Serum-free methods for derivation of stem cell-derived retinal pigment epithelial cells and related cells, compositions, methods, and systems

Abstract

Provided herein are serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods and systems that in several embodiments allow efficient, differentiation, isolation, and / or maturation of SC-RPE from a variety of different mammalian stem cells. In particular, provided herein serum free media related method and systems for maturation, enrichment and / or expansion of SC-RPE cells, as well as additional or alternative method and system for preparation of freezing stock, preparation of SC-RPE patch or cells suspension and related uses to transplant the SC-RPE in the sub-retinal space of an individual.

Description

Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT SERUM-FREE METHODS FOR DERIVATION OF STEM CELL-DERIVED RETINAL PIGMENT EPITHELIAL CELLS AND RELATED CELLS, COMPOSITIONS, METHODS, AND SYSTEMS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.63 / 510,095, entitled “Serum-Free Methods for the Derivation of Stem Cell-Derived Retinal Pigment Epithelial Cells and Related Cells, Compositions, Methods, and Systems” filed on June 23, 2023, with docket number P2765-USP, the contents of which is incorporated by reference in its entirety. FIELD

[0002] The present disclosure relates to cells which are stem cell-derived, and in particular to stem cell-derived retinal pigment epithelial cells. More particularly, the present disclosure relates to serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells and related cells, compositions, methods, and systems. BACKGROUND

[0003] Stem cells are at the center of many efforts to develop a technology capable of offering efficient and effective methods for cell generation and regeneration as well as for treatment and / or prevention of a variety of conditions.

[0004] In particular, stem cell technology has been applied to retinal pigment epithelium (RPE) to address RPE degeneration and loss of retinal pigment epithelial cells associated with a number of degenerative retinal diseases.

[0005] Challenges however remain due to a generally low and / or variable yield of RPE derivation procedures, as well as to relatively high complexity which impact the efficiency of RPE derivation as would be understood by a skilled person. SUMMARY

[0006] Provided herein are serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods, and systems that in several embodiments allow efficient, differentiation, isolation, and / or maturation from a variety of different mammalian stem cells.

[0007] In particular, according to a first aspect, serum-free SC-RPE methods and related cells,Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT compositions, methods and systems are based on the unexpected finding that stem cell line- specific combinations of serum-free cell culture differentiation media and cell culture substrates, can be used to provide an efficient SC-RPE differentiation in a stem cell line specific manner.

[0008] Accordingly, a toolbox method and systems and related serum-free SC-RPE differentiation media are herein described, in which combinations of serum-free SC-RPE differentiation medium and SC-RPE cell culture substrate are selected depending on the starting stem cell line, to perform SC-RPE differentiation by generating precursor cells capable of obtaining a mature RPE phenotype in accordance with the present disclosure.

[0009] In the SC-RPE differentiation method of the disclosure, the method comprises contacting stem cells of a starting stem cell line with an SC-RPE differentiation medium, and an SC-RPE cell culture substrate, the SC-RPE differentiation medium, and SC-RPE cell culture substrate selected depending on the starting stem cell line. In the SC-RPE differentiation method of the disclosure, the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cells.

[0010] In the SC-RPE differentiation system of the disclosure, the system comprises at least one serum-free SC-RPE differentiation medium and at least one SC-RPE cell culture substrate. In SC-RPE differentiation system of the disclosure, the at least one cell culture medium and the at least one cell culture substrate are included for combined use to provide a cell culture environment (herein also indicated as support) configured to allow introduction and / or maintenance of stem cells cultured in the cell culture support to obtain a differentiated SC-RPE cells according to methods of the disclosure.

[0011] Combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate configured for the SC-RPE differentiation of a starting stem cells line can be identified by a method comprising contacting the starting stem cell line with one or more candidate combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate, the contacting performed for a time and under condition allowing SC-RPE differentiation and selecting a candidate combination of the one or more candidate combinations which following the contacting results in SC-RPE differentiation of the starting stem cell line.

[0012] According to a second aspect, the serum-free SC-RPE methods and related cells, compositions, methods, and systems are also based on the unexpected finding that the yieldTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and maturation of differentiated SC-RPE cells can be promoted and / or enhanced by a serum- free RPE Maturation Medium of the disclosure.

[0013] The RPE Maturation Medium of the disclosure is a cell culture medium comprising a buffered physiological salt solution, vitamins, essential lipids, aerobic and anerobic energy sources, essential amino acids (EAA) and nonessential amino acids (NEAA), insulin, selenite and transferrin / Fe+3, progesterone, taurine, triiodothyronine (T3), corticosteroid, retinoid (technically included in the before mentioned vitamin class), and anti-oxidants in an effective amount to promote maturation of differentiated SC-RPE cells.

[0014] The SC-RPE maturation method of the disclosure is a method to maturate a differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cell, the method comprising contacting differentiated SC-RPE cells with a cell culture maturation medium of the present disclosure. For a time and under conditions to obtain a mature SC-RPE cell.

[0015] According to a third aspect, the serum-free SC-RPE methods and related cells, compositions, methods, and systems are also based on the unexpected finding that mature SC- RPE can be identified by the presence of marker CD57, the presence of the marker CD104, and / or the absence of marker CD49b.

[0016] Accordingly, an SC-RPE enrichment / purification method and system in accordance with the third aspect are described.

[0017] The SC-RPE enrichment method of the disclosure is a method to enrich mature SC- RPE cells, the method comprising providing differentiated and / or mature stem cell-derived retinal pigment epithelial cells; and contacting the cells with a probe specific for a marker selected from CD57, CD104, and CD49b for a time and under condition to obtain cells stained for CD57, CD104, and / or CD49b markers, if any is present. The SC-RPE enrichment method of the disclosure also comprises sorting the stained cells to select the cells stained for CD57 and / or CD104 markers, and discard cells stained for CD49b marker, to obtain a population of cells enriched in mature SC-RPE cells.

[0018] In addition or in the alternative to detection performed through marker selection in SC- RPE methods of the disclosure, the SC-RPE enrichment method provides for selection of mature SC-RPE based on detection of pigmentation of the differentiated and / or mature stem cells to be performed with one or more sorting devices configured to perform detection of pigmentation through absorption of light by melanin and / or scatter of light by melanosomesTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT present in the cells. In some embodiments selection of differentiated and / or mature stem cells through light absorption and / or light-scattering by the pigment of the cells can be performed in combination with selection based on marker selection as will be understood by a skilled person.

[0019] The SC-RPE enrichment system of the disclosure is a system to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising combination of one or more probes specific for a marker selected from CD57, CD104, and / or CD49b, reagents for the detection of the marker, and / or stem cell-derived retinal pigment epithelial (SC-RPE) cells for combined use to stain differentiated and or mature SC-RPE cells in a method to obtain a population of cells enriched in SC-RPE cells. In addition or in the alternative to probes for detection of SC-RPE, the system can comprise cell sorting devices to perform selection based on pigmentation and obtain a population of cells enriched based on detection of pigmentation in addition or in the alternative to enrichment based on presence of one or more markers.

[0020] According to a fourth aspect, the serum-free SC-RPE methods and related cells, compositions, methods, and systems are also based on the unexpected finding that an RPE Maintenance Medium of the present disclosure can be used to perform efficient expansion of SC-RPE cells that retain the capacity to obtain a mature RPE phenotype and function.

[0021] The RPE Maintenance Medium of the disclosure comprises an RPE Maturation Medium of the disclosure supplemented with basic fibroblast growth factor (bFGF), a Rho- associated coiled-coil containing protein kinase (ROCK) inhibitor and / or transforming growth factor beta receptor (TGFBR / ALK5) kinase inhibitor, and dimethyl sulfoxide (DMSO) in an effective amount to promote expansion of SC-RPE cells that retain the capacity to obtain a mature RPE phenotype and function. In some embodiments, ROCK inhibitors can comprise Y-27632, Thiazovivin, or RevitaCell, and the TGFBR kinase inhibitors can comprise A-83-01, RepSox (ALK5-inibitor), SB431542, or SB525334.

[0022] The SC-RPE expansion method of the disclosure is a method for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells that retain the capacity to obtain a mature RPE phenotype and function, the method comprising providing mature stem cell-derived retinal pigment epithelial (SC-RPE) cells; and contacting the SC-RPE cells with the RPE Maintenance Medium of the disclosure on an SC-RPE substrate of the disclosure, the contacting for a time and under condition to obtain a target cell density of the SC-RPE cells.

[0023] The SC-RPE expansion system of the disclosure is a system for expansion of stem cell-Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT derived retinal pigment epithelial (SC-RPE) cell, the system comprising the RPE Maintenance Medium, and an SC-RPE substrate of the disclosure for combined use in a method to perform primary expansion of the SC-RPE cells of the disclosure.

[0024] According to further aspects of the disclosure, the methods, and systems and related medium according to any one of the first to the fourth aspect can be used in connection with methods, systems and compositions related to various stages of the SC-RPE derivation process.

[0025] For example, according to a fifth aspect an SC-RPE frozen stock preparation method and system and SC-RPE obtainable or obtained thereby are described based on the use of the RPE Maintenance Medium of the disclosure.

[0026] The SC-RPE frozen stock preparation method according to the fifth aspect is method to prepare frozen stock of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising performing expansion of SC-RPE cells with the expansion method of the disclosure to obtain SC-RPE cells having a target cell density. The SC-RPE freezing stock preparation method further comprise harvesting the SC-RPE cells having the target cell density to obtain harvested SC-RPE cells, and freezing the harvested SC-RPE cells.

[0027] The SC-RPE frozen stock preparation system according to the fifth aspect is a system for frozen stock preparation of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising the RPE Maintenance Medium of the present disclosure, the SC-RPE substrate of the present disclosure, and freezing media for combined use in a method to provide frozen stock of the present disclosure.

[0028] According to a sixth aspect an SC-RPE method and systems are described for the production of RPE cells on a physical support, and SC-RPE cells monolayers obtainable and / or obtained thereby (herein also SC-RPE Patch production method), which are based on the use of the RPE Maintenance Medium of the disclosure. The SC-RPE Patch production method according to the sixth aspect is directed to production of functional RPE cells on a physical support that can be selected in view of the use of the product obtained thereby (e.g., experimental study or clinical use). Accordingly, the support can be on a physical non- permeable, permeable, or porous support (such as TC plastic, 2D permeable film or membrane, 2D-porous support, 3-D membrane / matrix, porous or solid microcarriers, and additional identifiable by a skilled person) coated with or modified with a suitable RPE substrate as willTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT be understood by a skilled person. In some cases the patch production method can be used to repair a region of the retinal pigment epithelium where the RPE is dysfunctional, degenerated, or missing as will be understood by a skilled person.

[0029] The SC-RPE Patch production method for the production of RPE cells on a physical support of the disclosure is a method to manufacture stem cell-derived retinal pigment epithelial (SC-RPE) cells on a physical support, the method comprising performing expansion of SC-RPE cells according to the method of the disclosure and to obtain SC-RPE cells having a target cell density. The SC-RPE method of the production of RPE cells on a physical support further comprises contacting the SC-RPE cells having the target cell density with a membrane support and the RPE Maturation Medium or an RPE Maintenance Medium of the disclosure to obtain a population of quiescent partially mature or mature SC-RPE cells monolayers.

[0030] According to a seventh aspect, a method to manufacture an SC-RPE cell suspension is described, the method comprises obtaining the SC-RPE cell suspension from any one of the SC-RPE cells of the present disclosure following performing the SC-RPE enrichment method of the disclosure.

[0031] According to an eighth aspect SC-RPE method and systems are described for transplanting an individual with SC-RPE monolayers on a flexible solid support, and / or the SC-RPE cells suspension obtainable and / or obtained with the methods and systems of the present disclosure (herein SC-RPE Transplantation method). The SC-RPE transplantation method comprises placing the SC-RPE monolayers on a flexible solid support, and / or the SC- RPE cells suspension of the disclosure in the subretinal space of the individual thus transplanting the individual with the of SC-RPE cells in accordance with methods of the of the disclosure.

[0032] The SC-RPE systems are described for transplanting SC-RPE in an individual, comprises SC-RPE monolayers on a flexible solid support, and / or the SC-RPE cells suspension together with reagents and devices for performing the placing according to a method transplanting SC-RPE in an individual of the present disclosure.

[0033] According to a ninth aspect a toolbox SC-RPE derivation method and system are described in which a user selects the steps and / or component in accordance with the starting stem cells and the experimental design.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0034] The SC-RPE derivation method of the disclosure is a method to derive retinal pigment epithelial (RPE) cell from a starting stem cell (SC), comprising differentiating the stem cell (SC) to obtain differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells and maturating the differentiated SC-RPE cells to obtain mature SC-RPE cells.

[0035] The SC-RPE derivation methods of the disclosure can optionally further comprise performing enrichment of mature SC-RPE stem cells, preparing a frozen stock of the SC-RPE, performing expansion of the SC-RPE, manufacturing SC-RPE cells for use in the SC-RPE Transplantation method and system of the disclosure and / or manufacturing SC-RPE cells on a physical support to obtain a population of quiescent and at least partially mature up to fully mature SC-RPE cells monolayers depending on the percentage of mature cells in the monolayer as will be understood by a skilled person upon reading of the present disclosure.

[0036] In the SC-RPE derivation methods of the disclosure at least one of the differentiating, the maturating, the performing enrichment, the preparing a frozen stock, the performing expansion, and the preparation of SC-RPE suspensions or the preparation of SC-RPE patches is performed according to method of the present disclosure.

[0037] The SC-RPE derivation system of the disclosure comprises at least one of the SC-RPE differentiation medium, the SC-RPE substrate, the RPE Maturation Medium, at least one probe specific for a marker selected from CD57, CD104, and CD49b, the RPE Maintenance Medium, at least one SC-RPE freezing medium, and at least one SC-RPE scaffold of the present disclosure.

[0038] According to a tenth aspect SC-RPE cells are described obtainable or obtained by the SC-RPE differentiation method, the SC-RPE maturation method, the SC-RPE enrichment method, the SC-RPE expansion method, or the SC-RPE derivation of method of the disclosure. In particular SC-RPE cell populations obtained with the methods and systems of the disclosure can have an percentage purity level of SC-RPE differentiated cells and / or SC-RPE mature cells, in the obtained cell population increased with respect to SC-RPE cells obtained in outcome corresponding differentiation methods, maturation methods, enrichment methods expansion methods, and / or derivation methods as will be understood by a skilled person upon reading of the present disclosure.

[0039] According to an eleventh aspect, any of the matured SCE-RPE cells herein described,Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT possibly obtained following maturation method, enrichment method, expansion method, derivation methods possibly following thawing from a frozen stock of the present disclosure can be used to treat and / or prevent retinal conditions in the individual.

[0040] The serum-free differentiation methods and systems herein described allow to decrease the variability in differentiation efficiency known to be associated with stem cell differentiation in with particular reference to differentiation performed with serum-free protocols. In particular, serum free protocols can provide varying levels of RPE differentiation yields efficiency due for example to issues such as disaggregation of embryoid bodies (EBs) in protocols employing EBs, insufficient pigmentation and / or insufficient RPE epithelial properties as will be understood by a skilled person.

[0041] In particular, the serum free differentiation methods and systems herein described allow one of skill to consistently perform differentiation of SC-RPE across a range of different starting stem cells to obtain yields of differentiated SC-RPE cells that can be higher than 50% as will be understood by a skilled person.

[0042] [The serum-free RPE Maturation medium and related methods and systems herein described allow obtaining in several embodiments a population of mature SC-RPE cells having RPE marker genes expression and corresponding a quiescent state, an RPE morphology, and / or functionality comparable to functional RPE in vivo, with surprising increased in yields and a decreased variability among starting stem cells compared to existing serum free media methods and systems for maturation of RPE cells.

[0043] In particular it is expected that, serum-free RPE Maturation medium and related methods and systems herein described allow in some embodiments to consistently obtain a population of mature SC-RPE cells having RPE marker genes expression and a corresponding quiescent state, an RPE morphology, and functionality comparable to functional RPE in vivo, in percentage equal to or greater than 40% of the starting differentiated cells in the cell population, and in preferred and most preferred embodiments herein described up to or greater than 95%, or up to or greater than 99% of the starting differentiated cells in the cell population.

[0044] The serum-free RPE Maturation Medium and related methods and systems herein described also comprise standardized serum-free protocols which allow production of mature SC-RPE cells with reliable and reproducible results as will be understood by a skilled personTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT upon reading of the present disclosure.

[0045] The serum-free RPE Enrichment methods and systems herein described also allow enrichment of a starting population of differentiated SC-RPE and / or mature RPE of at least 95% (mean = 98% + / - 1, n=22).

[0046] The serum-free RPE Maintenance medium and related methods and systems herein described also allow efficient proliferation with growth rates comparable to that seen with serum supplemented medium and a greater than 50-fold increase in number of RPE that can obtain a quiescent monolayer, having RPE marker expression, morphology, and functionality comparable to RPE in vivo compared to serum-free medium without the additional components of RPE-MMM using standardized protocols, which is unexpected in view of variability, and technical complexity of existing serum-free media.

[0047] The serum-free RPE Maintenance medium and related frozen stocks methods and systems herein described also allow compared to existing serum-free methods a consistent cryopreservation as well as decreased cellular stress and damage with corresponding increased quality of recovered cells following thawing and replating allowing for the attainment of quiescent RPE monolayers with up to and greater than 95% of the cells having RPE marker expression, morphology, and functionality comparable to RPE in vivo.

[0048] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments to allow for a substantial improvement in yield, purity, and quality, as well as a significant shortening of the time required to complete the process compared to existing procedures.

[0049] In particular, the serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in some embodiments to obtain a final SC-RPE yield of about 15,000 SC-RPE per starting stem cell for cases with differentiation efficiencies >50%, a purity >97% expected, often >99% with top-producing stem cell lines, and quality (uniform pigmentation, characteristic RPE morphology and phenotype, RPE signature gene expression comparable to cultured fetal RPE, minimal evidence of mesenchymal RPE gene expression) compared to existing procedures while using a complete processing time of only about 90 day.

[0050] In addition, the serum-free methods for the derivation of SC-RPE cells and related SC-Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE cells, compositions, methods, and systems in embodiments comprising the SC-RPE differentiation methods and systems herein described, is expected to provide increased yields in connection with a greater number of stem cell lines than existing derivation methods employing a single differentiation method.

[0051] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in some embodiments comprising enrichment methods of the disclosure, particularly when using cell sorter-based enrichment and standard 2-D culture methods, to allow a substantial reduction in labor and hence increased SC-RPE manufacture productivity in comparison to existing methods employing manual based enrichment or embryoid body culture. For example, it is expected that 1-2 persons working in a non-cGMP setting can produce sufficient frozen stocks of SC-RPE to generate as many as one trillion SC-RPE in about three months using stem cell lines and differentiation methods with differentiation efficiencies of 50% or greater, in the preferred embodiments, and with maximal SC-RPE expansion.

[0052] Accordingly, the serum-free methods for the derivation of SC-RPE cells and related cells, compositions, methods, and systems can be used in several embodiments to achieve high yield RPE derivation from many different starting stem cell lines. In some embodiments when stem cell lines are used that have differentiation efficiencies greater than 50%, final yields in excess of 15,000 SC-RPE cells per single stem cell are possible. With stem cell lines that have lower differentiation efficiencies or when the user chooses to reduce the extent of culture expansion the yield will be reduced proportionally, as will be understood by a skilled person upon reading of the disclosure.

[0053] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems can be used in several embodiments to achieve large-scale SC-RPE production (>30 billion SC-RPE per 50 cm2starting differentiation culture, with differentiation efficiencies >50% and maximal SC-RPE expansion) in a single run in the time span of approximately four months using standard culture methods. In clinical applications where the common dosages range from 100,000 to 200,00 cells this equates to enough cells to produce over 150,000 doses.

[0054] The serum-free methods for the derivation of SC-RPE cells and related cells, compositions, methods, and systems can be used in several embodiments to produce clinicalTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT grade stem cell-derived mammalian cells since the components of the compositions used in the derivation of SC-RPE methods described herein are usable with xeno-free cGMP compliant reagents.

[0055] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems can be used in several embodiments to select an efficient protocol for a given starting stem cell from a collection of spontaneous and semi-directed differentiation protocols which employ the use of serum-free SC-RPE differentiation and / or serum-free RPE Maturation Medium of the disclosure.

[0056] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments for culture expansion and / or identification and / or testing of a drug product that promotes RPE maturation and limits persistent RPE-to-mesenchymal transition.

[0057] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments to create intermediate frozen SC-RPE stocks for on-demand production of final product.

[0058] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems can be used in several embodiments to enact an automation compatible method for the isolation of pure populations of RPE cells from mixed cell populations based on the expression the cell surface antigens CD57 and / or CD104, lack of expression of CD49b, and / or light absorbance or light scattering of melanin / melanosomes.

[0059] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems can be used in several embodiments to provide cell-based therapeutic compliant pipeline for large-scale manufacture of high purity (^99%), high fidelity SC-RPE for use in the treatment of degenerative eye disorders involving RPE degeneration an / or loss of function. Fidelity refers to homogeneity of phenotype (pigmentation, shape, RPE gene expression relative to that of primary differentiated primary fetal RPE) and lack of or minimal expression of non-RPE genes or genes associated with RPE wound response as will be understood by a skilled person.

[0060] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods, and systems herein described can be used in any field where efficientTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT production of SC-RPE and / or production of high-quality SC-RPE cells are desired. Exemplary applications comprise medical and in particular clinical applications related to treatment and / or prevention of retinal degenerative conditions such as macular degeneration.

[0061] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein described can also be used for the manufacture of SC-RPE capable of delivering specific biologics (gene products) when used with genetically engineered stem cells or when the SC-RPE are genetically engineered during the production process.

[0062] The serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein described can further be used in drug research and / or to develop diagnostic and / or therapeutic approaches and / or tools (such as diagnostic and / or therapeutic cellular agents), to counteract conditions involving the retinal epithelium, such as monitoring of therapeutic cell / agent efficacy and safety during developmental stages and clinical usage.

[0063] Additional exemplary applications include uses of the serum-free methods for the derivation of SC-RPE cells and related SC-RPE cells, compositions, methods and systems herein described in several fields including basic biology research, applied biology, bio- engineering, bio-energy, medical research, medical diagnostics, therapeutics, and in additional fields identifiable by a skilled person upon reading of the present disclosure.

[0064] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure.

[0066] Figure 1 shows a set of whole well incident illumination images of exemplary induced pluripotent (iPSC) and embryonic (ESC) stem cell lines following incubation with exemplary combinations of SC-RPE differentiation media and SC-RPE extracellular matrix andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT subsequent incubation with to RPE Maturation Media (iPSC: #032411, #110211, and #120111; ESC: Shef1, H1, and H9). The iPSC lines were developed in-house from late passage donor fetal RPE cells using an RNA-based reprogramming method (ReproRNA™-OKSGM, STEMCELL Technologies, Inc.). The day that the image was collected, which corresponds to the day the culture was harvested for SC-RPE enrichment or discarded due to poor yields is indicated at the bottom of each image. Black image borders indicate cell line:media:substrate combinations that gave rise to acceptable quantities of pigmented SC-RPE to allow for production of substantial quantities of enriched SC-RPE. Each well image is a representative image selected from 3-6 replicate wells.

[0067] Figure 2 shows the beneficial effect of sequential incubation with the differentiation media and the RPE Maturation Medium on SC-RPE differentiation. Top Row: Whole well incident light images of Shef1 differentiation cultures where the step of incubation of with the RPE Maturation Medium was omitted and the cells remained in the exemplary differentiation for the entire culture period after the initial switch to the differentiation media. Second Row: Whole well incident light images of Shef1 cells differentiated using the standard procedure where the cells were incubated with the exemplary differentiation medium from Day-3 through Day-30 and thereafter with RPE Maturation Medium. Third Row: Whole well incident light images of iPSC #110211 cultures where the switch to the exemplary differentiation media was omitted and the cells were only incubated with RPE Maturation Medium beginning at Day-3 of the differentiation process. Bottom Row: Whole well incident light images of iPSC #110211 cells that were differentiated using the standard procedure where the cells are incubated with the exemplary differentiation media from Day-3 through Day-30 and thereafter with RPE Maturation Medium. The day that the image was collected is indicated at the bottom of each image.

[0068] Figure 3 shows that switching to RPE Maturation Medium after Day-31 of the differentiation protocol does not substantially improve the yield of pigmented SC-RPE. Whole well incident light images of cultures plated on the three exemplary substrates and switched from stem cell culture medium directly to RPE Maturation Medium at Day-3 (Top Row) or to CTS-KO-DMEM / 20%CTS-KOSR Medium at Day-3 and then switched to RPE Maturation Medium at Day-31 or Day-59 (Middle Rows). The bottom row shows whole well incident light images of negative control cultures where the cells were switched to CTS-KO- DMEM / 20%CTS-KOSR Medium at Day-3 and maintained in CTS-KO-DMEM / 20%CTS-Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT KOSR Medium for the entire differentiation time course. The day that the image was collected is indicated at the bottom of each image.

[0069] Figure 4 shows the substantial improvement in pigmented SC-RPE yield and homogeneity that can be achieved by passaging the cells prior to cell surface marker-based enrichment. Shef1 cells were plated on rhVTN-N or LAM521 coated plates and differentiated in CTS-KO-DMEM / 20%CTS-KOSR Medium using the optional Activin A treatment and then switched to RPE Maturation Medium on Day-31. On Day-87 the two cultures were harvested by protease digestion and a portion of the cells were plated at 100,000 cell / cm2on LAM521 culture wells and maintained in RPE Maintenance Medium for 42 additional days. Top Row: Whole well incident images of the cultures immediately before and 42-days after passage. Bottom Four Rows: Brightfield images of the corresponding cultures at different magnification. Regions of the culture with pigmentation were selected for the collection of the 10-100X images of the pre-passage culture to evaluate the nature of the SC-RPE. Note the marked improvement in the proportion of pigmented cells and normal RPE morphologies after passage.

[0070] Figure 5 shows results of experiments performed to evaluate how each of the RPE Maturation Medium components contribute to RPE phenotype maturation and pigmentation. Figure 5 Panel A shows 2×210X brightfield stitched images of one of three replicate wells in which Shef1-RPE produced using the CTS-KO-DMEM / 20%CTS-KOSR:PC-VTN or X- VIVO™ 10 / XF-B27:rhVTN-N exemplary differentiation and sorting protocols were grown in complete RPE Maturation Medium (RPE-MM) or RPE Maturation Medium missing one of the components as indicated. Figure 5 Panel B shows a diagram illustrating the quantitation of the amount of pigment for cells maintained in the complete RPE Maturation Medium (RPE-MM) and the n-1 formulations. The relative amount of pigmentation was determined based on the absorbance of light at 510 nm using a 3×3 multipoint scan of each well (mean + / - SEM, n=3).

[0071] Figure 6 shows results of experiments demonstrating the superiority of RPE Maturation Medium to facilitate RPE maturation and pigmentation compared to the other media formulations. Figure 6 Panel A shows 2×210X brightfield stitched images of one of three replicate wells in which Shef1-RPE produced using the CTS-KO-DMEM / 20%CTS- KOSR:PC-VTN (RPE-DM1 SC-RPE) or X-VIVO™ 10 / XF-B27:rhVTN-N (RPE-DM2 SC- RPE) exemplary differentiation and sorting protocols grown in RPE Maturation MediumTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (RPE-MM), CTS-KO-DMEM / 20%CTS-KOSR (RPE-DM1), or X-VIVO™ 10 / XF-B27 (RPE- DM2) or RPE-DM1, RPE-DM2, CTS-KO-DMEM (KO-DMEM), or CTS-KO-DMEM / F12 (KO-DMEM) with the addition of the RPE-MM non-base medium components (NEAA, N1, B27, taurine, and hydrocortisone). Figure 6 Panel B shows 20X brightfield images of the same cells maintained in RPE Maturation Medium, X-VIVO™ 10 / XF-B27, and X-VIVO™ 10 / XF- B27 supplemented with the RPE-MM non-base medium components (NEAA, N1, B27, taurine, and hydrocortisone).

[0072] Figure 7 shows a schematic representation of an exemplary method of stem cell expansion and SC-RPE differentiation.

[0073] Figure 8 shows a schematic representation of exemplary methods and system for SC- RPE differentiation of the present disclosure.

[0074] Figure 9 shows a schematic representation of an exemplary method and system for SC-RPE Fluorescence Activated Cell Sorting (FACS) Enrichment of the present disclosure.

[0075] Figure 10 shows three FACS scatter plots illustrating results of an exemplary SC-RPE gating strategy to sort SC-RPE. Pigmented SC-RPE are identified based on their relatively high side scatter (SSC) to forward scatter (FSC) ratio (Figure 10 left side diagram), positive expression of CD104 and CD57 (Figure 10 middle diagram), and no expression of CD49b (Figure 10 right side diagram).

[0076] Figure 11 shows results of cell sorting based on pigmentation and cell surface markers on the enrichment in populations of SC-RPE. Figure 11 Panel A Top Row: shows results of cells identified based on forward and side light scattering (FSC & SSC) profiles and SC-RPE sorting based on expression of both CD104 and CD57 and lack of expression of CD49b. The far-right panel shows the light scatter profiles of the CD104 Positive / CD57 Positive / CD49b Negative cells. Figure 11 Panel A Bottom Row: shows results of cells sorting based on pigmentation and cell surface marker expression. Figure 11 Panel B shows diagrams illustrating SC-RPE Purity analysis based on the expression of the RPE marker gene product PMEL of cells sorted based on pigmentation and surface marker expression.

[0077] Figures 12A and 12B show the results of experiments directed to detect the effect on pigmentation and the establishment and maintenance of RPE barrier function of basic FGF (bFGF) plus DMSO. Figure 12A shows a diagram illustrating the amount of pigmentation ofTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Shef1-RPE manufactured using the CTS-KO-DMEM / CTS-20%KOSR:rhVTN-N differentiation protocol and RPE Maturation Medium with and without 2 ng / ml bFGF + 0.1% DMSO according to the standard protocol. In the diagram of Figure 12A the amount of pigmentation of the Shef1-RPE was determined by measuring the absorbance of 510 nm light using 3×3 multipoint scan (background subtracted absorption value, n=1) at the indicated timepoints. Figure 12B shows a diagram illustrating RPE barrier function of the cells of Figure 12A determined by measuring the transepithelial electrical resistance of the macroporous insert cultures (mean + / - SEM, n=3).

[0078] Figure 13 shows results of experiments directed to measure effects of the addition of DMSO and bFGF to RPE Maturation Medium on SC-RPE pigmentation. Figure 13 Panel A shows representative 4X brightfield images of three replicate wells of SC-RPE cultured in RPE Maturation Medium supplemented with 0.1% DMSO (DMSO), 2 ng / mL bFGF (bFGF), or 0.1% DMSO + 2 ng / mL bFGF (DMSO bFGF) when plated at a density (4500 cell / cm2) at which they will not mature and pigment without additional treatment. Figure 13 Panel B show Whisker plots of the level of pigmentation detected in the of SC-RPE cultured of Figure 13 panel A. The middle two boxes indicate the middle data quartiles and the whiskers indicate the outer quartiles, the “X” denotes the mean, and any dots indicate outliers. The level of pigmentation was quantified by measuring the absorbance of 510 nm light using a 3×3 multipoint scan of each replicate well.

[0079] Figures 14A to 14D show results of exemplary expansion by continual treatment of SC-RPE with varying combinations of 0.1% DMSO, 2 ng / mL bFGF, and 5 ^M Y-27632. Figure 14A shows a schematic illustration of a workflow for the expansion of a frozen stock vial of iPSC-RPE. Figure 14B shows representative 4X brightfield images of a replicate well of SC-RPE cultured at a density conducive for non-passaged SC-RPE maturation (80,000 cell / cm2) in each of the media (DMSO, DMSO+bFGF, DMSO+Y27632, DMSO+Y27632+bFGF) schematically reported in Figure 14A as a function of passage. The day post plating of imaging is indicated for each condition. Figure 14C shows a diagram illustrating the relative level of pigmentation at Day-34 plotted versus the cumulative number of total population doublings up to the point of each passage (mean + / - SEM, n=3). Figure 14D shows a table of calculated yields at a level of pigmentation equal to 90% of the level observed at Passage 1.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0080] Figure 15 shows a schematic representation of an exemplary method and system for SC-RPE Primary Expansion method and system in combination with an exemplary method and system for Preparation of Frozen SC-RPE Stocks of the present disclosure.

[0081] Figure 16 shows a schematic representation of an exemplary method and system for thawing of Frozen Stocks, in combination with an exemplary method and system for SC-RPE secondary Expansion and an exemplary method and system for SC-RPE Patch Production of the present disclosure.

[0082] Figure 17 shows whole well incident light images for a set of different RPE differentiation and maturation methods obtained using two iPSC stem cell lines taken at the end of RPE maturation. The stem cells were plated on Matrigel coated culture wells and differentiated using a X-VIVO™ 10 / SM1 semi-directed differentiation method and maturated with RPE-MM with different combinations of supplements. Top Row: iPSC #110211 maturated with RPE-MM supplemented with N1 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). Second Row: iPSC #110211 maturated with RPE-MM supplemented with N2 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). Third row: iPSC #082809 maturated with RPE-MM supplemented with N1 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus). Bottom row: iPSC #110211 maturated with RPE-MM supplemented with N2 and 5%FBS, SM1 Neurocult minus vitamin A (Vitamin A Minus), or SM1 Neurocult (Vitamin A Plus).

[0083] Figure 18 shows the analysis of RPE barrier function by measurement of the transepithelial electrical resistance (TEER). Fetal RPE (n=5) and enriched SC-RPE #1102111 (n=4). The SCRPE #110201 cells were obtained from the cultures shown in Figure 17. Cells were grown on microporous transwell membranes using RPE-MM:N1 with SM1 minus vitamin A (grey bars) or RPE-MM:N1 with SM1 plus vitamin A (black bars). For the SC-RPE the cells were grown in the media used for differentiation. At one-month the TEER for each culture set of cells and culture conditions was measured and the Ohm•cm2(mean + / - SEM) values were plotted.

[0084] Figure 19 shows the average transcriptome profiles for 6 Enriched SC-RPE cell lines obtained by differentiation using a X-VIVO™ 10 / SM1 semi-directed differentiation method and maturated with RPE-MM:N1 medium supplemented with SM1 with (Plus) and withoutTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT (Minus) vitamin A. After enrichment and a post-sort maturation period of one-month using their corresponding RPE-MM medium their transcriptome profiles were determined by RNA AmpliSeq and the log2 transformed Plus Vitamin A / Minus Vitamin A ratio values plotted after alphabetical sorting of the gene symbols. The gene expression for profiles for several gene sets are shown. Top Chart: All Expressed Genes. Second chart down: Top-500 RPE Genes, up- regulated genes associated with RPE maturation. Third chart down: RPE Signature Genes, a curated set of 90 RPE marker genes. Fourth chart down: Top-500 induced genes associated with reversible RPE wound response. Top-500 induced genes associated with persistent EMT.

[0085] Figure 20 shows the log2 transformed Vitamin A Plus / Vitamin A Minus expression ratios for a select set of genes related to key RPE functions for SC-RPE differentiated and maintained using RPE-MM prepared using the B27-based supplement SM1 Neurocult with or without vitamin A (black bars) along with the Vitamin A Plus / Vitamin A Minus expression ratios obtained from fetal RPE cultured in the same two media (grey bars).

[0086] Figure 21 shows the gene expression levels of a select set of disease-associated genes obtained from enriched SC-RPE that were derived and matured for one-month using RPE-MM prepared with the B27-based supplement SM1 Neurocult with (Minus) or without (Plus) vitamin A, along with the values obtained when the SC-RPE were switched and matured in their corresponding opposite RPE-MM medium (Plus to Minus, Minus to Plus). The gene expression values are the trimmed mean of the M-value normalized reads per million (mean + / - SEM, Minus and Plus, n=6, Plus to Minus and Minus to Plus, n=3).

[0087] Figure 22 shows the vitamin A dose response curves for the expression of eight select genes obtained from fetal RPE matured for one month in RPE-MM prepared using a 2-fold dilution series (100-0%) of RPE-MM:SM1 Neurocult plus vitamin A diluted with RPE- MM:SM1 Neurocult minus vitamin A. Gene expression values are the trimmed mean of the M-value normalized reads per million as determined by RNA AmpliSeq. The 0% vitamin A values are plotted on the y-axis.

[0088] Figures 23A and Figure 23B show a comparison of the gene expression profiles of SC-RPE contrasted to pluripotent iPSC and fetal RPE. Enriched SC-RPE were obtained from eight ESC-RPE differentiations depicted in Figure 1: Shef1:CTS-KO-DMEM / 20%KOSR: PC- VTN, rhVTN-N, and LAM521; Shef1: X-VIVO™ 10 / XF-B27:LAM521; Shef1: X-VIVO™ 10 / XF-B27+Activin A:PC-VTN and rhVTN-N; H1: X-VIVO™ 10 / XF-B27:LAM521; andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT H9:CTS-KO-DMEM / 20%KOSR: rhVTN-N. After enrichment, the SC-RPE along with primary fetal RPE (fRPE) cells were matured for one month using RPE-MM:N1:CTS-B27 and their transcriptome profiles were determined by RNA AmpliSeq. In particular in Figure 23A shows scatter plots are shown comparing the average gene expression profiles of the eight SC- RPE lines, four pluripotent stem cell lines and four primary cultured fetal RPE lines. Figure 23B shows scatter plots comparing the expression of a Top-500 up-regulated gene set associated with RPE maturation for iPSC and fetal RPE (top-left) and comparing each of the eight SC-RPE with fRPE.

[0089] Figures 24A and Figure 24B shows the expression of RPE-signature genes and select RPE function genes for SC-RPE and fetal RPE. In particular Figure 24A shows expression levels of a curated gene set comprised of 90 RPE signature genes for all SC-RPE (black bars, mean, n=8) and all fRPE (grey bars, mean=4). The expression values are the trimmed mean of the M-values normalized reads per million. Figure 24B shows Average expression levels of a select set of RPE function genes in SC-RPE and fetal RPE. The gene expression levels are expressed relative to the average values obtained from four pluripotent iPSC line cultures.

[0090] Figure 25 shows a schematic representation of a cell sorter usable in the methods and systems of the present disclosure.

[0091] Figure 26 shows a schematic representation of devices configured to transplant RPE cells in sub-retinal space of an individual and usable in the methods and systems of the present disclosure.

[0092] Figure 27 shows a chart depicting the results of examining the effect of the timing, split ratios, and supplementation of RPE-MM with bFGF and DMSO on corrected SC-RPE differentiation yields when an optional intermediate passage is performed during the maturation phase of SC-RPE differentiation and maturation. DETAILED DESCRIPTION

[0093] Provided herein are serum-free methods for the derivation of stem cell-derived retinal pigment epithelial cells (SC-RPE) and related cells, compositions, methods, and systems that in several embodiments allow efficient, differentiation and / or maturation from a variety of different mammalian stem cells.

[0094] The wording “stem cell” as used herein indicates an undifferentiated cell of aTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT multicellular organism which is capable of giving rise to indefinitely more cells of the same type, and from which certain other kinds of cells arise by differentiation [1]. In particular stem cells encompass undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell [2]. Distinguished from progenitor cells, stem cells in the sense of the disclosure can be categorized as adult stem cells, embryonic stem cells and induced stem cells.

[0095] The wording “adult stem cells” or “somatic stem cells” in the sense of the disclosure indicate undifferentiated cells, found throughout the body of an individual such as animals mammals and humans after development, that multiply by cell division to replenish dying cells and regenerate damaged tissues [3].

[0096] The wording “embryonic stem cells” or “ESCs” as used herein indicate pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. In particular, embryonic stem cells in the sense of the disclosure comprise human embryonic stem cells or hESC as well as ESC from other mammals. ESC can be obtained by isolating the inner cell mass (embryoblast) of an embryo stage 4–5 days post fertilization, when the blastocyst consists of 50–150 cells using methods such as immunosurgery, mechanical dissection, microdissection, laser dissection, or minimized trophoblast cell proliferation as will be understood by a skilled person [4], [5].

[0097] In some embodiments ESC in the sense of the disclosure are ESC derived using non- embryo-destructive methods. Methods for making ESC using non-embryo destructive methods are identifiable to a skilled person (see e.g. [6], [7], [8]).

[0098] The wording “induced stem cells” or “iSC” in the sense of the disclosure indicates stem cells derived from somatic, reproductive, pluripotent, or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotent – iMSC, also called an induced multipotent progenitor cell – iMPC) or unipotent – (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation are also called induced somatic stem cells as will be understood by a skilled person [9].

[0099] In particular, it is expected that suitable iSC can give rise to all three germ layer lineages (ectoderm, endoderm and mesoderm). Therefore iMSC / iMPC cells which have this propertyTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT are expected to be suitable iSC. Moreover, it is also expected that a some RPE progenitors do not need to go through the differentiation step and can go straight to RPE maturation medium. For those RPE progenitor cells cell sorting is expected to be optional. Also, it is expected that for iTC, an additional step would be required to first generate pluripotent cells.

[0100] In some embodiments, the starting stem cells of the disclosure are from stem cell lines. In some embodiments, the starting cells are primary RPE as will be understood by a skilled person upon reading for the present disclosure. In particular, some aspects of the process can be used for the culture, enrichment, freezing, and / or expansion of primary RPE as will be understood by a skilled person.

[0101] The wording “stem cell line” in the sense of the disclosure indicates a group of stem cells that is cultured in vitro and can be propagated indefinitely. Stem cell lines are derived from either animal or human tissues and come from one of three sources: embryonic stem cells, adult stem cells, or induced stem cells. Accordingly, stem cell line refers to a group of identical stem cells that can be grown and nurtured in a lab dish. A line originates from a single cell or group of cells of common genetic origin. All resulting cells in the line are replicates of the original cells and stem cell lines can be propagated in culture to produce large numbers of cells

[0010] ,

[0011] . Stem cell lines can become heterogenous at the genetic and / or epigenetic level over time due to culture related artifacts. Therefore, replicates of the original cells of a stem cell lines can be subjected to a genetic or epigenetic drift over time that might alter their propensity to give rise to each of the three different germ layer cells as will be understood by a skilled person

[0012]

[0013]

[0014]

[0015]

[0016] .

[0102] In some embodiments herein described, serum- and xeno-free methods and systems and related composition are used to perform efficient derivation from starting stem cells to retinal pigment epithelial (RPE) cells.

[0103] The wording “serum-free” as used herein indicates the absence of the use of whole serum, plasma, or complex undefined mixtures of blood-derived products. It does not exclude the use of the use of defined serum components (such as and not limited to, serum albumin, insulin, transferrin, vitronectin, or growth factors) derived from serum, plasma, blood, or recombinant gene expression technologies.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0104] The wording “xeno-free” as used herein indicates the use of protein components derived from the same species as the stem cell used for the derivation of RPE or derived by recombinant technologies and having the same amino acid sequence as that of the stem cell species.

[0105] The wording “retinal pigment epithelial”, “retinal pigment epithelium”, or “RPE” as used herein indicates the pigmented cell layer of an eye of individual located outside the neurosensory retina firmly attached to the underlying choroid and overlying retinal visual cells or cells isolated from such cell layer. Key RPE characteristics are an epithelial morphology (monolayer of hexagonal shaped cells with apical / basal polarization and tight lateral junctions), expression of genes products involved in the retinoid cycle, phagocytosis of photoreceptors, polarized secretion of factors that support the function and maintenance of the retina and choroid, transport of metabolites and catabolites between the retina and choroid, and establishment of the retina:blood barrier, as will be understood by a skilled person. RPE in the sense of the disclosure can be subjected to various disfunction due to degeneration of the RPE which can results in conditions of the retina such as retinal degenerative diseases (RDD), including age-related macular degeneration (AMD), Stargardt’s macular dystrophy (SMD), Best’s disease (BEST), and retinitis pigmentosa (RP). RPE are also involved in diabetic retinopathy and in Gardner syndrome which is characterized by FAP (familial adenomatous polyps), osseous and soft tissue tumors, retinal pigment epithelium hypertrophy and impacted teeth.

[0017] ,

[0018] .

[0106] The wording “derivation” as used herein indicates a process by which a stem cell loses pluripotency and specializes into a specific cell type. The derivation process comprises a differentiation and a maturation stage where the cell type is determined, and the cell phenotype acquired. In some contexts, not referring specifically to stem cells, it can also refer to as being isolated from.

[0107] The term “differentiation” when referred to stem cells indicates the process of changing of a stem cell to a more specialized cell type, which involves a switch from proliferation to specialization. This involves a succession of alterations of gene expression, cell morphology, membrane potential, metabolic activity and signal responsiveness as will be understood by a skilled person

[0019] .

[0108] Differentiation of stem cells can be a spontaneous differentiation or a directedTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation.

[0109] The wording “spontaneous differentiation” in the sense of the disclosure indicates to a default differentiation process that occurs naturally upon removal of medium components that sustain the pluripotent state or switching to a medium that does not contain these components. In general, spontaneous differentiation relies on the presence or absence of signals derived from other cells in the culture of different germ layer origin and cell type to proceed. Accordingly, RPE can be derived from stem cells by removing factors that act to promote and maintain pluripotency, such as and not limited to TGFB1 and bFGF, in stem cell medium or by switching to a medium that does not have these factors. The yield of RPE from removal of factors is often < 20% and highly variable from line to line, presumably due to line specific preponderances to generate different ratios of ectoderm, endoderm, and mesoderm germ layer cells. Based on current understanding of RPE development, lines that generate substantial quantities of ectoderm cells with sufficiently low levels of mesoderm and endoderm cells to allow for neuroectoderm and eye field precursor cell differentiation, but not so low as to prevent their further development into RPE progenitors would be expected to result in the best yields. Increased yields can be obtained by screening multiple lines using a specific protocol to find a line with a desired yield. Alternatively, the initial culture conditions can be altered to affect the germ layer distribution.

[0110] The wording “directed differentiation” in the sense of the disclosure indicates the addition of components, such as small molecules or biologics, to the medium that promote differentiation; usually in conjunction with the removal of components that promote the maintenance of the pluripotent state. Accordingly, directed differentiation of a starting stem cell can be performed by using various biologics and small molecules to direct the differentiation toward RPE. In the case of RPE differentiation, directed differentiation protocols typically use media that favor ectoderm growth and inhibitors that block mesoderm- derived signals that promote ectodermal-to-epidermal differentiation and medium that support neuroectoderm cells, followed later by addition of mesoderm / endoderm derived factors that promote RPE progenitor development. In some instances, directed differentiation is associated with increased complexity of the procedure and / or requires a selection or adjustments of a specific differentiation method for optimal RPE differentiation from different stem cell lines.

[0111] The wording “semi-directed differentiation” in the sense of the disclosure refers toTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation methods that rely primarily on spontaneous differentiation processes for cell fate differentiation and employ the use of small molecules or biologics to direct a single or limited subset of differentiation steps. Semi-directed differentiation methods for a given cell type typically have complexity intermediate to that of spontaneous and directed differentiation methods.

[0112] Differentiation of SC- RPE cells is marked by the appearance of intracellular golden- brown to black pigment, and preferably also by the SC-RPE cell morphology (polygonal shape and / or formation of monolayer with tight junctions) as will be understood by a skilled person. The appearance of pigment in differentiated SC-RPE cells can be detected with various methods such as i) macroscopic observation on a suitable background (e.g. on a white background) using indirect illumination (see Example 12 and Figure 1), ii) observation using brightfield microscopy, and / or iii) image analysis or by scanning light absorbance readings at wavelength between 480-600 nm to quantitively detect pigmented culture area, and additional methods as will be understood by a skilled person upon reading of the present disclosure. Differentiated SC-RPE cells in the sense of the disclosure comprise partially differentiated SC- RPE which are marked by qualitatively detectable pigment and fully differentiated SC-RPE which comprise detectable pigment in increased quantity compared to the qualitative detectable pigment of the partially differentiated SC-RPE. Accordingly, specific quantitative detection threshold to distinguish between partially differentiated SC-RPE cells and fully differentiated SC-RPE cells can be set in connection to a specific imager configuration, specific image collection settings, and specific image analysis software.

[0113] The term “maturation” as used herein with respect to stem cells indicates the process of cells maturing and specializing, and eventually losing their ability to divide and become new cells. Pluripotent stem cells have the ability to become any cell in the human body. These cells form other cells, and through development, and maturation become specialized to a specific task

[0020] .

[0114] The term “maturation” as used herein with respect to RPE or SC-RPE refers to any step in the process where a proliferating cell with a determined RPE cell fate establishes a confluent monolayer, becomes quiescent, acquires a characteristic RPE morphology, increases expression of RPE marker genes, decreases expression of genes associated with proliferation and wound repair, develops melanosomes, and acquires the characteristic functionality ofTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT native RPE in vivo

[0021] .

[0115] The term “passage” as used herein with respect to the culturing of cells refers to the collecting of cells from one culture device and transferring the cells to a new culture device. For adherent cell culture this generally entails the use of cell dissociation agents or mechanical means to remove the cells from the culture surface and generate a suspension of single cells and / or cell clumps. The resulting suspensions are then seeded or plated in the new culture device such as, a culture container (dish, flask microplate, roller bottle) or bioreactor; typically at a lower density than they were at in the originating culture. When they are plated at the same or higher density the process can be referred to as “replating”.

[0116] The term “confluence” as used herein with respect the culturing of cells refers to the complete (100%) coverage of the culture surface with cells with exception of any areas where cells are absent due to plating artifacts, substrate defects, or incidental injury as evident by clear regions absent of any cells, often with clearly defined borders or defined areas where cells appear to have been removed or damaged by scrapping or excessively forceful pipetting.

[0117] The term “near confluence” as used herein with respect to the culturing of cells refers to the approximately 24-hour period preceding the time that they are estimated to become confluent; in other words, the day before confluence is estimated to occur based on known or observed doubling rate of the cell line in the cell culture on a selected substrate. For example, one of skill can follow culture growth on a daily basis and based on this observation estimate the culture doubling rate. With that knowledge reasonable predictions of whether confluence occurs in the next 24 hours can be made. For example, if the culture doubles every day, then near confluence would correspond to having confluence of 50-99%. If desired, one can validate the prediction by continuing to grow the culture with the lowest cell density and determine its state of confluence the next day.

[0118] The term “just confluence” as used herein with respect to the culturing of cells refers to anytime within the 24- to 32-hour period after confluence occurs. Identification of just confluence can be performed by daily monitoring of the cultures and detection of just confluent cells starting from the time when the cells are first observed to be confluent.

[0119] With respect to mature SC-RPE, it is generally accepted that the SC-RPE should be qualitatively indistinct from primary RPE in that, they should express all key RPE genes andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT few non-RPE genes as will be understood by a skilled person. Accordingly, maturation can be detected by example in the form of gene expression analysis where SC-RPE detected genes are compared to genes detected in fetal RPE.

[0120] Accordingly, a skilled person will understand that “differentiation” in methods of the disclosure refer to the case of a cell type changing to another cell type (i.e. cell type determination) and the term “maturation” in methods of the disclosure refer to the change in phenotype that occurs when a determined RPE cell becomes confluent, exits the cell cycle, and obtains a prototypical functional phenotype. The term “dedifferentiation” describes the process where RPE undergo an “irreversible switch to a mesenchymal phenotype due to accumulative rounds of cell division or suboptimal culture conditions”. These cells can be called RPE- derived mesenchymal cells or mesenchymal-RPE. Whenever RPE are in a state of proliferation but still have the potential to turn into mature RPE they are generally referred as RPE unless otherwise indicated.

[0121] In some embodiments, herein described serum-free methods are described and related cells compositions methods and systems that allow to obtain an efficient SC-RPE differentiation in a stem cell line specific manner.

[0122] In those embodiments, methods and systems of the disclosure, an alternative approach is used based on the unexpected finding that the differentiation media and substrate act as a functional unit to alter RPE cell fate determination and different cell lines have unique preference profiles across a collection of different media and substrate combinations as can be most easily observed after a process of SC-RPE maturation that enables and / or enhances SC- RPE pigmentation or by using immunohistology methods to identify immature SC-RPE prior to maturation.

[0123] In particular, the differentiation methods and system of the present disclosure are based, in part, on the unexpected finding that, some cells have preferences for specific media:substrate combinations where for a certain cell line some media:substrate combinations yield more SC-RPE than do other media:substrate combinations and where for another cell line other media:substrate combinations yield more SC-RPE.

[0124] For example, for cell lines H1, H9 Shef1, iPSC #. 110211, and iPSC #032411, differentiation performed on exemplary media:substrate combinations provided little to no SC-Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE yield, or yields well in excess of 50%, in a cell line specific manner (see Example 13 and Figure 1). Additionally, for those cell lines and media:substrate combinations, it has been surprisingly found that the cells lines have varying degrees of media:substrate dependency. In particular, it has been found that one line gave SC-RPE yields greater than about 50% on 66% of the tested media:substrate pairs, and another line gave only yields of greater than 50% in 11% of the same media:substrate combinations (see Example 13 and Figure 1).

[0125] Consequently, the methods and systems for SC-RPE cell differentiation described herein can be used by the skilled artisan to select a stem cell line and corresponding preferred differentiation medium:substrate combination to obtain an efficient generation of a population of partially differentiated SC-RPE, presumptive RPE progenitors, and immature SC-RPE across a greater number of stem cell lines than is possible using a single differentiation medium and substrate.

[0126] Accordingly, a “toolbox” method and systems and related serum-free SC-RPE differentiation media are herein described, wherein the term “toolbox” indicates a method where the specific steps and reagents used are selected by the user in view of the starting material and the result desired, as well as experimental design.

[0127] In particular, in method and systems of the disclosure and related serum-free SC-RPE differentiation media herein described combinations of serum-free SC-RPE differentiation medium and SC-RPE cell culture substrate are selected depending on the starting stem cell line, to perform SC-RPE differentiation in accordance with the present disclosure.

[0128] The wording “culture medium” or “medium” as used herein in connection with cell culture indicates a liquid or semi-solid (e.g., gel) designed to support the survival, growth, and cellular functions of the referenced cell. Accordingly, culture media typically comprise a carbon source such as glucose, water, various salts, a source of amino acids and nitrogen as well as any other component required for the survival growth and cellular function of the cultured cells. Cell culture media can also contain pure protein additives like growth factors as well as vitamins, cholesterol, amino acids, and fatty acids as well as other components from various sources as will be understood by a skilled person. Culture media can comprise components from natural animal sources (such as tissue extracts and biological fluids like plasma, serum, and amniotic fluid) as well as from artificial or synthetic sources.

[0129] Accordingly, culture media can be categorized as media from natural sources andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT synthetic or artificial media which can contain nutrients, vitamins, salts, oxygen and carbon dioxide gas phases, blood serum proteins, carbohydrates and cofactors in various combinations depending on the cultured cell

[0022] . Cell culture media can also be categorized as serum containing media and serum-free media. Bovine calf serum, newborn calf serum, fetal bovine serum, horse serum and human serum are used in cell culture to provide hormones, growth factors, protective protease inhibitors, chelators or carriers for nutrients, essential nutrients, components that promote cell adherence, as well as to bind and neutralize toxins. Serum-free media or ‘defined culture media’, are media which lack serum but have defined quantities of growth factors, lipoproteins and other proteins normally found in serum. Stem cells can be typically cultured in media containing serum or serum-free media with specific growth factors and additives which either promote stem cell self-renewal or promote differentiation into specialized cell types. The factor bFGF (basic fibroblast growth factor) is added to regulate stem cell-renewal for human embryonic stem cells (ESCs), induced pluripotent stem cells and neural stem cells. Leukemia Inhibitory Factor (LIF) inhibits differentiation and promotes the expansion of mouse but not human ESCs. Culture medium used for the propagation of human stem cells most typically includes the addition of transforming growth factor beta 1 (TGFB1) or nodal to maintain pluripotency. Retinoic acid, ascorbic acid, hormones and DNA demethylating agents, intracellular cAMP elevating IBMX, growth factors, or small molecule regulators of different extracellular or intracellular pathways can induce stem cells to differentiate down defined pathways

[0023] .

[0130] A “serum free SC-RPE differentiation medium” in the sense of the disclosure is a cell culture medium which lacks serum but have a combination of components configured to allow for the establishment of an effective ratio of ectoderm, endoderm, and mesoderm progenitor cells within the stem cell culture. The optimal ratio can vary depending on stem cell line and culture substrate, but in general it should skew towards the production of ectodermal cells yet with sufficient numbers of endoderm and mesoderm cells to provide the proper cues to enable the natural development of RPE. Aspects of the differentiation medium that affect cell growth rates or the relative length of time in different stages of the cell cycle such as the concentration or composition of the carbon energy sources, essential and non-essential nutrients (amino acids, lipids, and vitamins), metal salts, trace minerals, growth factors or those that might affect cell signaling, such as divalent cations, pH or specific amino acids can all act alone or in concert as will be understood by a skilled person.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0131] The term “cell culture substrate” or “substrate” as used herein indicates a material on which a cell or organism grows or is attached. Exemplary cell culture substrate comprise extracellular matrices composed mainly of proteins which are configured to facilitate attachment of cells through binding to specific cell surface matrix receptors and to provide chemical cues that affect or guide the behavior of cells interactive with each matrix. Cell substrates are commonly used to coat the inert physical supports that the cells are grown on such as plastic cultureware, porous or nonporous films or membranes, or porous or non-porous microcarriers. Additional exemplary substrate is the basement membrane, also referred to as basal laminae, which comprise extracellular sheets of proteins that surround tissues, providing structural support, a filtration function, and a surface for cell attachment, migration, and differentiation. In some instances, substrates can be used directly to produce 2- or 3- dimensional supports on which the cells may be grown on or in

[0024] .

[0132] An “SC-RPE cell culture substrate” in the sense of the disclosure is a substance configured to support stem cell attachment and growth and allow SC-RPE differentiation of stem cells in presence of a serum-free SC-RPE differentiation medium of the disclosure. In particular, the substrate facilitates the establishment of the optimal ratio of ectodermal, endodermal and mesodermal progenitor cells (ectoderm greater than endoderm and mesoderm) to allow for the natural development of RPE as will be understood by a skilled person upon reading of the present disclosure. Two-dimensional culture indicates the process of growth of cells on solid surface on which cells are seeded onto and that cannot be penetrated by the cells. Exemplary SC-RPE cell culture substrate which can be used in methods and systems herein described comprise laminin 521, such as truncated or full-length vitronectin, laminin 111, laminin 211, laminin 221, laminin 332, laminin 411, laminin 511, and soluble basement membrane matrix extract such as MatrigelTMand additional SC-RPE cells culture substrate identifiable by a skilled person.

[0133] In embodiments herein described a “serum-free SC-RPE differentiation medium” refers to a cell culture medium comprising a defined combination of essential components (e.g. serum albumin, insulin, transferrin, selenium), salts (Na, K, Ca, Mg, Cl, SO4, PO4of composition similar to serum), pH buffer(s), carbohydrate energy source(s), vitamins, amino acids, lipids and trace minerals) that support cell growth and absent of factors or agents that maintain pluripotency such as bFGF, TGFB1, or nodal. For optimal efficiency of spontaneous SC-RPE differentiation, the differentiation medium in conjunction with a specific cellTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT substrate, results in the preferential production of ectoderm germ layer cells in the immediate days following its addition to allow for subsequent efficient production of neuroectoderm cells, yet with sufficient quantities of mesoderm and endoderm germ layer cells to later direct the production of RPE progenitors. In addition, the differentiation medium supports the maintenance and growth of RPE progenitors. As each stem cell line is unique due to different genetics and culture history, the preferred medium for a given substrate according to the present disclosure is typically identified empirically, as it is expected that the medium that works best with one cell line may or may not work well with another cell line.

[0134] Accordingly in some preferred embodiments assessing different differentiation media or modified media formulations is performed by carrying out a complete RPE differentiation process of a candidate differentiation medium on tested RPE cells with methods identifiable by a skilled person upon reading of the present disclosure, determining the final efficiency of SC-RPE differentiation based on detected relative proportion of pigmented cells or the production of RPE gene products such as the highly expressed and secreted protein, pigment epithelial-derived factor (PEDF) according to methods identifiable by a skilled person; and selecting the tested RPE cells having a final efficiency above a set threshold indicative of a target efficiency based on the experimental design. However, for faster or larger scale screening of differentiation media it is possible to monitor performance by determining the absolute quantity and percentage of desired cell types which can be performed by detection of germ layer markers (e.g. SOX17, GATA4, T, NODAL, HAND1, CDX2) in the days following transition to the differentiation medium and neuroectoderm / eye field / RPE markers (e.g. PAX6, RAX, LHX2, MITF, SOX9, SOX10) in the days and weeks following the switch to differentiation medium using appropriate methods.

[0135] In some embodiments, an SC-RPE differentiation media herein described is from the RPE Differentiation Medium 1 (RPE-DM1) group of media of the disclosure and comprises the commercially available base medium KnockOut™ DMEM supplemented with KnockOut™ Serum Replacement Medium, GlutaMAX™, nonessential amino acid supplement, ^-mercaptoethanol, and normocin. A distinguishing feature of RPE-DM1 is the use of KnockOut™ Serum Replacement (KOSR) as the source of additional supplements required for cell growth and maintenance.

[0136] KnockOut™ DMEM (KO-DMEM) is a basal medium based on the formulation of DMEM and optimized for growth of undifferentiated embryonic and induced pluripotent stemTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT cells. DMEM (Dulbecco’s Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells. The composition of standard DMEM is reported in the following Table 1.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 1 DMEM composition

[0025] .

[0137] DMEM is unique from other media as it contains 4 times the concentration of amino acids and vitamins than the original Eagle’s Minimal Essential Medium. DMEM was originally formulated with low glucose (1 g / L) and sodium pyruvate, but is often used with higher glucose levels, with or without sodium pyruvate. The KO-DMEM used in the preferred formulation of RPE-DM1 of this disclosure is based on the high glucose plus pyruvate DMEM formulation. DMEM contains no proteins, lipids, or growth factors. Therefore, DMEM requires supplementation, commonly with 10% Fetal Bovine Serum (FBS). DMEM uses a sodium bicarbonate buffer system (3.7 g / L), and therefore requires a 5–10% CO2 environment to maintain physiological pH

[0026] .

[0138] In KnockOut™ DMEM, which contains no L-glutamine, the osmolarity of original DMEM is optimized to approximate that of mouse embryonic tissue (~320 mOsm / L). High glucose plus pyruvate DMEM, which KnockOut™ DMEM is based on, has a calculated osmolarity of approximately 360 mOsm / L. It is expected that the osmolarity of KnockOut™ DMEM has been adjusted with respect to DMEM by reducing the amount of sodium chloride and / or the amount of sodium bicarbonate in the original DMEM formulation by about 40 mOsm / L (see Table 1), as these are two major contributors to the final osmolarity of the medium. Alternatively, the osmolarity of DMEM may be reduced by reduction of the concentration of all medium components (except glucose and pyruvate whose concentration in KO-DMEM are stated) by about 10%. KnockOut™ DMEM has been used in literature in theTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT culture of stem cells. (see e.g.

[0027] ,

[0028] ,

[0029] ).

[0139] KnockOut™ DMEM or the cell therapy manufacturing grade CTS™ KnockOut™ DMEM with phenol red and without glutamine is available by the manufacturer ThermoFisher with product code: Gibco™ 10829018 and A1286101^

[0030] at the filing date of the present disclosure.

[0140] KnockOut™ Serum Replacement (KOSR) is a FBS-free formulation designed to replace FBS in feeder-dependent ESC and iPSC cultures. It has been cited in more than 2,000 publications on various PSC-related applications. The preferred composition and expected usable concentration ranges, formulation methods, and guidelines for its use are reported in patent publication WO1998030679A1

[0031] incorporated herein by reference in its entirety. Based on this publication it is expected that KnockOut™ Serum Replacement is comprised of the proteins lipid-rich albumin (AlbuMAX), insulin, and holo-transferrin; the amino acids glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; the vitamins and antioxidants thiamine, L-Ascorbic acid 2-phosphate, reduced glutathione; and the trace elements Ag+, Al+3, Ba+2, Cd+2, Co+2, Cr+3, Ge+4, Se+4, Br−, I−, F−, Mn+2, Si+4, V+5, Mo+6, Ni+2, Rb+, Sn+2, Zr+4. For ease of reference, the preferred concentration of each of these components is provided in Table 2. KnockOut™ Serum Replacement is commercially available and sold by manufacturer ThermoFisher with catalog numbers 10828010 for 100 mL and 10828028 for 500 mL or 12618013 for the CTS™ KnockOut™ SR XenoFree Medium which is the cell therapy manufacturing grade equivalent.

[0032] at the filing date of the present disclosure. ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^ ^^ ^^ ^^ ^^^^^^^^ ^^^^^ ^^ ^^ ^^ ^^^^^^ ^^^!" ^^^ #^^#^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ $^%^^^"^ ^^^&^ ^^ ^^ ^^ ^^^^^'^^^^"^ ^^&^^ ^^ ^^ ^^ ^^^%^ ^^%^ ^^^^"^ ^()^ ^^ ^^ ^^ ^^^^^^"^^^^"^ ^*^^^^ ^^ ^^ ^^ ^^^"'^^^^^^"^ (#)^ ^^ ^^ ^^ ^^+^"^%^^^^^^^"^ ^#&^^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^ ^^ ^^+ ^ ^^ ^^ ^^ ^^," ^^"^ ^(^)^ ^^ ^^ ^^ ^^^^ "^^^^"^ ^&^(^^ ^^ ^^ ^^ ^^^ %^'^^^^^^ ^^*^^ ^^ ^^ ^^ ^^^% ^^^^"^ ^^)^ ^^ ^^ ^^ ^^-^^^^"^ ^#^&(^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ ^^^^^^ ^^^^^^^^^^^^^^^^^^'"^ ^^^&^ ^^ ^^ ^^ ^^^^^^^"^ ^^^&^ ^^ ^^ ^^ $^^'^'^^^^"^^ "^^^"^^^ )^^*^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^. / 0)^1^.23^ #^##*)#^ ^^ ^^ ^^ ^^4^)^5^(^^0^1^^)23^ #^#^^^^ ^^ ^^ ^^ 6^^4^^^0^^^^16^^23^ #^#)^*^ ^^ ^^ ^^ 4^,0^^5^&^^0^14^^23^ #^^#^^ ^^ ^^ ^^ 4^4^^^5^^^0^14^^23^ #^#(^(^ ^^ ^^ ^^ 4 ^^,0^^)^5^^^0^14)23^ #^##^)^^ ^^ ^^ ^^ $"0^^1$"^23^ #^#^&^^ ^^ ^^ ^^ / ^^,"0)^1,"^23^ #^^^*^ ^^ ^^ ^^ ^^,"0)^1,"^23^ #^^**^ ^^ ^^ ^^ 76 ^1683^ #^##*#^^ ^^ ^^ ^^ 7^^1^83^ #^##*^^^ ^^ ^^ ^^ ^^4^^^5^^^^0^1^^^23^ #^#^#^^ ^^ ^^ ^^ / ^9^1983^ #^^^(^ ^^ ^^ ^^ / ^^,^0)^5^^^^0^1,^^23^ )^*^^ ^^ ^^ ^^ / ^-0)^1-*23^ #^#^^^^ ^^ ^^ ^^ ^ / ^^^(^^^0^^^5^^^^0^1^^(23^ #^#^&*^ ^^ ^^ ^^ / ^,0^^5^(^^0^1 / ^^23^ #^##)&^^ ^^ ^^ ^^ ^^4^^1^^23^ #^#*^^^ ^^ ^^ ^^ ,^4^^^1,^^23^ #^##^*&^ ^^ ^^ ^^ : 04^^^5^&^^0^1:^23^ #^#&^^^ ^^ + "!" "^^*#;^,'^^<^! ^^^+^'"^'^ ^^ =0^^^&#)#(^^^^^ ^^ ^^^^"^^^'^>"^"^"^"^'^^^^^^^^^^'"^^^^^^ ^^<"'^^^0'^" ^^^^'^!^ ^^^ ^^%^^"^^^^^'^'^'"^^

[0141] The KnockOut Serum Replacement composition reported in Table 2 can be used in methods and systems herein described as Embryonic Stem Cell Serum Replacement (ESCSR) as will be understood by a skilled person upon review of the present disclosure.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0142] GlutaMAX™ supplement is an improved cell culture supplement that can be used as a direct substitute for L-glutamine in cell culture media. GlutaMAX™ Supplement is an alternative to L-glutamine, with increased stability that improves cell health

[0033] . GlutaMAX™ Supplement is suitable for both adherent and suspension culture of mammalian cells, with no adaptation required. GlutaMAX™ Supplement is offered as a 200 mM solution (100X) of L- alanyl-L-glutamine dipeptide in 0.85% NaCl. GlutaMAX™ Supplement is also included in a variety of media formulations. Compared to L-glutamine, GlutaMAX™ Supplement minimizes toxic ammonia build-up, improves cell viability and growth, and remains stable across a wide range of temperatures. GlutaMAX™ is commercially available from manufacturer ThermoFisher with catalog numbers 35050061 for 100 mL or 3505007920×100 mL or A1286001 for 100 mL of the clinical manufacture grade equivalent. Alternatively, the solid L-alanyl-L-glutamine dipeptide can be obtained from multiple alternative vendors and used as would be clear to a skilled person based on the above general description at the filing date of the present disclosure.

[0143] In its most preferred embodiment, about 500 mL of RPE-DM1 Medium is formulated by combining 389 mL of CTS-KnockOut™ DMEM, 100 mL of CTS-KnockOut™ Serum Replacement, 5 mL of 200 mM CTS-GlutaMAX™ solution, 5 mL of 100X MEM Non- essential Amino Acids solution (100 mM alanine, 100 mM asparagine, 100 mM aspartic acid, 100 mM glutamic acid, 100 mM glycine, 100 mM proline, and 100 mM serine; ThermoFisher Scientific catalog numbers 11140050 or 11140076), and 909 ^L of 55 mM ^-mercaptoethanol (ThermoFisher Scientific catalog number 21985023). In addition, in applications where their use is allowed antibiotic or antimycotics such as penicillin streptomyocin, and normocin can be included in the medium, but their use is optional, and they are not believed to substantially impact RPE differentiation when used at their recommended concentrations. The concentration of the RPE-DM1 Medium components including the most preferred and the least preferred concentrations, are listed in Table 3.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0144] In each of Set-1, Set-2, Set-3, Set-4 and Set-5 reported in Table 3, each concentration within the indicated range for a component of the RPE-DM1 composition can be combined with each concentration within the indicated range of another component of the RPE-DM1 composition. Therefore, for example in Set 4 SnCl2 [Sn2+] can be comprised at a concentration of 0.000179 um in combination with ZrOCl2[Zr4+] at a concentration of 0.016 uM and in combination with any other concentrations comprised within each range of the other components as will be understood by a skilled person, providing that the osmolarity remains within the prescribed ranges.

[0145] For xeno-free applications of the method the protein components of the media should be derived from the same species as the stem cell line. Alternatively, nonprotein-based substitutes can be employed as will be understood by a skilled person.

[0146] Variations in the exemplified RPE-DM1 Medium of similar composition are expected to give rise to RPE differentiation. For example, alternative medium formulations can be made by changing the percentage of the KnockOut™ Serum Replacement from the most preferred concentration of 20% to within the range of 15 to 20% (preferred), 10 to 25% (less preferred), or 5 to 30% (least preferred). Additionally, in some cases lipid-rich serum albumin or transferrin substitutes can be used in place of or in addition to these two protein components as will be understood by a skilled person. For example, in some embodiments, lipid-richTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT albumin is substituted with its lipid components, and in particular with lysophosphatidic acid and sphingosine-1-phosphate, either by themselves or in combination with lipid-depleted (Fraction V) albumin

[0034] . In some embodiments, transferrin can be substituted with a complex of a lipophilic iron chelator, such as pyridoxal isonicotinoyl hydrazone, and ferric iron

[0035] . Similarly, the concentration of the nonessential amino acids, GlutaMAX™ and ^- mercaptoethanol may be altered by adjusting their relative ratios of their solutions and glutamine can be used as a direct 1:1 substitute for GlutaMAX™ (see Table 3 for allowable concentration ranges, see any one of sets 1 to 5). Other base medium such as, DMEM, KnockOut™ DMEM:F12, DMEM:F12 mixtures, ^-MEM, RPMI 1640, and Iscove’s Modified Dulbecco’s Medium and their variations can be substituted for KnockOut™ DMEM providing that the concentration of the individual medium components and final medium osmolarity remain in the indicated ranges in Table 3 (see any one of sets 1 to 5). The concentration of the medium components can be calculated based on the published alternative base medium formulations and the preferred composition of Knockout Serum Replacement (International Patent application PCT / US1998 / 000467 Publication No. WO1998030679A1, incorporated herein by reference in its entirety)

[0031] used as an Embryonic Stem Cell Serum Replacement (ESCSR) supplement. RPE-DM1 formulation variants are denoted by their base medium and percent volume of KnockOutTMSerum Replacement with additional changes to the other components (NEAA, GlutaMAX™, ^ME, or KOSR composition) addended (e.g., KO- DMEM / 20%KOSR, CTS-KO-DMEM / 20%KOSR, KO-DMEM / 15%KOSR, CTS-KO- DMEM / 20%KOSR / Glutamine, KO-DMEM:F12 / 20%KOSR). Media formulations that meet the requirements listed in Table 3 are expected to support stem cell differentiation to some degree, although differences in yield and substrate preference between media for a given stem cell line are expected. Their utility for derivation of RPE from specific stem cell lines can be assessed by monitoring stem cell survival and growth as well as the expression of germline, retinal progenitor, and RPE signature genes after transition to the differentiation medium using simple experimentation as would be understood by a skilled person in the field.

[0147] A representative example of an SC-RPE differentiation media formed by the most preferred formulation and allowable concentration ranges of RPE-DM1 Medium (Table 3) in accordance with the present disclosure and related method of preparation is reported in Example 2.

[0148] Providing that the individual components and final medium osmolarity remain in theTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ranges listed in Table 3, in some embodiments RPE-DM1 Medium formulations not possible by simple combination of commercially available base medium and KnockOut™ Serum Replacement can be made using custom base medium formulations, diluted or concentrated base medium, alteration of the concentration of the individual components in base medium by supplementation or subtraction of individual medium components, blended mixtures of base medium, or modified KnockOut Serum Replacement as described in International Patent application PCT / US1998 / 000467 Publication No. WO1998030679A1

[0031] .

[0149] In general, changes of the composition are expected to affect media:substrate preference and / or RPE yield as will be understood by a skilled person upon reading of the present disclosure. The rate of growth is expected to affect a stem cells propensity into the different germ layer cell types, either directly by changing the relative and absolute time in various stages of the cell cycle or by affecting the cell density and timing of events in that are cell density dependent. Furthermore, different stem cell lines have different inherent growth rates; possibly due to common genetic variation which is another prime determinant of differentiation potential

[0036] . Hence, changes to the medium composition that would be expected to alter growth rates such as altering the concentration and ratio of the two primary carbon energy sources (glucose and pyruvate), the concentration of the amino acids, substantial reduction in the concentration of the vitamins, changes to the pH (carbonate / CO2 and HEPES), the inorganic salt concentrations and relative ratios, and the concentration of the protein components (insulin, lipid rich serum albumin, and transferrin), and redox potential would have an expected effect on cell growth that would be clear to a skilled person in the field and could be tested by simple measurement of growth rates in the alternative medium formulations. In addition, some medium components may have effects on RPE differentiation that are not mediated by effects on cell growth. For example, it is expected that at least in some cases calcium signaling plays an essential role in ectoderm-to-neuroectoderm differentiation and calcium plays an essential role in cell:substrate and cell:cell adhesion. As such, altering the calcium concentration is expected to affect the yield and possibly the substrate preference in a stem cell line dependent fashion. The suitability of alternative medium in accordance with the present disclosure is preferably ultimately validated experimentally using several different stem cell lines with different propensities as would be understood by skilled person in the field.

[0150] In some embodiments, an SC-RPE differentiation media of the disclosure is a medium comprising X-VIVO™ 10 Medium supplemented one or more of the following supplements:Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT insulin, transferrin, sodium selenite, progesterone, putrescine, serum albumin, catalase, superoxide dismutase, corticosterone or hydrocortisone, d-galactose, ethanolamine, glutathione (reduced), L-carnitine, linoleic acid, linolenic acid, triiodo-I-thyronine (T3), DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A (retinyl acetate, retinol acetate) along with the optional ingredient biotin and lipoic acid. The medium resulting from the combination of X-VIVO™ 10 medium and the listed supplements is herein referred to a RPE Differentiation Medium 2 (RPE-DM2). The distinguishing feature of media classified as being RPE-DM2 is the use of the complete medium X-VIVOTM10 (or medium of similar composition and functionality) that is preferably further augmented with a B27-based supplement.

[0151] The X-VIVO™ 10 Medium is a commercially available serum-free Hematopoietic Cell Medium, with L-glutamine, and with or without gentamicin and phenol red that was designed to support the generation of LAK cells in a serum-free environment. X-VIVO™ 10 Media formulations are designed to support slower growing, less mature cells such as hematopoietic stem cells and natural killer cells. X-VIVOTM10 also supports the generation of lymphokine activated killer (LAK) cells and peripheral blood lymphocytes (PBL). Key applications for the use of X-VIVO™ 10 medium are: proliferation of peripheral blood lymphocytes, proliferation of tumor infiltrating lymphocytes, cryopreservation of human tissue, cultivation of human monocytes and macrophages, cultivation of dendritic cells, cultivation of CAR-T cells, and cultivation of hematopoetic stem cells. X-VIVO™ 10 Media is available from Lonza Biosciences as a 1X liquid in three different formulations: research grade X-VIVOTM10, which contains L-glutamine, gentamicin, and phenol red (catalog number BE04-380Q), cGMP grade TheraPeak X-VIVOTM10, which contains L-glutamine and without gentamicin, or phenol red and uses native human transferrin (catalog number BEBP04-743Q), and cGMP grade TheraPeak X-VIVOTM10, which contains L-glutamine and without gentamicin, or phenol red and uses recombinant human transferrin [catalog number (BEBP02- 055Q)

[0037] .

[0152] X-VIVOTM10 is a medium formulation comprised of a base medium, human serum albumin, human insulin, human transferrin, and possibly unknown additional supplements such as essential lipids and antioxidants. Iscove’s Modified Dulbecco’s Medium (IMDM), RPMI 1640, and DMEM are the most common base medium (in decreasing order) used in serum supplemented hematopoietic cell culture media

[0038] and IMDM is known to be the base medium used in two other commercially available serum-free media that are used for the sameTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT applications as X-VIVOTM10 (StemSpan Media family SFEM, SFEMII, H3000, and ACF from STEMCELL Technologies and QBSF-60 from Quality Biological). It is therefore expected that IMDM and possibly RPMI 1640 and DMEM can be comprised within or equivalent to the base medium of X-VIVOTM.

[0153] The additional supplements are a collection of compounds commonly used in neuronal culture. The supplement mixture is a defined yet complex mixture of antioxidant enzymes, proteins, vitamins, and fatty acids that are combined in optimized ratios to support neuronal survival in culture.

[0154] In some embodiments of the disclosure RPE-DM2 is prepared using the base culture media Iscove's Modified Dulbecco's Medium (IMDM) and / or RPMI 1640. In particular, RPE- DM2 can be prepared using IMDM, liquid, with phenol red (#12440053, 500 mL ThermoFisher), and RPMI 1640 liquid formulation, with glutamine, with phenol red (#11875093, 500 mL, ThermoFisher), The composition of preferred formulations of IMDM and RPMI 1640 is reported in Table 4 below.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0155] Other formulations of IMDM are also available (such as powder, with GlutaMax, orTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT with phenol red that can also be used as well as would be understood by a skilled person.

[0156] Other formulations of RPMI 1640 are also available (such as powder, or with GlutaMax, or with HEPES, or with phenol red etc.) that can also be used as well as would be understood by a skilled person.

[0157] In some embodiments RPE-DM2 is prepared by blending the IMDM and RPMI 1640 in various proportions to obtain unique RPE-DM2 base media formulations in the sense of the present disclosure. In particular, the resulting general formulation of an RPE-DM2 medium is reported in Table 4a below.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0158] In the RPE-DM1 formulation of Table 4a, each concentration within the indicated range for a component of the RPE-DM1 composition can be combined with each concentration within the indicated range of another component of the RPE-DM1 composition within the indicate osmolarity. Due to the substantial differences due to the use of HEPES and increased amounts of glucose in RPMI 1640 and the compensatory differences in inorganic salt composition (primarily NaCl and NaHCO3), when comprising a media using the individual components the medium resulting from the combination of various components at any one ofTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the concentration within the indicated ranges, it is preferred that the medium has an osmolarity about 290-325 mOsm / L, and is preferably 300-310 mOsm / L by adjusting the amount of NaCl accordingly. In addition for CaCl2and Ca(NO3)2the two should be considered together and when using Ca(NO3)2it is preferred that the amount of CaCl2be reduced. The same principle holds for mono- and dibasic sodium phosphate.

[0159] In some embodiments, RPE-DM2 medium can be provided by combining IMDM:RPMI1640 in various proportions as will be understood by a skilled person. In particular, in some embodiments the base medium of RPE-DM2 can be provided by mixing fixed proportions of each media (IMDM:RPMI = (10:0), (8:2), (6:4), (4:6), (2:8), and (0:10)) with ranges between adjoining the fixed mixtures IMDM:RPMI (10:0) to (8:2), (8:2) to (6:4), (6:4) to (4:6), (4:6) to (2:8), and (2:8) to (0:10). The related composition including ranges of possible concentrations of each component, comprised between the fixed dilution ratios is reported in Tables 4b to 4f below.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0160] In each of the RPE-DM2 media reported in Tables 4a to 4f, each concentration within the indicated range for a component of the RPE-DM2 composition can be combined with eachTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT concentration within the indicated range of another component of the RPE-DM2 composition providing that the osmolarity remains in the specified range, as will be understood by a skilled person.

[0161] In some embodiments a custom RPE-DM2 medium can be prepared based on the concentration ranges of the individual components provided in Tables 4a to 4f with the proviso that the calculated osmolarity of the custom formulation is 290-330 mOsm / L, preferably 300- 320, and more preferably 300-310. This can be generally achieved by adjusting the sodium chloride concentration to a value in the range of 113-139 mM as necessary.

[0162] In some embodiments of RPE-DM2 medium of the disclosure, IMDM, RPMI 1640, or the IMDM:RPMI blended media of any one of Tables 4a to 4f can then be supplemented with insulin, selenite, transferrin, ethanolamine, and albumin (collectively known as ITSE+A) to generate a minimal RPE-DM2 (IMDM-ITSE+A, RPMI-ITSE+A, or IMDM:RPMI- ITSE+A).

[0163] In some embodiments of RPE-DM2 medium of the disclosure, xeno-free ITSE+A can be obtained as a 100X cocktail from InVitra (catalog number 777ITS092). ITSE+A is comprised of 1.00 g / L recombinant human insulin, 0.55 g / L recombinant human transferrin, 0.00067 g / L sodium selenite, 0.20 g / L ethanolamine, and 20.00 g / L recombinant human albumin.

[0164] In some embodiments of RPE-DM2 medium of the disclosure, ITSE+A used at the preferable strength of 1X, less preferably at 0.25-0.5X, 0.5-0.75X, 0.75-1X, 1-1.5X, 1.5-2.0X, or 2-3X. To gain further control of the concentration of individual components ITSE+A they can be obtained individually, or as sub-cocktails (ITS and ITSE) and custom formulations can be prepared where the concentration of one or more of the individual components is adjusted within the forementioned range.

[0165] In some embodiments, an SC-RPE differentiation media RPE-DM2 of the disclosure is supplemented with a B27-based supplement. The original serum-free neuronal culture supplement composition, B27, was developed by Dr. Gregory Brewer and colleagues and is described in

[0039] and

[0040] and a modified B27-based composition, named NS21, is described in

[0041] . Typically, these supplements are prepared as concentrated stocks which are then added to the base medium. The formulations of B27 and NS21 at 1X strength based on an onlineTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT published protocol

[0042] and the original publications are provided in Table 5. Suitable B27- based 50X liquid stock solutions are commercially available from ThermoFisher Gibco (B- 27™ Supplement (50X), serum free [catalog numbers 17504001 and 17504044]; B-27™ Plus Supplement (50X), [catalog number A3582801]; B-27™ Supplement, XenoFree, [catalog number A1486701], CTS™ B-27™ Supplement XenoFree (50X) [catalog number A5047501]) and STEMCELL Technologies (Neurocult SM1, catalog number 05711). NS21- based medium supplements can be obtained from Millipore Sigma (N21 Medium Supplement (50X), catalog number SCM081) and R & D Systems (N21-MAX Media Supplement (50X), catalog number AR008). These commercial stocks have the same composition of compounds listed in Table 5, but the exact formulations are proprietary and the source (e.g., species, recombinant) and concentration of some of the components may vary from the original published versions and between different commercial products. ^^^^^^*^^+^,^^^^^^-^^^^^^^^^^^^^^^^.^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^ / ^^^^^^^^ ^^ ^^ ^^ .^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ?^^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ 6^>^^"^," ^^^^^^^^^^@^9 ^^'^^^^-^ )^^(^ ^^ ^^ ^^ ^^^^^^^@^^^>^^"^ #^(^&^ ^^ ^^ ^^ ^^^^^^ ^^^!" ^^@^^^^^^^ #^#(^*^ ^^ ^^ ^^ 4^'^^^^"@^^^>^^"^ #^#^#^^ ^^ ^^ ^^ ,^^" ^^^^"^A^^^^'^^"@^^^>^^"^ #^#^(^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^0^^^^^1^^^^^^^^^ ^^ ^^ ^^ ^^ $^^'^'^^^^"^^ "^^^"^^^ )^^*^ ^^ ^^ ^^ ,^^^^^^,"^"^^'"^ #^#^^*^@^#^#&)#^^ ^^ ^^ ^^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^^^^ ^^ ^^ ^^ A^^^^^^^^^^^^^^" ^^^ ^^^#^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ B'^^^^^^^^^"^ ^(^^^ ^^ ^^ ^^ ^^4^ ^^'^^"^^ ^#^^^ ^^ ^^ ^^ ^^^^^"^^^^^^^^ )^*^^ ^^ ^^ ^^ ^^^^^"^^^^^^^^^ )^*^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^3^^^^^^^^ ^^ ^^ ^^ ^^ A^6^^'^^^ ^#^^^^ ^^ ^^ ^^ ^"'^^^^^^^"'^'"^ #^)#^^ ^^ ^^ ^^ ^"'^^^^^ #^)^^^^ ^^ ^^ ^^ 4^ '^^^^'" ^^"^ #^#*^^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^ ^^ + ^."^'" ^^"^ #^#^##^ ^^ ^^ ^^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^##^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^4^^^ ^^ ^^ ^^ ^^ A^$^^^^'^^"^ &)^)^ ^^ ^^ ^^ ^^^^^^^^^^^^ #^^^&^^ ^^ ^^ ^^ +^' "^^^^"^ ^##^ ^^ ^^A" ^>"^^! ^^^6 "C" ^"'^^^^@^^^^)1)^3^D^6 "C" ^^^^^4^'^^^@^^^&^1^#3^^ ^^ ^^4^"^^"'^^^^@^^##&1^^3^^ ^ ^^E^^F^"^6^^^4^^^"^' ^'^^^^ ^^ ^^ ^^E^^F^"^ / ,^^^4^^^"^' ^'^^^^ ^^ ^^

[0166] In some embodiments the RPE-DM2 supplemented with B27 is a medium comprising X-VIVO™ 10 Medium. In some embodiments the RPE-DM2 supplemented with B27 is a medium comprising a composition of any one of Tables 4a to 4f as will be understood by a skilled person, upon reading of the present disclosure.

[0167] In preferred embodiment of RPE-DM2 medium of the disclosure, ITSE+A supplemented RPE-DM2 media are further supplemented with any of the B27-type supplements of the disclosure at a strength of 1X, less preferably 0.25-0.5X, 0.5-0.75X, 0.75- 1X, 1-1.5X, 1.5-2X, 1.5-2X, and 2-3X.

[0168] In preferred embodiments of RPE-DM2 medium of the disclosure, the B27-type supplements are with vitamin A.

[0169] In some embodiments of RPE-DM2 medium of the disclosure, the B27-type supplements are without vitamin A.

[0170] In some embodiments of RPE-DM2 medium of the disclosure, since all of the components of ITSE+A are also provided by the B27 supplements, supplementation of IMDM, RPMI 1640, or IMDM:RPMI blends with ITSE+A any one or more of ITSE+A’s individual components can be omitted and the base media can be then supplemented with B27. In this case it is expected that higher strengths of the B27 supplements can be required to achieve a desired outcome.

[0171] In some embodiments of RPE-DM2, supplementation with ITSE+A can be omitted, and the base media can be supplemented with a B27-base supplement alone.

[0172] A skilled person will understand that the specific IMDM, RPMI, or IMDM:RPMITitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT based RPE-DM2 differentiation media can be evaluated first by assessing the differentiation cultures in the immediate day or two following the switch from stem cell media to RPE-DM2 to insure that there is not excessive loss of cells ( roughly >20%). In the following days the onset of expression of eye-field transcription factors (such as PAX6, OTX2, LHX2, and RAX can be evaluated.

[0173] Expression of transcription factor can be performed for example with quantitative PCR, immunohistochemistry, or other equivalent methods as would be understood by a skilled person. In the following weeks the expression of the pigmented cell transcription factor MITF, RPE genes such as TYR and PMEL can be determined and at later times near the time of or after initiating RPE maturation successful outcomes can be quantified based on the percentage of pigmented culture area. In most preferred embodiments all these measures be done relative to a cell and differentiation method known to work. In the alternative, since a feature of the prototype RPE-DM2 based media X-VIVO 10 is that it supports at least some degree of RPE maturation it may be possible to prescreen novel formulations of RPE-DM2 to determine if they also support some degree of RPE maturation. It is expected that RPE-DM2 formulations that support the maturation of RPE would be good candidates for SC-RPE differentiation. This analysis can be carried out using previously obtained SC-RPE or with primary RPE cells.

[0174] In a most preferred embodiment RPE-DM2 is prepared by adding the appropriate amount of any of the commercially available or custom made 50X B27-based supplement stocks prepared according to the composition provided in Table 5 to one of the three X- VIVOTM10 formulations to result in a 1X concentration of the supplements in medium. In addition, in applications where their use is allowed antibiotic or antimycotics such as penicillin, streptomyocin, and normocin can be included in the medium, but their use is optional and they are not believed to substantially impact RPE differentiation when used at their recommended concentrations. In some embodiments of RPE-DM2 the concentration of the additional B27- based supplements can be 0.25-3X, more preferably between 0.5-2X, and even more preferably between 0.75-1.25X.

[0175] In some embodiments the concentration of the B27-based supplements can be in the range of 0-0.25X. This is generally less preferred as the yields of RPE are typically reduced, but for some lines it may be advantageous. The identification of optimum RPE-DM2 medium formulations can be determined by simple experimentation to determine their effect on germTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT layer bias and RPE differentiation as would be understood by a skilled person upon reading of the present disclosure.

[0176] A representative example of one of the most preferred embodiments of RPE-DM2 Medium formulation of the disclosure (TheraPeak X-VIVOTM10 with recombinant transferrin + 1X B-27™ Supplement, XenoFree) and related method of preparation is reported in Example 3. Variations in the exemplified X-VIVOTM10 + XF-B27 RPE-DM2 Medium are expected as will be understood by a skilled person upon reading of the present disclosure. For example, other grades of B27 or other B27-based supplement formulations such as NeuroCult SM1 (StemCell Technologies), N21 (Sigma / Millipore), N21-Max (R & D Systems) can be substituted directly for B27 as can custom made preparations of B27 and N21 based on the formulations provided in Table 5, thus providing alternative RPE-DM2 media such as X- VIVOTM10 / B-27, X-VIVOTM10 / B-27+, X-VIVOTM10 / CTS-B27, X-VIVOTM10 / NeuroCultSM1, X-VIVOTM / N21, X-VIVOTM10 / N21-Max, X-VIVOTM10 / custom B27, and X-VIVOTM10 / custom N21 and where the term X-VIVOTM10 denotes any X-VIVOTM10 of similar composition to the previously stated X-VIVOTM10 formulation.

[0177] The choice of which of the most preferred RPE-DM2 variants to use is largely dependent on the intended use of the cells. To produce SC-RPE that are intended to be used as a clinical therapeutic the media and supplements should preferably be xeno- and animal-free and certified for the manufacture of a cell-based clinical therapeutic. For purely research purposes any of the media and supplement choices are suitable, but non-clinical grade materials are generally less costly and easier to source. However, the source or specific derivation of B27 can affect media:substrate germline bias profile in a cell line-dependent manner. For example, it has been observed that some stem lines give higher yields of SC-RPE with research grade B27, whereas others give better yields with clinical grade B27. Hence, once a media and B27- based supplement version or source is selected for a given cell line it should not be changed.

[0178] As previously stated, in some embodiments, B27-based supplements can be omitted in its entirety, albeit with the possible reduction of the yields of SC-RPE. As such, in some embodiments of RPE-DM2, custom B27 or N21 formulations with varying concentrations of the individual components within the ranges that result from the previously described RPE- DM2 compositions in which the concentration of the individual components of the

[0166] and the concentrations listed in Table 4 or missing one or more of the components can be used. ForTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT example, since biotin and lipoic acid are unique to B27 and N21 respectively, yet B27- and N21-based supplements can be used interchangeably, it follows that biotin and lipoic acid supplementation may not be essential. Similarly, since serum albumin, insulin, transferrin, and selenite are also present in X-VIVOTM10, it would be expected that additional supplementation with these three components may not be essential or that their concentration in the supplement mixture may be reduced. In addition, the source of the components may be altered from what is used in the original supplement formulations. For example, lipid rich serum albumin can be substituted for lipid poor albumin or iron chelators can be substituted for transferrin. As each stem cell line is unique and there is no method to predict how a given cell line may behave, assessment of the utility of custom B27-derived formulations should be determined by simple experimentation using the stem cell lines of interest, as will be understood by a skilled person upon reading of the present disclosure.

[0179] In addition to X-VIVOTM10, it is expected that other serum-free medium commonly used for the culture of hematopoietic cells can be used as a serum-free base medium for the formulation of RPE-DM2 according to the fore described guidelines. Examples of suitable commercially available alternative serum-free base medium include, StemSpanTM-SFEM, StemSpanTM-SFEM, StemSpanTM-H3000, and StemSpanTM-ACF from STEMCELL Technologies; AIM VTM, CTS AIM VTMand StemProTM-34 SFM from ThermoFisher Gibco; and QBSF-60 from Quality Biological. Alternatively, custom serum-free media that use IMDM, RPMI 1640, DMEM:Ham’s F12 mixtures, or DMEM as described in US Patents 5,945,337 and 6,224,860B1 can be used. Finally, it is expected that the base media, IMDM, RPMI1640, DMEM, KnockOutTMDMEM, KnockOutTMDMEM:F12, and DMEM:F12 mixtures each supplemented with insulin, transferrin, and selenium, and albumin (lipid-rich or -free) (ITSA) can be substituted for X-VIVOTM10. Each of these X-VIVOTM10 alternatives can then be combined with a B27-based supplement to create a RPE-DM2 medium variant as would be understood by skilled person up reading this disclosure. Alternative RPE-DM2 formulations are denoted by their base medium and type of B27-based supplement. When the final concentration of the B27 supplement is not 1X that is also indicated (e.g., X-VIVOTM10 / CTS-B27, X-VIVOTM10 / 2X-CTS-B27, X-VIVOTM10 / N21-MAX, X-VIVOTM10 / NC- SM1, StemSpan-ACF / CTS-B27, IMDM / CustomB27, RPMI / CustomB27, and IMDM / ITSA / CustomB27, etc.). While the use of these alternative serum-free base media is expected to support SC-RPE differentiation, they are also expected to have unique cell lineTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT specific germ layer / substrate profiles. As with all SC-RPE differentiation media of the disclosure, the suitability of these alternatives for a given cell line can be determined experimentally as would be understood by skilled person in the field and upon reading of the present disclosure.

[0180] In some embodiments, the various SC-RPE differentiation media of the disclosure are further supplemented with Activin A (herein also RPE-DM1+ActivinA and RPE- DM2+ActivinA). Embodiments that employ the use of Activin A are considered to be semi- directed differentiation methods.

[0181] The wording “Activin A” as used herein indicates the dimeric protein product of the inhibin, beta A gene (INHBA) gene identifiable by a skilled person. Activin A is a member of the transforming growth factor beta (TGFB) superfamily and plays a role in the regulation of multiple cellular functions including, but not limited to, cell proliferation, differentiation, wound responses, apoptosis, and metabolism. In vivo, Activin A produced by neighboring extraocular mesenchyme promotes the development of RPE from retinal / RPE precursors. For the case of in vitro spontaneous SC-RPE differentiation, it is believed that efficient generation of RPE requires a sufficiently low percentage of mesenchymal germ layer to allow for ectoderm to neuroectoderm differentiation, but enough mesenchymal cells to subsequently promote ocular neuroectoderm to RPE differentiation. Supplementation of the differentiation medium with Activin A after the period of ocular neuroectoderm differentiation is expected to increase the yield of SC-RPE when there are insufficient mesenchymal cells to promote that transition by natural means and embodiments that employ the use of Activin A are considered to semi-directed differentiation methods.

[0182] In some embodiments of the differentiation methods of this disclosure RPE- DM1+ActivinA and RPE-DM2+ActivinA can be prepared freshly on the day of use by adding Activin A to any of the various embodiments of RPE-DM1 or RPE-DM2 medium to a final concentration of 140 ng / mL (most preferred), 70-210 ng / mL (preferred), or 20-280 ng / mL (less preferred). Lower concentrations than indicated are not expected to increase the efficiency of SC-RPE differentiation and higher concentrations than indicated, can possibly result in adverse effects due to increase binding to non-Activin A TGF^ superfamily receptors. For stem cell lines where the addition of Activin A to the differentiation medium is beneficial there is typically an earlier onset of pigmentation, an increase in the number of pigmented cells, anTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT apparent intensity pigmentation, and an increase in the expression of RPE signature genes.

[0183] A representative example of a preferred embodiment of the RPE-DM2+ActivinA SC- RPE differentiation medium of the disclosure and related method of preparation is reported in Example 4. Variations in the exemplified RPE-DM2+ActivinA and RPE-DM1+ActivinA SC- RPE differentiation media are expected as will be understood by a skilled person upon reading of the present disclosure. For example, lower dosages of Activin A are expected to result in a decrease in RPE differentiation. Significantly higher dosages can result in poor yields due to an increased potential for cross-reactivity with non-Activin A receptors. Other members of the TGFB superfamily that are expected to be able to substitute for Activin A, such as but not limited to, are TGFB1, GDF11, and GDF8. Addition of a ROCK inhibitor (such as Y-27632) is expected to augment the activity of Activin A as is further supplementation of the medium with nicotinamide.

[0184] Addition of Activin A is expected to result in a functional SC-RPE differentiation medium for all possible embodiments of RPE-DM1 and RPE-DM2, as will be understood by a skilled person upon reading of the present disclosure, but the benefit of activin supplementation will vary depending on the specific cell line / substrate / differentiation medium combination. The decision as to whether use an Activin A supplemented SC-RPE differentiation medium is generally determined empirically for a given cell line / substrate / medium combination by quantifying the SC-RPE yield as would be understood by skilled worker upon reading of the disclosure. Alternatively, the potential for its use can be assessed by determining the relative amounts of mesenchymal cells in the culture after the period of neuroectoderm differentiation (about one week) when not using Activin A. It would be expected that cell line / substrate / medium combinations that have low levels of mesenchymal cells compared to cultures that are known to have efficient SC-RPE differentiation without the use of Activin A supplementation might benefit from the use of Activin A. In the SC-RPE differentiation methods of the disclosure the Activin A supplemented SC-RPE differentiation media of the disclosure can be used in combination with the matching SC-RPE differentiation medium without Activin A. Typically, the period of its use is between day-6 and -14 of the RPE differentiation phase of the protocol after which the Activin A is medium is replaced with differentiation medium without Activin A. In some instances, the start of its use can be adjusted depending on the growth rate of the stem cells and timing of the switch from stem cell medium to SC-RPE differentiation medium (RPE-DM1 or RPE-DM2) and the cells can continue to beTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT maintained in RPE-DM+ActivinA until the RPE Maturation phase of the protocol.

[0185] An SC-RPE cell culture substrate is a substance configured to support stem cell attachment and growth and allow SC-RPE differentiation of stem cells in the presence of a serum-free SC-RPE differentiation medium of the disclosure. SC-RPE cell culture substrate of the disclosure are substrates configured to effect cell division times, alter cell morphology, and differentially effect gene expression through their specific receptors to provide an optimum ratio of three germ layer precursors. More specifically, in combination with an RPE-DM an optimum substrate should favor the production of ectoderm cells yet allow for the production of sufficient mesoderm and endoderm cells to provide the necessary signals for the development of ocular ectoderm and RPE progenitors. Different cell lines can have different substrate preferences and the preference can vary in an RPE-DM dependent manner.

[0186] In some embodiments, SC-RPE substrates are comprised of extracellular matrix or basement membrane proteins, derivatives, or mimics that allow for the attachment of stem cells by binding to specific receptors on the stem cell surface. Substrates with documented history of promoting the development of ectoderm-derived cell types are preferred. At an effective concentration, candidate substrates should support the attachment of essentially all viable stem cells. For a given cell line and RPE-DM combination, prospective substrates can be evaluated by comparing germ layer and progenitor cell profiles during the early stages of differentiation to that of cell line / RPE-DM / substrate concentration with known history of promoting SC-RPE differentiation. Alternatively, a substrate can be evaluated by its ability to generate SC-RPE based on the appearance of pigment and the expression of RPE signature genes.

[0187] In some embodiments, SC-RPE substrates comprise laminin 521, recombinant truncated vitronectin, or full-length plasma vitronectin (see e.g., Examples 5 and 6 and also e.g., Example 9, Example 19 and Example 21).

[0188] The wording laminin 521 as used here indicates a heterotrimeric protein comprised of ^5, ^2, and ^1 laminin subunits which are coded by the laminin family genes LAMA5, LAMB2, and LAMC1, respectively.

[0189] The wording recombinant truncated vitronectin as used here indicates a a recombinant protein having a vitronectin portion limited to, and possibly consisting of, amino acid residues 62-478 of the protein coded by the vitronectin gene, VTN.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0190] The wording plasma vitronectin as used here indicates the mature vitronectin protein coded by the vitronectin gene that results after cleavage of the signal peptide and that has been isolated from serum.

[0191] The wording full-length vitronectin as used here indicates the mature vitronectin protein coded by the vitronectin gene that results after cleavage of the signal peptide. Full- length vitronectin can be derived from natural sources such as plasma or recombinant methodologies.

[0192] Substrates that can also be used as SC-RPE differentiation substrate comprise soluble basement membrane matrix extract purified from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell line (commercially available as Matrigel, Gentex, and Cultrex BME), Collagen IV, or other members of the laminin protein family (e.g., LAM 111 (^1^1^1), LAM211 (^2^1^1), LAM221 (^2^2^1), LAM332 (^3^3^2), LAM411 (^4^1^1), and LAM511 (^5^1^1)). The synthetic substrate coating, Synthemax, which is a short synthetic vitronectin- derived peptide containing the arginine-glycine-asparagine amino acid domain that mediates binding to cellular integrin receptors has also been used for the derivation of RPE from stem cells and can also be used as a substrate. Similar laminin-derived peptides or fragments of laminin that retain laminin receptor binding activity can also be used as a substrate. When any substrates produced by recombinant methodologies are used can have amino acid sequence modifications so long as those modifications do not prevent binding to their specific receptors as will be understood by a skilled person. Custom combinations of any one of these substrates can also be used. While typically used for the derivation of endo- and mesodermal cells, the proteins collagen type 1 and fibronectin can be suitable substrates for stem cell lines that produce very low levels of these germ layer cells and fail to produce significant quantities of RPE using the previously described substrates.

[0193] The SC-RPE differentiation media and SC-RPE differentiation substates herein described can be used in the SC-RPE differentiation methods of the disclosure. In particular, in embodiments herein described the method comprises contacting stem cells growing on an SC-RPE substrate with one of the SC-RPE differentiation media. The optimal substrate- differentiation combination is stem cell line dependent. In the SC-RPE differentiation method of the disclosure, the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cells.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0194] In some embodiments, the stem cells are from an embryonic stem cell line, which can be a hESC. In some of those embodiments the stem cells can be from an ESC line selected from H1 (WA01) or H9 (WA09). In some of those embodiments the stem cells can be from an ESC line selected from Shef3, and Shef6. H1 and H9 are available from WiCell Research Institute and Shef3 and Shef6 can be obtained from the University of Sheffield Centre for Stem Cell Biology.

[0195] In some embodiments, the stem cells are from an iPSC stem cell line, which can be a human iPSC. In some of those embodiments the stem cells comprise stem cells from an iPSC line selected from CW70207, CW70066, CW70203, CW70316 which are from the Blinding Eye Disease Control Group of the California Institute for Regenerative Disease Repository and are available through FUJIFILM Cellular Dynamics.

[0196] In some embodiments, the stem cells are expected to be from human adult stem cells derived from bone marrow, umbilical cord blood, or adipose tissue all available from the American Type Culture Collection (ATCC).

[0197] In some embodiments, the stem cells are from the stem cell line Shef1, a human embryonic cell line derived by the University of Sheffield Centre for Stem Cell Biology.

[0198] In some embodiments of the SC-RPE differentiation method of the disclosure, starting stem cells are cultured in appropriate stem cell culture media in cultureware comprising one or more of the SC-RPE substrate of the disclosure.

[0199] The term “cultureware” in the sense of the disclosure indicates any device for aseptic cell culture that contains the cell culture medium, provides a surface that can be coated with the SC-RPE substrate on which cells can be grown on, and allows for gas exchange. Examples of suitable cultureware are tissue culture dishes, flasks, multiwell plates, or bioreactors. The surface on which the cells grow upon can be solid, porous, membranes, permeable films, or microcarriers. Most commonly the cultureware are plastic multiwell plates or tissue culture flasks and the support is the solid bottom surface of the plate or flask.

[0200] In embodiments of the SC-RPE differentiation method of the disclosure, stem cells can be thawed and plated on proper cultureware coated with an SC-RPE substrate of the disclosure (see e.g., the protocol of Example 7).Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0201] In embodiments of the SC-RPE differentiation method of the disclosure, stem cells can be propagated and / or cultured on SC-RPE differentiation substrate coated cultureware using standard methodologies that support the maintenance of a nondifferentiated state in serum-free conditions. In general, the medium should at a minimum be comprised of a physiological salt solution, essential and non-essential amino acids, essential vitamins, carbon energy source and growth factors that maintain pluripotency such as TGF^1 and bFGF. Examples of suitable medium are E8

[0043] or TeSR1-based media such as mTeSR1, mTeSR-Plus, and TeSR-AOF

[0044]

[0045] . Other media such as the KnockOut™ Serum replacement-DMEM or DMEM / F12 media supplemented with bFGF and TGF^1 can be used. The substrates must enable efficient stem cell attachment to the culture surface and can also play a role in maintenance of pluripotency. Examples of suitable substrates are rh-VTN-N, laminin 521, plasma vitronectin, recombinant derived full-length vitronectin, and soluble basement membrane extracts (e.g., Matrigel). Passage of the stem cells is done at subconfluence using mechanical, calcium chelator-based and / or protease-based cell dissociation reagents to detach the cells from the substrate and create single cells and / or cell clumps and cells are plated using a dilution factor of about 1:6 in stem cell medium with or without inhibitors of epithelial-to-mesenchymal transition (see e.g., culture medium of Example 1 and procedure of Example 5). While any serum-free media that supports stem cell growth and maintains pluripotency is expected to work, stem cells will have different growth rates and phenotype which can affect the substrate / RPE-DM preference and SC-RPE yield and may require adjustments to the differentiation method such as altering the plating density or the timing of the media switches.

[0202] In some embodiments of the SC-RPE differentiation method of the disclosure, cultureware can be precoated with one or more of the SC-RPE differentiation substrate of the disclosure (e.g., by the manufacturer). In some embodiments, the method can further include the optional additional step of substrate coating which can be performed in accordance with the manufacturer’s recommended concentrations and conditions for the selected substrates (see e.g., Example 6).

[0203] In some embodiments the method can further comprise screening a plurality of combinations of candidate SC-RPE differentiation medium and SC-RPE cell culture substrate by contacting the plurality of combinations with the starting stem cell, the contacting performed to select a combination of SC-RPE differentiation medium and SC-RPE cell culture substrate configured to provide differentiated SC-RPE cells from the starting stem cell, thus providingTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the selected SC-RPE differentiation medium and SC-RPE cell culture substrate.

[0204] In the representative embodiments of the Examples section and related differentiation screening protocols, cell cultureware was coated with 1 ^g / cm2of Cultrex Human Vitronectin, CTS-rhVTN-N, or Biolaminin 521 CTG. Adjusting the concentration of SC-RPE differentiation substrate is a variable in the method that can be altered and is expected to influence outcomes by affecting cell shape (flattening), rates of proliferation, and gene expression as will be understood by a skilled person. Such an adjustment of the concentration SC-RPE differentiation substrate can also affect the extent that cells remain attached to the dish early in the process when medium changes occur and later when the cells have differentiated. The optimal substrate concentration varies among cell lines and can be determined once the best candidate medium:substrate combination is determined as will be understood by a skilled person upon reading of the disclosure. The minimum possible substrate concentration is that which allows for attachment of at least 75% of viable stem cells. The optimal concentration can be further assessed by monitoring the expression of germ layer, neuroectoderm, ocular ectoderm and RPE marker gene expression at the appropriate times of differentiation.

[0205] In some embodiments, propagating and / or culturing the starting stem cells can be performed for a time period of 1 to 5 days or up to 6-7 days for slow growing cultures. The optimal timing will vary depending on the viability of the plated cells, the intrinsic growth rate of the specific cell line, stem cell medium, and substrate as will be understood by a skilled person. For the purpose of simple propagation of pluripotent stem cells the determination of the appropriate timing is based on the degree of cell confluence which should preferably be in the range of 60-90% (see e.g., the protocol of Example 8). For the rare, particularly slow growing culture, times of longer than 5 days can be necessary to achieve the desired degree of confluence and desired number of cells. Alternatively, to achieve the desired number of cells the seeding density can be increased to keep the passage times in line with the preferred timing, more individual cultures can be used, or additional passages can be performed to achieve the desired number of cells In preferred embodiments, passaging of stem cells, if any is performed, is performed before the stem cells reach confluence to minimize premature differentiation in particular with reference to stem cell lines that have a propensity to lose pluripotency during routine propagation, as would be understood by a skilled person

[0206] In some embodiments of the SC-RPE differentiation method of the disclosure, stemTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT cell culture can be performed under feeder-free conditions using Essential 8 Medium (Example 7) and truncated vitronectin (rh-VTN-N) coated cultureware (Example 6). Other standard feeder-free stem cell media such as mTESR and cultureware coatings such as laminin (LAM111, LAM211, LAM221, LAM332, and LAM411, LAM511, and LAM521), full-length vitronectin, or Matrigel can also be used. In an exemplary procedure stem cells are generally passaged as a mixture of single cells and cell clumps of roughly 2-20 cells per clump using EDTA based solutions (Versene), but enzymatic methods such as protease digestion (e.g., TrypLE™, Accutase™) or physical methods such as scraping and trituration can be used as well (Example 8). Transient addition of the ROCK inhibitors (e.g., Y-27632, thiazovivin, RevitaCellTMSupplement) to the medium can also be used at the time of passage to promote survival and limit the loss of pluripotency and is preferred as will be understood by a skilled person, but their use is not essential in particular, when the suspension contains minimal numbers of single cells (Example 8). Because the choice of stem cell culture medium and substrate coating effects growth rate and other properties it will likely effect the final differentiation results. Cell lines with slow to normal growth rates are expected to benefit from the use of Essential 8TMmedium, whereas faster growing cell lines are expected to benefit from the use of mTeSRTMbased media which typically result in a lower rate of growth.

[0207] In embodiments the of the SC-RPE differentiation method of the disclosure, the stem cells are contacted with an SC-RPE differentiation media in combination with an SC-RPE differentiation substrate (see e.g., the protocols of Example 9, Example 10, Example 19, Example 21, and Example 23).

[0208] In some embodiments, the SC-RPE differentiation media can be one of the variants of the KnockOutTMSerum Replacement-based RPE-DM1 formulated with different base media and amounts of KnockOutTMSerum Replacement: CTS-KO-DMEM / 5-30%KOSR, KO- DMEM / 5-30%KOSR, DMEM / 10-30%KOSR, CTS-KO-DMEM:F12 / 5-30%KOSR, KO- DMEM:F12 / 5-30%KOSR, DMEM:F12 / 5-30%KOSR, ^MEM / 5-30%KOSR, RPMI1640 / 5- 20%KOSR, and IMDM: / 5-20%KOSR where the KOSR is certified for clinical manufacturing (CTS grade), research grade, or ESCSR as provided in Table 2 of the present disclosure.

[0209] In some embodiments, the SC-RPE differentiation media can be one of the X-VIVO™ 10-based RPE-DM2 variants formulated with one of the B27-derived supplements at 0X to 2X strength: X-VIVO™ 10 / CTS-B27, X-VIVO™ 10 / XF-B27, X-VIVO™ 10 / B27-Plus, X- VIVO™ 10 / NC-SM1, X-VIVO™ 10 / cGMP-N21-MAX, and X-VIVO™ 10 / N21-MAX whereTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT X-VIVO™ 10 is any of the three different formulations of X-VIVO™ 10 or an SC-RPE differentiation media where the supplement is made from the individual components of B27 as provided as described in Table 5.

[0210] In some embodiments, the SC-RPE differentiation media RPE-DM1 and RPE-DM2 variants described in the present disclosure, can be supplemented with Activin A.

[0211] In some embodiments, wherein spontaneous differentiation of the starting stem cells is desired the SC-RPE differentiation media can be an RPE-DM1 variant formulated with different base media and amounts of KnockOutTMSerum Replacement selected from CTS-KO-DMEM / 5-30%KOSR, KO- DMEM / 5-30%KOSR, DMEM / 10-30%KOSR, CTS-KO-DMEM:F12 / 5-30%KOSR, KO- DMEM:F12 / 5-30%KOSR, DMEM:F12 / 5-30%KOSR, ^MEM / 5-30%KOSR, RPMI1640 / 5- 20%KOSR, and IMDM: / 5-20%KOSR where KOSR is certified for clinical manufacturing (CTS grade),research grade, or ESCSR as provided in Table 2 of the present disclosure. an RPE-DM2 variant formulated with one of the B27-derived supplements at 0X to 2X strength selected from X-VIVO™ 10 / CTS-B27, X-VIVO™ 10 / XF-B27, X-VIVO™ 10 / B27- Plus, X-VIVO™ 10 / NC-SM1, X-VIVO™ 10 / cGMP-N21-MAX, and X-VIVO™ 10 / N21- MAX where X-VIVO™ 10 is any of the three different formulations of X-VIVO™ 10 and where the supplement is made from the individual components of B27 as provided as described in Table 5.

[0212] In some embodiments, wherein semi-directed differentiation of the starting stem cells is desired the SC-RPE differentiation media can be an RPE-DM1 variant formulated with different base media and amounts of KnockOutTMSerum Replacement selected from CTS-KO-DMEM / 5-30%KOSR, KO- DMEM / 5-30%KOSR, DMEM / 10-30%KOSR, CTS-KO-DMEM:F12 / 5-30%KOSR, KO- DMEM:F12 / 5-30%KOSR, DMEM:F12 / 5-30%KOSR, ^MEM / 5-30%KOSR, RPMI1640 / 5- 20%KOSR, and IMDM: / 5-20%KOSR where KOSR is certified for clinical manufacturing (CTS grade),research grade, or ESCSR as provided in Table 2 of the present disclosure that is further supplemented with Activn A. an RPE-DM2 variant formulated with one of the B27-derived supplements at 0X to 2X strength selected from: X-VIVO™ 10 / CTS-B27, X-VIVO™ 10 / XF-B27, X-VIVO™ 10 / B27- Plus, X-VIVO™ 10 / NC-SM1, X-VIVO™ 10 / cGMP-N21-MAX, and X-VIVO™ 10 / N21- MAX where X-VIVO™ 10 is any of the three different formulations of X-VIVO™ 10 or anTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT RPE-DM2 variant where the supplement is made from the individual components of B27 as provided as described in Table 5 that is further supplemented with Activn A.

[0213] In particular, in some embodiments, RPE-DM1 formulated with CTS-KnockOutTMDMEM and CTS-20% KnockOutTMSerum Replacement (CTS-KO-DMEM / 20%CTS-KOSR) and RPE-DM2 formulated with cGMP grade TheraPeak X-VIVOTM10, without gentamicin or phenol red and 1X XenoFree-B27 (TP-X-VIVO™ 10 / XF-B27) are used for spontaneous differentiation and RPE-DM2 formulated with cGMP grade TheraPeak X-VIVOTM10 without gentamicin or phenol red and 1X XenoFree-B27 supplemented with Activin A (X-VIVO™ 10 / XF-B27+Activin A) are used as a simple semi-directed differentiation media. In some of those embodiments, these three media are then used in conjunction with three different substrates, preferably full-length human serum vitronectin (hsVTN), recombinant human truncated vitronectin (rhVTN-N), and recombinant human laminin 521 (LAM521) in all possible combinations: CTS-KO-DMEM / 20%CTS-KOSR:hsVTN, CTS-KO- DMEM / 20%CTS-KOSR:rh-VTN-N, CTS-KO-DMEM / 20%CTS-KOSR:LAM521, TP-X- VIVO™ 10 / XF-B27: hsVTN, TP-X-VIVO™ 10 / XF-B27:rhVTN-N, TP-X-VIVO™ 10 / XF- B27:LAM521, TP-X-VIVO™ 10 / XF-B27+ActivinA: hsVTN, TP-X-VIVO™ 10 / XF- B27+ActivinA:rhVTN-N, TP-X-VIVO™ 10 / XF-B27+ActivinA:LAM521. Each of these media were then contacted with the embryonic stem cell lines Shef1, H1, H9 and the induced pluripotent stem cell lines iPSC #032411, iPSC #110211, and iPSC #120111.

[0214] The use of other pluripotent stem cell lines and other combinations of medium and substrate using the media formulations such as any one of an RPE-DM1 variant formulated with different base media and amounts of KnockOutTMSerum Replacement selected from CTS-KO-DMEM / 5-30%KOSR, KO- DMEM / 5-30%KOSR, DMEM / 10-30%KOSR, CTS-KO-DMEM:F12 / 5-30%KOSR, KO- DMEM:F12 / 5-30%KOSR, DMEM:F12 / 5-30%KOSR, ^MEM / 5-30%KOSR, RPMI1640 / 5- 20%KOSR, and IMDM: / 5-20%KOSR where KOSR is commercially available CTS or research grade or made from the individual components of KOSR as described in Table 2 of the present disclosure, or an RPE-DM2 variant formulated with one of the B27-derived supplements at 0X to 2X strength selected from: X-VIVO™ 10 / CTS-B27, X-VIVO™ 10 / XF-B27, X-VIVO™ 10 / B27- Plus, X-VIVO™ 10 / NC-SM1, X-VIVO™ 10 / cGMP-N21-MAX, and X-VIVO™ 10 / N21- MAX where X-VIVO™ 10 is any one of the three different X-VIVO™ 10 formulations orTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT where the supplement is or made from the individual components of B27 as described in Table 5 of the present disclosure in combination with alternative substrates selected from Vitronectin, CTS-rhVTN-N, or Biolaminin 521 are possible as would be understood by a skilled person.

[0215] Additional combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate configured for the SC-RPE differentiation of a starting stem cells line can be identified by contacting the starting stem cell line with one or more candidate combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate, the contacting performed for a time and under condition allowing SC-RPE differentiation and selecting a candidate combination of the one or more candidate combinations which following the contacting results in SC-RPE differentiation of the starting stem cell line.

[0216] For “spontaneous differentiation” stem cells are generally contacted with differentiation medium 2-10 days (for example, 2-5 days, 3-4 days, 2-6 days, 3-6 days, 2-8 days, or 3-8 days) after plating in stem cell medium, at which time the majority of the replicate cultures are typically 60-90% confluent with some replicates possibly being of lesser confluence and / or some replicates being in the range of 90% to just confluent. Depending on the inherent cell line growth rate, choice of stem cell medium, substrate choice, plating density, and post-plating survival the timing of the first contact with differentiation medium can be extended to 1-7 days after plating in stem cell medium. The cells are contacted with differentiation medium for a total of about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days. Longer contact times in differentiation medium are possible, but minimal to no significant increase in the yield of SC-RPE is expected. At the end of the differentiation medium contact period the cultures are contacted without passage with RPE Maturation Medium.

[0217] For “semi-directed differentiation stem cells are generally contacted with differentiation medium 2-10 days (for example, 2-5 days, 3-4 days, 2-6 days, 3-6 days, 2-8 days, or 3-8 days) after plating in stem cell medium, at which time the majority of the replicate cultures are typically 60-90% confluent with some replicates possibly being of lesser confluence and / or some replicates being in the range of 90% to just confluent. Depending on the inherent cell line growth rate, choice of stem cell medium, substrate choice, of plating density, and post-plating survival the timing of first contact with differentiation medium can be extended to 1-7 days after plating. The cells are contacted with differentiation medium forTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT a total of 2-6 days (preferably 3 days) after which they are contacted with the corresponding differentiation medium supplemented with Activin A, for 4-28 days, more preferably 6-21 days, and most preferably 8 days. At the end of the Activin A treatment the cells are contacted with differentiation medium without Activin A for 0-60 days, more preferably 7-35 days, most preferably 14-21 days, after which time the differentiation medium without Activin A are contacted without passage with RPE Maturation Medium.

[0218] For both the spontaneous and semi-directed differentiation methods best results with respect to final yields the contacting with RPE Maturation Medium is carried out until the percentage of mature SC-RPE in the culture plateaus (generally 30-60 days). Cultures can be maintained in RPE Maturation Medium beyond this point; however, the yield can decrease due to difficulties in dissociating cultures for downstream processing.

[0219] The efficacy of the differentiation medium:substrate combinations for a given cell line can be assessed by determining the relative expression level of germ layer, neuroectoderm, retinal / RPE progenitors (e.g. by detection of germ layer markers (e.g. SOX17, GATA4, T, NODAL, HAND1, CDX2)) and retinal / RPE progenitors (e.g. (LHX2, OTX2, RAX, PAX6, MITF, SOX9, SOX10) in the days following transition to the differentiation medium and RPE marker genes in the days and weeks following the switch to RPE Maturation Medium (e.g. by detection of genes associated with pigmentation (e.g. PMEL, TYR, TYRP1, DCT), retinoid cycle (e.g. LRAT, RPE65, ALDH1A3, RBP1, RDH5, RDH10), RPE:RPE cell adhesion (e.g. TJP1, CDH19, CLDN19), ion channel (e.g. BEST1, TRPM1, TRPM3), or phagocytosis (e.g. MERTK, ITGAV)) as a function of time as would be understood by a skilled worker in the field. Alternatively, the differentiation process can be taken to its conclusion and RPE yield can be assessed based on the percentage of pigmented cells or the relative concentration of PEDF secreted into the medium can be monitored in real-time. Conditions that give rise to greater than 50% pigmented cells would be regarded as highly successful. Lower efficiencies of differentiation down to approximately 10% can also result in substantial quantities of final product, albeit with a corresponding reduction in final yield.

[0220] Accordingly in those embodiments, the differentiation protocols can be used with candidate combinations to act as screening kit. Using each candidate media:substrate combination on a cell of interest leads to selection of the combination that better provides for the desired results. As an outcome of this method six different stem cell lines have been identified, 3 human embryonic stem cell lines discussed in the Examples section (see e.g.,Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Examples 9, 10, and 12) and 3 additional human iPSC lines. H1 and H9 cells can be obtained from the WiCell Research Institute, Madison, WI, USA. The Shef1 cell line was isolated by the University of Sheffield Centre for Stem Cell Biology. The iPSC lines were produced by the inventors of this disclosure from primary fetal RPE obtained from three different donors. The fetal RPE were passaged until they became persistently mesenchymal, after which the cells were then reprogrammed using ReproRNA (STEMCELL Technologies) and iPSC were isolated based on the expression of Tra-1-60. The iPSC lines are available from the inventors of this disclosure.

[0221] In some embodiments, following identification and selection of candidate combination of choice, it is possible to modify the additional variables such as plating density, clump size, and the timing of contacting as well as media switches to provide a further customized protocol for the selected combination of SC-RPE differentiation medium and an SC-RPE cell culture substrate in connection with the specific starting stem cells according to embodiments of the methods herein described.

[0222] In some embodiments of the differentiation method, in particular those using rhVTN- N and laminin 521 as the substrate, the expanded stem cells are most preferably seeded at a plating density of approximately 20,000 cell / cm2of coated substrate surface area, preferably 15,000 to 25,000 cell / cm2, less preferably 10,000 cell / cm2to 40,00 cell / cm2, and least preferably, 5,000 to 60,000 cell / cm2. The preferred concentration of 20,000 cell / cm2is recommended starting density for any cell line, but the optimal density can vary depending on the particular line and choice of stem cell medium. In general, media:substrate:cell line combinations that result in larger cells and less compact colonies or faster growth rates, can benefit from lower seeding densities. Additionally, it is expected that the density will affect the preferred timing of the switch to RPE differentiation medium which is reflected by the extent of confluence. Determination of the optimal plating density and its associated timing of the contacting with differentiation medium for a cell line can be determined experimentally based on the expression of cell type markers at different time points during the differentiation process, and / or based on the accumulation of pigmentation after SC-RPE maturation. On this basis one can then select the condition that provides the highest yield of SC-RPE according to the experimental design, as would be understood by a skilled worker upon reading of this disclosure.

[0223] In some embodiments of the differentiation method, in particular those using full-Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT length vitronectin as the substrate, the expanded stem cells are most preferably seeded at a plating density of approximately 40,000 cell / cm2of coated substrate surface area, preferably 30,000 to 50,000 cell / cm2, less preferably 20,000 cell / cm2to 60,00 cell / cm2, and least preferably, 10,000 to 70,000 cell / cm2. In general, cells plated on full-length vitronectin are smaller and less flattened than when plated on rhVTN-N or laminin 521; hence the optimum density is often higher. The preferred concentration of 40,000 cell / cm2is a preferred starting density for any cell line, but the optimal density is expected to vary depending on the particular line and choice of stem cell medium. Consideration for the choice of cell density on VTN coated cultureware and methods of evaluation are as described in the present disclosure.

[0224] In all embodiments of the differentiation protocol, stem cells are plated as a mixture of single cells and clumps of varying cell numbers. While the relationship between clump sizes is not clearly understood, in many instances the pattern of pigmentation that is observed after differentiation suggest that SC-RPE develop at the borders of colonies or where different cell masses contact. Since smaller clumps sizes should result in an increase in the colony circumference:area ratio and a greater extent of colony:colony contacts it follows that they should result in a greater efficiency of RPE differentiation. It further follows that single cells are expected to give the best yields of RPE. However, stem cells can be sensitive single cell passage, and even in the presence of compounds such as ROCK inhibitors that act to promote stem cell survival after single cell passage there can be variable extents of survival. Therefore, as compromise a clump size distribution is in the range of 1-10 cells per clump and is a preferred starting point. Once a preferred differentiation medium:substrate combination is identified for specific cell line, further optimization of SC-RPE yield can be performed by varying the range of clump sizes. Further note, that clump size and plating density are expected to be inter-connected, hence a 2-dimensional matrix experimental design can assist in determining the optimal clump size and plating density combination by assessing the relative efficiency of RPE differentiation-based cell type specific marker gene expression as would be understood by skilled person upon reading this disclosure.

[0225] In some embodiments the stem cells are plated in stem cell medium containing the ROCK inhibitor, Y-27632, at a concentration of 10 ^M (most preferred), 7.5-12.5 ^M (preferred), 5-15 ^M less preferred, or 2.5-25 ^M. Thiazovivin, when used at a 5-fold lower concentration range or RevitaCell Supplement (ThermoFisher Scientific, catalog numbers A2644501 and A4238401) used in accordance with the manufactures instructions can beTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT substituted for Y-27632. In certain embodiments, other ROCK1 / ROCK2 inhibitors (e.g., GSK429286A, RKI-1477, Ripasudil, Netrasudil, Y-39983, Fasudil, Hydroxyfasudil, H-1152, Azaindole 1, WAY-624704) can be substituted for Y-27632. Guidance as to an appropriate concentration for an alternative ROCK inhibitor can be obtained by comparing the Ki values for ROCK1 and ROCK2 to that of Y-27632 and empirical testing using single cell stem cell passage to determine the minimal dosage required to enable cell survival without inhibiting growth rates.

[0226] In some embodiments, the contacting of the starting stem cell line with one or more candidate combinations of an SC-RPE differentiation medium and an SC-RPE cell culture substrate, is performed for a time and under conditions to provide differentiated SC-RPE cells.

[0227] In particular in some embodiments of the “spontaneous” differentiation protocol cells are generally contacted with differentiation medium 2-5 days after plating in stem cell medium, at which time the majority of the replicate cultures are typically 60-90% confluent with some replicates possibly being of lesser confluence and / or being in the range of 90% to just confluent, for a total of about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days, with the optimal time being determined by the one that gives rise to the highest level of expression of RPE marker genes or the highest percentage of cells expressing RPE marker genes in the shortest length of time. Alternatively, the optimal period of contact with differentiation medium can be determined based on the percentage of pigmented cells in the culture after transitioning the culture to RPE Maturation Medium and a sufficient period of culture (> 2 weeks) to allow for visualization of pigmentation. Contact times beyond 56 days are possible however, with little to no benefit in final yields of SC-RPE.

[0228] In all embodiments of the SC-RPE differentiation protocol cultures are fed with sufficient volumes of medium as necessary to maintain the composition of the medium. This is typically every 1-2 days with 2-3 mL of medium per 10 cm2of culture surface area and a roughly 90% media exchange depending on the protocol stage, the medium choice, and extent of medium acidification. Yellowish medium when using media containing phenol, a measured pH below 7.0. or cell death not associated with changes in type of media (e.g., stem cell medium to SC-RPE differentiation medium) is indicative of a need to feed the cells more frequently or increase the feeding volume. More frequent than necessary feeding schedules or greater than recommended volumes are possible, but those protocol changes are expected to affectTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT outcomes due to dilution of secreted factors that regulate the differentiation process as will be understood by a skilled person.

[0229] In some embodiments, during the period of stem cell growth post-plating in stem cell medium (e.g., Essential 8TMor mTeSRTM1) cells are fed every day until the cells reach 60-90% confluence with 2 mL / 10 cm2of stem cell medium. When media that employ the use of stabilized bFGF and with increased pH buffering (e.g., Essential 8TMFlex or mTeSRTMPlus) are employed, a feeding frequency of every other day can be used. Increasing the frequency and / or volume of feeding can also provide higher nutrient levels beneficial to the cells during this period of rapid growth as will be understood by a skilled person.

[0230] In “spontaneous” or “semi-directed” differentiation embodiments, which employ the use of RPE-DM1, and in particular those that use RPE-DM1 formulated with KnockOutTMDMEM, the preferred protocol is that cells be fed daily with 2 mL / 10 cm2at all times during its use. On occasion, a feeding frequency of every other day is possible especially in the later stages of differentiation. In this case it is preferred that the volume be increased by 50-100%.

[0231] In “spontaneous” or “semi-directed” differentiation embodiments which employ the use of RPE-DM2, the cultures are preferably fed daily with 2 mL / 10 cm2for the first feedings after the switch from stem cell medium to RPE-DM2. Thereafter, in embodiments that use RPE-DM2 formulated with X-VIVO™ 10 the cells can be fed every other day with 2 mL / 10 cm2of RPE-DM2. The use of RPE-DM2 formulated with other complete or base media can require more frequent feeding depending on its pH buffering capacity as will be understood by a skilled person.

[0232] In embodiments of the disclosure, the media, substrate, cultureware and / or other reagents used to perform the SC-RPE differentiation method of the disclosure can be comprised within corresponding SC-RPE differentiation system of the disclosure.

[0233] The SC-RPE differentiation system of the disclosure comprises at least one serum-free SC-RPE differentiation medium and at least one SC-RPE cell culture support coated or uncoated with a substrate of the present disclosure In the SC-RPE differentiation system of the disclosure, the at least one cell culture medium and the at least one cell culture substrate in combination with an appropriate cultureware or device are included for combined use to provide a cell culture system configured to allow introduction and / or maintenance of stem cells culture within the system to obtain a differentiated SC-RPE cells according to methods of theTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT disclosure.

[0234] In some embodiments, SC-RPE differentiation methods and system of the disclosure allow efficient SC differentiation which can be readily scaled up for large-scale cGMP manufacturing when cGMP compliant reagents are employed.

[0235] In some embodiments, SC-RPE differentiation methods and system of the disclosure allow efficient SC differentiation in a time period of ranging from 2 to 8 weeks with the preferred methods requiring a total of 4-5 weeks.

[0236] In some embodiments, SC-RPE differentiation methods and system of the disclosure provide a toolbox approach that allows differentiation of different stem cell lines.

[0237] In some embodiments, SC-RPE differentiation methods and system of the disclosure provide simplified approach minimizing requirement of multiple manipulations or steps.

[0238] In embodiments of the disclosure SC-RPE differentiated cells can be matured using an RPE Maturation Medium of the disclosure (herein RPE-MM), which has been unexpectedly found to promote and / or enhance yield and maturation of differentiated SC-RPE cells.

[0239] In some embodiments, the ability of the RPE Maturation Medium of the disclosure to promote and / or enhance yield and maturation of differentiated SC-RPE cell can occur even when the differentiation medium gives rise to minimal amounts of discernable RPE.

[0240] In some embodiments, in addition to supporting the establishment of a mature RPE phenotype, the RPE Maturation Medium of the disclosure appears to also play a role in earlier stages of SC-RPE development with at least some stem cell line:differentiation medium:substrate combinations, based on the observation that passaging during the time of contacting with RPE Maturation Medium can result in significant increases in SC-RPE differentiation efficiencies when cultures are passaged at later times (about 2-weeks) during the period of maturation compared to passaging after just a few days (3-days, Example 46 and Figure 27).

[0241] In some embodiments, the SC-RPE Maturation Medium allows for improved isolation of SC-RPE cells based on the presence of pigmentation, by enabling or enhancing pigment accumulation and melanosome maturation.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0242] The RPE Maturation Medium of the disclosure is a cell culture maturation medium comprising a buffered balanced salt solution, essential amino acids and nonessential amino acids, vitamins, hormones, essential components for lipid metabolism, carbon energy sources, serum albumin, insulin, transferrin, selenite, and antioxidants in an effective amount to promote maturation of differentiated SC-RPE cells.

[0243] In some embodiments, the RPE Maturation Medium of this disclosure uses a common serum-free medium formulation comprised of a base medium supplemented with the components of B27 as will be understood by a skilled person upon reading of the present disclosure.

[0244] In some embodiments of the disclosure RPE-MM can further be supplemented by additional nonessential amino acids (NEAA), insulin, transferrin, selenite, putrescine, progesterone, and hydrocortisone to obtain a RPE Maturation Medium of the disclosure according to additional embodiments of the disclosure.

[0245] In some embodiments of the disclosure insulin, transferrin, selenite, putrescine, and progesterone supplementation is obtained using a N1-based supplement mixture.

[0246] In embodiments of the RPE-MM of the disclosure, compositions of the RPE-MM can be made by modifying the amount of related base medium, B27 components and / or by adding further components in accordance with the indications of the present disclosure as will be understood by a skilled person.

[0247] The effects of the changes in composition on the effect of the resulting RPE-MM of the disclosure can be monitored through detection of pigmentation and / or expression of marker genes associated with mature RPE phenotype (see Example 43 and Figures 24A and 24B).

[0248] In outcome of the detection of pigmentation it has been surprisingly found that in embodiments of RPE-MM herein described, neither taurine nor additional supplementation with non-essential amino acids (NEAA), N1-based supplement mixture, or hydrocortisone, by themselves, is essential, to obtain mature RPE with typical RPE morphology. However, when maturation competent SC-RPE are plated at suboptimal density, such as 40,000 cell / cm2, omission of any one of them individually results in an a 50-60% reduction in the percentage of pigmented SC-RPE and cells with a mesenchymal phenotype. (See Example 18 and Figure 5).Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0249] In particular, it will be understood by a skilled person upon reading of the present disclosure that, when culturing minimally expanded SC-RPE under optimal conditions, supplementation with additional hydrocortisone is not necessary, with no overt differences in SC-RPE quality being observed because of its omission. However, its addition can have some benefits for RPE expansion due to its ability to minimize wound response. Using serum-based RPE medium it has been found that it can be eliminated in its entirety with no obvious overt effects on pigmentation or cell morphology.

[0250] It has also surprisingly been found that in preferred embodiments the RPE Maturation Medium, that removal of retinol and / or retinol acetate (collectively known as vitamin A) through the of use of B27 supplements without vitamin A results in significant changes in the expression of many RPE genes; yet the differentiation efficiencies are unchanged and the obtained SC-RPE have a normal pigment epithelial morphology and retain at least some RPE function. It has further been found that by varying the concentration of the vitamin A in RPE- MM by using B27 with and without vitamin A the expression the vitamin A-responsive genes can be altered in a dose-dependent and reversible manner.

[0251] In particular, the inventors have unexpectedly found that in embodiments of RPE-MM of the disclosure the presence / absence and concentrations of Vitamin A are a result effective variable. Presence of Vitamin A is not essential to obtain high yields of differentiated, readily identifiable, pigmented SC-RPE that express RPE-signature genes or for the culture RPE that retain at least some RPE function, as based on the formation tight junctions. (See Example 42, Figures 17 and 18). However, in the absence of vitamin A RPE express highly reduced levels of a several key RPE genes relating to regulation of RPE gene expression and function (see Example 42, Figure 19 and 20). In addition, in the absence of vitamin A there is an elevated expression of genes associated with wound response. The expression levels of the genes can be regulated in a dose-dependent manner and the effect on gene expression is reversable. Accordingly, embodiments of the RPE-MM comprising vitamin A are preferred.

[0252] In more preferred embodiments, the RPE-MM of the disclosure can comprise an amount of vitamin A selected in view of the features of the mature SC-RPE to be obtained as will be understood by a skilled person upon reading of the present disclosure.

[0253] Accordingly, in some embodiments of the RPE-MM of the disclosure the B27 supplement is a commercially available B27 supplement without vitamin A or is made fromTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the individual components of B27 or NS21 without retinol or retinol acetate, to which retinol and / or retinol acetate can be added to achieve a desired concentration of vitamin A (e.g. all- trans-retinol, catalog number 95144; retinol acetate, also referred to as retinyl acetate, catalog number R7882 Sigma-Aldrich) to achieve the desired RPE phenotype as can be assessed using gene expression profiling or in the alternative, quantitative PCR to determine the expression level of LRAT, EYA2, RAX, and SPP.

[0254] In some embodiments of the RPE-MM of the disclosure when vitamin A is provided from B27, at the highest concentration of vitamin A provided by B27 when used at its preferred 1X concentration, the expression levels for some of the RPE genes has been observed not to have reached their maximum based on the vitamin A dose-response analysis. This data supports the conclusion, that the quality of SC-RPE obtained using the serum-free RPE Maturation Medium can be advantageously improved by providing more vitamin A. Accordingly, in some preferred embodiments of the RPE-MM of the disclosure comprise B27 supplement, the concentration of vitamin A can be further increased by increasing the concentration of the B27 supplement in RPE Minimal Medium, using the B27-derived NS21 supplement which has twice the concentration of vitamin A, or preferably by further supplementation of RPE Maturation medium with retinol and / or retinyl acetate. The effect of altering the concentration of vitamin A and the selection of a desired concentration vitamin A can be determined using gene expression profiling or in the alternative, quantitative PCR to determine the expression level of LRAT, EYA2, RAX, and SPP1 as will be understood by a skilled person. Exemplary techniques for detection of gene expression comprise quantitative Real-Time PCR (qRT-PCR) RNA Sequencing (RNA-Seq), Microarray analysis involves hybridizing cDNA to a chip containing probes for thousands of genes in situ hybridization, immunohistochemistry and additional techniques identifiable by a skilled person.

[0255] In some embodiments, the desired phenotype is obtained at a concentration of vitamin A lower than what is provided by B27 supplement with vitamin A. In those embodiments, the concentration of vitamin A in RPE-MM can be reduced by first diluting B27 supplement with vitamin using a B27 supplement without vitamin A (NeuroCult™ SM1 Without Vitamin A (05731 STEMCELL Technologies) B-27 Supplement Minus Vitamin A (A1370701, ThermoFisher), CTS B-27 Supplement Xeno-Free Minus Vitamin A (A5047601, ThermoFisher), or N21-MAX Vitamin A Free Media Supplement (AR012 R & D Systems) prior to preparing the base media to obtain the desired concentration. In the alternative, RPE-Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT MM with and without vitamin A can be prepared with the respective B27 supplements and the media can be mixed to achieve the desired concentration.

[0256] In some embodiments of the RPE-MM of the disclosure, where it is desired to further increase B27 concentration, but not alter the vitamin A concentration any of the B27 supplements without vitamin A can be added and then adjusting the total concentration of Vitamin A to the desired concentration, preferably by adding a supplement from a same supplier (e.g. SM1 Neurocult Minus Vitamin A added to SM1 Neurocult (without vitamin A).

[0257] All-trans-retinol is processed by the RPE cells during the visual (retinoid) cycle to regenerate the photosensitive visual pigment 11-cis-retinal. In addition to being essential to vision, dysregulation of the retinoid cycle has been linked to a number of retinal and / or RPE diseases and disorders.

[0258] Beyond the effects of Vitamin A addition with regard to obtaining high quality SC- RPE the ability to regulate the RPE phenotype by manipulating the related concentration, RPE obtained with RPE-MM comprising vitamin A concentration have specific useful applications, such as providing disease models for both basic research or drug screening and drug development.

[0259] In some embodiments, the RPE maturation medium of the disclosure can comprise ^- MEM as base medium supplemented with additional nonessential amino acids (NEAA), N1 or N2 supplement, taurine, hydrocortisone, and a B27-based supplement.

[0260] The term “^-MEM” or “alpha-MEM” as used herein indicates Eagle’s Minimum Essential Medium, Alpha Modification suitable for cell culture with L-glutamine and sodium bicarbonate, and without ribonucleosides and deoxyribonucleosides.

[0261] The Minimum Essential Medium (MEM), developed by Harry Eagle, is one of the most widely used of all synthetic cell culture media. Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed they had specific nutritional requirements that could not be met by Eagle’s Basal Medium (BME). Subsequent studies using these and other cells in culture indicated additions to BME could be made to aid growth of a wider variety of fastidious cells. MEM, which incorporates these modifications, includes higher concentrations of amino acids so the medium more closely approximates the composition of cultured mammalian cells. Optional supplementation of non-essential aminoTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT acids to the formulations that incorporate either Hanks’ or Earle’s salts has broadened the usefulness of this medium.

[0262] The Alpha modification of MEM with Earle’s Balanced Salts, commonly referred to as alpha-MEM, contains non-essential amino acids, sodium pyruvate, and additional vitamins. These modifications were first described by Stanners for use in growing hybrid mouse and hamster cells. The preferred formulation of this disclosure is without the deoxyribonucleosides and ribonucleosides originally used in Stanners’ studies

[0046] ,

[0047] .

[0263] The Minimum Essential Medium Eagle (MEM) Alpha Modifications without nucleosides is commercially available medium in powder and liquid form from Corning Life Sciences (Product Numbers: 50-012-PC, powder and 15-012, liquid). The powdered form is provided without sodium bicarbonate, which must be added separately. The liquid form is provided without glutamine, which must be added separately. The formulation, concentration of the components and specification of the powder form (50-012-PC) is provided in Table 6 below.

[0264] Table 6. ^MEM Formulation and Specification^^ ^^ ^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ .^^^^^5^^^6^^^^^^^^^^^^^7*%8%^^8^.^ ^^ ^^ ^^ ^^ ^^ ^592^ ^:^ ^^^ ^^ ^^ ^^ ^^^5^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ / ^4^^ (&##^ *&^^^ ^^()*^^ ^^ ^^ ^^ ^^ / ^^40)^^^^^^^ ^^##^ &^^#^ ^(^&&^ ^^ ^^ ^^ ^^ 74^^ ^##^ ^^^(^ *)(*^ ^^ ^^ ^^ ^^ / ^^^+0^5^^0^ ^^#^ ^)&^#^ ^#^*^ ^^ ^^ ^^ ^^ 4^4^^^ ^##^ ^^^^#^ ^&#^^ ^^ ^^ ^^ ^^ ^.,0^^ ^^^^^ ^^#^^^ &^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^"^ ^*^ &^^^^ ^&^^ ^^ ^^ ^^ ^^ ^^^ .^^^^"5^4^^ ^^(^^^ ^^#^^^ (##^ ^^ ^^ ^^ ^^ ^^^^^^ ^.^^"5^^0^ *#^ ^*#^^^ )))^ ^^ ^^ ^^ ^^ ^^^^^^ '^^^^^^^^ )#^ ^))^^^ ^^*^ ^^ ^^ ^^ ^^ ^^4%^'"^^"5^4^5^^0^ ^##^ ^^*^(^ *(^^ ^^ ^^ ^^ ^^ ^^4%^'^^"5^^4^^ )^^^^ )^)^^^ ^^^(^ ^^ ^^ ^^ ^^ ^^$^^'^^^^^^^^^^ ^*^ ^^^^^^ *^#^ ^^ ^^ ^^ ^^ ^^$^^'^^^^"^ ^^^^ ^^(^^^ ^^^&^ ^^ ^^ ^^ ^^ $^%^^^"^ *#^ ^*^^^ (((^ ^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 6. ^MEM Formulation and Specification ^^ ^^ ^^ ^^^^ ^^ ^^ ^^^^ ^^ ^^^^ ^^ ^^ .^^^^^5^^^6^^^^^^^^^^^^^7*%8%^^8^.^ ^^ ^^^^ ^^ ^^ ^^^^^'^^^^"5^4^5^^0^ ^^^^^ ^#^^(^ ^##^ ^^ ^^ ^^ ^^ ^^^^^^"^^^^"^ *^^*^ ^)^^^^ ^##^ ^^ ^^ ^^ ^^ ^^^"^^^^"^ *^^*^ ^)^^^^ ^##^ ^^ ^^ ^^ ^^ ^^^%^^^"5^4^^ ^^^*^ ^&^^^^ )^^^ ^^ ^^ ^^ ^^ ^^^"'^^^^^^"^ ^*^ ^^^^^^ ^#^^ ^^ ^^ ^^ ^^ ^^+^"^%^^^^^^^"^ )^^*^ ^(*^^^ ^^^^ ^^ ^^ ^^ ^^ ^^+ ^^^^"^ ^#^ ^^*^^^ )^^^ ^^ ^^ ^^ ^^ ^^," ^^"^ ^*^ ^#*^^^ ^)&^ ^^ ^^ ^^ ^^ ^^^^ "^^^^"^ ^^^(^ ^^^^^^ ^##^ ^^ ^^ ^^ ^^ ^^^ %^'^^^^^^ ^#^ ^#^^^^ ^^^#^ ^^ ^^ ^^ ^^ ^^^% ^^^^"5^ / ^5^^^0^ *^^^^ ^()^^^ ^^^^ ^^ ^^ ^^ ^^ ^^-^^^^"^ ^(^&^ ^^^^^^ )^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^ ^^^^^^^^^ *#^ ^^(^^^ ^&^^ ^^ ^^ ^^ ^^ 6^^'^^^ #^^^ ^^^^)^ #^^#^^ ^^ ^^ ^^ ^^ 4^^^^^"^^^^^ ^^"^ ^^ ^)^^(^ ^^^(^ ^^ ^^ ^^ ^^ 9^^^^^^^^^^ ^^ ^^^^^^ ^^^^^ ^^ ^^ ^^ ^^ ^^^^^^^'^^^ ^^ ^&#^^^ ^^^^#^ ^^ ^^ ^^ ^^ / ^^^'^^^^^^"^ ^^ ^^^^^^ &^^^^ ^^ ^^ ^^ ^^ A^4^^^^^^^^^^'^'^"^^'"^ ^^ ^^(^*^ ^^^#^ ^^ ^^ ^^ ^^ +% ^^^^^^"5^4^^ ^^ ^#*^(^ ^^&(^ ^^ ^^ ^^ ^^ ^^^^!^^>^^^ #^^^ )^(^^^ #^^((^ ^^ ^^ ^^ ^^ ^^^^^^^"5^4^^ ^^ ))^^)^ ^^^(^ ^^ ^^ ^^ ^^ -^'^^^^^6^^^ ^^)(^ ^)**^^^ ^^##^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^4^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ A^$^^^^^"^ ^###^ ^&#^^^ ***^^ ^^ ^^ ^^ ^^ ,^^^^^^^% ^>^'"^ ^^#^ ^^#^#^ ^###^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ #^^^ ^#(^)^ #^^(^^ ^^ ^^ ^^ ^^ +^"^^^^^"^5 / ^^ ^#^ )^(^^^ ^(^(^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ,^"^^!^^^'^^^^ ^^ ^^ ^^ ^^^C^'^^^'^ / ^^40)^ ^^ )^*^2^#^*^ ^^ ^^ ^^ ^^ ^^^C^'^^ / ^^40)^G^^'^^^^^" ^^^40^^ ^^#^2^#^*^ ^^ ^^ ^^ ^^ 0^^^^^^^'%^C^'^^ / ^^40)^ ^^ ^^#^2^)#^^0^^H<.^^^0^ ^^ ^^ ^^ ^^ B^^^'^^^^^ ^^ I^#^^*^BEH^^^G^9=^ ^^ ^^ ^^ ^^ ^%^^^^^^^^^ ^^ / ".^'^>"^ ^^ ^^ ^^ ^^ 4"^^^$ ^C'^^^-B^0@^+^7@^ / $^^#&^^ +^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0265] In some embodiments the alpha-MEM formulation Table 6 can comprise one or more of the nucleosides (adenosine, cytidine, guanosine, adenosine, thymidine, uridine, 2’- deoxyadenosine, 2’-deoxycytidine, 2’-deoxyadenosine, each at a concentration of about 40 ^M.

[0266] Alpha-MEM of Table 6 without nucleosides is also available in powder form from ThermoFisher (catalog number: 12000) and Sigma (catalog number: M0894) and in liquid form from ThermoFisher (catalog number: 12561), Sigma (catalog number: M0200) and STEMCELL Technologies (catalog number: 36453). However, all of these products have slight differences in formulation compared to the Corning ^-MEM formulation. ThermoFisher powdered ^-MEM has 23% lower amounts of L-cystine, 17% higher amounts of tyrosine, and it uses pyridoxal•HCl as opposed to pyridoxine•HCl as its source of vitamin B6. ThermoFisher liquid medium has the additional difference that the amount of L-arginine is reduced by 17%. By comparison powdered and liquid Sigma ^-MEM has 25% lower amounts of CaCl2, 12% lower amounts of ascorbic acid, 2-fold greater amounts of panthothenate, 25% higher amounts of pyruvate, and it uses pyridoxal•HCl as opposed to pyridoxine•HCl as its source of vitamin B6. STEMCELL Technologies medium has 25% lower amounts of CaCl2, 23% lower amounts of L-cystine, 15% higher amounts of NaH2PO4, and it uses pyridoxal•HCl as opposed to pyridoxine•HCl as its source of vitamin B6. For example, the ThermoFisher powdered and liquid formulations have been found to support RPE maturation with minimal demonstrable differences based on the observance of pigmentation and morphology compared to the Corning ^-MEM formulation. While it is expected that the ^-MEM formulations from other suppliers will support RPE maturation, when using alternative sources of ^-MEM, the concentration of the components with reduced amounts compared to Corning® ^-MEM can be increased by supplementation. Formulations of ^-MEM with nucleosides are advantageous in cGMP application because they allow use of commercially available phenol-free media to provide the components of the RPE-MM of the disclosure.

[0267] In embodiments of the disclosure, other base cell culture media of formulation similar to alpha-MEM are expected to be usable in providing a base medium for RPE-MM of the disclosure, but in some cases commonly used media such as DMEM or DMEM:F12 (50:50) have been shown not to be able to substitute for alpha-MEM. (Example 18 Figure 6).

[0268] In some embodiments, the alpha-MEM of Table 6 is supplemented with a B27Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT supplement made from the individual components of B27 or NS21 without retinol or retinol acetate.

[0269] In some embodiments, the alpha-MEM of Table 6 is supplemented by NeuroCult™ SM1 Without Vitamin A (05731 STEMCELL Technologies) B-27 Supplement Minus Vitamin A is (A1370701, ThermoFisher), CTS B-27 Supplement XenoFreeMinus Vitamin A (A5047601, ThermoFisher), or N21-MAX Vitamin A Free Media Supplement (AR012 R & D Systems).

[0270] In some embodiments, the alpha-MEM of Table 6 is supplemented with retinol and / or retinol acetate (e.g. all-trans-retinol, catalog number 95144; retinol acetate, also referred to as retinyl acetate, catalog number R7882 Sigma-Aldrich).

[0271] In some embodiments, the alpha-MEM of Table 6 is supplemented with B27 supplement made from the individual components of B27 or NS21 and retinol and / or retinol acetate (e.g. all-trans-retinol, catalog number 95144; retinol acetate, also referred to as retinyl acetate, catalog number R7882 Sigma-Aldrich).are added to achieve a desired vitamin A concentration.

[0272] In some embodiments, the alpha-MEM of Table 6 is supplemented by NeuroCult™ SM1 Without Vitamin A, B-27 Supplement Minus Vitamin A, CTS B-27 Supplement XenoFreeMinus Vitamin A, or N21-MAX Vitamin A Free Media Supplement and retinol and / retinol acetate are added to achieve a desired concentration of vitamin A.

[0273] In some embodiments, the alpha-MEM of Table 6 is supplemented with any the B27 supplements with vitamin A of the disclosure is further supplemented with by NeuroCult™ SM1 without Vitamin A (05731 STEMCELL Technologies) B-27 Supplement Minus Vitamin A is (A1370701, ThermoFisher), CTS B-27 Supplement XenoFreeMinus Vitamin A (A5047601, ThermoFisher), or N21-MAX Vitamin A Free Media Supplement (AR012 R & D Systems).

[0274] In some embodiments, the alpha-MEM of Table 6 is supplemented with any the B27 supplements with vitamin A of the disclosure and retinol and / retinol acetate are added to achieve a desired concentration of vitamin A.

[0275] In some embodiments of the RPE-MM of the disclosure, ^-MEM is supplemented withTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT additional nonessential amino acids (NEAA).100X MEM Non-essential Amino Acids solution (10 mM alanine, 10 mM asparagine, 10 mM aspartic acid, 10 mM glutamic acid, 10 mM glycine, 10 mM proline, and 10 mM serine) is available from ThermoFisher Scientific (catalog numbers 11140050 or 11140076). In some embodiments of RPE-MM, the NEAA solution is added at a final strength of 1X (most preferred), 0.75 to 1.25X (preferred), 0.5 to 1.5X (less preferred), 0.25 to 2X (least preferred). While supplementation with additional NEAA has been associated with improved RPE phenotype on average (see e.g., Example 18 and Figure 5), it is not essential for RPE cells to pigment and obtain a normal RPE morphology. The addition of supplemental NEAAs is considered to be optional; although, some reduction in RPE quality is expected. Strengths higher than 2X is expected to be possible, but no further improvement in RPE phenotype and function is expected.

[0276] In some embodiments of the RPE-MM of the disclosure, ^-MEM is supplemented with B27- or NS21-based supplements which were derived from the defined serum-free medium, B18, of Brewer et al., 1989

[0040] which was developed as a supplement to facilitate neuronal cell culture. B27 and NS21 provide the additional components of serum albumin, insulin, transferrin, selenite, putrescine, progesterone, galactose, catalase, superoxide dismutase, D,L- alpha-tocopherol, D,L-alpha-tocopherol acetate, reduced glutathione, ethanolamine, linoleic acid, linolenic acid, carnitine, triiodo-I-thyronine (T3) and vitamin A (retinol acetate (B27 and NS21) and retinol (NS21))

[0039] ,

[0041] . B27 also includes biotin and NS21 also includes lipoic acid, both of which are also present in ^-MEM. Typically, these supplements are prepared as concentrated stocks which are then added to the base medium. The formulations of B27 and NS21 at 1X strength based on an online published protocol

[0042] and the original publications are provided in Table 5. 50X stock solutions of the supplements can be prepared using the reagent lists and instructions of the publications. Alternatively, suitable B27-based 50X liquid stock solutions are commercially available from ThermoFisher Gibco (B-27™ Supplement (50X), serum free [catalog numbers 17504001 and 17504044]; B-27™ Plus Supplement (50X), [catalog number A3582801]; B-27™ Supplement, XenoFree, [catalog number A1486701]; CTS™ B-27™ Supplement XenoFree (50X) [catalog number A5047501]); and STEMCELL Technologies (NeuroCultTMSM1, catalog number 05711). NS21-based medium supplements can be obtained from Millipore Sigma-Aldrich (N21 Medium Supplement (50X), catalog number SCM081) and R & D Systems (N21-MAX Media Supplement (50X), catalog number AR008). These commercial stocks have the same composition of compounds listed in TableTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 5, but the source (e.g., species, recombinant) and concentration of some of the components can vary from the original published versions and between different commercial products from that listed in Table 5. B-27™ Supplement (50X), serum free; B-27™ Supplement, XenoFree; CTS™ B-27™ Supplement XenoFree (50X); NeuroCultTM SM1; and N21-MAX Media Supplement (50X) have all found to be interchangeable when formulating RPE-MM, albeit with some differences in differentiation media:substrate preferences and yields. Based on this observation it is expected that RPE-MM formulated based on the individual B27 / N21 components and concentrations in Table 5 will yield a functional RPE-MM.

[0277] In some embodiments of RPE-MM, 50X B27-based supplements are most preferably used at a final concentration of 1X, preferably at 0.75 to 1.25X, less preferably at 0.5 to 1.5X, and least preferably at 0.25 to 2X.

[0278] In some embodiments of the RPE-MM of the disclosure, ^-MEM is supplemented with N1 supplement

[0048] . The N1 supplement is a 100X solution comprised of human insulin (0.5 mg / mL), partially iron saturated human transferrin (0.5 mg / mL), sodium selenite (0.5 ^g / mL), putrescine (1.6 mg / mL), and progesterone (0.73 ^g / mL) in Earle’s Balanced Salt Solution. It is typically used at 1X strength. Insulin, transferrin, and selenite are referred to as IST and are required for serum-free cell culture. Insulin, transferrin, selenite, and putrescine are also added as individual media additives in the serum supplemented RPE medium of Hu and Bok [49,]. All five components of N1 are also present in B27 supplement. The observation that the omission of addition of N1 supplements to RPE-MM results in diminished RPE pigmentation (see Example 18 and Figure 5) suggests that the concentration of at least one of these components in either supplement is limiting for RPE maturation. In context of the changes in RPE energy metabolism with maturation, insulin is expected to be possibly critical. N1 Medium Supplement (100×) can be obtained from Millipore Sigma-Aldrich (Catalog N6530) or alternatively it can be made from the individual components which are available from numerous suppliers.

[0279] In some embodiments, the RPE-MM of the disclosure uses N-2 supplement as an alternative to N1 supplement. N2 differs from N1 with respect to the concentration of some of the components and the use of holo-transferrin instead of partially iron saturated transferrin. It is also available in grades suitable for clinical manufacturing. N-2 supplement is a 100X solution comprised of human insulin (0.5 mg / mL), holo-human transferrin (10 mg / mL),Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT sodium selenite (0.52 ^g / mL), putrescine (1.611 mg / mL), and progesterone (0.63 ^g / mL) in water available from Thermofisher Scientific (N-2 Supplement (100X) Catalog 17502001 and 17502048, CTS™ N-2 Supplement. (100X) Catalog A1370701 or A13707-01). In some embodiments, N1 and N2 have been found to be interchangeable with respect to supporting RPE maturation with minimal to no overt differences as evident based on pigmentation and morphology (see Example 42 and Figure 17).

[0280] In some embodiments, the RPE-MM of the disclosure uses High Insulin N-2 (HI-N-2) supplement as an alternative to N1 supplement. High Insulin N-2 supplement has been shown to provide optimal conditions for neural stem cell expansion

[0051] and differs from normal N-2 in that the concentration of insulin in the 100X supplement stock solution is 2.5 mg / L compared to 0.5 mg / L. High Insulin N-2 is available from R & D Systems (N-2 Plus Media Supplement (100X, N-2+), catalog number AR003 with bovine insulin; N-2-MAX, catalog number AR009, with recombinant human insulin and human transferrin; and GMP N-2 MAX Media Supplement (100X), Animal-free, catalog number AR016, with recombinant human insulin and recombinant human transferrin.

[0281] 100X N1 supplement and its related 100X N-2, and 100X HI-N-2 supplements are most preferably used at final concentration of 1X, preferably 0.75 to 1.25X, less preferably 0.5 to 1.5X, and least preferably 0.25 to 2.0X. When RPE-MM is formulated using greater than 1X B27-based supplements, which contain all of the components of N1, N-2, or HI-N-2 at a similar concentration, the amount of the N1-related supplement can be reduced accordingly.

[0282] N1 type supplementation can also be achieved by custom formulation using the individual components and concentration ranges of the preceding described commercial preparations of N1, N-2, and HI-N-2 as would be understood by skilled person. The individual components or mixtures of subsets of the components (e.g., IST) suitable for cell culture are available from numerous vendors. Custom formulated N1-type supplements are expected to work in the same concentration range as the commercially available supplements.

[0283] In the RPE-MM of the disclosure ^-MEM can be supplemented with taurine . Taurine is not a component of alpha-MEM. Taurine is the most abundant amino acid in the retina and the retina has the highest concentration of taurine in the body. Much of that taurine is delivered to the retina from the vasculature via active transport by the RPE. Furthermore, in highly aerobic cells such as the RPE, taurine is concentrated in mitochondria where it reducesTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT superoxide generation and in the RPE and photoreceptor it also serves to sequester retinaldehydes, which are toxic intermediates of the visual cycle. Given the apparent importance of taurine in retina / RPE physiology, taurine may play a role in normal RPE maturation and function in addition to providing further protection against oxidative stress.

[0284] RPE-MM medium is most preferably supplemented with taurine to final concentration of 2.0 mM (0.25 g / L), preferably 1.5 to 2.5 mM, less preferably, 0.5 to 3 mM, least preferably 0 to 4 mM. Taurine inclusion in the medium is not essential for the production of pigmented RPE, however its addition increases the percentage of mature pigmented RPE in a given culture when cells are plated at lower than optimal seeding density (75,000-150,000 cell / cm2) (see Example 18 and Figure 5). Concentrations greater than 4 mM are expected to be possible, but no further improvement in RPE quality is expected. Taurine suitable for cell culture can be obtained from Millipore Sigma-Aldrich (catalog number T8691). Other high quality reagent grades, such as high purity molecular biology grade, which are available from numerous vendors would be expected to substitute for cell culture grade (USP) when the intended use of the SC-RPE is for non-clinical purposes.

[0285] In the RPE-MM of the disclosure ^-MEM is supplemented with hydrocortisone. Hydrocortisone an anti-inflammatory gluco- and mineral-corticoid that can affect cellular differentiation and has been shown to promote the upregulation of genes involved epithelial polarization, establishment of lateral tight junctions, and downregulates genes involved in epithelial-to-mesenchymal transition (EMT).

[0286] When culturing minimally expanded SC-RPE under optimal conditions, supplementation of RPE-Minimal Medium with additional hydrocortisone is not necessary, with no overt differences in SC-RPE quality being observed because of its omission. However, its addition can have some benefits for RPE expansion due to its ability to minimize wound response. Using serum-based RPE medium it has been found that hydrocortisone can be eliminated in its entirety with no obvious overt affects.

[0287] On that basis, hydrocortisone can, in some embodiments, be a desirable RPE medium additive, even if a non-essential one, for high quality RPE (see Example 18 and Figure 5). Small molecule inhibitors of EMT such as ROCK inhibitors or TGFBR kinase inhibitors have a similar, but more potent effect, although they do so through a different mechanism. Note that B27 supplement contains corticosterone which is the prodrug form of hydrocortisone,Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT suggesting that the concentration of corticosterone in B27 is insufficient on its own when SC- RPE are plated and suboptimal seeding densities(generally <75,000 cell / cm2).

[0288] RPE-MM is most preferably supplemented with hydrocortisone to a final concentration of 20 ^g / L (55.2 nM), preferably 15 to 25 ^g / L, less preferably 10 to 30 ^g / L, and least preferably 0 to 40 ^g / L using water soluble, cyclodextrin encapsulated hydrocortisone (Millipore Sigma-Aldrich, catalog number H0396). Concentrations greater than 40 ^g / L are expected to be possible but are not expected to result in further improvements in RPE phenotype and function. It is expected that other forms of hydrocortisone such as the sodium salt of hydrocortisone 21-hemisucinate (e.g., STEMCELL Technologies, catalog numbers 74142 or 74144), hydrocortisone stock solutions (e.g., STEMCELL Technologies, catalog number 07925 or 07926), hydrocortisone solubilized in ethanol (Millipore Sigma-Aldrich, catalog number H0888) can be used as an alternative. It is also expected the corticosterone (Millipore Sigma Aldrich, catalog number C2505) could be substituted for hydrocortisone; however, slightly higher concentrations are expected to be possibly required due to a lower anti-inflammatory potential.

[0289] In some embodiments of the disclosure RPE-MM can be the formulation RPE-MM which comprises the base medium ^-MEM supplemented with additional non-essential amino acids (glycine, alanine, asparagine, aspartate, glutamate, proline, and serine), insulin, transferrin, selenite, putrescine, progesterone, serum albumin, galactose, catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, ethanolamine, linoleic acid, linolenic acid, biotin, carnitine, vitamin A, taurine, triiodothyronine, corticosterone, and hydrocortisone, wherein the components in italics are expected to be non- essential but to possibly result in more homogenous pigmentation and epithelial morphology.

[0290] In the most preferred embodiment, the RPE Maturation Medium (RPE-MM) can be prepared as follows: 10.08 g of powdered medium ^-MEM medium and 2.2 g of sodium bicarbonate in tissue culture grade H2O to a final volume of 950 mL. For each 950 mL of dissolved ^-MEM medium 10 mL of 100X penicillin / streptomycin solution, 1 mL of normocin solution, 10 mL of N1 Supplement, 10 mL of 100X non-essential amino acids solution (NEAA), 20 mL of CTS-B27 Supplement, 0.25 g taurine, and 8 ^L of 2.5 mg / ml hydrocortisone are added and the complete medium is filter sterilized (see Example 11).

[0291] The concentration of all components of the most preferred formulation of RPE-MMTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT and allowable (general) concentration ranges of RPE-MM buffered balanced salt solution, essential amino acids and nonessential amino acids, vitamins, hormones, essential components for lipid metabolism, carbon energy sources, serum albumin, insulin, transferrin, selenite, and antioxidants in an effective amount to promote maturation of differentiated SC-RPE cells are provided in Tables 7A to 7E. For xeno-free culture the protein components should be from the same species as the stem cell line. It is expected that lipid-rich albumin, such as and not limited to albumin purified from serum using non-denaturing chromatography methods or the commercially available AlbuMAX, can be substituted for Serum Albumin, Fraction V, which is lipid-poor, at the concentrations indicated in Table 7A to 7E. When using lipid poor albumin, some benefits such as improved growth and RPE phenotype, and function can be expected by the addition the lipids found in lipid rich albumin associated

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"^^ ^^ ^^^^^^ ^ ^!"^ ^^ ^^ ^?^^^^ ^^ ^^^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^^^^^^^^"^ )&^^ ^^ ^^^ .^^^^"^ (##^ ^^ ^^^^^^ ^.^^"^ ^))^ ^^ ^^^^^^ '^^^^^^^^ )^*^ ^^ ^^4%^'"^^"^ *(^^ ^^ ^^4%^'^^"^ ^##^ ^^ ^^$^^'^^^^^^^^^^ (^#^ ^^ ^^$^^'^^^^"^ ^^^&^ ^^ $^%^^^"^ ^((^ ^^ ^^^^^'^^^^"^ ^##^ ^^ ^^^^^^"^^^^"^ ^##^ ^^ ^^^"^^^^"^ ^##^ ^^ ^^^%^^^"^ )^^^ ^^ ^^^"'^^^^^^"^ ^#^^ ^^ ^^+^"^%^^^^^^^"^ ^^^^ ^^ ^^+ ^^^^"^ ^^^^ ^^ ^^," ^^"^ ))&^ ^^ ^^^ ^^"^ ^###^ ^^ ^^^^ "^^^^"^ ^##^ ^^ ^^^ %^'^^^^^^ ^^^ ^^ ^^^% ^^^^"^ ^^^^ ^^ ^^-^^^^"^ )^^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ 4^^^^^"^^^^^ ^^"^ ^^^(^ ^^ B'^^^^^^^^^"^ ^(^^^ ^^ ^^^^^^^'^^^ ^^^^^ ^^ ^^4^ ^^'^^"^^ ^#^^^ ^^ ^^^^^"^^^^^^^^ )^*^^ ^^ ^^^^^"^^^^^^^^^ )^*^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 7A. RPE Maturation Medium ^^^^^'^ ^^ ^^ + "!" "^^ ^^ ^^^^^^ ^ ^!"^ ^^^^ ^?^^^^ ^^ ^^ ^^^^ ^^^^^^^^^ ^^ 6^^'^^^ ^#^(^ ^^ A^4^^^^^^^^^^'^'^"^^'"^^^ ^^^#^ ^^ 9^^^^^^^^^^ ^^^^^ ^^ ^%^^^^^'^^^"^ ^^^ ^^ / ^^^'^^^^^^"^ &^^^^ ^^ +^' "^^^^"^ ^^^^ ^^ +% ^^^^^^"^^^ ^^&(^ ^^ ^"'^^^^^ #^ ^^ ^"'^^^^^^^"'^'"^ #^)#^#^ ^^ ^^^^!^^>^^^ #^^((^ ^^ ^^^^^^^"^ ^^^(^ ^^ -^'^^^^^6^^^ ^^##^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ #^^^^^^ ^^ + ^."^'" ^^"^ #^#^)^^ ^^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^##)#^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ A^$^^^^'^^"^ &)^)###^ ^^ A^$^^^^^"^ ***^^ ^^ +% ^>^^^^^^^^ ^###^ ^^ 0^^^^^^"'^'"^ ^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ," ^^^^^^^^^^^ )^^(^ ^^ ^^^^^^^^ ^^*(^ ^^ ^ ^^^!" ^^^ #^^^*^ ^^ 4^'^^^^"^ #^#^#^^ ^^ ,^^" ^^^^"^A^^^^'^^"^ #^#^(^^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ )^^*^ ^^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^^^^ ^^ A^^^^^^^^^^^^^^" ^^^ ^^^#^ ^^ ^^^^^^^^^^^^ #^^(^^ ^^ ^^ ^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT Table 7A. RPE Maturation Medium^^^^^'^ ^^ ^^ + "!" "^^ ^^ ^^^^^^ ^ ^!"^ ^^ ^^ ^?^^^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^^"^^^^^"^ ^^^ ^^ 4%'^^^^"^ ^^^ ^^ $^^^^^^^"^ ^^^ ^^ ^^%^^^^^"^ ^^^ ^^ E ^^^^"^^^^%^ ^^^^^ ^^^ ^^ ^J^A"^^%^^"^^^^^"^ ^^^ ^^ ^J^A"^^%^%'^^^^"^ ^^^ ^^ ^J^A"^^%.^^^^^^^"^ ^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ +^"^^^^ "^^ ^(^(^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ / ^ ^^^^^^ *#^^##^?.H^^^ +"^^^^^^^^^ ^##^EH^^^ ^^ ,' "^'^^%^^^^^ ^##^?.H^^^ ^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ ^^ ^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^ ^ ^^'^^^ / ^2^ ^^ ^^^#^*^ ^^ ^^(&^)^ ^^'^^^72^ ^^ ^^^^^ ^^ *^^)^ ^^'^^^4^2^^ ^^ ^*^^^ ^^ ^&^#^ ^^'^^^^.2^^ ^^ ^^^^ ^^ &^&^ ^^'^^^^^2^^ ^^ #^ ^^ #^###^^*^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^'^^^9"2^^ ^^ #^ ^^ #^)^)^ ^^'^^^9"2)^ ^^ #^ ^^ #^#(^^ ^^'^^^4^2^^ ^^ #^ ^^ #^##^)#^ ^^'^^^:^2^^ ^^ #^ ^^ #^)^^^ ^^'^^^4^8^ ^^ ^^^)*(^ ^^ ^^^&^&^ ^^'^^^+0^8)^ ^^ ^^#^ ^^ ^#^(^ ^^'^^^,0^8^^ ^^ ^#)^ ^^ &^&^ ^^'^^^ / #)8^ ^^ #^ ^^ #^^&*^ ^^'^^^,"0)8^^^ ^^ #^#^^^^ ^^ #^^*^^ ^^ ^^ ^^ ^^ ^^ / ^4^^ ^^ ^^^#^^^ ^^ ^^&(&(^ / ^^40)^ ^^ ^*^#)^ ^^ ^&)(^^ 74^^ ^^ ^^^^^ ^^ *^^)^ / ^^^+0^^ ^^ ^^#^ ^^ ^#^(^ 4^4^^^ ^^ ^*^*^ ^^ ^&)&^ ^.,0^^ ^^ ^^^^ ^^ &^&^ / ^^,"0)^^ ^^ #^#^^^^ ^^ #^^*^^ 9"^ / 0)^)^ ^^ #^ ^^ #^ 9",0^^ ^^ #^ ^^ #^ :^,0^^ ^^ #^ ^^ #^ 4^,0^^ ^^ #^ ^^ #^ ^^4^^^ ^^ #^ ^^ #^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^ ^ ^^^^^^^^"^ ^^ )*(^ ^^ ^#(^ ^^^ .^^^^"^ ^^ *(*^ ^^ (^^^ ^^^^^^ ^.^^"^ ^^ ^#&^ ^^ ^*&^ ^^^^^^ '^^^^^^^^ ^^ )##^ ^^ )*#^ ^^4%^'"^^"^ ^^ ^(&^ ^^ *&^^ ^^4%^'^^"^ ^^ &^^#^ ^^ ^^#^ ^^$^^'^^^^^^^^^^ ^^ *&*^ ^^ ()*^ ^^$^^'^^^^"^ ^^ ^^(^^ ^^ ^^^#^ $^%^^^"^ ^^ ^^^^ ^^ ^^^^ ^^^^^'^^^^"^ ^^ ^&*^ ^^ ^#^^ ^^^^^^"^^^^"^ ^^ )^^^ ^^ ^^^^ ^^^"^^^^"^ ^^ )^^^ ^^ ^^^^ ^^^%^^^"^ ^^ )^#^ ^^ ^*^^ ^^^"'^^^^^^"^ ^^ ^&^^^ ^^ ^^)^ ^^+^"^%^^^^^^^"^ ^^ ^^^^ ^^ ^^^^ ^^+ ^^^^"^ ^^ ^^^^ ^^ ^^^^ ^^," ^^"^ ^^ )^)^ ^^ )()^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^^ ^^"^ ^^ ^*##^ ^^ ^^*#^ ^^^^ "^^^^"^ ^^ )^^^ ^^ ^^(^ ^^^ %^'^^^^^^ ^^ ^(^^^ ^^ *^^^^ ^^^% ^^^^"^ ^^ ^^^^ ^^ ^^^^ ^^-^^^^"^ ^^ )^^^ ^^ ^^^^ ^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ ^ ^ 4^^^^^"^^^^^ ^^"^ ^^ ^^#)^ ^^ ^^^^^ B'^^^^^^^^^"^ ^^ ^^^)^ ^^ ^#^*^ ^^^^^^^'^^^ ^^ ^#^^^ ^^ ^*^^^ ^^4^ ^^'^^"^^ ^^ ^^*&^ ^^ ^^^(^ ^^^^^"^^^^^^^^ ^^ ^^(&^ ^^ ^^*#^ ^^^^^"^^^^^^^^^ ^^ ^^(^^ ^^ ^^^^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ ^ ^ ^^^^ ^^^^^^^^^ ^^ ^^)^ ^^ ^^#^ 6^^'^^^ ^^ ^^^(^ ^^ ^)^^^ A^4^^^^^^^^^^'^'^"^^'"^^^ ^^ ^^#(^ ^^ )^^^^ 9^^^^^^^^^^ ^^ ^^^^^ ^^ )^*#^ ^%^^^^^'^^^"^ ^^ #^ ^^ ^^)^^ / ^^^'^^^^^^"^ ^^ &^#^^ ^^ ^^^^^ +^' "^^^^"^ ^^ ^*#^ ^^ ^^^^ +% ^^^^^^"^^^ ^^ ^^^&^ ^^ ^^#(^ ^"'^^^^^ ^^ #^ ^^ #^)^^^ ^"'^^^^^^^"'^'"^ ^^ #^^^&^ ^^ #^)&#^ ^^^^!^^>^^^ ^^ #^^(^^ ^^ #^)^^^ ^^^^^^^"^ ^^ ^^^^^ ^^ ^^)(^ -^'^^^^^6^^^ ^^ #^^&*^ ^^ ^^#^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ ^^ #^#&^^^ ^^ #^^^^^^ + ^."^'" ^^"^ ^^ #^#)^^^ ^^ #^#*^#^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ ^^ #^##^^)^ ^^ #^##)^^^ ^^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ ^ ^ A^$^^^^'^^"^ ^^ (^^*^ ^^ ^#^^ A^$^^^^^"^ ^^ *^^&^ ^^ ^^(^^ +% ^>^^^^^^^^ ^^ &^*^ ^^ ^#&^^ 0^^^^^^"'^'"^ ^^ #^ ^^ ^&)^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,+^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ + "!" "^^ ^^^^^^ ^ ^^ ^#$^ %^ ^^&^ ^^ ^^ ^?^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ^ ^ ," ^^^^^^^^^^^ ^^ ^&^^^ ^^ ^^^#^ ^^^^^^^^ ^^ ^^^^^ ^^ )^^(^ ^ ^^^!" ^^^ ^^ #^#^^#^ ^^ #^^)^^ 4^'^^^^"^ ^^ #^##^&#^ ^^ #^#^)#^ ,^^" ^^^^"^A^^^^'^^"^ ^^ #^#*^^^ ^^ #^#^(^^ ^^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ ^^ ^^^^^ ^^ ^^#(^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^ ^^*^^ ^^ ^^(*^ A^^^^^^^^^^^^^^" ^^^ ^^ ^^&#^ ^^ )^##^ ^^^^^^^^^^^^ ^^ #^^*^^ ^^ ^^^^^^ ^^ ^^ ^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^ ^ ^^"^^^^^"^ ^^ #^ ^^ ^^^)^ 4%'^^^^"^ ^^ #^ ^^ ^)^(^ $^^^^^^^"^ ^^ #^ ^^ ^^^^^ ^^%^^^^^"^ ^^ #^ ^^ ^)^(^ E ^^^^"^^^^%^ ^^^^^ ^^ #^ ^^ ^)^*^ ^J^A"^^%^^"^^^^^"^ ^^ #^ ^^ ^)^^^ ^J^A"^^%^%'^^^^"^ ^^ #^ ^^ ^)^&^ ^J^A"^^%.^^^^^^^"^ ^^ #^ ^^ ^^^)^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ ^ ^ +^"^^^^ "^^ ^^ ^#^^^ ^^ ^^^&^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ ^ ^ / ^ ^^^^^^ ^^ ^#^ ^^ ^^##^?.H^^^ +"^^^^^^^^^ ^^ ^#^ ^^ ^^##^EH^^^ ,' "^'^^%^^^^^ ^^ ^#^ ^^ ^^##^?.H^^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ ^^ ^^ ^^ ^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^^^5^^^^^^^^^^^ ^ ^ ^ ^^'^^^ / ^2^ ^)&^(&^ ^^ ^^^^(^^ ^^'^^^72^ ^(^^^ ^^ **&#^ ^^'^^^4^2^^ ^)^^^ ^^ ^&^(^ ^^'^^^^.2^^ ^))^ ^^ &^^^ ^^'^^^^^2^^ #^ ^^ #^###^^^^ ^^'^^^9"2^^ #^ ^^ #^^^^^ ^^'^^^9"2)^ #^ ^^ #^^^)^ ^^'^^^4^2^^ #^ ^^ #^##^*^^ ^^'^^^:^2^^ #^ ^^ #^^^&^ ^^'^^^4^8^ ^^^^^#^ ^^ ^)^)^^^ ^^'^^^+0^8)^ ^(*^ ^^ ^^)^^ ^^'^^^,0^8^^ *^*^ ^^ &^^^ ^^'^^^ / #)8^ #^ ^^ #^)^#^ ^^'^^^,"0)8^^^ #^#(#^^ ^^ #^^&^^ ^^ ^^ ^^ ^^ / ^4^^ ^^^^)#^ ^^ ^^^#^)^ / ^^40)^ ^*^^&^ ^^ )#*^#^ 74^^ ^(^^^ ^^ **&#^ / ^^^+0^^ ^(*^ ^^ ^^)^^ 4^4^^^ ^)&^^ ^^ ^&^^^ ^.,0^^ ^))^ ^^ &^^^ / ^^,"0)^^ #^#(#^^ ^^ #^^&^^ 9"^ / 0)^)^ #^ ^^ #^^^)^ 9",0^^ #^ ^^ #^^^^^ :^,0^^ #^ ^^ #^^^&^ 4^,0^^ #^ ^^ #^##^*^^ ^^4^^^ #^ ^^ #^###^^^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^ ^ ^ ^ ^^^^^^^^"^ ))^^ ^^ ^)^^ ^^^ .^^^^"^ *)^^ ^^ (^^^ ^^^^^^ ^.^^"^ )&)^ ^^ ^&)^ ^^^^^^ '^^^^^^^^ ^^*^ ^^ )^*^ ^^4%^'"^^"^ )(^^ ^^ *^^^ ^^4%^'^^"^ (^^)^ ^^ ^^#^ ^^$^^'^^^^^^^^^^ *(#^ ^^ ((#^ ^^$^^'^^^^"^ ^^^^^ ^^ ^*^^^ $^%^^^"^ ^^(^ ^^ &^(^ ^^^^^'^^^^"^ ^(^^ ^^ ^#&^ ^^^^^^"^^^^"^ )&^^ ^^ ^&^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^^^"^^^^"^ )&^^ ^^ ^^^^ ^^^%^^^"^ )&^^ ^^ *#^^ ^^^"'^^^^^^"^ ^(^&^ ^^ ^^*^ ^^+^"^%^^^^^^^"^ ^&&^ ^^ ^^^^ ^^+ ^^^^"^ )^^^ ^^ ^^^^ ^^," ^^"^ ^&&^ ^^ )&&^ ^^^ ^^"^ ^###^ ^^ ^*##^ ^^^^ "^^^^"^ )&*^ ^^ ^^)^ ^^^ %^'^^^^^^ ^^^^^ ^^ *)^)^ ^^^% ^^^^"^ ^^#^ ^^ ^^^^ ^^-^^^^"^ )&^^ ^^ ^^^^ ^^ ^^ ^^ ^^ 2^^^^^^^^^^^^^^^^ ^ ^ ^ 4^^^^^"^^^^^ ^^"^ (^^#^ ^^ )*^(^ B'^^^^^^^^^"^ &^^#^ ^^ ^^^(^ ^^^^^^^'^^^ ^#^^^ ^^ ^#^^^ ^^4^ ^^'^^"^^ *^#*^ ^^ ^*^^^ ^^^^^"^^^^^^^^ ^^^^^ ^^ *^^)^ ^^^^^"^^^^^^^^^ ^^&#^ ^^ *^)^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^ -^^^^^^^^^ ^ ^ ^ ^^^^ ^^^^^^^^^ ^^^^ ^^ ^^*^ 6^^'^^^ *^)^^ ^^ ^*^^^ A^4^^^^^^^^^^'^'^"^^'"^^^ ^^#^^ ^^ ^^^)^ 9^^^^^^^^^^ ^^^&^ ^^ ^^^)^ ^%^^^^^'^^^"^ #^ ^^ &^^^^ / ^^^'^^^^^^"^ ^^&^^ ^^ ^*^(^ +^' "^^^^"^ ^##^ ^^ ^^^^ +% ^^^^^^"^^^ ^^^^^ ^^ ^^^(^ ^"'^^^^^ #^ ^^ #^^)(^ ^"'^^^^^^^"'^'"^ #^^*^^ ^^ #^^*(^ ^^^^!^^>^^^ #^^(^ ^^ #^*^^ ^^^^^^^"^ ^^&*^ ^^ *^^*^ -^'^^^^^6^^^ #^^((^ ^^ ^^#^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ 3^^^^^^^ ^ ^ ^ 4^ '^^^^'" ^^"H^%^ ^^^ '^^^^"^ #^#*(^^ ^^ #^^(^)^ + ^."^'" ^^"^ #^#^^(^ ^^ #^#(^&^ ^ ^^^^^^^^'^% ^^^^"^^^)^^ #^##^^^^ ^^ #^##^^(^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^^ ^^ ^^ ^^ .^^^^^^^^^^5;^^^^^^^^ ^ ^ ^ A^$^^^^'^^"^ ^^^^^ ^^ ^^*^ A^$^^^^^"^ *)^*^ ^^ ^^^^^^ +% ^>^^^^^^^^ (*#^ ^^ ^^^*^ 0^^^^^^"'^'"^ #^ ^^ *((^ ^^ ^^ ^^ ^^ ^^^^^^^^^ ^ ^ ^ ," ^^^^^^^^^^^ ^&^&^ ^^ *(^^^ ^^^^^^^^ #^^^^^ ^^ *^)*^ ^ ^^^!" ^^^ #^#(^^^ ^^ ^^)*^ 4^'^^^^"^ #^##*^#^ ^^ #^#^*(^ ,^^" ^^^^"^A^^^^'^^"^ #^#)&*^ ^^ #^^^*^^ ^^ ^^ ^^ ^^ <^^^^^^^^^^^^ ^^^1^^5^^59^^^^^0^^^^^^ ^ ^ ^ $^^'^'^^^^"^^ "^^^"^^^ ^^()^ ^^ ^^&&^ A^^^^^^^^^^^^^^" ^^^^^"'^'"^ ^^#(^ ^^ )^^&^ A^^^^^^^^^^^^^^" ^^^ ^^^#^ ^^ )^(#^ ^^^^^^^^^^^^ #^^))^ ^^ ^^^^^ ^^ ^^ ^^ ^^ -^^^^^^^^^^ ^ ^ ^ ^^"^^^^^"^ #^ ^^ ^^^^^ 4%'^^^^"^ #^ ^^ ^^^^^ $^^^^^^^"^ #^ ^^ ^)^)^ ^^%^^^^^"^ #^ ^^ ^^^^^ E ^^^^"^^^^%^ ^^^^^ #^ ^^ ^^^#^ ^J^A"^^%^^"^^^^^"^ #^ ^^ ^(^)^ ^J^A"^^%^%'^^^^"^ #^ ^^ ^^^*^ ^J^A"^^%.^^^^^^^"^ #^ ^^ ^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^3^ ^^^^^^^^^&^^^^^^^^'^^ ^ ^ ^ +^"^^^^ "^^ ^)^^^ ^^ ^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^^^^^^^^^^^&^^^^^^^^'^ ^ ^ ^ / ^ ^^^^^^ ^#^ ^^^ ^^##^?.H^^^ +"^^^^^^^^^ ^#^ ^^^ ^^##^EH^^^ ,' "^'^^%^^^^^ ^#^ ^^^ ^^##^?.H^^^ A^+^^'^^'^"^^^^^^^^5K4^^^^^^ ^^ ^^ ^^Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^^^^^^,.^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^^+ "!" "^^ ^^^^^^ ^ ^#&^ %^ ^^$^ ^^ ^?^^^^ ^^ ^"^^^^^'^'^'"^^!^ ^A^4^^^^^^^ ^^^'^'^"^^'"^^ +% ^^^^^^^^^^^^"^^^^^'^'^'"^^ !^ ^+% ^^^^^^"^ ^^ ^^ ^^^^^^,!^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^"^^'^+ "!" 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[0292] In each of the RPE-MM reported in Tables 7A to 7E each concentration within the indicated range for a component can be combined with each concentration within the indicated range of another component with the proviso that the osmolarity of the media remains within the prescribed range, as will be understood by a skilled person.

[0293] Variations in the composition of Table 7A to 7E are expected to be functional when performed within the configuration of the RPE Maturation Medium of the disclosure. For example, ascorbic acid is an essential nutrient and hence it is expected to be an essential component of SC-RPE Medium. Low glucose plus pyruvate is expected to be beneficial byTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT forcing the RPE to become more dependent of aerobic energy production, which is their predominant mode of energy production in vivo and results in production of oxaloacetate. Data supporting this expectation comprise the finding that using low glucose DMEM plus pyruvate as the base medium for RPE-MM results in improved epithelial morphology compared to high glucose DMEM; while, neither DMEM nor pyruvate support RPE pigmentation. Oxaloacetate, is also expected to be possibly used as an alternative to pyruvate or in addition to pyruvate.

[0049] ^Use of pyruvate or oxalacetate is considered optional. Additional supplementation of RPE Maturation Medium with zinc, copper, or manganese ions is expected to yield slight improvements in RPE phenotype even if none of those metals are essential. Hypoxanthine is a common cell culture medium supplement that can promote cell growth it also is not found in alpha-MEM and its addition is optional.

[0294] From the experiment shown in Example 18 and Figures 5 and 6, it has been determined that each of the base media additions and the base media itself contribute to RPE phenotype maturation. However, except in the cases of the base media and B27 the effects of the individual media components on their own was merely quantitative and while they each result in improved RPE phenotype, they are not by themselves essential. From a comparison of the various base media, an examination of the constituents of the N1 and B27 supplements, the role of the individual components in cell function, and a knowledge of the unique aspects of RPE metabolism and function some conclusions as to the essential media requirements for RPE maturation are expected.

[0295] Accordingly in some embodiments of the RPE Maturation Medium of the disclosure, variations based on modification of the B27 components can be made. Serum albumin is a nonspecific carrier protein for hydrophobic steroids and fatty acids which is expected to be essential for RPE maturation. Galactose is both an energy source and a necessary substrate for protein glycosylation, which is not considered to be essential for RPE maturation but can be advantageous during periods of rapid growth and development. In certain embodiments, galactose is omitted during long term maintenance of RPE in culture. Inclusion of catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, and taurine all play a role in protecting against oxidative stress. Individually, none of these are expected to be essential, but inclusion of one or more of these is expected to improve RPE quality in culture and is expected to be essential for RPE maturation. Ethanolamine, linoleic acid, linolenic acid, biotin, and carnitine all participate in fatty acid and lipid biosynthesis andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT are all expected to be essential but for carnitine which is expected to be conditionally essential. It is expected that concentration of some of the components related to lipid biosynthesis can be reduced by the use of lipid-rich serum albumin.

[0296] Vitamin A is non-essential for RPE differentiation, pigmentation, and epithelial morphology, but its inclusion is considered to be beneficial based on an observed effect on visual cycle and complement pathway gene expression as well as subtle improvements to morphology (See Example 42 and Figures 20, 21, and 22).

[0297] Hydrocortisone and possibly corticosterone are non-essential and are expected not to be necessary for culture of minimally expanded cultures of RPE, but are expected to result in improved RPE quality, especially in instances where the cells have undergone significant cell doublings during the course of culture expansion. Moreover, hydrocortisone and corticosterone are expected to be interchangeable, albeit with some changes to the concentration to account for differences in potency. Progesterone has been shown to regulate calcium signaling in RPE and to up-regulate vascular endothelial growth factor by RPE. Hence its inclusion is predicted to be beneficial for the production of quality RPE.

[0298] In some embodiments of RPE-MM, the complete B27-based supplement is replaced with a modified B27-based supplement with altered concentration of specific components or missing one or more of the complete B27-based supplement components. This can be achieved by custom formulation based on the guidelines for production of these supplements in the previously cited protocols and publications relating to B27 and N21 and referencing the concentration ranges listed in Tables 7A to 7E as would be understood by a skilled person. B27-based supplements without antioxidants, vitamin A, and insulin are also available from ThermoFisher Scientific and STEMCELL Technologies.

[0299] In some embodiments of the RPE maturation medium of the disclosure, variations based on the use of any one of a N1-type 100X supplements (e.g., N1, N-2, CTS-N-2, N-2- Plus, N-2-MAX, GMP-N-2-MAX, Custom N1) and any one of a B27 50X supplement equivalent or derivative, (e.g., B27, B27 Plus (B27+), B27 XenoFree (XF-B27), CTS-B27 XenoFree (CTS-B27) NeuroCult SM1, NS21, N21-MAX, GMP-N21-MAX, and Custom B27) in any combination with the most preferred ^-MEM base medium and additional maturation medium supplements (NEAA, taurine, and hydrocortisone) can be used to provide a RPE Maturation Medium (RPE-MM). The resulting different RPE-MM variants can be denotedTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT using the naming convention, RPE-MM:“N1” / “B27”, where RPE-MM denotes the use of one of the commercially available powdered ^-MEM without nucleosides formulations and the most preferred concentrations of the additional maturation medium supplements, “N1” denotes the type of N1 supplement, and “B27” denotes the type of B27 supplement at the preferred strength of 1X. For example, when using N1 supplement and B27 XenoFree, the medium would be labeled RPE-MM:N1 / XF-B27. It is expected that when used at 1X strength the concentration of the components derived from commercial B27-based supplements will fall within the concentration ranges in Tables 7A to 7C. When alternative amounts of N1 or B27 supplements are used they can be noted by parenthetical amendment of the concentration to the supplement names, (e.g., RPE-MM:CTS-N-2(0.5X) / CTS-B27(1.5X)). When using strengths other than 1X of either N1 or B27 compensatory changes in the amounts of the other supplement is expected to possibly be necessary or desirable to stay within the acceptable concentration ranges due to the shared components in the two supplements.

[0300] In some embodiments of the RPE Maturation Medium of the disclosure can be made based on the expectation that since the components of N1 and hydrocortisone are all contained within B27, their addition can be omitted by increasing the amount of B27 Supplement in an RPE Maturation Medium. In some embodiments of the SC-RPE maturation medium of the disclosure, the maturation medium does not comprise antibiotics.

[0301] In some embodiments the formulation of RPE-MM can be advantageously described from a manufacturing standpoint as the result of a combination of an alpha-MEM medium and a B27 supplement which can be variated to provide different version of the RPE-MM of the disclosure.

[0302] Additionally, embodiments where the RPE-MM are derived from alpha-MEM are known or expected to provide a high percentage of pigmented cells with a mature RPE phenotype and morphology (generally> 95%, often >99 %) Additionally, RPE-MM are derived from alpha-MEM enables or enhances SC-RPE enrichment based on pigmentation non-manual automation capable cell sorting as will be understood by a skilled person.

[0303] In those embodiments a set of core components derived from the combination of alpha- MEM and B27 is reported in Table 8 belowTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0304] The core components of the RPE-MM of Table 8, can be used to obtain maturation of RPE cell or be variated to provide additional embodiments of buffered balanced salt solution, essential amino acids and nonessential amino acids, vitamins, hormones, essential components for lipid metabolism, carbon energy sources, serum albumin, insulin, transferrin, selenite, and antioxidants in an effective amount to promote maturation of differentiated SC-RPE cells. As will be understood by a skilled person.

[0305] In some embodiments, the core set of components of RPE-MM of Table 8 can be variated in accordance with the indications of the present disclosure to optimize performance of the RPE-MM on the RPE cells to be monitored through detection of pigmentation and / or preferably expression of marker genes of RPE maturation as will be understood by a skilled person.

[0306] In those embodiments the variations can be provided in terms of variations of base component alpha-MEM and B27 inclusive of modification of the related components and additional supplementation with advantage in related manufacturability as will be understood by a skilled person.

[0307] For example in preferred embodiments of RPE Maturation Medium of Table 8 obtained by combining alpha-MEM at 100% as described in Table 6, can be variated to include alpha-MEM in an amount of less than 100%, for example by using alpha-MEM at 97-100%, 94-97%, 90-94%, or 85-90% of the concentration reported in Table 6.

[0308] In some embodiments, when using powdered alpha-MEM, the RPE Maturation Medium of Table 8 can be variated, by providing powdered alpha-MEM at 100-103%, 103- 106%, or 106-110% of its normal concentration reported in Table 6.

[0309] In preferred embodiments, the RPE Maturation Medium of Table 8 can be variated byTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT combining alpha-MEM in any variation herein described with a B27-based supplement with vitamin A such as the B27 formulation of Table 5 is used at 1X, less preferably 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2.X, or 2-3X.

[0310] In some embodiments, the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with the B27-based supplement without vitamin A, preferably used at 1X, less preferably 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2.X, or 2-3X.

[0311] In some preferred embodiments, the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described and with taurine used at preferred concentrations of 2 mM, less preferably at 0-2 mM, 0.5-1 mM, 1-1.5 mM, 1.5-2 mM, 2-2.5 mM, 2.5-3 mM or 3-4 mM.

[0312] The preferred embodiment the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described, and further with the supplemental N1-based cocktail concentration at 1X, less preferably 0-0.25X, 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2X. In some of those embodiments the N1- based cocktail is made using the individual constituents (insulin, apo- or holo-transferrin, selenite, progesterone, and putrescine) at any of the concentrations found in N1, N2, or High Insulin N-2. These custom blended N1-based cocktails are used at 1X, less preferably 0-0.25X, 0.25-0.5X, 0.5-1X, 1-1.5X, 1.5-2.X.

[0313] In some embodiments the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described with N1-based cocktail, missing one or more of the individual components and the included components are used at any of the concentrations found in N1, N2, or High Insulin N-2. These custom blended N1-based cocktails are used at 1X, less preferably 0-0.25X, 0.25- 0.5X, 0.5-1X, 1-1.5X, 1.5-2.X.

[0314] In preferred embodiments the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described, and further supplementing the medium with a cocktail of NEAA (alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine) to increase the concentration of the non-essential amino acids in the medium by100 ^M, less preferably 0-25Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT ^M, 25-50 ^M, 50-100 ^M, 100-150 ^M, 150-250 ^M, or 250-300 ^M. In some of those embodiments of the RPE Maturation Medium is supplemented with a cocktail of NEAA missing one or more of alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine to increase the concentration of the non-essential amino acids in the medium by100 ^M, less preferably 0-25 ^M, 25-50 ^M, 50-100 ^M, 100-150 ^M, 150-250 ^M, or 250-300 ^M.

[0315] In some embodiments of the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described.

[0316] In preferred embodiments the RPE Maturation Medium of Table 8 can be variated by combining alpha-MEM in any variation herein described with B27 in any variation herein described further supplemented with hydrocortisone at a concentration is 50 nM, less preferably 0-10 nM, 10-20 nM, 30-40 nM, 30-60 nM, 60-80 nM, or 80-100 nM, 100-150 nM, or 150-200 nM.

[0317] In embodiments wherein the RPE Maturation Medium of Table 8 is variated , the outcome of reducing or increasing the concentration of alpha-MEM from its preferred strength is a reduction in the percentage of pigmented cells beginning about 1-2 weeks after becoming confluent a decrease in the level of RPE marker genes as provided elsewhere in the disclosure. In the extreme slow growth and cell death would be expected. The same is true for the expected outcomes of altering the amount of the B27.

[0318] For taurine, and additional supplementation of NEAA, N1, and hydrocortisone complete removal of any one of them individually is expected to reduce the percentage of pigmented cells in the culture by as much as 50% or more depending on the seeding densities (<40,000 cell / cm2) and the passage history of the cells. When more than one of these is removed it is expected that the effect would be combinatorial, with respect to RPE phenotype, but the cells are expected to propagate, albeit at a possibly reduced rate. When all four are eliminated it is still expected that the cells will grow. When culturing cells with higher concentrations of the supplements some improvement in culture phenotype can be expected, but at the high end of the concentrations some toxicity and accompanied loss or RPE might be observed.

[0319] The outcome of variating the concentrations of the RPE Maturation Medium of Table 7 using the supplements at non-preferred concentrations or amounts can most simply beTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT achieved by comparing to cells cultured in the preferred embodiment. Either by eye to qualitatively assess phenotype and pigment, by absorbance to quantitatively measure pigmentation, by qPCR using RPE marker probes, by quantification of PEDF secretion into the culture medium, or more preferably by gene expression profiling to fully assess the cell phenotype as would be understood by a skilled person upon reading of the disclosure.

[0320] In preferred embodiments of the disclosure a variated RPE Maturation Medium of Table 8 can be supplemented with vitamin A via B27- or NS21-based supplements that contain retinyl acetate (B27) or retinyl acetate and retinol (NS21). When the supplements are used at their preferred strength of 1X, retinyl acetate is provided to the medium at a final concentration of about 300 nM and retinol is provided at a final concentration of 350 nM. Since vitamin A is a potent regulator of RPE gene expression, when it is desired to reduce the amount of the B27 or NS21 supplement, addition of retinyl acetate and / or retinol should be considered and when the amount of these supplements are increased use of a B27 supplement without Vitamin A to increase the B27 concentration should be considered. Since NS21 has effectively twice the concentration of vitamin A compared to B27 some changes in gene expression are expected between the two supplements.

[0321] In additional preferred embodiments a variated RPE Maturation of Table 8 can comprise 1. Alpha-MEM based on the formulation in (Table 6, at 90-92%, 92-94%, 94-96%, 96- 98%, 98-100%, 100%, or up to 110%) which allows for use of liquid formulations when necessary or convenient (see e.g. cGMP embodiments) or a. In any of the available formulations b. With or without nucleosides 2. B27 / NS21(based on the formulation in Table 5), with possible variations herein described a. Plus / Minus Vitamin A (B27 / NS21 formulations are available commercially with and without retinol / retinyl acetate in both research and cGMP grade), b. Plus / Minus Insulin (B27 / NS21 formulations are available commercially with and without insulin in both research and cGMP grade) allows for adjustment of insulin (available in both research and cGMP grade) concentration, possible alternative to using N1 supplement to provide further control over process. When RPE is matured it is expected that being able to alter the insulinTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT concentration can allow an improved control of RPE maturation and phenotype at least in some embodiments. c. Plus / Minus Antioxidants (available commercially without^DL alpha-tocopherol acetate, DL alpha-tocopherol, superoxide dismutase, catalase, and glutathione) The presence of the antioxidants provided by B27 supplement is not expected to be essential in for obtaining SC-RPE but there addition may lead to improved RPE health. Omission of antioxidants can reduce medium complexity which is beneficial in clinical manufacture and omission of antioxidants are of particular relevance with embodiments entailing oxidative stress studies, d. Plus / Minus Triiodo-I-thyronine (T3) T3 has been implicated in RPE maturation proliferation and migration and is expected to regulate genes related to RPE function. The use of T3-minus B27 is expected to allow for further control to obtain a desired RPE phenotype.

[0322] The concentration of all components of the most preferred formulation of RPE-MM and allowable (general) concentration ranges of RPE-MM obtained by combination of Alpha- MEM and B27 of Table 8 variated and supplemented in accordance with the present disclosure is reported in Tables 8a to 8f. For xeno-free culture the protein components should be from the same species as the stem cell line.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0323] In each of the RPE-MM media reported in Tables 8a to 8h, each concentration within the indicated range for a core component of the RPE-MM can be combined with each concentration within the indicated range of another core component, of the composition, with the provision that the osmolarity also remains in the specified range as will be understood by a skilled person. For example when choosing a high glucose and high bicarbonate concentration, one would need to compensate by choosing a lower concentration of NaCl to maintain the osmolarity in the prescribed range.

[0324] In preferred embodiments of the disclosure the RPE media are prepared using powdered medium. This is done for two main reasons. First it allows for the ability to make different media formulations comprised of varying volumes of different liquid supplements and maintain the desired concentrations of all components. Secondly, it allows for the possibility of preparing more concentrated base media, the reason for which is that cultures subject greater evaporation, such as the outer wells in a multi-well plate or cultures in incubator with poor humidity control have been observed to have increased pigmentation and higher percentages of pigmented cells. By using powder media one can make more concentrated media with higher osmolarity if desired by adding more medium powder or reducing the amount of water used to dissolve it, as will be understood by a skilled person.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0325] In some embodiments, any one of the RPE Maturation Media of the disclosure use a powder form of ^-MEM without nucleosides.

[0326] In some embodiments, any one of the RPE Maturation Media of the disclosure can use a liquid form of ^-MEM. In some of those embodiments the addition of sodium bicarbonate can be omitted and glutamine is expected to possibly be needed to be added, depending on the specific medium formulation. When using liquid media some slight reduction in the quality of RPE phenotype can be expected due to dilution of the base medium components by the RPE- MM liquid supplements (RPE-MM) as will be understood by a skilled person.

[0327] When using glutamine-free base medium GlutaMAX (alanyl-L-glutamine dipeptide) can be directly substituted for glutamine.

[0328] The use of antibiotics or antimycotics is not essential to the function of RPE-MM. In some embodiments, where the use of antibiotics and / or antimycotics is not desirable (e.g., the production of SC-RPE for clinical use) the addition of penicillin, streptomycin, and / or normocin can be omitted.

[0329] Any media formulation where the concentration of the individual components, the inorganic ions, and osmolarity falls within the ranges provided in Tables 7A to 7E and more preferably in Tables 8A to 8g are all considered to be RPE Maturation Media. For example, while some components by themselves, such as additional NEAAs, taurine and hydrocortisone, can be omitted with modest reduction in RPE quality, it is expected that reduction of the concentration of such components in combination can result in a failure of the RPE to mature. The efficacy of alternative RPE-MM formulations can be assessed by simple experimentation as would be understood by skilled person in the field and upon reading of this disclosure. Using SC-RPE produced by the methods of this disclosure, primary RPE, or SC-RPE obtained from commercial sources the various RPE-MM formulations can be evaluated for their ability to promote RPE maturation by quantitative measurement of pigment accumulation, RPE gene expression, transepithelial electrical resistance, and photoreceptor outer segment phagocytosis; as well as by qualitative assessment of RPE morphology. The results obtained with the alternative media can be compared to those obtained using the most preferred formulation of RPE-MM.

[0330] For simple deviations of the formulation the medium name is modified to indicate theTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT change by simple amendment of the name with a suitable qualifier (e.g., RPE-MM, taurine- free; RPE-MM, hydrocortisone-free; RPE-MM, without additional NEAA; RPE-MM, without Pen / Strep / Normocin; RPE-MM, low calcium; RPE-MM, liquid; RPE-MM, with nucleosides; RPE-MM, without vitamin A; RPE-MM, without anti-oxidants; RPE-MM, high insulin; RPE- MM, without Vitamin E; RPE-MM, with Fe3+; etc.). When using alternative commercially available sources of ^-MEM with slight variations to the most preferred formulation (Table 6), the media name is amended to indicate the vendor and / or product identification. For alternative media where many changes are made, such as the case of the use of alternative base media such as DMEM:F12 mixtures or custom base media that meet the criteria of Tables 7A to 7E and more preferably in Tables 8A to 8g, they can be identified using a numbering scheme, i.e., RPE-MM1, RPE-MM2, RPE-MM3, and so on) and referencing a separate detailed description of the multiple changes.

[0331] In some embodiments, any one of the RPE Maturation Media of the disclosure can be used in the SC-RPE Maturation Method of the disclosure which is a method comprising contacting differentiated SC-RPE cells with a cell culture maturation medium of the present disclosure for a time and under conditions to obtain mature stem cell-derived retinal pigment epithelial (SC-RPE) cells.

[0332] In some embodiments of the SC-RPE maturation method of the disclosure coupled with the “spontaneous” differentiation method where the contacting of the cells with RPE differentiation medium begins 4 to 5 days after the initial plating, the contacting with RPE- MM is performed beginning between about 7 to 60 days after the initial plating; most preferably on about Day- 32, preferably between 21 and 37 days, less preferably between, 14 and 49 days, and least preferably between 7 and 60 days. After Day-37 there is typically no increase in mature SC-RPE yield and after Day-60 there can be a decrease in yield due to decreased isolation efficiency. Initial contact times substantially prior to 30 days are expected to be associated with decreased yields of RPE. (See Figures 1, 2, and 3). For embodiments where the contacting with RPE differentiation medium begins prior to or after 4-5 days after stem cell plating the timing and ranges of contacting with RPE-MM can be changed accordingly.

[0333] In some embodiments of the SC-RPE maturation method of the disclosure coupled with the “semi-directed” differentiation method, the contacting with RPE-MM follows the same guidelines as the differentiation protocol without Activin A. In embodiments of the “semi-directed” differentiation protocol where the contacting with RPE differentiation mediumTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT starts 4-5 days after first plating the stem cells and the last day of contacting with Actin A occurs on Days-15 or -16 this translates to a range of times to begin the contacting with RPE- MM between about 15 and 60 days after the initial plating, with the most preferable time being about Day-32, preferably between 21 and 37 days, and less preferably between 15 and 49 days, and least preferably between 15 and 60 days. For contact times beginning after Day-37 there is typically no increase in mature SC-RPE yield and after Day-60 there can be a decrease in yield due to decreased isolation efficiency. Initial contact times substantially prior to 30 days are expected to be associated with decreased yields of RPE. For embodiments where the contacting with RPE differentiation medium begins prior to or after 4-5 days after stem cell plating the timing and ranges of contacting with RPE-MM can be changed accordingly. For embodiments where the contacting with Activin A ends before Day-14 or continues after Day- 15 after stem cell plating the same guidelines for the initiation of contacting with RPE-MM apply with the proviso that the contacting cannot start until the after the last day of contacting with Activin A in RPE differentiation medium. (See Figures 1 and 2).

[0334] In a preferred embodiment the length of SC-RPE maturation for a given cell line:medium:substrate combination is determined empirically by monitoring the extent of pigmented cell accumulation and PEDF secretion as will be understood by a skilled person.

[0335] In embodiments of the SC-RPE maturation method of the disclosure coupled with the “spontaneous” or “semi-directed” differentiation method, the length of the period of contacting with RPE-MM is best determined empirically by regular assessment of the extent of pigment accumulation. Alternatively, it is expected that the extent of RPE maturation can be determined by measuring the amount pigment epithelium derived factor (PEDF) in the cell culture medium at the time of the regular culture feedings. PEDF is a highly abundant mature RPE secreted protein product of the SERPINF1 gene. Detection of pigmentation and PEDF secretion can be performed with methods known to a skilled person. In particular, pigmentation accumulation can most readily be assessed by collecting photographs of the cultures on a white background using incident light illumination or by collecting images using a common document scanner as function of time in culture and visually comparing the resulting images to determine when the extent of pigmentation in the culture reaches a plateau. Additional methods such as automated image analysis of culture images, measurement of light absorption to quantify pigmentation, as well as use AI based high resolution image analysis to quantify the degree of pigment accumulation are also envisioned as will be understood by a skilled person. The amount ofTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT PEDF secreted into the medium over a prescribed period of time as a function of the RPE maturation time course can be determined using standard analytical methods, such as using commercially available enzyme-linked immunosorbent assays (ELISA). When the amount of PEDF secretion per unit time plateaus minimal increase in SC-RPE yield is expected with increased RPE maturation time.

[0336] The optimal length of contact time in RPE-MM to result in maximal yields of SC-RPE is generally in the range of 30-60 days. Shorter times are expected to result in correspondingly reduced yields of SC-RPE and contact times greater than 90 days can result in decreased yields due to increasing difficulty in the physical isolation of the SC-RPE due to tight junction formation. Shorter contact times often being optimal for media of the RPE-DM2 family and protocols employing Activin-directed differentiation. Longer contact times are often required for media of the RPE-DM1 family. In addition, cell lines that appear to have different rates of RPE maturation have been observed. In several embodiments, with different cell lines, contacting with RPE Maturation Medium beginning at about Day-30 after stem cell plating and ending between Days-60 and -90 from the start of the SC-RPE differentiation method of the disclosure gave optimal results as defined as the time required for the percentage of pigmented cells to plateau.

[0337] In some embodiments of the SC-RPE maturation method of the disclosure, the frequency of feeding during the contacting can be based on the extent of medium acidification. With normal volumes of culture medium (about 3 mL / 10 cm2) this is typically at least every 3 days for the first week or two after the medium switch. Thereafter feeding twice a week (every 3-4 days) is possible.

[0338] In some embodiments of the SC-RPE maturation method of the disclosure, the contacting is performed on differentiated SC-RPE differentiated cells obtained with one of the SC-RPE differentiation methods of the disclosure (see e.g. protocols of Examples 12, 13, 16, 19, 20, 21, and 24).

[0339] In particular, in some embodiments of the SC-RPE maturation method of the disclosure, following completion of the SC-RPE differentiation method of the disclosure the differentiation media are replaced with one of the RPE Maturation Medium herein described. In particular the differentiation media can be replaced with one of the RPE Maturation media prepared combining any one of a N1-type 100X supplements and any one of a B27 50X supplement equivalent or derivative, in any combination with the most preferred ^-MEM baseTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT medium and additional maturation medium supplements. such as the media specifically described in

[0299] .

[0340] In some of those embodiments the replacement of the SC-RPE differentiation medium of the disclosure with the RPE Maturation Medium of the disclosure can be performed after about 30 days from the beginning of the SC-RPE differentiation method. The selection of 30 days as being the default timing of the beginning of the SC-RPE maturation method was based on the empirical finding that this is the general time when the first hints of pigmentation are often observed in differentiation media that have the capacity to support some degree of RPE maturation on their own (i.e., X-VIVO™ 10-based RPE-DM2). Interestingly, switching directly to RPE-MM from stem cell medium results in the emergence of numerous foci of pigmentation in the first few weeks of culture, but the foci fail to enlarge with time; suggesting that some presumptive RPE form quite quickly in the process, but RPE-MM inhibits the differentiation of additional RPE precursors when applied at these early stages of RPE differentiation. In the case of some cell line / substrate / media combinations hints of RPE pigmentation prior to 30 days in RPE-differentiation media have also been noted. It follows that in these cases earlier beginning contact times with RPE-MM is expected to be possible and would thus allow for shortening of the method. Furthermore, the observation that major improvements in yield are not seen after Day-30 would imply the earlier times are likely possible. That said, with respect to final RPE yield there is no downside to waiting to day-30 to initiate the beginning of the RPE other than the increase in time and costs. In addition to using the first onset of pigmentation as an indicator of the time to initiate contact with RPE- MM, it is expected that similar indication could be obtained by monitoring the onset and extent of expression RPE precursor marker gene, such as MITF, LHX2, and OTX2 and chose the timing of timing of the initiation or RPE-MM contact accordingly.

[0341] In some embodiments, in addition to the actual combinations of media and substrates, another dominant determinant of SC-RPE yield appears to be density, colony size, and extent of confluence of the stem cell colonies at the time of switching from stem cell medium to the RPE determination medium based on the observed patterns SC-RPE differentiation, as will be understood by a skilled person. Preferred conditions vary depending on the specific cell line:medium:substrate combination as will be understood by a skilled person upon reading of the present disclosure. Changes or adjustments to the protocol, as would be understood by skilled person, that affect the global and local cell density are expected to alter SC-RPETitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT differentiation efficiency. For example, cell line:media:substrate combinations that result in substantial pigmentation in regions known to often have lower initial cell densities or on the side wall of the culture dish or flask are expected to possibly benefit from lower density plating or switching to the differentiation medium at earlier times, whereas those that have more prominent pigmentation in the center of the well or outer third of the well can benefit from higher density plating or initiating the switch to differentiation medium at a later time.

[0342] In some embodiments of the SC-RPE maturation method of the disclosure, the cells will spontaneously detach as sheets depending on cell line:medium:substrate combinations (For example, see Example 13 and Figure 1, culture H9:X-VIVO™ / XF-B27+ActivinA:PC- VTN for one of several cases of a partially detached sheet). In those embodiments, while it is possible to continue to maintain the cultures as floating sheets the yield of RPE is often poor, even when there is a significant amount of presumptive RPE. In this case it is possible to rescue the culture at the time of detachment by dissociating the sheets using proteases (e.g., trypsin, TrypLE™, Accutase™, or Accumax™) as would be understood by a skilled person, and culturing the resulting cell suspensions on a substrate of choice (e.g., laminin or vitronectin, Matrigel, etc.) using RPE Maturation Medium. In some embodiments some cell lines give rise to 2-dimensional tissue like sheets of cells after RPE maturation that are highly resistant to dissociation and hence reduced the yield of SC-RPE at the time of enrichment. In this case the protocol can be adjusted by passaging the cells at the time of switching to RPE-MM or preferably in the days following the switch. Alternatively, it is possible to routinely proactively passage the cells at the time of the switch to RPE-MM or in the days following the switch with any or all cell lines and embodiments of the RPE differentiation method. Improved efficiencies of SC-RPE derivation have been associated with increased times of contact with RPE-MM prior to passage, supporting the conclusion that contact with RPE-MM prior to differentiation culture dissociation contributes to the generation RPE-progenitors or presumptive RPE as well as RPE maturation. While routinely proactively passage the cells can allow for the use of differentiation medium:substrate combinations where substrate detachment or lower than desired differentiation yield is an issue, it does come at the expenses of increased materials costs, an increase in the time required for SC-RPE derivation, and increases the amount of material that must be processed for enrichment of the SC-RPE (see e.g. Example 17 and Figure 4).

[0343] In some embodiments of the present disclosure, SC-RPE enrichment methods andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT systems are described to obtain an enriched population of mature SC-RPE cells, based on detecting the presence of markers CD57, the presence of CD104, and / or the absence of marker CD49b (see e.g., Example 26, 27, 28, and 29).

[0344] In some embodiments of the of the SC-RPE enrichment methods and systems a population of mature SC-RPE cells, is obtained based on detecting the presence of markers CD57, the presence of CD104, and / or the absence of marker CD49b and the presence of pigmentation (see e.g., Example 26, 27, 28, and 29).

[0345] The terms “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a target in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and / or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified, such as presence or absence. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A quantitative detection in the sense of the disclosure comprises detection performed semi-quantitatively, such as, above / below a certain amount of nucleic acid molecules as will be understood by a skilled person.

[0346] The wording “CD57” (Cluster of Differentiation 57) as used herein indicates an antigen also known as HNK1 (human natural killer-1) or LEU7. It is expressed as a carbohydrate epitope that contains a sulfoglucuronyl residue in several adhesion molecules of the nervous system. The B3GAT1 gene codes for an enzyme that plays a key role in a glucuronyl transfer reaction during the biosynthesis of the carbohydrate CD57 epitope. These enzymes exhibit strict acceptor specificity, recognizing nonreducing terminal sugars and their anomeric linkages.

[0052] .

[0347] The wording “CD104” (Cluster of Differentiation 104), as used herein indicates the gene product of integrin beta 4 (ITGB4). When associated with integrin alpha 6, the twoTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT integrin subunits combine to act as laminin receptor.

[0348] The wording “CD49b” (Cluster of Differentiation 49b), as used herein indicates the protein product of the integrin alpha 2 gene (ITGA2). When associated with integrin beta 1 (ITGB1), the two integrin subunits combine to form a functional receptor that binds to laminin, collagen, thrombospondin, E-cadherin, and tenascin.

[0349] In some embodiments of the of the SC-RPE methods an enriched population of mature SC-RPE cells is obtained based on the presence of pigmentation alone as determined by the absorption of light by melanin and / or the scatter of light by melanosomes

[0350] The word “pigmentation” as used herein indicates a colored material that is completely or nearly insoluble in water which is found within cells. In particular, pigmentation of a mature SC-RPE generally indicates the material within the pigment granules of SC-RPE cells which comprise melanin as will be understood by a skilled person. These pigmented granular cell organelles are also referred to as melanosomes. In immature SC-RPE, some diffuse and slight darkening of cells can often be observed in the absence of visibly discernable pigment granules using light microscopy. Melanin has a broad light absorption spectral profile with peak absorption at 320 nm that decreases by 50% by about 400 nm and then decreases to near zero by about 750 nm. Because of the broad spectral absorbance in the visual light spectrum pigmented cells appear golden brown to black under white light illumination depending on its concentration. In cells melanin can readily be detected and quantified by measuring light absorption in the range of 350-650 nm, a spectral range where few other cellular molecules absorb light. Best signal to noise ratios will typically be achieved using light wavelengths in 350-550 nm range. In addition, melanosomes can also be detected based on light scattering. When light is shined on a cell some of the light will be deflected or scattered at narrow angles (forward scatter, FSC), some will be scattered to the side (side scatter, SSC), and some will be reflected or scattered backwards (back scatter, BSC). In the case of cells the amount of FSC is mostly indicative of cell size whereas the amount of SSC and BSC are indicative of the amount of intracellular granularity (melanosomes, mitochondria, lysosomes, vesicles, vacuoles, etc.) Due to the relationship between particle size and scatter, light scatter analysis of cells is often done with laser light wavelengths from 405 to 488 nm, but wavelengths outside of this range can be useful depending on cell and particle size. While side and back light scatter are not exclusive to melanosomes, well pigmented cells can readily be distinguished from most, butTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT not all, non-pigmented cells of similar size based on their relatively high SSC:FSC or BSC:FSC ratios due to highly effective light scatter by melanosomes and the amount of melanosomes per SC-RPE.

[0351] Melanin has a broad light absorption profile and pigmented cells can be readily detected and distinguished from non-pigmented cells using light wavelengths in the range of 350-550 nm light since non-pigmented cells have minimal light absorbance in this range. In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are detected using a cell sorter configured with a 488 nm light absorption detector. Guidance for determining the appropriate sorting thresholds (or gates) can be obtained by determining the light absorption values of known non-pigmented cells, such as stem cells, fibroblasts, human embryonic kidney cells, or non-RPE in the culture as identified based on their CD49b, CD104, and / or CD57 staining profile. Alternatively, as would be understood by a person skilled in the field of cell sorting, in many cases it is possible to identify pigmented cells using scatter or contour plots where the light absorption values are plotted versus one of the light scatter values (forward, side, or back) and the pigment cells can identified as clear population of cells with the highest absorbance values and set gates to select that population. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained.

[0352] The scatter of light by melanosomes is a function of melanosome size and the light wavelength. In general, laser wavelengths suitable for the detection of cells and intracellular particles such as melanosomes using light scatter detection are in the range of 405-488 nm, with 488 nm being the most commonly used. In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are identified using a cell sorter configured with 488 nm laser light side scatter and forward scatter detectors and the values for both parameters for each cell is plotted using a scatter, contour, or ratio plot. Forward scatter signal intensity is proportional to cell size and side scatter is proportional to the amount of intracellular particles, like mitochondria, lysosomes, and melanosomes. Typically, a clear group of cells (comprised largely of pigmented cells) with relatively high side scatter to forward scatter signal intensity ratios compared to a group of cells (comprised largely of non-pigmented and minimally pigmented cells) with a lower side scatter to forward scatter ratio can readily be identified. As would be understood by a person skilled in cell sorting, appropriate side and forward scatterTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT gates can be set to effectively select the group of cells with high side versus forward scatter ratios. Further, guidance for determining the appropriate sorting thresholds (or gates) can be obtained by identifying non-RPE in the culture based on their CD49b, CD104, and / or CD57 staining profile. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained.

[0353] The SC-RPE enrichment method entails the use of a device to separate SC-RPE from non-RPE cells herein also indicated as cell sorters. A cell sorter in the sense of the disclosure has a detector capable of measuring the parameter of interest (fluorescence, light scatter, and / or light absorbance) and has the capability to then separate the desired cell from the unwanted cells. Typically flow cytometer-based instruments are used, but imaging-based devices, such as laser microdissection instruments, can be used to enrich SC-RPE as well. In preferred embodiments of the SC-RPE enrichment method of the disclosure the sorting device is a fluorescence activated cell sorter (FACS). For sorting based on marker staining of all three markers the FACS should have light scatter detectors capable of identifying single cells (forward and side and / or back scatter) and it must be capable of 3-color sorting using fluorescence detectors. For sorting based light absorption by melanin, the sorter must have a detector capable of measuring light absorption at wavelength within the 350-650 nm range and preferably in the range of 350-550 nm, more preferably 405-550 nm, even more preferably 405-488 nm, and most preferably 488 nm. Typically the light source is a laser, but light emitting diodes, band pass filtered light, or spectrometer-type light sources and detectors are possible. In addition, it is preferred that the light absorption enabled sorter is also capable of detecting single cells based on light scatter. For sorting based on the light scattering properties of melanosomes, the sorter must have a detector to measure cell size (typically forward light scatter) and a side and / or back light scatter detector to assess the degree of cell granularity (i.e. melanosome content). The scatter detectors use laser light in the range of 405-650 nm (more preferably 405-550 nm, even more preferably 405-488 nm, and most preferably 488 nm). For sorting based on all parameters it is preferred that a sorter capable of all detection mode is used, but a combination of multiple sorters can be used. For SC-RPE enrichment for the purpose of preparing material for clinical use it is desirable the sorter uses a closed fluidics system and preferably a uses a disposable, self-contained sorting chamber or cartridge An exemplary cellTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT sorter in the sense of the disclosure is described in Example 44 and schematically shown in Figure 25.

[0354] In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are enriched using a FACS configured with 405-650 nm (more preferably 405-550, even more preferably 405-488 nm, and most preferably 488 nm) laser light side scatter and forward scatter detectors and the values for both parameters for each cell is plotted using a dot, contour, or ratio plot. Typically, a clear group of cells with relatively high side scatter to forward scatter signal intensity ratios (comprised largely of pigmented cells) can readily be distinguished from a group of cells with a lower side scatter to forward scatter ratio (comprised largely of non- pigmented and minimally pigmented cells). As would be understood by a person skilled in cell sorting, appropriate side and forward scatter gates can be set to effectively select the group of cells with high side versus forward scatter ratios. Further, guidance for determining the appropriate sorting thresholds (or gates) can be obtained by identifying non-RPE in the culture based on their CD49b, CD104, and / or CD57 staining profile. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained. Alternatively, on suitably configure cell sorters images of the sorted cells can be collected in real-time and the pigmented cell purity can be calculated (also in real-time) using machine learning or artificial intelligence based image analysis. Additional cell sorting methods and devices can be identified by a skilled person.

[0355] In some embodiments of the SC-RPE enrichment method of the disclosure pigmented cells are enriched using a FACS most configured most preferably with a 488 nm light absorption detector (preferably 405-488 nm, less preferably 405-550 nm, and least preferably 350-650 nm) and pigmented SC-RPE cells are identified based on their relatively light absorption compared to non-pigmented cells. Guidance for determining the appropriate sorting thresholds (or gates) can be obtained by determining the light absorption values of known non- pigmented cells, such as stem cells, fibroblasts, human embryonic kidney cells, or non-RPE in the culture as identified based on their CD49b, CD104, and / or CD57 staining profile. Alternatively, as would be understood by a person skilled in the field of cell sorting, in many cases it is possible to identify pigmented cells using scatter or contour plots where the light absorption values are plotted versus one of the light scatter values (forward, side, or back) andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the pigment cells are then identified as a clear population of cells with similar light scatter to each other and the highest absorbance values compared to the other cells, Sorting gates can then be to select pigmented SC-RPE population. Further guidance for gate selection can be got based on known non-pigmented cells as previously described. For best results, the gate selection should be confirmed by sorting a small number of cells and determining the percentage of observable pigment cells by microscopy and cell counting. Depending on the results the gates can be adjusted until the desired purity and yield is obtained. Alternatively, on suitably configure cell sorters images of the sorted cells can be collected in real-time and the pigmented cell purity can be calculated (also in real-time) using machine learning or artificial intelligence based image analysis. Additional cell sorting methods and devices can be identified by a skilled person.

[0356] In some embodiments of the SC-RPE enrichment method of the disclosure SC-RPE are cells are enriched based on their CD49b, CD104, and / or CD57 staining profile using a FACS configured for the detection pigmentation by light absorption and / or light scatter. After identifying single cells based on light scatter, as would be understood by a skilled person, CD49b negative, CD104 positive and / or CD57 positive cells are collected. Guidance for selecting gates can be gotten using negative isotype control antibodies and by comparing the staining profiles of the pigmented and non-pigmented populations as well as by examining dot plots of all possible pairwise marker combination for both the pigmented and non-pigmented cell populations, as would be understood by skilled person.

[0357] It has been found that mature pigmented SC-RPE cells have a specific pattern of CD57, CD104 and CD49b marker expression and that extracellular epitopes of these markers can be recognized by specific probes which survive the process of cell dissociation required for enrichment. In particular they are marked by the presence of CD57 and CD104, and the absence of CD49b. This is consistent with published gene expression profiling of differentiated and mesenchymal RPE where it has been shown that the expression of B3GAT1 (CD57) and ITGB4 CD104) increase as RPE obtain a mature phenotype and that there is minimal expression of ITGA2 (CD49b) in differentiated RPE and elevated expression in mesenchymal RPE

[0021] . The inventors have found that the proteins expressed by B3GAT1 (CD57), ITGB4 (CD104), and ITGA2 (CD49b) are expressed in sufficient levels for detection, do survive the extensive protease digestion required to generate single cells, and therefore can be used as marker. It has also been found that some nonpigmented cells, which have the same markerTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT profile, in most cases can give rise to mature pigmented RPE upon subsequent culture. Cells in the enriched immature RPE population that fail to pigment upon further culture can be cells in the early stage of dedifferentiation to being a mesenchymal RPE, non-RPE with slightly overlapping marker staining profile or aberrant SC-RPE that failed to differentiate properly.

[0358] Accordingly, in some embodiments detection of mature SC-RPE cells can be performed as part of an SC-RPE enrichment method of the disclosure to enrich for a population of mature SC-RPE by selecting pigment cells with an RPE marker profile and to enrich for immature SC-RPE cells by selecting nonpigmented cells with a RPE-marker profile using non- manual methods that are less prone to user bias and skill and that can be readily automated. Enrichment based on RPE marker profile alone will result in the enrichment of a mixed population of mature and immature SC-RPE, many or most of which will mature on further culture. Enriched populations including immature SC-RPE will most always have lower purity but can be useful for some studies and can be cultured and resorted including pigmentation as a selection parameter to obtain more mature SC-RPE cells than in case where the SC-RPE immature cells comprising with an RPE marker profile were simply discarded.

[0359] The method comprises providing differentiated stem cell-derived retinal pigment epithelial (SC-RPE) cells and staining the differentiated SC-RPE cells with a probe specific for a marker selected from CD57, CD104, and / or CD49b to obtain stained differentiated SC-RPE cells stained for CD57 and / or CD104, and not stained for CD49b markers (see e.g. Examples 27 and 28).

[0360] The SC-RPE enrichment method of the disclosure further comprises sorting the stained SC-RPE cells to select the differentiated SC-RPE cells stained for CD57 and / or CD104 markers, and / or to discard cells stained for the CD49b marker, to obtain a population of SC- RPE cells enriched in mature SC-RPE cells.

[0361] In preferred embodiments of the SC-RPE enrichment method, the cells are sorted based on their marker staining profile and the presence of pigment relative to known or identifiable nonpigmented cells as a skilled person would understand upon reading of the disclosure.

[0362] In some embodiments of the SC enrichment, the cells can be isolated based on the presence of pigment alone.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0363] The wording “staining” as used herein in connection with cell detection indicates a series of techniques that can be used to improve or enable detection such as optical detection of the cells as will be understood by a skilled person. In particular, the word staining as used herein indicates techniques that can be used to enable or improve detection of living cells; in particular living SC-RPE cells following contacting with a probe specific for any one of the CD57, CD104, and / or CD49b markers as will be understood by a skilled person.

[0364] The term “probe” as described herein indicates a molecule capable of specifically detecting a target molecule such as one of the markers herein described. The wording “specific”, “specifically” or “specificity” as used herein with reference to the binding of a first molecule to second molecule refers to the recognition, contact and formation of a stable complex between the first molecule and the second molecule, with substantially less to no recognition, contact and formation of a stable complex between each of the first molecule and with other molecules that can be present. Exemplary specific bindings are antibody-antigen interaction, cellular receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions, and additional interactions identifiable by a skilled person.

[0365] In some embodiments, staining can be performed with probes specific for any one of the CD57, CD104, and / or CD49b such as antibodies, and or aptamers with the further requirement that the probe must recognize an epitope in the markers that survives the procedure to obtain single cells such as protease digestion during the process of producing single cells after SC-RPE differentiation and maturation, or any other culture or tissue containing SC-RPE or RPE. (e.g. Examples 27 and 28).

[0366] Depending on the selected mode of cell isolation, the probe can be coupled to any molecule that allows for sorting of the cells with appropriate staining pattern such as a fluorescent molecule, a paramagnetic bead or particle, a solid support, or a ligand or receptor.

[0367] In some embodiments the probe specific for CD57, CD104, and CD49b comprise anti- CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE / Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies.

[0368] The wording “sorting” as used herein in connection with cells, indicates the process through which a particular cell type is separated from others contained in a sample based on its physical or biological properties, such as size, morphological parameters, viability and bothTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT extracellular and intracellular protein expression

[0053] .

[0369] Examples of methods to perform cell sorting in the sense of the disclosure comprise Fluorescence Activated Cell Sorting (FACS), magnetic cell sorting, or solid phase affinity selection as will be understood by a skilled person

[0053] .

[0370] In some embodiments, the sorting can be performed based on a relative high side scatter (SSC) to forward scatter (FSC) ratio to identify melanosome containing pigmented cells and negative CD49b expression, positive CD104, and positive CD57 expression (see e.g., Example 29).

[0371] In some embodiments, light absorbance-based sorting and marker-based enrichment of SC-RPE cells can be performed by detecting light absorbance from a target cell culture and selecting cells of the target cell culture having absorbance of light between 350-650 nm (more preferably between 405-550 nm, even more preferably 405-488, and most preferrable 488 nm) as compared to known nonpigmented negative control cells or identifiable nonpigmented cells in the population and by negative CD49b expression, positive CD104, and / or positive CD57 expression. In some embodiments, the method can further comprise calibrating a light absorption threshold setting by sorting a small number of test cells, quantifying the percentage of pigmented cells in the sorted population, and then adjusting the threshold setting as necessary to achieve a desired yield and pigmented cells purity. Alternatively, on a suitably configured cell sorter, images of the sorted cells can be collected in real-time and the pigmented cell purity can be calculated (also in real-time) using machine learning or artificial intelligence based image analysis as will be understood by a skilled person. Additional cell sorting methods and devices can be identified by a skilled person.

[0372] In some embodiments, the sorting can be performed by selecting a population with a relative high absorbance of light within the range of 350-650 nm and with a relative high side scatter (SSC) to forward scatter (FSC) ratio, as compared to known nonpigmented cells, to identify pigmented cells and with negative CD49b expression, positive CD104, and / or positive CD57 expression.

[0373] In some embodiments, the sorting can be performed based on a relative high side scatter (SSC) to forward scatter (FSC) ratio, as compared to known nonpigmented cells, to identify melanosome containing pigmented cells.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0374] In some embodiments, the sorting can be performed by selecting a population with a relative high absorbance of light in the range of 350-650 nm, as compared to known nonpigmented cells, to identify pigmented cells.

[0375] In some embodiments, the sorting can be performed based by selecting a population with a relative high absorbance of light in the range of 350-650 nm, as compared to known nonpigmented cells, to identify pigmented cells using microfluidics, microchip-based FACS.

[0376] Methods to perform cell sorting in the sense of the disclosure further comprise affinity selection of SC-RPE cells. For example, a suspension of RPE cells can be first stained with an anti-CD49b antibody coupled to a paramagnetic particle and CD49b positive cells can be removed by magnetic selection. The resulting CD49b negative cells can then be stained with anti-CD104 antibodies coupled to a paramagnetic particle and with biotin conjugated anti- CD57 antibody. CD104 positive cells can then be captured by magnetic selection and after release the CD57 positive cells can be collected using a streptavidin coupled solid support. Other similar strategies are possible as would be understood by a person skilled in the field.

[0377] The homogeneous cell population obtained after sorting, which is optionally subjected to post-sorting recovery period (see e.g. Example 30), can be used for a variety of applications including research, diagnosis, and therapy as will be understood by a skilled person

[0053] .

[0378] In embodiments of the enrichment method of the disclosure positive selection can be performed based on the cell surface markers CD57 and / or CD104, which have been determined to be expressed on mature SC-RPE, and / or negative selection based on the absence of CD49b, which has been determined to be expressed on RPE that have undergone epithelial-to- mesenchymal transition and presumably by some non-SC-RPE.

[0379] In embodiments of the enrichment method of the disclosure, each of the positive detection of any one cell surface markers CD57 and / or CD104, and negative detection of CD49b can be used to select mature SC-RPE cells. Preferably selection is performed following multiple detection of at least two of CD57, CD104, and CD49b. More preferably detection of all three markers is performed.

[0380] Accordingly, in some embodiments the staining is performed to label cells presenting CD57 and CD104 and the selected cells are SC-RPE cells positive for at least one and preferably both of CD57 and CD104.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT

[0381] In some embodiments the staining is performed to label cells presenting CD57 and CD49b and the selected cells are SC-RPE cells positive for CD57, SC-RPE cells negative for CD49b and preferably SC-RPE cells each positive for CD57 and negative for and CD49b.

[0382] In some embodiments the staining is performed to label cells presenting CD104 and CD49b and the selected cells are SC-RPE cells positive for CD104, SC-RPE cells negative for CD49b and preferably SC-RPE cells each positive for CD104 and negative for and CD49b.

[0383] In some embodiments the staining is performed to label cells presenting CD57, CD104 and CD49b and the selected cells are SC-RPE cells positive for CD57, SC-RPE cells positive for CD104, and / or SC-RPE cells negative for CD49b. In preferred embodiments, the selected cells are SC-RPE cells are each positive for at least one and more preferably both of CD57 and CD104 or positive for CD104 and negative for and CD49b, or positive for CD57 and negative for CD49b. In most preferred embodiments, the selected cells are SC-RPE cells are each positive for both of CD57 and CD104 and negative for CD49b.

[0384] In embodiments of the enrichment method and system of the present disclosure while it is possible to achieve a large degree of enrichment based on a single cell surface marker alone, better levels of enrichment are achieved using multiple markers as will be understood by a skilled person upon reading of the present disclosure.

[0385] In addition, the enrichment method of the present disclosure can further comprise detecting RPE pigmentation of the SC-RPE cells based on light scattering by melanosome and / or or absorbance of light by melanosomes and melanin and selecting the SC-RPE cells having pigmentation associated with all or a subset of the mature SC-RPE markers (see e.g. Example 29).

[0386] In some embodiments, sorting of cells can be performed by FACS based on multiple criteria. In those embodiments, the choice of fluorescent labels will be dependent on the specifications of the cell sorter, but as much as possible dyes with high quantum yield and fluorescent intensity should be used to best discriminate labeled from unlabeled cells.

[0387] When sorting by FACS based on multiple criteria (more than one of pigmentation, CD57, CD104 and CD49b) the order of gating or gating protocol is irrelevant. However, the proper selection of the gate locations or windows can be aided by comparing the fluorescent labeling patterns of the pigmented and nonpigmented cell populations using all possibleTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT pairwise marker comparisons (i.e., CD57 vs. CD104, CD57 vs CD49b, and CD104 vs CD49b) to achieve the best level of purity and yield.

[0388] In some embodiments of the SC-RPE enrichment method and related system of the disclosure, the sorting is preferably based on detection of CD57, CD104 and CD49b as well as based on the detection of pigmentation of the SC-RPE cells to increase the final purity of mature SC-RPE cells obtained by the method.

[0389] In those preferred embodiments, the sorting of SC-RPE cells in the enrichment method of the disclosure, can further comprise sorting SC-RPE cells having a detectable pigmentation to further select mature SC-RPE cells based on presence or absence of pigmentation. In those embodiments, detecting SC-RPE cells having a detectable pigmentation can be performed after marker staining and the pigment-based sorting can be done in conjunction with sorting based on presence or absence of markers CD57, CD104 and / or CD49b. Alternatively, the pigmentation-based sorting can be done prior to staining. The resulting enriched population of pigmented cells are then stained follow by a round of sorting based on the presence or absence of markers CD57, CD104 and / or CD49b in conjunction with or without a second round of pigment sorting.

[0390] In those preferred embodiments, the enrichment method and systems of the disclosure can be performed by a combination of any method to perform marker-based selection with a cell sorting method based on absorbance of light by melanin and / or side-scatter of light by melanosomes to discriminate pigmented RPE from non-pigmented cells as will be understood by a skilled person upon reading of the present disclosure. For detecting absorbance wavelengths from 350 to 650 nm are possible, but the best signal to noise ratios are expected in the range from at least 400 nm, preferably from 405 to 550 nm. The same general guidance regarding wavelength is true for use for detection of melanosome by determination of side- scatter (see e.g., Example 29).

[0391] Accordingly in some embodiments, the detection of pigmented mature SC-RPE can be performed by measuring light absorbance readings at wavelength of at least 400 nm, preferably between 480-600 nm to quantitively detect pigmented culture area. ^

[0392] In some embodiments, detection of pigmented mature SC-RPE can be performed by by light scattering, preferably by detecting relative light side scatter to forward scatter ratioTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT following directing a light beam towards the cell culture are.

[0393] In some embodiments of the SC-RPE enrichment method and related system of the disclosure, sorting can be performed using a subset of markers to yield satisfactory results in cases of cell lines that have high differentiation efficiencies, where yield is a priority over purity, or where it is desirable to reduce cost and complexity as will be understood by a skilled person upon reading of the disclosure.

[0394] In some of the embodiments where the use antibodies or probes is not desired and the cells are enriched based on physical properties and the presence of melanin or melanosomes alone, improvements in purity can be expected by staining a portion of the total population for one or more of the markers CD49b, CD104, and CD57 and use the resulting staining profile to aid in the selection of the optimal scatter and pigment sorting gates. The resulting gates can then be used to sort the reserved unstained portion of the cell population. A similar strategy can be used when only a subset of marker probes is used for the actual sorting process.

[0395] In some embodiments sorting using multiple sort modalities can be combined to result in shortened sorting processing times. As an example, cells can be stained with an anti-CD57- paramagnetic microbead (Miltenyi BioTec catalog number 130-092-073), anti-CD104-PE, and anti-CD49b / APC. After capture and release of the CD57 positive cells by magnetic selection, the resulting CD57 positive cells can be further enriched by FACS based on, positive CD104 staining, negative CD49b staining, and / or pigmentation. Similarly, unwanted CD49b positive cells can be removed first by magnetic bead separation and the remaining cells can be sorted by FACS based on CD57, CD104, and / or pigmentation. Furthermore, when selection of CD57 or CD104 cells is carried out by methods such as paramagnetic bead selection, the cells can be dual stained with fluorescent labeled antibodies and paramagnetic beads directed at the same antigen. In this way, a bulk separation of CD57 or CD104 positive cells is possible while still allowing for isolation based quantitative levels of expression of all three markers and pigmentation. This can be especially advantageous with populations of cells with heterogenous expression of the RPE specific markers and allows for both decreased sorting times and enrichment of an SC-RPE population with enhanced homogeneity and purity.

[0396] In addition, or in the alternative, in some preferred embodiments, the enrichment method and systems of the disclosure, can be performed in combination with detection and separation of pigmented cells based on the light scattering or absorbance property ofTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT melanosomes or melanin.

[0397] In particular, in some embodiments, SC-RPE with the desired phenotype can then then isolated using fluorescence activated cell sorting (FACS, see e.g., Example 29).

[0398] In addition, in some embodiments the enrichment method and systems of the disclosure can be performed in combination with staining with viable or dead cell dyes or reagents to allow for the selection of viable SC-RPE.

[0399] In some embodiments of the SC-RPE enrichment method and related system of the disclosure, additional consideration for selection of the set of markers and / or pigmentation to be used for the staining and sorting steps, comprise the observation that nonpigmented cells that have the proper CD antigen expression profile can in some cases yield substantial number of cells that will give rise to pigmented SC-RPE upon re-culture, even though such a result is expected to generally lead to lesser purity due to the preponderance of non-SC-RPE in this population and overlapping labeling profiles. A skilled person will thus be able to identify the proper set of markers to use in specific application based on the specific starting cells, and experimental design.

[0400] In some embodiments of the enrichment methods and systems of the disclosure the staining and sorting can be preceded by a cell harvesting in particular in embodiments where the starting SC-RPE cells are SC-RPE differentiated cells of the disclosure (see e.g., Example 27). The harvesting is preferably performed at a time when the increase in the number pigmented cells and their degree of pigmentation begins to plateau. This time will vary depending on the specific stem cell line:media:substrate combination, but it is typically between 60-90 days after the initial plating for differentiation. Enrichment of SC-RPE at times prior to the plateau in the percentage of pigmented cells in the culture will result in correspondingly lower yields due to decreased numbers of SC-RPE, reduced or heterogeneous levels of marker expression, and reduced levels of pigmentation. After the plateau of pigmentation is reached no further increases in yields or lasting improvement in RPE phenotype are expected. Enrichment at times up to differentiation day 120 are generally acceptable, but with increasing time in culture decreased yields can result due to increasing difficulty in preparing single cell suspensions.

[0401] In some embodiments of the enrichment methods of the disclosure and systems andTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT related systems, the method comprises a cell harvesting step, the cell harvesting can be performed by dissociating the cultures by protease digestion using standard cell culture methods appropriate for the chosen protease. Preferred protease employed in this embodiment of the enrichment method comprise trypsin, TrypLE™, Accutase™ and Accumax™. Additional protease such as papain or dispase are expected to be possibly used. The proteases can be used alone, in combination, or sequentially. The preferred method is to use sequential digestion with trypsin or TrypLE™ in conjunction with Accutase™ or Accumax™. Beyond being able to dissociate the cultures into viable single cells that can survive the rigors of the enrichment process, the primary requirement directing the choice of protease is that it does not destroy the epitope recognized by the detection reagent, as will be understood by a skilled person.

[0402] In some embodiments of the enrichment methods of the disclosure and systems and related systems, the method comprises a post-sort recovery step, based on the observation that after sorting the cells can be used directly, but higher quality and yields are generally obtained if one allows them to recover and reenter the cell cycle before downstream use. In those embodiments, the post recovery step is preferably performed by allowing cells to grow to near or just confluence. Shorter periods of recovery are acceptable but will be associated with reduced yields. Substantially longer periods of time that allow for the cells to establish a mature phenotype generally result in difficulties with subsequent harvesting of the cells and are therefore less preferred embodiments.

[0403] In some embodiments of the enrichment method post-sort recovery step, cells are plated on substrate coated cultureware, at a preferrable density of about 75,000 to 150,000 cell / cm2. Lower plating density down to about 10,000 cell / cm2are possible and can result in higher final yields, but at an increased risk of reduced RPE quality due to epithelial-to- mesenchymal transition (EMT). Higher densities up to about 250,000 are possible but will reduce the final yield of SC-RPE.

[0404] Th...

Claims

Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT CLAIMS 1. An RPE Maturation Medium comprising the base medium ^-MEM supplemented with additional non-essential amino acids (glycine, alanine, asparagine, aspartate, glutamate, proline, and serine), insulin, transferrin, selenite, putrescine, progesterone, serum albumin, galactose, catalase, superoxide dismutase, D,L-alpha-tocopherol, D,L-alpha-tocopherol acetate, glutathione, ethanolamine, linoleic acid, linolenic acid, biotin, carnitine, vitamin A, taurine, triiodothyronine, corticosterone, hydrocortisone and optional antibiotics and antimycotics in an effective amount to promote differentiation and maturation of partially differentiated SC-RPE cells, presumptive SC-RPE progenitors, and immature SC-RPE.

2. The RPE Maturation Medium of claim 1, wherein the maturation medium has compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 3. The RPE Maturation Medium of claim 1, wherein the maturation medium is comprised of the base medium ^-MEM, additional nonessential amino acids, taurine, hydrocortisone, one of the N1-based supplements (N1, N-2, CTS-N-2, N-2-Plus, N-2-MAX, GMP-N-2-MAX, Custom N1) and one of the B27-based supplements, optionally comprising B27, B27+, XF- B27, CTS-B27, NeuroCult SM1, NS21, N21-MAX, GMP N21-MAX and Custom B27 and optional antibiotics and antimycotics at amounts and concentration ranges as defined in the disclosure.

4. The RPE Maturation Medium of claim 1, wherein the maturation medium is comprised of the base medium ^-MEM, additional nonessential amino acids, taurine, hydrocortisone, one of the B27-based supplements (B27, B27+, XF-B27, CTS-B27, NeuroCult SM1, NS21, N21- MAX, GMP N21-MAXand Custom B27) and optional antibiotics and antimycotics at amounts and concentration ranges as defined in the disclosure.

5. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

6. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 7. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

8. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

9. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

10. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

11. The RPE Maturation Medium of claim 1, wherein the maturation medium has the compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

12. A method to maturate a differentiated stem cell-derived retinal pigment epithelial (SC- RPE) cell, the method comprising contacting differentiated SC-RPE, with a cell culture maturation medium of the present disclosure for a time and under conditions to obtain matured stem cell-derived retinal pigment epithelial (SC-RPE) cell.

13. The method of claim 12, wherein the cell culture maturation medium is the RPE Maturation Medium of any one of claims 1 to 11.

14. The method of claim 12 or 13, wherein the contacting is performed for a time selected to obtain a target percentage of mature SC-RPE cells.

15. The method of claim 12 or 13, wherein the contacting is performed for a time selected to obtain a target quality of mature SC-RPE cells.

16. The method of claim 12 or 13, wherein the contacting is performed for a time selected to obtain a target percentage of mature SC-RPE cells in combination with a target quality of mature SC-RPE cells.

17. The method of any one of claims 12 to 16, wherein the contacting is performed for a time ranging from 15 to 90 days.

18. The method of any one of claims 12 to 16, wherein the contacting is performed for a time of about 35 days.

19. The method of any one of claims 12 to 18, wherein the differentiated stem cells are SC- RPE differentiated cells obtained byTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT contacting stem cells of a starting stem cell line with a cell culture support comprising an SC-RPE differentiation medium an SC-RPE cell culture substrate wherein the SC-RPE differentiation medium and the SC-RPE cell culture substrate are selected depending on the starting stem cell line, and wherein the contacting is performed for a time and under conditions to obtain the differentiated SC-RPE cell.

20. The method of claim 19, wherein SC-RPE differentiated cells are obtained by spontaneous differentiation methods and / or with a system configured for spontaneous differentiation, and wherein contacting differentiated SC-RPE cells with a cell culture maturation medium is performed beginning between and about 7 to 60 days after the initial plating; preferably from 14 to 50 days, more preferably from 21 to 37 days and even most preferably on about day 30.

21. The method of claim 19 wherein SC-RPE differentiated cells of claim are obtained by semi- directed differentiation methods and / or with a system configured for semi-directed differentiation, and wherein contacting differentiated SC-RPE cells with a cell culture maturation medium begin from 7 to 60 days after the initial plating, preferably from about 14 to 60 days after the initial plating, more preferably from 14 to 50 days after the initial plating, even more preferably between 21 and 37 days after the initial plating, most preferably about 30 days after the initial plating.

22. A matured SC-RPE cell population, obtained by the method of any one of claims 12 to 19 and / or by using the RPE Maturation Medium of any one of claims 1 to 11.

23. A method to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing the matured stem cell-derived retinal pigment epithelial (SC-RPE) cells population of claim 22; contacting the matured SC-RPE cells population with a probe specific for a marker selected from CD57, CD104, and / or CD49b to obtain stained mature SC-RPE cells stained for CD57, CD104, and / or CD49b markers; and sorting the stained SC-RPE cells to select the mature SC-RPE cells stained for CD57 and / or CD104 markers, and discard cells stained for CD49b marker, to obtain a population ofTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT SC-RPE cells enriched in mature SC-RPE cells.

24. The method of claim 23, wherein the providing comprises harvesting SC-RPE cells by dissociating the cultures by protease digestion.

25. The method of claim 23 or 24 wherein the probes specific for CD57, CD104, and / or CD49b comprise one or more of anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57- PE / Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies.

26. The method of any one of claims 23 to 24 wherein the sorting can be performed by Fluorescence Activated Cell Sorting or FACS and / or Immunomagnetic cell sorting.

27. The method of any one of claims 23 to 26, wherein the contacting is performed with one or more probes specific for CD57 and / or CD104 and the sorting is performed to select SC-RPE cells positive for at least one of CD57 and CD104, and / or SC-RPE cells each positive for both of CD57 and CD104.

28. The method of any one of claims 23 to 27, wherein the contacting is performed with one or more probes specific for CD57 and / or CD49b and the sorting is performed to select SC-RPE cells positive for CD57, SC-RPE cells negative for CD49b and / or SC-RPE cells each positive for CD57 and negative for CD49b.

29. The method of any one of claims 23 to 27, wherein the contacting is performed with one or more probes specific for CD104 and / or CD49b and the sorting is performed to select SC- RPE cells positive for CD104, SC-RPE cells negative for CD49b, and / or SC-RPE cells each positive for CD104 and negative for CD49b.

30. The method of any one of claims 23 to 29, wherein the contacting is performed with one or more probes specific for CD57, CD104 and / or CD49b and the sorting is performed to select SC-RPE cells positive for CD57, SC-RPE cells positive for CD104, and / or SC-RPE cells negative for CD49b.

31. The method of any one of claims 23 to 29, wherein the contacting is performed with one or more probes specific for CD57, CD104 and / or CD49b and the sorting is performed to select SC-RPE cells positive for at least one of CD57 and CD104, SC-RPE cells positive for both of CD57 and CD104, SC-RPE cells each positive for CD104 and negative for CD49b, SC-RPETitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT cells each positive for CD57 and negative for CD49b, and / or SC-RPE cells each positive for both of CD57 and CD104 and negative for CD49b.

32. The method of any one of claims 23 to 31, further comprising detecting pigmentation in stained mature SC-RPE cells to identify pigmented stained mature SC-RPE cells and sorting the stained SC-RPE cells to select the stained mature SC-RPE cells with detected pigmentation and discard the stained SC-RPE cells with no detected pigmentation. 33 The method of claim 32, wherein the detection is performed by qualitative macroscopic detection of pigmented cells on a suitable background. 34 The method of claim 32 or 33, wherein the detection is performed by performing by image analysis, such analysis of images by performing macroscopic and / or light microscopy.

35. The method of any one of claims 32 to 34, wherein the detection is performed by measuring light absorbance readings at wavelength of at least 400 nm, preferably between 480-600 nm to quantitively detect pigmented culture area. ^ 36 The method of any one of claims 32 to 35, wherein the detection is performed by light scattering, preferably by detecting relative light side scatter to forward scatter ratio following directing a light beam towards the cell culture area.

37. A method to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing the matured stem cell-derived retinal pigment epithelial (SC-RPE) cells population of claim 22; detecting pigmentation in SC-RPE cells of the mature SC-RPE cell and sorting the mature SC-RPE cell populations to select pigmented mature SC-RPE cells and discard mature SC-RPE cells with no detected pigmentation.

38. The method of claim 37, wherein the providing comprises harvesting SC-RPE cells by dissociating the cultures by protease digestion.

39. The method of claim 37 or 38, further comprising contacting the pigmented mature SC-RPE cells. with a probe specific for a markerTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT selected from CD57, CD104, and / or CD49b to obtain stained pigmented mature SC-RPE cells stained for CD57, CD104, and / or CD49b markers; and sorting the stained SC-RPE cells to select the pigmented mature SC-RPE cells stained for CD57 and / or CD104 markers, and discard pigmented cells stained for CD49b marker, to obtain a population of SC-RPE cells enriched in mature SC-RPE cells.

40. The method of any one of claims 23 to 39, wherein the method further comprises post- sorting cell recovery of the population of SC-RPE cells enriched in mature SC-RPE cells.

41. A system to enrich mature stem cell-derived retinal pigment epithelial (SC-RPE) cell of claim 22, the system comprising at least two probes each specific for at least one marker selected from CD57, CD104, and / or CD49b for combined use to stain differentiated SC-RPE cells in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of any one of claims 23 to 31.

42. The system of claim 41, further comprising an imaging device and / or a cell sorter for combined use to detect pigmented mature SC-RPE cells in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of any one of claims 32 to 40.

43. The system of claim 41 or 42, wherein the probe specific for at least one of CD57, CD104, and CD49b comprise one or more of anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti- CD57-PE / Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b-APC (Clone: REA188, Miltenyi Biotec) antibodies.

44. A system to enrich the mature stem cell-derived retinal pigment epithelial (SC-RPE) cell of claim 20, the system comprising mature stem cell-derived retinal pigment epithelial (SC- RPE) cell of claim 22, in combination with an imaging device and / or a cell sorter for combined use in a method to obtain a population of SC-RPE cells enriched in mature SC-RPE cells of any one of claims 32 to 40.

45. An enriched SC-RPE mature cell population obtained with the method of any one of claims 23 to 40 and / or with the system of claims 41 to 44.

46. An RPE Maintenance Medium comprising the RPE Maturation Medium of any one of claims 12 to 21 supplemented with basic fibroblast growth factor (bFGF), a protein kinase inhibitor, and dimethyl sulfoxide (DMSO) in an effective amount to promote expansion of mature SC-RPE cells.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 47. The RPE Maintenance Medium of claim 46, wherein the protein kinase inhibitor is Rho- Associated Coiled-Coil Containing Protein Kinase (ROCK) inhibitor.

48. The RPE Maintenance Medium of claim 47, wherein the ROCK inhibitor is Y-27632, Thiazovivin, or RevitaCell.

49. The RPE Maintenance Medium of claim 46, wherein the protein kinase inhibitor is any one of the transforming growth factor beta receptor (TGF^R) kinase inhibitors RepSox, A-83-01, or SB-431542.

50. The RPE Maintenance Medium of claim 46, wherein the protein kinase inhibitor is any one of the group of ROCK inhibitors Y-27632, Thiazovivin, or RevitaCell in combination with any one of the TGF^R kinase inhibitors RepSox, A-83-01, or SB-431542.

51. A method for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising providing increased numbers of stem cell-derived retinal pigment epithelial (SC-RPE) cells; and contacting the SC-RPE cells with the RPE Maintenance Medium of any one of claims 46 to 50 on an SC-RPE substrate, the contacting performed for a time and under condition to obtain a target cell density of the SC-RPE cells.

52. The method of claim 51, wherein the providing comprises providing SC-RPE cells thawed and plated from frozen SC-RPE stocks.

53. The method of claim 51 or 52, wherein the method further comprises harvesting the SC- RPE cells and replating the harvested SC-RPE cells until the cultures reach the desired density.

54. The method of any one of claims 51 to 53, wherein the method is further performed to expand the SC-RPE cells.

55. The method of any one of claims 51 to 54, wherein the SC-RPE cell culture substrate is selected from laminin, Vitronectin, Matrigel, Geltrex, Cultrex BME, Collagen IV and combinations thereof.

56. The method of any one of claims 51 to 55, wherein the SC-RPE cell culture substrate isTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin, laminin 332, and laminin 411.

57. The method of any one of claims 51 to 56, wherein the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof.

58. The method of any one of the claims 51 to 57, wherein the culture medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and / or TGF^R kinase inhibitor.

59. The method of any one of claims 51 to 58, wherein the SC-RPE cells are SC-RPE differentiated cells, the mature SC-RPE cells of claim 22 and / or the enriched SC-RPE cell population of claim 45.

60. An SC-RPE expansion system for expansion of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the system comprising the RPE Maintenance Medium any one of claims 46 to 50, and an SC-RPE substrate of for combined use in a method to perform expansion of the SC- RPE cells of any one of claims 51 to 59.

61. The system of claim 60, wherein the RPE Maintenance Medium comprises the RPE Maturation Medium of any one of claims 1 to 9.

62. The system of claim 60 or 61, wherein the culture medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and / or TGF^R kinase inhibitor.

63. The system of any one of claims 60 to 62, wherein the SC-RPE cell culture substrate is selected from laminin, Vitronectin, Matrigel, Geltrex, Cultrex BME, Collagen IV and combinations thereof.

64. The system of any one of claims 60 to 63, wherein the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin 332, and laminin 411.

65. The system of any one of claims 60 to 64, wherein the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin and combinations thereof.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 66. A method to prepare frozen stock of stem cell-derived retinal pigment epithelial (SC-RPE) cells, the method comprising - performing expansion of SC-RPE cells with the method of any one of claims 51 to 59 to obtain SC-RPE cells having a target cell density - harvesting the SC-RPE cells having the target cell density to obtain harvested SC-RPE cells; and - freezing the harvested SC-RPE cells.

67. A system for frozen stock preparation of stem cell-derived retinal pigment epithelial (SC- RPE) cell, the system comprising the SC-RPE maturation medium of any one of claims 1 to 11 and / or preferably the RPE Maintenance Medium of any one of claim 46 to 50 and / or an SC-RPE substrate of the present disclosure and freezing media, for combined use in a method to provide frozen stock of claim 66.

68. The system for frozen stock preparation of claim 67, wherein the RPE Maintenance Medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and / or TGF^R kinase inhibitor.

69. A frozen stock preparation of (SC-RPE) cell obtainable or obtained with the method of claim 66, or with the system of claims 67 or 68.

70. A method to perform the production of stem cell-derived retinal pigment epithelial (SC- RPE) patches (SC-RPE Patch), the method comprising - providing enriched SC-RPE cell population preferably the enriched cell population of claim 45, or a frozen SC-RPE stock, preferably the SC-RPE frozen stock of claim 69; - performing optional expansion of SC-RPE cells to obtain cells having a target cell density; and - contacting the SC-RPE cells having the target cell density with a physical support, RPE Maturation Medium of any one of claims 1 to 11, and / or preferably the RPE Maintenance Medium of any one of claims 46 to 50 to obtain a population of quiescent partially mature and / or mature SC-RPE cell monolayers.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 71. A system to perform the production of stem cell-derived retinal pigment epithelial (SC- RPE) patches (SC-RPE Patch), the method comprising enriched SC-RPE of claim 45 or frozen SC-RPE stock of claim 69, performing optional expansion of SC-RPE cells with the method of any one of claims 51 to 59 to obtain SC-RPE cells having the potential to mature and having a target cell density; contacting the SC-RPE cells having the target cell density with a physical support, for example a membrane support and / or a microcarrier support) and at least one of the RPE Maturation Media of any one of claims 1 to119, and / or preferably the RPE Maintenance Medium of any one of claims 46 to 49 to obtain a population of quiescent partially mature and / or mature SC-RPE cells monolayers.

72. The system for SC-RPE Patch production of claim 71, wherein the RPE Maintenance Medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and / or TGF^R kinase inhibitor.

73. A method to derive retinal pigment epithelial (RPE) cell, from a starting stem cell (SC), the method comprising differentiating the stem cell (SC) to obtain differentiated stem cell-derived retinal pigment epithelial (SC-RPE); and maturating the differentiated SC-RPE cells to obtain matured SC-RPE cells, the method optionally further comprising at least one of performing enrichment of matured SC-RPE stem cells, preparing a frozen stock of the SC-RPE, performing expansion of the SC-RPE and / or providing the matured SC-RPE on a physical support to obtain a population of quiescent partially matured or matured SC-RPE cells monolayers wherein the maturating, is performed by the method of any one of claims 12 to 21, the performing enrichment is performed by the method of any one of claims 23 to 40, the performing expansion is performed by the method of any one of claims 51 to 59,Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT the preparing a frozen stock is performed by the method of claim 66, and / or the providing the quiescent partially mature and / or mature SC-RPE on a physical support is performed according to method of claim 70.

74. The method of claim 73, wherein the differentiating is performed by contacting stem cells of a starting stem cell line with a cell culture support comprising a SC-RPE differentiation medium an SC-RPE cell culture substrate wherein the SC-RPE differentiation medium and the SC-RPE cell culture substrate are selected depending on the starting stem cell line, and wherein the contacting is performed for a time and under conditions to obtain a differentiated SC-RPE cell.

75. The method of claim 74, wherein the SC-RPE differentiation medium is a RPE-DM1 culture medium KnockOut™ DMEM supplemented with KnockOut™ Serum Replacement Medium, GlutaMAX™, nonessential amino acid supplement, ^-mercaptoethanol, and normocin.

76. The method of claim 75, wherein the concentrations of the RPE-DM1 medium has compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

77. The method of claim 75, wherein the RPE-DM1 has compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

78. The method of claim 75, wherein the concentration of the RPE-DM1 medium has compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

79. The method of claim 75, wherein the concentration of the RPE-DM1 medium has compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 80. The method of claim 75, wherein the concentration of the RPE-DM1 medium has compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT.

81. The method of claim 75, wherein the RPE-DM1 medium is comprised of a base medium, KOSR medium supplement in a concentration from 5 to 30%, from 0.25 to 4X NEAA supplement, ^-mercaptoethanol in a concentration from 25 to 200 mM, and glutamine and / or by L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM.

82. The method of claim 81, wherein the KOSR medium supplement is clinical grade (CTS- certified for manufacture of cells for clinical use).

83. The methods of claims 81 or 82, wherein the concentration of glutamine is 2mM.

84. The methods of any one of claims 81 to 83, wherein the RP-DM1 medium comprises glutamine in a concentration from 1.35 to 3.71 mM, or L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM, or a combination of glutamine and L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM.

85. The methods of any one of claims 81 to 84, wherein the concentration of NEAA supplement is preferably 0.5-2X, more preferably from 0.75-1.25X, or even more preferably 1X.

86. The methods of any one of claims 81 to 84, wherein the concentration of ^-mercaptoethanol is preferably from 50 to 150 mM, more preferably from 75 to 125 mM and even moreTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT preferably 100 mM.

87. The methods of any one of claims 81 to 86, wherein the concentration of the KOSR medium supplement is preferably 10-25%, more preferably between 15-20%, and even more preferably 20%.

88. The methods of any one of the claims 81 to 87, wherein the base medium is KO-DMEM or CTS-KO DMEM.

89. The methods of any one of the claims 81 to 88, wherein the base medium is KO- DMEM:F12 or CTS-KO-DMEM:F12.

90. The methods of any one of the claims 81 to 87, wherein the base medium is ^-MEM.

91. The methods of any one of the claims 81 to 87, wherein the base medium is RPMI1640.

92. The methods of any one of claims 81 to 87, wherein the base medium is IMDM.

93. The methods of claims 91 and 92, wherein the concentration of the KOSR medium supplement is preferably from 15 to 20%, more preferably from 10 to 20%, even more preferably 20%.

94. The method of claim 74, wherein the SC-RPE differentiation medium is an RPE-DM2 culture medium having compositionTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCTTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT possibly comprising or consisting of the cell culture medium, X-VIVO 10, preferably supplemented with a B27-based supplement at a 0.25 to 3X strength, more preferably 0.5 to 2X strength, and even more preferably 0.75 to 1.25X strength and most preferably at 1X strength.

95. The method of claim 94, wherein the B27-based supplement is selected from any one of CTS-B27, B27, B27+, XF-B27, NeuroCult SM1, NS21, N21, N21-MAX, GMP N21-MAX, Custom B27, or Custom N21.

96. The method of claim 94, wherein the RPE-DM2 culture medium is solely comprised of the cell culture medium, X-VIVO 10 and thus consists of cell culture medium, X-VIVO 10.

97. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced by any one of StemSpan SFEM, StemSpan H3000, or StemSpan ACF culture medium.

98. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced with AIM V or CTS-AIM V culture medium.

99. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced with IDMM supplemented with insulin, transferrin, selenate, and albumin.

100. The methods of claims 94 to 96, wherein the culture medium X-VIVO 10 is replaced with RPMI1640 supplemented with insulin, transferrin, selenate, and albumin.

101. The method of any one of claims 75 to 100, wherein the contacting is performed to obtain a spontaneous differentiation of SC-RPE cells.

102. The method of claim 100 wherein the contacting is performed for a time from about 11 to 56 days, more preferably 21-42 days, and most preferably 28-35 days.

103. The method of any one of claims 77 to102, wherein the SC-RPE differentiation medium is a culture medium selected from the RPE-DM1 group of media supplemented with Activin A.

104. The methods of claim 103, wherein the RPE-DM1 medium is supplemented with 20-280 ng / mL Activin A, more preferably 70- 210 ng / mL, even more preferably 140 ng / mL Activin A.

105. The method of any one of claims 77 to 102, wherein the SC-RPE differentiation medium is a culture medium selected from the RPE-DM2 group of media supplemented with Activin A.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 106. The methods of claim 105, wherein the RPE-DM2 medium is supplemented with 20-280 ng / mL Activin A, more preferably 70-210 ng / mL Activin A, even more preferably 140 ng / mL Activin A.

107. The method of any one of claims 102 to 106, wherein the contacting is performed to obtain a semi-directed differentiation of SC-RPE cells.

108. The method of claim 107, wherein the contacting is initiated from 1 to 14 days after reaching 60-90% confluence, preferably 2-6 days after reaching 60-90% confluence, more preferably 2-4 days after reaching 60-90% confluence, and even more preferably 3 days after reaching 60-90% confluence.

109. The method of claim 108, wherein the contacting is performed for a time from for 4-28 days, more preferably 6-21 days, and most preferably 8 days.

110. The method of any one of claims 102 to 109, wherein the method further comprises following the contacting, the step of treating the cells with the differentiation medium without Activin A.

111. The method of claim 110, wherein the treating is performed for up to 60 days, more preferably 7-35 days, most preferably 14-21 days, after which time the medium is replaced with differentiation medium without Activin A or RPE Maturation Medium.

112. The method of any one of claims 74 to 111, wherein the SC-RPE cell culture substrate is selected from laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV, and combinations thereof.

113. The method of any one of claims 74 to 111, wherein the SC-RPE cell culture substrate is selected from laminin 511, laminin 521, laminin 111, laminin 211, laminin 221, laminin 332, and laminin 411.

114. The method of any one of claims 74 to 113, wherein the SC-RPE cell culture substrate is selected from laminin 521, truncated vitronectin, full-length vitronectin, and combinations thereof.

115. The method of any one of claims 74 to 114, wherein the contacting is preceded by propagating and / or culturing the stem cells.

116. The method of claim 115, wherein the propagating and / or culturing is performed for 1 to 7 days, possibly 2 to 5 days and preferably 3 to 4 or 5 days.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 117. The method of claim 116, wherein the propagating and / or culturing is performed for 1 to 4 days, or 5 to 7 days.

118. The method of any one of claims 74 to 117, wherein the stem cells are embryonic stem cells, adult stem cells, or induced stem cells.

119. The method of any one of claims 74 to 118, wherein the starting stem cell line is a human stem cell line.

120. The method of any one of claims 74 to 119, wherein the starting stem cell line is H1, H9 or Shef1.

121. The method of any one of claims 74 to 120, wherein the starting stem cell line is Shef1.

122. A derivation system comprising at least two of at least one serum-free RPE Maturation Medium of any one of claims 1 to 11 and / or at least one serum-free RPE Maintenance Medium of any of claims 46 to 50 optionally in combination with at least one serum-free SC-RPE differentiation medium at least one SC-RPE cell culture substrate at least one probes each specific for a marker selected from CD57, CD104, and / or CD49b for combined use in a method to derive retinal pigment epithelial (RPE) cell, from a starting stem cell (SC), of claim of any one of 73 to 121.

123. The SC-RPE derivation system of claim 122, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of RPE-DM1 comprised of a base medium, KOSR medium supplement in a concentration from 5 to 30%, from 0.25 to 4X NEAA supplement, ^-mercaptoethanol in a concentration from 25 to 200 mM, and glutamine and / or by L-alanyl-L-glutamine dipeptide in a concentration from 1.35 to 3.71 mM.

124. The SC-RPE derivation system of claim 122 or 123, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 is comprised or consists of the cell culture medium, X-VIVO 10, preferably supplemented with a B27-based supplement at a 0.25 to 3X strength, more preferably 0.5 to 2X strength , and even more preferably 0.75 to 1.25X strength and most preferably at 1X strength.Title: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT 125. The SC-RPE derivation system of any one of claims 122 to 124, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM1 group of media supplemented with Activin A. 126.The SC-RPE derivation system of any one of claims 122 to 125, wherein the at least one serum-free SC-RPE differentiation medium comprises at least one of the RPE-DM2 group of media supplemented with Activin A.

127. The SC-RPE derivation system of any one of claims 122 to 126, wherein the at least one of RPE-DM1 family of media, comprises one or more RPE-DM1 media identified in any one of claims 75 to 93.

128. The SC-RPE derivation system of claim 123 to 127 wherein the at least one of RPE-DM2 family of media, comprises one or more of the RPE-DM2 type of medium identified in any one of claims 94 to 100.

129. The SC-RPE derivation system of the claim 125 to 128, wherein the RPE-DM1 medium supplemented with Activin is any one of the RPE-DM1 of claims 75 to 93 supplemented with Activin A; preferably at a concentration of 20-280 ng / mL, more preferably 70-210 ng / mL, and most preferably 140 ng / ml.

130. The SC-RPE derivation system of the claim 127 to 121, wherein the RPE-DM2 medium supplemented with Activin is any one of the RPE-DM2 of claims 94 to 100 supplemented with Activin A; preferably at 20-280 ng / mL, more preferably 70-210 ng / mL, and most preferably 140 ng / ml.

131. The SC-RPE derivation system of any one of claims 122 to 130, wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin, vitronectin, Matrigel, Geltrex, Cultrex BME, collagen IV and combinations thereof.

132. The SC-RPE derivation system of any one of claims 122 to 130, wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin 511, laminin 521, laminin 111, laminin 211, laminin 332 and laminin 411.

133. The SC-RPE derivation system of any one of claims 122 to 132, wherein the at least one SC-RPE cell culture substrate comprises at least one of laminin 521, truncated vitronectin, full- length vitronectin and combinations thereof.

134. The SC-RPE derivation system of any one of claims 122 to 133 wherein the at least oneTitle: “Serum-free Methods for Derivation…” Inventors: Monte Radeke et al Attorney Docket: P2675-PCT maturation medium comprises the maturation medium RPE-MM of any one of claims 1 to 11.

135. The SC-RPE derivation system of any one of claims 122 to 134, wherein the at least one maturation medium comprises the maturation medium of claim 11.

136. The SC-RPE derivation system of any one of claims 122 to 135, wherein the at least one maturation medium comprises a maturation medium of claim 4.

137. The SC-RPE derivation system of any one of claims 122 to 136, wherein the at least one maturation medium comprises a maturation medium of any one of claims 2 to 11.

138. The SC-RPE derivation system of any one of claims 122 to 137, wherein the at least one maintenance medium comprises a maintenance medium of any one of claims 46 to 50.

139. The SC-RPE derivation system of any one of claims 112 to 138, wherein the at least one maintenance medium is a variant of RPE Maintenance Medium omitting any one or combination of DMSO, bFGF, a ROCK inhibitor and / or TGF^R kinase inhibitor.

140. The SC-RPE derivation system of any one of claims 12 to 139, wherein the at least one probe specific for CD57 CD104, and / or CD49b comprise anti-CD104-PE (Clone: REA236, Miltenyi Biotec), anti-CD57-PE / Vio770 (Clone: REA739, Miltenyi Biotec) and anti-CD49b- APC (Clone: REA188, Miltenyi Biotec) antibodies.

141. A matured SC-RPE cell population obtained by the derivation method of any one of claims 74 to 121 and / or by using the RPE Derivation System of any one of claims 122 to 140.

142. A matured SC-RPE cell population of claim 22, for use in transplanting an individual with the SC-RPE mature cell to treat and / or prevent retinal conditions in the individual.

143. The enriched SC-RPE mature cell population of claim 45, for use in transplanting an individual with the SC-RPE mature cell to treat and / or prevent retinal conditions in the individual.

144. The matured SC-RPE cell population of claim 141, for use in transplanting an individual with the SC-RPE mature cell to treat and / or prevent retinal conditions in the individual.

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