WO2023146477A2 - A method of differentiating an induced pluripotent stem cell into a retinal pigment epithelial cell, a retinal pigment epithelial cell and methods of using the retinal pigment epithelial cell - Google Patents

A method of differentiating an induced pluripotent stem cell into a retinal pigment epithelial cell, a retinal pigment epithelial cell and methods of using the retinal pigment epithelial cell Download PDF

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WO2023146477A2
WO2023146477A2 PCT/SG2023/050051 SG2023050051W WO2023146477A2 WO 2023146477 A2 WO2023146477 A2 WO 2023146477A2 SG 2023050051 W SG2023050051 W SG 2023050051W WO 2023146477 A2 WO2023146477 A2 WO 2023146477A2
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rpe
medium
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WO2023146477A3 (en
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Xinyi SU
Regha KAKKAD
Mayuri BHARGAVA
Binxia YANG
Walter Hunziker
Zengping LIU
Haofei WANG
Bhav Harshad PARIKH
Qingfeng Chen
Fritz Shien Choong LAI
Chou CHAI
Kah Leong Lim
Toan Thang Phan
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Agency for Science Technology and Research Singapore
Singapore Health Services Pte Ltd
CellResearch Corp Pte Ltd
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Singapore Health Services Pte Ltd
CellResearch Corp Pte Ltd
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Priority to EP23747458.0A priority patent/EP4469561A4/en
Priority to JP2024544656A priority patent/JP2025507265A/ja
Priority to AU2023212665A priority patent/AU2023212665A1/en
Priority to CN202380031502.8A priority patent/CN119497749A/zh
Priority to US18/833,821 priority patent/US20250197803A1/en
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Definitions

  • the present invention relates to a method of generating an induced pluripotent stem cell.
  • the present invention concerns 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 of differentiating the induced pluripotent stem cell of this invention.
  • a pharmaceutical composition comprising a differentiated induced pluripotent stem cell obtained by the method is also concerned.
  • the present invention concerns a method of treating a congenital or acquired degenerative disorder in a subject, comprising administering to a subject a target cell differentiated from pluripotent stem cell.
  • the present invention also relates to a method of differentiating said induced pluripotent stem cell into a retinal pigment epithelial cell. Additionally, 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 concerns a retinal pigment epithelium consisting of or comprising a retinal pigment epithelial cell culture obtainable or obtained by the differentiation method. The present invention also relates to a pharmaceutical composition comprising a retinal pigment epithelial cell culture obtained by the differentiation method.
  • the present invention concerns a method of treating a retinal degenerative disease in a subject, comprising administering to a subject a retinal pigment epithelial cell differentiated from the induced pluripotent stem cell by the method.
  • the present invention also refers to an in vivo method of detecting the survival rate of a retinal pigment epithelial cell differentiated from an induced pluripotent stem cell by the defined method in a subject and an in vitro method of determining the immunogenicity of said retinal pigment epithelial cell differentiated from an induced pluripotent stem cell by the defined method in said subject, to whom said differentiated RPE cell has been pre-delivered.
  • Stem cells are a cell population possessing the capacities to self-renew indefinitely and to differentiate in multiple cell or tissue types.
  • the ability of stem cells to self-renew is critical to their function as reservoir of primitive undifferentiated cells and the "plasticity" of stem cells relies on their ability to trans-differentiate into tissues different from their origin and, perhaps, across embryonic germ layers.
  • most somatic cells have a limited capacity for selfrenewal due to telomere shortening (reviewed, for example, in Dice, J.F. (1993) Physiol. Rev. 73, 149-159).
  • Stem cell-based therapies thus have the potential to be useful for the treatment of a multitude of human and animal diseases.
  • Embryonic stem cells proliferate indefinitely and can differentiate spontaneously into all tissue types: they are thus termed pluripotent stem cells (reviewed, for example, in Smith, A.G. (2001) Anna. Rev. Cell. Dev. Biol. 17, 435-462). Even though the potential of embryonic stem cells is enormous, their use implies many ethical problems. Therefore, non-embryonic stem cells have been proposed as alternative sources.
  • Adult stem cells are more tissue-specific and may have less replicative capacity: they are thus termed multipotent stem cells (reviewed, for example, in Paul, G. et al. (2002) Drug Discov. Today 7, 295-302). These cells can be derived from the bone marrow stroma, fat tissue and dermis and have the ability to differentiate inter alia into chondrocytes, adipocytes, osteoblasts, myoblasts, cardiomyocytes, astrocytes, and tenocytes. In many cases, however, the number of stem cells extracted from the bone marrow stroma, fat tissue, dermis and umbilical cord blood is rather low.
  • a comprehensive source for very young and adaptable adult stem cells is the umbilical cord blood or tissue or the placenta.
  • a large amount of stem cells can be derived from umbilical cord tissue, namely from Wharton's jelly, the matrix of umbilical cord (Mitchell, K.E. et al. (2003) Stem Cells 21, 50-60; U.S. Patent 5,919,702; US Patent Application 2004/0136967). These cells have been shown to have the capacity to differentiate, for example, into a neuronal phenotype and into cartilage tissue, respectively.
  • Mesenchymal stem cells have also been isolated from the subendothelial layer of the umbilical cord vein, one of the three vessels (two arteries, one vein) found within the umbilical cord (Romanov, Y.A. et al. (2003) Stem Cells 21, 105-110; Covas, D.T. et al. (2003) Bra ⁇ . J. Med. Biol. Res. 36, 1179-1183). Further, mesenchymal stem cells as well as epithelial stem cells have successfully been isolated from the amniotic tissue of the umbilical cord (US2006/0078993).
  • mesenchymal stem cells can undergo differentiation in vitro and in vivo, making these stem cells promising candidates for mesodermal defect repair and disease management, the use of adult stem cells is limited by their multipotency.
  • non-embryonic cells can be reprogrammed to pluripotent stem cells: the so called induced pluripotent stem cells (iPS).
  • iPS induced pluripotent stem cells
  • IPS were generated for the first time by Takahashi and Yamanaka, who reprogrammed non-embryonic cells to a pluripotent state through overexpression of the 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).
  • Takahashi and Yamanaka used mouse embryonic fibroblasts and introduced the Yamanaka factors via retroviral transduction, thereby allowing the overexpression of the transcription factors and thus generating cells exhibiting the morphology and growth properties of embryonic cells.
  • iPS technology has entered the clinical translation stage with first-in-human trials being conducted for age-related macular degeneration (AMD; Mandai, M., et al,. N Engl J Med, 2017. 376(11): p. 1038-1046) and Parkinson’s Disease (PD; Reardon, S. and Cyranoski, D. (2014) ‘Japan stem-cell trial stirs envy’, Nature. England, pp. 287-288. doi: 10.1038/513287a).
  • AMD age-related macular degeneration
  • PD Parkinson’s Disease
  • the greatest promise of iPS technology lies in its potential for enabling autologous cell therapy, which may circumvent the need for long-term immunosuppression or histocompatibility matching to prevent rejection of transplanted cells.
  • iPS retinal pigment epithelial
  • iPS induced pluripotent stem cells
  • ES embryonic stem cells
  • iPS cells Age-related abnormalities increased in mitochondrial DNA as well, with fibroblast-derived iPS cells of elderly subjects harbouring significantly higher mutations than younger subjects (Kang et al., 2016, Cell Stem Cell 18, 625-636, May 5, 2016). iPS cells, derived from adults, may also require immune-suppression if derived from an allogenic host. The use of ES cells are associated with ethical issues and immune -rejection requiring use of immunosuppression. Skin cells are widely used for generation iPS cells due to ease of tissue collection, however they have higher changes of developing mutations due to long-term exposure to UV from sunlight (Apalla Z. et al., 2017, Dermatol Pract Concept. 2017 Apr; 7(2): 1-6).
  • the invention relates to a method of generating an induced pluripotent stem (iPS) cell as described herein, a resulting induced pluripotent stem cell, a method of differentiating a resulting induced pluripotent stem cell and a method of treating a disorder in a subject with a differentiated cell derived from an induced pluripotent stem cell.
  • iPS induced pluripotent stem
  • the invention provides a method of generating an induced pluripotent stem cell, wherein the method comprises expressing exogenous nucleic acid encoding the proteins OCT3/4, SOX2, KLF4, LIN 28 and L-MYC and the p53-shRNA in a stem cell of the amniotic membrane of the umbilical cord under conditions suitable to reprogram the stem cell, thereby generating the induced pluripotent stem cell.
  • stem cell of the amniotic membrane of the umbilical cord is a mesenchymal stem cell of the amniotic membrane of the umbilical cord or an epithelial stem cell of the amniotic membrane of the umbilical cord.
  • the invention also provides an induced pluripotent stem cell population obtainable by the method as well as an induced pluripotent stem cell population obtained by the method.
  • the induced pluripotent stem cell population can either be an induced pluripotent stem cell population that is derived from a mesenchymal stem cell (population) of the amniotic membrane of umbilical cord or an induced pluripotent stem cell population that is derived from an epithelial stem cell (population) of the amniotic membrane of the umbilical cord.
  • the invention also provides a pharmaceutical composition comprising an induced pluripotent stem cell of the present invention.
  • the invention provides a method of differentiating an induced pluripotent stem cell of the present invention into a target cell, wherein the induced pluripotent stem cell is differentiated into the target cell under conditions suitable for differentiation. Consequently, the invention also provides a pharmaceutical composition comprising a differentiated induced pluripotent stem cell obtained by the present invention.
  • the invention also provides a method of treating a congenital or acquired degenerative disorder in a subject, comprising administering to a subject a target cell differentiated from a pluripotent stem cell obtained by the present invention.
  • the invention provides an extracellular membranous vesicle produced by an induced pluripotent stem cell population of the invention or produced by a cell obtained by differentiation of an induced pluripotent stem cell of the invention.
  • the invention further comprises the use of such an extracellular membranous vesicle of the invention as delivery carrier of a therapeutic agent.
  • the invention also provides a cell culture medium comprising Mammary Epithelial Basal Medium MCDB 170, EpiLife medium, DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium) and FBS (Fetal Bovine Serum).
  • a cell culture medium comprising Mammary Epithelial Basal Medium MCDB 170, EpiLife medium, DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium) and FBS (Fetal Bovine Serum).
  • the invention further relates to a method of differentiating an induced pluripotent stem (iPS) cell as described herein into a retinal pigment epithelial (RPE) cell, a resulting RPE cell, a retinal pigment epithelium consisting of or comprising the RPE cell as described elsewhere herein, a method of treating a retinal degenerative disease in a subject with a RPE cell differentiated from an iPS cell by the method as described herein as well as a pharmaceutical composition comprising a RPE cell obtained by the method as described herein. Further, the invention also relates to an in vivo and an in vitro method using the RPE cell obtained by the method as described herein.
  • iPS induced pluripotent stem
  • the inventors describe the use of iPS cells derived from umbilical cord lining cells (short: CLiPS) to generate RPE cells for clinical use.
  • CLiPS umbilical cord lining cells
  • the inventors differentiated CLiPS to RPEs using the method of the present invention in comparion to ES and skin iPS cells and generated RPEs with consistently increased RPE differentiation efficiency.
  • CLiPS-derived RPEs had higher pigmentation than ES-derived RPEs based on increased expression levels of pigmentation specific genes such as MITF, PMEL17, and TRYP2 and RPE-specific genes such as BEST1, RPE65, MERTK, RLBP1.
  • CLiPS derived from different stem cells
  • the invention provides a method of differentiating an iPS cell into a RPE cell, the method comprising culturing the iPS cell derived from a stem cell of the amniotic membrane of the umbilical cord in a differentiation medium under conditions suitable for the differentiation into a RPE cell, thereby differentiating the iPS cell into the RPE cell.
  • the invention provides a RPE cell culture obtainable by the method as well as a RPE cell culture obtained by the method.
  • the invention also provides a retinal pigment epithelium consisting of or comprising said RPE cell culture obtainable by the method as well as consisting of or comprising said RPE cell culture obtained by the method.
  • the invention also provides a pharmaceutical composition comprising a RPE cell culture obtained by the method of the present invention.
  • the invention also provides a method of treating a retinal degenerative disease in a subject, comprising administering to a subject a RPE cell differentiated from an iPS cell by the method of the present invention.
  • the invention provides an in vivo method of detecting the survival rate of a RPE cell differentiated from an iPS cell by the method as defined herein in a subject, the method comprising a) introducing a RPE cell differentiated from an iPS cell by the method as defined into a subject, wherein said RPE cell comprises a bioluminescence label; b) detecting the bioluminescence signal of said RPE cell over time using an imaging method, thereby collecting imaging data; c) comparing the imaging data received in step b) to reference imaging data.
  • the invention provides an in vitro method of determining the immunogenicity of a RPE cell differentiated from an iPS cell by the method as defined in a subject, to whom said differentiated RPE cell has been pre-delivered, the method comprising: a) detecting pro-inflammatory cytokine levels using an imaging method in a sample obtained from said subject, the sample comprising said differentiated RPE cell, thereby collecting imaging data; b) comparing the imaging data received in step a) to reference imaging data.
  • FIG. 1 shows a flow-diagram schematically representing the experimental steps of an illustrative embodiment of a method of generating an induced pluripotent stem cell of the present invention.
  • the stem cells used herein are isolated from the amniotic membrane of the umbilical cord - also referred to as cord lining stem cells (CLSC).
  • CLSC cord lining stem cells
  • the cells pellet is resuspended in a buffer suitable for electroporation before the plasmids encoding for the Yamanaka factors are added to the cells-buffer mixture.
  • the electroporation is carried out with 1 pulse having a duration time of about 20ms and a voltage of about 1600V or with 2 pulses having a duration time of 30ms and a voltage of about 1350V for cord lining mesenchymal cells (CLMC) and cord lining epithelial cells (CLEC), respectively.
  • CLMC mesenchymal cells
  • CLEC cord lining epithelial cells
  • 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 the medium suitable for recovery and a second cell culture medium.
  • the media mixture is replaced with the same mixture of cell culture media about 4 days after electroporation. Thereby, colonies of cord lining induced pluripotent stem cells - also referred herein to as CLiPS - are generated.
  • the 1:1 mixture of two different cell culture media is replaced with the second cell culture medium. This medium is also replaced about every second day to keep the medium fresh.
  • the CLiPS colonies are picked and transferred to a coated cell culture vessel suitable for cell cultivation and proliferation.
  • the cell culture medium is replaced regularly with the same medium.
  • the CLiPS colonies are detached from the coated culture device and transferred to another cell culture vessel suitable for cell cultivation and proliferation. This way, the CLiPS colonies are further dissociated.
  • the CLiPS are passaged in a ratio of about 1:3 (v/v), wherein the passaging in a ratio of about 1:3 (v/v) is performed by contacting 1 volume dissociated CLiPS to 2 volume of fresh culture medium.
  • the CLiPS are then cultivated in a medium containing a substance enhancing the survival of the cells until reaching a confluence of about 30-60%. At this point, the CLiPS are capable to be differentiated into any desired target cells.
  • FIG. 2 shows an exemplary comparison of the reprogramming efficiency of individual CLSC populations.
  • the stem cells have been subjected to different electroporation settings to transfect the exogenous nucleic acid into the cells.
  • the electroporation has been carried out using the electroporation parameters indicated in Okita et al, supra, (1650V, 10ms, 3 pulses) and the respective parameters used in the present invention for transfection of epithelial stem cells of the amniotic membrane of the umbilical cord (also referred herein as “cord lining epithelial stem cell” or CLEC, 1350V, 30ms, 2 pulses) and mesenchymal stem cells of the amniotic membrane of the umbilical cord (also referred herein to as cord lining mesenchymal stem cell or CLMC, (1600V, 20ms, 1 pulse), respectively. 200K transfected cells were plated in triplicates in 6-well plates. About 21 days after transfection, the percentage reprogramming efficiency has been calculated as Col
  • Figure 3 shows exemplary colony development of induced pluripotent stem cells from human CLMC.
  • Figure 3a-f show a representative time course of colony development, wherein Figure 3a depicts a typical morphology of human CLMC cultured in its maintenance medium at Day 0 of cultivation.
  • Figure 3b depicts a typical morphology of human CLMC cultured in its maintenance medium at Day 15 of cultivation.
  • Figure 3c depicts a typical morphology of human CLMC cultured in its maintenance medium at Day 24 of cultivation.
  • Figure 3d depicts a typical morphology of human CLMC cultured in its maintenance medium at Day 29 of cultivation.
  • Figure 3e shows a 4x magnification of the typical morphology of an iPS colony first passage and 3f shows a lOx magnification of the typical morphology of an iPS colony at first passage.
  • Figure 3g-l depict an exemplary immunofluorescence staining of iPS derived from human cord lining cells showing the activation of endogenous expression of pluripotent embryonic stem cell markers, wherein Figure 3g shows the expression of KLF4, Figure 3h shows the expression of NANOG, Figure 3i shows the expression of OCT3/4, Figure 3j shows the expression of SOX2, Figure 3k shows the expression of SSEA4 and Figure 31 shows the expression of Tra-1-60.
  • Figure 3m shows an exemplary karyotype analysis demonstrating normal chromosomal numbers and G-banding patterns of CLiPS in the 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 emerging 10 days of reprogramming magnified 20x
  • Figure 3o shows exemplary a morphology of expanding human CLMSC-DTHN cultured on laminin-511 substrate magnified 4x.
  • Figure 3p shows exemplary a morphology of expanding human CLMSC-DTHN cultured on laminin-511 substrate magnified lOx.
  • Figure 3q shows exemplary a morphology of expanding human CLMSC-DTHN cultured on laminin-511 substrate magnified 20x.
  • Figure 3r shows an exemplary expression of the human pluripotent marker NANOG in CLMSC-DTHN iPS at passage No.3.
  • Figure 3s shows an exemplary expression of the human pluripotent marker OCT3/4 in CLMSC-DTHN iPS at passage No.3.
  • Figure 3t shows an exemplary expression of the human pluripotent marker SOX2 in CLMSC- DTHN iPS at passage No.3.
  • Figure 3u shows an exemplary expression of the human pluripotent marker NTRA-1-81 in CLMSC-DTHN iPS at passage No.3. Scale bars: all 100 pm.
  • Figure 3v shows an exemplary RT-PCR analyses of reprogramming gene expression and pluripotent gene expression in primary parental cells, parental cells 11 days after vector transfection (Dl l transfected cells) and in established iPS clones (CLiPS).
  • Vec denotes amplification specific for vector derived sequences.
  • Glycerinaldehyd-3-phosphat-Dehydrogenase (GAPDH) was used as an internal control.
  • PCR of homo sapiens (Hl) total RNA without reverse transcription was used to control for genomic contamination for all primer pairs.
  • Figure 4 shows an exemplary histological analysis of a teratoma formed by immunocompromised non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice after CLiPS injection.
  • NOD-SCID immunocompromised non-obese diabetic severe combined immunodeficiency
  • the teratoma formation assay reveals the formation of all three germ layers.
  • Figure 4a-inset shows a teratoma obtained from human CLEC-derived iPS 3 months after subcutaneous injection. Sections of the teratoma are further analyzed by a hematoxylin and eosin staining.
  • Figure 4a shows the presence of respiratory-like epithelium in the teratoma.
  • Figure 4b shows the presence of glandular structures representing the endoderm in the teratoma.
  • the arrowhead shows the presence of cartilage in the teratoma.
  • the arrowhead shows the presence of bone representing the mesoderm in the teratoma.
  • Figure 4e shows the presence of renal tissue in the teratoma.
  • the filled arrowheads indicate glomeruli and the hollow arrowheads indicate renal tubules.
  • Figure 4f the arrowhead shows the presence of neural epithelium representing the ectoderm in the teratoma.
  • CLiPS were induced to differentiate into specific tissues.
  • Figure 4g shows CLiPS differentiated into hepatocytes visualized with alpha-fetoprotein (AFP) and 4',6-diamidino-2-phenylindole (DAPI).
  • Figure 4h shows CLiPS differentiated into hepatocytes visualized with human serum albumin (HAS), cytokeratin 18 (CK18) and DAPI.
  • Figure 4i shows CLiPS differentiated into hepatocytes visualized with Oil Red O.
  • Figure 4j shows CLiPS differentiated into cardiomyocytes visualized with alpha-actinin (aACT), cardiac troponin I (cTnl), myosin regulatory light chain 2a (MLC2a) and DAPI.
  • Figure 4k shows CLiPS differentiated into dopaminergic neurons visualized with the floor-plate marker FOXA2, the roof plate marker LMX1A and DAPI.
  • Figure 41 shows CLiPS differentiated into dopaminergic neurons visualized with neuron-specific class III beta-tubulin (TUJI) and tyrosine Hydroxylase (TH).
  • Figure 4m shows CLiPS differentiated into oligodendrocyte progenitor cells visualized with OLIG2 and DAPI.
  • Figure 4n shows CLiPS differentiated into oligodendrocyte progenitor cells visualized with 04 and DAPI.
  • Figure 4o shows an electrophysiological analysis of mature human CLiPS-derived dopaminergic neurons at Day 45 of differentiation.
  • the human CLiPS-derived dopaminergic neurons fire trains of action potential with injected currents.
  • Scale bars 200 pm in Figure 4a, Figure 4c and Figure 4d; 100 pm in Figure 4b, Figure 4e and Figure 4f; 50 pm in Figure 4g, Figure 4h, Figure 4i, Figure 4k, Figure 41, Figure 4m; 25 pm in Figure 4j, Figure 4n.
  • Figure 5 shows an exemplary directed differentiation of human CLiPS into various different cell types, wherein Figure 5a depicts human CLiPS-derived neurons visualized with TH, Tuik and DAPI, Figure 5b depicts human CLiPS-derived hepatocytes visualized with CK18, HAS and DAPI, Figure 5c depicts human CLiPS-derived cardiomyocytes visualized with cTnl, aAct and DAPI and Figure 5d shows an electrophysiological analysis of contracting human CLiPS-derived cardiomyocytes illustrating the cells generating spontaneous action potentials.
  • Figure 6 shows an exemplary flow cytometric analysis of major histocompatibility complex (MHC) Class I and II, and T-cell co-stimulatory protein expression on iPS and dopaminergic neuroprogenitors differentiated from them.
  • Figure 6a shows a flow cytometric profile of immune-related gene expression on undifferentiated iPS.
  • Figure 6b shows a flow cytometric analysis of neural cell adhesion molecule (NCAM)-positive populations. These populations were gated for an analysis of immune-related protein expression.
  • Figure 6c shows an analysis of immune-related protein expression on Day25 differentiated dopaminergic neuroprogenitors .
  • MHC major histocompatibility complex
  • Figure 7 shows an in vivo comparison of engraftment of dopaminergic neuronal progenitor cells (NPCs) derived from human CLiPS and human adult fibroblast-iPS (asF-iPS) in NOD-SCID mice.
  • NPCs dopaminergic neuronal progenitor cells
  • AsF-iPS human adult fibroblast-iPS
  • Figure 7a shows in vivo engraftment of day 25 dopaminergic NPCs derived from human asF-iPS.
  • Figure 7b shows in vivo engraftment of day 25 dopaminergic NPCs derived from human CLEC-iPS (EC23-CLiPS).
  • Figure 7c shows in vivo engraftment of day 25 dopaminergic NPCs derived from CLMC-iPS (MC23-CLiPS).
  • Figure 7d shows an antibody staining of the grafted hemisphere of a Parkinson’s Disease (PD) mouse model created in an immunocompetent C57BL/6NTac mouse 1 month after transplantation with human CLEC-iPS-derived dopaminergic NPCs.
  • PD Parkinson’s Disease
  • Figure 7e shows long neuronal processes originating from the graft site projected along the forceps major of the corpus callosum to distal regions of the brain.
  • Arrowheads in Figure 7f indicate the human NCAM and TH double positive neurons, which are present in abundance in the injected site, as shown by the arrowheads.
  • Figure 7g shows the contralateral non-transplanted hemisphere of the same section as shown in Figure 7d.
  • Figure 7h illustrates that no surviving cells are visible in striatum transplanted with human adult asF-iPS-derived NPCs suggesting immune rejection.
  • Figure 7i indicates abundant microglia/macrophage aggregation in the transplanted hemisphere.
  • Figure 7j shows absence of microglia/macrophage aggregation in the non-transplanted hemisphere.
  • Figure 7k shows a higher magnification of Figure 7i It can be seen that microglia located proximal to and inside graft display a more amoeboid morphology characteristic of activated microglia.
  • Figure 71 shows a higher magnification of Figure 7k indicating an expression of CD68, which is an activation marker for microglia. Scale bars: 100 pm in Figure 7a-c and Figure 7k; 200 pm in Figure 7d, Figure 7g and Figure 7h; 50 pm in Figure 7e, Figure 7f and Figure 71.
  • Figure 8 shows the survival of human CLEC derived (EC23-CLiPS) dopaminergic neurons in mouse PD model 9 months after transplantation.
  • Figure 8a indicates HuNu+/hNCAM+/TH+ neurons present in the transplanted hemisphere.
  • Figure 8b is an overlay of Figures 8c -f and shows a higher magnification of the boxed area in Figure 8a.
  • Figure 8c indicates hNCAM-i- neurons present in the transplanted hemisphere.
  • Figure 8d indicates HuNu-i- neurons present in the transplanted hemisphere.
  • Figure 8e indicates TH+ neurons present in the transplanted hemisphere.
  • Figure 8f indicates nuclei of the neurons present in the transplanted hemisphere.
  • Figure 8g illustrates schematically the experimental steps starting from the induction of PD lesion by 6-hydroxydopamine (6-OHDA) injection into the striatum of C57BL/6NTac mice.
  • Pre-transplantation rotation behavioral assays were performed one and two weeks prior to NPC transplantation.
  • Figure 8h shows the results of an Apomorphine-induced rotational asymmetry assay in mice transplanted with dopaminergic NPCs derived from human EC23-CLiPS and asF-iPS, and sham control. The assays were performed every two weeks up to 22weeks after transplantation.
  • Figure 8h shows a representative in vivo Positron Emission Tomography (PET) imaging of the uptake of [18F]PE- P2I ligand to evaluate recovery of dopamine transporter (DAT) function in striatal dopaminergic neurons 6 months following transplantation.
  • PET Positron Emission Tomography
  • DAT dopamine transporter
  • FIG. 9 shows an exemplary in vivo PET imaging of striatal dopamine production in engrafted mice.
  • the PET illustrates the uptake of [18F]PE-P21 ligand to evaluate recovery of dopamine transporter (DAT) function in striatal dopaminergic neurons 6 months after iPS-derived NPCs transplantation.
  • Mice transplanted with human CLEC-iPS-derived NPCs show apparent recovery of DAT activity compared to those transplanted with human adult iPS- derived NPCs or sham transplanted controls.
  • DAT dopamine transporter
  • Figure 10 illustrates the in vivo maintenance of graft derived from human CLiPS 6 and 9 months after implantation into mice brains.
  • the graft is stained positive for human antigen NCAM and TH dopaminergic marker. A formation of tumors has not been recorded. Scale bars: 50pm.
  • Figure 11 shows the results of a histological and functional analysis of transplanted human EC23-CLiPS dopaminergic NPCs in a Medial Forebrain Bundle (MFB) lesion model of PD created in fully-immunocompetent Wistar Hannover rats.
  • Figure Ila shows engraftment of human EC23-CLiPS neurons in striatal region of a rat brain 3 months after transplantation demonstrated by positive double-staining for human cytoplasm (STEM 121) and human nuclear antigen (HuNu) antibodies. The staining indicates functional recovery.
  • MFB Medial Forebrain Bundle
  • Figure 11b indicates colocalization of Synapsin 1 immunoreactivity with hNCAM+/TH+ neurons suggesting possible integration of transplanted human CLiPS-derived cells with host tissues 3 months after transplantation.
  • Figure 11c shows a retrograde lesioning of the dopaminergic system in the substantia nigra in a rat brain.
  • Figure lid shows an unlesioned rat brain confirming the retrograde lesioning of the dopaminergic system in the substantia nigra of Figure 11c by tyrosine hydroxylase (TH) immunostaining.
  • Figure lie shows the result of an Apomorphine- induced rotational asymmetry assay in rats transplanted with dopaminergic NPCs derived from human CLEC23-iPS.
  • Figure 13 shows CLiPS differentiate to RPEs: Images of differentiation cultures from different stem cells, human ES cell (H9), iPS cell lines derived from skin (Asf5, AGO, HDFA), umbilical cord-lining mesenchymal cells (CLMC23, CLMC30, CLMC44) and umbilical cordlining ectodermal cells (CLEC23). Darker patches on the cell culture plates correspond to presence of pigmented RPE cells.
  • Figure 14 shows CLMCs have consistently high differentiation efficiency:
  • Figure 14a shows that a visual grading system for estimating RPE differentiation efficiency based on percentage area of the well occupied by pigmented cells, RPE differentiation efficiency is graded as 0, 1, 2 or 3 for no pigmentation, ⁇ 30%, 30-60% or >60% pigmentation, respectively.
  • Figure 14b shows RPE differentiation efficiency estimated by visual grading of pigmented cell area of differentiation plates. Each bar represents grading of one differentiation plate, numbers on the bars denotes percentage of wells on the plate with different grades of pigmentation indicated by different shades of brown. Numbers 1-3 represent biological replicates.
  • Figure 14c shows RPE differentiation efficiency of different stem cells estimated by Pmell7 by flow cytometry; cells from 3 wells were pooled for FACS analysis.
  • Figure 15 shows CLiPS-derived RPEs have more pigmentation compared to ES-derived:
  • Figure 15a shows images of differentiation plates taken at identical conditions on day 30 of differentiation, using ChemiDoc Touch gel imaging system (Bio-Rad laboratories).
  • Figure 15b shows phase-contrast Images of RPE from different stem cells exhibiting weak pigmentation of H9.
  • H9 human ES cell-derived RPE
  • CLMC23, CLMC30, CLMC44, CLEC23 RPE derived from CLiPS
  • AGO HDFA
  • Asf5 RPE derived from skin-iPS cells.
  • Figure 15c shows CLiPS- derived RPEs have more pigmentation compared to ES-derived.
  • FIG. 15d shows pigmentation- related and RPE specific genes are higher in CLiPs: RT-qCPR analysis of genes involved in pigmentation at day 18 and day 35 of differentiation: MITF, PMEL17, TYROSINASE, TRYP2.
  • Figure 16 shows CLiPS expressing RPE specific genes at day 18 and day 35 of differentiation: RT-qCPR analysis of RPE-specific RPE65 and MERTK.
  • Figure 17 shows CLiPS derived-RPEs are functional.
  • Figure 17a shows tight junctions are formed by in vitro generated RPEs, similar to native RPEs: Trans-epithelial electrical resistance (TEER), a measure of tight-junction integrity, in RPEs derived from different stem cells measured over a period of 4 months using Epithelial Volt Ohm meter, EV0M2TM.
  • Figure 17b shows in vitro generated RPEs are highly phagocytic: Percentage phagocytosis of FITC- labelled photoreceptor outer segments (POS) by RPEs derived from different stem cells.
  • POS photoreceptor outer segments
  • Figure 18 shows CLiPS derived-RPEs showing protein expression similar to ES-derived. CLiPS-RPEs showing apical expression of Mertk, junctional expression of ZO-1 and cytoplasmic expression of RPE65.
  • Figure 19 shows the original method of RPE differentiation and the modifications:
  • Figure 19a shows the schematic of the original method showing the differentiation mediums used at different stages and their composition.
  • Figure 19b shows the modifications introduced to the differentiation protocol, showing gradual increase in CHIR99021 concentration and replacement of FGF inhibitor, SU5402 with PD173074.
  • Figure 19c shows a photo of CLMC30 plate differentiated using the published protocol using SU5402 or modified protocol using PD173074 showing similar degree of RPE differentiation and pigmentation.
  • DM1-DM5 differentiation mediums 1-5.
  • Modified RPE differentiation protocol using PD173074 yields functional RPEs. Functionality of RPEs derived from differentiation methods using SU5405 or PD173074 tested for TEER ( Figure 19d) and phagocytosis of FITC-labelled POS particles ( Figure 19e).
  • Figures 20a and b show comparison of RPE yield by different purification methods.
  • Figures 20a and b show the schematic representation of different methods of RPE purification: Differentiation cultures containing RPE and non-RPE were purified (i) Manual purification: identification of non-RPE cells based on their morphology and lack of pigmentation and manual removal of them by scraping by observing under a dissection microscope, (ii) TrypLE purification: removal of majority of weakly attached non-RPE clusters by partial TrypLE treatment, (iii) TrypLE+ Manual: elimination of majority of weakly attached non-RPE clusters by partial TrypLE treatment followed by manual removal of few non-RPE clusters that escaped TrypLE treatment by observing under a dissection microscope (iv) TrypLE + scatter sorting: removal of weakly attached non-RPE clusters by partial TrypLE treatment followed by scatter sorting, (v) Scatter sorting: separation of all cells from mixed differentiation culture based on their relative light scatter, as scatter
  • Figures 20c and d show the original and modified scatter soring method to more accurately select the scatter high RPE cells.
  • Figure 20c shows arbitrarily chosen gates for scatter high (cyan) and low gate (magenta) as in the original protocol.
  • Figure 20d shows the modified gate selection using weakly attached non-RPE cells dissociated by partial TryPLE treatment to set scatter low gate (magenta) to more accurately select scatter high gate (cyan).
  • Figure 20e shows yield of RPE obtained from different methods of purification.
  • Figure 20f shosw purity of RPE from different methods of purification assessed by Pmell7 flow cytometry.
  • Figure 20g shows TEER of RPE obtained from different methods of purification; M: Manual purification, T: TrypLE purification, T+M: TrypLE+manual purification, T+Sc: TrypLE+Scatter sorting, Sc: Scatter sorting, T (loose): weakly attached non- RPE cells easily detached by TrypLE treatment, Sc low: scatter low non-RPE cells from scatter sorting.
  • Figure 20h shows phagocytic capacity of RPE from different methods of purification assessed by photoreceptor outer segment (POS) phagocytosis assay.
  • Figure 20i shows table comparing different methods of RPE purification.
  • Figure 20j shows quantitative PCR comparison of RPE specific gene expression in CLMC23 and H9.
  • qPCR results showing relative expression of RPE-specific genes such as BEST1, RPE65, RLBP1, MERTK, MITF, PMEL17 and TRYP2 normalized to GAPDH.
  • Figure 20k shows comparison of gene expression in CLMC23 and H9, expressed as fold change of CLMC23 over H9.
  • FIG. 21 shows that CLiPs-RPE (CLEC23-RPE) has bioenergetic profile similar to native RPE (AHRPE). CLiPsRPE (CLEC23-RPE) also show higher glycolysis and oxidative phosphorylation compared to both skiniPSC-RPE (ASF5-RPE) and hESC-RPE (H9-RPE).
  • Figure 21a shows that for OCR curve basal respiration, ATP production, maximal capacity and spare respiratory capacity are higher in CLiPs-RPE by 38%, 40%, 35% and 36% respectively compared to H9-RPE.
  • Figure 21b shows that for ECAR curve glycolysis, glycolytic capacity and glycolytic reserve are higher in CLiPs-RPE by 25%, 37% and 50% respectively compared to H9-RPE.
  • Figures 21c-f show that CLiPs-RPE show increased resistance to oxidized low-density lipoprotein (oxLDL) as evidenced by no decrease in maximal capacity after exposure to oxLDL dotted curve) for CLEC23-RPE (c) vs. 27% reduction in ASF5-RPE (d) and 43% reduction in H9-RPE (e).
  • oxLDL oxidized low-density lipoprotein
  • CLiPs-RPE cells response to oxidative stress is similar to that seen in native RPE (AHRPE - f) making them functionally closer to primary RPE compared to other differentiated RPE.
  • Figures 21g-j show that CLiPs-RPE show increased resistance to hydrogen peroxide (H2O2) as evidenced by no decrease in maximal capacity after exposure to H2O2 (dotted curve) for CLEC23-RPE (g) vs. 27% reduction in ASF5-RPE (h) and 99% reduction in H9-RPE (i).
  • CLiPs-RPE cells’ response to oxidative stress is similar to that seen in native RPE (AHRPE) (f & j) making them functionally closer to primary RPE compared to other differentiated RPE.
  • Figure 22 shows absence of immune system clearance of all stem cell-derived retinal pigment epithelial (SC-RPE) cell lines.
  • Figures 22a and b show in-vivo bioluminescence measurements (total radiance) of injected luciferase-expressing SC-RPEs embedded in matrigel plugs at indicated time points in both humanized and NOD-SCID IL2Ry-/- (immunodeficient) mice.
  • Figure 22c shows representative images of all SC-RPE lines showing RPE65, Ki67 and Hoechst staining from the matrigel plugs engrafted in humanized mice at the endpoint of 2 months. Scale bar, 50 pm.
  • Figure 23 shows that CLEC23-RPE group has reduced levels of pro-inflammatory cytokines involved in induction of cellular immune response.
  • Figures 23a and b show serum cytokines (IFN-y and IL- 18) at the end-point being analyzed.
  • Figure 23c shows representative images showing OTX2, human CD45 (hCD45) and Hoechst staining of the RPE-matrigel plugs indicating immune cell infiltration.
  • Figures 23d and e show cellular immune response grading (0 to 3) based on hCD45 positive cells in the RPE-matrigel plugs. Scale bar, 50 pm.
  • Figure 24 shows CLEC23-RPE may suppress CD8 T cell activation.
  • Figures 24a and b show serum cytokines (IL-23 and IL-17A) at the end-point being analyzed.
  • Figure 24c shows that T cell (CD3) to B cell (CD19) ratio was calculated after flow cytometry analysis.
  • Figure 24d shows that CD3-positive cells were further gated into Helper T (CD4) and Cytotoxic T (CD8) cells to analyze T cell differentiation.
  • Figures 24e and f show that CD4-positive and CD8-positive cells were gated into four groups of differing T cell activation status based on specific surface markers.
  • the present invention is inter alia directed to a method of generating an induced pluripotent stem cell, from a stem cell of the amniotic membrane of the umbilical cord under conditions suitable to reprogram the stem cell, thereby generating the induced pluripotent stem cell (iPS).
  • iPS induced pluripotent stem cell
  • both mesenchymal and epithelial stem cells of the amniotic membrane of the umbilical cord - also jointly referred to herein as cord lining stem cells (CLSC) are used to generate iPS - also referred to herein as cord lining-derived induced pluripotent stem cells or “CLiPS”. It has been surprisingly found that cord lining-derived induced pluripotent stem cells of the present invention are robust and homogenous stem cells capable to differentiate into functional target cells of different lineages (cf., Examples 3 and 4).
  • cord lining-derived induced pluripotent stem cells have the capacity to differentiate in multiple cell types and can, for example, be differentiated into various cells types such as hepatocytes representing endodermal tissue (cf., Example 8), cardiomyocytes representing mesodermal tissue (cf., Example 9), and dopaminergic neurons (cf., Example 7) and oligodendrocytes (cf., Example 10) representing ectodermal tissue.
  • hepatocytes representing endodermal tissue
  • cardiomyocytes representing mesodermal tissue
  • dopaminergic neurons cf., Example 7
  • oligodendrocytes cf., Example 10 representing ectodermal tissue.
  • the cord lining-derived induced pluripotent stem cells of the present invention can potentially be used as a universal source of cell for allogeneic cell transplantation in humans without the need for immunosuppression, and this making them ideal candidates for such cell based therapies.
  • the cord lining-derived induced pluripotent stem cells of the invention can be generated by an integration- and feeder free method, thereby allowing an iPS production under current good manufacturing practice (cGMP) conditions.
  • the present invention provides an ideal platform to produce iPS for subsequent cell-based therapy in humans or animals.
  • CLiPS derived from very young tissue are less likely to carry genetic, epigenetic and mitochondrial DNA mutations as they are derived from a young tissue. Because of these advantages, CLiPS are a potentially superior stem cell source for generating differentiated cells for regenerative medicine. Hence, they are superior over iPS cells derived from skin or blood which require invasive procedures of tissue collection. They are also free from ethical issues associated with ES cells. Thus, CLiPS are a better source of stem cell for regenerative medicine. The inventors found that such CLiPS robustly differentiate to retinal pigment epithelial (RPE) cells, also called RPEs, by the method of the present invention.
  • RPE retinal pigment epithelial
  • the inventors compared different stem cell resources: Human ES cells (ES), iPS cells derived from skins (skin- iPS) and cord-lining cells (CLiPS), for their ability to generate RPEs in-vitro.
  • CLiPS can be of either mesenchymal (CLMC) or ectodermal (CLEC) origin.
  • CLMC mesenchymal
  • CLEC ectodermal
  • the inventors then compared RPE differentiation efficiency of CLiPS with ES and skin-iPS cells. Compared to skin-iPS, CLiPS gave consistently high RPE differentiation efficiency than skin-iPS cells by visual grading and flow cytometry estimation. Comparison of pigmentation of differentiation cultures visually and by image analysis also showed that CLiPS -derived RPEs have higher pigmentation that ES- derived RPEs.
  • the RPE generated from CLiPS also displayed functional characteristics of RPE after maturation in-vitro suggesting they are a superior source of RPE cells. Additionally, the inventors found by comparing bioenergetics of RPE derived from different stem cells that CLiPS-RPE have higher glycolytic and mitochondrial respiration than ES-derived RPEs.
  • the method of the present invention used of differentiating an induced pluripotent stem (iPS) cell derived from a stem cell of the amniotic membrane of the umbilical cord (CLiPS or CLSC) into a RPE cell has been particularly modified as described herein, which achieves maximum RPE yield.
  • this method may comprise expressing exogenous nucleic acid encoding the proteins OCT3/4, SOX2, KLF4, LIN28 and L-MYC and the p53-shRNA.
  • the nucleic acid encoding OCT3/4 (Sequence ID No: 1), also sometimes referred to as POU5FL, OCT3 or OCT4, encodes for the octamer-binding transcription factor 4.
  • OCT3/4 (Sequence ID No: 2) forms a heterodimer with SOX2 to regulate pluripotency factors in a cell.
  • SOX2 (Sequence ID No: 3), also sometimes referred to as SEY, encodes for the sex determining region Y-box 2 transcription factor (Sequence ID No: 4). When bound to OCT3/4, SOX2 binds to a non-palindromic genomic sequence thus activating the transcription of pluripotent factors in a cell.
  • KLF4 (Sequence ID No: 5), also sometimes referred to as GKLF, encodes for the Krueppel-like factor 4.
  • KLF4 (Sequence ID No: 6) is a zinc finger transcription factor, which functions as a tumor suppressor controlling the Gl-to-2 transition of the cell cycle by mediating the tumor suppressor p53.
  • L-MYC (Sequence ID No: 7) encodes for a transcription factor (Sequence ID No: 8) activating the expression of proliferative genes.
  • LIN28 (Sequence ID No: 9) encodes for the RNA-binding protein Lin-28 homolog A (Sequence ID No: 10), which regulates the self-renewal of stem cells.
  • the p53-shRNA (Sequence ID No: 11) encodes for a small hairpin RNA directed to p53, a protein that may regulate the cell cycle by stopping it when the protein accumulates in the cell. To avoid a stopping of the cell cycle by p53, p53-shRNA may silence the expression of p53 posttranscriptional.
  • the exogenous nucleic acids encoding OCT3/4, SOX2, KLF4, LIN28, L MYC and p53-shRNA may be transferred into the CLSC for expression.
  • the proteins OCT3/4, SOX2, KLF4, LIN28, L-MYC and the p53 shRNA may be transferred directly into a CLSC.
  • an induced pluripotent stem cell population of the present invention is obtainable by reprogramming stem cells of the amniotic membrane of umbilical cord.
  • the stem cell of the umbilical cord may be an (isolated) mesenchymal stem cell of the amniotic membrane of the umbilical cord, also referred to as cord lining mesenchymal stem cell (CLMC), or an (isolated) epithelial stem cell of the amniotic membrane of the umbilical cord, also referred to as cord lining epithelial stem cell (CLEC).
  • CLMC cord lining mesenchymal stem cell
  • CLMC cord lining epithelial stem cell
  • the CLEC and CLMC used to generate the iPS of the present invention may be derived of any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, with stem cells of human origin being preferred in one embodiment. Accordingly, also the iPS of the present invention can be derived of any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, with stem cells of human origin being preferred in one embodiment. In a preferred embodiment CLEC is used to generate the iPS of the present invention.
  • epithelial stem cells of the amniotic membrane of the umbilical cord are used as starting material
  • these epithelial stem cells can, for example, be obtained as described in US patent application 2006/0078993 (leading to granted US patents 9,085,755 and 9,737,568) or the corresponding International patent application W02006/019357.
  • mesenchymal stem cells of the amniotic membrane of the umbilical cord are used as starting material, they can also be obtained as described in US patent application 2006/0078993 (leading to US patents 9,085,755 and 9,737,568) or the corresponding International patent application W02006/019357.
  • mesenchymal stem cell population as described in the published US application 2018/127721 or the corresponding International Application WO 2018/067071.
  • the mesenchymal stem cell population of International Application WO 2018/067071 has the advantage that 99 % or more of the stem cells of this population are positive for the three mesenchymal stem cell markers CD73, CD90 and at the same lack expression of CD34, CD45 and HLA-DR, meaning 99 % or even more cells of the mesenchymal stem population
  • International Application WO 2018/067071 express the stem cell markers CD73, CD90 and CD 105 while not expressing the markers CD34, CD45 and HLA-DR.
  • mesenchymal stem population International Application WO 2018/067071 is the ideal starting material for producing the CLiPS of the present invention under GMP conditions.
  • CLMCs transfected with a transgene will maintain their sternness 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.
  • mesenchymal stem cell markers such as CD73, CD90 and CD105
  • negative markers such as CD34, CD45 or HLA-DR.
  • a CLiPS of the present invention that has been generated by reprogramming of a CLMC described herein and isolated from the amniotic membrane of the umbilical cord, may 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, 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 cells of the CLIPS population may express each of the following markers: CD73, CD90 and CD 105.
  • such a CLMC derived population of induced pluripotent stem cells of the invention may be a 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, 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 % may lack expression of each of CD34, CD45 and HLA-DR.
  • CLMC derived population of induced pluripotent stem cells of the invention may be a population, in which at least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more cells of the CLMC population express each of CD73, CD90 and CD105 and lack expression of each of CD34, CD45 and HLA-DR.
  • an induced pluripotent stem cell population of the invention
  • an induced pluripotent stem cell is obtainable by any suitable method that reprograms a stem cell (population) of the amniotic membrane of umbilical cord into such an induced pluripotent stem cell (population).
  • While one method of generating such an induced pluripotent stem cell comprises expressing exogenous nucleic acids encoding the proteins OCT3/4, SOX2, KLF4, LIN28 and L-MYC and the p53-shRNA in a stem cell of the amniotic membrane of the umbilical cord under conditions suitable to reprogram the stem cell, thereby generating the induced pluripotent stem cell, the invention is by no means restricted to CLiPS obtained by this method.
  • the CLiPS can be obtained by any suitable method as, for example described in the review of Cieslar-Probuda et al "Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences” BBA - Molecular Cell Research, Volume 1864, Issue 7, July 2017, Pages 1359-1369.
  • the reprogramming may be performed in the present invention also chemically by using small molecules or biologically by expressing exogenous nucleic acids encoding for reprogramming factors within a cell.
  • the exogenous nucleic acids encoding the proteins OCT3/4, SOX2, KLF4, LIN28, L-MYC and the p53 shRNA may be provided as any suitable nucleic acid for expression.
  • the nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) comprising messenger RNA (mRNA) and microRNA (miRNA).
  • the exogenous nucleic acids may be transferred as such or the exogenous nucleic acids may be incorporated into one or more vector(s) suitable to be transferred into a cell.
  • any vector suitable to be transferred into CLSC can be used.
  • An illustrative example for such a vector may be a plasmid.
  • the exogenous nucleic acids may be provided by one, two, three or four vectors suitable to be transferred into a stem cell.
  • three vectors may provide the exogenous nucleic acids for reprogramming CLSC into CLiPS, wherein the vectors may 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).
  • a viral vector may be used to transfer the exogenous nucleic acid into the CSLC.
  • An example for such a viral vector may be a retrovirus, a lentivirus, an inducible lentivirus, a sendai virus or an adeno virus.
  • transfection may be performed to transfer the exogenous nucleic acids into CSLC.
  • transfection may comprise electroporation, microinjection, liposome- and non- liposome-mediated transfection and sonoporation.
  • the CLSC may be subjected to electroporation, wherein the electric parameters may be adjusted depending on the type of CLSC being used, as a CLMC may require different electroporation conditions than a CLEC.
  • the electric parameters may comprise the number of electric pulses applied to the stem cell, the duration time of the applied electric pulse(s) and the voltage of the applied electric pulse(s).
  • Each electric parameter may be adjustable to further optimize the electroporation of the present invention. When so doing, each electric parameter may be adjusted independently or in combination with one or more of the other electric parameter(s) (cf. Example 1).
  • any parameter setting suitable for allowing the transfer of exogenous nucleic acid into CLSC may be applied.
  • a CLMC may be subjected to electroporation.
  • the electroporation may be carried out with 1 electric pulse which may have a duration time of about 15 milliseconds (ms) to about 25 ms and a voltage of about 1550 V to about 1650 V.
  • a CLMC may be subjected to electroporation with 1 electric pulse, which may have a duration time of about 20 ms and a voltage of about 1600 V.
  • electroporation yielding usable amounts/numbers of CLiPS derived from CLMC depends on the ratio of each vector (plasmid) DNA transfected to the number of CLMC used for the transfection.
  • This ratio is expressed herein by the amount of each vector (plasmid) DNA (in pg) that is used to the number of CLMC (in IxlO 6 cell) subjected to electroporation.
  • the ratio of the amount of vector (plasmid) DNA for each vector to the number of cells may be in the range of 1.5pg DNA to about IxlO 6 CLMC to about 2.5 pg DNA to about IxlO 6 CLMC.
  • this ratio may be about 2.5pg DNA to about IxlO 6 CLMC, about 2.25pg DNA to about 1X10 6 CLMC, about 1.8pg DNA to about IxlO 6 CLMC, about 1.7pg DNA to about 1X10 6 CLMC, about 1.67pg DNA to about 1X10 6 CLMC, about 1.6pg DNA to about IxlO 6 CLMC, or about 1.5pg DNA to about IxlO 6 CLMC (cf. Table 1 showing that using a ratio of the amount of vector (plasmid) DNA for each plasmid to the number of cells of about 1.67pg DNA to about IxlO 6 CLMC yielded an effective transformation yield).
  • each of the vectors is used in the same amount in the electroporation of the CLMC.
  • a CLEC may be subjected to electroporation to yield CLiPS of the invention.
  • the electroporation may be carried out with 2 electric pulses, which may each have a duration time of about 25 ms to about 35 ms and a voltage of about 1300 V to about 1400 V each.
  • a CLEC may be subjected to electroporation with 2 electric pulses, which may have a duration time of about 30 ms and a voltage of about 1350 V each.
  • CLMC it has also been found for CLiPS derived from CLEC that electroporation yielding usable amounts/numbers of CLiPS derived from CLEC depends on the ratio of the amount of each plasmid DNA transfected to the number of CLEC used for the transfection. Also this ratio is expressed herein by the amount of vector (plasmid) DNA (in pg) that is used for transfection to the number of CLEC (in IxlO 6 cells) which is to be transfected. In illustrative examples, the ratio of the amount of vector (plasmid) DNA to the number of cells may be in the range of about 1.5pg DNA to about IxlO 6 CLEC to about 2.5pg DNA to about IxlO 6 CLEC.
  • the ratio may be about 1.5pg DNA to about IxlO 6 CLEC, about 1.6 pg DNA to about IxlO 6 CLEC, about 1.67pg DNA to about IxlO 6 CLEC, about 1.7pg DNA to about IxlO 6 CLEC, about 1.8 pg DNA to about IxlO 6 CLEC, about 1.9pg DNA to about IxlO 6 CLEC, about 2.0pg DNA to about IxlO 6 CLEC, or about 2.5pg DNA to about IxlO 6 CLEC (cf. Table 1 showing that using a ratio of the amount of plasmid DNA for each vector to the number of cells of about 1.67pg DNA to about IxlO 6 CLEC provided an effective transformation yield).
  • each of the vectors is used in the same amount in the electroporation of the CLEC.
  • the electroporation of both CLEC and CLMC may be performed in the method of the invention in a uniform electrical field. Thereby, critical consequences of the electroporation such as pH change, ion formation or heat generation may be minimized.
  • the uniform electric field may be generated by maximizing the gap between the electrodes while minimizing the surface area of each electrode.
  • An illustrative example for a system providing such a uniform electric field is the NeonTM Transfection System of ThermoFisher Scientific.
  • transfection can be carried using any suitable electroporation buffer.
  • the respective electroporation buffer provided by the manufacturer of the transfection system is typically used for electroporation.
  • the stem cells may be transferred into a medium suitable for cell recovery and cell cultivation.
  • any cell culture medium suitable for cell recovery and/or proliferation can be used.
  • Illustrative examples for such a suitable cell culture medium may be commonly used media for cultivation (propagation) of human induced pluripotent stem cell such as mTeSRl, StemMACSTM iPS-Brew XF, TeSRTM-E8, mTeSRTMPlus, TeSRTM2, mTeSRTMl.It is also possible to use for the cell recovery cultivation any medium that capable of supporting proliferation (without differentiation)/healthy growth of CLEC or CLMC.
  • suitable media for this cultivation of CLEC are, for example, described in US patent application 2006/0078993 and include EpiLife medium, Medium 171, MEGM-Mammary Epithelial Cell Medium or mixtures of such media such as the medium PTT- e3 (that has been used herein for the generation of CLiPS derived from CLEC and that is described herein in detail below).
  • suitable media for this cultivation of CLMC are, for instance, described in US patent applications 2006/0078993 and 2018/127721 as well as in International Patent Application W02007/046775 and include DMEM/10% FBS, DMEM:F12 culture medium (a 1:1 mixture of DMEM and Ham's F-12 medium), or a media such as PPT-6 (a culture medium comprising DMEM, F12-mediim, Medium 171 and FBS, see US application 2018/127721) or PTT4 (wherein the latter has been used in the Example Section herein for the generation of CLiPS derived from CLMC). It is also possible to use for this cell recovery cultivation mixtures of these media (for example a mixture of mTeSRl with the medium PTTe-3 or the medium PTT-4).
  • the medium suitable for cell recovery of transfected CLEC or CLMC as described herein may further contain growth factors, which may stimulate cellular growth and proliferation.
  • the growth factors may be added to the cell culture medium as such.
  • the recovery medium may contain serum such as, for example, fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the medium suitable for cell recovery after transfection may be a serum-free or a serum-containing medium.
  • composition of the medium suitable for cell recovery may differ, depending on the CLSC being used.
  • the medium suitable for the recovery of a transfected CLMC may consist of a (chemically) defined medium and FBS. Accordingly, the medium suitable for the recovery of a transfected CLMC 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.
  • CLMC are cultivated in medium PTT-4 for cell recovery after transfection, wherein medium PTT-4, as described in International Patent Application W02007/046775, consists of 90 % (v/v) CMRL-1066 and 10 % (v/v) FBS.
  • a medium suitable for the recovery of a transfected CLEC may be a serum-free medium, wherein the medium may contain cytokines and growth factors.
  • the medium suitable for the recovery of a transfected CLEC may be a defined medium.
  • a recovery medium may comprise Mammary Epithelial Basal Medium MCDB 170, EpiLife medium, DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium) and FBS (Fetal Bovine Serum).
  • such a medium comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 10 to about 30 % (v/v), EpiLife medium in a final concentration of about 20 to about 40 % (v/v), F12 in a final concentration of about 5 to about 15 % (v/v), DMEM in a final concentration of about 30 to about 45 % (v/v) and FBS in a final concentration of about 0.1 to 2 % (v/v).
  • One such medium may comprise Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 15 to about 25 % (v/v), EpiLife medium in a final concentration of about 25 to about 35 % (v/v), F12 in a final concentration of about 7.5 to about 13 % (v/v), DMEM in a final concentration of about 35 to about 40 % (v/v) and FBS in a final concentration of about 0.5 to 1.5 % (v/v).
  • Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 15 to about 25 % (v/v)
  • EpiLife medium in a final concentration of about 25 to about 35 % (v/v)
  • F12 in a final concentration of about 7.5 to about 13 % (v/v)
  • DMEM in a final concentration of about 35 to about 40 % (v/v)
  • FBS in a final concentration of about 0.5 to 1.5 % (v/v).
  • Another such medium may comprise Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 20 % (v/v), EpiLife medium in a final concentration of about 30 % (v/v), Fl 2 in a final concentration of about 12.5 (v/v), DMEM in a final concentration of about 37.5 % (v/v) and FBS in a final concentration of about 1.0 % (v/v).
  • % (v/v) refers to the volume of the individual component relative to the final volume of the culture medium.
  • DMEM fetal calf serum
  • 1 liter of culture medium contains about 350 ml to 400 ml DMEM.
  • the medium suitable for the recovery of a transfected CLEC cell is obtained by mixing to obtain a final volume of 1000 ml culture medium:
  • the growth factors in the medium suitable for the recovery of a transfected CLEC may an insulin like growth factor (IGF) such as IGF-1 or IGF-2, an epidermal growth factor (EGF) such as HB-EGF or EPR, a transforming growth factor (TGF) such as TGF-a or TGF-P 1, an activin, a bone morphogenic protein (BMP), a platelet derived growth factor (PDGF), transferrin and insulin.
  • IGF insulin like growth factor
  • EGF epidermal growth factor
  • TGF transforming growth factor
  • BMP bone morphogenic protein
  • PDGF platelet derived growth factor
  • CLEC are cultivated in medium PTTe-3 for cell recovery after transfection, wherein medium PTTe-3 contains human epidermal growth factor (EGF), one or more transforming Growth Factors such as TGF-alpha and/or TGF-beta (TGF-beta 1, TGF-beta 2 and/or TGF-beta 3), or insulin.
  • EGF epidermal growth factor
  • TGF-beta 1, TGF-beta 2 and/or TGF-beta 3 insulin.
  • the medium suitable for the recovery of a transfected CEEC may comprise human epidermal growth factor (EGF) in a final concentration of about 1 to about 15ng/ml.
  • the recovery medium may also comprise insulin in a final concentration of about 1 to about 7.5 pg/ml.
  • This recovery medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-Triiodo-E-thyronine sodium salt (T3).
  • the medium comprises all three of adenine, hydrocortisone, and 3,3',5-Triiodo-E- thyronine sodium salt (T3).
  • the medium may comprise adenine in a final concentration of about 0.05 to about 0.1 mM adenine, hydrocortisone in a final concentration of about 0.1 to 0.5 pM hydrocortisone and 3,3',5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.1 to about 5 ng/ml.
  • the recovery medium may comprise one of more transforming growth factors (TGF), for example transforming growth factor beta 1 (TGF-beta 1) and/or transforming growth factor alpha (TGF-alpha.
  • TGF-beta 1 may be present in a final concentration of about 0.1 to about 5 ng/ml and TGF-alpha may be present in a final concentration of about 1.0 to about 10 ng/ml.
  • the medium of recovery of CEEC may comprise Cholera Toxin from Vibrio cholerae (which is commercially available, for example, from Sigma Aldrich under catalogue number C8052. If cholera toxin from Vibrio cholerae is used, it may be present in a final concentration of about 1 x 10 -11 M to about 1 x 10" 10 M.
  • DMEM Dulbecco’s modified eagle medium which was developed in 1969 and is a modification of basal medium eagle (BME) (cf. Fig.l showing the data sheet of DMEM available from Lonza).
  • BME basal medium eagle
  • the original DMEM formula contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells.
  • DMEM has since then become a standard medium for cell culture that is commercially available from various sources such as ThermoFisher Scientific (catalogue number 11965-084), Sigma Aldrich (catalogue number D5546) or Lonza, to name only a few suppliers.
  • any commercially available DMEM can be used in the present invention.
  • the DMEM used herein is the DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g/L glucose and L-glutamine. In another preferred embodiment the DMEM used herein is the DMEM medium of Sigma Aldrich catalogue number D5546 that contains 1000 mg/L glucose, and sodium bicarbonate but is without L-glutamine. [069] By ‘ ‘ ‘ ‘F12” medium is meant Ham’s F12 medium. This medium is also a standard cell culture medium and is a nutrient mixture initially designed to cultivate a wide variety of mammalian and hybridoma cells when used with serum in combination with hormones and transferrin.
  • Ham’s F12 medium any commercially available Ham’s F12 medium (for example, from ThermoFisher Scientific (catalogue number 11765-054), Sigma Aldrich (catalogue number N4888) or Lonza, to name only a few suppliers) can be used in the present invention.
  • Ham’s F12 medium from Lonza is used.
  • DMEM/F12 or “DMEM:F12” is meant a 1:1 mixture of DMEM with Ham’s F12 culture medium.
  • DMEM/F12 (1:1) medium is a widely used basal medium for supporting the growth of many different mammalian cells and is commercially available from various supplier such as ThermoFisher Scientific (catalogue number 11330057), Sigma Aldrich (catalogue number D6421) or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention.
  • the DMEM:F12 medium used herein is the DMEM/F12 (1:1) medium available from Lonza under catalog number 12-719F (which is DMEM: F12 with L-glutamine, 15 mM HEPES, and 3.151 g/L glucose).
  • M171 is meant culture medium 171, which has been developed as basal medium for the culture of for the growth of normal human mammary epithelial cells. Also this basal medium is widely used and is commercially available from supplier such as ThermoFisher Scientific or Life Technologies Corporation (catalogue number M171500), for example. Any commercially available M171 medium can be used in the present invention. In preferred embodiments, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalogue number M171500.
  • MBD 170 a basal nutrient medium that is used for the growth of mammary epithelial cells and that is commercially available in powder form, for example, from United States Biological, Salem Massachusetts USA under catalogue number M2162 or from Bio-Connect B.V., Huissen, The Netherlands, under catalogue number (MBS652676_101)
  • EpiLife medium a HEPES and bicarbonate buffered liquid medium that is prepared without calcium chloride and that is commonly used for the 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.
  • CMRL medium is meant the medium that was originally developed by Connaught Medical Research Laboratories for the growth of Earle's 'L' cells under serum-free conditions. CMRL medium is known to be also especially useful for cloning monkey kidney cells and for growth of other mammalian cell lines when supplemented with horse or calf serum. CMRL medium is commercially available, for example, from ThermoFisher Scientific (catalogue number 11530037)
  • FBS fetal bovine serum
  • fetal calf serum i.e. the blood fraction that remains after the natural coagulation of blood, followed by centrifugation to remove any remaining red blood cells.
  • Fetal bovine serum is the most widely used serumsupplement for in vitro cell culture of eukaryotic cells because it has a very low level of antibodies and contains more growth factors, allowing for versatility in many different cell culture applications.
  • the FBS is preferably obtained from a member of the International Serum Industry Association (ISIA) whose primary focus is the safety and safe use of serum and animal derived products through proper origin traceability, truth in labeling, and appropriate standardization and oversight.
  • ISIA International Serum Industry Association
  • FBS FBS
  • ISIA members include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific and Life Science Production, to mention only a few.
  • the FBS is obtained from GE Healthcare under catalogue number A15- 151.
  • the medium suitable for cell recovery may also contain a compound, which may suppress an inflammatory response and/or may also enhance cell survival and proliferation after transfection.
  • a compound which may be a glucocorticoid.
  • Glucocorticoids are steroid hormones, which are able to up-regulate the expression of anti-inflammatory proteins in the nucleus and repress the expression of pro-inflammatory proteins in the cytosol.
  • the glucocorticoid used herein may be prednisolone, methylprednisolone, dexamethasone, betamethasone, corticosterone or hydrocortisone, to name only a few illustrative examples of suitable glucocorticoids.
  • glucocorticoids can be used in any suitable concentration, for example, in a concentration of about 0.1 pM to about 2.5 pM or to about 5pM.
  • the glucocorticoid in the medium suitable for the recovery of transfected CLSC may be hydrocortisone used in a concentration of about 0.1 pM to about 2.5 pM.
  • the hydrocortisone concentration in the medium suitable for the recovery of transfected CLSC is about 0.5 pM to about 2 pM. In one such illustrative example, the hydrocortisone concentration is about 1 pM.
  • the recovery of the transfected CLSC may be carried out in a cell culture device such as a cell culture vessel.
  • the cell culture vessel may be, but is not limited to, a cultivation flask, a petri dish, a roller bottle and a multiwall plate. Further, the cell culture vessel may be coated to provide a layer, which may facilitate the cell growth by supplying the cells with metabolites.
  • the coating of the cell culture vessel may be serum-derived or serum-free.
  • An example for a serum-derived coating may be a coating with gelatinous proteins from the basement membranelike matrix such as Matrigel.
  • a serum-free coating of the cell culture vessel may instead be characterized by being animal and xeno-free thus allowing a cell cultivation under cGMP conditions.
  • a serum-free coating of the cell culture vessel may be a coating with recombinant proteins or parts thereof such as, for example, a coating with a extracellular matrix protein such as collagen, fibronectin, elastin, laminin, including, for instance, the laminin-511 E8 fragment, or laminin 521, vitronectin, for example, in the form of commercially available citronectin XFTM, CELLstart or the SynthemaxTM vitronectin substrate.
  • transfected CLEC may be preferably cultivated in a cell culture vessel with a serum-derived coating
  • CLMC may be preferably cultivated in a cell culture vessel with a serum-free coating.
  • the medium suitable for the recovery of the transfected CLSC may be replaced with another cell culture medium after a suitable period of time.
  • the suitable period of time may, for example, be about 1, about 2 or about 3 days after transfection.
  • the medium replacement may be carried out about 2 days after transfection.
  • Another cell culture medium used for the medium replacement may also be a mixture of different cell culture media.
  • any cell culture medium or cell culture medium mixture suitable for yielding of iPS can be used.
  • the suitable cell culture medium or cell culture media mixture may contain a compound, which may suppress inflammatory response and enhance cell survival.
  • the medium suitable for cell recovery after transfection may be replaced with a mixture of two different cell culture media after a suitable period of time to ensure a proper supply of nutrients and a suitable blend of growth factors to the cells as they transition from their native state into a more pluripotent state when undergoing somatic reprogramming.
  • the cell culture media mixture of the present invention may consist of the medium suitable for cell recovery, which may contain hydrocortisone, and a second cell culture medium.
  • the two different cell culture media are mixed in a ratio of about 1:1 (v/v), wherein the mixture may be prepared by contacting 1 volume medium suitable for cell recovery to 1 volume second cell culture medium.
  • the two different cell culture media are mixed in a ratio of about 1:2 (v/v) or 2:1, wherein the mixture may be prepared by contacting 1 volume medium suitable for cell recovery to 2 volumes second cell culture medium (or 2 volumes medium suitable for cell recovery to 1 volumes second cell culture medium)
  • the second cell culture medium used for generating the cell culture mixture may be any cell culture medium suitable to enhance or maintain iPS proliferation (such medium is also termed “maintenance medium” herein).
  • a mixture such a 1 : 1 mixture of the medium used for cell recovery and the maintenance medium provides the advantage of allowing the CLiPS cells to transition gradually from their cognate culture medium to the ES/iPSC medium, instead of a sudden switch that might compromise their viability.
  • a suitable cell culture medium include, but are not limited to, commercially maintenance media such as mTeSRl, StemMACSTM iPS-Brew XF, TeSRTM-E8, mTeSRTMPlus, TeSRTM2 or mTeSRTMl, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co.
  • commercially maintenance media such as mTeSRl, StemMACSTM iPS-Brew XF, TeSRTM-E8, mTeSRTMPlus, TeSRTM2 or mTeSRTMl, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co.
  • mTeSRl since being manufactured under GMP conditions may be preferably used, if the iPS colonies are cultivated under animal- and xeno-free GMP conditions.
  • mTeSRl may be the second cell culture medium used for generating the cell culture mixture.
  • the 1:1 (v/v) cell culture media mixture may be replaced with the same mixture of cell culture media within a suitable period of time. This suitable period of time may be about 3, about 4, about 5 or about 6 days after transfection.
  • the 1:1 (v/v) cell culture media mixture may be replaced with the same mixture 4 days after transfection.
  • the 1 : 1 (v/v) cell culture media mixture may be further replaced with the second cell culture medium used for generating the cell culture mixture only.
  • a suitable period of time may be about 4, about 5, about 6 or about 7 days after transfection.
  • the 1:1 (v/v) cell culture media mixture may be replaced with the second cell culture medium 6 days after transfection.
  • the 1:1 (v/v) cell culture media mixture may be replaced with mTeSRl and mTeSRTMl, respectively, 6 days after transfection.
  • the regular cell culture media changes and replacements may contribute to an increase of surviving CLiPS.
  • CLiPS colonies may grow and proliferate.
  • the CLiPS may be further cultivated.
  • the cell culture medium may also be replaced regularly with the same medium to ensure a proper supply of nutrients and a suitable blend of growth factors to the cells.
  • the cell culture medium may be replaced daily or every second day, every third day or every fourth day.
  • the cell culture medium may be replaced every second day. Consequently, CLiPS colonies may further grow and proliferate.
  • CLiPS colonies may become visible to the naked eye about 10, 11, 12, 13, 14, 15, or 16 days after transfection (cf., Example 2).
  • a suitable colony size may comprise a length of about 0.1mm to about 2mm in diameter.
  • the CLiPS colony may be selected when reaching a length of about 0.5mm to about 1.5mm in diameter, wherein the CLiPS colonies may reach this size about 20 days after transfection.
  • the CLiPS colony may be picked. This may be carried out manually, if wanted.
  • a device allowing an enlarged view of the colonies may be used. Examples for such a device may be a magnifier or a microscope.
  • the CLiPS may be selected and picked under bright field microscopy.
  • the picked CLiPS colonies may be transferred to another cell culture vessel, wherein the coating of the cell culture vessel may vary from the coating of the cell culture vessel used for the recovery of the transfected CLSC or it may be the same.
  • the coating of the culture vessel is the same, since CLMC-derived CLiPS thus far cultivated under cGMP suitable conditions may be maintained animal- and xeno-free, thereby preserving cGMP conditions.
  • a CLMC-derived CLiPS colony may be transferred in a cell culture vessel coated with a serum-free substance such as the laminin-511 E8 fragment for further cultivation (cf., Example 3).
  • a CLEC- and/or CLMC-derived CLiPS colony may be transferred in a cell culture vessel coated with a serum-derived substance such as Matrigel for further cultivation.
  • the cell culture medium may preferably be the same as used before the colony picking.
  • the cell culture medium may be also replaced regularly after colony picking. For example, medium may be replaced daily, every second day or every third day. In a preferred example of the present invention, the cell culture medium may be replaced daily after colony picking.
  • the CLiPS colonies or a cell population formed from the colonies are typically detached from the coated cell culture vessel and transferred to a larger cell culture vessel for further cultivation under the same cultivation conditions used directly after the colony picking.
  • a suitable confluence may be at least about 40%, at least about 45 %, at least about 50%, at least about 55%, at least about 60% and at least about 65% confluence. It is noted in this context that the term “cell population” when used in relation to the propagation of CLiPS forming colonies is more suitable as the CLiPS cells do not take on a colony-like appearance when they reach a confluence of about 70 % to about 80%.
  • any dissociation agent suitable to disrupt cell adhesion or hydrolyze peptide bonds can be used.
  • An example for such a suitable dissociation agent may be a solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or a solution containing an enzyme such as trypsin or dispase (see the experimental section of the present application, in which dispase has been used to detach a CLiPS colony from the coated cell culture vessel).
  • the cell culture medium may also be replaced regularly, for example, daily, every second day or every third day. In a preferred example of the present invention, the cell culture medium may be replaced daily. This way, the CLiPS may further grow and proliferate.
  • a CLiPS colony or a cell population formed from a colony may be passaged when reaching a suitable size.
  • the suitable size 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.
  • the CLiPS colony or the cell population formed therefrom may be passaged when the culture reaches about 60-90 % confluence.
  • the CLiPS colony or the cell population formed therefrom may be passaged when reaching about 70-80 % confluence.
  • CLiPS may be passaged in a suitable ratio, wherein one volume CLiPS may be contacted with multiple volumes of cell culture medium.
  • CLiPS may be passaged in 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 passaging may be performed by dividing 1 volume dissociated CLiPS into about 2, or about 3, or about 4 or about 5 volumes of dissociated CLiPS, respectively.
  • CLiPS may be passaged in a ratio of about 1:3 (v/v).
  • any enzyme suitable to detach the cells from the culture vessel can be used.
  • dispase may be used for this purpose.
  • any chemical suitable to remove cell-to-cell adhesion can be used for CLiPS passaging in the context of the present invention, wherein the concentration of the chemical may be suitable to remove cell-to-cell adhesion without harming the cells.
  • An illustrative example for such a chemical may be EDTA. Since EDTA may kill cells at higher concentrations, a suitable EDTA concentration of the present invention may be about 0.5mM.
  • the cell culture medium used for passaging may be supplemented with a substance suitable for enhancing the survival of the CLiPS when dissociated.
  • any substance suitable for enhancing the survival of the CLiPS when dissociated may be used.
  • An example of such a suitable substance may be an inhibitor of a signaling pathway such as the rho-associated protein kinase (ROCK) signalling pathway.
  • the RHO/ROCK pathway inhibitor Y-27632 may be an illustrative example for a substance suitable for enhancing the survival of dissociated CLiPS.
  • a defined supplement for single-cell cloning of human iPS cells such as CloneRTM (available from StemCell Technologies) may also be used for enhancing the survival of the dissociated cells.
  • the passaged CLiPS may be cultivated in a medium supplemented with the substance suitable for enhancing the survival of the dissociated CLiPS for a suitable period of time before getting differentiated into a target cell.
  • a master cell bank containing (primary) isolated CLiPS By cultivating CLiPS after passaging, a master cell bank containing (primary) isolated CLiPS can be obtained.
  • CLiPS cells obtained by the process as described herein can be seeded in a cultivation vessel such as a cell culture plate.
  • CLiPS can, for this purpose, be suspended and cultured in any suitable medium, typically a maintenance medium for iPS cells such as commercially media mentioned above such as mTeSRl, StemMACSTM iPS-Brew XF, TeSRTM E8, mTeSRTMPlus, TeSRTM2 or mTeSRTMl, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium (ThermoFisher Scientific), StemFlex (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co. Inc), or PluriSTEM (Merck Millipore). Both CLiPS derived from CLMC and CliPS derived from CLEC can be cultivated in such a iPS maintenance medium.
  • a maintenance medium for iPS cells such as commercially media mentioned above such as mTeSRl, StemMACSTM iPS-Brew XF, TeSRTM E8, mTeSRTMPlus, TeSRTM2 or
  • the CLiPS cells (of both CLMC- and CLEC derived CLiPS) can be seeded at any suitable concentration, for example, or a concentration of about 0.5 x 10 6 cells/ml to about 5.0 x 10 6 cells/ml. In one example, the cells are suspended for subcultivation at a concentration of about 1.0 x 10 6 cells/ml.
  • the subculturing can be carried by cultivation either in simple culture flasks but also, for example, in a multilayer system such as CellSTACK (Corning, NY, USA) or Cell Factory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA) that can be stacked in incubators.
  • the subculturing can also be carried out in a closed self-contained system such as a bioreactor.
  • a bioreactor Different designs of bioreactors are known to the person skilled in the art, for example, parallelplate, hollow-fiber, or micro-fluidic bioreactors. See, for example, Sensebe et al. “Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra.
  • Quantum® Cell Expansion System (Terumo BCT, Inc), that has, for example, been used for the expansion of bone marrow mesenchymal stem cells for clinical trials (cf., Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058) and for the expansion of the highly pure cord ling mesenchymal stem cell population described in International Patent Application WO 2018/067071.
  • Another example of commercially available bioreactors that can be used for the subculturing of the CLiPS population of the present invention is the Xuri Cell Expansion System available from GE Healthcare.
  • the cultivation of the CLiPS population in an automated system such as the Quantum® Cell Expansion System is of particular benefit if a working cell bank for therapeutic application is to be produced under GMP conditions and a high number of cells is wanted.
  • CLiPS can be cultured till a suitable amount of cells have grown.
  • CLiPS are subcultivated till the CLiPS reach about 70% to about 80% confluency.
  • the isolation/cultivation of the population of CLiPS can be carried out under standard condition for the cultivation of mammalian cells.
  • the cells are harvested by removing them from the cultivation vessel used for the subcultivation.
  • the CLiPS harvesting is typically carried out by enzymatic treatment.
  • the isolated CLiPS are subsequently collected and are either be directly used or preserved for further use.
  • preserving is carried out by cryo-preservation.
  • cryo-preservation is used herein in its regular meaning to describe a process where here CLiPS are preserved by cooling to low sub-zero temperatures, such as (typically) -80°C or- 196°C (the boiling point of liquid nitrogen).
  • Cryopreservation can be carried out as known to the person skilled in the art and can include the use of cryo-protectors such as dimethylsulfoxide (DMSO) or glycerol, which slow down the formation of ice-crystals in the CLiPS cells.
  • cryo-protectors such as dimethylsulfoxide (DMSO) or glycerol
  • the present invention is also directed to CLiPS obtainable by the method as described herein and to CLiPS obtained by the method as described herein.
  • CLiPS obtainable/obtained by the present invention may grow and proliferate robustly (cf. Example 2 and Example 3). Thereby, CLiPS cultivation may be more efficient in comparison to a cultivation of iPS derived from, for example, the bone marrow stroma, fat tissue, the dermis or the Wharton's jelly.
  • Analysis of CLiPS functionality reveals expression of human embryonic stem cell markers indicating self-renewal properties and a normal karyotype (cf. Example 4 and Example 5).
  • CLiPS are capable to differentiate into multiple cell types (functional target cells) in vitro and in vivo indicating pluripotency (cf. Example 6). Therefore, CLiPS are highly suitable for medical and therapeutic applications. Consequently, the present invention is also directed to a pharmaceutical composition comprising an iPS obtainable/obtained by the method described herein.
  • the present invention is further directed to a method of differentiating a CLiPS into a target cell under conditions suitable for differentiation.
  • a suitable target cell include, but are by no means limited to, a neuronal cell, dopaminergic neuronal cell, an oligodentrocyte, an astrocyte, a cortical neuron, a hepatocyte, a cartilage cell, a muscle cell, a bone cell, a dental cell, a hair follicle cell, an inner ear hair cell, a skin cell, a melanocyte, a cardiomyocyte, a hematopoietic progenitor cell, a blood cell, an immune cell, a T- or B -lymphocyte, a microglia, a natural killer cell or a motor neuron, to mention only a few.
  • the CLiPS may be exposed to a priming substance, typically under conditions that are known to the skilled artesian from the differentiation of iPS derived from other sources into the target cell.
  • the exposure may be carried out under suitable conditions, which may comprise a cultivation in a cell culture vessel filled with a cell culture medium suitable for priming the CLiPS differentiation and for subsequent cultivation.
  • any cell culture medium suitable for priming, proliferating and differentiating iPS can be used, wherein the medium composition and thus the method of differentiation may depend on the target cell and may be taken from known protocols for the differentiation of iPS into the desired target cell (see in this respect, the reviews of Hirschi et al “Induced Pluripotent Stem Cells for Regenerative Medicine” Annu Rev Biomed Eng. 2014 July 11; 16: 277-294) or Shi et al “Induced pluripotent stem cell technology: a decade of progress” Nat Rev Drug Discov. 2017 February; 16(2): 115-130).
  • CEiPS may be cultivated in a medium adapted for proliferation and differentiation of the CLiPS into a dopaminergic neuronal cell.
  • the medium may be a Neurobasal medium supplemented with a growth factor such as B-27 minus vitamin A, transforming growth factor 3-P (TGFP3), a glial cell line-derived neurotrophic factor (GDNF), a brain-derived neurotrophic factor (BDNF), ascorbic acid, dibutyl cAMP, an inhibitor for glycogen synthase kinase 3 such as CHIR99021 and a y-secretase inhibitor such as (2S)-N-[(3,5-Difluorophenyl)acetyl]-E-alanyl-2-phenyl]glycine 1,1 -dimethylethyl ester (DAPT), which induces neuronal differentiation.
  • a growth factor such as B-27 minus vitamin A, transforming growth factor 3-P (TGFP3), a glial cell line-derived neurotrophic factor (GDNF), a brain-derived neurotrophic factor (BDNF), ascorbic acid, dibutyl cAMP
  • CLiPS differentiation into a dopaminergic neuronal cell is exemplary shown in Example 7.
  • CLiPS may be cultivated in a medium adapted for proliferation and differentiation of the CLiPS into a hepatocyte.
  • the medium may be a protein, lipid and growth factor-free medium supplemented with a compound inducing differentiation into a mesoendodermal fate.
  • RPMI 1640-B27 supplemented with Activin A may be an illustrative example for a suitable medium for CLiPS differentiation into a hepatocyte.
  • a CLiPS differentiation into a hepatocyte is exemplary shown in Example 8.
  • CLiPS may be cultivated in a medium adapted for proliferation and differentiation of the CLiPS into a cardiomyocyte.
  • the medium may be a protein, lipid and growth factor-free medium supplemented with an inhibitor for glycogen synthase kinase 3 such as CHIR99021.
  • RPMI/2%-B27 minus insulin may be an example for a suitable medium for CLiPS differentiation into a hepatocyte.
  • a CLiPS differentiation into a cardiomyocyte is exemplary shown in Example 9.
  • CLiPS may be differentiated into an oligodendrocyte using a chemically defined, growth factor-rich medium allowing a differentiation into paired box 6-positive (PAX6+) neural stem cells, which then give rise to oligodendrocyte transcription factor positive (OLIG2+) progenitors (cf. Example 10).
  • PAX6+ paired box 6-positive neural stem cells
  • OLIG2+ oligodendrocyte transcription factor positive
  • the differentiation of CLiPS into target cells may also be carried out under conditions suitable for cGMP production.
  • the present invention also includes a pharmaceutical composition comprising a differentiated CLiPS obtained by the method as described herein.
  • a pharmaceutical composition comprising differentiated CLiPS is an injection solution or any kind of graft suitable for implanting the differentiated CLiPS.
  • a graft may comprise differentiated CLiPS-derived multilayered tissue such as an organ or parts thereof.
  • the graft suitable for implanting the differentiated CLiPS may comprise an implantable matrix coated with differentiated CLiPS.
  • the pharmaceutical composition may be formulated/adapted for parenteral application.
  • the parenteral application may comprise a sterile preparation intended for injection, infusion or implantation in the human or animal body.
  • Transplantation of CLiPS-derived dopaminergic neurons in fully immunocompetent mice and rat Parkinson’s Disease models exhibited functional engraftment and even significant restoration of dopamine reuptake function (cf. Example 12 and Example 13).
  • the present invention further includes a method of treating a congenital or acquired degenerative disorder in a subject, wherein the subject may be selected from the group comprising a mouse, a rat, a rabbit, a pig, a dog, a cat, a non-human primate or a human.
  • the subject is human.
  • treating may comprise administering to a subject a target cell differentiated from CLiPS by the method as described herein.
  • the disease may any known disease which has been considered to be treated by means of cell-based therapy, see in this context, for example, Shi et al “Induced pluripotent stem cell technology: a decade of progress” supra.
  • the congenital or acquired degenerative disorder may have different origins.
  • such a congenital or acquired degenerative disorder may be a neural disorder such as, for example, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), Spinocerebellar ataxia (SCA) and batten disease.
  • a hepatic degenerative disorder may be inter alia liver failure, liver cirrhosis and viral hepatitis.
  • the congenital or acquired degenerative disorder may also be a cardiac disorder, inter alia comprising acute Danon disease, short-QT syndrome, Brugada syndrome, myocardial infarction, Jervell and Lange-Nielsen syndrome.
  • the disorder may also be an auto-immune disease such as multiple sclerosis.
  • the present invention is also directed to extracellular membranous vesicles that may be produced by CLiPS or the differentiated derivatives of CLiPS.
  • vesicles may include but not exclusively, vesicles ranging from 30 to 150 nanometres (nm) in diameter, also known as exosomes.
  • exosomes are now known to be involved in various important biological processes such as cell-cell communication, cellular senescence, proliferation, and differentiation, tissue homeostasis, tissue repair and regeneration, antigen presentation and immune modulation (see, for example, Pegtel, D.M. and S.J. Gould, Exosomes. Annu Rev Biochem, 2019. 88: p.
  • Exosomes have been implicated in a broad range of diseases including cancers (see, for example, Visan, K.S., R.J. 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.
  • 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 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 A.F. Hill, The role of extracellular vesicles in neurodegenerative diseases.
  • AD Alzheimer’s disease
  • PD Parkinson’s disease
  • prion disease prion disease
  • ALS amyotrophic lateral sclerosis
  • Exosome cargoes have been shown to consist of various biomolecules including proteins, lipids and nucleic acids.
  • RNA species such as tRNA, mRNA, IncRNA, circular RNA and miRNA can potentially regulate gene expression in target cells and tissue.
  • Exosomes produced by certain cell types have been shown to possess therapeutic properties.
  • mesenchymal stem cells (MSCs) isolated from different sources such as bone marrow, adipose tissue, and umbilical cord have emerged as particularly favourable.
  • MSC-derived exosomes to have shown potential therapeutic effects in animal models of cornea, cardiovascular, Alzheimer’s, Parkinson’s and inflammatory bowel diseases, among others.
  • pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) have been shown to produce exosomes (Song, Y.H., 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.
  • iPS-derived exosomes are considered to be safer than iPS-derived cells due to the risk of tumour formation from residual undifferentiated cells (Riazifar, M., et al., Stem Cell Extracellular Vesicles: Extended Messages of Regeneration. Annu Rev Pharmacol Toxicol, 2017. 57: p. 125-154).
  • therapeutic properties have also been demonstrated for exosomes isolated from differentiated derivatives of iPS.
  • treatment with exosomes purified from iPS -derived cardiomyocytes enhanced cardiac recovery in mouse model of myocardial infarction, with significant reduction in apoptosis and fibrosis compared to untreated animals.
  • exosomes from iPS-derived MSCs exosomes isolated from iPS- derived MSCs accelerated the proliferation of human dermal fibroblasts and human keratinocytes, and enhanced wound healing in in vitro scratch assays. There was no significant difference in the effects of these exosomes compared to those isolated from primary MSCs ( Kim, S., et al., Exosomes Secreted from Induced Pluripotent Stem Cell-Derived
  • extracellular membranous vesicles or exosomes produced by CLiPS are considered useful for the treatment of diseases including the above-mentioned exemplary disease such as cancer, osteoarthritis, diseases of the central nervous system such as such as stroke, Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease, and amyotrophic lateral sclerosis (ALS), mental disorders or metabolic diseases.
  • diseases including the above-mentioned exemplary disease such as cancer, osteoarthritis, diseases of the central nervous system such as such as stroke, Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease, and amyotrophic lateral sclerosis (ALS), mental disorders or metabolic diseases.
  • diseases including the above-mentioned exemplary disease such as cancer, osteoarthritis, diseases of the central nervous system such as such as stroke, Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease, and amyotrophic lateral sclerosis (ALS), mental disorders or metabolic diseases.
  • AD Alzheimer’s
  • exosomes are actively pursued as delivery carriers for facilitating cellular uptake of various therapeutic agents such as microRNA, drugs, and peptides (see Antimisiaris, S.G., 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.
  • extracellular membranous vesicles or exosomes produced by CLiPS can also be uses as delivery carriers for facilitating cellular uptake of therapeutic agents. Accordingly, the invention also encompasses the use of CLiPS or the differentiated derivatives of CLiPS for the purpose of delivery of exogenously loaded or transgenically expressed molecules.
  • Extracellular membranous vesicles and exosomes produced by CLiPS can be isolated using respective methods described in the literature. Typically, exosomes are purified from the extracellular milieu into which they are secreted. Known methods for the isolation of exosomes include ultracentrifugation, ultrafiltration, size-exclusion chromatography, field-flow fractionation, polymer coprecipitation, immunoaffinity, microfluidics, or acoustic nanofilter. All these methods can be used for the isolation of exosomes produced by CLiPS or the differentiated derivatives of CLiPS described here.
  • the present invention further relates a particular method of differentiating an iPS cell, which is derived from a stem cell of the amniotic membrane of the umbilical cord as defined elsewhere herein and which refers to CLiPS as also defined herein, into a RPE cell.
  • Said differentiation method comprises culturing said CLiPs in a differentiation medium under conditions suitable for the differentiation into a RPE cell.
  • a “retinal pigment epithelial (short: RPE) cell” refers to a cell derived from / from / taken from the retinal pigment epithelium. In other words, such cell is comprised by said retinal pigment epithelium and will be defined in more detail below.
  • RPE differentiation from CLiPs was achieved using the rapid, directed and modified differentiation method according to the present invention.
  • the CLiPS as used herein for differentiating into a RPE cell may be derived from umbilical cord-lining mesenchymal cells (such as CLMC23, CLMC30, CLMC44) and/or from umbilical cord-lining ectodermal cells (such as CLEC23).
  • the CLiPS as used herein for differentiating into a RPE cell is any one of CLMC23, CLMC30, CLMC44 or CLEC23.
  • the CLiPS as used herein for differentiating into a RPE cell is CLMC23.
  • the CLiPS as used herein for differentiating into a RPE cell is CLMC30.
  • the CLiPS as used herein for differentiating into a RPE cell is CLMC44.
  • the CLiPS as used herein for differentiating into a RPE cell is CLEC23.
  • a RPE cell differentiated from a CLiPS as defined herein by the differentiation method as described herein may refer to a CLiPS-derived RPE cell or to a CLiPS-RPE.
  • the differentiation of a RPE cell derived from CLiPs may be compared to the differentiation of a RPE cell derived from a ES cell such as H9 ES cell (also called ES derived RPE when referring to such RPE cell) and/or to the differentiation of a RPE cell derived from an iPS cell derived from the skin (also called skin iPS; thus skin iPS derived RPE when referring to such RPE cell) such as Asf5, AGO, or HDFA cell using the differentitation method according to the present invention (see the Example section).
  • the differentiation medium as used in the differentiation method of differentiating an iPS cell into a RPE cell comprising culturing iPS cells derived from a stem cell of the amniotic membrane of the umbilical cord is preferably a DMEM (Dulbecco’s modified eagle medium) medium as defined herein comprising N2 supplement, B27 supplement and non-essential amino acid (NEAA), even more preferably a DMEM (Dulbecco’s modified eagle medium) / F12 (Ham’s F12 medium) medium as defined elsewhere herein comprising N2 supplement, B27 supplement and non-essential amino acid (NEAA).
  • the DMEM/F12 medium used in the differentiation method of differentiating an iPS cell into a RPE cell comprises lx N2 supplement, lx B27 supplement, and lx NEAA.
  • lx N2 supplement, lx B27 supplement and lx NEAA in said medium means that the final concentration is lx as can also be seen in the embodiment below.
  • the differentiation medium, preferably DMEM medium, even more preferably DMEM/F12 medium as defined herein, is obtained by mixing to obtain a final volume of 1000 ml culture medium:
  • the differentiation medium as defined herein may further comprise / being supplemented with various growth factors and/or cytokines as defined elsewhere herein.
  • Such differentiation medium as defined above may refer to a base medium for iPS culturing.
  • Such base differentiation medium may then further be modified / supplemented for culturing iPS cells as defined herein in order for said cells to differentiate into RPE cells by using the method of differentiating an iPS cell into a RPE cell according to the present invention.
  • the differentiation medium used in the method of differentiating an iPS cell into a RPE cell according to the present invention for culturing iPS cells so that said cells differentiate into RPE cells may comprise a first differentiation medium additionally comprising at least any one of IGF1, DKK1, nicotinamide or LDN-193189.
  • Said first differentiation medium is based on said base medium comprising DMEM medium, preferably DMEM/F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM/F12 medium comprising lx N2 supplement, lx B27 supplement and lx NEAA.
  • the first differentiation medium as defined herein additionally comprises IGF1, DKK1, nicotinamide and LDN-193189.
  • the differentiation medium used in the method of differentiating an iPS cell into a RPE cell according to the present invention for culturing iPS cells so that said cells differentiate into RPE cells may additionally or alternatively comprise a second differentiation medium additionally comprising at least any one of IGF1, DKK1, nicotinamide, LDN-193189 or b-FGF.
  • Said second differentiation medium is also based on said base medium comprising DMEM medium, preferably DMEM/F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM/F12 medium comprising lx N2 supplement, lx B27 supplement and lx NEAA.
  • the second differentiation medium as defined herein additionally comprises IGF1, DKK1, nicotinamide, LDN-193189 and b-FGF.
  • the differentiation medium used in the method of differentiating an iPS cell into a RPE cell according to the present invention for culturing iPS cells that said cells differentiate into RPE cells may additionally or alternatively comprise a third differentiation medium additionally comprising at least any one of IGF1, DKK1, or Activin A.
  • Said third differentiation medium is also based on said base medium comprising DMEM medium, preferably DMEM/F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM/F12 medium comprising lx N2 supplement, lx B27 supplement and lx NEAA.
  • the third differentiation medium as defined herein additionally comprises IGF1, DKK1 and Activin A.
  • the differentiation medium used in the method of differentiating an iPS cell into a RPE cell according to the present invention for culturing iPS cells that said cells differentiate into RPE cells may additionally or alternatively comprise a fourth differentiation medium additionally comprising Activin A and SU5402 or Activin A and PD17307.
  • Said fourth differentiation medium is also based on said base medium comprising DMEM medium, preferably DMEM/F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM/F12 medium comprising lx N2 supplement, lx B27 supplement and lx NEAA.
  • the fourth differentiation medium as defined herein additionally comprises Activin A and PD17307.
  • PD17307 is applied in lower concentrations compared to the fibroblast growth factor inhibitor SU5402 which reduces undesirable changes in gene exypression caused by the application of a higher concentration when SU5402 is used, PD17307 is preferred by the differentiation method of the present invention ( Figure 19).
  • the differentiation medium used in the method of differentiating an iPS cell into a RPE cell according to the present invention for culturing iPS cells that said cells differentiate into RPE cells may additionally or alternatively comprise a fifth differentiation medium additionally comprising at least any one of Activin A, CHIR99021 or SU5402; or comprising at least any one of Activin A, CHIR99021 or PD17307.
  • Said fifth differentiation medium is also based on said base medium comprising DMEM medium, preferably DMEM/F12 medium comprising N2 supplement, B27 supplement and NEAA, even more preferably DMEM/F12 medium comprising lx N2 supplement, lx B27 supplement and lx NEAA.
  • the fifth differentiation medium as defined herein additionally comprises Activin A, CHIR99021 and PD17307 (using PD17307 for the same reasons as defined above).
  • the fifth differentiation medium preferably comprises Activin A, SU5402 or PD17307, preferably PD17307, and a first concentration of CHIR99021, which is below 3 pM.
  • the concentration of CHIR99021, an activator of Wnt signalling pathway, is then increased, when the fifth differentiation medium is again used to culture iPS cells to differentiate into RPE cells.
  • the gradual increase of the concentration prevents excessive cell death caused by the high concentration of CHIR when used. This improved the yield of pigmented RPE cells.
  • the fifth differentiation medium is then subsequently applied it preferably comprises Activin A, SU5402 or PD17307, preferably PD17307, and a second concentration of CHIR99021, which is about 3 pM.
  • IGF1 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 10 ng/ml.
  • IGF1 is used in the first differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 ng/ml, of about 6 to about 14 ng/ml, of about 7 to about 13 ng/ml, of about 8 to about 12 ng/ml, of about 9 to about 11 ng/ml, most preferably in a final concentration of about 10 ng/ml.
  • IGF1 is used in the second differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 ng/ml, of about 6 to about 14 ng/ml, of about 7 to about 13 ng/ml, of about 8 to about 12 ng/ml, of about 9 to about 11 ng/ml, most preferably in a final concentration of about 10 ng/ml.
  • IGF1 is used in the third differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 ng/ml, of about 6 to about 14 ng/ml, of about 7 to about 13 ng/ml, of about 8 to about 12 ng/ml, of about 9 to about 11 ng/ml, most preferably in a final concentration of about 10 ng/ml.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein IGF1 is applied in said differentiation medium as 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 for about 6 days, even more preferably for about 6 consecutive days, most preferably used for about 6 (consecutive) days in a final concentration of about 10 ng/ml.
  • IGF1 When IGF1 is applied in the first differentiation medium as defined herein, IGF1 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 0 to day 2 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 ng/ml.
  • IGF1 When IGF1 is applied in the second differentiation medium as defined herein, IGF1 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 2 to day 4 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 ng/ml.
  • IGF1 When IGF1 is applied in the third differentiation medium as defined herein, IGF1 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 4 to day 6 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 ng/ml.
  • DKK1 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 10 ng/ml.
  • DKK1 is used in the first differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 ng/ml, of about 6 to about 14 ng/ml, of about 7 to about 13 ng/ml, of about 8 to about 12 ng/ml, of about 9 to about 11 ng/ml, most preferably in a final concentration of about 10 ng/ml.
  • DKK1 is used in the second differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 ng/ml, of about 6 to about 14 ng/ml, of about 7 to about 13 ng/ml, of about 8 to about 12 ng/ml, of about 9 to about 11 ng/ml, most preferably in a final concentration of about 10 ng/ml.
  • DKK1 is used in the third differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 ng/ml, of about 6 to about 14 ng/ml, of about 7 to about 13 ng/ml, of about 8 to about 12 ng/ml, of about 9 to about 11 ng/ml, most preferably in a final concentration of about 10 ng/ml.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein DKK1 is applied in said differentiation medium as 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 for about 6 days, even more preferably for about 6 consecutive days, most preferably used for about 6 (consecutive) days in a final concentration of about 10 ng/ml.
  • DKK1 When DKK1 is applied in the first differentiation medium as defined herein, DKK1 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 0 to day 2 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 ng/ml.
  • DKK1 When DKK1 is applied in the second differentiation medium as defined herein, DKK1 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 2 to day 4 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 ng/ml.
  • DKK1 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 4 to day 6 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 ng/ml.
  • nicotinamide When nicotinamide is applied in the differentiation medium as defined elsewhere herein, a final concentration of at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM; or within a range of about 5 to about 15 mM, of about 6 to about 14 mM, of about 7 to about 13 mM, of about 8 to about 12 mM, of about 9 to about 11 mM is used. In a preferred embodiment, nicotinamide is used in the differentiation medium as defined elsewhere herein in a final concentration of about 10 mM.
  • nicotinamide is used in the first differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 mM, of about 6 to about 14 mM, of about 7 to about 13 mM, of about 8 to about 12 mM, of about 9 to about 11 mM, most preferably in a final concentration of about 10 mM.
  • nicotinamide is used in the second differentiation medium as defined elsewhere herein in 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 within a range of about 5 to about 15 mM, of about 6 to about 14 mM, of about 7 to about 13 mM, of about 8 to about 12 mM, of about 9 to about 11 mM, most preferably in a final concentration of about 10 mM.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein nicotinamide is applied in said differentiation medium as defined herein for at least about 2 days, at least about 3 days, at least about 4 days, preferably for about 4 days, even more preferably for about 4 consecutive days, most preferably used for about 4 (consecutive) days in a final concentration of about 10 mM.
  • nicotinamide When nicotinamide is applied in the first differentiation medium as defined herein, nicotinamide is used for about 2 days, preferably for about 2 consecutive days, meaning on day 0 to day 2 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 mM.
  • nicotinamide When nicotinamide is applied in the second differentiation medium as defined herein, nicotinamide is used for about 2 days, preferably for about 2 consecutive days, meaning on day 2 to day 4 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 10 mM.
  • a final concentration of at least about 0.1 pM, at least about 0.2 pM, at least about 0.3 pM, at least about 0.4 pM, at least about 0.5 pM, at least about 0.6 pM, at least about 0.7 pM, at least about 0.8 pM, at least about 0.9 pM, or at least about 1 pM is used.
  • LDN-193189 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 1 pM.
  • LDN-193189 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 0.2 pM.
  • LDN-193189 is used in the first differentiation medium as defined elsewhere herein in a final concentration within a range of about 0.5 to about 1.5 pM, of about 0.6 to about 1.4 pM, of about 0.7 to about 1.3 pM, of about 0.8 to about 1.2 pM, of about 0.9 to about 1.1 pM, most preferably in a final concentration of about 1 pM.
  • LDN-193189 is used in the second differentiation medium as defined elsewhere herein in a final concentration within a range of about 0.1 to about 0.3 pM, of about 0.11 to about 0.29 pM, of about 0.12 to about 0.28 pM, of about 0.13 to about 0.27 pM, of about 0.14 to about 0.26 pM, of about 0.15 to about 0.25 pM, most preferably in a final concentration of about 0.2 pM.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein LDN-193189 is applied in said differentiation medium as defined herein for at least about 2 days, at least about 3 days, at least about 4 days, preferably for about 4 days, even more preferably for about 4 consecutive days, most preferably used for about 4 (consecutive) days in a concentration of at least about 0.1 pM as defined elsewhere herein.
  • LDN-193189 When LDN-193189 is applied in the first differentiation medium as defined herein, LDN-193189 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 0 to day 2 of the culture, even more preferably LDN-193189 is used for about 2 (consecutive) days in a concentration of about 1 pM.
  • LDN-193189 When LDN-193189 is applied in the second differentiation medium as defined herein, LDN-193189 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 2 to day 4 of the culture, even more preferably LDN-193189 is used for about 2 (consecutive) days in a concentration of about 0.2 pM.
  • b-FGF is used in the differentiation medium as defined elsewhere herein in a final concentration of about 5 ng/ml.
  • b-FGF is used in the second differentiation medium as defined elsewhere herein in a final concentration of at least about 2.5 ng/ml, at least about 3 ng/ml, at least about 3.5 ng/ml, at least about 4 ng/ml, at least about 4.5 ng/ml, or at least about 5 ng/ml; or within a range of about 2.5 to about 7.5 ng/ml, of about 3 to about 7 ng/ml, of about 3.5 to about 6.5 ng/ml, of about 4 to about 6 ng/ml, of about 4.5 to about 5.5 ng/ml, most preferably in a final concentration of about 5 ng/ml.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein b-FGF is applied in said differentiation medium as defined herein for at least about 1 day, at least about 2 days, preferably for about 2 days, even more preferably for about 2 consecutive days, most preferably used for about 2 (consecutive) days in a final concentration of about 5 ng/ml.
  • b-FGF When b-FGF is applied in the second differentiation medium as defined herein, b-FGF is used for about 2 days, preferably for about 2 consecutive days, meaning on day 2 to day 4 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 5 ng/ml.
  • Activin A When Activin A is applied in the differentiation medium as defined elsewhere herein, a final concentration of at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml; or within a range of about 50 to about 150 ng/ml, of about 60 to about 140 ng/ml, of about 70 to about 130 ng/ml, of about 80 to about 120 ng/ml, of about 90 to about 110 ng/ml is used. In a preferred embodiment, Activin A is used in the differentiation medium as defined elsewhere herein in a final concentration of about 100 ng/ml.
  • Activin A is used in the third differentiation medium as defined elsewhere herein in 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 within a range of about 50 to about 150 ng/ml, of about 60 to about 140 ng/ml, of about 70 to about 130 ng/ml, of about 80 to about 120 ng/ml, of about 90 to about 110 ng/ml, most preferably in a final concentration of about 100 ng/ml.
  • Activin A is used in the fourth differentiation medium as defined elsewhere herein in 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 within a range of about 50 to about 150 ng/ml, of about 60 to about 140 ng/ml, of about 70 to about 130 ng/ml, of about 80 to about 120 ng/ml, of about 90 to about 110 ng/ml, most preferably in a final concentration of about 100 ng/ml.
  • Activin A is used in the fifth differentiation medium as defined elsewhere herein in 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 within a range of about 50 to about 150 ng/ml, of about 60 to about 140 ng/ml, of about 70 to about 130 ng/ml, of about 80 to about 120 ng/ml, of about 90 to about 110 ng/ml, most preferably in a final concentration of about 100 ng/ml.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein Activin A is applied in said differentiation medium as 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, at least about 8 days, at least about 10 days, at least about 12 days, preferably for about 12 days, even more preferably for about 12 consecutive days, most preferably used for about 12 (consecutive) days in a final concentration of about 100 ng/ml.
  • Activin A When Activin A is applied in the third differentiation medium as defined herein, Activin A is used for about 2 days, preferably for about 2 consecutive days, meaning on day 4 to day 6 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 100 ng/ml.
  • Activin A When Activin A is applied in the fourth differentiation medium as defined herein, Activin A is used for about 2 days, preferably for about 2 consecutive days, meaning on day 6 to day 8 of the culture, even more preferably used for about 2 (consecutive) days in a final concentration of about 100 ng/ml.
  • Activin A When Activin A is applied in the fifth differentiation medium as defined herein, Activin A is used for about 8 days, preferably for about 8 consecutive days, meaning on day 8 to day 16 of the culture, even more preferably used for about 8 (consecutive) days in a final concentration of about 100 ng/ml.
  • SU5402 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 10 pM.
  • SU5402 is used in the fourth differentiation medium as defined elsewhere herein in a final concentration of at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, or at least about 10 pM; or within a range of about 5 to about 15 pM, of about 6 to about 14 pM, of about 7 to about 13 pM, of about 8 to about 12 pM, of about 9 to about 11 pM, most preferably in a final concentration of about 10 pM.
  • SU5402 is used in the fifth differentiation medium as defined elsewhere herein in a final concentration of at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, or at least about 10 pM; or within a range of about 5 to about 15 pM, of about 6 to about 14 pM, of about 7 to about 13 pM, of about 8 to about 12 pM, of about 9 to about 11 pM, most preferably in a final concentration of about 10 pM.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein SU5402 is applied in said differentiation medium as defined herein for about 10 days, even more preferably for about 10 consecutive days, most preferably used for about 10 (consecutive) days in a concentration of about 10 pM as defined elsewhere herein.
  • SU5402 is applied in the fourth differentiation medium as defined herein, SU5402 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 6 to day 8 of the culture, even more preferably SU5402 is used for about 2 (consecutive) days in a concentration of about 10 pM.
  • SU5402 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, meaning on day 8 to day 16 of the culture, even more preferably used for about 8 (consecutive) days in a final concentration of about 10 pM.
  • PD17307 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 1 pM. In an even more preferred embodiment, PD17307 is used in the fourth differentiation medium as defined elsewhere herein in a final concentration of at least about 0.5 pM, at least about 0.6 pM, at least about 0.7 pM, at least about 0.8 pM, at least about 0.9 pM, or at least about 1 pM; or within a range of about 0.5 to about 1.5 pM, of about 0.6 to about 1.4 pM, of about 0.7 to about
  • PD17307 is used in the fifth differentiation medium as defined elsewhere herein in a final concentration of at least about 0.5 pM, at least about 0.6 pM, at least about 0.7 pM, at least about 0.8 pM, at least about 0.9 pM, or at least about 1 pM; or within a range of about 0.5 to about 1.5 pM, of about 0.6 to about 1.4 pM, of about 0.7 to about 1.3 pM, of about 0.8 to about 1.2 pM, of about 0.9 to about 1.1, most preferably in a final concentration of about 1 pM.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein PD 17307 is applied in said differentiation medium as defined herein for about 10 days, even more preferably for about 10 consecutive days, most preferably used for about 10 (consecutive) days in a concentration of about 1 pM as defined elsewhere herein.
  • PD17307 is applied in the fourth differentiation medium as defined herein, PD17307 is used for about 2 days, preferably for about 2 consecutive days, meaning on day 6 to day 8 of the culture, even more preferably PD17307 is used for about 2 (consecutive) days in a concentration of about 1 pM.
  • PD17307 When PD 17307 is applied in the fifth differentiation medium as defined herein, PD17307 is used for about 8 days, preferably for about 8 consecutive days, meaning on day 8 to day 16 of the culture, even more preferably used for about 8 (consecutive) days in a final concentration of about 1 pM.
  • CHIR99021 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 1.5 pM.
  • CHIR99021 as used in the differentiation medium as defined herein may be applied for culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell, for about 3 consecutive culture days.
  • CHIR99021 is used in the differentiation medium as defined elsewhere herein in a final concentration of about 1.5 pM for culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell, for about 3 consecutive culture days.
  • CHIR99021 is used in the fifth differentiation medium as defined elsewhere herein in a final concentration of at least about 1 pM and less than about 3 pM, of at least about 1.1 pM and less than about 3 pM, of at least about 1.2 pM and less than about 3 pM, of at least about 1.3 pM and less than about 3 pM, of at least about 1.4 pM and less than about 3 pM, of at least about 1 pM and less than about 2.5 pM, of at least about 1 pM and less than about 2 pM, of at least about 1 pM and less than about 1.9 pM, of at least about 1 pM and less than about 1.8 pM, of at least about 1 pM and less than about 1.7 pM, of at least about 1 pM and less than about 1.6 pM, most preferably in a final concentration of about 1.5 pM.
  • CHIR99021 is used in the fifth differentiation medium as defined elsewhere herein in a final concentration of at least about 1 pM and less than about 3 pM, of at least about 1.1 pM and less than about 3 pM, of at least about 1.2 pM and less than about 3 pM, of at least about 1.3 pM and less than about 3 pM, of at least about 1.4 pM and less than about 3 pM, of at least about 1 pM and less than about 2.5 pM, of at least about 1 pM and less than about 2 pM, of at least about 1 pM and less than about 1.9 pM, of at least about 1 pM and less than about 1.8 pM, of at least about 1 pM and less than about 1.7 pM, of at least about 1 pM and less than about 1.6 pM for culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell
  • CHIR99021 is applied to the differentiation medium, preferably to the fifth differentiation medium, as defined above, CHIR99021 is again applied in the differentiation medium for subsequently culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell, preferably subsequently culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell, for about 5 consecutive culture days.
  • CHIR99021 is then used in a final concentration of about 3 pM for subsequently culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell, even more preferably subsequently culturing the cell, namely the iPS cell of the invention which differentiates or has already been differentiated into a RPE cell, for about 5 consecutive culture days (meaning on day 11 to day 16 of the culture).
  • the yield of pigmented RPE cells improves.
  • the present invention further comprises the method of differentiating into a RPE cell, the method comprising culturing said iPS cell in a differentiation medium as defined elsewhere herein, wherein CHIR99021 is used for about 8 culture days, preferably for about 8 consecutive culture days, even more preferably wherein during the first 3 (consecutive) days of the about 8 days of culturing with CHIR99021, CHIR99021 is used in a final concentration of at least about 1 pM and less than about 3 pM, of at least about 1.1 pM and less than about 3 pM, of at least about 1.2 pM and less than about 3 pM, of at least about 1.3 pM and less than about 3 pM, of at least about 1.4 pM and less than about 3 pM, of at least about 1 pM and less than about 2.5 pM, of at least about 1 pM and less than about 2 pM, of at least about 1 pM and less than about 1.9 pM, of
  • the differentiation medium comprises a first differentiation medium comprising about 1 pM LDN-193189, about 10 ng/ml DKK1, about 10 ng/ml IGF1 and about 10 m nicotinamide.
  • the differentiation medium comprises a second differentiation medium comprising about 0.2 pM LDN-193189, about 10 ng/ml DKK1, about 10 ng/ml IGF1, about 10 mM nicotinamide and about 5 ng/ml b-FGF.
  • 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.
  • the differentiation medium comprises a fourth differentiation medium comprising about 100 ng/ml Activin A and about 10 pM SU5402, preferably comprising about 100 ng/ml Activin A and about 1 pM PD17307.
  • the differentiation medium comprises a fifth differentiation medium comprising about 100 ng/mL Activin A, about 10 pM SU5402, and about 1.5 pm CHIR99021, preferably comprising about 100 ng/mL Activin A, about 1 pM PD17307, and about 1.5 pm CHIR99021.
  • the differentiation medium comprises another fifth differentiation medium being applied after the first fifth differentiation medium has been applied in the differentiation method comprising about 100 ng/mL Activin A, about 10 pM SU5402, and about 3 pM CHIR99021, preferably comprising about 100 ng/mL Activin A, about 1 pM PD17307, and about 3 pM CHIR99021.
  • the iPS cell in the differentiation method when culturing the iPS cell in the differentiation method, it comprises culturing for about 2 days in the first differentiation medium as defined elsewhere herein, preferably for about 2 consecutive days in the first differentiation medium as defined elsewhere herein. This means that the iPS cell was first exposed to the first differentiation medium as defined herein from day 0 to day 2.
  • the iPS cell in the method of the invention when culturing the iPS cell in the method of the invention, it comprises culturing for about 2 days in the second differentiation medium as defined elsewhere herein, preferably it comprises culturing for about 2 days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the second differentiation medium as defined elsewhere herein, even more preferably it comprises culturing for about 2 consecutive days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the second differentiation medium as defined elsewhere herein.
  • the iPS cell may be exposed to the second differentiation medium from day 2 to day 4.
  • the iPS cell in the method of the invention when culturing the iPS cell in the method of the invention, it comprises culturing the iPS cell for about 2 days in the third differentiation medium as defined elsewhere herein, preferably it comprises culturing the iPS cell for about 2 days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the second differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the third differentiation medium as defined elsewhere herein, even more preferably it comprises culturing the iPS cell for about 2 consecutive days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the second differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the third differentiation medium as defined elsewhere herein.
  • the iPS cell may be exposed to the third differentiation medium from day 4 to day 6.
  • the iPS cell in the method of the invention when culturing the iPS cell in the method of the invention, it comprises culturing the iPS cell for about 2 days in the fourth differentiation medium as defined elsewhere herein, preferably it comprises culturing the iPS cell for about 2 days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the second differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the third differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the fourth differentiation medium as defined elsewhere herein, even more preferably it comprises culturing the iPS cell for about 2 consecutive days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the second differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the third differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the fourth differentiation medium as defined elsewhere herein.
  • the iPS cell may be exposed to the fourth differentiation medium from day 6 to day 8.
  • it comprises culturing for about 8 days in the fifth differentiation medium as defined elsewhere herein, preferably it comprises culturing for about 2 days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the second differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the third differentiation medium as defined elsewhere herein, subsequently culturing for about 2 days in the fourth differentiation medium as defined elsewhere herein, and subsequently culturing for about 8 days in the fifth differentiation medium as defined elsewhere herein, even more preferably it comprises culturing for about 2 consecutive days in the first differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive days in the second differentiation medium as defined elsewhere herein, subsequently culturing for about 2 consecutive
  • the iPS cell in the method of the invention when culturing the iPS cell in the method of the invention, it comprises culturing for about 4 days in the fifth differentiation medium as defined elsewhere herein comprising CHIR99021 used in a concentration of at least about 1 pM and less than about 3 pM as defined elsewhere herein, followed by subsequently culturing for another about 4 days in the fifth differentiation medium comprising CHIR99021 used in a concentration of about 3 pM, most preferably it comprises culturing for about 2 days in the first differentiation medium, subsequently culturing for about 2 days in the second differentiation medium, subsequently culturing for about 2 days in the third differentiation medium, subsequently culturing for about 2 days in the fourth differentiation medium, and subsequently culturing for about 4 days in the fifth differentiation medium comprising CHIR99021 used in a concentration of at least about 1 pM and less than about 3 pM as defined elsewhere herein, followed by subsequently culturing for another about 4 days in the fifth differentiation medium comprising
  • days for culturing in a particular medium may also be exchanged with the term “consecutive days”.
  • the cell may be exposed to the fifth differentiation medium comprising CHIR99021 used in a concentration of at least about 1 pM and less than about 3 pM as defined elsewhere herein from day 8 to day 11, followed by exposing the cells on day 11 to the fifth differentiation medium comprising CHIR99021 used in a concentration of about 3 pM from day 11 to day 16.
  • the present invention thus also comprises the method of differentiating an iPS cell into a RPE cell, wherein the iPS cell is cultured in the differentiation medium in total for about 11 to about 21 days, for about 12 to about 20 days, for about 13 to about 19 days, for about 14 to about 18 days, for about 15 to about 17 days, preferably for about 16 days in total, even more preferably for about 11 to about 21 consecutive days, for about 12 to about 20 consecutive days, for about 13 to about 19 consecutive days, for about 14 to about 18 consecutive days, for about 15 to about 17 consecutive days, most preferably for about 16 consecutive days.
  • the method of differentiating an iPS cell into a RPE cell further preferably comprises culturing the iPS cell in a mTESRl medium before culturing the iPS cell in the differentiation medium as defined herein.
  • CLiPs and as a reference cell line human ES cells may be grown on Matrigel coated tissue culture plate in mTeSRl medium. When the cells attain about 90 to about 95% confluence, they are then exposed to the differentiation medium as defined elsewhere herein, preferably to the first, even more preferably to the first followed by the second, third, fourth and fifth differentiation medium as defined herein.
  • the method of differentiating an iPS cell into a RPE cell further comprises culturing the iPS cell in a mTESRl medium for about 1 to about 4 culture days before culturing the iPS cell in the differentiation medium as defined herein, more preferably before culturing the iPS cell in the first differentiation medium as defined herein, most preferably before culturing the iPS cell in the first differentiation medium as defined herein, followed by the second, third, fourth and fifth differentiation medium as defined herein.
  • the method of differentiating an iPS cell into a RPE cell preferably further comprises culturing the RPE cell in a retinal pigment epithelial maintenance (short: RPEM) medium.
  • RPEM retinal pigment epithelial maintenance
  • the differentiation medium may be replaced by RPEM medium as will be defined below.
  • culturing RPE cell in said RPEM medium may start after day 16 of the culture (in particular after culturing the cells in the fifth differentiation medium as defined elsewhere herein).
  • said RPEM medium of the differentiation method of the invention comprises about 50 % DMEM/F12 and about 50 % minimum essential medium (MEM) comprising 0.5x N1 supplement and lx NEAA.
  • said RPEM medium of the differentiation method of the invention further comprises at least any one of a heat-inactivated fetal bovine serum (FBS), Glutamax, taurine, hydrocortisone, 3,3',5-Triiodo-L-thyronine, penicillin/streptomycin, nicotinamide, or sodium pyruvate.
  • said RPEM medium of the differentiation method of the invention further comprises a heat-inactivated fetal bovine serum (FBS), Glutamax, taurine, hydrocortisone, 3,3',5-Triiodo-L-thyronine, penicillin/streptomycin, nicotinamide, and sodium pyruvate.
  • FBS heat-inactivated fetal bovine serum
  • said RPEM medium of the differentiation method of the invention further comprises about 2 % heat-inactivated fetal bovine serum (FBS), lx Glutamax, about 0.25 mg/mL taurine, about 0.02 pg/mL hydrocortisone, about 0.013 ng/mL 3,3',5-Triiodo-L- thyronine, lx penicillin/streptomycin, about 10 rnM nicotinamide and lx sodium pyruvate.
  • FBS heat-inactivated fetal bovine serum
  • lx Glutamax about 0.25 mg/mL taurine
  • hydrocortisone about 0.02 pg/mL hydrocortisone
  • lx penicillin/streptomycin about 10 rnM nicotinamide and lx sodium pyruvate.
  • the present invention also comprises said differentiation method as defined elsewhere herein, wherein the RPE cell is cultured in said RPEM medium as defined elsewhere herein for about 9 to about 29 days, for about 10 to about 28 days, for about 11 to about 27 days, for about 12 to about 26 days, for about 13 to about 25 days, for about 14 to about 24 days, for about 15 to about 23 days, for about 16 to about 22 days, for about 17 to about 21 days, for about 18 to about 20 days, preferably for about 19 days, even more preferably for about 19 consecutive days.
  • the RPEM medium may be changed every about 2 to about 3 days during the culturing of said RPE cells in said medium, preferably every about 2 to about 3 days during the culturing of said RPE cells for about 9 to about 29 days, for about 10 to about 28 days, for about 11 to about 27 days, for about 12 to about 26 days, for about 13 to about 25 days, for about 14 to about 24 days, for about 15 to about 23 days, for about 16 to about 22 days, for about 17 to about 21 days, for about 18 to about 20 days, more preferably during the culturing of said RPE cells for about 19 days, even more preferably for about 19 consecutive days.
  • the present invention also comprises said differentiation method as defined elsewhere herein, wherein culturing the iPS cell in the differentiation medium as defined herein and culturing the RPE cell in the 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, in particular having about 16 days of culturing iPS cells in the differentiation medium as defined elsewhere herein and having about 19 days of culturing the differentiated RPE cells in the RPEM medium as defined elsehwhere herein.
  • the present invention also comprises said differentiation method as defined elsewhere herein further preferably comprising purifying the RPE cell in the RPEM medium after culturing said RPE cell in said RPEM medium. After differentiation there is a mixture of RPE cells and non-RPE cells, why a further purifying step can be helpful, so that differentiation plate may have only pure RPE cells ( Figure 20).
  • the additional purification step of the differentiation method preferably comprises a) manually identifying the RPE cell according to their pigmentation, wherein manually identifying the RPE cell according to their pigmentation preferably comprises selecting by microscopy, more preferably selecting by bright field microscopy as known to a person skilled in the art. This step may refer to a manual purification of RPE cells.
  • manually identifying the RPE cell according to their pigmentation may be performed by removing non-RPE cells, which have lesser pigmentation and different cell morphology than RPE cells, manually by scraping with a tip attached to the pipette, while observing through microscopy, such as bright field microscopy. It may further comprise washing, in particular with PBS for about 3 times, to remove all non-RPE cells.
  • the additional purification step of the differentiation method preferably comprises b) passaging the RPE cell, wherein passaging the RPE cells preferably comprises treating the RPE cell with Accutase or TrypLE, most preferably with TrypLE. This step may refer to a passaging purification of RPE cells.
  • passaging the RPE cell may be performed by detaching non-RPE cells and removing them by treatment with a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE.
  • the RPE cells may still be attached to the culture plate. It may then further comprise treating the remaining RPE cells again with a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE and further passaging said RPE cells.
  • the term initiallypassaging RPE cell“ refers to plating of the remaining RPE cell after having detached and removed non-RPE cell with a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE and treating said remaining RPE cell again with a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE. If TrypLE is applied, said step may refer to a TrypLE purification of RPE cells. Additionally or alternatively, the additional purification step of the differentiation method preferably comprises c) the combination of manually identifying the RPE cell according to their pigmentation as defined elsewhere herein and passaging the RPE cell as defined elsewhere herein.
  • the additional purification step of the differentiation method preferably comprises d) the combination of passaging the RPE cell and scatter sorting the RPE cell according to their pigmentation.
  • This purification step may be performed by removing non-RPE cells as defined above and further treating said remaining RPE cell with a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE and further processing the RPE cells in scatter sorting, wherein non-RPE cells removed by a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE may be used to set the gate for scatter low cells.
  • the additional purification step of the differentiation method preferably comprises e) the combination of scatter sorting the RPE cell according to their pigmentation. This step may refer to a scatter sorting purification of RPE cells.
  • scatter sorting RPE cell may be performed by resuspending cell pellet of dissociated single cells (being dissociated by using a gentle dissociation agent such as Accutase or TrypLE, preferably TrypLE) in any FACS buffer and passing it through a filter to get single cells and separating into scatter high and low fractions using e.g. any FACS cell sorter known to a person skilled.
  • a gentle dissociation agent such as Accutase or TrypLE, preferably TrypLE
  • passaging the RPE cell comprising treating the RPE cell with Accutase or TrypLE, preferably with TrypLE as defined above, followed by manually identifying the RPE cell according to their pigmentation as also defined elsewhere herein, is also the easiest to perform as partial TrypLE treatment removs majority of the non-RPE cells in a short amount of time needed for purification.
  • the purification of passaging the RPE cell comprising treating the RPE cell with Accutase or TrypLE, preferably with TrypLE, in combination with manually identifying RPE cells, involves an additional manual purification step to remove any non-RPE cells that might have escaped the treatment with a gentle dissociation agent such as Accutase or TrypLE, preferably with TrypLE and is mostly preferred as purification of RPE cells in the method of the present invention.
  • the present invention also comprises said differentiation method as defined elsewhere herein, wherein the iPS cell, which is derived from a stem cell of the amniotic membrane of the umbilical cord and used in the method of differentiating into a RPE cell, is particularly generated by expressing exogenous nucleic acids encoding the proteins OCT3/4, SOX2, KLF4, LIN28 and L-MYC and the p53-shRNA in said stem cell of the amniotic membrane of the umbilical cord under conditions suitable to reprogram the stem cell as defined elsewhere herein for the method of generating an induced pluripotent stem cell also comprised by the present invention.
  • the present invention also comprises said differentiation method as defined elsewhere herein, wherein the exogenous nucleic acids encoding the proteins OCT3/4, SOX2, KLF4, LIN28 and L-MYC and the p53-shRNA in said stem cell of the amniotic membrane of the umbilical cord are provided by one, two or three vectors, wherein preferably a first vector encodes the protein OCT3/4 and the 53-shRNA, a second vector encodes the proteins SOX2 and KLF4 and a third vector encodes the proteins L-MYC and LIN28.
  • each disclosure regarding the method of generating an induced pluripotent stem cell as defined elsewhere herein, which iPS cell is then differentiated into a RPE cell according to the described differentiation method, may be applicable, where necessary also to the method of differentiating an iPS cell into a RPE cell.
  • the present invention is also directed to a RPE cell culture / a RPE cell obtainable by the differentiation method as described herein and to a RPE cell culture / a RPE cell obtained by the differentiation method as described herein.
  • the present invention is also directed to a retinal pigment epithelium consisting of or comprising a retinal pigment epithelial cell culture / a RPE cell obtainable or obtained by the differentiation method as described herein.
  • the present invention is also directed to a pharmaceutical composition comprising a RPE cell culture / a RPE cell obtainable/obtained by the differentiation method as described herein.
  • a pharmaceutical composition comprising differentiated RPE cell / RPE cell culture comprising differentiated RPE cell is an injection solution or any kind of graft suitable for implanting the differentiated RPE cell.
  • said composition may comprise the RPE cell culture obtainable/obtained by the differentiation method as described herein.
  • said composition may comprise an implantable matrix, preferably a polyester matrix, even more preferably a polyester matrix in a transwell, said matrix coated with said differentiated RPE cells obtainable/obtained by the differentiation method as described herein which may have been grown on said matrix.
  • the RPE cells, grown on said matrix as defined herein may refer to said RPE cell culture obtainable/obtained by the differentiation method as described herein.
  • the pharmaceutical composition may be formulated/adapted for parenteral or local application as known to a person skilled. In such case, the parenteral application may comprise a sterile preparation intended for injection, infusion or implantation in the human or animal body.
  • Local application as used herein preferably refers to subretinal application.
  • the pharmaceutical composition refers to a graft as defined above with regard to the implantable matrix
  • said composition may be formulated/adapted for subretinal (under the retina of the eye) application, in other words may be transplanted subretinally.
  • the present invention also comprises a diagnostic composition for research purposes comprising the RPE cells (culture) and Matrigel.
  • Said composition also refers to a graft suitable for implanting into a subject as defined herein, wherein said RPE cells differentiated by the method as defined herein are mixed up with Matrigel and then the graft (cells in the Matrigel) is implanted into the subject.
  • the diagnostic composition refers to a graft as defined above with regard to the Matrigel
  • said composition may be formulated/adapted for subcutaneous application, in other words may be transplanted subcutaneously.
  • a RPE cell culture obtainable/obtained by the differentiation method as described herein, also comprised in a retinal pigment epithelium, and/or also comprised by a pharmaceutical composition as defined elsewhere herein, may refer to a plurality of a RPE cells, which are obtainable / obtained by the differentiation method and preferably comprising a culture medium for said RPE cells.
  • the term “population” may also be used interchangeably with the term “culture”.
  • Said differentiated RPE cell obtainable/obtained by the differentiation method of the present invention, which is comprised in said RPE cell culure can be further characterized: Said differentiated RPE cell firstly may comprise higher % areas of pigmentation compared to skin iPS-derived RPE.
  • all fours CLiPS tested by the invention which may be selected from the group consisting of CLMC23, CLMC30, CLMC44 and CLEC23 developed about 30 to about 100%, about 50 to about 100%, about 70 to about 100 % pigmented RPE cells using the differentiation medium as defined herein, compared to only 30% of the skin iPS cells (such as Asf5, AGO and/or HDFA) achieving similar pigmentation ( Figure 13).
  • said differentiated RPE cell additionally comprised in said culture, may express at least any one of BEST1, PMEL17, MITF, TYROSINASE, TRYP2, ZO-1, RPE65, RLBP1 or MERTK or a combination of all of the protein markers listed ( Figures 15, 16, and 18).
  • said differentiated RPE cell may express the protein marker BEST1.
  • said differentiated RPE cell, additionally comprised in said culture may express the protein marker PMEL17.
  • said differentiated RPE cell, additionally comprised in said culture may express the protein marker MITF.
  • said differentiated RPE cell, additionally comprised in said culture may express the protein marker TYROSINASE.
  • said differentiated RPE cell, additionally comprised in said culture may express the protein marker TRYP2.
  • said differentiated RPE cell, additionally comprised in said culture may express the protein marker ZO-1.
  • said differentiated RPE cell, additionally comprised in said culture may express the protein marker RPE65.
  • said differentiated RPE cell additionally comprised in said culture, may express the protein marker RLBP1.
  • said differentiated RPE cell additionally comprised in said culture, may express the protein marker MERTK.
  • CLiPS-derived RPE cell is thus more highly pigmented compared to ES-derived RPE cells such as H9 cells used herein. This is associated with higher expression of pigmentation associated genes such as for example MITF, PMEL17, TYROSINASE and TRYP2 ( Figure 15).
  • said differentiated RPE cell additionally comprised in said culture, can be characterized by lacking or having a reduced expression of the cell cycle proliferation marker Ki67. The expression of the mature RPE marker RPE65 and Ki67 as proliferation marker being absent, confirms the mature and quiescent state which reflects the survival rate of such RPE cells differentiated from CLiPS according to the method as defined elsewhere herein ( Figure 22).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing BEST1 with a fold change of at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3 or more, preferably of about 3 relative to a RPE cell differentiated from an embryonic stem cell (ES) (cell or culture from which it has been generated).
  • ES embryonic stem cell
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing PMEL17 with a fold change of at least about 0.9, at least about 0.91, at least about 0.92, at least about 0.93, at least about 0.94, at least about 0.95, at least about 1, at least about 1.1, at least about 1.2, at least about 1.3 or more relative, preferably of about 1.3 to a RPE cell differentiated from an ES (cell or culture from which it has been generated).
  • CLiPS-RPE thus comprise a higher % of PMEL17 positive cells after differentiation.
  • the CLiPS used (such as CLMC23, CLMC30 and CLEC23) in the differentiation method comprises about 89 % to about 95% purity of RPE cells.
  • the 3 skin iPS cells used such as Asf5, AGO and/or HDFA had above about 90% purity ( Figure 14).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing MITF with a fold change of at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8 or more, preferably of about 6.8 relative to a RPE cell differentiated from an ES (cell or culture from which it has been generated).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing TRYP2 with a fold change of at least about 2.9, at least about 3, at least about 3.5, at least about 4, at least about 4.1, at least about 4.2, at least about 4.3 or more, preferably of about 4.3 relative to a RPE cell differentiated from an ES (cell or culture from which it has been generated).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing RPE65 with a fold change of at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 0.91, at least about 0.92, at least about 0.93, at least about 0.94, at least about 0.95, at least about 0.96 or more, preferably of about 0.96 relative to a RPE cell differentiated from an ES (cell or culture from which it has been generated).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing RLBP1 with a fold change of at least about 17.5, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 26.1, at least about 26.2 or more, preferably of about 26.2 relative to a RPE cell differentiated from an ES (cell or culture from which it has been generated).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by expressing MERTK with a fold change of at least about 6, at least about 6.5, at least about 7, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.1 or more, preferably of about 9.1 relative to a RPE cell differentiated from an ES (cell or culture from which it has been generated) ( Figure 20k).
  • ES which is used for comparison and where a RPE cell has also been differentiated from, may refer to a H9 ES cell.
  • the term “relative to” or “to” can also be replace with the term “compared to”, bringing two cells (for example CLMC23 as tested cell and H9 as reference cell) in comparison to one another, e.g. with regard to gene expression expressed as fold change of the tested cell to the reference cell.
  • a “fold change” as used herein is a measure describing how much a quantity changes going from an initial to a final value. For example, an initial value of 30 and a final value of 60 correspond to a fold change of 2, or in common terms, a two-fold increase. Fold change is calculated simply as the ratio of the final value to the initial value, i.e. if the initial value is A and final value is B, the fold change is B/A.
  • the fold-change can be obtained with respect to mRNA levels of the markers as described herein. Such fold-changes may be measured using RT-qPCR.
  • CLiPS-derived RPEs may achieve trans-epithelial electrical resistance (short: TEER) similar to a RPE cell differentiated from an ES (cell or culture from which it has been generated) and/or to a RPE cell differentiated from a skin iPS (cell or culture from which it has been generated).
  • CLiPS-RPE also showed high phagocytosis similar to a RPE cell differentiated from an ES (cell or culture from which it has been generated) and/or to a RPE cell differentiated from a skin iPS (cell or culture from which it has been generated) (Figure 17).
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by comprising an increased oxygen consumption rate (OCR) and/or extracellular acidification rate (ECAR) relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS.
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • OCR may comprise basal respiration, ATP production, maximal capacity and/or spare respiratory capacity.
  • the term dealtincreased“ in this context with regard to OCR means that the OCR of RPE cell is increased by at least about 30 %, at least about 31 %, at least about 32 %, at least about 33 %, at least about 34 %, at least about 35 %, preferably at least about 35 %; or by between about 30 to about 45 %, by between about 31 to about 44 %, by between about 32 to about 43 by between about 33 to about 42 %, by between about 35 to about 40 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS.
  • the basal respiration of RPE cell is increased by about 38 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS;
  • the ATP production of RPE cell is increased by about 40 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS;
  • the maximal capacity of RPE cell is increased by about 35 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS;
  • the spare respiratory capacity of RPE cell is increased by about 36 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS.
  • ECAR may comprise glycolysis, glycolytic capacity and/or glycolytic reserve.
  • the term dealtincreased“ in this context with regard to ECAR means that the ECAR of RPE cell is increased by at least about 20 %, at least about 21 %, at least about 22 %, at least about 23 %, at least about 24 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %; or by between about 20 to about 55 %, by between about 25 to about 55 %, by between about 25 to about 50 relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS.
  • the glycolysis of RPE cell is increased by about 25 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS; ii) the glycolytic capacity of RPE cell is increased by about 37 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS; iii) the glycolytic reserve of RPE cell is increased by about 50 % relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS.
  • the RPE cell obtainable/obtained by the method as defined herein can further be characterized by having / comprising less immunogenicity as defined in more detail below relative to a RPE cell differentiated from an ES (cell or culture from which it has been generated) and/or relative to a RPE cell differentiated from a skin iPS (cell or culture from which it has been generated).
  • less immunogenicity refers to the RPE cell, preferably predelivered to a subject from whom a sample comprising said RPE cell has been obtained from for further analysis of said cells, has a reduced systemic immune response which may refer to (a) reduced level(s) of (a) pro-inflammatory cytokine(s) involved in induction of cellular immune response, preferably of IFN-y and/or IL- 18 (as a surrogate for cellular immune response) and/or of IL-23 and/or IL17A, cytokines involved in T cell activation, said redcued cytokine level being generated by said subject to whom said RPE cell has been pre-delivered before, in particular by said immune cells present in said subject and which are comprised also within said sample.
  • a reduced systemic immune response which may refer to (a) reduced level(s) of (a) pro-inflammatory cytokine(s) involved in induction of cellular immune response, preferably of IFN-y and/or IL- 18 (a
  • Such reduced systemic immune response in said RPE cell may also refer to a decreased accumulation of immune cells, preferably at the site of injection as defined in the Example section.
  • a reduced systemic immune response may thus comprise the RPE cell as defined herein having reduced systemic T cell activation, preferably reduced / suppressed CD8 cytotoxic T cell activation relative to a RPE cell differentiated from an ES and/or relative to a RPE cell differentiated from a skin iPS.
  • the present invention further includes a method of treating a retinal degenerative disease in a subject, wherein the subject may be selected from the group comprising a mouse, a rat, a rabbit, a pig, a dog, a cat, a non-human primate (monkey) or a human.
  • the subject is human.
  • treating may comprise administering to a subject as defined herein a RPE cell differentiated from CLiPS by the differentiation method as described herein and/or said RPE cell culture obtained by the method of the invention and/or said pharmaceutical composition as defined herein.
  • said differentiated cell / culture obtainable/obtained by the method as defined herein is suitable for administration in said particular treatment of a retinal degenerative disease, is demonstrated by the fact that said RPE cell comprises hypoimmunogenic properties such as that reduced levels of pro-inflammatory cytokines, preferably of IFN-y and/or IL- 18 as a surrogate are generated, and having reduced cellular immune response, preferably after pre-injection of said differentiated RPE cells into a subject as defined herein (Figure 23).
  • said RPE cells of the present invention may reduce immune cell infiltration at the localized site of RPE cell injection into a subject.
  • the sample obtained from a subject to whom said differentiated RPE cell has been predelivered before the cytokine analysis comprises decreased levels of IL-23 and/or IL17A, cytokines involved in T cell activation, relative to a sample obtained from a reference subject to whom RPE cell differentiated from an ES has been pre-delivered before and/or relative to a sample obtained from a reference subject to whom a RPE cell differentiated from a skin iPS has been pre-delivered before.
  • said RPE cell as defined herein may be capable that reduced levels of IL-23 and/or IL17A as another preferable example of pro-inflammatory cytokines are generated as defined herein.
  • T cell activation in particular CD8 cytotoxic T cell activation
  • said RPE cell may suppress T cell activation, in particular CD8 cytotoxic T cell activation ( Figure 24).
  • the degenerative disease being treated is a disease of the retina known to a person skilled, preferably the retinal degenerative disease is age-related macular degeneration (AMD) or retinal dystrophy.
  • the present invention refers to a method of treating AMD in a subject as defined herein, comprising administering to a subject a RPE cell differentiated from an iPS cell obtained by the method as defined herein.
  • the present invention refers to a method of treating retinal dystrophy in a subject as defined herein, comprising administering to a subject a RPE cell differentiated from an iPS cell obtained by the method as defined herein.
  • Administration of said RPE cell differentiated from CLiPS in the method of treatment may include parenteral or local (preferably subretinal) application as known to a person skilled.
  • the present invention further includes an in vivo method of detecting the survival rate of a RPE cell differentiated from an iPS cell by the differentiation method as defined elsewhere herein in a subject, the method comprising a step a) introducing a RPE cell differentiated from an iPS cell by the method as defined herein into a subject, wherein said RPE cell comprises a bioluminescence label.
  • the term “survival rate” refers to the RPE cell not having died and still being mature and/or comprising a quiescent state, which may be confirmed by detecting the expression of RPE65 as mature RPE marker and by detecting no expression of Ki67 as proliferation marker, after said cells have been introduced into said subject as defined herein and detected over an amount of time as further described. Monitoring the survival rate can also be used interchangeably herein with regard to the in vivo method as defined.
  • the term “introduce” or “introducing” in step a) refers to bringing said RPE cell as defined herein into said subject, preferably by transplanting said RPE cell into said subject, even more preferably by transplanting said RPE cell subcutaneously into said subject with regard to using a mouse as a subject and a Matrigel plug assay as defined elsewhere herein.
  • the term “subject” when used herein according to the in vivo method and also to the in vitro screening method includes mammalian and non-mammalian subjects. Preferably, the subject is an animal.
  • the subject of the in vivo and in vitro method may refer to a mammal, including human, domestic and farm animals, non-human primates, and any other animal that has mammary tissue.
  • the mammal is a mouse. In some embodiment the mammal is a rat. In some embodiment the mammal is a guinea pig. In some embodiment the mammal is a rabbit. In some embodiment the mammal is a cat. In some embodiment the mammal is a dog. In some embodiment the mammal is a monkey. In some embodiment the mammal is a horse. In a preferred embodiment the mammal / the animal as the subject used in said methods of the present invention is a mouse. In a most preferred embodiment the mammal / the animal as the subject used in said methods is a humanized mouse.
  • the RPE cell being differentiated according to the present invention and being introduced into said subject as defined herein within the in vivo method comprises a bioluminescence label.
  • label may be a fluorescent label or an enzyme suitable for bioluminescence.
  • the label is fluorescent label it can be a fluorophore (also called fluorochrome or chromophore).
  • Such fluorophore may be any one of a 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 a fluorescent protein such as R-PE or APC, or an expressed fluorescent protein such as CFP, EGFP, GFP or RFP.
  • FITC Fluorescein
  • the label when it is an enzyme it can be, but is not limited to, a luciferase, preferably selected from the group consisting of bacterial luciferase ( ⁇ uxAB), photinus luciferase, ren/7/a luciferase, and firefly luciferase.
  • said RPE cell being differentiated according to the present invention and being introduced into said subject as defined herein within the in vivo method, comprises a bioluminescence enyzme gene-encoding vector, preferably tagged with an expressed fluorescent protein as defined herein.
  • said RPE cell being differentiated according to the present invention and being introduced into said subject as defined herein within the in vivo method comprises a luciferase enyzme gene-encoding vector, preferably tagged with GFP.
  • step a) of said in vivo method as defined herein is the introduction of said RPE cell differentiated by the method as defined herein into a subject, the RPE cell being comprised in a RPE-matrigel plug as known to a person skilled in the art.
  • Being comprised in a Matrigel plug means a mixture/composition of said RPE cells with Matrigel as known to a person skilled in the art.
  • the in vivo method as defined herein further comprises step b) detecting the bioluminescence signal of said RPE cell over time using an imaging method, thereby collecting imaging data.
  • detecting or detection may also refer to crizoleukinic senor“) when used herein with regard to the in vivo method refers to the visualization and the qualitative analysis of the bioluminescence of said RPE cells in vivo using any known imaging method, preferably if the bioluminescence label is luciferase using the bioluminescent imaging method.
  • Said detection of a bioluminescence signal of said RPE cell is according to step b) of the in vivo method performed over time which may refer to at least about 2 days, about 7 days, about 10 days, about 14 days, about 17 days, about 21 days, about 24 days, about 28 days, about 35 days, about 42 days, about 49 days, or at least about 56 days; or between about 2 to about 56 days, or between about 2 to about 49 days.
  • Said detection of the bioluminescence is preferably monitored at regular intervals over said time course as defined herein.
  • the detection of bioluminescence may refer to the detection of total radiance of said cells expressed in p/s/cm2/sr as known to a person skilled in the art. By said detection of said bioluminescence signal over time imaging data are collected.
  • the in vivo method as defined herein further comprises step c) comparing the imaging data received in step b) to reference imaging data.
  • the reference imaging data refer to the bioluminescence signal(s) (“imaging signal”) of RPE cells being differentiated from an ES cell (preferably H9 cell) and/or differentiated from a skin iPS (preferably HDFA or ASF5) which has/have also been detected over time as defined herein.
  • said RPE cells differentiated from an ES cell and/or differentiated from a skin iPS have also been introduced as defined herein into a subject (e.g. different mouse), which is not the exact same subject as the subject to whom the RPE cells have been pre-delivered as defined herein (e.g. mouse), and wherein said RPE cells being differentiated from an ES cell and/or differentiated from a skin iPS also comprise the bioluminescence label as defined herein as for the CLiPS derived RPE cells.
  • the present invention also comprises the in vivo method as defined herein, wherein no difference in the bioluminescence signal in the imaging data as compared to the reference imaging data indicates survival of said RPE cell in said subject. No difference also comprises slight, but insignificant decrease in the bioluminescence signal over time in the imaging data compared to the reference imaging data of said RPE cells differentiated from an ES cell and/or differentiated from a skin iPS.
  • the present invention also comprises an animal comprising said RPE cell obtained/obtainable by the differentiation method as defined elsewhere herein.
  • said “animal” refers to any mammal as defined herein, preferably a mouse, most preferably a humanized mouse.
  • the animal comprising said RPE cell it may be meant that said RPE cell obtained/obtainable by the differentiation method as defined elsewhere herein is introduced to said animal as defined herein, preferably by transplanting said RPE cell subcutaneously.
  • the present invention also comprises an in vitro (screening) method of determining the immunogenicity of a RPE cell differentiated from an iPS cell by the method as defined herein in a subject, to whom said differentiated RPE cell has been pre-delivered, the method comprising a step a) detecting a pro-inflammatory cytokine level using an imaging method, in a sample which is obtained from said subject defined herein, wherein the sample comprises said differentiated RPE cell, thereby collecting imaging data.
  • Pre-delivered includes in this regard, that the differentiated RPE cell of the present invention has been delivered to the subject as defined herein prior to the in vitro screening method. After that the sample comprising the differentiated RPE cell is obtained from said subject and the sample further analyzed, e.g. for particular cytokine levels.
  • the term reiteratepre-introduced“ may also be used interchangeably.
  • detecting refers to the visualization and the qualitative analysis of said cytokine levels as defined herein in vitro using any known imaging method suitable for detecting cytokines such as flow cytometry.
  • Said cytokine level being detected as defined herein for the in vitro method refers, but is not limited to, a level of a cytokine associated with cell-mediated immunity, preferably to the level of the pro-inflammatory cytokine IFN-y, IL- 18, IL-23 and/or IL17A.
  • the level of said cytokine as defined herein may be expressed in pg/ml.
  • the sample being obtained from said defined subject in step a) of the in vitro method may be any biological sample taken from said subject, preferably blood serum sample.
  • the in vitro method may comprise as an additional or as an alternative step within said method detecting immune cell infiltration in a sample obtained from said subject as defined herein, the sample comprising said differentiated RPE cell, thereby further collecting imaging data.
  • the method further comprises a step b) comparing the imaging data received in step a), and/or received from the detection step with regard to the infiltration, to reference imaging data.
  • said reference imaging data refer to imaging data of the same detected pro-inflammatory cytokine level as defined herein within a sample from a subject to whom RPE cell differentiated from an ES cell and/or differentiated from a skin iPS as reference cell has been pre-delivered and/or to imaging data of the same immue cell(s) being infiltrated and detected within a sample from a subject to whom RPE cell differentiated from an ES cell and/or differentiated from a skin iPS as reference cell has been pre-delivered.
  • Said reference cell may be comprised also in the same kind of sample (e.g. blood sample, but different blood sample) as the sample which comprises the RPE cell, but the reference sample may be obtained from a different subject (e.g. a different mouse) compared to the test subject from which the sample comprising the RPE cell has been obtained from.
  • sample e.g. blood sample, but different blood sample
  • the reference sample may be obtained from a different subject (e.g. a different mouse) compared to the test subject from which the sample comprising the RPE cell has been obtained from.
  • a decreased cytokine level and/or reduced/decreased immue cell infiltration / accumulation in the imaging data as compared to reference imaging data indicates reduced immunogenicity of said RPE cells in said subject meaning the ability of said RPE cell according to the invention to provoke an immune response in the subject is lower / redcued compared to the immunogenicity of a RPE cell differentiated from an ES cell and/or differentiated from a skin iPS.
  • the results shown in Table 1 and Figure 2 indicate that suitable electroporation conditions could be found for both CLMC and CLEC.
  • the optimal electroporation setting for CLEC found here comprises 2 electric pulses each of 30ms and 1350 V using an amount of 1.67 g (plasmid) DNA of each of the three vectors (pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL) for a number of IxlO 6 cells.
  • Four individual CLEC lines (CLEC42, CLEC44, CLEC23 and CLEC30) transfected with these settings exhibited a survival rate of 4.67 %, 7.33 %, 9.33% and 7.50%, respectively.
  • the electroporation settings used here differ rather significantly from conditions reported for successful electroporation of epithelial cells such as corneal epithelial cells (1 electric pulse of 30ms and 1300 V and a ratio of the amount of plasmid DNA (pg) to the number of cells (IxlO 6 cells) of 1:1 (cf. Png, E. et al. (2011), Journal of Cellular Physiology. United States, 226(3), pp. 693-699),
  • the resulting transgenic cells exhibited a survival rate of 6.17 %, 7.50 %, 5.00% and 7.33%, respectively.
  • electroporation/transfection conditions found here to be the optimum for the generation of CLiPS from CLMC are different from electroporation conditions reported so far.
  • Sprangers, A. J., Freeman, B. and Ogle, B. M. (2011), pp. 62-66 for example, who examined possible negative effects of electroporation of human embryonic stem cell (hESC)-derived mesenchymal stem cells. So doing, Sprangers et al.
  • the present invention provides a unique and efficient protocol for CLEC and CLMC electroporation, respectively.
  • the variations in the transfection efficiencies across the four individual CLSC lines (cells from different donors) are inter-individual variabilities being an inherent and documented feature of iPS derivation.
  • a PCR amplification was performed with gene-specific primers on genomic DNA isolated from individual CLSC lines to determine the presence or absence of the DYS439 and SRY loci, which are both present on the Y chromosome.
  • aSF4 adult skin fibroblasts which is confirmed to be obtained from a male donor, was used as a positive control.
  • Example 2 Derivation of transgene integration- and feeder-free human iPS
  • Cord lining epithelial cells (CLEC) and 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 medium PTT-e3 and PTT-4, respectively.
  • Adult skin fibroblasts from a healthy, 78 years old male Asian donor were purchased from CellResearch Corporation Pte Ltd and cultured in DMEM/10% FBS.
  • the culture medium PTT-4 consists of 90 % (v/v) CMRL-1066 and 10 % (v/v) FBS, while the medium PTTe-3 has the following composition:
  • Somatic reprogramming was performed using the conditions established in Example 1 and was further in a feeder-independent manner.
  • Log-phase cultures were harvested by dissociation with TrypLE Express (ThermoFisher Scientific) and 0.72 million cells were pelleted in a 1.5ml centrifuge tube. The cell pellet was resuspended in 120 pL of Buffer R (NeonTM Transfection System 100 pL Kit, Thermo Fisher Scientific MPK10096).
  • a cocktail containing 1.2 pg each of episomal vectors pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL (Addgene plasmids #27077 (SEQ ID NO: 12), #27078 (SEQ ID NO: 13), #27080 (SEQ ID NO: 14), respectively) was added to the cells and mixed thoroughly (each vector was used in an amount of 1.67pg (plasmid) DNA for a number of IxlO 6 cells).
  • Neon® Tip The cell suspension was loaded into a 100 pL Neon® Tip and Neon electroporation was performed with the following parameters: adult skin fibroblasts - 1,650 V, 10 ms, 3 pulses; CLEC - 1350V, 30ms, 2 pulses; CLMC - 1600V, 20ms, 1 pulse.
  • Cells were immediately transferred into 6 ml of CLEC or CLMC medium containing 1 pM hydrocortisone (StemCell Technologies) and distributed equally into 3 wells of a Matrigel-coated 6-well plate. Two days later, the medium was switched to a 1:1 mixture of CLEC or CLMC medium and mTeSRl supplemented with 1 pM hydrocortisone.
  • the cell pellet is resuspended in 120pL of Buffer R (NeonTM Transfection System 100 pL Kit, Thermo Fisher Scientific MPK10096). A cocktail containing 1.2pg each of pCXLE-hUL, pCXLE-hSK, and pCXLE-hOCT3/4-shp53-F is added and mixed thoroughly. 4. The cell suspension is loaded into a lOOpL Neon® Tip. Electroporation is performed with the following parameters for CLEC: 1350V, 30ms, 2 pulses and the following parameters for CLMC: 1600V, 20ms, 1 pulse.
  • Cells are immediately transferred into 4 ml of CLEC or CLMC medium (PTTe-3 and PTT-4, respectively) containing IpM hydrocortisone and then distributed into 3 wells of a Matrigel coated 6-well plate.
  • the medium is changed to a 1:1 (v/v) mixture of CLEC or CLMC medium (PTT-e3 and PTT-4, respectively) and mTeSRl supplemented with 1 pM hydrocortisone.
  • iPS colonies may start appearing as early as 2 weeks after transfection. When iPS colonies reach about 0.5mm to about 1mm in diameter (around Day 20 onward), they are manually picked under bright field microscopy and each colony is placed in a single well of Matrigel coated 24- well plate (Nunc).
  • cells are passaged 1:3 by dissociation using 0.5mM EDTA when they reach about 70%-80% confluence. Newly passaged cells are cultured overnight in medium containing lOpM ROCK inhibitor Y-27632.
  • iPS were generated from a cGMP-grade CLMC line designated CLMSC-DTHN using the protocol described in WO2018/067071 for the production of the mesenchymal stem population of which 99 % of the stem cell express the markers CD73, CD90 and CD 105 while not expressing the markers CD34, CD45 and HLA-DR) cGMP quality reagents wherever possible.
  • the reprogramming protocol is the same protocol described for CLMC in Example 2 but Matrigel, an extracellular matrix substrate prepared from Engelbreth- Holm-Swarm (EHS) mouse sarcoma cells, was replaced with recombinant human laminin-511 E8 fragment (iMatrix-511 SILK, ReproCELL), which is a defined, animal- and xeno-free substrate for coating cell culture vessels.
  • EHS Engelbreth- Holm-Swarm
  • iMatrix-511 SILK recombinant human laminin-511 E8 fragment
  • mTeSRl used for reprogramming and subsequent maintenance of CLiPS clones was replaced with cGMP mTeSRTMl (StemCell Technologies).
  • Sub-culturing of CLiPS is performed when cultures reached -90% confluence. Spent culture medium is aspirated off along with any overtly differentiated areas that may be present. Caution should be taken not to allow cells to be exposed to air for too long. The culture is rinsed once with prewarmed (37°C) Dulbecco’s Phosphate Buffered Saline (DPBS).
  • DPBS Phosphate Buffered Saline
  • Appropriate volume of rewarmed (37°C) 0.5mM EDTA solution is added to the culture according to the culture vessel - 0.5ml/well of a 24-well dish, Iml/well of a 6-well dish or 2ml for a 6cm dish.
  • the culture is placed in an incubator at 37°C for 5 min following which it is observed under a microscope. Cells should appear rounded but not detached from the surface.
  • the duration of incubation at 37°C varies with different CLiPS lines and may range from about 5 - 10 min. Incubation duration will be largely based on prior experience with each line.
  • the EDTA solution is gentle aspirated off taking care not to dislodge the cells.
  • a medium such as mTeSRl or TeSR-E8 containing ROCK inhibitor Y-27632 is dispensed directly onto the cells to dislodge them.
  • the volume of medium used is dependent on the vessel size used - 0.5ml/well of a 24-well dish, Iml/well of a 6-well dish or 2ml for a 6cm dish.
  • gentle pipetting is repeated until most of the cells have been dislodged.
  • the cell suspension is then transferred to a 15 ml Falcon tube.
  • the culture vessel is rinsed with fresh medium and the rinse combined with the cell suspension in the Falcon tube.
  • the cells in the tube are diluted to the appropriate volume for plating on new Matrigel-coated vessels.
  • Split ratio may range from 1:3 to 1:10, depending on the density of the initial culture and also the growth rate of individual CLiPS lines.
  • DMSO tissue culture-grade dimethyl sulfoxide
  • DMSO tissue culture-grade dimethyl sulfoxide
  • the density of cells per aliquot is dependent on the desired rate at which cell confluence is achieved upon thawing and culturing of the aliquot.
  • Cryo vials are then transferred to a slow freezing apparatus such as Mr. FrostyTM Freezing Container (Thermo Scientific) or CoolCell® Cell Freezing Containers (BioCision LLC) and placed overnight at - 80°C.
  • cryovials are transferred to liquid nitrogen storage. It is not advisable to leave CLiPS aliquots at -80°C for more than 24hr.
  • Several commercial freezing medium such as mFreSRTM (StemCell Technologies) and CryoStor® CS10 (Biolife Solutions) are also available for cry opreservation and may be used according to manufacturers’ instructions.
  • CLiPS functionality was determined by subjecting colony developing CLiPS to an immunofluorescent staining after electroporation. Thereby, the expression of pluripotent embryonic stem cell markers (OCT4, SOX2, KLF4, NANOG, SSEA-4, TRA-1-81) was analyzed. For this purpose, cells were fixed with 4% formaldehyde in phosphate buffer saline (PBS) for 15 min and subsequently washed 3 times for 5 min with PBS. For staining of intracellular or nuclear markers (OCT4, SOX2, KLF4, NANOG), cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with FDB (5% FCS/1% NGS/1% BSA) for 1 h.
  • PBS phosphate buffer saline
  • PCR reactions were set up as follows: 0.5pl cDNA, 5 pl 2x MyTaq HS Mix (Bioline), 0.2pl forward primer (lOpM), 0.2pl reverse primer (lOpM), 4.2pl PCR water.
  • Thermal cycling was performed in an MJ Mini Thermal Cycler (Bio-Rad) with the following conditions: lx 95°C Imin, 30x (95 °C 15s, Tm 15s, 72°C 15s), 72°C Imin.
  • Primer sequences and annealing temperature used (Tm) are provided in Table 2 below.
  • a qualitative expression analysis was performed by an agarose gel analysis, wherein the samples were loaded on a 2% agarose gel incorporated with SYBR Safe DNA stain (Thermo Fisher Scientific) in lx TAE buffer and electrophoresed at 80V for 30 min. The gel images were captured using a ChemiDoc Imaging System (Bio-Rad).
  • CLMSC-DTHN express the pluripotent stem cell markers NANOG, OCT4, SOX2 and TRA-1-81 (Figure 3r-u) at levels indistinguishable from their non- GMP counterparts.
  • CLMSC-DTHN may provide the same embryonic properties non-GMP-derived CLiPS entail.
  • iPS were plated at a density of 3.5-4.0xl0 4 cells per cm 2 on Matrigel (Corning) coated dishes and cultured for 5 days in knockout serum replacement medium (KSR) containing Knock-Out DMEM, 15% knockout serum replacement, 1 X GlutaMAX and 10 mM P-mercaptoethanol. From Day5, KSR medium was transitioned stepwise to N2 medium as described in Tomishima “Midbrain dopamine neurons from hESCs.” 2012 Jun 10. In: StemBook. Cambridge (MA): Harvard Stem Cell Institute; 2008-.
  • KSR knockout serum replacement medium
  • NB27 medium composed of Neurobasal medium, 2% B27 minus vitamin A and lx 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), TGFP3 (transforming growth factor type P3, 1 ng/ml; R&D), dibutyryl cAMP (0.5mM; Santa Cruz Biotechnology), and DAPT (10 nM; Tocris,) for 9 days.
  • 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
  • TGFP3 transforming growth factor type P3, 1 ng/m
  • cells were dissociated using Accutase (Gibco) and replated at high cell density (3-4x10 5 cells per cm 2 ) on dishes pre-coated with poly-L-ornithine (PLO; 15 mg/ml)/laminin (1 pg/ml)/fibronectin (2 pg/ml) in NB27 medium supplemented with 10 pM ROCK inhibitor Y-27632. Cultures were maintained in NB27 medium with medium replacement every other day until the desired endpoint. Differentiated cells were analysed for expression of cell specific markers at this stage.
  • PLO poly-L-ornithine
  • cryosectioning was performed, wherein slides containing the sections were dehydrated by incubation at 37°C for 30 min, cooled to room temperature and washed 3 times with TBST. Section permeabilization, blocking, antibody staining and mounting were performed as described in Example 4. Primary antibodies from the same host species were used, a fluorochrome- conjugated monovalent antibody (Jackson ImmunoResearch) was used to saturate the first primary antibody before sequential incubation with the second primary antibody and conjugated secondary antibody.
  • L-15 maturation and maintenance medium (Leibovitz L-15 medium, 8.3% tryptose phosphate broth, 8.3% heat inactivated FBS, 10 pM hydrocortisone 21 -hemisuccinate, 1 pM Insulin (bovine pancreas), 1% L-Glutamine, 0.2% ascorbic acid) supplemented with 10 ng/mL hHGF and 20 ng/mL OSM for 9 days (changing medium every 48 hours). Differentiated cells were again analysed for expression of cell specific markers at this stage. For this purpose, cryosectioning was performed as described in Example 7.
  • hepatocyte-like cells were obtained from CLiPS and asF5-iPS using this protocol.
  • the antibody staining revealed the expression of the hepatocyte markers alphafetoprotein (AFP; Figure 4g, g’, g”), Cytokeratin 18 (CK18) and Human Serum Albumin (HSA; Figure 4h, h’, h”) after 17 days of differentiation.
  • AFP alphafetoprotein
  • CK18 Cytokeratin 18
  • HSA Human Serum Albumin
  • a majority of the differentiated cells exhibited a polygonal shape characteristic of hepatocytes.
  • staining with Oil Red O showed abundant lipid droplet accumulation in the cells, a hallmark of cultured hepatocytes ( Figure 4i, i’, i”).
  • iPS maintained on Matrigel in mTeSRl were dissociated into single cells with StemPro Accutase (Thermo Fisher Scientific) at 37 °C for 5 min and then seed onto a Matrigel-coated cell-culture dish at 1X10 5 -2X10 5 cell/cm 2 (5xl0 5 cells per 24-well) in mTeSRl supplemented with 5 pM ROCK inhibitor (Y-27632; Stemgent) for 24 h.
  • Y-27632 pM ROCK inhibitor
  • the concentration of CHIR99021 was lowered to 5pM from the original 12 pM at this stage.
  • HLA-E and HLA-G staining cells were permeabilized with BD Phosflow Perm/Wash Buffer I (BD Biosciences) according to manufacturer’s instructions prior to staining. Following staining, cells were washed 2x in lx Ca 2+ - and Mg 2+ - free DPBS/5mM EDTA, fixed with 1% paraformaldehyde for 1 hr in the dark and then were washed 2x in lx Ca 2+ - and Mg 2+ - free DPBS/5mM EDTA.
  • BD Phosflow Perm/Wash Buffer I BD Biosciences
  • HLA-ABC HLA-ABC was expressed across all iPS samples but a markedly reduced level was observed for EC23-CLiPS ( Figure 6a). HLA-DR expression was absent in all iPS samples ( Figure 6b), consistent with previous reports of negligible HLA-II expression in iPS (Saljb, K., et al., Sei Rep, 2017. 7(1): p. 13072 and Chen, H.F., 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 alloimmune response.
  • 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 6a).
  • 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.
  • HLA-ABC expression was reduced upon its differentiation to NPCs.
  • CD40 expression was downregulated across all NPC samples, with only EC23-iPS- and EC44-iPS-derived NPCs displaying slight expression.
  • HLA-E expression was absent in all NPC samples but slight upregulation of HLA-G was observed in asF-iPS- and EC23-iPS- derived NPCs.
  • 6-hydroxydopamine 6-hydroxydopamine
  • the unilateral 6-OHDA lesion is an established method for rodents and comprises the injection of 6-OHDA in the rodent brain causing motoric dysfunctions characterized by rotational asymmetry with the degree (Bagga, V., Dunnett, S. B. and Fricker, R. A. (2015) Behavioural Brain Research. Elsevier B.V., 288, pp. 107-117).
  • 6-OHDA lesions were induced in NOD/MrkBomTac-Prkdc scld mice (4 weeks old) purchased from InVivos Pte Ltd and maintained under SPF conditions at the Animal Research Facility, NNI and male C57BL/6NTac mice (6-8 weeks old) purchased from InVivos Pte Ltd., wherein the mice used for this experiment were fully immunocompetent and no immunosuppression was administered prior to or following transplantation.
  • NPCs derived from asF- iPS, EC23-CLiPS and MC23-CLiPS were transplanted into the striatum of immunocompetent, 6-OHDA lesioned C57BL/6 mice by stereotaxic injection was performed on mice models considered as successfully lesioned.
  • apomorphine-induced rotations were scored and mice displaying >6 rotations per min were used for transplantation.
  • Day25 dopaminergic progenitor cells were harvested by dissociation and resuspended to ⁇ 1.25xl0 5 cells/pl in HBSS supplemented with lOng/mL of BDNF, lOng/mL of GDNF.
  • mice Six months after transplantation, NPC-transplanted, sham-injected and non-manipulated mice were evaluated for striatal dopamine transporter (DAT) activity by positron-emission tomography (PET) imaging using the radioligand (2- [18F] Fluoroethyl 8-[(2E)-3-iodoprop-2-en- l-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 sessions. Animals were kept warm during scanning with integrated hot-air channels from the imaging bed.
  • DAT striatal dopamine transporter
  • PET positron-emission tomography
  • VOI volume of interest
  • the accuracy of the manual registration was accessed and verified by two different persons.
  • the combined transformation matrix was applied to transform the PET images to the MRI mouse template.
  • VOIs for left and right striatum and cerebellum were used for the analysis.
  • 3D erosion with one voxel was applied to the obtained VOIs.
  • [18F]FE-PE2I binding was quantified using the non-invasive reference tissue models, since they are equally accurate as compared to the kinetic analyses with the arterial input function (Varrone, A., et al., Nucl Med Biol, 2012. 39(2): p. 295-303).
  • the binding potential (BPnd) values were calculated using the simplified reference tissue model (SRTM) (Lammertsma, A.A. and S.P. Hume, 1996. 4(3 Pt 1): p. 153-8) with the cerebellum as the reference.
  • the regional time activity curves (TACs) were also extracted from the VOIs of striatum and cerebellum. Anesthesia was induced with 5% isoflurane in 100% O2 and maintained with 1.5-2% isofluorane during the imaging.
  • mice were analyzed for the presence of microglia/macrophages, as these cells are known to play important roles in allograft and xenograft rejection in the CNS (Hoornaert, C.J., et al., Stem Cells Transl Med, 2017. 6(5): p. 1434-1441),
  • an immunostaining for the microglia/macrophages-specific marker Ibal was performed after transcardial perfusion with 4% PFA.
  • PFA perfused brains were post-fixed overnight in 4% PFA followed by equilibration in 15% and 30% w/v sucrose solution in PBS until they settled to the bottom of the tubes. Brains were embedded in OCT freezing medium and 18 pm sections were cut on a CM3050 S cryostat (Leica Biosystems) and collected on BOND Plus Slides (Leica Microsystems).
  • microglia/macrophages-specific marker Ibal revealed an abundance of microglia/macrophage in the injected hemisphere compared to the non-injected hemisphere ( Figure 7i and Figure 7j).
  • Microglia/macrophages that infiltrated into the core of the graft displayed an amoeboid morphology characteristic of activated microglia compared to those at the periphery of the grafts which showed a ramified morphology typical of quiescent cells.
  • infiltrated microglia stained positively for CD68, a marker for activated microglia.
  • DAT dopamine transporter
  • Example 13 Transplantation of CLiPS-derived dopaminergic neurons in fully immunocompetent rodent rat models of Parkinson’s Disease
  • MFB lesions are known to cause a more complete depletion of the dopamine system compared to striatal lesions and are therefore presumed to be less likely to lead to spontaneous recovery (Torres, E.M. and S.B. Dunnett, Animal Models of Movement Disorders: Volume I, E.L. Lane and S.B. Dunnett, Editors. 2012, Humana Press: Totowa, NJ. p. 267-279).
  • the rats were fully immunocompetent and no immunosuppression was administered prior to or following transplantation.
  • female Wistar rats of ⁇ 8 weeks old were purchased from InVivos Pte Ltd.
  • Unilateral lesion was induced by stereotaxic injection of 20 pg of 6-OHDA in 4 pl 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 dura.
  • MBB medial forebrain bundle
  • apomorphine-induced rotations were scored as described in Example 12. Rats displaying >6 rotations/min were transplanted with 3 pl of about 1.25xl0 5 cells/pl Day25 dopaminergic progenitors into the left striatum at the following coordinates with reference to Bregma: AP +0.8 mm; ML -2.5 mm; and DV -5 mm from dura.
  • Example 14 Methods of differentiating and characterizing RPE cells
  • RPE differentiation from CLiPs was achieved using a rapid and directed differentiation protocol. Differentiated RPE cells were purified and plated on transwells for further characterization, such as immunostaining and gene expression assays using quantitative reverse transcriptase polymerase chain reaction (q-RT-PCR). Functionality of the cells was analysed using transepithelial electric resistance (TEER) and phagocytosis of photoreceptor outer segments (POS).
  • TEER transepithelial electric resistance
  • POS phagocytosis of photoreceptor outer segments
  • CLiPs were cultured on Matrigel (Corning) coated tissue culture plate (Corning Costar) in mTESRl (Stem Cell Technologies) medium.
  • CLiPs and human ES cells were grown on Matrigel coated tissue culture plate in mTeSRl medium. When cells attained 90-95% confluence they were exposed to various differentiation media, containing base medium (DMEM/F12 with lx B27 and lx N2 supplements and non-essential amino acids) supplemented with various growth factors.
  • base medium DMEM/F12 with lx B27 and lx N2 supplements and non-essential amino acids
  • Differentiation medium 1 from days 0 to 2, 1 pM LDN-193189, 10 ng/ml Dkkl, 10 ng/ml IGF1 and 10 rnM nicotinamide.
  • Differentiation medium 2 from days 2 to 4, 0.2 pM LDN- 193189, 10 ng/ml Dkkl, 10 ng/ml IGF1, 10 mM nicotinamide and 5 ng/ml b-FGF.
  • Differentiation medium 3 from days 4 to 6, 10 ng/ml Dkkl and 10 ng/ml IGF1 and 100 ng/ml Activin A.
  • Differentiation medium 4 From days 6 to 8, 100 ng/ml Activin A and 10 pM SU5402.
  • Differentiation medium 5 A From days 8-11, the basal medium contained 100 ng/mL Activin A, 10 pM SU5402, and 1.5 pm CHIR99021.
  • Differentiation medium 5B from days 11- 16, 100 ng/mL Activin A, 10 pM SU5402, and 3 pM CHIR99021.
  • RPE maintenance medium 50% DMEM/F12, 50% minimum essential medium Eagle, Alpha Modification, 10 mM nicotinamide, penicillin/streptomycin, sodium pyruvate, MEM non-essential amino acids, GlutaMAX (all 1:100), N1 supplement (1:200), 0.25 mg/ml taurine, 0.02 pg/ml hydrocortisone, and 0.013 ng/ml 3,3',5-Triiodo-L-thyronine supplemented with 2% heat-inactivated fetal bovine serum (FBS).
  • FBS heat-inactivated fetal bovine serum
  • 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 pg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR was done in technical triplicates (10 pl reactions) in a 96-well plate using KAPA SYBR FAST on a Quant Studio 3 Real-Time PCR Systems (Thermo Fischer).
  • Gene specific primers designed to create 75-200 base pair PCR product included OCT4, NANOG and SOX2 for pluripotency markers, OTX2, LHX2, RAX and SIX3 as early eye field markers, PAX6, MITF, VSX2 and SOX10 as early RPE markers, BEST- 1, PMEL 17, MERTK, TYROSINASE, TRYP2, and RPE65 as mature RPE markers.
  • the data were normalized to the “housekeeping” gene glyceraldehyde phosphate dehydrogenase (GAPDH).
  • rabbit zona-occludens 1 ZO-1, 1:200, Invitrogen
  • rabbit Occludin (1:125, Invitrogen)
  • mouse Retinal Pigment Epithelium-Specific 65 KDa Protein RPE65, 1:125, Abeam
  • mouse cellular retinaldehyde-binding protein CRALBP, 1:1000, Abeam
  • mouse bestrophin BEST-1, 1:125, Abeam
  • mouse Na+/ATPase (1:250, Invitrogen
  • mouse Ezrin (1:200, Abeam
  • mouse claudin 19 (1: 125).
  • TEER development of epithelial barrier properties and tight junction formation between the RPE cells reflecting integrity and polarity of RPE monolayer was determined by measuring TEER. For this, cells were cultured on permeable 0.4-pm 24-well Transwell inserts (Corning) coated with Synthemax-II (Corning). TEER measurements were taken every week, using the Epithelial Volt Ohm meter - EVOM2 (World Precision Instruments) following the manufacturer’s instructions. Briefly, electrodes were sterilized with 70% ethanol, air dried and equilibrated in RPE medium and placed in the transwell filter with the longer electrode positioned in the lower chamber touching the bottom of the dish and the shorter electrode in the upper chamber. Net TEER (Q.cm2) was calculated by subtracting the resistance values of experimental trans wells from those of controls, trans wells in which no cells were plated, and multiplying net values by the area of the filter membrane.
  • POS was isolated from porcine eyes collected from a local abattoir. They were halved with blade and the retinas were removed using forceps in a dark room under red light. Retinas were placed in the homogenisation medium, thoroughly mixed and filtered. The retina suspension was layered on top of a sucrose gradient (25-60%) and centrifuged at 112,398x g for 1 hour in an ultracentrifuge (Optima ultracentrifuge, Beckman). The pink POS layer was collected and POS were labelled with fluorescein isothiocyanate (FITC, Invitrogen) in 0.1M Sodium bicarbonate buffer (pH 9.5) for 1 hour at RT.
  • FITC fluorescein isothiocyanate
  • the labelled POS was washed and stored as aliquots in -80 °C till use.
  • the RPE cells growing on transwells were challenged with FITC labelled POS for 2 hours at 37°C in 5% CO2 incubator or 4°C for control. Cells challenged with unlabelled POS were used as controls. The wells were then washed thrice with PBS to remove unbound POS and dissociated to single cells using TrypLE (Gibco). FITC fluorescence was measured using BD LSR II Flow Cytometer to determine POS phagocytosis.
  • Non-RPE cells in differentiation cultures were removed manually by scraping with a 10 pl tip attached to the pipette by observing the culture plate under a dissection microscope . The plate was washed with PBS, 3 times to remove all the non-RPE cells. The remaining cells, highly enriched in RPEs, were dissociated by adding fresh TrypLE and incubation for 5-10 minutes.
  • the plate was thoroughly washed with PBS, 3 times to remove all the non-RPE cells and the tightly attached RPE cells were dissociated by incubating with fresh TrypLE for 5-10 minutes. Both tightly attached RPE and loosely attached non-RPE cells were plated for experiments.
  • the inventors used an RPE differentiation method to generate RPE from CLiPS, which can be of either mesenchymal (CLMC) or ectodermal (CLEC) origin (Table 4).
  • Table 4 Different stem cells used in this study [244] The inventors used ES cell and skin iPS cells as controls in RPE differentiation (Table 4). Using this method, CLiPS robustly differentiated into RPEs ( Figure 13).
  • Example 16 CLiPS have consistently high RPE differentiation efficiency than skin iPS cells
  • Example 17 CLiPS-derived RPEs have more pigmentation than ES-derived RPE
  • CLiPS-RPEs were determined by measuring Trans-epithelial electrical resistance (TEER) and phagocytosis of FITC labelled photoreceptor outer segments.
  • TEER Trans-epithelial electrical resistance
  • phagocytosis Figure 17b
  • Example 20 CLiPS-RPEs express protein similar to that of ES-derived RPEs
  • RPE purification was done with 30-35 day old differentiation cultures, by when the RPEs have gained pigmentation.
  • Methods to purify RPE from mixed differentiation cultures include manual removal of non-RPE cells at day 14 of differentiation based on the morphological differences between RPE and non-RPE cells (Foltz and Clegg, 2017), selective removal of non- RPE cells which are weakly attached to the culture dish by short treatment with a weak dissociation agent such as TrypLE or Accutase (Nazari et al., 2015, Yuko Iwasaki et al., 2016) and scatter sorting based on the higher scatter of light by melanosomes in the RPE cells (Shih et al., 2017).
  • a weak dissociation agent such as TrypLE or Accutase
  • the inventors performed RPE purification with 30-35 day old differentiation cultures unlike 14 day old cultures descried previously (Foltz and Clegg, 2017). By 30-35 days, RPE cells in differentiation cultures acquire brown pigmentation which helps to distinguish RPE cells from non-RPE cells in manual purification. The inventors compared different methods ( Figures 20a and b) to identify which method would give functional RPE of highest purity and yield.
  • the inventors introduced a control for low scatter using the weakly attached non-RPE cells in the differentiation culture (collected from partial TrypLE treatment). RPE cells was sorted and selected based on scatter high gate ( Figure 20d). This two- step purification involving the removal of non-RPE cells by TrypLE first before flow cytometry, helped to reduce sorting time. Percentage yield of RPE cells was calculated by number of RPE cells obtained after purification / total number of cells in mixed population of RPE cells prior to purification x 100%.
  • RPE cells Since preferential dissociations are expected to remove non-RPE cells, all cells obtained after purification were considered as RPE cells. Percentage yield of RPEs after purification was calculated from total number of cells present in differentiation cultures and the number of cells obtained after purification. For purification the inventors used a differentiation culture from HDFA iPS cells that contained 51% Pmell7 positive cells by flow cytometry, suggesting it contained 51% RPE cells. Hence the maximum achievable RPE yield with this culture was 51%. TrypLE purification yielded 50% RPE cells, very close to the maximum expected yield of 51%. Manual and TrypLE + manual gave slightly lower yields, 47.7 and 43.4% respectively. Methods involving scatter sorting gave low yield (21-22%) ( Figure 20e).
  • TrypLE purification would be an efficient way to generate RPE cells for most general research purposes because of its ease of purification, high yield, purity and functionality. TrypLE+ manual method, which involves an additional manual purification step to remove any non-RPE cells that might have escaped TrypLE treatment, would be ideal for generating RPE cells for transplantation.
  • the inventors compared the expression of BEST1, RPE65, MERTK, MITF, PMEL17, RLBP1 and TRYP2 in purified CLMC23 and H9 (Figure 20j). Majority of these genes showed an increased expression in CLMC23 compared to the ES-derived H9 RPE ( Figure 20k).
  • CLiPs-RPE also show higher glycolysis and oxidative phosphorylation compared to both skin-iPSC-RPE (ASF5-RPE) and hESC-RPE (H9-RPE) ( Figures 21a and b).
  • ASF5-RPE skin-iPSC-RPE
  • H9-RPE H9-RPE
  • CLiPs-RPE have higher bioenergetic profile compared to hESC-cell derived RPE. Healthy RPEs exhibit higher glycolysis and mitochondrial function compared to RPEs from AMD patients (Ferrington et al, 2017). Higher OCR and ECAR of CLiPS-RPE suggest they could be superior to hESC-derived RPE for clinical use.
  • Bioluminescent RPE lines were established using a luciferase (Luc) gene-encoding vector tagged with GFP, delivered via lentiviral infection. Stable expression of Luc in these lines was confirmed by analyzing the bioluminescent intensity.
  • the matrigel plug assay was adopted from previous publication (PMID: 15780993). The RPE-matrigel plugs were transplanted subcutaneously in humanized mice. The bioluminescence of RPE-matrigel plugs were monitored at regular intervals over a time course of 2 months using the bioluminescent imaging system.
  • the graft was extracted at end-point and immunofluorescence analysis was conducted using a mature RPE marker (RPE65) and a proliferation marker (Ki67) (Figure 22c). In all RPE lines tested, the expression of RPE65 was observed and Ki67 was absent, confirming the mature and quiescent state.
  • Example 25 Monitoring of pro-inflammatory cytokines as a surrogate for cellular immune response
  • Immunofluorescence analysis was conducted to observe immune cell infiltration using human CD45 (hCD45) marker.
  • OTX2 a RPE-specific transcription factor, was used to distinguish the RPE cells (Figure 23c). Congruent with the low levels of IFN-y and IL-18 cytokines, immune cell infiltration was indeed absent in CLEC23-RPE.
  • CLEC23-RPE had the lowest immune cell infiltration.
  • Example 26 CLEC23-RPE may modulate T cell activation to confer hypoimmunogenicity
  • the term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
  • the term “about” means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, most preferably plus or minus 1%.
  • a method of generating an induced pluripotent stem cell comprising expressing exogenous nucleic acids encoding proteins OCT3/4, SOX2, KLF4, LIN28 and L-MYC and p53-shRNA in a stem cell of the amniotic membrane of the umbilical cord under conditions suitable to reprogram the stem cell, thereby generating the induced pluripotent stem cell.
  • stem cell of the amniotic membrane of the umbilical cord is a mesenchymal stem cell of the amniotic membrane of the umbilical cord or an epithelial stem cell of the amniotic membrane of the umbilical cord.
  • mesenchymal stem of the amniotic membrane of the umbilical cord is a mesenchymal stem cell population, wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105.
  • exogenous nucleic acids encoding the proteins OCT3/4, SOX2, KLF4, LIN28 and L-MYC and the p53-shRNA are provided by one, two or three vectors, wherein preferably a first vector encodes the protein OCT3/4 and the 53-shRNA, a second vector encodes the proteins SOX2 and KLF4 and a third vector encodes the proteins L-MYC and LIN28.
  • the stem cell of the amniotic membrane of the umbilical cord is subjected to transfection to transfer the exogenous nucleic acids into the stem cell.
  • the ratio of the amount of vector (plasmid) DNA for each vector to the number of mesenchymal stem cells of the amniotic membrane of the umbilical cord subjected to electroporation is in the range of about 1.5pg plasmid DNA to about IxlO 6 CLMC to of about 2.5 pg DNA to about IxlO 6 CLMC, wherein the ratio is, for example, about 2.5 pg plasmid DNA : IxlO 6 cells, about 2.25 pg plasmid DNA : IxlO 6 cells, about 1.8 pg plasmid DNA: IxlO 6 cells, about 1.7 pg plasmid DNA : IxlO 6 cells, about 1.6 pg plasmid DNA : IxlO 6 cells, about 1.5 pg plasmid DNA : IxlO 6 cells, or preferably about 1.67 : IxlO 6 cells.
  • the ratio of the amount of vector (plasmid) DNA for each vector to the number of epithelial stem cells of the amniotic membrane of the umbilical cells subjected to electroporation is in the range of about 1.5pg DNA to about IxlO 6 cells to about 2.5pg DNA to about IxlO 6 cells, wherein the ratio is, for example, about 1.5 pg plasmid DNA : IxlO 6 cells, about 1.6 pg plasmid DNA : IxlO 6 cells, about 1.7 pg plasmid DNA : IxlO 6 cells, about 1.8 pg plasmid DNA : IxlO 6 cells, about 1.9 pg plasmid DNA : IxlO 6 cells, about 2.0 pg plasmid DNA : IxlO 6 cells, about 2.5 pg plasmid DNA : IxlO 6 cells, preferably about 1.67 pg plasmid DNA : IxlO 6 cells.
  • the method of item 13, wherein the medium suitable for the recovery of a transfected mesenchymal stem cell of the amniotic membrane of the umbilical cord consists of about 85 to 95 % (v/v) defined medium and 5 to 15 % (v/v) fetal bovine serum.
  • the medium of item 15, wherein the medium suitable for the recovery of a transfected mesenchymal stem cell of the amniotic membrane of the umbilical cord consists of about 90% (v/v) chemically defined medium and about 10% (v/v) fetal bovine serum.
  • the method of item 13 or 14, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises Mammary Epithelial Basal Medium MCDB 170, EpiLife medium, DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium) and FBS (Fetal Bovine Serum).
  • the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 10 to about 30 % (v/v), EpiLife medium in a final concentration of about 20 to about 40 % (v/v), F12 in a final concentration of about 5 to about 15 % (v/v), DMEM in a final concentration of about 30 to about 45 % (v/v) and FBS in a final concentration of about 0.1 to 2 % (v/v).
  • the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 15 to about 25 % (v/v), EpiLife medium in a final concentration of about 25 to about 35 % (v/v), F12 in a final concentration of about 7.5 to about 13 % (v/v), DMEM in a final concentration of about 35 to about 40 % (v/v) and FBS in a final concentration of about 0.5 to 1.5 % (v/v).
  • the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 20 % (v/v), EpiLife medium in a final concentration of about 30 % (v/v), F12 in a final concentration of about 12.5 (v/v), DMEM in a final concentration of about 37.5 % (v/v) and FBS in a final concentration of about 1.0 % (v/v).
  • the method of any of items 18 to 21, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord is obtained by mixing to obtain a final volume of 1000 ml culture medium:
  • the method of any of items 18 to 22, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises insulin in a final concentration of about 1 to about 7.5 pg/ml.
  • the method of any of items 18 to 24, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises human epidermal growth factor in a final concentration of about 1 to about 15ng/ml.
  • the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord further comprises at least one of the following supplements: adenine, hydrocortisone, and 3, 3', 5- Triiodo-L-thyronine sodium salt (T3).
  • the method of item 25, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises all three of adenine, hydrocortisone, and 3, 3',5-Triiodo-L-thyronine sodium salt (T3).
  • the method of any of items 18 to 26, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord further comprises one of more Transforming Growth Factors (TGF).
  • TGF Transforming Growth Factors
  • the method of item 27, wherein the medium comprises Transforming Growth Factor beta (TGF-beta) and/or transforming growth factor alpha.
  • TGF-beta Transforming Growth Factor beta
  • the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord further comprises Cholera Toxin from Vibrio cholerae.
  • the method of any one of items 14 to 29, wherein the medium suitable for cell recovery contains a compound suppressing inflammatory response and enhancing cell survival.
  • the method of item 30, wherein the compound is a glucocorticoid.
  • the method of item 31, wherein the glucocorticoid is selected from the group consisting of prednisolone, methylprednisolone, dexamethasone, betamethasone, corticosterone and hydrocortisone.
  • the method of item 31 or 32, wherein the hydrocortisone concentration is about 0.5pM to about 2pM.
  • the method of any one of items 13 to 33, wherein the cultivation is carried out in a coated cell culture vessel, wherein the cell culture vessel is preferably coated with a serum-derived substrate or a serum-free substrate.
  • any one of items 14 to 34 wherein the medium suitable for cell recovery is replaced with a mixture of two different cell culture media about 1, 2 or 3 days after transfection, preferably about 2 days after transfection, thereby yielding an induced pluripotent stem cell colony.
  • the method of item 35 wherein the two different cell culture media are the medium suitable for cell recovery and a second cell culture medium.
  • the method of item 35 or 36 wherein the two different cell culture media are mixed in a ratio of about 1:1 (v/v) prepared by contacting 1 volume medium suitable for cell recovery to 1 volume second cell culture medium.
  • the second cell culture medium is a maintenance medium for cultivation of induced pluripotent stem cells, wherein the medium is preferably selected from the group consisting of mTeSRl, StemMACSTM iPS-Brew XF, TeSRTM E8, mTeSRTMPlus, TeSRTM2, mTeSRTMl, Corning® NutriStem® hPSC XF Medium, Essential 8 Medium, StemFlex, StemFit Basic02 and PluriSTEM.
  • any one of items 35 to 39 wherein the mixture of cell culture media is replaced with the second cell culture medium within about 5, 6 or 7 days after transfection, preferably about 6 days after transfection.
  • the method of item 40 wherein the second cell culture medium is changed daily or every second day, third day, preferably every second day.
  • the method of item 40 or 41 wherein an induced pluripotent stem cell colony is selected when reaching a size of about 0.5mm to about 1.5mm in diameter, and the selected induced pluripotent stem colony is transferred to a coated cell culture vessel for cultivation and proliferation.
  • the method of item 42 wherein the induced pluripotent stem cell colony is selected under bright field microscopy.
  • the method of item 42 or 43 wherein the cell culture medium is changed daily or every second day, preferably every day.
  • the method of item 45 or 46 wherein a cell population formed from the induced pluripotent stem cell colony is passaged when reaching about 60-90% confluence, preferably when reaching 70-80% confluence.
  • the method of item 47 wherein the cell population formed from the induced pluripotent stem cell colony is passaged in a ratio of about 1:3 (v/v), wherein the passaging in a ratio of about 1:3 (v/v) is performed by dividing about 1 volume dissociated induced pluripotent stem cells into about 2 volumes of dissociated induced pluripotent stem cells.
  • An induced pluripotent stem cell population obtainable by the method as defined in any of items 1 to 51.
  • a pharmaceutical composition comprising an induced pluripotent stem cell as defined in item 52 or 53.
  • the target cell is selected from the group consisting of a dopaminergic neuronal cell, an oligodentrocyte, a hepatocyte, a cardiomyocyte, a hematopoietic progenitor cell, a blood cell, a neuronal cell, a motor neuron, a cartilage cell, a muscle cell, a bone cell, a dental cell, a hair follicle cell, an inner ear hair cell, a skin cell, a melanocyte, an immune cell, an astrocyte, a reproductive cell, a corneal cell, an intestinal cell, a lung cell, a kidney cell, a stomach cell, a mesenteric cell, and a fat cell.
  • the method of item 56 wherein the immune cell is selected from the group consisting of a T-lympocyte, a B-lymphocyte, a microglia, and a natural killer cell.
  • the method of item 56 wherein the induced pluripotent stem cell is cultivated in a medium adapted for proliferation and differentiation of the induced pluripotent stem cell into a dopaminergic neuronal cell.
  • the method of item 56 wherein the induced pluripotent stem cell is cultivated in a medium adapted for proliferation and differentiation of the induced pluripotent stem cell into a hepatocyte.
  • the method of item 56 wherein the induced pluripotent stem cell is cultivated in a medium adapted for proliferation and differentiation of the induced pluripotent stem cell into a cardiomyocyte.
  • the method of item 60 wherein the induced pluripotent stem cell is cultivated in a medium adapted for proliferation and differentiation of the induced pluripotent stem cell into an oligodentrocyte.
  • a pharmaceutical composition comprising a differentiated induced pluripotent stem cell obtained by the method as defined in any of items 56 to 61.
  • the pharmaceutical composition of item 62 wherein the pharmaceutical composition is adapted for parenteral application.
  • a method of treating a congenital or acquired degenerative disorder in a subject comprising administering to a subject a target cell differentiated from pluripotent stem cell by the method as defined in any of items 56 to 61.
  • the method of item 64 wherein the disorder is a neural disorder.
  • the method of item 65, wherein the disease is neural disorder is selected from the group consisting of Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Amyotrophic lateral sclerosis, multiple sclerosis and batten disease.
  • the method of item 64, wherein the disorder is a hepatic disorder.
  • a cell culture medium comprising Mammary Epithelial Basal Medium MCDB 170, EpiLife medium, DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium) and FBS (Fetal Bovine Serum).
  • the cell culture medium of item 71 wherein the medium comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 10 to about 30 % (v/v), EpiLife medium in a final concentration of about 20 to about 40 % (v/v), Fl 2 in a final concentration of about 5 to about 15 % (v/v), DMEM in a final concentration of about 30 to about 45 % (v/v) and FBS in a final concentration of about 0.1 to 2 % (v/v).
  • the medium comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 10 to about 30 % (v/v), EpiLife medium in a final concentration of about 20 to about 40 % (v/v), Fl 2 in a final concentration of about 5 to about 15 % (v/v), DMEM in a final concentration of about 30 to about 45 % (v/v) and FBS in a final concentration of about 0.1 to 2 % (v/v).
  • the cell culture medium of item 72 wherein the medium comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 15 to about 25 % (v/v), EpiLife medium in a final concentration of about 25 to about 35 % (v/v), Fl 2 in a final concentration of about 7.5 to about 13 % (v/v), DMEM in a final concentration of about 35 to about 40 % (v/v) and FBS in a final concentration of about 0.5 to 1.5 % (v/v).
  • the medium comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 15 to about 25 % (v/v), EpiLife medium in a final concentration of about 25 to about 35 % (v/v), Fl 2 in a final concentration of about 7.5 to about 13 % (v/v), DMEM in a final concentration of about 35 to about 40 % (v/v) and FBS in a final concentration of about 0.5 to 1.5 % (v/v).
  • the cell culture medium of item 73 wherein the medium comprises Mammary Epithelial Basal Medium MCDB 170 in a final concentration of about 20 % (v/v), EpiLife medium in a final concentration of about 30 % (v/v), F12 in a final concentration of about 12.5 (v/v), DMEM in a final concentration of about 37.5 % (v/v) and FBS in a final concentration of about 1.0 % (v/v).
  • the cell culture medium of any of items 71 to 77, wherein the medium suitable for the recovery of a transfected epithelial stem cell of the amniotic membrane of the umbilical cord comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-Triiodo-L-thyronine sodium salt (T3).
  • the cell culture medium of item 78 wherein the medium comprises all three of adenine, hydrocortisone, and 3,3',5-Triiodo-L-thyronine sodium salt (T3).
  • the cell culture medium of item 79 wherein the culture medium comprises adenine in a final concentration of about 0.05 to about 0.1 mM adenine, hydrocortisone in a final concentration of about 0.1 to 0.5 pM hydrocortisone and/or 3,3',5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.1 to about 5 ng/ml.
  • TGF Transforming Growth Factors
  • TGF-beta 1 Transforming Growth Factor beta 1
  • TGF-alpha transforming growth factor alpha

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PCT/SG2023/050051 2022-01-27 2023-01-27 A method of differentiating an induced pluripotent stem cell into a retinal pigment epithelial cell, a retinal pigment epithelial cell and methods of using the retinal pigment epithelial cell Ceased WO2023146477A2 (en)

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CA3249352A CA3249352A1 (en) 2022-01-27 2023-01-27 METHOD FOR DIFFERENTIATING AN INDUCED MULTIPOTENTED STEM CELL INTO A RETINAL PIGMENTED EPITHELIAL CELL, RETINAL PIGMENTED EPITHELIAL CELL AND METHODS FOR USING THE RETINAL PIGMENTED EPITHELIAL CELL
EP23747458.0A EP4469561A4 (en) 2022-01-27 2023-01-27 METHOD FOR DIFFERENTIATING AN INDUCED MULTIPOTENTED STEM CELL INTO A RETINAL PIGMENTED EPITHELIAL CELL, RETINAL PIGMENTED EPITHELIAL CELL AND METHODS FOR USING THE RETINAL PIGMENTED EPITHELIAL CELL
JP2024544656A JP2025507265A (ja) 2022-01-27 2023-01-27 人工多能性幹細胞を網膜色素上皮細胞に分化させる方法、網膜色素上皮細胞、および網膜色素上皮細胞を使用する方法
AU2023212665A AU2023212665A1 (en) 2022-01-27 2023-01-27 A method of differentiating an induced pluripotent stem cell into a retinal pigment epithelial cell, a retinal pigment epithelial cell and methods of using the retinal pigment epithelial cell
CN202380031502.8A CN119497749A (zh) 2022-01-27 2023-01-27 一种使诱导多能干细胞分化为视网膜色素上皮细胞的方法、视网膜色素上皮细胞和使用该视网膜色素上皮细胞的方法
US18/833,821 US20250197803A1 (en) 2022-01-27 2023-01-27 A method of differentiating an induced pluripotent stem cell into a retinal pigment epithelial cell, a retinal pigment epithelial cell and methods of using the retinal pigment epithelial cell

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EP4306634A4 (en) * 2021-03-09 2025-03-26 Riken METHOD FOR PRODUCING HYPOIMMUNOGENIC RETINAL PIGMENT EPITHELIAL CELLS

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EP2951290B1 (en) * 2013-02-01 2017-11-29 The United States of America, as represented by The Secretary, Department of Health and Human Services Method for generating retinal pigment epithelium (rpe) cells from induced pluripotent stem cells (ipscs)
CN108531443A (zh) * 2018-04-04 2018-09-14 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 小分子诱导多能性干细胞分化为视网膜色素上皮细胞的方法
CN110205283B (zh) * 2018-04-18 2021-04-23 浙江大学 一种诱导人羊膜上皮细胞向视网膜色素上皮细胞分化的方法及其应用

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EP4306634A4 (en) * 2021-03-09 2025-03-26 Riken METHOD FOR PRODUCING HYPOIMMUNOGENIC RETINAL PIGMENT EPITHELIAL CELLS

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