Methods for Differentiating Induced Pluripotent Stem Cells into Retinal Pigment Epithelial Cells, ... and Methods for Using Retinal Pigment Epithelial Cells

Abstract

The present invention relates to a method for differentiating induced pluripotent stem cells into retinal pigment epithelial cells. Furthermore, the present invention relates to a retinal pigment epithelial cell culture obtainable by the differentiation method and a retinal pigment epithelial cell culture obtained by the differentiation method. Furthermore, the present invention relates to a retinal pigment epithelium consisting of or comprising a retinal pigment epithelial cell culture obtainable or obtained by the differentiation method. The present invention also relates to a pharmaceutical composition comprising a retinal pigment epithelial cell culture obtained by the differentiation method. The present invention also relates to a method for treating a retinal degenerative disease in a subject, comprising administering to the subject retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the method. Finally, the present invention also relates to an in vivo method for detecting the viability of retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the defined method in a subject, and an in vitro method for determining the immunogenicity of the retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the defined method in the subject to which the differentiated RPE cells have been previously delivered. TIFF2025507265000008.tif19278

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 303,849, filed January 27, 2022, the contents of which are incorporated by reference in their entirety for all purposes. [Background technology]

[0002] 2. Background of the Invention The present invention relates to a method for producing induced pluripotent stem cells. Furthermore, the present invention relates to an induced pluripotent stem cell population obtainable by the method, and an induced pluripotent stem cell population obtained by the method. The present invention also relates to a pharmaceutical composition comprising the induced pluripotent stem cells of the present invention. The present invention also relates to a method for differentiating the induced pluripotent stem cells of the present invention. Furthermore, the present invention also relates to a pharmaceutical composition comprising the differentiated induced pluripotent stem cells obtained by the method. Furthermore, the present invention also relates to a method for treating a congenital or acquired degenerative disorder in a subject, comprising administering to the subject a target cell differentiated from a pluripotent stem cell. The present invention also relates to a method for differentiating an induced pluripotent stem cell into a retinal pigment epithelial cell. Furthermore, the present invention also relates to a retinal pigment epithelial cell culture obtainable by the differentiation method, and a retinal pigment epithelial cell culture obtainable by the differentiation method. Furthermore, the present invention also relates to a retinal pigment epithelium consisting of a retinal pigment epithelial cell culture obtainable or obtained by the differentiation method, or comprising a retinal pigment epithelial cell culture obtainable or obtained by the differentiation method. The present invention also relates to a pharmaceutical composition comprising a retinal pigment epithelial cell culture obtainable by the differentiation method. The present invention relates to a method for treating a retinal degenerative disease in a subject, comprising administering to the subject retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the method of the present invention.Finally, the present invention also relates to an in vivo method for detecting the viability of retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the defined method in a subject, and an in vitro method for determining the immunogenicity of said retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the defined method in said subject to which said differentiated RPE cells have previously been delivered.

[0003] FIELD OF THEINVENTION Stem cells are cell populations that possess the ability to self-renew indefinitely and differentiate into multiple cell or tissue types. The ability of stem cells to self-renew is important for their function as a reservoir of primitive undifferentiated cells, and the "fitness" of stem cells depends on their ability to transdifferentiate into tissues different from their origin, possibly beyond the embryonic germ layer. In contrast, most somatic cells have limited self-renewal capacity due to telomere shortening (reviewed, for example, in Dice, JF (1993) Physiol. Rev. 73, 149-159). Thus, stem cell-based therapeutics may be useful for the treatment of many human and animal diseases.

[0004] Embryonic stem cells (approximately 3-5 days after fertilization) can proliferate indefinitely and differentiate spontaneously into all tissue types: they are therefore referred to as pluripotent stem cells (reviewed, for example, in Smith, AG (2001) Annu. Rev. Cell. Dev. Biol. 17, 435-462). Although the potential of embryonic stem cells is considerable, their use is associated with many ethical issues. Therefore, non-embryonic stem cells have been proposed as alternative sources.

[0005] Adult stem cells may be more tissue specific and have a lower replicative capacity: therefore, they are referred to as pluripotent stem cells (reviewed, for example, in Paul, G. et al. (2002) Drug Discov. Today 7, 295-302). These cells can be derived from bone marrow stroma, adipose tissue and dermis and have the capacity to differentiate into, among others, chondrocytes, adipocytes, osteoblasts, myoblasts, cardiomyocytes, astrocytes and tenocytes. However, in most cases, the number of stem cells extracted from bone marrow stroma, adipose tissue, dermis and umbilical cord blood is rather low.

[0006] The comprehensive source of very young and adaptable adult stem cells, also called neonatal stem cells, is umbilical cord blood or tissue or placenta.For example, a large amount of stem cells can be obtained from umbilical cord tissue, i.e., Wharton's jelly, which is the matrix of the umbilical cord (Mitchell, KE et al. (2003) Stem Cells 21, 50-60 (Non-Patent Document 4); U.S. Patent No. 5,919,702 (Patent Document 1); U.S. Patent Application No. 2004 / 0136967 (Patent Document 2)). These cells have been shown to have the ability to differentiate into, for example, neuronal phenotype and cartilage tissue, respectively. Mesenchymal stem cells have also been isolated from the subendothelial layer of the umbilical vein, one of the three blood vessels (two arteries, one vein) found in the umbilical cord (Romanov, YA et al. (2003) Stem Cells 21, 105-110 (Non-Patent Document 5); Covas, DT et al. (2003) Braz. J. Med. Biol. Res. 36, 1179-1183 (Non-Patent Document 6)). Furthermore, mesenchymal stem cells as well as epithelial stem cells have been successfully isolated from the amniotic tissue of the umbilical cord (US2006 / 0078993 (Patent Document 3)). For example, mesenchymal stem cells can undergo differentiation in vitro and in vivo, making these stem cells promising candidates for mesodermal defect repair and disease management, but the use of adult stem cells is limited by their pluripotency. To overcome this limitation, non-embryonic cells can be reprogrammed into pluripotent stem cells, so-called induced pluripotent stem cells (iPS).

[0007] IPS were first generated by Takahashi and Yamanaka, who reprogrammed non-embryonic cells to a pluripotent state through overexpression of four transcription factors, OCT3 / 4, SOX2, KLF4 and C-MYC, also known as Yamanaka factors (Takahashi, K. and Yamanaka, S. (2006), Cell, 126(4), pp. 663-676). In detail, Takahashi and Yamanaka used mouse embryonic fibroblasts and introduced Yamanaka factors via retroviral transduction, thereby allowing overexpression of the transcription factors, resulting in cells that exhibited morphology and growth characteristics of embryonic cells. Although this method represented a major breakthrough, the transduction process could allow the transferred DNA to be integrated into the genome of the host cell, making iPS essential for therapeutic treatment in humans. In 2011, Okita, K. et al., Nature methods, 8(5), pp. 409-412, established a non-integration alternative method for generating iPS. Okita et al. used electroporation to transfer three episomal plasmid vectors encoding Yamanaka factors and p53-shRNA for p53 suppression into human skin fibroblasts and dental pulp, thus allowing overexpression of exogenous DNA and thereby generating integration-free human iPS. To support the growth and maintenance of integration-free human iPS, Okita et al., cultured iPS on a feeder layer consisting of STO cell line or mouse embryonic fibroblasts (MEFs), which have been transformed with neomycin resistance and mouse LIF gene (SNL). However, culturing on a feeder layer may entail the risk of contaminating iPS with foreign DNA. Therefore, the integration-free iPS by Okita et al. may also be essential for therapeutic treatment in humans.

[0008] A decade after its conception, iPS technology has entered the stage of clinical application, with first-in-human trials being conducted for age-related macular degeneration (AMD) (Mandai, M., et al,. N Engl J Med, 2017. 376(11): p. 1038-1046) and Parkinson's disease (PD) (Reardon, S. and Cyranoski, D. (2014) 'Japan stem-cell trial stirs envy', Nature. England, pp. 287-288. doi: 10.1038 / 513287a). The greatest promise of iPS technology lies in its potential to enable autologous cell therapy, which may circumvent the need for long-term immunosuppression or tissue compatibility matching to prevent rejection of transplanted cells. This paradigm has been demonstrated in fibroblast and bone marrow derived iPS cells in non-human primate models (Morizane, A., et al., Stem Cell Reports, 2013. 1(4): p. 283-92 (Non-Patent Document 11); Hallett, PJ, et al., Cell Stem Cell, 2015. 16(3): p. 269-74 (Non-Patent Document 12); Wang, S., et al., Cell Discov, 2015. 1: p. 15012 (Non-Patent Document 13); Shiba, Y., et al., Nature, 2016. 538(7625): p. 388-391 (Non-Patent Document 14)) and is the basis for the first human trials of iPS-based cell therapy for AMD (Mandai, M., et al., N Engl J Med, 2017. 376(11): p. 1038-1046 (Non-Patent Document 9). However, the significant time and expense involved in producing clinical grade iPS makes it unlikely to be implemented on a large scale for human therapy. Furthermore, there are situations in which the generation of autologous iPS from patients is not practical. For example, in the case of patients carrying disease-causing mutations, these mutations must first be corrected before it is possible to use iPS derived from these patients.This is achievable when the mutations are tractable, but when they are intractable, such as those underlying sporadic forms of many diseases, gene correction strategies may not be tenable. Therefore, there remains a need for alternative methods to generate iPS cells, where the resulting iPS cells can be differentiated into target cells, such as retinal pigment epithelial (RPE) cells, suitable for therapeutic treatment in humans. The stem cells currently used to generate RPE, namely induced pluripotent stem cells (iPS) and embryonic stem cells (ES), have several drawbacks. It is well established that an individual's genome accumulates mutations throughout life, which underlie age-related diseases such as cancer (Stratton MR et al., 2009, Nature 458, 719-7249 2001). iPS cells obtained from younger individuals harbor fewer mutations compared to iPS cells obtained from older individuals. When comparing DNA sequences from subjects aged 21-100 years, genetic and epigenetic mutations found in iPS cells increased with increasing donor age (Lo Sardo et al., 2017, Nat Biotechnol. 35(1):69-74). Age-related abnormalities also increased in mitochondrial DNA, with fibroblast-derived iPS cells from older subjects carrying significantly higher mutations than younger subjects (Kang et al., 2016, Cell Stem Cell 18, 625-636, May 5, 2016). iPS cells derived from adults may also require immunosuppression if derived from an allogeneic host. The use of ES cells is associated with ethical issues and immune rejection necessitating the use of immunosuppression. Skin cells are widely used to generate iPS cells because they are easy to extract, but they undergo significant changes that can lead to mutations due to long-term exposure to UV rays from sunlight (Apalla Z. et al., 2017, Dermatol Pract Concept. 2017 Apr; 7(2): 1-6 (Non-Patent Document 18)).

[0009] Consequently, it is an object of the present invention to provide a method for the generation of iPS cells and the differentiation of said particular iPS cells into RPE cells that meets these demands. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 5,919,702 [Patent Document 2] U.S. Patent Application No. 2004 / 0136967 [Patent Document 3] US2006 / 0078993 [Non-patent literature]

[0011] [Non-Patent Document 1] Dice, JF (1993) Physiol. Rev. 73, 149-159 [Non-Patent Document 2] Smith, AG (2001) Annu. Rev. Cell. Dev. Biol. 17, 435-462 [Non-Patent Document 3] Paul, G. et al. (2002) Drug Discov. Today 7, 295-302 [Non-Patent Document 4] Mitchell, KE et al. (2003) Stem Cells 21, 50-60 [Non-Patent Document 5] Romanov, YA et al. (2003) Stem Cells 21, 105-110 [Non-Patent Document 6] Covas, DT et al. (2003) Braz. J. Med. Biol. Res. 36, 1179-1183 [Non-Patent Document 7] Takahashi, K. and Yamanaka, S. (2006), Cell, 126(4), pp. 663-676 [Non-licensed document 8] Okita, K. et al., Nature methods, 8(5), pp. 409-412

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[0012] The present invention relates to methods of generating induced pluripotent stem cells (iPS) as described herein, the resulting induced pluripotent stem cells, methods of differentiating the resulting induced pluripotent stem cells, and methods of treating disorders in a subject using differentiated cells derived from the induced pluripotent stem cells.

[0013] Therefore, the present invention provides a method for producing induced pluripotent stem cells, comprising expressing exogenous nucleic acid encoding protein OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in umbilical cord amniotic stem cells under suitable conditions for reprogramming stem cells, thereby producing induced pluripotent stem cells.In this embodiment, umbilical cord amniotic stem cells are umbilical cord amniotic mesenchymal stem cells or umbilical cord amniotic epithelial stem cells.

[0014] Furthermore, the present invention also provides an induced pluripotent stem cell population obtainable by this method, and an induced pluripotent stem cell population obtained by this method.The induced pluripotent stem cell population can be either an induced pluripotent stem cell population derived from the mesenchymal stem cell (population) of the amniotic membrane of the umbilical cord, or an induced pluripotent stem cell population derived from the epithelial stem cell (population) of the amniotic membrane of the umbilical cord.

[0015] In addition, the present invention also provides a pharmaceutical composition comprising the induced pluripotent stem cells of the present invention.

[0016] Furthermore, the present invention provides a method for differentiating the induced pluripotent stem cells of the present invention into target cells, wherein the induced pluripotent stem cells are differentiated into the target cells under conditions suitable for differentiation. As a result, the present invention also provides a pharmaceutical composition comprising the differentiated induced pluripotent stem cells obtained by the present invention.

[0017] The present invention also provides a method for treating a congenital or acquired degenerative disorder in a subject, comprising the step of administering to the subject target cells differentiated from the pluripotent stem cells obtained by the present invention.

[0018] In addition, the present invention provides extracellular membrane vesicles produced by the induced pluripotent stem cell population of the present invention or produced by cells obtained by differentiation of the induced pluripotent stem cells of the present invention. The present invention further includes the use of such extracellular membrane vesicles of the present invention as delivery carriers for therapeutic agents.

[0019] The present invention also provides cell culture media including Mammary Epithelial Basal Medium MCDB 170, EpiLife Medium, DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), and FBS (Fetal Bovine Serum).

[0020] The invention further relates to methods of differentiating induced pluripotent stem (iPS) cells as described herein into retinal pigment epithelium (RPE) cells, the resulting RPE cells, retinal pigment epithelium consisting of or comprising RPE cells as described elsewhere herein, methods of treating retinal degenerative diseases in a subject using RPE cells differentiated from iPS cells by the methods described herein, and pharmaceutical compositions comprising RPE cells obtained by the methods described herein. Additionally, the invention also relates to in vivo and in vitro methods using RPE cells obtained by the methods described herein.

[0021] We describe the generation of clinical RPE cells using iPS cells (abbreviated CLiPS) derived from umbilical cord superficial cells. We differentiated CLiPS into RPE using the method of the present invention compared to ES and dermal iPS cells, and consistently generated RPE with increased RPE differentiation efficiency. CLiPS-derived RPE had higher pigmentation than ES-derived RPE based on increased expression levels of pigmentation-specific genes such as MITF, PMEL17 and TRYP2, as well as RPE-specific genes such as BEST1, RPE65, MERTK, RLBP1. Also on the functional level by comparing the bioenergetics of RPE derived from different stem cells (CLiPS, ES and dermal iPS), it was demonstrated that CLiPS-RPE contained increased glycolysis and mitochondrial respiration levels compared to ES-derived RPE.

[0022] Thus, in a first aspect of the present invention, there is provided a method for differentiating iPS cells into RPE cells, the method comprising culturing iPS cells derived from stem cells of the amniotic membrane of an umbilical cord in a differentiation medium under conditions suitable for differentiation into RPE cells, thereby differentiating the iPS cells into RPE cells.

[0023] In a second aspect, the invention provides an RPE cell culture obtainable by the method and an RPE cell culture obtained by the method.

[0024] In a third aspect of the invention, the invention also provides a retinal pigment epithelium consisting of an RPE cell culture obtainable by the method or comprising an RPE cell culture obtainable by the method, and consisting of an RPE cell culture obtainable by the method or comprising an RPE cell culture obtainable by the method.

[0025] In a fourth aspect of the invention, the invention also provides a pharmaceutical composition comprising an RPE cell culture obtained by the method of the invention.

[0026] In a fifth aspect of the present invention, the present invention also provides a method for treating a retinal degenerative disease in a subject, comprising administering to the subject RPE cells differentiated from iPS cells by the method of the present invention.

[0027] In a sixth aspect of the present invention, the present invention provides an in vivo method for detecting the viability of RPE cells differentiated from iPS cells by the method defined herein in a subject, the method comprising: (a) introducing into the subject RPE cells differentiated from iPS cells by the method defined herein, the RPE cells comprising a bioluminescent label; (b) detecting a bioluminescent signal of the RPE cells over time using an imaging method, thereby collecting imaging data; and (c) comparing the imaging data received in step (b) with reference imaging data.

[0028] In a seventh aspect of the present invention, the present invention provides an in vitro method for determining the immunogenicity of RPE cells differentiated from iPS cells by a defined method in a subject to which the differentiated RPE cells have previously been delivered, the method comprising: (a) detecting pro-inflammatory cytokine levels in a sample obtained from the subject comprising the differentiated RPE cells using an imaging method, thereby collecting imaging data; (b) comparing the imaging data received in step (a) with reference imaging data. [Brief description of the drawings]

[0029] The invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and drawings.

[0030] [Figure 1]A flow chart is shown that shows the experimental steps of an exemplary embodiment of the method for producing induced pluripotent stem cells of the present invention. The stem cells used herein are isolated from the amniotic membrane of the umbilical cord, also referred to as umbilical cord lining stem cells (CLSCs). This embodiment begins with the recovery of isolated CLSCs by dissociating the cells from a cell culture device (note, however, that CLSCs can also be provided in isolated form for the method of the present invention). The CLSCs are then counted, and approximately 700,000 cells are dispensed and pelleted into a microcentrifuge tube. The cell pellet is resuspended in a buffer suitable for electroporation before the plasmid encoding Yamanaka factors is added to the cell-buffer mixture. Electroporation is performed with one pulse with a duration of approximately 20 ms and a voltage of approximately 1600 V, or two pulses with a duration of 30 ms and a voltage of approximately 1350 V, for umbilical cord lining mesenchymal cells (CLMCs) and umbilical cord lining epithelial cells (CLECs), respectively. After electroporation, the stem cells are immediately transferred into a medium suitable for recovery, where the medium includes a compound that suppresses the inflammatory response and enhances cell survival. After a suitable recovery time, the medium suitable for recovery is replaced with a 1:1 mixture of two different cell culture media, where the two different cell culture media are a medium suitable for recovery and a second cell culture medium. To refresh the cell culture medium, the medium mixture is replaced with the same mixture of cell culture media about 4 days after electroporation. This creates colonies of umbilical cord lining induced pluripotent stem cells, also referred to herein as CLiPS. After about another 2 days, the 1:1 mixture of the two different cell culture media is replaced with the second cell culture medium. This medium is also replaced about every 2 days to keep the medium fresh. Once the CLiPS colonies reach a size of about 0.5 mm to 1.5 mm in diameter, they are picked and transferred to a coated cell culture vessel suitable for cell culture and growth. Again, the cell culture medium is replaced periodically with the same medium. After reaching approximately 50% confluency, the CLiPS colonies are detached from the coated culture device and transferred to another cell culture vessel suitable for cell culture and proliferation, thus further dissociating the CLiPS colonies.Upon reaching approximately 70-80% confluency, CLiPS are passaged at a ratio of approximately 1:3 (v / v), where passaging at a ratio of approximately 1:3 (v / v) is performed by contacting one volume of dissociated CLiPS with two volumes of fresh medium. CLiPS are then cultured in medium containing substances that enhance cell survival until they reach approximately 30-60% confluency. At this point, CLiPS can be differentiated into any desired target cells. [Diagram 2] Figure 1 shows an exemplary comparison of the reprogramming efficiency of individual CLSC populations. Stem cells were subjected to different electroporation settings to transfect cells with exogenous nucleic acid. Electroporation was performed using the electroporation parameters shown in Okita et al., supra (1650 V, 10 ms, 3 pulses) and the parameters used in the present invention for transfection of amniotic epithelial stem cells of umbilical cord (also referred to herein as "umbilical cord lining epithelial stem cells" or CLECs; 1350 V, 30 ms, 2 pulses) and amniotic mesenchymal stem cells of umbilical cord (also referred to herein as umbilical cord lining mesenchymal stem cells or CLMCs (1600 V, 20 ms, 1 pulse)). 200K transfected cells were plated in triplicate in 6-well plates. Approximately 21 days after transfection, the percentage of reprogramming efficiency was calculated as the number of colonies / 200,000x10. [Figure 3-1]3 shows exemplary colony development of induced pluripotent stem cells from human CLMCs. Figures 3a-f: A representative time course of colony development, where Figure 3a depicts typical morphology of human CLMCs cultured in the maintenance medium at day 0 of culture. Figure 3b: depicts typical morphology of human CLMCs cultured in the maintenance medium at day 15 of culture. Figure 3c: depicts typical morphology of human CLMCs cultured in the maintenance medium at day 24 of culture. Figure 3d: depicts typical morphology of human CLMCs cultured in the maintenance medium at day 29 of culture. Figure 3e: shows a 4x magnification of typical morphology of a first iPS colony passage, and Figure 3f: shows a 10x magnification of typical morphology of an iPS colony at first passage. Figures 3g-l: depict exemplary immunofluorescence staining of iPS derived from human umbilical cord lining cells showing activation of endogenous expression of pluripotent embryonic stem cell markers, where Figure 3g: shows expression of KLF4, Figure 3h: shows expression of NANOG, Figure 3i: shows expression of OCT3 / 4, Figure 3j: shows expression of SOX2, Figure 3k: shows expression of SSEA4, and Figure 3l: shows expression of Tra-1-60. Figure 3m: shows exemplary karyotype analysis demonstrating normal chromosome count and G-banding pattern of CLiPS in individual cell lines CLEC23 (EC23-CLiPS), CLMC23 (MC23-CLiPS), CLEC44 (EC44-CLiPS), and CLMC44 (MC44-CLiPS). Figure 3n: shows exemplary human CLMSC-DTHN cultures emerged at 10 days of reprogramming, magnified 20x. Figure 3o: Typical morphology of expanded human CLMSC-DTHN cultured on laminin-511 substrate at 4x magnification. Figure 3p: Typical morphology of expanded human CLMSC-DTHN cultured on laminin-511 substrate at 10x magnification. Figure 3q: Typical morphology of expanded human CLMSC-DTHN cultured on laminin-511 substrate at 20x magnification. Figure 3r: Exemplary expression of human pluripotency marker NANOG in passage 3 CLMSC-DTHN iPS. Figure 3s: Exemplary expression of human pluripotency marker OCT3 / 4 in passage 3 CLMSC-DTHN iPS.Figure 3t: Exemplary expression of human pluripotency marker SOX2 in CLMSC-DTHN iPS at passage 3. Figure 3u: Exemplary expression of human pluripotency marker NTRA-1-81 in CLMSC-DTHN iPS at passage 3. Scale bars: all 100 μm. Figure 3v: Exemplary RT-PCR analysis of reprogramming and pluripotency gene expression in primary parental cells, parental cells 11 days after vector transfection (D11 transfected cells) and established iPS clones (CLiPS). "Vec" indicates amplification specific to vector-derived sequences. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. PCR of homo sapiens (H1) total RNA without reverse transcription was used to control for genomic contamination of all primer pairs. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Diagram 3-4] See description of Figure 3-1. [Figure 3-5] See description of Figure 3-1. [Figure 4-1]Exemplary histological analysis of teratomas formed by immunodeficient non-obese diabetic severe combined immunodeficient (NOD-SCID) mice after CLiPS injection. Teratoma formation assay reveals the formation of all three germ layers. Figure 4a inset: Teratoma obtained from human CLEC-derived iPS 3 months after subcutaneous injection. Sections of teratomas are further analyzed by hematoxylin-eosin staining. Figure 4a: Presence of respiratory-like epithelium in teratomas. Figure 4b: Presence of glandular structures representing endoderm in teratomas. Figure 4c in: Arrows indicate the presence of cartilage in teratomas. Figure 4d in: Arrows indicate the presence of bone representing mesoderm in teratomas. Figure 4e: Presence of kidney tissue in teratomas. Solid arrows indicate glomeruli and hollow arrows indicate renal tubules. Figure 4f in: Arrows indicate the presence of neuroepithelium representing ectoderm in teratomas. Using directed differentiation protocols, CLiPS were induced to differentiate into specific tissues. Figure 4g: CLiPS differentiated into hepatocytes visualized with α-fetoprotein (AFP) and 4',6-diamidino-2-phenylindole (DAPI). Figure 4h: CLiPS differentiated into hepatocytes visualized with human serum albumin (HAS), cytokeratin 18 (CK18) and DAPI. Figure 4i: CLiPS differentiated into hepatocytes visualized with Oil Red O. Figure 4j: CLiPS differentiated into cardiomyocytes visualized with α-actinin (αACT), cardiac troponin I (cTnl), myosin regulatory light chain 2a (MLC2a) and DAPI. Figure 4k: CLiPS differentiated into dopaminergic neurons visualized with floor plate marker FOXA2, tectal plate marker LMX1A and DAPI. Figure 4l: CLiPS differentiated into dopaminergic neurons visualized with neuron-specific class III β-tubulin (TUJI) and tyrosine hydroxylase (TH). Figure 4m: CLiPS differentiated into oligodendrocyte precursor cells visualized with OLIG2 and DAPI. Figure 4n: CLiPS differentiated into oligodendrocyte precursor cells visualized with O4 and DAPI. Figure 4o: Electrophysiological analysis of mature human CLiPS-derived dopaminergic neurons at day 45 of differentiation.Human CLiPS-derived dopaminergic neurons fire trains of action potentials with injected current. Scale bars: 200 μm for Figure 4a, c and d; 100 μm for Figure 4b, e and f; 50 μm for Figure 4g, h, i, k, l and m; 25 μm for Figure 4j and n. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5-1] Exemplary directed differentiation of human CLiPS into a variety of different cell types is shown here, where FIG. 5a: depicts human CLiPS-derived neurons visualized with TH, Tuik and DAPI, FIG. 5b: depicts human CLiPS-derived hepatocytes visualized with CK18, HAS and DAPI, FIG. 5c: depicts human CLiPS-derived cardiomyocytes visualized with cTnl, αAct and DAPI, and FIG. 5d: electrophysiological analysis of contracting human CLiPS-derived cardiomyocytes illustrating cells generating spontaneous action potentials. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] Exemplary flow cytometry analysis of major histocompatibility complex (MHC) class I and II, and T cell costimulatory protein expression in iPS and dopaminergic neural progenitor cells differentiated therefrom. Figure 6a: Flow cytometry profile of immune-related gene expression in undifferentiated iPS. Figure 6b: Flow cytometry analysis of neural cell adhesion molecule (NCAM) positive populations. These populations were gated for analysis of immune-related protein expression. Figure 6c: Analysis of immune-related protein expression in differentiated dopaminergic neural progenitor cells at day 25. [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 6-4] See description of Figure 6-1. [Figure 6-5] See description of Figure 6-1. [Figure 6-6]See description of Figure 6-1. [Figure 6-7] See description of Figure 6-1. [Figure 6-8] See description of Figure 6-1. [Figure 6-9] See description of Figure 6-1. [Figure 6-10] See description of Figure 6-1. [Figure 7]Figure 7 shows a comparison of in vivo engraftment of dopaminergic neural progenitor cells (NPCs) derived from human CLiPS and human adult fibroblast iPS (asF-iPS) in NOD-SCID mice. Day 25 dopaminergic NPCs were injected into the striatum of NOD-SCID mice to assess the engraftment and differentiation potential of the cells in an immunodeficient environment. TH-immunoreactive dopaminergic neurons are present among abundant human NCAM-positive engrafted neurons. Figure 7a: shows in vivo engraftment of day 25 dopaminergic NPCs derived from human asF-iPS. Figure 7b: shows in vivo engraftment of day 25 dopaminergic NPCs derived from human CLEC-iPS (EC23-CLiPS). Figure 7c: shows in vivo engraftment of day 25 dopaminergic NPCs derived from CLMC-iPS (MC23-CLiPS). Figure 7d: Antibody staining of the transplanted hemisphere of a Parkinson's disease (PD) mouse model generated in immune-competent C57BL / 6NTac mice 1 month after human CLEC-iPS-derived dopaminergic NPC transplantation. Human NCAM (green) and TH (red) double positive neurons are abundant at the injection site. Figure 7e: Long neurites originating from the transplantation site project along the forceps major of the corpus callosum to distal regions of the brain. Arrow in Figure 7f: Human NCAM and TH double positive neurons are abundant at the injection site as indicated by the arrow. Figure 7g: Contralateral non-transplanted hemisphere of the same section shown in Figure 7d. Figure 7h: Illustrates the absence of viable cells in the striatum transplanted with human adult asF-iPS-derived NPCs, suggesting immune rejection. Figure 7i: Abundant microglia / macrophage aggregation in the transplanted hemisphere. Figure 7j: Absence of microglia / macrophage aggregation in the non-transplanted hemisphere. Figure 7k: Higher magnification of Figure 7i. Microglia located proximal and internal to the graft can be seen to adopt a more amoeboid morphology characteristic of activated microglia. Figure 7l: Higher magnification of Figure 7k, showing expression of CD68, a marker of microglial activation. Scale bars: 100 μm for Figures 7a-c and 7k; 200 μm for Figures 7d, 7g and 7h; 50 μm for Figures 7e, 7f and 7l. [Figure 8-1] Survival of human CLEC-derived (EC23-CLiPS) dopaminergic neurons in a mouse PD model 9 months after transplantation. Figure 8a: HuNu+ / hNCAM+ / TH+ neurons present in the transplanted hemisphere. Figure 8b: Overlay of Figures 8c-f, showing higher magnification of the boxed area in Figure 8a. Figure 8c: hNCAM+ neurons present in the transplanted hemisphere. Figure 8d: HuNu+ neurons present in the transplanted hemisphere. Figure 8e: TH+ neurons present in the transplanted hemisphere. Figure 8f: Nuclei of neurons present in the transplanted hemisphere. Figure 8g: Schematic illustration of the experimental steps starting from induction of PD lesions by 6-hydroxydopamine (6-OHDA) injection into the striatum of C57BL / 6NTac mice. Pre-transplant rotational behavior assays were performed 1 and 2 weeks before NPC transplantation. Figure 8h: Results of apomorphine-induced rotational asymmetry assay in mice transplanted with dopaminergic NPCs derived from human EC23-CLiPS and asF-iPS, as well as sham controls. Assays were performed every 2 weeks until 22 weeks after transplantation. Animals in the human EC23-CLiPS group showed statistically significant recovery of rotation compared to the asF-iPS group from 20 weeks after transplantation (n=5, p<0.05). No recovery was observed in the sham-treated group. Figure 8h: Representative in vivo positron emission tomography (PET) imaging of [18F]PE-P2I ligand uptake to assess recovery of dopamine transporter (DAT) function in striatal dopaminergic neurons 6 months after transplantation. Mice transplanted with human EC23-iPS NPCs showed recovery of DAT activity compared to mice transplanted with asF-iPS NPCs or sham-treated controls. Scale bars: 200 μm in Fig. 8a; 100 μm in Fig. 8b–f. [Figure 8-2] See description of Figure 8-1. [Figure 9]1 shows exemplary in vivo PET imaging of striatal dopamine production in transplanted mice. PET illustrates the incorporation of [18F]PE-P2l ligand to assess the recovery of dopamine transporter (DAT) function in striatal dopaminergic neurons 6 months after iPS-derived NPC transplantation. Mice transplanted with human CLEC-iPS-derived NPCs show clear recovery of DAT activity compared to mice transplanted with human adult iPS-derived NPCs or sham-transplanted controls. [Figure 10] FIG. 1 illustrates the in vivo maintenance of grafts derived from human CLiPS 6 and 9 months after transplantation into mouse brain. The grafts stain positive for the human antigen NCAM and TH dopaminergic marker. No tumor formation was recorded. Scale bar: 50 μm. [Figure 11] Figure 11 shows the results of histological and functional analysis of transplanted human EC23-CLiPS dopaminergic NPCs in a medial forebrain bundle (MFB) lesion model of PD generated in fully immunocompetent Wistar Hannover rats. Figure 11a: shows the engraftment of human EC23-CLiPS neurons in the striatal region of the rat brain 3 months after transplantation, demonstrated by positive double staining for human cytoplasmic (STEM 121) and human nuclear antigen (HuNu) antibodies. Staining indicates functional recovery. Figure 11b: shows colocalization of synapsin 1 immunoreactivity with hNCAM+ / TH+ neurons, suggesting possible integration of transplanted human CLiPS-derived cells with the host tissue 3 months after transplantation. Figure 11c: shows retrograde lesion of the dopaminergic system in the substantia nigra of the rat brain. Figure 11d: shows a non-lesioned rat brain confirming retrograde lesion of the dopaminergic system in the substantia nigra of Figure 11c by tyrosine hydroxylase (TH) immunostaining. Figure 11e: Results of an apomorphine-induced rotational asymmetry assay in rats transplanted with dopaminergic NPCs derived from human CLEC23-iPS. The results show that transplantation of CLiPS-NPCs mediated restoration of functional motor deficits in the rat MFB model of PD over the 6-month study period. Scale bars: 100 μm in Figures 11a and 11b; 200 μm in Figures 11c and 11d. [Figure 12a]12a, b, and c respectively show exemplary colonies of induced pluripotent stem cells derived from human CLECs generated by using PTTe-3 medium as the recovery medium. [Figure 12b] See legend to Figure 12a. [Figure 12c] See legend to Figure 12a. [Figure 13] Showing that CLiPS differentiate into RPE: Images of differentiation cultures from different stem cells, human ES cells (H9), skin-derived iPS cell lines (Asf5, AGO, HDFA), umbilical cord superficial mesenchymal cells (CLMC23, CLMC30, CLMC44) and umbilical cord superficial ectodermal cells (CLEC23). Dark patches on the cell culture plate correspond to the presence of pigmented RPE cells. [Figure 14a] We show that CLMCs have consistently high differentiation efficiency. Figure 14a: Visual grading system for estimating RPE differentiation efficiency based on the percentage of the area of ​​the well occupied by pigmented cells; RPE differentiation efficiency is graded as 0, 1, 2 or 3 for no pigmentation, <30%, 30-60% or >60% pigmentation, respectively. Figure 14b: RPE differentiation efficiency estimated by visual grading of the pigmented cell area of ​​the differentiation plate. Each bar represents the grading of one differentiation plate, and the numbers on the bar indicate the percentage of wells on the plate with different grades of pigmentation, indicated by different shades of brown. Numbers 1-3 represent biological replicates. Figure 14c: RPE differentiation efficiency of different stem cells estimated by Pmel17 by flow cytometry; cells from 3 wells were pooled for FACS analysis. [Figure 14b] See legend to Figure 14a. [Figure 14c] See legend to Figure 14a. [Figure 15a]Figure 15a: Images of differentiation plates taken under identical conditions at day 30 of differentiation using a ChemiDoc Touch gel imaging system (Bio-Rad laboratories). Figure 15b: Phase contrast images of different stem cell derived RPE showing weak pigmentation of H9: H9 (human ES cell derived RPE), CLMC23, CLMC30, CLMC44, CLEC23 (CLiPS derived RPE), AGO, HDFA, Asf5 (dermal iPS cell derived RPE). Figure 15c: Graph showing pigmentation intensity analyzed from images of differentiation plates taken using Biorad's ChemiDoc Touch system at different time points along the differentiation of H9: (human ES cell derived RPE), CLMC23, CLMC30 and CLEC23 (CLiPS derived RPE). FIG. 15d: Pigmentation-related and RPE-specific genes are elevated in CLiP: RT-qCPR analysis of genes involved in pigmentation at days 18 and 35 of differentiation: MITF, PMEL17, TYROSINASE, TRYP2. [Figure 15b] See legend to Figure 15a. [Figure 15c] See legend to Figure 15a. [Figure 15d-1] See legend to Figure 15a. [Figure 15d-2] See legend to Figure 15a. [Figure 16] CLiPS expressing RPE-specific genes at days 18 and 35 of differentiation: RT-qCPR analysis of RPE-specific RPE65 and MERTK. [Figure 17]We show that CLiPS-derived RPE are functional. Figure 17a: We show that in vitro generated RPE form tight junctions similar to native RPE: Transepithelial Electrical Resistance (TEER), a measure of tight junction integrity, in different stem cell derived RPE measured over 4 months using an Epithelial Volt Ohm meter EVOM2™. Figure 17b: We show that in vitro generated RPE are highly phagocytic: Phagocytosis rate of FITC-labeled photoreceptor outer segments (POS) by different stem cell derived RPE. [Figure 18] CLiPS-derived RPE exhibit similar protein expression as ES-derived RPE. CLiPS-RPE exhibit apical expression of Mertk, junctional expression of ZO-1, and cytoplasmic expression of RPE65. [Figure 19-1] The original method of RPE differentiation and its modifications are shown. Figure 19a: A schematic diagram of the original method is shown, showing the differentiation media used at different stages and their composition. Figure 19b: Modifications introduced in the differentiation protocol are shown, showing the gradual increase in CHIR99021 concentration and the replacement of the FGF inhibitor SU5402 with PD173074. Figure 19c: Photographs of CLMC30 plates differentiated by the published protocol with SU5402 or the modified protocol with PD173074, showing comparable RPE differentiation and pigmentation. DM1-DM5: Differentiation media 1-5. The modified RPE differentiation protocol with PD173074 results in functional RPE. The functionality of RPE derived from the differentiation method with SU5405 or PD173074 was tested for TEER (Figure 19d) and phagocytosis of FITC-labeled POS particles (Figure 19e). [Figure 19-2] See description of Figure 19-1. [Figure 19-3] See description of Figure 19-1. [Figure 20a]Figure 20a and b: Comparison of RPE yields by different purification methods. Specifically, Figure 20a and b show a schematic diagram of the different methods of RPE purification: Differentiated cultures containing RPE and non-RPE were purified by: (i) manual purification: identification of non-RPE cells based on morphology and lack of pigmentation by observation under a dissecting microscope and their manual removal by scraping, (ii) TrypLE purification: removal of most of the weakly adherent non-RPE clusters by partial TrypLE treatment, (iii) TrypLE + manual: removal of most of the weakly adherent non-RPE clusters by partial TrypLE treatment followed by manual removal of the few non-RPE clusters that escaped TrypLE treatment by observation under a dissecting microscope, (iv) TrypLE + scatter sorting: removal of weakly adherent non-RPE clusters by partial TrypLE treatment followed by scatter sorting, (v) scatter sorting: Total cells from mixed differentiation cultures were separated into high (pigmented RPE cells) and low (non-pigmented non-RPE cells) scattering populations based on their relative light scatter. Figures 20c and d show the original and modified scatter sorting methods to more accurately select high scattering RPE cells. Figure 20c shows the arbitrarily selected gates for the high scattering gate (cyan) and low scattering gate (magenta) as in the original protocol. Figure 20d shows the modified gate selection to set the low scattering gate (magenta) using weakly adherent non-RPE cells dissociated by partial TryPLE treatment to more accurately select the high scattering gate (cyan). Figure 20e shows the yield of RPE obtained from the different purification methods. Figure 20f shows the purity of RPE from the different purification methods assessed by Pmel17 flow cytometry. Figure 20g: TEER of RPE from different purification methods; M: manual purification, T: TrypLE purification, T+M: TrypLE+manual purification, T+Sc: TrypLE+scatter sorting, Sc: scatter sorting, T (Loose): weakly adherent non-RPE cells that were easily detached by TrypLE treatment, Sc Low: low scatter non-RPE cells from scatter sorting. Figure 20h: Phagocytic capacity of RPE from different purification methods assessed by photoreceptor outer segment (POS) phagocytosis assay.Figure 20i: Table comparing different RPE purification methods. Figure 20j: Quantitative PCR comparison of RPE specific gene expression in CLMC23 and H9. qPCR results show relative expression of RPE specific genes such as BEST1, RPE65, RLBP1, MERTK, MITF, PMEL17 and TRYP2 normalized to GAPDH. Figure 20k: Comparison of gene expression in CLMC23 and H9, expressed as fold change of CLMC23 over H9. [Figure 20b] See legend to Figure 20a. [Figure 20c] See legend to Figure 20a. [Figure 20d] See legend to Figure 20a. [Figure 20e] See legend to Figure 20a. [Fig. 20f] See legend to Figure 20a. [Figure 20g] See legend to Figure 20a. [Figure 20h] See legend to Figure 20a. [Figure 20i] See legend to Figure 20a. [Figure 20j] See legend to Figure 20a. [Figure 20k] See legend to Figure 20a. [Figure 21a]Figure 21a: OCR curves showing that basal respiration, ATP production, maximal capacity and spare respiratory capacity are 38%, 40%, 35% and 36% higher in CLiPs-RPE compared to H9-RPE. Figure 21b: ECAR curves showing that glycolysis, glycolytic capacity and glycolytic reserve are 25%, 37% and 50% higher in CLiPs-RPE compared to H9-RPE. Figure 21c-f: CLiPs-RPE demonstrate increased resistance to oxidized low-density lipoprotein (oxLDL) as evidenced by the lack of reduction in maximal capacity following exposure to oxLDL (dotted curve) for CLEC23-RPE (c) compared to a 27% reduction in ASF5-RPE (d) and a 43% reduction in H9-RPE (e). (The response of CLiPs-RPE cells to oxidative stress is similar to that seen in native RPE (AHRPE - f), making them functionally closer to primary RPE compared to other differentiated RPE. Figure 21g-j: CLiPs-RPE show increased resistance to hydrogen peroxide (H2O2) as evidenced by the lack of reduction in maximal capacity following exposure to H2O2 (dotted curve) for CLEC23-RPE (g) compared to a 27% reduction in ASF5-RPE (h) and a 99% reduction in H9-RPE (i). The response of CLiPs-RPE cells to oxidative stress is similar to that seen in native RPE (AHRPE) (f and j), making them functionally closer to primary RPE compared to other differentiated RPE. [Figure 21b] See legend to Figure 21a. [Figure 21c] See legend to Figure 21a. [Figure 21d] See legend to Figure 21a. [Figure 21e]See legend to Figure 21a. [Fig. 21f] See legend to Figure 21a. [Fig. 21g] See legend to Figure 21a. [Fig. 21h] See legend to Figure 21a. [Figure 21i] See legend to Figure 21a. [Figure 21j] See legend to Figure 21a. [Figure 22a] All stem cell-derived retinal pigment epithelial (SC-RPE) cell lines show a lack of immune system clearance. Figure 22a and b: In vivo bioluminescence measurements (total luminescence) of injected luciferase-expressing SC-RPE embedded in Matrigel plugs at the indicated time points in both humanized and NOD-SCID IL2Rγ- / - (immunodeficient) mice. Figure 22c: Representative images of all SC-RPE lines showing RPE65, Ki67 and Hoechst staining from Matrigel plugs implanted in humanized mice at the 2-month endpoint. Scale bar, 50 μm. [Figure 22b] See legend to Figure 22a. [Figure 22c] See legend to Figure 22a. [Figure 23a] Figure 23a and b: Serum cytokines (IFN-γ and IL-18) at analyzed endpoints. Figure 23c: Representative images showing OTX2, human CD45 (hCD45) and Hoechst staining of RPE-Matrigel plugs showing immune cell infiltration. Figure 23d and e: Grading of cellular immune response (0-3) based on hCD45 positive cells within RPE-Matrigel plugs. Scale bar, 50 μm. [Figure 23b] See legend to Figure 23a. [Figure 23c] See legend to Figure 23a. [Figure 23d] See legend to Figure 23a. [Figure 23e]See legend to Figure 23a. [Figure 24a] Figure 24a and b: Serum cytokines (IL-23 and IL-17A) at analyzed end points. Figure 24c: T cell (CD3) to B cell (CD19) ratios were calculated after flow cytometry analysis. Figure 24d: To analyze T cell differentiation, CD3 positive cells were further gated into helper T (CD4) and cytotoxic T (CD8) cells. Figure 24e and f: CD4 positive and CD8 positive cells were gated into four groups with different T cell activation status based on specific surface markers. [Figure 24b] See legend to Figure 24a. [Figure 24c] See legend to Figure 24a. [Fig. 24d] See legend to Figure 24a. [Figure 24e] See legend to Figure 24a. [Fig.24f] See legend to Figure 24a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description of the Invention The present invention is particularly directed to methods of generating induced pluripotent stem cells from umbilical cord amniotic stem cells under conditions suitable for reprogramming the stem cells, thereby generating induced pluripotent stem cells (iPS).

[0032] In the present invention, both mesenchymal and epithelial stem cells of the amniotic membrane of the umbilical cord, collectively referred to herein as umbilical cord lining stem cells (CLSC), are used to generate iPS, also referred to herein as umbilical cord lining-derived induced pluripotent stem cells or "CLiPS". Surprisingly, the umbilical cord lining-derived induced pluripotent stem cells of the present invention are robust and homogeneous stem cells that can differentiate into functional target cells of different lineages (see Examples 3 and 4). For example, the umbilical cord lining-derived induced pluripotent stem cells have the ability to differentiate into multiple cell types, and can differentiate into various cell types, such as hepatocytes (see Example 8), which represent endodermal tissue, cardiomyocytes (see Example 9), which represent mesodermal tissue, and dopaminergic neurons (see Example 7) and oligodendrocytes (see Example 10), which represent ectodermal tissue. Even more surprising and important is the finding that, for example, human CLiPS-derived dopaminergic neurons can functionally engraft in different species, surviving up to 9 months in a mouse Parkinson's disease (PD) model without immunosuppression and 6 months in a rat PD model without immunosuppression (see Examples 12 and 13). Therefore, in summary, the inventors have created a hypoimmunogenic cell source capable of engrafting, integrating, and mediating therapeutic recovery in a fully immunocompetent host. The umbilical cord lining-derived induced pluripotent stem cells of the present invention can potentially be used as a universal cell source for allogeneic cell transplantation in humans without the need for immunosuppression, making them ideal candidates for such cell-based therapies. As an additional advantage, it has been found herein that the umbilical cord lining-derived induced pluripotent stem cells of the present invention can be generated by an integrative and feeder-free method, thus allowing the production of iPS under current Good Manufacturing Practice (cGMP) conditions. Since GMP processes have been recently established for the large-scale production of umbilical cord amniotic mesenchymal stem cells (see International Application WO 2018 / 067071 or US Patent Application US2018127721), the present invention provides an ideal platform to produce iPS cells for subsequent cell-based therapies in humans or animals. In summary, CLiPS derived from very young tissues are less likely to carry genetic, epigenetic and mitochondrial DNA mutations because they are obtained from young tissues. Due to these advantages, CLiPS are potentially a good stem cell source for generating differentiated cells for regenerative medicine. Therefore, CLiPS are superior to iPS cells obtained from skin or blood, which require invasive procedures of tissue collection. Also, CLiPS are free of the ethical issues associated with ES cells. Therefore, CLiPS are a better stem cell source for regenerative medicine. The inventors found that such CLiPS can be reliably differentiated into retinal pigment epithelial (RPE) cells, also called RPE, by the method of the present invention. In this invention, the inventors compared different stem cell sources: human ES cells (ES), iPS cells derived from skin (skin-iPS) and cord-lining cells (CLiPS) for their ability to generate RPE in vitro. CLiPS can be of either mesenchymal (CLMC) or ectodermal (CLEC) origin. We next compared the RPE differentiation efficiency of CLiPS with ES cells and skin iPS cells. Compared with skin-iPS, CLiPS consistently showed higher RPE differentiation efficiency than skin-iPS cells by visual grading and flow cytometry estimation. Comparing the pigmentation of differentiation cultures visually and by image analysis also showed that CLiPS-derived RPE had higher pigmentation than ES-derived RPE. RPE generated from CLiPS also exhibited functional characteristics of RPE after in vitro maturation, suggesting that it is a superior source of RPE cells. Furthermore, by comparing the bioenergetics of RPE derived from different stem cells, we found that CLiPS-RPE had higher glycolysis and mitochondrial respiration than ES-derived RPE. The method of the present invention used to differentiate induced pluripotent stem (iPS) cells derived from umbilical cord amniotic stem cells (CLiPS or CLSC) into RPE cells has been specifically modified as described herein, thereby achieving maximum RPE yield.

[0033] Here, we first describe a method for producing iPS cells of the present invention. This method may include expressing exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28, and L-MYC, and p53-shRNA. Nucleic acids encoding OCT3 / 4 (SEQ ID NO:1), sometimes referred to as POU5FL, OCT3, or OCT4, encode octamer-binding transcription factor 4. OCT3 / 4 (SEQ ID NO:2) forms a heterodimer with SOX2 to regulate pluripotency factors in cells. SOX2 (SEQ ID NO:3), sometimes referred to as SEY, encodes sex-determining region Y-box 2 transcription factor (SEQ ID NO:4). When SOX2 binds to OCT3 / 4, it binds to non-palindromic genomic sequences and activates the transcription of pluripotency factors in cells. KLF4 (SEQ ID NO:5), sometimes referred to as GKLF, encodes Krueppel-like factor 4. KLF4 (SEQ ID NO:6) is a zinc finger transcription factor, which functions as a tumor suppressor controlling the G1 to G2 transition of the cell cycle by mediating the tumor suppressor p53. L-MYC (SEQ ID NO:7) encodes a transcription factor (SEQ ID NO:8) that activates the expression of proliferative genes. LIN28 (SEQ ID NO:9) encodes the RNA-binding protein Lin-28 homolog A (SEQ ID NO:10) that regulates stem cell self-renewal. p53-shRNA (SEQ ID NO:11) encodes a small hairpin RNA directed against p53, a protein that can regulate the cell cycle by arresting the cell cycle when the protein accumulates in the cell. To avoid cell cycle arrest by p53, p53-shRNA may post-transcriptionally silence the expression of p53. To generate CLiPS, exogenous nucleic acids encoding OCT3 / 4, SOX2, KLF4, LIN28, LMYC and p53-shRNA may be transferred into CLSCs for expression. Alternatively, the proteins OCT3 / 4, SOX2, KLF4, LIN28, L-MYC and p53 shRNA may be directly transferred into CLSCs.

[0034] As explained above, the induced pluripotent stem cell population of the present invention can be obtained by reprogramming the stem cells of the amniotic membrane of the umbilical cord. The stem cells of the umbilical cord can be the (isolated) mesenchymal stem cells of the amniotic membrane of the umbilical cord, also called umbilical cord lining mesenchymal stem cells (CLMC), or the (isolated) epithelial stem cells of the amniotic membrane of the umbilical cord, also called umbilical cord lining epithelial stem cells (CLEC). The CLEC and CLMC used to generate the iPS of the present invention can be derived from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, and in one embodiment, stem cells of human origin are preferred. Thus, the iPS of the present invention can also be derived from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, and in one embodiment, stem cells of human origin are preferred. In a preferred embodiment, CLEC are used to generate the iPS of the present invention.

[0035] When using the epithelial stem cells of the amniotic membrane of the umbilical cord as starting material, these epithelial stem cells can be obtained, for example, as described in US Patent Application No. 2006 / 0078993 (which leads to the granted US Patent Nos. 9,085,755 and 9,737,568) or the corresponding international patent application WO2006 / 019357.When using the mesenchymal stem cells of the amniotic membrane of the umbilical cord as starting material, they can be obtained, for example, as described in US Patent Application No. 2006 / 0078993 (which leads to the granted US Patent Nos. 9,085,755 and 9,737,568) or the corresponding international patent application WO2006 / 019357.

[0036] As starting material it is also possible to use mesenchymal stem cell populations as described in published US patent application 2018 / 127721 or corresponding international application WO 2018 / 067071. The mesenchymal stem cell population of international application WO 2018 / 067071 has the advantage that 99% or more of the stem cells of this population are positive for the three mesenchymal stem cell markers CD73, CD90 while lacking expression of CD34, CD45 and HLA-DR, meaning that 99% or more of the cells of the mesenchymal stem population of international application WO 2018 / 067071 express the stem cell markers CD73, CD90 and CD105, while not expressing the markers CD34, CD45 and HLA-DR. This highly homogeneous and well-defined cell population is an ideal candidate for clinical trials and cell-based therapies, since they fully meet the generally accepted criteria for human mesenchymal stem cells to be used for cell therapy, as defined, for example, by Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al.,. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a, review", Stem Cell Research & Therapy 2013, 4:66), Vonk et al., Stem Cell Research & Therapy (2015) 6:94, or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75-79. The mesenchymal stem population of international application WO 2018 / 067071 is therefore an ideal starting material for producing the CLiPS of the invention under GMP conditions.

[0037] In this regard, it is noted that transgene-transfected CLMCs, while maintaining their stemness and stem cell properties, may show a decreased proportion of cells expressing mesenchymal stem cell markers such as CD73, CD90 and CD105, but at the same time, may show an increased proportion of cells expressing negative markers such as CD34, CD45 or HLA-DR. See Yap et al., Malaysian J Pathol 2009; 31(2): 113-120); see also Madeira et al, Journal of Biomedicine and Biotechnology. Volume 2010, Article ID 735349, 12 pages. In light of this, it is possible that CLiPS of the present invention generated by reprogramming of CLMCs as described herein and isolated from the amniotic membrane of an umbilical cord may be a stem cell population in which at least about 81% or more, about 82% or more, at least 83% or more, at least 84% or more, at least about 85%, or about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the CLiPS population may express each of the following markers: CD73, CD90 and CD105.Furthermore, such CLMC-derived populations of induced pluripotent stem cells of the present invention may be populations in which at least about 81% or more, about 82% or more, at least 83% or more, at least 84% or more, at least about 85%, or about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% lack expression of each of CD34, CD45 and HLA-DR. A preferred example of such a CLMC-derived population of induced pluripotent stem cells of the present invention may be a population in which at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells in the CLMC population express each of CD73, CD90 and CD105, and lack expression of each of CD34, CD45 and HLA-DR.

[0038] Turning again to the creation of the induced pluripotent stem cell (population) of the present invention, it is important to note again that such induced pluripotent stem cell can be obtained by any suitable method of reprogramming the stem cell (population) of the amniotic membrane of umbilical cord into such induced pluripotent stem cell (population).One method of creating such induced pluripotent stem cell includes expressing exogenous nucleic acid encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in the stem cell of the amniotic membrane of umbilical cord under suitable conditions for reprogramming stem cell, thereby creating induced pluripotent stem cell, but the present invention is by no means limited to the CLiPS obtained by this method. Rather, CLiPS can be obtained by any suitable method, for example as described in the review article by Cieslar-Probuda et al. "Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences" BBA - Molecular Cell Research, Volume 1864, Issue 7, July 2017, Pages 1359-1369. For example, reprogramming can be performed in the present invention either chemically, by using small molecules, or biologically, by expressing exogenous nucleic acids encoding reprogramming factors in cells. Alternatively, exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28, L MYC and p53 shRNA can be provided as any nucleic acid suitable for expression. For example, the nucleic acid can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger RNA (mRNA) and microRNA (miRNA). Exogenous nucleic acid may be directly transferred, or exogenous nucleic acid may be incorporated into one or more vectors suitable for transfer into cells. In this regard, any vector suitable for transfer into CLSC may be used. An example of such a vector may be a plasmid. In the present invention, exogenous nucleic acid may be provided by one, two, three or four vectors suitable for transfer into stem cells.As an example, three vectors may provide exogenous nucleic acids for reprogramming CLSCs to CLiPS, which may be pCXLE-hOCT3 / 4-shp53-F (Addgene plasmid no. 27077; SEQ ID NO: 12), pCXLE-hSK (Addgene plasmid no. 27078, SEQ ID NO: 13) and pCXLE-hUL (Addgene plasmid no. 27080; SEQ ID NO: 14).

[0039] According to the above, any suitable method can be used to transfer exogenous nucleic acid or protein into CSLC. In one example, viral vector can be used to transfer exogenous nucleic acid into CSLC. Examples of such viral vectors can be retrovirus, lentivirus, inducible lentivirus, Sendai virus or adenovirus. Alternatively, transfection can be carried out to transfer exogenous nucleic acid into CSLC. In the present invention, transfection can include electroporation, microinjection, liposome-mediated and non-liposome-mediated transfection and sonoporation.

[0040] In a preferred example, CLSCs may be subjected to electroporation, where electrical parameters may be adjusted according to the type of CLSCs used, since CLMCs may require different electroporation conditions than CLECs. Electrical parameters may include the number of electrical pulses applied to the stem cells, the duration of the electrical pulses applied, and the voltage of the electrical pulses applied. Each electrical parameter may be adjustable to further optimize the electroporation of the present invention. In doing so, each electrical parameter may be adjusted independently or in combination with one or more of the other electrical parameters (see Example 1). In the present invention, any parameter setting suitable for allowing the transfer of exogenous nucleic acid into CLSCs may be applied. In one example of the present invention, CLMCs may be subjected to electroporation. In such a case, electroporation may be performed with one electrical pulse, which may have a duration of about 15 milliseconds (ms) to about 25 ms and a voltage of about 1550 V to about 1650 V. Thus, in one example, CLMCs may be subjected to electroporation with one electric pulse that may have a duration of about 20 ms and a voltage of about 1600 V. Furthermore, it has been found herein that electroporation resulting in a usable amount / number of CLiPS derived from CLMCs depends on the ratio of each vector (plasmid) DNA transfected and the number of CLMCs used for transfection. This ratio is defined herein as the number of CLMCs subjected to electroporation (1×10 cells) 6 It is expressed by the amount (in μg) of each vector (plasmid) DNA used relative to the number of cells (units). For example, the ratio of the amount of vector (plasmid) DNA of each vector to the number of cells is approximately 1 × 10 6 1.5 μg of DNA for 1 x 10 CLMCs 6 This ratio can range from about 2.5 μg of DNA to 1 CLMC. Thus, this ratio is approximately 1×10 6 Approximately 2.5 μg of DNA for each CLMC, approximately 1 × 10 6 Approximately 2.25 μg of DNA for each CLMC, approximately 1 × 10 6Approximately 1.8 μg of DNA for each CLMC, approximately 1 × 10 6 Approximately 1.7 μg of DNA for each CLMC, approximately 1 × 10 6 Approximately 1.67 μg of DNA for each CLMC, approximately 1 × 10 6 Approximately 1.6 μg of DNA per CLMC, or approximately 1 × 10 6 This can be approximately 1.5 μg of DNA per CLMC (approximately 1 × 10 6 See Table 1, which shows that an effective transformation yield was obtained using a ratio of the amount of vector (plasmid) DNA of each vector to the number of cells of about 1.67 μg DNA per CLMC). Thus, in one embodiment of generating CLiPS derived from CLMC, it is preferable to use the same amount of each vector in the electroporation of CLMC. CLECs may also be subjected to electroporation to obtain CLiPS of the present invention. For CLiPS derived from CLECs, electroporation may be performed with two electric pulses, each of which may have a duration of about 25 ms to about 35 ms and a voltage of about 1300 V to about 1400 V. Thus, in one example, CLECs may be subjected to electroporation with two electric pulses, each of which may have a duration of about 30 ms and a voltage of about 1350 V. As for CLMCs, it was also found that for CLEC-derived CLiPS, electroporation resulting in a usable amount / number of CLEC-derived CLiPS depends on the ratio between the amount of each transfected plasmid DNA and the number of CLECs used for transfection. This ratio is also defined herein as the number of CLECs transfected (1×10 cells). 6 It is expressed as the amount (in μg) of vector (plasmid) DNA used for transfection to the number of cells (units). For example, the ratio of the amount of vector (plasmid) DNA to the number of cells is approximately 1 × 10 6 Approximately 1.5 μg of DNA to approximately 1 × 10 6 The ratio can range from about 2.5 μg of DNA per CLEC. Thus, this ratio is about 1×10 6Approximately 1.5 μg of DNA per CLEC, approximately 1 × 10 6 Approximately 1.6 μg of DNA per CLEC, approximately 1 × 10 6 Approximately 1.67 μg of DNA per CLEC, approximately 1 × 10 6 Approximately 1.7 μg of DNA per CLEC, approximately 1 × 10 6 Approximately 1.8 μg of DNA per CLEC, approximately 1 × 10 6 Approximately 1.9 μg of DNA per CLEC, approximately 1 × 10 6 Approximately 2.0 μg of DNA per CLEC, or approximately 1 × 10 6 Approximately 2.5 μg of DNA per CLEC (approximately 1 × 10 6 (See Table 1, which shows that an effective transformation yield was obtained using a ratio of the amount of plasmid DNA of each vector to the number of cells of about 1.67 μg DNA per CLEC). Therefore, in one embodiment of generating CliPS derived from CLECs, it is preferable to use the same amount of each vector in the electroporation of CLECs. Electroporation of both CLECs and CLMCs can be performed with a uniform electric field in the method of the present invention. Thereby, important effects of electroporation such as pH change, ion formation or heat generation can be minimized. A uniform electric field can be created by maximizing the gap between the electrodes while minimizing the surface area of ​​each electrode. An example of a system that provides such a uniform electric field is the Neon™ Transfection System from ThermoFisher Scientific. Another example of a suitable commercially available transfection system is The Gene Pulser MXcell electroporation system available from Bio-Rad. Finally, transfection can be performed using any suitable electroporation buffer. When a commercially available transfection system is used, such as the Neon™ transfection system, the respective electroporation buffer provided by the transfection system manufacturer is typically used for electroporation.

[0041] After transfection, the stem cells may be transferred to a medium suitable for cell recovery and cell culture. In the present invention, any cell culture medium suitable for cell recovery and / or proliferation may be used. Examples of such suitable cell culture medium may be media commonly used for the culture (proliferation) of human induced pluripotent stem cells, such as mTeSR1, StemMACS™ iPS-Brew XF, TeSR™-E8, mTeSR™ Plus, TeSR™2, mTeSR™1, etc. Any medium capable of supporting proliferation (without differentiation) / healthy growth of CLECs or CLMCs may also be used for cell recovery culture. Examples of media suitable for the culture of the CLECs are described, for example, in US Patent Application No. 2006 / 0078993 and include EpiLife medium, Medium 171, MEGM-Mammary Epithelial Cell Medium or mixtures of such media, such as medium PTT-e3 (used herein for the generation of CLiPS derived from CLECs and described in detail herein below). Examples of media suitable for this culture of CLMCs are described, for example, in US Patent Application Nos. 2006 / 0078993 and 2018 / 127721 and International Patent Application WO2007 / 046775, and include media such as DMEM / 10% FBS, DMEM:F12 medium (a 1:1 mixture of DMEM and Ham's F-12 medium), or PPT-6 (a medium containing DMEM, F12-medium, Medium 171, and FBS, see US Patent Application No. 2018 / 127721) or PTT4 (wherein the latter was used in the Examples section of this specification for the generation of CLiPS derived from CLMCs). It is also possible to use mixtures of these media for this cell recovery culture (e.g., a mixture of mTeSR1 and medium PTTe-3 or medium PTT-4). The medium suitable for cell recovery of the transfected CLEC or CLMC described herein may further comprise growth factors that can stimulate cell growth and proliferation. The growth factors may be added directly to the cell culture medium. In addition, the recovery medium may comprise serum, such as fetal bovine serum (FBS).Thus, a suitable medium for cell recovery after transfection can be a serum-free medium or a serum-containing medium.

[0042] In line with the above disclosure, the composition of the medium suitable for cell recovery may vary depending on the CLSC used.

[0043] For example, a medium suitable for the recovery of transfected CLMCs may consist of a (chemically) defined medium and FBS. Thus, a medium suitable for the recovery of transfected CLMCs may consist of about 80% (v / v), about 85% (v / v), about 90% (v / v) or about 95% (v / v) of the chemically defined medium and about 20% (v / v), about 15% (v / v), about 10% (v / v) or about 5% (v / v) of FBS, respectively. In a preferred example, CLMCs are cultured in medium PTT-4 for cell recovery after transfection, where medium PTT-4, as described in International Patent Application WO2007 / 046775, consists of 90% (v / v) CMRL-1066 and 10% (v / v) FBS. A medium suitable for the recovery of transfected CLECs may be a serum-free medium, where the medium may contain cytokines and growth factors.

[0044] Also, the medium suitable for recovery of transfected CLECs may be a defined medium. Such recovery medium may include Mammary Epithelial Basal Medium MCDB 170, EpiLife Medium, DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), and FBS (Fetal Bovine Serum).

[0045] In an illustrative example, such a medium comprises mammary epithelial basal medium MCDB 170 at a final concentration of about 10 to about 30% (v / v), EpiLife medium at a final concentration of about 20 to about 40% (v / v), F12 at a final concentration of about 5 to about 15% (v / v), DMEM at a final concentration of about 30 to about 45% (v / v), and FBS at a final concentration of about 0.1 to 2% (v / v). One such medium may comprise mammary epithelial basal medium MCDB 170 at a final concentration of about 15 to about 25% (v / v), EpiLife medium at a final concentration of about 25 to about 35% (v / v), F12 at a final concentration of about 7.5 to about 13% (v / v), DMEM at a final concentration of about 35 to about 40% (v / v), and FBS at a final concentration of about 0.5 to 1.5% (v / v). Another such medium may comprise mammary epithelial basal medium MCDB 170 at a final concentration of about 20% (v / v), EpiLife medium at a final concentration of about 30% (v / v), F12 at a final concentration of about 12.5 (v / v), DMEM at a final concentration of about 37.5% (v / v), and FBS at a final concentration of about 1.0% (v / v). The value "% (v / v)" used herein refers to the volume of the individual component relative to the final volume of the medium. This means that if DMEM is present in the medium at a final concentration of, for example, about 35 to about 40% (v / v), then 1 liter of medium contains about 350 ml to 400 ml of DMEM. In one embodiment, a medium suitable for recovery of transfected CLEC cells is prepared by adding 100 ml of DMEM to obtain a final volume of 1000 ml of culture medium. 200 ml of Mammary Epithelial Basal Medium MCDB 170, 300 ml of EpiLife medium, 250 ml DMEM, 250 ml of DMEM / F12, and 1% fetal bovine serum It is obtained by mixing

[0046] The growth factors in the medium suitable for recovery of transfected CLECs may be insulin-like growth factors (IGFs) such as IGF-1 or IGF-2, epidermal growth factors (EGFs) such as HB-EGF or EPR, transforming growth factors (TGFs) such as TGF-α or TGF-β1, activin, bone morphogenetic proteins (BMPs), platelet-derived growth factors (PDGFs), transferrin, and insulin. In one example, CLECs are cultured in medium PTTe-3 for cell recovery after transfection, where medium PTTe-3 contains one or more transforming growth factors such as human epidermal growth factor (EGF), TGF-α and / or TGF-β (TGF-β1, TGF-β2, and / or TGF-β3), or insulin.

[0047] In accordance with the above, a medium suitable for recovery of transfected CLECs may contain human epidermal growth factor (EGF) at a final concentration of about 1 to about 15 ng / ml. The recovery medium may contain insulin at a final concentration of about 1 to about 7.5 μg / ml. The recovery medium may further contain at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In one embodiment, the medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In this case, the medium may contain adenine at a final concentration of about 0.05 to about 0.1 mM adenine, hydrocortisone at a final concentration of about 0.1 to 0.5 μM hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.1 to about 5 ng / ml. The recovery medium may contain one or more transforming growth factors (TGFs), such as transforming growth factor β1 (TGF-β1) and / or transforming growth factor α (TGF-α). In such a medium, TGF-β1 may be present at a final concentration of about 0.1 to about 5 ng / ml, and TGF-α may be present at a final concentration of about 1.0 to about 10 ng / ml. Additionally, the recovery medium for CLECs may contain cholera toxin from Vibrio cholerae, which is commercially available, for example, from Sigma Aldrich under catalog number C8052. When cholera toxin from Vibrio cholerae is used, it is present at a concentration of about 1×10 -11 M ~ approx. 1×10 -10 M final concentration.

[0048] "DMEM" refers to Dulbecco's Modified Eagle's Medium, developed in 1969 and a modification of Basal Medium Eagle (BME) (see FIG. 1 showing the data sheet of DMEM available from Lonza). The first DMEM formulation contained 1000 mg / L glucose and was first reported for the culture of embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture and is commercially available from a variety of sources, such as ThermoFisher Scientific (catalog number 11965-084), Sigma Aldrich (catalog number D5546), or Lonza, to name just a few suppliers. Thus, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is Sigma Aldrich catalog number D5546 DMEM medium, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0049] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed to allow the cultivation of a wide variety of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin. Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (catalog number 11765-054), Sigma Aldrich (catalog number N4888), or Lonza, to name just a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used. "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 medium. DMEM / F12 (1:1) medium is also a widely used basal medium for supporting the growth of many different mammalian cells, and is commercially available from various suppliers, such as ThermoFisher Scientific (catalog number 11330057), Sigma Aldrich (catalog number D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium (DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose) available from Lonza under catalog number 12-719F.

[0050] "M171" refers to medium 171, which was developed as a basal medium for culturing the proliferation of normal human mammary epithelial cells. This basal medium is also widely used and is commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is M171 medium available from Life Technologies Corporation under catalog number M171500.

[0051] "Mammary epithelial basal medium MCDB 170" refers to a basal nutrient medium used for the growth of mammary epithelial cells and commercially available in powder form, for example, from United States Biological, Salem Massachusetts USA under catalog number M2162, or from Bio-Connect BV, Huissen, The Netherlands under catalog number (MBS652676_10l).

[0052] EpiLife medium refers to a HEPES and bicarbonate buffered medium formulated without calcium chloride and commonly used for serum-free long-term culture of human epidermal keratinocytes and human corneal epithelial cells, designed for use in an incubator with an atmosphere of 5% CO2 and 95% air, available from ThermoFisher Scientific, catalog number MEPICF500, or from Sigma Aldrich under Product Code E 0151.

[0053] "CMRL medium" refers to a medium originally developed by Connaught Medical Research Laboratories for the growth of Earle's "L" cells under serum-free conditions. CMRL medium is also known to be particularly useful for cloning monkey kidney cells and for the growth of other mammalian cell lines when supplemented with horse serum or bovine serum. CMRL medium is commercially available, for example, from ThermoFisher Scientific (catalog number 11530037).

[0054] "FBS" refers to fetal bovine serum (also called "fetal bovine serum"), i.e., the blood fraction remaining after natural blood clotting followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells because it has very low levels of antibodies, contains more growth factors, and allows versatility in many different cell culture applications. FBS is preferably obtained from a member of the International Serum Industry Association (ISIA), whose main focus is the safety and safe use of serum and animal-derived products through proper origin traceability, authenticity of labeling, and proper standardization and oversight. Suppliers of FBS that are ISIA members include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In a currently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0055] A medium suitable for cell recovery may contain compounds that may suppress inflammatory responses and / or enhance cell survival and proliferation after transfection. Examples of such compounds may be glucocorticoids. Glucocorticoids are steroid hormones that may upregulate the expression of anti-inflammatory proteins in the nucleus and suppress the expression of pro-inflammatory proteins in the cytosol. The glucocorticoids used herein may be prednisolone, methylprednisolone, dexamethasone, betamethasone, corticosterone or hydrocortisone, to name just a few examples of suitable glucocorticoids. It is also possible to use two or more of such glucocorticoids together, for example a mixture of corticosterone and hydrocortisone. The glucocorticoids may be used at any suitable concentration, for example, at a concentration of about 0.1 μM to about 2.5 μM or 0.1 μM to about 5 μM. In one example, the glucocorticoid in the medium suitable for recovery of transfected CLSCs can be hydrocortisone used at a concentration of about 0.1 μM to about 2.5 μM. In one example, the hydrocortisone concentration in the medium suitable for recovery of transfected CLSCs is about 0.5 μM to about 2 μM. In one such example, the hydrocortisone concentration is about 1 μM.

[0056] The recovery of transfected CLSCs can be carried out in a cell culture device such as a cell culture vessel. The cell culture vessel can be, but is not limited to, a culture flask, a Petri dish, a roller bottle, and a multi-wall plate. Furthermore, the cell culture vessel can be coated to provide a layer that can promote cell growth by supplying the cells with metabolic products. The coating of the cell culture vessel can be serum-derived or serum-free. An example of a serum-derived coating can be a coating with gelatinous proteins from a basement membrane-like matrix such as Matrigel. Alternatively, the serum-free coating of the cell culture vessel can be characterized as animal-free and xeno-free, thus allowing cell culture under cGMP conditions. Examples of serum-free coatings of cell culture vessels can be coatings with recombinant proteins or parts thereof, such as coatings with extracellular matrix proteins such as collagen, fibronectin, elastin, laminins, including e.g. laminin-511 E8 fragment or laminin 521, vitronectin, e.g. in the form of commercially available Citronectin XF™, CELLstart or Synthemax™ vitronectin substrates. In one example of the invention, transfected CLECs can be preferably cultured in cell culture vessels with serum-derived coatings, whereas CLMCs can be preferably cultured in cell culture vessels with serum-free coatings.

[0057] The medium suitable for recovery of transfected CLSCs may be replaced with another cell culture medium after a suitable period of time. The suitable period of time may be, for example, about 1 day, about 2 days or about 3 days after transfection. Thus, in one example, the medium exchange may be performed about 2 days after transfection. The other cell culture medium used for medium exchange may be a mixture of different cell culture media. In the present invention, any cell culture medium or cell culture medium mixture suitable for iPS production may be used. Furthermore, the suitable cell culture medium or cell culture medium mixture may contain compounds that may suppress inflammatory responses and enhance cell survival. In the present invention, the medium suitable for cell recovery after transfection may be replaced with a mixture of two different cell culture media after a suitable period of time to ensure an adequate supply of nutrients and an appropriate blend of growth factors to the cells so that the cells transition from their native state to a more pluripotent state when somatic cell reprogramming occurs. Thus, the cell culture medium mixture of the present invention may consist of a medium suitable for cell recovery, which may contain hydrocortisone, and a second cell culture medium. In a preferred example, the two different cell culture media are mixed in a ratio of about 1:1 (v / v), where the mixture can be prepared by contacting one volume of a medium suitable for cell recovery with one volume of a second cell culture medium. In another preferred example, the two different cell culture media are mixed in a ratio of about 1:2 (v / v) or 2:1, where the mixture can be prepared by contacting one volume of a medium suitable for cell recovery with two volumes of the second cell culture medium (or two volumes of a medium suitable for cell recovery with one volume of the second cell culture medium). The second cell culture medium used to create the cell culture mixture can be any cell culture medium suitable for enhancing or maintaining iPS proliferation (such a medium is also referred to herein as a "maintenance medium"). Using a mixture such as a 1:1 mixture of a medium used for cell recovery and a maintenance medium has the advantage that it allows the CLiPS cells to be gradually transitioned from the allogeneic medium to the ES / iPSC medium, rather than an abrupt switch that may compromise their viability.Without wishing to be bound by theory, it is assumed that about 2 days after transfection, some of the successfully transfected umbilical cord lining stem cells begin to acquire the characteristics of pluripotent stem cells and simultaneously acquire the nutrient requirements of PSCs. Examples of such suitable cell culture media include, but are not limited to, commercially available maintenance media such as mTeSR1, StemMACS™ iPS-Brew XF, TeSR™-E8, mTeSR™Plus, TeSR™2 or mTeSR™1, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co. Inc), or PluriSTEM (Merck Millipore). Since the medium mTeSR™1 is manufactured under GMP conditions, it can be preferably used when iPS colonies are cultured under animal-free and xeno-free GMP conditions. Thus, in one preferred example, mTeSR1 can be the second cell culture medium used to make the cell culture mixture. In the present invention, the 1:1 (v / v) cell culture medium mixture can be replaced with the same cell culture medium mixture within a suitable period of time. This suitable period of time can be about 3 days, about 4 days, about 5 days or about 6 days after transfection. Thus, in one example, the 1:1 (v / v) cell culture medium mixture can be replaced with the same mixture 4 days after transfection. After a suitable period of time, the 1:1 (v / v) cell culture medium mixture can be further replaced with the second cell culture medium used to make the cell culture mixture only. In this context, the suitable period of time can be about 4 days, about 5 days, about 6 days or about 7 days after transfection. In one example, the 1:1 (v / v) cell culture medium mixture can be replaced with the second cell culture medium 6 days after transfection. In a preferred example, the 1:1 (v / v) cell culture medium mixture can be replaced with mTeSR1 and mTeSR™1, respectively, 6 days after transfection.Regular changes and replacement of cell culture medium may contribute to an increase in surviving CLiPS, thus allowing CLiPS colonies to grow and proliferate.

[0058] After changing the cell culture medium mixture to one cell culture medium, CLiPS can be further cultured. For this purpose, the cell culture medium can be periodically replaced with the same medium to ensure adequate supply of nutrients and appropriate blend of growth factors to cells. For example, the cell culture medium can be replaced every day or every 2, 3 or 4 days. In one example of the present invention, the cell culture medium can be replaced every 2 days. As a result, CLiPS colony can further grow and proliferate.

[0059] CLiPS colonies may become visible to the naked eye about 10, 11, 12, 13, 14, 15 or 16 days after transfection (see Example 2). Once they reach a suitable size, the CLiPS may be selected and transferred to another coated culture vessel for further culture and growth. In this context, suitable colony sizes may include a diameter of about 0.1 mm to a length of about 2 mm. In one example of the present invention, CLiPS colonies may be selected when they reach a diameter of about 0.5 mm to a length of about 1.5 mm, where the CLiPS colonies may reach this size about 20 days after transfection. In order to transfer CLiPS colonies having a suitable size to another culture vessel, the CLiPS colonies may be picked. This may be performed manually, if necessary. To facilitate picking of the colonies, a device may be used that allows a magnified view of the colonies. Examples of such devices may be a magnifying glass or a microscope. In the present invention, the CLiPS may be selected and picked under a bright field microscope. Turning to the cell culture vessel, the picked CLiPS colony may be transferred to another cell culture vessel, where the coating of the cell culture vessel may be different or the same as the coating of the cell culture vessel used for the recovery of the transfected CLSC. In a preferred example, the coating of the culture vessel is the same, since the CLiPS derived from CLMCs previously cultured under suitable conditions of cGMP are maintained animal-free and xeno-free, thereby allowing the cGMP conditions to be respected. As a result, for example, the CLiPS colony derived from CLMCs may be transferred to a cell culture vessel coated with a serum-free substance such as laminin-511 E8 fragment for further culture (see Example 3). Alternatively, the CLiPS colony derived from CLECs and / or derived from CLMCs may be transferred to a cell culture vessel coated with a serum-derived substance such as Matrigel for further culture. The cell culture medium is preferably the same as that used before colony picking. In the present example, the cell culture medium may be changed periodically after colony picking. For example, the medium may be changed every day, every 2 days or every 3 days.In a preferred embodiment of the present invention, the cell culture medium may be changed daily after colony picking.

[0060] Once a suitable confluency has been reached, the CLiPS colonies or cell populations formed from the colonies are typically detached from the coated cell culture vessel and transferred to a larger cell culture vessel for further culture under the same culture conditions used immediately after colony picking. Suitable confluencies may be at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% and at least about 65% confluency. In this regard, it is noted that the term "cell population" when used in relation to the growth of colony-forming CLiPS cells is more appropriate, since CLiPS cells do not take on a colony-like appearance when they reach about 70% to about 80% confluency. To detach the CLiPS colonies or cell populations formed from the colonies from the coated cell culture vessel, any dissociation agent suitable for disrupting cell adhesion or for hydrolyzing peptide bonds can be used. Examples of such suitable dissociation agents may be solutions containing chelating agents such as ethylenediaminetetraacetic acid (EDTA) or solutions containing enzymes such as trypsin or dispase (see the experimental section of this application, where dispase has been used to detach CLiPS colonies from coated cell culture vessels). The cell culture medium may be changed periodically, for example, every day, every 2 days or every 3 days. In a preferred embodiment of the present invention, the cell culture medium may be changed every day. In this way, CLiPS can further grow and proliferate.

[0061] In the present invention, CLiPS colonies or cell populations formed therefrom may be passaged when they reach a suitable size. The suitable size may correspond to a confluency of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% and about 95%. In an example of the present invention, CLiPS colonies or cell populations formed therefrom may be passaged when the culture reaches a confluency of about 60-90%. Thus, in a preferred example, CLiPS colonies or cell populations formed therefrom may be passaged when they reach a confluency of about 70-80%. When passaged, CLiPS may be passaged at a suitable ratio, where one volume of CLiPS may be contacted with multiple volumes of cell culture medium. In the present invention, CLiPS may be passaged at a ratio of about 1:3 (v / v) or about 1:4 (v / v) or about 1:5 (v / v) or about 1:6 (v / v), where passaging may be performed by splitting one volume of dissociated CLiPS into about 2 or about 3 or about 4 or about 5 volumes of dissociated CLiPS, respectively. In a preferred example, CLiPS may be passaged at a ratio of about 1:3 (v / v). To allow passaging of cultured CLiPS of the present invention, again any enzyme suitable for detaching cells from the culture vessel may be used. For example, dispase may be used for this purpose. Furthermore, any chemical suitable for removing cell-cell adhesion may be used for CLiPS passaging in the context of the present invention, where the concentration of the chemical may be appropriate for removing cell-cell adhesion without harming the cells. An example of such a chemical may be EDTA. Since EDTA may kill cells at higher concentrations, the suitable EDTA concentration of the present invention may be about 0.5 mM. In the present invention, the cell culture medium used for passage may be supplemented with a substance suitable for enhancing the survival of CLiPS when dissociated. For this purpose, any substance suitable for enhancing the survival of CLiPS when dissociated may be used. An example of such a suitable substance may be an inhibitor of a signaling pathway, such as the rho-associated protein kinase (ROCK) signaling pathway.Thus, the RHO / ROCK pathway inhibitor Y-27632 may be an example of a suitable substance for enhancing the survival of dissociated CLiPS. Alternatively, supplements defined for single cell cloning of human iPS cells, such as CloneR™ (available from StemCell Technologies), may be used to enhance the survival of dissociated cells. In the present invention, the passaged CLiPS may be cultured in a medium supplemented with a suitable substance for enhancing the survival of dissociated CLiPS for an appropriate period of time before being differentiated into target cells.

[0062] By culturing the CLiPS after passage, a master cell bank containing (primary) isolated CLiPS can be obtained. To generate a master cell bank of CLiPSs, the CLiPS cells obtained by the process described herein can be seeded in a culture vessel such as a cell culture plate. For this purpose, the CLiPS can be suspended and cultured in any suitable medium, typically a maintenance medium for iPS cells, such as the above-mentioned commercially available medium such as mTeSR1, StemMACS™ iPS-Brew XF, TeSRTM E8, mTeSRTMPlus, TeSRTM2 or mTeSRTM1, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co. Inc), or PluriSTEM (Merck Millipore). In such an iPS maintenance medium, both CLiPS derived from CLMCs and CLiPS derived from CLECs can be cultured. For subculture, CLiPS cells (both CLMC and CLEC derived CLiPS) can be cultured at any suitable concentration, for example, at or about 0.5×10 6 Cells / ml ~ approx. 5.0 x 10 6 The cells can be seeded at a concentration of about 1.0×10 cells / ml. In one example, the cells are seeded at about 1.0×10 6The cells are suspended for subculture at a concentration of 10000 cells / ml. Subculture can be performed by culture in simple culture flasks, but also in multi-layer systems such as CellSTACK (Corning, NY, USA) or Cell Factory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA), which can be stacked in an incubator. Alternatively, subculture can be performed in closed self-contained systems such as bioreactors. Different designs of bioreactors are known to those skilled in the art, such as parallel plate, hollow fiber, or microfluidic bioreactors. See, for example, Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra. An example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc), which has been used, for example, for the expansion of bone marrow mesenchymal stem cells for clinical trials (see Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058) and for the expansion of highly pure umbilical cord lining mesenchymal stem cell populations as described in International Patent Application WO 2018 / 067071. Another example of a commercially available bioreactor that can be used for subculturing the CLiPS populations of the invention is the Xuri Cell Expansion System available from GE Healthcare. Culturing CLiPS populations in an automated system such as the Quantum® Cell Expansion System is particularly useful when large numbers of cells are required to produce working cell banks for therapeutic applications under GMP conditions.In the case of subculture, CLiPS can also be cultured until a suitable amount of cells has grown. As an example, CLiPS are subcultured until they reach about 70% to about 80% confluency. Isolation / culturing of a population of CLiPS can be performed under standard conditions for the culture of mammalian cells. Once a desired / suitable number of CLiPS are obtained from the subculture, the cells are harvested by removing the CLiPS from the culture vessel used for the subculture. The recovery of CLiPS is typically performed by enzymatic treatment. The isolated CLiPS are then collected and used directly or stored for further use. Typically, storage is performed by cryopreservation. The term "cryopreservation" is used herein in its ordinary sense to describe a process in which CLiPS are preserved by cooling to subzero temperatures, such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be carried out as known to those of skill in the art and can include the use of cryoprotectants such as dimethylsulfoxide (DMSO) or glycerol, which retard the formation of ice crystals in the CLiPS cells.

[0063] The present invention is also directed to CLiPS obtainable by the method described herein and to CLiPS obtained by the method described herein. CLiPS obtainable / obtained by the present invention can grow and proliferate robustly (see Example 2 and Example 3). Thereby, CLiPS culture can be more efficient compared to the culture of iPS derived from, for example, bone marrow stroma, adipose tissue, dermis or Wharton's jelly. Analysis of CLiPS functionality reveals the expression of human embryonic stem cell markers, indicating self-renewal properties and normal karyotype (see Example 4 and Example 5). Furthermore, CLiPS can differentiate into multiple cell types (functional target cells) that show pluripotency in vitro and in vivo (see Example 6). Therefore, CLiPS are highly suitable for medical and therapeutic applications. As a result, the present invention is also directed to pharmaceutical compositions comprising iPS obtainable / obtained by the method described herein.

[0064] The present invention is further directed to a method for differentiating CLiPS into target cells under suitable conditions for differentiation. Examples of suitable target cells include, but are in no way limited to, neuronal cells, dopaminergic neuronal cells, oligodendrocytes, astrocytes, cortical neurons, hepatocytes, chondrocytes, muscle cells, bone cells, dental cells, hair follicle cells, inner ear hair cells, skin cells, melanocytes, cardiomyocytes, hematopoietic progenitor cells, blood cells, immune cells, T or B lymphocytes, microglia, natural killer cells or motor neurons, to name just a few. To promote directed differentiation into target cells, CLiPS may be exposed to a priming agent, typically under conditions known to those skilled in the art from the differentiation of iPS from other sources into target cells. Exposure may be carried out under suitable conditions, which may include culturing in a cell culture vessel filled with a cell culture medium suitable for priming and subsequent culturing of CLiPS differentiation. In the present invention, any cell culture medium suitable for priming, proliferation and differentiation of iPS can be used, where the medium composition and therefore the method of differentiation can depend on the target cell and can be adopted from known protocols for differentiation of iPS into desired target cells (in this respect, see the reviews of Hirschi et al "Induced Pluripotent Stem Cells for Regenerative Medicine" Annu Rev Biomed Eng. 2014 July 11; 16: 277-294) or Shi et al "Induced pluripotent stem cell technology: a decade of progress" Nat Rev Drug Discov. 2017 February; 16(2): 115-130). For example, CLiPS can be cultured in a medium adapted for proliferation and differentiation of CLiPS into dopaminergic neuronal cells.In such a case, the medium may be Neurobasal medium supplemented with growth factors that induce neural differentiation, such as B-27 minus vitamin A, transforming growth factor 3-β (TGFβ3), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ascorbic acid, dibutyl cAMP, inhibitors of glycogen synthase kinase 3 such as CHIR99021, and gamma secretase inhibitors such as (2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT). An example of such a medium is NB27. Differentiation of CLiPS into dopaminergic neuronal cells is illustratively shown in Example 7. As another example, CLiPS may be cultured in a medium adapted for the proliferation of CLiPS and their differentiation into hepatocytes. In this case, the medium may be a protein-, lipid- and growth factor-free medium supplemented with compounds that induce differentiation into a mesendoderm fate. RPMI 1640-B27 supplemented with activin A may be an example of a medium suitable for CLiPS differentiation into hepatocytes. Differentiation of CLiPS into hepatocytes is exemplarily shown in Example 8. As another example, CLiPS may be cultured in a medium adapted for CLiPS proliferation and differentiation into cardiomyocytes. In such a case, the medium may be a protein-, lipid- and growth factor-free medium supplemented with an inhibitor of glycogen synthase kinase 3, such as CHIR99021. RPMI / 2%-B27 minus insulin may be an example of a medium suitable for CLiPS differentiation into hepatocytes. Differentiation of CLiPS into cardiomyocytes is exemplarily shown in Example 9. As a further example, CLiPS may be differentiated into oligodendrocytes using a chemically defined growth factor-rich medium that allows differentiation into paired box 6-positive (PAX6+) neural stem cells, thereby resulting in oligodendrocyte transcription factor-positive (OLIG2+) progenitor cells (see Example 10). In this regard, it is noted that differentiation of CLiPS into target cells may also be carried out under conditions suitable for cGMP production.

[0065] The present invention also includes pharmaceutical compositions comprising differentiated CLiPS obtained by the methods described herein. Analysis of the immunogenicity of CLiPS and their neural derivatives revealed reduced immunogenicity (Example 11). Examples of pharmaceutical compositions comprising differentiated CLiPS are injection solutions or any kind of grafts suitable for transplanting differentiated CLiPS. In one example, such grafts may include multi-layered tissues derived from differentiated CLiPS, such as organs or parts thereof. In one example, grafts suitable for transplanting differentiated CLiPS may include transplantable matrices coated with differentiated CLiPS. The pharmaceutical compositions may be formulated / adapted for parenteral application. In such cases, parenteral application may include sterile preparations intended for injection, infusion or implantation in the human or animal body. Transplantation of CLiPS-derived dopaminergic neurons in fully immunocompetent mouse and rat Parkinson's disease models showed functional engraftment and even significant recovery of dopamine reuptake function (see Example 12 and Example 13).

[0066] The present invention further includes a method of treating a congenital or acquired degenerative disorder in a subject, which may be selected from the group including mice, rats, rabbits, pigs, dogs, cats, non-human primates, or humans. In a preferred example, the subject is a human. In this context, treating may include administering to the subject target cells differentiated from CLiPS by the methods described herein. The disease may be any known disease that has been considered to be treated by cell-based therapy, see, for example, Shi et al. "Induced pluripotent stem cell technology: a decade of progress" supra. Congenital or acquired degenerative disorders may have different origins. For example, such congenital or acquired degenerative disorders may be neurological disorders such as, for example, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA) and Batten's disease. Examples of liver degenerative disorders may be liver failure, liver cirrhosis, and viral hepatitis, among others. The congenital or acquired degenerative disorder may be a cardiac disorder, including acute Danon disease, short QT syndrome, Brugada syndrome, myocardial infarction, Jarvell and Lange-Nielsen syndrome, among others. The disorder may be an autoimmune disease, such as multiple sclerosis.

[0067] The present invention is also directed to extracellular membrane vesicles that may be produced by CLiPS or differentiated derivatives of CLiPS, which may include, but are not limited to, vesicles having diameters ranging from 30 to 150 nanometers (nm), also known as exosomes. Although initially thought to have a primarily excretory function, exosomes are now known to be involved in a variety of important biological processes, such as cell-cell communication, cellular senescence, proliferation and differentiation, tissue homeostasis, tissue repair and regeneration, antigen presentation, and immune regulation (see, for example, Pegtel, DM and SJ Gould, Exosomes. Annu Rev Biochem, 2019. 88: p. 487-514 or Kalluri, R. and VS LeBleu, The biology, function, and biomedical applications of exosomes. Science, 2020. 367(6478). Exosomes have also been implicated in a number of important roles in cancer (see, for example, Visan, KS, RJ Lobb, and A. Moller, The role of exosomes in the promotion of epithelial-to-mesenchymal transition and metastasis. Front Biosci (Landmark Ed), 2020. 25: p. 1022-1057, or Zhang, L. and D. Yu, Exosomes in cancer development, metastasis, and immunity. Biochim Biophys Acta Rev Cancer, 2019. 1871(2): p. 455-468), osteoarthritis (Asghar, S., et al., Exosomes in intercellular communication and implications for osteoarthritis. Rheumatology (Oxford), 2020. 59(1): p.57-68), diseases of the central nervous system such as stroke, Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and amyotrophic lateral sclerosis (ALS) (see, for example, Liu, W., et al., Role of Exosomes in Central Nervous System Diseases. Front Mol Neurosci, 2019. 12: p. 240 or Quek, C. and A. F. Hill, The role of extracellular vesicles in neurodegenerative diseases. Biochem Biophys Res Commun, 2017. 483(4): p. 1178-1186), psychiatric disorders (Saeedi, S., et al., The emerging role of exosomes in mental disorders. Transl Psychiatry, 2019. 9(1): p. 122), cardiovascular diseases (Wang, Y., et al., Exosomes: An emerging factor in atherosclerosis. Biomed Pharmacother, 2019. 115: p. 108951), metabolic diseases (see, for example, Dini, L., et al., Microvesicles and exosomes in metabolic diseases and inflammation. Cytokine Growth Factor Rev, 2020. 51: p. 27-39 or Soazig, LL, A. Ramaroson, and MM Carmen, Exosomes in metabolic syndrome, in Exosomes: A Clinical Compendium, LR Edelstein, et al., Editors. 2020, Academic Press. p. 343 - 356), and many more.

[0068] The cargo of exosomes is known to consist of various biomolecules including proteins, lipids and nucleic acids. RNA species such as tRNA, mRNA, lncRNA, circular RNA and miRNA can potentially regulate gene expression in target cells and tissues. Exosomes produced by certain cell types have been shown to possess therapeutic properties. In this regard, mesenchymal stem cells (MSCs) isolated from different sources such as bone marrow, adipose tissue and umbilical cord have emerged as particularly preferred. MSC-derived exosomes have shown potential therapeutic effects in animal models of corneal, cardiovascular, Alzheimer's, Parkinson's and inflammatory bowel diseases, among others. In addition to endogenous cells, in vitro cultured pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS), have been shown to produce exosomes (see YH, et al., Exosomes Derived from Embryonic Stem Cells as Potential Treatment for Cardiovascular Diseases. Adv Exp Med Biol, 2017. 998: p. 187-206. or Jeske, R., et al., Human Pluripotent Stem Cell-Derived Extracellular Vesicles: Characteristics and Applications. Tissue Eng Part B Rev, 2020. 26(2): p. 129-144. Due to the risk of tumor formation from residual undifferentiated cells, administration of cell-free iPS-derived exosomes is considered safer than iPS-derived cells (Riazifar, M., et al., Stem Cell Extracellular Vesicles: Extended Messages of Regeneration. Annu Rev Pharmacol Toxicol, 2017. 57: p. 125-154). Of note, therapeutic properties have also been demonstrated for exosomes isolated from differentiated derivatives of iPS cells.For example, treatment with exosomes purified from iPS-derived cardiomyocytes enhanced myocardial recovery in a mouse model of myocardial infarction, with a significant reduction in apoptosis and fibrosis compared to untreated animals. The exosomes also rescued in vitro cultures of iPS-cardiomyocytes from hypoxia and exosome biogenesis inhibition (Liu, B., et al., Cardiac recovery via extended cell-free delivery of extracellular vesicles secreted by cardiomyocytes derived from induced pluripotent stem cells. Nat Biomed Eng, 2018. 2(5): p. 293-303). In another study, exosomes from iPS-derived MSC-derived exosomes isolated from iPS-derived MSCs accelerated the proliferation of human dermal fibroblasts and human keratinocytes and enhanced wound healing in an in vitro scratch assay. There was no significant difference in the effects of these exosomes compared to those isolated from primary MSCs (Kim, S., et al., Exosomes Secreted from Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Accelerate Skin Cell Proliferation. Int J Mol Sci, 2018. 19(10).

[0069] Thus, according to these reports, the extracellular membrane vesicles or exosomes produced by the CLiPS of the present invention (either CLMC or CLEC derived) or differentiated derivatives of CLiPS are believed to be useful for treating diseases including the above exemplary diseases such as cancer, osteoarthritis, stroke, diseases of the central nervous system such as Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and amyotrophic lateral sclerosis (ALS), psychiatric disorders or metabolic diseases.

[0070] In addition, exosomes are being actively pursued as delivery vehicles to facilitate cellular uptake of various therapeutic agents, such as microRNAs, drugs and peptides, taking advantage of their efficient cargo delivery capabilities (see Antimisiaris, SG, S. Mourtas, and A. Marazioti, Exosomes and Exosome-Inspired Vesicles for Targeted Drug Delivery. Pharmaceutics, 2018. 10(4); Liao, W., et al., Exosomes: The next generation of endogenous nanomaterials for advanced drug delivery and therapy. Acta Biomater, 2019. 86: p. 1-14 or Wang, X., et al., Cell-derived Exosomes as Promising Carriers for Drug Delivery and Targeted Therapy. Curr Cancer Drug Targets, 2018. 18(4): p. 347-354. In line with this, the CLiPS of the present invention It is believed that extracellular membrane vesicles or exosomes produced by (either CLMC or CLEC derived) or differentiated derivatives of CLiPS can also be used as delivery vehicles to facilitate cellular uptake of therapeutic agents. Thus, the present invention also encompasses the use of CLiPS or differentiated derivatives of CLiPS for the purpose of delivery of exogenously loaded or transgenically expressed molecules.

[0071] Extracellular membrane vesicles and exosomes produced by CLiPS (either from CLMC or CLEC) or differentiated derivatives of CLiPS can be isolated using methods described in the literature. Typically, exosomes are purified from the extracellular environment in which they are secreted. Known methods for the isolation of exosomes include ultracentrifugation, ultrafiltration, size-exclusion chromatography, field-flow fractionation, polymer co-precipitation, immunoaffinity, microfluidics, or acoustic nanofilters. All of these methods can be used to isolate exosomes produced by CLiPS or differentiated derivatives of CLiPS described herein.

[0072] The present invention further relates to a specific method of differentiating iPS cells, derived from umbilical cord amniotic stem cells as defined elsewhere herein, referred to as CLiPS as also defined herein, into RPE cells, which comprises culturing the CLiPs in a differentiation medium under conditions suitable for differentiation into RPE cells.

[0073] "Retinal pigment epithelial (abbreviated RPE) cells" refers to cells obtained / from / taken from the retinal pigment epithelium. In other words, such cells are comprised by the retinal pigment epithelium and are defined in more detail below. RPE differentiation from CLiPs was achieved using the rapid, directed and modified differentiation method according to the present invention. The CLiPS used herein for differentiation into RPE cells can be derived from umbilical cord superficial mesenchymal cells (such as CLMC23, CLMC30, CLMC44) and / or from umbilical cord superficial ectodermal cells (such as CLEC23). In a preferred embodiment, the CLiPS used herein for differentiation into RPE cells is any one of CLMC23, CLMC30, CLMC44 or CLEC23. In a preferred embodiment, the CLiPS used herein for differentiation into RPE cells is CLMC23. In another preferred embodiment, the CLiPS used herein for differentiation into RPE cells is CLMC30. In another preferred embodiment, the CLiPS used herein for differentiation into RPE cells is CLMC44.In another preferred embodiment, the CLiPS used herein for differentiation into RPE cells is CLEC23.The RPE cells differentiated from the CLiPS defined herein by the differentiation method described herein can be referred to as CLiPS-derived RPE cells or CLiPS-RPE.The differentiation of RPE cells derived from CLiPs can be compared with the differentiation of RPE cells derived from ES cells such as H9 ES cells (when referring to such RPE cells, also referred to as ES-derived RPE), and / or with the differentiation of RPE cells derived from skin-derived iPS cells such as Asf5, AGO or HDFA cells (also referred to as skin iPS, and therefore also referred to as skin iPS-derived RPE) using the differentiation method according to the present invention (see Examples).

[0074] The differentiation medium used in the differentiation method for differentiating iPS cells into RPE cells, which comprises culturing iPS cells derived from amniotic stem cells of the umbilical cord, is preferably DMEM medium (Dulbecco's Modified Eagle Medium) as defined herein, containing N2 supplement, B27 supplement and non-essential amino acids (NEAA), and even more preferably DMEM medium (Dulbecco's Modified Eagle Medium) / F12 medium (Ham's F12 medium) as defined elsewhere herein, containing N2 supplement, B27 supplement and non-essential amino acids (NEAA). In a preferred embodiment, the DMEM / F12 medium used in the differentiation method for differentiating iPS cells into RPE cells comprises 1x N2 supplement, 1x B27 supplement and 1x NEAA. Including 1x N2 supplement, 1x B27 supplement and 1x NEAA in the medium means that the final concentration is 1x, as also seen in the following embodiment. In said embodiment, the differentiation medium, preferably DMEM medium, and even more preferably DMEM / F12 medium as defined herein, is added to the medium at a concentration of 1x, so as to obtain a final volume of 1000 ml of culture medium. 10 mL of 100x N2 supplement; · 20 mL of 50x B27 supplement; 10 mL of 100×NEAA; 960 mL of DMEM, preferably DMEM / F12 It is obtained by mixing

[0075] The differentiation medium defined herein may further comprise / be supplemented with various growth factors and / or cytokines as defined elsewhere herein.Such differentiation medium defined above may refer to the basic medium for iPS culture.Then, such basic differentiation medium may be further modified / supplemented to culture iPS cells defined herein so that iPS cells differentiate into RPE cells by using the method of differentiating iPS cells into RPE cells according to the present invention.

[0076] In particular, the differentiation medium used for culturing iPS cells in the method of differentiating iPS cells into RPE cells according to the present invention can comprise a first differentiation medium further comprising at least one of IGF1, DKK1, nicotinamide or LDN-193189.The first differentiation medium is based on a basal medium comprising DMEM medium, preferably DMEM / F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM / F12 medium comprising 1xN2 supplement, 1xB27 supplement and 1xNEAA.In a preferred embodiment, the first differentiation medium defined herein further comprises IGF1, DKK1, nicotinamide and LDN-193189.

[0077] Furthermore, the differentiation medium used in the method of differentiating iPS cells into RPE cells according to the present invention for culturing the iPS cells to differentiate into RPE cells can additionally or alternatively comprise a second differentiation medium further comprising at least one of IGF1, DKK1, nicotinamide, LDN-193189 or b-FGF.The second differentiation medium is also based on a basal medium comprising DMEM medium, preferably DMEM / F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM / F12 medium comprising 1xN2 supplement, 1xB27 supplement and 1xNEAA.In a preferred embodiment, the second differentiation medium defined herein further comprises IGF1, DKK1, nicotinamide, LDN-193189 and b-FGF.

[0078] Furthermore, the differentiation medium used in the method of differentiating iPS cells into RPE cells according to the present invention can additionally or alternatively comprise a third differentiation medium, which further comprises at least one of IGF1, DKK1 or activin A. The third differentiation medium is also based on a basal medium, which comprises DMEM medium, preferably DMEM / F12 medium comprising N2 supplement, B27 supplement and NEAA, and even more preferably DMEM / F12 medium comprising 1xN2 supplement, 1xB27 supplement and 1xNEAA. In a preferred embodiment, the third differentiation medium defined herein further comprises IGF1, DKK1 and activin A.

[0079] Furthermore, the differentiation medium used to culture iPS cells in the method of differentiating iPS cells into RPE cells according to the present invention may additionally or alternatively comprise a fourth differentiation medium further comprising activin A and SU5402 or activin A and PD17307. The fourth differentiation medium is also based on a basal medium comprising DMEM medium, preferably DMEM / F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM / F12 medium comprising 1xN2 supplement, 1xB27 supplement and 1xNEAA. In a preferred embodiment, the fourth differentiation medium defined herein further comprises activin A and PD17307. PD17307 is preferred for the differentiation method of the present invention because PD17307 is applied at a lower concentration compared to the fibroblast growth factor inhibitor SU5402, which reduces the undesirable changes in gene expression caused by the application of high concentrations when SU5402 is used (Figure 19).

[0080] Furthermore, the differentiation medium used for culturing iPS cells in the method of differentiating iPS cells into RPE cells according to the present invention can additionally or alternatively comprise a fifth differentiation medium, which further comprises at least one of activin A, CHIR99021 or SU5402, or at least one of activin A, CHIR99021 or PD17307. The fifth differentiation medium is also based on a basal medium, which comprises DMEM medium, preferably DMEM / F12 medium containing N2 supplement, B27 supplement and NEAA, even more preferably DMEM / F12 medium containing 1xN2 supplement, 1xB27 supplement and 1xNEAA. In a preferred embodiment, the fifth differentiation medium defined herein further comprises activin A, CHIR99021 and PD17307 (PD17307 is used for the same reason as defined above). The fifth differentiation medium preferably contains activin A, SU5402 or PD17307, preferably PD17307, and a first concentration of CHIR99021 that is less than 3 μM. Then, when the fifth differentiation medium is used again to culture iPS cells and differentiate them into RPE cells, the concentration of the Wnt signaling pathway activator CHIR99021 is increased. By gradually increasing the concentration, excessive cell death caused by high concentrations of CHIR during use is suppressed. This improves the yield of pigmented RPE cells. When the fifth differentiation medium is subsequently applied, it preferably contains activin A, SU5402 or PD17307, preferably PD17307, and a second concentration of CHIR99021 that is about 3 μM.

[0081] When IGF1 as a supplement is applied in the differentiation medium defined elsewhere herein, a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml is used. In a preferred embodiment, IGF1 is used in the differentiation medium defined elsewhere herein at a final concentration of about 10 ng / ml. In even more preferred embodiments, IGF1 is used in the first differentiation medium as defined elsewhere herein at a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or at a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml. In another even more preferred embodiment, IGF1 is used in the second differentiation medium as defined elsewhere herein at a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or at a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml.In another even more preferred embodiment, IGF1 is used in the third differentiation medium as defined elsewhere herein at a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or at a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml. The present invention further comprises a method for differentiation into RPE cells, comprising culturing iPS cells in a differentiation medium as defined elsewhere herein, wherein IGF1 is applied in the differentiation medium as defined herein at a final concentration of about 10 ng / ml for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, preferably about 6 days, even more preferably about 6 consecutive days, and most preferably used for about 6 consecutive days.When IGF1 is applied in the first differentiation medium as defined herein, IGF1 is applied in a final concentration of about 10 ng / ml for about 2 days, preferably about 2 consecutive days, which means use from day 0 to day 2 of culture, and even more preferably used for about 2 consecutive days.When IGF1 is applied in the second differentiation medium as defined herein, IGF1 is applied in a final concentration of about 10 ng / ml for about 2 days, preferably about 2 consecutive days, which means use from day 2 to day 4 of culture, and even more preferably used for about 2 consecutive days. When IGF1 is applied in the third differentiation medium defined herein, IGF1 is used at a final concentration of about 10 ng / ml for about 2 days, preferably for about 2 consecutive days, which means use from day 4 to day 6 of culture, and even more preferably for about 2 (consecutive) days.

[0082] When DKK1 is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, is used. In a preferred embodiment, DKK1 is used in a differentiation medium as defined elsewhere herein at a final concentration of about 10 ng / ml. In an even more preferred embodiment, DKK1 is used in the first differentiation medium as defined elsewhere herein at a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or at a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml. In another even more preferred embodiment, DKK1 is used in the second differentiation medium as defined elsewhere herein at a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or at a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml.In another even more preferred embodiment, DKK1 is used in a third differentiation medium as defined elsewhere herein at a final concentration of at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml or at least about 10 ng / ml, or at a final concentration within the range of about 5 to about 15 ng / ml, about 6 to about 14 ng / ml, about 7 to about 13 ng / ml, about 8 to about 12 ng / ml, about 9 to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml. The present invention further comprises a method for differentiation into RPE cells, comprising culturing iPS cells in differentiation medium as defined elsewhere herein, wherein DKK1 is applied in the differentiation medium as defined herein at a final concentration of about 10 ng / ml for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, preferably about 6 days, even more preferably about 6 consecutive days, and most preferably used for about 6 consecutive days.When DKK1 is applied in the first differentiation medium as defined herein, DKK1 is applied in the final concentration of about 10 ng / ml for about 2 days, preferably about 2 consecutive days, which means that it is used from day 0 to day 2 of culture, and even more preferably used for about 2 consecutive days.When DKK1 is applied in the second differentiation medium as defined herein, DKK1 is applied in the final concentration of about 10 ng / ml for about 2 days, preferably about 2 consecutive days, which means that it is used from day 2 to day 4 of culture, and even more preferably used for about 2 consecutive days. When DKK1 is applied in the third differentiation medium defined herein, DKK1 is used at a final concentration of about 10 ng / ml for about 2 days, preferably for about 2 consecutive days, which means use from day 4 to day 6 of culture, and even more preferably for about 2 (consecutive) days.

[0083] When nicotinamide is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM or at least about 10 mM, or a final concentration in the range of about 5 to about 15 mM, about 6 to about 14 mM, about 7 to about 13 mM, about 8 to about 12 mM, about 9 to about 11 mM is used. In a preferred embodiment, nicotinamide is used in a differentiation medium as defined elsewhere herein at a final concentration of about 10 mM. In an even more preferred embodiment, nicotinamide is used in a first differentiation medium as defined elsewhere herein at a final concentration of at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM or at least about 10 mM, or a final concentration in the range of about 5 to about 15 mM, about 6 to about 14 mM, about 7 to about 13 mM, about 8 to about 12 mM, about 9 to about 11 mM, most preferably at a final concentration of about 10 mM. In another even more preferred embodiment, nicotinamide is used in the second differentiation medium as defined elsewhere herein at a final concentration of at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM or at least about 10 mM, or at a final concentration in the range of about 5 to about 15 mM, about 6 to about 14 mM, about 7 to about 13 mM, about 8 to about 12 mM, about 9 to about 11 mM, most preferably at a final concentration of about 10 mM. The present invention further includes a method of differentiation into RPE cells, comprising culturing iPS cells in a differentiation medium as defined elsewhere herein, wherein nicotinamide is applied in a differentiation medium as defined herein at a final concentration of about 10 mM for at least about 2 days, at least about 3 days, at least about 4 days, preferably for about 4 days, even more preferably for about 4 consecutive days, and most preferably used for about 4 (consecutive) days. When nicotinamide is applied in the first differentiation medium defined herein, nicotinamide is used at a final concentration of about 10 mM for about 2 days, preferably for about 2 consecutive days, meaning use from day 0 to day 2 of culture, and even more preferably for about 2 (consecutive) days.When nicotinamide is applied in the second differentiation medium defined herein, nicotinamide is used at a final concentration of about 10 mM for about 2 days, preferably for about 2 consecutive days, which means use from day 2 to day 4 of culture, and even more preferably for about 2 (consecutive) days.

[0084] When LDN-193189 is applied in the differentiation medium defined elsewhere herein, a final concentration of at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM or at least about 1 μM is used.In a preferred embodiment, LDN-193189 is used in the differentiation medium defined elsewhere herein at a final concentration of about 1 μM.In another preferred embodiment, LDN-193189 is used in the differentiation medium defined elsewhere herein at a final concentration of about 0.2 μM. In an even more preferred embodiment, LDN-193189 is used in a first differentiation medium as defined elsewhere herein at a final concentration in the range of about 0.5 to about 1.5 μM, about 0.6 to about 1.4 μM, about 0.7 to about 1.3 μM, about 0.8 to about 1.2 μM, about 0.9 to about 1.1 μM, and most preferably at a final concentration of about 1 μM. In another even more preferred embodiment, LDN-193189 is used in a second differentiation medium as defined elsewhere herein at a final concentration in the range of about 0.1 to about 0.3 μM, about 0.11 to about 0.29 μM, about 0.12 to about 0.28 μM, about 0.13 to about 0.27 μM, about 0.14 to about 0.26 μM, about 0.15 to about 0.25 μM, and most preferably at a final concentration of about 0.2 μM. The present invention further includes a method of differentiating iPS cells into RPE cells comprising culturing iPS cells in a differentiation medium as defined elsewhere herein, wherein LDN-193189 is applied in the differentiation medium as defined elsewhere herein at a concentration of at least about 0.1 μM for at least about 2 days, at least about 3 days, at least about 4 days, preferably for about 4 days, even more preferably for about 4 consecutive days, and most preferably used for about 4 (consecutive) days.When LDN-193189 is applied in the first differentiation medium as defined herein, it is used at a concentration of about 1 μM for about 2 days, preferably for about 2 consecutive days, meaning use from day 0 to day 2 of culture, and even more preferably, LDN-193189 is used for about 2 (consecutive) days.When LDN-193189 is applied in the second differentiation medium as defined herein, it is used at a concentration of about 0.2 μM for about 2 days, preferably for about 2 consecutive days, meaning use from day 2 to day 4 of culture, and even more preferably, LDN-193189 is used for about 2 (consecutive) days.

[0085] When b-FGF is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 2.5 ng / ml, at least about 3 ng / ml, at least about 3.5 ng / ml, at least about 4 ng / ml, at least about 4.5 ng / ml or at least about 5 ng / ml, or a final concentration within the range of about 2.5 to about 7.5 ng / ml, about 3 to about 7 ng / ml, about 3.5 to about 6.5 ng / ml, about 4 to about 6 ng / ml, about 4.5 to about 5.5 ng / ml, is used. In a preferred embodiment, b-FGF is used in a differentiation medium as defined elsewhere herein at a final concentration of about 5 ng / ml. In even more preferred embodiments, b-FGF is used in the second differentiation medium as defined elsewhere herein at a final concentration of at least about 2.5 ng / ml, at least about 3 ng / ml, at least about 3.5 ng / ml, at least about 4 ng / ml, at least about 4.5 ng / ml or at least about 5 ng / ml, or at a final concentration in the range of about 2.5 to about 7.5 ng / ml, about 3 to about 7 ng / ml, about 3.5 to about 6.5 ng / ml, about 4 to about 6 ng / ml, about 4.5 to about 5.5 ng / ml, most preferably at a final concentration of about 5 ng / ml. The present invention further includes a method of differentiation into RPE cells, comprising culturing iPS cells in a differentiation medium as defined elsewhere herein, wherein b-FGF at a final concentration of about 5 ng / ml is applied in the differentiation medium as defined herein for at least about 1 day, at least about 2 days, preferably about 2 days, even more preferably about 2 consecutive days, and most preferably used for about 2 (consecutive) days. When b-FGF is applied in the second differentiation medium defined herein, it is used at a final concentration of about 5 ng / ml for about 2 days, preferably for about 2 consecutive days, which means use from day 2 to day 4 of culture, and even more preferably for about 2 (consecutive) days.

[0086] When Activin A is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 50 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml or at least about 100 ng / ml, or a final concentration within the range of about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 70 to about 130 ng / ml, about 80 to about 120 ng / ml, about 90 to about 110 ng / ml, is used. In a preferred embodiment, Activin A is used in a differentiation medium as defined elsewhere herein at a final concentration of about 100 ng / ml. In even more preferred embodiments, Activin A is used in a third differentiation medium as defined elsewhere herein at a final concentration of at least about 50 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml or at least about 100 ng / ml, or at a final concentration within the range of about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 70 to about 130 ng / ml, about 80 to about 120 ng / ml, about 90 to about 110 ng / ml, and most preferably at a final concentration of about 100 ng / ml. In another even more preferred embodiment, Activin A is used in a fourth differentiation medium as defined elsewhere herein at a final concentration of at least about 50 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml or at least about 100 ng / ml, or at a final concentration within the range of about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 70 to about 130 ng / ml, about 80 to about 120 ng / ml, about 90 to about 110 ng / ml, most preferably at a final concentration of about 100 ng / ml.In another even more preferred embodiment, Activin A is used in a fifth differentiation medium as defined elsewhere herein at a final concentration of at least about 50 ng / ml, at least about 60 ng / ml, at least about 70 ng / ml, at least about 80 ng / ml, at least about 90 ng / ml or at least about 100 ng / ml, or at a final concentration within the range of about 50 to about 150 ng / ml, about 60 to about 140 ng / ml, about 70 to about 130 ng / ml, about 80 to about 120 ng / ml, about 90 to about 110 ng / ml, most preferably at a final concentration of about 100 ng / ml. The present invention further comprises a method for differentiating into RPE cells, comprising culturing iPS cells in differentiation medium as defined elsewhere herein, wherein Activin A is applied in the differentiation medium as defined herein at a final concentration of about 100ng / ml for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 8 days, at least about 10 days, at least about 12 days, preferably about 12 days, even more preferably about 12 days in succession, and most preferably about 12 days in succession.When Activin A is applied in the third differentiation medium as defined herein, Activin A is applied in the final concentration of about 100ng / ml for about 2 days, preferably about 2 days in succession, which means that it is applied from the 4th day to the 6th day of culture, and even more preferably it is used for about 2 days in succession. When Activin A is applied in the fourth differentiation medium defined herein, Activin A is used at a final concentration of about 100 ng / ml for about 2 days, preferably for about 2 consecutive days, meaning from day 6 to day 8 of culture, and even more preferably for about 2 consecutive days. When Activin A is applied in the fifth differentiation medium defined herein, Activin A is used at a final concentration of about 100 ng / ml for about 8 days, preferably for about 8 consecutive days, meaning from day 8 to day 16 of culture, and even more preferably for about 8 consecutive days.

[0087] When SU5402 is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM or at least about 10 μM, or a final concentration within the range of about 5 to about 15 μM, about 6 to about 14 μM, about 7 to about 13 μM, about 8 to about 12 μM, about 9 to about 11 μM, is used. In a preferred embodiment, SU5402 is used in a differentiation medium as defined elsewhere herein at a final concentration of about 10 μM. In even more preferred embodiments, SU5402 is used in a fourth differentiation medium as defined elsewhere herein at a final concentration of at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM or at least about 10 μM, or at a final concentration within the range of about 5 to about 15 μM, about 6 to about 14 μM, about 7 to about 13 μM, about 8 to about 12 μM, about 9 to about 11 μM, and most preferably at a final concentration of about 10 μM. In another even more preferred embodiment, SU5402 is used in a fifth differentiation medium as defined elsewhere herein at a final concentration of at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM or at least about 10 μM, or at a final concentration in the range of about 5 to about 15 μM, about 6 to about 14 μM, about 7 to about 13 μM, about 8 to about 12 μM, about 9 to about 11 μM, most preferably at a final concentration of about 10 μM. The present invention further includes a method of differentiation into RPE cells comprising culturing iPS cells in a differentiation medium as defined elsewhere herein, wherein SU5402 is applied in the differentiation medium as defined elsewhere herein at a concentration of about 10 μM for about 10 days, even more preferably for about 10 consecutive days, most preferably for about 10 (consecutive) days. When SU5402 is applied in the fourth differentiation medium defined herein, SU5402 is used at a concentration of about 10 μM for about 2 days, preferably for about 2 consecutive days, which means use from day 6 to day 8 of culture, and even more preferably SU5402 is used for about 2 (consecutive) days.When SU5402 is applied in the fifth differentiation medium defined herein, SU5402 is used at a concentration of about 10 μM for about 8 days, preferably for about 8 consecutive days, which means use from day 8 to day 16 of culture, and even more preferably SU5402 is used for about 8 (consecutive) days.

[0088] When PD17307 is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM or at least about 1 μM, or a final concentration within the range of about 0.5 to about 1.5 μM, about 0.6 to about 1.4 μM, about 0.7 to about 1.3 μM, about 0.8 to about 1.2 μM, about 0.9 to about 1.1 μM, is used. In a preferred embodiment, PD17307 is used in a differentiation medium as defined elsewhere herein at a final concentration of about 1 μM. In even more preferred embodiments, PD17307 is used in a fourth differentiation medium as defined elsewhere herein at a final concentration of at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM or at least about 1 μM, or at a final concentration within the range of about 0.5 to about 1.5 μM, about 0.6 to about 1.4 μM, about 0.7 to about 1.3 μM, about 0.8 to about 1.2 μM, about 0.9 to about 1.1 μM, and most preferably at a final concentration of about 1 μM. In another even more preferred embodiment, PD17307 is used in a fifth differentiation medium as defined elsewhere herein at a final concentration of at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM or at least about 1 μM, or at a final concentration in the range of about 0.5 to about 1.5 μM, about 0.6 to about 1.4 μM, about 0.7 to about 1.3 μM, about 0.8 to about 1.2 μM, about 0.9 to about 1.1 μM, most preferably at a final concentration of about 1 μM. The present invention further includes a method of differentiation into RPE cells, comprising culturing iPS cells in a differentiation medium as defined elsewhere herein, wherein PD17307 is applied in a differentiation medium as defined elsewhere herein at a concentration of about 1 μM for about 10 days, even more preferably for about 10 consecutive days, and most preferably used for about 10 (consecutive) days.When PD17307 is applied in the fourth differentiation medium as defined herein, PD17307 is used at a concentration of about 1 μM for about 2 days, preferably for about 2 consecutive days, meaning use from day 6 to day 8 of culture, and even more preferably, PD17307 is used for about 2 (consecutive) days.When PD17307 is applied in the fifth differentiation medium as defined herein, PD17307 is used at a final concentration of about 1 μM for about 8 days, preferably for about 8 consecutive days, meaning use from day 8 to day 16 of culture, and even more preferably, PD17307 is used for about 8 (consecutive) days.

[0089] When CHIR99021 is applied in a differentiation medium as defined elsewhere herein, a final concentration of at least about 1 μM and less than about 3 μM, at least about 1.1 μM and less than about 3 μM, at least about 1.2 μM and less than about 3 μM, at least about 1.3 μM and less than about 3 μM, at least about 1.4 μM and less than about 3 μM, at least about 1 μM and less than about 2.5 μM, at least about 1 μM and less than about 2 μM, at least about 1 μM and less than about 1.9 μM, at least about 1 μM and less than about 1.8 μM, at least about 1 μM and less than about 1.7 μM, at least about 1 μM and less than about 1.6 μM is used. In a preferred embodiment, CHIR99021 is used in a differentiation medium as defined elsewhere herein at a final concentration of about 1.5 μM. CHIR99021 used in the differentiation medium defined herein can be applied to culture cells, i.e., iPS cells of the present invention that are differentiated or have already differentiated into RPE cells, for about 3 consecutive days of culture. In a more preferred embodiment, CHIR99021 is used in the differentiation medium defined elsewhere herein at a final concentration of about 1.5 μM to culture cells, i.e., iPS cells of the present invention that are differentiated or have already differentiated into RPE cells, for about 3 consecutive days of culture.

[0090] In even more preferred embodiments, CHIR99021 is used in a fifth differentiation medium as defined elsewhere herein at a final concentration of at least about 1 μM and less than about 3 μM, at least about 1.1 μM and less than about 3 μM, at least about 1.2 μM and less than about 3 μM, at least about 1.3 μM and less than about 3 μM, at least about 1.4 μM and less than about 3 μM, at least about 1 μM and less than about 2.5 μM, at least about 1 μM and less than about 2 μM, at least about 1 μM and less than about 1.9 μM, at least about 1 μM and less than about 1.8 μM, at least about 1 μM and less than about 1.7 μM, at least about 1 μM and less than about 1.6 μM, and most preferably at a final concentration of about 1.5 μM. In an even more preferred embodiment, CHIR99021 is at a final concentration of at least about 1 μM and less than about 3 μM, at least about 1.1 μM and less than about 3 μM, at least about 1.2 μM and less than about 3 μM, at least about 1.3 μM and less than about 3 μM, at least about 1.4 μM and less than about 3 μM, at least about 1 μM and less than about 2.5 μM, at least about 1 μM and less than about 2 μM, at least about 1 μM and less than about 1 μM and less than about 1.9 μM, at least about 1 μM and less than about 1.8 μM, at least about 1 μM and less than about 1.7 μM, at least about 1 μM and less than about 1.6 μM for culturing cells, i.e., iPS cells of the present invention, which are to be differentiated or which have already been differentiated into RPE cells, for about 3 consecutive days of culture (which means from day 8 to day 11 of culture). The fifth differentiation medium is used as defined elsewhere herein at a final concentration of 1 μM.

[0091] When CHIR99021 is applied to the differentiation medium, preferably the fifth differentiation medium, as defined above, CHIR99021 is applied again in the differentiation medium for the subsequent culture of the cells, i.e., the iPS cells of the present invention, which are differentiated into RPE cells or have already been differentiated, preferably for about 5 consecutive days of culture. In a preferred embodiment, CHIR99021 is then used at a final concentration of about 3 μM for the subsequent culture of the cells, i.e., the iPS cells of the present invention, which are differentiated into RPE cells or have already been differentiated, even more preferably for about 5 consecutive days of culture (meaning from day 11 to day 16 of culture). By gradually increasing the final concentration to 1.5 μM for 3 days, followed by 3 μM for 5 days, the yield of pigmented RPE cells is improved.

[0092] The present invention further relates to a method of differentiation into RPE cells, wherein CHIR99021 is used during about 8 days of culture, preferably during about 8 consecutive days of culture, and even more preferably, CHIR99021 is used at a final concentration of at least about 1 μM and less than about 3 μM, at least about 1.1 μM and less than about 3 μM, at least about 1.2 μM and less than about 3 μM, at least about 1.3 μM and less than about 3 μM, at least about 1.4 μM and less than about 3 μM, at least about 1 μM and less than about 2.5 μM, at least about 1 μM and less than about 2 μM, at least about 1 μM and less than about 1.9 μM, at least about 1 μM and less than about 1.8 μM, at least about 1 μM and less than about 1.7 μM, at least about 1 μM and less than about 1.6 μM, and most preferably at about 1.5 μM and less than about 1.5 μM during the first 3 (consecutive) days of the about 8 days of culture with CHIR99021. The method includes culturing iPS cells in differentiation medium as defined elsewhere herein, in which CHIR99021 is used at a final concentration of about 1 μM, and during the next (consecutive) 5 days of about 8 days of culture with CHIR99021, CHIR99021 is used at a final concentration of about 3 μM.

[0093] In a most preferred embodiment, the differentiation medium comprises a first differentiation medium comprising about 1 μM LDN-193189, about 10 ng / ml DKK1, about 10 ng / ml IGF1 and about 10 mM nicotinamide. In another most preferred embodiment, the differentiation medium comprises a second differentiation medium comprising about 0.2 μM LDN-193189, about 10 ng / ml DKK1, about 10 ng / ml IGF1, about 10 mM nicotinamide and about 5 ng / ml b-FGF. In another most preferred embodiment, the differentiation medium comprises a third differentiation medium comprising about 10 ng / ml DKK1, about 10 ng / ml IGF1 and about 100 ng / ml activin A. In another most preferred embodiment, the differentiation medium comprises a fourth differentiation medium comprising about 100 ng / ml activin A and about 10 μM SU5402, preferably a fourth differentiation medium comprising about 100 ng / ml activin A and about 1 μM PD17307. In another most preferred embodiment, the differentiation medium comprises a fifth differentiation medium comprising about 100 ng / mL activin A, about 10 μM SU5402 and about 1.5 μM CHIR99021, preferably a fifth differentiation medium comprising about 100 ng / mL activin A, about 1 μM PD17307 and about 1.5 μM CHIR99021. In another most preferred embodiment, the differentiation medium comprises about 100 ng / mL activin A, about 10 μM SU5402 and about 3 μM CHIR99021, preferably comprising another fifth differentiation medium applied after the initial fifth differentiation medium has been applied in the differentiation method comprising about 100 ng / mL activin A, about 1 μM PD17307 and about 3 μM CHIR99021.

[0094] In a preferred embodiment of the present invention, the step of culturing the iPS cells in the differentiation method comprises culturing in a first differentiation medium as defined elsewhere herein for about 2 days, preferably in a first differentiation medium as defined elsewhere herein for about 2 consecutive days, meaning that the iPS cells are initially exposed to a first differentiation medium as defined elsewhere herein from day 0 to day 2.

[0095] In another preferred embodiment, the step of culturing the iPS cells in the method of the present invention comprises culturing in the second differentiation medium as defined elsewhere herein for about 2 days, preferably culturing in the first differentiation medium as defined elsewhere herein for about 2 days followed by culturing in the second differentiation medium as defined elsewhere herein for about 2 days, and even more preferably culturing in the first differentiation medium as defined elsewhere herein for about 2 consecutive days followed by culturing in the second differentiation medium as defined elsewhere herein for about 2 consecutive days. This means that on the second day of culturing the iPS cells in the method of the present invention, the iPS cells can be exposed to the second differentiation medium from day 2 to day 4.

[0096] In another preferred embodiment, in the case of the step of culturing iPS cells in the method of the present invention, the step comprises culturing iPS cells in the third differentiation medium as defined elsewhere herein for about 2 days, preferably comprising culturing iPS cells in the first differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in the second differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in the third differentiation medium as defined elsewhere herein for about 2 days, and even more preferably comprising culturing iPS cells in the first differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in the second differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in the third differentiation medium as defined elsewhere herein for about 2 consecutive days. This means that on the fourth day of culturing iPS cells in the method of the present invention, the iPS cells can be exposed to the third differentiation medium from day 4 to day 6.

[0097] In another preferred embodiment, in the case of the step of culturing the iPS cells in the method of the present invention, the step comprises culturing the iPS cells in the fourth differentiation medium as defined elsewhere herein for about 2 days, preferably the step comprises culturing the iPS cells in the first differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in the second differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in the third differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in the fourth differentiation medium as defined elsewhere herein for about 2 days, and even more preferably the step comprises culturing the iPS cells in the first differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in the second differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in the third differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in the fourth differentiation medium as defined elsewhere herein for about 2 consecutive days. This means that on the 6th day of the culture of the iPS cells in the method of the present invention, the iPS cells can be exposed to the fourth differentiation medium from day 6 to day 8.

[0098] In another preferred embodiment, in the case of the step of culturing the iPS cells in the method of the present invention, the step comprises culturing the iPS cells for about 8 days in the fifth differentiation medium as defined elsewhere herein, preferably comprising culturing for about 2 days in the first differentiation medium as defined elsewhere herein, followed by culturing for about 2 days in the second differentiation medium as defined elsewhere herein, followed by culturing for about 2 days in the third differentiation medium as defined elsewhere herein, followed by culturing for about 2 days in the fourth differentiation medium as defined elsewhere herein, followed by culturing for about 8 days in the fifth differentiation medium as defined elsewhere herein, and even more preferably comprising culturing for about 2 consecutive days in the first differentiation medium as defined elsewhere herein, followed by culturing for about 2 consecutive days in the second differentiation medium as defined elsewhere herein, followed by culturing for about 2 consecutive days in the third differentiation medium as defined elsewhere herein, followed by culturing for about 2 consecutive days in the fourth differentiation medium as defined elsewhere herein, followed by culturing for about 8 consecutive days in the fifth differentiation medium as defined elsewhere herein. This means that on day 8 of the culture of iPS cells in the method of the present invention, the iPS cells may be exposed to the fifth differentiation medium from day 8 to day 16.

[0099] In an even more preferred embodiment of the present invention, the step of culturing the iPS cells in the method of the present invention comprises culturing for about 4 days in a fifth differentiation medium as defined elsewhere herein comprising CHIR99021 used at a concentration of at least about 1 μM and less than about 3 μM as defined elsewhere herein, followed by culturing for another about 4 days in a fifth differentiation medium comprising CHIR99021 used at a concentration of about 3 μM, and most preferably comprises culturing for about 2 days in a first differentiation medium comprising CHIR99021 used at a concentration of at least about 1 μM and less than about 3 μM as defined elsewhere herein, followed by culturing for about 2 days in a second differentiation medium, followed by culturing for about 2 days in a third differentiation medium, followed by culturing for about 2 days in a fourth differentiation medium, followed by culturing for about 4 days in a fifth differentiation medium, followed by culturing for another about 4 days in a fifth differentiation medium comprising CHIR99021 used at a concentration of about 3 μM. In this context, the term "days" for culturing in a particular medium may be replaced with the term "consecutive days", meaning that on day 8 of culturing in the methods of the invention, the cells may be exposed to a fifth differentiation medium comprising CHIR99021 used at a concentration of at least about 1 μM and less than about 3 μM as defined elsewhere herein from day 8 to day 11, followed by exposing the cells on day 11 to a fifth differentiation medium comprising CHIR99021 used at a concentration of about 3 μM from day 11 to day 16.

[0100] Thus, the present invention also includes a method of differentiating iPS cells into RPE cells, wherein the iPS cells are cultured in a differentiation medium for a total of about 11 to about 21 days, about 12 to about 20 days, about 13 to about 19 days, about 14 to about 18 days, about 15 to about 17 days, preferably a total of about 16 days, even more preferably about 11 to about 21 consecutive days, about 12 to about 20 consecutive days, about 13 to about 19 consecutive days, about 14 to about 18 days, about 15 to about 17 days, and most preferably about 16 consecutive days.

[0101] In a further embodiment of the present invention, the method for differentiating iPS cells into RPE cells preferably further comprises culturing the iPS cells in mTESR1 medium before culturing the iPS cells in the differentiation medium defined herein. Human ES cells as CLiP and reference cell lines may be grown on tissue culture plates coated with Matrigel in mTeSR1 medium. Once the cells reach about 90 to about 95% confluence, they are then exposed to a differentiation medium as defined elsewhere herein, preferably the first differentiation medium, even more preferably the first differentiation medium followed by the second, third, fourth and fifth differentiation medium as defined herein. In a preferred embodiment, the method of differentiating iPS cells into RPE cells further comprises the step of culturing the iPS cells in mTESR1 medium for about 1 to about 4 days of culture prior to culturing the iPS cells in a differentiation medium as defined herein, more preferably prior to culturing the iPS cells in the first differentiation medium as defined herein, most preferably prior to culturing the iPS cells in the first differentiation medium as defined herein, followed by the second, third, fourth and fifth differentiation medium as defined herein.

[0102] In a further embodiment of the present invention, the method for differentiating iPS cells into RPE cells preferably further comprises culturing RPE cells in retinal pigment epithelium maintenance (abbreviated as RPEM) medium. According to the present invention, after culturing iPS cells in differentiation medium and differentiating them into RPE cells, the differentiation medium can be replaced with RPEM medium as defined below. Preferably, culturing RPE cells in RPEM medium can be started after the 16th day of culture (particularly after culturing cells in the fifth differentiation medium as defined elsewhere herein). In a preferred embodiment, the RPEM medium of the differentiation method of the present invention comprises about 50% DMEM / F12 and about 50% minimum essential medium (MEM) containing 0.5xN1 supplement and 1xNEAA. In a more preferred embodiment, the RPEM medium of the differentiation method of the present invention further comprises at least one of heat-inactivated fetal bovine serum (FBS), Glutamax, taurine, hydrocortisone, 3,3',5-triiodo-L-thyronine, penicillin / streptomycin, nicotinamide, or sodium pyruvate. In an even more preferred embodiment, the RPEM medium of the differentiation method of the present invention further comprises heat-inactivated fetal bovine serum (FBS), Glutamax, taurine, hydrocortisone, 3,3',5-triiodo-L-thyronine, penicillin / streptomycin, nicotinamide, and sodium pyruvate. In a most preferred embodiment, the RPEM medium of the differentiation method of the present invention further comprises about 2% heat-inactivated fetal bovine serum (FBS), 1× Glutamax, about 0.25 mg / mL taurine, about 0.02 μg / mL hydrocortisone, about 0.013 ng / mL 3,3′,5-triiodo-L-thyronine, 1× penicillin / streptomycin, about 10 mM nicotinamide and 1× sodium pyruvate.

[0103] The present invention also includes differentiation methods as defined elsewhere herein, wherein RPE cells are cultured in RPEM medium as defined elsewhere herein for about 9 to about 29 days, about 10 to about 28 days, about 11 to about 27 days, about 12 to about 26 days, about 13 to about 25 days, about 14 to about 24 days, about 15 to about 23 days, about 16 to about 22 days, about 17 to about 21 days, about 18 to about 20 days, preferably for about 19 days, and even more preferably for about 19 consecutive days. The RPEM medium may be replaced about every 2 to 3 days during the culture of RPE cells in the medium, preferably every about 9 to about 29 days, about 10 to about 28 days, about 11 to about 27 days, about 12 to about 26 days, about 13 to about 25 days, about 14 to about 24 days, about 15 to about 23 days, about 16 to about 22 days, about 17 to about 21 days, about 18 to about 20 days during the culture of RPE cells, more preferably every about 19 days during the culture of RPE cells, and even more preferably every about 2 to about 3 days for about 19 consecutive days.

[0104] The present invention also includes a differentiation method as defined elsewhere herein, wherein the culture of iPS cells in the differentiation medium as defined elsewhere herein and the culture of RPE cells in RPEM medium as defined elsewhere herein comprise about 20 to about 50 days, about 25 to about 45 days, about 30 to about 40 days, preferably about 30 to about 35 days, most preferably about 35 days, specifically comprising the culture of iPS cells in the differentiation medium as defined elsewhere herein for about 16 days and the culture of differentiated RPE cells in RPEM medium as defined elsewhere herein for about 19 days.

[0105] The present invention also includes the differentiation method as defined elsewhere herein, which preferably further comprises a step of purifying the RPE cells in RPEM medium after culturing the RPE cells in the RPEM medium. After differentiation, a mixture of RPE cells and non-RPE cells is present, but a further purification step may be useful so that there are only pure RPE cells in the differentiation plate (Figure 20). The additional purification step of the differentiation method preferably comprises (a) manually identifying the RPE cells according to their pigmentation, where manually identifying the RPE cells according to their pigmentation preferably comprises selecting by microscopy, more preferably by bright field microscopy, as known to those skilled in the art. This step may refer to manual purification of the RPE cells. Specifically, manually identifying the RPE cells according to their pigmentation may be performed by manually removing non-RPE cells, which have less pigmentation and different cell morphology than the RPE cells, by scraping with a tip attached to a pipette, while observing by microscopy, such as bright field microscopy. It may further comprise washing, specifically about three times with PBS, to remove all non-RPE cells. Additionally or alternatively, the additional purification step of the differentiation method preferably comprises (b) passaging RPE cells, wherein passaging RPE cells preferably comprises treating RPE cells with Accutase or TrypLE, most preferably with TrypLE. This step may be referred to as the passaging purification of RPE cells. Specifically, passaging RPE cells may be performed by detaching non-RPE cells and removing them by treating with a mild dissociation agent such as Accutase or TrypLE, preferably with TrypLE. RPE cells may still be attached to the culture plate. Then, it may further comprise treating the remaining RPE cells again with a mild dissociation agent such as Accutase or TrypLE, preferably with TrypLE, and further passaging the RPE cells.In this context, the term "passaging RPE cells" refers to plating the remaining RPE cells after detaching and removing non-RPE cells with a mild dissociation agent such as Accutase or TrypLE, preferably TrypLE, and treating the remaining RPE cells again with a mild dissociation agent such as Accutase or TrypLE, preferably TrypLE. When TrypLE is applied, this step may be referred to as TrypLE purification of RPE cells. Additionally or alternatively, the additional purification step of the differentiation method preferably includes a combination of (c) manually identifying RPE cells according to their pigmentation as defined elsewhere herein and passaging RPE cells as defined elsewhere herein. Purification carried out by passaging RPE cells, preferably with TrypLE purification as described above, may remove most of the non-RPE cells / cell clumps, but some small clumps may still be present, which may be removed by manual purification as defined herein. Additionally or alternatively, the additional purification step of the differentiation method preferably comprises a combination of (d) passaging RPE cells and scattering sorting RPE cells according to their pigmentation. This purification step may be carried out by removing non-RPE cells as defined above and further treating the remaining RPE cells with a mild dissociation agent such as Accutase or TrypLE, preferably with TrypLE, and further treating the RPE cells with scattering sorting, where the non-RPE cells removed by a mild dissociation agent such as Accutase or TrypLE, preferably with TrypLE, may be used to set a gate for cells with low scattering. Additionally or alternatively, the additional purification step of the differentiation method preferably comprises a combination of (e) scattering sorting RPE cells according to their pigmentation. This step may be referred to as scattering sorting purification of RPE cells.Specifically, scatter sorting of RPE cells can be performed by resuspending a cell pellet of dissociated single cells (dissociated by using a gentle dissociation agent such as Accutase or TrypLE, preferably TrypLE) in any FACS buffer and passing it through a filter to obtain single cells and separate them into high scatter and low scatter fractions using, for example, any FACS cell sorting device known to one of skill in the art.

[0106] By comparing the different RPE purification methods defined herein, it was found that passaging RPE cells comprising treating RPE cells with Accutase or TrypLE, preferably with TrypLE, as defined above, results in a high RPE yield (about 47%) and is easier and faster than manual purification of RPE cells, and therefore may be preferred. Passaging RPE cells comprising treating RPE cells with Accutase or TrypLE, preferably with TrypLE, as defined above, followed by manually identifying RPE cells according to their pigmentation, as also defined elsewhere herein, not only results in a high RPE yield (about 43%), but also in the highest cell purity (about 99% PMEL17 positive cells), which may be of utmost importance for transplantation. Alternatively, passaging RPE cells, which includes treating RPE cells with Accutase or TrypLE, preferably TrypLE, as defined above, followed by manually identifying RPE cells according to their pigmentation, as similarly defined elsewhere herein, is also the easiest to perform, since partial TrypLE treatment removes most of the non-RPE cells in the short time required for purification. In summary, purification of passaging RPE cells, which includes treating RPE cells with Accutase or TrypLE, preferably TrypLE, in combination with manually identifying RPE cells, with an additional manual purification step to remove any non-RPE cells that may have escaped treatment with a mild dissociation agent such as Accutase or TrypLE, preferably TrypLE, is mostly preferred as purification of RPE cells in the method of the present invention.

[0107] The present invention also includes the differentiation method as defined elsewhere herein, in which the iPS cells used in the method of differentiation from amniotic stem cells of the umbilical cord into RPE cells are notably produced by expressing exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC, and p53-shRNA in said amniotic stem cells of the umbilical cord under conditions suitable for reprogramming the stem cells as defined elsewhere herein for the method of producing induced pluripotent stem cells which are also included by the present invention. In a preferred embodiment, the present invention also includes a differentiation method as defined elsewhere herein, in which the exogenous nucleic acid encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC, and p53-shRNA in umbilical cord amniotic stem cells are provided by one, two or three vectors, preferably a first vector encoding proteins OCT3 / 4 and 53-shRNA, a second vector encoding proteins SOX2 and KLF4, and a third vector encoding proteins L-MYC and LIN28. In summary, each disclosure as defined elsewhere herein regarding the method of producing induced pluripotent stem cells iPS cells, which are then differentiated into RPE cells according to the differentiation method described, may also be applicable to the method of differentiating iPS cells into RPE cells, if necessary.

[0108] The present invention also relates to RPE cell cultures / RPE cells obtainable by the differentiation methods described herein and to RPE cell cultures / RPE cells obtained by the differentiation methods described herein.The present invention also relates to retinal pigment epithelium consisting of or comprising retinal pigment epithelium cell cultures / RPE cells obtainable and obtained by the differentiation methods described herein.

[0109] The present invention also relates to pharmaceutical compositions comprising RPE cell cultures / RPE cells obtainable / obtained by the differentiation methods described herein. Examples of pharmaceutical compositions comprising RPE cell cultures comprising differentiated RPE cells / differentiated RPE cells are injections or any type of graft suitable for transplanting differentiated RPE cells. When the pharmaceutical composition is an injection, the composition may comprise RPE cell cultures obtainable / obtained by the differentiation methods described herein. When the pharmaceutical composition is a graft suitable for transplantation, the composition may comprise an implantable matrix, preferably a polyester matrix, even more preferably a polyester matrix in a transwell, coated with differentiated RPE cells obtainable / obtained by the differentiation methods described herein that can be grown on the matrix. In this context, RPE cells grown on a matrix as defined herein may refer to RPE cell cultures obtainable / obtained by the differentiation methods described herein. The pharmaceutical composition may be formulated / adapted for parenteral or topical application, as known to those skilled in the art. In such a case, parenteral application may include a sterile preparation intended for injection, infusion or implantation in the human or animal body. Topical application as used herein preferably refers to subretinal application. When the pharmaceutical composition refers to a graft as defined above with respect to a graftable matrix, the composition may be formulated / adapted for subretinal (under the retina of the eye) application, in other words, may be implanted subretina. The present invention also includes a diagnostic composition for research purposes, comprising RPE cells (culture) and Matrigel. The composition also refers to a graft suitable for implantation into a subject as defined herein, in which RPE cells differentiated by the method as defined herein are mixed with Matrigel, and then the graft (cells in Matrigel) is implanted into the subject. When the diagnostic composition refers to a graft as defined above with respect to Matrigel, the composition may be formulated / adapted for subcutaneous application, in other words, may be implanted subcutaneously.

[0110] The RPE cell culture, which can be obtained / obtained by the differentiation method described herein, also included in the retinal pigment epithelium, and / or also included by the pharmaceutical composition defined elsewhere herein, may refer to a plurality of RPE cells, which can be obtained / obtained by the differentiation method, and preferably includes a medium for the RPE cells. The term "population" may be used interchangeably with the term "culture". The differentiated RPE cells, which can be obtained / obtained by the differentiation method of the present invention and are included in the RPE cell culture, may be further characterized: First, the differentiated RPE cells may include a higher pigmented area % compared to skin iPS-derived RPE. All four CLiPS tested according to the present invention, which may be selected from the group consisting of CLMC23, CLMC30, CLMC44 and CLEC23, developed about 30 to about 100%, about 50 to about 100%, about 70 to about 100% pigmented RPE cells using the differentiation medium defined herein, compared to only 30% of skin iPS cells (such as Asf5, AGO and / or HDFA) that achieved similar pigmentation (Figure 13). Furthermore, the differentiated RPE cells additionally included in the culture may express at least one of BEST1, PMEL17, MITF, TYROSINASE, TRYP2, ZO-1, RPE65, RLBP1 or MERTK, or any combination of all of the listed protein markers (Figures 15, 16 and 18). In one embodiment, the differentiated RPE cells additionally included in the culture may express the protein marker BEST1. In another embodiment, the differentiated RPE cells additionally contained in the culture may express the protein marker PMEL17. In another embodiment, the differentiated RPE cells additionally contained in the culture may express the protein marker MITF. In another embodiment, the differentiated RPE cells additionally contained in the culture may express the protein marker TYROSINASE. In another embodiment, the differentiated RPE cells additionally contained in the culture may express the protein marker TRYP2. In another embodiment, the differentiated RPE cells additionally contained in the culture may express the protein marker ZO-1.In another embodiment, the differentiated RPE cells additionally contained in the culture can express the protein marker RPE65. In another embodiment, the differentiated RPE cells additionally contained in the culture can express the protein marker RLBP1. In another embodiment, the differentiated RPE cells additionally contained in the culture can express the protein marker MERTK. Thus, CLiPS-derived RPE cells are more highly pigmented compared to ES-derived RPE cells, such as H9 cells used herein. This is associated with high expression of pigmentation-related genes, such as MITF, PMEL17, TYROSINASE and TRYP2 (Figure 15). Furthermore, the differentiated RPE cells additionally contained in the culture can be characterized by lack of or reduced expression of cell cycle proliferation marker Ki67. The lack of expression of mature RPE marker RPE65 and Ki67 as proliferation marker confirms the maturation and quiescence that reflects the viability of such RPE cells differentiated from CLiPS by the method defined elsewhere herein (Figure 22).

[0111] More specifically, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by expressing BEST1 with a fold change of at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3 or more, preferably about 3, compared to RPE cells differentiated from embryonic stem cells (ES) (cells or cultures from which they were generated). Additionally or alternatively, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by expressing PMEL17 with a fold change of at least about 0.9, at least about 0.91, at least about 0.92, at least about 0.93, at least about 0.94, at least about 0.95, at least about 1, at least about 1.1, at least about 1.2, at least about 1.3 or more, preferably about 1.3, compared to RPE cells differentiated from ES (cells or cultures from which they were generated). Thus, CLiPS-RPE contains higher PMEL17 positive cell% after differentiation. The CLiPS (such as CLMC23, CLMC30 and CLEC23) used in the differentiation method was found to contain about 89% to about 95% purity of RPE cells. In contrast, only one of the three skin iPS cells used, such as Asf5, AGO and / or HDFA, had a purity of more than about 90% (Figure 14). Additionally or alternatively, the RPE cells obtainable / obtained by the method defined herein can be further characterized by expressing MITF with a fold change of at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8 or more, preferably about 6.8, compared to RPE cells differentiated from ES (cells or cultures from which it was generated).Additionally or alternatively, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by expressing TRYP2 with a fold change of at least about 2.9, at least about 3, at least about 3.5, at least about 4, at least about 4.1, at least about 4.2, at least about 4.3 or more, preferably about 4.3, compared to RPE cells differentiated from ES (cells or cultures from which they were generated). Additionally or alternatively, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by expressing RPE65 with a fold change of at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 0.91, at least about 0.92, at least about 0.93, at least about 0.94, at least about 0.95, at least about 0.96 or more, preferably about 0.96, compared to RPE cells differentiated from ES (cells or cultures from which they were generated). Additionally or alternatively, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by expressing RLBP1 with a fold change of at least about 17.5, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 26.1, at least about 26.2 or more, preferably about 26.2, compared to RPE cells differentiated from ES (cells or cultures from which they are generated). Additionally or alternatively, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by expressing MERTK with a fold change of at least about 6, at least about 6.5, at least about 7, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.1 or more, preferably about 9.1, compared to RPE cells differentiated from ES (cells or cultures from which they are generated) (Figure 20k). In the present context, such ES cells, used for comparison, from which RPE cells are also differentiated, may be referred to as H9 ES cells.

[0112] In this context, the term "compared to" or "to" can also be replaced with the term "compared to", where two cells (e.g., CLMC23 as test cell and H9 as reference cell) are compared to each other, for example, in terms of gene expression expressed as fold change of test cell relative to reference cell. As used herein, "fold change" is a measure that describes how much a quantity changes from an initial value to a final value. For example, an initial value of 30 and a final value of 60 corresponds to a 2-fold change, or in general terms, a 2-fold increase. Fold change is simply calculated as the ratio of final value to initial value, i.e., if the initial value is A and the final value is B, the fold change is B / A. Fold change can be obtained for the mRNA levels of markers described herein. Such fold change can be measured using RT-qPCR.

[0113] It was also found that CLiPS-derived RPE could achieve a transepithelial electrical resistance (TEER) similar to that of RPE cells differentiated from ES (cells or cultures) and / or RPE cells differentiated from dermal iPS (cells or cultures from which they were produced). CLiPS-RPE also showed high phagocytosis similar to that of RPE cells differentiated from ES (cells or cultures from which they were produced) and / or RPE cells differentiated from dermal iPS (cells or cultures from which they were produced) (Figure 17).

[0114] Additionally or alternatively, the RPE cells obtainable / obtained by the methods defined herein can be further characterized by comprising an increased oxygen consumption rate (OCR) and / or extracellular acidification rate (ECAR) compared to RPE cells differentiated from ES and / or compared to RPE cells differentiated from dermal iPS. These characteristics refer to RPE bioenergetics, which indicates enhanced glycolysis and / or mitochondrial respiration. Glycolysis function was quantified by ECAR and oxidative phosphorylation (oxPhos) by OCR (Figure 21).

[0115] In this context, OCR may include basal respiration, ATP production, maximal capacity and / or spare respiratory capacity. The term "increased" in this context with respect to OCR means that the OCR of RPE cells is increased by at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, preferably by at least about 35%, or by about 30 to about 45%, by about 31 to about 44%, by about 32 to about 43%, by about 33 to about 42%, by about 35 to about 40%, compared to RPE cells differentiated from ES and / or compared to RPE cells differentiated from dermal iPS cells. More specifically, (i) basal respiration of RPE cells is increased by approximately 38% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells; (ii) ATP production of RPE cells is increased by approximately 40% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells; (iii) maximum capacity of RPE cells is increased by approximately 35% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells; iv) spare respiratory capacity of RPE cells is increased by approximately 36% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells.

[0116] Further, in the present context, ECAR can include glycolysis, glycolytic capacity and / or glycolytic reserve. The term "increased" in this context with respect to ECAR means that the ECAR of an RPE cell is increased by at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or by about 20 to about 55%, by about 25 to about 55%, by about 25 to about 50% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells. More specifically, (i) glycolysis of RPE cells is increased by about 25% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells; (ii) glycolytic capacity of RPE cells is increased by about 37% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells; (iii) glycolytic reserve of RPE cells is increased by about 50% compared to RPE cells differentiated from ES cells and / or compared to RPE cells differentiated from dermal iPS cells.

[0117] Furthermore, additionally or alternatively, the RPE cells obtainable / obtained by the method defined herein can be further characterized by having / comprising a lower immunogenicity as defined in more detail below, compared to RPE cells differentiated from ES (cells or cultures from which they are generated) and / or compared to RPE cells differentiated from skin iPS (cells or cultures from which they are generated). In this context and with respect to the in vivo method defined below, lower immunogenicity preferably means that the RPE cells previously delivered to the subject from which a sample comprising said cells was obtained for further analysis of said cells have (a) a reduced systemic immune response, which may refer to a reduction in the levels of pro-inflammatory cytokines involved in the induction of a cellular immune response, preferably reduced levels of IFN-γ and / or IL-18 (as a surrogate for a cellular immune response) and / or reduced levels of cytokines IL-23 and / or IL17A involved in T cell activation, which are generated by the subject to which said RPE cells were previously delivered, in particular by immune cells present in said subject and also contained in said sample. Such reduction of systemic immune response in RPE cells may also refer to the reduction of immune cell accumulation, preferably at injection site, as defined in the Examples section.Thus, reduction of systemic immune response may include that RPE cells as defined herein have reduced systemic T cell activation, preferably reduced / suppressed CD8 cytotoxic T cell activation, compared to RPE cells differentiated from ES and / or compared to RPE cells differentiated from skin iPS.

[0118] The present invention further includes a method for treating retinal degenerative diseases in a subject, which may be selected from the group including mice, rats, rabbits, pigs, dogs, cats, non-human primates (monkeys) or humans. In a preferred example, the subject is a human. In this context, treating may include administering RPE cells differentiated from CLiPS by the differentiation method described herein and / or the RPE cell culture obtained by the method of the present invention and / or the pharmaceutical composition defined herein to a subject defined herein. The suitability of the differentiated cells / cultures obtainable / obtained by the method defined herein for administration in the specific treatment of retinal degenerative diseases is demonstrated by the fact that RPE cells include low immunogenicity, such as resulting in reduced levels of pro-inflammatory cytokines, preferably reduced levels of IFN-γ and / or IL-18 as substitutes, and having reduced cellular immune response, preferably after prior injection of differentiated RPE cells into a subject defined herein (Figure 23). This demonstrates that the RPE cells of the present invention may reduce immune cell infiltration at the local site of injection of RPE cells into a subject. It was also demonstrated that samples obtained from subjects to which differentiated RPE cells were previously delivered prior to cytokine analysis contain reduced levels of the cytokines IL-23 and / or IL17A involved in T cell activation, compared to samples obtained from reference subjects to which RPE cells differentiated from ES cells were previously delivered, and / or compared to samples obtained from reference subjects to which RPE cells differentiated from skin iPS cells were previously delivered. In summary, RPE cells as defined herein may be capable of producing reduced levels of IL-23 and / or IL17A, as another preferred example of pro-inflammatory cytokines as defined herein. Also, T cell activation, particularly CD8 cytotoxic T cell activation, may be suppressed in samples from subjects comprising RPE cells of the present invention. In other words, RPE cells may suppress T cell activation, particularly CD8 cytotoxic T cell activation (Figure 24). The degenerative disease to be treated is a retinal disease known to those skilled in the art, and preferably, the retinal degenerative disease is age-related macular degeneration (AMD) or retinal dystrophy.In one embodiment, the present invention refers to the method for treating AMD in a subject as defined herein, comprising administering to the subject RPE cells differentiated from iPS cells obtained by the method as defined herein.In another embodiment, the present invention refers to the method for treating retinal dystrophy in a subject as defined herein, comprising administering to the subject RPE cells differentiated from iPS cells obtained by the method as defined herein.The administration of RPE cells differentiated from CLiPS in the treatment method can include parenteral or topical (preferably subretinal) application, as known to those skilled in the art.

[0119] The present invention further includes an in vivo method for detecting the viability of RPE cells differentiated from iPS cells by the differentiation methods defined elsewhere in this specification in a subject, the method comprising: (a) introducing into a subject RPE cells differentiated from iPS cells by the methods defined herein, wherein the RPE cells comprise a bioluminescent label.

[0120] In this context, the term "viability" refers to RPE cells not dying, remaining mature and / or quiescent, which can be confirmed by detecting the expression of RPE65 as a mature RPE marker and not detecting the expression of Ki67 as a proliferation marker after the cells are introduced into a subject as defined herein and detected over a period of time as further described. Viability monitoring can also be used interchangeably herein with respect to the in vivo method as defined.

[0121] The term "introducing" or "introducing" in step (a) refers to bringing RPE cells as defined herein into a subject, preferably by implanting RPE cells into the subject, even more preferably by subcutaneous implantation of RPE cells into the subject in relation to using a mouse as the subject and the Matrigel plug assay as defined elsewhere herein. The term "subject" as used herein according to the in vivo method and similarly according to the in vitro screening method includes mammalian and non-mammalian subjects. Preferably, the subject is an animal. The subject of the in vivo and in vitro methods may refer to a mammal, including humans, domestic and farm animals, non-human primates, and any other animal having mammary tissue. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a rat. In some embodiments, the mammal is a guinea pig. In some embodiments, the mammal is a rabbit. In some embodiments, the mammal is a cat. In some embodiments, the mammal is a dog. In some embodiments, the mammal is a monkey. In some embodiments, the mammal is a horse. In a preferred embodiment, the subject mammal / animal used in the method of the present invention is a mouse. In a most preferred embodiment, the mammal / animal subject used in the method is a humanized mouse.

[0122] The RPE cells differentiated according to the present invention and introduced into a subject as defined herein in the in vivo method comprise a bioluminescent label.In this context, the term "label" can be a fluorescent label or an enzyme suitable for bioluminescence.When the label is a fluorescent label, it can be a fluorophore (also called a fluorochrome or chromophore). Such fluorophores can be any one of, but are not limited to, fluorescent dyes such as Fluorescein (FITC), Alexa Fluor 350, 405, 488, 532, 546, 555, 568, 594, 647, 680, 700, 750, Pacific Blue, Coumarin, Pacific Green, Cy3, Texas Red, PE, PerCP-Cy5, PE-Cy7, Pacific Orange, or fluorescent proteins such as R-PE or APC, or expressed fluorescent proteins such as CFP, EGFP, GFP or RFP. When the label is an enzyme, it can be a luciferase, preferably selected from the group consisting of bacterial luciferase (\uxAB), photinus luciferase, ren / 7 / a luciferase, and firefly luciferase, but is not limited thereto. In a preferred embodiment, the RPE cells differentiated according to the present invention and introduced into a subject as defined herein in an in vivo method comprise a vector encoding a bioluminescent enzyme gene, preferably tagged with an expressed fluorescent protein as defined herein. In a most preferred embodiment, the RPE cells differentiated according to the present invention and introduced into a subject as defined herein in an in vivo method comprise a vector encoding a luciferase enzyme gene, preferably tagged with GFP.Alternatively or additionally, also encompassed by step (a) of the in vivo methods defined herein is the introduction into a subject of RPE cells differentiated by the methods defined herein, said RPE cells being comprised in an RPE-Matrigel plug as known to those of skill in the art, by which is meant a mixture / composition of RPE cells and Matrigel, as known to those of skill in the art.

[0123] The in vivo method defined herein further comprises step (b) detecting bioluminescent signals of the RPE cells over time using an imaging technique, thereby collecting imaging data.

[0124] The term "detecting or detection" (sometimes referred to as "monitoring") as used herein with respect to in vivo methods refers to visualization and qualitative analysis of bioluminescence of RPE cells in vivo using any known imaging method, preferably using bioluminescence imaging methods, where the bioluminescent label is luciferase. Detection of bioluminescent signal of RPE cells is by step (b) of the in vivo method carried out over a period of time that may refer to at least about 2 days, about 7 days, about 10 days, about 14 days, about 17 days, about 21 days, about 24 days, about 28 days, about 35 days, about 42 days, about 49 days, or at least about 56 days; or between about 2 and about 56 days, or between about 2 and about 49 days. Detection of bioluminescence is preferably monitored at regular intervals over a time course as defined herein. Detection of bioluminescence may refer to detection of total luminescence of cells expressed in p / s / cm2 / sr as known to those skilled in the art. Imaging data is collected upon detection of bioluminescent signal over time.

[0125] The in vivo method as defined herein further comprises step (c) of comparing the imaging data received in step (b) with reference imaging data.In this context, reference imaging data refers to the bioluminescence signal ("imaging signal") of RPE cells differentiated from ES cells (preferably H9 cells) and / or from skin iPS (preferably HDFA or ASF5) that is similarly detected over time as defined herein.In this context, the RPE cells differentiated from ES cells and / or from skin iPS are also introduced into a subject (e.g. a different mouse) that is not exactly the same subject as the subject (e.g. a mouse) that the RPE cells are previously delivered to as defined herein, and the RPE cells differentiated from ES cells and / or from skin iPS also comprise bioluminescence label as defined herein for CLiPS-derived RPE cells.

[0126] The present invention also includes the in vivo method defined herein, wherein no difference in the bioluminescence signal in imaging data compared with reference imaging data indicates the survival of RPE cells in subject.No difference also includes a slight but insignificant decrease in the bioluminescence signal over time in imaging data compared with reference imaging data of RPE cells differentiated from ES cells and / or differentiated from skin iPS cells.

[0127] The present invention also includes animals comprising RPE cells obtained / obtainable by the differentiation method defined elsewhere herein. In this context, "animal" refers to any mammal as defined herein, preferably mouse, most preferably humanized mouse. By animal comprising RPE cells, it may be meant that RPE cells obtained / obtainable by the differentiation method defined elsewhere herein are introduced into an animal as defined herein, preferably by subcutaneous implantation of said RPE cells.

[0128] The introduction of RPE cells differentiated from iPS cells by in vivo differentiation methods into a subject as defined herein and subsequent in vitro analysis of a sample containing said RPE cells obtained from said subject may be particularly envisaged for the analysis of such cells in various models for research and development purposes.

[0129] Therefore, the present invention also includes an in vitro (screening) method for determining the immunogenicity of RPE cells differentiated from iPS cells by the method defined herein in a subject to which differentiated RPE cells have been delivered in advance, comprising: step (a) detecting pro-inflammatory cytokine levels in a sample obtained from the subject defined herein, the sample comprising differentiated RPE cells, by using an imaging method, thereby collecting imaging data. In this respect, "delivered in advance" includes that the differentiated RPE cells of the present invention are delivered to the subject defined herein before the in vitro screening method. Then, a sample comprising differentiated RPE cells is obtained from the subject, and the sample is further analyzed, for example, for specific cytokine levels. The term "pre-introduced" may be used interchangeably.

[0130] In this context, detecting refers to visualizing and quantitatively analyzing the cytokine levels as defined herein in vitro using any known imaging method suitable for detecting cytokines, such as flow cytometry.The cytokine levels detected as defined herein for in vitro methods refer to, but are not limited to, the levels of cytokines related to cellular immunity, preferably the levels of pro-inflammatory cytokines IFN-γ, IL-18, IL-23 and / or IL17A.The cytokine levels as defined herein can be expressed in pg / ml.

[0131] In step (a) of the in vitro method, the sample obtained from the defined subject may be any biological sample taken from said subject, preferably a serum sample.

[0132] The in vitro method may include, as an additional or alternative step within the method, detecting immune cell infiltration in a sample obtained from a subject as defined herein, the sample comprising differentiated RPE cells, thereby further collecting imaging data.

[0133] The method further comprises step (b) of comparing the imaging data received in step (a) and / or from the detection step with reference imaging data. Again, the reference imaging data refers to the imaging data of the same detected pro-inflammatory cytokine levels as defined herein in a sample from a subject to which RPE cells differentiated from ES cells and / or RPE cells differentiated from skin iPS cells are previously delivered as reference cells, and / or the imaging data of the same infiltration and detection of immune cells in a sample from a subject to which RPE cells differentiated from ES cells and / or RPE cells differentiated from skin iPS cells are previously delivered as reference cells. The reference cells can also be included in the same type of sample (e.g., a blood sample, but a different blood sample) as the sample containing RPE cells, but the reference sample can be obtained from a different subject (e.g., a different mouse) compared to the test subject from which the sample containing RPE cells is obtained. Preferably, reduced cytokine levels and / or reduced / reduced immune cell infiltration / accumulation in the imaging data compared to reference imaging data indicates reduced immunogenicity of the RPE cells in the subject, meaning that the ability of the RPE cells of the present invention to induce an immune response in the subject is lower / reduced compared to the immunogenicity of RPE cells differentiated from ES cells and / or RPE cells differentiated from skin iPS cells.

[0134] The invention is further illustrated by the following non-limiting experimental examples. EXAMPLES

[0135] Experimental Example Example 1: Development of electroporation parameters suitable for CLiPS Electroporation according to the protocol described in Okita et al., supra, was found to be completely ineffective. When the episomal vectors pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL were electroporated into the reaction mixture of CLMCs according to the protocol described in Okita et al., supra, no IPS colonies were detected. For CLECs, the episomal vectors pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL (Addgene plasmid no. 27077 (including specifically SEQ ID NO:12-SEQ ID NO:11), no. 27078 (SEQ ID NO:13), no. 27080 (SEQ ID NO:14)) were electroporated according to the protocol described in Okita et al., supra. A mean reprogramming efficiency (expressed in terms of IPS colony counts) of only 0.2% was found after electroporation of CLMCs with (SEQ ID NO:14). It was therefore necessary to develop from scratch an electroporation protocol suitable for the CLMC-derived CLiPS method or, in the case of CLECs, to provide a significantly improved electroporation protocol. For this purpose, the electrical parameters constituting the electroporation, such as the number of electrical pulses, duration and voltage, were varied to develop electroporation conditions that could be used with CLSCs. In this experiment, a number of different electroporation settings were tested for each individual CLMC and CLEC sample cultured under cell-specific conditions as described herein. After each electroporation, approximately 200.000 cells were plated in triplicate in 6-well plates for culture. Approximately 21 days after electroporation, the CLSC colonies that had arisen up to that point were counted to determine the viability. The viability was used to draw conclusions about the electroporation efficiency. The percentage efficiency was calculated as colony count / 200,000 x 100%.

[0136] The results shown in Table 1 and Figure 2 indicate that suitable electroporation conditions could be found for both CLMCs and CLECs. The optimal electroporation settings for CLECs found here were 1 x 10 6 The electroporation settings used for CLECs increased the electroporation efficiency by approximately 23.35% in the case of CLEC42 and 36.65% in the case of CLEC44, compared to Okita et al., 2011. Thus, these electroporation parameters / settings were surprisingly found to increase the electroporation efficiency by approximately 30% on average for CLECs, compared to the conditions used by Okita et al. for the electroporation of human dermal fibroblasts. Of note, the electroporation settings used herein are consistent with conditions reported for successful electroporation of epithelial cells such as corneal epithelial cells (one electric pulse of 30 ms and 1300 V and a cell number of 1 × 10 6 This is quite different from the ratio of the amount of plasmid DNA (μg) to the number of cells (1:1) (see Png, E. et al. (2011), Journal of Cellular Physiology. United States, 226(3), pp. 693-699).

[0137] As mentioned above, the effect of optimizing the electroporation protocol is even more significant for CLMC, since electroporation according to Okita et al., supra, did not result in any survival of CLMC. Four individual CLMC lines (CLMC42, CLMC44, CLMC23, and CLMC30) were electroporated with one electric pulse of 20 ms and 1600 V, and approximately 1 × 10 6 It was found herein that a ratio of plasmid DNA amount of each of the three episomal vectors (pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL) to the number of cells of 1.67 μg (plasmid) DNA per CLMC was successfully transfected. The resulting transgenic cells showed viability of 6.17%, 7.50%, 5.00% and 7.33%, respectively. Of note, the electroporation / transfection conditions found herein to be optimal for the generation of CLiPS from CLMC are also different from previously reported electroporation conditions. In this context, see for example Sprangers, AJ, Freeman, B. and Ogle, BM (2011), pp. 62-66, which investigated possible negative effects of electroporation of mesenchymal stem cells derived from human embryonic stem cells (hESC). In doing so, Sprangers et al. reported that using one electrical pulse of 20 ms and 1400 V, 1 × 10 6A total of 4 μg (plasmid) DNA transfected into 10 mesenchymal stem cells was found to be optimal for MSC transfection. Thus, the present invention provides unique and efficient protocols for CLEC and CLMC electroporation, respectively. The variability in transfection efficiency across the four individual CLSC lines (cells from different donors) is an inherent and documented feature of iPS derivation, individual-to-individual variability. To confirm the gender of the donor CLSC lines and the CLiPS derived therefrom, PCR amplification was performed using gene-specific primers on genomic DNA isolated from the individual CLSC lines to confirm the presence or absence of the DYS439 and SRY loci, both of which are located on the Y chromosome. aSF4 adult dermal fibroblasts, confirmed to be derived from a male donor, were used as a positive control.

[0138] Table 1. Optimized electroporation conditions for the generation of CLiPS. TIFF2025507265000002.tif114153

[0139] Example 2: Transgene integration and derivation of feeder-free human iPS Umbilical cord lining epithelial cells (CLEC) and umbilical cord lining mesenchymal cells (CLMC) were isolated and supplied by CellResearch Corporation Pte Ltd, Singapore. CLEC and CLMC were thawed and expanded in their media PTT-e3 and PTT-4, respectively. Adult dermal fibroblasts from a healthy 78-year-old Asian male donor were purchased from CellResearch Corporation Pte Ltd and cultured in DMEM / 10% FBS.

[0140] The medium PTT-4 consisted of 90% (v / v) CMRL-1066 and 10% (v / v) FBS, and the medium PTTe-3 had the following composition: TIFF2025507265000003.tif66140

[0141] Somatic cell reprogramming was performed using the conditions established in Example 1, and further performed in a feeder-independent manner. Log-phase cultures were harvested by dissociation with TrypLE Express (ThermoFisher Scientific) and 720,000 cells were pelleted in a 1.5 ml centrifuge tube. The cell pellet was resuspended in 120 μL of Buffer R (Neon™ Transfection System 100 μL Kit, Thermo Fisher Scientific MPK10096). A cocktail containing 1.2 μg each of the episomal vectors pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL (Addgene plasmids no. 27077 (SEQ ID NO:12), no. 27078 (SEQ ID NO:13), no. 27080 (SEQ ID NO:14), respectively) was added to the cells and mixed thoroughly (1×10 each of the vectors). 6A (plasmid) DNA amount of 1.67 μg per cell number was used). The cell suspension was loaded onto a 100 μL Neon® Tip and Neon electroporation was performed with the following parameters: adult dermal fibroblasts - 1,650 V, 10 ms, 3 pulses; CLEC - 1350V, 30 ms, 2 pulses; CLMC - 1600 V, 20 ms, 1 pulse. Cells were immediately transferred into 6 ml of CLEC or CLMC medium containing 1 μM hydrocortisone (StemCell Technologies) and evenly distributed into 3 wells of a 6-well plate coated with Matrigel. After 2 days, the medium was switched 1:1 between CLEC or CLMC medium and mTeSR1 supplemented with 1 μM hydrocortisone. A medium change was performed with the same medium on the 4th day after transfection. On the 6th day after transfection, the medium was switched to complete mTeSR1 without hydrocortisone thereafter. Thereafter, medium changes were performed every 2 days with mTeSR1. When the iPS colonies reached a diameter of approximately 1-2 mm (around day 20 onwards), they were manually picked under a bright-field microscope, and each colony was placed into a single well of a 24-well plate (Nunc) coated with Matrigel. When the cells in each well reached approximately 50% confluency, they were detached with dispase (StemCell Technologies) and transferred to a well of a 6-well plate coated with Matrigel. The cells were then passaged 1:3 when they reached near confluency by dissociation with 0.5 mM EDTA. The newly passaged cells were cultured overnight in medium containing 10 μM ROCK inhibitor Y-27632. In addition to mTeSR1, other commercially available ES / iPS media such as StemMACS™ iPS-Brew XF (Miltenyi Biotec) and TeSR-E8 (StemCell Technologies) were used to maintain the culture of iPS.

[0142] Protocol for preparing CLiPS: 1. Actively dividing CLECs or CLMCs cultured in T-75 flasks in their maintenance media PTTe-3 and PTT-4, respectively, are harvested by dissociation using TrypLE Express (ThermoFisher Scientific). 2. Count the cells and aliquot 720,000 cells into a microcentrifuge tube and pellet. 3. Resuspend the cell pellet in 120 μL of Buffer R (Neon™ Transfection System 100 μL Kit, Thermo Fisher Scientific MPK10096). Add a cocktail containing 1.2 μg each of pCXLE-hUL, pCXLE-hSK and pCXLE-hOCT3 / 4-shp53-F and mix thoroughly. 4. Load the cell suspension into a 100 μL Neon® Tip. Electroporation is performed with the following parameters for CLEC: 1350 V, 30 ms, 2 pulses and for CLMC: 1600 V, 20 ms, 1 pulse. 5. The cells are immediately transferred to 4 ml of CLEC or CLMC medium (PTTe-3 and PTT-4, respectively) containing 1 μM hydrocortisone and then distributed into 3 wells of a Matrigel-coated 6-well plate. 6. Two days after electroporation, replace the medium with a 1:1 (v / v) mixture of CLEC or CLMC medium (PTT-e3 and PTT-4, respectively) and mTeSR1 supplemented with 1 μM hydrocortisone. 7. Four days after electroporation, perform a medium change with the same 1:1 (v / v) medium mixture. 8. Six days after electroporation, replace the medium with mTeSR1 only, without hydrocortisone. 9. Change medium every 2 days 10. iPS colonies may begin to appear as early as 2 weeks after transfection. When the iPS colonies reach a diameter of about 0.5 mm to about 1 mm (around day 20 onwards), they are manually picked under a bright-field microscope and each colony is placed into a single well of a Matrigel-coated 24-well plate (Nunc). 11. After colony picking, perform medium changes for isolated colonies every day. 12. When the cells in each well occupy approximately 50% of the culture surface, they are detached with dispase (StemCell Technologies) and transferred to wells of a Matrigel-coated 6-well plate. 13. After that, when the cells reach approximately 70%-80% confluency, they are passaged 1:3 by dissociation with 0.5 mM EDTA. The newly passaged cells are cultured overnight in medium containing 10 μM ROCK inhibitor Y-27632.

[0143] Following the protocol above, small clusters of cells that appeared morphologically distinct from the parent cells began to appear around day 10. By day 15, the cell clusters acquired clear edges (Figure 3b), and from day 20 onwards, individual embryonic stem cell-like colonies appeared (Figure 3c and Figure 3d). Colonies were picked when they reached 1-2 mm in diameter and expanded for characterization and storage. Expanded CLiPS exhibited cell morphology indistinguishable from that of adult dermal fibroblast-derived iPS or human embryonic stem cells (ES), with characteristic large nuclei and thin cytoplasm (Figure 3e and Figure 3f).

[0144] Example 3: Derivation of cGMP-Compliant CLiPS (CLMSC-DTHN) To provide proof of concept that CLiPS can be produced under conditions compatible with human therapeutic use, iPS were generated from a cGMP grade CLMC line, designated CLMSC-DTHN, using the protocol described in WO2018 / 067071 for the production of mesenchymal stem populations in which 99% of the stem cells express the markers CD73, CD90 and CD105, but not the markers CD34, CD45 and HLA-DR, and cGMP quality reagents whenever possible. The reprogramming protocol was the same as described for CLMC in Example 2, but with the replacement of Matrigel, an extracellular matrix substrate prepared from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, with recombinant human laminin-511 E8 fragment (iMatrix-511 SILK, ReproCELL), a defined animal-free and xeno-free substrate for coating cell culture vessels. Additionally, mTeSR1 used for reprogramming and subsequent maintenance of CLiPS clones was replaced with cGMP mTeSR™1 (StemCell Technologies).

[0145] Under the conditions described herein, CLMSC-DTHN were reprogrammed with comparable kinetics and efficiency to CLMC (data not shown). At 10 days after transfection with the reprogramming vector, small clusters of cells with compact morphology could be observed (Figure 3n). These clusters grew into isolatable colonies from day 20 onwards. The expanded colonies exhibited the characteristic cell morphology of human pluripotent stem cells (Figure 3n-q).

[0146] Growth and cryopreservation of CLiPS Subculture of CLiPS (again using a medium suitable for maintaining iPS cells, such as mTeSR1 or TeSR-E8) is performed when the cultures reach approximately 90% confluence. Aspirate off the spent medium along with any clearly differentiated areas that may be present. Be careful not to expose the cells to air for long periods of time. Rinse the cultures once with pre-warmed (37°C) Dulbecco's phosphate-buffered saline (DPBS). Add to the cultures the appropriate amount of pre-warmed (37°C) 0.5 mM EDTA solution according to the culture vessel - 0.5 ml / well for 24-well dishes, 1 ml / well for 6-well dishes or 2 ml for 6 cm dishes. Place the cultures in a 37°C incubator for 5 min and then observe under a microscope. The cells should appear round but not detached from the surface. The duration of the incubation at 37°C depends on the CLiPS line and can range from about 5 to 10 min. The duration of incubation depends mainly on previous experience with each strain. After incubation, gently aspirate off the EDTA solution, taking care not to dislodge the cells. Using a 1 ml pipettor, dispense medium such as mTeSR1 or TeSR-E8 containing the ROCK inhibitor Y-27632 directly onto the cells to dislodge the cells. The amount of medium used depends on the size of the vessel used, 0.5 ml / well for 24-well dishes, 1 ml / well for 6-well dishes or 2 ml for 6 cm dishes. Repeat gentle pipetting until the majority of the cells are dislodged. The cell suspension is then transferred to a 15 ml Falcon tube. Rinse the culture vessel with fresh medium and combine the rinse with the cell suspension in the Falcon tube. Dilute the cells in the tube to an appropriate volume for plating into a new Matrigel-coated vessel. The split ratio can range from 1:3 to 1:10, depending on the density of the initial culture and the growth rate of the individual CLiPS line.

[0147] For cryopreservation, cells are suspended in mTeSR1 or TeSR-E8 (or any other suitable medium) supplemented with 10% v / v tissue culture grade dimethyl sulfoxide (DMSO; e.g., Hybri-Max™, Sigma-Aldrich). This cell suspension is then dispensed into an appropriate number of cryovials. The cell density per aliquot depends on the desired rate at which cell confluency is achieved when the aliquot is thawed and cultured. The cryovials are then transferred to a slow-freezing device such as Mr. Frosty™ Freezing Container (Thermo Scientific) or CoolCell® Cell Freezing Containers (BioCision LLC) and placed at −80° C. overnight. The next day, the cryovials are transferred to liquid nitrogen storage. It is not recommended to leave CLiPS aliquots at −80° C. for more than 24 hours. Several commercially available freezing media, such as mFreSR™ (StemCell Technologies) and CryoStor® CS10 (Biolife Solutions), are also available for cryopreservation and may be used according to the manufacturer's instructions.

[0148] Example 4: Analysis of CLiPS functionality The functionality of CLiPS was determined by subjecting colony-forming CLiPS to immunofluorescence staining after electroporation, whereby the expression of pluripotent embryonic stem cell markers (OCT4, SOX2, KLF4, NANOG, SSEA-4, TRA-1-81) was analyzed. For this purpose, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 15 min and then washed three times for 5 min with PBS. For staining of intracellular or nuclear markers (OCT4, SOX2, KLF4, NANOG), cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with FDB (5% FCS / 1% NGS / 1% BSA) for 1 h. For staining of surface markers (SSEA-4, TRA-1-81), the permeabilization step was omitted. Cells were incubated overnight at 4 °C with primary antibodies appropriately diluted in FDB, followed by a 2 h incubation at room temperature with secondary antibodies conjugated with the appropriate fluorochromes. Stained samples were mounted in ProLong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific).

[0149] Furthermore, the number and structure of chromosomes within individual CLiPS strains were assessed by performing karyotyping and G-banding analysis, where the G-banding analysis was performed by the Cytogenetics Laboratory, KK Women's and Children's Hospital Pte. Ltd., Singapore.

[0150] In addition, RT-PCR analysis was performed to analyze the expression of reprogramming and pluripotency genes in primary parental cells, parental cells 11 days after vector transfection (D11 transfected cells) and CLiPS. For this purpose, total RNA was isolated from cell pellets using RNeasy Mini or Plus Mini kits (Qiagen). 2 μg of total RNA was treated with DNase I and used for cDNA synthesis with RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific). PCR reactions were set up as follows: 0.5 μl cDNA, 5 μl 2×MyTaq HS Mix (Bioline), 0.2 μl forward primer (10 μM), 0.2 μl reverse primer (10 μM), 4.2 μl PCR water. Thermal cycling was performed in an MJ Mini Thermal Cycler (Bio-Rad) under the following conditions: 1× 95° C. 1 min, 30× (95° C. 15 sec, Tm 15 sec, 72° C. 15 sec), 72° C. 1 min. The primer sequences and annealing temperatures (Tm) used are shown in Table 2 below.

[0151] Qualitative expression analysis was performed by agarose gel analysis, where samples were loaded onto 2% agarose gels incorporating SYBR Safe DNA stain (Thermo Fisher Scientific) in 1× TAE buffer and electrophoresed for 30 min at 80 V. Gel images were captured using a ChemiDoc Imaging System (Bio-Rad).

[0152] (Table 2) Primer sequences TIFF2025507265000004.tif124138TIFF2025507265000005.tif158138

[0153] Results reveal that CLiPS showed robust expression of human embryonic stem cell (hES) markers KLF4, NANOG, OCT4, SOX2, SSEA4 and TRA-1-60 as demonstrated by antibody staining (Fig. 3g-l). G-banding analysis showed that CLiPS maintained normal karyotypes from colony picking up to 17 passages (Fig. 3m). RT-PCR analysis of gene expression in parental cells, cells at day 11 post-transfection and expanded iPS clones revealed that activation of endogenous OCT4, SOX2, KLF4, LIN28 and L-MYC genes replaced the role of vector-driven expression of these genes for pluripotency maintenance in fully reprimed CLiPS (Fig. 3v). Induction of the endogenous NANOG locus, a gene critical for somatic cell repriming, was evident at day 11 post-transfection. The absence of detectable levels of EBNA-1 transcripts in CLiPS clones suggests that the plasmid vector was lost from these cells. Expression of additional hES-specific genes GDF3, DPPA5, DNMT3, FGF4 and REX-1 in CLiPS further confirms their hES-like molecular phenotype. TERT, encoding the catalytic reverse transcriptase subunit of telomerase, essential for regulating self-renewal and maintaining pluripotency, is expressed in CLiPS at levels similar to H1 hES.

[0154] Example 5: Expression analysis of pluripotent embryonic stem cell markers in CLiPS-DTHN To analyze the expression of pluripotent embryonic stem cell markers (Oct4, Sox2, Klf4, Nanog) indicative of pluripotency, the developing CLMSC-DTHN were subjected to immunofluorescence staining after electroporation. The immunofluorescence staining protocol was the same as that described for CLiPS in Example 4.

[0155] The results reveal that CLMSC-DTHN express pluripotent stem cell markers NANOG, OCT4, SOX2 and TRA-1-81 at levels indistinguishable from their non-GMP counterparts (Figure 3r-u). Thus, CLMSC-DTHN may provide the same embryonic characteristics associated with non-GMP-derived CLiPS.

[0156] Example 6: Determination of CLiPS pluripotency The pluripotency of CLiPS and aSF-iPS was assessed by teratoma formation assay in NOD-SCID mice. For this purpose, 1 × 10 6 CLiPS cells were pelleted and resuspended in 0.1 ml ice-cold Matrigel and cultured in 6-8 week old NOD / MrkBomTac-Prkdc scid Mice were injected into the dorsal flank. Mice were sacrificed 3 months later and teratomas were harvested for histological analysis, in which paraffin wax sectioning and hematoxylin-eosin staining were performed using standard techniques.

[0157] Results show that some mice developed palpable tumors one month after subcutaneous injection of iPS cells into the dorsal flank. Histological analysis of teratomas isolated 3 months after injection revealed that CLiPS cells spontaneously differentiated into endodermal, mesodermal, and ectodermal tissues (Figure 4a-f).

[0158] Example 7: Differentiation of CLiPS into dopaminergic neurons As an important prerequisite for potential future therapeutic applications of CLiPS, their ability to differentiate into specific tissue types under defined in vitro conditions needs to be demonstrated. In the case of dopaminergic neuronal differentiation, the midbrain floor plate induction protocol described in Kriks, S., et al, Nature, 2011. 480(7378): p. 547-51 was used for differentiation of iPS into dopaminergic neural precursors and neurons. Briefly, iPS were plated in Matrigel (Corning)-coated dishes at 1 cm 2 3.5~4.0×10 4The cells were plated at a density of 1 / 4 cells and cultured for 5 days in Knock-Out Serum Replacement Medium (KSR) containing Knock-Out DMEM, 15% Knock-Out Serum Replacement, 1× GlutaMAX, and 10 mM β-mercaptoethanol. From day 5, the KSR medium was gradually changed to N2 medium as described in Tomishima "Midbrain dopamine neurons from hESCs." 2012 Jun 10. In: StemBook. Cambridge (MA): Harvard Stem Cell Institute; 2008- Available at: https: / / www.ncbi.nlm.nih.gov / books / NBK133274 / doi: 10.3824 / stembook.1.70.1. On day 11, the medium was changed to NB27 medium composed of Neurobasal medium, 2% B27 minus vitamin A and 1× GlutaMAX, supplemented with CHIR (until day 13), BDNF (brain-derived neurotrophic factor, 20 ng / ml; Miltenyi), ascorbic acid (0.2 mM, Sigma), GDNF (glial cell line-derived neurotrophic factor, 20 ng / ml; Miltenyi), TGFβ3 (transforming growth factor type β3, 1 ng / ml; R&D), dibutyryl cAMP (0.5 mM; Santa Cruz Biotechnology) and DAPT (10 nM; Tocris,) for 9 days. On day 20, cells were dissociated using Accutase (Gibco) and plated at high cell density (1 cm) on dishes precoated with poly-L-ornithine (PLO; 15 mg / ml) / laminin (1 μg / ml) / fibronectin (2 μg / ml) in NB27 medium supplemented with 10 μM ROCK inhibitor Y-27632. 2 3~4×10 per 5The cultures were maintained in NB27 medium with medium changes every other day until the desired end point. Differentiated cells were analyzed at this stage for the expression of cell-specific markers. For this purpose, cryosectioning was performed in which slides containing sections were dehydrated by incubation at 37° C. for 30 min, cooled to room temperature and washed three times with TBST. Permeabilization, blocking, antibody staining and mounting of sections were performed as described in Example 4. Primary antibodies from the same host species were used, and fluorochrome-conjugated monovalent antibodies (Jackson ImmunoResearch) were used to saturate the first primary antibody before sequential incubation with the second primary antibody and the conjugated secondary antibody.

[0159] The results demonstrate that dopaminergic neurons were obtained from CLiPS and asF5-iPS cells using this protocol. Antibody staining revealed that nearly 90% of cells co-expressed the floor plate marker FOXA2 and the tectal plate marker LMX1A (Figure 4k, k', k''), a defining feature of midbrain DA neuron precursors. Further differentiation yielded abundant mature neurons as shown by TUJ1 staining, of which approximately 30-50% co-expressed the dopaminergic marker tyrosine hydroxylase (TH) (Figure 4l, l', l''). Electrophysiological analysis of CLiPS-derived neurons at day 45 of differentiation demonstrated that the cells exhibited mature functional properties, and injection of hyperpolarizing current demonstrated that trains of action potentials displayed a voltage sag response characteristic of mature midbrain DA neurons (Figure 4m).

[0160] Example 8: Differentiation of CLiPS into hepatocytes As an important prerequisite for potential future therapeutic applications of CLiPS, its ability to differentiate into desired target cell types, or into specific tissue types, under defined in vitro conditions must be demonstrated. For hepatic differentiation, a protocol originally developed for human embryonic stem (ES) cell differentiation on mouse feeder layers (Medine, CN, et al., J Vis Exp, 2011(56): p. e2969) was adapted for CLiPS and asF-iPS differentiation in mTeSR1 on Matrigel. The modification was that when the iPS cultures reached 20-30% confluency, they were supplemented with DMSO to 2% and incubated for 24 h. When the cultures reached approximately 30%-60% confluency, definitive endoderm formation was induced by replacing mTeSR1 with priming medium (RPMI 1640-B27 supplemented with 100 ng / mL activin A and 50 ng / mL Wnt3a). Cultures were maintained in priming medium for 3 days, with medium changes every 24 hours. After 72 hours in priming medium, medium was switched to SR-DMSO (80% KO-DMEM, 20% KO-SR, 0.5% L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol and 1% DMSO) for 5 days, with medium changes every 48 hours). On day 8, cultures were switched to L-15 maturation maintenance medium (Leibovitz L-15 medium, 8.3% tryptose phosphate broth, 8.3% heat-inactivated FBS, 10 μM hydrocortisone 21-hemisuccinate, 1 μM insulin (bovine pancreas), 1% L-glutamine, 0.2% ascorbic acid) supplemented with 10 ng / mL hHGF and 20 ng / mL OSM for 9 days (medium changes every 48 hours). The differentiated cells were again analyzed at this stage for the expression of cell-specific markers. For this purpose, cryosectioning was performed as described in Example 7.

[0161] The results demonstrate that hepatocyte-like cells were obtained from CLiPS and asF5-iPS using this protocol. Antibody staining revealed the expression of hepatocyte markers alpha-fetoprotein (AFP; Figure 4g, g', g''), cytokeratin 18 (CK18) and human serum albumin (HSA; Figure 4h, h', h'') after 17 days of differentiation. The majority of differentiated cells exhibited a polygonal shape characteristic of hepatocytes. Furthermore, staining with Oil Red O showed abundant intracellular lipid droplet accumulation, a characteristic of cultured hepatocytes (Figure 4i, i', i'').

[0162] Example 9: Differentiation of CLiPS into cardiomyocytes As an important prerequisite for potential future therapeutic applications of CLiPS, its ability to differentiate into specific tissue types under defined in vitro conditions needs to be demonstrated. For cardiomyocyte differentiation, the protocol for cardiomyocyte differentiation of iPS was adapted from the protocol described in Lian, X., et al., Proc Natl Acad Sci USA, 2012. 109(27), p. E1848-57. iPS maintained on Matrigel in mTeSR1 were dissociated into single cells with StemPro Accutase (Thermo Fisher Scientific) for 5 min at 37 °C and then plated on Matrigel-coated cell culture dishes at 1 × 10 cells in mTeSR1 supplemented with 5 μM ROCK inhibitor (Y-27632; Stemgent). 5 ~2×10 5 Cells / cm 2 (5 × 10 per 24 wells 5Cells were seeded with 100 μM (cells) for 24 h. As a modification, when the cells reached approximately 80% confluency, the medium was switched to mTeSR1 supplemented with 2% DMSO. Once the cells reached confluency, they were treated with CHIR99021 in RPMI / B27-insulin for 24 h. As another modification, the concentration of CHIR99021 was reduced from the original 12 μM to 5 μM at this stage. The next day, the medium was changed to RPMI / 2% B27 without insulin. Two days later, half of the old medium was combined with an equal volume of fresh medium containing 10 μM IWP2 (Tocris). The remaining medium in the wells was discarded and the mixture was added to the cultures. After two days, the medium was switched to RPMI / 2% B27 without insulin only. After 48 h, the cultures were maintained in RPMI / 2% B27 medium with medium changes every 3 days until the desired endpoint. Beating cardiomyocytes were fixed and stained for cell-specific markers as described in Example 7.

[0163] The results demonstrate that cardiomyocytes were obtained from CLiPS and asF5-iPS using this protocol. Antibody staining revealed that the expression of spontaneously contracting cardiomyocytes was observed from day 8 of differentiation. Immunofluorescent antibody staining against functional cardiac markers myosin regulatory light chain 2a (MLC2a), cardiac troponin I (cTnI) and α-actinin (αACT) revealed sarcomere structures in differentiated cardiomyocytes (Figure 4j,j',j''). No significant difference in differentiation efficiency was observed between them.

[0164] Example 10: Differentiation of CLiPS into oligodendrocytes To further demonstrate the ability of the induced pluripotent stem cells of the present invention to differentiate into a given target cell type, CLiPS were differentiated into oligodendrocytes.The oligodendrocyte differentiation of CLiPS and asF-iPS was carried out according to the protocol of Douvaras, P. and V. Fossati, Nat Protoc, 2015.10(8): p.1143-54.Furthermore, to analyze the expression of cell-specific markers, cryosectioning was carried out as described in Example 7.

[0165] At day 75 of differentiation, clusters of Olig2-positive oligodendrocyte precursor cells (OPCs; Fig. 4n) or O4-positive late OPCs were obtained (Fig. 4o).

[0166] Example 11: Immunogenicity analysis To gain insight into the immunogenicity of CLiPS and their neural derivatives, the expression of a panel of immunogenicity-associated markers by these cells was assessed by flow cytometric analysis. For this purpose, primary cells and differentiated DA NPCs at day 25 were harvested by dissociation with TrypLE Express, while iPS cultures were harvested by dissociation with 0.5 mM EDTA. Cells were cultured in 1x Ca2+ buffer containing 0.1% bovine serum albumin (BSA). 2+ Free and Mg 2+ Cells were resuspended at 5 million cells / ml in Ca-free DPBS. 100 μl of cells were stained with the appropriate conjugated antibody or its isotype control for 30 min on ice in the dark. For HLA-E and HLA-G staining, cells were permeabilized with BD Phosflow Perm / Wash Buffer I (BD Biosciences) prior to staining according to the manufacturer's instructions. After staining, cells were resuspended in 1× Ca2+ buffer and washed with 1× PBS. 2+ Free and Mg 2+ The cells were washed twice in DPBS / 5 mM EDTA-free and fixed in 1% paraformaldehyde for 1 h in the dark, followed by 1× Ca 2+ Free and Mg 2+ The cells were washed twice in DPBS / 5 mM EDTA-free. 2+ Free and Mg 2+ The cells were resuspended in free DPBS / 5 mM EDTA and analyzed on a flow cytometer. Stained primary cells and iPS were analyzed on a FACSCalibur, while stained dopaminergic neural progenitor cells (NPCs) were analyzed on a FACSCanto II instrument (both from BD Biosciences). Data were analyzed using the FlowJo software package (FlowJo LLC). Antibodies used are listed in Table 3.

[0167] Table 3: Antibodies used in flow cytometry TIFF2025507265000006.tif117130

[0168] MHC class I HLA-A, HLA-B, and HLA-C as well as MHC class II HLA-DR molecules are known to be important for alloimmune responses. Results reveal that HLA-ABC is expressed across all iPS samples, but significantly reduced levels are observed in EC23-CLiPS (Fig. 6a). HLA-DR expression was absent in all iPS samples (Fig. 6b), consistent with previous reports of negligible HLA-II expression in iPS (Saljo, K., et al., Sci Rep, 2017. 7(1): p. 13072 and Chen, HF, et al., Cell Transplant, 2015. 24(5): p. 845-64.). T cell costimulatory molecules CD40, CD80, and CD86 play important roles in T cell activation during alloimmune responses. Of the three molecules examined, only CD40 was expressed on iPS, at the lowest level by asF-iPS and the highest level by MC23-CLiPS compared to the others (Figure 6a). As tolerogenic HLA-E and HLA-G have been reported to be expressed in CLMC (Deuse, T., et al., Cell Transplant, 2011. 20(5): p. 655-67) and CLEC (Zhou, Y., et al., Cell Transplant, 2011. 20(11-12): p. 1827-41), we also examined the expression of these antigens by CLiPS. Analysis of permeabilized cells revealed that expression of HLA-E was negligible in MC23-CLiPS and EC44-CLiPS, and below detectable levels in the other samples. We then repeated the expression profiling of the full panel of markers in day 25 DA differentiation cultures. Analysis was performed on neural cell populations gated on positive staining for NCAM. The NCAM+ fraction was greater than 97% in all samples, with asF-iPS and EC23-CLiPS showing comparable differentiation efficiencies of 99.5% (Figure 6b). HLA-ABC was expressed by all NPC samples, but generally at lower levels compared to their parental iPS (Figure 6c).EC23-CLiPS-derived NPCs expressed the lowest levels of HLA-ABC among the samples, reflecting the trend shown by their parental iPS. HLA-ABC expression levels in MC23-CLiPS were reduced upon their differentiation into NPCs. CD40 expression was downregulated across all NPC samples, with only EC23-iPS- and EC44-iPS-derived NPCs showing slight expression. HLA-E expression was absent in all NPC samples, whereas slight upregulation of HLA-G was observed in asF-iPS- and EC23-iPS-derived NPCs. These results indicate reduced immunogenicity of CLiPS.

[0169] Example 12: Transplantation of CLiPS-derived dopaminergic neurons in a fully immunocompetent mouse model of Parkinson's disease Previous studies have demonstrated that dopaminergic neurons generated from human embryonic stem cells and iPS cells using various protocols can be effectively used in rodents with Parkinson's disease (PD) (Kriks, S., et al, Nature, 2011. 480(7378): p. 547-51; Hargus, G., et al., Proc Natl Acad Sci USA, 2010. 107(36): p. 15921-6; Doi, D., et al., Stem Cell Reports, 2014. 2(3): p. 337-50; Grealish, S., et al., Cell Stem Cell, 2014. 15(5): p. 653-65; Kirkeby, A., et al., Cell Rep, 2012. 1(6): p. 703-14; Qiu, L., et al., Stem Cells Transl Med, 2017. 6(9): p. 1803-1814; Rhee, YH, et al., J Clin Invest, 2011. 121(6): p. 2326-35; Samata, B., et al., Nat Commun, 2016. 7: p. 13097; Wakeman, DR, et al., Stem Cell Reports, 2017. 9(1): p. 149-161) and non-human primates (Kriks, S., et al, Nature, 2011. 480(7378): p. 547-51; Hargus, G., et al., Proc Natl Acad Sci USA, 2010. 107(36): p. 15921-6; Wakeman, DR, et. al., Stem Cell Reports, 2017. 9(1): p. 149-161; Daadi, MM, et al., PLoS One, 2012. 7(7): p. e41120; Kikuchi, T., et al., Nature, 2017. 548(7669): p. 592-596) and have been shown to be able to engraft in these models.In all these studies, animals were either rendered immunodeficient or pharmacologically immunosuppressed to prevent graft rejection. The need for immunodeficient or immunosuppressed animals is justified by the fact that transplants must be autologous (Morizane, A., et al., Stem Cell Reports, 2013. 1(4): p. 283-92; 4. Hallett, PJ, et al., Cell Stem Cell, 2015. 16(3): p. 269-74; Wang, S., et al., Cell Discov, 2015. 1: p. 15012; Emborg, ME, et al Cell Rep, 2013. 3(3): p. 646-50; Sundberg, M., et al., Stem Cells, 2013. 31(8): p. 1548-62) or MHC-matched allogeneic (Morizane, A., et al., 2017. 8(1): p. 385) It was only unnecessary when performed using iPS-derived cells.

[0170] To demonstrate the engraftment potential of CLiPS-derived DA NPCs differentiated using the method of the present invention, 25-day-old NPCs differentiated from asF-iPS, EC23-CLiPS and MC23-CLiPS were transplanted into immunodeficient NOD-SCID mice (n=3). In this regard, it should be noted that all animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the National Neuroscience Institute (NNI), Singapore.

[0171] To test the immunogenicity of CLiPS-derived DA NPCs, transplantation needs to be performed in PD models. For this purpose, a 6-hydroxydopamine (6-OHDA) unilateral lesion mouse model was generated. Unilateral 6-OHDA lesion is an established method for rodents, which consists of injecting 6-OHDA into the rodent brain to induce motor dysfunction characterized by angular rotational asymmetry (Bagga, V., Dunnett, SB and Fricker, RA (2015) Behavioural Brain Research. Elsevier BV, 288, pp. 107-117). In the present invention, NOD / MrkBomTac-Prkdc mice purchased from InVivos Pte Ltd and maintained under SPF conditions at the Animal Research Facility, NNI were used. scid 6-OHDA lesions were induced in mice (4 weeks old) and male C57BL / 6NTac mice (6–8 weeks old) purchased from InVivos Pte Ltd., where the mice used in this experiment were fully immunocompetent and no immunosuppression was administered before or after transplantation.

[0172] To generate the mouse PD model, 7.5 μg of 6-OHDA (Sigma, Merck-Millipore; dissolved at 2.5 mg / ml in 0.9% NaCl containing 0.2% ascorbic acid) was delivered to the left striatum by stereotaxic injection at the following coordinates: anterior-posterior (AP) +0.5 mm; medial-lateral (ML) -1.8 mm from bregma; dorsal-ventral (DV) -3.0 mm from cranium. After 2 weeks of acclimation, three NPC samples (i.e., NPCs derived from asF-iPS, EC23-CLiPS and MC23-CLiPS) were transplanted by stereotaxic injection into the striatum of immune-competent 6-OHDA-lesioned C57BL / 6 mice, making the mouse model considered to be successful in injury.

[0173] To determine the model suitable for transplantation, apomorphine-induced rotation was scored, and mice with more than 6 rotations per minute were used for transplantation. For transplantation, dopaminergic progenitor cells were harvested by dissociation at day 25 and diluted to approximately 1.25 × 10 in HBSS supplemented with 10 ng / mL BDNF, 10 ng / mL GDNF. 5 Cells were resuspended at 1000 cells / μl. 2 μL of cell suspension was injected into lesioned mice at the following coordinates: AP +0.5 mm; ML -2.0 mm, and DV -2.8 mm from the skull. To evaluate whether transplanted NPCs could integrate and mediate functional benefits in lesioned animals, rotational asymmetry tests were performed at 2-week intervals. Rotation assays were performed every 2 weeks up to 9 months by intraperitoneally injecting mice with 0.05 mg / kg apomorphine dissolved in 0.9% NaCl containing 0.1% w / v ascorbic acid. Rotations were recorded using a digital camera and counted manually. Batches of animals were killed by terminal anesthesia at 1, 6, and 9 months after transplantation.

[0174] Six months after transplantation, NPC-transplanted, sham-injected and non-operated mice were assessed for striatal dopamine transporter (DAT) activity by positron emission tomography (PET) using the radioligand (2-[18F]fluoroethyl 8-[(2E)-3-iodoprop-2-en-1-yl]-3-(4-methylphenyl)-8-azabicyclo[3.2.1]octane-2-carboxylate) ([18F]FE-PE2I). Animals were fasted for 3 h before the imaging session. Animals were kept warm during scanning by integrating a hot air channel from the imaging bed. Respiratory rate and temperature were monitored throughout the scanning session to ensure sufficient anesthesia levels. Mice were imaged using a nanoScan PET / MRI scanner (Mediso Ltd., Hungary) at the SingHealth Experimental Medicine Centre (SEMC). The scanner is equipped with 12 detector modules with axial field of view (FOV) 94 mm and transaxial FOV 94 mm or 120 mm diameter with 1:3 and 1:5 coincidence modes, respectively. After intravenous injection of 3.57–10.61 MBq of [18F]FE-PE2I via the tail vein in a maximum volume of 0.1 ml, animals were placed in a lateral recumbent position and 3D dynamic PET scans were performed with frames of increasing duration, i.e., 4 frames in 10 s, 4 frames in 20 s, 3 frames in 1 min, 7 frames in 3 min, and 6 frames in 6 min, for 62 min. [18F]PE-PE2I was synthesized at Singapore Radiopharmaceuticals Pte Ltd. MRI images were used for attenuation correction of PET scans and as a structural reference for PET images in data analysis. Thus, T1-weighted MRI images were acquired using the MRI component of the Nanoscan PET / MRI scanner. An integrated mouse head coil covers the entire brain during MRI scans. 3D GRE EXT sequence: 0.6 mm slices were acquired using a 64 mm square FOV, 128 × 128 matrix, 20 ms repetition time (TR), 2.3 ms echo time (TE), and 25° flip angle.All image and kinetic analyses of [18F]FE-PE2I PET images were performed using PMOD (version 3.5; PMOD Technologies). All PET images were first automatically registered to the MRI images using the FUSION tool in PMOD. The MRI images were then manually registered to a T2-weighted mouse template (M. Mirrione, C57BL / 6J mice; Ma, Y., et al., Neuroscience, 2005. 135(4): p. 1203-15; Mirrione, MM, et al., Neuroimage, 2007. 38(1): p. 34-42), including a volume-of-interest (VOI) template with 20 regions. The accuracy of the manual registration was accessed and verified by two different individuals. Finally, a joint transformation matrix was applied to convert the PET images to the MRI mouse template. The left and right striatum and cerebellum VOIs were used for the analysis. To reduce errors due to misregistration and misdefinition (He, B. and EC Frey, Phys Med Biol, 2010. 55(12): p. 3535-44), a one-voxel 3D erosion was applied to the obtained VOIs. [18F]FE-PE2I binding was quantified using a non-invasive reference tissue model, as it gives comparable accuracy compared to kinetic analysis using an arterial input function (Varrone, A., et al., Nucl Med Biol, 2012. 39(2): p. 295-303). Binding potential (BPnd) values ​​were calculated using the simplified reference tissue model (SRTM) (Lammertsma, AA and SP Hume, 1996. 4(3 Pt 1): p. 153-8) with the cerebellum as reference. Regional time activity curves (TACs) were also extracted from the striatal and cerebellar VOIs. Anesthesia was induced with 5% isoflurane in 100% O2 and maintained with 1.5–2% isoflurane during imaging.

[0175] Mouse brain sections were analyzed for the presence of microglia / macrophages, since these cells are known to play an important role in allograft and xenograft rejection in the CNS (Hoornaert, CJ, et al., Stem Cells Transl Med, 2017. 6(5): p. 1434-1441). To this end, immunostaining for the microglia / macrophage specific marker Iba1 was performed after transcardial perfusion with 4% PFA. For this purpose, PFA-perfused brains were fixed overnight in 4% PFA and then equilibrated in 15% and 30% w / v sucrose solutions in PBS until they sank to the bottom of the tube. Brains were embedded in OCT freezing medium and 18 μm sections were cut on a CM3050 S cryostat (Leica Biosystems) and collected on BOND Plus Slides (Leica Microsystems).

[0176] Results revealed the presence of hNCAM+ / TH+ neurons in all three groups 1 month after transplantation (Fig. 7a-c), suggesting that NPCs could differentiate into mature neurons and survive in the host environment. However, no signs of engraftment were observed in the asF-iPS (Fig. 7h) or MC23-iPS (data not shown) groups. hNCAM / TH+ fibers could be seen extending from neurons within the graft core in the EC23-CLiPS group along the axonal tracts in the corpus callosum (Fig. 7d and Fig. 7e). Immunostaining for the microglia / macrophage-specific marker Iba1 revealed the abundant presence of microglia / macrophages in the injected hemisphere compared to the non-injected hemisphere (Fig. 7i and Fig. 7j). Microglia / macrophages infiltrating the core of the graft exhibited an amoeboid morphology characteristic of activated microglia, compared to those at the periphery of the graft, which displayed a ramified morphology typical of quiescent cells. Furthermore, the infiltrated microglia stained positive for CD68, a marker of activated microglia. At 1 month after transplantation, no microglial accumulation was observed at the injection site in asF5-iPS and MC23-CLiPS NPC-transplanted brains. This is presumably because microglia dispersed and returned to a quiescent state after clearance of the xenografts. Human TH+ neurons survived up to 9 months in some animals transplanted with EC23-CLiPS NPCs, as confirmed by human nuclear antigen (HuNu) and human NCAM staining (Figure 8a-f). Rotational asymmetry testing revealed that upon exposure to apomorphine, a dopamine agonist, injured animals exhibited opposite-directed rotations due to hypersensitivity of postsynaptic D2 dopamine receptors on the injured striatum as a result of dopamine depletion. The effectiveness of the administered intervention would be manifested as an improvement in this rotational asymmetry. Only animals transplanted with EC23-CLiPS NPCs showed improved rotational behavior in both species, in contrast to asF-iPS NPC- or sham-transplanted animals (Figure 8h). In these mice, the reduction in rotation reached significance (p<0.05) from 20 weeks after transplantation, decreasing to 18.2 ± 24.7% and 11.1 ± 20.8% at 20 and 22 weeks, respectively.This model showed a latency of recovery, with deterioration following transplantation initially observed. This is likely due to the inflammatory response resulting from the stereotactic injection and the time required for NPCs to mature, integrate with, and innervate the host tissue. The functional improvement of Parkinson's motor symptoms in EC23-CLiPS NPC-transplanted animals suggests functional recovery of dopaminergic function in the transplanted striatum. To investigate this further, we performed PET imaging with the dopamine transporter (DAT) ligand [18F]FE-PE2I in transplanted mice (Bang, JI, et al., Nucl Med Biol, 2016. 43(2): p. 158-64; Sasaki, T., et al.,. J Nucl Med, 2012. 53(7): p. 1065-73). DAT is a presynaptic transmembrane protein primarily responsible for the reuptake of dopamine released at synapses, and molecular imaging of DAT is a well-established tool for studying dopaminergic function. PET imaging 6 months after transplantation showed that DAT activity in the transplanted injured hemisphere was restored to approximately 71.4 ± 10.3% (n=3) of that in the non-injured hemisphere of EC23-iPS NPC-transplanted mice (Figure 8i). In contrast, in asF-iPS-NPC-transplanted mice, recovery was only 16.4 ± 4.0%. These results clearly demonstrate a significant recovery of dopamine reuptake function in EC23-iPS NPC-transplanted mice.

[0177] Example 13: Transplantation of CLiPS-derived dopaminergic neurons in a fully immunocompetent rodent rat model of Parkinson's disease Our transplantation results show that EC23-CLiPS-derived NPCs are tolerated when transplanted into the striatum of C56BL / 6 mice. To exclude the possibility of species-specific bias of this phenomenon, we replicated the transplantation study in another species, Wistar rats. Parkinson's disease was induced in these rats by injecting 6-OHDA into the MFB to damage the nigrostriatal pathway. In this context, it should be noted that all animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the National Neuroscience Institute (NNI), Singapore. Additional approval for rat experiments was provided by the IACUC of the National University of Technology (NTU), Singapore. MFB lesions are known to cause a more complete depletion of the dopamine system compared to striatal lesions, and therefore are assumed to be less likely to lead to spontaneous recovery (Torres, EM and SB Dunnett, Animal Models of Movement Disorders: Volume I, EL Lane and SB Dunnett, Editors. 2012, Humana Press: Totowa, NJ. p. 267-279). Rats were fully immunocompetent and no immunosuppression was administered before or after transplantation. For analysis, approximately 8-week-old female Wistar rats were purchased from InVivos Pte Ltd. Unilateral lesions were induced by stereotactically injecting 20 μg of 6-OHDA in 4 μl into the left medial forebrain bundle (MFB) at the following coordinates: AP -4.4 mm from dura; ML -1.2 mm; and DV -8.6 mm. To determine the model suitable for transplantation, apomorphine-induced rotations were scored as described in Example 12. Rats showing more than 6 rotations per minute were injected with 3 μl of approximately 1.25 × 10 5Cells / μl of day 25 dopaminergic progenitor cells were transplanted: AP +0.8 mm; ML -2.5 mm; and DV -5 mm from the dura. To assess whether transplanted NPCs could integrate and mediate functional benefits in lesioned animals, rotational asymmetry studies were performed at monthly intervals as described in Example 12. Rats were sacrificed at 6 months by terminal anesthesia and brains were harvested for immunohistological analysis after transcardial perfusion with 4% PFA as described in Example 12. Several animals that did not meet the lesioning criteria were similarly transplanted and sacrificed at 1 and 3 months post-transplantation to assess cell survival and engraftment.

[0178] The results reveal that unilateral depletion of dopaminergic neurons in the substantia nigra as a result of retrograde transport of 6-OHDA via the MFB was confirmed in the model by DAB staining of TH in midbrain sections (Fig. 11d). Animals showing at least 5 rotations / min upon apomorphine exposure were transplanted with asF-iPS-, EC23-CLiPS- and MC23-CLiPS-derived NPCs. Histological analysis 3 months after transplantation showed the presence of hCyto+ / HuNu+ and hNCAM+ / TH+ cells only in the EC23-CLiPS group. In addition, TH+ neurons in the grafts showed expression of synapsin 1, suggesting integration with host neurons (Fig. 11b). Furthermore, only animals transplanted with EC23-CLiPS NPCs showed improvement in rotational behavior in both species, in contrast to asF-iPS NPC or sham-transplanted animals (Fig. 11e). The rat model also showed a latency of recovery, with a deterioration initially observed after transplantation. This is likely due to the inflammatory response caused by the stereotactic injection and the time required for NPCs to mature, integrate with, and innervate the host tissue.The results also show significant restoration of dopamine reuptake function in CLiPS-derived NPC-transplanted rats.

[0179] Example 14: Methods for Differentiating and Characterizing RPE Cells Using a rapid and directed differentiation protocol, RPE differentiation from CLiPs was achieved. Differentiated RPE cells were purified and plated in transwells for further characterization, including immunostaining and gene expression assays using quantitative reverse transcriptase polymerase chain reaction (q-RT-PCR). Transepithelial electrical resistance (TEER) and phagocytosis of photoreceptor outer segments (POS) were used to analyze cell functionality.

[0180] Umbilical cord surface induced pluripotent stem cells (CLiPs) culture CLiPs were cultured in mTESR1 (Stem Cell Technologies) medium on tissue culture plates (Corning Costar) coated with Matrigel (Corning).

[0181] Differentiation into retinal pigment epithelium We used the directed differentiation protocol by Foltz and Clegg (2017) with different modifications. CLiPs and human ES cells were grown in mTeSR1 medium on tissue culture plates coated with Matrigel. Once the cells reached 90-95% confluence, they were exposed to different differentiation media containing basal medium (DMEM / F12 with 1x B27 and 1x N2 supplements and non-essential amino acids) supplemented with different growth factors.

[0182] Differentiation medium 1: from day 0 to day 2, 1 μM LDN-193189, 10 ng / ml Dkk1, 10 ng / ml IGF1 and 10 mM nicotinamide. Differentiation medium 2: from day 2 to day 4, 0.2 μM LDN-193189, 10 ng / ml Dkk1, 10 ng / ml IGF1, 10 mM nicotinamide and 5 ng / ml b-FGF. Differentiation medium 3: from day 4 to day 6, 10 ng / ml Dkk1 and 10 ng / ml IGF1 and 100 ng / ml activin A. Differentiation medium 4: from day 6 to day 8, 100 ng / ml activin A and 10 μM SU5402. Differentiation medium 5A: From day 8 to day 11, basal medium contained 100 ng / mL activin A, 10 μM SU5402 and 1.5 μ m CHIR99021. Differentiation medium 5B: from days 11 to 16, 100 ng / mL activin A, 10 µM SU5402 and 3 µM CHIR99021. On day 16, the basal medium was replaced with the following RPE maintenance medium: 50% DMEM / F12, 50% Minimum Essential Medium Eagle, Alpha Modification, 10 mM nicotinamide, penicillin / streptomycin, sodium pyruvate, MEM non-essential amino acids, GlutaMAX (all 1:100), N1 supplement (1:200), 0.25 mg / ml taurine, 0.02 µg / ml hydrocortisone, and 0.013 ng / ml 3,3',5-triiodo-L-thyronine supplemented with 2% heat-inactivated fetal bovine serum (FBS). The medium was changed every 2-3 days. In a modified protocol, Su5402 in differentiation media 4, 5A and 5B was replaced with 1 µM PD173074.

[0183] Characterization of CLiPs-derived RPE · Transcription Assay – Quantitative Reverse Transcriptase Polymerase Chain Reaction Immunocytochemistry to assess RPE-specific proteins Transepithelial resistance (TEER) Photoreceptor outer segment (POS) phagocytosis assay

[0184] Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) RNA samples were collected on days 0, 2, 4, 6, 8, 12, 16, and 30 (D30). Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR was performed in technical triplicates (10 μl reactions) in 96-well plates using KAPA SYBR FAST on a Quant Studio 3 Real-Time PCR System (Thermo Fischer). Gene-specific primers designed to generate PCR products of 75-200 base pairs included pluripotency markers OCT4, NANOG, and SOX2, early eye field markers OTX2, LHX2, RAX, and SIX3, early RPE markers PAX6, MITF, VSX2, and SOX10, and mature RPE markers BEST-1, PMEL17, MERTK, tyrosinase, TRYP2, and RPE65. Data were normalized to the "housekeeping" gene glyceraldehyde phosphate dehydrogenase (GAPDH).

[0185] immunocytochemistry Six weeks after seeding in transwells, cells were washed with PBS and fixed with 4% paraformaldehyde (pH 7.4) for 20 min at room temperature (RT). Fixed cells were then washed with PBS, permeabilized with 0.2% Triton X-100 for 3–5 min at RT, and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at RT. Cells were then probed with primary antibodies in 1% BSA overnight at 4 °C. After three washes to remove the primary antibodies, cells were incubated for 45 min at RT with the appropriate Alexa Fluor-conjugated secondary antibodies (1:1000; Life Technologies), DAPI to stain nuclei, and Alexa-conjugated phalloidin to stain actin. Cells were washed with PBS and mounted with Fluorsave (Calbiochem). Cells were imaged using a 40x or 63x oil immersion objective on an LSM 700 confocal microscope (Carl Zeiss, Jena, Germany).

[0186] The following antibodies were used: rabbit zona-occludens 1 (ZO-1, 1:200, Invitrogen), rabbit occludin (1:125, Invitrogen), mouse retinal pigment epithelium-specific 65 KDa protein (RPE65, 1:125, Abcam), mouse cellular retinaldehyde-binding protein (CRALBP, 1:1000, Abcam), mouse bestrophin (BEST-1, 1:125, Abcam), mouse Na+ / ATPase (1:250, Invitrogen), mouse Ezrin (1:200, Abcam) and mouse claudin-19 (1:125).

[0187] Transepithelial resistance (TEER) TEER was measured to determine the formation of tight junctions between RPE cells and the development of epithelial barrier properties, which reflect the integrity and polarity of the RPE monolayer. For this, cells were cultured on permeable 0.4 μm 24-well Transwell inserts (Corning) coated with Synthemax-II (Corning). TEER measurements were performed weekly using an Epithelial Volt Ohm meter - EVOM2 (World Precision Instruments) according to the manufacturer's instructions. Briefly, electrodes were sterilized with 70% ethanol, air-dried, equilibrated in RPE medium, and placed in the Transwell filter with the longer electrode in the lower chamber against the bottom of the dish and the shorter electrode in the upper chamber. Net TEER (Ω.cm2) was calculated by subtracting the resistance of the experimental Transwell from the resistance of the control Transwell without cells plated on it, and multiplying the net value by the area of ​​the filter membrane.

[0188] Photoreceptor outer segment (POS) phagocytosis assay POS were isolated from porcine eyeballs collected from a local slaughterhouse. Pig eyeballs were cut in half with a blade and the retina was removed using forceps under red light in a dark room. The retina was placed in homogenization medium, mixed thoroughly, and filtered. The retinal suspension was layered on top of a sucrose gradient (25–60%) and centrifuged at 112,398 × g for 1 h in an ultracentrifuge (Optima Ultracentrifuge, Beckman). The pink POS layer was collected and POS was labeled with fluorescein isothiocyanate (FITC, Invitrogen) in 0.1 M sodium bicarbonate buffer (pH 9.5) for 1 h at RT. The labeled POS was washed and stored in aliquots at -80 °C until use. For the phagocytosis assay, RPE cells growing on transwells were exposed to FITC-labeled POS for 2 h at 37 °C in a 5% CO2 incubator or at 4 °C in the case of controls. Cells exposed to unlabeled POS were used as controls. Wells were then washed three times with PBS to remove unbound POS and dissociated into single cells using TrypLE (Gibco). POS phagocytosis was determined by measuring FITC fluorescence using a BD LSR II flow cytometer.

[0189] Flow cytometry Four- to six-week-old cells grown on 24-well plates were stained with Pmel17 antibody (Dako, M0634) using IntraPrep Permeabilization Reagent (Beckman Coulter, IM2389). Briefly, cells were dissociated with TrypLE and fixed and permeabilized according to the instructions provided in the kit. Approximately 50,000 cells were transferred to a 15 ml centrifuge tube and incubated overnight with 2–4 μl of Pmel or RPE65 antibody on a roller shaker in a cold room. After washing with PBS, cells were incubated with Alexa-labeled 488 / 647 secondary antibody and analyzed on a FACS LSR5 II instrument (BD).

[0190] RPE purification method (i). Manual purification: Non-RPE cells in differentiation cultures were manually removed by scraping with a 10 μl tip attached to a pipette while observing the culture plate under a dissecting microscope. The plate was washed three times with PBS to remove all non-RPE cells. The remaining cells, highly enriched for RPE, were dissociated by adding fresh TrypLE and incubating for 5–10 min.

[0191] (ii). TrypLE purification: RPE cells are more strongly attached to tissue culture plates compared to non-RPE cells. Non-RPE cells are weakly attached to the growth surface and can be easily removed by treatment with a gentle dissociation agent such as TrypLE. After aspirating the medium, loosely attached non-RPE clumps were removed by adding phosphate-buffered saline to the wells and vigorously pipetting with a 1 ml pipette. After removal of the detached clumps, the plates were incubated with TrypLE for 5–10 min and the non-RPE cells were detached by gentle tapping and pipetting to release the non-RPE cells. The detached non-RPE cells were collected, dissociated, and used as the “loose” fraction. The plates were washed thoroughly 3 times with PBS to remove all non-RPE cells, and firmly attached RPE cells were dissociated by incubation with fresh TrypLE for 5–10 min. Both firmly attached and loosely attached non-RPE cells were plated for the experiments.

[0192] (iii). TrypLE + manual purification: The TrypLE purification above removed most of the large non-RPE aggregates, but some small aggregates were still present, which were removed by manual purification as described in Method 1.

[0193] (iv) TrypLE + scatter sorting: After aspirating the medium, loosely attached RPE cells were removed as described in method 3. The remaining cells were dissociated by TrypLE treatment for an additional 5-10 min and processed as described in scatter sorting. During sorting, the weakly attached non-RPE cells removed by TrypLE treatment were used to set the gate for low scatter cells. The high scatter population present only in the tightly attached fraction served to set the gate for sorting RPE cells.

[0194] (v) Scatter sorting: All cells on the differentiation plate were dissociated into single cells by TrypLE treatment and pelleted. The cell pellet was resuspended in FACS buffer, passed through a 70 μm filter to obtain single cells, and separated into high and low scatter fractions using a BD FACS Aria II cell sorter.

[0195] Analysis of mitochondrial and glycolytic functions Analysis of mitochondrial and glycolytic function was performed on CLiPs-RPE, skin-iPSC-RPE and H9-RPE cells using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience) with assay conditions described by the manufacturer. Cells were plated at 6 × 10 in Synthemax-II coated 96-well plates. 4Cells were plated at a seeding density of 100 μM and grown for 48 h. Oxygen consumption rate (OCR) was detected using the Cell Mito Stress Test under basal conditions followed by the sequential addition of oligomycin (2 μM), FCCP (1.5 μM), and rotenone (0.5 μM) and antimycin A (0.5 μM), allowing the measurement of the following parameters: basal respiration, OCR, ATP production, maximal respiration, spare respiratory capacity and non-mitochondrial respiration. Extracellular acidification rate (ECAR) was measured using the glycolysis stress test under basal conditions followed by the sequential addition of 10 mM glucose, 5 μM oligomycin and 100 mM 2-DG, allowing the measurement of glycolytic capacity and glycolytic reserve parameters. Assay results were normalized to cell number.

[0196] Image analysis to assess pigmentation Black and white 16-bit images of the differentiation plates were taken at regular intervals from day 17 using a Chemidoc touch system (Biorad). Based on background intensity threshold, the black area of ​​RPE cells and their luminescence were measured using Matlab software. The pigmentation brightness obtained from the analysis was subtracted from the maximum possible luminescence intensity (65535) of the 16-bit image to obtain the pigmentation darkness.

[0197] Example 15: CLiPS differentiate into RPE cells We used RPE differentiation methods to generate RPE from CLiPS that can be of either mesenchymal (CLMC) or ectodermal (CLEC) origin (Table 4).

[0198] Table 4. Different stem cells used in this study TIFF2025507265000007.tif51128

[0199] We used ES cells and skin iPS cells as controls in RPE differentiation (Table 4). Using this method, CLiPS were reliably differentiated into RPE (Figure 13).

[0200] Example 16: CLiPS have consistently higher RPE differentiation efficiency than skin iPS cells To compare the RPE differentiation efficiency of different types of stem cells (Table 4), we developed a visual grading system (Figure 14a) by estimating the percentage of the area occupied by pigmented cells in each well of the differentiation plate. RPE differentiation efficiency is graded as 0, 1, 2 or 3 for no pigmentation, <30%, 30-60% or >60% pigmentation, respectively. A stacked column chart was plotted (Figure 14b), showing different grades of pigmentation in different colors for each differentiation plate. In biological replicates of differentiation, iPSCs derived from ES cells and umbilical cord superficial mesenchymal cells (CLMCs) consistently showed higher RPE differentiation efficiency compared to skin iPSCs. The high RPE differentiation of CLiPS was further confirmed by estimating the percentage of cells expressing the RPE-specific protein Pmel17 by flow cytometry (Figure 14c).

[0201] Example 17: CLIP-derived RPE is more pigmented than ES-derived RPE To compare the pigmentation intensity and pigmentation acquisition kinetics of RPE generated from different cell lines, images of the differentiation plates were taken at regular intervals from day 17 of differentiation using a ChemiDoc Touch gel imaging system (Bio-Rad laboratories). CLiPS had darker pigmentation than ES cells in the differentiation plate images (Figure 15a) and phase contrast images taken at day 30 (Figure 15b). Analysis of pigmentation intensity at different days of differentiation showed that CLiPS-RPE had higher pigmentation than ES-derived RPE throughout differentiation (Figure 15c). Consistent with this, CLMC23 differentiation cultures showed higher expression of pigmentation-related genes MITF, PMEL17, TYROSINASE, and TRYP2 (Figure 15d).

[0202] Example 18: CLiPS-derived RPE expresses RPE-specific genes We confirmed the expression of RPE-specific genes in CLiPS-derived RPE by quantitative PCR on differentiation days 18 and 35. Robust expression of the RPE-specific genes RPE65 and MERTK ( FIG. 16 ) was observed, with levels higher in CLiPS-derived RPE on day 35, suggesting that they may be more mature.

[0203] Example 19: CLiPS-derived RPE have functional tight junctions and are capable of phagocytosis The functionality of CLiPS-RPE was determined by measuring transepithelial electrical resistance (TEER) and phagocytosis of FITC-labeled photoreceptor outer segments. TEER (FIG. 17a) and phagocytosis (FIG. 17b) of CLiPS-RPE were similar to those of ES-derived RPE.

[0204] Example 20: CLiPS-RPE expresses similar proteins as ES-derived RPE Polarization of the RPE is crucial for apical-basal specific functions, and proteins show localized expression in polarized RPE monolayers. To examine whether CLiPS show similar protein expression to previously reported RPE, we immunostained RPE for various proteins. ZO-1 was expressed at cell-cell junctions, Mertk was expressed apically, and RPE-65 was expressed in the cytoplasm (Figure 18), similar to native, ES-, and iPS-derived RPE.

[0205] Example 21: Modification of RPE Differentiation Protocol We developed a slightly modified RPE differentiation protocol based on the method developed by Foltz and Clegg 2017 (J Vis Exp. 2017; (128): 56274). (A) The use of CHIR at 3 μM as described in the original Clegg protocol (Figure 19a) led to excessive cell death around days 11-12 of differentiation, resulting in a dramatic reduction in RPE yield. To prevent this, we modified the protocol by gradually increasing CHIR in the medium in the following manner: starting with 1.5 μM for 3 days (days 8-11 of differentiation), followed by 3 μM for 5 days (days 12-17). (B) A new FGF inhibitor for RPE differentiation was also identified: We replaced SU5402 with PD173074 (Figure 19b) and achieved a similar degree of RPE differentiation and pigmentation (Figure 19c). PD173074 was used at a much lower concentration than SU5402 (1 μM instead of 10 μM), which reduced the undesirable changes in gene expression induced by higher concentrations of the chemical (Waldmann T et al., 2014, Chem Res Toxicol, . 2014 Mar 17;27(3):408-20). RPE differentiated with the different FGF inhibitors, SU5402 or PD173074, had comparable TEER and phagocytic activity (Figure 19d, e).

[0206] Example 22: Development of RPE purification method RPE purification was performed on 30-35 day old differentiated cultures, by which time RPE achieves pigmentation. Methods for purifying RPE from mixed differentiated cultures include manual removal of non-RPE cells on day 14 of differentiation based on morphological differences between RPE and non-RPE cells (Foltz and Clegg, 2017), selective removal of non-RPE cells that are weakly attached to the culture dish by brief treatment with weak dissociation agents such as TrypLE or Accutase (Nazari et al., 2015, Yuko Iwasaki et al., 2016) and scatter sorting based on the high scattering of light by melanosomes in RPE cells (Shih et al., 2017). We performed RPE purification using 30-35 day old differentiated cultures, unlike the 14 day old cultures previously described (Foltz and Clegg, 2017). By days 30-35, RPE cells in differentiation cultures acquire brown pigmentation, which helps distinguish RPE from non-RPE cells during manual purification. We compared different methods (Figure 20a and b) to identify which method yields functional RPE with the highest purity and yield. These included: (i) manual purification: identification of non-RPE cells based on morphology and lack of pigmentation by observation under a dissecting microscope and their manual removal by scraping; (ii) TrypLE purification: removal of the majority of weakly adherent non-RPE clusters by partial TrypLE treatment; (iii) TrypLE + manual: removal of the majority of weakly adherent non-RPE clusters by partial TrypLE treatment followed by manual removal of the few non-RPE clusters that survived TrypLE treatment by observation under a dissecting microscope; (iv) TrypLE + scatter sorting: removal of weakly adherent non-RPE clusters by partial TrypLE treatment followed by scatter sorting; (v) scatter sorting: separation of all cells from mixed differentiation cultures into high (pigmented RPE cells) and low (non-pigmented non-RPE cells) populations based on their relative light scatter (Figure 20c). To make gate selection more precise, we introduced a low-scattering control using weakly adherent non-RPE cells (harvested from partial TrypLE treatment) in differentiated cultures.RPE cells were sorted and selected based on the high scatter gate (Figure 20d). This two-step purification, which first involved removal of non-RPE cells by TrypLE before flow cytometry, helped to reduce sorting time. The yield of RPE cells was calculated by the number of RPE cells obtained after purification / total number of cells in the mixed population of RPE cells before purification × 100%.

[0207] Since preferential dissociation is expected to remove non-RPE cells, all cells obtained after purification were considered as RPE cells. The yield of RPE after purification was calculated from the total number of cells present in the differentiation culture and the number of cells obtained after purification. For purification, we used a differentiation culture from HDFA iPS cells that contained 51% Pmel17 positive cells by flow cytometry, suggesting that it contained 51% RPE cells. Hence, the maximum RPE yield achievable with this culture was 51%. TrypLE purification yielded 50% RPE cells, very close to the expected maximum yield of 51%. Manual and TrypLE + manual gave slightly lower yields, 47.7% and 43.4%, respectively. Methods with scatter sorting gave lower yields (21–22%) (Figure 20e). All purification methods achieved a purity of >95% by Pmel17 flow cytometry (Figure 20f). By 10 weeks, TEER values ​​of RPE purified by the different methods were comparable (Figure 20g). In phagocytosis assays, all purification methods yielded RPE cells with phagocytosis rates above 90% (Figure 20h).

[0208] In conclusion, RPE cells purified by any method had comparable purity, TEER and POS phagocytosis. In terms of yield, manual, TrypLE and TrypLE+scatter resulted in the highest RPE yield. TrypLE and TrypLE+manual were the easiest to perform, as partial TrypLE treatment removed most of the non-RPE cells (Figure 20i). TrypLE purification would be an efficient method to generate RPE cells for general research purposes due to its ease of purification, high yield, purity and functionality. The TrypLE+manual method, with an additional manual purification step to remove any non-RPE cells that may have escaped the TrypLE treatment, would be ideal to generate RPE cells for transplantation. After purification, we compared the expression of BEST1, RPE65, MERTK, MITF, PMEL17, RLBP1 and TRYP2 in purified CLMC23 and H9 (Figure 20j). The majority of these genes showed increased expression in CLMC23 compared to ES-derived H9 RPE (FIG. 20k).

[0209] Example 23: CLiPS-derived RPE have high glycolysis and mitochondrial respiration Measurement of glycolysis and mitochondrial respiration are indicators of bioenergetics and cell health. Bioenergetics of RPE cells derived from different stem cells was measured using the Cell Mito Stress Test assay conditions with an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience). Glycolysis was quantified by extracellular acidification rate (ECAR) and oxidative phosphorylation (oxPhos) by oxygen consumption rate (OCR). Figure 21a and b demonstrate that among differentiated RPE, CLiPs-RPE cells have the closest bioenergetic profile to primary RPE (AHRPE), making them more physiologically close to native RPE. Furthermore, CLiPs-RPE also show higher glycolysis and oxidative phosphorylation compared to both skin-iPSC-RPE (ASF5-RPE) and hESC-RPE (H9-RPE) (Figure 21a and b). These results suggest that CLiPs-RPE has a higher bioenergetic profile compared to hESC-derived RPE. Healthy RPE exhibits higher glycolysis and mitochondrial function compared to AMD patient-derived RPE (Ferrington et al., 2017). The higher OCR and ECAR of CLiPS-RPE suggest that they may be superior to hESC-derived RPE for clinical use. This is also evident from the increased resistance to oxidative stress exhibited by CLiPs-RPE upon exposure to oxidized low-density lipoprotein (Figure 21c) and hydrogen peroxide (Figure 21g) compared to skin-iPSC-RPE (Figure 21d and h) and hESC-RPE (Figure 21e and i), which are susceptible to oxidative stress. This indicates that CLiPs-RPE may survive better after transplantation. The response of CLiPs-RPE cells to oxidative stress is similar to that seen in native RPE (AHRPE) (Figure 21f and j), making them functionally closer to primary RPE compared to other differentiated RPE.

[0210] Example 24: CLiPS RPE has potential immune privilege properties in a humanized mouse model Bioluminescent RPE lines were established using a GFP-tagged luciferase (Luc) gene-encoding vector delivered by lentiviral infection. Stable expression of Luc in these lines was confirmed by analyzing bioluminescence intensity. To test the immunogenicity of the different RPE lines, we adapted the Matrigel plug assay from a previous publication (PMID: 15780993). RPE-Matrigel plugs were subcutaneously implanted in humanized mice. The bioluminescence of the RPE-Matrigel plugs was monitored at regular intervals over a period of two months using a bioluminescence imaging system. The total luminescence (bioluminescence) of all RPE lines in humanized mice showed a slight, but insignificant, decrease in signal over time (Figure 22a). This indicated that RPE cells did not obviously proliferate but remained in the expected quiescent state, typical of mature RPE. To distinguish whether the slight decrease observed over time was due to clearance by the immune system, RPE-Matrigel plugs were implanted into NOD-SCID IL2Rγ- / - (NSG) immunodeficient mice. A similar decrease in total luminescence was observed, ruling out clearance of RPE cells by the immune system (Figure 22b). Otherwise, this decrease could be due to a lack of nutrient supply to the area, which gradually led to cell death over the experimental period. To confirm the survival of RPE cells within the Matrigel-RPE plugs and determine their status, the grafts were extracted at the end point and subjected to immunofluorescence analysis using a mature RPE marker (RPE65) and a proliferation marker (Ki67) (Figure 22c). In all RPE lines tested, expression of RPE65, but not Ki67, was observed, confirming a mature and quiescent state.

[0211] Example 25: Monitoring pro-inflammatory cytokines as a surrogate for cellular immune responses After confirming the survival of mature RPE cell grafts in all groups, serum samples collected at the end point of humanized mice were tested for the presence of the main pro-inflammatory cytokines (IFN-γ and IL-18), which are central effectors of cell-mediated immunity (PMID: 29856726). All RPE lines tested showed cytokine levels in the picogram range, which was below the threshold for inducing systemic immune system activation (Figure 23a and b). Unexpectedly, for both cytokines, CLEC23-RPE consistently showed the lowest levels compared to the other lines. Next, to detect localized immune responses, we investigated the immune response at the local level, the RPE-Matrigel plug. Immunofluorescence analysis was performed to observe immune cell infiltration using the human CD45 (hCD45) marker. RPE cells were differentiated using OTX2, an RPE-specific transcription factor (Figure 23c). Consistent with the low levels of IFN-γ and IL-18 cytokines, immune cell infiltration was indeed absent in CLEC23-RPE. Qualitative grading (grades 0–3) based on hCD45-positive cells was performed to determine the severity of immune infiltration from all groups (at least n=3) and plotted (Figure 23d and e). CLEC23-RPE had the lowest immune cell infiltration. These data suggest that CLEC23-RPE potentially has immune privileged properties.

[0212] Example 26: CLEC23-RPE can regulate T cell activation and confer low immunogenicity Transplant rejection is primarily driven by cell-mediated immune responses. As observed from the previous figures, CLEC23-RPE had the least infiltration of immune cells. Therefore, we hypothesized that CLEC23-RPE may affect the activation of T cells, a component involved in cell-mediated immunity. The known cytokines IL-23 and IL17A involved in T cell activation were analyzed in humanized mouse serum (Fig. 24a and b) (PMID: 26252407). CLEC23-RPE showed the lowest levels of both cytokines compared to the other groups. As these cytokines affect T cell activation, we used flow cytometry to calculate the relative ratio of T cells (CD3) to B cells (CD19) (Fig. 24c). Only the CLEC23-RPE group had a lower number of T cells relative to B cells, indicating that T cell activation may be suppressed. Additional analysis of T cell subsets, helper T (CD4) cells and cytotoxic T (CD8) cells, was performed, showing that CLEC23-RPE had the fewest cytotoxic T cells relative to helper T cells (Fig. 24d). The activation states of these two subsets were then further classified into four groups: naive (resting), central memory (CM; pre-activation), and two activation states, effector memory (EM) and effector memory re-expressed CD45RA (TEMRA). There was no clear difference in the activation state of the CD4 helper T cell subset (Fig. 24e). However, in the CD8 cytotoxic T cell subset, CLEC23-RPE showed a clearly higher naive population (Fig. 24f). Therefore, we infer that the potential immune privileged state of CLEC23-RPE may result from suppression of activation of CD8 cytotoxic T cells.

[0213] It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0214] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0215] The terms "less than" or "more than" or "less than" do not include a specific number. "A" and "an" and "the" and similar references used in the context of describing the present invention (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. The term "and / or", wherever used herein, includes the meanings of "and", "or" and "all or any other combination of the elements connected by the term". The term "including" means "including but not limited to". "Including" and "including but not limited to" are used interchangeably. The term "about" means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, and most preferably plus or minus 1%.

[0216] The invention illustratively described herein may be suitably practiced in the absence of any element or elements, or limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," "containing," and the like, shall be read inclusively and without limitation. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the element of the claim. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Furthermore, the terms and expressions used herein are used as terms of description, not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, although the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention embodied therein disclosed herein may be left to those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of the invention. This includes generic descriptions of the invention using conditional or negative limitations that exclude any subject matter from the genus, whether or not the excluded subject matter is specifically recited herein. In addition, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of that Markush group. Further aspects of the invention will become apparent from the following claims.

[0217] The present invention is further characterized by the following: 1. expressing exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in the amniotic stem cells of the umbilical cord under conditions suitable for reprogramming the stem cells, thereby generating induced pluripotent stem cells. A method for producing induced pluripotent stem cells, comprising: 2. 2. The method of item 1, wherein the umbilical cord amniotic stem cells are umbilical cord amniotic mesenchymal stem cells or umbilical cord amniotic epithelial stem cells. 3. 3. The method of item 1 or 2, wherein the mesenchymal stem cells of the amniotic membrane of the umbilical cord are a mesenchymal stem cell population, and at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105. 4. 4. The method of item 3, wherein at least about 90% or more of the cells of the mesenchymal stem cell population lack expression of the following markers: CD34, CD45 and HLA-DR. 5. 5. The method of item 3 or 4, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the mesenchymal stem cell population express each of CD73, CD90 and CD105, and lack expression of each of CD34, CD45 and HLA-DR. 6. 6. The method of any of items 1 to 5, wherein the exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and the p53-shRNA are provided by one, two or three vectors, preferably a first vector encoding the proteins OCT3 / 4 and 53-shRNA, a second vector encoding the proteins SOX2 and KLF4, and a third vector encoding the proteins L-MYC and LIN28. 7. 7. The method of any of items 1 to 6, wherein the stem cells of the amniotic membrane of the umbilical cord are subjected to transfection to transfer the exogenous nucleic acid to the stem cells. 8. The method of item 7, wherein the stem cells of the amniotic membrane of the umbilical cord are subjected to electroporation to transfer the exogenous nucleic acid to the stem cells. 9. 9. The method of item 8, wherein the mesenchymal stem cells of the amniotic membrane of the umbilical cord are subjected to one pulse of electroporation having a duration of about 15-25 ms and a voltage of about 1550-1650 V, preferably one pulse of electroporation having a duration of about 20 ms and a voltage of about 1600 V. 10. The ratio of the amount of vector (plasmid) DNA of each vector to the number of mesenchymal stem cells in the amniotic membrane of the umbilical cord subjected to electroporation was approximately 1 × 10 6 Approximately 1.5 μg of plasmid DNA to approximately 1 × 10 6 The ratio ranges from about 2.5 μg of DNA to 1×10 CLMCs, for example, about 2.5 μg of plasmid DNA:1×10 6 cells, approximately 2.25 μg of plasmid DNA: 1×10 6 cells, approximately 1.8 μg of plasmid DNA: 1×10 6 cells, approximately 1.7 μg of plasmid DNA: 1×10 6 cells, approximately 1.6 μg of plasmid DNA: 1×10 6 cells, approximately 1.5 μg of plasmid DNA: 1×10 6 cells, or preferably about 1.67:1×10 6 Item 9. The method of item 9, wherein the cells are 11. The method of item 8, wherein the epithelial stem cells of the amniotic membrane of the umbilical cord are subjected to electroporation with two pulses having a duration of about 25-35 ms and a voltage of about 1300-1400 V, preferably electroporation with two pulses having a duration of about 30 ms and a voltage of about 1350 V. 12. The ratio of the amount of vector (plasmid) DNA of each vector to the number of amniotic epithelial stem cells of umbilical cord cells subjected to electroporation was approximately 1 × 10 6Approximately 1.5 μg of DNA per cell to approximately 1 × 10 6 The ratio ranges from about 2.5 μg of DNA per cell, for example, about 1.5 μg of plasmid DNA:1×10 6 cells, approximately 1.6 μg of plasmid DNA: 1×10 6 cells, approximately 1.7 μg of plasmid DNA: 1×10 6 cells, approximately 1.8 μg of plasmid DNA: 1×10 6 cells, approximately 1.9 μg of plasmid DNA: 1×10 6 cells, approximately 2.0 μg of plasmid DNA: 1×10 6 cells, approximately 2.5 μg of plasmid DNA: 1×10 6 cells, preferably about 1.67 μg of plasmid DNA: 1×10 6 The method of item 11, wherein the cells are 13. 13. The method of any of items 7 to 12, wherein the transfected stem cells are cultured in a medium suitable for cell recovery. 14. 14. The method of item 13, wherein the medium suitable for cell recovery is a serum-free medium. 15. 14. The method of item 13, wherein the medium suitable for recovery of transfected umbilical cord amniotic mesenchymal stem cells consists of about 85-95% (v / v) defined medium and 5-15% (v / v) fetal bovine serum. 16. The medium of item 15, wherein the medium suitable for recovery of transfected umbilical cord amniotic mesenchymal stem cells consists of about 90% (v / v) chemically defined medium and about 10% (v / v) fetal bovine serum. 17. The medium of item 14 or 15 containing approximately 85-95% (v / v) CMRL 1066 and approximately 5-15% (v / v) FBS. 18. The method of item 13 or 14, wherein the medium suitable for recovery of transfected umbilical cord amniotic epithelial stem cells comprises Mammary Epithelial Basal Medium MCDB 170, EpiLife Medium, DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), and FBS (Fetal Bovine Serum). 19. 19. The method of item 18, wherein the medium suitable for recovery of transfected umbilical cord amniotic epithelial stem cells comprises Mammary Epithelial Basal Medium MCDB 170 at a final concentration of about 10 to about 30% (v / v), EpiLife Medium at a final concentration of about 20 to about 40% (v / v), F12 at a final concentration of about 5 to about 15% (v / v), DMEM at a final concentration of about 30 to about 45% (v / v), and FBS at a final concentration of about 0.1 to 2% (v / v). 20. 20. The method of item 19, wherein the medium suitable for recovery of transfected umbilical cord amniotic epithelial stem cells comprises Mammary Epithelial Basal Medium MCDB 170 at a final concentration of about 15 to about 25% (v / v), EpiLife Medium at a final concentration of about 25 to about 35% (v / v), F12 at a final concentration of about 7.5 to about 13% (v / v), DMEM at a final concentration of about 35 to about 40% (v / v), and FBS at a final concentration of about 0.5 to 1.5% (v / v). twenty one. 21. The method of item 20, wherein the medium suitable for recovery of the transfected umbilical cord amniotic epithelial stem cells comprises Mammary Epithelial Basal Medium MCDB 170 at a final concentration of about 20% (v / v), EpiLife Medium at a final concentration of about 30% (v / v), F12 at a final concentration of about 12.5% ​​(v / v), DMEM at a final concentration of about 37.5% (v / v), and FBS at a final concentration of about 1.0% (v / v). twenty two. A medium suitable for the recovery of transfected umbilical cord amniotic epithelial stem cells was prepared by adding 100 ml of medium to obtain a final volume of 1000 ml of culture medium. 200 ml Mammary Epithelial Basal Medium MCDB 170, 300 ml EpiLife medium, 250 ml DMEM, 250 ml DMEM / F12, 1% fetal bovine serum 22. The method according to any one of items 18 to 21, wherein the method is obtained by mixing twenty three. 23. The method of any of items 18 to 22, wherein the medium suitable for recovery of the transfected umbilical cord amniotic epithelial stem cells comprises insulin at a final concentration of about 1 to about 7.5 μg / ml. twenty four. 25. The method of any of items 18 to 24, wherein the medium suitable for recovery of the transfected umbilical cord amniotic epithelial stem cells comprises human epidermal growth factor at a final concentration of about 1 to about 15 ng / ml. twenty five. 26. The method of any of items 18 to 25, wherein the medium suitable for recovery of the transfected umbilical cord amniotic epithelial stem cells further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 26. 26. The method of item 25, wherein the medium suitable for recovery of transfected umbilical cord amniotic epithelial stem cells comprises all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 27. 27. The method of any of items 18 to 26, wherein the medium suitable for recovery of transfected umbilical cord amniotic epithelial stem cells further comprises one or more transforming growth factors (TGFs). 28. 28. The method of item 27, wherein the medium comprises transforming growth factor beta (TGF-β) and / or transforming growth factor alpha. 29. 29. The method of any of items 18 to 28, wherein the medium suitable for recovery of the transfected umbilical cord amniotic epithelial stem cells further comprises cholera toxin derived from Vibrio cholerae. 30. 30. The method of any of items 14 to 29, wherein the medium suitable for cell recovery comprises a compound that suppresses inflammatory responses and enhances cell survival. 31. 31. The method of item 30, wherein the compound is a glucocorticoid. 32. 32. The method of item 31, wherein the glucocorticoid is selected from the group consisting of prednisolone, methylprednisolone, dexamethasone, betamethasone, corticosterone and hydrocortisone. 33. 33. The method of item 31 or 32, wherein the hydrocortisone concentration is about 0.5 μM to about 2 μM. 34. 34. The method according to any of items 13 to 33, wherein the culturing is carried out in a coated cell culture vessel, which cell culture vessel is preferably coated with a serum-derived or serum-free substrate. 35. 35. The method of any of items 14 to 34, wherein the medium suitable for cell recovery is replaced with a mixture of two different cell culture media about 1, 2 or 3 days after transfection, preferably about 2 days after transfection, thereby obtaining induced pluripotent stem cell colonies. 36. 36. The method of item 35, wherein the two different cell culture media are a medium suitable for cell recovery and a second cell culture medium. 37. 37. The method of item 35 or 36, wherein the two different cell culture media are mixed in a ratio of about 1:1 (v / v) prepared by contacting one volume of a medium suitable for cell recovery with one volume of a second cell culture medium. 38. 38. The method of item 36 or 37, wherein the second cell culture medium is a maintenance medium for culturing induced pluripotent stem cells, said medium being preferably selected from the group consisting of mTeSR1, StemMACS™ iPS-Brew XF, TeSR™-E8, mTeSR™ Plus, TeSR™2, mTeSR™1, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium, StemFlex, StemFit Basic02 and PluriSTEM. 39. 39. The method of any of items 35 to 38, wherein the mixture of cell culture media is replaced with the same mixture of cell culture media within about 3, 4 or 5 days from transfection, preferably within about 4 days from transfection. 40. 40. The method of any of items 35-39, wherein the mixture of cell culture media is replaced with the second cell culture medium within about 5, 6 or 7 days after transfection, preferably within about 6 days after transfection. 41. 41. The method of item 40, wherein the se...

Claims

1. A method for differentiating induced pluripotent stem (iPS) cells into retinal pigment epithelial (RPE) cells, comprising culturing iPS cells derived from stem cells of the amniotic membrane of an umbilical cord in a differentiation medium under conditions suitable for differentiation into RPE cells, thereby differentiating the iPS cells into RPE cells.

2. 2. The method of claim 1, wherein the differentiation medium is DMEM (Dulbecco's Modified Eagle Medium) / F12 (Ham's F12 Medium) medium containing N2 supplement, B27 supplement and non-essential amino acids (NEAA).

3. 3. The method of claim 2, wherein the DMEM / F12 medium comprises 1x N2 supplement, 1x B27 supplement, and 1x NEAA.

4. To obtain a final volume of 1000 ml of culture medium, 10 mL of 100x N2 supplement; 20 mL of 50x B27 supplement; 10 mL of 100× NEAA; 960 mL of DMEM / F12 3. The method of claim 2, wherein the differentiation medium is obtained by mixing:

5. The differentiation medium (i) a first differentiation medium further comprising at least one of IGF1, DKK1, nicotinamide, or LDN-193189, preferably comprising IGF1, DKK1, nicotinamide, and LDN-193189; (ii) a second differentiation medium further comprising at least one of IGF1, DKK1, nicotinamide, LDN-193189, or b-FGF, preferably comprising IGF1, DKK1, nicotinamide, LDN-193189, and b-FGF; (iii) a third differentiation medium further comprising at least one of IGF1, DKK1, or activin A, preferably comprising IGF1, DKK1, and activin A; (iv) a fourth differentiation medium further comprising activin A and either SU5402 or PD173074, preferably comprising activin A and PD173074; and / or (v) A fifth differentiation medium further comprising at least one of activin A, CHIR99021, or either SU5402 or PD173074, preferably comprising activin A, CHIR99021 and PD173074.

2. The method of claim 1, comprising:

6. 6. The method of claim 5, wherein IGF1 of (i), (ii) and / or (iii) is used at a final concentration of at least 5 ng / ml, preferably IGF1 of (i), (ii) and / or (iii) is used at a final concentration of about 10 ng / ml.

7. The method of claim 5, wherein the DKK1 of (i), (ii) and / or (iii) is used at a concentration of at least 5 ng / ml, preferably the DKK1 of (i), (ii) and / or (iii) is used at a concentration of about 10 ng / ml.

8. 6. The method of claim 5, wherein (i) and / or (ii) nicotinamide is used at a concentration of at least 5 mM, preferably (i) and / or (ii) nicotinamide is used at a concentration of about 10 mM.

9. 6. The method of claim 5, wherein the LDN-193189 in (i) and / or (ii) is used at a concentration of at least 0.1 μM, preferably the LDN-193189 in (i) is used at a concentration of about 1 μM and / or the LDN-193189 in (ii) is used at a concentration of about 0.2 μM.

10. 6. The method of claim 5, wherein the b-FGF in (ii) is used at a concentration of at least 2.5 ng / ml, preferably the b-FGF in (ii) is used at a concentration of about 5 ng / ml.

11. The method of claim 5, wherein activin A (iii), (iv) and / or (v) is used at a concentration of at least 50 ng / ml, preferably activin A (iii), (iv) and / or v) is used at a concentration of about 100 ng / ml.

12. The method of claim 5, wherein SU5402 in (iv) and / or (v) is used at a concentration of at least 5 μM, preferably SU5402 in (iv) and / or (v) is used at a concentration of about 10 μM.

13. 6. The method of claim 5, wherein PD173074 in (iv) and / or (v) is used at a concentration of at least 0.5 μM, preferably PD173074 in (iv) and / or (v) is used at a concentration of about 1 μM.

14. 6. The method of claim 5, wherein CHIR99021 in (v) is used at a concentration of at least 1 μM and less than about 3 μM to culture the cells, preferably for about 3 days of continuous culture.

15. 15. The method of claim 14, wherein CHIR99021 in (v) is used at a concentration of about 3 μM for subsequent culturing of the cells, preferably for a period of about 5 days of continuous culture.

16. The method of claim 5, wherein the step of culturing the iPS cells comprises culturing the iPS cells in the first differentiation medium for about 2 days.

17. 6. The method of claim 5, wherein the step of culturing the iPS cells comprises culturing the iPS cells in the first differentiation medium for about 2 days, followed by culturing the iPS cells in the second differentiation medium for about 2 days.

18. 6. The method of claim 5, wherein the step of culturing the iPS cells comprises culturing in a first differentiation medium for about 2 days, followed by culturing in the second differentiation medium for about 2 days, and then culturing in the third differentiation medium for about 2 days.

19. 6. The method of claim 5, wherein the step of culturing the iPS cells comprises culturing the iPS cells in the first differentiation medium for about 2 days, followed by culturing the iPS cells in the second differentiation medium for about 2 days, followed by culturing the iPS cells in the third differentiation medium for about 2 days, and followed by culturing the iPS cells in the fourth differentiation medium for about 2 days.

20. 6. The method of claim 5, wherein the step of culturing the iPS cells comprises culturing in the first differentiation medium for about 2 days, followed by culturing in the second differentiation medium for about 2 days, followed by culturing in the third differentiation medium for about 2 days, followed by culturing in the fourth differentiation medium for about 2 days, and followed by culturing in the fifth differentiation medium for about 8 days.

21. 6. The method of claim 5, wherein LDN-193189 is used for at least 2 days of culture.

22. The method of claim 5, wherein DKK1 is used for at least 2 days of culture.

23. 6. The method of claim 5, wherein SU5402 or PD173074 is used for about 10 days of culture.

24. 6. The method of claim 5, wherein CHIR99021 is used for about 8 days of culture.

25. The method of claim 1, wherein the iPS cells are cultured in the differentiation medium for 11 to 21 days, preferably for about 16 days.

26. 2. The method of claim 1, further comprising culturing the iPS cells in mTESR1 medium prior to culturing the iPS cells in the differentiation medium, preferably culturing the iPS cells in mTESR1 medium for 1 to 4 days of culture.

27. 10. The method of claim 1, further comprising culturing the RPE cells in retinal pigment epithelium maintenance (RPEM) medium.

28. 28. The method of claim 27, wherein the RPEM medium comprises about 50% DMEM / F12 and about 50% Minimum Essential Medium (MEM) containing 0.5x N1 supplement and 1x NEAA.

29. 29. The method of claim 28, wherein the RPEM medium further comprises at least one of heat-inactivated fetal bovine serum (FBS), Glutamax, taurine, hydrocortisone, 3,3',5-triiodo-L-thyronine, penicillin / streptomycin, nicotinamide, or sodium pyruvate.

30. 29. The method of claim 28, wherein the RPEM medium further comprises about 2% heat-inactivated fetal bovine serum (FBS), 1× Glutamax, about 0.25 mg / mL taurine, about 0.02 μg / mL hydrocortisone, about 0.013 ng / mL 3,3',5-triiodo-L-thyronine, 1× penicillin / streptomycin, about 10 mM nicotinamide, and 1× sodium pyruvate.

31. 28. The method of claim 27, wherein the RPE cells are cultured in the RPEM medium for 9 to 29 days, preferably for about 19 days.

32. 28. The method of claim 27, wherein the step of culturing the iPS cells in differentiation medium and the step of culturing the RPE cells in RPEM medium comprises 20 to 50 days, preferably 30 to 35 days, and most preferably about 35 days.

33. 28. The method of claim 27, further comprising purifying the RPE cells in the RPEM medium after culturing the RPE cells in the RPEM medium.

34. The refining step is a. manually identifying the RPE cells according to their pigmentation; b. passaging the RPE cells; c. manually identifying the RPE cells according to their pigmentation and passaging the RPE cells; d. passaging the RPE cells and scatter sorting the RPE cells according to their pigmentation; and / or e. Scatter sorting of RPE cells according to their pigmentation 34. The method of claim 33, comprising:

35. 34. The method of claim 34, wherein manually identifying the RPE cells according to their pigmentation in (a) and / or (c) comprises selecting by microscopic examination.

36. 36. The method of claim 35, wherein the microscopy is bright-field microscopy.

37. 34. The method of claim 34, wherein passaging the RPE cells of (b), (c) and / or (d) comprises treating the RPE cells with Accutase or TrypLE, preferably with TrypLE.

38. The method of claim 1, wherein the iPS cells are produced by expressing exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC, and p53-shRNA in the stem cells of the amniotic membrane of the umbilical cord under conditions suitable for reprogramming the stem cells.

39. 39. The method of claim 38, wherein the stem cells of the amniotic membrane of the umbilical cord are subjected to transfection to transfer the exogenous nucleic acid into the stem cells, and the transfected stem cells are cultured in a medium suitable for cell recovery, wherein the medium suitable for cell recovery contains a compound that suppresses inflammatory responses and enhances cell survival.

40. The method of claim 1 , wherein the stem cells of the amniotic membrane of the umbilical cord are mesenchymal stem cells of the amniotic membrane of the umbilical cord or epithelial stem cells of the amniotic membrane of the umbilical cord.

41. The method of claim 40, wherein the mesenchymal stem cells of the amniotic membrane of the umbilical cord are a mesenchymal stem cell population, and at least 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90, and CD105.

42. 42. The method of claim 41, wherein at least 90% or more of the cells of said mesenchymal stem cell population lack expression of the following markers: CD34, CD45 and HLA-DR.

43. The method of claim 41, wherein at least 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the cells of said mesenchymal stem cell population express each of CD73, CD90 and CD105, and lack expression of each of CD34, CD45 and HLA-DR.

44. The method of claim 38, wherein the exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC, and the p53-shRNA are provided by one, two or three vectors, preferably wherein a first vector encodes the proteins OCT3 / 4 and the p53-shRNA, a second vector encodes the proteins SOX2 and KLF4, and a third vector encodes the proteins L-MYC and LIN28.

45. A retinal pigment epithelial (RPE) cell culture obtainable by the method defined in any of claims 1 to 44.

46. A retinal pigment epithelial (RPE) cell culture obtained by the method defined in any one of claims 1 to 44.

47. A retinal pigment epithelium consisting of or comprising a retinal pigment epithelial cell culture obtainable by a method as defined in any of claims 1 to 44.

48. A retinal pigment epithelium consisting of or comprising a retinal pigment epithelial cell culture obtained by a method as defined in any one of claims 1 to 44.

49. 45. A pharmaceutical composition comprising a retinal pigment epithelial (RPE) cell culture obtained by the method defined in any one of claims 1 to 44.

50. 50. The pharmaceutical composition of claim 49, adapted for parenteral or topical application.

51. 46. ​​The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express at least one of BEST1, PMEL17, MITF, TYROSINASE, TRYP2, ZO-1, RPE65, RLBP1 or MERTK.

52. 46. ​​The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express BEST1 with a fold change of at least 2 compared to RPE cells differentiated from embryonic stem cells (ES).

53. 46. ​​The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express PMEL17 with a fold change of at least 0.9 compared to RPE cells differentiated from ES cells.

54. 46. ​​The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express MITF with a fold change of at least 4.5 compared to RPE cells differentiated from ES cells.

55. 46. ​​The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express TRYP2 with a fold change of at least 2.9 compared to RPE cells differentiated from ES cells.

56. The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express RPE65 with a fold change of at least 0.6 compared to RPE cells differentiated from ES cells.

57. The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express RLBP1 with a fold change of at least 17.5 compared to RPE cells differentiated from ES cells.

58. The RPE cell culture of claim 45, wherein the RPE cells contained in the culture express MERTK with a fold change of at least 6 compared to RPE cells differentiated from ES cells.

59. The RPE cell culture of claim 45, wherein the RPE cells contained in the culture have an increased oxygen consumption rate (OCR) and / or extracellular acidification rate (ECAR) compared to RPE cells differentiated from ES cells.

60. 45. A method of treating a retinal degenerative disease in a subject, comprising administering to the subject retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem (iPS) cells by the method defined in any one of claims 1 to 44.

61. 61. The method of claim 60, wherein the retinal degenerative disease is age-related macular degeneration (AMD) or retinal dystrophy.

62. 1. An in vivo method for detecting the viability of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem (iPS) cells by the method defined in any one of claims 1 to 44 in a subject, the method comprising: (a) introducing RPE cells differentiated from iPS cells by the method defined in any one of claims 1 to 44 into a subject, wherein the RPE cells comprise a bioluminescent label; (b) detecting bioluminescent signals of the RPE cells over time using an imaging method, thereby collecting imaging data; (c) comparing the imaging data received in step (b) with reference imaging data.

63. 63. The method of claim 62, wherein the absence of a difference in bioluminescence signal in the imaging data from the subject compared to reference imaging data indicates survival of the RPE cells in the subject.

64. 10. An in vitro method for determining the immunogenicity of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem (iPS) cells by the method defined in any one of claims 1 to 44 in a subject to whom the differentiated RPE cells have previously been delivered, the method comprising: (a) detecting pro-inflammatory cytokine levels in a sample obtained from the subject, the sample comprising the differentiated RPE cells, using an imaging method, thereby collecting imaging data; (b) comparing the imaging data received in step (a) with reference imaging data;

65. 65. The method of claim 64, wherein a decrease in cytokine levels in the imaging data compared to reference imaging data indicates a decrease in immunogenicity of the RPE cells in the subject.

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