Process for obtaining functional lymphocytes

Abstract

The present invention relates to the use of the following compounds: Activin A, BMP4, CHIR99, EGF, FGF8, FGF10, IGF1, LY3, Noggin, retinoic acid, and Y27, for carrying out the differentiation process of induced pluripotent stem cells, i.e., iPSc, into functional thymic epithelial progenitor cells, i.e., TEP, preferably in vitro or ex vivo.

Description

Technical Field

[0001] The present invention relates to a process for obtaining functional lymphocytes and to such cells.

[0002] The thymus provides a microenvironment essential for the development and maturation of T cells. Thymic epithelial cells (TECs), which are composed of thymic cortical epithelial cells (cTECs) and thymic medullary epithelial cells (mTECs), have been well demonstrated to be important for these tightly regulated processes.

[0003] The TEC population is known to be an important regulator of different T cell developmental programs, but recent studies have revealed significant new TEC heterogeneity that needs to be considered in the context of understanding microenvironmental control of T cell development. Importantly, therapeutic interventions can also be detrimental to thymic function. Such clinical procedures include ablative preconditioning used in the treatment of cancer, which then impairs T cell-mediated immune reconstitution after bone marrow transplantation. As a result, studying thymic epithelial cells in both healthy and diseased states is important for understanding how thymic function is regulated and how it can be manipulated for therapeutic benefit. Recently, significant progress has been made in understanding the biology of thymic epithelium, including the developmental pathways that give rise to different cortical and medullary epithelial lineages. Furthermore, there has been progress in mapping how newly identified heterogeneity in thymic epithelium can be related to functional specialization in the thymic microenvironment.

[0004] In vitro generation of functional thymic epithelial cells (TECs) or TEC progenitors (TEPs) from human pluripotent stem cells (hPSCs) can generate cells, tissues, or organs that assist in T cell reconstitution in patients with congenital disorders such as DiGeorge syndrome, resulting in thymic dysfunction, and acquired dysfunction due to long-term immunosuppressive therapy combined with HIV infection, high-dose chemotherapy and radiotherapy treatments, graft-versus-host disease, and aging (which itself results in insufficient thymopoietic function). The number of TECs in the human adult thymus is limited, and it is difficult to define a reliable method to expand them from the postnatal thymus; thus, generating TECs from pluripotent stem cells (PSCs) is an important goal. Creating an in vitro protocol for tightly controlled differentiation of hPSCs into TECs requires precise knowledge and application of developmental and cytokine cues. Generation of functional TEPs from mouse or human PSCs that support mouse or human T cell development has been described following transplantation of TEPs under the renal capsule in mice, but high-level in vitro reconstitution of naive human T cells by potent PSC-derived TECs has not been demonstrated. Therefore, there is a need in the art for methods to generate TEPs and TECs that can support efficient T cell development in vitro.

[0005] If functional TECs can be generated from human pluripotent stem cells, they would have important applications in modeling the human immune response in mice and in modeling and treating thymic deficiency syndromes such as immunodeficiencies that complicate bone marrow transplantation for DiGeorge syndrome, Nude syndrome, and leukemia. The cells could also be clinically used for cell therapy and transplanted into patients to achieve T cell reconstitution, generate immune tolerance to prevent graft rejection after organ transplantation, or restore thymic function impaired by injury or aging.

[0006] International Application No. 2020205859 discloses a method for inducing in vitro differentiation of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into thymic epithelial cell precursors (TEC precursors), where thymic epithelial cells (TECs) or thymic epithelial cell precursors (TEPs) can generate thymic organs and T cells in vivo. This application teaches a differentiation culture medium supplemented with noggin, retinoic acid, FGF8b, sonic hedgehog, BMP, and YM155. In this application, the inventors show that the generation of TEC precursors and subsequent maturation in TECs can occur after transplantation into mice. International Application No. 2020220040 also teaches a process for obtaining TEPs and TECs.

[0007] However, in vitro functional thymic lymphopoiesis for clinical use and therapy, i.e., T cell generation, still cannot be easily achieved considering the essential animal transplantation.

[0008] Therefore, there is a need to provide an in vitro process for producing functional T cells from iPSCs for later free use in therapy. One object of the present invention is to provide a method for producing thymic epithelial cells and thymic epithelial precursors from pluripotent cells, specifically from iPSCs.

[0009] Another object of the present invention is to provide a method for treating conditions associated with thymic deficiency.

[0010] The present invention relates to the use of a composition comprising the following compounds: activin A, BMP4, CHIR99, EGF, FGF8, FGF10, IGF1, LY3, noggin, retinoic acid, and Y27 for performing a differentiation process of induced pluripotent stem cells, i.e., iPSCs, into functional thymic epithelial precursors, i.e., TEPs (thymic epithelial progenitor), preferably in vitro or ex vivo.

[0011] More preferably, the present invention relates to a set of the following compounds for carrying out the differentiation process of induced pluripotent stem cells, i.e., iPSc, into functional thymic epithelial precursors, i.e., TEP, preferably in vitro or ex vivo: - A first set containing a Y27 compound, - A second set containing activin A and CHIR99, - A third set containing activin A, - A fourth set containing Y27 and activin A, - A fifth set containing retinoic acid and FGF8, - A sixth set containing noggin, retinoic acid, LY3, and FGF8, - A seventh set containing retinoic acid, LY3, CHIR99, BMP4, and FGF8, - An eighth set containing retinoic acid, CHIR99, BMP4, and FGF8, and - A ninth set containing retinoic acid, BMP4, FGF8, FGF10, IGF1, EGF, and RANKL.

[0012] More preferably, the present invention relates to the use of the set of compounds described above, wherein the iPSC is - Incubated with a first set containing a Y27 compound, then - Incubated with a second set containing activin A and CHIR99, then - Incubated with a third set containing activin A, then - Incubated with a fourth set containing Y27 and activin A, then - Incubated with a fifth set containing retinoic acid and FGF8, then - Incubated with a sixth set containing noggin, retinoic acid, LY3, and FGF8, then - Incubated with a seventh set containing retinoic acid, LY3, CHIR99, BMP4, and FGF8, then - Incubated with an eighth set containing retinoic acid, CHIR99, BMP4, and FGF8, and then - Incubated with a ninth set containing retinoic acid, BMP4, FGF8, FGF10, IGF1, EGF, and RANKL.

[0013] The present invention is based on the unexpected observation made by the inventors that a specific combination of growth factors enables the differentiation of induced pluripotent stem cells into functional thymic epithelial precursors, namely TEP. Compared with the art, the TEP obtained by the present invention is fully functional and can easily and efficiently differentiate into mature TEC, namely medullary TEC (mTEC) and cortical TEC (cTEC). The thymic gland can also be obtained by using the above-described composition in addition to the differentiation process.

[0014] As used herein, the terms "differentiation" and "cell differentiation" refer to the process by which less specialized cells (i.e., stem cells) develop or mature or differentiate into more specialized cells or differentiated cells (i.e., thymic epithelial cells) having a more distinct form and / or function.

[0015] As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably, and all such names include progeny. Thus, the terms "transformant" and "transformed cell" include the culture derived therefrom regardless of the number of primary target cells and transfers. It is also understood that due to intentional or accidental mutations, not all progeny will have exactly the same DNA content. Mutant progeny having the same function or biological activity as screened in the originally transformed cell are included. If a different name is intended, it will be apparent from the context.

[0016] As used herein, the term "induced pluripotent stem cell" is generally abbreviated as iPS cell or iPSC or IPSc, and refers to a type of pluripotent stem cell artificially generated from non-pluripotent cells, typically adult somatic cells, or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, and the like.

[0017] IPSc are non-natural cells that have been genetically modified to force the expression of several genes that restore the differentiation program. As a result, the specialized acquired functions of the differentiated cells are lost, but pluripotency is acquired.

[0018] Pluripotency refers to the ability of cells to develop into the three primary germ cell layers of the early embryo and thus into all cells of the adult body, but not into extra-embryonic tissues such as the placenta. Embryonic stem cells and induced pluripotent stem cells are characterized by their pluripotency. Only totipotent cells can give rise to all differentiated cells. These totipotent cells are not included in the present invention.

[0019] iPSCs are typically induced by introducing the products of a specific set of pluripotency-related genes or "reprogramming factors" into a given cell type. The original set of reprogramming factors (also called Yamanaka factors) is the transcription factors Oct4 (Pou5f1), Sox2, Klf4, and cMyc. This combination is the most common in the production of iPSCs, but each of the factors can also be functionally replaced by unrelated genes such as related transcription factors, miRNAs, small molecules, or lineage specifiers.

[0020] iPSC differentiation is typically a slow and inefficient process, taking 1-2 weeks in mouse cells and 3-4 weeks in human cells, with an efficiency of about 0.01-0.1%. However, significant progress has been made in improving the efficiency and the time required to obtain iPSCs. When the reprogramming factors are introduced, the cells begin to form colonies similar to pluripotent stem cells, which can be isolated based on their morphology, the conditions selected for their growth, or through the expression of surface markers or reporter genes. Further details are shown below.

[0021] In the present invention, the following compounds are used.

[0022] Activin A is a member of the TGF-β superfamily and is recognized as a multifunctional cytokine that is expressed in a wide range of tissues and cells with roles in the regulation of inflammation, fibrosis, and wound repair, and promotes atherogenesis by inhibiting foam cell formation and neointimal hyperplasia and HF.

[0023] BMP4, or bone morphogenetic protein 4, is a member of the bone morphogenetic protein family, which is part of the transforming growth factor-beta superfamily. The superfamily includes a large family of growth factors and differentiation factors. BMP4 is highly conserved evolutionarily. BMP4 is found in the ventral marginal zone and in the eye, heart blood, and otic vesicle during early embryonic development.

[0024] CHIR99, or CHIR99021, or -[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile is an aminopyrimidine derivative of the following formula

[0025]

Chemical formula

[0026] EGF, epidermal growth factor, is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. In humans, EGF has 53 amino acids (sequence NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR, SEQ ID NO: 1) and a molecular weight of approximately 6 kDa. It contains three disulfide bridges (Cys6-Cys20, Cys14-Cys31, Cys33-Cys42).

[0027] FGF8, fibroblast growth factor 8, is important and necessary for setting and maintaining the midbrain / hindbrain boundary (or mesencephalon / met-encephalon boundary), which plays an important role of "organizer" in development, like the Spemann "organizer" of the gastrulating embryo. FGF8 is expressed in the region where Otx2 and Gbx2 mutually cross-inhibit each other, and its expression is maintained by this interaction. Once expressed, FGF8 induces other transcription factors to form an intercellular cross-regulatory loop, thus establishing the boundary. Throughout development, FGF8 regulates the growth and differentiation of progenitor cells in this region to bring about the final structures of the midbrain and hindbrain. It has been demonstrated that FGF8 is sufficient to induce the re-patterning of midbrain and hindbrain structures.

[0028] FGF10, fibroblast growth factor 10, is a member of the fibroblast growth factor (FGF) family. FGF family members have broad mitogenic and cell survival activities and are involved in various biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion. Fibroblast growth factor 10 is a paracrine signaling molecule first found in limb bud and organogenesis development. FGF10 initiates limb development and is involved in the branching of morphogenesis in multiple organs such as the lung, skin, ear, and salivary gland. FGF10 signaling is necessary for epithelial branching. Therefore, all branched morphogenetic organs such as the lung, skin, ear, and salivary gland require a certain level of FGF10 expression. This protein shows no activity against fibroblasts.

[0029] IGF-1, insulin-like growth factor 1, also called somatomedin C, is a hormone with a molecular structure similar to insulin that plays an important role in childhood growth and has anabolic effects in adults.

[0030] LY3, that is, LY364947, also called 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, is a compound of the following formula,

[0031]

Chem.

[0032] Noggin, also known as NOG, is a protein involved in the development of many tissues including nervous tissue, muscle, and bone. The amino acid sequence of human Noggin is highly homologous to those of rat, mouse, and Xenopus laevis (aquatic frog genus). Noggin is an inhibitor of several bone morphogenetic proteins (BMPs), and inhibits at least BMP2, BMP4, BMP5, BMP6, BMP7, BMP13, and BMP14.

[0033] Retinoic acid (all-trans retinoic acid, also called RA (Retinoic acid)) is a metabolite of vitamin A1 (all-trans retinol) that mediates the functions of vitamin A1 necessary for growth and development. All-trans retinoic acid is required in chordates including all higher animals from fish to humans. During early embryonic development, all-trans retinoic acid produced in specific regions of the embryo acts as an intercellular signaling molecule that induces the development of the posterior part of the embryo, thereby helping to determine the position along the anterior / posterior axis of the embryo. RA acts via Hox genes, which ultimately control anterior / posterior patterning during the early developmental stages.

[0034] Y-27632, hereinafter Y27, (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride is a compound of the following formula:

[0035]

Chem.

[0036] Advantageously, the above compounds are used simultaneously, separately, or sequentially.

[0037] It is more advantageous for the compounds used to induce the differentiation of iPSCs into TEP to be used sequentially.

[0038] Advantageously, the present invention relates to the use as defined above, wherein the differentiation process is carried out for at least 14 days.

[0039] It is essential to use the composition as defined above for at least 14 days to carry out the differentiation process from iPSCs to TEP.

[0040] More advantageously, the compounds contained in the composition as defined above can be used continuously at least once during the above-mentioned at least 14 days.

[0041] For example, the Y27 compound can be used at the start of the differentiation process, and then the CHIR99 compound and actinomycin A, further retinoic acid and FGF8, further BMP4 and LY3 compound, and then FGF10, IGF1, and EGF can be used.

[0042] Advantageously, the present invention relates to the use as defined above, wherein the iPSc expresses the OCT4 and NANOG genes.

[0043] The generation of induced pluripotent cells largely depends on the transcription factors used for induction. Certain products of the Oct-3 / 4 and Sox gene families (Sox1, Sox2, Sox3, and Sox15) have been identified as important transcriptional regulators involved in the induction process whose absence renders induction impossible. However, additional genes including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28 have been identified to increase the induction effect.

[0044] Oct-3 / 4 (Pou5f1): Oct-3 / 4 is one of the family of octamer ("Oct") transcription factors and plays an important role in maintaining pluripotency. The absence of Oct-3 / 4 in Oct-3 / 4+ cells such as blastomeres and embryonic stem cells leads to spontaneous trophoblast differentiation, and thus the presence of Oct-3 / 4 gives rise to the pluripotency and differentiation potential of embryonic stem cells. Various other genes of the "Oct" family, including Oct1 and Oct6, which are closely related to Oct-3 / 4, cannot induce induction, thus demonstrating the exclusivity of Oct-3 / 4 for the induction process. However, the team led by Hans Scholer (who discovered the Oct4 gene in 1989) showed that overexpression of Oct4 during reprogramming causes epigenetic changes that deteriorate the quality of iPSCs.

[0045] Sox Family: The Sox family of transcription factors is associated with maintaining pluripotency, similar to Oct-3 / 4. In contrast to Oct-3 / 4, which is exclusively expressed in pluripotent stem cells, it is associated with multipotent and unipotent stem cells. Sox2 was the first gene used in induction, but other transcription factors of the Sox family have also been found to function similarly in the induction process. Sox1 produces iPS cells with the same efficiency as Sox2, and the genes Sox3, Sox15, and Sox18 also generate iPS cells, but with reduced efficiency.

[0046] Klf Family: Klf4 of the Klf family of transcription factors was first identified. It has been demonstrated as a factor for the generation of mouse iPS cells and as a factor for the generation of human iPS cells. Klf2 and Klf4 have been found to be factors capable of generating iPS cells, and the related genes Klf1 and Klf5 were also the same, but with low efficiency.

[0047] Myc Family: The Myc family of transcription factors is an oncogene involved in cancer. c-myc has been demonstrated to be a factor involved in the generation of mouse iPS cells and a factor involved in the generation of human iPS cells. However, the use of the "myc" family of genes in the induction of iPS cells is a problem for the fate of iPS cells as a clinical therapy because 25% of the mice transplanted with c-myc-induced iPS cells developed lethal teratomas. N-myc and L-myc have been identified to induce pluripotency with similar efficiency instead of c-myc.

[0048] Nanog: In embryonic stem cells, Nanog is necessary to promote pluripotency together with Oct-3 / 4 and Sox2. It has also been reported that it is possible to generate iPS cells using Nanog as one of the factors.

[0049] LIN28: LIN28 is an mRNA-binding protein that is expressed in embryonic stem cells and embryonic cancer cells related to differentiation and proliferation. LIN28 has been demonstrated to be a factor in iPSC generation in combination with OCT4, SOX2, and NANOG.

[0050] Glis1: Glis1 is a transcription factor that can be used together with Oct-3 / 4, Sox2, and Klf4 to induce pluripotency. It brings many advantages when used in place of C-myc.

[0051] Advantageously, the present invention relates to the use of the following compounds: Activin A, BMP4, CHIR99, EGF, FGF8, FGF10, IGF1, LY3, Noggin, Retinoic acid Y27, RANKL, IL-7, FLT3, SCF, and Glutamax for carrying out the differentiation process of induced pluripotent stem cells, i.e., iPSc, into functional thymic epithelial cells, preferably in vitro or ex vivo.

[0052] In other words, the present invention relates to the use of a composition comprising BMP4, FGF8, FGF10, IGF1, EGF, RANKL, IL7, FLT3, SCF, and glutamax for carrying out the differentiation process of TEP cells (i.e., obtained from TEP differentiated from iPSc) into TEC, preferably in vitro or ex vivo.

[0053] The inventors have discovered that the TEP obtained as defined above can be efficiently differentiated into thymic organoids or TEC by using the above-described composition.

[0054] RANKL, receptor activator of nuclear factor-kappa B ligand, is known as a type II membrane protein and is a member of the tumor necrosis factor (TNF) superfamily. RANKL has been identified to affect the immune system and control bone regeneration and remodeling. RANKL is the ligand of receptor RANK, the binding partner of the apoptosis regulatory gene osteoprotegerin (OPG), and controls cell proliferation by modifying the protein levels of Id4, Id2, and cyclin D1. RANKL is expressed in several tissues and organs including skeletal muscle, thymus, liver, colon, small intestine, adrenal gland, osteoblasts, mammary epithelial cells, prostate, and pancreas. The variation in the concentration levels of RANKL across several organs reaffirms the importance of RANKL in tissue growth (especially bone growth) and immune function in the body.

[0055] IL7, interleukin 7, i.e., IL-7, stimulates the differentiation of pluripotent (multipotent) hematopoietic stem cells into lymphoid progenitor cells. IL7 also stimulates the proliferation of all cells (B cells, T cells, and NK cells) in the lymphoid lineage. IL7 is important for B cell maturation, the survival, development, and proliferation during certain stages of T and NK cell homeostasis.

[0056] FTL3, or FLT3-L, is the ligand of the tyrosine kinase receptor FMS-like tyrosine kinase 3, also called FLT3. FTL3 is a hematopoietic cytokine that regulates the proliferation of early hematopoietic cells. It has been shown to synergize with various cytokines to stimulate the growth and differentiation of early hematopoietic cells. FLT3 ligand also stimulates the expansion of monocytes and immature dendritic cells and induces early B cell lineage differentiation and NK cell growth.

[0057] Stem cell factor (SCF), also known as kit ligand (KL), steel factor, binds to the c-KIT receptor (CD117) and is a cytokine that plays an important role in hematopoiesis during embryonic development. All sites where hematopoiesis occurs, such as the fetal liver and bone marrow, express SCF. SCF can function as an inductive cue that directs hematopoietic stem cells (HSCs) to their stem cell niches (the microenvironments where stem cells reside) and plays an important role in HSC maintenance. SCF has been shown to increase the survival of HSCs in vitro and contributes to the self-renewal and maintenance of HSCs in vivo. HSCs at all stages of development express the same level of the SCF receptor (c-KIT). Stromal cells surrounding HSCs are components of the stem cell niche and secrete several ligands, including SCF.

[0058] Glutamax is a L-glutamine substitute. Glutamax is the dipeptide L-alanyl-L-glutamine in 0.85% NaCl.

[0059] Regarding the compositions used to enable the differentiation of iPSCs into TEP cells, the compounds contained in the compositions that enable the differentiation of TEP into TEC can be used simultaneously, separately, or sequentially.

[0060] Advantageously, RANKL, IL7, FTL3, SCF, and Glutamax are all used simultaneously during all differentiation processes. These compounds can associate with BMP4, FGF8, FGF10, IGF1, and EGF for at least the first step of the differentiation process from TEP to TEC.

[0061] The present invention relates to a method for differentiating iPSCs into functional TEP cells in vitro or ex vivo, the method comprising: a) incubating iPSCs in a culture medium supplemented with a Y27 compound for 24 hours to obtain a Y27 culture medium; b) Removing the Y27 compound from the Y27 compound, adding the activin A growth factor and the CHIR99 compound for 24 hours to obtain an ActA / CHIR99 culture medium; c) Removing the CHIR99 growth from the ActA / CHIR99 culture medium, maintaining the culture with activin A for 48 hours to obtain an ActA culture medium; d) Adding the Y27 compound to the ActA culture for 24 hours to obtain a Y27 / ActA culture medium; e) Removing ActA and the Y27 compound from the Y27 / Act1 culture medium, adding the FGF8 growth factor and retinoic acid to the Y27 / Act1 culture medium for 24 hours to obtain an RA / F8 culture medium; f) Adding the noggin growth factor and the LY3 compound to the RA / F8 culture medium for 48 hours to obtain an RA / F8 / NOG / LY3 culture medium; g) Removing the noggin growth factor from the RA / F8 / NOG / LY3 culture medium, adding the CHIR99 compound and the BMP4 growth factor for 48 hours to obtain an RA / F8 / LY3 / CHIR99 / BMP culture medium; h) Removing the LY3 compound from the RA / F8 / LY3 / CHIR99 / BMP culture for 24 hours to obtain an RA / F8 / CHIR99 / BMP culture medium; i) Modifying the concentrations of the BMP4 growth factor and the FGF8 growth factor in the RA / F8 / CHIR99 / BMP culture medium for 24 hours; j) Removing the CHIR99 compound from the RA / F8 / CHIR99 / BMP culture medium, adding the FGF10 growth factor, the IGF1 growth factor, and the EGF growth factor for 72 hours to obtain an RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Recovering the functional TEP from the RA / F8 / BMP / F10 / IGF1 / EGF culture medium after 72 hours.

[0062] In addition to certain studies in which the inventors collected compounds and growth factors involved in the thymus differentiation process, it was shown that an 11-step sequence is required to obtain TEP cells that are fully functional and can be further used to initiate another differentiation process to obtain TEC cells.

[0063] From step b) to step j), mention is made of "adding" or "removing" one or more compounds or growth factors. This means that the compound or growth factor is - added to the previous culture, or a new culture medium containing the corresponding compound and / or growth factor is used with the new compound or growth factor by replacing the previous one, - removed from the previous culture medium, or a new culture medium containing the corresponding compound and / or growth factor is used without the "removed" compound or growth factor by replacing the previous one.

[0064] In other words, the present invention relates to the above method, which method is a) incubating iPSc in a first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium, b) removing the Y27 culture medium from the cells and incubating the cells with a second culture medium supplemented with activin A growth factor and CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium, c) removing the ActA / CHIR99 culture medium from the cells and incubating the cells with a third culture medium supplemented with actin A growth factor for 48 hours to obtain cells in the ActA culture medium, d) removing the ActA culture medium from the cells and incubating the cells in a fourth medium supplemented with actin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium, e) Removing the Y27 / ActA culture medium from the cells and incubating the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Removing the RA / F8 culture medium from the cells and incubating the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Removing the RA / F8 / NOG / LY3 culture medium from the cells and incubating the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Removing the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Removing the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0065] The culture medium defined in the method defined above is an aqueous liquid containing the minimum essential nutrients necessary for cell survival and growth, and more generally for maintaining cell homeostasis. The culture medium contains salts, nutrients such as amino acids, nucleotides, and ribonucleotides, vitamins, etc. In particular, for performing the differentiation process, several specific culture media are available from various manufacturers, and these specific media contain several compounds that maintain or assist the differentiation process. For example, mTeSR™1 or mTeSR™Plus medium commercially available from StemCell Biotechnologies, or X-VIVO™10 medium available from Lonza can be used. Those skilled in the art will be able to select the most appropriate medium for performing the above-described steps. Preferably, step a) is performed using a first culture medium, and the other steps are performed using a second culture medium different from the first culture medium.

[0066] All steps are performed in a cell incubator maintained at a temperature of about 37°C, with 5% CO2 and constant humidity. These conditions are classical conditions for culturing cells and cell lines and for performing the differentiation process.

[0067] Advantageously, the present invention relates to the above-described method, wherein the activin A growth factor is used at a concentration of 100 ng / mL in step b) and at a concentration of 50 ng / mL in steps c) and d).

[0068] Accordingly, the present invention advantageously relates to the above-described method, which method comprises a) incubating iPSc in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium; b) removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and a CHIR99 compound to obtain cells in an ActA / CHIR99 culture medium; c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of actin A growth factor and the Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with the FGF8 growth factor and the retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with the FGF8 growth factor, the retinoic acid compound, the noggin growth factor, and the LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with the FGF8 growth factor, the retinoic acid compound, the BMP4 growth factor, the LY3 compound, and the CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with the FGF8 growth factor, the retinoic acid compound, the BMP4 growth factor, and the CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with the FGF8 growth factor, the retinoic acid compound, the BMP4 growth factor, and the CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0069] More preferably, the present invention relates to the method defined above, wherein the Y27 compound and the FGF10, IGF1, and EGF growth factors are used at a concentration of 10 μM, i.e., 10 μmol / L or 10−6 mol / L. -6 mol / L.

[0070] Accordingly, the present invention relates to the method described above, which method comprises: a) Incubating iPSc in the first culture medium supplemented with 10 μM of the Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Removing the Y27 culture medium from the cells and incubating the cells with the second culture medium supplemented with activin A growth factor and CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Removing the ActA / CHIR99 culture medium from the cells and incubating the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Removing the ActA culture medium from the cells and incubating the cells in the fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Removing the Y27 / ActA culture medium from the cells and incubating the cells in the fifth medium supplemented with FGF8 growth factor and retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0071] Accordingly, the present invention advantageously relates to the above method, which method a) Incubate iPSc for 24 hours in a first culture medium supplemented with 10 μM of Y27 compound to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain the cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain the cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0072] Advantageously, the present invention relates to the method as defined above, wherein the CHIR99 growth factor is used at a concentration of 5 μM.

[0073] More advantageously, the present invention relates to the method as defined above, wherein the noggin growth factor is used at a concentration of 100 ng / mL.

[0074] Advantageously, the present invention relates to the method as described above, and the method a) Incubate iPSc for 24 hours in the first culture medium supplemented with Y27 compound to obtain the cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with the second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound to obtain the cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with the third culture medium supplemented with actinin A growth factor to obtain the cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with actin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, 100 ng / mL of 100 ng / mL of noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0075] More preferably, the present invention relates to the method defined above, which method comprises a) incubating iPSc in a first culture medium supplemented with 10 μM of the Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with activin A growth factor and 5 μM of the CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium; c) removing the ActA / CHIR99 culture medium from the cells and incubating the cells for 48 hours with a third culture medium supplemented with actinin A growth factor to obtain cells in the ActA culture medium; d) removing the ActA culture medium from the cells and incubating the cells for 24 hours in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound to obtain cells in the Y27 / ActA culture medium; e) removing the Y27 / ActA culture medium from the cells and incubating the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium; f) removing the RA / F8 culture medium from the cells and incubating the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) removing the RA / F8 / NOG / LY3 culture medium from the cells and incubating the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and 5 μM of the CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium; h) Removing the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Removing the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0076] The present invention more preferably relates to the above method, which method a) Incubating iPSc for 24 hours in a first culture medium supplemented with 10 μM of Y27 compound to obtain cells in the Y27 culture medium, and b) Removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and 5 μM of CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Removing the ActA / CHIR99 culture medium from the cells and incubating the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0077] Advantageously, the present invention relates to the method as defined above, wherein the BMP4 growth factor is used at a concentration of 10 ng / mL in steps g) to h) and at a concentration of 50 ng / mL in steps i) to j).

[0078] Advantageously, the present invention relates to the method as described above, which method a) incubating iPSc in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with an activin A growth factor and a CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium; c) removing the ActA / CHIR99 culture medium from the cells and incubating the cells for 48 hours with a third culture medium supplemented with an activin A growth factor to obtain cells in the ActA culture medium; d) removing the ActA culture medium from the cells and incubating the cells for 24 hours in a fourth medium supplemented with an activin A growth factor and a Y27 compound to obtain cells in the Y27 / ActA culture medium; e) removing the Y27 / ActA culture medium from the cells and incubating the cells for 24 hours in a fifth medium supplemented with an FGF8 growth factor and a retinoic acid compound to obtain cells in the RA / F8 culture medium; f) removing the RA / F8 culture medium from the cells and incubating the cells for 48 hours in a sixth medium supplemented with an FGF8 growth factor, a retinoic acid compound, a noggin growth factor, and an LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) Removing the RA / F8 / NOG / LY3 culture medium from the cells and incubating the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, h) Removing the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, i) Removing the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP. This includes the above steps.

[0079] More preferably, the present invention preferably relates to the above method, and the method is a) Incubating iPSc for 24 hours in a first culture medium supplemented with Y27 compound to obtain cells in the Y27 culture medium, b) Removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of activin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of activin A growth factor and the Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with the FGF8 growth factor and the retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with the FGF8 growth factor, the retinoic acid compound, the noggin growth factor, and the LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with the FGF8 growth factor, the retinoic acid compound, 10 ng / mL of the BMP4 growth factor, the LY3 compound, and the CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with the FGF8 growth factor, the retinoic acid compound, 10 ng / mL of the BMP4 growth factor, and the CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with the FGF8 growth factor, the retinoic acid compound, 50 ng / mL of the BMP4 growth factor, and the CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells in the tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0080] Therefore, the present invention relates to the above method, which method a) Incubating iPSc in the first culture medium supplemented with 10 μM of Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Removing the Y27 culture medium from the cells and incubating the cells with the second culture medium supplemented with activin A growth factor and CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Removing the ActA / CHIR99 culture medium from the cells and incubating the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Removing the ActA culture medium from the cells and incubating the cells in the fourth medium supplemented with actinin A growth factor and 10 μM of Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Removing the Y27 / ActA culture medium from the cells and incubating the cells in the fifth medium supplemented with FGF8 growth factor and retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Removing the RA / F8 culture medium from the cells and incubating the cells in the sixth medium supplemented with FGF8 growth factor, retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) Removing the RA / F8 / NOG / LY3 culture medium from the cells and incubating the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, h) Removing the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, i) Removing the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP. This includes the above steps.

[0081] Therefore, the present invention advantageously relates to the above method, which method is: a) Incubating iPSc for 24 hours in a first culture medium supplemented with 10 μM of Y27 compound to obtain cells in the Y27 culture medium, b) Removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of activin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of activin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0082] Advantageously, the present invention relates to the above method, which method a) Incubate the iPSc in the first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with actinin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells in the sixth medium supplemented with FGF8 growth factor, retinoic acid compound, 100 ng / mL of 100 ng / mL of noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0083] More preferably, the present invention relates to the method defined above, which method a) Incubate iPSc for 24 hours in a first culture medium supplemented with 10 μM of Y27 compound to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with actin A growth factor and 10 μM of Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells in the tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0084] The present invention more preferably relates to the above method, which a) Incubating iPSc in the first culture medium supplemented with 10 μM of Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Removing the Y27 culture medium from the cells and incubating the cells with the second culture medium supplemented with 100 ng / mL of activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Removing the ActA / CHIR99 culture medium from the cells and incubating the cells with the third culture medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Removing the ActA culture medium from the cells and incubating the cells in the fourth medium supplemented with 50 ng / mL of actinin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Removing the Y27 / ActA culture medium from the cells and incubating the cells in the fifth medium supplemented with FGF8 growth factor and retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Removing the RA / F8 culture medium from the cells and incubating the cells in the sixth medium supplemented with FGF8 growth factor, retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP, and includes.

[0085] Advantageously, the present invention relates to a method as defined above, wherein retinoic acid is used at a concentration of 0.75 μM in steps e) and f).

[0086] More advantageously, the present invention relates to a method as defined above, wherein retinoic acid is used at a concentration of 0.1 μM in steps g) to j).

[0087] Advantageously, the present invention relates to a method as defined above, and the method is a) Incubate iPSc in a first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Remove the Y27 culture medium from the cells and incubate the cells with a second culture medium supplemented with activin A growth factor and CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with a third culture medium supplemented with actin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Remove the ActA culture medium from the cells and incubate the cells in a fourth medium supplemented with actin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound for 48 hours to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium; h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium; i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0088] Advantageously, the present invention relates to the method defined above, which method a) Incubate iPSc in the first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with 50 ng / mL of actin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound for 48 hours to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0089] Advantageously, the present invention relates to the method defined above, which method a) Incubate iPSc in a first culture medium supplemented with 10 μM Y27 compound for 24 hours to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with activin A growth factor and CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with actin A growth factor and 10 μM of Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of retinoic acid compound, noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Removing the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 24 hours in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain the cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells for 72 hours in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain the cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP.

[0090] Advantageously, the present invention relates to the method defined above, which method a) Incubating iPSc for 24 hours in the first culture medium supplemented with 10 μM of Y27 compound to obtain the cells in the Y27 culture medium; b) Removing the Y27 culture medium from the cells and incubating the cells for 24 hours with the second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound to obtain the cells in the ActA / CHIR99 culture medium; c) Removing the ActA / CHIR99 culture medium from the cells and incubating the cells for 48 hours with the third culture medium supplemented with 50 ng / mL of actinin A growth factor to obtain the cells in the ActA culture medium; d) Removing the ActA culture medium from the cells and incubating the cells for 24 hours in the fourth medium supplemented with 50 ng / mL of actinin A growth factor and Y27 compound to obtain the cells in the Y27 / ActA culture medium; e) Removing the Y27 / ActA culture medium from the cells and incubating the cells for 24 hours in the fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain the cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0091] Advantageously, the present invention relates to the method defined above, which method comprises a) Incubating iPSc for 24 hours in a first culture medium supplemented with Y27 compound to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with actinin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with actinin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of retinoic acid compound, 100 ng / mL of 100 ng / mL of noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound for 24 hours to obtain the cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain the cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0092] More preferably, the present invention relates to the method defined above, and the method a) Incubate iPSc in the first culture medium supplemented with 10 μM of Y27 compound for 24 hours to obtain the cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain the cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain the cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with actinin A growth factor and 10 μM of Y27 compound for 24 hours to obtain the cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound for 24 hours to obtain the cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, 100 ng / mL noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and 5 μM CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0093] The present invention more preferably relates to the above method, and the method a) Incubate iPSc for 24 hours in a first culture medium supplemented with 10 μM Y27 compound to obtain cells in the Y27 culture medium, b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and 5 μM of CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and 5 μM CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, and 10 μM FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0094] Advantageously, the present invention relates to the above method, which a) Incubate iPSc in the first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with activin A growth factor and CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with actinin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and 0.75 μM retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, noggin growth factor, and LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL BMP4 growth factor, LY3 compound, and CHIR99 compound for 48 hours to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0095] More preferably, the present invention preferably relates to the above method, which method is a) Incubate iPSc in a first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of retinoic acid compound, noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and CHIR99 compound for 24 hours to obtain the cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain the cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0096] Therefore, the present invention relates to the above method, and the method a) Incubate iPSc in the first culture medium supplemented with 10 μM of Y27 compound for 24 hours to obtain the cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with activin A growth factor and CHIR99 compound for 24 hours to obtain the cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain the cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with actinin A growth factor and 10 μM of Y27 compound for 24 hours to obtain the cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound for 24 hours to obtain the cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, 10 ng / mL retinoic acid compound, BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL BMP4 growth factor, and CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL BMP4 growth factor, and 10 μM FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0097] Accordingly, the present invention advantageously relates to the above method, which method a) Incubate iPSc for 24 hours in a first culture medium supplemented with 10 μM Y27 compound to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of activin A growth factor and CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of retinoic acid compound, noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0098] Advantageously, the present invention relates to the above method, which method a) Incubate iPSc in the first culture medium supplemented with Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with actinin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with actinin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium, k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP, and the method includes the above steps.

[0099] More preferably, the present invention relates to the method defined above, and the method is a) Incubate the iPSc in a first culture medium supplemented with 10 μM of Y27 compound for 24 hours to obtain cells in the Y27 culture medium, and b) Remove the Y27 culture medium from the cells and incubate the cells for 24 hours with a second culture medium supplemented with activin A growth factor and 5 μM of CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium, and c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells for 48 hours with a third culture medium supplemented with actin A growth factor to obtain cells in the ActA culture medium, and d) Remove the ActA culture medium from the cells and incubate the cells for 24 hours in a fourth medium supplemented with actin A growth factor and 10 μM of Y27 compound to obtain cells in the Y27 / ActA culture medium, and e) Remove the Y27 / ActA culture medium from the cells and incubate the cells for 24 hours in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound to obtain cells in the RA / F8 culture medium, and f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of retinoic acid compound, 100 ng / mL of noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium, and g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells in the tenth medium supplemented with FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0100] The present invention more preferably relates to the above method, and the method a) Incubate iPSc in the first culture medium supplemented with 10 μM of Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) Remove the Y27 culture medium from the cells and incubate the cells with the second culture medium supplemented with 100 ng / mL of activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) Remove the ActA / CHIR99 culture medium from the cells and incubate the cells with the third culture medium supplemented with 50 ng / mL of actin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) Remove the ActA culture medium from the cells and incubate the cells in the fourth medium supplemented with 50 ng / mL of actin A growth factor and Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) Remove the Y27 / ActA culture medium from the cells and incubate the cells in the fifth medium supplemented with FGF8 growth factor and 0.75 μM of retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) Remove the RA / F8 culture medium from the cells and incubate the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, 0.75 μM retinoic acid compound, 100 ng / mL noggin growth factor, and LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL BMP4 growth factor, LY3 compound, and 5 μM CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium; h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL BMP4 growth factor, and 5 μM CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium; i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, 50 ng / mL BMP4 growth factor, and 5 μM CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, 50 ng / mL BMP4 growth factor, and 10 μM FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP.

[0101] More preferably, the present invention relates to the method defined above, wherein the FGF8 growth factor is used at a concentration of 50 ng / mL in steps e) and f), at a concentration of 20 ng / mL in steps g) and h), and at a concentration of 10 ng / mL in step j).

[0102] Finally, more preferably, the present invention relates to the method defined above, which method comprises a) incubating iPSc for 24 hours in a first culture medium supplemented with 10 μM of the Y27 compound to obtain cells in the Y27 culture medium; b) removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with 100 ng / mL of the activin A growth factor and 5 μM of the CHIR99 compound to obtain cells in the ActA / CHIR99 culture medium; c) removing the ActA / CHIR99 culture medium from the cells and incubating the cells for 48 hours with a third culture medium supplemented with 50 ng / mL of the actin A growth factor to obtain cells in the ActA culture medium; d) removing the ActA culture medium from the cells and incubating the cells for 24 hours in a fourth medium supplemented with 50 ng / mL of the actin A growth factor and 10 μM of the Y27 compound to obtain cells in the Y27 / ActA culture medium; e) removing the Y27 / ActA culture medium from the cells and incubating the cells for 24 hours in a fifth medium supplemented with 50 ng / mL of the FGF8 growth factor and 0.75 μM of the retinoic acid compound to obtain cells in the RA / F8 culture medium; f) removing the RA / F8 culture medium from the cells and incubating the cells for 48 hours in a sixth medium supplemented with 50 ng / mL of the FGF8 growth factor, 0.75 μM of the retinoic acid compound, 100 ng / mL of the noggin growth factor, and 10 μM of the LY3 compound to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) Remove the RA / F8 / NOG / LY3 culture medium from the cells and incubate the cells for 48 hours in a seventh medium supplemented with 20 ng / mL of FGF8 growth factor, 0.1 μM of retinoic acid compound, 10 ng / mL of BMP4 growth factor, 5 μM of LY3 compound, and 5 μM of CHIR99 compound to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium, and h) Remove the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in an eighth medium supplemented with 20 ng / mL of FGF8 growth factor, 0.1 μM of retinoic acid compound, 10 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium, and i) Remove the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 24 hours in a ninth medium supplemented with 10 ng / mL of FGF8 growth factor, 0.1 μM of retinoic acid compound, 50 ng / mL of BMP4 growth factor, and 5 μM of CHIR99 compound to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium, and j) Remove the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubate the cells for 72 hours in a tenth medium supplemented with 10 ng / mL of FGF8 growth factor, 50 ng / mL of BMP4 growth factor, and 10 μM of FGF10 growth factor, IGF1 growth factor, and EGF growth factor to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium and k) Remove the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recover the cells that are functional TEP. This includes.

[0103] More preferably, the present invention relates to a method as defined above, wherein the functional TEP cells express the FOXN1 and PAX9 genes and are EPCAM+CD205+.

[0104] The TEP cells obtained by the process defined above express the specific membrane markers EPCAM and CD205 and the genes FOXN1 and PAX9.

[0105] The FOXN1 gene encodes forkhead box protein N1, a DNA-binding transcription factor that regulates keratin gene expression. Depletion or knockout of the FOXN1 gene results in an athymic phenotype. FOXN1 has been shown to bind to and regulate genes involved in T cell maturation and antigen presentation in the thymus.

[0106] The PAX9 gene encodes the paired box gene 9 protein, also known as PAX9. Thymic lymphocyte formation has been shown to require Pax9 function in thymic epithelial cells.

[0107] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that mediates Ca2+-independent homotypic cell-cell adhesion in epithelia.

[0108] CD205 is an endocytosis receptor that is highly expressed by thymic cortical epithelial cells and a subset of dendritic cells (DC).

[0109] The present invention also relates to functional TEP cells that can be obtained by or directly obtained by the method defined above, which cells express the FOXN1 and PAX9 genes, are EPCAM+CD205+, and the TEP expresses NTRK2, CDH11, FLRT3 proteins, preferably TBX3, NTRK2, MEIS2, CDH11, PRSS23, and FLRT3 proteins.

[0110] These cells are novel because they are derived from non-natural stem cells (i.e., iPSCs), even if they have the same characteristics and differentiation potential as natural TECs obtained from the differentiation of cells derived from fertilized eggs. Furthermore, as demonstrated in the examples, the TEP obtained by the process defined above expresses cell markers that are not expressed in either natural TEP or TEP obtained from processes disclosed in the art.

[0111] Despite the specific markers defined above, cells cannot be comprehensively described except by the processes that enable their acquisition.

[0112] In another aspect of the present invention, a method for obtaining thymic epithelial cells from the functional TEP defined above is disclosed herein, the method comprising a. Incubating the functional TEP for 96 hours in a culture medium supplemented with a first composition containing L-glutamine and the BMP4, FGF8, FGF10, IGF1, and EGF growth factors and a second composition containing the RANKL, IL7, FTL3, and SCF growth factors to obtain a first TEP differentiation culture; b. Removing the first composition from the first TEP differentiation culture for 120 hours to obtain a second TEP differentiation culture; c. Recovering thymic epithelial cells from the second TEP differentiation culture.

[0113] Advantageously, in step b, RANKL can be added twice a week.

[0114] In other words, the present invention relates to a method for obtaining thymic epithelial cells from the functional TEP defined above, the method comprising a. Incubating the functional TEP for 96 hours in a culture medium supplemented with a first composition containing L-glutamine and the BMP4, FGF8, FGF10, IGF1, and EGF growth factors and a second composition containing the RANKL, IL7, FTL3, and SCF growth factors to obtain cells in a first TEP differentiation culture medium; a. Removing cells from the first TEP differentiation culture and contacting the cells with a culture medium supplemented with the second composition for 120 hours to obtain cells in a second TEP differentiation culture medium; b. Removing the second TEP differentiation culture from the cells and recovering the cells that are functional thymic epithelial cells.

[0115] Advantageously, the present invention relates to a method for obtaining thymic epithelial cells from functional TEP as defined above, the method comprising: a) incubating the functional TEP for 96 hours in a culture medium supplemented with a first composition comprising L-glutamine and BMP4, FGF8, FGF10, IGF1, and EGF growth factors and a second composition comprising RANKL, IL7, FTL3, and SCF growth factors to obtain a first TEP differentiation culture; b) removing the first TEP differentiation culture from the cells and incubating the cells incubated with the first TEP differentiation culture for 120 hours with a culture medium supplemented with a second composition comprising L-glutamine and RANKL, IL7, FTL3, and SCF growth factors to obtain a second TEP differentiation culture; c) recovering thymic epithelial cells from the second TEP differentiation culture.

[0116] More advantageously, the present invention relates to a method as defined above, the method comprising: a. incubating the functional TEP for 96 hours in a culture medium supplemented with L-glutamine and a first composition comprising 50 ng / mL BMP4, 10 ng / mL FGF8, 10 ng / mL FGF10, 10 ng / mL IGF1, and 10 ng / mL EGF growth factors and a second composition comprising 50 ng / mL RANKL, 5 ng / mL IL7, 5 ng / mL FTL3, and 10 ng / mL SCF growth factors to obtain cells in a first TEP differentiation culture medium; a. removing the cells from the first TEP differentiation culture and contacting the cells with a culture medium supplemented with the second composition (the second composition comprising 50 ng / mL RANKL, 5 ng / mL IL7, 5 ng / mL FTL3, and 10 ng / mL SCF growth factors) for 120 hours to obtain cells in a second TEP differentiation culture medium; b. removing the second TEP differentiation culture from the cells and recovering the cells that are functional thymic epithelial cells.

[0117] More preferably, the present invention relates to a method for differentiating iPSc into functional TECs in vitro or ex vivo, the method comprising: a) incubating the iPSc in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in the Y27 culture medium; b) removing the Y27 culture medium from the cells and incubating the cells with a second culture medium supplemented with an activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in the ActA / CHIR99 culture medium; c) removing the ActA / CHIR99 culture medium from the cells and incubating the cells with a third culture medium supplemented with an activin A growth factor for 48 hours to obtain cells in the ActA culture medium; d) removing the ActA culture medium from the cells and incubating the cells in a fourth medium supplemented with an activin A growth factor and a Y27 compound for 24 hours to obtain cells in the Y27 / ActA culture medium; e) removing the Y27 / ActA culture medium from the cells and incubating the cells in a fifth medium supplemented with an FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in the RA / F8 culture medium; f) removing the RA / F8 culture medium from the cells and incubating the cells in a sixth medium supplemented with an FGF8 growth factor, a retinoic acid compound, a noggin growth factor, and a LY3 compound for 48 hours to obtain cells in the RA / F8 / NOG / LY3 culture medium; g) removing the RA / F8 / NOG / LY3 culture medium from the cells and incubating the cells in a seventh medium supplemented with an FGF8 growth factor, a retinoic acid compound, a BMP4 growth factor, a LY3 compound, and a CHIR99 compound for 48 hours to obtain cells in the RA / F8 / LY3 / CHIR99 / BMP culture medium; h) removing the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells and incubating the cells in an eighth medium supplemented with an FGF8 growth factor, a retinoic acid compound, a BMP4 growth factor, and a CHIR99 compound for 24 hours to obtain cells in the first RA / F8 / CHIR99 / BMP culture medium; i) Removing the first RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells in the ninth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound for 24 hours to obtain cells in the second RA / F8 / CHIR99 / BMP culture medium; j) Removing the second RA / F8 / CHIR99 / BMP culture medium from the cells and incubating the cells in the tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF culture medium; k) Removing the RA / F8 / BMP / F10 / IGF1 / EGF culture medium from the cells and recovering the cells that are functional TEP; l) Incubating the functional TEP in a culture medium supplemented with L-glutamine etc., a first composition containing 50 ng / mL of BMP4, 10 ng / mL of FGF8, 10 ng / mL of FGF10, 10 ng / mL of IGF1, and 10 ng / mL of EGF growth factor, and a second composition containing 50 ng / mL of RANKL, 5 ng / mL of IL7, 5 ng / mL of FTL3, and 10 ng / mL of SCF growth factor for 96 hours to obtain cells in the first TEP differentiation culture medium; m) Removing the cells from the first TEP differentiation culture and contacting the cells with a culture medium supplemented with the second composition (the second composition contains 50 ng / mL of RANKL, 5 ng / mL of IL7, 5 ng / mL of FTL3, and 10 ng / mL of SCF growth factor) for 120 hours to obtain cells in the second TEP differentiation culture medium; n) Removing the second TEP differentiation culture from the cells and recovering the cells that are functional thymic epithelial cells.

[0118] The present invention also relates to thymic epithelial cells that can be obtained or directly obtained by the method defined above.

[0119] Advantageously, the present invention relates to thymic epithelial cells as defined above, which cells express the AIRE, PSMB11, and HLA-DRA genes, together with the FOXN1 and PAX9 genes, and are EPCAM+CD205+.

[0120] Since these cells are derived from non-natural-occurring novel TEP, they are novel to the art.

[0121] The present invention also relates to a method of doing so in a subject in need of treating or preventing a thymic disease, the method comprising administering, transplanting, or grafting a therapeutically effective amount of the TEP or TEC as defined above, a solution comprising the TEP or TEC as defined above, a composition comprising the TEP or TEC as defined above in the present disclosure, or a pharmaceutical composition comprising the TEP or TEC as defined above, to a subject in need thereof. The subject is preferably a mammal, most preferably a human.

[0122] A further embodiment is a method of doing so in a subject in need of treating or preventing an autoimmune disease, the method comprising administering, transplanting, or grafting a therapeutically effective amount of the TEP or TEC as defined above, a solution comprising the TEP or TEC as defined above, a composition comprising the TEP or TEC as defined above, or a pharmaceutical composition comprising the TEP or TEC as defined above, to a subject in need thereof. The subject is preferably a mammal, most preferably a human.

[0123] Another embodiment is a method of doing so in a subject in need of restoration or repair of thymus function, the method comprising administering, transplanting, or grafting to the subject in need thereof a therapeutically effective amount of TEP or TEC as defined above, a solution comprising TEP or TEC as defined above, a composition comprising TEP or TEC as defined above, or a pharmaceutical composition comprising TEP or TEC as defined above. The subject is preferably a mammal, most preferably a human. In some embodiments, the disorder is due to injury. In some embodiments, the disorder is due to aging. In some embodiments, the disorder is due to congenital abnormality.

[0124] Yet another embodiment is a method of doing so in a subject in need of T cell reconstitution after bone marrow transplantation, the method comprising administering, transplanting, or grafting to the subject in need thereof a therapeutically effective amount of TEP or TEC as defined above, a solution comprising TEP or TEC as defined above, a composition comprising the cells of the present disclosure, or a pharmaceutical composition comprising TEP or TEC as defined above. The subject is preferably a mammal, most preferably a human.

[0125] Another aspect of the invention is - the TEP cells as defined above, or - the TEC cells as defined above, or both thereof, for use in: - treating or preventing a thymic disease, - or treating or preventing an autoimmune disease, - or for use in any of T cell reconstitution after bone marrow transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] The invention will be better understood in view of the following examples and the following drawings.

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Example

[0127] Example 1 - Differentiation of induced pluripotent stem cells (iPSc) in thymic epithelial precursors (TEP) 1. Purpose This is a protocol that describes the acquisition of TEP from the differentiation of iPSc.

[0128] The protocol provides technical details regarding the steps necessary to generate TEP.

[0129] This is used in experiments that require research on TEP.

[0130] 1. Process Note: The "day" of differentiation is defined by a 24-hour duration. The process is time-dependent, and it is important to consider the exact duration.

[0131] 1. - Day 1 Prepare p culture wells by depositing 500 μL of Matrigel diluted at 1% in DMEM:F12 (1:1 mixture of DMEM and Ham F-12 - Thermo Fisher Scientifc) and incubate at 37 °C for at least 3 hours.

[0132] Use iPSc cultures that are within 60 - 80% confluent and do not undergo spontaneous differentiation.

[0133] Wash the iPSc with DPBS- / - (without potassium chloride (KCl) 200 mg / L, potassium dihydrogen phosphate (KH2PO4) 200.0 mg / L, sodium chloride (NaCl) g / L, and disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) 2.160 g / L, without calcium and magnesium), remove the DPBS- / - , add 500 μL of TrypLE per well, and incubate at 37 °C for 5 minutes.

[0134] Add 500 μL of mTeSER1 (StemCell Technologies) per well. Flash multiple times to dissociate all cells.

[0135] Centrifuge at 200 g for 5 minutes, remove the supernatant, and resuspend the cell pellet in 1 mL of mTE SR1.

[0136] Count the cells.

[0137] Prepare the D-1 cell suspension: 130,000 * Pipette the volume of the cell suspension containing p cells, make it into p mL of mTESR1, and add Y27 to a final concentration of 10 μM.

[0138] Put 1 mL of the D-1 cell suspension per well, homogenize, and place it in an incubator at 37 °C and 5% CO2.

[0139] 1. Day 0 Prepare the D0 culture medium: Base XVIVO10, 5 μM of CHIR99, 100 ng / mL of activin A.

[0140] Remove the culture medium in the well.

[0141] Wash with DPBS- / - .

[0142] Add 1 mL of the D0 culture medium per well.

[0143] 1. Day 1 Prepare D1 culture medium: Base XVIVO10, 50 ng / mL activin A.

[0144] Remove the culture medium in the wells.

[0145] Wash with DPBS- / -

[0146] Add 1 mL of D1 culture medium per well.

[0147] 1. Day 2 Prepare D1 culture medium: Base XVIVO10, 50 ng / mL activin A.

[0148] Remove the culture medium in the wells.

[0149] Wash with DPBS- / -

[0150] Add 1 mL of D1 culture medium per well.

[0151] Prepare q culture wells by depositing 500 μL of Matrigel diluted to 1% in DMEM:F12 and incubate at 37 °C for at least 3 hours.

[0152] The cells must reach confluence.

[0153] 1. Day 3 Wash the cells with DPBS- / - and remove the DPBS- / - and add 500 μL of TrypLE per well and incubate at 37 °C for 5 minutes.

[0154] Add 500 μL of TrypLE per well. Flash multiple times to dissociate all cells.

[0155] Centrifuge at 200 g for 5 minutes, remove the supernatant and resuspend the cell pellet in 1 mL of XVIVO10.

[0156] Count the cells.

[0157] Prepare the D3 cell suspension (see the table): 50,000 * Pipette the volume of the cell suspension containing q cells and make it q mL in XVIVO10, add activin A to a final concentration of 50 ng / mL, and add Y27 to a final concentration of 10 μM.

[0158] Add 1 mL of the D3 cell suspension per well, homogenize, and place it in an incubator at 37°C and 5% CO2.

[0159] 1. Day 4 Prepare the D4 medium: base XVIVO10, 0.75 μM retinoic acid, and 50 ng / mL FGF8.

[0160] Remove the culture medium in the well.

[0161] Wash with DPBS- / -

[0162] Add 1 mL of the D4 culture medium per well.

[0163] 1. Day 5 Prepare the D5 medium: main component XVIVO10, 0.75 μM retinoic acid, 50 ng / mL FGF8, 10 μM LY3, 100 ng / mL noggin.

[0164] Remove the culture medium in the well.

[0165] Wash with DPBS- / -

[0166] Add 1 mL of the D5 culture medium per well.

[0167] 1. Day 7 Prepare the D7 medium: main component XVIVO10, 0.1 μM retinoic acid, 5 μM CHIR99, 5 μM LY3, 10 ng / mL BMP4, and 20 ng / mL FGF8.

[0168] Remove the culture medium in the wells.

[0169] Wash with DPBS- / - .

[0170] Add 1 mL of D7 culture medium per well.

[0171] 1. Day 9 Prepare D9 medium: Base XVIVO10, 0.1 μM retinoic acid, 5 μM CHIR99, 10 ng / mL BMP4, and 20 ng / mL FGF8.

[0172] Remove the culture medium in the wells.

[0173] Wash with DPBS- / - .

[0174] Add 1 mL of D7 culture medium per well.

[0175] Prepare r culture wells by depositing 500 μL of Matrigel diluted to 1% in DMEM:F12 and incubate at 37 °C for at least 3 hours.

[0176] 1. Day 10 If the cells have not yet reached confluence, wait before proceeding.

[0177] Add 500 μL of TrypLE per well. Flash several times to dissociate all cells.

[0178] Centrifuge at 200 g for 5 minutes, remove the supernatant, and resuspend the cell pellet in 1 mL of XVIVO10.

[0179] Count the cells.

[0180] Prepare the D10 cell suspension (see table): 100 000 *Pipette the volume of the cell suspension containing r cells and make it r mL in XVIVO10, and add 0.1 μM retinoic acid, 5 μM CHIR99, 50 ng / mL BMP4, and 20 ng / mL FGF8.

[0181] Put 1 mL of the D10 cell suspension per well, homogenize, and place it in an incubator at 37 °C and 5% CO2.

[0182] 1. On day 11 Prepare D11 medium: base XVIVO10, 0.1 μM retinoic acid, 50 ng / mL BMP4, 10 ng / mL FGF8, 10 ng / mL FGF10, 10 ng / mL IGF1, 10 ng / mL EGF.

[0183] Remove the culture medium in the well.

[0184] Wash with DPBS- / -

[0185] Add 1 mL of D11 culture medium per well.

[0186] 1. On day 13 Prepare D13 medium: base XVIVO10, 0.1 μM retinoic acid, 50 ng / mL BMP4, 10 ng / mL FGF8, 10 ng / mL FGF10, 10 ng / mL IGF1, 10 ng / mL EGF.

[0187] Remove the culture medium in the well.

[0188] Wash with DPBS- / -

[0189] Add 1 mL of D14 culture medium per well.

[0190] 1. On day 14 TEP is in a state where it can be collected.

[0191] Perform lysis in the culture well and verify FOXN1 and PAX9 expression by RT-qPCR.

[0192] Verify the differentiation yield using culture wells: >50% EPCAM+CD205+ cells.

[0193] The above protocol enables the production of TEP from iPSc for the maturation tracking process.

[0194] The following table summarizes the above steps.

[0195]

Table 1

[0196] Example 2 - Maturation of thymic epithelial precursors (TEP) and formation of thymic organoids in functional thymic epithelial cells (TEC) 1. Purpose The following protocol describes the maturation of TEC in functional thymic organoids obtained from iPSc-derived TEP, as disclosed in Example 1.

[0197] The protocol provides technical details on the steps necessary to generate thymic organoids. This is used in experiments that require research on thymic organoids.

[0198] 2. Process Note: Early thymic progenitor (ETP) is here derived from a primary sample of thymus from a young patient who underwent heart surgery on the same day.

[0199] ETP is a bone marrow-derived hematopoietic stem cell that gives rise to the T cell lineage and has the phenotype: CD45+CD3-CD4-CD8-CD14-CD19-CD56-CD34+CD7+.

[0200] 2.1. Day 0 Verify the quality of the D14 TEP culture. The cells should be in a confluent state, forming a high-density monolayer with protrusions and bulges.

[0201] Prepare the washing buffer: PBS, 0.5% BSA, 2 mM EDTA.

[0202] Prepare the FACS buffer: PBS, 2% FBS, 5 mM EDTA.

[0203] Prepare the beads for ETP isolation: Pipette 1 mL of anti-mouse Dynabeads, wash with 2 mL of washing buffer for 1 minute, and place on the Dynabead Magnet for 2 minutes. Remove the supernatant and resuspend the beads in 500 μL of washing buffer. Label the beads with mouse anti-human CD3 antibody while stirring at 40 °C for 45 minutes. Wash 3 times with 20 mL of washing buffer on the Dynabeads magnet.

[0204] Resuspend in 20 mL of washing buffer.

[0205] Isolate ETP from the donor primary thymus sample: Cut the thymus in 50 mL Falcon in RPMI medium into 1 cubic millimeter pieces. Let it settle, collect the supernatant, and filter through a 70 μm mesh. Repeat 3 times. Centrifuge at 200 g for 5 minutes. Resuspend the pellet in 10 mL of red blood cell lysis buffer. Incubate at room temperature for 5 minutes. Add 20 mL of PBS and centrifuge at 200 g for 5 minutes. Resuspend the pellet in 10 mL of washing buffer. Count the cells. Collect 1 billion cells and adjust the volume to 20 mL with washing buffer.

[0206] Add to the Dynabeads and incubate at 40 °C for 30 minutes with stirring.

[0207] Place the tube on the magnet for 2 minutes. Collect the supernatant and centrifuge at 200 g for 5 minutes. Resuspend the pellet in 1 mL of FACS buffer. Label the cells with Lin (CD3 CD4 CD8 CD14 CD19 CD56), CD7, and CD34.

[0208] Sort cells using a flow cytometer. Centrifuge at 200 g for 5 minutes. Resuspend the pellet in 1 mL of XVIVO10 (LONZA ref#04-380Q) buffer. Count the cells.

[0209] Wash the TEP with DPBS- / - and remove the DPBS- / - . Add 500 μL per well of TrypLE (ThermoFischer Scientific; recombinant trypsin, free of any cell and animal contaminants) and incubate at 37 °C for 5 minutes.

[0210] Add 500 μL per well of XVIVO10. Flush multiple times to dissociate all cells.

[0211] Spin at 200 g for 5 minutes, remove the supernatant, and resuspend the cell pellet in 1 mL of XVIVO10.

[0212] Count the cells.

[0213] Pool two cell suspensions and adjust the volume with XVIVO10 to concentrations of 200,000 TEP / mL and 50,000 ETP / mL.

[0214] Add D14 supplement at the desired concentrations (see Table 2), namely, 50 ng / mL of BMP4, 10 ng / mL of FGF8, 10 ng / mL of FGF10, 10 ng / mL of IGF1, 10 ng / mL of EGF, 50 ng / mL of RANKL, 5 ng / mL of IL-7, 5 ng / mL of FLT3, 10 ng / mL of SCF, and 1% Glutamax.

[0215] Homogenize and plate at 100 μL per well in a Low Binding 96-well plate. Place in an incubator at 37 °C, 5% CO2.

[0216] 2.2. Day 1 Preparing the hydrogel: Thaw aliquots of thrombin (10 U / mL) and aprotinin (26000 U / mL) solution on ice. Do not vortex. Thaw fibrinogen (8 mg / mL) in a 37 °C water bath. Do not vortex.

[0217] Gently pipette up and down to homogenize the aliquots. Prepare 0.25×o Eppendorf tubes (where o is the number of organoids seeded the previous day). Add 150 μL of fibrinogen and 10 μL of aprotinin to each tube. Add 150 μL of thrombin to the tubes, quickly pipette up and down twice without generating bubbles, then pipette 150 μL and place it into the insert of a 24-well Hanging Insert plate. Quickly repeat this for the remaining 150 μL in a second well. Avoid forming bubbles by handling the pipette carefully.

[0218] Once all wells of the plate are filled, incubate at 37 °C for 1 hour. The clear liquid solution will solidify and become opaque.

[0219] Prepare D15 solution, namely, 50 ng / mL of BMP4, 10 ng / mL of FGF8, 10 ng / mL of FGF10, 10 ng / mL of IGF1, 10 ng / mL of EGF, 50 ng / mL of RANKL, 5 ng / mL of IL-7, 5 ng / mL of FLT3, 10 ng / mL of SCF, and 1% Glutamax.

[0220] Seeding the organoids: Verify the quality of the re-aggregation process. The organoids should form spherical cell masses with a compact core surrounded by a low-density thymocyte crown (see [Figure 16]).

[0221] Carefully collect the organoids using a tip-cut P200 cone pre-washed with anti-adhesion solution and seed one by one on top of the hydrogel at a rate of 2 organoids per well. Ensure that no cells remain in the P96 well.

[0222] Add 300 μL of D15 solution slowly on top of the hydrogel without direct contact. Add 700 μL of D15 solution to the bottom of each well. Place it in an incubator at 37 °C and 5% CO₂.

[0223] 2.3. Day 2 Check if the organoids are sufficiently seeded: The hydrogel should stay in place, and the organoids should not sediment to the bottom of the insert. Leave the D15 solution until then.

[0224] 2.4. Day 5 From this point on, the culture medium can be changed daily or twice a week (the organoids can be maintained in culture for up to 6 weeks).

[0225] Add D19 solution, namely 50 ng / mL of RANKL, 5 ng / mL of IL-7, 5 ng / mL of FTL3, 10 ng / mL of SCF, and 1% Glutamax.

[0226] 2.5. Day 14 TEP is in a state where it can be harvested.

[0227] Perform lysis (in RTL Buffer, Qiagen ref#79216) in the culture wells and verify FOXN1 and PAX9 expression by RT-qPCR.

[0228] Verify the differentiation yield using the culture wells: >50% EPCAM+CD205+ cells by flow cytometry using appropriate antibodies

[0229] The following table summarizes the above steps.

[0230]

Table 2

[0231] 3. Results [Fig. 17] shows the structural changes of thymic organoids cultured in hydrogel. The organoids shift from a compact spherical morphology [Fig. 16] to a larger and lower-density structure formed by elongated cells. This structural change reflects the low-density sponge-like mesh of the in vivo 3D structure of thymic epithelium. By using IF and 3D imaging, the inventors visualized this structure in the organoids using cytokeratin 8 (KRT8) to label TEC.

[0232] The obtained TEP expresses predicted markers of thymic epithelial identity such as FOXN1 and PAX9. Furthermore, the TEP morphology is similar to primary cells with large polygonal cells [Fig. 15]. Therefore, this process enables the generation of primary-like TEP from iPSc.

[0233] Example 3 - Identification of a set of biomarkers specific to TEP generated by iPSc differentiation The aim was to identify a set of biomarkers that are highly specifically expressed in TEP generated by the inventors' protocol versus controls and public in vivo data.

[0234] Method: Seven bulk samples of 14-day-old thymic epithelial precursors (TEP) derived from iPSc using the inventors' protocol were sequenced using DGEseq. Three samples of D0 cells (iPSc) at the start of differentiation were included as negative controls. Five samples of primary human postnatal TEC were included as positive controls.

[0235] To exclude the possibility of marker contamination from unwanted differentiation products, D14 TEP was purified by FACS (BD FACS ARIA) with their most advanced phenotype EPCAMhi CD205+ using antibodies EPCAM-PE (Miltenyi, 130-113-263) and CD205-FITC (Biolegend, 11-2059-82). Six samples from independent differentiations were sorted and named TEP_puri hereinafter.

[0236] RNA extraction Cell samples were lysed using RLT lysis buffer (Qiagen, 79216). RNA extraction was performed on the purified TEP samples using the RNAeasy mini kit (Qiagen, 74104) and the micro kit (Qiagen, 74004). The purified TEP samples were sorted directly into RLT lysis buffer.

[0237] RNA sequencing The DEG sequencing protocol was carried out according to the inventors' implementation of a previously developed protocol (1). 10 ng of total RNA was used for library preparation. mRNA was tagged using a poly(A) tail-specific adapter, a well-specific barcode, and a universal molecular identifier (UMI) with template switching reverse transcription. cDNA preparations were pooled, amplified, tagged by transposon fragmentation, and the 3' end was enriched. Sequencing was performed on an Illumina® HiSeq 2500 using the Hiseq Rapid SBS Kit. The kits used were the Zymo purification kit Researche D4004-1-L, Kit Advantage 2 PCR Enzyme System (Clontech, 639206), QIAquick Gel Extraction, Kit AgencourtAMPure XP magnetic beads, Nextera DNA (FC-121-1031).

[0238] The purified TEP samples were sequenced using the SMART-seq protocol on the GENO’MIC platform of the Cochin Institute.

[0239] RNAseq data analysis The read pairs used for the analysis met the following criteria: all 16 bases of the first read had a quality score of at least 10, and the first 6 bases corresponded exactly to the designed well-specific barcode. The second read was aligned to the RefSeq human mRNA sequence (hg19) using bwa version 0.7.17. Reads that mapped to multiple transcripts of different genes or contained more than three mismatches with the reference sequence were excluded from the analysis. For each sample, a DGE profile was generated by counting the number of unique UMIs associated with each RefSeq gene. Sequenced samples with at least 50000 counts and 6000 expressed genes were retained for further analysis. Batch correction and differential expression (DE) were performed using the DEseq2 R package with a threshold of 1 log2 fold change and a Benjamini Hochberg p-value of 0.05 to classify genes as differentially expressed versus control.

[0240] Data visualization was performed with a custom pipeline based on the ComplexHeatmap and ggplot2 R packages.

[0241] Filtering of the DE gene list for marker identification Two lists of DE genes generated by DE analysis of bulk TEP samples against negative control (D0 iPSc) and positive control (primary TEC) were restricted to their common part in R using the data manipulation package tidyr. The resulting marker list was further filtered to include only positive markers, i.e., markers upregulated in TEP.

[0242] The top 20% of markers were selected based on Log2FoldChange and average expression. To further exclude markers that are also expressed in vivo, the marker expression on the limited list was manually verified against two publicly available scRNAseq datasets including TEP from Park et al. and Magaletta et al., as well as the thymus reference scRNAseq dataset from the Human Protein Atlas (https: / / www.proteinatlas.org / ). Markers were discarded if observable expression was detected in the TEC cluster. The demonstrated functionality of the remaining markers in pharyngeal organogenesis and their link to epithelial gene ontology were the next criteria for marker selection to reduce the false positive risk. Final marker selection was performed using purified TEP samples, and markers highly expressed in the purified TEP data were selected as the final markers.

[0243] Results: Sample transcriptome similarity: Heatmaps of Pearson correlation and clustering of samples reveal a clear separation of the TEC and iPSc groups from the TEP ([Figure 19]).

[0244] Quality control and verification of differentiation: The expression of the main markers of the target lineages (i.e., iPSc, TEC, and TEP markers) was analyzed to confirm the quality of the samples and verify the marker analysis. It can be clearly seen that the iPSc gene clusters (NANOG, POU5F1) and the mature TEC gene clusters (HLA-DR, CD80) are highly expressed in samples from the negative control group and the positive control group, respectively. This verifies that these samples are valid controls. Bulk and purified TEP samples show the expression of classical TEP markers (EYA1, KRT8), which confirms the efficiency of differentiation in these samples and verifies their use as a basis for TEP marker research ([Figure 20]).

[0245] The final limited list of markers is reported below.

[0246]

Table 3

[0247] The confirmation of the low expression of this limited marker list is carried out on the dataset from Magaletta of thymic organogenesis. For these markers in the Magaletta dataset, it is possible to detect from low expression to complete absence of expression, and it was confirmed that these are specific to the TEP differentiation products of the inventors ([Figure 21]).

[0248] To further limit the marker list, the criterion of cellular localization can be applied. Cell membrane markers are of particular interest as they enable the purification of live cells. Based on this one criterion, the final set of markers that are specific to the TEP differentiation products of the inventors and not expressed in in vivo TEP can be limited to NTRK2, CDH11, FLRT3. In conclusion, the TEP obtained through the described protocol shows more similar functionality than natural TEP, but can be identified through the expression of the set of markers NTRK2, CDH11, FLRT3.

[0249] Example 4: Identification of a set of biomarkers specific to mature TECs in an organoid culture system from TEP generated according to the process of the present invention The aim was to identify a set of biomarkers that are highly specifically expressed in TECs generated by the inventors' protocol versus controls and public in vivo data.

[0250] Method: Data acquisition and preprocessing: Single-cell RNA sequencing (scRNAseq) data for the study were obtained from publicly available datasets or generated in-house. Public datasets of human primary TECs used as controls were reanalyzed from the raw data of the study by Park et al. The inventors generated their dataset from D21 organoids cultured according to their protocol. The organoids were incubated in 0.5 mg / mL collagenase / dispase solution at 37 °C for 30 minutes, mechanically disrupted, and then dissociated by incubation with TrypLE at 37 °C for 5 minutes. The resulting cell suspension was washed with cold dPBS and filtered through a 100 μm mesh. Cells were stained with 1 μg / mL anti-EPCAM PE, anti-CD45 APC-Cy7, anti-HLA-DR PeCy5, and anti-CD205 APC antibodies, and DAPI. Cells were sorted using the CD45-EPCAMhi phenotype on a BD FACS ARIA. The cell suspension was washed and loaded onto a Chromium 10X cassette for library generation. Count matrix data files were downloaded and preprocessed using the Seurat package in R. To ensure high-quality data for downstream analysis, a quality control step was performed by excluding cells with more than 10% mitochondrial genes, less than 500, or more than 5000 counts.

[0251] Data integration and dimensionality reduction: For the data of Park et al., multiple dataset integration was performed using the integration workflow of Seurat by canonical correlation analysis (CCA). This step enables the integration of datasets into a common space and reconciles technical variations across different experiments. Following data integration, dimensionality reduction was performed by PCA by retaining the first 15 dimensions.

[0252] Cell clustering and TEC identification: Unsupervised cell clustering was performed using Seurat's graph-based clustering algorithm with Louvain parameters. The clustering results were visualized using UMAP or other visualization techniques to identify distinct clusters representing different cell populations. To identify the TEC cluster, marker genes known to be specific to TEC (EPCAM, FOXN1, PAX9) were used, and differential expression analysis was performed to find genes highly expressed in the TEC cluster compared to other clusters.

[0253] Pseudobulk generation and differential expression analysis: Once the TEC clusters were identified, pseudobulk transcriptomes were generated for each TEC cluster by summing the expression values of the cells within each cluster. To identify genes differentially expressed between TEC clusters, differential expression analysis was performed using the DESeq2 package in R. The pseudobulk transcriptomes from each TEC cluster were compared using the statistical framework of DESeq2. Genes with significant fold changes and adjusted p-values (i.e., Benjamini-Hochberg adjusted p-values, with a threshold set at 0.05) were considered to be differentially expressed.

[0254] Identification of marker genes The top 20% of markers were selected based on Log2FoldChange and mean expression. To further exclude markers that are also expressed in vivo, marker expression on the restricted list was manually confirmed against two publicly available scRNAseq datasets containing TEC from the paper by Magaletta et al. and the Human Protein Atlas ( / / www.proteinatlas.org / ). Markers were discarded if observable expression was detected in the TEC cluster. The demonstrated functionality of the remaining markers in pharyngeal organogenesis and their link to epithelial gene ontology were the next criteria for marker selection to reduce the risk of false positives.

Claims

1. The use of a composition comprising the following compounds: activin A, BMP4, CHIR99, EGF, FGF8, FGF10, IGF1, LY3, noggin, retinoic acid, and Y27, Use for carrying out the differentiation process of induced pluripotent stem cells, i.e., iPSScs, into functional thymic epithelial precursors, i.e., TEP, preferably in vitro or ex vivo.

2. The use according to claim 1, wherein the differentiation process is carried out for 14 days.

3. The use according to claim 1, wherein the iPSc expresses the OCT4 and NANOG genes.

4. A method for differentiating iPSScs into functional TEPs in vitro or ex vivo, a. A step of incubating the iPSc for 24 hours in a culture medium supplemented with the (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y27) compound to obtain a Y27 culture medium, b. The steps of removing the Y27 culture medium from the cells and incubating the cells for 24 hours with a second culture medium supplemented with activin A growth factor and the -[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitride (CHIR99) compound to obtain the cells in ActA / CHIR99 culture medium, c. The step of removing the ActA / CHIR99 culture medium from the cells and incubating the cells for 48 hours with a third culture medium supplemented with actinin A growth factor to obtain the cells in ActA culture medium, d. The step of removing the ActA culture medium from the cells and incubating the cells for 24 hours in a fourth medium supplemented with actinin A growth factor and Y27 compound to obtain the cells in Y27 / ActA culture medium, e. A step of removing the Y27 / ActA culture medium from the cells and incubating the cells for 24 hours in a fifth medium supplemented with fibroblast growth factor 8 (FGF8) growth factor and retinoic acid (RA) compound to obtain the cells in RA / F8 culture medium, f. The step of removing the RA / F8 culture medium from the cells and incubating the cells for 48 hours in a sixth medium supplemented with FGF8 growth factor, retinoic acid compound, noggin growth factor, and 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-quinoline (LY3) compound to obtain the cells in RA / F8 / NOG / LY3 culture medium, g. The step of removing the RA / F8 / NOG / LY3 culture medium from the cells, incubating the cells for 48 hours in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, bone morphogenetic protein 4 (BMP4) growth factor, LY3 compound, and CHIR99 compound to obtain the cells in RA / F8 / LY3 / CHIR99 / BMP culture medium, h. The steps of removing the RA / F8 / LY3 / CHIR99 / BMP culture medium from the cells, incubating the cells for 24 hours in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, and CHIR99 compound, to obtain the cells in the first RA / F8 / CHIR99 / BMP culture medium, i. The first RA / F8 / CHIR99 / BMP culture medium is removed from the cells, and the cells are incubated for 24 hours in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, bone morphogenetic protein 4 (BMP4) growth factor, and CHIR99 compound to obtain the cells in a second RA / F8 / CHIR99 / BMP culture medium. j. The steps of removing the second RA / F8 / CHIR99 / BMP culture medium from the cells, incubating the cells for 72 hours in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, fibroblast growth factor 10 (FGF10) growth factor, insulin-like growth factor 1 (IGF1) growth factor, and epidermal growth factor (EGF) growth factor to obtain the cells in RA / F8 / BMP / F10 / IGF1 / EGF culture medium, k. A method comprising the step of recovering the functional TEP from the RA / F8 / BMP / F10 / IGF1 / EGF culture medium after 72 hours.

5. The method according to claim 4, wherein the activin A growth factor is used at a concentration of 100 ng / mL in step b) and at a concentration of 50 ng / mL in steps c) and d).

6. The method according to claim 4, wherein the Y27 compound and the FGF10, IGF1, and EGF growth factors are used at a concentration of 10 μM.

7. The method according to claim 4, wherein CHIR99 growth factor is used at a concentration of 5 μM, and the noggin growth factor is used at a concentration of 100 ng / mL.

8. The method according to claim 4, wherein the BMP4 growth factor is used at a concentration of 10 ng / mL in steps g) to h) and at a concentration of 50 ng / mL in steps i) to j).

9. The method according to step 4, wherein the retinoic acid is used in steps e) and f) at a concentration of 0.75 μM.

10. The method according to step 4, wherein the FGF8 growth factor is used at a concentration of 50 ng / mL in steps e) and f), at a concentration of 20 ng / mL in steps g) and h), and at a concentration of 10 ng / mL in step j).

11. The method according to claim 4, wherein the functional TEP cells express the FOXN1 and PAX9 genes and are EPCAM+CD205+.

12. Functional TEP cells that can be obtained by the method of claim 4 or directly obtained, wherein the cells express the FOXN1 and PAX9 genes, are EPCAM+CD205+, and express the NTRK2, CDH11, and FLRT3 proteins.

13. A method for obtaining thymic epithelial cells from functional TEP according to claim 12, a. A step of obtaining a first TEP differentiated culture by incubating functional TEP for 96 hours in a culture medium supplemented with a first composition comprising L-glutamine, bone morphogenetic protein family 4 (BMP4), fibroblast growth factor 8 (FGF8), fibroblast growth factor 10 (FGF10), insulin-like growth factor 1 (IGF1), and epidermal growth factor (EGF) growth factor, and a second composition comprising nuclear factor kappa-B activating receptor ligand (RANKL), interleukin 7 (IL7), FMS-like tyrosine kinase 3 (FTL3) ligand, and stem cell factor (SCF), b. The first TEP differentiated culture is removed from the cells, and the cells incubated with the first TEP differentiated culture are incubated for 120 hours in a culture medium supplemented with L-glutamine and a second composition containing RANKL, IL7, FTL3, and SCF to obtain a second TEP differentiated culture. c. A method comprising the step of recovering the thymic epithelial cells from a second TEP differentiated culture.

14. Thymic epithelial cells that can be obtained by the method of claim 13 or directly obtained, wherein the cells express the AIRE, PSMB11, and HLA-DRA genes together with the FOXN1 and PAX9 genes, and are EPCAM+CD205+.

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