WO2005070011A2 - Improved modalities for the treatment of degenerative diseases of the retina - Google Patents
Improved modalities for the treatment of degenerative diseases of the retina Download PDFInfo
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Definitions
- This invention relates generally to methods for improved cell-based therapies for retinal degeneration and other visual disorders as well as treatment of Parkinson's disease and for differentiating mammalian embryonic stem cells and mammalian embryo-derived cells into retinal pigment epithelium (RPE) cells and other eye tissue including, but not limited to) rods, cones, bipolar, comeal, neural, iris epithelium, and progenitor cells.
- RPE retinal pigment epithelium
- CNS central nervous system
- ASD age-related macular degeneration
- RPE retinal pigment epithelium
- INL intemuncial
- Restoration of moderate-to-high acuity vision therefore, requires the functional replacement of some or all of the damaged cellular layers.
- retinitis pigmentosa a family of inherited retinal degenerations
- RP retinitis pigmentosa
- the underlying cellular mechanisms are diverse and can result from various mutations in many genes. Most involve mutations that alter the expression of photoreceptor-cell-specific genes, with mutations in the rhodopsin gene accounting for approximately 10% of these.
- the regulatory genes of apoptosis are altered (for example, Box and Pax2). AMD is a clinical diagnosis encompassing a range of degenerative conditions that likely differ in etiology at the molecular level.
- the retinal pigment epithelium is one of the most important cell types in the eye, as it is crucial to the support of the photoreceptor function. It performs several complex tasks, including phagocytosis of shed outer segments of rods and cones, vitamin A metabolism, synthesis of mucoploysacharides involved in the metabolite exchange in the subretinal space, transport of metabolites, regulation of angiogenesis, absorption of light, enhancement of 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 result in a condition known as choroidal neovascularization (CNN).
- CNN choroidal neovascularization
- This neovascular (“wet”) form of AMD is particularly destructive and seems to result from a loss of proper regulation of angiogenesis. Breaks in Bruch's membrane as a result of RPE dysfunction allows new vessels from the choroidal circulation access to the subretinal space, where they can physically disrupt outer-segment organization and cause vascular leakage or hemorrhage leading to additional photoreceptor loss. C ⁇ N can be targeted by laser treatment. Thus, laser treatment for the "wet" form of AMD is in general use in the United States. There are often undesirable side effects, however, and therefore patient dissatisfaction with treatment outcome.
- RPE plays an important role in photoreceptor maintenance, and regulation of angiogenesis
- various RPE malfunctions in vivo are associated with vision-altering ailments, such as retinitis pigmentosa, RPE detachment, displasia, atlrrophy, retinopathy, macular dystrophy or degeneration, including age-related macular degeneration, which can result in photoreceptor damage and blindness.
- the variety of other degenerative conditions affecting the macula include, but are not limited to, cone dystrophy, cone-rod dystrophy, malattia leventinese, Doyne honeycomb dystrophy, Sorsby's dystrophy, Stargardt disease, pattern/butterfly dystrophies, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, and toxic maculopathies.
- General retinal diseases that can secondarily effect the macula include retinal detachment, pathologic myopia, retinitis pigmentosa, diabetic retinopathy, CMV retinitis, occlusive retinal vascular disease, retinopathy of prematurity (ROP), choroidal rupture, ocular histoplasmosis syndrome (POHS), toxoplasmosis, and Leber's congenital amaurosis. None of the above lists is exhaustive. All of the above conditions involve loss of photoreceptors and, therefore, treatment options are few and insufficient. Because of its wound healing abilities, RPE has been extensively studied in application to transplantation therapy. In 2002, one year into the trial, patients were showing a 30-50% improvement.
- RPE cells derived from fetal tissue are another problem, as these cells have shown a very low proliferative potential.
- Emory University researchers performed a trial where they cultured RPE cells from a human eye donor in vitro and transplanted them into six patients with advanced Parkinson's Disease. Although a 30-50% decrease in symptoms was found one year after transplantation, there is a shortage of eye donors, this is not yet FDA approved, and there would still exist a need beyond what could be met by donated eye tissue.
- therapies using ectopic RPE cells have been shown to behave like fibroblasts and have been associated with a number of destructive retinal complications including axonal loss (Villegas-Perez, et.
- RPE delivered as a loose sheet tends to scroll up. This results in poor effective coverage of photoreceptors as well as a multilayered RPE with incorrect polarity, possibly resulting in cyst formation or macular edema.
- Delivery of neural retinal grafts to the subretinal (submacular) space of the diseased human eye has been described in Kaplan et. al. (1997), Humayun et. al. (2000), and del Cerro et. al. (2000). A serious problem exists in that the neural retinal grafts typically do not functionally integrate with the host retina.
- 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 and RPE-like cells from stem cells and to provide improved methods and therapies for the treatment of retinal degeneration, h 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 of generating cells for therapy for retinal degeneration using RPE cells, RPE-like cells, the progenitors of these cells or a combination of two or three of any of the preceding derived from mammalian embryonic stem cells in order to treat various conditions including but not limited to retinitis pigmentosa and macular degeneration and associated conditions.
- the cell types which can be produced using this invention include, but are not limited to, RPE, RPE-like cells, and RPE progenitors. Cells which may also be produced include iris pigmented epithelial (IPE) cells.
- Vision associated neural cells including intemuncial neurons (e.g.
- retinal neurons of the inner nuclear layer (INL)) and amacrine cells interneurons that interact at the second synaptic level of the vertically direct pathways consisting of the photoreceptor-bipolar- ganglion cell chain - they 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
- IPL inner plexiform layer
- retinal cells, rods, cones, and comeal cells can be produced.
- cells providing the vasculature of the eye can also be produced.
- the cells of the present invention may be transplanted into the subretinal space by using vitrectomy surgery.
- Non-limiting examples include the transplantation of these cells in a suspension, matrix, or substrate.
- Animal models of retinitis pigmentosa that may be treated include rodents (rd mouse, RPE-65 knockout mouse, tubby-like mouse, RCS rat, cats (Abyssinian cat), and dogs (cone degeneration "cd” dog, progressive rod-cone degeneration "prcd” dog, early retinal degeneration "erd” dog, rod-cone dysplasia 1, 2 & 3 "rcdl, rcd2 & rcd3" dogs, photoreceptor dysplasia "pd” dog, and Briard "RPE-65” (dog).
- Evaluation is performed using behavioral tests, fluorescent angiography, histology, or functional testing such as measuring the ability of the cells to perform phagocytosis (photoreceptor fragments), vitamin A metabolism, tight junctions conductivity, or evaluation using electron microscopy.
- phagocytosis photoreceptor fragments
- vitamin A metabolism vitamin A metabolism
- tight junctions conductivity or evaluation using electron microscopy.
- One of the many advantages to the methods presented here is the ability to produce and treat many more patients than it would be possible to treat if one were limited to using eye donor tissue.
- a further embodiment of the present invention provides methods for the spontaneous differentiation of hES cells into cells with numerous characteristics of RPE. These RPE preparations are capable of phenotypic changes in culture and maintaining RPE characteristics through multiple passages.
- the present invention also provides for methods of differentiation of established RPE cell lines into alternate neuronal lineages, comeal cells, retinal cells as a non-limiting example through the use of bFGF or FGF.
- Another embodiment of the present invention is a method for the derivation of new RPE lines and progenitor cells from existing and new ES cell lines.
- properties such as growth rate, expression of pigment, or de- differentiation and re-differentiation in culture, of RPE-like cells when they are derived from different ES cell lines.
- Cells which may also be derived from existing and new ES cell lines include iris pigmented epithelial (IPE) cells.
- vision associated neural cells including intemuncial neurons (e.g. "relay" neurons of the inner nuclear layer (INL)) and amacrine cells can also be produced using this invention. Additionally, retinal cells, rods, cones, and comeal cells can be produced.
- cells providing the vasculature of the eye can also be produced.
- Another embodiment of the present invention is a method for the derivation of RPE lines or precursors to RPE cells that have an increased ability to prevent neovascularization.
- Such cells can be produced by aging a somatic cell from a patient such that telomerase is shortened where at least 10% of the normal replicative lifespan of the cell has been passed, then the use of said somatic cell as a nuclear transfer donor cell to create cells that overexpress angiogenesis inhibitors such as Pigment Epithelium Derived Factor (PEDF/EPC-1).
- angiogenesis inhibitors such as Pigment Epithelium Derived Factor (PEDF/EPC-1).
- PEDF/EPC-1 Pigment Epithelium Derived Factor
- such cells may be genetically modified with exogenous genes that inhibit neovascularization.
- an additional embodiment of the present invention includes the characterization of ES-derived RPE-like cells.
- the ES-derived pigmented epithelial cells strongly resemble RPE by their morphology, behavior and molecular markers, their therapeutic value will depend on their ability to perform RPE functions and to remain non-carcinogenic. Therefore, the ES-derived RPE cells are characterized using one or more of the following techniques: (i) assessment of their functionality, i.e.
- RPE-like ES cells derivatives through animal model transplantations, (as a non-limiting example this can include SCID mice);
- phenoytping and karyotyping of RPE-like cells (iv) evaluation of ES cells-derived RPE-like cells and RPE tissue by gene expression profiling, (v) evaluation of the expression of molecular markers of RPE at the protein level, including bestrophin, CRALBP, RPE-65, PEDF.
- the cells can also be evaluated based on their expression of transcriptional activators normally required for the eye development, including rx/rax, chxlO/vsx-2/alx, ots-1, otx-2, six3/optx, six6/optx2, mitf, pax6/mitf, and pax6/pax2 (Fischer and Reh, 2001, Baumer et. al., 2003).
- An additional embodiment of the present invention is a method for the characterization of ES-derived RPE-like cells using at least one of the techniques selected from the group consisting of (i) assessment of the ES-derived RPE-like cells functionality; (ii) evaluation of the pluripotency of RPE-like ES cell derivatives through animal model transplantations; (iii) phenoytping and karyotyping of RPE- like cells; (iv) evaluation of gene expression profiling, (v) evaluation of the expression of molecular markers of RPE at the protein level; and (vi) the expression of transcriptional activators normally required for the eye development.
- these techniques may be used for the assessment of multiple hES cell- derived cell types.
- Another embodiment of the present invention is a method for the derivation of RPE cells and RPE precursor cells directly from human and non-human animal morula or blastocyst-staged embryos (EDCs) without the generation of ES cell lines.
- Embryonic stem cells ES
- hES human embryonic stem cells
- hES human embryonic stem cells
- Another embodiment of the present invention is a method of identifying cells such as RPE cells, hematopoietic cells, muscle cells, liver cells, pancreatic beta cells, neurons, endothelium, progenitor cells or other cells useful in cell therapy or research, derived from embryos, embryonic stem cell lines, or other embryonic cells with the capacity to differentiate into useful cell types by comparing the messenger RNA transcripts of such cells with cells derived in- vivo.
- This method facilitates the identification of cells with a normal phenotype and for deriving cells 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 pigment epithelium (RPE).
- RPE retinal pigment epithelium
- RPE is a densely pigmented epithelial monolayer between the choroid and neural retina. It serves as a part of a barrier between the bloodstream and retina, and it's functions include phagocytosis of shed rod and cone outer segments, absorption of stray light, vitamin A metabolism, regeneration of retinoids, and tissue repair. (Grierson et. al, 1994, Fisher and Reh, 2001, Marmorstein et. al, 1998). The RPE is easily recognized by its cobblestone cellular morphology of black pigmented cells.
- RPE retinaldehyde-binding protein
- CRALBP retinaldehyde-binding protein
- RPE65 a cytoplasmic protein involved in retinoid metabolism (Ma et. al., 2001, Redmond et. al., 1998); bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2, Marmorstein et. al, 2000), and pigment epithelium derived factor (PEDF) a 48kD secreted protein with angiostatic properties (Karakousis et. al., 2001, Jablonski et.
- CRALBP retinaldehyde-binding protein
- VMD2 Best vitelliform macular dystrophy gene
- PEDF pigment epithelium derived factor
- RPE cells are normally mitotically quiescent, but can begin to divide in response to injury or photocoagulation. RPE cells adjacent to the injury flatten and proliferate forming a new monolayer (Zhao et. al, 1997). Several studies have indicated that the RPE monolayer can produce cells of fibroblast appearance that can later revert to their original RPE morphology (Grierson et. al., 1994, Kirchhof et. al., 1988, Lee et. al, 2001). It is unclear whether the dividing cells and pigmented epithelial layer are from the same lineage as two populations of RPE cells have been isolated: epithelial and fusiforms.
- RPE can be maintained as an epithelium or rapidly dedifferentiate and become proliferative (Zhao 1997, Opas and Dziak, 1994). Interestingly, the epithelial phenotype can be reestablished in long-term quiescent cultures (Griersion et. al., 1994). In mammalian development, RPE shares the same progenitor with neural retina, the neuroepithelium of the optic vesicle. Under certain conditions, it has been suggested that RPE can transdifferentiate into neuronal progenitors (Opas and Dziak, 1994), neurons (Chen et.
- bFGF a process associated with the expression of transcriptional activators normally required for the eye development, including rx/rax, chxlO/vsx-2/alx, ots-1, otx-2, six3/optx, six6/optx2, mitf, and pax6/pax2 (Fischer and Reh, 2001, Baumer et. al, 2003).
- the present invention provides for the derivation of trabecular meshwork cells from hES and also for genetically modified trabecular meshwork cells for the treatment of glaucoma.
- the present invention also provides for the derivation of trabecular meshwork cells from RPE progenitors and RPE-like cells and also for genetically modified trabecular meshwork cells for the treatment of glaucoma.
- the present invention includes methods for the derivation of RPE cells and RPE precursor cells directly from human and non-human animal morula or blastocyst-staged embryos (EDCs) without the generation of ES cell lines, comprising a) maintaining ES cells in vitro in an undifferentiated state; b) differentiating the ES cells into RPE and RPE precursor cells; and, c) identifying cells the RPE cells by comparing the messenger RNA transcripts of such cells with cells derived in- vivo.
- EDCs blastocyst-staged embryos
- telomere lines or precursors to RPE cells that have an increased ability to prevent neovascularization
- said methods comprising: a) aging a somatic cell from an animal such that telomerase is shortened wherein at least 10% of the nonnal replicative lifespan of the cell has been passed; and, b) using the somatic cell as a nuclear transfer donor cell to create cells that overexpress angiogenesis inhibitors, wherein the angiogenesis inhibitors can be Pigment Epithelium Derived Factor (PEDF/EPC-1).
- PEDF/EPC-1 Pigment Epithelium Derived Factor
- Figure 1A is a photograph of pigmented regions in a 2.5 month old adherent culture, a well of a 6-well plate, scanned;
- Figure IB is a photograph of pigmented regions in a 2.5 month old cultured grown in EB, at 45x magnification;
- Figure IC is a photograph of a pigmented area of an adherent culture;
- Figure ID is a photograph of a pigmented region of an EB grown culture;
- Figure IE is a photograph of the boundary between pigmented region and the rest of the culture, x200;
- Figure F same as Figure E but at x400 magnification. Arrows in A and B point to pigmented regions Figure 2A-F.
- FIG. 2A is a photograph showing primary EB outgrowth, 1 week
- Figure 2B is a photograph showing the primary culture of cells, isolated by trypsin, 1 week
- Figure 2C is a photograph showing epithelial islet surrounded by proliferating cells
- Figure 2D is a photograph showing the regain of pigmentation and epithelial morphology in 1 month old culture
- Figure 2E is a photograph showing the culture after 3 passages, x200 magnification
- Figure 2F shows the same culture as in E, x400 magnification, Hoffman microscopy. Black arrows point to pigmented cells, white arrows show outgrowing cells with no pigment.
- Figure 3 Left Panel (A-D) and Right Panel is a series of photographs and one graph - these show markers of RPE in hES cells-derived pigmented epithelial cells.
- Figures 3 A and 3B are photographs showing immunolocalization of RPE marker, bestrophin and corresponding phase microscopy field, x200 magnification;
- Figures 3C and 3D are photographs showing CRALBP and corresponding phase contrast microscopy field, x400 magnification.
- FIG. 1 Shows the colocalization of bestrophin (A) and CRALBP (C) to pigmented cells (C,D); arrowheads point to the absence of staining for these proteins (A,B) in non-pigmented regions (C,D)
- Figure 3 shows a photograph and graph of western blot of cell lysates (line hES #36) with antibodies to bestrophin (a) and CRALBP (b); c,d - undifferentiated hES cells , c ⁇ control to anti-CRALBP antibody, d — control to anti- bestrophin antibody
- Figure 4 shows photographs which demonstrate the expression of markers of
- Pax6 (Figure 4A), Pax2 (Figure 4E) and mitf (Figure 4B, Figure 4F) in RPE-like cells in long-term quiescent cultures.
- Figure 4C Figure 4G - phase contrast
- Figure 4D Figure 4H - merged images of Pax6/mitf phase contrast
- Figure 4 A Figure 4B, Figure 4C
- Figure 4E Figure 4F, Figure 4G
- Figure 5A-B show photographs of RPE differentiation in the culture of human embryo-derived cells: bypassing the stage of derivation of ES cell lines.
- Figure 6 shows the transcriptional comparison of RPE preparations.
- FIG. 6A-F Based on the Ontological annotation, this table represents the expression patterns of RPE related genes for hES cell-derived retinal pigment epithelium (hES- RPE), hES cell derived transdifferentiated (hES-RPE-TD), ARPE- 19 and D407, and freshly isolated human RPE (fe-RPE).
- Figure 6G Further data mining revealed known RPE specific ontologies, such as melanin biosynthesis, vision, retinol- binding, only in fetal RPE and ES-RPE but not ARPE- 19.
- embryo or “embryonic” is meant a developing cell mass that has not implanted into 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 the two-cell stage, and aggregated blastomeres.
- embryo-derived cells refers to embryo-derived cells.
- hES cells human embryonic stem cells
- hES human embryo-derived cells
- hES refers to cells isolated from the inner cell mass of human blastocysts or morulae and that have been serially passaged as cell lines and can also include blastomeres and aggregated blastomeres.
- human embryo-derived cells refers to morula-derived cells, blastocyst-derived cells including those of the inner cell mass, embryonic shield, or epiblast, or other totipotent or pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm, and mesoderm and their derivatives, also including blastomeres and cell masses from aggregated single blastomeres or embryos from varying stages of development, but excluding human embryonic stem cells that have been passaged as cell lines.
- Embryonic stem (ES) cells which have the ability to differentiate into virtually any tissue of a human body can provide a limitless supply of rejuvenated and histocompatible cells for transplantation therapy, as the problem of immune rejection can be overcome with nuclear transfer and partheno genetic technology.
- the recent findings of Hirano et. al. (2003) have shown that mouse ES cells can produce eye-like structures in differentiation experiments in vitro. Among those, pigmented epithelial cells were described, resembling retinal pigment epithelium. Preliminary experiments carried out at Advanced Cell Technology with primate and human ES cell lines show that a in a specialized culture system these cells differentiate into RPE-like cells that can be isolated and passaged.
- RPE Human and mouse NT, Cyno parthenote ES cell derivatives have multiple features of RPE: these pigmented epithelial cells express four molecular markers of RPE - bestrophin, CRALBP, PEDF, and RPE65; like RPE, their proliferation in culture is accompanied by dedifferentiation - loss of pigment and epithelial morphology, both of which are restored after the cells form a monolayer and become quiescent.
- RPE-like cells can be easily passaged, frozen and thawed, thus allowing their expansion.
- the inventors have further shown that human ES cells also produce multiple eye (vitreous body)-like structures in differentiation experiments in vitro.
- RPE Transplantation At present, chronic, slow rejection of the RPE allo grafts prevents scientists from determining the therapeutic efficacy of this RPE transplantation.
- 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 patient's own RPE, i.e. homografts, but this has the drawback of using old, diseased RPE to replace even more diseased RPE.
- a third approach is to use iris epithelium (IPE) from the same patient but this has the drawback that IPE may not perform all the vision related functions of RPE.
- IPE iris epithelium
- Nuclear transfer and parthenogenesis facilitate histocompatibility of grated RPE cells and progenitors.
- RPE defects in Retinitis Pigmentosa Retinitis pigmentosa is a hereditary condition in which the vision receptors are gradually destroyed through abnormal genetic programming. Some forms cause total blindness at relatively young ages, where other forms demonstrate characteristic "bone spicule" retinal changes with little vision destruction. This disease affects some 1.5 million people worldwide.
- One of the patients improved from barely seeing light to being able to count fingers held at a distance of about six feet from the patient's face.
- vision improved to ability to see letters through tunnel vision.
- the transplants in these studies were performed by injection, introducing the new retinal cells underneath the existing neural retina. Not all of the cells survived since the transplanted fetal cells were allogeneic (i.e. not genetically- matched), although those that did survive formed comiections with other neurons and begin to function like the photoreceptors around them. Approximately a year after the first eight people received the transplants, four have recovered some visual function and a fifth shows signs of doing so.
- Three newly derived human embryonic stem cell lines are similar in properties to those described earlier (Thomson et. al. 1998, Reibunoff et. al., 2000, Richards et. al., 2000, Lanzendorf et. al., 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 through 45 passages in culture or over 130 population doublings. All hES cell lines differentiate into derivatives of three germ layers in EB or long term adherent cultures and in teratomas.
- hES cells are similar to retinal pigment epithelium by the following criteria: morphologically, they have a typical epithelial cobblestone monolayer appearance and contain dark brown pigment in their cytoplasm, which is known to be present in the human body only in melanocytes, keratinocytes, retinal and iris pigment epithelium (IPE). Melanocytes, however, are non-epithelial cells, and keratynocytes don't secrete but only accumulate melanin. The set of RPE- specific proteins — bestrophin, CRALBP, PEDF - present in these cells indicates that they are likely to be similar to RPE and not IPE.
- the inventors have observed the same pattern of tubulin beta III localization in primary and passaged cultures of RPE and RPE-like cells which can reflect a dedifferentiation of such cells in culture or indicate a separate population of cells committed to a neuronal fate, that were originally located next to pigmented cells through differentiation of hES cells in long-term cultures and could have been co- isolated with RPE-like cells.
- RPE and the neural retina share the same bipotential neuroepithelial progenitor, and their fate was shown to be determined by Pax2, Pax6, and Mitf (Baumer et. al, 2003), the latter being a target of the first two.
- Pax6 at earlier stages acts as an activator of proneural genes and is downregulated in the RPE in further development, remaining in amacrine and ganglion cells in mature retina (reviewed by Ashery -Padan and Grass, 2001). In goldfish, it is also found in mitotically active progenitors of regenerating neurons (Hitchcock et. al., 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 that surround pigmented epithelial islands in long term cultures or in cells with a "partial" RPE phenotype (lightly pigmented and loosely packed).
- progenitors can in turn coexist with mature RPE-like cells in culture or appear as a result of dedifferentiation of RPE-like cells.
- cells of neural retina can transdifferentiate into RPE in vitro (Opas et.
- tubulin beta III and Pax6 positive cells could represent a transient stage of such transdifferentiation of co-isolated neural cells or neural progenitors into RPE-like cells.
- Differentiation of hES cells into RPE-like cells happened spontaneously when using methods described in the Examples below, and the inventors noticed that pigmented epithelial cells reliably appeared in cultures older than 6-8 weeks and their number progressed overtime — in 3-5 months cultures nearly every EB had a large pigmented region.
- six more newly derived hES lines turned into RPE-like cells, which suggests that since neural fate is usually chosen by ES cells spontaneously, RPE-like cells can arise by default as an advanced stage of such pathway.
- permissive and/or instructive differentiation signals come from extracellular matrix and growth factors produced by differentiating derivatives of hES cells.
- the model of differentiation of hES cells into RPE-like cells could be a useful tool to study how such microenvironment orchestrates RPE differentiation and transdifferentiation.
- RPE plays an important role in photoreceptor maintenance, and various RPE malfunctions in vivo are associated with a number of vision-altering ailments, such as RPE detachment, displasia, athrophy, retinopathy, retinitis pigmentosa, macular dystrophy or degeneration, including age-related macular degeneration, which can result in photoreceptor damage and blindness. Because of its wound healing abilities, RPE has been extensively studied in application to transplantation therapy. It has been shown in several animal models and in humans (Gouras et. al., 2002, Stanga et. al., 2002, Binder et. al., 2002, Schraermeyer et. al., 2001, reviewed by Lund et.
- Example 1 Spontaneous differentiation into pigmented epithelial cells in long term cultures When hES cell cultures are allowed to overgrow on MEF in the absence of
- LIF, FGF and Plasmanate they form a thick multilayer of cells.
- dark islands of cells appear within the larger clusters (Figl). These dark cells are easily seen with the naked eye and looked like "freckles" in a plate of cells as shown in Fig 1 A. At higher magnification these islands appear as tightly packed polygonal cells in a cobblestone monolayer, typical of epithelial cells, with brown pigment in the cytoplasm (Fig. IC).
- Fig 1, E,F There are differences in the amount of pigment in the cells with cells in the central part of the islands having the most pigment and those near the edges the least.
- EB embryoid bodies
- fig IB pigmented epithelial cells
- Morphology of the cells in the pigmented regions of EBs was very similar to that of adherent cultures (fig ID).
- Example 2 Isolation and culture of pigmented epithelial cells The inventors isolated pigmented epithelial cells from both adherent hES cell cultures and from EBs.
- Pigmented polygonal cells were digested with enzymes (trypsin, and/or collagenase, and/or dispase), and the cells from these pigmented islands were selectively picked with a glass capillary. Although care was taken to pick only pigmented cells, the population of isolated cells invariably contained some non-pigmented cells. After plating cells on gelatin or laminin for 1-2 days, the cells were considered to be primary cultures (P0). Primary cultures contained islands of pigmented polygonal cells as well as some single pigmented cells. After 3-4 days in culture, non-pigmented cells that seemed to have lost epithelial morphology (flatter and cells with lamellipodia) appeared at the periphery of some islands (fig.2).
- enzymes trypsin, and/or collagenase, and/or dispase
- the epithelial structure and cobblestone morphology are not shared by other pigmented cells, e.g. melanocytes. It is also noteworthy that RPE cells have been shown to lose and regain their pigment and epithelial morphology when grown in culture (Zhao 1997, Opas and Dziak, 1994), and the pigmented cells behaved in a similar manner, so to test the hypothesis that the ES derived cells may be RPE, they were stained with antibodies to known markers for RPE: bestrophin and CRALBP. Figure 4 (left panel) shows membrane localization of bestrophin (A) and CRALBP (C), both are found in pigmented epithelial islands.
- FIG. 4 Another known PRE marker, RPE65, was found in the RPE-like cells by real-time RT-PCR ( Figure 4, right panel, bottom), the PEDF ELISA assay showed the presence of PEDF in cell lysates of all presumed RPE cultures, and Western blot showed a band of approximately 48 kD (not shown).
- Figure 4 shows localization of PAX-6, Pax2, mitf, and tubulin beta III in recently passaged and old cultures of hES cells-derived RPE.
- tubulin beta III had a similar pattern of distribution (not shown).
- mitf- positive and Pax6-negative cells located close to the periphery of pigmented islands (figure 4, A-C).
- Pax2 was found only in a very small subset of mitf-negative cells (Figure 4, E-H). No presence of either of these proteins was ever detected in the cells of "mature" pigmented epithelial islands.
- these markers in cells that only had some RPE features were often visible, i.e. either looked epithelial but had no pigment or in certain single pigmented cells away from pigmented epithelial islands.
- Example 4 Characterization of RPE-like cells derived from hES cell lines H9 and ACT J-l from Cyno-1 ES cells and derivation of RPE-like cells from existing hES cell lines HI and H7.
- An RPE-like cell line is expanded, tested for freezing and recovery, and characterized using the following methods and molecular markers of RPE cells: bestrophin and CRALBP by Western blot and immunofluorescence, PEDF by ELISA and Western blot, and REP65 by RT-PCR.
- the cells are injected in SCID mice with undifferentiated hES or Cyno-1 cells as a control to evaluate tumorigenicity.
- Karyotyping of RPE-like cells will be done by a clinical laboratory on a commercial basis.
- RPE-like cells Characterization of the functional properties of RPE-like cells and studies of their transplantation potential are then carried out as otherwise described in this application and also using those techniques known to those skilled in the art. Gene expression profiling experiments are done using Affymetrix human genome arrays. Gene expression is compared in RPE-like cells derived from ES cells and in retinal samples from autopsies. Several animal models can be used to verify the effectiveness of the transplanted RPE-like cells, including but not limited to, rhesus monkey, rat, and rabbit.
- ES cells are cultured on feeder cells or as embryoid bodies (EB) in the presence of bFGF, insulin, TGF-beta, IBMX, bmp-2, bmp-4 or their combinations, including stepwise addition.
- EB embryoid bodies
- ES cells are grown on various extracellular matrix-coated plates (laminin, fibronectin, collagen I, collagen IV, Matrigel, etc.) in evaluating the role of ECM in RPE formation.
- RPE progenitors Pax6, Pax2, mitf
- CRALBP bestrophin, PEDF, REP65
- RPE progenitors Pax6, Pax2, mitf
- CRALBP bestrophin, PEDF, REP65
- This approach can also be used to produce common progenitors of RPE and other eye tissues, such as photoreceptor or neural retina which can be isolated and further characterized for their differentiation potential and used in transplantation studies.
- Example 6 Derivation of RPE and other eye tissue progenitors from existing and new ES cell lines.
- RPE progenitor markers will be correlated with the expression of the surface proteins in order to find a unique combination of surface markers for RPE progenitor cells. If such markers are found, antibodies to surface proteins can be used to isolate a pure population of RPE progenitors that can be then cultured and further differentiated in culture or used in transplantation studies to allow their differentiation after grafting. If the data from the gene expression profiling experiments is insufficient, to isolate the RPE progenitors the following approach will be used. ES cells and RPE- like cells will be transfected with GFP under the control of a Pax6 promoter, and stable transfectants will be selected.
- GFP/Pax6-positive cells will be isolated by FACS and used as an antigen source for mouse injection to raise monoclonal antibodies to the surface molecules of Pax6 positive cells. Because Pax6 is present not only in RPE progenitors, screening will be done (by FACS) using several strategies: a) against proliferating RPE-like cells, b) against Pax2-positive RPE cells, c) against mitf-positive RPE cells. For b) and c) RPE cells will be transfected with GFP under the corresponding promoter; as a negative control, RPE or ES cells negative by these antigens will be used.
- RPE-like cells Three ES cell lines that already produced RPE-like cells (H9, ACT J-l, Cyno-1), RPE-like cells will be used to continue to derive RPE-like cells and their progenitors as described in Aims 1 and 2, and HI and H7 hES cell lines will be used to produce new RPE-like cell lines. After expansion and characterization for molecular markers of RPE, these lines will be single-cloned, and the resulting lines will be characterized as described in Aim 1. The lines meeting criteria for RPE cells will be used for transplantation studies.
- New human ES cell lines will be derived from unused INF embryos, from donated oocytes, stimulated to develop without fertilization (parthenote), and from generated developing blastocysts obtained from donated oocytes with the application of nuclear transfer technology. RPE-like cells and common eye progenitors will be derived from these lines using the approach in Aim 2, and the resulting lines will be characterized as in Aim 1. [Optional] new human ES cell lines will be derived in a virus-free system, characterized and submitted for clinical trials.
- Example 7 Therapeutic potential of RPE-like cells and progenitors in various animal models of retinitis pigmentosa & macular degeneration.
- Primate ES cells are tested in cynomologus monkeys (Macaques). Initially, vitrectomy surgery is performed and the cells are 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 transplantation.
- Example 8 Direct differentiation of RPE cells from human embryo-derived cells. Human blastocyst-staged embryos are plated in the presence of murine or chick embryo fibroblasts with or without immunosurgery to remove the trophectoderm or directly plates on extracellular matrix protein-coated tissue cultureware. Instead of culturing and passaging the cells to produce a human ES cell line, the cells are directly differentiated.
- hEDC cell cultures When hEDC cell cultures are allowed to overgrow on MEF in the absence of LIF, FGF and Plasmanate, they will form a thick multilayer of cells. (Alternate growth factors, media, and FBS can be used to alternate direct differentiation as is known to those skilled in the art.) About 6 weeks later, dark islands of cells will appear within the larger clusters. These dark cells are easily seen with the naked eye and looked like "freckles" in a plate of cells as shown in Fig 5B. At higher magnification these islands appear as tightly packed polygonal cells in a cobblestone monolayer, typical of epithelial cells, with brown pigment in the cytoplasm (Fig. 5A).
- hEDC cells When hEDC cells are directly differentiated they may, though typically have not, fonned embryoid bodies (EB). Pigmented epithelial cells appear in about 1-2% of these differentiated cells and/or EBs in the first 6-8 weeks. Over time more and more EBs develop pigmented cells, and by 3 months nearly every EB had a pigmented epithelial region. Morphology of the cells in the pigmented regions of EBs was very similar to that of adherent cultures.
- MEF medium high glucose DMEM, supplemented with 2mM GlutaMAX I, and 500 u/ml Penicillin, 500 ug/ml streptomycin (all from Invitrogen) and 16% FCS (HyCLone).
- hES Cells Growth medium knockout high glucose DMEM supplemented with 500 u/ml Penicillin, 500 ug/mlstreptomycin, 1 % non-essential amino acids solution, 2mM GlutaMAX I securely 0.1 mM beta-mercaptoethanol, 4 ng/ml bFGF (Invitrogen), 1 -ng/ml human LIF (Chemicon, Temecula, CA), 8.4% of Serum Replacement (SR, Invitrogen) and 8.4% Plasmanate (Bayer).
- Derivation medium contained the same components as growth medium except that it had lower concentration of SR and Plasmanate (4.2% each) and 8.4 % FCS and 2x concentration of human LIF and bFGF, as compared to growth medium.
- EB medium same as growth medium except bFGF, LIF, and Plasmanate; the SR concentration was 13%.
- RPE medium 50% EB medium and 50% MEF medium.
- hES cell lines The cell lines, hES 35, 36, 45, used for these studies were derived with modifications of previously reported procedures (Thomson et. al., 1998, Reubinoff et. al, 2000, Lanzendorf et. al., 2001).
- Immunostaining Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% NP- 40 for localization of intracellular antigens, and blocked with 10% goat serum, 10% donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) in PBS (Invitrogen) for at least one hour. Incubation with primary antibodies was carried out overnight at 4oC, the secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) were added for one hour. Between all incubations specimens were washed with 0.1% Tween-20 (Sigma) in PBS 3-5 times, 10-15 minutes each wash.
- Specimens were mounted using Nectashield with DAPI (Vector Laboratories, Burlingame, CA) and observed under fluorescent microscope (Nikon). Localization of alkaline phosphatase was done either by Vector Red (Vector Laboratories, Burlingame, CA) to live cells or after the second wash during immunostaining according to manufacturer's instructions.
- Trizol reagent Invitrogen
- Qiagen Quantitect Probe RT-PCR reagents
- hES cell lines Two female one male hES cell lines were used in these studies. Details on the derivation of these hES lines are reported elsewhere. All lines have been passaged more than 50 times during which time they maintain an undifferentiated colony morphology, high alkaline phosphatase activity, and express Oct-4, SSEA-3, SSEA- 4, TRA 1-60, and TRA 1-81 (data not shown). Two lines have normal karyotype (hES36, hES35), while there were both normal and aneuploid subpopulations in hES45. Upon spontaneous differentiation both in vitro and in teratomas all lines expressed the markers of three germ layers - muscle actin, alpha-fetoprotein, and tubulin beta III.
- Example 9 Use of transcript genomics to identify normal differentiated cells differentiated ex vivo.
- Transcriptomics - hES-cell derivatives are likely to play an important role in the future of regenerative medicine. Qualitative assessment of these and other stem cell derivatives remains a challenge that could be approached using functional genomics.
- the gene expression profile of our data set was compared to two human RPE cell lines (non-transformed ARPE-19 and transformed D407, Rogojina et. al., 2003) to determine whether hES-RPE have similar global transcriptional profiles.
- Example 10 Use of RPE cells for treatment of Parkinson 's Disease.
- hRPE can be used as an alternative source of cells for cell therapy of Parkinson's Disease because they secrete L-DOPA.
- One of the many advantages to the use of hES cell-derived RPE is that it circumvents the shortage of donor eye tissue. It also facilitates the use of gene therapy.
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