Progress In Stem Cells Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery (original) (raw)
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Stem cells (Dayton, Ohio), 2015
Stem cells provide a potentially unlimited source of cells for treating a plethora of human diseases. Regenerative therapies for retinal degenerative diseases are at the forefront of translation to the clinic, with stem cell-derived retinal pigment epithelium (RPE)-based treatments for age-related macular degeneration (AMD) already showing promise in human patients. Despite our expanding knowledge of stem cell biology, methods for deriving cells, including RPE have remained inefficient. Thus, there has been a push in recent years to develop more directed approaches to deriving cells for therapy. In this concise review, we summarize recent efforts that have been successful in improving RPE derivation efficiency by directing differentiation from human pluripotent stem cells using developmental cues important for normal RPE specification and maturation in vivo. In addition, potential obstacles for clinical translation are discussed. Finally, we review how derivation of RPE from human i...
2013
Age-related macular degeneration (AMD), the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries, is associated with the dysfunction and death of the retinal pigment epithelial (RPE) cells. As a result, there has been significant interest in developing RPE culture systems both to study AMD disease mechanisms and to provide substrate for possible cell-based therapies. Because of their indefinite self-renewal, human pluripotent stem cells (hPSCs) have the potential to provide an unlimited supply of RPE-like cells.
Stem Cell Research & Therapy
Retinal pigment epithelium (RPE) degeneration is the hallmark of age-related macular degeneration (AMD). AMD, as one of the most common causes of irreversible visual impairment worldwide, remains in need of an appropriate approach to restore retinal function. Wet AMD, which is characterized by neovascular formation, can be stabilized by currently available therapies, including laser photocoagulation, photodynamic therapy, and intraocular injections of anti-VEFG (anti-vascular endothelial growth factor) therapy or a combination of these modalities. Unlike wet AMD, there is no effective therapy for progressive dry (non-neovascular) AMD. However, stem cell-based therapies, a part of regenerative medicine, have shown promising results for retinal degenerative diseases such as AMD. The goal of RPE cell therapy is to return the normal structure and function of the retina by re-establishing its interaction with photoreceptors, which is essential to vision. Considering the limited source of...
Stem cells as source for retinal pigment epithelium transplantation
Progress in Retinal and Eye Research, 2014
Inherited maculopathies, age related macular degeneration and some forms of retinitis pigmentosa are associated with impaired function or loss of the retinal pigment epithelium (RPE). Among potential treatments, transplantation approaches are particularly promising. The arrangement of RPE cells in a well-defined tissue layer makes the RPE amenable to cell or tissue sheet transplantation. Different cell sources have been suggested for RPE transplantation but the development of a clinical protocol faces several obstacles. The source should provide a sufficient number of cells to at least recover the macula area. Secondly, cells should be plastic enough to be able to integrate in the host tissue. Tissue sheets should be considered as well, but the substrate on which RPE cells are cultured needs to be carefully evaluated. Immunogenicity can also be an obstacle for effective transplantation as well as tumorigenicity of not fully differentiated cells. Finally, ethical concerns may represent drawbacks when embryo-derived cells are proposed for RPE transplantation. Here we discuss different cell sources that became available in recent years and their different properties. We also present data on a new source of human RPE. We provide a protocol for RPE differentiation of retinal stem cells derived from adult ciliary bodies of post-mortem donors. We show molecular characterization of the in vitro differentiated RPE tissue and demonstrate its functionality based on a phagocytosis assay. This new source may provide tissue for allogenic transplantation based on best matches through histocompatibility testing.
Pigment Cell & Melanoma Research, 2011
Compared with neural crest-derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light-absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11-cis-retinal by re-isomerizing all-trans-retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors' outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age-related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE-like cells can be obtained relatively easily when stem cells are subjected to a two-step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.
Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells
Human Molecular Genetics, 2010
Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.
In vitro differentiation of cGMP-grade retinal pigmented epithelium from human embryonic stem cells
International Journal of Retina and Vitreous
Background: The World Health Organization (WHO) estimates that the number of individuals who lose their vision due to retinal degeneration is expected to reach 6 million annually in 2020. The retinal degenerative diseases affect the macula, which is responsible for central and detailed vision. Most macular degeneration, i.e., age-related macular degeneration (AMD) develops in the elderly; however, certain hereditary diseases, such as the Stargardt disease, also affect young people. This degeneration begins with loss of retinal pigmented epithelium (RPE) due to formation of drusen (atrophic) or abnormal vessels (exudative). In wet AMD, numerous drugs are available to successful treat the disease; however, no proven therapy currently is available to treat dry AMD or Stargardt. Since its discovery, human embryonic stem cells (hESCs) have been considered a valuable therapeutic tool. Some evidence has shown that transplantation of RPEs differentiated from hESCs cells can result in recovery of both RPE and photoreceptors and prevent visual loss. Methods: The human embryonic WA-09 stem cell lineage was cultured under current Good Manufacturing Practices (cGMP) conditions using serum-free media and supplements. The colonies were isolated manually and allowed to spontaneously differentiate into RPE cells. Results: This simple and effective protocol required minimal manipulation and yielded more than 10e8 RPE cells by the end of the differentiation and enrichment processes, with cells exhibiting a cobblestone morphology and displaying cellular markers and a gene expression profile typical of mature RPE cells. Moreover, the differentiated cells displayed phagocytic activity and only a small percentage of the total cells remained positive for the Octamer-binding transcriptions factor 4 (OCT-4) pluripotency cell marker. Conclusions: These results showed that functional RPE cells can be produced efficiently and suggested the possibility of scaling-up to aim at therapeutic protocols for retinal diseases associated with RPE degeneration.
Derivation of Functional Retinal Pigmented Epithelium from Induced Pluripotent Stem Cells
Stem Cells, 2009
Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age-related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on integrin avb5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC-RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential.
Investigative Opthalmology & Visual Science, 2015
PURPOSE. We tested what native features have been preserved with a new culture protocol for adult human RPE. METHODS. We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. RESULTS. Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 6 0.24 mV (mean 6 SEM, n ¼ 31), apical positive, and the mean transepithelial resistance (R T) was 178.7 6 9.9 XÁcm 2 (mean 6 SEM, n ¼ 31). Application of 100 lM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 6 0.53 lLÁcm 2 Áh À1 (mean 6 SEM, n ¼ 6) and TEP by 0.33 6 0.048 mV (mean 6 SEM, n ¼ 25). Gene expression of cultured RPE was comparable to native adult RPE (n ¼ 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 6 146 pg/mL/d, compared to an apical secretion of 1548 6 162 pg/mL/d (n ¼ 14, P < 0.01), while PEDF preferentially secreted apically 1487 6 280 ng/mL/d compared to a basolateral secretion of 864 6 132 ng/mL/d (n ¼ 14, P < 0.01). CONCLUSIONS. The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.
Stem Cells and Development, 2012
We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum-and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these diseasespecific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases.