, and it is now logical to consider targeting the IGF2 gene as part of our hopes to treat neurofibromatosis. In other words, the discovery of a long-range interacting sequence of DNA provides us with totally new targets for drug development. Several other groups have also shown that inter-chromosomal gene regulation can control immune genes and the genes that regulate our sense of smell. We believe that many, if not most, genes are controlled, as least in part, by long range inter-chromosomal interactions, and we have proposed a new method to try to define all of these interactions in cultured human cells. It is highly likely that many of these long range inter-chromosomal interactions are important in the regulation of a gene, and therefore, we plan to catalog all of these interactions in human embryonic stem cells, and see whether the interactions remain the same or are altered as the cells differentiate into fat cells, and, in the future, into other kinds of cells. This catalog will provide us with numerous new clues to the regulation of disease-related genes, and will suggest new drug or gene-therapy related targets to treat these diseases. By comparing the list of interacting genes in normal embryonic cells with those in cells harboring a genetic disease, we may develop new diagnostic tools in addition to potential therapeutic avenues.
Statement of Benefit to California: 
Our goal is to characterize how genes are regulated by distant DNA segments on different chromosomes. We will develop a complete catalog of these long range interactions, which will provide a list of how one gene may regulate another. This catalog will provide the framework for determining how disease-related genes are controlled and will allow investigators to develop new diagnostic tools and/or new therapeutic targets to treat various genetic diseases. These are targets that had never heretofore been considered because the long range interactions among these genes had not been known. Clinical translation of these studies into new drugs will be of great benefit to the people of California.
Progress Report: 
  • It is estimated that by 2020, over 450,000 Californians will suffer from vision loss or blindness due to the age-related macular degeneration (AMD), the most common cause of retinal degeneration in the elderly. A layer of cells at the back of the eye called the retinal pigment epithelium (RPE), provides support, protection, and nutrition to the light sensitive retina, and cooperates with other retinal cells to maintain normal visual function. The dysfunction and/or loss of these RPE cells play a critical role in the development of dry AMD, the most common form of that disease. RPE cells derived from human ES cells (hES-RPE) are a potentially unlimited resource for cell replacement therapy.
  • During this grant period we were able to develop methods to reproducibly derive RPE cells from established human ES (hES) cell lines.
  • Normal RPE cells have a polarized appearance, meaning that they have specialized appearances and functions on the top (retinal facing) and bottom of the cells. We were then able to induce the hES-derived RPE to develop a polarized appearance very similar to that found in the normal eye. These cells had specialized characteristics that were remarkable similar to normal RPE and showed ability to function like normal RPE cells. We then developed sheets of hES-derived RPE on substrates that could be used for surgical implantation into animal eyes.
  • We then performed studies to establish appropriate animal models in which to test the ability of hES-derived RPE to prevent or slow retinal degeneration in animal models that have some features of human AMD. We were not able to use standard mouse models of AMD because mouse eyes are too small to perform the surgical implantation of polarized RPE. We first looked at a rabbit model of RPE degeneration induced by a toxin (sodium iodate). We were able to establish reproducible RPE degeneration in these animals but whenever we tried to implant hES-derived RPE under the retinas of these animals, the RPE cells did not survive. We believe that the toxin that was used to induce this model was being taken up by the transplanted cells resulting in their death. We then tested a genetic model of retinal degeneration in rats (RCS rat). We were able to demonstrate reproducible retinal degeneration in these animals. When we transplanted hES-derived RPE under the retinas of these animals, we were able to show that the RPE were indeed human in origin and that they were able to survive for at least several weeks after transplantation. Importantly, we were able to show that in the areas where hES-derived RPE were transplanted, there was less retinal degeneration than in the areas in which there was no transplantation or "sham" transplantation.
  • Thus, in conclusion, we were able to show (1) the reproducible culture of RPE cells from human ES cell lines, (2) that these hES-derived RPE had the appearance and functional characterisitics of human RPE and could be grown on substrates as sheets or polarized cells, and (3) that when these sheets of polarized hES-derived RPE were implanted under the retinal of rats with retinal degeneration, that they were able to survive and showed the ability to slow the progression of retinal degeneration in these animals.
  • The results of this study suggest that hES cells may be an unlimited source of RPE cells suitable for transplantation into eyes with retinal degeneration and that subretinal implantation of polarized hES-derived RPE may represent a novel therapy for dry AMD.

© 2013 California Institute for Regenerative Medicine