Retinal Pigment Epithelium Derived From Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Retinal Pigment Epithelium Derived from Human Embryonic Stem Cells The long-term objective of this proposal is to develop applications of human embryonic stem (hES) cells in the treatment of eye diseases, including Age-Related Macular Degeneration, one of the leading causes of human blindness. This disease, as well as other retinal degenerations, is thought to involve the death of the retinal pigmented epithelium (RPE), a layer of pigmented cells in the back of the eye that nourish the photoreceptors of the retina. Preliminary work has shown that hES cells can differentiate into RPE-like cells, so a possible cellular therapy could involve transplantation of these cells in areas where native RPE has been lost. However, there is much to do before it is safe to begin human clinical trials with these cells. Currently only small numbers of cells can be produced, and it is not yet clear if they can fulfill all of the important functions carried out by the native RPE. Studies are proposed that focus on aspects of hES-RPE cell biology that will impact their potential application in ocular therapy. The first aim targets the identification of molecular mechanisms regulating the differentiation of hES-RPE cells, focusing on growth factors, extracellular matrix, matrix receptors, and transcription factors that have been implicated in RPE development in animal studies. Currently little is known about the factors that cause an undifferentiated hES cell to turn into an RPE cell. Ultimately, these studies should lead to the identification of optimal culture conditions that drive hES cells down the RPE differentiation pathway. The second aim is a comprehensive a molecular and cellular characterization of hES-RPE to assess the full extent of similarity to native RPE. Key RPE functions, including transport of nutrients, synthesis of pigment, and interaction with photoreceptors will be assessed. hES-RPE will be compared to native RPE via analysis of proteins, RNAs, and DNAs. Finally, the third aim proposes functional testing of hES-RPE in animal models to evaluate their key functions in vivo. While the proposed studies are considered high risk, they have an equally high potential gain. The experiments will provide a provisional assessment of the suitability of hES-RPE for ocular therapeutic applications.
Statement of Benefit to California: 
Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients in California. An estimated 30% of people over 75 years of age are diagnosed with AMD and the disease incidence is predicted to double over the next 25 years, which represents a significant public health challenge to the State. The disease leads to the deterioration of fine acuity vision in the middle of the visual field, and can eventually lead to total blindness. The pathogenesis of this disease is poorly understood and there are no effective treatments. Loss of vision is a result of photoreceptor death, which is thought to be a consequence of the death of the neighboring retinal pigment epithelial (RPE) cells. One strategy that holds promise for the treatment of AMD is the replacement of defective RPE cells via cellular transplantation, and recently, it was demonstrated that human embryonic stem cells can be coaxed to turn into RPE-like cells (hES-RPE). This proposal will assess the suitability of these cells for possible use in human therapy for AMD. The benefit to California will be the development of a potential therapy for a blinding disease that affects millions of its citizens.
Progress Report: 
  • Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
  • One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
  • To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
  • It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
  • Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).

© 2013 California Institute for Regenerative Medicine