Method for differentiating induced pluripotent stem cells into retinal pigment epithelial cells, retinal pigment epithelial cells, and method for using retinal pigment epithelial cells

Abstract

The invention relates to a method for differentiating induced pluripotent stem cells into retinal pigment epithelial cells. In addition, 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 retinal pigment epithelium consisting of or comprising a retinal pigment epithelium cell culture obtainable or obtained by said differentiation method. The invention also relates to a pharmaceutical composition comprising a retinal pigment epithelial cell culture obtained by said differentiation method. The present invention relates to a method of treating a retinal degenerative disease in a subject, the method comprising administering to the subject retinal pigment epithelial cells differentiated from induced pluripotent stem cells by the method. Finally, the invention also relates to an in vivo method for detecting the survival rate of retinal pigment epithelial cells in a subject, said retinal pigment epithelial cells being differentiated from induced pluripotent stem cells by a defined method, and to an in vitro method for determining the immunogenicity of retinal pigment epithelial cells in a subject, the retinal pigment epithelial cells are differentiated from induced pluripotent stem cells by a defined method, the differentiated RPE cells having been pre-delivered to the subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 303,849, filed on January 27, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Background Art

[0003] The present invention relates to a method for producing induced pluripotent stem cells. In addition, 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 cell of the present invention. The present invention also relates to a method for differentiating the induced pluripotent stem cell of the present invention. In addition, it also relates to a pharmaceutical composition comprising differentiated induced pluripotent stem cells obtained by the method. In addition, the present invention 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 the induced pluripotent stem cell into a retinal pigment epithelial cell. In addition, 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. In addition, the present invention relates to a retinal pigment epithelium, which consists of or comprises 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 relates to a method for treating a retinal degenerative disease in a subject, the method 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 survival rate of retinal pigment epithelial cells in a subject, the retinal pigment epithelial cells being differentiated from induced pluripotent stem cells by a defined method, and an in vitro method for determining the immunogenicity of the retinal pigment epithelial cells in the subject, the retinal pigment epithelial cells being differentiated from induced pluripotent stem cells by a defined method, and the differentiated RPE cells having been pre-delivered to the subject. Technical Field

[0004] Stem cells are a cell population with unlimited self-renewal and the ability to differentiate into a variety of cell or tissue types. The ability of stem cell self-renewal is critical for its function as a reservoir of original undifferentiated cells, and the "plasticity" of stem cells depends on its transdifferentiation into tissues different from its source tissue, and perhaps also on its ability to cross embryonic germ layers. In contrast, most somatic cells have limited self-renewal capacity due to telomere shortening (reviewed in, for example, Dice, JF (1993) Physiol. Rev. 73, 149-159). Therefore, stem cell-based therapy has the potential for treating a variety of human and animal diseases.

[0005] Embryonic stem cells (from about 3 to 5 days after fertilization) proliferate indefinitely and can spontaneously differentiate into all tissue types: they are therefore called pluripotent stem cells (reviewed in, for example, Smith, AG (2001) Annu. Rev. Cell. Dev. Biol. 17, 435-462). Although the potential of embryonic stem cells is enormous, their use implies many ethical issues. Therefore, non-embryonic stem cells have been proposed as an alternative source.

[0006] Adult stem cells are more tissue-specific and may have less replication capacity: they are therefore called multipotent stem cells (reviewed in, for example, 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 ability to differentiate into chondrocytes, adipocytes, osteoblasts, myoblasts, cardiomyocytes, astrocytes and tenocytes, etc. However, in many cases, the number of stem cells extracted from bone marrow stroma, adipose tissue, dermis and umbilical cord blood is quite small.

[0007] A wide source of very young and adaptive adult stem cells (also called newborn stem cells) is umbilical cord blood or tissue or placenta. For example, a large number of stem cells can be derived from umbilical cord tissue, i.e., from the umbilical cord matrix-Wharton's jelly (Mitchell, KE et al., (2003) Stem Cells 21, 50-60; US Patent 5,919,702; US Patent Application 2004 / 0136967). It has been shown that these cells have the ability to differentiate into, for example, neuronal phenotypes and cartilage tissue, respectively. Mesenchymal stem cells can also be isolated from the subendothelial layer of the umbilical cord vein, which is one of the three blood vessels (two arteries and one vein) found in the umbilical cord (Romanov, YA et al., (2003) Stem Cells 21, 105-110; Covas, DT et al., (2003) Braz. J. Med. Biol. Res. 36, 1179-1183). In addition, mesenchymal stem cells and epithelial stem cells have been successfully isolated from umbilical cord amniotic tissue (US2006 / 0078993). Although, for example, mesenchymal stem cells can undergo differentiation in vitro and in vivo, making these stem cells a promising candidate for mesoderm defect repair and disease management, the use of adult stem cells is limited by their pluripotency. In order to overcome this limitation, non-embryonic cells can be reprogrammed into pluripotent stem cells: so-called induced pluripotent stem cells (iPS).

[0008] IPS were first produced by Takahashi and Yamanaka, who reprogrammed non-embryonic cells to a pluripotent state by overexpressing 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 the Yamanaka factors by retroviral transduction, thereby allowing overexpression of the transcription factors and thus producing cells that exhibited the morphology and growth characteristics of embryonic cells. Although this method is a major breakthrough, the transduction process may result in the integration of the transferred DNA into the genome of the host cell, making iPS critical for therapeutic treatment of humans. In 2011, Okita, K. et al., Nature methods, 8 (5), pp. 409–412 have established a non-integrative alternative for generating iPS. Okita et al. used electroporation to transfer three additional plasmid vectors encoding Yamanaka factors and p53-shRNA for p53 inhibition into human skin fibroblasts and dental pulp, thus allowing overexpression of exogenous DNA, thereby producing human iPS without integration. In order to support the growth and maintenance of human iPS without integration, Okita et al. (supra) cultured iPS on a feeder layer composed of mouse embryonic fibroblasts (MEF) or STO cell lines, which had been transformed with neomycin resistance and mouse LIF gene (SNL). However, the culture on the feeder layer may bring the risk of exogenous DNA contamination of iPS. Therefore, the iPS without insertion according to Okita et al. (supra) is also critical for therapeutic treatment of humans.

[0009] Ten years after its conception, iPS technology entered the clinical translation phase, with the first human trials 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 is its potential to enable autologous cell therapy, which can avoid the need for long-term immunosuppression or tissue compatibility matching to prevent rejection of transplanted cells. This paradigm has been demonstrated in non-human primate models using fibroblasts and bone marrow derived iPS (Morizane, A. et al., Stem Cell Reports, 2013. 1(4): p. 283-92; Hallett, PJ et al., Cell Stem Cell, 2015. 16(3): p. 269-74; Wang, S., et al., Cell Discov, 2015. 1: p. 15012; Shiba, Y. et al., Nature, 2016. 538(7625): p. 388-391), and is the basis for the first human trial of iPS-based cell therapy for AMD (Mandai, M. et al., N Engl J Med, 2017. 376(11): p. 1038-1046). However, the significant time and cost associated with clinical-grade iPS production will make it impossible to implement it on a large scale for human treatment. In addition, there are situations where it may not be practical to generate autologous iPS from patients. For example, for patients carrying disease-causing mutations, these mutations must first be corrected before iPS cells from these patients can be used. This is achievable when the mutations are tractable, but in cases where the mutations are refractory, such as underlie many sporadic forms of the disease, gene correction strategies may not be feasible.

[0010] Therefore, there remains a need for alternative methods of generating iPS cells, wherein the resulting iPS cells are capable of differentiating into target cells suitable for therapeutic treatment in humans, such as retinal pigment epithelial (RPE) cells.

[0011] The stem cells currently used for RPE production, i.e. induced pluripotent stem cells (iPS) and embryonic stem cells (ES), have some disadvantages. It is well known that individual genomes accumulate mutations during the entire life span, which is the basis of age-related diseases such as cancer (Stratton MR et al., 2009, Nature 458, 719–7249 2001). Compared with those cells derived from elderly individuals, iPS cells derived from young individuals carry fewer mutations. Comparing the DNA sequences of subjects aged 21-100 years old, the genetic and epigenetic mutations found in iPS cells increase with the increase of donor age (Lo Sardo et al., 2017, Nat Biotechnol.35 (1): 69-74). Mitochondrial DNA abnormalities related to age also increase, and the iPS cells of fibroblast-derived iPS cells of elderly subjects have significantly higher mutations than young 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 requiring the use of immunosuppression. Skin cells are widely used to generate iPS cells due to easy tissue collection, however, they have a higher mutation rate due to long-term exposure to UV from sunlight (Apalla Z. et al., 2017, Dermatol Pract Concept. 2017Apr; 7(2): 1–6).

[0012] Therefore, it is an object of the present invention to provide a method for generating iPS cells and differentiating said specific iPS cells into RPE cells that meet these needs. Summary of the invention

[0013] The present invention relates to methods of producing induced pluripotent stem (iPS) cells as described herein, the resulting induced pluripotent stem cells, methods of differentiating the resulting induced pluripotent stem cells, and methods of treating a disorder in a subject with differentiated cells derived from induced pluripotent stem cells.

[0014] Therefore, the present invention provides a method for producing induced pluripotent stem cells, wherein the method comprises expressing exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in umbilical cord amniotic membrane stem cells under conditions suitable for reprogramming stem cells, thereby producing induced pluripotent stem cells. In an embodiment of the method, the umbilical cord amniotic membrane stem cells are umbilical cord amniotic membrane mesenchymal stem cells or umbilical cord amniotic membrane epithelial stem cells.

[0015] In addition, the present invention also provides an induced pluripotent stem cell population obtainable by the method, and an induced pluripotent stem cell population obtained by the method. The induced pluripotent stem cell population may be an induced pluripotent stem cell population derived from umbilical cord amniotic membrane mesenchymal stem cells (population), or an induced pluripotent stem cell population derived from umbilical cord amniotic membrane epithelial stem cells (population).

[0016] In addition, the present invention also provides a pharmaceutical composition comprising the induced pluripotent stem cells of the present invention.

[0017] Further, 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 target cells under conditions suitable for differentiation. Therefore, the present invention also provides a pharmaceutical composition comprising the differentiated induced pluripotent stem cells obtained by the present invention.

[0018] The present invention provides a method for treating a congenital or acquired degenerative disorder in a subject, the method comprising administering to the subject target cells differentiated from pluripotent stem cells obtained by the present invention.

[0019] In addition, the present invention provides an extracellular membrane vesicle 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 the extracellular membrane vesicle of the present invention as a delivery vehicle for a therapeutic agent.

[0020] The invention provides a cell culture medium, which comprises mammary epithelial basal culture medium MCDB 170, EpiLife culture medium, DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 culture medium) and FBS (fetal bovine serum).

[0021] The invention further relates to a method for making induced pluripotent stem (iPS) cells as described herein differentiate into retinal pigment epithelium (RPE) cells, the RPE cells obtained, the retinal pigment epithelium composed of or comprising the RPE cells as described elsewhere herein, a method for treating the retinal degenerative diseases of a subject using the RPE cells differentiated from iPS cells by the methods as described herein, and a pharmaceutical composition comprising the RPE cells obtained by the methods as described herein. In addition, the invention further relates to a method in vivo and in vitro using the RPE cells obtained by the methods as described herein.

[0022] The inventors have described the use of iPS cells derived from umbilical cord lining cells (abbreviation: CLiPS) to produce RPE cells for clinical use.Compared with ES and skin iPS cells, the inventors use the method of the present invention to be differentiated into RPE by CLiPS, and have produced RPE with RPE differentiation efficiency that increases all the time.Based on the increase of the expression level of pigmentation specific genes (such as MITF, PMEL17 and TRYP2) and RPE specific genes (such as BEST1, RPE65, MERTK, RLBP1), the RPE derived from CLiPS has higher pigmentation than the RPE derived from ES.Equally at the functional level, by comparing the bioenergetics of the RPE derived from different stem cells (CLiPS, ES and skin iPS), it is proved that CLiPS-RPE includes increased glycolysis and mitochondrial respiration levels compared with the RPE derived from ES.

[0023] Therefore, in a first aspect, the present invention provides a method for differentiating iPS cells into RPE cells, the method comprising culturing iPS cells derived from umbilical cord amniotic stem cells in a differentiation medium under conditions suitable for differentiation into RPE cells, thereby differentiating the iPS cells into RPE cells.

[0024] In a second aspect of the present invention, the present invention provides a RPE cell culture obtainable by the method and a RPE cell culture obtained by the method.

[0025] In a third aspect of the invention, the present invention also provides a retinal pigment epithelium consisting of or comprising the RPE cell culture obtainable by the method, and a retinal pigment epithelium consisting of or comprising the RPE cell culture obtainable by the method.

[0026] In a fourth aspect of the present invention, the present invention further provides a pharmaceutical composition comprising the RPE cell culture obtained by the method of the present invention.

[0027] In a fifth aspect, the present invention also provides a method for treating a retinal degenerative disease in a subject, the method comprising administering to the subject RPE cells differentiated from iPS cells by the method of the present invention.

[0028] In a sixth aspect of the invention, the present invention provides an in vivo method for detecting the survival rate of RPE cells in a subject, wherein the RPE cells are differentiated from iPS cells by the method as defined herein, the method comprising a) introducing RPE cells differentiated from iPS cells by the method as defined into the subject, wherein the RPE cells contain a bioluminescent marker; b) using an imaging method to detect the bioluminescent signals of the RPE cells over time, thereby collecting imaging data; c) comparing the imaging data received in step b) with reference imaging data.

[0029] In a seventh aspect of the invention, the present invention provides an in vitro method for determining the immunogenicity of RPE cells in a subject, wherein the RPE cells are differentiated from iPS cells by a method as defined, and the differentiated RPE cells have been pre-delivered to the subject, the method comprising: a) detecting the level of proinflammatory cytokines in a sample obtained from the subject using an imaging method, wherein the sample comprises the differentiated RPE cells, thereby collecting imaging data; b) comparing the imaging data received in step a) with reference imaging data. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings, in which:

[0031] Figure 1A flow chart schematically representing the experimental steps of an exemplary embodiment of the method for producing induced pluripotent stem cells of the present invention is shown. The stem cells used herein are isolated from the amniotic membrane of the umbilical cord - also referred to as umbilical cord lining stem cells (CLSC). This embodiment starts with harvesting isolated CLSC by dissociating cells from a cell culture device (however, it should be noted here that CLSC can also be provided in a separated form for use in the method of the present invention). Then, the CLSC is counted, and about 0.7 million cells are aliquoted into a microcentrifuge and precipitated. The cell pellet is resuspended in a buffer suitable for electroporation, and then a plasmid encoding the Yamanaka factor is added to the cell-buffer mixture. Electroporation is performed with 1 pulse (the duration of the pulse is about 20ms and the voltage is about 1600V) or with 2 pulses (the duration of the pulse is 30ms and the voltage is about 1350V) for umbilical cord lining mesenchymal cells (CLMC) and umbilical cord lining epithelial cells (CLEC), respectively. After electroporation, the stem cells are immediately transferred to a culture medium suitable for recovery, wherein the culture medium contains compounds that inhibit inflammatory responses and enhance cell survival. After an appropriate time of recovery, the medium suitable for recovery is replaced with a 1:1 mixture of two different cell culture media, wherein the two different cell culture media are a medium suitable for recovery and a second cell culture media. In order to update the cell culture media, about 4 days after electroporation, the culture medium mixture is replaced with the same cell culture medium mixture. Thus, colonies of umbilical cord-lined induced pluripotent stem cells, also referred to herein as CLiPS, are produced. About another 2 days later, a 1:1 mixture of two different cell culture media is replaced with a second cell culture medium. The culture medium is also replaced about every two days to keep the culture medium fresh. When a size of about 0.5 mm to 1.5 mm in diameter is reached, the CLiPS colonies are picked and transferred to a coated cell culture container suitable for cell culture and proliferation. Again, the cell culture medium is replaced regularly with the same culture medium. After reaching about 50% confluence, the CLiPS colonies are dissociated from the coated culture device and transferred to another cell culture container suitable for cell culture and proliferation. In this way, the CLiPS colonies are further dissociated. When about 70-80% confluence is reached, CLiPS are passaged at a ratio of about 1:3 (v / v), wherein the passage is performed by contacting 1 volume of dissociated CLiPS with 2 volumes of fresh culture medium. CLiPS are then cultured in a culture medium containing a substance that enhances cell survival until about 30-60% confluence is reached. At this point, CLiPS are able to differentiate into any desired target cells.

[0032] Figure 2An exemplary comparison of the reprogramming efficiency of a single CLSC group is shown. Stem cells have been subjected to different electroporation settings to transfect exogenous nucleic acids into cells. Electroporation parameters (1650V, 10ms, 3 pulses) and the corresponding parameters for transfecting umbilical cord amniotic epithelial stem cells (also referred to herein as "umbilical cord lining epithelial stem cells" or CLEC, 1350V, 30ms, 2 pulses) and umbilical cord amniotic mesenchymal stem cells (also referred to herein as umbilical cord lining mesenchymal stem cells or CLMC, (1600V, 20ms, 1 pulse) are used in the present invention for transfection. 200K transfected cells are laid in triplicate in 6-well plates. About 21 days after transfection, the reprogramming efficiency percentage is calculated with colony number / 200,000x10.

[0033] Figure 3 Shown is an exemplary colony development of induced pluripotent stem cells from human CLMCs. Figure 3 af shows a representative time course of colony development, where Figure 3 a Depicts the typical morphology of human CLMCs cultured in their maintenance medium at day 0 of culture. Figure 3 b Depicts the typical morphology of human CLMCs cultured in their maintenance medium at day 15 of culture. Figure 3 c Depicts the typical morphology of human CLMCs cultured in their maintenance medium on day 24 of culture. Figure 3 d Depicts the typical morphology of human CLMCs cultured in their maintenance medium on day 29 of culture. Figure 3 e shows a 4x magnification of the typical morphology of first generation iPS colonies, and 3f shows a 10x magnification of the typical morphology of first generation iPS colonies. Figure 3 gl depicts an exemplary immunofluorescence staining of iPS cells derived from human umbilical cord lining cells showing activation of endogenous expression of pluripotent embryonic stem cell markers, where Figure 3 g shows the expression of KLF4, Figure 3 h shows the expression of NANOG, Figure 3 i shows the expression of OCT3 / 4, Figure 3 j shows the expression of SOX2, Figure 3 k shows the expression of SSEA4, Figure 3 l shows the expression of Tra-1-60, Figure 3 m shows an exemplary karyotype analysis showing normal chromosome number 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 an exemplary human CLMSC-DTHN culture at 20x magnification appearing at 10 days of reprogramming. Figure 3 o shows an exemplary morphology of expanded human CLMSC-DTHN cultured on a laminin-511 substrate at 4x magnification. Figure 3 p shows the morphology of expanded human CLMSC-DTHN cultured on laminin-511 matrix at 10x magnification. Figure 3 q shows an exemplary morphology of expanded human CLMSC-DTHN cultured on a laminin-511 matrix at 20x magnification. Figure 3 r shows exemplary expression of the human pluripotency marker NANOG in CLMSC-DTHN iPS at passage 3. Figure 3 s shows exemplary expression of human pluripotency marker OCT3 / 4 in CLMSC-DTHN iPS at passage 3. Figure 3 t shows exemplary expression of the human pluripotency marker SOX2 in CLMSC-DTHN iPS at passage 3. Figure 3 u shows exemplary expression of human pluripotency marker NTRA-1-81 in CLMSC-DTHN iPS at passage 3. Scale bars: 100 μm for each. Figure 3 V shows an exemplary RT-PCR analysis of reprogramming gene expression 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) is used as an internal control. PCR using total RNA of Homo sapiens (H1) without reverse transcription is used to control genomic contamination of all primer pairs.

[0034] Figure 4 An exemplary histological analysis of teratomas formed in immunocompromised non-obese diabetic severe combined immunodeficient (NOD-SCID) mice following CLiPS injection is shown. The teratoma formation assay revealed the formation of all three germ layers. Figure 4 a The inset shows a teratoma obtained from human CLEC-derived iPS 3 months after subcutaneous injection. Sections of the teratoma were further analyzed by hematoxylin and eosin staining. Figure 4 a shows the presence of respiratory epithelium in teratoma. Figure 4 b shows the presence of glandular structures representing endoderm in teratoma. Figure 4 In c, the arrow shows the presence of cartilage in the teratoma. Figure 4 In d, the arrow shows the presence of bone representing the mesoderm in the teratoma. Figure 4e shows the presence of renal tissue in teratoma. Solid arrows indicate glomeruli, and hollow arrows indicate renal tubules.

[0035] exist Figure 4 In f, arrows show the presence of neuroepithelium representing ectoderm in teratoma. Using directed differentiation protocols, CLiPS were induced to differentiate into specific tissues. Figure 4 g shows CLiPS differentiated into hepatocytes visualized with alpha-fetoprotein (AFP) and 4',6-diamidino-2-phenylindole (DAPI). Figure 4 h shows CLiPS differentiated into hepatocytes visualized with human serum albumin (HAS), cytokeratin 18 (CK18) and DAPI. Figure 4 i shows CLiPS differentiated into hepatocytes visualized with Oil Red O. Figure 4 j shows CLiPS differentiated into cardiomyocytes visualized with α-actinin (αACT), cardiac troponin I (cTnl), myosin regulatory light chain 2a (MLC2a), and DAPI. Figure 4 k shows CLiPS differentiated into dopaminergic neurons visualized with floor-plate marker FOXA2, roof plate marker LMX1A and DAPI. Figure 4 l shows CLiPS differentiated into dopaminergic neurons visualized with neuron-specific class III β-tubulin (TUJI) and tyrosine hydroxylase (TH). Figure 4 m shows CLiPS differentiated into oligodendrocyte progenitor cells visualized with OLIG2 and DAPI. Figure 4 n shows CLiPS differentiated into oligodendrocyte progenitors visualized with O4 and DAPI. Figure 4 o shows electrophysiological analysis of mature human CLiPS-derived dopaminergic neurons at day 45 of differentiation. Human CLiPS-derived dopaminergic neurons fired trains of action potentials with injected current. Scale bar: Figure 4 a. Figure 4 c and Figure 4 200 μm in d; Figure 4 b. Figure 4 e and Figure 4 100 μm in f; Figure 4 g. Figure 4 h. Figure 4 i. Figure 4 k. Figure 4 1. Figure 4 m is 50μm; Figure 4 j. Figure 4 n is 25μm.

[0036] Figure 5Exemplary directed differentiation of human CLiPS into various cell types is shown, wherein Figure 5 a depicts human CLiPS-derived neurons visualized with TH, Tuik, and DAPI. Figure 5 b depicts human CLiPS-derived hepatocytes visualized with CK18, HSA, and DAPI, Figure 5 c depicts human CLiPS-derived cardiomyocytes visualized with cTnl, αAct, and DAPI, and Figure 5 d shows electrophysiological analysis of contracting human CLiPS-derived cardiomyocytes, demonstrating that the cells generate spontaneous action potentials.

[0037] Figure 6 Shown are exemplary flow cytometric analyses of major histocompatibility complex (MHC) class I and class II and T cell co-stimulatory protein expression on iPS cells and dopaminergic neural progenitor cells differentiated from them. Figure 6 a shows flow cytometry profiles of immune-related gene expression on undifferentiated iPS cells. Figure 6 b shows flow cytometric analysis of neural cell adhesion molecule (NCAM)-positive populations. These populations were gated for analysis of immune-related protein expression. Figure 6 c shows the analysis of immune-related protein expression on day 25 differentiated dopaminergic neural progenitor cells.

[0038] Figure 7 In vivo comparison of dopaminergic neuron progenitor cells (NPCs) derived from human CLiPS and human adult fibroblast-iPS (asF-iPS) engraftment in NOD-SCID mice is shown. Day 25 dopaminergic NPCs were injected into the striatum of NOD-SCID mice to evaluate the engraftment and differentiation potential of cells in an immunodeficient environment. TH immunoreactive dopaminergic neurons were present in abundant human NCAM-positive engrafted neurons. Figure 7 a shows in vivo engraftment of day 25 dopaminergic NPCs derived from human asF-iPS. Figure 7 b shows in vivo engraftment of day 25 dopaminergic NPCs derived from human CLEC-iPS (EC23-CLiPS). Figure 7 c shows in vivo engraftment of day 25 dopaminergic NPCs derived from CLMC-iPS (MC23-CLiPS). Figure 7 d shows antibody staining of the transplanted hemisphere of a Parkinson's disease (PD) mouse model generated in immunocompetent C57BL / 6NTac mice 1 month after transplantation with human CLEC-iPS-derived dopaminergic NPCs. Human NCAM (green) and TH (red) double-positive neurons are abundantly present at the injection site. Figure 7e shows long neuronal processes originating from the transplantation site and projecting along the forceps major of the corpus callosum to distal regions of the brain. Figure 7 The arrows in f indicate human NCAM and TH double-positive neurons, which are abundantly present at the injection site as indicated by the arrows. Figure 7 g shows the Figure 7 d shows the same section of the contralateral non-transplanted hemisphere. Figure 7 h shows no visible surviving cells in the striatum transplanted with human adult asF-iPS-derived NPCs, suggesting immune rejection. Figure 7 i shows abundant accumulation of microglia / macrophages in the transplanted hemisphere. Figure 7 j shows the absence of microglia / macrophage accumulation in the non-transplanted hemisphere. Figure 7 k shows Figure 7 Higher magnification of i. It can be seen that microglia located near and within the graft display more deformed morphology characteristic of activated microglia. Figure 7 l shows Figure 7 Higher magnification of k, showing the expression of CD68, an activation marker for microglia. Scale bar: Figure 7 ac and Figure 7 k is 100 μm; Figure 7 d. Figure 7 g and Figure 7 h: 200 μm; Figure 7 e. Figure 7 f and Figure 7 l is 50μm.

[0039] Figure 8 Shown is the survival of human CLEC-derived (EC23-CLiPS) dopaminergic neurons in a mouse PD model 9 months after transplantation. Figure 8 a shows the presence of HuNu+ / hNCAM+ / TH+ neurons in the transplanted hemisphere. Figure 8 b is Figure 8 cf overlap, and shows Figure 8 Higher magnification of the boxed area in a. Figure 8 c shows the presence of hNCAM+ neurons in the transplanted hemisphere. Figure 8 d shows the presence of HuNu+ neurons in the transplanted hemisphere. Figure 8 e shows the presence of TH+ neurons in the transplanted hemisphere. Figure 8 f shows the nuclei of neurons present in the transplanted hemisphere. Figure 8g Schematic representation of the experimental steps starting from the induction of PD lesions by injection of 6-hydroxydopamine (6-OHDA) into the striatum of C57BL / 6NTac mice. Pre-transplant rotational behavior assay was performed one and two weeks before NPC transplantation. Figure 8 h shows the results of apomorphine-induced rotational asymmetry assay in mice transplanted with dopaminergic NPCs derived from human EC23-CLiPS and asF-iPS and sham controls. Assays were performed every two weeks after transplantation until 22 weeks. Starting from the 20th week after transplantation, animals in the human EC23-CLiPS group showed statistically significant rotational recovery compared to the asF-iPS group (n=5, p<0.05). No recovery was observed in the sham group. Figure 8 h shows representative in vivo positron emission tomography (PET) imaging of [18F]PE-P2I ligand uptake to assess the restoration of dopamine transporter (DAT) function in striatal dopaminergic neurons 6 months after transplantation. Mice transplanted with human EC23-iPS NPCs showed restoration of DAT activity compared to mice transplanted with human asF-iPS NPCs or sham controls. Scale bar: Figure 8 200 μm in a; Figure 8 100 μm in bf.

[0040] Fig. 9 Exemplary in vivo PET imaging of striatal dopamine production in transplanted mice is shown. PET shows [18F]PE-P2I ligand uptake 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 showed a significant recovery of DAT activity compared to mice transplanted with human adult iPS-derived NPCs or sham transplanted controls.

[0041] Fig.10 In vivo maintenance of human CLiPS-derived grafts 6 and 9 months after implantation into the mouse brain is shown. The grafts stained positive for the human antigen NCAM and TH dopaminergic markers. Tumor formation was not recorded. Scale bar: 50 μm.

[0042] Fig.11 Shown are the results of histological and functional analysis of human EC23-CLiPS dopaminergic NPCs transplanted in the medial forebrain bundle (MFB) injury model of PD established in fully immunocompetent Wistar Hannover rats. Fig.11 a shows engraftment of human EC23-CLiPS neurons in the striatum region of the rat brain 3 months after transplantation, as confirmed by positive double staining with human cytoplasmic (STEM 121) and human nuclear antigen (HuNu) antibodies. Staining indicates functional recovery. Fig.11 b shows the co-localization of Synapsin 1 immunoreactivity with hNCAM+ / TH+ neurons, suggesting possible integration of transplanted human CLiPS-derived cells into host tissues 3 months after transplantation. Fig.11 c shows retrograde damage to the dopaminergic system in the substantia nigra in the rat brain. Fig.11 d shows an intact rat brain, confirmed by tyrosine hydroxylase (TH) immunostaining. Fig.11 Retrograde damage to the dopaminergic system in the substantia nigra of c. Fig.11 e shows the results of apomorphine-induced rotational asymmetry assay in rats transplanted with dopaminergic NPCs derived from human CLEC23-iPS. The results indicate that transplantation of CLiPS-NPCs mediates the restoration of functional motor deficits in the rat MFB model of PD over the 6-month study period. Scale bar: Fig.11 a and Fig.11 100 μm in b; Fig.11 c and Fig.11 200 μm in d.

[0043] Fig.12 a, b, and c each show an exemplary colony of induced pluripotent stem cells derived from human CLECs generated by using PTTe-3 medium as a recovery medium.

[0044] Fig.13 CLiPS differentiation into RPE is shown: images of differentiated cultures from different stem cells - human ES cells (H9), skin-derived iPS cell lines (Asf5, AGO, HDFA), umbilical cord lining mesenchymal cells (CLMC23, CLMC30, CLMC44), and umbilical cord lining ectoderm cells (CLEC23). The darker blocks on the cell culture plate correspond to RPE cells with pigmentation.

[0045] Fig.14 It is shown that CLMCs have consistently high differentiation efficiency: Fig.14 a shows the visual grading system used to assess RPE differentiation efficiency based on the percentage of well area occupied by pigmented cells, with RPE differentiation efficiency graded as 0, 1, 2, or 3 for no pigmentation, <30%, 30-60%, or >60% pigmentation, respectively. Fig.14 B shows the RPE differentiation efficiency assessed by the visual grading of the pigmented cell area of ​​differentiation plate. Each post represents the classification of a differentiation plate, and the numbers on the post represent the percentage of the pigmented wells on the plate represented by different grades of brown of different shades. Numbers 1-3 represent biological repetitions. Fig.14c shows the RPE differentiation efficiency of different stem cells estimated from Pmel17 by flow cytometry; cells from 3 wells were pooled for FACS analysis.

[0046] Fig.15 It is shown that CLiPS-derived RPE has more pigmentation compared to ES-derived RPE: Fig.15 a shows an image of a differentiation plate taken under the same conditions on day 30 of differentiation using the ChemiDoc Touch gel imaging system (Bio-Rad Laboratories). Fig.15 b shows phase contrast images of RPE from different stem cells, showing weak pigmentation of H9.H9: (human ES cell-derived RPE), CLMC23, CLMC30, CLMC44, CLEC23 (RPE from CLiPS), AGO, HDFA, Asf5 (RPE from skin-iPS cells). Fig.15 C shows that compared with ES derived RPE, CLiPS derived RPE has more pigmentation.Shown the figure of pigmentation darkness obtained by image analysis of the differentiation flat board taken at different points along differentiation H9: (RPE from human ES cell source), CLMC23, CLMC30 and CLEC23 (RPE from CLiPS) using the Cmmeidoc Touch system from Biorad. Fig.15 d shows that pigmentation-related and RPE-specific genes are higher in CLiPS: RT-qCPR analysis of pigmentation-related genes at day 18 and day 35 of differentiation: MITF, PMEL17, tyrosinase, TRYP2.

[0047] Fig.16 Shown are CLiPS expressing RPE-specific genes at days 18 and 35 of differentiation: RT-qCPR analysis of the RPE-specific RPE65 and MERTK.

[0048] Fig.17 It was shown that CLiPS-derived-RPE is functional. Fig.17 a shows the tight junctions formed by in vitro generated RPE, similar to native RPE: using the epithelial volt-ohmmeter EVOM2 TM Transepithelial electrical resistance (TEER), a measure of tight junction integrity, was measured in RPE derived from the different stem cells over a period of 4 months. Fig.17 b shows that in vitro generated RPE are highly phagocytic: percentage of FITC-labeled photoreceptor outer segments (POS) phagocytosed by RPE derived from different stem cells.

[0049] Fig.18CLiPS-derived RPE is shown, showing similar protein expression to ES-derived RPE. CLiPS-RPE shows apical expression of Mertk, junctional expression of ZO-1, and cytoplasmic expression of RPE65.

[0050] Figure 19 shows the original method and improvement of RPE differentiation: Figure 19a shows the schematic diagram of the original method, wherein shows the differentiation medium and its composition used in different stages. Figure 19b shows the improvement introduced into the differentiation scheme, wherein shows that CHIR99021 concentration gradually increases and replaces FGF inhibitor-SU5402 with PD173074. Figure 19c shows the photo of the CLMC30 plate differentiated using the public scheme adopting SU5402 or the improved scheme adopting PD173074, wherein shows the RPE differentiation and pigmentation of similar degree. DM1-DM5: Differentiation medium 1-5. The improved RPE differentiation scheme adopting PD173074 produces functional RPE. The functionality of the RPE derived from the differentiation method adopting SU5405 or PD173074, the phagocytosis (Figure 19e) of POS particles marked by TEER (Figure 19d) and FITC was tested.

[0051] Figures 20a and b show a comparison of RPE yields by different purification methods. In particular, Figure 20a and b show schematic diagrams of different RPE purification methods: differentiation cultures containing RPE and non-RPE are purified: (i) manual purification: non-RPE cells are identified based on morphology and lack of pigmentation, and they are manually removed by scraping by observation under a dissecting microscope, (ii) TrypLE purification: most of the weakly attached non-RPE clusters are removed by partial TrypLE treatment, (iii) TrypLE+ manual: most of the weakly attached non-RPE clusters are eliminated by partial TrypLE treatment, and a few non-RPE clusters that escape the TrypLE treatment are subsequently manually removed by observation under a dissecting microscope, (iv) TrypLE+ scatter sorting: weakly attached non-RPE clusters are removed by partial TrypLE treatment, followed by scatter sorting, (v) scatter sorting: all cells from mixed differentiation cultures are separated according to high scattering (pigmented RPE cells) and low scattering (non-pigmented non-RPE cells) populations based on their relative light scattering. Figure 20 c and d show original and improved scattering sorting method, to more accurately select high scattering RPE cells.Figure 20 c shows the gate of any selection for high scattering (cyan) and low scattering (magenta) as in the original scheme.Figure 20 d shows improved gate selection, wherein adopts the weakly attached non-RPE cell of dissociation by part TryPLE process to set low scattering gate (magenta) to more accurately select high scattering gate (cyan).Figure 20 e shows the productive rate of the RPE obtained from different purification methods.Figure 20 f shows the purity of the RPE from different purification methods assessed by Pmel17 flow cytometry. Figure 20g shows the TEER of RPE obtained from different purification methods; M: manual purification, T: TrypLE purification, T+M: TrypLE+ manual purification, T+Sc: TrypLE+ scattering sorting, SC: scattering sorting, T (loose): weakly attached non-RPE cells easily dissociated by TrypLE treatment, Sc low: low scattering non-RPE cells from scattering sorting. Figure 20h shows the phagocytic ability of RPE from different purification methods assessed by photoreceptor outer segment (POS) phagocytosis assay. Figure 20i shows a table comparing different RPE purification methods. Figure 20j shows the quantitative PCR comparison of RPE specific gene expression in CLMC23 and H9. qPCR results show the relative expression of RPE specific genes such as BEST1, RPE65, RLBP1, MERTK, MITF, PMEL17 and TRYP2 standardized with GAPDH. Figure 20k shows comparison of gene expression in CLMC23 and H9, expressed as fold change in CLMC23 relative to H9.

[0052] Figure 21 shows that CLiPs-RPE (CLEC23-RPE) has a bioenergetic profile similar to natural RPE (AHRPE). Compared with skin iPSC-RPE (ASF5-RPE) and hESC-RPE (H9-RPE), CLiPsRPE (CLEC23-RPE) also shows higher glycolysis and oxidative phosphorylation. Figure 21a shows that for OCR curve, basal respiration, ATP production, maximum capacity and spare respiratory capacity are respectively 38%, 40%, 35% and 36% higher in CLiPs-RPE than in H9-RPE. Figure 21b shows that for ECAR curve, glycolysis, glycolytic capacity and glycolytic reserve are respectively 25%, 37% and 50% higher in CLiPs-RPE than in H9-RPE. Figure 21c-f shows that CLiPs-RPE shows increased resistance to oxidized low-density lipoprotein (oxLDL), which is confirmed by the following: the maximum capacity is not reduced after CLEC23-RPE is exposed to oxLDL (dashed curve) (c) vs. 27% decrease in ASF5-RPE (d) and 43% decrease in H9-RPE (e). The response of CLiPs-RPE cells to oxidative stress is similar to that seen in natural RPE (AHRPE-f), making CLiPs-RPE cells functionally closer to primary RPE compared to other differentiated RPE. Figure 21g-j shows that CLiPs-RPE shows increased resistance to hydrogen peroxide (H2O2), which is confirmed by the following: the maximum capacity is not reduced after CLEC23-RPE is exposed to H2O2 (dashed curve) (g) vs. 27% decrease in ASF5-RPE (h) and 99% decrease in H9-RPE (i). The response of CLiPs-RPE cells to oxidative stress is similar to that seen in native RPE (AHRPE) (f & j), making CLiPs-RPE cells functionally closer to primary RPE than other differentiated RPE.

[0053] Figure 22 shows that the immune system does not remove all stem cell-derived retinal pigment epithelial (SC-RPE) cell lines. Figure 22a and b show in humanization and NOD-SCID IL2 Rγ- / - (immunodeficient) mice, at the time point of indication, the in vivo bioluminescence measurement (total radiance) of the SC-RPE of the expression luciferase embedded in the matrigel plug of injection. Figure 22c shows the representative images of all SC-RPE systems, wherein showing RPE65, Ki67 and Hoechst staining from the matrigel plug implanted in humanized mice at the end of 2 months. Scale bar, 50 μm.

[0054] Figure 23 shows that CLEC23-RPE group has the proinflammatory cytokines that participate in the cellular immune response induction of reduced level. Figure 23a and b show the serum cytokines (IFN-γ and IL-18) when analyzing the end point. Figure 23c shows the representative images of OTX2, human CD45 (hCD45) and Hoechst staining showing RPE-matrigel plugs, indicating immune cell infiltration. Figure 23d and e show the cellular immune response classification (0 to 3) based on hCD45 positive cells in RPE-matrigel plugs. Scale, 50 μm.

[0055] Fig.24 It was shown that CLEC23-RPE can inhibit CD8 T cell activation. Fig.24 a and b show serum cytokines (IL-23 and IL-17A) at the end point of the analysis. Fig.24 c shows the ratio of T cells (CD3) to B cells (CD19) calculated after flow cytometry analysis. Fig.24 d shows that CD3-positive cells were further gated into helper T (CD4) and cytotoxic T (CD8) cells to analyze T cell differentiation. Fig.24 e and f show gating of CD4-positive and CD8-positive cells into four groups of different T cell activation states based on specific surface markers. DETAILED DESCRIPTION

[0056] In particular, the present invention relates to a method for producing induced pluripotent stem cells (iPS) by generating induced pluripotent stem cells from umbilical cord amniotic stem cells under conditions suitable for reprogramming stem cells.

[0057] In the present invention, umbilical cord amniotic mesenchymal and epithelial stem cells - also collectively referred to herein as umbilical cord lining stem cells (CLSCs) for the generation of iPS - are also referred to herein as umbilical cord lining-derived induced pluripotent stem cells or "CLiPS". It has been surprisingly found that the umbilical cord lining-derived induced pluripotent stem cells of the present invention are robust and homogeneous stem cells that are capable of differentiating into functional target cells of different lineages (see Examples 3 and 4). For example, umbilical cord lining-derived induced pluripotent stem cells have the ability to differentiate into multiple cell types, and can, for example, differentiate into various cell types, such as hepatocytes representing endodermal tissue (see Example 8), cardiomyocytes representing mesoderm tissue (see Example 9), and dopaminergic neurons representing ectoderm tissue (see Example 7) and oligodendrocytes (see Example 10). More surprisingly and importantly, it was found that, for example, human CLiPS-derived dopaminergic neurons were able to functionally engraft in different species and survived up to 9 months in a mouse Parkinson's disease (PD) model without immunosuppression and up to 6 months in a rat PD model without immunosuppression (see Examples 12 and 13). Therefore, in summary, the inventors have generated a low immunogenic cell source that can be transplanted, integrated and mediated therapeutic recovery in a fully immunocompetent host. The induced pluripotent stem cells derived from the umbilical cord lining of the present invention can potentially be used as a universal source of cells for allogeneic cell transplantation in humans without the need for immunosuppression, and this makes them ideal candidates for such cell-based therapies. As a further advantage, it is found that the induced pluripotent stem cells derived from the umbilical cord lining of the present invention can be produced by a non-integrated and feeder-free method, thereby allowing the production of iPS under current good manufacturing practice (cGMP) conditions. Since a GMP method for producing large quantities of umbilical cord amniotic mesenchymal stem cells has recently been established (see International Application WO 2018 / 067071 or U.S. Patent Application US2018127721), the present invention provides an ideal platform for producing iPS for subsequent cell-based therapies in humans or animals.

[0058] In summary, CLiPS from very young tissues are less likely to carry genetic, epigenetic and mitochondrial DNA mutations because they are from young tissues. Due to these advantages, CLiPS are a potentially superior stem cell source for generating differentiated cells for regenerative medicine. Therefore, CLiPS are superior to iPS cells from skin or blood that require invasive tissue collection procedures. They also do not have the ethical issues associated with ES cells. Therefore, CLiPS are a better stem cell source for regenerative medicine.

[0059] The inventors found that, by the method of the present invention, this CLiPS is robustly differentiated into retinal pigment epithelial (RPE) cells, also referred to as RPE. For the present invention, the inventors compared the ability of different stem cell sources to produce RPE in vitro: human ES cells (ES), iPS cells (skin-iPS) of skin origin and umbilical cord lining cells (CLiPS). CLiPS can be mesenchymal (CLMC) or ectoderm (CLEC) derived. Then, the inventors compared the RPE differentiation efficiency of CLiPS with ES and skin-iPS cells. By visual grading and flow cytometry estimation, compared with skin-iPS, CLiPS always provides high RPE differentiation efficiency than skin-iPS cells. Visual comparison and image analysis of the pigmentation of differentiation culture also show that CLiPS-derived RPE has higher pigmentation than ES-derived RPE. The RPE produced by CLiPS also shows the functional characteristics of RPE after in vitro maturation, suggesting that they are excellent sources of RPE cells. In addition, the inventor found that CLiPS-RPE has higher glycolysis and mitochondrial respiration than ES-derived RPE by comparing the bioenergy of the RPE derived from different stem cells.The method for the induced pluripotent stem (iPS) (CLiPS or CLSC) cell differentiation of RPE cells derived from umbilical cord amniotic membrane stem cells of the present invention has been specially improved as described herein, and it realizes maximum RPE yield.

[0060] Now first describe the method of producing iPS of the present invention, which may include expressing exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA. Nucleic acid encoding OCT3 / 4 (sequence ID No: 1), sometimes also referred to as POU5FL, OCT3 or OCT4, encodes octamer binding transcription factor 4. OCT3 / 4 (sequence ID No: 2) forms a heterodimer with SOX2 to regulate pluripotency factors in cells. SOX2 (sequence ID No: 3), sometimes also referred to as SEY, encodes sex determining region Y-box 2 transcription factor (sequence ID No: 4). When SOX2 binds to OCT3 / 4, it binds to non-palindromic genomic sequences, thereby activating the transcription of pluripotency factors in cells. KLF4 (sequence ID No: 5), sometimes also referred to as GKLF, encodes Krueppel-like factor 4. KLF4 (Sequence ID No: 6) is a zinc finger transcription factor that acts as a tumor suppressor, controlling the G1 to G2 transition of the cell cycle by mediating the tumor suppressor p53. L-MYC (Sequence ID No: 7) encodes a transcription factor that activates the expression of proliferation genes (Sequence ID No: 8). LIN28 (Sequence ID No: 9) encodes an RNA binding protein Lin-28 homolog A (Sequence ID No: 10), which regulates the self-renewal of stem cells. p53-shRNA (Sequence ID No: 11) encodes a small hairpin RNA against p53, a protein that can regulate the cell cycle by terminating it when the protein accumulates in the cell. In order to avoid the termination of the cell cycle by p53, p53-shRNA can silence the expression of p53 after transcription. In order to produce CLiPS, exogenous nucleic acids encoding OCT3 / 4, SOX2, KLF4, LIN28, L MYC and p53-shRNA can be transferred into CLSCs for expression. Alternatively, proteins OCT3 / 4, SOX2, KLF4, LIN28, L-MYC, and p53-shRNA can be directly transferred to CLSCs.

[0061] As described above, the induced pluripotent stem cell population of the present invention can be obtained by reprogramming umbilical cord amniotic membrane stem cells. Umbilical cord stem cells can be (isolated) mesenchymal stem cells of umbilical cord amniotic membrane, also referred to as umbilical cord lining mesenchymal stem cells (CLMC), or (isolated) epithelial stem cells of umbilical cord amniotic membrane, also referred to as umbilical cord lining epithelial stem cells (CLEC). CLEC and CLMC for producing iPS of the present invention can be from any mammalian species, such as mice, rats, guinea pigs, rabbits, goats, horses, dogs, cats, sheep, monkeys or humans, preferably stem cells of human origin in one embodiment. Therefore, the iPS of the present invention can also be from any mammalian species, such as mice, rats, guinea pigs, rabbits, goats, horses, dogs, cats, sheep, monkeys or humans, preferably stem cells of human origin in one embodiment. In a preferred embodiment, CLEC is used to produce iPS of the present invention.

[0062] In the case of using umbilical cord amniotic epithelial stem cells as starting material, these epithelial stem cells can be obtained, for example, as described in U.S. Patent Application 2006 / 0078993 (granted U.S. Patents 9,085,755 and 9,737,568) or the corresponding International Patent Application WO2006 / 019357. If umbilical cord amniotic mesenchymal stem cells are used as starting material, they can also be obtained as described in U.S. Patent Application 2006 / 0078993 (granted U.S. Patents 9,085,755 and 9,737,568) or the corresponding International Patent Application WO2006 / 019357.

[0063] Mesenchymal stem cell populations as described in published U.S. application 2018 / 127721 or the corresponding international application WO 2018 / 067071 can also be used as starting materials. The advantage of the mesenchymal stem cell population of international application WO 2018 / 067071 is that 99% or more of the stem cells in the population are positive for three mesenchymal stem cell markers CD73, CD90, while lacking the expression of CD34, CD45 and HLA-DR, which means that 99% or even more cells in the mesenchymal stem cell population of international application WO 2018 / 067071 express stem cell markers CD73, CD90 and CD105, but do not express markers CD34, CD45 and HLA-DR. This very homogeneous and well-defined cell population is an ideal candidate for clinical trials and cell-based therapies because they, for example, fully meet the generally accepted criteria for human mesenchymal stem cells for cell therapy, as defined by Dominii 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. Therefore, the mesenchymal stem cell population of international application WO 2018 / 067071 is an ideal starting material for producing CLiPS of the present invention under GMP conditions.

[0064] In this context it is noted that CLMCs transfected with a transgene will retain their stemness and stem cell characteristics, but may show a decrease in the percentage of cells expressing mesenchymal stem cell markers such as CD73, CD90 and CD105, while at the same time may also show an increase in the percentage 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, page 12. In view of this, the CLiPS of the present invention produced by reprogramming of CLMCs isolated from umbilical cord amniotic membrane as described herein can be a stem cell population wherein 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 more, 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 can express each of the following markers: CD73, CD90 and CD105. In addition, such a CLMC-derived induced pluripotent stem cell population of the present invention may be a 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 more, 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, or about 99% may lack expression of each of CD34, CD45, and HLA-DR. A preferred example of such a CLMC-derived induced pluripotent stem cell population of the present invention may be a population wherein 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 of the CLMC population express each of CD73, CD90 and CD105, and lack the expression of each of CD34, CD45 and HLA-DR.

[0065] Returning to the generation of the induced pluripotent stem cell (group) of the present invention, it is important to note that such induced pluripotent stem cells are obtainable by any suitable method of reprogramming umbilical cord amniotic stem cells (group) into such induced pluripotent stem cells (group). Although one method of producing such induced pluripotent stem cells includes expressing exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in umbilical cord amniotic stem cells under conditions suitable for reprogramming stem cells, thereby producing induced pluripotent stem cells, the present invention is by no means limited to CLiPS obtained by this method. On the contrary, CLiPS can be obtained by any suitable method, such as described in the review of Cieslar-Probuda et al. "Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences" BBA-Molecular Cell Research, Vol. 1864, No. 7, July 2017, pp. 1359-1369. For example, in the present invention, reprogramming can also be carried out chemically 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 suitable nucleic acid for expression. For example, nucleic acid can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and microRNA (miRNA) including messenger RNA (mRNA). Exogenous nucleic acid can be transferred as is, or exogenous nucleic acid can be incorporated into one or more vectors suitable for transfer to cells. In this context, any vector suitable for transfer to CLSC can be used. An illustrative example of this vector can be a plasmid. In the present invention, exogenous nucleic acid can be provided by one, two, three or four vectors suitable for transfer to stem cells. In one illustrative example, three vectors can provide exogenous nucleic acids for reprogramming CLSCs into CLiPSs, wherein the vectors can be pCXLE-hOCT3 / 4-shp53-F (Addgene plasmid #27077; sequence ID No: 12), pCXLE-hSK (Addgene plasmid #27078, sequence ID No: 13), and pCXLE-hUL (Addgene plasmid #27080; sequence ID No: 14).

[0066] According to the above, any method suitable for transferring exogenous nucleic acids or proteins into CSLCs can be used. In one example, a viral vector can be used to transfer exogenous nucleic acids into CSLCs. Examples of such viral vectors can be retroviruses, lentiviruses, inducible lentiviruses, Sendai viruses, or adenoviruses. Alternatively, transfection can be performed to transfer exogenous nucleic acids into CSLCs. In the present invention, transfection can include electroporation, microinjection, liposome- and non-liposome-mediated transfection, and sonoporation.

[0067] In a preferred example, CLSC can be subjected to electroporation, wherein electrical parameters can be adjusted according to the type of CLSC used, because CLMC may require electroporation conditions different from CLEC. Electrical parameters can include the number of electric pulses applied to stem cells, the duration of the electric pulses applied, and the voltage of the electric pulses applied. Each electrical parameter can be adjustable to further optimize the electroporation of the present invention. When doing so, each electrical parameter can be adjusted independently or in combination with one or more other electrical parameters (see Example 1). In the present invention, any parameter setting suitable for allowing exogenous nucleic acids to be transferred to CLSC can be applied. In one example of the present invention, CLMC can be subjected to electroporation. In this case, electroporation can be performed with 1 electric pulse, the duration of the electric pulse can be about 15 milliseconds (ms) to about 25ms, and the voltage can be about 1550V to about 1650V. Therefore, in one example, CLMC can be subjected to electroporation of 1 electric pulse, the duration of the electric pulse can be about 20ms, and the voltage can be about 1600V. In addition, it has been found herein that electroporation to produce a usable amount / number of CLiPS derived from CLMCs depends on the ratio of each vector (plasmid) DNA transfected to the number of CLMCs used for transfection. This ratio is determined herein by the amount of each vector (plasmid) DNA used (in μg) to the number of CLMCs subjected to electroporation (in 1×10 6 In an illustrative embodiment, the ratio of the amount of vector (plasmid) DNA to the number of cells for each vector can be 1.5 μg DNA to about 1×10 6 CLMC to about 2.5 μg DNA ratio of about 1 × 10 6 Therefore, the ratio can be about 2.5 μg DNA to about 1×10 6 CLMC, about 2.25 μg DNA, about 1×10 6 CLMC, about 1.8 μg DNA, about 1 × 10 6 CLMC, about 1.7 μg DNA, about 1×10 6 CLMC, about 1.67 μg DNA, about 1×10 6CLMC, about 1.6 μg DNA, about 1×10 6 CLMC, or about 1.5 μg DNA to about 1 × 10 6 CLMC (see Table 1, which shows the amount of vector (plasmid) DNA used for each plasmid to the number of cells of about 1.67 μg DNA ratio of about 1×10 6 The ratio of CLMCs to each other produces an effective conversion yield). Therefore, in one embodiment of producing CLiPS derived from CLMCs, it is preferred that each vector is used in the same amount in the electroporation of CLMCs. CLECs may also be subjected to electroporation to produce CLiPS of the present invention. In the case of deriving CLiPS from CLECs, electroporation may be performed with 2 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. Therefore, in one embodiment, CLECs may be subjected to electroporation with 2 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 for CLiPS derived from CLECs that the electroporation to produce a usable amount / number of CLiPS derived from CLECs depends on the ratio of the amount of each plasmid DNA transfected to the number of CLECs used for transfection. This ratio is also expressed herein by the amount of vector (plasmid) DNA used for transfection (in μg) to the number of CLECs to be transfected (in 1×10 6 In an illustrative embodiment, the ratio of the amount of vector (plasmid) DNA to the number of cells can be about 1.5 μg DNA to about 1×10 6 CLEC to about 2.5 μg DNA ratio of about 1 × 10 6 Therefore, the ratio can be about 1.5 μg DNA to about 1×10 6 CLEC, about 1.6 μg DNA, about 1 × 10 6 CLEC, about 1.67 μg DNA, about 1×10 6 CLEC, about 1.7 μg DNA to about 1 × 10 6 CLEC, about 1.8 μg DNA, about 1 × 10 6 CLEC, about 1.9 μg DNA, about 1 × 10 6 CLEC, about 2.0 μg DNA to about 1 × 10 6 CLEC, or about 2.5 μg DNA to about 1 × 10 6 CLEC (see Table 1, which shows the amount of plasmid DNA used for each vector and the cell number of about 1.67 μg DNA to about 1×10 6The ratio of CLECs to each other provides an effective conversion yield). Therefore, in one embodiment of producing CliPS derived from CLECs, it is preferred to use each vector in the same amount in the electroporation of CLECs. Electroporation of both CLECs and CLMCs can be performed in a uniform electric field in the method of the present invention. Thus, key consequences of electroporation such as pH changes, ion formation, or heat generation can be minimized. A uniform electric field can be generated by maximizing the gap between electrodes while minimizing the surface area of ​​each electrode. An illustrative example of a system that provides such a uniform electric field is the Neon ELECTROTECHNICAL® from ThermoFisher Scientific. TM Transfection system. Another example of a suitable commercial transfection system is the Gene Pulser MXcell electroporation system, available from Bio-Rad. Finally, any suitable electroporation buffer may be used for transfection. TM In the case of a transfection system, the corresponding electroporation buffer provided by the transfection system manufacturer is usually used for electroporation.

[0068] After transfection, the stem cells can be transferred to a culture medium suitable for cell recovery and cell culture. In the present invention, any cell culture medium suitable for cell recovery and / or proliferation can be used. Illustrative examples of such suitable cell culture medium can be a culture medium commonly used for culturing (proliferation) human induced pluripotent stem cells, such as mTeSR1, StemMACS TM iPS-Brew XF, TeSR TM -E8, mTeSR TM Plus, TeSR TM 2. mTeSR TM1. For cell recovery culture, any culture medium capable of supporting proliferation (without differentiation) / healthy growth of CLECs or CLMCs may also be used. Examples of suitable culture media for such culture of CLECs are described, for example, in U.S. Patent Application 2006 / 0078993, and include EpiLife medium, medium 171, MEGM-mammary epithelial cell medium, or mixtures of such culture media, such as medium PTT-e3 (which has been used herein to produce CLiPS derived from CLECs, and which is described in detail below). Examples of suitable culture media for such culture of CLMCs are described, for example, in U.S. patent applications 2006 / 0078993 and 2018 / 127721 and international patent application WO2007 / 046775, and include DMEM / 10% FBS, DMEM:F12 medium (a 1:1 mixture of DMEM and Ham's F12 medium), or a culture medium such as PPT-6 (a culture medium comprising DMEM, F12-Mediim, culture medium 171, and FBS, see U.S. application 2018 / 127721) or PTT4 (where the latter has been used in the Examples section herein to produce CLiPS derived from CLMCs). For such cell recovery culture, mixtures of these culture media (e.g., a mixture of mTeSR1 with culture medium PTTe-3 or culture medium PTT-4) may also be used. The culture medium suitable for cell recovery of transfected CLECs or CLMCs as described herein may further contain growth factors that may stimulate cell growth and proliferation. The growth factors may be added to the cell culture medium as is. Additionally, the recovery medium may contain serum, such as, for example, fetal bovine serum (FBS). Thus, a medium suitable for cell recovery after transfection may be a serum-free or serum-containing medium.

[0069] In light of the above disclosure, the composition of culture medium suitable for cell recovery may vary, depending on the CLSCs used.

[0070] For example, a culture medium suitable for recovering transfected CLMCs may consist of a (chemically) defined medium and FBS. Thus, a culture medium suitable for recovering transfected CLMCs may consist of about 80% (v / v), about 85% (v / v), about 90% (v / v) or about 95% (v / v) chemically defined medium and about 20% (v / v), about 15% (v / v), about 10% (v / v) or about 5% (v / v) FBS, respectively. In a preferred embodiment, CLMCs are cultured in culture medium PTT-4 to recover cells after transfection, wherein culture medium PTT-4, as described in international patent application WO2007 / 046775, consists of 90% (v / v) CMRL-1066 and 10% (v / v) FBS. A culture medium suitable for recovering transfected CLECs may be a serum-free culture medium, wherein the culture medium may contain cytokines and growth factors.

[0071] The medium suitable for recovering transfected CLECs can also be a defined medium. This recovery medium can include mammary epithelial basal medium MCDB 170, EpiLife medium, DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium) and FBS (fetal bovine serum).

[0072] In an illustrative embodiment, such a culture medium comprises a mammary epithelial basal medium MCDB 170 at a final concentration of about 10% to about 30% (v / v), an EpiLife medium at a final concentration of about 20% to about 40% (v / v), a F12 at a final concentration of about 5% to about 15% (v / v), a DMEM at a final concentration of about 30% to about 45% (v / v), and a FBS at a final concentration of about 0.1% to 2% (v / v). One such culture medium may comprise a mammary epithelial basal medium MCDB 170 at a final concentration of about 15% to about 25% (v / v), an EpiLife medium at a final concentration of about 25% to about 35% (v / v), a F12 at a final concentration of about 7.5% to about 13% (v / v), a DMEM at a final concentration of about 35% to about 40% (v / v), and a FBS at a final concentration of about 0.5% to 1.5% (v / v). Another such culture medium may comprise mammary epithelial basal medium MCDB170 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). As used herein, the value of "% (v / v)" refers to the volume of a single component relative to the final volume of the culture medium. This means that if there is, for example, DMEM at a final concentration of about 35% to about 40% (v / v) in the culture medium, 1 liter of culture medium contains about 350ml to 400ml DMEM. In one embodiment, a culture medium suitable for recovering transfected CLEC cells is obtained by mixing the following to obtain a final volume of 1000ml of culture medium:

[0073] -200 ml mammary epithelial basal medium MCDB 170,

[0074] -300 ml EpiLife medium,

[0075] -250ml DMEM,

[0076] -250 ml DMEM / F12, and

[0077] -1% fetal bovine serum.

[0078] Growth factors in a culture medium suitable for recovering transfected CLECs can 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, activins, bone morphogenetic proteins (BMPs), platelet-derived growth factors (PDGFs), transferrin, and insulin. In one example, CLECs are cultured in a culture medium PTTe-3 to recover cells after transfection, wherein the culture medium PTTe-3 contains human epidermal growth factor (EGF), one or more transforming growth factors such as TGF-α and / or TGF-β (TGF-β1, TGF-β2, and / or TGF-β3), or insulin.

[0079] According to the above, the culture medium suitable for recovering transfected CLEC can include human epidermal growth factor (EGF) with a final concentration of about 1 ng / ml to about 15 ng / ml. The recovery culture medium can also include insulin with a final concentration of about 1 μg / ml to about 7.5 μg / ml. This recovery culture medium can further include at least one of the following supplements: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3). In one embodiment, the culture medium includes adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3) in all three. In this case, the culture medium can include adenine with a final concentration of about 0.05 mM to about 0.1 mM adenine, hydrocortisone with a final concentration of about 0.1 μM to 0.5 μM hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3) with a final concentration of about 0.1 ng / ml to about 5 ng / ml. The recovery medium may include 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 ng / ml to about 5 ng / ml, and TGF-α may be present at a final concentration of about 1.0 ng / ml to about 10 ng / ml. In addition, the medium for recovering CLECs may include cholera toxin from Vibrio cholerae (which may be commercially available from SigmaAldrich, for example, under catalog number C8052). If cholera toxin from Vibrio cholerae is used, it may be present at a final concentration of about 1×10 -11 M is about 1×10 - 10 The final concentration of M was present.

[0080] "DMEM" refers to Dulbecco's Modified Eagle's Medium, which was developed in 1969 and is a modification of Basal Medium Eagle (BME) (see Figure 1, which shows a data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L of glucose and was first reported for culturing 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 No. 11965-084), Sigma Aldrich (Catalog No. D5546), or Lonza, to name just a few suppliers. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is a DMEM medium available from Lonza at Catalog No. 12-604F. The medium is a DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is a DMEM medium from Sigma Aldrich Catalog No. D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0081] "F12" culture medium refers to Ham's F12 culture medium. This culture medium is also a standard cell culture medium, and is a nutrient mixture originally designed to cultivate various mammals and hybridoma cells when used in combination with serum and hormones and transferrin. Any commercially available Ham's F12 culture medium (for example, from ThermoFisher Scientific (Catalog Number 11765-054), Sigma Aldrich (Catalog Number N4888) or Lonza, only a few suppliers are listed) can be used in the present invention. In a preferred embodiment, the Ham's F12 culture medium from Lonza is used. "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 culture medium. In addition, DMEM / F12 (1:1) culture medium is a widely used basal culture medium that supports the growth of many different mammalian cells, which can be commercially obtained 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, available from Lonza at catalog number 12-719F (which is DMEM:F12 containing L-glutamine, 15mM HEPES and 3.151g / L glucose).

[0082] "M171" refers to culture medium 171, which has been developed as a basal medium for the growth and culture of normal human mammary epithelial cells. This basal medium is also widely used and can be commercially available, for example, from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (Cat. No. M171500). Any commercially available M171 culture medium can be used in the present invention. In a preferred embodiment, the M171 culture medium used herein is the M171 culture medium available from Life Technologies Corporation under Catalog No. M171500.

[0083] "Mammary Epithelial Basal Medium MCDB 170" refers to a basal nutrient medium for growing mammary epithelial cells, which is commercially available in powder form, for example, from Biological, Salem Massachusetts USA, under catalog number M2162 or from Bio-Connect BV, Huissen, The Netherlands, under catalog number (MBS652676_101).

[0084] EpiLife medium refers to a HEPES and bicarbonate buffered liquid medium that is prepared without calcium chloride and is typically used for long-term serum-free culture of human epidermal keratinocytes and human corneal epithelial cells and is designed for use in an incubator with an atmosphere of 5% CO2 and 95% air. It can be obtained from ThermoFisherScientific under catalog number MEPICF500 or from Sigma Aldrich under product code E 0151.

[0085] "CMRL medium" refers to a medium originally developed by Connaught Medical Research Laboratories for growing Earle's 'L' cells under serum-free conditions. CMRL medium is also known to be particularly useful for cloning monkey kidney cells and for growing other mammalian cell lines when supplemented with horse or calf serum. CMRL medium is commercially available, for example, from ThermoFisher Scientific (Catalog No. 11530037).

[0086] "FBS" refers to fetal bovine serum (also referred to as "fetal calf serum"), i.e., the blood fraction remaining after the natural coagulation of blood followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for eukaryotic cell in vitro cell culture, because it has very low levels of antibodies and contains more growth factors, allowing versatility in many different cell culture applications. FBS is preferably obtained from members of the International Serum Industry Association (ISIA), which mainly focuses on the safety and safe use of serum and animal-derived products by appropriate source traceability, authenticity of labeling and appropriate standardization and supervision. FBS suppliers as ISIA members include Gibco of Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Thermo Fisher Scientific and Life Science Production, only mentioning a few. In a currently preferred embodiment, FBS is obtained from GE Healthcare with catalog number A15-151.

[0087] The culture medium suitable for cell recovery can also contain compounds, which can inhibit inflammatory reactions and / or can also enhance cell survival and proliferation after transfection. An illustrative example of this compound can be a glucocorticoid. A glucocorticoid is a steroid hormone that can upregulate the expression of anti-inflammatory proteins in the nucleus and inhibit the expression of pro-inflammatory proteins in the cytoplasm. The glucocorticoid used herein can be prednisolone, methylprednisolone, dexamethasone, betamethasone, corticosterone or hydrocortisone, and only some illustrative examples of suitable glucocorticoids are listed. Two or more such glucocorticoids can also be used together, such as a mixture of corticosterone and hydrocortisone. A glucocorticoid can be used at any suitable concentration, for example, with a concentration of about 0.1 μM to about 2.5 μM or to about 5 μM. In an illustrative embodiment, the glucocorticoid suitable for recovering the culture medium of the transfected CLSC can be the hydrocortisone used at a concentration of about 0.1 μM to about 2.5 μM. In one embodiment, the concentration of hydrocortisone in the culture medium suitable for recovering transfected CLSCs is about 0.5 μM to about 2 μM. In one such illustrative embodiment, the concentration of hydrocortisone is about 1 μM.

[0088] The recovery of transfected CLSC can be carried out in a cell culture device such as a cell culture container. The cell culture container can be, but is not limited to, a culture bottle, a culture dish, a roller bottle and a multilayer plate. In addition, the cell culture container can be coated to provide a layer that can promote cell growth by providing metabolites to the cells. The coating of the cell culture container can be serum-derived or serum-free. An example of a serum-derived coating can be a coating with a gel-like protein from a basement membrane-like matrix such as Matrigel. The serum-free coating of the cell culture container can be characterized by being free of animals and xeno, thereby allowing cells to be cultured under cGMP conditions. An example of a serum-free coating of a cell culture container can be a coating with a recombinant protein or a portion thereof, such as, for example, a coating with an extracellular matrix protein, such as collagen, fibronectin, elastin, laminin, including, for example, laminin-511E8 fragments, or laminin 521, vitronectin, for example, with commercially available citronectin XF TM , CELLstart or Synthemax TM In one example of the present invention, the transfected CLECs may be preferably cultured in a cell culture vessel having a serum-derived coating, while the CLMCs may be preferably cultured in a cell culture vessel having a serum-free coating.

[0089] After the suitable time period, another cell culture medium can be replaced with a culture medium suitable for recovering the transfected CLSC. The suitable time period can be, for example, about 1, about 2 or about 3 days after transfection. Therefore, in one embodiment, culture medium replacement can be performed about 2 days after transfection. Another cell culture medium for culture medium replacement can also be a mixture of different cell culture media. In the present invention, any cell culture medium or cell culture medium mixture suitable for producing iPS can be used. In addition, suitable cell culture medium or cell culture medium mixture can contain compounds that can suppress inflammatory response and enhance cell survival. In the present invention, the culture medium suitable for cell recovery after transfection can be replaced with a mixture of two different cell culture media after the suitable time period, to ensure that when the cell undergoes somatic cell reprogramming, when it is changed from its native state to a more energetic state, the suitable blend of nutrients and growth factors is to the appropriate supply of cells. Therefore, the cell culture medium mixture of the present invention can be composed of a culture medium suitable for cell recovery and a second cell culture medium that can contain hydrocortisone. In a preferred embodiment, two different cell culture media are mixed at a ratio of about 1: 1 (v / v), wherein the mixture can be prepared by contacting 1 volume of a culture medium suitable for cell recovery with 1 volume of a second cell culture medium. In another preferred embodiment, two different cell culture media are mixed at a ratio of about 1: 2 (v / v) or 2: 1, wherein the mixture can be prepared by contacting 1 volume of a culture medium suitable for cell recovery with 2 volumes of a second cell culture medium (or 2 volumes of a culture medium suitable for cell recovery with 1 volume of a second cell culture medium). The second cell culture medium used to produce a cell culture mixture can be any cell culture medium suitable for enhancing or maintaining iPS proliferation (such a culture medium is also referred to herein as a "maintenance culture medium"). Using a mixture, such as a 1: 1 mixture of a culture medium for cell recovery and a maintenance culture medium, provides the advantage of allowing CLiPS cells to gradually transition from their homologous culture medium to ES / iPSC culture medium, rather than a sudden transition that may impair their viability. Without wishing to be bound by theory, it is assumed that about two days after transfection, some successfully transfected umbilical cord lining stem cells will begin to acquire pluripotent stem cell characteristics, accompanied by the acquisition of PSC nutritional requirements. Illustrative examples of such suitable cell culture media include, but are not limited to, commercial maintenance media such as mTeSR1, StemMACS TM iPS-BrewXF、TeSRT M -E8, mTeSR TM Plus, TeSR TM 2 or mTeSR TM 1. hPSC XFMedium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisherScientific), StemFit Basic02 (Ajinomoto Co. Inc), or PluriSTEM (Merck Millipore). If iPS colonies are cultured under animal- and xeno-free GMP conditions, the medium mTeSR can be preferably used. TM 1, because it is prepared under GMP conditions. Therefore, in a preferred embodiment, mTeSR1 can be the second cell culture medium for producing a 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 time period. This suitable time period can be about 3, about 4, about 5 or about 6 days after transfection. Therefore, in one embodiment, 4 days after transfection, the 1:1 (v / v) cell culture medium mixture can be replaced with the same mixture. After a suitable time period, the 1:1 (v / v) cell culture medium mixture can be further replaced with a second cell culture medium used only for producing a cell culture mixture. In this case, the suitable time period can be about 4, about 5, about 6 or about 7 days after transfection. In one embodiment, 6 days after transfection, the 1:1 (v / v) cell culture medium mixture can be replaced with a second cell culture medium. In a preferred embodiment, 6 days after transfection, mTeSR1 and mTeSR TM 1 Replacement of 1:1 (v / v) cell culture medium mixture. Regular cell culture medium replacement and replacement can help increase surviving CLiPS. Therefore, CLiPS colonies can grow and proliferate.

[0090] After the cell culture medium mixture is replaced with a cell culture medium, the CLiPS can be further cultured. To this end, the cell culture medium can also be replaced with the same culture medium regularly to ensure the proper supply of the cells with a suitable mixture of nutrients and growth factors. For example, the cell culture medium can be replaced every day or every two days, every three days or every four days. In one embodiment of the present invention, the cell culture medium can be replaced every two days. Therefore, the CLiPS colonies can further grow and proliferate.

[0091] At about 10, 11, 12, 13, 14, 15 or 16 days after transfection, CLiPS colonies may become visible to the naked eye (see Example 2). When a suitable size is reached, CLiPS can be selected and transferred to another coated culture container for further culture and proliferation. In this case, a suitable colony size may include a length of about 0.1 mm to about 2 mm in diameter. In one embodiment of the present invention, when a length of about 0.5 mm to about 1.5 mm in diameter is reached, CLiPS colonies can be selected, wherein about 20 days after transfection, CLiPS colonies can reach this size. In order to transfer CLiPS colonies of suitable size to another culture container, CLiPS colonies can be picked. If necessary, this can be performed manually. In order to facilitate colony picking, a device that allows an enlarged view of the colony can be used. An example of such a device can be a magnifying glass or a microscope. In the present invention, CLiPS can be selected and picked under a bright field microscope. Turning to the cell culture container, the CLiPS colony picked can be transferred to another cell culture container, wherein the coating of the cell culture container can be different from the coating of the cell culture container for recovering the transfected CLSC, or it can be the same. In a preferred embodiment, the coating of the culture container is the same, because the CLiPS derived from CLMC cultured under cGMP suitable conditions so far can remain free of animals and xenobiotics, thereby maintaining cGMP conditions. Therefore, the CLiPS colony derived from CLMC can be, for example, transferred to a cell culture container coated with a serum-free substance such as laminin-511E8 fragment for further cultivation (see Example 3). Alternatively, the CLiPS colony derived from CLEC and / or CLMC can be, for example, transferred to a cell culture container coated with a serum-derived substance such as Matrigel for further cultivation. The cell culture medium can preferably be the same as that used before the colony is picked. In an embodiment of the present invention, the cell culture medium can also be replaced regularly after the colony is picked. For example, the culture medium can be replaced every day, every two days or every three days. In a preferred embodiment of the present invention, after colony picking, the cell culture medium may be changed every day.

[0092] When a suitable confluence is reached, CLiPS colonies or cell groups formed by colonies are typically dissociated from the coated cell culture container and transferred to a larger cell culture container for further culture under the same culture conditions used directly after colony picking. Suitable confluence can 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% confluence. It is noted in this context that the term "cell group" is more suitable when used in connection with CLiPS proliferation that forms colonies, because when they reach about 70% to about 80% confluence, CLiPS cells do not present a colony-like appearance. In order to dissociate CLiPS colonies or cell groups formed by colonies from coated cell culture containers, any dissociation agent suitable for disrupting cell adhesion or 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 part of the present application, where dispase has been used to dissociate CLiPS colonies from coated cell culture vessels). The cell culture medium may also be replaced regularly, for example every day, every two days or every three days. In a preferred embodiment of the present invention, the cell culture medium may be replaced every day. In this way, CLiPS can further grow and proliferate.

[0093] In the present invention, CLiPS colonies or cell groups formed by colonies can be passaged when a suitable size is reached. Suitable sizes may correspond to about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95% confluence. In one embodiment of the present invention, CLiPS colonies or cell groups formed therefrom can be passaged when the culture reaches about 60-90% confluence. Therefore, in a preferred embodiment, CLiPS colonies or cell groups formed therefrom can be passaged when about 70-80% confluence is reached. For passage, CLiPS can be passaged at a suitable ratio, in which one volume of CLiPS can be in contact with multiple volumes of cell culture medium. In the present invention, CLiPS can 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), wherein the passage can be performed by dividing 1 volume of dissociated CLiPS into about 2, or about 3, or about 4 or about 5 volumes of dissociated CLiPS, respectively. In a preferred embodiment, CLiPS can be passaged at a ratio of about 1:3 (v / v). In order to allow the passage of the cultured CLiPS of the present invention, any enzyme suitable for separating cells from the culture container can also be used. For example, dispase can be used for this purpose. In addition, any chemical suitable for removing cell-cell adhesion can be used for CLiPS passage in the context of the present invention, wherein the concentration of the chemical can be suitable for removing cell-cell adhesion without damaging the cells. An illustrative example of such a chemical can be EDTA. Since EDTA can kill cells at higher concentrations, a suitable EDTA concentration of the present invention can be about 0.5 mM. In the present invention, the cell culture medium used for passage can be supplemented with a substance suitable for enhancing the survival of CLiPS during dissociation. For this purpose, any substance suitable for enhancing the survival of CLiPS during dissociation can be used. Examples of such suitable substances can be inhibitors of signal transduction pathways such as rho-associated protein kinase (ROCK) signal transduction pathways. Therefore, the RHO / ROCK pathway inhibitor Y-27632 can be an illustrative example of a substance suitable for enhancing the survival of dissociated CLiPS. Alternatively, a supplement with defined components for single-cell cloning of human iPS cells, such as CloneR TM (available from StemCell Technologies) to enhance survival of dissociated cells. In the present invention, passaged CLiPS may be cultured in a medium supplemented with a substance suitable for enhancing survival of dissociated CLiPS for a suitable period of time prior to differentiation into target cells.

[0094] By culturing CLiPS after passaging, a master cell bank containing (primary) isolated CLiPS can be obtained. In order to produce a master cell bank of CLiPS, the CLiPS cells obtained by the method described herein can be seeded in a culture container such as a cell culture plate. For this purpose, CLiPS can be suspended and cultured in any suitable culture medium, typically a maintenance medium for iPS cells, such as the commercial culture medium mentioned above, such as mTeSR1, StemMACS TM iPS-Brew XF, TeSRTME8, mTeSRTMPlus, TeSRTM2 or mTeSRTM1, hPSC XF Medium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co. Inc), or PluriSTEM (Merck Millipore). CLiPS derived from CLMC and CLiPS derived from CLEC can be cultured in such iPS maintenance medium. For subculture, CLiPS cells (CLMC and CLEC-derived CLiPS) can be seeded at any suitable concentration, for example, or about 0.5×10 6 cells / ml to about 5.0×10 6 In one embodiment, the concentration of about 1.0×10 6 Cells are suspended at a concentration of 10 cells / ml for subculture. Subculture can be carried out by culturing in a simple culture bottle, or in, for example, a multilayer system such as CellSTACK (Corning, NY, USA) or a cell factory (Nunc, part of ThermoFisher Scientific Inc., Waltham, MA, USA), which can be stacked in an incubator. Alternatively, subculture can also be carried out in a closed, self-sustaining system such as a bioreactor. Different designs of bioreactors are known to those skilled in the art, such as parallel plates, hollow fibers or microfluidic bioreactors. See, for example, Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review" (ibid.). An illustrative example of a commercial hollow fiber bioreactor is Cell Expansion System (Terumo BCT, Inc), which has been used, for example, for the expansion of bone marrow mesenchymal stem cells in clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 Aug; 16(8): 1048-1058) and the expansion of high-purity umbilical cord lining mesenchymal stem cell populations described in International Patent Application WO 2018 / 067071. Another example of a commercially available bioreactor that can be used for subculture of the CLiPS population of the present invention is the Xuri Cell Expansion System available from GE Healthcare. If a working cell bank for therapeutic applications is to be produced under GMP conditions and a high number of cells is required, the CLiPS population is grown in an automated system such as Cultivation in the Cell Expansion System is particularly beneficial. For subculture, CLiPS can also be cultured until the appropriate amount of cells have grown. In an illustrative embodiment, CLiPS is subcultured until CLiPS reaches about 70% to about 80% confluence. The separation / cultivation of CLiPS groups can be carried out under standard conditions for mammalian cell culture. Once the required / appropriate amount of CLiPS is obtained from the subculture, the cells are harvested by moving them out of the culture container for subculture. The harvesting of CLiPS is usually carried out by enzyme treatment. The separated CLiPS is then collected and used directly or stored for further use. Usually, it is preserved by cryopreservation. The term "cryopreservation" is used in its conventional meaning herein to describe a method in which CLiPS is preserved by cooling to a low subzero temperature such as (usually) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be performed as known to those skilled in the art, and may include the use of a cryoprotectant, such as dimethyl sulfoxide (DMSO) or glycerol, which slows down the formation of ice crystals in CLiPS cells.

[0095] The present invention also relates to CLiPS obtainable by the methods described herein and CLiPS obtained by the methods described herein. CLiPS obtainable / obtained by the present invention can grow and proliferate robustly (see Examples 2 and 3). Therefore, CLiPS culture may be more effective than culturing iPS derived from, for example, bone marrow stroma, adipose tissue, dermis or Wharton's jelly. CLiPS functional analysis reveals the expression of human embryonic stem cell markers, indicating self-renewal properties and normal karyotype (see Examples 4 and 5). In addition, CLiPS can differentiate into a variety of cell types (functional target cells) in vitro and in vivo, indicating pluripotency (see Example 6). Therefore, CLiPS is highly suitable for medical and therapeutic applications. Therefore, the present invention also relates to pharmaceutical compositions comprising iPS obtainable / obtained by the methods described herein.

[0096] The present invention further relates to a method for differentiating CLiPS into target cells under conditions suitable for differentiation. Examples of suitable target cells include, but are by no means limited to, neuronal cells, dopaminergic neuronal cells, oligodendrocytes, astrocytes, cortical neurons, hepatocytes, chondrocytes, myocytes, osteocytes, 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, only a few are mentioned. In order to promote directed differentiation into target cells, CLiPS can be exposed to an initiating substance, usually under conditions known to those skilled in the art that iPS derived from other sources will be differentiated into target cells. Exposure can be carried out under suitable conditions, which may include culturing in a cell culture vessel filled with a cell culture medium suitable for initiating CLiPS differentiation and subsequent culturing. In the present invention, any cell culture medium suitable for initiating, proliferating and differentiating iPS cells can be used, wherein the composition of the culture medium and thus the differentiation method can depend on the target cells and can be obtained in known protocols for differentiating iPS cells into desired target cells (in this regard, see Hirschi et al. "Induced Pluripotent Stem Cells for Regenerative Medicine" Annu Rev Biomed Eng. 2014 Jul 11; 16: 277-294 or Shi et al. "Induced pluripotent stem cell technology: a decade of progress" Nat Rev Drug Discov. 2017 Feb; 16(2): 115-130). For example, CLiPS cells can be cultured in a culture medium suitable for the proliferation and differentiation of CLiPS cells into dopaminergic neuronal cells. In this case, the culture medium can be a neurobasal medium supplemented with growth factors 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 γ-secretase inhibitors such as (2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT) that induce neuronal differentiation. An illustrative example of such a culture medium is NB27. The differentiation of CLiPS into dopaminergic neuronal cells is exemplarily shown in Example 7. As another example, CLiPS can be cultured in a culture medium suitable for the proliferation and differentiation of CLiPS into hepatocytes. In this case, the culture medium can be a protein-, lipid-, and growth factor-free culture medium supplemented with compounds that induce differentiation into a mesendodermal fate.RPMI 1640-B27 supplemented with activin A can be an illustrative example of a suitable culture medium for CLiPS differentiation into hepatocytes. Example 8 exemplifies the differentiation of CLiPS into hepatocytes. As another illustrative example, CLiPS can be cultured in a culture medium suitable for CLiPS proliferation and differentiation into cardiomyocytes. In this case, the culture medium can be a protein-free, lipid-free and growth factor-free culture medium supplemented with a glycogen synthase kinase 3 inhibitor such as CHIR99021. RPMI / 2%-B27 minus insulin can be an example of a suitable culture medium for CLiPS differentiation into hepatocytes. Example 9 exemplifies the differentiation of CLiPS into cardiomyocytes. As another illustrative example, CLiPS can be differentiated into oligodendrocytes using a chemically defined growth factor-rich culture medium that allows differentiation into paired box 6-positive (PAX6+) neural stem cells, which then produce oligodendrocyte transcription factor-positive (OLIG2+) progenitor cells (see Example 10). In this case, it can be noted that the differentiation of CLiPS into target cells can also be carried out under conditions suitable for cGMP production.

[0097] The present invention also includes pharmaceutical compositions comprising differentiated CLiPS obtained by the methods described herein. Analysis of the immunogenicity of CLiPS and its neural derivatives showed reduced immunogenicity (Example 11). Examples of pharmaceutical compositions comprising differentiated CLiPS are injection solutions or any type of transplants suitable for implanting differentiated CLiPS. In one example, such a transplant may include a multilayered tissue derived from differentiated CLiPS, such as an organ or part thereof. In one embodiment, a transplant suitable for implanting differentiated CLiPS may include an implantable matrix coated with differentiated CLiPS. Pharmaceutical compositions may be formulated / adjusted for parenteral application. In this case, parenteral application may include a sterile preparation intended for injection, infusion or implantation in a human or animal body. Transplantation of CLiPS-derived dopaminergic neurons in fully immunocompetent mouse and rat Parkinson's disease models showed significant recovery of functional transplantation and even dopamine reuptake function (see Example 12 and Example 13).

[0098] The present invention further includes a method for treating a congenital or acquired degenerative disorder in a subject, wherein the subject can be selected from mice, rats, rabbits, pigs, dogs, cats, non-human primates or humans. In a preferred embodiment, the subject is a human. In this context, treatment can include administering to the subject a target cell differentiated from CLiPS by the method as described herein. The disease can be any known disease that is considered to be treated by cell-based therapy, in this context, see, for example, Shi et al. "Induced pluripotent stem cell technology: a decade of progress" (ibid.). Congenital or acquired degenerative disorders may have different origins. For example, this congenital or acquired degenerative disorder can be a neurological disorder, such as, for example, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA) and Batten disease. Examples of liver degenerative disorders can especially be liver failure, cirrhosis and viral hepatitis. The congenital or acquired degenerative disorder may also be a cardiac disorder, including, among others, acute Danon's disease, short QT syndrome, Brugada syndrome, myocardial infarction, Jervell and Lange-Nielsen syndrome. The disorder may also be an autoimmune disease, such as multiple sclerosis.

[0099] The present invention also relates to extracellular membrane vesicles that can be produced by CLiPS or differentiated derivatives of CLiPS. Such vesicles can include, but are not limited to, vesicles with a diameter ranging from 30 nanometers (nm) to 150 nanometers (nm), also known as exosomes. Initially thought to be primarily responsible for excretion functions, exosomes are now known to participate in a variety of important biological processes, such as cell-cell communication, cell senescence, proliferation and differentiation, tissue homeostasis, tissue repair and regeneration, antigen presentation and immune regulation (see, e.g., Pegtel, DM and SJ Gould, Exosomes. Annu Rev Biochem, 2019. 88: p. 487-514 or Kalluri, R. and VS Le Bleu, The biology, function, and biomedical applications of exosomes. Science, 2020. 367 (6478). Exosomes are involved in a wide range of diseases, including cancer (see, e.g., 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 disease 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 AFHill, The role of extracellular vesicles in neurodegenerative diseases. Biochem Biophys Res Commun, 2017. 483(4): p. 1178-1186), mental 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, L.R. Edelstein et al., Editors. 2020, Academic Press. p. 343–356) and more. .

[0100] Exosome cargo has been shown to consist of a variety of 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 have therapeutic properties. In this regard, mesenchymal stem cells (MSCs) isolated from different sources such as bone marrow, adipose tissue, and umbilical cord have been shown to be particularly advantageous. MSC-derived exosomes have shown potential therapeutic effects in animal models of corneal, cardiovascular, Alzheimer's, Parkinson's, and inflammatory bowel diseases. 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 (Song, 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, the administration of cell-free iPS-derived exosomes is considered safer than the administration of iPS-derived cells (Riazifar, M. et al., Stem Cell Extracellular Vesicles: Extended Messages of Regeneration. Annu Rev Pharmacol Toxicol, 2017.57: p.125-154). Notably, the therapeutic properties of exosomes isolated from iPS differentiation derivatives have been demonstrated. For example, treatment with exosomes purified from iPS-derived cardiomyocytes enhanced cardiac recovery in a mouse model of myocardial infarction, with significant reductions in apoptosis and fibrosis compared to untreated animals.Exosomes also rescued iPS-cardiomyocytes in vitro culture 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 exosomes separated from iPS-derived MSCs accelerated the proliferation of human skin fibroblasts and human keratinocytes in an in vitro scratch assay, and enhanced wound healing. Compared with those isolated from primary MSCs, the effects of these exosomes were not significantly different (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).

[0101] Therefore, according to these reports, the extracellular membrane vesicles or exosomes produced by the CLiPS of the present invention (derived from CLMC or CLEC) or differentiated derivatives of CLiPS are believed to be useful for treating diseases, including the above-mentioned exemplary diseases such as cancer, osteoarthritis, central nervous system diseases such as stroke, Alzheimer's disease (AD), Parkinson's disease (PD), prion disease and amyotrophic lateral sclerosis (ALS), mental disorders or metabolic diseases.

[0102] In addition, taking advantage of their efficient cargo delivery capabilities, exosomes are actively sought as delivery vehicles for promoting the cellular uptake of various therapeutic agents such as microRNAs, drugs, and peptides (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 extracellular membrane vesicles or exosomes produced by the CLiPS (derived from CLMC or CLEC) or the differentiated derivatives of CLiPS of the present invention are also considered to be used as delivery vectors for promoting cellular uptake of therapeutic agents. Therefore, the present invention also includes the use of CLiPS or differentiated derivatives of CLiPS for the purpose of delivering exogenously loaded or transgenic expressed molecules.

[0103] The extracellular membrane vesicles and exosomes produced by CLiPS (derived from CLMC or CLEC) or differentiated derivatives of CLiPS can be separated using the corresponding methods described in the literature. Typically, exosomes are purified from the extracellular environment into which they are secreted. Known methods for isolating exosomes include ultracentrifugation, ultrafiltration, size exclusion chromatography, field flow fractionation, polymer coprecipitation, immunoaffinity, microfluidics or acoustic nanofilters. All of these methods can be used to separate exosomes produced by CLiPS or differentiated derivatives of CLiPS described herein.

[0104] The present invention further relates to a specific method for differentiating iPS cells into RPE cells, the iPS cells being derived from umbilical cord amniotic stem cells as defined elsewhere herein and also referred to as CLiPS as defined herein. The differentiation method comprises culturing the CLiPs in a differentiation medium under conditions suitable for differentiation into RPE cells.

[0105] "Retinal pigment epithelium (abbreviation: RPE) cell" refers to the cell derived from / from / taken from the retinal pigment epithelium. In other words, such cell is included by the retinal pigment epithelium, and will be defined in more detail below. Use the rapid, directed and improved differentiation method according to the present invention to achieve RPE differentiation from CLiPS. The CLiPS used to differentiate into RPE cells as used herein can be derived from umbilical cord lining mesenchymal cells (such as CLMC23, CLMC30, CLMC44) and / or be derived from umbilical cord lining ectoderm cells (such as CLEC23). In a preferred embodiment, the CLiPS used to differentiate into RPE cells as used herein is any one of CLMC23, CLMC30, CLMC44 or CLEC23. In a preferred embodiment, the CLiPS used to differentiate into RPE cells as used herein is CLMC23. In another preferred embodiment, the CLiPS used to differentiate into RPE cells as used herein is CLMC30. In another preferred embodiment, the CLiPS used to differentiate into RPE cells as used herein is CLMC44. In another preferred embodiment, the CLiPS used to be differentiated into RPE cell as used herein is CLEC23. The RPE cell differentiated by CLiPS defined herein by differentiation method as described herein can refer to CLiPS derived RPE cell or CLiPS-RPE.Use differentiation method according to the present invention, the differentiation of the RPE cell derived from CLiPS can be with the differentiation of the RPE cell (also referred to as ES derived RPE when referring to such RPE cell) derived from ES cell (such as H9 ES cell) and / or with the iPS cell (also referred to as skin iPS) derived from skin; Therefore when referring to such RPE cell, it is skin iPS derived RPE (such as Asf5, AGO or HDFA cell) The differentiation of the RPE cell is compared (referring to the embodiment part).

[0106] The differentiation medium used in the differentiation method for making iPS cells differentiate into RPE cells including the iPS cells derived from umbilical cord amniotic stem cells is preferably DMEM (Dulbecco's modified Eagle's medium) culture medium comprising N2 supplements, B27 supplements and non-essential amino acids (NEAA) defined herein, even more preferably DMEM (Dulbecco's modified Eagle's medium) / F12 (Ham's F12 culture medium) culture medium comprising N2 supplements, B27 supplements and non-essential amino acids (NEAA) defined elsewhere herein. In a preferred embodiment, the DMEM / F12 culture medium used in the differentiation method for making iPS cells differentiate into RPE cells comprises 1 × N2 supplements, 1 × B27 supplements and 1 × NEAA. Comprise 1 × N2 supplements, 1 × B27 supplements and 1 × NEAA in the culture medium to mean that final concentration is 1x, which can also be seen in the embodiments below. In the embodiment, differentiation medium is obtained by mixing the following to obtain a final volume of 1000ml culture medium, preferably DMEM culture medium, even more preferably DMEM / F12 culture medium as defined herein:

[0107] -10 ml of 100×N2 supplement;

[0108] -20 mL of 50× B27 supplement;

[0109] -10 mL of 100×NEAA;

[0110] -960 mL of DMEM, preferably DMEM / F12.

[0111] Differentiation medium as defined herein can further comprise / be supplemented with various growth factors and / or cytokines as defined elsewhere herein.This differentiation medium as defined above can refer to the basal medium for iPS cultivation.Then such basal differentiation medium can be further improved / supplemented for cultivating iPS cells as defined herein, so that the cell differentiation is made into RPE cells by using the method making the iPS cell differentiation into RPE cells according to the present invention.

[0112] In particular, the differentiation medium used in the method for making iPS cells differentiate into RPE cells of the present invention for cultivating iPS cells to differentiate the cells into RPE cells can include the first differentiation medium, which additionally includes at least any one of IGF1, DKK1, nicotinamide or LDN-193189. The first differentiation medium is based on the basal medium, and the basal medium includes DMEM culture medium, preferably includes DMEM / F12 culture medium containing N2 supplement, B27 supplement and NEAA, and even more preferably includes DMEM / F12 culture medium containing 1 × N2 supplement, 1 × B27 supplement and 1 × NEAA. In a preferred embodiment, the first differentiation medium as defined herein additionally includes IGF1, DKK1, nicotinamide and LDN-193189.

[0113] In addition, the differentiation medium used in the method for making iPS cells differentiate into RPE cells of the present invention for cultivating iPS cells to make the cells differentiate into RPE cells can additionally or alternatively include the second differentiation medium, which additionally includes at least any one of IGF1, DKK1, nicotinamide, LDN-193189 or b-FGF. The second differentiation medium is also based on the basal medium, and the basal medium includes DMEM culture medium, preferably includes DMEM / F12 culture medium containing N2 supplement, B27 supplement and NEAA, and even more preferably includes DMEM / F12 culture medium containing 1 × N2 supplement, 1 × B27 supplement and 1 × NEAA. In a preferred embodiment, the second differentiation medium as defined herein additionally includes IGF1, DKK1, nicotinamide, LDN-193189 and b-FGF.

[0114] In addition, the differentiation medium used in the method for making iPS cells differentiate into RPE cells of the present invention for cultivating iPS cells so that the cells are differentiated into RPE cells can additionally or alternatively include the 3rd differentiation medium, which additionally includes at least any one of IGF1, DKK1 or activin A. The 3rd differentiation medium is also based on the basal medium, and the basal medium includes the basal medium of DMEM culture medium, preferably includes the DMEM / F12 culture medium containing N2 supplement, B27 supplement and NEAA, and even more preferably includes the DMEM / F12 culture medium containing 1 × N2 supplement, 1 × B27 supplement and 1 × NEAA. In a preferred embodiment, the 3rd differentiation medium as defined herein additionally includes IGF1, DKK1 and activin A.

[0115] In addition, the differentiation medium used in the method for making iPS cells differentiate into RPE cells of the present invention for cultivating iPS cells to make the cells differentiate into RPE cells can additionally or alternatively include the 4th differentiation medium, which additionally includes activin A and SU5402 or activin A and PD17307. The 4th differentiation medium is also based on the basal medium, and the basal medium includes DMEM culture medium, preferably includes DMEM / F12 culture medium containing N2 supplement, B27 supplement and NEAA, and even more preferably includes DMEM / F12 culture medium containing 1 × N2 supplement, 1 × B27 supplement and 1 × NEAA. In a preferred embodiment, the 4th differentiation medium as defined herein additionally includes activin A and PD17307. Due to PD17307 being applied with a concentration lower than fibroblast growth factor inhibitor SU5402, this reduces the undesirable changes in gene expression caused by applying higher concentrations when using SU5402, so the differentiation method of the present invention preferably PD17307 (Figure 19).

[0116] In addition, the differentiation medium used in the method that makes iPS cell differentiate into RPE cell of the present invention for cultivating iPS cell so that described cell differentiation becomes RPE cell can additionally or alternatively include the 5th differentiation medium, it additionally includes at least any one of activin A, CHIR99021 or SU5402;Or include at least one of activin A, CHIR99021 or PD17307.Described 5th differentiation medium is also based on described basal medium, and described basal medium includes DMEM culture medium, preferably includes DMEM / F12 culture medium containing N2 supplement, B27 supplement and NEAA, even more preferably includes DMEM / F12 culture medium containing 1 × N2 supplement, 1 × B27 supplement and 1 × NEAA.In preferred embodiments, as defined herein, the 5th differentiation medium additionally includes activin A, CHIR99021 and PD17307 (for the same reason as defined above, PD17307 is used). The 5th differentiation medium preferably comprises activin A, SU5402 or PD17307, preferably PD17307, and the CHIR99021 of the first concentration, and this first concentration is lower than 3 μM.When using the 5th differentiation medium to cultivate iPS cell to be differentiated into RPE cell again, the concentration of CHIR99021 as the activator of Wnt signal transduction pathway increases subsequently.The gradual increase of concentration prevents the excessive cell death caused by it when using high concentration CHIR.This improves the productive rate of pigmentation RPE cell.When applying the 5th differentiation medium subsequently, it preferably comprises activin A, SU5402 or PD17307, preferably PD17307, and the CHIR99021 of the second concentration, and this second concentration is about 3 μM.

[0117] When IGF1 is applied as a supplement to a differentiation medium defined elsewhere herein, 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 is used; or at a final concentration in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml to about 11 ng / ml. In a preferred embodiment, IGF1 is used in a 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 in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml 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 in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml 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 in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml.The present invention further includes the method that is differentiated into RPE cell, and described method is included in the differentiation medium defined in other places herein and cultivates described iPS cell, wherein IGF1 applies 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 continuous days, most preferably use about 6 (continuous) days / day with the final concentration of about 10ng / ml in the described differentiation medium defined herein.When IGF1 is used in the first differentiation medium as defined herein, IGF1 uses about 2 days, preferably about 2 continuous days (this means the 0th day to the 2nd day of cultivation), even more preferably use about 2 (continuous) days with the final concentration of about 10ng / ml.When IGF1 is used in the second differentiation medium as defined herein, IGF1 uses about 2 days, preferably about 2 continuous days (this means the 2nd day to the 4th day of cultivation), even more preferably use about 2 (continuous) days with the final concentration of about 10ng / ml. When IGF1 is used in the third differentiation medium as defined herein, IGF1 is used for about 2 days, preferably for about 2 consecutive days (this means day 4 to day 6 of culture), even more preferably for about 2 (consecutive) days at a final concentration of about 10 ng / ml.

[0118] When DKK1 is applied to a differentiation medium as defined elsewhere herein, 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 is used; or a final concentration in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml 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 even more preferred embodiments, 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 in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml 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 in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml 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 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 in the range of about 5 ng / ml to about 15 ng / ml, about 6 ng / ml to about 14 ng / ml, about 7 ng / ml to about 13 ng / ml, about 8 ng / ml to about 12 ng / ml, about 9 ng / ml to about 11 ng / ml, most preferably at a final concentration of about 10 ng / ml.The present invention further includes the method for being differentiated into RPE cell, and the method described is included in the differentiation medium defined in other places herein, and cultivates the iPS cell, wherein DKK1 is applied in the differentiation medium defined herein 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 continuous days, most preferably about 6 (continuous) days / day with a final concentration of about 10ng / ml.When DKK1 is used in the first differentiation medium as defined herein, DKK1 uses about 2 days, preferably about 2 continuous days (this means the 0th day to the 2nd day of cultivation), and even more preferably about 2 (continuous) days are used with a final concentration of about 10ng / ml.When DKK1 is used in the second differentiation medium as defined herein, DKK1 uses about 2 days, preferably about 2 continuous days (this means the 2nd day to the 4th day of cultivation), and even more preferably about 2 (continuous) days are used with a final concentration of about 10ng / ml. When DKK1 is used in the third differentiation medium as defined herein, DKK1 is used for about 2 days, preferably for about 2 consecutive days (which means day 4 to day 6 of culture), even more preferably for about 2 (consecutive) days at a final concentration of about 10 ng / ml.

[0119] When nicotinamide is used in a differentiation medium as defined elsewhere herein, 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 is used; or a final concentration in the range of about 5 mM to about 15 mM, about 6 mM to about 14 mM, about 7 mM to about 13 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM. In a preferred embodiment, nicotinamide is used in a differentiation medium as defined elsewhere herein at a final concentration of about 10 mM. In even more preferred embodiments, nicotinamide is used in the 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 in the range of about 5 mM to about 15 mM, about 6 mM to about 14 mM, about 7 mM to about 13 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM, most preferably at a final concentration of about 10 mM. In another even more preferred embodiment, nicotinamide is with at least about 5mM, at least about 6mM, at least about 7mM, at least about 8mM, at least about 9mM or at least about 10mM; or at a final concentration in the range of about 5mM to about 15mM, about 6mM to about 14mM, about 7mM to about 13mM, about 8mM to about 12mM, about 9mM to about 11mM, most preferably used in the second differentiation medium as defined elsewhere herein with a final concentration of about 10mM. The present invention further includes a method for being differentiated into RPE cells, the method being included in cultivating the iPS cells in a differentiation medium as defined elsewhere herein, wherein nicotinamide is applied in the differentiation medium as defined herein for at least about 2 days, at least about 3 days, at least about 4 days, preferably about 4 days, even more preferably about 4 consecutive days, most preferably using about 4 (individual consecutive) days / days with a final concentration of about 10mM. When nicotinamide is used in the first differentiation medium as defined herein, nicotinamide is used for about 2 days, preferably for about 2 consecutive days (this means day 0 to day 2 of culture), and even more preferably for about 2 (consecutive) days at a final concentration of about 10 mM. When nicotinamide is used in the second differentiation medium as defined herein, nicotinamide is used for about 2 days, preferably for about 2 consecutive days (this means day 2 to day 4 of culture), and even more preferably for about 2 (consecutive) days at a final concentration of about 10 mM.

[0120] When LDN-193189 is applied in a differentiation medium as 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 a differentiation medium as defined elsewhere herein at a final concentration of about 1 μM. In another preferred embodiment, LDN-193189 is used in a differentiation medium as 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 μM to about 1.5 μM, about 0.6 μM to about 1.4 μM, about 0.7 μM to about 1.3 μM, about 0.8 μM to about 1.2 μM, about 0.9 μM to about 1.1 μM, 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 μM to about 0.3 μM, about 0.11 μM to about 0.29 μM, about 0.12 μM to about 0.28 μM, about 0.13 μM to about 0.27 μM, about 0.14 μM to about 0.26 μM, about 0.15 μM to about 0.25 μM, most preferably at a final concentration of about 0.2 μM. The present invention further includes the method that is divided into RPE cell, and the method described is included in the differentiation medium as defined elsewhere herein, and cultivates the iPS cell, wherein LDN-193189 is applied in the differentiation medium as defined elsewhere herein, at least about 2 days, at least about 3 days, at least about 4 days, preferably about 4 days, even more preferably about 4 continuous days, most preferably with the concentration of at least about 0.1 μM as defined elsewhere herein, use about 4 (continuous) days / day.When LDN-193189 is used in the first differentiation medium as defined herein, LDN-193189 uses about 2 days, preferably about 2 continuous days (this means the 0th day to the 2nd day of cultivation), and even more preferably LDN-193189 uses about 2 (continuous) days with the concentration of about 1 μM. When LDN-193189 is used in the second differentiation medium as defined herein, LDN-193189 is used for about 2 days, preferably for about 2 consecutive days (which means day 2 to day 4 of culture), even more preferably LDN-193189 is used at a concentration of about 0.2 μM for about 2 (consecutive) days.

[0121] When b-FGF is applied in a differentiation medium defined elsewhere herein, 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 ng / ml to about 7.5 ng / ml, about 3 ng / ml to about 7 ng / ml, about 3.5 ng / ml to about 6.5 ng / ml, about 4 ng / ml to about 6 ng / ml, about 4.5 ng / ml to about 5.5 ng / ml. In a preferred embodiment, b-FGF is used in a differentiation medium defined elsewhere herein at a final concentration of about 5 ng / ml. In even more preferred embodiments, b-FGF is with at least about 2.5ng / ml, at least about 3ng / ml, at least about 3.5ng / ml, at least about 4ng / ml, at least about 4.5ng / ml or at least about 5ng / ml;Or at about 2.5ng / ml to about 7.5ng / ml, about 3ng / ml to about 7ng / ml, about 3.5ng / ml to about 6.5ng / ml, about 4ng / ml to about 6ng / ml, about 4.5ng / ml to about 5.5ng / ml in the scope of final concentration, most preferably with the final concentration of about 5ng / ml in the second differentiation medium defined elsewhere herein.The present invention further includes the method that is differentiated into RPE cell, described method is included in the differentiation medium as defined elsewhere herein, cultivates described iPS cell, wherein b-FGF is applied in the described differentiation medium defined herein at least about 1 day, at least about 2 days, preferably about 2 days, even more preferably about 2 continuous days, most preferably with the final concentration of about 5ng / ml, use about 2 (individual continuous) days. When b-FGF is used in the second differentiation medium defined herein, b-FGF is used for about 2 days, preferably for about 2 consecutive days (this means day 2 to day 4 of culture), even more preferably for about 2 (consecutive) days at a final concentration of about 5 ng / ml.

[0122] When activin A is applied in a differentiation medium as defined elsewhere herein, 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 is used; or a final concentration in the range of about 50 ng / ml to about 150 ng / ml, about 60 ng / ml to about 140 ng / ml, about 70 ng / ml to about 130 ng / ml, about 80 ng / ml to about 120 ng / ml, about 90 ng / ml to about 110 ng / ml. 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 the 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 in the range of about 50 ng / ml to about 150 ng / ml, about 60 ng / ml to about 140 ng / ml, about 70 ng / ml to about 130 ng / ml, about 80 ng / ml to about 120 ng / ml, about 90 ng / ml 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 the 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 in the range of about 50 ng / ml to about 150 ng / ml, about 60 ng / ml to about 140 ng / ml, about 70 ng / ml to about 130 ng / ml, about 80 ng / ml to about 120 ng / ml, about 90 ng / ml to about 110 ng / ml, most preferably at a final concentration of about 100 ng / ml. In yet an even more preferred embodiment, activin A is used in the 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 in the range of about 50 ng / ml to about 150 ng / ml, about 60 ng / ml to about 140 ng / ml, about 70 ng / ml to about 130 ng / ml, about 80 ng / ml to about 120 ng / ml, about 90 ng / ml to about 110 ng / ml, most preferably at a final concentration of about 100 ng / ml.The present invention further includes the method that is divided into RPE cell, described method is included in the differentiation medium as defined elsewhere herein, cultivates described iPS cell, wherein activin A applies 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 continuous days, most preferably use about 12 (continuous) days with the final concentration of about 100ng / ml in the differentiation medium as defined herein.When activin A is applied in the 3rd differentiation medium as defined herein, activin A uses about 2 days, preferably about 2 continuous days (this means the 4th day to the 6th day of cultivation), even more preferably use about 2 (continuous) days / day with the final concentration of about 100ng / ml. When activin A is used in the fourth differentiation medium as defined herein, activin A is used for about 2 days, preferably for about 2 consecutive days (this means the 6th to 8th day of culture), and even more preferably for about 2 (consecutive) days / day at a final concentration of about 100 ng / ml. When activin A is used in the fifth differentiation medium as defined herein, activin A is used for about 8 days, preferably for about 8 consecutive days (this means the 8th to 16th day of culture), and even more preferably for about 8 (consecutive) days / day at a final concentration of about 100 ng / ml.

[0123] When SU5402 is applied in a differentiation medium as defined elsewhere herein, it is used 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 in the range of about 5 μM to about 15 μM, about 6 μM to about 14 μM, about 7 μM to about 13 μM, about 8 μM to about 12 μM, about 9 μM to about 11 μM. 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 the 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 in the range of about 5 μM to about 15 μM, about 6 μM to about 14 μM, about 7 μM to about 13 μM, about 8 μM to about 12 μM, about 9 μM to about 11 μM, most preferably at a final concentration of about 10 μM. In another even more preferred embodiment, SU5402 is with 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 μM to about 15 μM, about 6 μM to about 14 μM, about 7 μM to about 13 μM, about 8 μM to about 12 μM, about 9 μM to about 11 μM, most preferably with a final concentration of about 10 μM in the 5th differentiation medium defined elsewhere herein. The present invention further includes a method for differentiating into RPE cells, the method being included in culturing the iPS cells in a differentiation medium as defined elsewhere herein, wherein SU5402 is applied to the differentiation medium as defined elsewhere herein for about 10 days, even more preferably for about 10 consecutive days, most preferably using about 10 (continuous) days / days with a concentration of about 10 μM. When SU5402 is applied in the fourth differentiation medium as defined herein, SU5402 is used for about 2 days, preferably for about 2 consecutive days (this means the 6th to 8th day of culture), and even more preferably SU5402 is used for about 2 (consecutive) days / day at a concentration of about 10 μM. When SU5402 is applied in the fifth differentiation medium as defined herein, SU5402 is used for about 8 days, preferably for about 8 consecutive days (this means the 8th to 16th day of culture), and even more preferably for about 8 (consecutive) days / day at a final concentration of about 10 μM.

[0124] When PD17307 is applied in a differentiation medium as defined elsewhere herein, 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; or a final concentration in the range of about 0.5 μM to about 1.5 μM, about 0.6 μM to about 1.4 μM, about 0.7 μM to about 1.3 μM, about 0.8 μM to about 1.2 μM, about 0.9 μM to about 1.1 μM. 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 in a range of about 0.5 μM to about 1.5 μM, about 0.6 μM to about 1.4 μM, about 0.7 μM to about 1.3 μM, about 0.8 μM to about 1.2 μM, about 0.9 μM to about 1.1 μM, most preferably at a final concentration of about 1 μM. In another even more preferred embodiment, PD17307 is 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 μM to about 1.5 μM, about 0.6 μM to about 1.4 μM, about 0.7 μM to about 1.3 μM, about 0.8 μM to about 1.2 μM, about 0.9 μM to about 1.1 μM, most preferably with a final concentration of about 1 μM in the fifth differentiation medium as defined elsewhere herein. The present invention also includes a method for differentiating into RPE cells, the method being included in culturing the iPS cells in a differentiation medium as defined elsewhere herein, wherein PD17307 is applied in the differentiation medium as defined elsewhere herein for about 10 days, even more preferably for about 10 consecutive days, most preferably with a concentration of about 1 μM as defined elsewhere herein for about 10 days. When PD17307 is applied in the fourth differentiation medium as defined herein, PD17307 is used for about 2 days, preferably for about 2 consecutive days (this means day 6 to day 8 of culture, and even more preferably PD17307 is used for about 2 (consecutive) days / day at a concentration of about 1 μM. When PD17307 is applied in the fifth differentiation medium defined herein, PD17307 is used for about 8 days, preferably for about 8 consecutive days (this means day 8 to day 16 of culture), and even more preferably for about 8 (consecutive) days / day at a final concentration of about 1 μM.

[0125] When CHIR99021 is applied in a differentiation medium 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 defined elsewhere herein at a final concentration of about 1.5 μM. The CHIR99021 used in the differentiation medium as defined herein can be used for culturing cells and reaching about 3 continuous culture days, and the cell is the iPS cell of the present invention, and it is differentiated into or has been differentiated into RPE cells. In a preferred embodiment, CHIR99021 is used in the differentiation medium as defined elsewhere herein with a final concentration of about 1.5 μM to be used for culturing cells and reaching about 3 continuous culture days, and the cell is differentiated into or has been differentiated into the iPS cell of the present invention of RPE cells.

[0126] In even more preferred embodiments, CHIR99021 is used in the fifth differentiation medium 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 μM and less than about 1.5 μM. In even more preferred embodiments, CHIR99021 is present in an amount 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 1.8 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 1.8 μM, at least about 1 μM and less than about 1.8 μM, at least about 1 μM and less than about 1.9 μM, at least about 1 μM and less than about 1.8 ... A final concentration of about 1 μM and less than about 1.6 μM is used in the fifth differentiation medium defined elsewhere in this article for culturing cells for about 3 consecutive culture days, wherein the cells are differentiated into or have differentiated into the iPS cells of the present invention of RPE cells, and most preferably is used in the fifth differentiation medium defined elsewhere in this article at a final concentration of about 1.5 μM for culturing cells for about 3 consecutive culture days (meaning the 8th to 11th day of culture), wherein the cells are differentiated into or have differentiated into the iPS cells of the present invention of RPE cells.

[0127] When CHIR99021 is applied to differentiation medium, preferably applied in the 5th differentiation medium as defined above, CHIR99021 is applied in differentiation medium again, for culturing cells subsequently, the cell is differentiated into or has differentiated into the iPS cell of the present invention of RPE cell, preferably for culturing cells subsequently and reaching about 5 continuous culture days, the cell is differentiated into or has differentiated into the iPS cell of the present invention of RPE cell. In a preferred embodiment, CHIR99021 is subsequently used for culturing cells subsequently with a final concentration of about 3 μM, the cell is differentiated into or has differentiated into the iPS cell of the present invention of RPE cell, even more preferably for culturing cells subsequently and reaching about 5 continuous culture days (meaning the 11th day to the 16th day of cultivation), the cell is differentiated into or has differentiated into the iPS cell of the present invention of RPE cell. By gradually increasing 1.5 μM to reach 3 days, followed by a final concentration of 3 μM to reach 5 days, the yield of the RPE cell of pigmentation increases.

[0128] The present invention further includes a method for differentiating into RPE cells, the method comprising culturing the iPS cells in a differentiation medium as defined elsewhere herein, wherein CHIR99021 is used for about 8 culture days, preferably for about 8 consecutive culture days, and even more preferably wherein during the first 3 (consecutive) days / days of culturing for about 8 days with CHIR99021, CHIR99021 is at a 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 3μM, at least about 1.4μM and less than about 3μM, at least about 1μM and less than about 2.5μM, at least about 1μM and less than about 2μM, at least about 1μM and less than about 1.9μM, at least about 1μM and less than about 1.8μM, at least about 1μM and less than about 1.7μM, at least about 1μM and less than about 1.6μM, and most preferably used at a final concentration of about 1.5μM, and wherein CHIR99021 is used at a final concentration of about 3μM during the last 5 (consecutive) days / days of culturing with CHIR99021 for about 8 days.

[0129] 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 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 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 another fifth differentiation medium applied after applying the first fifth differentiation medium in the differentiation method, comprising about 100 ng / mL activin A, about 10 μM SU5402, and about 3 μM CHIR99021, preferably about 100 ng / mL activin A, about 1 μM PD17307, and about 3 μM CHIR99021.

[0130] In a preferred embodiment of the present invention, when culturing iPS cells in a differentiation method, it comprises culturing in a first differentiation medium as defined elsewhere herein for about 2 days, preferably culturing in a first differentiation medium as defined elsewhere herein for about 2 consecutive days. This means that the iPS cells are first exposed to the first differentiation medium as defined herein from day 0 to day 2.

[0131] In another preferred embodiment, when culturing iPS cells in the method of the present invention, it comprises culturing in a second differentiation medium as defined elsewhere herein for about 2 days, preferably it comprises culturing in a first differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a second differentiation medium as defined elsewhere herein for about 2 days, even more preferably it comprises culturing in a first differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a second differentiation medium as defined elsewhere herein for about 2 consecutive days. This means that on day 2 of culturing iPS cells in the method of the present invention, the iPS cells may be exposed to the second differentiation medium from day 2 to day 4.

[0132] In another preferred embodiment, when culturing iPS cells in the method of the present invention, it comprises culturing iPS cells in the third differentiation medium as defined elsewhere herein for about 2 days, preferably it comprises 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, even more preferably it comprises 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 4th 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.

[0133] In another preferred embodiment, when culturing iPS cells in the method of the present invention, it comprises culturing iPS cells in a fourth differentiation medium as defined elsewhere herein for about 2 days, preferably it comprises culturing iPS cells in a first differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a second differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a third differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a fourth differentiation medium as defined elsewhere herein for about 2 days, even more preferably it comprises culturing iPS cells in a first differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a second differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a third differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a fourth differentiation medium as defined elsewhere herein for about 2 consecutive days. This means that on day 6 of culturing iPS cells in the method of the present invention, iPS cells may be exposed to the fourth differentiation medium from day 6 to day 8.

[0134] In another preferred embodiment, when culturing iPS cells in the method of the present invention, it comprises culturing in a fifth differentiation medium as defined elsewhere herein for about 8 days, preferably it comprises culturing in a first differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a second differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a third differentiation medium as defined elsewhere herein for about 2 days, followed by culturing in a fourth differentiation medium as defined elsewhere herein for about 2 days, and then culturing in a fifth differentiation medium as defined elsewhere herein for about 8 days, even more preferably it comprises culturing in a first differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a second differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a third differentiation medium as defined elsewhere herein for about 2 consecutive days, followed by culturing in a fourth differentiation medium as defined elsewhere herein for about 2 consecutive days, and then culturing in a fifth differentiation medium as defined elsewhere herein for about 8 consecutive days. This means that on the 8th day of culturing iPS cells in the method of the present invention, the iPS cells can be exposed to the fifth differentiation medium from day 8 to day 16.

[0135] In an even more preferred embodiment of the present invention, when iPS cells are cultured in the method of the present invention, it 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, followed by culturing for about 4 additional days in a fifth differentiation medium comprising CHIR99021 used at a concentration of about 3 μM, most preferably, it comprises culturing for about 2 days in a first differentiation medium, 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, and then culturing for about 4 days in 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, followed by culturing for about 4 additional 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 specific culture medium may also be interchanged with the term "consecutive days". This means that on day 8 of culturing in the method of the present invention, the cells can be exposed from day 8 to day 11 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, and then on day 11 the cells are exposed from day 11 to day 16 to a fifth differentiation medium comprising CHIR99021 used at a concentration of about 3 μM.

[0136] Therefore, the present invention also includes methods for differentiating iPS cells into RPE cells, wherein the iPS cells are cultured in 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 about 16 days in total, 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 consecutive days, about 15 to about 17 consecutive days, and most preferably 16 consecutive days.

[0137] In a further embodiment of the present invention, the method for making iPS cells differentiate into RPE cells is further preferably included in cultivating iPS cells in differentiation medium as defined herein, and cultivating iPS cells in mTESR1 culture medium.CLiPs and human ES cells as reference cell lines can grow on tissue culture plates coated with matrix gel (Matrigel) in mTeSR1 culture medium.When cells reach about 90% to about 95% confluence, they are then exposed to differentiation medium as defined elsewhere herein, preferably exposed to the first differentiation medium, even more preferably exposed to the first differentiation medium, then exposed to the second, third, fourth and fifth differentiation medium as defined herein.In a preferred embodiment, the method for making iPS cells differentiate into RPE cells is also included in cultivating iPS cells in differentiation medium as defined herein, more preferably before cultivating iPS cells in the first differentiation medium as defined herein, most preferably in the first differentiation medium as defined herein, then before cultivating iPS cells in the second, third, fourth and fifth differentiation medium as defined herein, in mTESR1 culture medium, cultivate iPS cells about 1 to about 4 culture days.

[0138] In a further embodiment of the present invention, the method for making iPS cells differentiate into RPE cells is preferably further included in culturing RPE cells in retinal pigment epithelium maintenance (abbreviation: RPEM) culture medium. According to the present invention, iPS cells are cultured in differentiation medium, and then after iPS cells differentiate into RPE cells, differentiation medium can be replaced by RPEM culture medium as defined below. Preferably, culturing RPE cells in the RPEM culture medium can start after the 16th day of culture (particularly after culturing cells in the fifth differentiation culture medium as defined elsewhere herein). In a preferred embodiment, the RPEM culture medium of the differentiation method of the present invention comprises about 50% DMEM / F12 and about 50% minimum essential medium (MEM), and the minimum essential medium comprises 0.5×N1 supplement and 1×NEAA. In a more preferred embodiment, the RPEM culture medium of the differentiation method of the present invention also comprises heat-inactivated fetal bovine serum (FBS), Glutamax, taurine, hydrocortisone, 3,3', 5-triiodo-L-thyronine, penicillin / streptomycin, nicotinamide or at least one of 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.

[0139] The present invention also includes the differentiation method as defined elsewhere herein, wherein RPE cells are cultured in the 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 about 19 days, even more preferably about 19 consecutive days. During the cultivation of the RPE cells in the RPEM culture medium, the RPEM culture medium can be replaced every about 2 to about 3 days, preferably during the cultivation of the RPE cells in the RPEM culture medium 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, more preferably during the cultivation of the RPE cells in the RPEM culture medium for about 19 days, even more preferably during about 19 consecutive days, the RPEM culture medium can be replaced every about 2 to about 3 days.

[0140] The present invention also includes the differentiation method as defined elsewhere herein, wherein culturing iPS cells in a differentiation medium as defined herein and culturing RPE cells in a RPEM medium as defined herein comprises 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, particularly having culturing iPS cells in a differentiation medium as defined elsewhere herein for about 16 days and having culturing differentiated RPE cells in a RPEM medium as defined elsewhere herein for about 19 days.

[0141] The present invention also includes the described differentiation method as defined elsewhere herein, and it is further preferably included in after cultivating described RPE cells in described RPEM culture medium, purifying described RPE cells in described RPEM culture medium.After differentiation, there is a mixture of RPE cells and non-RPE cells, and this is why further purification step may be helpful, and differentiation plate can only have pure RPE cells (Figure 20) like this.The additional purification step of differentiation method preferably includes a) manually identifying RPE cells according to its pigmentation, wherein manually identifying RPE cells according to its pigmentation preferably includes selecting by microscopy, more preferably selecting by bright field microscope well known to persons skilled in the art.This step may refer to the manual purification of RPE cells.Especially, manually identifying RPE cells according to pigmentation can be carried out by manually removing non-RPE cells simultaneously by microscopy through adopting the tip scraping attached to pipette, and compared with RPE cells, described non-RPE cells have less pigmentation and different cell morphologies.It may further include washing, particularly washing about 3 times with PBS, to remove all non-RPE cells. In addition or alternatively, the extra purification step of the differentiation method preferably includes b) making RPE cell passage, wherein making RPE cell passage preferably includes using Accutase or TrypLE, and most preferably using TrypLE to process RPE cells.This step can refer to the passage purification of RPE cells.Especially, can be by using gentle dissociating agent such as Accutase or TrypLE, preferably with TrypLE process to dissociate non-RPE cells and remove them to make RPE cell passage.RPE cells can still be attached to culture plate.Then it can further include again using gentle dissociating agent such as Accutase or TrypLE, preferably with TrypLE process remaining RPE cells and further make described RPE cell passage. In this context, term " making RPE cell passage " refers to using gentle dissociating agent such as Accutase or TrypLE, preferably dissociate with TrypLE and remove non-RPE cells after, by remaining RPE cell bed plate and again use gentle dissociating agent such as Accutase or TrypLE, preferably process described remaining RPE cells with TrypLE.If TrypLE has been applied, described step can refer to the TrypLE purification of RPE cell.Additionally or alternatively, the extra purification step of differentiation method preferably includes c) as defined elsewhere herein according to pigmentation manual identification RPE cell and as defined elsewhere herein making the combination of RPE cell passage.Although the purification carried out by making RPE cell passage, preferably use above-mentioned TrypLE purification, most of non-RPE cells / cell agglomerates can be removed, but still there may be some small agglomerates, which can be removed by manual purification as defined herein.Additionally or alternatively, the extra purification step of differentiation method preferably includes d) making RPE cell passage and according to the combination of its pigmentation scattering sorting RPE cell.This purification step can be carried out by following: as defined above, remove non-RPE cell, and with gentle dissociating agent such as Accutase or TrypLE, preferably further process described remaining RPE cell with TrypLE, and further process RPE cell in scattering sorting, wherein by gentle dissociating agent such as Accutase or TrypLE, preferably the non-RPE cell removed with TrypLE can be used for arranging the gating of low scattering cells.Additionally or alternatively, the extra purification step of differentiation method preferably includes e) according to the pigmentation of RPE cell, RPE cell is carried out the combination of scattering sorting.This step can refer to the scattering sorting purification of RPE cell. In particular, scatter sorting of RPE cells can be performed by resuspending a cell mass of dissociated single cells (by using a gentle dissociating agent such as Accutase or TrypLE, preferably TrypLE dissociation) in any FACS buffer and passing it through a filter to obtain single cells and separating them into a high scatter fraction and a low scatter fraction using, for example, any FACS cell sorter known to the skilled person.

[0142] By comparing different RPE purification methods as defined herein, find that including as defined above with Accutase or TrypLE, preferably with TrypLE process RPE cell make RPE cell passage produce high RPE productive rate (about 47%) and compared with the manual purification of RPE cell, it is easier and faster, therefore it is preferred.Including as defined above with Accutase or TrypLE, preferably with TrypLE process RPE cell make RPE cell passage, also as defined in other places herein subsequently according to its pigmentation manual identification RPE cell, not only produced high RPE productive rate (about 43%), and obtain the cell of highest purity (about 99% PMEL17 positive cell), this may be the most important for transplanting.In addition, including as defined above with Accutase or TrypLE, preferably with TrypLE process RPE cell make RPE cell passage, also as defined in other places herein subsequently according to its pigmentation manual identification RPE cell, also be the easiest to carry out, because in the short time amount required for purification, part TrypLE handles and removes most of non-RPE cells. In summary, comprising passaging RPE cells with Accutase or TrypLE, preferably treating RPE cells with TrypLE and combining the purification of manually identifying RPE cells involves an extra manual purification step to remove any non-RPE cells that may escape mild dissociation agents such as Accutase or TrypLE, preferably TrypLE treatment, and is most preferred as the purification of RPE cells in the method for the present invention.

[0143] The present invention also includes the differentiation method as defined elsewhere herein, wherein the iPS cell derived from umbilical cord amniotic stem cells and used in the method for differentiating into RPE cells is specifically produced by expressing exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in the umbilical cord amniotic stem cells under conditions suitable for reprogramming stem cells as defined elsewhere herein, and the conditions are used in the method for producing induced pluripotent stem cells also included in the present invention. In a preferred embodiment, the present invention also includes the differentiation method as defined elsewhere herein, wherein the exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in the umbilical cord amniotic stem cells are provided by one, two or three vectors, wherein preferably the first vector encodes proteins OCT3 / 4 and 53-shRNA, the second vector encodes proteins SOX2 and KLF4 and the third vector encodes proteins L-MYC and LIN28. In summary, every disclosure concerning the method of generating induced pluripotent stem cells as defined elsewhere herein, wherein the iPS cells are subsequently differentiated into RPE cells according to said differentiation method, also applies where necessary to the method of differentiating iPS cells into RPE cells.

[0144] The invention still further relates to a kind of RPE cell culture / RPE cell obtainable by differentiation method as described herein, and relate to a kind of RPE cell culture / RPE cell obtained by differentiation method as described herein.The invention still further relates to a kind of retinal pigment epithelium, it is made up of retinal pigment epithelium cell culture / RPE cell obtainable by differentiation method as described herein / obtainable or comprises retinal pigment epithelium cell culture / RPE cell obtainable by differentiation method as described herein / obtainable.

[0145] The present invention further relates to a pharmaceutical composition, which comprises RPE cell culture / RPE cell obtainable by differentiation method as described herein / obtained. The example of the pharmaceutical composition comprising the RPE cell culture of differentiation / containing the RPE cell of differentiation is an injection solution or any type of transplant suitable for implanting the RPE cell of differentiation. When the pharmaceutical composition is an injection solution, the composition may comprise the RPE cell culture obtainable by differentiation method as described herein / obtained. When the pharmaceutical composition is a transplant suitable for implantation, the composition may comprise an implantable matrix, preferably a polyester matrix, even more preferably a polyester matrix in a transwell plate (transwell), and the matrix is ​​coated with the RPE cell of the differentiation obtainable by differentiation method as described herein / obtained that can be grown on the matrix. In this context, the RPE cell grown on the matrix as defined herein may refer to the RPE cell culture obtainable by differentiation method as described herein / obtained. Pharmaceutical composition may be formulated / adjusted for parenteral or topical application, as known to the technical staff. In this case, parenteral application may include the sterile preparation intended to be injected, infused or implanted in the human body or animal body. Topical application as used herein preferably refers to subretinal application.If pharmaceutical composition refers to the transplant as defined above about implantable matrix, then the composition can be prepared / adjusted for subretinal (under the retina of eyes) application, in other words, can be subretinal implanted.The present invention also includes a kind of diagnostic composition for research purpose, which comprises RPE cells (cultures) and matrigel.The composition also refers to the transplant suitable for implanting experimenter as defined herein, wherein the RPE cells differentiated by the method as defined herein are mixed with matrigel, and then the transplant (cell in matrigel) is implanted into experimenter.If diagnostic composition refers to the transplant as defined above about matrigel, then the composition can be prepared / adjusted for subcutaneous application, in other words, can be subcutaneously implanted.

[0146] Obtainable by differentiation method as herein described / obtained, also included in retinal pigment epithelium, and / or also included in the RPE cell culture in the pharmaceutical composition as defined elsewhere herein can refer to multiple RPE cells, it is obtainable by differentiation method / obtained and preferably comprises the culture medium for described RPE cell.Term " group / colony " also can be used interchangeably with term " culture ".The RPE cell of the described differentiation that is included in described RPE cell culture, obtainable by differentiation method of the present invention / obtained, can be further characterized: compared with the RPE in skin iPS-source, the RPE cell of described differentiation can first comprise higher pigmentation area%.By visual grading, compared with only 30% of the skin iPS cell (such as Asf5, AGO and / or HDFA) having achieved similar pigmentation, all four kinds of CLiPS (which are selected from CLMC23, CLMC30, CLMC44 and CLEC23) tested by the present invention use differentiation culture medium as defined herein to produce about 30% to about 100%, about 50% to about 100%, about 70% to about 100% pigmentation RPE cell ( Fig.13 ). In addition, the differentiated RPE cells further contained in the culture may express at least one of BEST1, PMEL17, MITF, TYROSINASE, TRYP2, ZO-1, RPE65, RLBP1 or MERTK or a combination of all the listed protein markers ( Fig.15 , 16In one embodiment, the RPE cells of the differentiation contained in the culture may express protein marker BEST1. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker PMEL17. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker MITF. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker TYROSINASE. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker TRYP2. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker ZO-1. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker RPE65. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker RLBP1. In another embodiment, the RPE cells of the differentiation contained in the culture may express protein marker MERTK. Therefore, CLiPS-derived RPE cells are more highly pigmented than ES-derived RPE cells (such as H9 cells used herein). This is associated with higher expression of pigmentation-related genes such as, for example, MITF, PMEL17, TYROSINASE, and TRYP2 ( Fig.15 ). In addition, the RPE cells of the differentiation included in the culture are characterized in that the cell cycle proliferation marker Ki67 lacks or has a reduction and expresses. The expression of mature RPE marker RPE65 and the expression loss of Ki67 as a proliferation marker have confirmed maturity and quiescent state, which reflects the survival rate (Figure 22) of this type of RPE cells differentiated from CLiPS according to the method defined elsewhere herein.

[0147] In more detail, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as: relative to the RPE cell that breaks up from embryonic stem cell (ES) (producing its cell or culture), with 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 higher, preferably about 3 fold changes and expresses BEST1. Additionally or alternatively, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as, relative to the RPE cell that breaks up from ES (producing its cell or culture), with 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 higher, preferably about 1.3 fold changes and expresses PMEL17. Therefore, after differentiation, CLiPS-RPE contains a higher % of PMEL17 positive cells. It was found that the CLiPS used in the differentiation method (such as CLMC23, CLMC30 and CLEC23) contained RPE cells with a purity of about 89% to about 95%. In contrast, only one of the three skin iPS cells used (such as Asf5, AGO and / or HDFA) had a purity higher than about 90% ( Fig.14). Additionally or alternatively, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as, relative to the RPE cell that breaks up from ES (producing its cell or culture), with 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 higher, preferably about 6.8 fold change expression MITF. Additionally or alternatively, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as, relative to the RPE cell that breaks up from ES (producing its cell or culture), with few 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 higher, preferably about 4.3 fold change expression TRYP2. Additionally or alternatively, the RPE cells obtainable / obtained by the method as defined herein can be further characterized as, relative to the RPE cells differentiated from ES (cells or cultures producing it), 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 higher, preferably about 0.96. Additionally or alternatively, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as, relative to the RPE cell that breaks up from ES (producing its cell or culture), with 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 higher, preferably about 26.2 fold change expression RLBP1. Additionally or alternatively, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as, relative to the RPE cell that breaks up from ES (producing its cell or culture), with 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 higher, preferably about 9.1 fold change expression MERTK (Figure 20k). In this context, such ES cells used for comparison and from which RPE cells are also differentiated may be referred to as H9 ES cells.

[0148] In this context, the term "relative to" or "for" can also be replaced by the term "compared to...", for example, in terms of gene expression expressed as a fold change of a test cell relative to a reference cell, two cells (e.g., CLMC23 as a test cell and H9 as a reference cell) are compared to each other. "Fold change" as used herein is a measure that describes the change in quantity from an initial value to a final value. For example, an initial value of 30 and a final value of 60 correspond to a 2-fold change, or, in layman's terms, a two-fold increase. Simply calculate the fold change as the ratio of the final value to the initial value, i.e., if the initial value is A and the final value is B, the fold change is B / A. As described herein, the fold change can be obtained based on the mRNA level of the marker. This fold change can be measured using RT-qPCR.

[0149] It was also found that CLiPS-derived RPE can achieve epithelial cell transmembrane electrical resistance (TEER) similar to RPE cells differentiated from ES (cells or cultures from which it is produced) and / or RPE cells differentiated from skin iPS (cells or cultures from which it is produced). Similar to RPE cells differentiated from ES (cells or cultures from which it is produced) and / or RPE cells differentiated from skin iPS (cells or cultures from which it is produced), CLiPS-RPE also showed high phagocytosis ( Fig.17 ).

[0150] Additionally or alternatively, the RPE cell that can obtain / obtained by the method as defined herein can be further characterized as, relative to the RPE cell differentiated from ES and / or relative to the RPE cell differentiated from skin iPS, comprising increased oxygen consumption rate (OCR) and / or extracellular acidification rate (ECAR).These features relate to the bioenergetics of described RPE, and it proves that glycolysis and / or mitochondrial respiration increase.By ECAR quantitative glycolysis function, by OCR quantitative oxidative phosphorylation (oxPhos) (Figure 21).

[0151] In this case, OCR can include basal respiration, ATP production, maximum capacity and / or spare respiratory capacity.Term " increase " in the context of about OCR refers to relative to the RPE cell differentiated from ES and / or relative to the RPE cell differentiated from skin iPS, the OCR of RPE cell increases 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 at least about 35%; Or increase by about 30% to about 45%, about 31% to about 44%, about 32% to about 43%, about 33% to about 42%, about 35% to about 40%. In more detail, i) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the basal respiration of RPE cells increased by about 38%; ii) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the ATP production of RPE cells increased by about 40%; iii) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the maximum capacity of RPE cells increased by about 35%; iv) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the spare respiratory capacity of RPE cells increased by about 36%.

[0152] In addition, in this context, ECAR can include glycolysis, glycolytic capacity and / or glycolytic reserve.Term " increase " in the context of about ECAR means relative to the RPE cell differentiated from ES and / or relative to the RPE cell differentiated from skin iPS, the ECAR of RPE cell increases 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 increase by about 20% to about 55%, about 25% to about 55%, about 25% to about 50%. In more detail, i) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the glycolysis of RPE cells increased by about 25%; ii) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the glycolytic capacity of RPE cells increased by about 37%; iii) relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, the glycolytic reserve of RPE cells increased by about 50%.

[0153] In addition, additionally or alternatively, the RPE cells obtainable / obtained by the method as defined herein can be further characterized as, relative to the RPE cells differentiated from ES (cells or cultures producing it) and / or relative to the RPE cells differentiated from skin iPS (cells or cultures producing it), having / comprising lower immunogenicity as defined in more detail below. In this context, and also in the context of in vivo method as defined below, lower immunogenicity refers to RPE cells, preferably the RPE cells pre-delivered to experimenter have the systemic immune response of reduction, described experimenter is the experimenter for the further analysis of described cell for obtaining the sample comprising described RPE cells therefrom, the systemic immune response of described reduction can refer to the proinflammatory cytokine (a) participating in inducing cellular immune response, preferably IFN-γ and / or IL-18 (as the indicator (surrogate) of cellular immune response) and / or IL-23 and / or IL17A, the level of the cytokine participating in T cell activation is reduced, the cytokine level of described reduction is produced by the described experimenter of previously pre-delivered described RPE cells, particularly produced by the described immunocyte being present in described experimenter and also included in described sample.The systemic immune response of this reduction in described RPE cells can also refer to immunocyte, preferably reduces at the accumulation of injection site as defined in the embodiments part. Thus, a reduced systemic immune response may comprise RPE cells as defined herein having reduced systemic T cell activation relative to RPE cells differentiated from ES and / or relative to RPE cells differentiated from skin iPS, preferably having reduced / inhibited CD8 cytotoxic T cell activation.

[0154] The present invention also includes a method for treating retinal degenerative diseases in a subject, wherein the subject can be selected from mice, rats, rabbits, pigs, dogs, cats, non-human primates (monkeys) or people. In a preferred embodiment, the subject is a person. In this context, treatment can include applying to the subject as defined herein by differentiation method as described herein from CLiPS differentiated RPE cells and / or the RPE cell culture obtained by the method of the present invention and / or the pharmaceutical composition as defined herein. Cells / cultures of the differentiation obtained by the method as defined herein are suitable for use in the specific treatment of retinal degenerative diseases, and this is proved by the following facts that the RPE cells include low immunogenic properties, such as the proinflammatory cytokines that produce a reduced level, preferably IFN-γ and / or IL-18 as indicators, and there is a reduced cellular immune response, preferably comprising the above-mentioned properties (Figure 23) after the RPE cells of the differentiation are pre-injected into the subject defined herein. This proves that the RPE cells of the present invention can reduce the immune cell infiltration at the local site where RPE cells are injected into the subject. In addition, it is demonstrated that relative to the sample obtained from the reference subject of the RPE cell differentiated from ES previously delivered and / or relative to the sample obtained from the reference subject of the RPE cell differentiated from skin iPS previously delivered, the sample obtained from the subject of the RPE cell differentiated previously delivered before cytokine analysis comprises the IL-23 and / or IL17A of reduced level, which are the cytokines participating in T cell activation. In a word, the RPE cell as defined herein can produce the IL-23 and / or IL17A of reduced level as defined herein, as another preferred embodiment of proinflammatory cytokines. In addition, the T cell activation in the sample comprising the RPE cell of the present invention from the subject, especially CD8 cytotoxic T cell activation is suppressed. In other words, the RPE cell can suppress T cell activation, particularly CD8 cytotoxic T cell activation ( Fig.24 ). The degenerative disease treated is a retinal disease known to a technician, preferably, the retinal degenerative disease is age-related macular degeneration (AMD) or retinal dystrophy. In one embodiment, the present invention relates to a method for treating AMD in a subject as defined herein, the method comprising administering to the subject RPE cells differentiated from iPS cells obtained by the method as defined herein. In another embodiment, the present invention relates to a method for treating retinal dystrophy in a subject as defined herein, the method comprising administering to the subject RPE cells differentiated from iPS cells obtained by the method as defined herein. In the method of treating, the RPE cells differentiated from CLiPS may include parenteral or topical (preferably subretinal) applications as known to technicians.

[0155] The present invention also includes an in vivo method for detecting the survival rate of RPE cells in a subject, wherein the RPE cells are differentiated from iPS cells by a differentiation method as defined elsewhere herein, the method comprising a) introducing RPE cells differentiated from iPS cells by a method as defined herein into a subject, wherein the RPE cells comprise a bioluminescent marker.

[0156] In this context, term " survival rate " refers to that RPE cells are not dead and are still mature and / or comprise quiescent state, this can be by after described cell is introduced in described experimenter as defined herein and as further described in a period of time detection, detection as the expression of RPE65 of mature RPE marker and detection as the non-expression of Ki67 of proliferation marker are confirmed.Monitoring survival rate also can be used interchangeably with as defined in vivo method in this article.

[0157] The term "introducing (introduce or introducing)" in step a) refers to bringing the RPE cells as defined herein into the subject, preferably by implanting the RPE cells to bring the RPE cells into the subject, even more preferably, by subcutaneously implanting the RPE cells to bring the RPE cells into the subject with respect to using mice as the subject and as defined elsewhere herein in terms of the matrigel plug assay. The term "subject" when used herein according to in vivo methods and in vitro screening methods includes mammals and non-mammalian subjects. Preferably, the subject is an animal. The subject of in vivo and in vitro methods can refer to mammals, including people, livestock and farm animals, non-human primates and any other animals with mammary tissue. In some embodiments, mammals are mice. In some embodiments, mammals are rats. In some embodiments, mammals are guinea pigs. In some embodiments, mammals are rabbits. In some embodiments, mammals are cats. In some embodiments, mammals are dogs. In some embodiments, mammals are monkeys. In some embodiments, mammals are horses. In preferred embodiments, the mammal / animal used as a subject in the method of the present invention is a mouse. In a most preferred embodiment, the mammal / animal used as a subject in the method is a humanized mouse.

[0158] The RPE cells that are introduced into the experimenter as defined herein according to the present invention that are differentiated and in vivo method comprise bioluminescent markers.In this context, term " marker " can be fluorescent marker or be suitable for bioluminescent enzyme.When marker is fluorescent marker, it can be fluorophore (also referred to as fluorescent dye or chromophore).This fluorophore can be any one in fluorescent dye, such as but not limited to 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 protein such as R-PE or APC, or the fluorescent protein of expression such as CFP, EGFP, GFP or RFP. When marker is enzyme, it can be but not limited to luciferase, preferably selected from bacterial luciferase (\uxAB), North American firefly luciferase, ren / 7 / a luciferase and firefly luciferase.In preferred embodiments, the RPE cell introduced into the experimenter as defined herein in vivo method according to the present invention is differentiated and comprises the carrier of encoding bioluminescent enzyme gene, preferably with the fluorescent protein marker of expression as defined herein.In most preferred embodiments, the RPE cell introduced into the experimenter as defined herein in vivo method according to the present invention is differentiated and comprises the carrier of encoding luciferase gene, preferably with GFP marker.Additionally or alternatively, the step a) of the method in vivo as defined herein also includes introducing the RPE cell differentiated by the method as defined herein into the experimenter, wherein the RPE cell is included in RPE-matrigel plug as known by persons skilled in the art.Included in matrigel plug refers to the mixture / composition of the RPE cell and matrigel known by persons skilled in the art.

[0159] The in vivo method as defined herein further comprises the step b) detecting the bioluminescent signal of said RPE cells over time using an imaging method, thereby collecting imaging data.

[0160] When used in this article about in vivo method, the term "detection (detecting / detection)" (also referred to as "monitoring") refers to using any known imaging method to visualize and qualitatively analyze the bioluminescence of the RPE cell in vivo, preferably if the bioluminescent marker is a luciferase using a bioluminescent imaging method. The detection of the bioluminescent signal of the RPE cell is carried out over time according to the step b) of the in vivo method, and the time can 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 about 2 to about 56 days, or about 2 to about 49 days. The detection of the bioluminescent is preferably monitored at regular intervals during the time course defined herein. The detection of bioluminescent can refer to the detection of the total radiation of the cell represented by P / s / cm2 / sr known to those skilled in the art. By the detection of the bioluminescent signal over time, imaging data is collected.

[0161] In vivo method as defined herein also includes step c): the imaging data received in step b) is compared with reference to imaging data.In this context, reference to imaging data refers to the bioluminescent signal (" imaging signal ") of the RPE cell that is detected over time from ES cell (preferably H9 cell) differentiation and / or from skin iPS (preferably HDFA or ASF5) differentiation as defined herein.In this context, the RPE cell that will be differentiated from ES cell and / or from skin iPS as defined herein is introduced into subject (for example, different mice), this subject is completely different from the subject (for example, mouse) that has pre-delivered RPE cell as defined herein, and wherein the RPE cell that is differentiated from ES cell and / or from skin iPS also comprises bioluminescent markers as defined herein as the RPE cell derived from CLiPS.

[0162] The present invention also includes in vivo method as defined herein, wherein compared with reference to imaging data, the bioluminescent signal of imaging data no difference indicates that the RPE cell described in the experimenter survives.No difference also includes compared with the reference imaging data of the RPE cell differentiated from ES cell and / or differentiated from skin iPS, and the bioluminescent signal of imaging data is slightly but not significantly reduced in time.

[0163] The present invention also includes the animal comprising the RPE cell obtained by the differentiation method defined as elsewhere herein / obtainable.In this context, the "animal" refers to any mammal as defined herein, is preferably a mouse, most preferably a humanized mouse.Mention the animal comprising the RPE cell, it may refer to the RPE cell obtained by the differentiation method defined as elsewhere herein / obtainable being imported into the animal defined herein, preferably by subcutaneous implantation of the RPE cell.

[0164] In particular it is envisaged that the introduction of said RPE cells differentiated from iPS cells by an in vivo differentiation method into said subject as defined herein and the subsequent in vitro analysis of samples comprising said RPE cells obtained from said subject for analysis of such cells in various models for research and development purposes.

[0165] Therefore, the present invention also includes a kind of in vitro (screening) method for determining the immunogenicity of RPE cells in an experimenter, the RPE cells are obtained from iPS cell differentiation by the method as defined herein, the RPE cells of the differentiation have been pre-delivered to the experimenter, and the method comprises: a) using imaging methods to detect the proinflammatory cytokine level in the sample obtained from the experimenter defined herein, wherein the sample comprises the RPE cells of the differentiation, thereby collecting imaging data." pre-delivery" is included in this respect before the in vitro screening method, and the RPE cells of the differentiation of the present invention have been delivered to the experimenter as defined herein. After this, obtain the sample of the RPE cells comprising differentiation from the experimenter, and further analyze the sample, for example, analyze specific cytokine levels.Term "pre-introduction" can also be used interchangeably.

[0166] In this context, detection refers to in vitro visualization and quantitative analysis of the cytokine levels defined herein using any known imaging method suitable for detecting cytokines, such as flow cytometry. The cytokine levels to be detected as defined herein for in vitro methods of detection refer to, but are not limited to, the levels of cytokines associated with cell-mediated immunity, preferably the levels of proinflammatory cytokines IFN-γ, IL-18, IL-23 and / or IL17A. The levels of the cytokines defined herein can be expressed in pg / ml.

[0167] The sample obtained from said defined subject in step a) of the in vitro method may be any biological sample obtained from said subject, preferably a serum sample.

[0168] The in vitro method may comprise detecting immune cell infiltration in a sample obtained from said subject as defined herein, said sample comprising said differentiated RPE cells, thereby further collecting imaging data, which may be an additional or alternative step in said method.

[0169] The method further comprises step b) will receive in step a) and / or the imaging data received from the detection step about infiltration and compare with reference imaging data.In addition, described reference imaging data refers to the imaging data of the proinflammatory cytokine level of identical detection as defined herein in the sample of the experimenter of the RPE cell of having delivered in advance from ES cell differentiation and / or from skin iPS differentiation, as reference cell, and / or described reference imaging data refers to the imaging data of infiltrating and detecting the same immune cell in the sample of the experimenter of the RPE cell of having delivered in advance from ES cell differentiation and / or from skin iPS differentiation, as reference cell.Described reference cell also can be included in the sample (such as blood sample, but different blood samples) of the same kind as the sample comprising RPE cell, but reference sample is obtained from the experimenter (such as different mice) different from the test subject obtained from the sample comprising RPE cell. Preferably, compared with the reference imaging data, the reduced cytokine levels and / or reduced / decreased immune cell infiltration / accumulation of the imaging data indicate that the immunogenicity of the RPE cells in the subject is reduced compared with the immunogenicity of RPE cells differentiated from ES cells and / or differentiated from skin iPS, which means that the ability of the RPE cells according to the present invention to stimulate an immune response in a subject is lower / reduced.

[0170] The present invention will be further illustrated by the following non-limiting experimental examples.

[0171] Experimental Examples

[0172] Example 1: Development of suitable electroporation parameters for CLiPS

[0173] It was found that electroporation according to the protocol described by Okita et al. (supra) did not work at all. When the reaction mixture of CLMCs was electroporated with the episomal vectors pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL according to the protocol of Okita et al. (supra), no IPS colonies were detected. For CLMCs, following the protocol of Okita et al. (supra), the episomal vectors pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL (Addgene plasmids #27077 (SEQ ID NO: 12 - particularly comprising SEQ ID NO: 11), #27078 (SEQ ID NO: 13), #27080 (SEQ ID NO:14) After electroporation of CLMC, the average reprogramming efficiency of CLEC (expressed in terms of IPS colony counts) is only 0.2%. Therefore, it is necessary to develop a suitable electroporation scheme for the CLiPS method derived from CLMC from scratch, or in the case of CLEC, to provide a significantly improved electroporation scheme. For this purpose, electrical parameters such as the number of electric pulses, duration and voltage constituting electroporation are changed to develop available electroporation conditions for CLSC. In this experiment, numerous different electroporation settings were performed for individual CLMC and CLEC samples, and the samples were cultured under cell-specific conditions as described herein, respectively. After each electroporation, about 200.000 cells were laid in triplicate in 6-well plates for cultivation. About 21 days after electroporation, the counting of the CLSC colonies that have then developed was performed to determine the survival rate. The survival rate is used to draw conclusions about the electroporation efficiency. The efficiency percentage was calculated with colony number / 200,000x 100%.

[0174] Table 1 and Figure 2 The results shown in Figure 2 indicate that both CLMC and CLEC can find suitable electroporation conditions. 6The number of cells was 1.67 μg (plasmid) DNA of each of the three vectors (pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK and pCXLE-hUL), and the optimal electroporation settings for CLEC found herein included 2 electric pulses, each of 30 ms and 1350 V. Four individual CLEC lines (CLEC42, CLEC44, CLEC23 and CLEC30) transfected with these settings showed 4.67%, 7.33%, 9.33% and 7.50% survival rates, respectively. Compared with Okita et al. (supra), the electroporation settings for CLEC increased the electroporation efficiency for CLEC42 by about 23.35% and the electroporation efficiency for CLEC44 by about 36.65%. Thus, it has been surprisingly found that these electroporation parameters / settings increase the electroporation efficiency by about 30% on average for CLECs compared to the conditions used by Okita et al. for electroporation of human skin fibroblasts. It is noteworthy that the electroporation settings used here are similar to the conditions reported for successful electroporation of epithelial cells such as corneal epithelial cells (1 electric pulse of 30 ms and 1300 V, amount of plasmid DNA (μg) and number of cells (1×10 6 The ratio of 1:1 in 10 cells (see Png, E et al. (2011), Journal of Cellular Physiology. United States, 226(3), pp. 693-699)) is significantly different.

[0175] The effect of optimizing the electroporation protocol was even more pronounced for CLMCs, since, as mentioned above, electroporation according to Okita et al. (supra) did not produce viable CLMCs at all. Here, it was found that the ratio of the amount of plasmid DNA of each of the three episomal vectors (pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, and pCXLE-hUL) to the number of cells was approximately 1×10 6Four separate CLMC lines (CLMC42, CLMC44, CLMC23 and CLMC30) were successfully transfected at a ratio of 1:10 CLMC. The resulting transgenic cells showed viability of 6.17%, 7.50%, 5.00% and 7.33%, respectively. It is worth noting that the optimal electroporation / transfection conditions for producing CLiPS from CLMCs found herein are also different from the electroporation conditions reported to date. In this context, see, for example, Sprangers, AJ, Freeman, B. and Ogle, BM (2011), pp. 62–66, who studied possible negative effects of electroporation of mesenchymal stem cells derived from human embryonic stem cells (hESCs). Thus, Sprangers et al. found that using 1 electric pulse of 20 ms and 1400 V at 1×10 6 Transfection of a total of 4 μg (plasmid) DNA in each mesenchymal stem cell provides optimal conditions for MSC transfection. Therefore, the present invention provides a unique and effective scheme for electroporation of CLEC and CLMC, respectively. The variation in transfection efficiency between four separate CLSC lines (cells from different donors) is an inter-individual variability, an inherent and documented feature of iPS derivation. In order to confirm the gender of the donor CLSC lines and the CLiPS derived from them, genomic DNA isolated from each CLSC line was PCR amplified with gene-specific primers to determine the presence of DYS439 and SRY loci, both of which are present on the Y chromosome. It was confirmed that aSF4 adult skin fibroblasts obtained from male donors were used as positive controls.

[0176] Table 1: Optimized electroporation conditions for generating CLiPS

[0177]

[0178]

[0179] Example 2: Derivation of human iPS cells without transgene integration and feeders

[0180] 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 propagated in their culture media PTT-e3 and PTT-4, respectively. Adult skin fibroblasts from a healthy 78-year-old male Asian donor were purchased from CellResearch Corporation Pte Ltd and cultured in DMEM / 10% FBS.

[0181] The medium PTT-4 consisted of 90% (v / v) CMRL-1066 and 10% (v / v) FBS, while the medium PTTe-3 had the following composition:

[0182]

[0183] Somatic cell reprogramming was performed using the conditions established in Example 1 and further in a feeder-independent manner. Logarithmic phase cultures were harvested by dissociation with TrypLE Express (ThermoFisher Scientific) and 0.72 million cells were pelleted in a 1.5 ml centrifuge tube. The cell pellet was resuspended in 120 μL of Buffer R (Neon TM Transfection System 100 μL Kit, Thermo Fisher Scientific MPK10096). A mixture containing 1.2 μg of each of the episomal vectors pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, and pCXLE-hUL (Addgene plasmids #27077 (SEQ ID NO: 12), #27078 (SEQ ID NO: 13), and #27080 (SEQ ID NO: 14), respectively) was added to the cells and mixed thoroughly (for 1×10 6 The cell suspension was placed in 100 μL Tip and Neon electroporation was performed using the following parameters: adult dermal fibroblasts—1,650 V, 10 ms, 3 pulses; CLEC—1350 V, 30 ms, 2 pulses; CLMC—1600 V, 20 ms, 1 pulse. Cells were immediately transferred to 6 ml of CLEC or CLMC medium containing 1 μM hydrocortisone (StemCell Technologies) and evenly distributed into 3 wells of a Matrigel-coated 6-well plate. Two days later, the medium was changed to a 1:1 mixture of CLEC or CLMC medium and mTeSR1 supplemented with 1 μM hydrocortisone. On day 4 after transfection, a medium change was performed with the same medium. On day 6 after transfection, the medium was changed to complete mTeSR1, and hydrocortisone was omitted here. Subsequently, medium changes were performed every two days with mTeSR1. When the diameter of the iPS colonies reached about 1-2mM (about day 20 forward), they were picked manually under a bright field microscope and each colony was placed in a single well of a 24-well plate (Nunc) coated with matrigel. When the cells in each well reached ~50% confluence, they were dissociated with dispase (StemCell Technologies) and transferred to the wells of a 6-well plate coated with matrigel. Subsequently, when the cells approached confluence, the cells were passaged at 1:3 by dissociation with 0.5mM EDTA. The newly passaged cells were cultured overnight in a medium containing 10μM ROCK inhibitor Y-27632. In addition to mTeSR1, other commercial ES / iPS culture media such as StermMACS TM iPS-Brew XF (Miltenyi Biotec) and TeSR-E8 (StemCell Technologies) have been used to maintain iPS cultures.

[0184] Protocol used to generate CLiPS:

[0185] 1. Actively dividing CLECs or CLMCs cultured in their maintenance medium PTTe-3 and PTT-4, respectively, in T-75 flasks were harvested by dissociation using TrypLE Express (ThermoFisher Scientific).

[0186] 2. Count cells and aliquot 0.72 million cells into microcentrifuge tubes and pellet.

[0187] 3. Resuspend the cell pellet in 120 μL buffer R (Neon TMTransfection System 100 μL Kit, Thermo Fisher Scientific MPK10096) was added with a mixture containing 1.2 μg each of pCXLE-hUL, pCXLE-hSK and pCXLE-hOCT3 / 4-shp53-F and mixed thoroughly.

[0188] 4. Load the cell suspension into 100 μL Tip. Electroporation was performed using the following parameters for CELC: 1350 V, 30 ms, 2 pulses, and for CLMC: 1600 V, 20 ms, 1 pulse.

[0189] 5. Immediately transfer cells into 4 ml of CLEC or CLMC medium (PTTe-3 and PTT-4, respectively) containing 1 μM hydrocortisone and distribute into 3 wells of the coated 6-well plate.

[0190] 6. Two days after electroporation, the culture medium was changed to a 1:1 (v / v) mixture of CLEC or CLMC culture medium (PTT-e3 and PTT-4, respectively) and mTeSR1 supplemented with 1 μM hydrocortisone.

[0191] 7. Four days after electroporation, perform a medium replacement with the same 1:1 (v / v) medium mixture.

[0192] 8. Six days after electroporation, the medium was changed to mTeSR1 only. Hydrocortisone was omitted at this time.

[0193] 9. Replace the culture medium every two days.

[0194] 10. iPS colonies may begin to appear as early as 2 weeks after transfection. When the diameter of iPS colonies reached about 0.5 mm to about 1 mm (about day 20 onwards), they were picked manually under a bright field microscope and each colony was placed in a single well of a 24-well plate (Nunc) coated with Matrigel.

[0195] 11. After colony picking, the culture medium of isolated colonies was changed every day.

[0196] 12. When the cells in each well occupied approximately 50% of the culture surface, they were dissociated with dispase (StemCell Technologies) and transferred to wells of a Matrigel-coated 6-well plate.

[0197] 13. Subsequently, when the cells reached about 70%-80% confluence, the cells were passaged at 1:3 by dissociation using 0.5 mM EDTA. The newly passaged cells were cultured overnight in medium containing 10 μM ROCK inhibitor Y-27632.

[0198] Following the above protocol, small clusters of cells that appear morphologically distinct from the parental cells begin to appear, beginning around day 10. By day 15, the clusters have acquired defined edges ( Figure 3 b), and from day 20 onwards discrete embryonic stem cell-like colonies appeared ( Figure 3 c and Figure 3 d). Colonies were picked when they reached 1-2 mm in diameter, expanded for characterization and storage. Expanded CLiPS exhibited a cell morphology indistinguishable from that of adult skin fibroblast-derived iPS or human embryonic stem (ES) cells, with characteristic large nuclei and thin cytoplasm ( Figure 3 e and Figure 3 f).

[0199] Example 3: Derivatization of cGMP-compatible CLiPS (CLMSC-DTHN)

[0200] To provide proof of concept that CLiPS can be generated under conditions compatible with human therapeutic applications, iPS were generated from a cGMP-grade CLMC line designated CLMSC-DTHN using the protocol described in WO2018 / 067071 for the generation of a population of mesenchymal stem cells in which 99% of the stem cells expressed the markers CD73, CD90, and CD105, but not the markers CD34, CD45, and HLA-DR) cGMP quality reagents were used whenever possible. The reprogramming protocol was identical to that described for CLMCs in Example 2, but Matrigel (an extracellular matrix prepared from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) was replaced by recombinant human laminin-511E8 fragment (iMatrix-511 SILK, ReproCELL), a defined, animal- and xeno-free matrix for coating cell culture vessels. In addition, mTeSR1 used for reprogramming and subsequent maintenance of CLiPS clones was replaced by cGMP mTeSR TM 1 (StemCell Technologies) instead.

[0201] Under the conditions described herein, CLMSC-DTHN reprogrammed with kinetics and efficiency comparable to CLMC (data not shown). At 10 days after transfection with the reprogramming vector, small clusters of cells with a compact morphology ( Figure 3n). These clusters developed into separable colonies from day 20 onwards. The expanded colonies showed the characteristic cell morphology of human pluripotent stem cells ( Figure 3 nq).

[0202] Propagation and cryopreservation of CLiPS

[0203] When the culture reaches ~90% confluence, CLiPS is subcultured (again using a medium suitable for maintaining iPS cells, such as mTeSR1 or TeSR-E8). The spent medium is aspirated along with any obvious differentiated areas that may be present. Care should be taken not to expose the cells to air for too long. Rinse the culture once with preheated (37°C) Dulbecco's phosphate-buffered saline (DPBS). Depending on the culture container - 0.5ml / well of a 24-well dish, 1ml / well of a 6-well dish, or 2ml for a 6cm dish, an appropriate volume of reheated (37°C) 0.5mM EDTA solution is added to the culture medium. The culture is placed in a 37°C incubator for 5 minutes and then observed under a microscope. The cells should appear round but not detached from the surface. The duration of incubation at 37°C varies with different CLiPS lines and can range from about 5-10 minutes. The duration of incubation will be largely based on previous experience with each cell line. After incubation, gently aspirate the EDTA solution, being careful not to remove the cells. Using a 1 ml pipette, culture medium such as mTeSR1 or TeSR-E8 containing the ROCK inhibitor Y-27632 is dispensed directly onto the cells to remove it. The volume of culture medium used depends on the size of the container used—0.5 ml / well for a 24-well culture dish, 1 ml / well for a 6-well culture dish, or 2 ml for a 6 cm culture dish. Repeat gentle pipetting until most of the cells are removed. The cell suspension is then transferred to a 15 ml Falcon tube. Rinse the culture container with fresh culture medium and merge the rinse with the cell suspension in the Falcon tube. Dilute the cells in the tube to a suitable volume for plating on a new matrix gel-coated container. The separation ratio can be in the range of 1:3 to 1:10, depending on the density of the initial culture and the growth rate of a single CLiPS system.

[0204] For cryopreservation, cells were suspended in PBS supplemented with 10% v / v tissue culture grade dimethyl sulfoxide (DMSO; e.g., Hybri-Max TM , Sigma-Aldrich) in mTeSR1 or TeSR-E8 (or any other suitable culture medium). The cell suspension is then aliquoted into an appropriate number of cryovials. The cell density of each aliquot depends on the desired rate at which cell confluence is achieved when thawing and culturing the aliquot. The cryovials are then transferred to a slow freezing apparatus such as Mr. Frosty TMFreezing Container (Thermo Scientific) or Cell Freezing Containers (BioCision LLC) and placed at -80°C overnight. The next day, transfer the cryovials to liquid nitrogen for storage. It is not recommended to store CLiPS aliquots at -80°C for more than 24 hours. Several commercial freezing media such as mFreSr TM (StemCell Technologies) and CS10 (Biolife Solutions) can also be used for cryopreservation and can be used according to the manufacturer's instructions.

[0205] Example 4: Analysis of CLiPS functionality

[0206] CLiPS functionality was determined by immunofluorescence staining of colony-forming CLiPS after electroporation. Thus, the expression of pluripotent embryonic stem cell markers (OCT4, SOX2, KLF4, NANOG, SSEA-4, TRA-1-81) was analyzed. To this end, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 15 minutes and then washed 3 times with PBS for 5 minutes each. For staining of intracellular or nuclear markers (OCT4, SOX2, KLF4, NANOG), cells were permeated with 0.1% Triton X-100 in PBS for 10 minutes and then blocked with FDB (5% FCS / 1% NGS / 1% BSA) for 1 hour. For staining of surface markers (SSEA-4, TRA-1-81), the permeation step was omitted. Cells were incubated overnight at 4°C with the first antibody appropriately diluted with FDB and then incubated with the second antibody coupled with the appropriate fluorescent dye for 2 hours at room temperature. The stained samples were mounted in ProLong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific).

[0207] In addition, the number and structure of chromosomes in individual CLiPS lines were assessed by karyotyping and G-banding analysis performed by the Cytogenetics Laboratory, KK Women's and Children's Hospital Pte. Ltd., Singapore.

[0208] In addition, RT-PCR analysis was performed to analyze the expression of reprogramming and pluripotent 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 PlusMini kits (Qiagen). 2 μg of total RNA was treated with DNase I and used for cDNA synthesis using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific). The PCR reaction was 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) with the following conditions: 1×95°C 1 min, 30×(95°C 15 s, Tm 15 s, 72°C 15 s), 72°C 1 min. The primer sequences and annealing temperatures (Tm) used are provided in Table 2 below.

[0209] Qualitative expression analysis was performed by agarose gel analysis, where samples were loaded onto 2% agarose gel spiked with SYBR Safe DNA stain (Thermo Fisher Scientific) in 1×TAE buffer and electrophoresed for 30 min at 80 V. Gel images were captured using the ChemiDoc Imaging System (Bio-Rad).

[0210] Table 2: Primer sequences

[0211]

[0212]

[0213] The results showed that CLiPS displayed robust expression of human embryonic stem cell (hES) markers KLF4, NANOG, OCT4, SOX2, SSEA4, and TRA-1-60 as demonstrated by antibody staining ( Figure 3 G-band analysis showed that CLiPS maintained a normal karyotype up to 17 generations after colony picking ( Figure 3 m). RT-PCR analysis of gene expression in parental cells, cells at day 11 after transfection, and expanded iPS clones revealed that activation of endogenous OCT4, SOX2, KLF4, LIN28, and L-MYC genes had replaced vector-driven expression of these genes in maintaining pluripotency in fully reprogrammed CLiPS ( Figure 3 v). At day 11 post-transfection, induction of the endogenous NANOG locus, a key gene for somatic cell reprogramming, was evident. The absence of detectable levels of EBNA-1 transcripts in CLiPS clones suggested that the plasmid vector had been lost from these cells. Expression of additional hES-specific genes GDF3, DPPA5, DNMT3, FGF4, and REX-1 in CLiPS further confirmed their hES-like molecular phenotype. TERT, which encodes the catalytic reverse transcriptase subunit of telomerase, which is essential for regulating self-renewal and maintaining pluripotency, was expressed in CLiPS at the same levels as in H1 hES.

[0214] Example 5: Expression analysis of pluripotent embryonic stem cell markers in CLiPS-DTHN

[0215] To analyze the expression of pluripotent embryonic stem cell markers (Oct4, Sox2, Klf4, Nanog) indicating pluripotency, immunofluorescence staining was performed on developing CLMSC-DTHN after electroporation. The immunofluorescence staining protocol was the same as that described in Example 4 for CLiPS.

[0216] Results showed that CLMSC-DTHN expressed pluripotent stem cell markers NANOG, OCT4, SOX2, and TRA-1-81 at levels indistinguishable from their non-GMP counterparts ( Figure 3 ru). Therefore, CLMSC-DTHN can provide the same desired embryonic properties of non-GMP derived CLiPS.

[0217] Example 6: Determination of CLiPS pluripotency

[0218] The pluripotency of CLiPS and aSF-iPS was evaluated in NOD-SCID mice by teratoma formation assay. For this purpose, 1 × 10 6 CLiPS cells were resuspended in 0.1 ml of ice-cold Matrigel and injected into 6-8 week old NOD / MrkBomTac-Prkdc scid Dorsal flank of mouse. Mice were sacrificed 3 months later and teratomas were harvested for histological analysis using standard techniques for paraffin sectioning and hematoxylin and eosin staining.

[0219] The results showed that palpable tumors formed in some mice starting 1 month after the iPS cells were injected subcutaneously into the dorsal flanks of mice. Histological analysis of teratomas isolated 3 months after injection showed that CLiPS cells spontaneously differentiated into tissues of the endoderm, mesoderm, and ectoderm lineages ( Figure 4 af).

[0220] Example 7: Differentiation of CLiPS into dopaminergic neurons

[0221] As an important prerequisite for the potential future therapeutic application of CLiPS, it is necessary to demonstrate their ability to differentiate into specific tissue types under defined in vitro conditions. For dopaminergic neuronal differentiation, iPS were differentiated into dopaminergic neural progenitor cells and neurons using the midbrain floor plate induction protocol described by Kriks, S. et al., Nature, 2011. 480(7378): p.547-51. Briefly, iPS were cultured at 3.5-4.0×10 4 cells / cm 2 The cells were plated at a density of 100 μg / cm2 on a culture dish coated with matrigel (Corning) and cultured for 5 days in a knockout serum replacement medium (KSR) containing Knock-Out DMEM, 15% knockout serum replacement, 1×GlutaMAX, and 10 mM β-mercaptoethanol. From the 5th day, KSR medium was gradually transitioned to N2 medium as described in Tomishima "Midbraindopamine neurons from hESCs." 2012Jun 10. In: StemBook. Cambridge (MA): Harvard Stem Cell Institute; 2008-. (available from 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, which consisted of Neurobasal medium, 2% B27 minus vitamin A, and 1× GlutaMAX and 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-like 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 cultured at high cell density (3–4×10 5 cells / cm 2) The cells were replated on culture dishes pre-coated 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. The cultures were maintained in NB27 medium with medium replacement every other day until the desired endpoint. The differentiated cells were analyzed for cell-specific marker expression at this stage. For this purpose, cryosections were performed, wherein the slides containing the sections were dehydrated by incubation at 37°C for 30 minutes, cooled to room temperature and washed 3 times with TBST. Section permeabilization, blocking, antibody staining and fixing were performed as described in Example 4. Using primary antibodies from the same host species, the first primary antibody was saturated using a fluorescent dye-conjugated monovalent antibody (Jackson ImmunoResearch) and then incubated sequentially with the second primary antibody and the conjugated secondary antibody.

[0222] The results showed that dopaminergic neurons were obtained from CLiPS and asF5-iPS using this protocol. Antibody staining revealed that almost 90% of the cells co-expressed the floor plate marker FOXA2 and the roof plate marker LMX1A ( Figure 4 k, k', k"), which is a clear marker of midbrain DA neuron precursors. Further differentiation produces abundant mature neurons, as shown by TUJ1 staining, of which approximately 30-50% co-express the dopaminergic marker tyrosine hydroxylase (TH) ( Figure 4 l, l', l"). Electrophysiological analysis of CLiPS-derived neurons at day 45 of differentiation confirmed that the cells displayed mature functional properties, with trains of action potentials displaying voltage dip responses characteristic of mature midbrain DA neurons upon injection of hyperpolarizing current ( Figure 4 m).

[0223] Example 8: Differentiation of CLiPS into hepatocytes

[0224] As an important prerequisite for the potential future therapeutic application of CLiPS, it is necessary to demonstrate their ability to differentiate into desired target cell types or specific tissue types under determined in vitro conditions. For liver differentiation, the protocol originally developed for differentiation of human embryonic cells (ES) on mouse feeder layers (Medine, CN et al., J Vis Exp, 2011 (56): p.e2969) is applicable to CLiPS and asF-iPS differentiation in mTeSR1 on matrigel. An improvement is that when iPS culture reaches 20-30% confluence, DMSO is supplemented to 2% and incubated for 24 hours. When the culture reaches ~ 30%-60% confluence, mTeSR1 is replaced by initiation culture medium (RPMI 1640-B27 supplemented with 100ng / mL activin A and 50ng / mL Wnt3a), inducing definitive endoderm formation. The culture is maintained in the initiation culture medium for 3 days, and the culture medium is replaced every 24 hours. After 72 hours in the priming medium, the medium was changed 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, the cultures were switched to L-15 maturation and maintenance medium (Leibovitz L-15 medium, 8.3% tryptose broth, 8.3% heat-inactivated FBS, 10 μM 21-hemisuccinate hydrocortisone, 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 analyzed again at this stage for cell-specific marker expression. For this purpose, cryosections were performed as described in Example 7.

[0225] The results showed that hepatocyte-like cells were obtained from CLiPS and asF5-iPS using this protocol. Antibody staining showed that the hepatocyte marker alpha-fetoprotein (AFP; Figure 4 g, g′, g″), cytokeratin 18 (CK18), and human serum albumin (HSA; Figure 4 h, h', h"). Most of the differentiated cells showed polygonal characteristics of hepatocytes. In addition, staining with Oil Red O showed a large amount of lipid droplets accumulated in the cells, which is a hallmark of cultured hepatocytes ( Figure 4 i, i', i'').

[0226] Example 9: Differentiation of CLiPS into cardiomyocytes

[0227] As an important prerequisite for the potential future therapeutic application of CLiPS, it is necessary to demonstrate their ability to differentiate into specific tissue types under defined in vitro conditions. For cardiomyocyte differentiation, the cardiomyocyte differentiation protocol of iPS was modified from the protocol described in Lian, X. et al., Proc Natl Acad Sci USA, 2012.109(27), p.E1848-57. iPS maintained on matrix gel in mTeSR1 was dissociated into single cells with StemPro Accutase (Thermo Fisher Scientific) at 37°C for 5 minutes and then 1×10 5 -2×10 5 cells / cm 2 (5×10 5 Cells / 24 wells) were seeded onto Matrigel-coated cell culture dishes in mTeSR1 supplemented with 5 μM ROCK inhibitor (Y-27632; Stemgent) for 24 hours. In a modification, when the cells reached ~80% confluence, the medium was changed to mTeSR1 supplemented with 2% DMSO. When the cells reached confluence, they were treated with CHIR99021 in RPMI / B27-insulin for 24 hours. In another modification, the concentration of CHIR99021 was reduced to 5 μM from the original 12 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 mixed 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 culture. After two days, the medium was changed to RPMI / 2% B27 without insulin only. After 48 hours, the cultures were maintained in RPMI / 2% B27 with medium changes every 3 days until the desired endpoint. As described in Example 7, beating cardiomyocytes were fixed and stained for cell-specific markers.

[0228] The results showed 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 8 days of differentiation. Immunofluorescence antibody staining of functional cardiac markers myosin regulatory light chain 2a (MLC2a), cardiac troponin I (cTnI), and α-actinin (αACT) revealed sarcomere structures within differentiated cardiomyocytes ( Figure 4 j, j', j"). No significant difference in differentiation efficiency was observed among them.

[0229] Example 10: Differentiation of CLiPS into oligodendrocytes

[0230] 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. Oligodendrocyte differentiation of CLiPS and asF-iPS was performed according to the protocol of Douvaras, P. and V. Fossati, Nat Protoc, 2015.10(8): p.1143-54. In addition, cryosections were performed as described in Example 7 to analyze cell-specific marker expression.

[0231] On day 75 of differentiation, Olig2-positive oligodendrocyte precursor cell clusters (OPCs; Figure 4 n) and O4-positive late OPCs clusters ( Figure 4 o).

[0232] Example 11: Immunogenicity Analysis

[0233] To gain some insight into the immunogenicity of CLiPS and their neural derivatives, these cells were evaluated for expression of a panel of immunogenicity-related markers by flow cytometric analysis. For this purpose, primary cells and day 25 differentiated DA NPCs were harvested by dissociation with TrypLE Express, while iPS cultures were harvested by dissociation with 0.5 mM EDTA. Cells were resuspended in 1× Ca-free medium containing 0.1% bovine serum albumin (BSA). 2+ and Mg 2+ 5 million cells / ml in DPBS. 100 μl of cells were stained with the appropriate conjugated antibody or its isotype control on ice in the dark for 30 min. For HLA-E and HLA-G staining, cells were permeabilized with BD Phosflow Perm / Wash Buffer I (BD Biosciences) according to the manufacturer's instructions before staining. After staining, cells were incubated in 1× Ca-free 2+ and Mg 2+ Wash 2× in DPBS / 5 mM EDTA, fix with 1% paraformaldehyde for 1 h in the dark, and then in 1× Ca-free 2+ and Mg 2+ Wash 2× in DPBS / 5 mM EDTA. Resuspend the cells in 0.5 ml 1× Ca-free 2+ and Mg 2+ The cells were fixed with 50% PBS / 5 mM EDTA and analyzed on a flow cytometer. The primary cells and iPS were analyzed on a FACSCalibur, while the dopaminergic neuron progenitor cells (NPC) were analyzed on a FACSCanto II instrument (both from BD Biosciences). The data were analyzed using the FlowJo software package (FlowJoLLC). The antibodies used are listed in Table 3.

[0234] Table 3: Antibodies used for flow cytometry

[0235]

[0236] MHC class I HLA-A, -B and -C and MHC class II HLA-DR molecules are known to be important for allogeneic immune responses. The results showed that HLA-ABC was expressed in all iPS samples, but significantly reduced levels were observed for EC23-CLiPS ( Figure 6 a). HLA-DR expression was absent in all iPS samples ( Figure 6 b), which is consistent with previous reports that HLA-II expression in iPS cells is negligible ( 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 co-stimulatory molecules CD40, CD80 and CD86 play an important role in activating T cells during allogeneic immune responses. Of the three molecules evaluated, only CD40 was expressed on iPS, with the lowest level expressed by asF-iPS and the highest level expressed by MC23-CLiPS compared to the rest ( Figure 6 a). Since tolerogenic HLA-E and HLA-G have been reported to be expressed on 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), the expression of these antigens by CLiPS was also investigated. Analysis of permeabilized cells showed only minor expression of HLA-E in MC23-CLiPS and EC44-CLiPS, which was below detectable levels in other samples. Next, expression analysis of the entire marker panel was repeated on day 25 DA differentiated cultures. The neural cell population gated for NCAM positive staining was analyzed. The NCAM+ fraction of all samples exceeded 97%, with asF-iPS and EC23-CLiPS showing comparable differentiation efficiencies of 99.5% ( Figure 6 b) HLA-ABC was expressed by all NPC samples, but at generally 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 on MC23-CLiPS decreased as they differentiated into NPCs. CD40 expression was downregulated in all NPC samples, with only EC23-iPS-derived and EC44-iPS-derived NPCs showing slight expression. HLA-E expression was absent in all NPC samples, but slight upregulation of HLA-G was observed in asF-iPS-derived and EC23-iPS-derived NPCs. These results indicate reduced immunogenicity in CLiPS.

[0237] Example 12: Transplantation of CLiPS-derived dopaminergic neurons in a fully immunocompetent mouse model of Parkinson's disease Jingyuan

[0238] Previous studies have shown that dopaminergic neurons generated from human embryonic stem cells and iPS using various protocols can be implanted into rodents (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) models of Parkinson's disease (PD). In all these studies, animals were immunocompromised or pharmacologically immunosuppressed to prevent transplant rejection.The need for immunocompromised or immunosuppressed animals is eliminated only when 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) iPS-derived cells are used for transplantation.

[0239] To demonstrate the implantability of CLiPS-derived DA NPCs differentiated using the methods of the present invention, transplanted day 25 NPCs differentiated from asF-iPS, EC23-CLiPS, and MC23-CLiPS were transplanted into immunocompromised NOD-SCID mice (n=3). In this case, it was 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.

[0240] In order to test the immunogenicity of CLiPS-derived DA NPCs, transplantation on a PD model is required. 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 involves injecting 6-OHDA into the rodent brain, causing motor dysfunction characterized by a certain degree of 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 purchased from InVivos Pte Ltd and maintained under SPF conditions at the Animal Research Facility, NNI scid6-OHDA lesions were induced in WT mice (4 weeks old) and male C57BL / 6NTac mice (6-8 weeks old) purchased from InVivos Pte Ltd, where the mice used for this experiment were fully immunocompetent and no immunosuppression was administered before or after transplantation.

[0241] To generate a mouse PD model, 7.5 μg of 6-OHDA (Sigma, Merck-Millipore; dissolved in 0.9% NaCl containing 0.2% ascorbic acid at 2.5 mg / ml) 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 the skull. After two weeks of adaptation, the three NPC samples (i.e., NPCs derived from asF-iPS, EC23-CLiPS, and MC23-CLiPS) were transplanted into the striatum of immunocompetent 6-OHDA-lesioned C57BL / 6 mice, which were considered to be successfully injured by stereotaxic injection for the mouse model.

[0242] To determine an appropriate model for transplantation, apomorphine-induced rotations were scored and mice displaying >6 rotations per minute were used for transplantation. For transplantation, day 25 dopaminergic progenitor cells were harvested by dissociation and resuspended in HBSS supplemented with 10 ng / mL BDNF, 10 ng / mL GDNF to a concentration of ∼1.25 × 10 5 Cells / μl. 2 μL of cell suspension was injected into injured mice at the following coordinates: AP + 0.5 mm; ML - 2.0 mm, DV - 2.8 mm from the skull. To evaluate whether transplanted NPCs can integrate and mediate functional benefits in injured animals, rotational asymmetry tests were performed at 2-week intervals. Rotational assays were performed every 2 weeks until 9 months, in which mice were injected intraperitoneally 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. The batches of animals were sacrificed by terminal anesthesia at 1, 6, and 9 months after transplantation.

[0243] Six months after transplantation, NPC-transplanted, sham-injected and unmanipulated mice were evaluated for striatal dopamine transporter (DAT) activity by positron emission tomography (PET) imaging using a 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 hours prior to the imaging session. During the scan, animals were kept warm with an integrated hot air channel from the imaging bed. Respiratory rate and temperature were monitored throughout the scan to ensure adequate anesthesia levels. Mice were imaged using a nanoScan PET / MRI scanner (Mediso Ltd., Hungary) at SingHealth Experimental Medicine Centre (SEMC). The scanner is equipped with 12 detector modules with an axial field of view (FOV) of 94 mm and a transverse FOV of 94 mm or 120 mm diameter in a 1:3 and 1:5 overlap mode, respectively. The animal's head was first placed in a prone position, and after an intravenous injection of 3.57-10.61 MBq of [18F]FE-PE2I with a maximum volume of 0.1 ml through the tail vein, a 62-minute 3D dynamic PET scan was performed with frames of increasing duration, i.e., 4 times of 10 seconds, 4 times of 20 seconds, 3 times of 1 minute, 7 times of 3 minutes and 6 times of 6 minutes. [18F]PE-PE2I was synthesized at Radiopharmaceuticals Pte Ltd., Singapore. MRI images were used for attenuation correction of PET scans and as structural references for PET images in data analysis. Therefore, T1-weighted MRI images were obtained using the MRI component of the nanoScanPET / MRI scanner. During the MRI scan, the integrated mouse head coil covered the entire brain. 0.6 mm slices were acquired using a 3D GRE EXT sequence: 64 mm square FOV, 128 × 128 matrix, 20-ms repetition time (TR), 2.3-ms echo time (TE), 25 degree flip angle. All images and dynamic analyses of [18F]FE-PE2I PET images were performed using PMOD (version 3.5; PMOD Technologies). All PET images were first automatically registered to MRI images using the FUSION tool in PMOD. The MRI images were then manually registered to a T2-weighted mouse template (Mirrimone, C57BL / 6J mouse; Ma, Y. et al., Neuroscience, 2005. 135(4): p.1203-15; Mirrione, MM et al., Neuroimage, 2007. 38(1): p.34-42), which contained a volume of interest (VOI) template with 20 regions.The accuracy of manual registration was accessed and verified by two different people. Finally, the combined transformation matrix was applied to transform the PET images into MRI mouse templates. The VOIs of the left and right striatum and the cerebellum were used for analysis. In order to reduce the errors caused by misregistration and missense (He, B. and E.C. Frey, Phys Med Biol, 2010.55 (12): p.3535-44), a 3D erosion with one voxel was applied to the obtained VOI. [18F] FE-PE2I binding was quantitatively performed using a non-invasive reference tissue model because they were equally accurate (Varrone, A. et al., Nucl Med Biol, 2012.39 (2): p.295-303) compared with the kinetic analysis using arterial input function. The binding potential (BPnd) value was calculated using a simplified reference tissue model (SRTM) (Lammertsma, AA and SP Hume, 1996.4 (3Pt 1): p.153-8) with the cerebellum as a reference. Regional time activity curves (TACs) were also extracted from the VOIs of the striatum and cerebellum. During imaging, anesthesia was induced with 5% isoflurane in 100% O2 and maintained with 1.5-2% isoflurane.

[0244] Mouse brain sections were analyzed for the presence of microglia / macrophages, as 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). For this purpose, immunostaining of microglia / macrophage-specific marker Iba1 was performed after transcardial perfusion with 4% PFA. For this purpose, the PFA-perfused brain was post-fixed overnight in 4% PFA and then continued to balance in 15% and 30% w / v sucrose solutions in PBS until they settled to the bottom of the test tube. The brain was 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).

[0245] The results showed that hNCAM+ / TH+ neurons were present in all three groups 1 month after transplantation ( Figure 7 ac), indicating that NPCs can differentiate into mature neurons and survive in the host environment. Figure 7 h) or MC23-iPS (data not shown) groups. hNCAM / TH+ fibers can be seen extending from neurons in the transplanted core of the EC23-CLiPS group along the axonal bundles of the corpus callosum ( Figure 7d and Figure 7 e). Immunostaining for the microglia / macrophage-specific marker Iba1 revealed an abundance of microglia / macrophages in the injected hemisphere compared to the non-injected hemisphere ( Figure 7 i and Figure 7 j). Microglia / macrophages infiltrating the core of the grafts showed deformed morphology characteristic of activated microglia, while those microglia surrounding the grafts showed a branched morphology typical of quiescent cells. In addition, the infiltrating microglia stained positive for CD68, a marker for activated microglia. No accumulation of microglia was observed at the injection site of asF5-iPS- and MC23-CLiPS NPC-transplanted brains 1 month after transplantation, presumably because they had dispersed and returned to a quiescent state after xenograft clearance. As confirmed by human nuclear antigen (HuNu) and human NCAM staining, human TH+ neurons survived for up to 9 months in some animals transplanted with EC23-CLiPS NPCs ( Figure 8 af). Rotational asymmetry testing showed that when stimulated with the dopamine agonist apomorphine, injured animals will exhibit contraversive rotations due to hypersensitivity of postsynaptic D2 dopamine receptors on the injured striatum caused by dopamine depletion. The efficacy of the given intervention will be manifested as an improvement in this rotational asymmetry. Compared to asF-iPS NPCs or sham-transplanted animals, only animals transplanted with EC23-CLiPS NPCs showed improved rotational behavior in both species ( Figure 8h). In these mice, the reduction in rotation reached significance (p<0.05) starting at week 20 after transplantation, decreasing to 18.2±24.7% and 11.1±20.8% at weeks 20 and 22, respectively. The model showed delayed recovery, with deterioration initially observed after transplantation. This may be due to the time required for the stereotaxic injection to induce an inflammatory response and for the NPCs to mature, integrate with, and innervate the host tissue. The functional improvement of Parkinson's disease motor symptoms in EC23-CLiPS NPC-transplanted animals suggests functional restoration of dopaminergic function in the transplanted striatum. To further investigate this, we performed PET imaging in transplanted mice using the dopamine transporter (DAT) ligand [18F]FE-PE2I (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 that is primarily responsible for the reuptake of dopamine released at synapses, and molecular imaging of DAT is an established tool for studying dopaminergic function. PET imaging 6 months after transplantation showed that DAT activity in the transplanted injured hemisphere recovered to approximately 71.4±10.3% of that in the uninjured hemisphere of EC23-iPS NPC-transplanted mice (n=3) ( Figure 8 i). In contrast, the recovery rate of asF-iPS-NPC transplanted mice was only 16.4±4.0%. These results clearly indicate a significant recovery of dopamine reuptake function in EC23-iPS NPC transplanted mice.

[0246] Example 13: Transplantation of CLiPS-derived DOPA in a fully immunocompetent rodent rat model of Parkinson's disease Aminergic neurons

[0247] Our transplantation results show that EC23-CLiPS-derived NPCs are tolerated when transplanted into the striatum of C56BL / 6 mice. In order to exclude possible species-specific biases of this phenomenon, transplantation studies were repeated in different species, Wistar rats. Parkinson's syndrome was induced in these rats by injecting 6-OHDA into the MFB to damage the nigrostriatal pathway. In this case, it was noted that all animal experiments were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the National Neuroscience Institute (NNI) of Singapore. Additional approval for rat experiments was provided by the IACUC of the National Technological University (NTU) of Singapore. It is known that MFB damage causes a more complete depletion of the dopamine system compared to striatal damage, and therefore it is speculated that it is less likely to lead to spontaneous recovery (Torres, EM and SB Dunnett, Animal Models of Movement Disorders: Volume I, ELLane and SB Dunnett, Editors. 2012, Humana Press: Totowa, NJ. p. 267-279). The rats have full immunocompetence and no immunosuppression was administered before or after transplantation. For analysis, female Wistar rats aged 8 weeks were purchased from InVivos Pte Ltd. Unilateral damage was induced by stereotaxic injection of 20 μg 6-OHDA in 4 μl into the left medial forebrain bundle (MFB) at the following coordinates: AP-4.4 mm; ML-1.2 mm; and DV-8.6 mm from the dura. In order to determine a suitable transplantation model, apomorphine-induced rotation was scored as described in Example 12. Rats that showed >6 rotations / min were transplanted with 3 μl of approximately 1.25 × 10 5 Cells / μl dopaminergic progenitors were transplanted into the left striatum at day 25: AP + 0.8 mm; ML - 2.5 mm; and DV - 5 mm from the dura. To evaluate whether transplanted NPCs can integrate and mediate functional benefits in injured animals, rotational asymmetry tests were performed at 1 month intervals as described in Example 12. Rats were sacrificed by terminal anesthesia at 6 months after transcardial perfusion with 4% PFA as described in Example 12 and brains were harvested for immunohistological analysis. Some animals that did not pass the injury criteria were also transplanted and sacrificed 1 and 3 months after transplantation to evaluate cell survival and engraftment.

[0248] The results showed that unilateral depletion of dopaminergic neurons in the substantia nigra caused by retrograde transport of 6-OHDA via the MFB was confirmed in the model by DAB staining of TH in midbrain sections. Fig.11 d). Animals that showed at least 5 revolutions per minute under apomorphine stimulation 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 transplants showed expression of protrudin 1, indicating integration with host neurons ( Fig.11 b). Furthermore, only animals transplanted with EC23-CLiPS NPCs showed improved rotational behavior in both species compared with asF-iPS NPCs or sham-transplanted animals ( Fig.11 e). The rat model also showed delayed recovery, with deterioration initially observed after transplantation. This may be due to the inflammatory response induced by stereotaxic injection and the time required for NPCs to mature, integrate with host tissues, and innervate host tissues. The results also showed a significant restoration of dopamine reuptake function in rats with CLiPS-derived NPC transplants.

[0249] Example 14: Methods of Differentiating and Characterizing RPE Cells

[0250] Rapid and directed differentiation scheme was used to achieve RPE differentiation from CLiPs. Differentiated RPE cells were purified and plated on transport well plates for further characterization, such as immunostaining and using quantitative reverse transcriptase polymerase chain reaction (q-RT-PCR) gene expression assays. The function of cells was analyzed using epithelial cell transmembrane electrical resistance (TEER) and phagocytosis of photoreceptor outer segments (POS).

[0251] Umbilical Cord Lining Induced Pluripotent Stem Cell (CLiPS) Culture

[0252] CLiPs were cultured on Matrigel (Corning)-coated tissue culture plates (Corning Costar) in mTESR1 (Stem Cell Technologies) medium.

[0253] Differentiate into retinal pigment epithelium

[0254] The inventors used the directed differentiation protocol of Foltz and Clegg (2017) with different modifications. CLiPs and human ES cells were grown on Matrigel-coated tissue culture plates in mTeSR1 medium. When the cells reached 90-95% confluence, they were exposed to different differentiation media containing basal medium (DMEM / F12 with 1×B27 and 1×N2 supplements and non-essential amino acids) supplemented with different growth factors.

[0255] Differentiation Medium 1: 0 to 2 days, 1 μM LDN-193189, 10 ng / ml Dkk1, 10 ng / ml IGF1, and 10 mM nicotinamide. Differentiation Medium 2: 2 to 4 days, 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: 4 to 6 days, 10 ng / ml Dkk1 and 10 ng / ml IGF1 and 100 ng / ml Activin A. Differentiation Medium 4: 6 to 8 days, 100 ng / ml Activin A and 10 μM SU5402. Differentiation Medium 5A: 8–11 days, basal medium containing 100 ng / mL Activin A, 10 μM SU5402, and 1.5 μM CHIR99021. Differentiation medium 5B: Days 11–16, 100 ng / mL Activin A, 10 μM SU5402, and 3 μM CHIR99021. On day 16, the basal medium was replaced with RPE maintenance medium: 50% DMEM / F12, 50% Minimum Essential Medium Eagle, α-modified, 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 medium 4, 5A, and 5B was replaced by 1 μM PD173074.

[0256] Characterization of CLiPs-derived RPE

[0257] ●Transcription assay-quantitative reverse transcriptase polymerase chain reaction

[0258] ●Immunocytochemistry for evaluation of RPE-specific proteins

[0259] ● Epithelial cell transmembrane electrical resistance (TEER)

[0260] Photoreceptor outer segment (POS) phagocytosis assay

[0261] Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)

[0262] RNA samples were collected on days 0, 2, 4, 6, 8, 12, 16, and D30. Total RNA was isolated using the RNeasy Mini kit (Qiagen). cDNA was synthesized from 1 μg RNA using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR was performed in triplicate (10 μl reaction) 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 75–200 base pair PCR products included OCT4, NANOG, and SOX2 as pluripotency markers, OTX2, LHX2, RAX, and SIX3 as early eye field markers, PAX6, MITF, VSX2, and SOX10 as early RPE markers, and BEST-1, PMEL 17, MERTK, TYROSINASE, TRYP2, and RPE65 as mature RPE markers. Data were normalized to the "housekeeping" gene glyceraldehyde phosphate dehydrogenase (GAPDH).

[0263] Immunocytochemistry

[0264] After 6 weeks of seeding in the transport well plates, cells were washed with PBS and fixed with 4% paraformaldehyde (pH 7.4) at room temperature (RT) for 20 minutes. The fixed cells were then washed with PBS, permeabilized with 0.2% Triton X-100 for 3-5 minutes at room temperature, and blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. The cells were then probed with primary antibodies in 1% BSA at 4°C overnight. After washing 3 times to remove the primary antibodies, the cells were incubated with appropriate Alexa Fluor-conjugated secondary antibodies (1:1000; Life Technologies), DAPI for staining nuclei, and Alexa-conjugated phalloidin for staining actin for 45 minutes at room temperature. The cells were washed with PBS and fixed using Fluorsave (Calbiochem). The cells were imaged using a LSM 700 confocal microscope (Carl Zeiss, Jena, Germany) using a 40x or 63x oil immersion objective.

[0265] The following antibodies were used: rabbit zona-occludens 1 (ZO-1, 1:200, Invitrogen), rabbit occludin (1:125, Invitrogen), mouse retinal pigment epithelium-specific 65KDa protein (RPE65, 1:125, Abcam), mouse cellular retinal 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).

[0266] Transepithelial electrical resistance (TEER)

[0267] The development of epithelial barrier properties and tight junction formation between RPE cells reflecting the integrity and polarity of the RPE monolayer were determined by measuring TEER. For this reason, cells were cultured on permeability 0.4 μm 24-well transport plate inserts (Corning) coated with Synthemax-II (Corning). According to the manufacturer's instructions, TEER measurements were performed weekly using an epithelial volt-ohmmeter-EVOM2 (World Precision Instruments). In brief, the electrodes were sterilized with 70% ethanol, air dried, balanced in RPE culture medium, and placed in a transport plate filter, wherein the longer electrode was located in the lower chamber contacting the bottom of the culture dish, and the shorter electrode was located in the upper chamber. Net TEER (Ω cm2) was calculated by subtracting the resistance value of the experimental transport plate from the resistance value of the control (transport plate without plated cells), and the net value was multiplied by the filter membrane area.

[0268] Photoreceptor outer segment (POS) phagocytosis assay

[0269] POS is isolated from pig eyes collected from a local slaughterhouse. The pig eyes were cut in half with a blade under red light in a dark room, and the retina was removed with forceps. The retina was placed in a homogenization medium, mixed thoroughly and filtered. The retinal suspension was layered on a sucrose gradient (25-60%) and centrifuged at 112,398 × g for 1 hour in an ultracentrifuge (Optima ultracentrifuge, Beckman). The pink POS layer was collected and POS was labeled with fluorescein isothiocyanate (FITC, Invitrogen) in 0.1M sodium bicarbonate buffer (pH 9.5) for 1 hour at room temperature. The labeled POS was washed and stored in aliquots at -80°C until use. For phagocytosis assays, RPE cells grown on transport well plates were stimulated with POS labeled with FITC at 37°C in a 5% CO2 incubator or at 4°C for controls for 2 hours. Cells stimulated with unlabeled POS were used as controls. The wells were then washed three times with PBS to remove unbound POS and dissociated into single cells using TrypLE (Gibco). FITC fluorescence was measured using a BD LSR II flow cytometer to determine POS phagocytosis.

[0270] Flow cytometry

[0271] The 4-6 week-old cells grown on 24-well plates were stained with Pmel17 antibody (Dako, M0634) using IntraPrep permeabilization reagent (Beckman Coulter, IM2389). In brief, cells were dissociated with TrypLE, and fixed and permeabilized according to the instructions provided in the test kit. About 50,000 cells were transferred to a 15ml centrifuge tube, and incubated overnight with 2-4 μl Pmel or RPE65 antibodies on a roller shaker in a cold room, washed with PBS subsequently, cells were incubated with the 488 / 647 secondary antibody labeled with Alexa, and analyzed in a FACS LSR5 II instrument (BD).

[0272] RPE purification method

[0273] (i) Manual purification: Non-RPE cells in the differentiation culture were manually removed by scraping with a 10 μl tip of a pipette by observing the culture plate under a dissecting microscope. The plate was washed 3 times with PBS to remove all non-RPE cells. The remaining cells highly enriched in RPE were dissociated by adding fresh TrypLE and incubating for 5-10 minutes.

[0274] (ii) TrypLE purification: Compared with non-RPE cells, RPE cells have stronger attachment to tissue culture plates. Due to the weaker attachment to the growth surface, non-RPE cells can be easily removed by processing with a gentle dissociation agent such as TrypLE. After sucking out the culture medium, phosphate buffered saline is added to the well, and the loosely attached non-RPE agglomerates are removed by strong pipetting with a 1ml pipette. After removing the separated clusters, the plate is incubated with TrypLE for 5-10 minutes, and the non-RPE cells are separated to release the non-RPE cells by gently knocking (tapping) and pipetting (pipetting). The non-RPE cells separated are collected, dissociated and used as "loose" fractions. The plate is thoroughly washed 3 times with PBS to remove all non-RPE cells, and the tightly attached RPE cells are dissociated by incubating 5-10 minutes with fresh TrypLE. Both tightly attached RPE and loosely attached non-RPE cells are plated for experiment.

[0275] (iii). TrypLE + manual purification: Although the above TrypLE purification removed most of the large non-RPE aggregates, some small aggregates were still present, which were removed by manual purification as described in Method 1.

[0276] (iv) TrypLE+ scatter sorting: After aspirating the culture medium, loosely attached RPE cells were removed as described in method 3, and the remaining cells were dissociated by further TrypLE treatment for 5-10 minutes, and processed as described in scatter sorting. During sorting, the weakly attached non-RPE cells removed by TrypLE treatment were used to set the gating of low scatter cells. The high scattering group present only in the strongly attached fraction helped to set the gating of sorting RPE cells.

[0277] (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 scatter fraction and low scatter fraction using a BD FACS Aria II cell sorter.

[0278] Analysis of mitochondrial and glycolytic function

[0279] CLiPs-RPE, skin-iPSC-RPE, and H9-RPE cells were analyzed for mitochondrial and glycolytic function using an XFe96 extracellular flux analyzer (Seahorse Bioscience) using assay conditions described by the manufacturer. Cells were plated at 6 × 10 4The inoculation density of 1000 μg / mL was plated in a 96-well plate coated with Synthemax-II, and grown for 48 hours. Oxygen consumption rate (OCR) was detected under basal conditions, and oligomycin (2 μM), FCCP (1.5 μM) and rotenone (0.5 μM) and antimycin A (0.5 μM) were added successively using a cell mitochondrial stress test (Cell Mito Stress Test). This allows the following parameters to be measured: basal respiration, OCR, ATP production, maximum respiration, spare respiratory capacity and non-mitochondrial respiration. Extracellular acidification rate (ECAR) was measured under basal conditions, and 10 mM glucose, 5 μM oligomycin and 100 mM 2-DG were added successively using a glycolytic stress test. This measured the parameters of glycolytic capacity and glycolytic reserve. The assay result was standardized for cell number.

[0280] Image analysis to assess pigmentation

[0281] Use Chemidoc Touch system (Biorad) to shoot the black and white 16-bit image of differentiation plate at regular intervals from the 17th day.Based on background intensity threshold, use Matlab software to measure the black area and brightness of RPE cell.From the maximum possible brightness intensity (65535) of 16-bit images, deduct the brightness of the pigmentation obtained from analysis, to obtain the darkness of pigmentation.

[0282] Example 15: Differentiation of CLiPS into RPE cells

[0283] The inventors used an RPE differentiation method to generate RPE from CLiPS, which could be of mesenchymal (CLMC) or ectodermal (CLEC) origin (Table 4).

[0284] Table 4: Different stem cells used in this study

[0285] Cell lines Cell Type Donor age Donor gender Donor's ethnic background AsF5 Skin iPS 78 male Asia AG04148 Skin iPS 56 male White HDFA Skin iPS 31 female White CLEC23 CLEC 0 male Asia CLMC23 CLMC 0 male Asia CLMC30 CLMC 0 male Asia CLMC44 CLMC 0 female Asia H9 cells People 0 female

[0286] In the RPE differentiation, the inventors used ES cells and skin iPS cells as controls (Table 4). Using this method, CLiPS cells were robustly differentiated into RPE ( Fig.13 ).

[0287] Example 16: CLiPS cells consistently have high RPE differentiation efficiency compared to skin iPS cells

[0288] To compare the RPE differentiation efficiency of different stem cell types (Table 4), the inventors developed a visual grading system by estimating the percentage of area occupied by pigmented cells in each well of the differentiation plate ( Fig.14a). RPE differentiation efficiency was graded as 0, 1, 2, or 3, corresponding to no pigmentation, <30%, 30-60%, or >60% pigmentation, respectively. A stacked bar chart of each differentiation plate was plotted with different colors to indicate different levels of pigmentation ( Fig.14 b). In biological replicates of differentiation, ES cells and iPS cells derived from umbilical cord lining mesenchymal cells (CLMCs) consistently showed high RPE differentiation efficiency compared to skin iPS cells. The high RPE differentiation of CLiPS was further confirmed by estimating the percentage of cells expressing the RPE-specific protein Pmel17 by flow cytometry ( Fig.14 c).

[0289] Example 17: CLiPS-derived RPE has higher pigmentation than ES-derived RPE

[0290] To compare the intensity of pigmentation of RPE produced by various cell lines and the kinetics of pigmentation acquisition, images of differentiation plates were taken at regular intervals starting from differentiation day 17 using the ChemiDoc Touch gel imaging system (Bio-Rad Laboratories). Images of differentiation plates taken on day 30 ( Fig.15 a) and phase contrast images ( Fig.15 b), CLiPS has darker pigmentation than ES cells. Analysis of pigmentation intensity at different days of differentiation showed that CLiPS-RPE has higher pigmentation than ES-derived RPE throughout the differentiation process ( Fig.15 c). Consistent with this, differentiated cultures of CLMC23 showed higher expression of pigmentation-related genes MITF, PMEL17, TYROSINASE, and TRYP2 ( Fig.15 d).

[0291] Example 18: CLiPS-derived RPE expresses RPE-specific genes

[0292] The inventors examined the expression of RPE-specific genes in CLiPS-derived RPE by quantitative PCR at differentiation days 18 and 35. Strong expression of RPE-specific genes RPE65 and MERTK was observed ( Fig.16 ), and the expression levels were higher in CLiPS-derived RPE at day 35, suggesting that they might be more mature.

[0293] Example 19: CLiPS-derived RPE has functional tight junctions and is capable of phagocytosis

[0294] The functionality of CLiPS-RPE was determined by measuring the transepithelial electrical resistance (TEER) of the epithelial cells and the phagocytosis of FITC-labeled photoreceptor outer segments. Fig.17 a) and phagocytosis ( Fig.17 b) Similar to ES-derived RPE.

[0295] Example 20: CLiPS-RPE expresses proteins similar to ES-derived RPE

[0296] Polarization of the RPE is critical for apico-basal-specific functions, and proteins show localized expression in polarized RPE monolayers. To test whether CLiPS show similar protein expression to previously reported RPE, the inventors immunostained RPE for different proteins. Similar to native, ES, and iPS-derived RPE, ZO-1 is expressed at cell-cell junctions, Mertk is expressed on the apical side, and RPE-65 is expressed in the cytoplasm ( Fig.18 ).

[0297] Example 21: Improvement of RPE differentiation protocol

[0298] The present inventors have developed a slightly improved RPE differentiation protocol based on the method developed by Foltz and Clegg 2017 (J Vis Exp. 2017; (128): 56274). (A) As described in the original Clegg protocol, using CHIR at 3 μM (Figure 19a) resulted in excessive cell death around the 11th to 12th day of differentiation, resulting in a significant reduction in RPE yield. In order to prevent this, the present inventors improved the protocol by gradually increasing CHIR in the culture medium in the following manner: starting from 1.5 μM for 3 days (the 8th to 11th day of differentiation), followed by 3 μM for 5 days (the 12th to 17th day). (B) A new FGF inhibitor for RPE differentiation was also identified: the present inventors replaced SU5402 (Figure 19b) with PD173074 to achieve similar degrees of RPE differentiation and pigmentation (Figure 19c). PD173074 was used at a much lower concentration than SU5402 (1 μM instead of 10 μM), which can reduce the undesirable changes in gene expression induced by high concentrations of chemicals (Waldmann T et al., 2014, Chem Res Toxicol, . 2014 Mar 17; 27(3): 408-20). The RPE obtained by differentiation with different FGF inhibitors SU5402 or PD173074 had comparable TEER and phagocytosis (Figure 19d, e).

[0299] Example 22: Method developed for RPE purification

[0300] RPE purification is carried out with the differentiation culture of 30-35 days old, and now RPE has obtained pigmentation.The method of purifying RPE from mixed differentiation culture is included in the morphological difference based on RPE and non-RPE cells at the 14th day of differentiation and manually removes non-RPE cells (Foltz and Clegg, 2017), by selectively removing non-RPE cells (Nazari et al., 2015, Yuko Iwasaki et al., 2016) weakly attached to culture dish by short-time treatment with weak dissociation agents such as TrypLE or Accutase, and scattering sorting (Shih et al., 2017) of the higher scattering of melanosomes to light in RPE cells. Different from the culture of 14 days old described previously (Foltz and Clegg, 2017), the inventor carries out RPE purification with the differentiation culture of 30-35 days old. By 30-35 days, the RPE cells in the differentiation culture acquire brown pigmentation, which helps to distinguish RPE cells from non-RPE cells in manual purification. The inventors compared different methods (Figure 20 a and b) to identify which method will produce functional RPE with the highest purity and yield. These methods include (i) manual purification: based on its form and lack of pigmentation, identify non-RPE cells, and manually remove them via scraping by observing under a dissecting microscope, (ii) TrypLE purification: remove most of the weakly attached non-RPE clusters by part TrypLE processing, (iii) TrypLE+ is manual: eliminate most of the weakly attached non-RPE clusters by part TrypLE processing, then manually remove a few non-RPE clusters that escape TrypLE processing by observing under a dissecting microscope, (iv) TrypLE+ scattering sorting: remove weakly attached non-RPE clusters by part TrypLE processing, then carry out scattering sorting, (v) scattering sorting: based on their relative light scattering (Figure 20 c), all cells from mixed differentiation culture are separated according to high scattering (pigmented RPE cells) and low scattering (non-pigmented non-RPE cells) groups.In order to make the selection of gating more accurate, the inventor uses the weakly attached non-RPE cells (collected from part TrypLE processing) in differentiation culture to introduce the control of low scattering. Based on high scattering gate sorting and selection RPE cells (Figure 20 d). This two-step purification involves first removing non-RPE cells by TrypLE before flow cytometry, which helps to reduce sorting time. The yield percentage of RPE cells is calculated as follows: the total number of cells × 100% in the mixed colony of RPE cells before the number / purification of the RPE cells obtained after purification.

[0301] Since non-RPE cells are removed by expected pre-dissociation, all cells obtained after purification are considered as RPE cells. The yield percentage of RPE after purification is calculated by the total number of cells present in the differentiation culture and the cell number obtained after purification. In order to purify, the inventors used the differentiation culture from HDFA iPS cells, and the culture was shown to contain 51% Pmel17 positive cells by flow cytometry, indicating that it contained 51% RPE cells. Therefore, the maximum RPE yield obtainable with this culture is 51%. TrypLE purification produces 50% RPE cells, which is very close to the maximum expected yield of 51%. Manual and TrypLE+ manual yields are slightly lower, 47.7% and 43.4% respectively. The method related to scattering sorting produces low yield (21-22%) (Figure 20e). All purification methods were shown to obtain a purity of more than 95% (Figure 20f) by Pmel17 flow cytometry. By 10 weeks, the TEER value of the RPE purified by different methods was similar (Figure 20g). In the phagocytosis assay, all purification methods yielded RPE cells with more than 90% phagocytosis (Fig. 20h).

[0302] In a word, the RPE cells purified by all methods all have suitable purity, TEER and POS phagocytosis.With regard to yield, manual, TrypLE and TrypLE+ scattering have obtained the highest RPE yield.Because part of TrypLE processes and removes most of non-RPE cells, TrypLE and TrypLE+ are the easiest to carry out manually (Figure 20i).Because it is easy to purify, high yield, purity and functionality, therefore for most of general research purposes, TrypLE purification will be an effective way to produce RPE cells.TrypLE+ manual method, it includes extra manual purification step to remove any non-RPE cells that may escape TrypLE processing, which will be ideal for producing RPE cells for transplanting.After purification, the inventors compared the expression (Figure 20j) of BEST1, RPE65, MERTK, MITF, PMEL17, RLBP1 and TRYP2 in CLMC23 and H9 of purification. Most of these genes showed increased expression in CLMC23-derived RPE compared to ES-derived H9 RPE ( FIG. 20k ).

[0303] Example 23: CLiPS-derived RPE has high glycolysis and mitochondrial respiration

[0304] The measurement of glycolysis and mitochondrial respiration provides the indication of bioenergetics and cell health.Using XFe96 extracellular flux analyzer (Seahorse Bioscience), the bioenergetics of the RPE cells derived from different stem cells are measured by cell mitochondrial stress test determination conditions.Carry out quantitative glycolysis function and quantitative oxidative phosphorylation (oxPhos) by extracellular acidification rate (ECAR) and by oxygen consumption rate (OCR).Figure 21a and b prove that in the RPE of differentiation, CLiPS-RPE cells have the bioenergetics spectrum closest to primary RPE (AHRPE), so that they are closer to natural RPE physiologically.In addition, compared with skin-iPSC-RPE (ASF5-RPE) and hESC-RPE (H9-RPE), CLiPS-RPE also shows higher glycolysis and oxidative phosphorylation (Figure 21a and b).These results suggest that compared with the RPE derived from hESC cells, CLiPs-RPE has higher bioenergetics spectrum. Healthy RPE exhibits higher glycolysis and mitochondrial function compared to RPE from AMD patients (Ferrington et al., 2017). The higher OCR and ECAR of CLiPS-RPE suggest that they may be superior to hESC-derived RPE in clinical applications. This is demonstrated by the following: CLiPs-RPE exposed to oxidized low-density lipoprotein (Figure 21c) and hydrogen peroxide (Figure 21g) have increased resistance to oxidative stress compared to skin iPSC-RPE (Figures 21d and h) and hESC-RPE (Figures 21e and i), which are susceptible to oxidative stress. This suggests that CLiPs-RPE can survive better after transplantation. The response of CLiPs-RPE cells to oxidative stress is similar to that seen in natural RPE (AHRPE) (Figures 21f and j), making them functionally closer to primary RPE than other differentiated RPE.

[0305] Example 24: CLiPS RPE has potential immune privilege in a humanized mouse model

[0306] Bioluminescent RPE system was established using luciferase (Luc) gene encoding vectors marked with GFP delivered by lentiviral infection. By analyzing the bioluminescent intensity, the stable expression of Luc in these cell lines was confirmed. In order to test the immunogenicity of different RPE systems, the matrigel plug analysis from previous publications (PMID: 15780993) was adopted. RPE-matrigel plugs were subcutaneously transplanted into humanized mice. The bioluminescence of RPE-matrigel plugs was monitored at regular intervals over a 2-month time course using a bioluminescent imaging system. The total radiation (bioluminescence) of all RPE systems in humanized mice showed a slight but not significant decrease in signal over time (Figure 22a). This shows that RPE cells do not have obvious proliferation and remain in the typical quiescent state of mature RPE expected by them. In order to distinguish whether the slight decrease observed over time is caused by the removal of the immune system, RPE-matrigel plugs were transplanted into NOD-SCID IL2Rγ- / - (NSG) immunodeficient mice. The same total radiation reduction was observed, excluding the removal of RPE cells by the immune system (Figure 22b). This reduction may also cause the cell death during the experiment due to the lack of nutrition supply in this region.In order to confirm the survival of RPE cells and determine its state in matrigel-RPE plug, transplant is extracted at the terminal and immunofluorescence analysis (Figure 22 c) is carried out using mature RPE marker (RPE65) and proliferation marker (Ki67).In the RPE system of all tests, the expression of RPE65 is observed, and there is no Ki67, which has confirmed maturity and quiescent state.

[0307] Example 25: Monitoring of pro-inflammatory cytokines as indicators of cellular immune responses

[0308] After confirming the survival of mature RPE cell transplants in all groups, the presence of key proinflammatory cytokines (IFN-γ and IL-18) collected at the endpoint of humanized mice was tested, which are key effectors of cell-mediated immunity (PMID: 29856726). All RPE lines tested showed cytokine levels in the picogram range, which are below the threshold value (Figure 23a and b) for inducing systemic immune system activation. Unexpectedly, for two cytokines, CLEC23-RPE always showed the lowest level compared with other cell lines. Then at the local level, immune response was studied in RPE-matrix glue plugs to detect local immune response. Immunofluorescence analysis was performed using human CD45 (hCD45) markers to observe immune cell infiltration. OTX2 (a kind of RPE-specific transcription factor) was used to distinguish RPE cells (Figure 23c). Consistent with low levels of IFN-γ and IL-18 cytokines, there was no immune cell infiltration in CLEC23-RPE. Qualitative grading (0-3 scale) based on hCD45 positive cells was performed to determine the severity of immune infiltration in 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.

[0309] Example 26: CLEC23-RPE can modulate T cell activation to confer low immunogenicity

[0310] Transplant rejection is mainly caused by cell-mediated immune response. As observed from the previous figure, CLEC23-RPE has the lowest immune cell infiltration. Therefore, we hypothesized that CLEC23-RPE may affect the activation of T cells, an element involved in cell-mediated immunity. IL-23 and IL17A, which are known cytokines involved in T cell activation, were analyzed in humanized mouse serum. Fig.24 a and b) (PMID: 26252407). CLEC23-RPE showed the lowest levels of both cytokines compared to the other groups. Since these cytokines affect T cell activation, the relative ratio of T cells (CD3) to B cells (CD19) was calculated using flow cytometry ( Fig.24 c). Only the CLEC23-RPE group had lower T cell counts relative to B cells, suggesting that T cell activation may be inhibited. Additional analysis of T cell subsets - helper T cells (CD4) and cytotoxic T cells (CD8) was performed and demonstrated that CLEC23-RPE had the lowest cytotoxic T cells relative to helper T cells ( Fig.24d). The activation status of these two subsets was then further divided into four groups, naive (quiescent), central memory (CM; pre-activated), and two activation states, effector memory (EM) and effector memory re-expressing CD45RA (TEMRA). There were no significant differences between the activation states of the CD4 helper T cell subsets ( Fig.24 e). However, in the CD8 cytotoxic T cell subset, CLEC23-RPE clearly showed a higher naive population ( Fig.24 f). Therefore, the inventors hypothesized that the potential immune privileged state of CLEC23-RPE might be caused by the inhibition of CD8 cytotoxic T cell activation.

[0311] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0312] All patents and publications mentioned in this specification are indicative of the level of ordinary technicians in the field to which the invention belongs. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0313] The term "less than" or "greater than" or "lower than" does not include specific numbers. The terms "a" and "an" and "said" and similar references used in the context of describing the present invention (especially in the context of the claims) are interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. The statements of the range of values ​​herein are intended only to be used as a shorthand method for referring to each independent value within the range individually. Unless otherwise indicated herein, each independent value is incorporated into the specification as if the value were stated individually herein. Wherever the term "and / or" is used herein, it includes the meaning of "and", "or" and "all or any other combination of elements connected by the term". The term "includes" means "including but not limited to". "Includes" and "including but not limited to" are used interchangeably. The term "about" refers to plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, and most preferably plus or minus 1%.

[0314] The present invention described exemplarily here can be properly implemented without any elements, restrictions not specifically disclosed here. Therefore, for example, the terms "comprise", "include", "contain", etc. should be understood broadly and not restrictively. When "consisting of..." is used herein, any elements, steps or components not specified in the claim elements are not included. When used herein, "consisting essentially of..." does not exclude materials or steps that have no substantial impact on the basic and novel features of the claims. In addition, the terms and expressions used herein have been used as descriptive terms rather than restrictive terms, and when using these terms and expressions, it is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and changes of the invention implemented therein disclosed herein can be adopted by those skilled in the art, and such modifications and changes are considered to be within the scope of the present invention. The present invention has been described broadly and generally here. Each narrower category and sub-generic grouping falling into the general disclosure also forms a part of the present invention. This includes the generic description of the invention, with the proviso or negative limitation removing any subject matter from the genus, regardless of whether the removed material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will become apparent from the following claims.

[0315] The present invention is also characterized by the following items:

[0316] 1. A method for producing induced pluripotent stem cells, wherein the method comprises expressing exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in umbilical cord amniotic membrane stem cells under conditions suitable for reprogramming the stem cells, thereby producing the induced pluripotent stem cells.

[0317] 2. The method according to item 1, wherein the umbilical cord amniotic membrane stem cells are umbilical cord amniotic membrane mesenchymal stem cells or umbilical cord amniotic membrane epithelial stem cells.

[0318] 3. The method according to item 1 or 2, wherein the umbilical cord amniotic membrane mesenchymal stem cells are a mesenchymal stem cell population, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105.

[0319] 4. The method according to item 3, wherein at least about 90% or more of the cells in the mesenchymal stem cell population lack expression of the following markers: CD34, CD45 and HLA-DR.

[0320] 5. The method of any one of items 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, or 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.

[0321] 6. The method according to any one of items 1 to 5, wherein the exogenous nucleic acids encoding the proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA are provided by one, two or three vectors, wherein preferably the first vector encodes the proteins OCT3 / 4 and 53-shRNA, the second vector encodes the proteins SOX2 and KLF4, and the third vector encodes the proteins L-MYC and LIN28.

[0322] 7. The method according to any one of items 1 to 6, wherein the umbilical cord amniotic stem cells are subjected to transfection to transfer the exogenous nucleic acid into the stem cells.

[0323] 8. The method according to item 7, wherein the umbilical cord amniotic stem cells are subjected to electroporation to transfer the exogenous nucleic acid into the stem cells.

[0324] 9. The method according to item 8, wherein the umbilical cord amniotic membrane mesenchymal stem cells are subjected to 1 pulse of electroporation, the pulse duration is about 15-25 ms, and the voltage is about 1550-1650V, preferably subjected to 1 pulse of electroporation, the pulse duration is about 20 ms, and the voltage is about 1600V.

[0325] 10. The method according to item 9, wherein the ratio of the amount of vector (plasmid) DNA of each vector to the number of the umbilical cord amniotic membrane mesenchymal stem cells subjected to electroporation is about 1.5 μg plasmid DNA to about 1×10 6 CLMC to about 2.5 μg DNA ratio of about 1 × 10 6 The ratio is, for example, about 2.5 μg plasmid DNA: 1×10 6 cells, about 2.25 μg plasmid DNA: 1×10 6 cells, about 1.8 μg plasmid DNA: 1×10 6cells, about 1.7 μg plasmid DNA: 1×10 6 cells, about 1.6 μg plasmid DNA: 1×10 6 cells, about 1.5 μg plasmid DNA: 1×10 6 cells, or preferably about 1.67:1×10 6 cells.

[0326] 11. The method according to item 8, wherein the umbilical cord amniotic epithelial stem cells are subjected to electroporation with 2 pulses, the duration of the pulses being about 25-35 ms and the voltage being about 1300-1400 V, preferably, subjected to electroporation with 2 pulses, the duration of the pulses being about 30 ms and the voltage being about 1350 V.

[0327] 12. The method according to item 11, wherein the ratio of the amount of vector (plasmid) DNA of each vector to the number of amniotic epithelial stem cells of the umbilical cell subjected to electroporation is about 1.5 μg DNA to about 1×10 6 cells to about 2.5 μg DNA to about 1×10 6 The ratio is, for example, about 1.5 μg plasmid DNA: 1×10 6 cells, about 1.6 μg plasmid DNA: 1×10 6 cells, about 1.7 μg plasmid DNA: 1×10 6 cells, about 1.8 μg plasmid DNA: 1×10 6 cells, about 1.9 μg plasmid DNA: 1×10 6 cells, about 2.0 μg plasmid DNA: 1×10 6 cells, about 2.5 μg plasmid DNA: 1×10 6 cells, preferably about 1.67 μg plasmid DNA: 1×10 6 cells.

[0328] 13. The method according to any one of items 7 to 12, wherein the transfected stem cells are cultured in a medium suitable for cell recovery.

[0329] 14. The method according to item 13, wherein the culture medium suitable for cell recovery is a serum-free culture medium.

[0330] 15. The method according to item 13, wherein the culture medium suitable for recovering transfected umbilical cord amniotic membrane mesenchymal stem cells consists of about 85% to 95% (v / v) defined medium and 5% to 15% (v / v) fetal bovine serum.

[0331] 16. The culture medium according to item 15, wherein the culture medium suitable for recovering transfected umbilical cord amniotic membrane mesenchymal stem cells consists of about 90% (v / v) chemically defined medium and about 10% (v / v) fetal bovine serum.

[0332] 17. The culture medium according to any one of items 14 or 15, wherein the culture medium contains about 85% to 95% (v / v) CMRL 1066 and about 5% to 15% (v / v) FBS.

[0333] 18. The method according to item 13 or 14, wherein the culture medium suitable for recovering transfected umbilical cord amniotic epithelial stem cells comprises mammary epithelial basal medium MCDB 170, EpiLife medium, DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium) and FBS (fetal bovine serum).

[0334] 19. A method according to item 18, wherein the culture medium suitable for recovering 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).

[0335] 20. A method according to item 19, wherein the culture medium suitable for recovering 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).

[0336] 21. The method according to item 20, wherein the culture medium suitable for recovering transfected umbilical cord amniotic epithelial stem cells comprises mammary epithelial basal medium MCDB 170 with a final concentration of about 20% (v / v), EpiLife medium with a final concentration of about 30% (v / v), F12 with a final concentration of about 12.5 (v / v), DMEM with a final concentration of about 37.5% (v / v) and FBS with a final concentration of about 1.0% (v / v).

[0337] 22. The method according to any one of items 18 to 21, wherein the culture medium suitable for recovering transfected umbilical cord amniotic epithelial stem cells is obtained by mixing the following to obtain a final volume of 1000 ml of culture medium:

[0338] 200 ml Mammary Epithelial Basal Medium MCDB 170

[0339] 300 ml EpiLife medium

[0340] 250ml DMEM

[0341] 250ml DMEM / F12

[0342] 1% fetal bovine serum.

[0343] 23. The method according to any one of items 18 to 22, wherein the culture medium suitable for recovering transfected umbilical cord amniotic epithelial stem cells comprises insulin at a final concentration of about 1 μg / ml to about 7.5 μg / ml.

[0344] 24. The method according to any one of items 18 to 24, wherein the culture medium suitable for recovering the transfected umbilical cord amniotic epithelial stem cells comprises human epidermal growth factor at a final concentration of about 1 ng / ml to about 15 ng / ml.

[0345] 25. A method according to any one of items 18 to 25, wherein the culture medium suitable for recovering 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).

[0346] 26. The method according to item 25, wherein the culture medium suitable for recovering transfected umbilical cord amniotic epithelial stem cells comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0347] 27. The method according to any one of items 18 to 26, wherein the culture medium suitable for recovering the transfected umbilical cord amniotic epithelial stem cells further comprises one or more transforming growth factors (TGF).

[0348] 28. The method according to item 27, wherein the culture medium comprises transforming growth factor β (TGF-β) and / or transforming growth factor α.

[0349] 29. The method according to any one of items 18 to 28, wherein the culture medium suitable for recovering the transfected umbilical cord amniotic epithelial stem cells further comprises cholera toxin from Vibrio cholerae.

[0350] 30. The method according to any one of items 14 to 29, wherein the medium suitable for cell recovery contains compounds that inhibit inflammatory responses and enhance cell survival.

[0351] 31. The method according to item 30, wherein the compound is a glucocorticoid.

[0352] 32. The method according to claim 31, wherein the glucocorticoid is selected from prednisolone, methylprednisolone, dexamethasone, betamethasone, corticosterone and hydrocortisone.

[0353] 33. The method according to item 31 or 32, wherein the concentration of hydrocortisone is about 0.5 μM to about 2 μM.

[0354] 34. The method according to any one of items 13 to 33, wherein the culturing is performed in a coated cell culture vessel, wherein the cell culture vessel i...

Claims

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

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

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

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

5. The method according to any one of claims 1 to 4, wherein the differentiation medium comprises i) a first differentiation medium, which further comprises at least one of IGF1, DKK1, nicotinamide or LDN-193189, preferably comprises IGF1, DKK1, nicotinamide and LDN-193189; ii) a second differentiation medium, which further comprises at least one of IGF1, DKK1, nicotinamide, LDN-193189 or b-FGF, preferably comprises IGF1, DKK1, nicotinamide, LDN-193189 and b-FGF; iii) a third differentiation medium, which further comprises at least one of IGF1, DKK1 or Activin A, preferably comprises IGF1, DKK1 and Activin A; iv) a fourth differentiation medium, which further comprises activin A and SU5402 or PD17307, preferably comprises activin A and PD17307; and / or v) a fifth differentiation medium, which further comprises Activin A, CHIR99021, or at least one of SU5402 or PD17307, and preferably comprises Activin A, CHIR99021 and PD17307.

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

7. The method according to claim 5 or 6, wherein the DKK1 of claim 5 i), ii) and / or iii) is used in a concentration of at least about 5 ng / ml, preferably wherein the DKK1 of claim 5 i), ii) and / or iii) is used in a concentration of about 10 ng / ml.

8. The method according to any one of claims 5 to 7, wherein the nicotinamide of claim 5 i) and / or ii) is used in a concentration of at least about 5 mM, preferably wherein the nicotinamide of claim 5 i) and / or ii) is used in a concentration of about 10 mM.

9. The method according to any one of claims 5 to 8, wherein the LDN-193189 of claim 5 i) and / or ii) is used at a concentration of at least about 0.1 μM, preferably wherein the LDN-193189 of claim 5 i) is used at a concentration of about 1 μM and / or wherein the LDN-193189 of claim 5 ii) is used at a concentration of about 0.2 μM.

10. The method according to any one of claims 5 to 9, wherein the b-FGF of claim 5ii) is used at a concentration of at least about 2.5 ng / ml, preferably wherein the b-FGF of claim 5ii) is used at a concentration of about 5 ng / ml.

11. The method according to any one of claims 5 to 10, wherein activin A of claims 5iii), iv) and / or v) is used at a concentration of at least about 50 ng / ml, preferably wherein activin A of claims 5iii), iv) and / or v) is used at a concentration of about 100 ng / ml.

12. The method according to any one of claims 5 to 11, wherein SU5402 according to claim 5 iv) and / or v) is used in a concentration of at least about 5 μM, preferably wherein SU5402 according to claim 5 iv) and / or v) is used in a concentration of about 10 μM.

13. The method according to any one of claims 5 to 11, wherein PD17307 according to claim 5 iv) and / or v) is used at a concentration of at least about 0.5 μM, preferably wherein PD17307 according to claim 5 iv) and / or v) is used at a concentration of about 1 μM.

14. The method according to any one of claims 5 to 13, wherein: The CHIR99021 of claim 5v) is used at a concentration of at least about 1 μM and less than about 3 μM for culturing the cells, preferably culturing the cells for about 3 consecutive culture days.

15. The method according to claim 14, wherein CHIR99021 according to claim 5v) is used at a concentration of about 3 μM for subsequent culturing of the cells, preferably for subsequent culturing of the cells for about 5 consecutive culture days. 16 . The method according to claim 5 , wherein culturing the iPS cells comprises culturing in the first differentiation medium for about 2 days. 17 . The method according to any one of claims 5 to 16 , wherein culturing the iPS cells comprises culturing in a first differentiation medium for about 2 days, followed by culturing in a second differentiation medium for about 2 days.

18. The method according to any one of claims 5 to 17, wherein culturing the iPS cells comprises culturing in a first differentiation medium for about 2 days, followed by culturing in a second differentiation medium for about 2 days, followed by culturing in a third differentiation medium for about 2 days.

19. The method according to any one of claims 5 to 18, wherein culturing the iPS cells comprises culturing in a first differentiation medium for about 2 days, followed by culturing in a second differentiation medium for about 2 days, followed by culturing in a third differentiation medium for about 2 days, and followed by culturing in a fourth differentiation medium for about 2 days.

20. The method according to any one of claims 5 to 19, wherein culturing the iPS cells comprises culturing in a first differentiation medium for about 2 days, followed by culturing in a second differentiation medium for about 2 days, followed by culturing in a third differentiation medium for about 2 days, followed by culturing in a fourth differentiation medium for about 2 days, and followed by culturing in a fifth differentiation medium for about 8 days.

21. The method of any one of claims 5 to 20, wherein LDN-193189 is used for at least about 2 culture days.

22. The method of any one of claims 5 to 21, wherein DKK1 is used for at least about 2 culture days.

23. The method according to any one of claims 5 to 22, wherein SU5402 or PD173074 is used for about 10 culture days.

24. The method according to any one of claims 5 to 23, wherein CHIR99021 is used for about 8 culture days.

25. The method according to any one of claims 1 to 24, wherein the iPS cells are cultured in the differentiation medium for about 11 to about 21 days, preferably about 16 days.

26. The method according to any one of claims 1 to 25, wherein the method further comprises culturing the iPS cells in mTESR1 medium before culturing the iPS cells in the differentiation medium, preferably culturing the iPS cells in mTESR1 medium for about 1 to about 4 culture days.

27. according to the method described in any one in claim 1 to 26, wherein said method further is included in retinal pigment epithelium and maintains (RPEM) culture medium and cultivates described RPE cell.

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

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. The method of claim 28 or 29, 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. according to the method described in any one in claim 27 to 30, wherein described RPE cell is cultivated in RPEM culture medium for about 9 to about 29 days, preferably about 19 days.

32. according to the method described in any one in claim 27 to 31, wherein in described differentiation medium, cultivating described iPS cell and in described RPEM culture medium, cultivating described RPE cell comprise about 20 to about 50 days, preferably about 30 to about 35 days, most preferably about 35 days.

33. according to the method described in any one in claim 27 to 32, wherein said method further comprises purifying the RPE cells in the described culture medium after cultivating the described cell in the described RPEM culture medium.

34. The method of claim 33, wherein purifying comprises: a. Manual identification of RPE cells based on their pigmentation; b. passaging RPE cells; c. Manually identify and passage RPE cells based on their pigmentation; d. passaging the RPE cells and scatter sorting the RPE cells according to their pigmentation; and / or e. Scatter sorting of RPE cells based on their pigmentation.

35. according to the described method of claim 34, wherein claim 34a) and / or c) described manual identification RPE cell according to its pigmentation comprises and selects by microscopy.

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

37. according to any one of the methods in claim 34 to 36, wherein claim 34b), c) and / or d) making RPE cell passage comprise using Accutase or TrypLE, preferably process described RPE cell with TrypLE.

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

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

40. The method according to any one of the preceding claims, wherein the umbilical cord amniotic membrane stem cells are umbilical cord amniotic membrane mesenchymal stem cells, or umbilical cord amniotic membrane epithelial stem cells.

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

42. The method of claim 41, 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.

43. The method of any one of claims 41 to 42, 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.

44. The method according to any one of claims 38 to 43, wherein the exogenous nucleic acids encoding proteins OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA are provided by one, two or three vectors, wherein preferably the first vector encodes proteins OCT3 / 4 and 53-shRNA, the second vector encodes proteins SOX2 and KLF4 and the third vector encodes proteins L-MYC and LIN28.

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

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

50. The pharmaceutical composition of claim 49, wherein the pharmaceutical composition is suitable for parenteral or topical administration.

51. according to the RPE cell culture described in any one in claim 45 to 46, the epithelium described in any one in claim 47 to 48, and / or the pharmaceutical composition described in any one in claim 49 to 50, wherein the RPE cells included in the culture express at least one of BEST1, PMEL17, MITF, TYROSINASE, TRYP2, ZO-1, RPE65, RLBP1 or MERTK.

52. according to any one of claim 45 to 46 and / or the RPE cell culture of claim 51, any one of claim 47 to 48 and / or the epithelium of claim 51 and / or any one of claim 49 to 50 and / or the pharmaceutical composition of claim 51, wherein, The RPE cells contained in the culture express BEST1 at a fold change of at least about 2 relative to RPE cells differentiated from embryonic stem (ES) cells.

53. according to any one of claim 45 to 46 and / or any one of claim 51 to 52 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 52 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 52 pharmaceutical composition, wherein, The RPE cells contained in the culture express PMEL17 at a fold change of at least about 0.9 relative to RPE cells differentiated from ES.

54. according to any one of claim 45 to 46 and / or any one of claim 51 to 53 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 53 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 53 pharmaceutical composition, wherein, The RPE cells contained in the culture express MITF at a fold change of at least about 4.5 relative to RPE cells differentiated from ES.

55. according to any one of claim 45 to 46 and / or any one of claim 51 to 54 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 54 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 54 pharmaceutical composition, wherein, The RPE cells contained in the culture express TRYP2 at a fold change of at least about 2.9 relative to RPE cells differentiated from ES.

56. according to any one of claim 45 to 46 and / or any one of claim 51 to 55 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 55 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 55 pharmaceutical composition, wherein, The RPE cells contained in the culture express RPE65 at a fold change of at least about 0.6 relative to RPE cells differentiated from ES.

57. according to any one of claim 45 to 46 and / or any one of claim 51 to 56 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 56 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 56 pharmaceutical composition, wherein, The RPE cells contained in the culture express RLBP1 at a fold change of at least about 17.5 relative to RPE cells differentiated from ES.

58. according to any one of claim 45 to 46 and / or any one of claim 51 to 57 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 57 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 57 pharmaceutical composition, wherein relative to the RPE cell differentiated from ES, the RPE cell included in the culture expresses MERTK with at least about 6 multiple changes.

59. according to any one of claim 45 to 46 and / or any one of claim 51 to 58 RPE cell culture, any one of claim 47 to 48 and / or any one of claim 51 to 58 epithelium and / or any one of claim 49 to 50 and / or any one of claim 51 to 58 pharmaceutical composition, wherein, The RPE cells contained in the culture comprise an increased oxygen consumption rate (OCR) and / or extracellular acidification rate (ECAR) relative to RPE cells differentiated from ES.

60. A method of treating a retinal degenerative disease in a subject, the method 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. The method of claim 60, wherein the retinal degenerative disease is age-related macular degeneration (AMD) or a retinal dystrophy.

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

63. according to the method for claim 62, wherein the imaging data from the experimenter is compared with reference to the imaging data and the survival of the RPE cell described in the experimenter is indicated by bioluminescent signal without difference.

64. 1 kinds of in vitro methods for determining the immunogenicity of retinal pigment epithelial (RPE) cells in an experimenter, wherein the retinal pigment epithelial (RPE) cells are obtained from the differentiation of induced pluripotent stem (iPS) cells by the method defined as any one of claims 1 to 44, the RPE cells of the differentiation have been pre-delivered to the experimenter, and the method comprises: a) detecting the level of pro-inflammatory cytokines in a sample obtained from the subject using an imaging method, wherein the sample comprises the differentiated RPE cells, thereby collecting imaging data; b) comparing the imaging data received in step a) with reference imaging data.

65. according to the method for claim 64, wherein compared with reference to imaging data, the cytokine level indication of the reduction of described imaging data reduces the immunogenicity of described RPE cell in described experimenter.

Images