Genetic analysis and modification of hES cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Vision Loss
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Although some hESC lines have been reported to be stable in culture, we and others have found that the stability is variable for different lines. Furthermore, small DNA rearrangements that cannot be detected by chromosome analysis, which has been used as a key measure of hESC stability, exist in many cell lines. It is of crucial importance to detect any potential genetic abnormalities in hESC, and hESC genomes must be stable if they are to become useful clinical reagents. Our long-term goal is to generate genetically stable hESC lines that can be eventually used safely for therapeutic purposes. However, knowledge of long-term genetic stability of these cell lines is necessary to ensure the safe therapeutic use of these cells, and it is essential for the generation of new cell lines in the future. We believe that the groundwork for improving the genetic stability of the existing cell lines should be done before we derive new cell lines. Therefore, we would like to use the existing hESC lines to explore culture conditions for improving genomic stability of hESC upon long-term culture. We propose to follow the molecular features of existing cell lines, both federally- and non-federally-approved lines, over time to identify differences potentially caused by culturing methods and conditions. There is very little information on the non-federally approved cell lines due to the lack of funding. It is important to compare these cell lines with the federally approved cell lines because they are derived and cultured under different conditions and methods. The common genetic and epigenetic features of these cells may also provide insights into the characteristics and plasticity of embryonic stem cells. We also propose to study how genetic changes such as permanent removal or addition of a gene to the hESC will impact these cells, especially the genes involved in DNA repair and modification. We have already begun to design new ways to manipulate these cells for both the understanding of basic biology and the future therapeutic application.
Statement of Benefit to California: 
Similar to many other proposals, the work proposed here would help California in general ways such as biotechnology development and human health improvement. The work proposed here would also provide several specific benefits for the California community. The strength of the research team is genetics and functional analysis, and the approach is focused on fundamental understanding of stem cell genetics and is hypothesis-driven. Therefore, the work proposed will not only serve as a stepping stone for clinical application of embryonic cells, it will also provide a solid understanding of the biological function of these cells. Much work on embryonic stem cells has been focused on the potential clinical applications of these cells and not as much on genetic stability and fundamental biology. Also, most of the work has been done using the federally-approved cell lines due to funding constraints. The stability of these cells is of key importance for their clinical application, so improving genetic stability in culture and increasing the genetic adaptability of these cells are essential. Comparing federally-approved and non-federally-approved cell lines is also important since they were derived and cultured under different conditions. Therefore, we have begun work in these two directions with cell lines from both sources. Experiments designed to manipulate these cells based on critical genetic information may provide important insight into the function and genetic adaptability of human embryonic stem cells. The research reagents generated in this proposal will make many other experiments in normal human cells possible. The unique aspect of this proposal may move basic scientific understanding of normal cells forward and give California a leading edge in both clinical application and basic biology of human embryonic stem cells.
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