MXPA06008299A - Improved modalities for the treatment of degenerative diseases of the retina - Google Patents

Abstract

This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium(RPE) cells and other retinal progenitor cells.

Description

MOPALIPAPES MEJORAPAS FOR THE TREATMENT PE ENFERMEPAPES PEGENERATIVAS PE LA RETINA This application is being filed as an international TCP patent application on January 24, 2005, in the journal of Advanced Cell Technology, Inc., a national corporation of the United States, applicant for the designation of all countries, except the United States. , and Irima V. Klimanskaya, a US citizen, applicant for the designation of the United States only, and claims priority for the provisional US application serial number 60/538/964, filed on January 23, 2004.

FIELD PE INVENTION This invention relates generally to methods for improved cell-based therapies for retinal degeneration and other visual disorders, as well as for the treatment of Parkinson's disease and for the differentiation of mammalian embryonic stem cells and embryo-derived cells. mammal in retinal pigmented epithelial cells (RPE) or other tissues of the eye, including, without limitation, rods, cones, bipolar, corneal, neural cells, iris epithelium and progenitor cells.

ANTECEPENT IS OF THE INVENTION Many parts of the central nervous system (CNS) show laminal organization, and neuropathological processes generally involve more than one of these multiple cell layers. CNS diseases often include neuronal cell loss, and due to the absence of endogenous repopulation, effective recovery of function after CNS-related disease is extremely limited or absent. In particular, the known common retinal condition is age-related macular degeneration (AMD), which is the result of the loss of photoreceptors together with retinal pigmented epithelium (RPE), with additional variable involvement of internuncial neurons ("transmission"). ") of the inner nuclear layer (INL). The restoration of moderate to high visual acuity, therefore, requires the functional replacement of some of the damaged cell layers. Anatomically, retinitis pigmentosa (RP), a family of hereditary retinal degenerations, is a continuous decrease in the number of photoreceptor cell nuclei, which leads to loss of vision. Although the phenotype is similar in most forms of RP, the underlying cellular mechanisms are diverse, and may be the result of several mutations in many genes. Most involve mutations that alter the expression of specific genes for photoreceptor cells, and mutations in the rhodopsin gene make up approximately 10% of these. In other forms of the disease, the regulatory genes of apoptosis are altered (eg, Bax and Pax2). AMD is a clinical diagnosis that includes a range of degenerative conditions that probably differ in etiology at the molecular level. All cases of AMD share the characteristic of photoreceptor cell loss within the central retina. However, this common endpoint seems to be a secondary consequence of early abnormalities at the RPE level, neovascularization, and underlying Bruch membrane. The latter may refer to difficulties with the replacement of the photoreceptor membrane, whose understanding is still poor. Additionally, retinal pigmented epithelium is one of the most important types of cells in the eye, since it is crucial for the support of photoreceptor function. It performs several complex tasks, including phagocytosis of exterior segments detached from canes and cones, metabolism of vitamin A, synthesis of mucopollsaccharides involved in the exchange of metabolites in the subretinal space, transport of metabolites, regulation of angiogenesis, absorption of light, improvement of the resolution of images, and the regulation of many other functions in the retina through secreted proteins, such as proteases and protease inhibitors. An additional feature present in some cases of AMD is the presence of aberrant blood vessels, which results in a condition known as choroidal neovascularization (CNV). This neovascular ("wet") form of AMD is particularly destructive, and appears to be the result of a loss of proper regulation of angiogenesis. Brunch membrane disruptions as a result of RPE dysfunction allow new vessels of the choroidal circulation to access the subretinal space, where they can physically interrupt the organization of the outer segment and cause vascular leakage or hemorrhage, leading to further loss of photoreceptors. The CNV can be targeted by laser treatment. Thus, laser treatment for the "wet" form of AMD is commonly used in the United States. Often there are undesirable side effects, however, and therefore, the result of the treatment will be patient dissatisfaction. This is due to the fact that laser burns, if they occur, are associated with the death of the photoreceptor and with absolute, irreparable blindness within the corresponding part of the visual field. In addition, laser treatment does not repair the underlying predisposition towards the development of CNV. Undoubtedly, laser burns have been used as a practical method for induction of CNV in monkeys (Archer and Gardiner, 1981). Macular laser treatments for CNV are used to a much greater extent in other countries, such as the United Kingdom. Generally, there is no recognized treatment for the more common "dry" form of AMD, in which there is loss of photoreceptor over irregular patches of RPE atrophy in the macula, and associated extracellular material called drusen. Since RPE plays an important role in the maintenance of the photoreceptor, and the regulation of angiogenesis, various flaws with RPE in vivo are associated with diseases that disrupt vision, such as retinitis pigmentosa, RPE detachment, dysplasia, atrophy, retinopathy, dystrophy or macular degeneration, including macular degeneration related to age, which can result in photoreceptor damage and blindness. Specifically and in addition to the AMD, the variety of other degenerative conditions that affect the macula, include, without limitation, cone dystrophy, cone-rod dystrophy, malattia leventinese, Doyne honeycomb dystrophy, Sorsby's dystrophy, Stargardt disease, pattern / butterfly dystrophies, Best viteliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid striae, and toxic maculopathies. General retinal diseases that can secondarily affect the macula include retinal detachment, pathological myopia, retinitis pigmentosa, diabetic retinopathy, CMV retinitis, occlusive retinal vascular disease, premature retinopathy (ROP), choroidal rupture, ocular histoplasmosis syndrome ( POHS), toxoplasmosis, and congenital amaurosis of Leber. None of the previous lists is exhaustive. All of the above conditions involve loss of photoreceptors, and therefore, the treatment options are few and insufficient.

Due to its ability to heal wounds, RPE has been studied extensively in relation to its application for transplant therapy. In 2002, one year into the trial, patients showed an improvement of 30 to 50%. It has been demonstrated in several animal models and in humans (Gouras and co-authors, 2002, Stanga and co-authors, 2002, Binder and co-authors, 2002, Schraermeyer and co-authors, 2001, reviewed by Lund and co-authors, 2001 ) that RPE transplantation has a good potential for vision restoration. However, even in an immunologically privileged site, such as the eye, there is a problem with graft rejection, which hinders the progress of this approach if allogeneic transplantation is used. Although new photoreceptors (PRC) have been introduced experimentally by transplantation, the grafted PRCs show a marked reluctance to link with the surviving neurons of the host retina. The dependence of RPE cells derived from fetal tissue is another problem, since these cells have demonstrated a very low proliferative potential. Researchers from Emory University conducted a test where they cultured RPE cells from a human eye donor in vitro and transplanted it into six patients with advanced Parkinson's disease. Although a decrease in symptoms of 30 to 50% was found one year after transplantation, there is a shortage of eye donors, this is not yet approved by the FDA, and there may still be a need beyond what could be achieved with the donated eye tissue.

Until now, therapies using ectopic RPE cells have shown that they behave like fibroblasts and have been associated with a number of destructive retinal complications including axonal loss (Viliegas-Perez and co-authors, 1998) and proliferative vitreoretinopathy (PVR) with retinal detachment (Cleary and Ryan, 1979). The RPE supplied as a loose blade tends to run. This results in poor effective photoreceptor coverage, as well as an RPE with multiple layers with incorrect polarity, possibly resulting in the formation of cysts or macular edema. The supply of neural retina grafts to the subretinal (submacular) space of the diseased human eye has been described in Kaplan and co-authors (1997), Humayun and co-authors (2000), and del Cerro and co-authors (2000). There is a serious problem in which neural retina grafts typically do not functionally integrate with the host retina. In addition, the absence of an RPE monolayer means that the dysfunction of the RPE or interruption of the Brunch membrane has not been rectified. Both are fundamental antecedents of visual loss. Thus, there are no effective means to reconstitute RPE in any of the current therapies, and deficiencies remain in each, particularly the essential problem of a functional disconnection between the graft and the host retina. Therefore, there is a need for improved retinal therapy.

BRIEF DESCRIPTION OF THE INVENTION The purpose of the present invention is to provide improved methods for the derivation of eye cells, including, but not limited to, neural cells, including horizontal cells and amacrine cells, retinal cells such as rods and cones, corneal cells, vascular cells, and RPE cells and RPE-like of stem cells, and provide improved methods and therapies for the treatment of retinal degeneration. In particular, these methods involve the use of RPE and RPE-like cells derived from human embryonic stem cells. One embodiment of the present invention provides an improved method for generating cells for use in therapy to treat degeneration using RPE cells, cells similar to those of RPE, progenitors of these cells or a combination of two or three of any of the Embryonic precedent stem cells derived from mammal, in order to treat various conditions, including, without limitation, retinitis pigmentosa and macular degeneration and associated conditions. The types of cells that can be produced using the invention, include, without limitation, RPE, cells similar to those of the RPE, and progenitors of RPE. Cells that can also be produced include iris pigmented epithelial cells (IPE). The neural cells associated with vision include internuncial neurons (for example, "transmission" neurons of the inner nuclear layer (INL) and amacrine cells (interneurons that interact on the second synaptic level of vertically direct pathways, constituted by the chain of Photoreceptor-bipolar-ganglion cells, which are synaptically active in the inner plexiform layer (IPL) and serve to integrate, modulate and interpose a temporal domain to the visual message presented to the ganglion cell), can also be produced using this invention. The cells of the present invention can be transplanted into the subretinal space using vitrectomy surgery.Non-limiting examples include the transplantation of these cells into a suspension, matrix or substrate.The animal models of retinitis pigmentosa that can be treated include rodents (mouse rd , RPE-65 knockout mouse, Tubby protein-like mouse, RCS rat, cats (Abi cat) sinio) and dogs (dog with "cd" cone degeneration), dog with progressive degeneration of rods and cones "prcd", dog with early retinal degeneration "erd", dogs with dysplasia 1, 2 and 3 of rods and cones, dogs "redi, rcd2 and rcd3", dog with dysplasia of the photoreceptor "pd", and dog Briard "RPE-65". The evaluation is performed using behavioral tests, fluorescence angiography, histology, or functional tests such as measuring the ability of cells to perform phagocytosis (photoreceptor fragments), vitamin A metabolism, tight junction conductivity, or evaluation using electron microscopy . One of the many advantages of the methods presented here is the ability to produce and treat many more patients than would be possible if one were limited to using tissue from the donor eye. A further embodiment of the present invention provides methods for the spontaneous differentiation of hES cells into cells with numerous characteristics of the RPE. These RPE preparations are capable of making phenotypic changes in culture, and of maintaining the characteristics of the RPE during multiple steps. The present invention also provides methods of differentiation of established RPE cell lines, in alternative neuronal lineages, corneal cells, retinal cells as a non-limiting example by the use of bFGF or FGF. Another embodiment of the present invention is a method for the derivation of new lines of RPE and progenitor cells from existing and new ES cell lines. There may be variations in the properties, such as rate of growth, expression or pigment, or de-differentiation and re-differentiation in culture, or cells similar to those of the RPE when they come from different ES cell lines. There may be certain variations in their functionality and karyotypic stability, so it is desirable to provide methods for the derivation of new RPE lines and new ES cell lines, which could allow choosing the lines having the desired properties that can be be selected clonally to produce a pure population of cells similar to those of high quality RPE. Cells that can also come from existing and new ES cell lines include pigmented iris epithelial cells (IPE). In a further embodiment, neural cells associated with vision may also be produced, including internuncial neurons (eg, "transmission" neurons of the inner nuclear layer (INL) and amacrine cells)., using this invention. Additionally, retinal cells, rods, cones and corneal cells can be produced. In a further embodiment of the present invention, one can also produce cells that provide the vasculature of the eye. Another embodiment of the present invention is a method for the derivation of RPE lines or precursors of RPE cells that have an increased capacity to prevent neovascularization. These cells can be produced by aging a somatic cell of a patient, such that the telomerase is reduced where at least 10% of the reproductive life span of the cell has passed, then the use of said somatic cell as a donor cell for nuclear transfer in order to create cells that overexpress angiogenesis inhibitors such as Pigmented Epithelium Derivative Factor (PEDF / EPC-1). Alternatively, these cells can be genetically modified with exogenous genes that inhibit neovascularization. Another embodiment of the present invention uses an ES bank or embryo derived cells with homozygosity in the HLA region, such that said cells have reduced complexity of their HLA antigens. Therefore, a further embodiment of the present invention includes the characterization of cells similar to those of RPE from ES. Although the pigmented epithelial cells derived from ES are very similar to those of RPE due to their morphology, behavior and molecular markers, their therapeutic value will depend on their ability to perform RPE functions and to remain non-carcinogenic. Therefore, RPE cells derived from ES are characterized using one or more of the following techniques: (i) evaluation of their functionality, i.e. phagocytosis of photoreceptor fragments, vitamin A metabolism, wound healing potential; (ii) evaluation of the pluripotency of ES cell derivatives similar to those of RPE through transplants in animal models, (as a non-limiting example, this may include SCID mice); (iii) determination of phenotype and karyotype of cells similar to those of RPE; (iv) evaluation of cells similar to those of RPE derived from ES cells and RPE tissue by elaboration of expression profile, (v) evaluation of the expression of molecular markers of RPE at the protein level, including bestrophin, CRALB, RPE -65, PEDF. The cells can also be evaluated based on their expression of transcriptional activators normally required for eye development, including rx / rax, chx10 / vsx-2 / alx, ots-1, otx-2, six3 / optx, six6 / optx2, mitf, pax6 / mitf and pax6 / pax2 (Fischer and Reh, 2001, Baumer and co-researchers, 2003).

A further embodiment of the present invention is a method for the characterization of cells similar to those of RPE, from ES, using at least one of the techniques selected from the group consisting of (i) evaluation of the functionality of cells similar to those of RPE, from ES; (ii) evaluation of the pluripotency of ES cell derivatives similar to those of RPE through transplants in animal models; (iii) elaboration of phenotype and karyotype of cells similar to those of RPE; (iv) evaluation of the gene expression profile, (v) evaluation of the expression of molecular markers of RPE at the protein level; and (vi) the expression of transcriptional activators normally required for eye development. In a further embodiment, these techniques can be used for the evaluation of multiple cell types derived from hES cells. Another embodiment of the present invention is a method for the derivation of RPE cells and RPE precursor cells directly from embryos developed up to the morula or blastocyst (EDC) stage of human and non-human animals without the generation of cell lines IS. Embryonic stem cells (ES) can be maintained indefinitely in vitro, in an undifferentiated state and are still capable of differentiating in virtually any cell type. Human embryonic stem cells (hES) are useful for studies on the differentiation of human cells, and can be considered as a potential source for transplant therapies. To date, the differentiation of human and mouse ES cells has been reported in numerous cell types (reviewed by Smith, 2001) including cardiomyocytes [Kehat and coinvestigators 2001, Mummery and co-investigators, 2003 Carpenter and co-investigators, 2002 ], neurons and neural precursors (Reubinoff and co-investigators 2000, Carpenter and co-investigators 2001, Schuldiner and co-investigators, 2001), adipocytes (Bost and coinvestigators, 2002, Aubert and co-investigators, 1999), cells similar to hepatocytes (Rambhatla and co-investigators, 2003), hematopoietic cells (Chadwick and co-investigators, 2003). oocytos (Hubner and co-investigators, 2003), thymocyte-like cells (Lin RY and co-investigators, 2003), pancreatic islet cells (Kahan, 2003), and osteoblasts (Zur Nieden and coinvestigators, 2003). Another embodiment of the present invention is a method for identifying cells such as RPE cells, hematopoietic cells, muscle cells, liver cells, pancreatic beta cells, neurons, endothelium, progenitor cells or other cells useful in therapy or in cell research. , from embryos, embryonic stem cell lines, or other embryonic cells with the ability to differentiate into useful cell types by comparing the messenger RNA transcripts of these cells with cells derived in vivo. This method facilitates the identification of cells with a normal phenotype and for cell derivation optimized for cell therapy for research. The present invention provides for the differentiation of human ES cells into a specialized cell in the neuronal lineage, the retinal pigmented epithelium (RPE). The RPE is a densely pigmented epithelial monolayer between the choroid and the neural retina. It serves as part of a barrier between the bloodstream and the retina, and its functions include phagocytosis of detached rod segments and detached cones, deviated light absorption, vitamin A metabolism, retinoid regeneration, and tissue repair (Grierson et al. co-researchers, 1994, Fisher and Reh, 2001, Marmorstein and co-researchers, 1998). The RPE is easily recognized by its cobbled cell morphology of black pigmented cells. In addition, there are several known markers of RPE, including retinaldehyde-binding cellular protein (CRALBP), a cytoplasmic protein that is also found in apical microvilli (Bunt-Milam and Saap, 1983).; RPE65, a cytoplasmic protein involved in retinoid metabolism (Ma and co-investigators, 2001, Redmond and co-investigators, 1998); bestrophin, the product of the Best Viteliform Macular Dystrophy gene (VMD2, Marmorstein and co-investigators, 2000), and pigmented epithelium-derived factor (PEDF), a secreted 48kD protein with angiostatic properties (Karakousis and coinvestigators, 2001, Jablonski and co-investigators, 2000). An unusual feature of the RPE is its obvious plasticity. RPE cells are usually mitotically quiescent, but may begin to divide in response to damage or photocoagulation. RPE cells adjacent to the damage flatten and proliferate, forming a new monolayer (Zhao and coinvestigators, 1997). Several studies have indicated that the RPE monolayer can produce fibroblast-like cells that can be reverted to their original RPE morphology (Grierson and co-investigators, 1 994, Kirchhof and co-investigators, 1988, Lee et al. researchers, 2001). It is not clear whether the cells that divide and the pigmented epithelial layer come from the same lineage, given that two populations of RPE cells have been isolated: epithelial and fusiform (McKay and Burke, 1994). In vitro, depending on the combination of growth factors and substrate, the RPE can be maintained as an epithelium or rapidly de-diffuse and become prollferative (Zhao 1997, Opas and Dziak, 1994). Interestingly, the epithelial phenotype can be reestablished in long-term inactive cultures. (Griersion and co-researchers, 1994). In the development of mammals, the RPE shares the same progenitor with the neural retina, the neuroepithelium of the optic vesicle. Under certain conditions, it has been suggested that RPE can be transdifferentiated into neuronal progenitors (Opas and Dziak, 1 994), neurons (Chen and co-investigators, 2003, Vinores and coinvestigators, 1,995), and lens epithelium (Eguchl, 1986). ). One of the factors that can stimulate the change of RPE in neurons is bFGF (Opaz and Dziak, 1994, a process associated with the expression of transcriptional activators normally required for eye development, including rx / rax, chx10 / vsx-2 / alx, ots-1, otx-2, six3 / optx, six6 / optx2, mitf, and pax6 / pax2 (Fischer and Reh, 2001, Baumer and co-researchers, 2003) Recently, it has been shown that the margins of the Chicken retina contain neural stem cells (Fischer and Reh, 2000) and that cells pigmented in that area, which express paxd / mitf, can form neuronal cells in response to FGF (Fisher and Reh, 2001). the derivation of trabecular cells genetically modified for the treatment of glaucoma The present invention also provides for the derivation of cells from the trabecular network of progenitors of RPE and cells similar to those of RPE and also cells of the trabecular network modified gene for the treatment of glaucoma. The present invention includes methods for the derivation of RPE cells and RPE precursor cells directly from embryos in the morula or blastocyst development stage (EDC) without the generation of ES cell lines, comprising: a) maintaining the cells It is in vitro in undifferentiated state; b) differentiate the ES cells in RPE cells and RPE precursors and c) identify the RPE cells by comparing the messenger RNA transcripts of these cells with cells obtained in vivo. Additionally, this invention provides methods for the derivation of RPE cell lines or RPE precursors that have an increased capacity to prevent neovascularization, said methods comprising: a) aging a somatic cell of an animal, such that telomerase is reduces, where at least 10% of the amplitude of normal reproductive life of the cell has passed; and b) using the somatic cell as a donor cell for nuclear transfer, in order to create cells that overexpress angiogenesis inhibitors., wherein the inhibitors of angiogenesis can be Pigmented Epithelium Derivative Factor (PEDF / EPC-1). The present invention provides methods for the treatment of Parkinson's disease, with RPE from hES cells, cells similar to those of RPE and / or progenitor cells of RPE. These can be delivered by intrastriatal stereotactic implantation with microcarriers. Alternatively, they can be supplied without the use of microcarriers. The cells can also be expanded in culture, and used in the treatment of Parkinson's disease by any method familiar to those skilled in the art. Other features and advantages of the invention will be apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-F is a series of photographs showing the appearance of pigmented areas (characteristic of RPE cells) in hES cells with spontaneous differentiation. Figure 1A is a photograph of regions pigmented in an adherent culture of 2.5 months of maturation, a receptacle of a plate with 6 receptacles, scanned with scanner; Figure 1B is a photograph of pigmented regions in a 2.5 month maturation culture grown in EB, with an increase of 45x; Figure 1C is a photograph of a pigmented area of an adherent culture; Figure 1D is a photograph of a pigmented region of a culture grown in EB; Figure 1E is a photograph of the boundary between the pigmented region and the rest of the crop, x200; Figure F is the same as figure E, but with an increase of x400. The arrows in A and B point to the pigmented regions. Figure 2A-F, is a series of photographs showing the loss and regain of pigmentation and epithelial morphology in the culture. Figure 2A is a photograph showing the outer growth of primary ECs, 1 week; Figure 2B is a photograph showing the primary culture of the cells, isolated by trypsin, 1 week; Figure 2C is a photograph showing the epithelial islet surrounded by proliferating cells; Figure 2D is a photograph showing the new obtaining of pigmentation and epithelial morphology in a culture of 1 month of maturation; Figure 2E is a photograph showing the crop after 3 passes, increase of x200; Figure 2F shows the same culture as in E, with an increase of x400, with Hoffman's microscope. The black arrows point to pigmented cells, the white arrows show cells with exterior growth without pigment. Figure 3, left panel (A-D) and right panel, is a series of photographs and a graphic. These show RPE markers in pigmented epithelial cells from hES cells. Figures 3A and 3B are photographs showing the inmolocation of RPE marker, bestrophin and the corresponding phase in the microscopic field, magnification of x200; Figures 3C and 3D are photographs showing CRELBP and the corresponding phase of contrast in the microscopic field, increase of x400. The arrows show the co-localization of bestrophin (A) and CRALBP (C) in pigmented cells (C, D); the arrows point to the absence of coloration for these proteins (A, B) in the non-pigmented regions (C, D). Figure 3, right panel, shows a photograph and a Western detection graph of cell lysates (hES # 36 line) with antibodies to bestrophin (a) and CRELBP (b); c, d - undifferentiated h ES cells; c- control for anti-CRALB P antibody; d-control for anti-bestrophin antibody. Figure 4 shows photographs demonstrating the expression of Pax6 (Figure 4A), Pax2 (Figure 4E) and mitf (Figure 4B, Figure 4F) markers in RPE-like cells in long-term inactive cultures. Figures 4C, 4G - phase contrast, Figures 4D, 4H - combined images of Pax6 / mitf / phase contrast (Figure 4A, Figure 4B, Figure 4C) and Pax2 / mitf / phase contrast (Figure 4E, Figure 4F , figure 4G). Figure 5A-B shows photographs of differentiation in RPE in the culture of cells from human embryos; skipping the stage of derivation of ES cell lines. Figure 6 shows the transcriptional comparison of RPE preparations. Figure 6A-F - based on the ontological annotation, this table represents the expression patterns of related RPE genes for retinal pigmented epithelium derived from hES cells (hES-RPE), transdifferentiated derived hES cells (hES-RPE-TD), ARPE -19 and D-407, and recently isolated human RPE (fe-RPE). Figure 6G- Additional data mining revealed specific ontologies for known RPEs, such as melanin biosynthesis, vision, retinol binding, only in fetal RPE and in ES-RPE, but not in ARPE-19.

DETAILED DESCRIPTION PE THE INVENTION Various embodiments of the invention are described in detail, and may be further illustrated by the examples provided. As used in this description, and through the claims that follow, the meaning of "a", "an", and "the" includes reference to the plural, unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "over," unless the context clearly dictates otherwise. The terms used in this specification generally have their ordinary meanings in the subject, within the context of the invention, and in the specific context in which each term is used. Certain terms that are used to describe the invention are described below, or elsewhere in the specification, to provide additional guidance to the practitioner, in the description of compositions and methods of the invention, and in how to make and use them. For convenience, certain terms may be highlighted, for example, using italics and / or quotation marks. The use of highlights has no influence on the scope and meaning of a term; The scope and meaning of a term is the same, in the same context, whether it is highlighted or not. It may be noted that the same thing can be said in more than one sense. Consequently, alternative language and synonyms can be used for any or more of the terms discussed here, and it also has no special meaning if a term is developed or discussed here. Synonyms are provided for certain terms. A mention of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any of the terms discussed herein, is illustrative only, and in no way limits the scope of the invention, while the data is processed, sampled, converted, or the like, according to the invention without considering any theory or scheme of action in particular.

DEFINITIONS By "embryo" or "embryo" is meant a mass of developing cells that has not been implanted in the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained in an embryo. This also includes blastomeres, obtained as early as in the two-cell stage, and aggregated blastomeres. The term "embryonic stem cells" refers to cells derived from embryos. More specifically, it refers to cells isolated from the inner cell mass of blastocysts or morulae, and which have been passed in series as cell lines. The term "human embryonic stem cells" (hES cells) refers to cells derived from human embryos. More specifically, hES refers to cells isolated from the inner cell mass of blastocysts or human morulae, and which have been serially passed as cell lines, and may also include aggregated blastomeres and blastomeres. The term "human embryonic stem cells" (hEDC) refers to morula-derived cells, blastocyst-derived cells, including those of the inner cell mass, embryonic envelope or epiblast, or other totipotent or pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm and mesoderm and its derivatives, including also blastomeres and aggregated simple blastomere cell masses or embryos of various stages of development, but excluding human embryonic stem cells that have been passed as cell lines.

Embryonic stem cells (ES) that have the ability to differentiate into virtually any tissue in a human body can provide an unlimited supply of rejuvenated and histocompatible cells for transplant therapy, since the problem of immune rejection can be solved by transfer nuclear and parthenogenetic technology. The recent findings of Hirano and coinvestigators (2003) have shown that mouse ES cells can produce structures similar to the eye in in vitro differentiation experiments. Between these, pigmented epithelial cells were described, similar to those of the retinal pigmented epithelium. Preliminary experiments carried out in Advanced Cell Technology with primate and human ES cell lines show that in a specialized culture system these cells differentiate into RPE-like cells that can be isolated and transitioned. The human and mouse NTs, derived from ES parthenote Cyno cells have multiple RPE characteristics: these pigmented epithelial cells express four molecular markers of RPE - biostrofine, CRALBP, PEDF, and RPE65; Like the RPE, its proliferation in culture is accompanied by dedifferentiation - loss of pigment and epithelial morphology, both restored after the cells form a monolayer and become inactive. These cells similar to those of RPE can be easily transitioned, frozen and thawed, thus allowing their expansion. The inventors have further demonstrated that human ES cells also produce multiple structures similar to those of the eye (vitreous body) in in vitro differentiation experiments. Histological analysis of these structures shows a pattern of cells consistent with early retinal development, including rod and cones-like cell aggregates.

RPE TRANSPLANT Currently, slow rejection of RPE allografts prevents scientists from determining the therapeutic efficacy of this RPE transplant. Several methods are being considered to overcome this obstacle. The easiest way is to use systemic immunosuppression, which is associated with serious side effects, such as cancer and infection. A second approach is to transplant the RPE of the patient himself, that is, homografts, but this has the disadvantage of using old, sick RPE to replace RPE even more ill. Still, a third approach is to use the iris epithelium (IPE) of the same patient, but this has the disadvantage that the IPE can not perform all the functions related to the vision of the RPE. Finally, it will be necessary to find a method to eliminate the rejection, and then the scientists will be able to determine the real efficiency of the RPE transplant in AMD and in ARMD. Nuclear transfer and parthenogenesis facilitate the histocompatibility of RPE cells and grafted progenitors.

DEFECTS PEL RPE IN RETINITIS PIGM ENTOSA Retinitis pigmentosa is a hereditary condition in which vision receptors are destroyed gradually due to abnormal genetic programming. Some forms cause total blindness at relatively young ages, other forms show Retinal changes of characteristic "bone spicule" on the retina with little destruction of vision. This disease affects approximately 1.5 million people around the world. Two genetic defects that cause autosomal recessive RP have been found in genes expressed exclusively in RPE: one is due to an RPE protein involved in the metabolism of vitamin A (cis retinaldehyde binding protein), the second involves another protein unique to RPE , RPE65. Once the rejection has been conquered, both of these RP forms must be immediately treatable by RPE transplantation. This treatment was inconceivable a few years ago when PR was a form of blindness that was not treatable and poorly understood. New research in RPE transplantation suggests that there is a promise for the treatment of retinal degeneration, including macular degeneration. In addition, a number of patients with advanced RP have regained some useful vision after transplantation of fetal retinal cells. One of the patients, for example, improved from being able to see the light until he was able to count the fingers held at a distance of approximately 1.80 meters (6 feet) from the patient's face. In a second case, vision improved to achieve the ability to see letters through tunnel vision. The transplants in these studies were done by injection, introducing the new retinal cells below the existing neural retina. Not all the cells survived, since the transplanted fetal cells were allogeneic (that is, they were not genetically coincident), although those that survived formed connections with other neurons and began to function as the photoreceptors around them. Approximately one year after the first eight people received the transplants, four had regained some visual function, and a fifth showed symptoms of recovering. Three lines of recently obtained human embryonic stem cells are similar in their properties to those described above (Thomson and co-investigators 1998, Reibunoff and co-investigators, 2000, Richards and co-investigators, 2000, Lanzendorf and co-investigators, 2001) : they maintain undifferentiated phenotype and express known markers of undifferentiated hES cells, Oct-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-I-60, TRA-I-81 up to 45 steps in culture or during 130 population doublings, All hES cell lines differ in derivatives of three germ layers in EB or long-term adherent cultures and in teratomas. One of the derivatives of the differentiation of hES cells is similar to the retinal pigmented epithelium, by the following criteria: morphologically, they have a typical epithelial monolayer with cobblestone appearance, and contain dark brown pigment in their cytoplasm, which is known to be present in the human body only in melanocytes, keratinocytes, pigmented epithelium of the retina and the iris (IPE). Melanocytes, however, are non-epithelial cells, and keratocytes do not secrete melanin, but only accumulate it. The set of RPE-specific proteins -bestrophin, CRALBP, PEDF- present in these cells indicates that they are probably similar to those of the RPE and not those of the IPE. Another similarity is the behavior of the pigmented cells isolated in culture, when no pigment was observed or very little was observed in the proliferating cells, but was maintained in tightly packed epithelial islands or reexpressed in newly established cobble monolayer after the cells were they became inactive. This behavior was described for cultured RPE cells (reviewed by Zhao and co-investigators, 1997), and it was previously reported (Vinores and co-investigators, 1995) that a neuronal marker tubulin beta II was located specifically in the cells of RPE in the in vitro de-differentiation, and not in the cells with the typical RPE morphology, suggesting that this reflects the plasticity of the RPE and its ability to de-differentiate into a neural lineage. The inventors have observed the same localization pattern of tubulin beta III in primary and transformed cultures of RPE and cells similar to those of RPE, which may reflect a de-differentiation of such cells in culture, or indicate a separate population of cells compromised for a neuronal fate, which were originally located near the pigmented cells by differentiation of hES cells in long-term cultures and may have been co-isolated with cells similar to those of RPE. In the growth of the optic vesicle, the RPE and the neural retina share the same bipotential neuroepithelial progenitor, and it was demonstrated that their fate is determined by Pax2, Pax6 and Mitf (Baumer and co-investigators, 2003), the latter being an objective of the first two. Pax6 in early stages acts as an activator of proneural genes and is down-regulated in the RPE in further development, remaining in the mature and gangllonar cells in the mature retina (reviewed by Ashery-Padan and Gruss, 2001). In the golden fish, it is also found in mitotically active progenitors, or in regenerating neurons (Hitchcock and co-researchers, 1996). The inventors have found that many of the RPE-like cells expressed mitf and Pax6 in a pattern similar to tubulin beta III and were found only in non-pigmented cells of non-epithelial morphology surrounding pigmented epithelial islands in long-term cultures or in cells with a "partial" RPE phenotype (slightly pigmented and loosely packed). In proliferating cells in recently transformed cultures, all these markers were found near each cell, suggesting either an inversion of cells similar to those of RPE to the progenitor stage the onset of proliferation, or massive proliferation of retired progenitors. Interestingly, in teratomas where islands of pigmented cells with epithelial morphology were also found, Pax6 was expressed in non-pigmented cells adjacent to the pigmented regions (data not shown). Multiple studies have previously shown the de-differentiation of the RPE in culture and its transdifferentiation in neuronal phenotype cells (Reh and Gretton, 1987, Skaguchi and co-investigators, 1997, Vinores and co-investigators, 1995, Chen and co-investigators, 2003), neuronal, amacrine and photoreceptor cells (Zhao and co-investigators, 1995), glla (Skaguchi and co-investigators, 1997), neuronal retina (Galy and co-investigators)., 2002), and in neuronal progenitors (Opaz and Dziak, 1993). These progenitors can coexist in turn with cells similar to those of mature RPE in culture or appear as a result of de-differentiation of cells similar to those of RPE. At the same time, neural retina cells can be transdifferentiated in RPE in vitro (Opas and coinvestigators, 20019), so that alternatively, cells positive to tubulin beta II and Pax6 could represent a transitory stage of this transdifferentiation of neural cells co- isolated or neural progenitors in cells similar to those of RPE.

Differentiation of hES cells into cells similar to those of RPE occurred spontaneously when methods described in the following examples were used, and the inventors noted that pigmented epithelial cells appeared reliably in cultures with maturity greater than 6.8 weeks and their amount progressed over time - in cultures of 3 to 5 months, almost every EB had a large pigmented region. In addition to the described hES lines, six more recently derived hES lines were transformed into cells similar to those of RPE, suggesting that since neural fate is usually chosen by ES cells spontaneously, RPE-like cells may appear by default as an advanced stage of this way. It is also possible that these long-term cultures, in which the differentiated hES cells form a multi-layered environment, the signals of permissive and / or instructive differentiation come from the extracellular matrix and from growth factors produced by cell differentiation derivatives. hES. The differentiation model of hES cells in cells similar to those of RPE could be a useful tool to study how much of this microenvironment orchestrates the differentiation and transdifferentiation in RPE. RPE plays an important role in the maintenance of the photoreceptor, and various RPE dysfunctions in vivo are associated with a number of diseases with impaired vision, such as RPE detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, dystrophy or degeneration. macular, including age-related macular degeneration, which can result in photoreceptor damage and blindness. Due to its capabilities for wound healing, RPE has been studied extensively in the application to transplant therapy. It has been demonstrated in several animal models and in humans (Goures and co-investigators, 2002, Stanta and coinvestigadores, 2002, Binder and co-investigators, 2002, Schraermeyer and co-investigators, 2001, reviewed by Lund and coinvestigadores, 2001) , that the transplant of RPE has a good potential for the restoration of vision. Recently, another prospective niche for RPE transplantation was proposed, and the clinical trials phase was still reached: since these cells secrete dopamine, they could be used for the treatment of Parkinson's disease (Subramanian, 2001). However, even in an immunologically privileged eye, there is a problem of graft rejection, which hinders the progress of this approach if allogeneic transplantation is used. The other problem is that it rests on fetal tissue, since the adult RPE has a very low proliferative potential. As a source of immunologically compatible tissues, h ES cells hold promise for transplant therapy, since the problem of immune rejection can be solved with nuclear transfer technology. The new derivative of differentiation of human ES cells, cells similar to those of the retinal pigmented epithelium and the reliability and simplicity of this differentiation system can offer a supply of RPE cells with attractive power for transplantation.

EXAMPLES EXAMPLE 1 SPONTANEOUS DIFFERENTIATION IN EPITHELIAL CELLS PIGMENTS IN LONG-TERM CROPS When the hES cell cultures are allowed to grow in MEF in the absence of LIF, FGF and Plasmanate, they form multiple thin layers of cells. Approximately 6 weeks later, dark islands of cells appear within the larger groups (figure 1). These dark cells are more easily seen with the naked eye and are seen as "freckles" on a plate with cells such as the one shown in Figure 1A. With a higher magnification, these islands resemble polygonal cells packed tightly in a cobble monolayer, typical of epithelial cells, with brown pigment in the cytoplasm (Figure 1C). There are differences in the amount of pigment in the cells with the cells in the central part of the islands with most of the pigment and with those near the less important edges (Figure 1)., E, F). When the hES cells form embryoid bodies (EB), pigmented epithelial cells appear in approximately 1 to 2% of EB in the first 6 to 8 weeks (Figure 1B). Over time, more and more pigmented cells develop EB, and in about 3 months each EB has a pigmented epithelial region (Figure 1D). The morphology of the cells in the pigmented regions of the EB was very similar to that of adherent cultures (figure 1D).

EXAMPLE 2 ISOLATION AND CULTURE OF PIGMENTED EPITHELIAL CELLS The inventors isolated pigmented epithelial cells from cultures of hES cells and EBs. The pigmented polygonal cells were digested with enzymes (trypsin and / or collagenase, and / or dispase), and the cells of these pigmented islands were selectively taken with a glass capillary. While care was taken to take only the pigmented cells, the population of isolated cells invariably contained some non-pigmented cells. After placing the cells in plates on gelatin or laminin for 1 to 2 days, the cells were considered primary cultures (PO). The primary cultures contained islands of pigmented polygonal cells, as well as some pigmented cells alone. After 3 to 4 days of culture, non-pigmented cells appeared that appeared to have lost their epithelial morphology (more flat and lamellar cells) in the periphery of some islands (figure 2). The amount of these peripheral cells increased with time, suggesting that these cells were proliferating, and after 2 weeks, most of the cells in the newly formed monolayer did not contain pigment or contained very little. After continuous culture, for another 2 to 3 weeks, pigmented epithelial cells began to reappear, visibly indistinguishable from those of the original cultures (Figure 2).

EXAMPLE 3 DETECTION OF RPE MARKERS The characterization of these human cells differentiated as RPE is based on their similarity with previously described RPE cultures; mainly, its epithelial morphology and possession of pigment. There are three types of pigmented epithelial cells in the human body: pigmented retinal and iris epithelium, and keratinocytes, but the latter do not secrete pigment. The epithelial structure and cobble morphology are not shared by other pigmented cells, for example, melanocytes. It is also noteworthy that it has been shown that RPE cells lose and regain their pigment and epithelial morphology when grown in culture (Zhao 1997, Opas and Dziak, 1994), and the pigmented cells were compared in a similar way, such that to test the hypothesis that cells from ES can be RPE, they were stained with antibodies to known markers for RPE: bestrophin and CRALBP. Figure 4 (left panel) shows the location of bestrophin (A) and CRALBP (C) in the membrane, both were found in pigmented epithelial islands. Not all cells with these antibodies and intensity of staining were correlated with the expression of pigment and the "tightening" of the colonies - the edges of each pigmented island where the cells were larger and were packaged more loosely, showed lower expression of both proteins. To further characterize the cells presumably of RPE, analysis of the expression of bestrophin, CRALBP by Western detection was performed. Figure 4 (right panel) shows the bands corresponding to bestrophin, 68 kD (a), CRALBP, 36 kD (b) in cell lysates. All these proteins were found both in the primary cultures and in the subsequent steps. Another known PRE marker, RPE65, was found in the RPE-like cells by real-time RT-PCR (Figure 4, right panel, below), the ELISA assay for PDEF showed the presence of PEDF in cell lysates of all cultures which were presumed to be RPE, and Western detection showed a band of approximately 48 kD (not shown). DETECTION OF MARKERS OF NEURONAL AND RETINAL PROGENITORS IN CROPS SIMILAR TO RPE Figure 4 shows localization of PAX-6, Pax2, mitf, and tubulin beta II in recently transformed and old cultures of RPE from hES cells. In proliferating cultures (day 3 after trypsinization, not shown), where the morphology similar to that of the RPE of the proliferating cells is lost, almost every cell showed the presence of mitf, Pax6, tubulin beta II and nestin (not shown). Only Pax2 was found in a small subset of cells, which appeared to be negative to mitf, whereas there was a strong degree of co-localization of Pax6 / mitf, mitf / tubulin beta III, and Pax6 / tubulin beta III. In 21 days the old inactive cultures were re-established after the pigmented epithelial islands, groups of PAX-6 and mitf were found mostly in non-pigmented cells with non-epithelial morphology between pigmented epithelial islands (Figure 4, AC), and tubulin beta lll had a similar distribution pattern (not shown). However, there were populations of cells positive for mitf and negative for Pax6, located near the periphery of pigmented islands (figure 4, A-C). Only Pax2 was found only in a very small subset of mitf-negative cells (Figure 4, E-H). No presence of any of these proteins was detected in the cells of "mature" pigmented epithelial islands. However, these markers in cells that only had some RPE characteristics were frequently visible, that is, they appeared epithelial but did not have pigment, or in certain pigmented cells alone they were outside the pigmented epithelial islands.

EXAMPLE 4 CHARACTERIZATION OF CELLS SIMILAR TO RPE A DEPARTING FROM CELL LINES HES H9 AND ACT -J-1 FROM CELLS IS CYNO-1 AND DERIVATION OF CELLS SIMILAR TO THOSE OF RPE FROM EXISTING H1 H1 AND H7 CELL LINES A cell line similar to those of RPE is expanded, tested for freezing and recovery, and characterized using the following molecular markers and methods of RPE cells: bsetrophin and CRALBP by Western detection and immunofluorescence, PEDF by ELISA and Western detection , and REP65 by RT-PCR. Cells are injected into SCID mice with undifferentiated hES cells or Cyno-1 as a control to assess tumorigenicity. The determination of karyotype of cells similar to those of RPE will be carried out by a clinical laboratory on a commercial basis. The characterization of the functional properties of the cells similar to those of RPE and the studies of their transplantation potential are then carried out as described in another way in this application, and also using those techniques that are familiar to the connoisseurs of the invention. matter. The gene expression profile determination experiments are performed using Affymetrix human genome arrays.

Genetic expression is compared in cells similar to those of RPE that come from ES cells and in retinal autopsy samples. Various animal models can be used to verify the effectiveness of cells similar to transplanted RPE cells, including, without limitation, rhesus monkey, rat and rabbit.

EXAMPLE 5 OPTIMIZATION OF THE CULTIVATION SYSTEM FOR DIFFERENTIATION ENSURING HIGH PERFORMANCE OF CELLS SIMILAR TO RPE ES cells are grown in alimanator cells or as embryoid bodies (EB) in the presence of bFGF, insulin, TGF-beta, IBMX, bmp-2, bmp-4 or combinations thereof, including step-by-step addition. Alternatively, the ES cells are cultured in various plates coated with extracellular matrix (laminin, fibronectin, collagen I, collagen IV, Matrigel, etc.), evaluating the role of the ECM in the formation of RPE. The expression of molecular markers of early RPE progenitors (Pax6, Pax2, mitf) and RPE cells (CRALBP, bestrophin, PEDF, REP65), is evaluated at various time intervals by RT-PCR to verify and determine successful combinations of the agents mentioned above and the stepwise procedure that produces enrichment in cells similar to those of RPE and other ocular tissues, such as photoreceptor or neural retina, which can be isolated and further characterized by their differentiation potential and used in studies of transplant.

EXAMPLE 6 DERIVATION OF RPE AND OTHER TISSUE PROGENITORS OCULAR FROM CELL LINES IS EXISTING AND NEW Using the genetic expression profile data, the expression of RPE progenitor markers will be correlated with the expression of surface proteins, in order to find a unique combination of surface markers for RPE progenitor cells. If these markers are found, antibodies from surface proteins can be used to isolate a pure population of RPE progenitors that can then be cultured and further differentiated in culture, or used in transplant studies to allow their differentiation after grafting. If the data from the experiments for the determination of the gene expression profile are insufficient, the following approach will be used to isolate the RPE progenitors. ES cells and cells similar to those of RPE with GFP will be transfected under the control of a Pax6 promoter, and stable transfectants will be selected. From a culture of ES cells with transfected differentiation or ES cells or proliferating (de-differentiated) RPE cells, GFP / pax6 positive cells will be isolated by FACS and used as a source of antigen for injection to mice, to elevate the monoclonal antibodies to the surface molecules of Pax6-positive cells. Because Pax6 is present not only in the RPE progenitors, exploration (by FACS) will be performed using several strategies: a) against cells similar to those of proliferating RPE, b) against RPE cells positive for Pax2, c) against RPE cells positive for mitf. For b) and c) the RPE cells will be transfected with GEP under the corresponding promoter; As a negative control, RPE or ES cells negative for these antigens will be used. After the expansion of positive clones selected by the three strategies, antibodies against all types of cells used in the examination will be tested and further analyzed: since this strategy can produce antibodies that recognize the antigens on the surface of the cell, specific and non-specific for RPE progenitors, the cells of the total population in differentiation of ES cells or RPE cells selected with these antibodies, will be evaluated with respect to molecular markers of RPE progenitors and with respect to their ability to produce RPE. Using the defined optimized step procedures, to produce RPE or other early progenitors of ocular tissues and antibodies to their unique surface markers, these progenitors will be isolated from differentiated ES cells and grown in vitro. Their ability to differentiate into various tissues of the eye will be investigated using the strategy described in objective 2. Three ES cell lines already produced cells similar to those of RPE (H9, ACT J-1, Cyno-1). Cells similar to those of RPE will be used to continue producing cells similar to those of RPE and their progenitors, as described in objectives 1 and 2, and hES cell lines H1 and H7 will be used to produce new cell lines similar to those of RPE. After the expansion and characterization of molecular markers of RPE, these lines will be cloned alone, and the resulting lines will be characterized as described in objective 1. The lines that meet the criteria for RPE cells will be used for transplant studies . New human ES cell lines will be derived from unused IVF embryos, from donated oocytes, stimulated to develop without fertilization (parthenote), and from developing blastocysts generated from donated oocytes with the application of nuclear transfer technology. Cores similar to those of RPE and common eye progenitors will come from these lines using the objective 2 approach, and the resulting lines will be characterized as in objective 1. [Optional] new human ES cell lines will be developed in a free system of virus, characterized and subjected to clinical tests.

EXAMPLE 7 THERAPEUTIC POTENTIAL OF CELLS SIMILAR TO THOSE OF RPE AND PROGENITORS IN VARIOUS ANIMAL MODELS PIGMENTOUS RETINITIS AND MACULAR DEGENERATION Primate ES cells were tested in cynomologus monkeys (Macaques). Initially, vitrectomy surgery was performed and the cells were transplanted into the subretinal space of the animals. The first step is the transplantation of the cells in the suspension format, after which a substrate or matrix is used to produce a monolayer transplant. This can also be done in immunosuppressed rabbits using cells from human ES cells, and also in various other animal models using cells derived from human ES cells, and also in various other animal models of retinitis pigmentosa, including rodents (mouse rd, RPE knockout mouse -65, Tubby protein-like mouse, RCS rat, cats (abyssinian cat) and dogs (dog with "cd" cones degeneration), dog with progressive degeneration of "prcd" canes and cones, dog with early "retinal" degeneration "erd" , dogs with dysplasia 1, 2 and 3 of canes and cones, dogs "redi, rcd2 and rcd3", dog with dysplasia of the photoreceptor "pd", and dog Briard "RPE-65" .The evaluation is carried out using behavioral tests, fluorescent angiography, histology (whether or not there is restoration of the photoreceptor and possibly ERG.) Functional testing will also be carried out, including phagocytosis (photoreceptor fragments), vitamin metabolism a A, conductivity of narrow junctions, and electron microscope.

EXAMPLE 8 DIRECT DIFFERENTIATION OF CELL RPE CELLS DERIVED FROM HUMAN EMBRYO CELLS Human embryos are placed in the blastocyst stage of development, in the presence of murine fibroblasts or chicken embryo with or without immunosurgery to eliminate the trofectoderm or directly on the tissue culture utensil coated with extracellular matrix protein. Instead of culturing and transforming the cells to produce a human ES cell line, the cells differentiate directly. When the cultures of hEDC cells are allowed to grow further in MEF in the absence of LIF, FGF and Plasmanato, these will form a thick multiple layer of cells. (Alternate growth factors, media and FBS can be used to alternate direct differentiation as knowledgeable by the subject know), approximately 6 weeks later, dark islands of cells will appear within the larger groups. These dark cells can be seen more easily with the naked eye and are seen as "freckles" on a cell plate, as shown in Figure 5B. With a higher magnification, these islands resemble polygonal cells packed tightly in a cobble monolayer, typical of epithelial cells, with brown pigment in the cytoplasm (Figure 5A). There are differences in the amount of pigment in the cells with the cells in the central part of the islands that have most of the pigment, and in those that are close to the edges that have less (figure 5B). When hEDC cells are directly differentiated, although they typically do not, they can form embryoid bodies (EB). Pigmented epithelial cells appear in approximately 1 to 2% of these differentiated cells and / or EB in the first 6 to 8 weeks. Over time, more and more pigmented cells develop EB, and in 3 months almost all EBs had a pigmented epithelial region. The morphology of the cells in the pigmented regions of EB was very similar to that of the adherent cultures.

MATERIALS AND METHODS Medium MEF: DMEM high in glucose, supplemented with 2 mM of GlutaMAXI, and 500 u / mL of penicillin, 500 ug / mL of streptomycin (all of Invitrogen), and 16% of FCS (HyCLone). Growth medium for hES cells: DMEM high in glucose for knockout supplemented with 500 u / mL of penicillin, 500 ug / mL streptomycin, 1% solution of non-essential amino acids, 2 mM GlutaMAX I, 0.1 mM beta-mercaptoethanol, 4 ng / mL bFGF (Invitrogen), 1 ng-mL human LIF (Chemicon, Temecula, CA), 8.4% serum replacement (SR, Invitrogen) , and 8.4% of plasmanate (Bayer). The derivation medium contained the same components as the growth medium, except that it had a lower concentration of SR and plasmanate (4.2% each) and 8.4% FCS and 2x concentration of human LIF and bFGF, compared to the Growth medium. Medium EB: same as the growth medium, except for bFGF, LIF and plasmanate; the SR concentration was 13%. RPE medium: 50% of EB medium and 50% of MEF medium.

CELLULAR LINES The hES 35, 36, 45 cell lines used for these studies were derived with modifications of previously reported procedures (Thomson and co-investigators, 1998, Reubinoff and co-investigators, 2000, Lanzendorf and co-investigators, 2001). The frozen human blastocysts (line hES35) or divided embryos (lines hES36 and hES45), were donated for the study, approved by two institutional review committees, by couples who had finished their fertility treatment. The differentiation experiments were performed with adherent hES cells or with embryoid bodies (EB). For adherent differentiation, the hES cells were allowed to grow in excess in MEF until the hES colonies lost their narrow borders, at which time the culture medium was replaced with EB medium (usually, 8 to 10 days after the transformation). The medium was changed every 1 to 2 days. For EB formation, hES cells were trypsinized and cultured in EB medium in plates with low adherence (Costar).

INM U NODETECTION Cells were fixed with 2% formaldehyde, permeabilized with 0.1% NP40 for localization of intracellular antigens, and blocked with 10% goat serum, 10% donkey serum (Jackson I mmunoresearch Laboratories, West Grove, PA) in PBS (I nvitrogen) for at least one hour. Incubation with primary antibodies was carried out overnight at 4 ° C, secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) were added for one hour. Among all incubations, samples were washed with 0.1% Tween-20 (Sigma) in PBS 3 to 5 times, 10 to 15 minutes each wash. Samples were mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA), and observed under a fluorescent microscope (N ikon). Alkaline phosphatase localization was performed either by Vector Red (Vector Laboratories, West Burlingame, CA) for live cells, or after the second washing during immunodetection according to the manufacturer's instructions. Antibodies used: bestrophin (Novus Biologicals, Littleton, CO), the anti-CRALBP antibody was a generous obseqio from Dr. Saari, University of Washington. Secondary antibodies were from Jackson Immunoresearch Laboratories, and Streptavidin-FITC was purchased at Amersham.

ISOLATION AND TRANSFORMATION OF SIMILAR CELLS TO THE PE RPE Adherent cultures of hES or EB cells were rinsed with PBS twice and incubated in 0.25% Trypsin / 1 mM EDTA (Invitrogen) at 37 ° C until the monolayer was loosened. The cells of the pigmented regions were detached by scraping with a glass capillary, transferred to MEF medium, centrifuged at 200X g, and placed in plates on gelatin-coated plates in RPE medium. The medium was changed after the cells were attached (usually in 1 to 2 days = and every 5 to 7 days after that, the cells were passed every 2 to 4 weeks with 0.05% Trypsin / 0.53 mM EDTA (Invitrogen ).

WESTERN DETECTION AND ELISA Samples were prepared in Laemmii regulator (Laemmii, 1970), supplemented with 5% mercaptoethanol and protease inhibitor cocktail (Roche), boiled for 5 minutes and loaded on a gradient gel of 8 to 16% (BioRad, Hercules, CA ), using a Mini-Protean device; the gels were run at 25-30 mA per gel; Proteins were transferred to a nitrocellulose 0.2 membrane (Schleicher and Shull, Keene, NH) at 20 volts overnight. The spots were stained briefly with Ponceau Red (Sigma) to visualize the bands, washed with Milli-Q water, and blocked for 1 hour with 5% dry milk without fat in 0.1% of TBST (Bio-Rad). The primary antibodies for bestrophin, CRALBP or PD F (Chemicon) were added for 2 hours followed by three 15 minute washes with TBST; secondary antibodies conjugated with peroxidase were added for 1 hour, and the washings were repeated. The spots were detected using the ECL system with Super-Signal reagent (Pierce). PEDF ELISA was performed on cell lysates using the PEDF ELISA kit (Chemicon) according to the manufacturer's procedure.

RT-PCR IN TI EMPO REAL Total RNA from ES cultures was purified in differentiation by a two-step procedure. Crude RNA was isolated using Trizol reagent (Invitrogen) and further purified in RNeazy minicolumns (Qiagen). The RPE65 transcript levels were monitored by real-time PCR using a commercial sensitizer set for the RPE65 (Demand on Demand # Hs00165642_ml, Applied Biosystems) and Quantitect Probe (Qiagen) RT-PCR reagents according to the established procedure. by the manufacturer (Qiagen).

TRAINING AND CHARACTERIZATION OF LINE OF CELLS HES NO DI FERENCIADAS In these studies two female and one male hES cell lines were used. The details of the transformation of these h ES lines are reported elsewhere. All lines had been passed more than 50 times, during which time they maintained an undifferentiated colony morphology, high alkaline phosphatase activity, and express Oct-4, SSEA-3, SSEA-4, TRA 1-60, and TRA 1-81 (the data is not shown). Two lines have normal karyotype (hES36, h ES35), while there were normal and aneuploid subpopulations in hES45. With spontaneous differentiation both in vitro and in teratomas, all the lines expressed the markers of three germ layers - muscle actin, alpha-fetoprotein, and tubulin beta l l l.

EJ EMPLO 9 USE OF PE TRANSC RIPTO GENOMICS TO IPENTIFY NORMAL CELLS PIFERENCIAPAS TRANSFORMAPAS EX VIVO The transcriptomic hES cell derivatives probably play an important role in the future of regenerative medicine. The qualitative evaluation of these and other stem cell derivatives remains a challenge that could be addressed using functional genomics. We compared the transcripclonal profile of hES-RPE against its counterpart in vivo, fetal RPE cells, which have been extensively investigated with respect to their transplant value. Then, both profiles were compared with previously published transcriptomic data (Rogojina and co-investigators, 2003) in human RPE cell lines. The gene expression profile of our data set was compared to two human RPE cell lines (untransformed ARPE-19 and transformed D407, Rogojina and coinvestigators, 2003) to determine if hES-RPE have similar global transcriptional profiles. To account for common household genes expressed in all cells, we used publicly available Affymetrix datasets from undifferentiated hES cells (line H1, h1-hES, SAto et al., 2003) and bronchial epithelial cells (BE, Wright and coinvestigators, 2004) as a control based on its common epithelial origin that could allow the exclusion of common house and epithelial genes and identify genes specific for RPE. There were similarities and differences between hES-RPE, hES-RPE-TD, ARPE-19, D407. The similutudes were further demonstrated by analyzing the exclusive intersection between those genes present in hES-RPE / ARPE-19, but not in BE (1026 genes). To count as antecedent, we compared this with the exclusive intersection of genes present in BE / hES-RPE, but not in ARPE-19 (186 genes), which results in a similarity of five to six times greater in hES-RPE and in ARPE-19 when compared to BE. C407 / ARPE19 appear to lose specific genes for RPE such as RPE65, bestrophin, CRALBP, PEDF, which is typical of long-term transformed cells (Figure 6). Additional data mining revealed specific ontologies for known RPEs, such as melanin biosynthesis, vision, retinyl binding, only in fetal RPE and in ES-RPE, but not in ARPE19. The comparison of hES-RPE, ARPE-19 and D407 with its in vivo counterpart, recently isolated human fetal RPE (feRPE) was in agreement with our previous data, demonstrating that the transcriptional identity of hES-RPE for feRPE is significantly greater than the of D407 for feRPE (difference of 2.3 times - 849 genes / 373 genes) and that of ARPE-19 for feRPE (difference of 1.6 times - 588 genes / 364 genes (figure 5c / 5d) .The specific markers for RPE identified above, which were present only in hES-RPE and not in ARPE-19 or in D407 were also present in feRPE, demonstrating a greater similutud of hES-RPE with its in vivo counterpart than that of cultivated RPE lines. Seven hundred and eighty-four genes present in hES-RPE were absent in the feRPE and ARPE data sets. Since the retention of originating genes could potentially result in the transformation of hES derivatives into malignant teratomas if transplanted into patients, we create genetic data with conservative "origin" potential using currently available Affymetrix microarray data sets. Abeyta and co-researchers, 2004. Sato, 2003). This resulted in a list of 3806 genes present in all sets of 12 data (including common household genes). Only 36 of the 784 genes present in the hES-RPE data, but not in feRPE-ARPE-19 were common with the 3806 potential originating genes. None of these original genes were known, such as Oct4, Sox2, TDGF1.

EXAMPLE 10 USE OF RPE CELLS FOR THE TREATMENT OF PARKINSON'S DISEASE HRPE can be used as an alternative source of cells for the cell therapy of Parkinson's disease because they secrete L-DOPA. Studies have shown that these single cells to gelatin coated microcarriers can be successfully transplanted into hemiparkinsonian monkeys and produce remarkable improvements (10-50 thousand cells per target), and in the FDA approved test initiated in 2000, patients received transplants from intraestriatal hRPE without adverse effects. One of the many advantages of using the RPE from hES cells is that it circumvents the shortage of ocular donor tissue. It also facilitates the use of gene therapy.

OTHER MODALITIES From the foregoing description, it will be evident that variations and modifications can be made to the invention described herein, to adapt it for different uses and conditions.

Claims (18)

1. A method for treating or preventing retinal degeneration, comprising the use of a cell selected from the group consisting of at least one of: RPE cells, cells similar to those of RPE or progenitors similar to RPE from mammalian embryonic stem cells . The method of claim 1, further characterized in that the condition of retinal degeneration is selected from the group consisting of at least one of: retinitis pigmentosa and macular degeneration. 3. The method of claim 1, further comprising transplantation of the cell by vitrectomy surgery into the subretinal space of the eye. 4. The method of claim 3, further characterized in that the cells are transplanted into a suspension, matrix, or substrate. The method of claim 2, further characterized in that retinitis pigmentosa is associated with an animal model. 6. The method of claim 5, wherein in the animal model it is selected from the group consisting of: rd mouse, RPE-65 knockout mouse, Tubby protein-like mouse, RCS rat, Abyssinian cat, dog with cone degeneration. cd ", dog with progressive degeneration of sticks and cones" prcd ", dog with early retinal degeneration" erd ", dogs with dysplasia 1, 2 and 3 of canes and cones, dogs" redi, rcd2 and rcd3", dog with dysplasia of the photoreceptor "pd", and dog Briard "RPE-65". The method of claim 6, further characterized in that the result of the therapy in the animal model is evaluated using one or more behavioral tests, fluorescent angiography, histology, and functional test such as measuring the ability of the cells to perform phagocytosis (photoreceptor fragments), vitamin A metabolism, tight junction conductivity, or evaluation using an electron microscope. 8. A method for the spontaneous differentiation of hES cells into RPE cells, similar to those of RPE, or progenitor cells of RPE, said method comprises: a) allowing the cultures of hES cells to grow in excess over MEF; b) allow the cultures of hES cells to form a thick multiple layer of cells; c) culturing the hES cells; d) isolation and culture of the RPE cells, similar to those of RPE, and / or pigmented RPE progenitors of the resulting cell cultures. The method of claim 8, further characterized in that the isolation and culture of RPE-like cells comprises: a) digesting the cultured hES cells or EB with an enzyme; b) selectively isolate the pigmented cells; c) plating the isolated cells on gelatin or laminin for 1 to 2 days to form primary cultures (PO); d) continue the cultivation of the primary culture for a period of up to 3 weeks; and e) isolating cells similar to those of RPE. The method of claim 9, further characterized in that the enzyme is selected from the group consisting of one or more of trypsin, collagenase, and dispase. The method of claim 8, further characterized in that the RPE cells are cultured to establish a new RPE cell line. The method of claim 11, further characterized in that the RPE cell line is differentiated into alternative lineages comprising the treatment of the RPE cell line in culture with bFGF or FGF. The method of claim 11, further characterized in that the new RPE cell lines vary from the RPE cell lines already established in at least one of the selected characteristics of the group consisting of: growth rate, pigment expression , de-differentiation in culture, and re-differentiation in culture, or cells similar to those of RPE when they come from different ES cell lines. 14. A method for the derivation of RPE lines or precursors of RPE cells that have an increased capacity to prevent neovascularization, said method comprises: a) maturing a somatic cell of an animal, in such a way that the telomerase decreases, where at least 10% of the amplitude of normal reproductive life of the cell has elapsed; and b) using the somatic cell as a donor cell for nuclear transfer in order to create cells that overexpress angiogenesis inhibitors, wherein the angiogenesis inhibitor can be a Pigmented Epithelium Derivative Factor (PEDF / EPC-1). 15. The method of claim 14, further characterized in that somatic cells are genetically modified with exogenous genes that inhibit neovascularization. 16. The method of claim 8, further characterized in that the cells similar to those of the RPE come from a bank of ES cells or embryo origin cells with homozygosity in the HLA region, such that ES cells have reduced complexity of its HLA antigens. 17. The method of claim 8, further characterized in that the ES cells come from a human being. 18. A method for the treatment of the disease of Parkinson's, which involves the transplantation of cells similar to those of the RPE or progenitor cells.