Methods for the generation of iPSCs

Abstract

Provided herein are methods for making and producing iPSCs, such as iPSCs that are essentially free of exogenous viral residues, and RPE cells.Such methods can include low-density seeding (e.g., single-cell passage) and / or elevated temperature cultured cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 329,097, filed April 8, 2022, the disclosure of which is incorporated by reference herein in its entirety for all purposes. [Background technology]

[0002] background Induced pluripotent stem cells (iPSCs) reprogrammed using non-integrating viruses or non-viral based reprogramming systems typically retain residual copies of vectors after the first reprogramming. For clinical applications, it is important to remove these exogenous vectors in iPSCs to reduce the number of passaging or subcloning procedures. Therefore, there remains a need for an efficient process to generate iPSCs that are free of exogenous materials. Summary of the Invention

[0003] overview The present invention is based in part on the development of a novel modified process for the generation and manufacture of iPSCs.

[0004] Thus, in one aspect, the present disclosure provides a method for producing a method for treating a pulmonary circulation comprising the steps of: (a) obtaining a starting population of iPSCs generated with a reprogramming vector; (b) seeding the iPSCs at low density and culturing them in a culture medium; (c) optionally repeating step (b) one or more times; (d) optionally culturing the iPSCs at an elevated temperature; (e) Selecting and passageing single iPSC colonies 1-8 times to generate populations of iPSCs essentially free of exogenous vector residues. The present invention provides a method for generating a clonal population of induced pluripotent stem cells (iPSCs) essentially free of exogenous vector residues, comprising:

[0005] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device, comprising the steps of: (a) obtaining a starting population of iPSCs generated with a reprogramming vector; (b) seeding the iPSCs at low density and culturing them in a culture medium; (c) optionally repeating step (b) one or more times; (d) optionally culturing the iPSCs at an elevated temperature; (e) Selecting and passageing single iPSC colonies 1-8 times to obtain iPSCs essentially free of exogenous vector residues. The present invention provides a method for producing induced pluripotent stem cells (iPSCs) essentially free of exogenous vector residues, comprising:

[0006] In some embodiments, the starting population of iPSCs is made from the somatic cells of a human individual.In some embodiments, the somatic cells are blood cells.In some embodiments, the blood cells are peripheral blood cells.In some embodiments, the blood cells are CD34+ or CD71+ enriched cells.

[0007] In some embodiments, the starting population of iPSCs is a polyclonal pool of iPSCs. In some embodiments, step (a) comprises: (i) providing a polyclonal pool of iPSCs produced using a reprogramming vector; (ii) optionally, passing the iPSCs one or more times; and (iii) optionally, cryopreserving the iPSCs generated by step (i) or step (ii), thereby providing a starting population of iPSCs.

[0008] In some embodiments, the starting population of iPSCs has not been passaged or has been passaged once prior to its use in the method.

[0009] In some embodiments, the reprogramming vector is a non-integrating viral vector. In some embodiments, the viral vector is a Sendai viral vector that contains one or more temperature-sensitive mutations.

[0010] In some embodiments, step (b) is repeated once. In some embodiments, step (b) is repeated twice.

[0011] In some embodiments, each time step (b) is performed, independently, comprises dissociating the iPSCs into essentially single cells prior to seeding. In some embodiments, each time step (b) is performed, independently, comprises dissociating the iPSCs into essentially single cells prior to seeding at about 1 to about 1500 cells / cm. 2 For example, about 140 to about 350 cells / cm 2 or about 470 to about 1150 cells / cm 2 This involves seeding iPSCs at a density of 100-1500 nm.

[0012] In some embodiments, step (b), when performed last, comprises seeding the iPSCs at a clonal density. In some embodiments, the clonal density is about 140 to about 350 cells / cm. 2 , for example, about 340 cells / cm 2 It is.

[0013] In some embodiments, each time step (b) is performed, it independently comprises seeding iPSCs in a culture medium supplemented with a Rho-associated protein kinase (ROCK) inhibitor. In some embodiments, the culture medium of step (b) is a fully defined medium. In some embodiments, the culture medium is Essential 8 medium.

[0014] In some embodiments, each time step (b) is performed, independently, comprises culturing the iPSCs at about 37.0°C to about 39.0°C.

[0015] In some embodiments, each time step (b) is performed, it independently comprises culturing about 140 to about 350 cells / cm in culture medium supplemented with a ROCK inhibitor.2 or about 470 to about 1150 cells / cm 2 The method includes seeding iPSCs at a density of 1000×1000 μg / ml, followed by culturing in culture medium at about 37.0° C. in a 5% CO 2 incubation atmosphere until single iPSC colonies appear.

[0016] In some embodiments, when last performed, step (b) comprises culturing the cells at about 140 to about 350 cells / cm in culture medium supplemented with a ROCK inhibitor. 2 The method includes seeding iPSCs at a density of 1000×, followed by culturing the iPSCs for 1-3 days in culture medium at approximately 37.0 °C in a 5% CO2 incubation atmosphere.

[0017] In some embodiments, step (d) is not performed. In some embodiments, step (d) is performed. In some embodiments, step (d) comprises culturing the iPSCs in Essential 8 medium in a 5% CO2 incubation atmosphere. In some embodiments, the elevated temperature is about 38.0°C to about 39.0°C. In some embodiments, step (d) comprises culturing the iPSCs at the elevated temperature for 5 to 8 days. In some embodiments, step (d) further comprises culturing the iPSCs at about 37.0°C for at least 1 day.

[0018] In some embodiments, step (d) comprises culturing the iPSCs in Essential 8 medium at about 38.0°C to about 39.0°C in a 5% CO2 incubation atmosphere for about 6 days, followed by 1 to 2 days at about 37.0°C in a 5% CO2 incubation atmosphere.

[0019] In some embodiments, essentially free of exogenous viral residues is determined by quantitative real-time polymerase chain reaction (qRT-PCR) or quantitative polymerase chain reaction (qPCR).

[0020] In another aspect, the present disclosure provides a method for producing a composition comprising the steps of: (a) obtaining a starting population of iPSCs generated using a Sendai virus vector containing a temperature-sensitive mutation; (b1) iPSCs were dissociated into essentially single cells at ~470–~1150 cells / cm in culture medium supplemented with ROCK inhibitor. 2 followed by culturing in culture medium at about 37.0° C. in a 5% CO2 incubation atmosphere until single iPSC colonies appear; (b2) iPSCs were dissociated into essentially single cells at ~140-~350 cells / cm in culture medium supplemented with ROCK inhibitor. 2 followed by culturing in a culture medium for about 3 days at about 37° C. in a 5% CO2 incubation atmosphere; (c) culturing the iPSCs in a culture medium at about 38.0° C. to about 39.0° C. in a 5% CO2 incubation atmosphere for about 6 days, followed by 1 to 2 days at about 37.0° C. in a 5% CO2 incubation atmosphere; and (d) selecting and culturing a single iPSC colony for less than eight passages to generate a population of iPSCs that is essentially free of exogenous viral residues. Provided is a method for generating a clonal population of induced pluripotent stem cells (iPSCs) essentially free of exogenous viral residues, comprising:

[0021] In another aspect, the present disclosure provides a method for producing a composition comprising the steps of: (a) obtaining a starting population of iPSCs generated using a Sendai virus vector containing a temperature-sensitive mutation; (b1) iPSCs were dissociated into essentially single cells at ~470–~1150 cells / cm in culture medium supplemented with ROCK inhibitor. 2 followed by culturing in culture medium at about 37.0° C. in a 5% CO2 incubation atmosphere until single iPSC colonies appear; (b2) iPSCs were dissociated into essentially single cells at ~140-~350 cells / cm in culture medium supplemented with ROCK inhibitor. 2followed by culturing in a culture medium for about 3 days at about 37.0° C. in a 5% CO2 incubation atmosphere; (c) culturing the iPSCs in a culture medium at about 38.0° C. to about 39.0° C. in a 5% CO2 incubation atmosphere for about 6 days, followed by 1 to 2 days at about 37.0° C. in a 5% CO2 incubation atmosphere; and (d) selecting and culturing a single iPSC colony for less than eight passages to obtain iPSCs that are essentially free of exogenous viral residues; A method for producing induced pluripotent stem cells (iPSCs) essentially free of exogenous viral residues is provided, comprising:

[0022] In another aspect, the present application provides a method for producing a method for treating a cancer cell comprising the steps of: (a) obtaining a population of iPSCs essentially free of exogenous viral residues produced according to any one of claims 1 and 3-32; (b) seeding and culturing the iPSCs in a retinal induction medium to induce differentiation of the cells into cells of a retinal lineage; (c) culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells; (d) culturing the cells in a retinal medium to form differentiated RPE cells; and (e) culturing the differentiated RPE cells in an RPE maturation medium, thereby generating human RPE cells. The present invention provides a method for producing human retinal pigment epithelial (RPE) cells, comprising:

[0023] In another aspect, the present application provides a method for producing a method for manufacturing a semiconductor device comprising: (a) obtaining a population of iPSCs essentially free of exogenous viral residues according to any one of claims 1 to 33; (b) seeding and culturing the iPSCs in a retinal induction medium to induce differentiation of the cells into cells of a retinal lineage; (c) culturing the retinal lineage cells in a retinal differentiation medium to further differentiate the retinal lineage cells; (d) culturing the cells in a retinal medium to form differentiated RPE cells; and (e) culturing the differentiated RPE cells in an RPE maturation medium, thereby obtaining human RPE cells. The present invention provides a method for producing human retinal pigment epithelial (RPE) cells, comprising:

[0024] In some embodiments, the methods provided do not include the formation of embryoid bodies.

[0025] In some embodiments, the iPSCs in step (a) are dissociated into single cells.

[0026] In some embodiments, step (b) comprises dissociating the iPSCs into essentially single cells prior to seeding. In some embodiments, step (b) comprises: (i) seeding at about 5,000 to about 40,000 cells / cm; 2 (ii) without a feeder layer; (iii) in a fully defined culture medium; and / or (iv) in a xeno-free culture medium. In some embodiments, (b) comprises culturing the iPSCs on a matrix. In some embodiments, the matrix comprises at least one recombinant cell adhesion protein, such as laminin, vitronectin, or fibronectin. In some embodiments, the cell adhesion protein is a human protein.

[0027] In some embodiments, the retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, and insulin growth factor 1 (IGF1). In some embodiments, the retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor, and IGF1.

[0028] In some embodiments, step (e) comprises dissociating differentiated RPE cells, reseeding RPE cells, and culturing RPE cells in RPE maturation medium, said RPE maturation medium comprising MEK inhibitor.In some embodiments, RPE cells are reseeded on degradable scaffold in RPE maturation medium.In some embodiments, RPE maturation medium comprises at least one primary cilia inducer, such as prostaglandin E2 (PGE2) or aphidicolin.

[0029] In some embodiments, the methods provided further comprise cryopreserving the human RPE cells.

[0030] In another aspect, the present disclosure provides a method for producing a composition comprising the steps of: (a) obtaining a population of iPSCs essentially free of exogenous viral residues produced according to any one of claims 1 and 3-32, and dissociating the iPSCs into essentially single cells in a fully defined medium; (b) seeding and culturing the iPSCs on laminin, vitronectin, or a combination thereof in retinal induction medium containing LDN193189, CKI-7, and SB431542 to induce differentiation of the cells into retinal lineage cells; (c) culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, CKI-7, SB431542, and PD0325901 to further differentiate the retinal lineage cells; (d) culturing the cells in a retinal medium containing nicotinamide and activin A to form differentiated RPE cells; and (e) culturing the differentiated RPE cells in an RPE maturation medium, thereby generating human RPE cells. A method is provided for producing human retinal pigment epithelial (RPE) cells, comprising:

[0031] In another aspect, the present disclosure provides (a) obtaining iPSCs essentially free of exogenous viral residues according to any one of claims 1 to 33 and dissociating the iPSCs into essentially single cells in a fully defined medium; (b) seeding and culturing the iPSCs on laminin, vitronectin, or a combination thereof in retinal induction medium containing LDN193189, CKI-7, and SB431542 to induce differentiation of the cells into retinal lineage cells; (c) culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, CKI-7, SB431542, and PD0325901 to further differentiate the retinal lineage cells; (d) culturing the cells in a retinal medium containing nicotinamide and activin A to form differentiated RPE cells; and (e) culturing the differentiated RPE cells in an RPE maturation medium, thereby obtaining human RPE cells. A method for producing human retinal pigment epithelial (RPE) cells is provided, which comprises the steps of:

[0032] In another aspect, provided herein is a pharmaceutical composition comprising human RPE cells produced or manufactured by the method described herein, a pharma- ceutically acceptable carrier, and optionally a scaffold. In some embodiments, the scaffold is a poly(lactic-co-glycolic acid) (PLGA) scaffold. [Brief description of the drawings]

[0033] [Figure 1A] Figures 1A, 1B, and 1C are phase-contrast microscopy images of early passage colonies of iPSCs reprogrammed with CytoTune Sendai viral vector at 37° C. (Figure 1A) or at 38.5° C. (Figures 1B and 1C). When cultured in the latter condition, a subset of colonies showed morphological signs of differentiation, which may serve as a selection event for clones that may be dependent on the viral vector to maintain pluripotency. [Figure 1B] Please see the legend to FIG. 1A. [Figure 1C] Please see the legend to FIG. 1A. [Diagram 2] Figures 2A and 2B show iPSCs cultured at a constant temperature of 37°C (Figure 2A) or at an elevated temperature of 38.5°C (Figure 2B) and stained with anti-Sendai antibody. Sendai virus vector was present in some iPSC colonies when cultured at 37°C but was eliminated from those treated with elevated temperatures. iPSC lines confirmed to be free of Sendai virus via RT-qPCR were used as controls. [Diagram 3] Figure 3A is a raw data plot showing the Sendai virus signal upon PCR amplification, and Figure 3B illustrates the Sendai virus copies of iPSC-derived clones cultured at increasing temperatures at passage 2 and measured at both passages 4 and 5, showing that Sendai virus was eliminated for all clones. [Figure 4] FIG. 4 shows copies of Sendai virus in various clones derived from iPSCs cultured by the method described in Example 3, and demonstrates that Sendai virus was removed from all clones. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0035] As used herein, "about" is understood to be within the normal tolerance range in the art, for example, within 2 standard deviations of the mean value. "About" can be understood to be within 10% of the value, for example, within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0036] "Allele" refers to one of two or more forms of a gene. Diploid organisms, such as humans, contain two copies of each chromosome, and therefore carry one allele on each chromosome. The term "homozygous" means to contain two identical alleles at a particular locus; the term "heterozygous" means to contain two different alleles at a particular locus.

[0037] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, when used in phrases such as "A and / or B," the term "and / or" is intended to include A and B, A or B, A (alone), and B (alone). Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0038] As used herein, "cell" refers to the structural and functional unit of an organism that can replicate independently, is enclosed by a membrane, and contains biological molecules and genetic material. As used herein, a cell can be a naturally occurring cell or an artificially modified cell (e.g., a fusion cell or a genetically modified cell).

[0039] As used herein, a "cell population" or a "population of cells" refers to a plurality or group of cells, typically of a common type. A cell population can be derived from a common precursor or may contain more than one cell type. A "clonal" cell population refers to a cell population from a single cell, such that all cells in the population arise from a single cell of origin. In some embodiments, a clonal population of cells can be obtained by plating starting cells at a "clonal density", which refers to a density where the starting cells are plated sparsely, such that each cell essentially divides alone during expansion and the resulting colonies essentially do not contact each other. An "enriched" cell population refers to a cell population derived from a starting cell population (e.g., an unfractionated heterogeneous cell population) that contains a higher percentage of a particular cell type than the percentage of the particular cell type in the starting population. A cell population may be enriched for one or more cell types and / or depleted of one or more cell types.

[0040] Where an embodiment is described with the term "comprising", other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also included.

[0041] As used herein, the term "defined" or "fully defined" when used in relation to a medium, extracellular matrix, or culture condition means a medium, extracellular matrix, or culture condition in which the chemical composition and amount of essentially all components are known. For example, a defined medium does not contain undefined factors, such as in fetal bovine serum, bovine serum albumin, or human serum albumin. In general, a defined medium includes a basal medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, which includes amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin. An exemplary fully defined medium is Essential 8™ medium.

[0042] As used herein, "differentiation", "differentiating" or "differentiating" refers to the process by which undifferentiated cells become more specialized types with changes in structural and / or functional characteristics. In some embodiments, "differentiation" refers to the process by which human stem cells acquire the cell type of retinal pigment epithelium (RPE) cells, which have characteristics that indicate that RPE cells are mature terminally differentiated cells. The term "differentiated cells" encompasses any somatic cell that is not multiplicity in its native form, as that term is defined herein. Thus, the term "differentiated cells" also encompasses partially differentiated cells, such as multipotent cells, or stable, non-pluripotent partially reprogrammed cells or partially differentiated cells, which are made using any of the compositions and methods described herein. In some embodiments, differentiated cells are cells that are stable intermediate cells, such as non-pluripotent partially reprogrammed cells. In some embodiments, the term "differentiated cell" also refers to a cell of a more specialized cell type (e.g., decreased developmental potential) derived from a cell of a less specialized cell type (e.g., increased developmental potential) (e.g., from an undifferentiated cell or a reprogrammed cell), where the cell has undergone a cell differentiation process. The term "terminally differentiated cell" or "mature cell" refers to a cell that does not undergo further differentiation in its native state without treatment or external manipulation. In some embodiments, a terminally differentiated cell has lost the ability to differentiate into a more specialized cell type. Mature cells typically have altered cell structure and tissue-specific proteins and are involved in specialized functions. The term "undifferentiated cell" refers to a cell other than a terminally differentiated cell. Thus, an undifferentiated cell exhibits characteristic markers and morphological features that clearly distinguish it from a terminally differentiated cell of embryonic or adult origin.

[0043] The term "embryo" refers to a mass of cells resulting from one or more divisions of a zygote or activated oocyte having an artificially reprogrammed nucleus.

[0044] The term "embryoid body" or "EB" refers to aggregates of pluripotent stem cells that can undergo differentiation into cells of the endodermal, mesodermal, and ectodermal germ layers. The aggregation of pluripotent stem cells forms spheroidal structures, allowing the EBs to be cultured non-adherently in suspension.

[0045] The term "embryonic stem (ES) cell" refers to an undifferentiated pluripotent cell obtained from an earlier stage embryo, e.g., the inner cell mass at the blastocyst stage, or created by artificial means (e.g., nuclear transfer), and which can give rise to any differentiated cell type, including germ cells (e.g., sperm and eggs), in the embryo or adult.

[0046] As used herein, "essentially" means nearly all or completely with respect to a given value, dimension, shape, element, material, or another aspect that it modifies.For example, in some embodiments, when used in conjunction with a value, "essentially" means, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of that value.In some embodiments, the methods provided herein involve dissociating iPSCs essentially into single cells, which means that iPSCs are almost entirely or completely dissociated into single cells, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of iPSCs are dissociated into single cells.

[0047] As used herein, "episomal vector" or "episome" refers to an extrachromosomal DNA molecule that can replicate autonomously and maintain itself in the cytoplasm of a cell. In some embodiments, an episomal vector does not integrate into the genome of a host cell. In some embodiments, an episomal vector is of viral origin. In some embodiments, an episomal vector is of non-viral origin.

[0048] As used herein, "essentially free" when used in relation to a given element or material in a composition means that the given element or material is not intentionally incorporated into the composition, is not desired in the composition, is not detected in the composition, and / or is present only as a contaminant or only in trace amounts.In some embodiments, the composition is free of a given element or material, where the given element or material is less than 0.05% by weight of the composition, preferably less than 0.01% by weight.In some embodiments, the composition is essentially free of a given element or material, where the given element or material cannot be detected using standard analytical methods for that element or material.

[0049] The term "exogenous" or "heterologous", when used in reference to a nucleic acid such as DNA, refers to a nucleic acid that originates from a source that is foreign to a particular host cell, or that is modified from its original form when derived from the same source, respectively. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell, but that is modified, for example, by using DNA shuffling. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. In some embodiments, an exogenous nucleic acid may be expressed to produce an exogenous polypeptide. A "homologous" nucleic acid sequence is a nucleic acid sequence that is naturally associated with the host cell into which it is introduced.

[0050] As used herein, "expanding" or "expansion" means culturing one or more cells with the goal of obtaining a large number of cells in culture.

[0051] As used herein, "feeder layer" or "feeder cells" refers to a coating layer of cells, such as on the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.

[0052] As used herein, "feeder-free" or "feeder-independent" refers to a culture supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, or LIF) as an alternative to a feeder layer. Thus, "feeder-free" or feeder-independent culture systems and culture media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. In some cases, feeder-free cultures utilize animal-based matrices (e.g., MATRIGEL™) or are grown on substrates such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to remain essentially undifferentiated without the need for a mouse fibroblast feeder layer.

[0053] As used herein, "haplotype" refers to the combination of alleles at multiple loci by a single chromosome.Haplotypes can be based on a set of single nucleotide polymorphisms (SNPs) on a single chromosome and / or on alleles within the major histocompatibility complex (MHC).The haplotype of a subject can be easily determined using assays well known in the art.

[0054] As used herein, the term "haplotype-matched" is defined as a cell (e.g., iPSC cell) and the subject treated with the cell or cell derivative share one or more MHC locus haplotypes.Haplotype-matched iPSC cell can be autologous or allogeneic.Autologous cells that grow in tissue culture and differentiate into, for example, RPE cells are haplotype-matched with the subject.

[0055] The term "induced pluripotent stem cell" or "iPSC" refers to cells that are produced by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (herein referred to as "reprogramming factors"). iPSCs can be produced using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0056] As used herein, an "isolated" cell is one that has been substantially separated or purified from other cells in an organism or culture. An isolated cell can be, for example, at least 99%, at least 98% pure, at least 95% pure, or at least 90% pure.

[0057] As used herein, the term "modified," when used in reference to a cell, e.g., a mammalian cell, means a non-naturally occurring cell in which one or both of the following have been manipulated by the hand of man (e.g., by overexpression, reduction, and / or inhibition): (1) the genetic content of the cell; or (2) the expression of one or more genes.

[0058] As used herein, "pluripotency" refers to the property of cells that differentiate into all other cell types in organisms, except extraembryonic or placental cells.Pluripotent stem cells can differentiate into all three germ layers of cell types (e.g., ectoderm, mesoderm, and endoderm cell types) even after long-term culture.In some embodiments, pluripotent stem cells are embryonic stem cells derived from the inner cell mass of blastocyst.In some embodiments, pluripotent stem cells are induced pluripotent stem cells derived by reprogramming somatic cells.

[0059] As used herein, "preconfluent" refers to a cell culture in which the percentage of the culture surface covered by cells is about 60-80%. Typically, preconfluent refers to a culture in which about 70% of the culture surface is covered by cells.

[0060] As used herein, "PSC culture medium" or "PCM" refers to any growth medium for culturing pluripotent stem cells. In some embodiments, the PCM comprises a Rock inhibitor.

[0061] The term "retina" refers to the light-sensitive layer of tissue that covers the inside of the eye. The term "retinal pigment epithelium" or "RPE" refers to the single layer of pigmented cells that lies between the retina and the blood vessel-filled layer, the choroid.

[0062] As used herein, "retinal lineage cell" refers to the cell that can produce or differentiate into retinal pigment epithelium (RPE) cell.In some embodiments, "mature" RPE cell refers to the RPE cell that downregulates the expression of immature RPE markers such as Pax6 and upregulates the expression of mature RPE markers such as RPE65.The "maturation" of RPE cell refers to the process in which RPE development pathway is regulated to produce mature RPE cell.For example, the regulation of cilia function can result in the maturation of RPE cell.

[0063] As used herein, "retinal induction medium" or "RIM" refers to a growth medium that contains a WNT pathway inhibitor and a BMP pathway inhibitor and can cause PSCs to differentiate into retinal lineage cells. In some embodiments, RIM also contains a TGFβ pathway inhibitor.

[0064] As used herein, "retinal differentiation medium" or "RDM" refers to a medium that contains a WNT pathway inhibitor, a BMP pathway inhibitor and a MEK inhibitor and differentiates retinal cells. In some embodiments, the RDM also contains a TGFβ pathway inhibitor.

[0065] As used herein, "retinal medium" or "RM" refers to a growth medium for the culture of retinal cells that contains activin A and nicotinamide.

[0066] As used herein, "RPE maturation medium" or "RPE-MM" refers to a medium for maturation of RPE cells, comprising taurine and hydrocortisone. In some embodiments, RPE-MM also comprises triiodothyronine. In some embodiments, RPE-MM also comprises PD0325901 or PGE2.

[0067] As used herein, "stem cells" refer to cells that, under appropriate conditions, can differentiate into a diverse range of specialized cell types, but under other appropriate conditions, can self-renew and remain in an essentially undifferentiated pluripotent state. The term "stem cells" also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells can be obtained from hematopoietic stem cells or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are called "induced pluripotent stem cells" or "iPSCs".

[0068] As used herein, "substantially the same HLA type" refers to the HLA type of the donor matching the HLA type of the subject to the extent that cells obtained by inducing differentiation of iPSCs derived from the donor's somatic cells can engraft in the subject when transplanted into the subject.

[0069] As used herein, "super donor" refers to an individual who is homozygous for a certain MHC class I and II gene.A homozygous individual can function as a super donor, and its cells, including tissues and other materials that contain the cells, can be transplanted into an individual who is either homozygous or heterozygous for that haplotype.A super donor can be homozygous for each of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus alleles.

[0070] A "vector", "plasmid" or "recombinant DNA construct" is generally understood to mean a nucleic acid resulting from human intervention, including recombinant means or direct chemical synthesis, with a set of specific nucleic acid elements that allow for transcription or translation of a particular nucleic acid, for example, in a host cell. An expression vector can be part of a plasmid, a virus, or a nucleic acid fragment. Typically, a vector can include a nucleic acid to be transcribed, operably linked to a promoter. A "reprogramming vector" includes one or more nucleic acid sequences encoding one or more reprogramming factors, such as Oct4, Sox2, c-Myc, Klf4, Nanog, and / or Lin28. In some embodiments, a reprogramming vector encodes at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors, and can reprogram somatic cells into pluripotent stem cells.

[0071] As used herein, the term "vector remnant" or "vector remainder" refers to any remaining vector or vector fragment within a host cell.

[0072] As used herein, the terms "virus vector", "virus-based vector" and "viral vector" are used interchangeably to describe a genetically modified virus that has been engineered by recombinant DNA technology such that its entry into a host cell results in a specific biological activity, such as the expression of one or more transgenes carried by the vector. In some embodiments, the transgene is a reprogramming factor. A viral vector may or may not be replication-competent in a targeted cell, tissue, or organism. In some embodiments, a viral vector does not alter the host genome. In some embodiments, a viral vector is a "non-integrating viral vector" whose genetic material does not essentially integrate into the host genome, but remains episomal in the cytoplasm. Thus, the expression and presence of the virus is transient and is not transmitted to daughter cells via the host cell genome. Meanwhile, generally, an integrating viral vector integrates a fragment of its genetic material into the host cell genome; such integrated material is referred to herein as a "viral vector integration remnant" or a "viral vector integration remnant residue".

[0073] The term "xeno-free" or "XF" when used in reference to a medium, extracellular matrix, or culture condition means a medium, extracellular matrix, or culture condition that is essentially free of heterogeneous animal-derived components. In the culture of human cells, any protein from a non-human animal, such as mouse, is a xeno-component. In certain aspects, a xeno-free matrix may be essentially free of any non-human animal-derived components, thus excluding mouse feeder cells or MATRIGEL™. MATRIGEL™ is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, including laminin (major component), collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0074] Generation and manufacturing of induced pluripotent stem cells (iPSCs) In various embodiments, the present disclosure provides iPSCs that are essentially free of exogenous vector residues and the methods of their creation and manufacture.In some embodiments, the methods disclosed herein comprise low-density seeding and / or cell culture at elevated temperature.In some embodiments, the methods disclosed herein further comprise single cell passage.In some embodiments, the methods disclosed herein allow for obtaining iPSCs that are essentially free of exogenous vector residues at early passage.

[0075] In various embodiments, the present disclosure provides a method for producing a population of iPSCs that is essentially free of exogenous vector residues.In some embodiments, the population of iPSCs is a clonal population.In various embodiments, the present disclosure provides a method for producing iPSCs that is essentially free of exogenous vector residues.

[0076] In some embodiments, the methods provided include (a) obtaining a starting population of iPSCs generated with a non-integrating reprogramming vector; (b) seeding the iPSCs at low density and culturing in PSC culture medium; (c) optionally repeating step (b) one or more times; (d) optionally culturing the iPSCs at elevated temperature; and (e) selecting single iPSC colonies and passaged 1-8 times. In some embodiments, the iPSCs essentially free of exogenous vector residues are cryopreserved after generation.

[0077] In some embodiments, the starting population of iPSCs is produced by a method comprising introducing exogenous polynucleotides encoding one or more reprogramming factors into somatic cells using a reprogramming vector.In some embodiments, the starting population of iPSCs is a polyclonal pool of iPSCs.In some embodiments, the starting population of iPSCs is not passaged before its use in the provided method.In some embodiments, the starting population of iPSCs is passaged once before its use in the provided method.

[0078] In some embodiments, step (a) comprises: (i) providing a polyclonal pool of iPSCs generated with a reprogramming vector; (ii) optionally passaging the iPSCs one or more times; and (iii) optionally cryopreserving the iPSCs generated by step (i) or step (ii), thereby providing a starting population of iPSCs.

[0079] In some embodiments, step (b) is not repeated. In some embodiments, step (b) is repeated once. In some embodiments, step (b) is repeated twice.

[0080] In some embodiments, step (b), each time performed, independently includes dissociating the iPSCs into essentially single cells prior to seeding. In some embodiments, the iPSCs are dissociated from the adhesive surface by incubation with a cell dissociation enzyme, e.g., trypsin or TrypLE™. In some embodiments, the iPSCs are dissociated into a suspension of essentially single cells by pipetting. In some embodiments, the essentially single cell iPSC suspension is counted prior to seeding, e.g., by a hemocytometer or an automated cell counter, e.g., VICELL™ or TC20. In some embodiments, the cells are diluted to a cell density of about 10,000 to about 500,000 cells / mL, about 50,000 to about 200,000 cells / mL, or about 75,000 to about 150,000 cells / mL. In some embodiments, the iPSCs are diluted in a fully defined culture medium, such as ESSENTIAL 8™ (E8™) medium. In some embodiments, ROCK inhibitor is added to culture medium to increase survival of iPSCs after dissociation into essentially single cells, without allowing cells to attach to culture vessel.In some embodiments, in step (b), iPSCs are seeded in culture medium supplemented with ROCK inhibitor.In some embodiments, blebbistatin is added to culture medium to increase survival of iPSCs after dissociation into essentially single cells, without allowing cells to attach to culture vessel.

[0081] In some embodiments, each time step (b) is performed, it independently includes seeding iPSCs into a suitable culture vessel, such as a tissue culture plate, a flask, a 6-well plate, a 24-well plate, or a 96-well plate. Suitable culture vessels for culturing cells, such as iPSCs, include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as the cells can be cultured therein. In some embodiments, cells can be cultured in a volume of at least or about 0.2 mL, 0.5 mL, 1 mL, 2 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, 550 mL, 600 mL, 800 mL, 1000 mL, 1500 mL, or any range derivable therein, depending on the needs of the culture. In some embodiments, the culture vessel can be a bioreactor, which can refer to any device or system ex vivo that supports a biologically active environment so that cells can grow. Bioreactors can be at least or about 2 L, 4 L, 5 L, 6 L, 8 L, 10 L, 15 L, 20 L, 25 L, 50 L, 75 L, 100 L, 150 L, 200 L, 500 L, 1 m 3 , 2 m 3 , 4 m 3 , 6 m 3 , 8 m 3 , 10 m 3 , 15 m 3 or any range of volumes derivable therein. In some embodiments, iPSCs are seeded into 6-well plates.

[0082] In some embodiments, each time step (b) is performed, it independently comprises seeding the iPSCs at a low density. In some embodiments, the iPSCs are seeded at a low density, e.g., about 1 to about 1500 cells / cm. 2 For example, about 1 to about 150 cells / cm 2 , about 140 to about 350 cells / cm 2 , about 340 to about 480 cells / cm 2 , about 470 to about 1150 cells / cm 2 or about 1000 to about 1500 cells / cm 2 In some embodiments, iPSCs are seeded into 6-well plates at a density of, for example, about 140 to about 350 cells / cm. 2 In some embodiments, iPSCs are seeded into 6-well plates at a density of, for example, about 470 to about 1150 cells / cm. 2 In some embodiments, the iPSCs are seeded into 6-well plates at a density of, for example, about 50 cells / cm. 2 , about 75 cells / cm 2 , about 100 cells / cm 2 , about 125 cells / cm 2 , about 150 cells / cm 2 , about 175 cells / cm 2 , about 200 cells / cm 2 , about 225 cells / cm 2 , about 250 cells / cm 2 , about 275 cells / cm 2 , about 300 cells / cm 2 , about 325 cells / cm 2 , about 350 cells / cm 2 , about 375 cells / cm 2 , about 400 cells / cm 2 , about 500 cells / cm 2 , about 600 cells / cm 2 , about 700 cells / cm 2 , about 800 cells / cm 2 , about 900 cells / cm 2 , about 1000 cells / cm 2 , about 1100 cells / cm 2 , about 1200 cells / cm 2 , about 1300 cells / cm 2 , about 1400 cells / cm 2, or approximately 1500 cells / cm 2 In some embodiments, iPSCs are seeded into 6-well plates at a density of, for example, about 340 cells / cm. 2 Cells are seeded into 6-well plates at a density of 1000×.

[0083] In some embodiments, step (b), when performed last, comprises seeding the iPSCs at a clonal density. In some embodiments, the iPSCs are seeded into a 6-well plate at a clonal density. In some embodiments, the clonal density is about 140 to about 350 cells / cm. 2 In some embodiments, the clonal density is about 140 cells / cm 2 , about 150 cells / cm 2 , about 160 cells / cm 2 , about 170 cells / cm 2 , about 180 cells / cm 2 , about 190 cells / cm 2 , about 200 cells / cm 2 , about 210 cells / cm 2 , about 220 cells / cm 2 , about 230 cells / cm 2 , about 240 cells / cm 2 , about 250 cells / cm 2 , about 260 cells / cm 2 , about 270 cells / cm 2 , about 280 cells / cm 2 , about 290 cells / cm 2 , about 300 cells / cm 2 , about 310 cells / cm 2 , about 320 cells / cm 2 , about 330 cells / cm 2 , about 340 cells / cm 2 , or approximately 350 cells / cm 2 In some embodiments, the clonal density is about 340 cells / cm 2 In some embodiments, the iPSCs are seeded into a 96-well plate at a clonal density. In some embodiments, the clonal density is about 0.5 to about 5 cells / cm. 2 In some embodiments, the clonal density is about 0.5 cells / cm 2, about 1 cell / cm 2 , about 1.5 cells / cm 2 , about 2 cells / cm 2 , about 2.5 cells / cm 2 , about 3 cells / cm 2 , about 3.5 cells / cm 2 , about 4 cells / cm 2 , about 4.5 cells / cm 2 , or approximately 5 cells / cm 2 In some embodiments, the clonal density is about 1.5 cells / cm 2 It is.

[0084] Cells, such as iPSCs, can be cultured with the nutrients necessary to support the growth of each particular cell population. Generally, cells are cultured in a growth medium that includes a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also include fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffering agents, and inorganic salts. Exemplary growth media include minimal essential media, such as Dulbecco's Modified Eagle's Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Eagle's Minimum Essential Medium (MEM) Alpha Medium, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. In addition, minimal essential media may be supplemented with additives, such as horse, calf, or fetal bovine serum. Alternatively, the medium may be serum-free. In other cases, the growth medium may contain "knock out serum replacement," referred to herein as a serum-free formulation optimized to grow and maintain undifferentiated cells, such as stem cells, in culture. In some embodiments, the iPSCs described herein are cultured in a fully defined and feeder-free medium. In some embodiments, the iPSCs described herein are cultured in Essential 8 medium.

[0085] In some embodiments, in step (b), the iPSCs dissociated into essentially single cells are cultured in a fully defined culture medium, such as Essential 8 medium, optionally supplemented with a ROCK inhibitor, after seeding. Thus, in some embodiments, the PSC culture medium in step (b) is Essential 8 medium. In some embodiments, about 40-48 hours after seeding, the medium is aspirated and fresh PSC culture medium, such as Essential 8 medium, is added to the culture. In some embodiments, the iPSCs dissociated into essentially single cells are cultured in PSC culture medium for about 1, 2, 3, 4, 5, 6, or 7 days after seeding. In some embodiments, the iPSCs are cultured to a preconfluent state, such as where each cell essentially divides individually during expansion. In some embodiments, the iPSCs are cultured to a preconfluent state, such that individual colonies are essentially not in contact with each other.

[0086] The cells, such as iPSCs, are cultured at an appropriate temperature. In some embodiments, the iPSCs described herein are cultured at about 37.0°C to about 39.0°C. In some embodiments, iPSCs are cultured at about 36.5°C, about 36.6°C, about 36.7°C, about 36.8°C, about 36.9°C, about 37.0°C, about 37.1°C, about 37.2°C, about 37.3°C, about 37.4°C, about 37.5°C, about 37.6°C, about 37.7°C, about 37.8°C, about 37.9°C, about 38.0°C, about 38.1°C, about 38.2°C, about 38.3°C, about 38.4°C, about 38.5°C, about 38.6°C, about 38.7°C, about 38.8°C, about 38.9°C, about 39.0°C, about 39.1°C, about 39.2°C, about 39.3°C, about 39.4°C, or about 39.5°C.

[0087] In some embodiments, each time step (b) is performed, independently, comprises culturing the iPSCs at about 37.0° C. to about 39.0° C. In some embodiments, in step (b), the iPSCs are cultured at about 37.0° C.

[0088] In some embodiments, the elevated temperature in step (d) is about 38.0°C to about 39.0°C. In some embodiments, the elevated temperature in step (d) is about 37.8°C, about 37.9°C, about 38.0°C, about 38.1°C, about 38.2°C, about 38.3°C, about 38.4°C, about 38.5°C, about 38.6°C, about 38.7°C, about 38.8°C, about 38.9°C, about 39.0°C, about 39.1°C, or about 39.2°C. In some embodiments, step (d) comprises culturing the iPSCs at the assessed temperature for about 5, 6, 7, or 8 days in a PSC culture medium, such as, for example, Essential 8 medium. In some embodiments, step (d) comprises culturing the iPSCs at the assessed temperature for about 6 days. In some embodiments, step (d) further comprises culturing the iPSCs at about 37.0° C. for at least 1 day, e.g., 1 day, 2 days, or 3 days, after culturing the iPSCs at the elevated temperature.

[0089] Other culture conditions can be further defined as appropriate. In some embodiments, for example, the CO2 concentration is about 1-10%, for example, about 2-5%, or any range derivable therein; the O2 concentration can be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

[0090] In some embodiments, the iPSC colonies are manually selected in step (e). In some embodiments, the manually selected iPSC colonies are passaged 1, 2, 3, 4, 5, 6, 7, or 8 times.

[0091] Any method known in the art can be used to determine and / or confirm that iPSCs are essentially free of exogenous vector residues.In some embodiments, essentially free of exogenous vector residues is determined by polymerase chain reaction (PCR), for example, quantitative PCR (qPCR), real-time PCR (RT-PCR) or quantitative real-time RT-PCR (aRT-PCR).In some embodiments, essentially free of exogenous vector residues can be determined by immunostaining.

[0092] somatic cell source In some embodiments, any cell other than germ cell can be used as the starting cell for reprogramming to generate iPSC.For example, in some embodiments, keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or stomach cells can be used for reprogramming to generate iPSC.In some embodiments, T cells can be used as the source of somatic cells for reprogramming.There is no limit to the degree of cell differentiation or the age of the animal from which cells are collected; therefore, in some embodiments, undifferentiated precursor cells (including somatic stem cells) or terminally differentiated mature cells can be used as the source of somatic cells for reprogramming to generate iPSC.

[0093] In some embodiments, somatic cells of human individuals are used to reprogram and generate iPSCs.In some embodiments, somatic cells are blood cells, such as peripheral blood cells.In some embodiments, blood cells are enriched with CD34+.In some embodiments, blood cells are enriched with CD71+.In one embodiment, somatic cells are retinal pigment epithelium (RPE) cells.RPE cells can be adult or fetal RPE cells.

[0094] In some embodiments, iPSCs can be generated from the somatic cells of the subject to be treated or another subject ("donor") that has the same or substantially the same HLA type as that of the subject ("recipient"). In some embodiments, the major HLA of the donor (e.g., the three major loci HLA-A, HLA-B and HLA-DR) is identical to the major HLA of the recipient. In some embodiments, the donor can be a super donor; thus, iPSCs derived from an MHC homozygous super donor can be used to generate, for example, RPE cells. Thus, iPSCs derived from a super donor can be transplanted in a subject that is homozygous or heterozygous for its haplotype. For example, iPSCs can be homozygous for two HLA alleles, e.g., HLA-A and HLA-B. Thus, iPSCs generated from a super donor can be used in the methods disclosed herein to generate RPE cells that can potentially "match" to a large number of potential recipients.

[0095] Reprogramming and Genetic Modification Somatic cells can be reprogrammed to generate iPSCs by any method known to those skilled in the art, for example, the method disclosed in US Patent Application Publication Nos. 2009 / 0246875; 2010 / 0210014; and 2012 / 0276636; US Patent Nos. 8,058,065; 8,129,187; 8,278,620; and PCT Publication No. WO 2007 / 069666.Generally, one or more nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells.In some embodiments, somatic cells are treated with one or more reprogramming substances (e.g., reprogramming factors) or one or more nucleic acids that code reprogramming substances (e.g., vectors that code reprogramming factors) to generate iPSCs. Methods for introducing one or more reprogramming agents, or nucleic acids encoding these reprogramming agents, are known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0196360 and U.S. Patent No. 8,071,369.

[0096] Exemplary reprogramming factor combinations are described, for example, in U.S. Patent Application Publication No. 2012 / 0196360 and include: (1) Oct3 / 4, Klf4, Sox2, and L-Myc (Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7, or Soxl8; Klf4 can be replaced with KM, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, and SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, and human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, and HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, and HPV16 E7; (6) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, and Bmil; (7) Oct3 / 4, Klf4, Sox2, L-Myc, and Lin28; (8) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, S, and V40LT; (9) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, and SV40LT; (10) Oct (11) Oct3 / 4, Klf4, Sox2, L-Myc, and SV40LT; (12) Oct3 / 4, Klf4, and Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, and SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, and HP. VI 6 E6;(15)Oct3 / 4, Klf4, Sox2, TERT, and HPV16 E7;(16)Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, and HPV16 E7;(17)Oct3 / 4, Klf4, Sox2, TERT, and Bmil;(18)Oct3 / 4, Klf4, Sox2, and Lin28;(19)Oct3 / 4, Klf4, Sox2, Lin28, and SV40LT;(20)Oct3 / 4, Klf4, Sox2, Lin28, TERT, and SV40LT;(21)Oct3 / 4, Klf4, Sox2, and SV40LT;or (22) Oct3 / 4, Esrrb, and Sox2 (Esrrb may be replaced by Esrrg);

[0097] In some embodiments, the reprogramming factors used in the methods described herein include Oct3 / 4, Klf4, and Sox2. In some embodiments, the reprogramming factors may also include L-Myc, c-Myc, Lin28, Lin28b, and / or Nanog. In some embodiments, reprogramming factors such as Klf4, c-Myc, Lin28, or Nanog can increase reprogramming efficiency. In some embodiments, the reprogramming factors include Oct4, Nanog, and Sox2. In some embodiments, the reprogramming factors include Oct3 / 4, Klf4, and Myc. In some embodiments, the reprogramming factors used in the methods described herein include at least three, or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28. In some embodiments, the reprogramming factors include Oct3 / 4, Sox2, c-Myc, and Klf4. In some embodiments, the reprogramming factors include Oct3 / 4, Klf4, Sox2, and Sal4.

[0098] In some embodiments, somatic cell reprogramming may further comprise contacting cell with one or more signal transduction regulators, said regulators can be small molecules, inhibitory nucleotides, expression cassettes, or protein factors.Non-limiting examples of such regulators include glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signal transduction inhibitors, leukemia inhibitory factor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof.

[0099] In some embodiments, the reprogramming factors are introduced into somatic cells using one or more reprogramming vectors that code for the reprogramming factors. Thus, in some embodiments, somatic cells are reprogrammed using a reprogramming vector that codes for one or more reprogramming factors to generate iPSCs, such as the starting population of iPSCs of the methods described herein. In some embodiments, the reprogramming vector is a viral vector, such as a retroviral vector, a lentiviral vector, or a Sendai virus vector. In some embodiments, the reprogramming vector is a non-integrating viral vector. Non-limiting examples of non-integrating viral vectors include adenoviral vectors, alphavirus vectors, baculovirus vectors, Epstein-Barr virus (EBV)-based vectors, picornavirus vectors, Sendai virus (SeV) vectors, and vaccinia virus vectors.

[0100] In some embodiments, the non-integrating virus vector is a SeV vector. In some embodiments, the SeV vector comprises one or more temperature-sensitive mutations. In some embodiments, the one or more temperature-sensitive mutations are in a polymerase-associated gene, such as phosphoprotein (P) and / or large protein (L). In some embodiments, the SeV vector is weakly expressed at temperatures above 37°C, for example at 38°C or higher. In some embodiments, the SeV vector is essentially not expressed at temperatures above 38°C.

[0101] In certain embodiments, the reprogramming factors are directly introduced into the somatic cells by protein transduction.

[0102] In some embodiments, the iPSCs can be genetically modified. In some embodiments, the iPSCs can be modified to express exogenous nucleic acids using genetic constructs (e.g., vectors).

[0103] In some embodiments, such a genetic construct includes a tyrosinase enhancer operably linked to a nucleic acid sequence encoding a promoter and a marker. The tyrosinase gene is disclosed, for example, in GENBANK® Accession No. 22173 available on January 1, 2013. This sequence aligns to positions 5286971-5291691 (reverse orientation) of chromosome 7 of mouse strain C57BL / 6. In some embodiments, the tyrosinase enhancer is a retinal pigment epithelium (RPE) specific enhancer, such as D-MITF, DCT, TYRP1, RPE65, VMD2, MERTK, MYRIP, RAB27A, or a regulatory element of 4721 base pair sequence sufficient for expression in RPE cells as described in Murisier et al.

[0104] In some embodiments, the promoter is any promoter expressed in RPE cells, such as the tyrosinase promoter. In some embodiments, the marker can be a protein (e.g., a secreted protein, a cell surface protein, or an internal protein), a nucleic acid (e.g., an mRNA or an enzymatically active nucleic acid molecule), or a polysaccharide. In some embodiments, the marker can be a fluorescent protein (e.g., green fluorescent protein or red fluorescent protein), an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, firefly / Renilla luciferase, or nanoluc), or another protein. In some embodiments, the marker can be a selectable marker (e.g., an antibiotic resistance marker). In some embodiments, the marker can be identified by biochemical or enzymatic assays, or biological reactions that rely on the function of the gene product. Included are any such cellular component determinants that can be detected by antibodies, lectins, probes, or nucleic acid amplification reactions that are specific for the marker of the cell type of interest.

[0105] In some embodiments, constructs may include other genes, such as genes that may affect stem cells on RPE differentiation, or RPE function, or physiology, or pathology.Thus, in some embodiments, constructs may include nucleic acids encoding one or more of MITF, PAX6, TFEC, OTX2, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOLV4, ADAM 10, CD55, CD59, and ARMS2.

[0106] In some embodiments, construct can also include other elements, such as ribosome binding site (internal ribosome binding sequence) for translation initiation and transcription / translation terminator.In some embodiments, iPSC is transfected with said construct.Construct suitable for stable transfection includes but is not limited to retroviral vector, lentiviral vector, and Sendai virus.

[0107] Culture and maintenance Once induced, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with various media and techniques developed for culturing pluripotent stem cells (e.g., embryonic stem cells), such as those described in U.S. Pat. No. 7,442,548 and U.S. Patent Application Publication No. 2003 / 0211603. In the case of mouse cells, the culture is usually performed with the addition of leukemia inhibitory factor (LIF) as a differentiation inhibitor to the culture medium. In the case of human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs, as known to those skilled in the art, can also be used.

[0108] In certain embodiments, undefined conditions may be used. For example, in some embodiments, pluripotent cells may be cultured on fibroblast feeder cells or on media exposed to fibroblast feeder cells to maintain stem cells in an undifferentiated state. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts treated with radiation or antibiotics to terminate cell division as feeder cells. In other embodiments, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using defined feeder-independent culture systems such as TESR™ medium or E8™ medium. In various embodiments, iPSCs as described herein are cultured in E8™ medium.

[0109] In some embodiments, iPSCs can be grown under conditions known to cause human ES cells to differentiate into specific cell types and express human ES cell markers such as SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0110] Generation and production of retinal pigment epithelial (RPE) cells In various aspects, provided herein are methods for differentiating iPSCs generated or produced by the methods disclosed herein, e.g., iPSCs that are essentially free of exogenous viral residues, into RPE cells.

[0111] The cells in the retina that respond directly to light are photoreceptor cells. Photoreceptors are light-sensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, photoreceptor cells convert the incoming light energy focused by the lens into an electrical signal that is then sent to the brain via the optic nerve. Vertebrates have two types of photoreceptor cells, including cones and rods. Cones are adapted to detect fine central vision and color vision and perform well in bright light. Rods are responsible for peripheral vision and vision in low light. The nerve signals from rods and cones are processed by other neurons in the retina.

[0112] The retinal pigment epithelium functions as a barrier between the bloodstream and the retina and interacts closely with the photoreceptors in maintaining visual function. It is composed of a single layer of hexagonal cells densely packed with granules of melanin that absorb the light energy reaching the retina. The main functions of specialized RPE cells include transport of nutrients such as glucose, retinol, and fatty acids from the blood to the photoreceptors; transport of water, metabolic end products, and ions from the subretinal space to the blood; light absorption and protection from photooxidation; reisomerization of all-trans-retinol to 11-cis-retinal; phagocytosis of detached photoreceptor membranes; and secretion of various essential factors for the structural integrity of the retina.

[0113] The retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, best vitelliform macular dystrophy gene (VMD2), and pigment epithelium-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with multiple vision-altering conditions, including retinal pigment epithelial detachment, dysplasia, atrophy, retinopathies, retinitis pigmentosa, macular dystrophies, or degeneration.

[0114] RPE cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually recognized by their cobblestone morphology and early appearance of pigment. In addition, differentiated RPE cells have a high transepithelial electrical resistance (TER) and transepithelial potential (TEP) across the monolayer (TER > 100 ohms cm 2 ; TEP>2 mV), transport fluid and CO2 from the apical to the basolateral side, and control the polarized secretion of cytokines.

[0115] RPE cells express multiple proteins that can serve as markers for detection by the use of methodologies such as immunocytochemistry, Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). For example, RPE-specific markers may include cellular retinaldehyde-binding protein (CRALBP), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP-1), retinal pigment epithelium-specific 65 kDa protein (RPE65), pre-melanosome protein (PMEL17), bestrophin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-2. Specifically, when assessed by quantitative RT-PCR, expression of these genes is approximately 100-1000-fold lower in RPE cells than in ES or iPSC cells.

[0116] RPE cell markers can be detected at the mRNA level, for example, by reverse transcription polymerase chain reaction (RT-PCR) using sequence-specific primers, Northern blot analysis, or dot blot hybridization analysis in standard amplification methods using publicly available sequence data (GENBANK®). Expression of tissue-specific markers detected at the protein or mRNA level is considered positive if its level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, more specifically, more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold, or higher than that of control cells such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0117] Dysfunction, damage and loss of RPE cells are factors in many ocular diseases and disorders, including age-related macular degeneration (AMD), hereditary macular degeneration including Best's disease, and retinitis pigmentosa. A potential treatment for such diseases is the transplantation of RPE cells into the retina of those in need of such treatment. It is speculated that the replenishment of RPE cells by such transplantation may slow, halt or reverse deterioration, improve retinal function, and prevent blindness resulting from such conditions. However, it is difficult to obtain RPE cells directly from human donors and embryos.

[0118] Derivation of RPE cells from essentially single cell PSCs In various embodiments, a method is provided for generating or producing RPE cells from an essentially single cell suspension of pluripotent stem cells (PSCs), such as iPSCs that are essentially free of exogenous viral residues, which are generated or produced by the methods disclosed herein.In some embodiments, the method includes: (a) obtaining a population of iPSCs that are essentially free of exogenous viral residues; (b) seeding and culturing the iPSCs in retinal induction medium to induce the differentiation of the cells into retinal lineage cells; (c) culturing the retinal lineage cells in retinal differentiation medium to further differentiate the retinal lineage cells; (d) culturing the cells in retinal medium to form differentiated RPE cells; and (e) culturing the differentiated RPE cells in RPE maturation medium, thereby generating RPE cells.In some embodiments, the iPSCs of step (a) are dissociated into essentially single cells before use in step (b).In some embodiments, the method does not include the formation of embryoid bodies.In some embodiments, the RPE cells are cryopreserved after generation.

[0119] In some embodiments, iPSCs are cultured to a pre-confluent state to prevent any cell aggregates. In some embodiments, iPSCs are dissociated by incubation with cell dissociation enzymes such as TRYPSIN™ or TRYPLE™. In some other embodiments, iPSCs can be dissociated into essentially single cell suspensions by pipetting. In some embodiments, blebbistatin (e.g., about 2.5 μM) can be added to the medium to increase the survival of iPSCs after dissociation into single cells without allowing the cells to attach to the culture vessel. In some embodiments, a ROCK inhibitor can be used instead of blebbistatin to increase the survival of iPSCs after dissociation into single cells.

[0120] In some embodiments, the efficiency of RPE differentiation from single cell iPSCs can be increased by accurately counting the seeding density. Thus, in some embodiments, the essentially single cell suspension of iPSCs is counted prior to seeding in step (b), for example, using a hemocytometer or an automated cell counter, such as a VICELL® or TC20. In some embodiments, the cells can be diluted to a cell density of about 10,000 to about 500,000 cells / mL, about 50,000 to about 200,000 cells / mL, or about 75,000 to about 150,000 cells / mL. In a non-limiting example, the essentially single cell suspension of iPSCs is diluted to a density of about 100,000 cells / mL in a fully defined culture medium, such as ESSENTIAL 8™ (E8™) medium.

[0121] Once a single cell suspension of iPSCs is obtained at a known cell density, the cells are generally seeded into a suitable culture vessel, such as a flask or tissue culture plate (e.g., a 6-well, 24-well, or 96-well plate). In some embodiments, the cells are cultured in a volume of at least or about 0.2 mL, about 0.5 mL, about 1 mL, about 2 mL, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 100 mL, about 150 mL, about 200 mL, about 250 mL, about 300 mL, about 350 mL, about 400 mL, about 450 mL, about 500 mL, about 550 mL, about 600 mL, about 800 mL, about 1000 mL, about 1500 mL, or any range derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel is a bioreactor, e.g., a bioreactor having a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0122] In some embodiments, the iPSCs are plated in step (b) at a cell density suitable for efficient differentiation. In some embodiments, the iPSCs are plated at a cell density of about 1,000 to about 75,000 cells / cm. 2 For example, about 5,000 to about 40,000 cells / cm 2 In some embodiments, the iPSCs are seeded in a 6-well plate at a cell density of about 50,000 to about 400,000 cells per well. In some embodiments, the iPSCs are seeded in a 6-well plate at a cell density of about 100,000, about 150,000, about 200,000, about 250,000, about 300,000, or about 350,000 cells per well, for example, about 200,000 cells per well.

[0123] In some embodiments, iPSCs (e.g., in steps (a) and (b)) are cultured on a matrix. PSCs, such as iPSCs, are generally cultured on a culture plate coated with one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins, such as vitronectin, laminin, collagen, and / or fibronectin, and can be used to coat culture surfaces as a means of providing a solid support for pluripotent cell growth. The term "extracellular matrix" is recognized in the art. The components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Extracellular matrix (ECM) proteins may be of natural origin and purified from human or animal tissues, or they may be genetically engineered recombinant proteins or synthetic in nature. ECM proteins may be in the form of whole proteins or peptide fragments, natural or engineered. Examples of ECM proteins that may be useful in matrices for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition comprises synthetically produced peptide fragments of fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is xeno-free. For example, xeno-free matrices for culturing human cells can use matrix components of human origin, in which any non-human animal components can be excluded.

[0124] In some embodiments, the iPSCs are cultured on a culture plate coated with a matrix. In some embodiments, the matrix comprises at least one recombinant cell adhesion protein, such as laminin, vitronectin, or fibronectin. In some embodiments, the cell adhesion protein is a human protein. In some embodiments, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In some embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0125] In some embodiments, the iPSCs are cultured without a feeder layer.

[0126] In some embodiments, the single cell iPSCs are cultured in fully defined culture medium after plating. In some embodiments, about 18-24 hours after plating, the medium is aspirated and fresh medium, such as E8™ medium, is added to the culture. In some embodiments, the iPSCs are cultured in fully defined culture medium for about 1, 2, or 3 days after plating. Preferably, the single cell PSCs are cultured in fully defined culture medium for about 2 days before proceeding with the differentiation process.

[0127] In some embodiments, iPSCs are cultured in xeno-free culture medium. In some embodiments, the medium may or may not contain any substitute for serum. Serum substitutes may include substances that suitably contain albumin (e.g., lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Serum substitutes may be prepared, for example, by methods disclosed in International Publication No. WO 98 / 30679. Any commercially available substance may be used, such as KNOCKOUT™ Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), or GLUTAMAX™ (Gibco).

[0128] In some embodiments, the retinal induction medium comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and insulin growth factor 1 (IGF1).

[0129] In some embodiments, the retinal differentiation medium comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, a MEK inhibitor, and IGF1.

[0130] Other culture conditions can be appropriately defined. For example, the culture temperature can be about 30-40°C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39°C. In some embodiments, the cells are cultured at 37°C. The CO2 concentration can be about 1-10%, for example, about 2-5%, or any range derivable therein. The oxygen partial pressure can be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

[0131] In some embodiments, RPE cells are dissociated after culturing in retinal medium and reseeded on the degradable scaffold in RPE maturation medium, thereby generating mature RPE cells. In some embodiments, the degradable scaffold is a poly(lactic-co-glycolic acid) (PLGA) scaffold.

[0132] Induction of RPE cells from embryoid bodies of PSCs In some embodiments, RPE cells are derived from iPSCs through a process of differentiating embryoid bodies (EBs) of iPSCs into cultures highly enriched for RPE cells. In some embodiments, EBs are generated from iPSCs by the addition of a rho-associated coiled-coil kinase (ROCK) inhibitor and cultured in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor. The EBs are plated on MATRIGEL™-coated tissue culture in a second medium lacking basic fibroblast growth factor (bFGF), containing a Nodal pathway inhibitor, containing about 20 ng to about 90 ng Noggin, and containing about 1 to about 5% knockout serum replacement to form differentiated RPE cells. The differentiated RPE cells are cultured in a third medium containing activin and WNT3a. The RPE cells are then cultured in RPE medium containing about 5% fetal serum, a canonical WNT inhibitor, a non-canonical WNT inhibitor, and an inhibitor of the sonic hedgehog and FGF pathways to generate human RPE cells.

[0133] Culture in retinal induction medium After the single cell iPSCs adhere to the culture plate, the cells are cultured in retinal induction medium (RIM) to initiate the differentiation process into retinal lineage cells. In some embodiments, the medium is aspirated and replaced with fresh RIM every day. In some embodiments, the iPSCs are cultured in RIM for about 1 to about 5 days, e.g., about 1, 2, 3, 4 or 5 days, e.g., about 2 days, to generate retinal lineage cells.

[0134] In some embodiments, RIM comprises a WNT pathway inhibitor and can cause PSC to differentiate into retinal lineage cells. In some embodiments, RIM further comprises a TGFβ pathway inhibitor and a BMP pathway inhibitor. One exemplary RIM medium is shown in Table 1. In some embodiments, RIM can comprise DMEM and F12 in about 1:1 ratio.

[0135] In some embodiments, the WNT pathway inhibitor is any inhibitor of a member of the WNT family of proteins, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11, and Wnt16. Examples of suitable WNT inhibitors known in the art include, but are not limited to, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydrazide (PNU 74654), 2,4-diamino-quinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH 535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK 715), Dickkopf-related protein 1 (DKK1), and secreted frizzled-related protein (SFRP1). In some embodiments, the inhibitor of WNT includes antibodies against WNT, dominant negative variants of WNT, and siRNA and antisense nucleic acids that suppress the expression of WNT. In some embodiments, inhibition of WNT can also be achieved using RNA-mediated interference (RNAi). In some embodiments, the WNT pathway inhibitor is CKI-7.

[0136] In some embodiments, the BMP pathway inhibitor can be a general inhibitor of BMP signaling or an inhibitor specific to BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Exemplary BMP inhibitors include, but are not limited to, 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[ Include 6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML 347).In some embodiments, the BMP pathway inhibitor is LDN193189.

[0137] In some embodiments, the TGFβ pathway inhibitor may include any inhibitor of TGFβ signal transduction in general.For example, in some embodiments, the TGFβ pathway inhibitor may be 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-ieri-butyl-3H-imidazol-4-yl)-6-methyl Pyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, Left-Right Determinant (Lefty), 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D 4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW 788388), 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY 364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R 268712), or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox). In some embodiments, the TGFβ pathway inhibitor is SB431542.

[0138] In some embodiments, the RIM includes a WNT pathway inhibitor, such as CKI-7, a BMP pathway inhibitor, such as LDN193189, and a TGFβ pathway inhibitor, such as SB431542. For example, in some embodiments, the RIM includes about 5 nM to about 50 nM, such as about 10 nM, LDN193189, about 0.1 μM to about 5 μM, such as about 0.5 μM, CKI-7, and about 0.5 μM to about 10 μM, such as about 1 μM, SB431542. In some embodiments, the RIM can include a knockout serum replacement, such as about 1% to about 5%, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin growth factor 1 (IGF1). In some embodiments, the IGF1 is animal-free IGF1 (AF-IGF1) and is contained in the RIM at about 0.1 ng / mL to about 10 ng / mL, for example, about 1 ng / mL.

[0139] Culture in retinal differentiation medium Retinal lineage cells can then be cultured in retinal differentiation medium (RDM) for further differentiation.In some embodiments, cells are fed with fresh RDM every day after aspirating the medium the day before.In some embodiments, cells are cultured in RDM for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 days, for example about 7 days, to induce differentiated retinal cells.

[0140] In some embodiments, the RDM comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. In some other embodiments, the RDM comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a bFGF inhibitor.

[0141] In some embodiments, the WNT pathway inhibitor is a WNT pathway inhibitor disclosed herein. In some embodiments, the WNT pathway inhibitor is CKI-7.

[0142] In some embodiments, the BMP pathway inhibitor is a BMP pathway inhibitor disclosed herein. In some embodiments, the BMP pathway inhibitor is LDN193189.

[0143] In some embodiments, the TGFβ pathway inhibitor is a TGFβ pathway inhibitor disclosed herein. In some embodiments, the TGFβ pathway inhibitor is SB431542.

[0144] In some embodiments, the MEK inhibitor is any chemical or drug that inhibits the mitogen-activated protein kinase enzyme MEK1 or MEK2.For example, in some embodiments, the MEK inhibitor includes N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7- Fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), and 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).In some embodiments, the MEK inhibitor is PD0325901.

[0145] In some embodiments, bFGF inhibitors include, but are not limited to, N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea (PD173074), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 98059), 1-tert-butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea (PD161570), 6-(2,6-dichlorophenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one dihydrochloride hydrate (PD166285), N-[2-amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)-urea (PD166866), and MK-2206.

[0146] In some embodiments, the RDM comprises a WNT pathway inhibitor, e.g., CKI-7, a BMP pathway inhibitor, e.g., LDN193189, a TGFβ pathway inhibitor, e.g., SB431542, and a MEK inhibitor, e.g., PD0325901. In some embodiments, the RDM comprises LDN193189, CKI-7, SB431542, and PD0325901. In some embodiments, the concentrations of the Wnt pathway inhibitor, the BMP pathway inhibitor, and the TGFβ pathway inhibitor are higher, e.g., about 9 to about 11 fold higher, e.g., about 10 fold higher, in the RDM compared to the RIM. In some embodiments, the RDM comprises about 50 nM to about 200 nM, e.g., about 100 nM, LDN193189, about 1 μM to about 10 μM, e.g., about 5 μM, CKI-7, about 1 μM to about 50 μM, e.g., about 10 μM, SB431542, and about 0.1 μM to about 10 μM, e.g., about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM, PD0325901. One exemplary RDM medium is shown in Table 1.

[0147] In some embodiments, the RDM comprises DMEM and F12 in about a 1:1 ratio, knockout serum replacement (e.g., about 1% to about 5%, e.g., about 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., about 1 ng / mL to about 50 ng / mL, e.g., about 10 ng / mL).

[0148] Culture in retinal medium Then, differentiated retinal cells can be further differentiated by culturing cells in retinal medium (RM).In some embodiments, medium is replaced with RM at room temperature every day.In some embodiments, cells are cultured in RM for about 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 days, for example about 10 days, to induce differentiated RPE cells.

[0149] In some embodiments, the RM comprises activin A and optionally nicotinamide. In some embodiments, the RM comprises about 50 to about 200 ng / mL, e.g., about 100 ng / mL, of activin A, and about 1 mM to about 50 mM, e.g., about 10 mM, of nicotinamide. In some other embodiments, the RM can comprise a TGF-β pathway activator, e.g., GDF1, and / or a WNT pathway activator, e.g., WAY-316606, IQ1, QS11, SB-216763, BIO(6-bromoindirubin-3'-oxime), or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. In some other embodiments, the RM can additionally comprise WNT3a. One exemplary RM medium is shown in Table 1.

[0150] In some embodiments, the RM can include DMEM and F12 in about a 1:1 ratio, about 1% to about 5%, e.g., about 1.5%, knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid.

[0151] Culture in RPE maturation medium For further differentiation of RPE cells, the cells can be cultured in RPE maturation medium (RPE-MM). Exemplary RPE-MM media are shown in Table 1. In some embodiments, the RPE-MM comprises about 100 μg / mL to about 300 μg / mL, e.g., about 250 μg / mL, taurine, about 10 μg / L to about 30 μg / L, e.g., about 20 μg / L, hydrocortisone, and about 0.001 μg / L to about 0.1 μg / L, e.g., about 0.013 μg / L, triiodothyronine. In some embodiments, the RPE-MM comprises MEM alpha, N-2 supplement, MEM non-essential amino acids (NEAA), sodium pyruvate, and / or fetal bovine serum (e.g., about 0.5% to about 10%, e.g., about 1% to about 5%). In some embodiments, the RPE-MM does not comprise a WNT pathway inhibitor.

[0152] In some embodiments, the medium is replaced with room temperature RPE-MM every other day. In some embodiments, the cells are cultured in RPE-MM for about 5 to about 10 days, e.g., about 5 days. Then, in some embodiments, the cells are dissociated, e.g., with a cell dissociation enzyme, reseeded, and cultured for an additional period, e.g., about 5 to about 30 days, e.g., about 15 to 20 days, to further differentiate into RPE cells.

[0153] The RPE cells can be cryopreserved at this stage.

[0154] RPE cell maturation The RPE cells can then be cultured in RPE-MM for a continuous period of time for maturation. In some embodiments, the RPE cells are grown on a culture plate, such as a 6-well, 12-well, 24-well, or 10 cm plate. In some embodiments, the RPE cells can be maintained in RPE-MM for about 4 to about 10 weeks, such as about 6 to 8 weeks, such as 6, 7, or 8 weeks.

[0155] In some embodiments of the continued maturation of RPE cells, cells are dissociated and reseeded on degradable scaffold assembly, and are cultured for a period of time.In some embodiments, the degradable scaffold assembly is formed by biodegradable polymer, such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), polyorthoester, polyanhydride, polyphosphazene, and combinations thereof.In some embodiments, the degradable scaffold assembly is formed by PLGA.

[0156] In some embodiments of continued maturation of RPE cells, cells can be dissociated by cell dissociation enzymes such as TRYPLE™ and reseeded on a degradable scaffold assembly such as a custom SNAPWELL™ design for approximately 1-2 weeks in RPE-MM supplemented with MEK inhibitors (e.g., PD0325901) or bFGF inhibitors. In some embodiments, methods for culturing RPE cells on degradable scaffolds are described in PCT Publication No. WO 2014 / 121077, which is incorporated herein by reference in its entirety. Briefly, the main components of the method are the CORNING® COSTAR® SNAPWELL™ plate, Bio-Insert 0-Ring, and a biodegradable scaffold. The SNAPWELL™ plate provides the structure and platform for the biodegradable scaffold. The microporous membrane that creates the apical and basal sides is ideal for not only providing support to the scaffold but also isolating separate sides of the polarized cell layer. The ability to detach the membrane of the SNAPWELL™ insert allows the support ring of the insert to be used as a scaffold anchor. The resulting monolayer of differentiated, polarized, confluent, and functional RPE cells can be cryopreserved at this stage (e.g., in xeno-free CS10 medium).

[0157] In some embodiments, mature RPE cells can be further developed into a functional RPE cell monolayer that behaves as an intact RPE tissue by continued culture in RPE-MM with additional chemicals or small molecules that promote RPE maturation. Examples of such small molecules include, but are not limited to, primary cilia inducers such as prostaglandin E2 (PGE2) or aphidicolin. In some embodiments, PGE2 can be added to the RPE-MM at a concentration of about 25 μM to about 250 μM, for example, about 50 μM to about 100 μM. In some embodiments, the RPE-MM can include a canonical WNT pathway inhibitor. Exemplary canonical WNT pathway inhibitors include, but are not limited to, N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), and 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). In some embodiments, the cells are cultured in such medium for an additional period of time, e.g., for an additional about 1 week to about 5 weeks, e.g., for an additional about 2 to 4 weeks, to result in a mature, functional RPE cell monolayer.

[0158] Thus, the presently disclosed method provides mature RPE cells from iPSCs that can be consistently reproduced on a large scale for clinical application.

[0159] Cryopreservation of RPE cells RPE cells produced by the methods disclosed herein can be cryopreserved, for example, as described in PCT Publication No. 2012 / 149484. The cells can be cryopreserved with or without a substrate. In some embodiments, the storage temperature ranges from about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and overlapping ranges thereof. In some embodiments, lower temperatures are used for preservation (e.g., maintenance) of the cryopreserved cells. In some embodiments, liquid nitrogen (or other similar liquid coolant) is used to preserve the cells. In some embodiments, the cells are preserved for greater than about 6 hours. In some embodiments, the cells are preserved for about 72 hours. In some embodiments, the cells are preserved for 48 hours to about 1 week. In still other embodiments, the cells are preserved for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. The cells can also be stored for longer periods. The cells can be cryopreserved separately or on a substrate, for example, on any substrate disclosed herein.

[0160] In some embodiments, additional cryoprotectants can be used. For example, in some embodiments, cells are cryopreserved in a cryopreservation solution that includes one or more cryoprotectants, for example, serum albumin, such as DM80, human or bovine serum albumin. In some embodiments, the solution includes about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% dimethyl sulfoxide (DMSO). In some embodiments, the solution includes about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% DMSO or albumin. In certain embodiments, the solution includes 2.5% DMSO. In another particular embodiment, the solution includes 10% DMSO.

[0161] In some embodiments, the cells can be cooled during cryopreservation, for example, at about 1° C. / min. In some embodiments, the cryopreservation temperature is about −80° C. to about −180° C., or about −125° C. to about −140° C. In some embodiments, the cells are cooled to 4° C. before cooling at about 1° C. / min. Cryopreserved cells can be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, once the cells reach about −80° C., they are transferred to a storage area of ​​liquid nitrogen. Cryopreservation can also be performed using a controlled rate freezer. Cryopreserved cells can be thawed, for example, at a temperature of about 25° C. to about 40° C., typically at a temperature of about 37° C.

[0162] Pharmaceutical Compositions Also provided herein are pharmaceutical compositions of RPE cells obtained by the methods disclosed herein. These compositions contain at least about 1×10 3 RPE cells, approximately 1 x 10 4 RPE cells, approximately 1 x 10 5 RPE cells, approximately 1 x 10 6 RPE cells, approximately 1 x 10 7 RPE cells, approximately 1 x 10 8 RPE cells, or approximately 1 x 10 9 In certain embodiments, the composition is a substantially purified (with respect to non-RPE cells) preparation comprising differentiated RPE cells produced by the methods disclosed herein.

[0163] Also provided is a composition comprising a scaffold, such as a polymer carrier and / or extracellular matrix, and an effective amount of RPE cells produced by the method disclosed herein.For example, the cells are provided as a monolayer of cells.The matrix material is generally physiologically acceptable and suitable for use in vivo applications.For example, physiologically acceptable materials include, but are not limited to, absorbable and / or non-absorbable solid matrix materials, such as small intestinal submucosa (SIS), cross-linked or non-cross-linked alginate, hydrocolloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) mesh, fleece, and bioadhesives.

[0164] Suitable polymer carriers include porous meshes or sponges formed from synthetic or natural polymers, as well as polymer solutions. For example, the matrix is ​​a polymer mesh or sponge, or a polymer hydrogel. Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; and polysaccharides such as polymers of alginic acid and hyaluronic acid. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include polymers of hydroxy acids, such as polylactic acid (PLA), polyglycolic acid (PGA) and polylactic-glycolic acid (PGLA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof. Non-biodegradable polymers include polyacrylic acid, polymethacrylic acid, ethylene vinyl acetate, and polyvinyl alcohol.

[0165] Polymers capable of forming malleable ionic or covalently crosslinked hydrogels can be used. Hydrogels are materials formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that traps water molecules to form a gel. Examples of materials that can be used to form hydrogels include polysaccharides such as alginate, polyphosphazine, and polyacrylic acid, which are ionically crosslinked, or block copolymers such as PLURONICS™ or TETRONICS™, polyethylene oxide-polypropylene glycol block copolymers, which are crosslinked by temperature or H, respectively. Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen.

[0166] In some embodiments, provided herein is a composition comprising a PLGA scaffold and an effective amount of RPE cells obtained by the methods disclosed herein.

[0167] In some embodiments, the pharmaceutical compositions provided can be packaged in a suitable container with instructions for a desired purpose, such as reconstituting RPE cell function to improve disease or abnormality of retinal tissue. In some embodiments, the RPE cells produced by the disclosed methods can be engineered to form RPE and can be used to replace degenerated RPE in subjects in need thereof.

[0168] Use of retinal pigment epithelial cells Also provided herein are methods for generating RPE or RPE-enriched cell populations that can be used for a number of important research, development, and commercial purposes.

[0169] In some embodiments, the methods disclosed herein provide for the production of at least or about 10 RPE cells that comprise at least or about 90% (e.g., at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivable therein) of RPE cells. 6 , 10 7 , 10 8 , 5×10 8 , 10 9 , 10 10 The resulting cell population is a population of cells (or any range derivable therein).

[0170] In some embodiments, the starting cells for the method include at least or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 In some embodiments, the starting cell population may include the use of at least or about 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The seeding density may be in cells / mL, or any range derivable therein.

[0171] RPE cells produced by the methods disclosed herein may be used in any of the methods and applications currently known in the art for RPE cells.

[0172] Compound Evaluation and Screening In some embodiments, the present invention provides a method for evaluating a compound, comprising the step of assaying the pharmacological or toxicological properties of the compound on RPE cells.The present invention also provides a method for evaluating a compound for its effect on RPE cells, comprising the steps of: (a) contacting the RPE cells provided herein with the compound; and (b) assaying the effect of the compound on RPE cells.

[0173] In some embodiments, RPE cells can be used commercially to screen factors (such as solvents, small molecule drugs, peptides, oligonucleotides, etc.) or environmental conditions (such as culture conditions or manipulations) that affect the characteristics of such cells and their various progeny. For example, test compounds can be chemical compounds, small molecules, polypeptides, growth factors, cytokines, or other biological agents.

[0174] In some embodiments, the method includes contacting RPE cells with a test agent and determining whether the test agent modulates the activity or function of RPE cells in the population. In some embodiments, the screening assay is used to identify agents that modulate the proliferation of RPE cells or change RPE cell differentiation. The screening assay can be performed in vitro or in vivo. Methods for screening and identifying ophthalmic or RPE agents include those suitable for high-throughput screening. For example, RPE cells can be placed or placed on culture dishes, flasks, roller bottles, or plates (e.g., single multi-well dishes or dishes, such as 8, 16, 32, 64, 96, 384 and 1536 multi-well plates or dishes) at any defined position for the identification of potential therapeutic molecules. Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0175] Other screening applications involve testing pharmaceutical compounds for their effect on the maintenance or repair of retinal tissue. Screening can be done because the compound is designed to have a pharmacological effect on cells, or because compounds designed to have other effects may have unintended side effects on cells of this tissue type.

[0176] Treatment and transplantation Other embodiments may provide for the use of RPE cells to enhance ocular tissue maintenance and repair for any condition in need thereof, including retinal degeneration or severe damage.

[0177] To determine the suitability of the cell composition for therapeutic administration, the cells can be first tested in a suitable animal model.In one aspect, RPE cells are evaluated for their ability to survive and maintain their phenotype in vivo.The cell composition is administered to an immunodeficient animal (e.g., nude mice, or animals that are made immunodeficient chemically or by irradiation).Tissue is harvested after a period of growth and evaluated for whether pluripotent stem cell-derived cells are still present.

[0178] Several animals are available for testing the suitability of RPE cell compositions. For example, the Royal College of Surgeon's (RCS) rat is a well-known model of retinal dystrophy (Lund et al., 2006). In addition, the suitability and survival of RPE cells can be determined by transplantation (e.g., subcutaneous or subretinal) with Matrigel in immunodeficient animals such as NOG mice (Kanemura et al., 2014).

[0179] The human RPE cells described herein, or pharmaceutical compositions comprising these cells, can be used for the manufacture of medicaments to treat conditions in patients in need thereof. The RPE cells can be cryopreserved in advance. In some embodiments, the disclosed RPE cells are derived from iPSCs, and can thus be used to provide "personalized medicine" to patients with ocular diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct disease-causing mutations, differentiated into RPE, and engineered to form RPE tissue. This RPE tissue can be used to replace endogenous degenerated RPE in the same patient. Alternatively, iPSCs derived from healthy donors or from HLA homozygous "super donors" can be used. RPE cells can be treated in vitro with certain factors, such as pigment epithelium-derived factor (PEDF), transforming growth factor (TGF) beta, and / or retinoic acid, to provide an anti-inflammatory and immunosuppressive environment in vivo.

[0180] By introducing RPE cells obtained by using the method disclosed herein, various ocular conditions can be treated or prevented. The conditions include retinal diseases or disorders that are generally related to retinal dysfunction or deterioration, retinal damage, and / or loss of retinal pigment epithelium. The conditions that can be treated include, but are not limited to, degenerative diseases of the retina, such as Stargardt's macular dystrophy, retinitis pigmentosa, macular degeneration (such as age-related macular degeneration), glaucoma, and diabetic retinopathy. Further conditions include Leber's congenital amaurosis, hereditary or acquired macular degeneration, Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, other dystrophies of RPE, and damage to RPE and retina caused by any one of light, laser, inflammation, infection, radiation, neovascularization, or traumatic injury. In certain embodiments, a method is provided for treating or preventing a condition characterized by retinal degeneration, comprising administering an effective amount of a composition comprising RPE cells to a subject in need thereof. These methods can include selecting a subject with one or more of these conditions, and administering a therapeutically effective amount of RPE cells sufficient to treat the condition and / or ameliorate the symptoms of the condition. RPE cells can be transplanted in various formats. For example, RPE cells can be introduced to the target site in the form of a cell suspension, or can be attached as a monolayer on a substrate such as a matrix, an extracellular matrix, or a biodegradable polymer, or can be combined. RPE cells can be transplanted together with other retinal cells such as photoreceptors (co-transplantation). In some embodiments, RPE cells are generated from iPSCs from the subject to be treated, and thus are autologous. In other embodiments, RPE cells are generated from an MHC-matched donor.

[0181] In some embodiments, RPE cells can be used for autologous RPE grafts into subjects suitable for undergoing regenerative medicine. RPE cells may be transplanted in combination with other retinal cells, such as photoreceptors. Transplantation of RPE cells made by the disclosed method can be performed by various techniques and methods known in the art, such as those described in U.S. Pat. Nos. 5,962,027 and 6,045,791. In some embodiments, transplantation is performed via pars plana vitrectomy followed by delivery of cells through a small retinal opening or by direct injection into the subretinal space. In some embodiments, RPE cells can be introduced to the target site in the form of a cell suspension, can be attached on a matrix, such as an extracellular matrix, or can be provided on a substrate, such as a biodegradable polymer. In some embodiments, RPE cells can also be transplanted together with other cells, such as retinal cells with photoreceptors (co-transplantation). Thus, compositions are provided that include RPE cells obtained by the methods disclosed herein. In some embodiments, these RPE cells include a tyrosinase enhancer operably linked to a promoter and a nucleic acid encoding a marker. In other embodiments, the RPE cells also contain a second constitutive promoter operably linked to a nucleic acid encoding a second marker.

[0182] kit In some embodiments, kits are provided that include one or more media and components for the generation of RPE cells, for example. The reagent system may be packaged in either aqueous media or lyophilized form, if necessary. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means in which the components may be placed, and preferably appropriately aliquoted. If there is more than one component in the kit, the kit generally also includes a second, third, or other additional container in which the additional components may be separately placed. However, various combinations of components may be included in the vial. The components of the kit may be provided as dry powders. If the reagents and / or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is also envisioned that the solvent may be provided in another container means. The kit typically also includes a means for containing the kit components in a closed state for commercial sale. Such containers may include injected or blow molded plastic containers in which the desired vials are retained. The kit can also include instructions for use, eg, in printed or electronic, such as digital, format. EXAMPLES

[0183] Example 1. Preparation of starting populations of iPSCs The starting population of iPSCs can be reprogrammed from somatic cells by any method known in the art. This example describes an exemplary method.

[0184] Collect blood from donors by peripheral blood collection and collect mononuclear cells (MNC). The isolated peripheral blood mononuclear cells (PBMC) are cryopreserved and stored in liquid nitrogen until use.

[0185] CD34+ or CD71+ cells are purified from PBMCs, for example, using magnetic beads, and expanded. Then, the cells are reprogrammed by introducing a reprogramming vector. In some embodiments, the cells are reprogrammed using the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit, following the manufacturer's protocol, resulting in iPSCs cultured on vitronectin-coated plates. After the introduction of the reprogramming vector, for example after transduction with Sendai reprogramming virus, the cells are monitored for the appearance of cell clumps / colonies, which represent reprogrammed cells. These colonies are then selected and optionally expanded for one passage to obtain the starting population of iPSCs for the next step.

[0186] Example 2. Preparation of starting populations of iPSCs This example describes further exemplary methods for generating a starting population of iPSCs.

[0187] Collect blood from donors by peripheral blood collection and collect mononuclear cells (MNC). The isolated peripheral blood mononuclear cells (PBMC) are cryopreserved and stored in liquid nitrogen until use.

[0188] CD34+ or CD71+ cells are purified from PBMCs, for example, using magnetic beads, and expanded. Then, the cells are reprogrammed by introducing a reprogramming vector. In some embodiments, the cells are reprogrammed using CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit, following the manufacturer's protocol, resulting in iPSCs cultured on vitronectin-coated plates. After the introduction of the reprogramming vector, for example after transduction with Sendai reprogramming virus, the cells are monitored for the appearance of cell clumps / colonies, which represent reprogrammed cells. This results in a pooled culture of iPSCs, which are optionally passaged once or multiple times, and optionally cryopreserved, resulting in a starting population of iPSCs for the next step.

[0189] Example 3. Generation of iPSCs essentially free of exogenous viral residues A starting population of iPSCs was obtained using Sendai virus reprogramming vectors as described in Example 1 or Example 2. iPSCs were dissociated into single cell suspensions. Cells were counted, diluted as necessary, and plated in vitronectin-coated 6-well plates at a density of approximately 5,000-10,000 cells / well in ESSENTIAL 8 (E8) medium and cultured at 37°C. The medium was replaced with fresh E8 medium every day or every other day until individual iPSC colonies appeared. The medium was supplemented with RevitaCell supplement containing ROCK inhibitor, which does not allow cells to attach to the plate but increases the survival of iPSCs after dissociation into single cells.

[0190] The wells with the least differentiated cells were selected. The cells in the selected wells were dissociated into a single cell suspension. The cells were replated in E8 medium at a density of approximately 1,500-3,000 cells / well on vitronectin-coated 6-well plates and cultured at 37°C until individual iPSC colonies appeared. The medium was replaced with fresh E8 medium every day or every other day. RevitaCell supplement was added to the medium, which does not allow the cells to attach to the plate but increases the survival of iPSCs after dissociation into single cells.

[0191] After 2–4 days of culture at 37 °C to allow the emergence of individual iPSC colonies, the culture temperature was increased to 38.5 °C for 5–6 days and then returned to 37 °C for an additional 1–2 days. In controls, the culture temperature was kept constant at 37 °C. Cells were not allowed to overgrow to prevent individual colonies from coalescing.

[0192] Subsequently, one iPSC colony per well was manually selected and passaged 1–8 times. The resulting iPSCs were subjected to immunostaining and RT-qPCR analysis of Sendai virus residues. The immunostaining assay used a fluorescently labeled rabbit polyclonal anti-Sendai virus antibody. For RT-qPCR, a TaqMan kit (Thermo Fisher, containing a labeled TaqMan probe and Sendai virus cDNA template) was used.

[0193] Figures 2B, 3A and 3B show that iPSCs derived from a starting iPSC population obtained as described in Example 1 and colonies treated at elevated temperatures were free of Sendai virus. Figure 4 shows that iPSCs derived from a starting iPSC population obtained as described in Example 2 and colonies treated at elevated temperatures were free of Sendai virus. The data show that early introduction of elevated temperature at an early stage before clonal selection can result in vector-free iPSCs at early passages (less than 8).

[0194] Example 4. Preparation of RPE cells Example 4.1. Preparation of single cell iPSCs A population of iPSCs essentially free of exogenous viral residues is obtained as described in Example 2. The iPSCs are dissociated into a single cell suspension in E8™ medium to remove any aggregates or embryoid bodies. Cells are counted and, if necessary (e.g., approximately 1×10 in E8 medium at room temperature), cultured. 5 Dilute the iPSCs in E8 medium (up to 100,000 cells / mL) and seed them at a density of approximately 200,000 cells / well in vitronectin-coated 6-well plates and place them in a humidified incubator at 37 °C. After approximately 18-24 h, aspirate the medium and add fresh E8 medium to the cultures. For proper attachment to the plate, culture the cells in E8 medium for approximately 2 days after seeding. Add blebbistatin (e.g., 2.5 μM) to the E8 medium, which does not allow the cells to attach to the culture vessel but increases the survival of iPSCs after dissociation into single cells.

[0195] Example 4.2. Differentiation of iPSCs into RPE cells The single-cell iPSCs are then cultured in various differentiation media to induce them into RPE cells.

[0196] On day 3, aspirate the E8™ medium and add retinal induction medium (RIM) at room temperature (e.g., RIM in Table 1). Culture the cells in RIM for approximately 2-4 days. Each day, aspirate the medium and add fresh RIM.

[0197] Next, culture the cells in retinal differentiation medium (RDM) (e.g., RDM in Table 1) for approximately 7-14 days. Each day, aspirate the medium and add room temperature RDM to the cells to produce differentiated retinal cells.

[0198] Subsequently, to induce RPE cells, culture the cells in retinal medium (RM) (e.g., RM in Table 1) for 7 to 10 days. Replace the medium with room temperature RM every day to obtain RPE cells.

[0199] For maturation of RPE cells, the cells are cultured in RPE maturation medium (RPE-MM) (e.g., RPE-MM in Table 1) for 5-10 days. The medium is replaced with room temperature RPE-MM every other day. The cells are then dissociated with cell dissociation enzymes and re-seeded on vitronectin-coated plates. At this stage, the induced PRE cells can be cryopreserved, for example, in xeno-free CS10 medium. Alternatively, to continue RPE maturation, the plated cells are cultured for approximately another 15 days.

[0200] Example 4.3. Continuous maturation of RPE cells For continued maturation of RPE cells, cells are dissociated with cell dissociation enzymes (e.g., TRYPLE™) and reseeded on top of the custom SNAPWELL™ designed degradable scaffold assembly in RPE-MM with MEK inhibitors (e.g., PD325901) for 1-2 weeks. This results in a differentiated, polarized, confluent monolayer of functional RPE cells, which can be cryopreserved at this stage in xeno-free CS10 medium.

[0201] Mature RPE cells can be further developed into a functional RPE cell monolayer that functions as an intact RPE tissue by continued culture in RPE-MM with additional small molecules such as primary ciliary inducers like PGE2 or aphidicolin. Without being bound by theory, these primary ciliary inducers suppress the canonical WNT pathway, induce cell cycle exit in cells, and induce apical-basal polarization in the RPE monolayer. RPE maturation can alternatively be induced by canonical WNT pathway inhibitors such as rWP2 and endo-rWRl, which also induce cell cycle exit in RPE cells to promote RPE maturation. Cells are cultured in this medium for an additional 2-3 weeks to obtain a mature, functional RPE cell monolayer.

[0202] Example 4.4. Depletion of contaminating cells The RPE cells obtained as described in Example 3 may have residual contaminating non-RPE cells as well as immature RPE cells (collectively referred to as "contaminating cells"), both of which can be dissociated and removed to obtain a mature RPE enriched cell population. The contaminating cells can be removed from the culture by any method known in the art, for example, magnetic cell dissociation (MACS®), fluorescence activated cell sorting (FACS), or single cell sorting.

[0203] In some embodiments, the MACS® method is used to dissociate different cell populations according to their surface antigens to dissociate contaminating cells from mature RPE-enriched cells.CD24, CD56, and / or CD90 are used as surface markers to dissociate / remove contaminating cells.These markers are expressed on PSCs, but are lost during the differentiation of stem cells into RPE cells.

[0204] In some embodiments, inclusion of the MEK inhibitor PD0325901 at a concentration of 1 μM in the medium for a certain time frame beginning from day 2 after iPSC plating, including culture after depletion of contaminating cells, through the end of the differentiation process, can improve both the purity of the RPE population (meaning reduced contaminating cells) and the resulting maturity of the RPE population. Inclusion of 1 μM PD0325901 has been shown to improve both the purity and maturity of the RPE population when included in the RDM as well as RPE-MM (approximately days 42-50) of the RPE process described herein.

[0205] In some embodiments, reducing the percentage of fetal bovine serum in the medium used during depletion of RPE-MM and contaminating cells from 5 percent to 0.5 to 1 percent can improve both the purity of the RPE population and the resulting maturity of the RPE population.

[0206] Example 4.5. Characterization of RPE cells Characterization of RPE cells for their purity (e.g., RPE-specific markers), maturity, and functionality (e.g., measurement of transepithelial potential / electrical resistance) is performed by various methods known in the art, such as flow cytometry, immunostaining, and electrophysiological techniques.

[0207] Table 1. Exemplary medium components TIFF2025511931000001.tif35155TIFF2025511931000002.tif231155TIFF2025511931000003.tif79155

[0208] Equivalents / Other Aspects Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the scope of the claims appended hereto.

Claims

1. In the following order: (a) A step of obtaining a starting population of induced pluripotent stem cells (iPSCs) produced using a reprogramming vector; (b) A step of seeding the iPSCs at a low density and culturing them in a culture medium; (c) Optionally, a step of repeating step (b) once or more times; (d) Optionally, a step of culturing the iPSC at an elevated temperature; (e) A process of selecting a single iPSC colony and passing it 1 to 8 times in order to create a population of iPSCs that are essentially free of exogenous vector residues. A method for producing a clonal population of induced pluripotent stem cells (iPSCs) that is essentially free of exogenous vector residues, including [specific exogenous vector residues].

2. In the following order: (a) A step of obtaining a starting population of induced pluripotent stem cells (iPSCs) produced using a reprogramming vector; (b) A step of seeding the iPSCs at a low density and culturing them in a culture medium; (c) Optionally, a step of repeating step (b) once or more times; (d) Optionally, a step of culturing the iPSC at an elevated temperature; (e) A process of selecting a single iPSC colony and passing it 1 to 8 times in order to obtain iPSCs that are essentially free of exogenous vector residues. A method for producing induced pluripotent stem cells (iPSCs) that are essentially free of exogenous vector residues, including [specific exogenous vector residues].

3. The method according to claim 1 or 2, wherein the starting population of iPSCs is produced from somatic cells of a human individual.

4. Step (a) is, (i) A process to obtain a polyclonal pool of iPSCs created using a reprogramming vector; (ii) optionally, the iPSC is passed through once or more times; and (iii) Optionally, a step of freezing and storing the iPSCs produced by step (i) or step (ii), thereby providing the starting population of iPSCs. The method according to claim 1 or 2, including the method described in claim 1 or 2.

5. The method according to claim 1 or 2, wherein the starting population of iPSCs has not been passaged or has been passaged once prior to its use in the method.

6. The method according to claim 1 or 2, wherein the reprogramming vector is a non-embedded viral vector.

7. The method according to claim 1 or 2, wherein step (b) is repeated once or twice.

8. Each time it is performed, process (b) is performed independently. (1) Dissociate the iPSCs into essentially single cells before seeding. (2) Seed the iPSCs at a density of approximately 1 to 1500 cells / cm². (3) Seeding the iPSCs in the culture medium to which a Rho-related protein kinase (ROCK) inhibitor has been added, wherein the culture medium is a fully defined medium, and / or (4) Cultivate iPSCs at approximately 37.0°C to 39.0°C. The method according to claim 1 or 2, including the method described in claim 1 or 2.

9. When the last step (b) is performed, The iPSCs are seeded at a clonal density, optionally such that the clonal density is approximately 140 to approximately 350 cells / cm². The method according to claim 1 or 2, including the method described in claim 1 or 2.

10. Each time it is performed, process (b) is performed independently. Approximately 140 to 350 cells / cm² were added to the culture medium to which the ROCK inhibitor had been added. 2 Or approximately 470 to 1150 cells / cm² 2 The iPSCs are seeded at the density mentioned above, and then 5% CO2 is added until a single iPSC colony appears. 2 The culture medium is incubated at approximately 37.0°C in an incubation atmosphere. The method according to claim 1 or 2, including the method described in claim 1 or 2.

11. When the last step (b) is performed, Approximately 140 to 350 cells / cm² were added to the culture medium to which the ROCK inhibitor had been added. 2 The iPSCs were sown at a density of 5% CO2, followed by 5% CO2. 2 The iPSCs are cultured in the culture medium at approximately 37.0°C in an incubation atmosphere for 1 to 3 days. The method according to claim 1 or 2, including the method described in claim 1 or 2.

12. The method according to claim 1 or 2, wherein step (d) is not performed.

13. The method according to claim 1 or 2, wherein step (d) is performed.

14. (1) Step (d) is 5% CO 2 This includes culturing the iPSCs in Essential 8 medium in an incubation atmosphere. (2) The increased temperature is approximately 38.0°C to approximately 39.0°C. (3) Step (d) includes culturing the iPSCs at the elevated temperature for 5 to 8 days, and / or (4) Step (d) further comprises culturing the iPSC at approximately 37.0°C for at least one day. The method according to claim 13.

15. Step (d) is, 5% CO 2 In an incubation atmosphere, the cultures were incubated in Essential 8 medium at approximately 38.0°C to 39.0°C for approximately 6 days, followed by 5% CO2. 2 The iPSCs are cultured in an incubation atmosphere at approximately 37.0°C for 1 to 2 days. The method according to claim 13, including the method described in claim 13.

16. In the following order: (a) A step to obtain a starting population of induced pluripotent stem cells (iPSCs) produced using a Sendai virus vector containing a temperature-sensitive mutation; (b1) Dissociate the iPSCs into essentially single cells, seed them at a density of about 470 to about 1150 cells / cm 2 in a culture medium supplemented with a ROCK inhibitor, and subsequently culture them in the culture medium at about 37.0 °C in a 5% CO 2 incubation atmosphere until single iPSC colonies appear; (b2) The iPSCs are dissociated into essentially single cells and cultured in a culture medium to which a ROCK inhibitor has been added, with approximately 140 to 350 cells / cm². 2 Seeds are sown at this density, followed by 5% CO2. 2 A step of culturing in the culture medium at approximately 37.0°C in an incubation atmosphere for approximately 3 days; (c) 5% CO 2 The culture medium was incubated at approximately 38.0°C to 39.0°C in an incubation atmosphere for approximately 6 days, followed by 5% CO2. 2 A step of culturing the iPSC in an incubation atmosphere at approximately 37.0°C for 1 to 2 days; and (d) A step of selecting a single iPSC colony and culturing it for less than 8 passages in order to obtain iPSCs that are essentially free of exogenous viral residues. The method according to claim 1 or 2, including the method described in claim 1 or 2.

17. (a) A step of obtaining a population of iPSCs that are essentially free of exogenous viral residues according to claim 1 or 2; (b) A step of seeding the iPSCs and culturing them in retinal induction medium in order to induce differentiation of the cells into retinal lineage cells; (c) A step of culturing the retinal lineage cells in a retinal differentiation medium in order to further differentiate the retinal lineage cells; (d) A step of culturing differentiated retinal pigment epithelial (RPE) cells in retinal culture medium; and (e) A step of culturing the differentiated RPE cells in RPE maturation medium to obtain human RPE cells. A method for producing or manufacturing human retinal pigment epithelial (RPE) cells, including [the specified element].

18. (1) The method does not involve the formation of an embryoid body, (2) The iPSCs of step (a) are dissociated into single cells, or step (b) includes dissociating the iPSCs into essentially single cells before seeding. (3) Step (b) is (i) At a cell density of approximately 5,000 to 40,000 cells / cm², (ii) Without a supporting cell layer, (iii) In a fully defined culture medium, and / or (iv) In xenofree culture medium The process includes sowing the iPSCs. (4) Step (b) includes culturing iPSCs on a matrix, (5) The retinal induction medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, and insulin growth factor 1 (IGF1), (6) The retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor, and IGF1. (7) Step (e) comprises dissociating the differentiated RPE cells, reseeding the RPE cells, and culturing the RPE cells in the RPE maturation medium, wherein the RPE maturation medium comprises a MEK inhibitor, and optionally, the RPE cells are reseeded on a degradable scaffold in the RPE maturation medium. (8) The RPE maturation medium contains at least one primary ciliary inducing factor, and / or (9) The method further includes the step of cryopreserving the human RPE cells, The method according to claim 17.

19. (a) A step of obtaining an iPSC that is essentially free of exogenous viral residues according to claim 1 or 2, and dissociating the iPSC into essentially single cells in a fully defined culture medium; (b) A step of seeding the iPSCs and culturing them on laminin, vitronectin, or a combination thereof in a retinal induction medium containing LDN193189, CKI-7, and SB431542 in order to induce differentiation of the cells into retinal lineage cells; (c) A step of culturing the retinal lineage cells in a retinal differentiation medium containing LDN193189, CKI-7, SB431542, and PD0325901 in order to further differentiate the retinal lineage cells; (d) A step of culturing differentiated retinal pigment epithelial (RPE) cells in a retinal medium containing nicotinamide and activin A in order to form RPE cells; and (e) A step of culturing the differentiated RPE cells in RPE maturation medium to obtain human RPE cells. A method for producing human retinal pigment epithelial (RPE) cells, which includes but does not involve embryoid body formation.

20. A pharmaceutical composition comprising human RPE cells prepared or manufactured according to claim 17, a pharmaceutically acceptable carrier, and optionally a scaffold.

21. A pharmaceutical composition comprising human RPE cells prepared or manufactured according to claim 19, a pharmaceutically acceptable carrier, and optionally a scaffold.

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