RPE Cell Populations and Generation Methods

专利摘要:

公开号:ES2774456T9
申请号:ES15832827T
申请日:2015-12-30
公开日:2020-11-05
发明作者:Osnat Bohana-Kashtan；Belmaker Lior Ann Rosenberg；Ofer Wiser
申请人:Cell Cure Neurosciences Ltd；
IPC主号:

专利说明:

[0002] RPE Cell Populations and Generation Methods
[0004] Field and background of the invention
[0006] The present invention, in some embodiments thereof, relates to retinal pigment epithelial cells and, more particularly, but not exclusively, to the evaluation of such cells as a therapeutic agent. The present invention also relates to the generation of retinal pigment epithelium cells from embryonic stem cells.
[0008] The retinal pigment epithelium (RPE) is a monolayer of pigmented cells, located between the neural and choriocapillary retina. RPE cells play crucial roles in the maintenance and function of the retina and its photoreceptors. These include the formation of the blood-retinal barrier, the absorption of parasitic light, the supply of nutrients to the neural retina, the regeneration of visual pigment, and the uptake and recirculation of photoreceptor-released outer segments.
[0010] Retinal tissue can degenerate for various reasons. Among them are: arterial or venous occlusion, diabetic retinopathy and retinopathy of prematurity, which are usually hereditary. Diseases such as retinitis pigmentosa, retinoschisis, lattice degeneration, Best's disease, and age-related macular degeneration (AMD) are characterized by progressive types of retinal degeneration.
[0012] RPE cells can potentially be used for degenerative RPE cell replacement therapy in the aforementioned retinal diseases. It can also be used as a vehicle for the introduction of genes for the treatment of retinal degenerative diseases. These cells can also serve as an in vitro model of retinal degenerative diseases, as a tool for massive analytical screening to determine a therapeutic effect of small molecules, and for the discovery and testing of new drugs for retinal degenerative diseases. RPE cells could also be used for basic research on the development, maturation, characteristics, properties, metabolism, immunogenicity, function, and interaction of RPE with other cell types.
[0014] Human fetal and adult RPE have been used as an alternative donor source for allogeneic transplantation. However, practical problems in obtaining sufficient tissue supply and ethical concerns regarding the use of tissues from aborted fetuses limit the widespread use of these donor sources. Given these limitations in the provision of adult and fetal RPE grafts, the potential of alternative donor sources has been studied. Human pluripotent stem cells provide significant advantages as a source of RPE cells for transplantation. Their pluripotent developmental potential can allow their differentiation into truly functional RPE cells, and given their infinite self-renewal potential, they can serve as an unlimited donor source of RPE cells. In fact, human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPS) have been shown to differentiate into RPE cells in vitro, attenuate retinal degeneration, and maintain visual function after subretinal transplantation with relative to the Royal College of Surgeons (RCS) rat model of retinal degeneration caused by RPE dysfunction. Thus, pluripotent stem cells can be a limitless source for RPE cell production.
[0016] Current protocols for the derivation of RPE cells from pluripotent stem cells provide mixed populations of pigmented and non-pigmented cells. However, pure pigmented cell populations are desired for the use of RPE cells in basic research, drug discovery, and cell therapy.
[0017] The prior art includes WO 2013/114360, WO 2008/129554 and WO 2013/184809.
[0019] Summary of the invention
[0021] The present invention is defined by the claims.
[0023] According to one aspect of some embodiments of the present invention, a population of human polygonal RPE cells is provided, in which at least 95% of the cells thereof jointly express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein. (CRALBP), in which the transepithelial electrical resistance of the cell population is greater than 100 ohms.
[0025] According to one aspect of some embodiments of the present invention, a population of human RPE cells is provided, in which at least 80% of the cells thereof jointly express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein ( CRALBP) and in which the cells of the population each secrete angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor a ( sTNF-R1).
[0027] According to embodiments of the invention, the cells of the population each secrete angiogenin, a tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor for tumor necrosis factor a (sTNF-R1).
[0028] According to embodiments of the invention, cells secrete angiogenin, TIMP2, sgp130 or sTNF-R1 in a polarized manner.
[0029] According to embodiments of the invention, cells each secrete angiogenin, TIMP2, sgp130, and sTNF-R1 in a polarized manner.
[0030] According to embodiments of the invention, the ratio of apical sgp130 secretion: basal sgp130 secretion is greater than 1.
[0031] According to embodiments of the invention, the apical sTNF-R1 secretion: basal sTNFR1 secretion ratio is greater than 1.
[0032] According to embodiments of the invention, the ratio of basal angiogenin secretion: apical angiogenin secretion is greater than 1.
[0033] According to embodiments of the invention, the apical TIMP2 secretion: basal TIMP2 secretion ratio is greater than 1. According to embodiments of the invention, the number of Oct4 + TRA-1-60 + cells in the population is below 1: 250,000.
[0034] According to embodiments of the invention, at least 80% of the cells express bestrophin 1, as measured by immunostaining.
[0035] According to embodiments of the invention, at least 80% of the cells express microphthalmia-associated transcription factor (MITF), as measured by immunostaining.
[0036] According to embodiments of the invention, more than 50% of cells express paired box gene 6 (PAX-6) as measured by FACS.
[0037] According to embodiments of the invention, cells secrete more than 750 ng of pigment epithelium derived factor (PEDF) per ml per day.
[0038] According to embodiments of the invention, cells secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner.
[0039] According to embodiments of the invention, the ratio of apical PEDF secretion: basal PEDF secretion is greater than 1. According to embodiments of the invention, the ratio remains greater than 1 after incubation for 8 hours at 2-8 ° C. According to embodiments of the invention, the transepithelial electrical resistance of the cell population is greater than 100 ohms.
[0040] According to embodiments of the invention, the transepithelial electrical resistance of the cells remains greater than 100 ohms after incubation for 8 hours at 2-8 ° C.
[0041] According to embodiments of the invention, the ratio of basal VEGF secretion: apical VEGF secretion is greater than 1. According to embodiments of the invention, the ratio remains greater than 1 after incubation for 8 hours at 2-8 ° C. According to embodiments of the invention, the cell population is capable of rescuing visual acuity in the SCR rat after subretinal administration.
[0042] According to embodiments of the invention, the cell population is capable of rescuing photoreceptors for at least 180 days after subretinal administration in the RCS rat.
[0043] According to embodiments of the invention, the cell population is generated by ex vivo differentiation of human embryonic stem cells.
[0044] According to embodiments of the invention, the cell population is generated by:
[0045] (a) culturing human embryonic stem cells in a medium comprising nicotinamide to generate differentiating cells, wherein the medium is devoid of activin A;
[0046] (b) culturing the differentiating cells in a medium comprising nicotinamide and activin A to generate cells that further differentiate towards the RPE lineage; Y
[0047] (c) culturing cells that further differentiate toward the RPE lineage in a medium comprising nicotinamide, wherein the medium is devoid of activin A.
[0048] According to embodiments of the invention, embryonic stem cells are propagated in a medium comprising bFGF and TGFp.
[0049] According to embodiments of the invention, embryonic stem cells are cultured in human umbilical cord fibroblasts.
[0050] According to embodiments of the invention, steps (a) - (c) are performed under conditions where the atmospheric oxygen level is less than about 10%.
[0051] According to embodiments of the invention, the method further comprises culturing the differentiated cells in a medium under conditions in which the atmospheric oxygen level is greater than about 10% in the presence of nicotinamide after step (c).
[0052] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical composition comprising the cell population described herein as an active agent and a pharmaceutically acceptable carrier.
[0053] According to one aspect of some embodiments of the present invention, a use of the cell population described herein is provided to treat a degeneration of the retina.
[0054] According to one aspect of some embodiments of the present invention, a method of generating RPE cells is provided comprising:
[0055] (a) culturing pluripotent stem cells in a medium comprising a differentiating agent to generate differentiating cells, wherein the medium is devoid of a member of the transforming growth factor p (TGF p) superfamily;
[0056] (b) culturing the differentiating cells in a medium comprising the transforming growth factor p (TGF p) superfamily member and the differentiating agent to generate cells that further differentiate toward the RPE lineage;
[0057] (c) culturing cells that further differentiate toward the RPE lineage in medium comprising a differentiating agent to generate RPE cells, wherein the medium is devoid of a member of the transforming growth factor superfamily p ( TGF p), wherein steps (a) - (c) are performed under conditions where the atmospheric oxygen level is less than about 10%.
[0058] According to embodiments of the invention, step (a) is carried out under non-stick conditions.
[0059] According to embodiments of the invention, the non-adherent conditions comprise a non-adherent culture plate. According to embodiments of the invention, step (a) comprises:
[0060] i) culturing the cultured population of human pluripotent stem cells in a medium comprising nicotinamide, in the absence of activin A; under non-adherent conditions to generate a cell cluster comprising differentiating cells; and subsequently;
[0061] ii) culturing the differentiating cells of (i) in a medium comprising nicotinamide, in the absence of activin A under adherent conditions.
[0062] According to embodiments of the invention, the method further comprises dissociating the cell cluster prior to step (ii) to generate groups of cells or a single cell suspension of cells.
[0063] According to embodiments of the invention, the method further comprises culturing the differentiated cells in a medium under conditions where the level of atmospheric oxygen is greater than about 10% in the presence of a differentiating agent after step (c).
[0064] According to embodiments of the invention, the transforming growth factor p (TGF p) superfamily member is selected from the group consisting of TGFp1, TGFp3, and activin A.
[0065] According to embodiments of the invention, the differentiating agent of step (a) and the differentiating agent of step (c) are identical.
[0066] According to embodiments of the invention, the differentiating agent of step (a) is nicotinamide (NA) or 3-aminobenzamide.
[0067] According to embodiments of the invention, the method further comprises selecting polygonal cells after step (c). According to embodiments of the invention, the method further comprises propagating the polygonal cells.
[0068] According to embodiments of the invention, the propagation is carried out on an adherent surface or an extracellular matrix.
[0069] According to embodiments of the invention, the pluripotent stem cells comprise embryonic stem cells. According to embodiments of the invention, embryonic stem cells are propagated in a medium comprising bFGF and TGFp.
[0070] According to embodiments of the invention, embryonic stem cells are cultured in human umbilical cord fibroblasts.
[0071] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein may be used in practice or testing embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
[0072] Brief description of the various views of the drawings
[0073] Some embodiments of the invention are described herein, by way of example only, with reference to the accompanying drawings. With specific reference now to the detailed drawings, it is emphasized that the data is shown by way of example and for purposes of illustrative comment on embodiments of the invention. In this regard, the description taken with the drawings makes it apparent to those skilled in the art how embodiments of the invention can be practiced.
[0074] In the drawings:
[0075] Figure 1 is a graph illustrating the linearity of the data.
[0076] Figure 2 is a FACS analysis of negative control hESC cells stained with anti-CRALBP antibody and anti-PMEL 17 antibody.
[0077] Figure 3 is a positive control FACS analysis of OpRegen® reference RPE line cells stained with anti-CRALBP antibody and anti-PMEL 17 antibody.
[0078] Figure 4 is a FACS analysis of OpRegen® 5C with known 25% spikes in hESC stained with anti-CRALBP antibody and anti-PMEL 17 antibody.
[0079] Figure 5 is a FACS analysis of OpRegen® 5C with known 50% spikes in hESC stained with anti-CRALBP antibody and anti-PMEL17 antibody.
[0080] Figure 6 is a FACS analysis of OpRegen® 5C with known 75% spikes in hESC stained with anti-CRALBP antibody and anti-PMEL17 antibody.
[0081] Figure 7 is a FACS analysis of OpRegen® 5C with known 95% spikes in hESC stained with anti-CRALBP antibody and anti-PMEL 17 antibody.
[0082] Figure 8 is a FACS analysis of hESCs stained with isotype controls.
[0083] Figure 9 is a FACS analysis of OpRegen® 5C cells stained with the isotype controls.
[0084] Figure 10: Co-immunostaining with PMEL17 differentiates RPE cells (CRALBP + PMEL17 +) from pigmented non-RPE cells (PMEL17 + CRALBP-; such as melanocytes).
[0085] Figure 11: morphology results for simulated production 4 and 5 at in-process control points (IPC) 5, and 8-10.
[0086] Figure 12: Manufacturing procedure, steps 1-3: Generation of human umbilical cord fibroblast feeder cell bank.
[0088] Figure 13: Manufacturing procedure, steps 4-5: hESC expansion.
[0090] Figure 14: manufacturing procedure, steps 6-13: differentiation to RPE cells (OpRegen®).
[0092] Figure 15: manufacturing procedure, steps 14-17: expansion of pigmented cells.
[0094] Figure 16: Detailed OpRegen® manufacturing procedure and in-process checkpoints (yellow stars, IPC 1-11). (NUTSPlus, Nutristem medium containing bFGF and TGFP; NUTSMinus, Nutristem medium without bFGF and TGFP; NIC, nicotinamide; SB, spheroidal bodies).
[0096] Figure 17: CRALBP + PMEL17 + RPE cell level in OpRegen® 4 and 5 mock production runs. Density plots of IPC points 8 and 11 (* IPC point 8 was tested after cryopreservation) and representative density plots of OpRegen® 5C positive control hESC and HAD-C102 negative control (the range of % CRALBP + PMEL17 + in the negative control was 0.02-0.17%). The numbers within each graph indicate the percentage of CRALBP + PMEL17 + cells out of the activated population of living single cells. Analysis was performed using FCS Express 4 software.
[0098] Figure 18: Immunofluorescence staining of IPC 7, 10 and 11 mock production sites 5 with antibodies specific for the RPE markers bestrophin 1, MITf, ZO-1, and CRALBP.
[0100] Figures 19A-C: representative color background photograph of group 2 (BSS +; figure 19A), group 5 against lateral untreated eyes (OD; figure 19B) and group 5, treated eyes (OS; figure 19C) at P60. Hyper and hypopigmented areas in high dose (OS) treated eyes are assumed to be indicative of transplanted cells.
[0102] Figure 20: Optokinetic tracking acuity thresholds measured at P60, P100, P150, and P200. The groups treated with cells (group 3-25,000, group 4-100,000 and group 5-200,000) outperformed all controls, reaching the dose of group 4 (100,000) and 5 (200,000) the best rescue. Contralateral unoperated eyes were equivalent to group 1 (untreated) and group 2 (vehicle control / BSS +) (not shown).
[0104] Figures 21A-B: graphs illustrating focal (Figure 21A) and full field (Figure 21B) results for a representative rat.
[0106] Figures 22A-B: Figure 22A illustrates a photomontage of individual images of cresyl violet stained sections from a representative cell-treated eye. The arrows illustrate the location of photoreceptor shielding and the presumed location of the grafted cells. Figure 22B illustrates the comparison between eyes injected with BSS + (group 2) and eyes injected with representative cells (multiple dose groups represented) at day 60, 100, 150 and 200 after birth. GCL: nerve ganglion cell layer; ONL: outer nuclear layer; RPE: retinal pigmented epithelium.
[0108] Figure 23: thickness of the outer nuclear layer measured in number of nuclei. Each point represents the count of each animal in each dose group for all ages.
[0110] Figure 24: Immunofluorescent images of positive control tissue and animals treated with representative experimental cells at P60, P100, P150 and P200 stained with anti-human nuclei marker (HNM, green), antipremelanosomic marker (PMEL17, red), anti-proliferation marker human (Ki67, red) and anti-rat cone arrestin (red). Dapi (blue) is used for background staining to highlight the nuclear layers. Human melanoma was used as positive control tissue for PMEL17, human amygdala for Ki67 and juvenile rat retina from RCS for cone arrestin. Down arrows indicate outer nuclear layer; Up arrows indicate positively stained human RPE cells (OpRegen®), generated as described herein.
[0112] Figure 25 is a graph illustrating cone quantification after subretinal transplantation of OpRegen® cells in the RCS rat. Cell-treated eyes were significantly older than control eyes at all ages.
[0114] Figures 26A-J: Immunofluorescent staining of OpRegen® cells in the subretinal space. Figure 26A represents an area of the retina with several cells of the RPE (red, arrows) central zone and no residue (seen using rat antirodopsin antibody, green; arrow), but where the cells are not (peripheral), the residue zone is reconstituted. At higher magnification (Figure 26B), some outer rhodopsin-stained segments rest along the grafted cells. Furthermore, the residue zone is reconstituted as the distance from the transplanted cells increases. Figures 26C-J are individual sections through the section showing rhodopsin positive tissue within the transplanted cells (arrows).
[0115] Figures 27A-C are photographs illustrating the biodistribution of cells after subretinal injection in NOD-SCID. Figure 27A illustrates the ability of OpRegen® cells to graft into the NOD-SCID subretinal space 9 months after transplantation. Pigmented cells stain positive for human nuclei and PMEL17. Figure 27B is a photograph illustrating clustered cells at the bleb site after injection. Figure 27C is a photograph illustrating the subsequent propagation of cells in a monolayer after injection.
[0117] Figure 28 is a graphic illustration of a Transwell assay that can be used to assay RPE cell potency.
[0119] Figure 29 are the results of FACS analysis illustrating PAX6 expression in RPE cells generated as described herein (P2-DP, finished product: mock production IV, mock production V, OpRegen® lot 2A; HuRPE: ScienCell normal human RPE) and in production (P0).
[0121] Figure 30 is a graph illustrating PAX6 expression in OpRegen® cells, as assessed by FACS (HES, human embryonic stem cells used as negative control).
[0123] Figure 31 is the results of FACS analysis illustrating the double staining of PAX6 and CRALBP.
[0125] Figures 32A-C are graphs illustrating ELISA evaluation of angiogenin secretion by OpRegen® cells. A. Increased secretion of angiogenin in mock production V. B. Secretion of angiogenin by three different batches of OpRegen® cells (step 3) and in a Transwell for 3 weeks (step 4) during which apical and basal secretion was assessed. C. Angiogenin secretion by RPE 7 cells (step 3).
[0127] Figures 33A-E illustrate the secretion of TIMP-1 and TIMP-2 by OpRegen® cells. A. Relative TIMP-1 and TIMP-2 protein levels detected by protein matrix. B. ELISA TIMP-2 levels at QC sites 3 and 4 of mock production V. C-D. ELISA TIMP-2 secretion levels by different batches of OpRegen® cells (step 3) and in a Transwell for 3 weeks during which apical and basal secretion was assessed (step 4). E. Levels of T iMp -2 secreted from control RPE 7 and HuRPE cells (step 3, days 4 and 14).
[0129] Figures 34A-D illustrate secretion of sgp130 by OpRegen® cells as measured by ELISA. A. Secretion levels of sgp130 at QC sites 3 and 4 of mock production V. B-C. Levels of sgp130 secreted by various batches of OpRegen® cells (step 3) and in a Transwell for 3 weeks during which apical and basal secretion were assessed (step 4). D. Levels of sgp130 secreted from control cells RPE 7 and HuRPE (step 3, days 4 and 14).
[0131] Figures 35A-D illustrate sTNF-R1 protein levels in OpRegen® cell supernatant as measured by ELISA. A. Levels of sTNF-R1 in cell supernatant from QC sites 3 and 4 of mock production V. B-C. STNF-R1 levels in OpRegen® batch supernatant (step 3) and in a Transwell for 3 weeks during which the apical and basal levels were assessed (step 4). D. Levels of sTNF-R1 from control HuRPE and RPE7 cell cultures on day 4 and day 14 (step 3).
[0133] Figure 36 illustrates the morphology of OpRegen® 5C (reference line), RPE1 and RPE7 in Transwell. OpRegen® 5C, RPE1 and RPE7 were imaged weekly (week 1-4) after seeding in Transwell. OpRegen® 5C generated a homogeneous polygonal monolayer from week 1 while RPE1 and RPE7 generated a different inhomogeneous morphology one week after seeding and holes began to appear at week 2. The RPE1 cells separated from the Transwell after 3 weeks in culture.
[0135] Figure 37 illustrates that RPE1 and RPE7 cells jointly express CRALBP and PMEL-17. The purity assay using FACs showed that 99.91% and 96.29% of the RPE1 and RPE7 cells, respectively, are double positive for the RPE markers CRALBP and PMEL-17, similar to the levels observed in the OpRegen® cells from mock production V (positive control). HAD-C 102 hESC were used as a negative control.
[0136] Description of specific embodiments of the invention
[0138] The present invention, in some embodiments thereof, relates to retinal pigment epithelial cells and, more particularly, but not exclusively, to the evaluation of such cells as a therapeutic agent. The present invention also relates to the generation of retinal pigment epithelial cells from human embryonic stem cells.
[0140] Before explaining at least one embodiment of the invention in detail, it should be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
[0142] The neural retina initiates vision and is supported by the underlying retinal pigment epithelium (RPE). RPE cell dysfunction, degeneration, and loss are prominent features of Best's disease, subtypes of retinitis pigmentosa (RP) and age-related macular degeneration (AMD), which is the leading cause of visual impairment in western countries. In these states, there is a progressive visual loss that often leads to blindness.
[0144] The retina and adjacent RPE both arise from the neural ectoderm. In lower species, RPE regenerates the retina, but in mammals, RPE-mediated regeneration is inhibited and regeneration occurs to a very limited extent by stem cells located in the peripheral retinal margin.
[0146] Some human embryonic stem cells (hESC) can serve as an unlimited donor source of RPE cells for transplantation. The potential for mouse, primate, and human ESCs to differentiate into RPE-like cells, to attenuate retinal degeneration, and to maintain visual function after subretinal transplantation has been demonstrated.
[0148] Various protocols have been developed for the differentiation of human embryonic stem cells into RPE cells (see, for example, WO 2008/129554).
[0150] The present inventors have now discovered a unique and simple way to qualify cell populations that have successfully differentiated into RPE cells based on the expression of particular polypeptides. Out of the myriad possible polypeptides expressed in these differentiated cells, the present inventors have found that a combination of two particular markers can be used to support successful differentiation.
[0151] The present inventors have also discovered that pigment epithelial derived factor (PEDF) secretion can be used as a marker to support early stages of the RPE differentiation process (see Table 4).
[0153] While further reducing the present invention to practice, the present inventors identified additional proteins that are secreted by RPE cells that can be used, in some embodiments, as a signature to define the cells.
[0155] Therefore, according to one aspect of the present description, a method of scoring whether a cell population is a suitable therapeutic agent for treating an ocular condition is provided, which comprises analyzing the co-expression of the premelanosome protein (PMEL 17) and at least a polypeptide selected from the group consisting of cellular retinaldehyde binding protein (CRALBP), lecithin-retinol acyltransferase (LRAT) and box 9 of sex determining region Y (SOX 9) in the cell population, in which when the number of cells that jointly express PMEL17 and the at least one polypeptide is above a predetermined level, the cell population is qualified as a suitable therapeutic agent for treating a retinal disorder.
[0157] According to another aspect, a method of scoring whether a cell population is a suitable therapeutic agent for treating an ocular condition is provided, comprising analyzing the co-expression of cellular retinaldehyde binding protein (CRALBP) and at least one polypeptide selected from the group consisting of premelanosome protein (PMEL17), lecithin-retinol acyltransferase (LRAT) and box 9 of sex determining region Y (SOX 9) in the cell population, in which when the number of cells that jointly express CRALBP and the at least one polypeptide is above a predetermined level, the cell population is qualified as a suitable therapeutic agent to treat an ocular condition.
[0159] As used herein, the phrase "suitable therapeutic agent" refers to the suitability of the cell population for treating ocular conditions. Cells that are therapeutic can exert their effect through any one of multiple mechanisms. An exemplary mechanism is the trophic support effect that promotes the survival of degenerating photoreceptors or other cells within the retina. Therapeutic RPE cells can also exert their effect through a regenerative mechanism that replenishes the malfunction and / or degenerates RPE host cells. According to one embodiment, the RPE cells are mature and have the functional ability to phagocytose outer released segments of photoreceptors including rhodopsin. According to another embodiment, the RPE cells are not fully mature.
[0161] Ocular conditions for which cell populations serve as a therapeutic agent include, but are not limited to, retinal diseases or disorders generally associated with retinal dysfunction, retinal injury, and / or loss of retinal pigment epithelium. A non-limiting list of conditions that can be treated according to the description includes retinitis pigmentosa, Leber congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), Best's disease, retinal detachment, gyratory atrophy, choroideremia, patterned dystrophy as well as other dystrophies of the RPE, Stargardt's disease, retinal and RPE damage due to damage caused by any one of photic, laser, inflammatory, infectious, radiation, neovascular or traumatic injury.
[0163] As mentioned, the method of this aspect of the description is carried out by measuring the amount (e.g., percentage of cells) that expresses premelanosome protein (PMEL17; SwissProt # P40967) and at least one polypeptide selected from the group that consists of cellular retinaldehyde binding protein (CRALBP; SwissProt no. P12271), lecithin-retinol acyltransferase (LRAT; SwissProt # 095327) and Y-sex determining region box 9 (SOX 9; P48436).
[0165] Alternatively, the method of this aspect is carried out by measuring CRALBP (CRALBP; SwissProt # P12271) and at least one polypeptide selected from the group consisting of lecithin-retinol acyltransferase (LRAT; SwissProt # 095327), box 9 of sex determining region Y (SOX 9; P48436) and PMEL17 (SwissProt # P40967).
[0167] Thus, for example, CRALBP and PMEL17 can be measured; PMEL17 and LRAT can be measured, or PMEL17 and SOX9 can be measured. Alternatively, CRALBP and LRAT can be measured, or CRALBP and SOX9 can be measured.
[0169] It will be appreciated that more than two of the polypeptides mentioned herein may be used, for example, three of the above-mentioned polypeptides or even all four of the above-mentioned polypeptides.
[0170] The methods for analyzing the expression of the aforementioned polypeptides normally involve the use of antibodies that specifically recognize the antigen. Commercially available antibodies that recognize CRALBP include, for example, those made by Abcam (eg, ab15051 and ab189329, clone B2). Commercially available antibodies that recognize PMEL17 include, for example, those made by Abcam (eg, ab137062 and ab189330, clone EPR4864). Commercially available antibodies that recognize LRAT include, for example, those manufactured by Millipore (eg, MABN644). Commercially available antibodies that recognize SOX9 include, for example, those made by Abcam (eg, ab185230). The analysis can be carried out using any method known in the art including flow cytometry, Western blot, immunocytochemistry, radioimmunoassay, PCR, etc.
[0172] For flow cytometry, the antibody can be bound to a fluorescent moiety and analyzed using fluorescence activated cell sorting (FACS). Alternatively, the use of secondary antibodies with fluorescent moieties is envisioned.
[0174] It will be appreciated that since the polypeptides being tested are intracellular polypeptides, cells are normally permeabilized so that the antibodies are able to bind to their targets. Cells can be fixed first to ensure the stability of soluble antigens or antigens with a short half-life. This should preserve the target protein in the original cell location. Antibodies can be prepared in permeabilization buffer to ensure that cells remain permeable. It will be appreciated that when selecting on cell populations, the light scattering profiles of the cells on the flow cytometer will change considerably after permeabilization and fixation.
[0176] Methods of permeabilization of the cell membrane are known in the art and include, for example:
[0178] 1. Formaldehyde followed by detergent: fixation in formaldehyde (eg, no more than 4.5% for 10-15 min (this will stabilize the proteins), followed by alteration of the membrane by detergent such as Triton or NP-40 ( 0.1 to 1% in PBS), Tween 20 (0.1 to 1% in PBS), Saponin, Digitonin and Leucoperm (eg 0.5% v / v in PBS);
[0180] 2. Formaldehyde (eg, no more than 4.5%) followed by methanol;
[0182] 3. Methanol followed by detergent (eg 80% methanol then 0.1% Tween 20);
[0184] 4. Fixation with acetone and permeabilization.
[0186] As used herein, the term "flow cytometry" refers to an assay in which the proportion of a material (eg, RPE cells that comprise a particular marker) in a sample is determined by labeling the material. (for example, attaching a labeled antibody to the material), causing a stream of fluid containing the material to pass through a beam of light, separating the emitted light from the sample into constituent wavelengths using a series of filters and mirrors , and detecting light.
[0188] A multitude of flow cytometers are commercially available including, for example, Becton Dickinson FACScan, Navios flow cytometer (Beckman Coulter, Serial # AT15119 RHE9266, and FACScalibur (BD Biosciences, Mountain Vista, CA). Antibodies that can be used for FACS analysis are taught in Schlossman S, Boumell L, et al., [Leucocyte Typing V. New York: Oxford University Press; 1995] and are widely commercially available.
[0190] It will be appreciated that the level of expression of the aforementioned polypeptides can be at the RNA level as well as the protein level. Exemplary methods for determining the expression of a polypeptide based on the RNA level include, but are not limited to, PCR, RT-PCR, Northern blotting, etc.
[0192] In order to qualify the cells as useful as a therapeutic agent, the amount of at least two of the co-expressed polypeptides in the cells must be increased above a statistically level. significant compared to non-RPE cells (eg, undifferentiated embryonic stem cells).
[0194] According to a particular embodiment, in order to qualify that the cells are useful as a therapeutic agent, at least 80% of the cells of the population must express detectable levels of PMEL17 and one of the aforementioned polypeptides (for example, CRALBP), more preferably at least 85% of the cells in the population should express detectable levels of PMEL17 and one of the polypeptides mentioned above (e.g. CRALBP), more preferably at least 90% of the cells in the population should express detectable levels of PMEL17 and one of the aforementioned polypeptides (eg CRALBP), more preferably at least 95% of the cells in the population should express detectable levels of PMEL17 and one of the aforementioned polypeptides (eg CRALBP), plus preferably 100% of the cells in the population should express detectable levels of PMEL17 and one of the aforementioned polypeptides (for example or, CRALBP) as tested by a method known to those of skill in the art (eg, FACS).
[0196] According to another embodiment, in order to qualify the cells as useful as a therapeutic agent, the level of co-expression of CRALBP and one of the aforementioned polypeptides (eg PMEL17) (eg, as measured by fluorescent intensity mean) should be increased by at least two times, more preferably at least 3 times, more preferably at least 4 times and even more preferably by at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 compared to undifferentiated ESC.
[0198] According to a particular embodiment, in order to qualify that the cells are useful as a therapeutic agent, at least 80% of the cells of the population must express detectable levels of CRALBP and one of the aforementioned polypeptides (for example, PMEL17), more preferably at least 85% of the cells in the population should express detectable levels of CRALBP and one of the polypeptides mentioned above (eg PMEL17), more preferably at least 90% of the cells in the population should express detectable levels CRALBP and one of the aforementioned polypeptides (eg PMEL17), more preferably at least 95% of the cells in the population should express detectable levels of CRALBP and one of the aforementioned polypeptides (eg PMEL17), plus preferably 100% of the cells in the population should express detectable levels of CrAlBP and one of the aforementioned polypeptides (for example or, PMEL17) as tested by a method known to those of skill in the art (eg FACS).
[0200] Furthermore, the cell can be scored in vivo in animal models. One such model is the Royal College of Surgeons (RCS) rat model. After transplantation, the therapeutic effect of cells can be analyzed using methods including fundus imaging, optokinetic monitoring thresholds (OKT), electroretinogram (ERG), histology, cone count, and rhodopsin ingestion. These methods are further described in Example 5, hereinafter.
[0202] Cells can be scored or characterized in additional ways including, for example, karyotype analysis, morphology, cell number and viability, potency (barrier function and polarized secretion of PEDF and VEGF), residual hESC level, Gram stain, and sterility. Exemplary tests that may be performed are described in Example 4.
[0204] In addition, cells can be analyzed for barrier function and their level of growth factor secretion in a polarized manner (eg, pigment epithelial derived factor (PEDF) or VEGF, cytokines, interleukins, and / or chemokines).
[0206] For the analysis of secreted PEDF, the culture supernatant of the cells is harvested, and the cells are harvested and counted. The amount of PEDF in cell culture supernatants can be quantified using a PEDF ELISA assay (such as the PEDF ELISAquant ™ Sandwich ELISA Antigen Detection Kit, BioProductsMD, PED613) according to the manufacturer's protocol.
[0208] Furthermore, the secretion direction of PEDF and VEGF can be analyzed in cells. This can be done using a Transwell assay as illustrated in Figure 28. Before or after qualification, cells can be preserved according to methods known in the art (eg, frozen or cryopreserved) or can be administered directly to the subject.
[0210] The present invention contemplates analyzing cell populations comprising retinal pigment epithelial (RPE) cells from any source. Thus, cell populations may comprise RPE cells obtained from a donor (i.e. native RPE cells of the retinal pigmented layer) or may comprise RPE cells that differentiated ex vivo from a population of stem cells (cells hSC-derived RPEs, such as pluripotent stem cells, eg, human embryonic stem cells). According to another embodiment, RPE cells are obtained by transdifferentiation, see, for example, Zhang et al., Protein Cell 2014, 5 (1): 48-58.
[0211] According to one embodiment, the RPE cells that are tested do not express Pax6.
[0212] According to another embodiment, the RPE cells that are tested express Pax6.
[0213] "Retinal pigment epithelium cells", "RPE cells", "RPE", which may be used interchangeably as the context allows, refers to cells of a cell type functionally similar to native RPE cells that form the cell layer of the retinal pigment epithelium (for example, after transplantation into the eye, they exhibit functional activities similar to those of native RPE cells).
[0214] According to one embodiment, the RPE cell expresses at least one, two, three, four, or five markers of mature RPE cells. Such markers include, but are not limited to, CARLBP, RPE65, PEDF, PMEL17, bestrophin, and tyrosinase. Optionally, RPE cells can also express a marker for an RPE progenitor, eg, MITf. In another embodiment, the RPE cells express PAX-6. In another embodiment, the RPE cells express at least one marker from a retinal progenitor cell including, but not limited to, OTX2, SIX3, SIX6, and LHX2.
[0215] According to yet another embodiment, RPE cells are those that are differentiated from embryonic stem cells according to the method described in the examples section hereinafter, the content of the examples being as included in the specification itself.
[0216] As used herein, the phrase "mature RPE cell markers" refers to antigens (eg, proteins) that are elevated (eg, at least 2 times, at least 5 times, at least 10 times ) in mature RPE cells relative to non-RPE cells or immature RPE cells.
[0217] As used herein, the phrase "RPE progenitor cell markers" refers to antigens (eg, proteins) that are elevated (eg at least 2 times, at least 5 times, at least 10 times ) in RPE progenitor cells relative to non-RPE cells.
[0218] According to another embodiment, the RPE cells have a morphology similar to that of native RPE cells that form the retinal pigment epithelial cell layer, i.e., pigmented and / or have a characteristic polygonal shape.
[0219] According to still another embodiment, the RPE cells are capable of treating diseases such as macular degeneration.
[0220] According to yet another embodiment, the RPE cells satisfy at least 1, 2, 3, 4, or all of the requirements listed hereinabove.
[0221] The term "hSC-derived RPE cells" is used herein to denote RPE cells that are obtained by targeted differentiation from hSC. According to a preferred embodiment, the hSC derived RPE cells are functional RPE cells as presented by parameters defined hereinbelow. The term "directed differentiation" is used interchangeably with the term "RPE-induced differentiation" and is to be understood to mean the process of manipulating hSC under culture conditions that induce / promote differentiation to give the RPE cell type.
[0222] According to a particular embodiment, RPE cells are obtained by directed differentiation of hSC in the presence of one or more members of the TGFp superfamily, and present at least one of the following characteristics: - during differentiation, the cultured cells respond to the TGFP signaling;
[0223] - RPE cells express markers indicative of terminal differentiation, for example bestrophin 1, CRALBP and / or RPE65;
[0224] - after transplantation (that is, in situ), RPE cells show a trophic effect to support photoreceptors adjacent to RPE cells;
[0225] - in addition, RPE cells in situ are capable of phagocytosis of outer segments of photoreceptors released as part of the normal renewal process of these photoreceptors;
[0226] - in addition, the RPE cells in situ are capable of generating a retinal function and barrier in the visual cycle.
[0227] As used herein, the phrase "stem cells" refers to cells that are capable of remaining in an undifferentiated state (eg, pluripotent or multipotent stem cells) for prolonged periods of time in culture until they are induced to differentiate into other cell types that have a particular, specialized function (eg, fully differentiated cells). Preferably, the phrase "stem cells" encompasses embryonic stem cells (ESC), induced pluripotent stem cells (iPS), adult stem cells, mesenchymal stem cells, and hematopoietic stem cells.
[0229] According to a particular embodiment, the RPE cells are derived from pluripotent stem cells including human embryonic stem cells or induced pluripotent stem cells.
[0231] The phrase "embryonic stem cells" refers to embryonic cells that are capable of differentiating into cells from all three embryonic germ layers (ie, endoderm, ectoderm, and mesoderm), or that remain in an undifferentiated state. For reference, the phrase "embryonic stem cells" may comprise cells that are obtained from embryonic tissue formed after gestation (eg, blastocysts) prior to embryo implantation (ie, a preimplantation blastocyst), enlarged blastocyst cells (EBC) that are obtained from a post-implantation / pre-gastrulation phase blastocyst (see WO2006 / 040763) and embryonic germ cells (EG) that are obtained from the genital tissue of a fetus at any time during the gestation, preferably before 10 weeks of gestation. The embryonic stem cells of some embodiments of the invention can be obtained using well known cell culture methods.
[0233] As a reference example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically derived from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, a single-celled human embryo can expand to the blastocyst stage. For isolation of human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by surgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then seeded in a tissue culture flask containing the appropriate medium that allows its overgrowth. After 9 to 15 days, the ICM-derived overgrowth is dissociated into clumps either by mechanical dissociation or by enzymatic degradation, and the cells are then re-seeded on fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette / stem cell tool, mechanically dissected into fragments / clusters and re-seeded. The resulting ES cells are then routinely divided every 4-7 days. For additional details on the methods of preparing human ES cells, see Reubinoff et al., Nat Biotechnol 2000, May: 18 (5): 559; Thomson et al., [US Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].
[0235] It will be appreciated that commercially available stem cells can also be used (for reference only). Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry [hypertext transfer protocol: //grants.nih.gov/stem_cells/registry/current.htm] and other European registries. Non-limiting reference examples of commercially available embryonic stem cell lines are HAD-C102, ESI, BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB- 6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13 ), WA14 (H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6 , BJNhem19, BJNhem20, SA001, SA001.
[0237] In addition, ES cells can also be obtained from other species, including mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, Dev Biol. 163: 288-92], rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves and Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], various species of domestic animals [Notarianni et al., 1991, J Reprod Fertil Supl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67] and non-human primate species (Indian macaque and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].
[0238] For reference, enlarged blastocyst cells (EBC) can be obtained from a blastocyst at least nine days post-fertilization in a phase prior to gastrulation. Before blastocyst culture, the zona pellucida is digested [eg, by Tyrode's acid solution (Sigma Aldrich, St. Louis, MO, USA)] to expose the inner cell mass. The blastocysts are then cultured as full embryos for at least nine and no more than fourteen days after fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culture methods.
[0240] Another method for preparing ES cells described in Chung et to the., Cell Stem Cell, volume 2, volume 2, 113-117, February 7, 2008. This method comprises removing a single cell of an embryo during in a process of fertilization vitro. The embryo is not destroyed in this procedure.
[0242] Still another method of preparing ES cells is by parthenogenesis. The embryo is also not destroyed in the procedure.
[0243] Currently practiced ES culture methods are mainly based on the use of feeder cell layers that secrete factors necessary for stem cell proliferation, while at the same time inhibiting their differentiation. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian tube epithelial cells, primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), murine fetal fibroblasts (MFF), human embryonic fibroblasts (HEF). , human fibroblasts obtained from the differentiation of human embryonic stem cells, human fetal muscle cells (HFM), human fetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF), human umbilical cord fibroblasts, cells obtained from the umbilical cord or placenta, and human bone marrow stromal cells (hMSC). Growth factors can be added to the medium to maintain the ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGFp. In another embodiment, agents can be added to the medium to maintain the hESCs in an initial undifferentiated state, see, for example, Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.
[0245] Feeder cell-free systems have also been used in ES cell culture, such systems use matrices supplemented with serum replacement, cytokines, and growth factors (including IL6 and soluble IL6 receptor chimera) as replacement for the feeder cell coat. Stem cells can be grown on a solid surface such as an extracellular matrix (eg MatrigelRTM or laminin) in the presence of a culture medium, eg the Lonza L7 system, mTeSR, StemPro, XFKSR, E8). In contrast to feeder-based cultures that require the simultaneous growth of feeder cells and stem cells and which can result in mixed cell populations, stem cells that have grown on feeder-free systems are easily detached from the surface. The culture medium used to grow stem cells contains factors that effectively inhibit differentiation and promote their growth such as bFGF and MEF-conditioned medium. However, commonly used feeder-free culture systems utilize an animal-based matrix (eg, MatrigelRTM) supplemented with mouse or bovine serum, or with MEF-conditioned medium [Xu C, et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19: 971-4] that present the risk of cross-transfer of animal pathogens to human ES cells thereby compromising future clinical applications.
[0247] Numerous methods are known to differentiate ESC towards the RPE lineage and include both directed differentiation protocols such as those described in WO 2008/129554, 2013/184809 and spontaneous differentiation protocols such as those described in US Patent No. 8,268,303 and US patent application 20130196369.
[0249] According to a particular embodiment, the RPE cells are generated from ESC cells using a directed differentiation protocol, for example, as disclosed in the examples section.
[0251] In an exemplary differentiation protocol, embryonic stem cells differentiate into the RPE cell line using a first differentiating agent and then further differentiate into RPE cells using a member of the transforming growth factor p superfamily. (TGFp), (eg, TGFp1, TGFp2, and TGFp3 subtypes, as well as homologous ligands including activin (eg, activin A, activin B, and activin AB), nodal, anti-Mullerian hormone (AMH), some bone morphogenetic proteins (BMP) , for example BMP2, BMP3, BMP4, BMP5, BMP6 and BMP7, and growth and differentiation factors (GDF)).
[0253] According to a particular embodiment, the member of the TGFp superfamily is selected from the group consisting of TGFp1, activin A and TGFp3.
[0255] According to a specific embodiment, the transforming growth factor p (TGFp) superfamily member is activin A, eg, between 20-200 ng / ml, eg 100-180 ng / ml.
[0257] The first agent of differentiation promotes differentiation towards the RPE lineage. For example, the first differentiating agent can promote the differentiation of pluripotent stem cells into neural progenitors. Such cells can express neural precursor markers such as PAX6.
[0259] According to a particular embodiment, the first differentiating agent is nicotinamide (NA), for example, between 1 100 mM, 5-50 mM, 5-20 mM, for example 10 mM.
[0261] NA, also known as "niacinamide," is the amide derivative form of vitamin B3 (niacin), which is thought to preserve and maintain beta cell function. NA has the chemical formula C ⁶ H ⁶ N ² O. NA is essential for growth and the conversion of food into energy, and has been used in the treatment of arthritis and the treatment and prevention of diabetes.
[0264] According to a particular embodiment, the nicotinamide is a nicotinamide derivative or a nicotinamide mimic. The term "nicotinamide derivative (NA)" as used herein denotes a compound that is a chemically modified derivative of natural NA. In one embodiment, the chemical modification may be a substitution of the pyridine ring of the basic NA backbone (via the carbon or nitrogen member of the ring), via the nitrogen atoms or the oxygen atoms of the amide moiety. When substituted, one or more hydrogen atoms can be replaced with a substituent and / or a substituent can be attached to an N atom to form a positively charged tetravalent nitrogen. Thus, the nicotinamide of the present invention includes a substituted or unsubstituted nicotinamide. In another embodiment, the chemical modification may be a deletion or replacement of an individual group, for example, to form a thiobenzamide analog of NA, all of this being appreciated by those skilled in organic chemistry. The derivative in the context of the invention also includes the nucleoside derivative of nA (eg, nicotinamide adenine).
[0266] A variety of NA derivatives are described, some also in relation to an inhibitory activity of the PDE4 enzyme (WO03 / 068233; WO02 / 060875; GB2327675A), or as inhibitors of VEGF receptor tyrosine kinase (WO01 / 55114) . For example, the process of preparing 4-aryl-nicotinamide derivatives (WO05 / 014549). Other exemplary nicotinamide derivatives are disclosed in WO01 / 55114 and EP2128244.
[0268] Nicotinamide mimics include modified forms of nicotinamide, and chemical analogs of nicotinamide that recapitulate the effects of nicotinamide on RPE cell differentiation and maturation from pluripotent cells. Exemplary nicotinamide mimics include benzoic acid, 3-aminobenzoic acid, and 6-aminonicotinamide. Another class of compounds that can act as nicotinamide mimics are poly (ADP-ribose) polymerase (PARP) inhibitors. Exemplary PARP inhibitors include 3-aminobenzamide, iniparib (BSI 201), olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, and BMN- 673.
[0270] According to a particular embodiment, the differentiation is carried out as follows:
[0272] a) culturing ESC in a medium comprising a first differentiating agent (eg nicotinamide); Y
[0273] b) culturing cells obtained from step a) in a medium comprising a member of the TGFp superfamily (eg activin A) and the first differentiating agent (eg nicotinamide).
[0275] Preferably, step (a) is carried out in the absence of the TGFp superfamily member.
[0277] The protocol described above can be continued by cultivating the cells obtained in step (b) in a medium comprising the first differentiating agent (eg nicotinamide), but devoid of a member of the TGFp superfamily (eg activin A) . This step is referred to herein as step (c).
[0279] The protocol described above is now described in more detail, with additional embodiments.
[0281] The differentiation process begins once sufficient amounts of ESC are obtained. They are normally removed from adherent cell culture (eg using collagenase A, dispase, TrypLE select, EDTA) and seeded on a non-adherent substrate (eg Hydrocell non-adherent cell culture plate) in the presence of nicotinamide (and absence of activin A). Exemplary concentrations of nicotinamide are between 1-100 mM, 5-50 mM, 5-20 mM, eg, 10 mM. Once the cells are seeded onto the non-adherent substrate, the cell culture can be referred to as cell suspension, preferably free-floating pools in suspension culture, i.e., aggregates of cells derived from human embryonic stem cells (hESCs). Cell clusters do not adhere to any substrate (eg, culture plate, carrier). Free-floating stem cell sources were previously described in WO 06/070370. This phase can be carried out for a minimum of 1 day, more preferably two days, three days, 1 week or even 10 days. Preferably, the cells are not cultured for more than 2 weeks in suspension together with the nicotinamide (and in the absence of the TGFp superfamily member, eg, activin A).
[0282] According to a preferred embodiment, when the cells are cultured on the non-adherent substrate, the atmospheric oxygen conditions are manipulated so that the percentage is equal to or less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%, less than about 7%, less than about 6%, and more preferably about 5% (for example, between 1% -20%, 1% -10 % or 0-5%).
[0284] Examples of non-adherent cell culture plates include those manufactured by Hydrocell (eg, Cat.
[0285] 174912), Nunc, etc.
[0287] Clusters typically comprise at least 50-500,000, 50-100,000, 50-50,000, 50-10,000, 50-5000, 50 1000 cells. According to one embodiment, the cells in the clusters are not layered and form irregular conformations. In one embodiment, the pools are devoid of pluripotent embryonic stem cells. In another embodiment, the pools comprise small amounts of pluripotent embryonic stem cells (eg, no more than 5%, or no more than 3% (eg, 0.01-2.7%) of cells that co-express OCT4. and TRA 1-60 at the protein level). Clusters typically comprise cells that have partially differentiated under the influence of nicotinamide. Such cells can express neural precursor markers such as PAX6. The cells also express markers of progenitors from other lineages such as, for example, alpha-feto protein, MIXL1 and Brachyuri.
[0289] Clusters can be dissociated using enzymatic or non-enzymatic (eg, mechanical) methods known in the art. According to one embodiment, the cells are dissociated so that they are no longer in clusters, for example aggregated or in groups of 2-100,000 cells, 2-50,000 cells, 2-10,000 cells, 2-5000 cells, 2-1000 cells, 2-500 cells, 2-100 cells, 2-50 cells. According to a particular embodiment, the cells are in a single cell suspension.
[0291] The cells (eg dissociated cells) are then seeded on an adherent substrate and cultured in the presence of nicotinamide, eg between 1-100 mM, 5-50 mM, 5-20 mM, eg 10 mM (and no activin A). This phase can be carried out for a minimum of 1 day, more preferably two days, three days, 1 week or even 14 days. Preferably, cells are not grown for more than 1 week in the presence of nicotinamide on adherent cell culture (and in the absence of activin).
[0293] Collectively, cells are normally exposed to nicotinamide (at concentrations between 1-100 mM, 5-50 mM, 5-20 mM, eg 10 mM) for about 2-3 weeks, and preferably no more than 4 weeks before the addition of the second differentiation factor (eg activin A).
[0295] Examples of adherent substrates include, but are not limited to, collagen, fibronectin, laminin (eg laminin 521).
[0297] Following the first phase of directed differentiation (i.e., culture in the presence of nicotinamide (e.g. 10 mM) under non-adherent culture conditions under low oxygen atmospheric conditions followed by cultivation on an adherent substrate in the presence of nicotinamide under atmospheric conditions of low oxygen), the semi-differentiated cells are then subjected to a further differentiation phase on the adherent substrate, culturing in the presence of nicotinamide (eg 10 mM) and activin A (eg 20-200 ng / ml, 100-200 ng / ml, for example 140 ng / ml, 150 ng / ml, 160 ng / ml or 180 ng / ml). This phase can be carried out for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks, one week to eight weeks, one week to four weeks, for example, for at least one week, at at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, or even eight weeks. Preferably, this phase is carried out for about two weeks. According to one embodiment, this differentiation phase is also carried out under low atmospheric oxygen conditions, that is, less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%. %, less than about 7%, less than about 6%, and more preferably about 5% (eg, between 1% -20%, 1% -10%, or 0-5%).
[0299] After the second phase of directed differentiation (i.e., culturing in the presence of nicotinamide and activin A on an adherent substrate), the additional differentiated cells can optionally undergo a subsequent phase of differentiation on the adherent substrate, culturing in the presence of nicotinamide (for example, between 1 100 mM, 5-50 mM, 5-20 mM, for example 10 mM), in the absence of activin A. This phase can be carried out for at least one day, 2 days, 3 days, 1 week, at least two weeks, at least three weeks, or even four weeks. Preferably, this phase is carried out for about a week. This differentiation phase can be effected under low atmospheric oxygen conditions (i.e., less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%, less than about 7%, less than about 6%, and more preferably about 5% (for example, between 1% -20%, 1% -10%, or 0-5%) or normal or a combination of both (that is, initially under low atmospheric oxygen conditions and later, when slightly pigmented cells are observed, under normal oxygen conditions).
[0300] According to a particular embodiment, when the atmospheric oxygen conditions return to normal atmospheric conditions, the cells are cultured for at least one more day (for example, up to two weeks) in the presence of nicotinamide (for example, 10 mM) and in the absence of activin A.
[0301] The basic medium according to the description is any cell culture medium known in the art to support cell growth in vitro, usually a medium comprising a defined base solution, including salts, sugars, amino acids and any other nutrient required for maintenance. of cells in culture in a viable state. Non-limiting examples of commercially available basic media that can be used as described include Nuristem (without bFGF and TGFp for ESC differentiation, with bFGF and TGFp for ESC expansion) Neurobasal ™, KO-DMEM, DMEM, DMEM / F12, system Lonza L7, mTeSR, StemPro, XF KSR, E8, Cellgro ™ Stem Cell Growth Medium, or XVivo ™. The basic medium can be supplemented with a variety of agents as is known in the art dealing with cell cultures. The following is a non-limiting reference to various supplements that may be included in the culture system to be used according to the present disclosure: - serum or with a medium containing serum replacement, such as, but not limited to, inactivating serum replacement ( KOSR), Nutridoma-CS, TCH ™, N2, derived from N2, or B27 or a combination;
[0302] - an extracellular matrix component (ECM), such as, but not limited to, fibronectin, laminin, collagen and gelatin. The ECM can then be used to carry the one or more members of the TGFP superfamily of growth factors;
[0303] - an antibacterial agent, such as, but not limited to, penicillin and streptomycin;
[0304] - non-essential amino acids (NEAA), neurotrophins known to play a role in promoting SC survival in culture, such as, but not limited to, BDNF, NT3, NT4.
[0305] According to a preferred embodiment, the medium used to differentiate ESCs is Nuristem medium (Biological Industries, 05-102-1A or 05-100-1A).
[0306] According to a particular embodiment, the differentiation of ESC is carried out under conditions free of foreign components. According to one embodiment, the proliferation / growth medium is devoid of foreign contaminants, ie, free of animal derived components such as serum, animal derived growth factors, and albumin. Therefore, according to this embodiment, the cultivation is carried out in the absence of foreign contaminants.
[0307] Other methods for growing ESC under conditions free of foreign components are provided in US Patent Application Publication No. 20130196369.
[0308] During the stages of differentiation, embryonic stem cells can be monitored to determine their differentiation status. Cellular differentiation can be determined upon examination of specific cell or tissue markers known to be indicative of differentiation.
[0309] Specific tissue / cell markers can be detected using immunological techniques well known in the art [Thomson JA et al., (1998). Science 282: 1145-7]. Examples include, but are not limited to, flow cytometry for membrane-bound or intracellular markers, immunohistochemistry for extracellular and intracellular markers, and enzyme immunoassay for secreted molecular markers (eg, PEDF).
[0310] Thus, according to another aspect of the present disclosure, a retinal epithelial cell generation method is provided comprising:
[0311] (a) cultivating pluripotent stem cells in medium comprising a differentiating agent to generate differentiating cells, wherein the medium is devoid of a member of the transforming growth factor P (TGF P) superfamily;
[0312] (b) culturing the differentiating cells in a medium comprising the transforming growth factor P (TGF P) superfamily member and the differentiating agent to generate cells that further differentiate toward the RPE lineage;
[0313] (c) analyzing pigment epithelium derived factor (PEDF) secretion from cells that further differentiate towards the RPE lineage; Y
[0314] (d) culturing cells that further differentiate toward the RPE lineage in medium comprising a differentiating agent to generate RPE cells, wherein the medium is devoid of a member of the transforming growth factor P superfamily ( TGF P), in which step (d) is carried out when the amount of PEDF is above a predetermined level.
[0315] Preferably, step (d) is carried out when the PEDF level is above 100 ng / ml / day, 200 ng / ml / day, 300 ng / ml / day, 400 ng / ml / day or 500 ng / ml /day.
[0316] Another method to determine the potency of cells during or after the differentiation process is by analyzing the barrier function and polarized secretion of PEDF and VEGF, as illustrated in Example 4, hereinafter.
[0317] Once the cells are promoted to RPE cells, they can be selected and / or expanded.
[0318] According to a particular embodiment, the selection is based on a negative selection, that is, the removal of non-RPE cells. This can be done mechanically by the removal of non-pigmented cells or the removal of non-polygonal cells or by the use of surface markers.
[0319] According to another embodiment, the selection is based on a positive selection, that is, selection based on morphology (eg, pigmented cells and / or polygonal cells). This can be done by visual analysis or the use of surface markers.
[0320] According to yet another embodiment, the selection is based first on a negative selection and then on a positive selection.
[0321] Cell expansion of the RPE can be effected in an extracellular matrix, for example gelatin, collagen or poly-D-lysine and laminin. For expansion, cells can be cultured in serum-free KOM, medium comprising serum (eg, DMEM 20%), or Nuristem medium (06-5102-01-1A from Biological Industries). Optionally, cells can be exposed to nicotinamide during the expansion phase, at concentrations between 1-100 mM, 50 mM, 5-20 mM, eg 10 mM. Under these culture conditions, the pigmented cells reduce pigmentation and acquire a fibroid-like morphology. After further prolonged cultivation and proliferation in high-density cultures, the cells regain the characteristic polygonal conformational morphology and preferably also the cell pigmentation of the RPE.
[0322] RPE cells can be expanded in suspension or in a monolayer. Expansion of RPE cells in monolayer cultures can be modified to large-scale expansion in bioreactors by methods well known to those of skill in the art.
[0323] The RPE cell population generated according to the methods described herein can be characterized according to several different parameters.
[0324] Thus, for example, the RPE cells obtained are polygonal in shape and pigmented.
[0325] According to one embodiment, at least 70%, 75%, 80%, 85%, 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% of the cells of the RPE cell populations obtained jointly express both premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP).
[0326] Upon administration, the cells described herein are capable of forming a monolayer (as illustrated in Figure 27C).
[0327] According to one embodiment, the transepithelial electrical resistance of cells in a monolayer is greater than 100 ohms. Preferably, the transepithelial electrical resistance of the cells is greater than 150, 200, 250, 300, 300, 400, 500, 600, 700, 800 or even greater than 900 ohms.
[0328] According to a particular embodiment, the TEER is between 100-1000 ohms, more preferably between 100-900 ohms, for example, between 200-900 ohms, 300-800 ohms, 300-700 ohms, 400-800 ohms or 400-700 ohms.
[0329] Devices for measuring transepithelial electrical resistance (TEER) are known in the art. An exemplary system for measuring TEER is illustrated in Figure 28.
[0330] It will be appreciated that the cell populations disclosed herein are devoid of undifferentiated human embryonic stem cells. According to one embodiment, less than 1: 250,000 cells are Oct4 + TRA-1-60 + cells, as measured, for example, by FACS. The cells also do not express or down-regulate the expression of GDF3 or TDGF relative to hESC as measured by PCR.
[0331] Another way to characterize the cell populations disclosed herein is by marker expression. Thus, for example, at least 80%, 85% or 90% of cells express bestrophin 1, as described measured by immunostaining. According to one embodiment, between 90-95% of the cells express bestrophin.
[0333] According to another embodiment, at least 80%, 85%, 87%, 89% or 90% of the cells express microphthalmia associated transcription factor (MITF), as measured by immunostaining. For example, between 85-95% of cells express MITF.
[0335] According to another embodiment, at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 87%, 89%, or 90% of the cells express gene paired box 6 (PAX-6) as measured by FACS.
[0337] The cells described herein can also be characterized according to the amount and / or type of factors they secrete. Thus, according to one embodiment, the cells preferably secrete more than 500, 750, 1000 or even 2000 ng of pigment epithelial derived factor (PEDF) per ml per day, (eg after 14 days in culture) as measured by ELISA.
[0339] It will be appreciated that the RPE cells generated herein secrete PEDF and vascular endothelial growth factor (VEGF) in a polarized manner. According to particular embodiments, the apical secretion ratio of PEDF: basal PEDF secretion is greater than 1. According to particular embodiments, the apical secretion ratio of PEDF: basal PEDF secretion is greater than 2. According to particular embodiments, the secretion ratio apical PEDF: basal secretion of PEDF is greater than 3. Furthermore, the ratio of basal secretion of VEGF: apical secretion of VEGF is greater than 1. According to particular embodiments, the ratio of basal secretion of VEGF: apical secretion of VEGF is greater 1.5, 2 or 2.5.
[0341] The cells of the present disclosure secrete additional factors including, for example, angiogenin, the immunomodulatory factors IL-6, sgp130, MIF, sTNF-R1, sTRAIL-R3, MCP-1 and osteoprotegerin, the extracellular matrix regulators TIMP-1. and TIMP-2 and the Axl protein.
[0343] According to another aspect, at least 80% of the cells of the cell population jointly express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP) and also a portion (at least 10%, 20%, 30 %, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of cells each secrete / release angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2) , soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor α (sTNF-R1).
[0345] It will be appreciated that in some cases all cells that jointly express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP) also secrete / release angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor a (sTNF-R1).
[0347] In other cases, the majority (more than 50%, 60%, 70%, 80, 90% of cells that jointly express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) also secrete / Free angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the receptor 1 for ubiquitous membrane tumor necrosis factor (sTNF-R1).
[0349] The RPE cells generated herein preferably secrete angiogenin, TIMP2, sgp130 and sTNF-R1 in a polarized manner.
[0351] According to particular embodiments, the ratio of apical sgp130 secretion: basal sgp130 secretion is greater than 1.
[0352] According to particular embodiments, the ratio of apical secretion of sgp130: basal secretion of sgp130 is greater than 2.
[0353] According to particular embodiments, the ratio of apical secretion of sgp130: basal secretion of sgp130 is greater than 3.
[0355] Furthermore, the apical ratio of sTNF-R1: baseline of sTNF-R1 is greater than 1. According to particular embodiments, the apical ratio of sTNF-R1: basal of sTNF-R1 is greater than 2. According to particular embodiments, the apical ratio of sTNF-R1: baseline sTNF-R1 is greater than 3.
[0357] Furthermore, the basal angiogenin secretion: apical angiogenin secretion ratio is greater than 1. According to particular embodiments, the basal angiogenin secretion: apical angiogenin secretion ratio is greater than 1.5, 2, 2.5, or 3.
[0359] Furthermore, the apical TIMP2 secretion ratio: basal TIMP2 secretion is greater than 1. According to particular embodiments, the apical TIMP2 secretion ratio: basal TIMP2 secretion is greater than 2. According to particular embodiments, the apical secretion ratio of TIMP2: baseline TIMP2 secretion is greater than 3.
[0361] Cell stability is another characteristic feature. Thus, for example, the amount of PEDF secretion remains stable in cells after incubation at 2-8 ° C for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours. Furthermore, the polarized secretion of PEDF and VEGF remains stable after incubation of cells at 2-8 ° C for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours. Furthermore, the TEER of cells remains stable in cells after incubation at 2-8 ° C for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours.
[0362] In another embodiment, the cells are characterized by their therapeutic effect. Thus, for example, the present inventors have demonstrated cell populations are capable of rescuing visual acuity in the rat from RCS after subretinal administration. Furthermore, cell populations are capable of rescuing photoreceptors (eg, cone photoreceptors) for up to 180 days (in some embodiments at least 180 days) after subretinal administration in the rat from RCS.
[0364] It will be appreciated by those skilled in the art that derivation of RPE cells is of great benefit. An in vitro model can be used for the development of new drugs to promote RPE cell survival, regeneration and function. RPE cells can serve for massive analytical screening for compounds that have a toxic or regenerative effect on RPE cells. They can be used to discover mechanisms, new genes, soluble or membrane-bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells.
[0366] RPE cells can also serve as an unlimited source of RPE cells for transplantation, replacement, and support of malfunctioning or degenerated RPE cells in retinal degenerations. Furthermore, the genetically modified RPE cells can serve as a vector to carry and express genes in the eye and retina after transplantation.
[0368] RPE cells produced by the method of the present disclosure can be used for large-scale and / or long-term culturing of such cells. For this purpose, the method of the description is to be carried out in bioreactors and / or cell culture systems suitable for large-scale cell production, and in which undifferentiated hSCs are to be cultured according to the description. General requirements for culturing cells in bioreactors and / or cell culture systems are well known to those skilled in the art.
[0370] Collection of the cells can be accomplished by various methods known in the art. Non-limiting examples include mechanical dissection and dissociation with papain or trypsin (eg, TrypLE select). Other methods known in the art are also applicable.
[0372] RPE cells generated as described herein can be transplanted to various target sites within the eye of a subject. According to one embodiment, the transplantation of the RPE cells is to the subretinal space of the eye, which is the normal anatomical location of the RPE (between the outer segments of the photoreceptor and the choroid). Furthermore, depending on the migratory capacity and / or positive paracrine effects of the cells, transplantation into additional ocular compartments can be considered to include the inner or outer retina, the retinal periphery, and within the choroid.
[0374] Retinal diseases that can be treated using the RPE cells described herein include, but are not limited to, retinitis pigmentosa, retinoschisis, reticular degeneration, Best's disease, and age-related macular degeneration (AMD).
[0376] Additionally, transplantation can be performed by various techniques known in the art. Methods for performing RPE transplants are described in, for example, US Patent Nos. 5,962,027, 6,045,791 and 5,941,250 and in Eye Graefes Arch Clin Exp Ophthalmol March 1997; 235 (3): 149-58; Biochem Biophys Res Commun 2000 Feb 24; 268 (3): 842-6; Opthalmic Surg 1991 Feb; 22 (2): 102-8. Methods for performing corneal transplants are described in, for example, US Patent No. 5,755,785, and in Eye 1995; 9 (Pt 6 Su): 6-12; Curr Opin Opthalmol 1992 Aug; 3 (4): 473-81; Ophthalmic Surg Lasers 1998 Apr; 29 (4): 305-8; Ophthalmology 2000 Apr; 107 (4): 719-24; and Jpn J Ophthalmol November-December 1999; 43 (6): 502-8. If primarily paracrine effects are to be used, the cells can also be delivered and kept in the eye encapsulated within a semi-permeable container, which will also reduce the cells' exposure to the host immune system (Neurotech USA CNTF Delivery System; PNAS 7 March 2006 vol. 103 (10) 3896-3901).
[0377] According to one embodiment, the transplantation is performed by means of vitrectomy surgery through the flat area followed by administration of the cells through a small retinal opening in the subretinal space or by direct injection. Alternatively, the cells can be delivered into the subretinal space by means of a transscleral, transchoroidal approach. In addition, direct transscleral injection into the vitreous space or administration to the anterior retinal periphery in proximity to the ciliary body can be performed.
[0379] RPE cells can be transplanted in a number of ways. For example, RPE cells can be introduced to the target site in the form of a cell suspension, or adhered to a matrix, extracellular matrix, or substrate such as a biodegradable polymer or a combination. RPE cells can also be transplanted (co-transplantation) with other retinal cells, such as photoreceptors.
[0381] Thus, the description also refers to RPE cell pharmaceutical compositions described herein. The composition is preferably one suitable for transplantation into the eye. Thus, for example, RPE cells can be formulated in an intraocular irrigating solution such as BSS plus ™.
[0382] It is expected that, during the life of a patent maturing from this application, many technologies relevant to the generation of RPE cells will be developed, and the term RPE cells is intended to include all of these new technologies a priori.
[0384] As used herein, the term "about" refers to ± 10%.
[0386] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".
[0388] The term "consisting of" means "including and limited to".
[0390] The term "consisting essentially of" means that the composition, method, or structure may include additional components, steps, and / or parts, but only if the additional components, steps, and / or parts do not substantially alter the new, basic characteristics. of the claimed composition, method or structure.
[0391] As used herein, the singular form "a", "an" and "the" includes plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.
[0393] Throughout this application, various embodiments of this invention may be presented in a slot format. It should be understood that the description in interval format is merely for convenience and conciseness and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of an interval should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that interval. For example, the description of a range such as from 1 to 6 should be considered to have specifically disclosed ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 up to 6, etc., as well as individual numbers within that range, for example 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.
[0395] As used herein, the term "method" refers to ways, means, techniques, and procedures to accomplish a given task including, but not limited to, those ways, means, techniques, and procedures either well known or readily known. developed from ways, means, techniques and procedures known to professionals in the chemical, pharmacological, biological, biochemical and medical techniques.
[0397] As used herein, the term "treating" includes nullifying, substantially inhibiting, slowing or reversing the evolution of a condition, substantially ameliorating clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of clinical or aesthetic symptoms of a condition. state.
[0399] It is appreciated that certain features of the invention, which are described, for clarity, in the context of different embodiments, may also be provided in combination in a single embodiment. Instead, various features of the invention, which are described, for clarity, in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as is suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
[0401] Various embodiments and aspects of the present invention as defined hereinbefore and as claimed in the claims section below, find experimental support in the following examples.
[0403] Examples
[0405] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.
[0407] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in US Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), third edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); Immunoassays available in the patent and scientific literature are extensively described, see, for example, US Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985); "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). Other general references are provided throughout this document. Procedures in this regard are believed to be well known in the art and are provided for the convenience of the reader.
[0409] Reference example 1
[0411] CRALBP / PMEL 17 Double Stain FACS Method Qualification
[0413] The objective of this study was to qualify the CRALBP / PMEL 17 double-stained FACS method by demonstrating the accuracy and precision of the method in a minimum of 6 independent known addition assays over at least 3 days of testing. Assay qualification was performed using the OpRegen® 5C batch as positive control cells and HAD-C 102-hESC as negative control cells. A calibration curve of known amounts of RPE (OpRegen® 5C) with known hESC additions was used to test the accuracy and precision at different points of known additions. Expected accuracy and precision were up to 25% at all points.
[0414] Staining protocol: Negative control hESC cells taken from a cryopreserved hESC bank (HAD-C 102 p48 4.5.2014) were thawed in Nutristem (containing HSA) according to sponsor protocols. Positive Control RPE Cell Pool: Cells from the OpRegen® 5C lot (reference line) were thawed in 20% DMEM-HS according to sponsor protocols. Thawed hESC OpRegen® 5C and HAD-C102 were centrifuged, resuspended in 1 ml of PBS (-), filtered through a 35 µm cell filter and counted with trypan blue. The cell concentration was adjusted to 0.73x106-106 cells / ml in PBS (-). 1 µl / ml FVS450 was added to each cell suspension followed by vortexing and incubation for 6 minutes at 37 ° C. FVS450 was washed with 0.1% BSA and resuspended in 0.1% BSA -Fc-block (5 min at RT) to block all Fc epitopes on the cells. The cells were then washed with PBS (-) and fixed in 80% methanol (5 min at 4 ° C). Fixed cells were washed once with PBS (-), once with 0.1% PBS-T, and permeabilized with 0.1% PBS-T (20 minutes at RT). The permeabilization solution was replaced with 10% normal goat serum (NGS) blocking solution (200,000 cells / 50 µl) for at least 30 minutes (max one hour) at RT. During the incubation time, quality sample tubes (QS) were prepared and, at the end of blocking, cells were divided and immunostained. Cells were incubated with primary antibodies for 30 min followed by 3 washes with 0.1% PBS-T and 30 min incubation with secondary antibodies and 3 washes with 0.1% PBS-T.
[0416] Negative control and positive control cells were stained with the FVS450 viability stain, fixed, blocked and permeabilized. A calibration curve of known numbers of cells of the positive control RPE (OpRegen® 5C) was then generated in negative control hESC, at 4 concentrations (25%, 50%, 75% and 95% RPE in hESC), based on the trypan blue viability cell count of each population. Negative and positive control cells and mixed populations were immunostained with monoclonal primary antibodies specific for the RPE markers CRALBP and PMEL 17, followed by staining with paired secondary antibodies (anti-mouse FITC antibody and anti-Alexa Fluor 647 antibody from rabbit, respectively). Stained cells were analyzed by FACS to measure the percentage of activated CRALBP + PMEL17 + cells of viable single cells.
[0418] RESULTS
[0420] Accuracy: Assay accuracy was determined from the test results of 4 levels of RPE with known additions (25%, 50%, 75%, and 95%). The accuracy of the RPE pool (OpRegen® 5C) was determined relative to being potentially 100% RPE cells. Each of the level values were analyzed by six independent series / determinations.
[0422] The 50% concentration level was considered to be the lower limit of quantification with an expected accuracy of up to 25% (the 50% level ranged from -8.41 to 20.14; the 75% and 99% levels , 5% ranged from -5.32 to 6.88).
[0424] These results meet the expected outcomes for relative bias of up to 25%, and indicate that the assay is accurate for the determination of double-positive CRALBP + PMEL17 + cells at concentrations ranging from 50-99.5%. Since OpRegen® 5C produces 99.5% of double positive CRALBP + PMEL17 + RPE cells, a relative bias of less than 25% cannot be guaranteed for a result> 99.5%.
[0425] Table 1
[0430] Intermediate precision: The intermediate precision of the assay was determined from the results of 6 tests carried out by one operator. In each trial, the percentage of individual viable RPEs was determined and the% CY was calculated from that. Table 2 summarizes the test results. As shown, the% CY for all concentration levels was below 20% and can be measured with adequate precision. The% CY for the 25%, 50%, 75%, 95% and 100% concentration levels of r Pe were 16.14%, 10.61%, 5.10% , 1.17% and 0.34%, respectively. These results meet the expected values for precision. The measured RPE percentage is within 20% of the expected value at all concentrations. These results indicate that the assay is accurate for the determination of RPE at concentrations ranging from 25-99.5%.
[0432] Table 2
[0435]
[0438] Repeatability: Sample repeatability was tested in 3 runs (# 2, # 3, and # 4) in which duplicate samples of SC OpRegen® were stained and acquired side by side. The results confirmed that the sample identity obtained within an experiment is repeatable and uniform throughout the samples.
[0440] Linearity / Range: As shown in Figure 1, linearity was measured using data that was found to be both accurate and precise. The regression coefficient between the target (with known additions) and measured results over the tested interval (50% -100%) was found to be 0.99. Therefore, the range of the method that demonstrates acceptable accuracy and precision and linearity is the range between 50% and 99.5% of RPE cells, which covers the expected range of samples tested.
[0442] Positive Control Cells: The provisional level of double positive CRALBP / PMEL17 cells was set equal to or greater than 95%.
[0444] Negative Control Cells: The provisional level of hESC double positive CRALBP / PMEL17 cells was set to equal to or less than 2%.
[0446] Stability: the results show that the stained samples are also stable at 4 ° C after one and 4 days, and the accuracy is maintained within the expected acceptance criteria, therefore data acquisition can be performed within 96 hours sample preparation.
[0448] conclusion
[0450] The results presented herein indicate that the disclosed method is qualified and suitable for its intended use of in vitro determination of RPE purity in the final OpRegen® product and at different stages in the OpRegen® production process, with Relative bias accuracy of <25% and% CV precision <20% in the 50% -99.5% range of RPE cells.
[0452] Reference example 2
[0454] OpRegen® Purity Level Assessment
[0456] A FACS-based method was developed to assess the level of purity of human retinal pigment epithelium (RPE) cells as well as non-RPE cell impurities in RPE cells. The cellular retinaldehyde binding protein (CRALBP), one of the components of the visual cycle, was bioinformatically identified as a unique marker for mature RPE cells. Preliminary studies using CRALBP-specific monoclonal antibody have shown a purity of above 98% in RPE cells generated according to methods described herein. These results were further supported by immunostaining for PMEL17, a melanosome marker found in RPE. Furthermore, unlike some specific RPE markers, CRALBP is not expressed in melanocytes, a possible cellular contamination of the neural crest.
[0457] Test sample and controls: Human primary melanocytes (ATCC, PCS-200-013) were used as negative control cells for CRALBP and as positive control cells for PMEL17, melanosome-enriched type I transmembrane glycoprotein (melanin granules). HADC102-hESC in P29 (OpRegen® parental line) were used, as negative control cells for both CRALBP and PMEL17. Clinical grade OpRegen® cells (lot 2A) and research grade OpRegen® cells (produced in GMP as sham production; IV D16 sham production) were used as samples tested. Cells were generated as described in Example 3.
[0459] Immunostaining and FACS analysis: cells were thawed and stained using fixable viability stain (FVS450) (BD 562247), fixed with 80% methanol, immunostained with mouse anti-CRALBP primary antibody (clone B2, Abcam ab15051), or its isotype control for mouse IgG2a (Abcam ab170191) and rabbit anti-human PMEL17 antibody (clone EPR4864, Abcam abl37062) followed by goat anti-mouse (Dako F0479) and goat anti-rabbit secondary antibodies (Jackson 111-606-144), respectively.
[0461] FACS data acquisition was performed using a validated Navios flow cytometer (Beckman Coulter) and analysis was performed using FlowJo 7.6.
[0463] RESULTS
[0465] Initial FACS data using anti-CRALBP monoclonal antibody showed that the purity level of OpRegen® is above 98%. Melanocytes, which are a potential neural crest contaminant, were found to be negative for the single RPE specific marker CRALBP (1.7%). The parenta1HADC102-hESC line was negative for CRALBP (0.2%), as expected.
[0466] The OpRegen® purity level remained above 98% after double staining with CRALBP and PMEL17 (Figure 10). The melanocytes stained positive for PMEL17, as expected, but negative for the double-labeled population (~ 1%). HADC102-hESC stained negative for CRALBP and PMEL17 (0.07%).
[0468] Reference example 3
[0470] Description of the manufacturing procedure and process controls
[0472] OpRegen® is manufactured from the foreign-free GMP quality hESC HAD-C 102 line grown in GMP-quality human umbilical cord fibroblast feeders free of irradiated foreign components. Human fibroblast feeder cell lines of clinical quality (CRD008; MCB) and working cell banks (WCB) were produced under Good Manufacturing Practice (GMP) conditions and free of foreign components, tested, characterized, and tested. deposited in banks appropriately. These were then used in the derivation of the clinical grade hESC line HAD-C 102 from surplus human blastocysts under GMP conditions and free of foreign components.
[0474] In the initial phase of production, hESCs are expanded in irradiated feeders as colonies. They are then transferred to suspension culture to initiate targeted differentiation. Spheroid bodies (SB) are formed and then plated as an adherent culture under conditions of directed differentiation continuing toward a neural target and subsequently into RPE cells. At the end of the differentiation phase, non-pigmented areas are physically removed and the pigmented cells are harvested, seeded, and enzymatically expanded. Purified hESC-derived RPE cells (DS) are harvested in step 2 and immediately processed to DP. The duration of the manufacturing process depends on the growth rate of hESC (~ 2 months from thawing) and in total it usually covers more than 4-5 months.
[0476] Each stage of the manufacturing procedure, including in-process quality control (QC) testing, is briefly described below.
[0478] Stages 1-3: Generation of human umbilical cord fibroblast ( WCB) feeder working cell bank . A human umbilical cord feeder master cell bank (MCB) vial (CRD008-MCB) was thawed in step 3-4, expanded in Dulbecco's Modified Eagle's Medium (DMEM, SH30081.01, Hyclone) supplemented with serum 20% human (14-498E, Lonza), was irradiated (gamma cell, 220 Exel, MDS Nordion 3,500 rads) and preserved in steps 7-8 to generate the working cell banks (WCB). Prior to cryopreservation, feeder cell culture samples were tested for sterility, mycoplasma, and Limulus amoebocyte lysate (LAL), morphology, karyotype, cell number, and viability. Furthermore, upon thawing, its identity to MCB, its inability to proliferate, and its ability to support the growth of HAD-C102-hESC was confirmed indifferently. If the WCB passed all the QC tests, the bank was released for hESC expansion.
[0480] Production steps 1-3 are depicted in Figure 12.
[0482] Stages 4-5: hECS expansion. A single vial of the human umbilical cord fibroblast WCB (either CRD008-WCB8 or CRD008-WCB9) was thawed and seeded in recombinant human gelatin coated center well plates (RhG100-001, Fibrogen) at a concentration of 70,000 -100,000 cells / ml / plate in DMEM (SH30081.01, Hyclone) supplemented with 20% human serum (14-498E, Lonza). Cells were incubated overnight at 37 ° C, 5% CO ² to allow fibroblasts to attach. 1-4 days later, a sample of the HAD-C102-hESC MCB was thawed and seeded for 6-7 days at 37 ° C, 5% CO ² on top of the feeder cells in Nutristem “Plus medium. ”(Which is GMP grade and free of foreign components) containing the growth factors bFGF and TGF-p (05-102-1A, Biological Industries, Israel). On day 6-7, the hESC culture was mechanically altered (using a sterile tip or disposable sterile stem cell tool; 14602 Swemed) and transferred to freshly prepared additional plates containing feeder cells at a concentration of 70,000-100,000. cells / plate. This was repeated weekly for several steps to achieve the required amount of hESC to initiate differentiation (Figure 13, steps 4-5). Prior to use, the expanded HAD-C102-hESCs were tested for sterility, mycoplasma, LAL, karyotype, and identity for MCB. Furthermore, its pluripotent morphological appearance as well as its unified expression of pluripotency markers (TRA-1-60, Oct4 and alkaline phosphatase) were confirmed (Figure 2, step 5). Production steps 4-5 are depicted in Figure 13.
[0484] Stages 6-13: Differentiation to RPE cells. Collagenase expanded HAD-C102-hESC (4152, Worthington) were enzymatically treated for further expansion in 6 cm cell culture plates (Figure 14, step 6). Expanded HADC102-hESCs were then used in the derivation of the OpRegen® DS.
[0486] Differentiation of each batch of OpRegen® was initiated by mechanical transfer of collected HAD-C102-hESC collagenase A pools from the stage 6 culture to feeder-free non-adherent 6 cm Hydrocell culture plates in the presence of medium. Nutristem “Minus” (which does not contain bFGF growth factors or TGF-P; 06-5102-01-1A Biological Industries, special order) supplemented with 10 mM nicotinamide (N-5535, Sigma) (Figure 14, step 7). The plates were then cultured for up to one week under conditions of a low oxygen atmosphere (5%) (37 ° C, 5% CO ² ) to allow the generation of spheroidal bodies. One week old suspended spheroidal bodies were then collected, gently dissociated by pipetting, and transferred to 6-well plates coated with human laminin (511, Biolamin) for an additional week of growth in a low oxygen atmosphere (5% ) in the presence of Nutristem "Minus" medium supplemented with 10 mM nicotinamide (Figure 14, step 8). Cells continued to grow in a low oxygen atmosphere (5%) for up to an additional 4 weeks; two weeks in the presence of Nutristem "Minus" medium supplemented with 10 mM nicotinamide and activin A 140 ng / ml (G-120-14E, Peprotech) (figure 14, step 9), followed by up to 2 weeks in the presence of Nutristem medium " Minus ”supplemented only with 10 mM nicotinamide (Figure 14, step 10). When areas of light pigmentation became evident in polygonal cell patches, the plates were transferred back to a normal oxygen atmosphere (20%) (37 ° C, 5% CO ² ) and grown for up to 2 weeks in the presence of Nutristem "Minus" medium with 10 mM nicotinamide (Figure 14, step 11). After up to 2 weeks, expanded polygonal patches with distinctive pigmentation were evident within areas of non-pigmented cells (Figure 14, step 12) and the remaining pigmented cells were detached and manually harvested after 15 minutes of TrypLE Select (12563- 011, Invitrogen) at 37 ° C (Figure 14, step 13). Production steps 6-13 are depicted in Figure 14.
[0488] Stages 14-17: OpRegen® cell expansion. Pigmented cells were then transferred to gelatin-coated 6-well plates (0.5-1x106 cells / plate; P0) for 2-3 days of growth in the presence of DMEM (SH30081.01, Hyclone) supplemented with 20% human serum. % (14-498E, Lonza) (figure 15, stage 14). DMEM was then replaced with Nutristem "Minus" medium and the cells were grown for 2-3 weeks until the plate was covered with lightly pigmented polygonal cells (Figure 15, step 14). These P0 cells were then expanded in gelatin-covered flasks for two additional steps (P1, P2). Cells in P0 and in P1 were harvested after treatment with TrypLE Select at 37 ° C, washed and cultured for 2-3 days in gelatin-coated flasks in the presence of DMEM supplemented with 20% human serum. DMEM was replaced with Nutristem "Minus" medium and the cells were grown for 2-3 weeks until the plate was covered with lightly pigmented polygonal cells (Figure 15, steps 15-16). Cells in P2 that were grown in T175 flasks were then harvested after TrypLE Select treatment at 37 ° C, resuspended in DMEM supplemented with 20% human serum, pooled, and counted.
[0490] A sample of the growth medium was taken from each batch to determine sterility, mycoplasma, and LAL tests. Cell morphology was observed and documented (Figure 15, step 17). Production steps 14-17 are depicted in Figure 15.
[0492] Reference example 4
[0494] Process control points
[0496] The IPC points are depicted in Figure 16. The sampling points chosen to evaluate hESC impurity and RPE purity in the production procedure are described below:
[0498] IPC point 1: HAD-C102 mechanically expanded hESC prior to differentiation having normal karyotype. This is the starting material where the highest level of hESC is expected. This point was added to assess the peak hESC level prior to differentiation.
[0500] IPC point 2: hESC HAD-C 102 expanded with collagenase before differentiation. At this stage, some differentiation is expected and therefore a reduction in the level of cells expressing Oct4 and TRA-1-60 as well as in the level of expression of GDF3 and TDGF. This point was added to evaluate an hESC impurity during the undirected differentiation phase.
[0502] IPC point 3: spheroid bodies produced one week after induction of hESC differentiation under feeder-free conditions in the presence of nicotinamide. At this earliest phase of differentiation, hESC impurity is expected during differentiation at the maximum level, and therefore this evaluation is expected to provide an indication of the highest level of safety concern.
[0504] IPC point 4: cells at the end of activin A treatment. Activin A directs differentiation towards RPE cells. At this point, a large decrease in hESC impurity and a large increase in the expression of RPE markers are expected. This point was added to monitor the differentiation of hESC to RPE.
[0506] IPC points 5-7: cells at the end of the differentiation process before and after the separation of the non-pigmented areas (IPC point 6) from the pigmented areas (IPC point 7). IPC points 5 and 6 are expected to contain cellular impurities, while sample 7 represents the product at the end of the differentiation process before expansion. Cell contaminations found in sample 6 can be found in small amounts in sample 7, and in smaller amounts in the product.
[0507] IPC point 8: pigmented cells at P0. Pigmented cells at the end of the differentiation process that expanded for 2-3 weeks. These cells represent the two phases of the product before the end of the production process.
[0509] IPC point 9: pigmented cells in P1. P0 cells that expanded for 2-3 weeks. These cells represent the product one phase before the end of the production process.
[0511] IPC point 10: cells pigmented in P2 before cryopreservation. P1 cells that expanded for 2-3 weeks are harvested and pooled. These cells represent the active principle (DS) before cryopreservation.
[0513] IPC point 11: P2 cryopreserved pigmented cells. These cells represent the finished product (DP). In all production, at all sampling points, cell culture medium was collected for evaluation of the secretion of pigment epithelium derived factor (PEDF), which is known to be secreted from RPE cells.
[0515] RESULTS
[0517] Quantification of TRA-1-60 + Oct4 + hESC. The level of hESC was determined in the various samples collected in the production procedure using a robust and highly sensitive Oct4 / TRA-1-60 double staining FACS method. One week after the withdrawal of feeders and growth factors that support the growth of pluripotent cells (TGFp and bFGF), under growth conditions that support early neural / ocular field differentiation, there was only 0.0106-2.7% of TRA-1-60 + Oct4 + cells (point of IPC 3, spheroid bodies). After the addition of activin A that promotes RPE differentiation, the level of TRA-1-60 + Oct4 + cells was further reduced to 0.00048-0.0168% (IPC point 4, activin end), and at At the end of differentiation after excision of non-pigmented cells, the level of TRA-1-60 + Oct4 + cells was 0.00033-0.03754% (IPC point 7, pigmented cells). At P0, two phases before the end of the production procedure, TRA-1-60 + Oct4 + cells were detected at levels of 0.00009-0.00108% (below the LOD-close to LLOQ) (IPC point 8) . The levels of TRA-1-60 + Oct4 + cells in P1 (IPC point 9), P2 before cryopreservation (active principle; IPC point 10) and P2 after cryopreservation (DP; IPC point 11) were below of the LLOQ of the assay (i.e. 0.00004-0.00047%, 0.00000-0.00016% and 0.00000-0.00020%, respectively).
[0519] Relative Expression of the hESC Pluripotency Markers GDF3 and TDGF: The relative expression of the GDF3 and TDGF pluripotency genes at the various IPC sites in the production procedure was analyzed. There was a gradual reduction in the level of expression of GDF3 and TDGF, which was correlated with the gradual reduction in the numbers of TRA-1-60 + Oct4 + cells, in the differentiation process. At the end of P0, two phases before the end of the production procedure, P1, and P2 before (active principle) and after (finished product) cryopreservation, the expression levels of GDF3 and TDGF were similar to the expression level observed in OpRegen® 5C negative control cells.
[0521] Quantification of CRALBP + PMEL17 + cells: CRALBP + PMEL17 + cells were evaluated for the measurement of RPE purity at the end of the differentiation phase, they were evaluated at P0 and P2 (IPC points 8 and 11, respectively). As can be seen in table 3 and in figure 17, the purity level of CRALBP + PMEL17 + of the RPE at P0 (IPC point 8), two phases before the end of the production procedure, was in the range of 98, 53-98.83%. A similar RPE purity level was detected in P2 after cryopreservation (99.61-99.76%; IPC point 11) (Table 3).
[0523] Table 3
[0527] Confocal imaging of cells immunostained with bestrophin 1, MITF, and CRALBP in sham production runs 4 and 5: cells were immunostained for RPE markers bestrophin 1, MITF, ZO-1, and CRALBP at the end of the differentiation phase (IPC point 7), at the end of the expansion phase (IPC point 10, DS) and after cryopreservation (IPC point 11, d P). Manually isolated non-pigmented cells (IPC point 6) were seeded for immunostaining, but during fixation they detached from the plate and thus could not be stained. Selected pigmented cells (IPC point 7) seeded for 12 days (in sham production 5 only, paralleling cells at P0 from ongoing production) and for 28 days stained positive for all RPE markers tested and the percentage of cells expressing bestrophin 1 and MITF was 93% and 93.3-96.5%, respectively. Similar levels of bestrophin 1 and MITF positive cells were detected in P0 (94.9% and 95.9%, respectively; tested only in sham production 4), P2 prior to cryopreservation, active ingredient (the 92.2-92.75% and 93.7-95.5%, respectively) and P2 after cryopreservation, finished product (91.1-95.7% and 83.8-94.9%, respectively; immunostaining with reduced MITF in sham production 5 demonstrates an outlier of the area randomly selected for analysis). CRALBP expression (as well as ZO-1) was detected in all IPC samples 7, 10 and 11 (Figure 18).
[0529] Relative expression of the RPE bestrophin 1, CRALBP and RPE65 markers in mock productions 2, 4 and 5: the relative expression of the RPE bestrophin 1, CRALBP and RPE65 genes was measured at the various IPC sites in the production procedure . There was a gradual increase in the level of relative expression of bestrophin 1, CRALBP and RPE65 in the production procedure. At the end of the treatment with activin A (IPC point 4), which directs differentiation towards RPE cells, the relative levels of bestrophin 1, CRALBP and RPE65 were 685, 36 and 325, respectively, times higher compared to their levels. Relative values in hESC that were mechanically passed prior to differentiation (IPC point 1; sham production 4). The relative expression levels of bestrophin 1, CRALBP and RPE65 peaked from the end of the differentiation phase (IPC 5 points) to the P1 phase (IPC 9 point). In these phases the respective expression levels were 5,838-11,841, 211-299 and 5,708-8,687 times higher compared to the levels in hESC that were mechanically passed before differentiation (IPC point 1).
[0531] Morphology evaluation in simulated productions 4 and 5: cells were analyzed for morphology at the end of the differentiation phase (IPC point 5) for the estimation of the relative areas of pigmented cells and, in the expansion phases P0-P2 (IPC points 8-10), to verify the confluent polygonal morphology. The estimated relative pigmented cell area at the end of the differentiation phase before excision of the non-pigmented areas (IPC point 5) was 32.5% ± 13.5% (mean ± SD, n = 7 wells of one 6-well plate) in sham production 4 and 60% ± 13% in sham production 5 (mean ± SD, n = 7 wells of a 6-well plate) (see representative images in Figure 11). Areas of pigmented cells were selected and expanded. The morphology at the end of the expansion phases P0 (point of IPC 8), P1 (point of IPC 9) and P2 (point of IPC 10) demonstrated a densely concentrated culture with an epithelial monolayer morphology with typical polygonal conformation (Figure 11 ).
[0533] PEDF secretion and potency measurement in mock productions 4 and 5: Pigment epithelium derived factor (PEDF), which is known to be secreted from RPE cells, was measured in cell culture medium at various points of IPC in simulated productions 4 and 5. As can be seen in table 4, very low levels of PEDF were secreted, in the range of 4-79 ng / ml / day, by hESC (IPC points 1 and 2) and by spheroidal bodies (IPC point 3; end of first week with nicotinamide). At the end of treatment with activin A (IPC point 4), which directs differentiation towards RPE cells, the level of secreted PEDF was in the range of 682-1038 ng / ml / day, 31-37 times higher compared to the level secreted by spheroid bodies. After incubation of cells under normal oxygen conditions with nicotinamide (IPC point 5), a further increase (2.2-4.6-fold) in PEDF secretion was observed to 1,482-4,746 ng / ml / day. During the expansion phase (P0-P2, IPC 8-10, respectively), the levels of secreted PEDF were in the range of 2,187-8,681 ng / ml / day, peaking at P0-P1.
[0535] Table 4: PEDF secretion in simulated productions 4 and 5.
[0537] The tight junctions generated between RPE cells allow the generation of the blood-retinal barrier and a polarized secretion of PEDF and VEGF. PEDF is secreted to the apical side where it acts as a neurotropic and antiangiogenic growth factor. VEGF is secreted primarily to the basal side, where it acts as a proangiogenic growth factor in the choroidal endothelium. RPE polarization (barrier function and polarized secretion of PEDF and VEGF) was measured in a Transwelll system at the end of P0 (IPC point 8), end of P2 before cryopreservation (IPC point 10) and end of P2 after cryopreservation (IPC point 11). As can be seen in Table 5, transepithelial electrical resistance / barrier function (TEER) and polarized secretion of PEDF and VEGF were demonstrated at all IPC sites.
[0539] Table 5
[0544] Batch release test of RPE cells produced in sham production runs 4 and 5: to verify that OpRegen® produced in sham production runs 4 and 5 is comparable to OpRegen® produced by BPF, abbreviated as OpRegen®, carried out a batch release test that included tests of morphology at the end of P2 before cryopreservation (IPC point 10, DS), and viability, total cell number / cryovial, identity (expression of bestrophin 1 and MITF) , hESC impurity and karyotyping at the end of P2 after cryopreservation (IPC point 11, DP). OpRegen® produced in simulated production runs 4 and 5 passed the batch release criteria. OpRegen® produced in Sham Production Run 2 were not cryopreserved and therefore could not be tested.
[0546] CONCLUSION
[0548] Three sham production runs (sham production runs 2, 4 and 5) were carried out under research quality conditions using the same GMP production methods, GMP quality cells free of foreign components (hESC HAD-C 102 which were grown on irradiated CRD008 feeders), quality GMP reagents free from foreign components, and quality GMP labware that were used in the production of GMP from clinical batches. Simulated productions 2, 4 and 5 aimed to assess the level of hESC impurity in production and simulated productions 4 and 5 also aimed to identify important in-process quality controls.
[0550] Using a qualified TRA-1-60 / Oct4 double stain FACS method (LOD 0.0004%, 1 / 250,000 and LLOQ 0.001%, 1 / 100,000) and a qualified flow cytometer, hESC impurity was observed at the level below the LOD of the assay at the end of the differentiation phase, in negatively selected pigmented cells, three phases before the end of the sham production procedure 5. In sham production runs 2 and 4, performed before the Assay qualification using a main facility flow cytometer, the hESC impurity level was below the assay LOD two stages before the end of the production procedure. In support of these data, quantitative RT-PCR analysis demonstrated down-regulated expression of the pluripotent hESC genes GDF3 and TDGF to levels similar to those of the negative control (OpRegen® 5C cells) two phases before the end of the production procedure. .
[0552] Identity tests performed three phases before the end of production (isolation of pigmented cells) showed expression of bestrophin 1 and MITF in 93% and 96.5% of the immunostained cells, respectively, as well as expression of CRALBP and ZO- 1 (not quantized). RPE purity tests performed at a later stage (i.e., P0, 2 stages before the end of the production procedure), after a cycle of expansion of the negatively selected pigmented cells, showed that> 98.5% of the cells were CRALBP + PMEL17 + double positive by FACS. A similar level of RPE purity (ie> 99.6%) was also detected in the finished product. These results were supported by morphology tests demonstrating epithelial monolayer morphology with typical polygonal conformation and by quantitative RT-PCR analysis demonstrating the upregulated expression of r Pe bestrophin 1, CRALBP and RPE65 genes to levels similar to those of the positive control (OpRegen® 5C cells).
[0554] PEDF, which is known to be secreted from RPE cells, was measured in cell culture medium at various stages in the mock production runs 4 and 5 procedure. At the end of activin A treatment (IPC point 4, previously shown by Idelson et al. 2009), to direct differentiation towards RPE cells, the level of secreted PEDF was greatly increased (31-fold in sham production 4 and 37-fold in sham production 5) relative to the stage of previous production (induction of spheroidal bodies). PEDF secretion levels continued to increase and peaked at P0-P1 (1.7-5.8-fold increase relative to levels after activin A). Assessment of the relative areas of pigmented cells at the end of the differentiation process (IPC point 5) was identified as another important quality control measure for the assessment of RPE differentiation. Using this measure, a 2-fold difference in pigmented cell yield was observed in sham production runs 4 and 5 (32.5% in sham production 4 and 60% in sham production 5), which was correlated with a similar difference observed in PEDF secretion at this stage (1,482 ng / ml / day in sham production 4 and 4,746 ng / ml / day in sham production 5).
[0556] In conclusion, no TRA-1-60 + Oct4 + hESC impurity was observed as early as 3 stages before the end of the production procedure. This correlated with low expression levels of GDF3 and TDGF, high expression levels of bestrophin 1, CRALBP and RPE65, and high levels of bestrophin 1 and MITF single positive cells, as well as high content of double positive CRALBP + PMEL17 + cells. (tested one phase later). Important safety and efficacy IPCs were identified in critical production phases.
[0558] Reference example 5
[0559] Effectiveness evaluation
[0561] Experimental system: the present inventors examined whether subretinal transplantation of RPE cells generated as described in Example 4 could delay RDD evolution in the Royal College of Surgeons (RCS) rat model.
[0563] 25,000, 100,000 or 200,000 RPE cells were transplanted into the subretinal space of a RCS rat eye on the day after birth (P) 21-23 (before photoreceptor death onset); BSS + (Alcon) -treated and untreated animals served as controls. The groups were separated into 4 survival ages: day after birth P60, P100, P150 and P200. Background photography was used to identify blister formation and monitor injection quality. Ophthalmoscopy was also performed on P60, P100, P150 and P200. Optomotor tracking was used to measure visual acuity of all animals at all time points (P60, P100, P150, P200).
[0565] Focal and full-field ERGs were evaluated in all study groups at P60 and P100. On the assigned sacrifice date for each animal, both eyes were excised, fixed in 4% paraformaldehyde, cryopreserved, embedded in optimum cut-off temperature (OCT) compound, and cryosectioned. Cresyl violet staining was used to identify and enumerate photoreceptor structural rescue. Immunofluorescent (IF) staining was used to identify transplanted cells, assess their fate, their proliferation status, and their ability to phagocytose outer photoreceptor segments. In addition, immunofluorescence was used in the measurement of host cone rescue.
[0567] The study design is summarized in Table 6 below in this document.
[0569] Table 6
[0574] MATERIALS AND METHODS
[0576] Cell Counts: Cells were counted before aliquoting at appropriate dosage concentrations. Cell viability prior to injection for all injection time points averaged 94.0% ± 0.03. Cell viability after injection averaged 92.4% ± 0.02.
[0578] Surgery: A small incision was made through the conjunctiva and sclera using gradually smaller gauge needles: 18, 22, 25 and 30. A puncture on the lateral margin of the cornea was used to reduce intraocular pressure, to reduce the output of the injected cells. The glass pipette was then inserted into the subretinal space and 2 µl of suspension was injected. The sclerotomy was then sutured. Successful injection of the cells or the buffer alone (b Ss) was first confirmed by manual visualization of a subretinal bleb, which was subsequently photographed using a fundus camera (Micron III).
[0580] Optokinetic follow-up thresholds: Optokinetic follow-up thresholds were measured and recorded blind. Repeated measures ANOVA or one-way ANOVA with Fisher's LSD post hoc analysis were used to analyze the OKT data.
[0582] Electroretinogram ( ERG): Two forms of ERG were measured: an exploratory form of focal ERG in which a small spot of light is used to stimulate a localized area of the retina, and a conventional style of full-field ERG in which it is stimulated entire visual field.
[0584] Histology and Immunohistochemistry: The two eyes of each animal were collected, fixed, cryoprotected, embedded and frozen. The frozen blocks were cryosectioned at 12 µm. Approximately 60 slides were obtained containing 4 sections per slide.
[0586] Cresyl Violet: Cresyl Violet stained sections were examined for: 1) injection site and suture, 2) evidence of photoreceptor rescue, 3) evidence of transplanted cells, 4) adverse pathology. For each slide, the maximum thickness of the outer nuclear layer was also recorded for quantification of salvage.
[0587] Immunofluorescence ( IF): RPE cell treated eye slides selected for IF were chosen from cresyl violet stained sections containing cells in the subretinal space according to the size and morphology of the transplanted human cells. Furthermore, protection of the host's ONL was used as a secondary criterion. All IF staining was performed as dual stains with DAPI serving as background nuclear staining. At least one slide from each cell-treated animal was used for each series.
[0589] Run # 1 was performed using rabbit anti-melanoma gp100 monoclonal antibody (PMEL17, clone EPR4864; human specific, Abcam cat # ab137062) stained in conjunction with mouse monoclonal marker anti-nuclei (HuNu, clone 3E1 .3, Millipore, cat # MAB4383) to detect human RPE and non-RPE cells.
[0591] Run # 2 was made using rabbit anti-Ki67 monoclonal antibody (Ki67; clone EPR3610, human specific, Abcam, cat # ab92742) and anti-nuclei marker to detect proliferating human cells.
[0593] Run # 3 was made using polyclonal rabbit anti-rat cone arrestin antibody (Millipore cat # ab15282) to evaluate sections for cone count (see section 6.8.3). In addition, selected slides were stained using mouse anti-rhodopsin monoclonal antibody (Rho 1D4 clone, Millipore, MAB5356) in combination with PMEL17 to identify transplanted human cells containing rhodopsin / host outer segments as a measure of their phagocytic activity.
[0595] Cone count: z-stack confocal images were acquired from retinal sections obtained from all cell transplanted eyes and from saline injected controls of the same age. Cell injected eye sections were chosen in the photoreceptor rescue area as defined using previously evaluated cresyl violet stained sections. 3 observers counted the cones blindly. They then averaged the three counts, and the counts were compared between the dosage and age groups.
[0596] Ingestion of rhodopsin: a possible rescue mechanism employed by transplanted cells is to ingest outer segments of photoreceptors and release residues. Removal of the debris site reduces toxic stress on photoreceptors and thus helps maintain photoreceptor survival. In this case, the present inventors selected specific animals for evaluation of rhodopsin ingestion by RPE cells based on indices of cell survival and photoreceptor protection. This evaluation was carried out by immunofluorescence.
[0598] RESULTS
[0600] Fundus imaging: fundus images collected at necropsy of cell-treated eyes revealed hyper- and hypopigmented areas of the retina corresponding to the location where the subretinal bullae formed during surgery; the location where the cells were deposited in the subretinal space (Figures 19A-C). These irregular areas were not evident in the eyes injected or not injected with BSS +.
[0602] Optokinetic follow-up thresholds: OKT thresholds were rescued in all cell-treated groups at all ages (Figure 20). Cell-treated groups outperformed saline-injected or non-operated eyes at all ages. There was a significant dose-dependent effect between the low dose (25K) and the two largest doses (100K (p <0.0001) and 200K (p <0.0001)), especially at later ages, but not observed no apparent benefit for the high dose (200K) over the intermediate dose (100K) OKT (p = 0.5646). Although OKT thresholds were rescued in all cell-treated groups, absolute visual acuity values slowly decreased over time. The OKT thresholds of the untreated and saline-injected animals continued to decrease over the course of the study. The BSS + injected eyes were not different from the untreated group (p = 0.6068) and the other untreated eyes.
[0604] Focal ERGs: Focal ERGs were measured in all (n = 252) experimental rats at ~ P60. Individual animals treated with RPE cells performed well and significantly outperformed controls, as illustrated in Figure 21A.
[0606] Full-field ERGs: The full-field ERGs of 125 RCS rats at P60 and 63 RCS rats were measured at PlOo. Individual animals treated with RPE cells performed well and significantly outperformed controls, as illustrated in Figure 21B.
[0608] Cresyl violet staining: An exemplary photomontage of a cresyl violet stained section is shown in Figure 22A. Representative images of BSS + injected and cell-treated eyes (multi-cluster images) are presented in Figure 22B.
[0610] The thickness of the outer nuclear layer (ONL) was measured as a primary indicator of photoreceptor rescue. The data they were recorded as the maximum number of photoreceptor nuclei present in each dose group over the ages (Figure 23). Cell-treated groups had significantly greater ONL thickness at P60, P100, and P150 (all p <0.0001) than BSS + treated eyes. Regarding the percentage of animals with evidence of photoreceptor rescue, 76-92% of the animals in P60, 80-90% in P100, 72-86% in P150 and 0-18% in P200 had evidence of photoreceptor.
[0612] Immunofluorescence: The transplanted RPE cells were positively identified by immunofluorescence in animals of each survival age (FIG. 24), however, the number of animals with identified cells decreased with increasing age. Repeating the staining of additional slides in animals that originally did not reveal transplanted cells resulted in additional animals identified with positive cells, but not in all cases.
[0614] Despite not finding transplanted cells in all animals by IF analysis, the results of the ONL thickness measurement indicated that 70-90% of the cell-treated animals had significant photoreceptor rescue, confirmed by rescue of OKT, suggesting that most of the treated eyes contained transplanted cells at some point. The proliferation marker Ki67 was used to identify proliferating human cells. No Ki67 positive human cells were observed (Figure 24).
[0616] Cone Count: Cone counts in animals that received cell transplants were significantly better than control eyes (Figure 25; p = <0.0001 for each comparison). Overall, there was no difference between cone counts at low, medium, and high cell dose. A representative image of each age is presented in figure 24.
[0618] Rhodopsin ingestion: In each case tested (n = 6), fluorescently labeled rhodopsin was observed within the transplanted RPE cells (Figures 26A-J). This confirms that the transplanted cells ingest residues from the outer segment after transplantation.
[0620] conclusion
[0622] When transplanted into the subretinal space of RCS rats, RPE cells rescued visual acuity in the RCS rat relative to controls at all ages tested. ERG responses were protected when the graft was large enough or in an area of the retina accessible for evaluation. Rod and cone photoreceptors were rescued in the graft area for up to 180 days after transplantation. Taken together, these data demonstrate that OpRegen® maintains the functional and structural integrity of the host's retina for extended periods. Thus, OpRegen® has significant potential for the treatment of disorders of human RPE cells such as rP and AMD.
[0624] Reference example 6
[0626] RPE cell stability
[0628] Short term stability
[0630] The RPE cells formulated (generated as described in Example 4) in BSS plus were prepared to a final volume of 600-1000 µl per vial. Short term stability was tested at time points 0, 4, 8 and 24 hours. The cells were stable at all time points.
[0632] Cell viability and RPE cell concentration were stable at the 8 hour incubation time point for all dose formulations; average viability percentage (± SD) for the following concentrations:
[0634] • Low concentration (70x103 per 100 µl of BSS plus) changed from 93% ± 5 at the 0 hour time point to 91% ± 1 at the 8 hour time point, a non-significant decrease.
[0636] • High concentration (70x103 per 100 µl of BSS plus) changed from 92% ± 3 at the 0 hour time point to 91% ± 2 at the 8 hour time point, a non-significant decrease.
[0638] For the mean concentration (250x103 per 100 µl BSS plus) that was tested, there were no significant changes over time points.
[0640] The global interval for all time points and formulated doses was between 88% - 97% from the 0 hour time point to 8 hours, obtaining the average of all the results for the 0 hour time point (93 % ± 3) and the time point 8 hours (91% ± 1) a decrease of 2% was found.
[0642] No significant changes were observed in cell concentration, neither in time points nor in formulated doses. Cell concentration did not change in the 3 studies apart from a small decrease observed in one batch in the high dose (2%).
[0644] The appearance of the different dosage formulations did not change over the time points tested; the cell suspension was free of foreign particles and undissociated aggregates.
[0646] The identity and purity of each dose of formulated RPE cells at all time points tested were stable up to 24 hours and were within the batch release criteria. At 8 hours (for all formulated RPE cell doses), the level of positive cells for MITF and bestrophin was in the range of 86-97% and 90-94%, respectively, and the level of CRALBP + cells Double positive PMEL17 + was in the range of 98.35-99.64%.
[0648] The formulated RPE cell doses maintained their potency at all time points tested (4, 8, 24 hours), both by secreting high levels of PEDF and by forming a polarized RPE monolayer with predominantly polarized PEDF secretion in the apical side and VEGF on the basal side. Results for time points tested 8 hours: TEER was in the range of 376 - 724 ohms, ratio of apical to basal PEDF in the range of 2.77 - 5.70 and ratio of basal to VEGF to apical in the range of 2.04-3.88.
[0650] Sterility was maintained at all incubation time points for all cell dose formulations.
[0652] These results support the cellular stability of OpRegen® in the final formulation at all clinical doses for at least 8 hours when kept at 2-8 ° C. There is a safety margin of up to 24 hours based on the partial data collected (identity, sterility and potency of mean dose).
[0654] The results of the short-term stability test are summarized in Table 7 below.
[0656] Table 7
[0661] Long-term stability: Three batches of RPE cells were frozen in liquid nitrogen in the vapor phase. Long-term stability testing in cryopreservation began after the freezing date. The results provided are after three years of freezing. The following parameters are being tested: viability, number of cells, RPE identity (% bestrophin 1 positive cells and% MITF), RPE purity (% FACS, CRALBP + p Me L17 + RPE cells), potency (polarization and secretion of PEDF), karyotype analysis and sterility. At each time point, the required number of vials are thawed and cells are prepared for assays as described herein.
[0662] The results of the long-term stability test are summarized in Table 8 below.
[0664] Table 8
[0669] RESULTS
[0671] Viability, total number of cells / vial, and RPE identity were maintained over the three-year period. Furthermore, as indicated, the data demonstrated potency and purity at levels similar to those collected prior to storage.
[0673] A normal karyotype was observed 4 years after cryopreservation. This indicates that long-term vapor-phase storage thus far had no detrimental effect on the genomic stability of RPE.
[0675] The sterility of the sample was demonstrated by testing for the absence of bacterial / fungal growth in all clinical lots at 3 months. Another batch was negative 4 years after cryopreservation. Based on these uniformly acceptable stability results, which cover a three-year stability test period so far, it is concluded that the RPE cellular product is stable for at least three years when stored at <-180 ° C. in the vapor phase of liquid nitrogen.
[0677] Reference example 7
[0679] Safety and biodistribution
[0681] The objectives of the study were to evaluate the survival, biodistribution and safety of RPE cells (generated as described in Example 4) after subretinal administration in male and female NOD-SCID mice during a study period of 6 months. .
[0683] NOD-SCID (NOD.CB17-Prkdcscid) mice, 5-6 weeks old at the time of injection, were injected with either BSS Plus (vehicle control) or two doses of RPE cells: 50x103 cells or 100x103 cells (maximum feasible dose), suspended in 1 µl of BSS Plus. The RPE was delivered to the subretinal via the transvitreal route (the proposed clinical route of administration) using a Hamilton 33G needle. A single dose of 50x103 cells or 100x103 cells was injected into one eye, while the other eye served as an internal control. Each dosing session contained mice (male and female) from each group. The mice included in the study after the preliminary test were randomly assigned to the various test groups. Two randomizations were performed. A measured value randomization procedure, by weight, was used for placement into treatment groups prior to vehicle / test article administration. Following administration, animals suitable for use in the study were transferred to the target study using sequential randomization for placement in the final treatment groups. Mice with ocular abnormalities, abnormal clinical observations, or weighing less than 16 grams in the preliminary test, and mice that underwent an unsuccessful subretinal injection of RPE were excluded from the study.
[0685] Study Measurements: RPE safety assessment in this study was based on mortality, clinical observations, body weight, ophthalmic examinations, clinical pathology (hematology and blood chemistry), gross evaluations of general pathology, organ weights (absolute and relative to body and brain weight), histopathological evaluation of eyes and various organs of animals. Survival assessment and RPE biodistribution was performed by histopathological and fluorescence immunostaining evaluations of eyes and various organs and qPCR analysis. The following measurements were made:
[0687] • clinical observation;
[0689] • body weight;
[0691] • ophthalmological examinations (including macroscopic and biomicroscopic examinations);
[0693] • quality surgical microscopic examination of the subretinal injection using the LEICA M80 Stereo microscope (ophthalmoscopy);
[0695] • complete blood count and blood chemistry;
[0697] • necropsy and general pathology;
[0699] • organ weight (absolute and relative to body and brain weights);
[0701] • collection, fixation and paraffin blocking of treated and untreated contralateral eyes, including the optic nerve;
[0703] • blind H&E histopathology of eyes and tissues (sternum bone with bone marrow, brain, heart, kidneys, liver, lung, mandibular lymph nodes, spinal cord, spleen, thymus, masses and general lesions);
[0705] • Blind semiquantification of pigmented cells on H&E stained slides;
[0707] • Blind immunostaining of selected slides adjacent to a representative H&E slide demonstrating pigmented cell graft to the eye for a human marker (human nuclei) plus an RPE marker (human PMEL17) and evaluation of human RPE and non-human cells. of the RPE, human marker (human nuclei) plus a proliferation marker (human Ki67) and evaluation of human and non-human proliferative cells, and RPE marker (RPE65) plus proliferation marker (human Ki67) and evaluation of human proliferative cells of the RPE and non-RPE;
[0709] • blind immunostaining of selected slides adjacent to a representative H&E slide demonstrating teratoma, tumor, abnormal cells and lesions for a human marker (human nuclei) to exclude human origin;
[0711] • collection and extraction of genomic DNA from blood, bone marrow (collection of femurs), brain, left and right eyes with optic nerves, heart, left and right kidneys, liver, lung, mandibular lymph nodes, ovaries, skeletal crural biceps muscle, spinal cord, spleen, testes, and thymus and human beta-globin qPCR analysis;
[0713] • histopathology with H&E in tissues (other than the above) was positive for human beta-globin in animals of the same group and time point.
[0715] RESULTS
[0717] There were no RPE-related toxicological findings in live examinations that included detailed clinical observation, body weight, ophthalmological examination, and clinical pathology composed of hematology and serum clinical chemistry. The observation of "discolored, dark eye" in the left eye with an albino background was found in mice treated with pigmented RPE cells at both dose levels on detailed clinical observation and ophthalmological examination. Ophthalmological examination of the surviving animals indicated that this observation consisted of medium-vitreous, dark pigmentation foci. The pigmented foci were randomly distributed along a line extending from the temporal posterior lens capsule to the nasal retinal surface. These foci were interpreted to be RPE cells that escaped from the injection cannula upon removal from the eye after injection, as supported by vitreous reflux observed during injection or RPE cells that leak into the vitreous after injection. subretinal implant.
[0719] All ocular injuries observed in this study were considered to have arisen as a consequence of anesthesia, the surgical injection procedure, or incidentally as age-related changes. The finding of multiple pigmented foci within the vitreous suggests that RPE cells may be viable within the vitreous body. The presence of pigmented cells in the vitreous body in some of the RPE-treated animals was confirmed at the microscopic level.
[0720] Regarding biodistribution as assessed by qPCR using a set of human beta-globin gene probes / primers, at intervals of 2 weeks, 2 months and 6 months, the left eyes treated with 100x103 OpRegen® cells were positive for RPE DNA in 8/12, 11/12, and 16/16 animals with group mean levels at 38, 47 and 249 copies / | ig of total ocular DNA, respectively, indicating an increasing trend with time. There was no significant difference between males and females. In these animals, RPE DNA was not detected in untreated right eyes and in all nonocular tissues, including blood, femoral bone marrow, brain, heart, kidneys, liver, lung, mandibular lymph nodes, ovaries, biceps muscle. skeletal crural, spinal cord, spleen, testes, and thymus, except the spinal cord (27 / | ig DNA copies) of a 2-week-old male animal and skeletal muscle (16 DNA copies / ^ g) and spinal cord ( below grade level) from a 2 week old female animal (probably due to inadvertent contamination by exogenous human DNA during DNA extraction from these tissues).
[0722] Gross changes related to RPE were limited to black discoloration or black foci in the left eye of some animals at intervals of 2 and 6 months, according to clinical observation and / or ophthalmological examination in life. These changes were correlated with pigmented cells and were not considered adverse as determined by microscopic examination of surviving animals in the high dose group and animals killed in extremis and found dead in both dose groups. Pigmented cells were present in the treated left eye in almost all surviving mice examined at each time point in the high dose group (in the subretinal space at 11/12, 12/12 and 16/16 at 2 week intervals , 2 months and 6 months), as well as animals slaughtered in extremis or found dead in both low and high dose groups. The most common locations of pigmented cells were the subretinal space and the vitreous body as confirmed by immunostaining for human cell-specific biomarkers and RPE. In the subretinal space, pigmented cells tended to be restricted to the injection site at earlier time points, while at later time points they were present at distant sites from injection sites, suggesting local cell spread. There was a slight increase in the average total number of pigmented cells per eye at the 6-month time point compared to the 2-week or 2-month time points in males. This increase in the number of pigmented cells of human origin was supported by qPCR analysis.
[0724] Long-term engraftment of RPE cells is illustrated in Figure 27A. Pigmented cells stained positive for human nuclei and PMEL17 in the subretinal space of NOD-SCID 9 months after transplantation.
[0726] Figure 27B is a photograph illustrating clumping at the blister site after injection. Figure 27C is a photograph illustrating the subsequent propagation of cells in a monolayer after injection.
[0727] RPE was not associated with any change in organ weight. There were no gross or microscopic changes in the untreated right eyes and non-ocular organs examined in this study, which included the brain, heart, kidneys, liver, lung, mandibular lymph nodes, spinal cord, spleen, and thymus. Anti-human nuclei biomarker antibody staining (human nuclei) was observed in 64%, 36%, and 73% of the left eyes tested at the time points of 2 weeks, 2 months, and 6 months, respectively. , in the animals examined in the high dose group.
[0729] The highest level of detection for human nuclei was observed in pigmented cell populations within the subretinal space followed by the vitreous body. RPE-specific anti-human biomarker antibody PMEL17 staining was observed in most of the animals tested, while another RPE-specific biomarker, RPE65, had varying levels of detection at different time points. These RPE-specific biomarkers were detected mainly in the subretinal space and less in the vitreous body. The human cell proliferation biomarker Ki67 was detected in only a few cells in a small number of animals, mainly in pigmented cells within the vitreous body and less within the subretinal space. The incidence of Ki67 positivity decreased over time with only one animal at 6 months. Ki67 positive cells were not associated with any abnormal morphology.
[0731] Various microscopic injection site changes were observed at all time points and all study groups, and were considered related to the surgical injection procedure. Some of these changes were slightly more prominent in the animals examined in the high dose group at 6 months. For example, retinal detachment was observed in one animal and the incidence or severity of retinal degeneration / atrophy or fibroplasia was slightly increased compared to the vehicle control group.
[0733] There were no RPE-dependent effects on the mortality and survival rate of the animals.
[0735] conclusion
[0737] No local or systemic toxicological, lethal or tumorigenic effects were observed in the NOD / SCID animal model during the 6 month study period after single injection of RPE at dose levels up to 100,000 cells / µl / eye. RPE cell biodistribution was restricted to the treated left eye with local spread of subretinal cells from the subretinal injection site as a function of time. RPE cells were predominantly present in the subretinal space followed by the vitreous body in most of the animals examined in the high dose group at intervals of 2 weeks, 2 months and 6 months, with variable positivity on immunostaining for antibodies against human nuclei and / or RPE-specific human biomarkers. The persistence of RPE cells in the eye was estimated to be at least 6 months with very limited cell proliferation. The limited proliferation took place mainly in the vitreous body and had no adverse effects. There was evidence that the number of RPE cells increased in the treated eye over time, although this was accompanied by a decrease in the incidence of proliferation in the subretinal population examined. The expression of both RPE-specific markers, RPE65 and PMEL17, was predominantly in RPE cells within the subretinal space compared to those of the vitreous body, where the majority of Ki67-positive cell incidences were found. The latter suggests that the increase in RPE cells over time is limited to the vitreous space and that the expression of specific RPE markers, RPE65 and PMEL17, may be regulated by the microenvironment. In conclusion, based on the data presented above, there are no serious safety concerns related to the injection of the currently described RPE cells compared to the vehicle control group.
[0738] Reference Example 8
[0739] Pax-6 expression in RPE cells
[0740] Objective: development of a method based on FACS to evaluate the level of PAX-6 in cells of the human retinal pigment epithelium (RPE).
[0741] MATERIALS AND METHODS
[0742] Frozen RPE cells (generated as described in Example 4) were thawed, centrifuged, resuspended in 1 ml of PBS minus, filtered through a 35 µm cell filter and counted with the NC-200 cells. Cell concentration was adjusted to ~ 1x10 6 cells / ml in minus PBS. 1 µl / ml FVS450 was added to each ml of cell suspension followed by vortexing and incubation for 6 minutes at 37 ° C. FVS450 was quenched with 0.1% BSA (- Ig) -PBS minus, and resuspended in 0.1% BSA (- Ig) -Fc-block (5 min at RT) to block all Fc epitopes in cells. The cells were then fixed and stained with anti-Pax-6 antibody (AF647, cat # 562249).
[0743] RESULTS
[0744] As can be seen in Figure 29, cells in P0 and P2 are positive for PAX6 (81.5% -82.5% in P0 and 91.3% -96.1% in P2). P2 is the step at the end of the production procedure and P0 is two previous expansion phases. The data was shown to be consistent between batches, as shown in Figures 29 and 30. Furthermore, the present inventors showed by FACS analysis that RPE cells double-stained for PAX-6 and CRALBP (Figure 31).
[0745] Reference example 9
[0746] Identification of proteins secreted by RPE cells
[0747] Objective: to identify a signature of proteins (known and new) secreted by OpRegen® (RPE cells) that can be used as a batch release potency assay, as well as a procedural control assay. The supernatants were collected from RPE cells (generated as described in example 3) that were cultured under different culture conditions indicated below. The supernatants were then selected using the RayBiotech G6 and G7 matrices according to the manufacturer's instructions after an overnight incubation of the supernatants with the related matrix.
[0748] 1. Post-thaw finished product RPE cells grown for 4 and 14 days in a 12-well plate (0.5x10 6 cells / well in step 3) (referred to herein as OpRegen®).
[0749] 2. Cells of the finished product RPE after thawing cultured for 14 days in a 12-well plate and then cultured for 3 weeks in a Transwell (according to AM-RPE-15) and demonstrated a TEER> 500 Q. The supernatants were taken of the apical and basal chambers.34
[0750] 3. Cells generated according to the protocol described in example 3, before (QC3) and after (QC4) treatment with activin A.
[0751] 4. Nutristem medium (Nut-) without addition of TGFp or FGF.
[0752] Supernatants were also collected from the following cell cultures and tested by ELISA:
[0753] 1. OpRegen® cells of finished product after thawing that were each cultured for 14 days in a 12-well plate and then cultured for 3 weeks in a Transwell (according to AM-RPE-15) and demonstrated a TEER of 355 Q and 505 Q, respectively. The supernatants were taken from day 14 (step 3) and from the apical and basal chambers.
[0754] 2. Post-thaw RPE 7 cells that were cultured for 14 days in a 12-well plate (0.5x10 6 cells / well in step 3).
[0755] 3. Mock production cells VI at the end of step 1 of the production procedure that were grown on Laminin-521 after enzymatic or mechanical isolation (as described in example 3). These cells were tested for potency according to AM-RPE-15 and cell supernatants were collected on day 14 in a 12-well plate (step 2) and cells after 3 weeks in a Transwell of the chambers. apical and basal.
[0756] 4. Fetal HuRPE cells at step 3, days 4 and 14 (0.5x106 cells / well).
[0757] Validation of the test by ELISA was performed according to the manufacturer's instructions related to each ELISA kit. In each protocol, the incubation with the supernatants was overnight.
[0758] Study design: supernatants were collected from cells that were grown under different culture conditions and kept at -80 ° C. After analysis of the protein matrix, the validation of the hits was measured by ELISA.
[0759] RESULTS
[0760] The results for matrix G7 are provided in Table 9 below.
[0761] Table 9
[0765] The results for matrix G6 are provided in Table 10 below. Table 10
[0766]
[0769] The proteins secreted by the RPE can be divided into 3 functional groups: 1) angiogenic proteins such as VEGF and angiogenin, 2) extracellular matrix regulators such as TIMP-1 and TIMP-2 and 3) immunomodulatory proteins such as IL-6, MIF, sgp130, sTNF-R1, sTRAIL-R3, MCP-1 and osteoprotegerin. The receptor tyrosine kinase Axl was also found to be secreted by RPE cells. 6 proteins that demonstrated high levels of secretion and / or demonstrated a polarized secretion pattern (apical / basal) were selected for validation by ELISA (angiogenin, TIMP-2, MIF, sgp130, stNf-R1 and sTRAIL-R3). The matrix data also demonstrated VEGF secretion as seen in the polarization assay.
[0771] Angiogenin: Protein matrix data demonstrated increased angiogenin secretion throughout the production procedure (Tables 9 and 10). These results were confirmed by ELISA demonstrating that the level of angiogenin secreted by the differentiating cells that were treated with nicotinamide before the addition of activin A was 0.52 ng / ml, while after 2 weeks of treatment with nicotinamide and activin A, the level of angiogenin secretion increased to 0.91 ng / ml (Figure 32A). RPE cells that were cultured for 2 weeks in a 12-well plate (0.5x10 6 cells / well; step 3) after thawing secreted angiogenin (Figure 32B). Polarized RPE cells (week 3 in Transwell; TEER> 350Q, apical / basal PEDF ratios and basal / apical VEGF> 1) secreted angiogenin in a polarized manner to the basal side with little or no secretion to the apical side (the levels of baseline were in the range of 0.1-0.25 ng / ml and apical angiogenin levels in the range of 0.05-0.12 ng / ml; Figure 32B). The RPE 7 cells generated according to Idelson et al., 2009 could not generate the barrier function in the Transwell system (TEER below 100 Q), although they could secrete VEGF and PEDF. The ability of RPE7 cells to secrete angiogenin was tested when placed in a 12-well plate for 14 days. RPE7 secreted angiogenin on day 14 of culture at a level that is within the range of RPE cells generated as described herein (Figure 32C).
[0773] TIMP-1 and TIMP-2 Secretion: Detection of the protein matrix demonstrated secretion of TIMP-1 and TIMP-2 from polarized and nonpolarized RPE cells (Figure 33A-E). Interestingly, the matrix data showed polarized secretion of TIMP-2 towards the apical side and TIMP-1 towards the basal side (Figure 33A). ELISA data confirmed that TIMP-2 is secreted primarily on the apical side by all batches of RPE tested so far (Figures 33C-D, apical range 69.9 - 113.3 ng / ml and baseline range of 11.9-43.7 ng / ml). T iMP-2 was also secreted by nonpolarized OpRegen® cells at levels similar to levels secreted by normal human fetal RPE cells (HuRPE, ScienCell) (Figures 33C-E). RPE 7 cells also secreted TIMP-2 at levels similar to OpRegen® cells (Figures 33C-E). Interestingly, very low levels of TIMP-2 were detected throughout the production procedure at the QC3 and QC4 checkpoints (Figure 33B).
[0774] Secretion of sgp130 by OpRegen® Cells: Protein matrix data demonstrated increased secretion of sgp130 throughout the OpRegen® production procedure as seen in IPC / QC checkpoints 3 and 4 (Tables 9 and 10). ELISA data confirmed higher levels of sgp130 secretion after 2 weeks of activin A treatment (IPC / QC4; 1.64 ng / ml) compared to levels secreted by cells after nicotinamide treatment before addition of activin A (IPC / QC3; 0.68 ng / ml) (Figure 34A). OpRegen® cells that were cultured for 2 weeks in a 12-well plate (0.5x106 cells / well; step 3) after thawing secreted sgp130 (Figures 34B-C). RPE 7 cells grown under similar conditions secreted sgp130 at levels that were within the range of OpRegen® cells (1.0 ng / ml on day 14; Figure 34D). Fetal HuRPE cells secreted low levels of sgp130 on both day 4 and day 14.
[0776] Polarized OpRegen® cells secreted sgp130 in a polarized manner towards the apical side with little or no secretion towards the basal side (apical sgp130 secretion levels were between 0.93-2.06 ng / ml and baseline sgp130 levels were in the range of 0-0.2 ng / ml; Figures 34B-C).
[0778] sTNF-R1 released: Very low levels of sTNF-R1 released were detected by ELISA in the supernatant of differentiating cells before (IPC / QC3 0.01 ng / ml) and after two weeks of treatment with nicotinamide and activin A ( IPC / QC4 0.02 ng / ml) (Figure 35A). OpRegen® cells that were cultured for 2 weeks in a 12-well plate (0.5x106 cells / well; step 3) after thawing contained sTNF-R1 in the culture day 14 supernatant (Figures 35B-C). HuRPE cells grown under similar conditions had similar levels of sTNF-R1 in their culture supernatant, while RPE 7 cells demonstrated relatively low levels of sTNF-R1 (Figure 35D).
[0780] Polarized OpRegen® cells secreted sTNF-R1 released at higher levels to the apical side (apical and basal sTNF-R1 levels were in the range of 0.22-1.83 ng / ml and 0.01-0.11 ng / ml, respectively; Figures 35C-D).
[0782] sTRAIL-R3: Protein array data detected sTRAIL-R3 in the supernatant of OpRegen® cells (Tables 9 and 10). The ELISA confirmed the presence of sTRAIL-R3 throughout the OpRegen® production procedure (493 pg / ml in QC3 and 238 pg / ml in Qc 4). In the fetal HuRPE culture there was no sTRAIL-R3 and in the RPE 7 culture, very low levels of sTRAIL-R3 (4 pg / ml).
[0784] Detection of MIF: Protein matrix data detected MIF in OpRegen® cell supernatant (Tables 9 and 10). The ELISA confirmed the presence of MIF throughout the OpRegen® production procedure (100.3 ng / ml in QC3 and 44.7 ng / ml in QC4). Polarized OpRegen® cells demonstrated higher levels of MIF on the apical side (apical MIF levels in the range of 26.6-138.3 ng / ml and baseline in the range of 1.9-30.5 ng / ml) .
[0785] Reference Example 10
[0787] OpRegen® compared to RPE1 and RPE7
[0789] Objective: to compare OpRegen® (RPE cells) with RPE cells generated according to the protocol of Idelson et al, 2009.
[0791] MATERIALS AND METHODS
[0793] OpRegen® (RPE cells) were generated as described in Example 3.
[0795] RPE cells were generated according to the protocol of Idelson et al, 2009 and named RPE1 and RPE7.
[0797] A Transwell system (as illustrated in Figure 28) was used to allow the development of a polarized RPE monolayer with stable barrier properties and polarized secretion of PEDF and VEGF. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of the RPE monolayer, and enzyme-linked immunosorbent assay (ELISA) was used to assess polarized secretion of PEDF and VEGF. The cells were thawed and cultured for 14 days in the presence of nicotinamide. PEDF secretion was tested on days 7 and 14. The cells were then transferred to a Transwell (Costar 3460, 0.4 | im) for an additional 4 weeks during which TEER was measured and medium was collected (for assessment of cytokine secretion) from the upper and lower Transwell chambers weekly up to 4 weeks. When cells are polarized, TEER must be above 100 Q and the ratio between apical to basal PEDF secretion and basal to apical VEGF secretion must be above 1.
[0799] All the OpRegen® batches that were tested demonstrated the ability to generate a barrier function (TEER range 368-688 Q) and secrete PEDF and VEGF in a polarized manner (the apical / basal PEDF ratio ranged from 3, 47-8.75 and basal / apical VEGF ratio of 1.39-2.74) (see Table 11).
[0801] T ab la 11
[0802]
[0805] ND: not determined since TEER was below 100 Q and large holes were observed in the culture.
[0806] RPE1 and RP7, which were produced under GMP conditions according to Idelson et al (2009), could not generate the barrier function (TEER <100 Q) in 3 independent studies. Cells seeded in the Transwell could not generate a homogeneous closed polygonal monolayer and large holes were observed (Figure 36). Although the cells could not generate the barrier function, RPE1 and RPE7 were able to secrete PEDF (see Table 11) and VEGF (not shown) at levels similar to OpRegen® and their CRALBP + PMEL17 + purity level was 99.91% and 96.29%, respectively, similar to OpRegen® (Figure 37).
[0808] Based on these data, it can be concluded that RPE1 and RPE7 are defective in their ability to generate tight junctions.
[0810] Although the invention has been described in conjunction with specific embodiments thereof, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art.
[0812] Furthermore, the citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

权利要求:
Claims (23)
[1]
i. Population of human polygonal retinal pigment epithelium (RPE) cells, in which at least 95% of the cells of the same cell express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) together, in which the resistance transepithelial electrical cell population is greater than 100 ohms.
[2]
2. Human retinal pigment epithelium (RPE) cell population, in which at least 80% of the cells of the same cell express premelanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP) jointly and in which the Population cells each secrete angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgpl30), and soluble form of the ubiquitous membrane receptor for tumor necrosis factor α (sTNF-R1).
[3]
3. The cell population according to claim 1, wherein the cells of the population each secrete angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgpl30) and soluble form of the ubiquitous membrane receptor 1 for the tumor necrosis factor a (sTNF-R1).
[4]
4. Cell population according to claim 2, wherein the cells secrete said angiogenin, said TIMP2, said sgpl30 or said sTNF-R1 in a polarized manner.
[5]
5. Cell population according to claim 4, wherein the ratio of apical sgpl30 secretion: basal sgpl30 secretion is greater than 1.
[6]
Cell population according to claim 4, wherein the ratio of basal angiogenin secretion: apical angiogenin secretion is greater than 1.
[7]
Cell population according to claim 4, in which the apical TIMP2 secretion: basal TIMP2 secretion ratio is greater than 1.
[8]
8. Cell population according to claim 1, wherein the number of Oct4 + TRA-1-60 + cells in the population is below 1: 250,000.
[9]
9. Cell population according to claim 1, wherein
(i) at least 80% of the cells express bestrophin 1, as measured by immunostaining;
(ii) at least 80% of the cells express microphthalmia-associated transcription factor (MITF), as measured by immunostaining;
(iii) more than 50% of cells express paired box gene 6 (PAX-6) as measured by FACS; (iv) cells secrete more than 750 ng of pigment epithelial derived factor (PEDF) per ml per day, and / or (v) the ratio of apical PEDF secretion: basal PEDF secretion is greater than 1.
[10]
10. Cell population according to claim 2, wherein the transepithelial electrical resistance of the cell population is greater than 100 ohms.
[11]
11. Cell population according to claim 1, which is generated by:
(a) cultivating human embryonic stem cells in a medium comprising nicotinamide to generate differentiating cells, wherein said medium is devoid of activin A;
(b) culturing said differentiating cells in a medium comprising nicotinamide and activin A to generate cells that further differentiate towards the RPE lineage; Y
(c) culturing said cells that further differentiate towards the RPE lineage in a medium comprising nicotinamide, wherein said medium is devoid of activin A.
[12]
A cell population according to claim 11, wherein steps (a) - (c) are performed under conditions where the atmospheric oxygen level is less than about 10%.
[13]
13. Pharmaceutical composition comprising the cell population according to claim 1 as an active agent and a pharmaceutically acceptable carrier.
[14]
14. Cell population according to claim 1, for use in the treatment of a degeneration of the retina.
[15]
15. Method of generation of retinal pigment epithelium (REP) cells, comprising:
(a) cultivating pluripotent stem cells in a medium comprising a differentiating agent to generate differentiating cells, wherein said medium is devoid of a member of the transforming growth factor p (TGF p) superfamily;
(b) culturing said differentiating cells in a medium comprising said member of the transforming growth factor p (TGF p) superfamily and said differentiating agent to generate cells that further differentiate toward the RPE lineage;
(c) culturing said cells that further differentiate toward the RPE lineage in a medium comprising a differentiating agent to generate RPE cells, wherein said medium is devoid of a member of the transforming growth factor p superfamily ( TGF p), wherein steps (a) - (c) are performed under conditions where the atmospheric oxygen level is less than about 10%; Y
(d) expanding said RPE cells.
[16]
16. Method according to claim 15, wherein said RPE cells are expanded in step (d) in suspension or in a monolayer.
[17]
Method according to any one of claims 15 or 16, in which the expansion of said RPE cells in step (d) is carried out in an extracellular matrix.
[18]
18. Method according to any one of claims 15 to 17, wherein the differentiating agent is selected from the group consisting of nicotinamide (NA) and 3-aminobenzamide.
[19]
19. Method according to claim 15, wherein step (a) comprises:
i) culturing said cultured population of human pluripotent stem cells in a medium comprising nicotinamide, in the absence of activin A; under non-adherent conditions to generate a cell cluster comprising differentiating cells; and subsequently
ii) culturing said cells in differentiating from (i) in a medium comprising nicotinamide, in the absence of activin A under adherent conditions.
[20]
20. The method of claim 19, further comprising culturing said differentiated cells in a medium under conditions in which the level of atmospheric oxygen is greater than about 10% in the presence of a differentiating agent after step (c).
[21]
21. A method according to any one of claims 15 to 20, wherein said member of the transforming growth factor p (TGF p) superfamily is selected from the group consisting of TGFp1, TGFp3 and activin A.
[22]
Method according to any one of claims 15 to 21, further comprising selecting polygonal cells after step (c).
[23]
23. The method according to claim 19, wherein said pluripotent stem cells comprise embryonic stem cells, and said embryonic stem cells are propagated in a medium comprising bFGF and TGFp.

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IL273405D0|2020-05-31|

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EA201791416A1|2017-11-30|

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JP2018502576A|2018-02-01|

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HK1246348A1|2018-09-07|

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KR20170115514A|2017-10-17|

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DK3240892T3|2020-02-17|

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EP3240892B9|2020-07-22|

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SG11201705379VA|2017-08-30|

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IL253238D0|2017-08-31|

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ES2774456T3|2020-07-21|

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BR112017014345A2|2018-01-02|

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US20210332325A1|2021-10-28|

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PH12017501218A1|2018-01-29|

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CA2972575A1|2016-07-07|

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EP3240892A1|2017-11-08|

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IL253238A|2020-04-30|

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WO2016108239A9|2017-08-10|

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JP6882981B2|2021-06-02|

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DK3240892T5|2020-10-19|

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EP3674397A1|2020-07-01|

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AU2015373050A1|2017-07-13|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201462097753P| true| 2014-12-30|2014-12-30|

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US201562116972P| true| 2015-02-17|2015-02-17|

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US201562195309P| true| 2015-07-22|2015-07-22|

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PCT/IL2015/051269|WO2016108239A1|2014-12-30|2015-12-30|Rpe cell populations and methods of generating same|

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