WO2015175504A1 - Differentiation of human pluripotent stem cells into retinal pigment epithelium using hif1 inhibitors - Google Patents

Abstract

The presently disclosed subject matter provides scalable methods for the differentiation of pluripotent stem cells (PSCs) into retinal pigment epithelium (RPE) cells using an agent that decreases the expression level and/or activity of hypoxia-inducible factor 1 (HIF1). Also provided are methods for treating a subject in need of RPE cells.

Description

DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS INTO RETINAL PIGMENT EPITHELIUM USING HIF1 INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

61/991,822, filed May 12, 2014, the entirety of which is incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This presently disclosed subject matter was made with government support under R21 EY023812 awarded by the National Institutes of Health (NIH). The government has certain rights in the presently disclosed subject matter.

BACKGROUND

The retinal pigment epithelium (RPE) abuts the photoreceptor cell layer and has many roles in visual function, including absorption of stray light, formation of the blood-retina barrier, transport of nutrients, secretion of growth factors, isomerization of retinol, and daily clearance of shed photoreceptor outer segments (Strauss (2005) Physiol. Rev. 85(3): 845-881).

Dysfunction and death of RPE have been observed in various human degenerative diseases that lead to blindness. For example, RPE dysfunction and cell death is associated with both the neovascular ("wet) and atrophic ("dry") forms of Age-related Macular Degeneration (AMD) (Curcio et al. (1996) Invest. Ophthalmol. Vis. Sci. 37(7): 1236-1249). AMD is the leading cause of irreversible blindness among people older than 60 years (Gehrs et al. (2006) Ann. Med. 38(7):450-71).

Currently, there are few if any available options to treat or replace diseased RPE cells. One approach being explored for the treatment of atrophic AMD is the transplantation of autologous RPE (da Cruz et al. (2007) Prog. Retin. Eye Res. 26(6): 598-635). However, although a potentially promising approach, harvesting autologous RPE involves complex surgery with possible sight-threatening complications (Caramoy (2010) Br. J. Ophthalmol. 94(8): 1040-1044; Degenring et al. (201 1) ^icto

Ophthalmol. 89(7): 654-659). In addition, RPE harvested from an AMD patient, even if peripheral RPE is used as the source, runs the risk of being already impaired, due to the environmental and/or genetic factors that initially predisposed the patient to AMD.

An alternative approach to obtain human RPE cells is to generate them from human pluripotent stem cells (hPSCs), either from embryonic stem cells (hESCs) (Thomson et al. (1998) Science 282: 1145-1147) or from induced pluripotent stem cells (hiPSCs) (Takahashi et al. (2007) Cell 131(5): 861-872; Yu et al. (2007) Science 318(5858): 1917-1920). Since hPSCs can indefinitely self-renew, they could provide an unlimited supply of RPE-like cells for transplantation in vivo (Lee and MacLaren (201 1) Br. J. Ophthalmol. 95(4): 445-449), as well as for research applications such as disease modeling and drug studies (Juuti-Uusitalo et al. (2012) PLoS One 7(1): e30089). These RPE-like cells derived from hPSCs are referred to as "hPSC-RPE cells". In several animal models, hPSC-RPE cells have been shown to maintain their function after subretinal transplantation and be able to attenuate retinal degeneration with partial preservation of visual function (Haruta et al. (2004) Invest. Ophthalmol. Vis. Sci. 45(3): 1020-1025; Lund et al. (2006) Cloning Stem Cells 8(3): 189-199;

Vugler et al. (2008) Exp. Neurol. 214(2): 347-361; Carr et al. (2009) PLoS One 4(12): e8152; Idelson et al. (2009) Cell Stem Cell 5(4): 396-408; Lu et al. (2009) Stem Cells 27(9): 2126-2135). In addition, a human clinical trial based on hESC-RPE transplantation is currently ongoing (Schwartz et al. (2012) Lancet 379(9817): 713- 720).

Using different cocktails of growth factors or small molecules, several subsequent studies reported RPE yields ranging from 25% to 40%, after several weeks, as assessed by marker expression or the presence of pigmented cells (Osakada et al. (2008) Nat. Biotechnol. 26(2): 215-224; Idelson et al. (2009) Cell Stem Cell 5(4): 396-408; Meyer et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106(39): 16698- 16703; Osakada et al. (2009) J. Cell Sci. 122(Pt 17): 3169-3179; Zahabi et al. (2012) Stem Cells Dev. 21(12):2262-72). To date, the highest yield of RPE cells observed after stepwise treatment with growth factors and small molecules is about 60-80% (Buchholz et al. (2013) Stem Cells Transl. Med. 2(5): 384-393). Despite the success obtained with the generation of human RPE cells from hPSCs, published protocols to date all rely on mechanical dissection of pigmented colonies for RPE purification

(Bharti et al. (2011) Pigment Cell Melanoma Res. 24(1): 21-34; Rowland et al. (2012) J. Cell. Physiol. 227(2): 457-466). Both procedures are potentially problematic since they require time- and labor-consuming manual steps, which present challenge for scale-up and therefore constitute bottlenecks for large-scale production of high quality and consistent hPSC-RPE.

SUMMARY

The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non- limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al, (eds.), Current Protocols in Molecular Biology,

Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning. A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies— A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/ Appleton & Lange 10^th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at

http://omia.angis.org.au/contact.shtml. The Kinetochore, Springer, 2009. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

In some aspects, the presently disclosed subject matter provides a method for differentiating pluripotent stem cells into retinal pigment epithelium (RPE) cells, the method comprising culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of hypoxia- inducible factor 1 (HIF1) until at least a portion of the population of pluripotent stem cells differentiate into RPE cells. In some embodiments, prior to culturing in the differentiation medium comprising the agent, the population of pluripotent stem cells is cultured in a medium that does not comprise the agent. In some embodiments, the medium comprises an mTeSRl medium. In some embodiments, the population of pluripotent stem cells is cultured in the medium for a period of between about 10 days and about 15 days. In some embodiments, prior to culturing in the medium, the population of pluripotent stem cells is produced by amplification using clonal propagation with a myosin inhibitor.

In some embodiments, the agent comprises chetomin. In some embodiments, the differentiation medium comprises the agent in a concentration of about 10 nM to about 100 nM. In some embodiments, the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent for a period of about 10 days. In some embodiments, the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent until at least 25% of the population of pluripotent stem cells differentiate into RPE cells. In some embodiments, the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent until at least 40% of the population of pluripotent stem cells differentiate into RPE cells. In some embodiments, the differentiation medium comprises a chemically defined medium. In some embodiments, the differentiation medium comprises a DMN2 medium.

In some embodiments, the method further comprises performing a single step of cell passaging to produce a subpopulation of cells comprising RPE cells. In some embodiments, prior to performing the single step of cell passaging, the population of pluripotent stem cells is cultured in a differentiation medium that does not comprise the agent for a period of about 15 days.

In some embodiments, the method further comprises culturing the

subpopulation of cells comprising RPE cells in an RPE medium until the

subpopulation of cells forms a homogenous monolayer of polygonal and pigmented cells characteristic of an endogenous RPE cell morphology. In some embodiments, at least 95% of the cells forming the homogenous monolayer comprise RPE cells. In some embodiments, the cells forming the homogenous monolayer express at least one marker indicative of an endogenous RPE cell. In some embodiments, at least one marker is selected from the group consisting oiMITF, ZO-1, BEST1, RLBP1, OTX2, and PMEL17. In some embodiments, the cells forming the homogenous monolayer phagocytose photoreceptor outer segments. In some embodiments, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the pluripotent stem cells are human cells.

In certain aspects, the presently disclosed subject matter provides a method for treating a subject in need of RPE cells, the method comprising: (a) providing a population of RPE cells produced by culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of hypoxia-inducible factor 1 (HIF1) until at least a portion of the population of pluripotent stem cells differentiate into RPE cells; and

(b) administering to the subject an effective amount of the RPE cells produced in (a). In some embodiments, dysfunction and/or death of RPE cells has been observed in the subject. In some embodiments, the subject has or is suspected of having age-related macular degeneration (AMD). In some embodiments, the AMD is dry AMD. In some embodiments, the pluripotent stem cells comprise hESCs or hiPSCs. In some embodiments, the hiPSCs comprise autologous cells produced by reprogramming normal cells obtained from the subject.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1A, FIG. IB and FIG. 1C show scalable RPE generation without chetomin: FIG. 1A is a schematic view of the differentiation process; FIG. IB shows morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm; and FIG. 1C shows results of a flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65- positive. Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; PI, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week;

FIG. 2 A and FIG. 2B show schematic views of embodiments of high throughput screening to identify small molecules that promote RPE differentiation;

FIG. 3 A and FIG. 3B show embodiments of qPCR results from high throughput screening;

FIG. 4A and FIG. 4B show key RPE marker expression (MITF, OTF-2, and

PMEL-17) during hPSC differentiation in 384-well plates (FIG. 4A); and hPSC differentiating in 384-well plates at week 1 (FIG. 4B);

FIG. 5A and FIG. 5B show results from two small molecules that were identified using the high throughput screen: retinoic acid (FIG. 5A); and 6- bromoindirubin-3 '-oxime (BIO; FIG. 5B);

FIG. 6A and FIG. 6B show the structure of chetomin (FIG. 6A); and the up- regulation of three key RPE markers (MITF, OTF-2, and PMEL-17) in response to a low concentration of chetomin (40 nM; FIG. 6B);

FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D show that treatment with chetomin induces up-regulation of RPE markers in a dose-response pattern: FIG. 7A is a schematic view of an embodiment of the presently disclosed differentiation process; FIG. 7B shows up-regulation of MITF; FIG. 7C shows up-regulation of OTF-2; and FIG. 7D shows up-regulation of PMEL-17; FIG. 8A, FIG. 8B and FIG. 8C show a dose-response pattern from ΙΟηΜ to 80nM of chetomin: FIG. 8A is a schematic view of an embodiment of the presently disclosed differentiation process; FIG. 8B shows results of flow cytometric analysis of the expression of PMEL-17; and FIG. 8C shows the percentage of PMEL-17+ cells found for each concentration of chetomin tested;

FIG. 9 shows green fluorescent protein expression in cells in the presence (40 nM) or absence (DMSO only) of chetomin;

FIG. 10A, FIG. 10B, FIG. IOC and FIG. 10D show the functionality of the RPE monolayer as seen by the phagocytosis of pH-Rhodo-labeled bioparticles: FIG. 10A is a schematic view of an embodiment of the differentiation process; FIG. 10B is a bright- field photomicrograph of the RPE monolayer; FIG. IOC shows the percentage of PMEL-17+ cells before and after cell passaging; and FIG. 10D shows flow cytometric analysis of the expression of PMEL-17;

FIG. 1 1A, FIG. 1 IB, FIG. 1 1C, FIG. 1 ID, FIG. 1 IE, FIG. 1 IF and FIG. 11G show chetomin directed differentiation of hPSCs into RPE: FIG. 1 1A is a schematic view of an embodiment of the differentiation protocol; FIG. 1 IB shows typical morphology of hPSCs after 35 days of differentiation. Note: CTM treatment seems to induce the death of many non-RPE cells, usually leaving large clusters enriched in RPE cells at d35; FIG. 1 1C shows the ratio of PMEL17+ cells after 35 days of differentiation in 3 different hPSC lines. Differences between control and CTM treated samples are significant with P-value < 0.05 (Student T-test); FIG. 1 ID is a flow cytometry histogram for PMEL17 after 35d of differentiation. In black, DMSO treated control; in red, CTM treated sample; FIG. 1 IE shows the morphology of differentiated hPSCs one month after passage into RPE medium; FIG. 1 IF shows the ratio of PMEL17+ cells one month after passage into RPE medium; and FIG. 1 1G is a flow cytometry histogram for PMEL17, one month after passage into RPE medium. In black, isotype control; in blue, control sample; in red, CTM treated sample;

FIG. 12 shows the effect of dimeric epidithiodiketopiperazine (ETP 2), another dimeric ETP in the same class as chetomin, on key RPE markers (MITF, OTF-2, and PMEL-17);

FIG. 13 A, FIG. 13B, FIG. 13C, FIG. 13D, and FIG. 13E show identification and validation of CTM as an inducer of RPE during hPSC differentiation. FIG. 13A shows HT qPCR screen design; FIG. 13B shows the chemical structure of CTM; FIG. 13C shows the measurement of the expression of key RPE markers by qPCR after 15d of differentiation; FIG. 13D shows flow cytometric analysis of PMEL17 expression after 35d of differentiation (black: isotype control, red: PMEL17 antibody); and FIG. 13E shows the percentage of PMEL17+ cells after 35d of differentiation in four different hPSC lines;

FIG. 14A, FIG. 14B, FIG. 14C, and FIG. 14D show RPE differentiation following NIC + CTM (NC) treatment. FIG. 14A shows cell culture morphology after 35d of differentiation, just before whole dish passage; FIG. 14B shows flow cytometric analysis of PMEL17 expression after 35d of differentiation (black: isotype control, red: PMEL17 antibody); FIG. 14C shows the percentage of PMEL17+ cells at d35 in 4 different hPSC lines; (N= 10 mM NIC, NC= 10 mM NIC,+50 nM CTM; and FIG. 14D shows RPE morphology and expression of key markers by immuno- staining;

FIG. 15 shows flow cytometric analysis of PMEL17 and OTX2 expression after 15d of NIC+CTM treatment in hiPSC IMR904;

FIG. 16 shows the expression of RPE markers by qPCR in DMN2 media;

FIG. 17 shows the experimental design for a single round of Ht qPCR screening. DMN2: DMEM/F12 + MEMNEAA + 1% N2 (Reichman et al. (2014) Proc. Natl. Acad. Sci. U.S.A. I l l, 8518-8523);

FIG. 18 shows siRNA knock-down. niRNA levels in hiPSCs 72h after siRNA treatment. NT: nontargeted siRNA;

FIG. 19A, FIG. 19B, and FIG. 19C show the generation of a cell marker reporter cell line using CRISPR-Cas9 gene editing. FIG. 19A shows a schematic of RPE cell marker targeting; FIG. 19B shows a schematic representation of the PMEL17/OTX2 dual reporter system; and FIG. 19C shows an example of a retinal reporter cell line SIX6-EGFP reporter during retinal differentiation; and

FIG. 20 shows quantitative real-time PCR analysis of key RPE markers expressed by hPSC-RPE cells obtained after manual picking or serial passage. For each histogram, expression levels with different letters were significantly different (p<0.05). Error bars represent standard deviation. Abbreviations: hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; RPE, retinal pigment epithelium; fRPE, fetal RPE cells; VN- PAS, vitronectin peptide-acrylate surface. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

In the current state of the art, human pluripotent stem cells (hPSCs) can be differentiated into retinal pigment epithelium (RPE), albeit at low efficiency and through a manual approach that is laborious and time consuming. The presently disclosed subject matter provides methods that use agents that decrease the expression level and/or activity of hypoxia- inducible factor 1 (HIF1), such as the small molecule chemotin, to produce a highly pure population of functional retinal pigment epithelium cells. Compared to the state of the art, in some embodiments, these methods are simpler yet induce RPE at efficiencies comparable to the best protocol to date, and do not require expensive growth factors. The presently disclosed methods allow for the efficient and large scale production of RPE from hPSCs and are useful for applications requiring large numbers of cells, such as high-throughput screening or regenerative medicine. I. METHOD FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO RETINAL PIGMENT EPITHELIUM (RPE) CELLS

In some embodiments, the presently disclosed subject matter provides a method for differentiating pluripotent stem cells into retinal pigment epithelium (RPE) cells, the method comprising culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of hypoxia-inducible factor 1 (HIF1) until at least a portion of the population of pluripotent stem cells differentiate into RPE cells.

The starting materials for the presently disclosed methods are pluripotent stem cells. As used herein, the term "pluripotent stem cell" or "pluripotent cell" is intended to indicate a cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or at least the ectoderm (epidermal tissues and nervous system). Thus, preferably, pluripotent stem cells can give rise to any fetal or adult cell type. The pluripotent stem cells are preferably of vertebrate, in particular mammalian, preferably human, primate or rodent origin. Preferred pluripotent cells are selected from embryonic stem cells, induced pluripotent stem cells, stem cells derived from the amniotic fluid or stem cells derived from fetuses, in particular embryonic germ cells derived from fetuses, germline stem cells (in particular from the testes) and pluripotent somatic stem cells.

In some embodiments, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). As used herein, an "embryonic stem cell" is a pluripotent cell that can be propagated in cell culture and stably maintains the ability to differentiate into all three embryonic germ layers. It also stably retains a normal karyotype, thus providing a powerful source for forming different cell types in vitro. As used herein, an "induced pluripotent stem cell" is a type of pluripotent stem cell that can be generated directly from an adult cell. In many respects, induced pluripotent stem cells possess the same properties as natural pluripotent stem cells, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. It should be appreciated, however, that induced pluripotent stem cells, as well as cells produced by directing their differentiation (i.e., RPE cells of the presently disclosed subject matter) may exhibit differences from natural pluripotent stem cells and native RPE cells, such as variations in expression levels of certain genes. That is, the RPE cells disclosed herein share many distinguishing features of native RPE cells, but are different in certain aspects (e.g., gene expression profiles). In some embodiments, the RPE cell is non-native. As used herein, "non-native" means that the RPE cells are markedly different in certain aspects from RPE cells which exist in nature, i.e., native RPE cells. It should be appreciated, however, that these marked differences typically pertain to structural features which may result in the RPE cells exhibiting certain functional differences, e.g., although the gene expression patterns of RPE cells may differ from native RPE cells, the RPE cells behave in a similar manner to native RPE cells but certain functions may be altered (e.g., improved) compared to native RPE cells. For example, it has been found that when hPSC-RPE cells obtained after serial passage were compared with cultured fRPE cells and Ml, a primary line of adult RPE cells, hPSC-RPE cells had overall lower mRNA levels for BEST1, whereas the opposite was true for TYR (FIG. 20; Maruotti et al. (2013) Stem Cells Translational Medicine 2:341-354). The presently disclosed subject matter provides a method for differentiating pluripotent stem cells into RPE cells. The RPE is a polarized monolayer of densely packed cells in the mammalian eye that separates the neural retina from the choroid. The cells in the RPE contain pigment granules and perform a crucial role in retinal physiology by forming a blood-retinal barrier and closely interacting with photoreceptors in the maintenance of visual function by absorbing the light energy focused by the lens on the retina, by transporting ions, water, and metabolic end products from the subretinal space to the blood and by taking up nutrients such as glucose, retinol, and fatty acids from the blood and delivering these nutrients to photoreceptors. As used herein, the term "retinal pigment epithelium cells" is intended to indicate the pigmented cell layer just outside the neural retina, and are firmly attached to the underlying choroid and overlying retinal visual cells.

In some embodiments, the agent decreases the expression level and/or activity of hypoxia- inducible factor 1 (HIF1). Examples of agents that decrease the expression level and/or activity of HIF1 are known in the art and include, but are not limited to, chetomin, topotecan, P 13 kinase inhibitors; LY294002; rapamycin; histone deacetylase inhibitors such as [(E)-(l S,4S, 10S,21R)-7-[(Z)-ethylidene]-4,21- diisopropyl-2-oxa-12, 13-dith-ia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene- 3,6,9, 19,22-pentanone (FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors such as geldanamycin, 17-allylamino-geldanamycin (17-AAG), and other geldanamycin analogs, and radicicol and radicicol derivatives such as KF58333; genistein; indanone; staurosporin; protein kinase- 1 (MEK-1) inhibitors such as PD98059 (2'-amino-3'-methoxyflavone); PX-12 (1-methylpropyl 2-imidazolyl disulfide); pleurotin PX478; quinoxaline 1,4-dioxides; sodium butyrate (NaB);

sodium nitropurruside (SNP) and other NO donors; microtubule inhibitors such as novobiocin, panzem (2 -methoxy estradiol or 2-ME2), vincristines, taxanes, epothilones, discodermolide, and derivatives of any of the foregoing; coumarins; barbituric and thiobarbituric acid analogs; camptothecins; and YC-1. (See U.S. Patent No. 6,979,675). Other examples include derivatives of phenanthrene-quinone, such as tanshinone IIA (T2A) ((Li et al. (2015) PLoS One 10(2): eOl 17440); analogues of tirapazamine, such as TX-402 (Nozawa-Suzuki et al. (2015) Biochem. Biophys. Res. Commun. 457(4):706-l l); curcumin and its analogues, such as EF31 and UBS109 (Nagaraju et al. (2015) Cancer Lett. 357(2):557-65); quassinoids, such as

glaucarubinone (Huynh et al. (2015) Biochim. Biophys. Acta 1853(1): 157-65);

flavonoids, such as nobiletin (Chen et al. (2014) BMC Pharmacol. Toxicol. 15:59); sorafenib; 2 -methoxy estradiol (Ma et al. (2014) Cancer Lett. 355(1):96-105);

phytoalexins, such as glyceollins (Lee et al. (2015) J. Cell. Physiol. 230(4):853-62); 3-(5-amino-2-methyl-4-oxo-4Hquinazolin-3-yl)-piperidine-2,6-dione (U.S. Patent No. 8,906,932); farnesyl transferase inhibitors, such as tipifarnib, lonafarnib, and

L744,832 (U.S. Patent No. 8,853,274); gamma-secretase inhibitors, such as

RO4929097, DAPT, compound E, MK-0752, and PF03084014 (U.S. Patent No. 8,853,274); and preexisting anticancer agents, such as taxol, rafamycin, 17-AAG (17- allylaminogeldanamycin), and YC-1 (guanylyl cyclase activator) (U.S. Patent No. 8,772,290).

In some embodiments, the agent decreases hypoxia-inducible gene expression induced by the activation of HIF1. In some embodiments, the agent inhibits p300 and/or CBP, coactivators of HIFla. Examples of p300/CBP inhibitors include, but are not limited to, garcinol, anacardic acid, curcumin, demethoxy curcumin, C646, histone acetyltransferase inhibitor II, L002, and 5-chloro-2-(4-nitrophenyl)-3(2H)- isothiazolone. In some embodiments, the agent comprises chetomin. Chetomin is a cell- permeable antimicrobial agent, having the chemical name (3S,5aRA0bSA laS)- 2,3,5a,6, 10b,l l-hexahydro-3-(hydroxymethyl)-10b-(3-[(15,45)-3-[[4- (hydroxymethyl)-5,7-dimethyl-6,8-dioxo-2,3-dithia-5,7-diazabicyclo[2.2.2]oct-l- yl)methyl]-lH-indol-l-yl]-2-methyl-3,l la-epidithio-1 laH- pyrazino[l \2': l,5]pyrrolo[2,3-£]indole-l,4-dione, and having the following structure:

Chetomin is one of the epidithiodiketopiperazine (ETP) family of fungal secondary metabolites. Chetomin is known to disrupt hypoxia-inducible factor 1 (HIF1) activity through direct targeting of the interactions between its a subunit and p300 coactivator or its orthologue, CREB-binding protein (CBP), thereby blocking transactivation of the hypoxia-inducible gene expression (Dubey et al. (2013) J. Am. Chem. Soc.

135(1 1):4537-49). It has been found herein that chetomin can also be used to help differentiate pluripotent stem cells into retinal pigment epithelium cells.

As used herein, "decreasing the expression level and/or activity of hypoxia- inducible factor 1" includes any decrease in expression, protein activity, or level of the HIF 1 gene or protein encoded by HIF 1 gene. The decrease may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of the HIF 1 gene or the activity or level of the HIF1 protein. In some embodiments, the agent may inhibit the interaction of HIF 1 with a coactivator, cofactor, or another molecule that interacts with the HIF 1 gene and/or protein. In some embodiments, the agent may target a coactivator, cofactor, or another molecule that interacts with the HIF1 gene and/or protein and/or is involved in the same pathway as the HIF gene and/or protein to modulate hypoxia-inducible gene expression. "Differentiation" or "differentiating" as used herein, refers to the process of switching the state of a cell from one cell type to another, and more specifically in the context of the present disclosure, indicates the process of a human stem cell acquiring the cell type of an RPE cell with at least one characteristic feature indicative that said RPE cell is a mature (terminally differentiated) cell. Characteristic features may include, but are not limited to, the ability of the cells to form a homogenous monolayer, a polygonal appearance, pigmented cells, and/or expression of a marker indicative of an endogenous RPE cell.

As used herein, a "differentiation medium" is a medium that is suitable for differentiation of pluripotent stem cells into RPE cells. Examples of differentiation media are given hereinbelow although other differentiation media also can be used with the presently disclosed methods. For example, the medium can be prepared using a medium conventionally used for mammalian cell culture as a basal medium. As the basal medium, for example, one or more kinds of media for mammalian culture, preferably media for pluripotent stem cell culture, can be used in

combination. As representative commercially available products, GMEM medium, DMEM medium, DMEM/F12 medium, F10 medium, ReproStem medium (Reprocell) and the like are available, and these may be used in combination or partly modified before use. The medium may contain serum and/or a serum alternative. As the serum, a serum derived from a mammal such as bovine and the like can be used, and fetal bovine serum (FBS) and the like are generally used. The concentration of serum or serum alternative can be appropriately set within the range of, for example, 0.5- 30% (v/v). The concentration may be constant or step wisely changed and, for example, the concentration may be step wisely decreased at about 1-3 day (preferably 2 day) intervals. For example, serum or a serum alternative can be added at three-step concentrations of 20%, 15% and 10%. One with skill in the art will be able to determine other examples of differentiation and RPE media to use in the presently disclosed methods. In some embodiments, the differentiation medium comprises a chemically defined medium in which the components of the medium are known. In some embodiments, the differentiation medium comprises a DM 2 medium.

In some embodiments, culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of HIF 1 until at least a portion of the population of pluripotent stem cells differentiate into RPE cells comprises culturing the population of pluripotent stem cells until at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, or at least 49% of the population of pluripotent stem cells differentiate into RPE cells. In some embodiments, the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent until at least 25% of the population of pluripotent stem cells differentiate into RPE cells. In some embodiments, the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent until at least 40% of the population of pluripotent stem cells differentiate into RPE cells.

In some embodiments, prior to culturing in the differentiation medium comprising the agent, the population of pluripotent stem cells is cultured in a medium that does not comprise the agent. In some embodiments, the medium comprises an mTeSRl medium. In some embodiments, the population of pluripotent stem cells is cultured in the medium for a period of between about 10 days and about 15 days.

In some embodiments, prior to culturing in the medium, the population of pluripotent stem cells is produced by amplification using clonal propagation with a myosin inhibitor. Examples of myosin inhibitors include, but are not limited to, blebbistatin, N-benzyl-p-toluene sulphonamide (BTS), 2,3-Butanedione monoxime (BDM), pentachloropseudilin (PCIP), pentabromopseudilin (PBP), 2,4,6- triiodophenol (TIP), and MyoVin-1. In other embodiments, the myosin inhibitor is blebbistatin, a small-molecule inhibitor with high affinity for myosin II.

In some embodiments, the differentiation medium comprises the agent that decreases the expression level and/or activity of HIF1 in a concentration of about 10 nM to about 100 nM, particularly about 20 nM to about 80 nM, more particularly about 40 nM to about 60 nM. In some embodiments, the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent for a period of about 8 days to about 12 days, such as for a period of about 10 days.

In some embodiments, the methods further comprise performing a single step of cell passaging to produce a subpopulation of cells comprising RPE cells. As used herein, "cell passaging" (also known as passaging, subculture or splitting cells) involves splitting the cells and transferring a small number into a new vessel. As used herein, this small number of cells taken from the larger population of cells is called a "subpopulation". Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached, and then a small number of detached cells can be used to seed a new culture, while the rest is discarded. Adherent cells are grown or cultured in petri dishes, multi-wells plates or culture flasks, for example. As used herein, "culturing" cells means the maintenance or growth of cells in or on a medium. The term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g. with an immortal phenotype), primary cell cultures, finite cell lines (e.g. non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos. As used herein, performing a "single step" of cell passaging means that the cells are transferred to fresh media only once. In some embodiments, prior to performing the single step of cell passaging, the population of pluripotent stem cells is cultured in a differentiation medium that does not comprise the agent for a period of about 10 to 20 days, such as about 15 days.

In general, the terms "cell culture" and "culture system" refer to a culture system suitable for the propagation of stem cells and/or RPE cells. The term denotes a combination of elements, at minimum including a basic medium (a cell culture medium usually comprising a defined base solution, which includes salts, sugars and amino acids). The culture system in accordance with the presently disclosed subject matter may further comprise other elements such as, without being limited thereto, a serum or serum replacement, a culture (nutrient) medium and other exogenously added factors, which together provide suitable conditions that support cell growth as well as other components typically used in cell culture systems. The above elements may be collectively classified as soluble elements. However, in the context of the present presently disclosed subject matter, the elements may also be associated to a carrier, i.e. non-soluble elements. The association may be by chemical or physical attachment/binding. For example, the element may be immobilized onto a matrix (e.g. extracellular matrix), presented by cells added to the system or bound to biodegradable material. Further, the element may be released from a carrier, the carrier may be a cell or a vesicle encapsulating or embedding the element. Thus, in the following text, elements supplementing the basic media to form the culture system comprise both soluble and non-soluble elements.

In some embodiments, the time of growth in medium used in the presently disclosed methods can vary from days to weeks, depending on the type of cell grown, the growth conditions, such as temperature and % (¾ and % CO₂ during growth, the type of medium used, such as solid or liquid, the ingredients in the medium, and the like. In some embodiments, the CO₂ concentration is, for example, about 1 to 10%, such as about 5%. In some embodiments, colonies of pluripotent cells are partially dissociated into clumps with suitable enzymes such as collagenase or dispase, if necessary.

At least one advantage of the presently disclosed methods, in some embodiments, is that RPE-inducing growth factors known in the art are not added to the pluripotent stem cells. This allows the presently disclosed methods to be performed in a cost-effective way. Examples of RPE-inducing growth factors that are not required in the media include, but are not limited to, basic fibroblast growth factor (bFGF), noggin, retinoic acid, Sonic hedgehog (Shh), insulin-like growth factor 1 (IGF1), Dkkl, nicotinamide, Activin A, SU5402, and vasoactive intestinal peptide (VIP).

In other embodiments, RPE-inducing growth factors are added to the media.

In some embodiments, a growth factor can be added at certain time points during growth of the cells. In some embodiments, the growth factor and the agent that decreases the expression level and/or activity of HIF 1 work synergistically to increase the resulting population of RPE cells. In some embodiments, the growth factor is nicotinamide.

A "cell" used in the presently disclosed methods in their many embodiments is desirably a human cell, although it is to be understood that the methods described herein are effective with respect to all cells derived from a vertebrate, which are intended to be included in the term "cell." Suitable animal cells include mammalian cells, including, but not limited to, cells from primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like;

porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like. In some embodiments, the pluripotent stem cell is a human cell.

In some embodiments, the method further comprises culturing the

subpopulation of cells comprising RPE cells in an RPE medium until the

subpopulation of cells forms a homogenous monolayer of polygonal and pigmented cells characteristic of an endogenous RPE cell morphology. As used herein, the term "homogenous monolayer" refers to an approximately single cell layer of similar cells. As used herein, the term "endogenous RPE cell" refers to an RPE cell that originates in a subject. In some embodiments, the homogenous layer comprises at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% RPE cells. In some embodiments, at least 95% of the cells forming the homogenous monolayer comprise RPE cells.

In some embodiments, the cells forming the homogenous monolayer express at least one marker indicative of an endogenous RPE cell. As used herein, a "marker" refers to a molecule indicative of a certain kind of cell. For example, a marker can include, but is not limited to, a nucleic acid, such as a transcript of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipid, a lipid, a lipoprotein or a small molecule. In some embodiments, at least one marker is selected from the group consisting oiMITF (microphthalmia-associated transcription factor, e.g., Entrez Gene ID 4286), ZO-1 (e.g., Entrez Gene ID 7082), BEST1 (bestrophin 1, e.g., Entrez Gene ID 7439), R 5 7(retinaldehyde binding protein 1, e.g., Entrez Gene ID 6017), OTX2

(orthodenticle homeobox 2, e.g., Entrez Gene ID 5015), and PMEL17

(premelanosome protein, e.g., Entrez Gene ID 6490).

Testing for a marker indicative of an endogenous RPE cell can be performed using any known method in the art. In some embodiments, the presence of absence of a marker can be tested by using quantitative PCR (qPCR) and/or flow cytometry.

In general, flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering. In flow cytometry, cells are suspended in a stream of fluid and then passed by an electronic detection apparatus. Flow cytometry is used to characterize cells by making measurements on each at rates up to thousands of events per second. The measurement consists of simultaneous detection of the light scatter and fluorescence associated with each event. Commonly, the fluorescence characterizes the expression of cell surface molecules or intracellular markers sensitive to cellular responses to drug molecules. The technique often permits homogeneous analysis such that cell associated fluorescence can often be measured in a background of free fluorescent indicator. The technique often permits individual particles to be sorted from one another.

In general, PCR refers to an in vitro method for amplifying a specific polynucleotide template sequence. The PCR reaction involves a repetitive series of temperature cycles. The reaction mix usually comprises dNTPs (each of the four deoxynucleotides dATP, dCTP, dGTP, and dTTP), primers, buffers, DNA polymerase, and target nucleic acid molecule or template. The PCR step can use a variety of thermostable DNA-dependent DNA polymerases, such as Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3 '-5' proofreading endonuclease activity. Two oligonucleotide primers are used to generate a PCR product. A third oligonucleotide, or probe, is designed to detect the PCR product.

Generally, primer design or determining which sequences to use for making a primer is well known in the art. Computer programs are available to determine if a set of nucleotides in a polynucleotide is optimal for initiating a PCR reaction.

Therefore, different primers can be used to initiate a PCR reaction and to detect a specific gene product. As such, the expression products of the presently disclosed subject matter can be detected using different primers and the presently disclosed subject matter is not limited to a specific set of primers. Variations on the general PCR method are known in the art.

In some embodiments, the presence of a marker is detected by using real-time or quantitative PCR (qPCR). Real-time PCR (RT-PCR) is used to clone expressed genes or parts of genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, in a qPCR reaction, the newly synthesized cDNA produced from the RT-PCR is amplified and simultaneously quantified.

In some embodiments, the cells forming the homogenous monolayer phagocytose photoreceptor outer segments. As used herein, the term "phagocytose" means the act of ingesting a smaller particle or cell fragment, such as a photoreceptor outer segment.

II. METHODS FOR TREATING A SUBJECT IN NEED OF RETINAL PIGMENT EPITHELIUM (RPE) CELLS

In some embodiments, the presently disclosed subject matter provides a method for treating a subject in need of RPE cells, the method comprising: (a) providing a population of RPE cells produced by culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of hypoxia- inducible factor 1 (HIF1) until at least a portion of the population of pluripotent stem cells differentiate into RPE cells; and (b) administering to the subject an effective amount of the RPE cells produced in (a).

As used herein, the term "treating" can include reversing, alleviating, inhibiting the progression of, preventing or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition (e.g., a disease or disorder that causes dysfunction and/or death of RPE cells). In some embodiments, the treatment reduces the dysfunction and/or death of RPE cells. For example, the treatment can reduce the dysfunction and/or death of RPE cells by at least 5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75%, 80%, 85%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more as compared to the dysfunction and/or death of RPE cells in a subject before undergoing treatment or in a subject who does not undergo treatment. In some embodiments, the treatment completely inhibits dysfunction and/or death of RPE cells in the subject.

In some embodiments, the dysfunction and/or death of RPE cells has been observed in the subject. In some embodiments, the subject has or is suspected of having a disease or disorder related to the dysfunction and/or death of RPE cells, such as age-related macular degeneration (AMD). In some embodiments, the AMD is dry AMD.

In some embodiments, the pluripotent stem cells comprise hESCs or hiPSCs.

In some embodiments, the hiPSCs comprise autologous cells produced by reprogramming normal cells obtained from the subject. A variety of suitable methods for reprogramming cells to hiPSCs are available to the skilled artisan. The terms "subject" and "patient" are used interchangeably herein. The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a condition or disease.

Aspects of the presently disclosed subject matter relate to RPE cells produced by the presently disclosed methods and formulated for local administration (e.g., ocular). The presently disclosed subject matter also contemplates the use of such RPE cell compositions for the treatment of a disease or disorder that causes dysfunction and/or death of RPE cells. III. GENERAL DEFINITIONS

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a subject" includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about" even though the term "about" may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term "about," when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term "about" when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range. Throughout the specification and claims, a given chemical formula, structure, and/or name shall encompass all tautomers, congeners, and optical- and

stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

Certain compounds for use within the methods of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds for use within the methods of the present disclosure do not include those which are known in art to be too unstable to synthesize and/or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds for use within the methods described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

Unless otherwise stated, structures depicted herein for use within the methods of the presently disclosed subject matter are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

It will be apparent to one skilled in the art that certain compounds for use within the methods of the presently disclosed subject matter may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. The term "tautomer," as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

Unless otherwise stated, structures depicted herein for use within the methods of the presently disclosed subject matter are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by C- or C-enriched carbon are within the scope of this disclosure.

The compounds for use within the methods of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

EXAMPLE 1

Differentiation of Human Pluripotent Stem Cells into Retinal Pigment Epithelium using Chetomin

Methods

Human pluripotent stem cell culture: Adapting a previously described method (Walker et al. (2010) Nat. Commun. 1 :71), the hESC line H7 (kind gift from Dr. Hai Quan Mao, Johns Hopkins University) and the hiPSC line IMR90-4 (WiCell Research Institute, Madison, WI) were maintained by clonal propagation either on growth factor-reduced Matrigel or on V -PAS Synthemax (Corning Enterprises, Corning, NY), in mTeSRl medium (StemCell Technologies, Vancouver, BC, Canada), in a 10% C(V5% O2 incubator. For passaging, hPSC colonies were first incubated with 5 M blebbistatin (Sigma-Aldrich, St. Louis, MO) in mTesRl and then collected after 5- 10 min of treatment with Accutase (Sigma-Aldrich). Cell clumps were gently dissociated into a single-cell suspension and pelleted by centrifugation. Thereafter, hPSCs were resuspended in mTeSRl with blebbistatin and plated at approximately 1,000 -1,500 cells per cm². Two days after passage, the medium was replaced with mTeSRl (without blebbistatin) and then changed daily.

Differentiation and culture of RPE from hPSCs (without CTM): In general, pluripotent stem cells were plated at 20,000 cells per cm² and maintained in TeSRl . Five days after passage, the cells formed a monolayer and were transferred to a 5% C(V20% O2 incubator. Three days later, the culture medium was replaced with differentiation medium (DM) consisting of Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F 12) (catalog no. 1 1330; Invitrogen, Carlsbad, CA), 15% knockout serum (Invitrogen), 2 mM glutamine (Invitrogen), IX nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and IX antibiotic-antimycotic (Invitrogen). Approximately 25-30 days later, pigmented foci became clearly visible and were grown for an additional 25 days. At that point, the whole monolayer of differentiating cells was passaged a first time (PI) by incubation for 4 hours in DM supplemented with 0.25% (wt/vol) collagenase IV (Gibco, Grand Island, NY). The loosened cell monolayer was thereafter broken into small clumps by vigorous pipetting. These clumps were collected by centrifugation, resuspended in Accumax (Sigma-Aldrich), and incubated for 20-30 minutes at 37°C. After vigorous pipetting, most clumps were dispersed into single cells, and the solution was filtered through a 40-um nylon mesh (BD Falcon, San Jose, CA). Differentiated cells were plated at 100,000 cells per cm² on Matrigel-coated or VN-PAS plates and allowed to grow for 15-20 days in RPE medium (Gamm et al. (2008) Invest. Ophthalmol. Vis. Sci. 49:788 -799) consisting of 70% DMEM (Invitrogen), 30% Ham's F-12 Nutrient Mix (catalog no. 11765; Invitrogen), IX B27 (Invitrogen), and IX antibiotic- antimycotic (Invitrogen). For routine passage after P I, hPSC-RPE cells were collected by using Accumax, pelleted by centrifugation, and replated at 100,000 cells per cm². Specific modifications in the days of growth are noted in the descriptions for each experiment.

In control experiments, hPSCs were cultured in mTeSRl according to the manufacturer's instructions (clump-passage method). After differentiation in DM for 50 days, the pigmented foci were isolated by manual picking as previously

(Klimanskaya (2006) Methods Enzymol. 418: 169 -194), resuspended in Accumax (Sigma- Aldrich), and incubated for 20-30 min in a 37°C water bath. Dissociated clumps were then directly seeded onto Matrigel-coated plates and further passaged as described above. Characterization of hPSC-RPE cells isolated through serial passage or by manual picking was performed at P2, after 50 days culture in RPE medium.

Differentiation and culture of RPE from hPSCs using CTM: hPSCs were treated with 5uM blebbistatin, dissociated to single cells and plated onto matrigel coated dishes at 20,000 cells/cm² in mTeSRl (StemCell Technologies), in 5% (¾, 10% CO₂ incubator, as previously described (Maruotti et al. (2013) Stem Cells Transl. Med. 2(5): 341-354). After 2 days, cells were transferred to a 20% 0₂, 5% C0₂ incubator and further cultured for 6 days in mTeSRl . On the 8th day of culture, mTeSRl was exchanged for Differentiation Medium (DM: DMEM/F 12, 15% Knock- Out Serum, IX NEAA and 2 mM Glutamine, all from Invitrogen, Carlsbad, CA). On the 9th day, 50nM CTM (Sigma-Aldrich) was added to DM until the 19th day.

Afterwards, cells were maintained in DM only until the 35th day of differentiation. According to the hPSC line considered, CTM treatment increased the percentage of PMEL17 positive cells (a key RPE marker) from <10% (DMSO control) to 25%- 50%.

At that point, whole dish cell passage was carried with treatment with 0.25% Collagenase IV (Invitrogen) followed by digestion with Accumax (Sigma-Aldrich), as previously described (Maruotti et al. (2013) Stem Cells Transl. Med. 2(5): 341-354). Cells were then plated on vitronectine coated plates (Synthemax, Corning Life Sciences, Corning, NY) and cultured in RPE medium (DMEM 70%, F12 30%, IX B27, IX Antibiotic -Antimycotic, all from Invitrogen). After one month in RPE medium, a pure population of RPE cells was obtained with >97% PMEL17 positive cells from the CTM treated wells, compared to 70% PMEL17 positive cells from the DMSO treated control wells.

Flow cytometry: For intracellular and nuclear markers, immunostaining was performed using the IntraPrep permeabilization kit (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. Primary antibody concentration was 1 g per 1 million cells for mouse anti-RPE65 (Abeam) and mouse anti-MITF (C5) (Thermo Fisher Scientific, Rockford, IL) and 0.035g per 1 million cells for rabbit anti-OCT4 (Abeam, Cambridge, MA). Secondary antibodies were either goat anti- mouse conjugated to Alexa 488 (Invitrogen) or goat anti-rabbit conjugated to Alexa 647 (Invitrogen). A nonspecific, species-appropriate isotype control was included in all flow cytometry experiments, and stained cells were analyzed using an Accuri C6 flow cytometer (BD Biosciences, San Diego, CA). For each marker, analyses were performed on three biological repeats.

Phagocytosis assay: The cells were incubated for 16 hours either at 4°C or

37°C in ambient air with 0.1 mg per cm² of pH-Rhodo-labeled bioparticles

(Invitrogen) resuspended in CCVindependent medium (Invitrogen) supplemented with 4 mM glutamine (Invitrogen) and 1 antibiotic-antimycotic (Invitrogen).

Afterwards, the cells either were fixed and immunostained or were dissociated and analyzed by flow cytometry.

Reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction: Total RNAs were extracted (RNeasy Mini Kit) and treated with RNase-free DNase I (both from Qiagen, Valencia, CA). Extracted RNAs were reverse-transcribed (High Capacity cDNA kit; Applied Biosystems, Foster City, CA), reverse transcription-polymerase chain reactions (RT-PCRs) were performed with PCR SuperMix (Invitrogen), and quantitative real-time PCRs (qPCRs) were performed with EvaGreen qPCR Mastermix (Abm, Richmond, BC, Canada).

Quantitative PCR samples were run in triplicate, and expression levels were normalized using the geometric mean of three reference genes: FBXL12, SRP72, and CREBBP (Synnergren et al. (2007) Stem Cells 25:473- 480). Gene-specific primers were obtained from published sequences or designed using Beacon Designer (PREMIER Biosoft, Palo Alto, CA). RNA from Ml, a primary culture of RPE derived from an adult donor eye, was a kind gift from Dr. Noriko Esumi (Johns Hopkins University).

Statistical Analysis: Analysis of variance (Tukey test) analysis was done with

Prism 6.01 (GraphPad Software, Inc., San Diego, CA).

Results

Differentiation of hPSC monolayers is associated with progressive expression of RPE markers: In order to induce differentiation of hPSCs, cells were plated at high density (20,000 cells per cm²) in TeSRl and cultured for 8 days, during which time the cells developed into a monolayer (FIG. 1A). At that point, the pluripotency maintaining medium was exchanged for differentiation medium (DM). Following this protocol, spontaneously differentiating monolayers of hPSCs reproducibly generated RPE cells after several weeks (Buchholz et al. (2009) Stem Cells 27:2427- 2434; Carr et al. (2009) PLoS One 4:e8152; Vugler et al. (2008) Exp. Neurol.

214:347-361). For the first weeks, the differentiating monolayer remained colorless, and pigmented colonies typically started to be visible 25-30 days after the switch to DM (dl). Approximately 50 days after switching to DM, numerous large clusters of pigmented cells were readily observed in the cultures (FIG. IB). In order to assess the extent of differentiation toward putative RPE, the cell monolayers were analyzed by flow cytometry for expression of RPE65, a retinal pigment epithelium-specific 65 kDa protein encoded by the RPE65 gene (Entrez Gene ID 6121). Because monolayers of differentiating cells are difficult to dissociate into single-cell suspensions required for flow cytometry procedures, they were first incubated for an extended period of time in DM supplemented with collagenase IV. Upon prolonged incubation with collagenase, the monolayers detached and formed many small clumps. These clumps were then easily dissociated to single cells by treatment with Accumax. Following this approach, monocellular solutions of differentiating cells suitable for flow cytometry analysis were reproducibly obtained. The fraction of RPE65 + hESC cells was found to be 16.6 + 2.5% (FIG. 1C).

High throughput screening to identify small molecules that promote RPE differentiation: It had been found that high efficiency (60-80%) RPE differentiation was achieved in 14 days with a combination of growth factors and small molecules (Buchholz et al. (2013) Stem Cells Transl. Med. 2:384-93). However, only a 26% efficiency at day 60 (d60) was achieved with a small molecule only approach (Osakada et al. (2009) J. Cell. Sci.122:3169 -3179).

Therefore, a high-throughput screen (HTS) was developed aimed at finding small molecules that could improve RPE differentiation in terms of efficiency and time course (FIGS. 2 and 3). For the screening, hPSCs were maintained by clonal propagation. Using automated liquid handlers, they were differentiated in 384-well format. In DMSO treated plates, uniformly differentiated cultures were observed. In addition, high-throughput quantitative real-time PCR (qPCR) for key RPE markers indicated similar expression levels across the plate (FIG. 4A). FIG. 4B shows hPSC differentiating in a 384- well plate. Therefore, HTS qPCR was found to be a realistic approach to identify primary hits. Three target genes, MITF, OTX2, and Pmell 7, were tested and one reference gene, CREBBP, was used. Small molecules were added to DM as described hereinabove. 303 small compounds were tested using 4 different concentrations (10 uM, 2 uM, 0.4 uM, 0.08 uM). For the bioinformatic analysis, compounds were eliminated if expression levels of CREBBP were reduced by > 2xSD, suggesting that those compounds induced cell death. The expression level target was (AACt) > 3xSD.

Out of the 303 compounds, the HTS qPCR screen results showed up- regulation of the MITF gene with 18 compounds (5.9%), up-regulation of the MITF and OTX2 genes with 5 compounds (1.7%), and up-regulation of MITF, OTX2, and PMEL17 with 1 compound (0.33%). There was no instance of OTX2 or PMEL17 up- regulation alone. Up-regulation of the MITF and OTX2 genes with two of the compounds, retinoic acid, a differentiation inducer (Panel A) and 6-bromoindirubin- 3'-oxime, a Wnt pathway activator (BIO; Panel B), is shown in FIG. 5.

Characterization of chetomin (CTM): The up-regulation oiMITF, OTX2, and PMELI 7 was found with one compound, chetomin (CTM), a disruptor of the CHI domain of p300/CBP (Kung et al. (2004) Cancer Cell. 6(l):33-43). The structure of CTM is shown in FIG. 6A. The dose-response profile of CTM showed that it induced 10-15 fold up-regulation of all three RPE markers, MITF, OTX2, and PMEL17, at low concentrations (40 nM; FIG. 6B). To validate the results, a dose-response profile of CTM was generated using 1.25nM to 80nM of CTM at 2-fold increments and qPCR was performed for the three RPE markers at day 15 (dl5) (FIG. 7A). Treatment with CTM induced up-regulation of the RPE markers in a dose-response pattern with a threshold of 10-20nM (FIGS. 7B-7D). To further validate the results, a dose-response profile of CTM was generated using ΙΟηΜ to 80nM of CTM at 2-fold increments and flow cytometry for PMELI 7 was performed at day 35 (d35) (FIGS. 8A-8B; FIG 8B: black: isotype; red: treatment). Treatment with CTM induced a 2-5 fold increase in the percentage of PMELI 7+ cells at d35 of differentiation in four different hiPSC and hESC lines (FIG. 8C; P value < 0.05, 20nM vs Control (ANOVA)).

TYR, another gene that plays a role in RPE function, encodes the enzyme responsible for the conversion of tyrosine to the pigment melanin. A reporter cell line with GFP (Green fluorescent protein) under the control of a TYR promoter and a RPE specific enhancer (kind gift from Dr. Bharti and Dr. Miller, NEI, Bethesda) was differentiated in the presence or absence of CTM and expression was determined after 20 days (FIG. 9). GFP could be seen in the cells in the presence of CTM, further validating that CTM can be used for the production of RPE.

An essential function assumed by RPE cells in vivo is the phagocytosis of outer segments shed by photoreceptors (Strauss (2005) Physiol. Rev. 85:845-881). In order to test whether hiPSC-RPE cells were capable of such phagocytosis, they were first incubated in the presence of pH-Rhodo-labeled bioparticles, fixed, and stained for the tight junction protein ZO-1, which marks the apical side of RPE cells. It is only upon entering low pH phagosomes that pH-Rhodo-labeled bioparticles become fluorescent, thus providing a convenient system to observe particles specifically engulfed by cells. FIG. 10 shows that after CTM treatment of cells, it was possible to obtain a pure and functional RPE monolayer within a single passage, as shown by the phagocytosis of pH-Rhodo-labeled bioparticles (black: 4°C negative control; red: 37°C).

FIGS. 1 lA-11C show that, in general, CTM treatment increased the percentage of PMEL17 positive cells (a key RPE marker) from <10% (DMSO control) to 25%-50%. After one month in RPE medium, a pure population of RPE cells was obtained (FIGS. 1 lD-1 IE), with >97% PMEL17 positive cells from the CTM treated wells, compared to 70% PMEL17 positive cells from the DMSO treated control wells.

To help decipher the CTM mode of action, cells were treated with ETP 2

(dimeric epidithiodiketopiperazine), which is also a dimeric ETP in the same class as chetomin. ETP 2 selectively disrupts the interaction of HIF la with p300/CBP coactivators (Dubey et al. (2013) J. Am. Chem. Soc. 135 (1 1):4537-4549). It was found that ETP 2 treatment did not reproduce the range of the effect of CTM. Only the MITF and OTX2 RPE markers were up-regulated and the increase was not as high as observed with CTM treatment, and the PMEL17 marker was unaffected (FIG. 12).

In conclusion, a non-biased HTS qPCR approach can identify new compounds capable of increasing differentiation of human pluripotent stem cells into retinal pigment epithelium. In addition, chetomin has been found to significantly up-regulate key RPE markers as shown by qPCR and flow cytometry. RPE cells produced by treatment with chetomin can be purified after only one month of differentiation and with a single passage. Chetomin can be used to generate hPSC derived RPE, for example for human clinical trials, and can be used as a molecular probe to understand RPE differentiation.

EXAMPLE 2

Development of a Small Molecule Protocol for the Directed Differentiation of Retinal Pigment Epithelium (RPE) from Human Pluripotent Stem Cells (hPSC)

Background

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss and blindness among the elderly in developed countries. Currently there are about 1.75 million Americans who have AMD. In addition, with the aging population of the United States, this devastating disease of the retina is expected to affect approximately 3 million people by 2020 (Friedman et al. (2004) Arch.

Ophthalmol. 122, 564-572).

Much research has been done over the past several years on the more advanced yet less common form of Age Related Macular Degeneration (AMD), the so called "wet" or neovascular form. As a result of this work, very effective anti- angiogenesis treatments have been developed. Unfortunately, for the majority (approximately 80-90%), of AMD patients, who suffer from the non-neovascular, atrophic or dry form of AMD, there are few therapeutic options available.

An early event associated with AMD is damage to the retinal pigmented- epithelial (RPE) cells. As the RPE deteriorates, there is progressive degeneration of photoreceptor cells. As this occurs primarily, although not exclusively, in the macular area of the retina and since the macula is responsible for high resolution and color vision, AMD is particularly destructive to a patient's functional vision and his/her quality of life.

A number of important challenges remain in the development of RPE transplantation as a viable therapeutic approach for large numbers of patients.

Amongst these is the availability of safe, efficient, and consistent protocols for the generation of large numbers of functional RPE cells from human pluripotent stem cells (hPSC). Recently, faster and more efficient protocols have been devised, with efficiencies as high as 60 to 80% (Buchholz et al.(2013) Curr. Protoc. Chem. Biol. 4,49-63; Zahabi et al. (2012) Stem Cells Dev.). However, despite these important advances, available protocols remain complex, with stepwise treatments using various small molecules and growth factors. In addition, the use of factors expressed in animals or bacteria is not conducive to protocols ultimately aimed at producing tissue for human transplantation. Therefore, the need for a high-efficiency protocol to generate RPE from hPSCs using exclusively small molecules as differentiation factors remains.

In previous studies, a pilot screen was performed of approximately 300 compounds. A molecule, chetomin (CTM, a disruptor of the CHI domain of p300) was identified that consistently increases the yield of RPE cells by 30-40% after one month of differentiation. Indicating the power of broad-based screening, CTM has never been described in the context of pluripotent stem cell differentiation. By combining CTM with nicotinamide (NIC), a small molecule previously reported to modestly increase RPE differentiation, up to 60% of differentiating hPSCs were converted into RPE. This efficiency is similar to the best growth factor-based differentiation protocols reported to date. These studies demonstrate that HT qPCR is a feasible approach that has the potential to identify new compounds that can drive RPE differentiation.

Specific Aims

The goal of these experiments is to develop a xeno-free, chemically defined differentiation protocol that yields >95% RPE cells without any purification step, and therefore is directly amenable to the generation of GMP-quality ES- and iPS-derived RPE cells for clinical trials.

The first aim of these experiments is to perform a HT qPCR screen for additional RPE differentiation promoting small molecules in chemically defined conditions. Despite the limited size of the chemical library used in the pilot screen, chetomin was identified and confirmed as a compound that can promote RPE differentiation. Identifying additional compounds that induce RPE differentiation from hPSC would offer more opportunities to design combined and/or serial treatments for promoting more efficient RPE production, as well as provide additional molecular probes that would be useful for analyzing mechanisms of RPE

differentiation. Therefore, the successful HT qPCR approach to screen large bioactive and diverse chemical collections can be expanded. For each RPE- promoting compound identified, dose-response validation can be performed in the hPSC line used for the screen, as well as in two additional hPSC lines to ensure the observed activity is not cell line-specific. Additionally, the use of a recent chemically defined medium formulation can be adopted to replace a proprietary supplement that had previously been used in the differentiation culturing protocol. Knowing precisely the composition of the differentiation medium can help to identify how its components may interact with compounds that promote RPE differentiation and may make the transition to xeno-free conditions more straightforward.

The second aim of these experiments is to explore the mechanisms by which the small molecules identified promote RPE differentiation. As was true in the preliminary screen, it is likely that RPE-differentiation-promoting compounds will be identified that were never described before in the context of hPSC differentiation. In order to begin to understand how these compounds function in this capacity, whole transcriptome expression can be compared between treated and non-treated hPSC lines using Next Generation Sequencing (NGS)-based RNA sequencing (RNA-seq). Transcriptome analysis at two time points through the course of the RPE

differentiation protocol can help to identify potential pathways by which the small molecule may promote RPE cell fate and differentiation. To confirm whether an identified putative pathway is involved in promoting RPE differentiation, gain- or loss-of-function experiments can be performed of candidate pathway(s) members using siRNA or plasmid transfection, respectively, to assess how modulation of the pathway affects the timing of expression of key RPE markers.

The third aim of the experiments is to optimize small molecule combination for RPE differentiation using a clustered regularly interspaced short palindromic repeats (CRISPR) generated hPSC reporter line. Using the compounds identified, combined and/or serial treatments can be tested to determine the optimal condition for induction of RPE differentiation. Treatment design can be driven by data from the first aim on RPE marker expression, and the knowledge of the pathway(s) each compound acts upon, either from the literature of from a previous aim. For this optimization step of the RPE differentiation protocol, a high-throughput compatible assay will be needed that can discern subtle differences in treatments and that can readily discriminate between an increase in expression of an RPE marker at the well- based level from an increase in the percentage of RPE cells. For these studies, CRISPR technology can be used to generate a dual-reporter line for OTX2 and PMEL17 that can be used to follow RPE differentiation by flow cytometry. Using a High Throughput Flow Cytometry (HTFC) platform, multiple compound treatment strategies can be assessed to develop an optimized protocol.

Results

Previously, a pilot study was initiated to identify compounds that promote RPE differentiation from hPSC. Based on a culture system previously developed for large-scale RPE generation (Maruotti et al. (2013) Stem Cells Transl. Med. 2,341- 354), a HT qPCR pilot screen was developed and performed with a small library of over 300 compounds that target diverse molecular pathways (StemSelect, Millipore). As indicated in FIG. 13, cells were plated in 384-well plates and cultured for 10 days (d) in mTeSRl, before switching at differentiation day 0 (dO) to Differentiation Medium (DM: DMEM/F12 + Knock-Out Serum). For the next 10 days, cells were treated with compounds from the StemSelect library at four different concentrations. Then on day 15 (dl5), RPE differentiation markers were assessed by qPCR

(FIG.13A). One compound, chetomin (CTM, a disruptor of the CHI domain of p300) (FIG. 13B), increased expression of the three key RPE markers, MITF, OTX2 and PMEL17, assessed in the screen, 10 to 15 fold relative to vehicle treated samples. The effect CTM had on RPE differentiation was validated by performing dose- response experiments. In the 25-50 nM range, it was found that CTM increased the expression of all three markers assayed by qPCR in four different hPSC lines (FIG. 13C). Additionally, flow cytometry experiments showed that 30-40% of the cell population expressed PMEL17 after 35 days of differentiation (FIGS. 13D and 13E) compared to just 15% in the vehicle treated condition. Next, the optimal length for CTM treatment was determined by identifying the condition in which the

greatest increase of expression of key RPE markers relative to vehicle treated samples was seen, and that CTM treatment regimen was combined with administration of NIC, a small molecule which as noted above is known to modestly increase RPE differentiation (Idelson et al. (2009) Cell Stem Cell, 5, 396-408). The combined treatment of CTM and NIC resulted in RPE yields between 40-60%> of total cells at day 35 (35d) of differentiation in five different hPSC lines (FIGS. 14A, 14B and 14C). The whole dish was passaged a single time into RPE medium (RPEM: DMEM 70% + F12 30% + B27) (Gamm et al. (2008) Invest. Ophthalmol Vis. Sci., 49, 788- 799) and a month later, cells had formed a homogenous monolayer of polygonal and pigmented cells, characteristic of RPE morphology. The hPSC-RPE cells expressed key RPE markers including MITF, ZO-1, BEST1 and RLBP1 (FIG. 14D) as assessed by immunostaining. By flow cytometric analysis of RPE65 expression, the cells were >95% pure, and they were functional, as over 80% of the cells were able to phagocytose photoreceptor outer segments. Based on these results, it has been demonstrated that HT qPCR is a viable approach to identify new compounds that promote RPE differentiation. Previous work additionally suggests that by combining compounds that promote RPE differentiation, RPE yields can be further optimized. In that regard, it is interesting to note that the RPE generation efficiency in the

NIC/CTM protocol for the common cell line hiPSC IMR904 (Maruotti et al, manuscript in preparation) is similar to previously published protocols that utilize growth factors and stepwise treatments (Buchholz et al. (2013) Stem Cells Transl. Med. 2(5): 384-393).

First aim: For the first aim, a HT qPCR screen can be performed for additional RPE-differentiation-promoting small molecules in chemically defined conditions (FIG. 17). A pilot study showed that treatment of ES and iPS cells with a

combination of NIC and CTM results in about 30% PMEL17+/OTX2+ cells after only 15 days (15d) of differentiation (FIG. 15). This indicates that RPE fate commitment can be induced within a period of two weeks of differentiation, as has been previously observed (Buchholz et al. (2013) Stem Cells Transl. Med. 2(5): 384- 393). Therefore, it is during this time window that cultures can be treated with the compounds being screened for maximizing RPE differentiation. The overall design of the screen can follow that of the pilot study. hiPSC IMR904 cells can be amplified by clonal propagation in E8 medium (Chen et al. (2011) Nat. Methods 8, 424-429) and plated at 5000 cells per well on four sets of 384 well plates pre-coated with synthetic vitronectin (Synthemax, Corning), using the ROCK pathway inhibitor blebbistatin to prevent cell death. hiPSCs can be cultured for a total of 10 days (lOd) in E8 until a confluent monolayer is obtained. Culture medium can be changed daily using an automated washer/dispenser (EL406, Biotek). Cells can then be switched to DMN2 media (see below) and small molecules at four different concentrations can be added with a 96 head pintool (Tango, Art Robbins). On each plate, in addition to DMSO treated negative control wells and wells to be used for reverse-transcriptase (RT) minus controls, CTM treated wells can be included as a positive control.

For the proposed expanded screen, DMN2 media can be used rather than Knock-Out Serum (KSR)-based differentiation media. KSR-media has been widely used since the first description of RPE generation from hPSC (Klimanskaya et al.( 2004) Cloning Stem Cells 6, 217-245), but since it is a proprietary reagent (from Invitrogen) its exact composition is not publicly available. Recently a

simple and chemically defined medium, DMN2, based on N2 supplement instead of KSR, has been described for RPE differentiation (Reichman et al. (2014) Proc. Natl. Acad. Set U.S.A. 111, 8518-8523). Knowing precisely the composition of this medium may help to identify how its components may interact with the hits, and it also can make the transition to xeno-free conditions more straightforward. The preliminary results indicate that CTM can potently induce RPE differentiation in DMN2, with an 8- and 12-fold increase for PMEL17 and MITF, respectively, although the up-regulation for OTX2 (approximately 3-fold) was more moderate compared to differentiation in KSR-based media (FIG. 16).

After the 10 days of DMN2 plus library compound treatment, cells can be cultured an additional 5 days in DMN2 without compound in order to allow the cells to further differentiate and mature to a point where RPE marker expression is high enough to clearly detect by qPCR. Cells can be lysed with the Real-Time Ready Cell Lysis kit (Roche), and the Transcriptor kit (Roche) can be used to generate cDNA. Next, qPCR can be performed with the CFX384 (BioRad) and expression of the following key RPE markers can be assessed: MITF, OTX2 and PMEL17. qPCR data can be normalized using the geometric mean of the reference genes CREBBP, FBXL12 and SRP72 (Synnergren et al. (2007) Stem Cells 25:473- 480), which proved very stable in the pilot screen. Data analysis can be performed using the AACt method to identify compounds that up-regulate all three key RPE markers (Bittker (2012) Curr. Protoc. Chem. Biol. 4, 49-63).

Using the same assay as described, primary hits can be validated in 8-point dose-response assays with both hiPSC IMR904, the line used for the screen, and an additional hESC line such as H7. In addition to confirming the screen results, this experiment can also give an optimal concentration range at which the compound exhibits its effect. Using this range, additional validation experiments can be performed using flow cytometry: IMR904 and H7 cultures can be treated for 10 days with the compound, and then assessed at day 15 (dl5) and day 30 (d30) for the expression of PMEL17 and OTX2. At each of these time points, a sample of differentiated cells can also be plated at high density onto synthemax coated plates in order to check if the putative RPE cells can survive passaging and produce pigmented and polygonal cell sheets. This step is important to ensure that the tested compounds allow further amplification of the RPE cells, for potential mass production. The most promising compounds can be prioritized for further analysis in the second aim.

One library that can be screened is the 1280 compound Library of

Pharmocologically Active Compounds (LOPAC, Sigma). This library is a collection of drug-like molecules that target diverse molecular pathways including

phosphorylation, ion channels, lipid signaling, G-proteins, and gene regulation. Other libraries that can be screened include the ICCB library (480 compounds), the

Prestwick library of FDA approved drugs (1200 compounds), the GSK protein kinase inhibitor set (367 compounds), and the Kinacore library (2037 compounds,

Chembridge).

High-throughput methods using robotic tools can be used for each round of screening. At the completion of each round, quality control of the cDNAs from each 384-well plate can be assessed by qPCR analysis of a single reference gene,

CREBBP, and one of the target genes, PMEL17, using the following criteria: CVs for the DMSO and CTM treated controls < 10%, Ct difference between DMSO controls and RT minus >5 for reference and target gene, and AACt between CTM and DMSO treated controls > 3SD of the DMSO controls for the target gene. Plates satisfying these criteria can be frozen at -80C and analyzed further upon completion of the screen. This preliminary evaluation can quickly identify plates that may have potential hits (based on PMEL17 expression), and these plates can be a priority for the complete qPCR analysis.

Second aim: It is possible that some of the potential compounds will have been already described in the literature for hPSC differentiation. For those for which no information is available, a combined transcriptomic and siRNA approach can be taken to gain insight into their mechanism of action. Hits can be prioritized for analysis based on their ability to induce the largest PMEL17+/OTX2+ population at day 15, highest increase in RPE marker expression, and best survival of RPE cells after passage. For transcriptomic analysis, hiPSC IMR904 can be differentiated in triplicate in the presence or absence of each of the selected compounds, and RNA can be extracted at the one week and two week time points. It seems that this is when the most dramatic increase in PMEL17 expression occurs following CTM treatment as seen by qPCR analysis. After one week, the cell populations in the treated and untreated samples are phenotypically still quite similar so the gene expression changes measured should mostly reflect changes in regulatory factors and pathways rather than the end results of differentiation or changes in the makeup of the cell population. At the two week time point, it is expected that the transcription profile changes reflect more of the actual RPE differentiation changes induced by the small molecule treatment. Additionally, once gene expression changes of potential interest are identified, qPCR can be used to test more time points (0, 2, and 12 hrs and 1, 4, 7, 14, and 28 days post small molecule treatment) to define in more detail the kinetics of the expression changes of the genes of interest. NGS libraries can be prepared with the TruSeq Stranded mRNA kit (Illumina). Samples can then be analyzed on a HiSeq 2500 (Illumina). For each sample, 30M (2xl50bp) reads can be obtained, which for a single cell type should be sufficient depth for reasonable assessment of differential gene expression (Tarazona et al. (201 1) Genome Research 21, 2213-2223).

After examining sequencing parameters such as per base sequence quality and alignment rates, the sequencing reads can be trimmed and aligned to the current version of the human genome (hgl9) using TopHat2. Then the aligned reads can be mapped to transcripts and summarized as transcription levels in FPKM (Fragments Per Kilobase of transcript per Million mapped reads) using Cufflink. To evaluate transcript/gene expression changes, Cuffdiff or SAM (significance analysis of microarray) can be used to obtain the p-value of each transcript expression level difference between normal and treated groups and also to determine the false discovery rate (FDR). For example, the focus can be on genes with a corrected p- value < .05, FDR < 5%, and a fold-change (FC) > 2. Another important factor for prioritization of putative differentially expressed genes can be what is known about their biology, such as transcription factors (activators and repressors), chromatin modifying proteins, and other regulatory factors.

Once the RNA seq data is confirmed, and genes of interest are prioritized, the activity of a small sub-set of genes can be tested through loss-of- function (LOF) and gain-of-function (GOF) experiments. For LOF experiments, siRNA experiments can be performed to knock-down (KD) putative regulatory factors that are up-regulated following small molecule treatment to determine if the KD reduces or eliminates the ability of the small molecule to promote RPE differentiation. hPSCs can be grown to confluence in E8, before differentiation is initiated in DMN2. Since siRNA KD typically lasts a week to 10 days in dividing cells (Bartlett and Davis (2006) Nucleic Acids Research 34, 322-333), cells can be transfected using Lipofectamine

RNAiMAX (Invitrogen) (Zhao et al. (2008) Mol. Biotech. 40, 19-26) at the time when medium is switched to DMN2 (dO). At one and two week time intervals, the KD efficiency of the targeted genes as well as the expression level of markers indicative of RPE differentiation can be determined. Results can be normalized to samples transfected with a non-targeting control siRNA. Preliminary experiments indicate that the protocol can provide potent KD, with up to 96% KD achieved in hPSCs 3 days post transfection (FIG. 18). The kinetics of siRNA KD during differentiation are currently being investigated, but it is likely, based on the efficiency of knockdown achieved at three days, that at least for some genes, the effects should last through the one week time point and potentially beyond. To complement these LOF studies, GOF experiments can additionally be performed in which an expression vector can be transfected to overexpress genes found to be up-regulated by the RPE-promoting compounds, to determine if such over-expression mimics the effect of the RPE differentiation-promoting small molecule. Results of the experiments proposed in the second aim can not only suggest pathways targeted by the selected compounds and maybe their mechanisms of action, but will hopefully also shed light on the RPE differentiation process itself and how it can be optimized. This information can be used to design the combinatorial assays presented in the third aim.

Third aim: Using the validated hits from the first aim and the knowledge of their targeted pathways from the second aim and/or the literature, RPE differentiation regimens can be designed that include combinations or serial additions of compounds in order to develop an optimized protocol for generating RPE cells. To assess all the combinations, a large number of conditions can be tested, and ultimately the precise timing of compound addition can be optimized. Since this aim focuses on optimizing conditions that maximize RPE yields, rather than assessing "whole well" expression of various RPE markers, the percentage of cells that express RPE markers can be assessed. This cell-level measurement can not only allow the detection of small differences in RPE yield, but can make the distinction between a few cells expressing high levels of RPE-associated genes compared to a large number of cells with modest increase in expression. In order to be able to assess expression at the cell level in a high-throughput manner, for these experiments a high-throughput flow cytometric assay can be established to follow RPE differentiation. Clustered Regularly

Interspaced Short Palindromic Repeats/Cas9 (CRISPR) technology can be used to create a PMEL17-NeonGreen (with membrane tag)/OTX2-mRuby2 (with H2B - nuclear- tag) dual RPE-reporter hiPSC line (FIGS.19A and 19B). Neon and Ruby fluorescent proteins have particularly good characteristics for flow cytometry.

Homology directed repair (HDR) can be used to guide the insertion of the appropriate DNA donor fragment into a target site at regions of homology between the donor fragment and the genomic DNA target. Constructs can be created that can direct the integration of a self-cleaving P2A peptide sequence (Kim et al.

(201 1) PLoS ONE 6(4)), targeted fluorescent protein cassette in frame at the stop codon of OTX2 and PMEL17. While PMEL17 is expressed in both melanocytes and RPE cells, only RPE also expresses OTX2. The P2A sequence engineered between the C-terminus of the endogenous protein and the fluorescent protein can minimize possible fusion protein functional defects. First the plasmids encoding the Cas9 nuclease, the OTX2 gRNA and the mRuby2 donor sequence can be introduced into hiPSC IMR904 using the Neon transfection system (Life Technology). Following electroporation, hiPSCs can be cultured at clonal density. Because OTX2 is strongly expressed in hiPSC in their pluripotent state, ruby-expressing transfected clones can be directly isolated and a dozen of them amplified for several passages to ensure stable integration of the construct. Stable cell lines can then be sequenced to validate the construct integration. Next, OTX2-mRuby2 hiPSC can be transfected with the PMEL17-NeonGreen construct using the same approach. Alternatively, since CRISPR-mediated gene editing can be multiplexed, this approach may be used along with PCR screening to identify doubly edited cells. Because PMEL17 is only weakly expressed in hPSC (Buchholz et al. (2013) Stem Cells Transl. Med. 2(5): 384-393), gDNA PCR can be used to identify transfected clones, and sequencing can be used to validate them. Three different PMEL17-NeonGreen/OTX2-mRuby2 clones can be selected and differentiated for two weeks in the presence of NIC and CTM (or other hit from the first aim). Immuno-staining and flow cytometry can be used to validate the correct spatio-temporal expression pattern of the dual reporter system, and ensure that the lines differentiate to form functional RPE. Once a faithful reporter line has been identified, it can be used to carry out the drug combinatorial assays described below. Depending on how long it takes to generate and validate the proposed reporter line, it may also be utilized for the siRNA KD experiments described in the second aim or even for single compound validation in the first aim. A PMEL17- NeonGreen/OTX2-mRuby2 hiPSC line can also be used to directly screen compound libraries instead of relying on the HT qPCR approach described in the first aim.

Based on the phenotype induced by the identified compounds and potential pathway by which the compounds act, combinatorial and/or serial regimens can be rationally designed and the PMEL17/OTX2 hiPSC line can be treated for four weeks. Reporter expression can be quantified by flow cytometry at the two week and four week time points. Treatments that induce the highest percentage of

PMEL17/OTX2 cells at either time point can be determined, with a target of >95% PMEL17+/OTX2+ cells. Although the ultimate goal is to develop a differentiation protocol that does not require a purification step, whole dish passage of differentiating cells from treatments with high percentages of PMEL17+/OTX2+ at two weeks can still be tested to determine whether culture in RPEM or DMN2 (Reichman et al. (2014) Proc. Natl. Acad. Sci. U.S.A. 1 1 1, 8518-8523) can lead to pure RPE culture two or three weeks later.

Once a treatment has been identified for high efficiency RPE differentiation, its reproducibility can be validated with an extended array of hPSC lines, and in depth characterization can be performed of the RPE cells obtained using assays developed previously (Maruotti et al. (2013) Stem Cells Transl. Med. 2(5): 341-354; Maruotti et al, in preparation). This can include assessment of culture purity by flow cytometry for additional markers (MITF, RPE65) ,and immuno-staining for key RPE markers (BEST1, RLBPl, RPE65, ZO-1, MITF),as well as functional characterization of the RPE, including measuring the ability of the cells to phagocytose photoreceptor outer segments, secrete growth factors in a polarized manner and convert all-trans retinol into 1 1-cis retinal (visual cycle).

At the completion of the third aim, a high efficiency, chemically defined process for RPE generation from hPSC that exclusively uses small molecules to drive differentiation is expected. If the optimized protocol doesn't require B27-based RPEM media, it will be also xeno-free. If RPEM is required, by replacing the BSA with human serum albumin (HSA) and using recombinant transferrin and insulin, xeno-free conditions can be achieved.

REFERENCES

All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A method for differentiating pluripotent stem cells into retinal pigment epithelium (RPE) cells, the method comprising culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of hypoxia- inducible factor 1 (HIFl) until at least a portion of the population of pluripotent stem cells differentiate into RPE cells.

2. The method of claim 1, wherein prior to culturing in the differentiation medium comprising the agent, the population of pluripotent stem cells is cultured in a medium that does not comprise the agent.

3. The method of claim 2, wherein the medium comprises an mTeSRl medium.

4. The method of claim 2, wherein the population of pluripotent stem cells is cultured in the medium for a period of between about 10 days and about 15 days.

5. The method of claim 2, wherein prior to culturing in the medium, the population of pluripotent stem cells is produced by amplification using clonal propagation with a myosin inhibitor.

6. The method of claim 1, wherein the agent comprises chetomin.

7. The method of claim 1, wherein the differentiation medium comprises the agent in a concentration of about 10 nM to about 100 nM.

8. The method of claim 1, wherein the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent for a period of about 10 days.

9. The method of claim 1, wherein the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent until at least 25% of the population of pluripotent stem cells differentiate into RPE cells.

10. The method of claim 1, wherein the population of pluripotent stem cells is cultured in the differentiation medium comprising the agent until at least 40% of the population of pluripotent stem cells differentiate into RPE cells.

11. The method of claim 1 , wherein the differentiation medium comprises a chemically defined medium.

12. The method of claim 1 1, wherein the differentiation medium comprises a DMN2 medium.

13. The method of claim 1, further comprising performing a single step of cell passaging to produce a subpopulation of cells comprising RPE cells.

14. The method of claim 13, wherein prior to performing the single step of cell passaging, the population of pluripotent stem cells is cultured in a differentiation medium that does not comprise the agent for a period of about 15 days.

15. The method of claim 13, further comprising culturing the

subpopulation of cells comprising RPE cells in an RPE medium until the

subpopulation of cells forms a homogenous monolayer of polygonal and pigmented cells characteristic of an endogenous RPE cell morphology.

16. The method of claim 15, wherein at least 95% of the cells forming the homogenous monolayer comprise RPE cells.

17. The method of claim 15, wherein the cells forming the homogenous monolayer express at least one marker indicative of an endogenous RPE cell.

18. The method of claim 15, wherein the at least one marker is selected from the group consisting oiMITF, ZO-1, BEST1, RLBP1, OTX2, and PMEL17.

19. The method of claim 15, wherein the cells forming the homogenous monolayer phagocytose photoreceptor outer segments.

20. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

21. The method of claim 1 , wherein the pluripotent stem cells are human cells.

22. A method for treating a subject in need of RPE cells, the method comprising:

(a) providing a population of RPE cells produced by culturing a population of pluripotent stem cells in a differentiation medium comprising an agent that decreases the expression level and/or activity of hypoxia- inducible factor 1 (HIF l) until at least a portion of the population of pluripotent stem cells differentiate into RPE cells; and (b) administering to the subject an effective amount of the RPE cells produced in (a).

23. The method of claim 22, wherein the dysfunction and/or death of RPE cells has been observed in the subject.

24. The method of claim 22, wherein the subject has or is suspected of having age-related macular degeneration (AMD).

25. The method of claim 24, wherein the AMD is dry AMD.

26. The method of claim 22, wherein the pluripotent stem cells comprise hESCs or hiPSCs.

27. The method of claim 26, wherein the hiPSCs comprise autologous cells produced by reprogramming normal cells obtained from the subject.