Method for obtaining functional lymphocytes

Abstract

The present invention relates to the use of a composition comprising the following compounds: activin A, BMP4, CHIR99, EGF, FGF 8, FGF 10, IGF1, LY3, Noggin, retinoic acid and Y27 for carrying out a process of inducing the differentiation of pluripotent stem cells or iPSc into functional thymic epithelial progenitor cells or TEP, preferably in vitro or ex vivo.

Description

[0001] The present invention relates to a method for obtaining functional lymphocytes and cells obtained by the method.

[0002] The thymus provides an essential microenvironment for T cell development and maturation. Thymic epithelial cells (TEC), composed of thymic cortical epithelial cells (cTEC) and thymic medullary epithelial cells (mTEC), have been well documented to be critical for these tightly regulated processes.

[0003] Although TEC populations are known to be key regulators of different T cell developmental programs, recent studies have revealed significant new TEC heterogeneity that must be considered in understanding the microenvironmental control of T cell development. Of note, therapeutic interventions may also be detrimental to thymic function. Such clinical treatments include ablative preconditioning used in cancer treatment, which then impairs T cell-mediated immune reconstitution after bone marrow transplantation. Therefore, studying thymic epithelial cells in healthy and diseased states is important for understanding how thymic function is controlled and how it can be manipulated for therapeutic benefit. Recently, important progress has been made in understanding the biology of the thymic epithelium, including the developmental pathways that give rise to different cortical and medullary epithelial lineages. In addition, there is progress in how the newly identified heterogeneity in the thymic epithelium can be mapped to functional specialization in the thymic microenvironment.

[0004] Producing functional TEC or TEC progenitors (TEP) in vitro from human pluripotent stem cells (hPSC) can produce cells, tissues or organs that contribute to T cell reconstruction in patients with thymic dysfunction caused by congenital disorders such as DiGeorge syndrome and acquired dysfunction caused by HIV infection, high-dose chemotherapy and radiotherapy treatment, graft-versus-host disease and long-term immunosuppressive therapy combined with advanced age (which itself leads to poor thymic production function). Since the number of TEC in the adult thymus is limited and a reliable method for amplifying TEC from the postpartum thymus has always been elusive, it is an important goal to produce TEC from pluripotent stem cells (PSC). Creating an in vitro protocol for strictly controlling the differentiation of hPSC to TEC requires precise understanding and application of developmental time and cytokine clues. Although it has been described that functional TEPs are produced from mice or human PSCs that support mouse or human T cell development after transplanting TEPs under the midrenal capsule of mice, it has not yet been demonstrated that high levels of original human T cells can be reconstructed in vitro by effective PSC-derived TECs. Therefore, there is a need in the art for methods of generating TEPs and TECs that can support efficient in vitro T cell development.

[0005] Being able to generate functional TECs from human pluripotent stem cells will have important applications in modeling human immune responses in mice and in modeling and treating thymic deficiency syndromes such as DiGeorge syndrome, Nude syndrome, and immune deficiencies that complicate bone marrow transplantation for leukemia. The cells can also be used clinically for cell therapy and transplanted into patients to achieve T cell reconstitution, or for generating immune tolerance to prevent transplant rejection after organ transplantation, or for restoring thymic function that is impaired due to injury or aging.

[0006] International application WO2020205859 discloses a method for inducing human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), to differentiate into thymic epithelial cell progenitors (TEC progenitors) in vitro, wherein the thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEPs) are capable of producing thymic organs and T cells in vivo. The application teaches a differentiation medium supplemented with Noggin, retinoic acid, FGF8b, sonic hedgehog, BMP and YM155. In the application, the inventors demonstrated that the generation of TEC progenitors and their subsequent maturation in TECs can be carried out after transplantation into mice. International application WO2020 / 220040 also teaches a method for obtaining TEPs and TECs.

[0007] However, in vitro functional thymopoieisis, ie, the generation of T cells for clinical applications and clinical therapy, is still not easily possible due to the mandatory animal engraftment.

[0008] Therefore, there is a need to provide an in vitro method for generating functional T cells from iPSCs for subsequent free use in therapy.An object of the present invention is to provide a method for generating thymic epithelial cells and thymic epithelial progenitor cells from pluripotent cells and in particular from iPSCs.

[0009] Another object of the present invention is to provide a method for treating pathologies involving thymic defects.

[0010] The present invention relates to the use of a composition comprising the following compounds: activin A, BMP4, CHIR99, EGF, FGF 8, FGF 10, IGF1, LY3, Noggin, retinoic acid and Y27,

[0011] The composition is used to implement a differentiation process of induced pluripotent stem cells or iPScs into functional thymic epithelial progenitor cells or TEPs, preferably an in vitro or ex vivo differentiation process.

[0012] More advantageously, the present invention relates to the use of the following groups of compounds for carrying out a differentiation process of induced pluripotent stem cells or iPScs into functional thymic epithelial progenitor cells or TEPs, preferably an in vitro or ex vivo differentiation process,

[0013] - the first group comprising Y27 compounds,

[0014] - A second group containing Activin A and CHIR99,

[0015] - A third group comprising activin A,

[0016] - A fourth group comprising Y27 and activin A,

[0017] - A fifth group comprising retinoic acid and FGF8,

[0018] - Group 6 containing noggin, retinoic acid, LY3 and FGF8,

[0019] - A seventh group comprising retinoic acid, LY3, CHIR99, BMP4 and FGF8,

[0020] - Group 8 comprising retinoic acid, CHIR99, BMP4 and FGF8, and

[0021] - A ninth group comprising retinoic acid, BMP4, FGF8, FGF10, IGF1, EGF and RANKL.

[0022] More advantageously, the present invention relates to the use of the above-mentioned groups of compounds, wherein iPSCs are incubated with:

[0023] - The first group contains Y27 compounds, followed by

[0024] - A second group containing Activin A and CHIR99, followed by

[0025] - A third group containing activin A, followed by

[0026] - A fourth group containing Y27 and activin A, followed by

[0027] - A fifth group containing retinoic acid and FGF8, followed by

[0028] - Group 6 containing noggin, retinoic acid, LY3 and FGF8, followed by

[0029] - Group 7 containing retinoic acid, LY3, CHIR99, BMP4, and FGF8, followed by

[0030] - The eighth group contained retinoic acid, CHIR99, BMP4 and FGF8, and then

[0031] - A ninth group comprising retinoic acid, BMP4, FGF8, FGF10, IGF1, EGF and RANKL.

[0032] The present invention is based on the unexpected observation of the inventors that a specific combination of growth factors allows differentiation of induced pluripotent stem cells into functional thymic epithelial progenitor cells or TEPs. Compared to the prior art, the TEPs obtained according to the present invention are fully functional and can be easily and efficiently differentiated into mature TECs, i.e. medullary TECs (mTECs) and cortical TECs (cTECs). In addition to the differentiation process, thymic bulks can also be obtained by using the above-mentioned composition.

[0033] As used herein, the terms "differentiation" and "cell differentiation" refer to the process by which a less specialized cell (ie, stem cell) develops or matures or differentiates into a more specialized or differentiated cell (ie, thymic epithelial cell) with a more distinct form and / or function.

[0034] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that not all progeny will have exactly the same DNA content, due to intentional or unintentional mutations. Included are mutant progeny screened for the same function or biological activity as the originally transformed cell. Where different names are intended, this will be clear from the context.

[0035] As used herein, the term "induced pluripotent stem cells" is often abbreviated as iPS cells or iPSCs or IPScs, and refers to a type of pluripotent stem cells artificially produced from non-pluripotent cells (usually adult somatic cells) or terminally differentiated cells (such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.).

[0036] IPSc are non-naturally occurring cells that have been genetically modified to carry out the expression of genes that restore the differentiation program, resulting in the loss of specialized acquired functions of differentiated cells and the acquisition of pluripotency.

[0037] Pluripotency describes that cells can develop into the three primary germ cell layers of the early embryo, and therefore develop into all cells of the adult, but do not develop into extraembryonic tissues such as the placenta. Embryonic stem cells and induced pluripotent stem cells are characterized by their pluripotency. Only totipotent cells can provide all differentiated cells. The present invention does not cover these totipotent cells.

[0038] iPSC is usually derived by introducing the product of a specific set of pluripotency-related genes or "reprogramming factors" into a given cell type. A group of initial reprogramming factors (also known as Yamanaka factors) are transcription factors Oct4 (Pou5f1), Sox2, Klf4 and cMyc. Although this combination is the most conventional in generating iPSC, each of these factors can be functionally replaced by related transcription factors, miRNA, small molecules or even non-related genes such as lineage-specific molecules (lineage specifier).

[0039] iPSC differentiation is usually a slow and inefficient process, requiring 1-2 weeks for mouse cells and 3-4 weeks for human cells, with an efficiency of about 0.01%-0.1%. However, considerable progress has been made in improving efficiency and the time required to obtain iPSCs. After the introduction of reprogramming factors, 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 by the expression of surface markers or reporter genes. More details are given below.

[0040] In the present invention, the following compounds are used:

[0041] Activin A is a member of the TGF-β superfamily and has been recognized as a multifunctional cytokine that is expressed in a variety of tissues and cells and plays a role in the regulation of inflammation, fibrosis, and wound repair. It promotes atherogenesis by inhibiting foam cell formation and neointimal hyperplasia and HF.

[0042] BMP4 or bone morphogenetic protein 4 is a member of the bone morphogenetic protein family that is part of the transforming growth factor-β 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 auditory bulla during early embryonic development.

[0043] 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 formula

[0044] [Chemical formula 1]

[0045]

[0046] Its CAS number is 252917-06-9. CHIR99021 promotes the self-renewal potential of mouse embryonic stem cells (ESCs) by inhibiting glycogen synthase kinase-3 (GSK-3) activity and enhances the upregulation of β-catenin and c-Myc functions. CHIR99021 promotes the self-renewal of ESCs by regulating transforming growth factor β (TGF-β), Notch and mitogen-activated protein kinase (MAPK) signaling pathways. CHIR99021 is an agonist of wingless / integrated (wnt) signaling, upregulating cyclin A expression and promoting cell proliferation in non-small cell lung cancer (NSCLC) cell lines.

[0047] 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 mass of about 6 kDa. It contains three disulfide bonds (Cys6-Cys20, Cys14-Cys31, Cys33-Cys42).

[0048] FGF 8, fibroblast growth factor 8 is important and necessary for establishing and maintaining the midbrain / hindbrain border (or mesencephalon / met-encephalon border), which plays an important role as an "organizer" in development, such as the Spemann "organizer" of the gastrulating embryo. FGF 8 is expressed in areas where Otx2 and Gbx2 cross-inhibit each other, and expression is maintained through this interaction. Once expressed, FGF 8 induces other transcription factors to form cross-regulatory loops between cells, thereby establishing boundaries. Through development, FGF 8 regulates the growth and differentiation of progenitor cells in this area to produce the final structure of the midbrain and hindbrain. It has been shown that FGF8 is sufficient to induce the remodeling of the midbrain and hindbrain structures.

[0049] FGF10, fibroblast growth factor 10 is a member of the fibroblast growth factor (FGF) family. FGF family members have extensive mitogenic and cell survival activities, and participate in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. Fibroblast growth factor 10 is the paracrine signaling molecule first seen in limb buds and organogenesis. FGF10 begins the development of limbs and participates in morphogenic branches in multiple organs such as lungs, skin, ears and salivary glands. Epithelial branching requires FGF10 signaling. Therefore, all branch morphogenesis organs such as lungs, skin, ears and salivary glands require constant expression of FGF10. This protein does not show activity to fibroblasts.

[0050] IGF-1, insulin-like growth factor 1, also known as somatomedin C, is a hormone similar in molecular structure to insulin that plays an important role in growth in children and has anabolic effects in adults.

[0051] LY3, LY364947, also known as 4-[3-(2-pyridyl)-1H-pyrazol-4-yl]-quinoline, is a compound of the formula

[0052] [Chemical formula 2]

[0053]

[0054] Its CAS number is 396129-53-6. This compound is a potent ATP-competitive inhibitor of TGFβR-I. In combination with valproic acid, this compound can replace SOX2 in the reprogramming of mouse embryonic fibroblasts transduced with OCT4, KLF4 and c-MYC.

[0055] 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 that of rat, mouse and Xenopus laevis (a genus of aquatic frog). Noggin is an inhibitor of several bone morphogenetic proteins (BMPs): it inhibits at least BMP2, BMP4, BMP5, BMP6, BMP7, BMP13 and BMP14.

[0056] Retinoic acid (also called all-trans retinoic acid or RA) is a metabolite of vitamin A1 (all-trans retinol) that mediates the functions of vitamin A1 required for growth and development. All-trans retinoic acid is required in chordates, which include all higher animals from fish to humans. During early embryonic development, all-trans retinoic acid produced in specific areas of the embryo helps determine the position along the embryo's anterior / posterior axis by acting as an intercellular signaling molecule that guides the development of the embryo's posterior. RA works through Hox genes, which ultimately control the front / posterior shaping in the early developmental stages.

[0057] Y-27632, hereinafter referred to as Y27, (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride is a compound of the formula:

[0058] [Chemical formula 3]

[0059]

[0060] Its CAS number is 129830-38-2. Y27 is a cell-permeable, efficient and selective inhibitor of Rho-related, coiled-coil-containing protein kinase (ROCK). Y27 has been described as enhancing the survival of human embryonic stem (ES) cells when dissociated into single cells by preventing dissociation-induced apoptosis (anoikis), thereby increasing their cloning efficiency, using a forced aggregation scheme to improve embryoid body formation, increase the survival of single human ES cells cryopreserved after thawing, and block the apoptosis of neural precursors derived from mouse ES after dissociation and transplantation. Y27 is also combined with CHIR99021 to participate in the direct lineage reprogramming of fibroblasts to mature neurons. Finally, it is known that Y27 improves the survival of human ES cell monolayers at the beginning of a differentiation scheme.

[0061] Advantageously, the abovementioned compounds are used simultaneously, individually or sequentially.

[0062] It is more advantageous that the compounds for inducing differentiation of iPSCs into TEPs are used sequentially.

[0063] Advantageously, the invention relates to the use as defined above, wherein the differentiation process is carried out for at least 14 days.

[0064] In order to carry out the differentiation process from iPSCs to TEPs, the composition as defined above has to be used during at least 14 days.

[0065] More advantageously, the compounds contained in the composition as defined above may be used sequentially at least once during the aforementioned period of at least 14 days.

[0066] For example, the differentiation process may be initiated with a Y27 compound, followed by a CHIR99 compound and actinin A, further followed by retinoic acid and FGF8, further followed by BMP4 and LY3 compounds, and then followed by FGF10, IGF1, and EGF.

[0067] Advantageously, the present invention relates to the use as defined above, wherein the iPSc expresses the OCT4 and NANOG genes.

[0068] The generation of induced pluripotent cells depends critically on the transcription factors used for induction. Oct-3 / 4 and certain products of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as key transcriptional regulators involved in the induction process, and their 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 efficiency of induction.

[0069] Oct-3 / 4 (Pou5f1): Oct-3 / 4 is a member of the octamer ("Oct") family of transcription factors and plays a key 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 the presence of Oct-3 / 4 thus induces pluripotency and differentiation potential in embryonic stem cells. Various other genes in the "Oct" family, including Oct-3 / 4's close relatives Oct1 and Oct6, failed to trigger induction, demonstrating the exclusivity of Oct-3 / 4 for the induction process. However, Hans A team led by Dr. Carolyn González-Gilchristina Rodriguez (who discovered the Oct4 gene in 1989) demonstrated that overexpression of Oct4 during reprogramming causes epigenetic changes that deteriorate the quality of iPSCs.

[0070] Sox family: The Sox family of transcription factors is associated with maintaining pluripotency similar to Oct-3 / 4, but is associated with multipotent stem cells and unipotent stem cells in contrast to Oct-3 / 4, which is exclusively expressed in pluripotent stem cells. Although Sox2 is the initial gene for induction, other transcription factors in the Sox family have been found to play a role in the induction process. Sox1 produces iPS cells with similar efficiency to Sox2, and the genes Sox3, Sox15, and Sox18 also produce iPS cells, but with reduced efficiency.

[0071] Klf family: Klf4 of the Klf family of transcription factors was first identified. As a factor for generating mouse iPS cells, it was proven to be a factor for generating human iPS cells. Klf2 and Klf4 were found to be factors capable of generating iPS cells, and related genes Klf1 and Klf5 were also found to be, but with reduced efficiency.

[0072] Myc Family: The Myc family of transcription factors are proto-oncogenes implicated in cancer. c-myc has been shown to be a factor involved in the generation of mouse iPS cells and is a factor involved in the generation of human iPS cells. However, the use of the "myc" gene family in the induction of iPS cells is troubling for the contingency of iPS cells as clinical therapy, as 25% of mice transplanted with c-myc-induced iPS cells develop lethal teratomas. N-myc and L-myc have been identified as alternatives to c-myc to induce pluripotency with similar efficiency.

[0073] Nanog: In embryonic stem cells, Nanog is essential together with Oct-3 / 4 and Sox2 in promoting pluripotency. There are also reports that iPS cells can be generated using Nanog as one of the factors.

[0074] LIN28: LIN28 is an mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells and is associated with differentiation and proliferation. LIN28 has been shown to be a factor in iPSC generation in combination with OCT4, SOX2, and NANOG.

[0075] Glis1: Glis1 is a transcription factor that can be used together with Oct-3 / 4, Sox2, and Klf4 to induce pluripotency. It has many advantages when used instead of C-myc.

[0076] Advantageously, the present invention relates to the use of the following compounds: Activin A, BMP4, CHIR99, EGF, FGF 8, FGF10, IGF1, LY3, Noggin, Retinoic acid Y27, RANKL, IL-7, FLT3, SCF and Glutamax

[0077] The compound is used for implementing a differentiation process of induced pluripotent stem cells or iPScs into functional thymic epithelial cells, preferably an in vitro or ex vivo differentiation process.

[0078] In other words, the present invention relates to the use of a composition comprising BMP4, FGF 8, FGF 10, IGF1, EGF, RANKL, IL7, FLT3, SCF and glutamax for carrying out a differentiation process of TEP cells as defined above (i.e., TEPs obtained by differentiation from iPSCs) into TECs, preferably an in vitro or ex vivo differentiation process.

[0079] The present inventors have found that by using the above composition, TEPs obtained as defined above can be efficiently differentiated into thymic organoids or TECs.

[0080] RANKL, the 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 a ligand of the receptor RANK, a binding partner of the apoptosis regulatory gene, osteoprotegerin (OPG), and controls cell proliferation by changing 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 glands, osteoblasts, mammary epithelial cells, prostate and pancreas. Changes in RANKL concentration levels in several organs once again confirm the importance of RANKL in tissue growth (especially bone growth) and immune function in vivo.

[0081] IL7, interleukin 7 or IL-7 stimulates the differentiation of multipotent (pluripotent) hematopoietic stem cells into lymphoid progenitor cells. IL7 also stimulates the proliferation of all cells in the lymphoid lineage (B cells, T cells and NK cells). IL7 is important for the proliferation of B cell maturation, survival of T cells and NK cells, development and certain stages of homeostasis.

[0082] FTL3 or FLT3-L is a ligand of the tyrosine kinase receptor FMS-like tyrosine kinase 3, also known as FLT3. FTL3 is a hematopoietic cytokine that regulates the proliferation of early hematopoietic cells. It has been shown that it works synergistically with a variety of 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.

[0083] SCF, stem cell factor (also known as SCF, KIT-ligand, KL or steel factor) is a cytokine that binds to the c-KIT receptor (CD117). SCF plays an important role in hematopoiesis during embryonic development. Sites where hematopoiesis occurs, such as fetal liver and bone marrow, express SCF. SCF can serve as a guiding cue that directs hematopoietic stem cells (HSCs) to their stem cell niches (the microenvironment where stem cells reside), and it plays an important role in HSC maintenance. It has been shown that SCF increases the survival of HSCs in vitro and contributes to the self-renewal and maintenance of HSCs in vivo. HSCs at all developmental stages express the same levels of the SCF receptor (c-KIT). The stromal cells that surround HSCs are components of the stem cell niche, and they release many ligands, including SCF.

[0084] Glutamax is an L-glutamine substitute. Glutamax is the dipeptide L-alanyl-L-glutamine in 0.85% NaCl.

[0085] As for the composition for allowing IPSCs to differentiate into TEP cells, the compounds contained in the composition for allowing TEPs to differentiate into TECs may be used simultaneously, individually or sequentially.

[0086] Advantageously, RANKL, IL7, FTL3, SCF and Glutamax are used simultaneously in all differentiation processes. These compounds can be combined with BMP4, FGF 8, FGF 10, IGF1 and EGF, at least for the first step of the differentiation process from TEP to TEC.

[0087] The present invention relates to a method for differentiating iPSc into functional TEP in vitro or ex vivo, the method comprising the following steps:

[0088] a) incubating iPScs in a medium supplemented with a Y27 compound for 24 hours to obtain a Y27 medium,

[0089] b) removing the Y27 compound from the Y27 compound and adding Activin A growth factor and CHIR99 compound for 24 hours to obtain ActA / CHIR99 medium,

[0090] c) removing CHIR99 growth from ActA / CHIR99 medium and maintaining the culture with Activin A for 48 hours to obtain ActA medium,

[0091] d) adding Y27 compound to the ActA culture for 24 hours to obtain Y27 / ActA culture medium,

[0092] e) removing ActA and Y27 compound from the Y27 / Act1 medium, and adding FGF8 growth factor and retinoic acid to the Y27 / Act1 medium for 24 hours to obtain RA / F8 medium,

[0093] f) adding Noggin growth factor and LY3 compound to RA / F8 medium for 48 hours to obtain RA / F8 / NOG / LY3 medium,

[0094] g) removing Noggin growth factor from RA / F8 / NOG / LY3 medium and adding CHIR99 compound and BMP4 growth factor for 48 hours to obtain RA / F8 / LY3 / CHIR99 / BMP medium,

[0095] h) removing the LY3 ​​compound from the RA / F8 / LY3 / CHIR99 / BMP culture for 24 hours to obtain the RA / F8 / CHIR99 / BMP culture medium,

[0096] i) Changing the concentrations of BMP4 and FGF8 growth factors in the RA / F8 / CHIR99 / BMP medium for 24 hours,

[0097] j) removing the CHIR99 compound from the RA / F8 / CHIR99 / BMP medium and adding FGF10 growth factor, IGF1 growth factor, and EGF growth factor for 72 hours to obtain a RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0098] k) Recovery of functional TEPs from RA / F8 / BMP / F10 / IGF1 / EGF medium after 72 hours.

[0099] The inventors have demonstrated, in addition to specific studies of a collection of compounds and growth factors involved in the thymic differentiation process, that this series of 11 steps is necessary to obtain TEP cells that are fully functional and can further be used to initiate another differentiation process in order to obtain TEC cells.

[0100] From step b) to step j), it is mentioned that one or more compounds or growth factors are added or removed. This means that the compound or growth factor

[0101] - is added to a previous culture, or by replacing the previous culture medium with a new medium containing the corresponding compound and / or growth factor together with the new compound or growth factor,

[0102] - removal from the previous culture medium, or by replacing the previous culture medium with a new medium containing the corresponding compound and / or growth factor, without "removing" the compound or growth factor.

[0103] In other words, the present invention relates to the above method, which comprises:

[0104] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0105] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0106] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0107] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0108] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0109] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0110] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0111] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0112] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0113] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0114] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0115] The culture medium as defined in the method defined above is an aqueous liquid containing the minimum essential nutrients required for cell survival and proliferation and more generally for maintaining cell homeostasis. The culture medium contains salts, nutrients (such as amino acids, nucleotides and ribonucleotides), vitamins. In particular, for the differentiation process, some specific culture media are available from different manufacturers, which contain some compounds that maintain or help the differentiation process. For example, mTeSR commercialized by StemCell Biotechnologies can be used. TM 1 or mTeSR TM Plus medium, or X-VIVO available from Lonza TM 10 Culture medium. A person skilled in the art can select the most relevant culture medium to carry out the above steps. Preferably, step a) is carried out with a first culture medium, and the other steps are carried out with a second culture medium different from the second culture medium.

[0116] All steps were performed in a cell incubator, where the temperature was maintained at about 37°C and with 5% CO2, with a constant humidity rate. These conditions are classical conditions for culturing cells and cell lines and for performing differentiation processes.

[0117] Advantageously, the present invention relates to the above method, wherein Activin A growth factor is used at a concentration of 100 ng / mL in step b) and at a concentration of 50 ng / mL at step c) and step d).

[0118] Therefore, the present invention advantageously relates to the above method, which comprises:

[0119] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0120] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0121] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0122] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0123] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0124] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0125] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0126] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0127] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0128] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0129] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0130] More advantageously, the present invention relates to the above method, wherein the Y27 compound and the FGF10, IGF1 and EGF growth factors are in an amount of 10 μM, i.e. 10 μmol / L or 10 10 -6 mol / L concentration was used.

[0131] Therefore, the present invention relates to the above method, which comprises:

[0132] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0133] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0134] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0135] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0136] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0137] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0138] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0139] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0140] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0141] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0142] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0143] Therefore, the present invention advantageously relates to the above method, which comprises:

[0144] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0145] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0146] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0147] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0148] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0149] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0150] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0151] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0152] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0153] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0154] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0155] Advantageously, the present invention relates to a method as defined above, wherein the CHIR99 growth factor is used at a concentration of 5 μM.

[0156] More advantageously, the present invention relates to a method as defined above, wherein Noggin growth factor is used at a concentration of 100 ng / mL.

[0157] Advantageously, the present invention relates to the above method, comprising:

[0158] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0159] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0160] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0161] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0162] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0163] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0164] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound and 5 μM CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0165] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor and 5 μM CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0166] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0167] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0168] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0169] More advantageously, the present invention relates to a method as defined above, comprising:

[0170] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0171] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0172] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0173] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0174] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0175] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0176] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound and 5 μM CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0177] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor and 5 μM CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0178] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0179] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0180] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0181] The present invention more advantageously relates to the above method, which comprises:

[0182] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0183] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0184] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0185] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0186] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0187] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0188] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound and 5 μM CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0189] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor and 5 μM CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0190] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0191] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0192] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0193] Advantageously, the present invention relates to a 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).

[0194] Advantageously, the present invention relates to the above method, comprising:

[0195] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0196] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0197] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0198] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0199] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0200] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0201] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0202] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0203] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0204] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0205] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0206] More advantageously, the present invention advantageously relates to the above method, which comprises:

[0207] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0208] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0209] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0210] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0211] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0212] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0213] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0214] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0215] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0216] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0217] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0218] Therefore, the present invention relates to the above method, which comprises:

[0219] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0220] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0221] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0222] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0223] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0224] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0225] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0226] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0227] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0228] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0229] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0230] Therefore, the present invention advantageously relates to the above method, which comprises:

[0231] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0232] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0233] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0234] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0235] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0236] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0237] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0238] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0239] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0240] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0241] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0242] Advantageously, the present invention relates to the above method, comprising:

[0243] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0244] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0245] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0246] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0247] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0248] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0249] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0250] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0251] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a second RA / F8 / CHIR99 / BMP medium,

[0252] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0253] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0254] More advantageously, the present invention relates to a method as defined above, comprising:

[0255] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0256] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0257] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0258] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0259] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0260] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0261] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0262] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0263] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a second RA / F8 / CHIR99 / BMP medium,

[0264] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0265] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0266] The present invention more advantageously relates to the above method, which comprises:

[0267] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0268] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0269] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0270] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0271] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0272] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0273] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0274] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0275] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a second RA / F8 / CHIR99 / BMP medium,

[0276] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0277] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0278] Advantageously, the invention relates to a method as defined above, wherein retinoic acid is used in step e) and step f) at a concentration of 0.75 μM.

[0279] More advantageously, the present invention relates to a method as defined above, wherein retinoic acid is used in steps g) to j) at a concentration of 0.1 μM.

[0280] Advantageously, the present invention relates to a method as defined above, comprising:

[0281] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0282] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0283] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0284] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0285] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0286] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0287] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0288] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0289] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0290] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0291] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0292] Advantageously, the present invention relates to a method as defined above, comprising:

[0293] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0294] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0295] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0296] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0297] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0298] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0299] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0300] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0301] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0302] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0303] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0304] Advantageously, the present invention relates to a method as defined above, comprising:

[0305] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0306] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0307] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0308] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0309] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0310] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0311] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0312] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0313] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0314] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0315] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0316] Advantageously, the present invention relates to a method as defined above, comprising:

[0317] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0318] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0319] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0320] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0321] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0322] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0323] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0324] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0325] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0326] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0327] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0328] Advantageously, the present invention relates to a method as defined above, comprising:

[0329] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0330] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0331] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0332] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0333] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0334] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, 100 ng / mL of a Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0335] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound and 5 μM CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0336] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor and 5 μM CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0337] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0338] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0339] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0340] More advantageously, the present invention relates to a method as defined above, comprising:

[0341] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0342] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0343] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0344] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0345] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0346] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, 100 ng / mL of a Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0347] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound and 5 μM CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0348] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor and 5 μM CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0349] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0350] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0351] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0352] The present invention more advantageously relates to the above method, which comprises:

[0353] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0354] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0355] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0356] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0357] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0358] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, 100 ng / mL of a Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0359] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor, LY3 compound and 5 μM CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0360] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, BMP4 growth factor and 5 μM CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0361] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0362] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0363] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0364] Advantageously, the present invention relates to the above method, comprising:

[0365] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0366] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0367] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0368] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0369] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0370] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0371] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0372] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0373] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0374] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0375] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0376] More advantageously, the present invention advantageously relates to the above method, which comprises:

[0377] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0378] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0379] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0380] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0381] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0382] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0383] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0384] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0385] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0386] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0387] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0388] Therefore, the present invention relates to the above method, which comprises:

[0389] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0390] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0391] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0392] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0393] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0394] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0395] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0396] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0397] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0398] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0399] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0400] Therefore, the present invention advantageously relates to the above method, which comprises:

[0401] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0402] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0403] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0404] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0405] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0406] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0407] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0408] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells in an eighth medium supplemented with FGF8 growth factor, retinoic acid compound, 10 ng / mL of BMP4 growth factor and CHIR99 compound for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0409] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a 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 a second RA / F8 / CHIR99 / BMP medium,

[0410] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0411] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0412] Advantageously, the present invention relates to the above method, comprising:

[0413] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0414] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0415] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0416] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0417] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0418] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, 100 ng / mL of a Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0419] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0420] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0421] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a second RA / F8 / CHIR99 / BMP medium,

[0422] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0423] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0424] More advantageously, the present invention relates to a method as defined above, comprising:

[0425] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0426] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and 5 μM of the CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0427] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0428] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0429] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0430] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, 100 ng / mL of a Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0431] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0432] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0433] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a second RA / F8 / CHIR99 / BMP medium,

[0434] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0435] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0436] The present invention more advantageously relates to the above method, which comprises:

[0437] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0438] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0439] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0440] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0441] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0442] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with FGF8 growth factor, 0.75 μM of a retinoic acid compound, 100 ng / mL of a Noggin growth factor, and a LY3 compound for 48 hours to obtain cells in a RA / F8 / NOG / LY3 medium,

[0443] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0444] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a first RA / F8 / CHIR99 / BMP medium,

[0445] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells 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 for 24 hours to obtain cells in a second RA / F8 / CHIR99 / BMP medium,

[0446] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0447] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0448] More advantageously, the present invention relates to the method as defined above, wherein the FGF8 growth factor is used at a concentration of 50 ng / mL in steps e) and f), 20 ng / mL in steps g) and h, and 10 ng / mL at step j).

[0449] Finally, more advantageously, the present invention relates to a method as defined above, comprising:

[0450] a) incubating iPScs in a first culture medium supplemented with 10 μM of a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0451] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with 100 ng / mL of Activin A growth factor and 5 μM of CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0452] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with 50 ng / mL of actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0453] d) removing the ActA medium from the cells, and incubating the cells in a fourth medium supplemented with 50 ng / mL of actinin A growth factor and 10 μM of the Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0454] e) removing the Y27 / ActA medium from the cells, and incubating the cells in a fifth medium supplemented with 50 ng / mL of FGF8 growth factor and 0.75 μM of a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0455] f) removing the RA / F8 medium from the cells, and incubating the cells in a sixth medium supplemented with 50 ng / mL of FGF8 growth factor, 0.75 μM of retinoic acid compound, 100 ng / mL of Noggin growth factor, and 10 μM of LY3 compound for 48 hours to obtain cells in RA / F8 / NOG / LY3 medium,

[0456] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells 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 for 48 hours to obtain cells in RA / F8 / LY3 / CHIR99 / BMP medium,

[0457] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 24 hours to obtain cells in the first RA / F8 / CHIR99 / BMP medium,

[0458] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0459] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells 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 for 72 hours to obtain cells in the RA / F8 / BMP / F10 / IGF1 / EGF medium, and

[0460] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering the cells that are functional TEPs.

[0461] More advantageously, 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+.

[0462] The TEP cells obtained according to the method defined above express the specific membrane markers EPCAM and CD205, and express the genes FOXN1 and PAX9.

[0463] The FOXN1 gene encodes forkhead box protein N1, a DNA-binding transcription factor that regulates keratin gene expression. Deletion or knockout of the FOXN1 gene produces an athymic phenotype. FOXN1 has been shown to bind and regulate genes involved in T cell maturation and antigen presentation in the thymus.

[0464] The PAX9 gene encodes the paired box gene 9 protein, also known as PAX9. It has been demonstrated that thymopoiesis requires Pax9 function in thymic epithelial cells.

[0465] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that mediates Ca2+-independent homogeneous cell-cell adhesion in epithelia.

[0466] CD205 is an endocytic receptor expressed at high levels by cortical thymic epithelial cells and subsets of dendritic cells (DCs).

[0467] The present invention also relates to functional TEP cells obtainable or directly obtainable by the method as defined above, said cells expressing FOXN1 and PAX9 genes and being EPCAM+CD205+, TEP expressing NTRK2, CDH11, FLRT3 proteins, preferably TBX3, NTRK2, MEIS2, CDH11, PRSS23 and FLRT3 proteins.

[0468] These cells are novel because they are derived from non-naturally occurring stem cells (i.e. iPSCs), even though they have the same properties and the same differentiation capacity compared to natural TECs obtained from differentiation of cells derived from fertilized eggs. In addition, as demonstrated in the examples, the TEPs obtained according to the method defined above express cell markers that are not expressed in natural PEPs or PEPs obtained from methods disclosed in the art.

[0469] Despite the specific markers defined above, cells cannot be exhaustively described except by methods that allow their acquisition.

[0470] In another aspect of the present invention, there is disclosed a method for obtaining thymic epithelial cells from the functional TEP as defined above, the method comprising

[0471] a. incubating the functional TEP in a medium supplemented with L-glutamine, etc. and a first composition comprising BMP4, FGF8, FGF10, IGF1 and EGF growth factors and a second composition comprising RANKL, IL7, FTL3 and SCF growth factors for 96 hours to obtain a first TEP differentiation culture,

[0472] b. removing the first composition from the first TEP differentiation culture for 120 hours to obtain a second TEP differentiation culture, and

[0473] c. Recovery of thymic epithelial cells from the second TEP differentiation culture.

[0474] Advantageously, in step b, RANKL may be added twice a week.

[0475] In other words, the present invention relates to a method for obtaining thymic epithelial cells from functional TEPs as defined above, the method comprising:

[0476] a. incubating the functional TEPs in a medium supplemented with L-glutamine, etc. and a first composition comprising BMP4, FGF8, FGF10, IGF1 and EGF growth factors and a second composition comprising RANKL, IL7, FTL3 and SCF growth factors for 96 hours to obtain cells in a first TEP differentiation medium,

[0477] a. removing the cells from the first TEP differentiation culture and contacting the cells with a medium supplemented with a second composition for 120 hours to obtain cells in a second TEP differentiation medium, and

[0478] b. Removing the second TEP differentiation culture from the cells and recovering cells that are functional thymic epithelial cells.

[0479] Advantageously, the present invention relates to a method for obtaining thymic epithelial cells from functional TEPs as defined above, the method comprising:

[0480] a) incubating the functional TEPs in a culture medium supplemented with L-glutamine and a first composition comprising BMP4, FGF8, FGF10, IGF1 and EGF growth factors and a second composition comprising RANKL, IL7, FTL3 and SCF growth factors for 96 hours to obtain a first TEP differentiation culture,

[0481] b) removing the first TEP differentiation culture from the cells and incubating the cells incubated with the first TEP differentiation culture with a medium supplemented with L-glutamine and a second composition comprising RANKL, IL7, FTL3 and SCF growth factors for 120 hours to obtain a second TEP differentiation culture, and

[0482] c) Recovering thymic epithelial cells from the second TEP differentiation culture.

[0483] More advantageously, the present invention relates to a method as defined above, comprising:

[0484] a. incubating the functional TEPs in a medium supplemented with L-glutamine, etc. and a first composition comprising 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 factors and a second composition comprising 50 ng / mL of RANKL, 5 ng / mL of IL7, 5 ng / mL of FTL3 and 10 ng / mL of SCF growth factors for 96 hours to obtain cells in the first TEP differentiation medium,

[0485] a. removing the cells from the first TEP differentiation culture and contacting the cells with a medium supplemented with a second composition for 120 hours to obtain cells in a second TEP differentiation medium, the second composition comprising 50 ng / mL of RANKL, 5 ng / mL of IL7, 5 ng / mL of FTL3 and 10 ng / mL of SCF growth factor, and

[0486] b. Removing the second TEP differentiation culture from the cells and recovering cells that are functional thymic epithelial cells.

[0487] More advantageously, the present invention relates to a method for differentiating iPSc into functional TEC in vitro or ex vivo, the method comprising the following steps:

[0488] a) incubating iPScs in a first culture medium supplemented with a Y27 compound for 24 hours to obtain cells in a Y27 culture medium,

[0489] b) removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with Activin A growth factor and a CHIR99 compound for 24 hours to obtain cells in ActA / CHIR99 medium,

[0490] c) removing the ActA / CHIR99 medium from the cells, and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain cells in ActA medium,

[0491] d) removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and a Y27 compound for 24 hours to obtain cells in a Y27 / ActA medium,

[0492] e) removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with FGF8 growth factor and a retinoic acid compound for 24 hours to obtain cells in RA / F8 medium,

[0493] f) removing the RA / F8 medium from the cells and incubating the cells 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 RA / F8 / NOG / LY3 medium,

[0494] g) removing the RA / F8 / NOG / LY3 medium from the cells, and incubating 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 RA / F8 / LY3 / CHIR99 / BMP medium,

[0495] h) removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 a first RA / F8 / CHIR99 / BMP medium,

[0496] i) removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating 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 a second RA / F8 / CHIR99 / BMP medium,

[0497] j) removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating 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 RA / F8 / BMP / F10 / IGF1 / EGF medium,

[0498] k) removing the RA / F8 / BMP / F10 / IGF1 / EGF medium from the cells and recovering cells that are functional TEPs,

[0499] l) incubating the functional TEPs in a culture medium supplemented with L-glutamine, etc. and a first composition comprising 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 factors and a second composition comprising 50 ng / mL of RANKL, 5 ng / mL of IL7, 5 ng / mL of FTL3 and 10 ng / mL of SCF growth factors for 96 hours to obtain cells in a first TEP differentiation culture medium,

[0500] m) removing the cells from the first TEP differentiation culture and contacting the cells with a medium supplemented with a second composition comprising 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 a second TEP differentiation medium, and

[0501] n) removing the second TEP differentiation culture from the cells and recovering cells that are functional thymic epithelial cells.

[0502] The present invention also relates to thymic epithelial cells obtainable or directly obtainable by a method as defined above.

[0503] Advantageously, the present invention relates to a thymic epithelial cell as defined above, said cell expressing the AIRE, PSMB11 and HLA-DRA genes and the FOXN1 and PAX9 genes and being EPCAM+CD205+.

[0504] These cells are novel in the art because they are derived from non-naturally occurring and novel TEPs.

[0505] The present invention also relates to a method for treating or preventing a thymic disease in a subject in need thereof, the method comprising the step of administering, transplanting or implanting a therapeutically effective amount of a TEP or TEC as defined above, a solution comprising a TEP or TEC as defined above, a composition comprising a TEP or TEC as defined above of the present disclosure, or a pharmaceutical composition comprising a TEP or TEC as defined above to a subject in need thereof. The subject is preferably a mammal, and most preferably a human.

[0506] Another embodiment is a method for treating or preventing an autoimmune disease in a subject in need thereof, the method comprising the step of administering, transplanting or implanting a therapeutically effective amount of a TEP or TEC as defined above, a solution comprising a TEP or TEC as defined above, a composition comprising a TEP or TEC as defined above, or a pharmaceutical composition comprising a TEP or TEC as defined above to a subject in need thereof. The subject is preferably a mammal, and most preferably a human.

[0507] Another embodiment is a method of restoring or repairing thymic function impairment in a subject in need thereof, the method comprising administering, transplanting or implanting a therapeutically effective amount of a TEP or TEC as defined above, a solution comprising a TEP or TEC as defined above, a composition comprising a TEP or TEC as defined above, or a pharmaceutical composition comprising a TEP or TEC as defined above to a subject in need thereof. The subject is preferably a mammal, and most preferably a human. In some embodiments, the impairment is due to damage. In some embodiments, the impairment is due to aging. In some embodiments, the impairment is due to congenital malformation.

[0508] Another embodiment is a method of reconstituting T cells after bone marrow transplantation in a subject in need thereof, the method comprising the step of administering, transplanting or implanting a therapeutically effective amount of a TEP or TEC as defined above, a solution comprising a TEP or TEC as defined above, a composition comprising cells of the present disclosure, or a pharmaceutical composition comprising a TEP or TEC as defined above to a subject in need thereof. The subject is preferably a mammal, and most preferably a human.

[0509] Another aspect of the present invention relates to a composition comprising

[0510] - a TEP cell as defined above, or

[0511] - a TEC cell as defined above,

[0512] or both

[0513] The composition is used for

[0514] - Treat or prevent thymus diseases,

[0515] - or to treat or prevent autoimmune diseases,

[0516] - or to reconstitute T cells after a bone marrow transplant. BRIEF DESCRIPTION OF THE DRAWINGS

[0517] The present invention will be better understood with reference to the following examples and the following drawings:

[0518] [ Figure 1 ][ Figure 1 ] represents a photograph showing the cell morphology at D0 following induction of iPSc differentiation.

[0519] [ Figure 2 ][ Figure 2 ] represents a photograph showing the cell morphology at D1 following induction of iPSc differentiation.

[0520] [ Figure 3 ][ Figure 3 ] represents a photograph showing the cell morphology at D2 following induction of iPSc differentiation.

[0521] [ Figure 4 ][ Figure 4 ] represents a photograph showing the cell morphology at D3 following induction of iPSc differentiation.

[0522] [ Figure 5 ][ Figure 5 ] represents a photograph showing the cell morphology at D4 following induction of iPSc differentiation.

[0523] [ Figure 6 ][ Figure 6 ] represents a photograph showing the cell morphology at D5 following induction of iPSc differentiation.

[0524] [ Figure 7 ][ Figure 7 ] represents a photograph showing the cell morphology at D6 following induction of iPSc differentiation.

[0525] [ Figure 8 ][ Figure 8 ] represents a photograph showing the cell morphology at D7 following induction of iPSc differentiation.

[0526] [ Fig. 9 ][ Fig. 9 ] represents a photograph showing the cell morphology at D8 following induction of iPSc differentiation.

[0527] [ Fig.10 ][ Fig.10 ] represents a photograph showing the cell morphology at D9 following induction of iPSc differentiation.

[0528] [ Fig.11 ][ Fig.11 ] represents a photograph showing the cell morphology at D10 following induction of iPSc differentiation.

[0529] [ Fig.12 ][ Fig.12 ] represents a photograph showing the cell morphology at D11 following induction of iPSc differentiation.

[0530] [ Fig.13 ][ Fig.13 ] represents a photograph showing the cell morphology at D14 following induction of iPSc differentiation.

[0531] [ Fig.14 ][ Fig.14 ] is a graph showing the FOXN1 gene expression level relative to GAPDH on day 0, day 10, and day 14 of iPSc differentiation.

[0532] [ Fig.15 ][ Fig.15] is a fluorescent micrograph of iPSCs differentiated during 14 days. The photograph shows that FOXN1 protein and DNA are colocalized in the nucleus. The cytoplasm is labeled with keratin 8.

[0533] [ Fig.16 ][ Fig.16 ] is a photograph of TEP cells after being cultured with the differentiation mixture for 1 day.

[0534] [ Fig.17 ][ Fig.17 ] is a photograph of TEP cells after being cultured with the differentiation mixture for 11 days.

[0535] [ Fig.18 ][ Fig.18 ] is a fluorescence micrograph of TEP cells after differentiation into TEC. The photograph shows that DNA (nucleus) and keratin 8 (cytoplasm) are not co-localized.

[0536] [ Fig.19 ] Heatmap of Pearson correlations between samples.

[0537] [ Fig. 20 ] Heat map of target marker repertoires for sample validation.

[0538] [ Fig.21 ]UMAP representation of the Magaletta et al. pharyngeal organogenesis dataset, and relative levels of marker expression. Example

[0539] Example 1 - Differentiation of induced pluripotent stem cells (iPSc) from thymic epithelial progenitor cells (TEPs)

[0540] 1. Purpose

[0541] This is a protocol describing the acquisition of TEPs from the differentiation of iPScs.

[0542] This protocol provides technical details on the steps required to produce TEP.

[0543] It is used in experiments that require manipulation of TEP.

[0544] 1. Methods

[0545] NOTE: A "day" of differentiation is defined as a 24 hour duration. This method is time dependent and the appropriate duration is critical.

[0546] 1. Day 1

[0547] Wells were prepared by depositing 500 μL of Matrigel diluted at 1% in DMEM:F12 (1:1 mixture of DMEM and Ham's F-12 - Thermo Fisher Scientific) and incubated at 37°C for at least 3 hours.

[0548] iPSc cultures within 60%-80% confluence and without spontaneous differentiation were used.

[0549] Wash iPScs with DPBS- / - (potassium chloride (KCl) 200 mg / L, potassium dihydrogen phosphate (KH2PO4) 200.0 mg / L, sodium chloride (NaCl) g / L, and disodium hydrogen phosphate (Na2HPO4-7H2O) 2.160 g / L, without calcium and magnesium), remove DPBS- / -, add 500 μL TrypLE to each well, and incubate at 37°C for 5 minutes.

[0550] Add 500 μL of mTeSER1 (StemCell Technologies) to each well and rinse several times to dissociate all cells.

[0551] Centrifuge at 200 g for 5', remove the supernatant and resuspend the cell pellet in 1 mL of mTESR1.

[0552] Count the cells.

[0553] Preparation of D-1 cell suspension: aspirate a certain volume of cell suspension containing 130 000*p cells, make up to pmL mTESR1, and add Y27 to a final concentration of 10 μM.

[0554] 1 mL of D-1 cell suspension was deposited per well, homogenized and placed in an incubator at 37°C and 5% CO2.

[0555] 1. Day 0

[0556] Prepare D0 medium: basal XVIVO10, 5 μM CHIR99, 100 ng / mL Activin A.

[0557] Remove the culture medium from the wells.

[0558] Wash with DPBS- / -.

[0559] Add 1 mL of D0 medium to each well.

[0560] 1. Day 1

[0561] Prepare D1 medium: basal XVIVO10, 50 ng / mL Activin A.

[0562] Remove the culture medium from the wells.

[0563] Wash with DPBS- / -.

[0564] Add 1 mL of D1 medium to each well.

[0565] 1. Day 2

[0566] Prepare D1 medium: basal XVIVO10, 50 ng / mL Activin A.

[0567] Remove the culture medium from the wells.

[0568] Wash with DPBS- / -.

[0569] Add 1 mL of D1 medium to each well.

[0570] q culture wells were prepared by depositing 500 μL of Matrigel diluted 1% in DMEM:F12 and incubated at 37°C for at least 3 hours.

[0571] Cells must reach confluence.

[0572] 1. Day 3

[0573] Wash cells with DPBS- / -, remove DPBS- / -, add 500 μL TrypLE per well, and incubate at 37°C for 5'.

[0574] Add 500 μL of TrypLE to each well. Rinse several times to dissociate all cells.

[0575] Centrifuge at 200 g for 5', remove the supernatant and resuspend the cell pellet in 1 mL XVIVO10.

[0576] Count the cells.

[0577] Preparation of D3 cell suspension (see table): aspirate a certain volume of cell suspension containing 50 000*q cells, make up to qmL XVIVO10, add Activin A to a final concentration of 50 ng / mL and add Y27 to a final concentration of 10 μM.

[0578] 1 mL of D3 cell suspension was deposited per well, homogenized and placed in an incubator at 37°C and 5% CO2.

[0579] 1. Day 4

[0580] Prepare D4 medium: basal XVIVO10, 0.75 μM retinoic acid, and 50 ng / mL FGF8.

[0581] Remove the culture medium from the wells.

[0582] Wash with DPBS- / -.

[0583] Add 1 mL of D4 medium to each well.

[0584] 1. Day 5

[0585] Prepare D5 medium: basal XVIVO10, 0.75 μM retinoic acid, 50 ng / mL FGF8, 10 μM LY3, 100 ng / mL Noggin.

[0586] Remove the culture medium from the wells.

[0587] Wash with DPBS- / -.

[0588] Add 1 mL of D5 medium to each well.

[0589] 1. Day 7

[0590] Prepare D7 medium: basal XVIVO10, 0.1 μM retinoic acid, 5 μM CHIR99, 5 μM LY3, 10 ng / mL BMP4, and 20 ng / mL FGF8.

[0591] Remove the culture medium from the wells.

[0592] Wash with DPBS- / -.

[0593] Add 1 mL of D7 medium to each well.

[0594] 1. Day 9

[0595] Prepare D9 medium: basal XVIVO10, 0.1 μM retinoic acid, 5 μM CHIR99, 10 ng / mL BMP4, and 20 ng / mL FGF8.

[0596] Remove the culture medium from the wells.

[0597] Wash with DPBS- / -.

[0598] Add 1 mL of D7 medium to each well.

[0599] Each well was prepared by depositing 500 μL of Matrigel diluted 1% in DMEM:F12 and incubated at 37°C for at least 3 hours.

[0600] 1. Day 10

[0601] If the cells have not yet reached confluence, wait before passaging them.

[0602] Add 500 μL of TrypLE to each well. Rinse several times to dissociate all cells.

[0603] Centrifuge at 200 g for 5', remove the supernatant and resuspend the cell pellet in 1 mL XVIVO10.

[0604] Count the cells.

[0605] Prepare D10 cell suspension (see table): take a certain volume of cell suspension containing 100 000 cells, make up to 1 mL XVIVO10, add 0.1 μM retinoic acid, 5 μM CHIR99, 50 ng / mL BMP4 and 20 ng / mL FGF8.

[0606] 1 mL of D10 cell suspension was deposited per well, homogenized and placed in an incubator at 37°C and 5% CO2.

[0607] 1. Day 11

[0608] Prepare D11 medium: basal 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.

[0609] Remove the culture medium from the wells.

[0610] Wash with DPBS- / -.

[0611] Add 1 mL of D11 medium to each well.

[0612] 1. Day 13

[0613] Prepare D13 medium: basal 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.

[0614] Remove the culture medium from the wells.

[0615] Wash with DPBS- / -.

[0616] Add 1 mL of D14 medium to each well.

[0617] 1. Day 14

[0618] Prepare TEP for harvest.

[0619] Lysis was performed in culture wells to verify FOXN1 and PAX9 expression by RT-qPCR.

[0620] Wells were used to verify differentiation yield: >50% EPCAM+CD205+ cells.

[0621] The above protocol allows the generation of TEPs from iPScs for subsequent maturation steps.

[0622] The following table summarizes the above steps:

[0623] [Table 1]

[0624] sky -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Culture medium M X X X X X X X X X X X X X X Compound unit Y27 μM 10 10 ActA ng / mL 100 50 50 50 NOG ng / mL 100 100 RA μM 0.75 0.75 0.75 0.1 0.1 0.1 0.1 0.1 0.1 0.1 LY3 μM 10 10 5 5 CHIR99 μM 5 5 5 5 BMP4 ng / mL 5 10 10 10 50 50 50 50 FGF8 ng / mL 50 50 50 20 20 20 10 10 10 10 FGF10 ng / mL 10 10 10 IGF1 ng / mL 10 10 10 EGF ng / m 10 10 10

[0625] M = mTESR1 medium; X = X-VIVO 10 medium.

[0626] Example 2 - Maturation of thymic epithelial progenitor cells (TEPs) and thymic organoids in functional thymic epithelial cells (TECs) The formation of officials

[0627] 1. Purpose

[0628] The following protocol describes the maturation of TECs in functional thymic organoids obtained from iPSc-derived TEPs as disclosed in Example 1.

[0629] This protocol provides technical details on the steps required to generate thymic organoids. It is intended for use in experiments requiring manipulation of thymic organoids.

[0630] 2. Methods

[0631] Note: Early thymic progenitors (ETPs) were derived here from primary samples of the thymus from young patients who underwent cardiac surgery on the same day.

[0632] ETPs are hematopoietic stem cells derived from the bone marrow that give rise to the T cell lineage, and their phenotype is: CD45+CD3-CD4-CD8-CD14-CD19-CD56-CD34+CD7+.

[0633] Day 0

[0634] Check the quality of D14 TEP cultures. Cells must be confluent and form a dense monolayer with ridges and projections.

[0635] Prepare wash buffer: PBS, 0.5% BSA, 2 mM EDTA.

[0636] Prepare FACS buffer: PBS, 2% FBS, 5 mM EDTA.

[0637] Prepare beads for ETP separation: Pipette 1 mL anti-mouse Dynabeads, wash 1' with 2 mL wash buffer, place on Dynabead magnet 2'. Remove supernatant and resuspend beads in 500 μL wash buffer. Label beads with mouse anti-human CD3 antibody for 45' with stirring at 40°C. Wash 3 times with 20 mL wash buffer on Dynabead magnet.

[0638] Resuspend in 20 mL of wash buffer.

[0639] Isolation of ETP from donor primary thymus samples: Dissect thymus in 50 mL of RPMI medium in Falcon into 1 mm3 pieces. Pellet, collect supernatant, filter through 70 μm mesh. Repeat 3 times. Centrifuge at 200 g for 5'. Resuspend pellet in 10 mL of red blood cell lysis buffer. Incubate at room temperature for 5'. Add 20 mL of PBS and centrifuge at 200 g for 5'. Resuspend pellet in 10 mL of wash buffer. Count cells. Collect 1 billion cells and adjust volume to 20 mL with wash buffer.

[0640] Add to Dynabeads and incubate at 40 °C with agitation for 30'.

[0641] Place the tube on the magnet for 2'. Collect the supernatant and centrifuge at 200g for 5'. Resuspend the pellet in 1 mL FACS buffer. Label the cells with Lin (CD3 CD4 CD8 CD14 CD19 CD56), CD7 and CD34.

[0642] Sort cells using flow cytometry. Centrifuge at 200 g for 5'. Resuspend the pellet in 1 mL of XVIVO10 (LONZA Reference No. 04-380Q) buffer. Count cells.

[0643] TEPs were washed with DPBS- / -, DPBS- / - was removed, 500 μL of TrypLE (Thermo Fischer Scientific; - recombinant trypsin, free of any cell and animal contaminants) was added to each well and incubated at 37°C for 5'.

[0644] Add 500 μL XVIVO 10 to each well. Rinse several times to dissociate all cells.

[0645] Centrifuge at 200 g for 5', remove the supernatant and resuspend the cell pellet in 1 mL XVIVO10.

[0646] Count the cells.

[0647] The two cell suspensions were combined and the volume was adjusted with XVIVO10 to a concentration of 200 000 TEP / mL and 50 000 ETP / mL.

[0648] D14 supplements were added at the required concentrations (see Table 2), namely: 50 ng / mL BMP4, 10 ng / mL FGF8, 10 ng / mL FGF10, 10 ng / mL IGF1, 10 ng / mL EGF, 50 ng / mL RANKL, 5 ng / mL IL-7, 5 ng / mL FLT3, 10 ng / mL SCF, and 1% Glutamax.

[0649] Homogenize and plate at 100 μL / well in a low binding 96-well plate. Place in an incubator at 37°C and 5% CO2.

[0650] Day 1

[0651] Prepare hydrogel: Thaw aliquots of thrombin (10 U / mL) and aprotinin (26000 U / mL) solutions on ice. Do not vortex. Thaw fibrinogen (8 mg / mL) in a 37°C hot water bath. Do not vortex.

[0652] Slowly rinse the aliquot to homogenize. Prepare 0.25×o eppendorfs tubes, where o is the number of organoids seeded the day before. Add 150 μL of fibrinogen and 10 μL of aprotinin to each tube. Add 150 μL of thrombin to the tube, rinse quickly 2 times without creating bubbles, then draw 150 μL and place in the insert of the 24-well hanging insert. Repeat quickly with the remaining 150 μL in the second well. Avoid forming bubbles by carefully operating the pipette.

[0653] When all wells of the plate are prepared, incubate for 1 hour at 37° C. The clear liquid solution solidifies and becomes opaque.

[0654] A D15 solution was prepared, namely 50 ng / mL BMP4, 10 ng / mL FGF8, 10 ng / mL FGF10, 10 ng / mL IGF1, 10 ng / mL EGF, 50 ng / mL RANKL, 5 ng / mL IL-7, 5 ng / mL FLT3, 10 ng / mL SCF and 1% Glutamax.

[0655] To verify the quality of the reaggregation step, organoids must form spherical cell clusters with a dense core surrounded by a less dense corona of thymocytes (see [ Fig.16 ]).

[0656] Using a top-cut P200 cone previously washed with anti-adhesion solution, delicately collect the organoids and seed them one by one on top of the hydrogel in an amount of 2 organoids per well. Check that no cells are left in the P96 wells.

[0657] Slowly add 300 μL of D15 solution on top of the hydrogels without directly touching them. Add 700 μL of D15 solution at the bottom of each well. Place in an incubator at 37°C and 5% CO2.

[0658] Day 2

[0659] Check that the organoids are well seeded: the hydrogel must stay in place and the organoids must not settle on the bottom of the insert. Leave the D15 solution.

[0660] Day 5

[0661] From this point on, the culture medium can be changed daily or twice a week (organoids can be maintained in culture for up to 6 weeks).

[0662] D19 solution, 50 ng / mL RANKL, 5 ng / mL IL-7, 5 ng / mL FTL3, 10 ng / mL SCF, and 1% Glutamax, was added.

[0663] Day 14

[0664] Prepare TEP for harvest.

[0665] Lysis was performed in culture wells (in RTL buffer, Qiagen Ref. 79216) to verify FOXN1 and PAX9 expression by RT-qPCR.

[0666] Use the culture wells to verify differentiation yield by flow cytometry using appropriate antibodies: >50% EPCAM+CD205+ cells

[0667] The following table summarizes the above steps:

[0668] [Table 2]

[0669] sky 14 15 16 17 18 19 20 21 22 23 Culture medium X X X X X X X X X X Compound unit Y27 μM ActA ng / mL NOG ng / mL RA μM LY3 μM CHIR99 μM BMP4 ng / mL 50 50 50 50 50 FGF8 ng / mL 10 10 10 10 10 FGF10 ng / mL 10 10 10 10 10 IGF1 ng / mL 10 10 10 10 10 EGF ng / mL 10 10 10 10 10 RANKL ng / mL 50 50 50 50 50 50 50 50 50 50 IL7 ng / mL 5 5 5 5 5 5 5 5 5 5 FTL3 ng / mL 5 5 5 5 5 5 5 5 5 5 SCF ng / mL 10 10 10 10 10 10 10 10 10 10 Glutamax 1％ 1％ 1％ 1％ 1％ 1％ 1％ 1％ 1％ 1％

[0670] X = X-VIVO 10 medium.

[0671] 3. Results

[0672] [ Fig.17 ] shows the structural changes of thymic organoids cultured in hydrogels. The organoids change from a dense spherical form to a Fig.16] to a larger and less dense structure formed by elongated cells. This structural change reflects the in vivo 3D structure of the thymic epithelium, a low-density, sponge-like meshwork. By using IF and 3D imaging, we highlighted this structure in organoids using cytokeratin 8 (KRT8) labeling of TECs.

[0673] The obtained TEPs expressed expected thymic epithelial identity markers, such as FOXN1 and PAX9. In addition, TEP morphology resembled primary cells with large polygonal cells [ Fig.15 ]. Thus, this method allows the generation of primary-like TEPs from iPScs.

[0674] Example 3 - Identification of a panel of biomarkers specific to TEPs differentiated from iPScs

[0675] The aim was to identify a panel of biomarkers expressed with high specificity in TEPs generated by our protocol relative to controls and published in vivo data.

[0676] method :

[0677] Seven large samples of day 14 thymic epithelial progenitors (PEPs) derived from iPScs using our protocol were sequenced using DGEseq. Three samples of D0 cells (iPScs) at the start of differentiation were included as negative controls. Five primary human postnatal TEC samples were included as positive controls.

[0678] To remove potential marker contamination from undesired differentiation products, D14 PEPs were purified by FACS (BD FACS ARIA) on their prior art 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 are referred to below as TEP_puri.

[0679] RNA extraction

[0680] Cell samples were lysed using RLT lysis buffer (Qiagen, 79216). RNA was extracted from purified TEP samples using RNAeasy Mini Kit (Qiagen, 74104) or Micro Kit (Qiagen, 74004). Purified TEP samples were sorted directly into RLT lysis buffer.

[0681] RNA sequencing

[0682] The DEG sequencing protocol was performed according to the implementation of our previously developed protocol (1). Library preparation was performed using 10 ng of total RNA. mRNA was labeled using template-switched reverse transcription using poly(A) tail-specific adapters, well-specific barcodes, and universal molecular identifiers (UMIs). cDNA preparations were pooled, amplified, and tag-fragmented by transposon fragmentation to enrich for 3' ends. The cDNA was purified using the HiSeq Rapid SBS kit in Sequencing was performed on HiSeq 2500. The kits used were Zymo purification kit Researche D4004-1-L, Kit Advantage 2 PCR enzyme system (Clontech, 639206), QIAquick gel extraction, kit Agencourt AMPure XP magnetic beads, and Nextera DNA (FC-121-1031).

[0683] The purified TEP samples were sequenced using the SMART-seq protocol on the GENO'MIC platform at Cochin Institute.

[0684] RNAseq data analysis

[0685] Read pairs for analysis met the following criteria: the quality score of all 16 bases of the first read was at least 10, and the first 6 bases corresponded accurately 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 several transcripts of different genes or contained more than 3 mismatches with the reference sequence were filtered out from the analysis. DGE maps were generated by counting the number of unique UMIs associated with each RefSeq gene for each sample. Sequencing samples with at least 50,000 counts and 6,000 expressed genes were retained for further analysis. Batch correction and differential expression (DE) were performed with the DEseq2 R package with a threshold of 1 log2 fold change and a 0.05 Benjamini Hochberg p value to classify genes as differentially expressed genes versus controls.

[0686] Data visualization based on ComplexHeatmap and ggplot2 R packages on a custom pipeline.

[0687] Filtering the DE gene list to identify markers

[0688] Using the data manipulation package tidyr, two lists of DE genes generated by DE analysis of a large number of TEP samples for negative (D0 iPSc) and positive (primary TEC) controls were restricted to their intersection in R. The resulting lists of markers were further filtered to only positive markers, i.e. markers upregulated in TEPs.

[0689] Markers in the top 20% of Log2 fold change and average expression were selected. To further filter out markers that are also expressed in vivo, the expression of markers on the restricted list was manually checked against 2 public scRNAseq datasets including TEP from Park et al. and Magaletta et al., and a 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 association with the epithelial gene ontology was the next criterion for marker selection to reduce the risk of false positives. Final marker selection was performed using purified TEP samples, where markers that were highly expressed in the purified TEP data were selected as final markers.

[0690] result :

[0691] Sample transcriptome similarity :

[0692] Pearson correlation heatmaps and clustering of samples revealed a clear separation of TEC and iPSc groups from TEPs ([ Fig.19 ]).

[0693] Quality control and validation of differentiation :

[0694] The expression of the main markers (i.e. iPSc, TEC and TEP markers) of the target pedigree is analyzed to confirm the quality of the sample and verify the marker analysis. It can be clearly found that the iPSc gene cluster (NANOG, POU5F1) and the mature TEC gene cluster (HLA-DR, CD80) are highly expressed in the samples from the negative control group and the positive control group, respectively. This verifies that these samples are effective controls. A large amount of and purified TEP samples show the expression of classical TEP markers (EYA1, KRT8), which confirms the efficiency of differentiation in those samples and verifies their use as the basis of TEP marker research ([ Fig. 20 ]).

[0695] The final restricted list of markers is reported as follows:

[0696] [Table 3]

[0697]

[0698] The low expression of this restricted list of markers was confirmed in the thymic organogenesis dataset from Magaletta. Low to no expression of these markers could be detected in the Magaletta dataset, confirming that they are unique to our TEP differentiation products ([ Fig.21 ]).

[0699] To further restrict the list of markers, a cellular localization criterion can be applied. Cell membrane markers are of particular interest because they allow purification of viable cells. Based on this criterion, the final set of markers that are unique to our TEP differentiation products and not expressed in TEPs in vivo can be limited to: NTRK2, CDH11, FLRT3. In summary, TEPs obtained by the described protocol exhibit functionality similar to native TEPs, but can be identified by the expression of the marker set NTRK2, CDH11, FLRT3.

[0700] Example 4: Identification of mature TEPs in an organoid culture system from TEPs produced according to the methods of the present invention A set of biomarkers unique to TEC.

[0701] The aim was to identify a panel of biomarkers expressed with high specificity in TECs generated by our protocol relative to controls and published in vivo data.

[0702] method :

[0703] Data acquisition and preprocessing :

[0704] Single-cell RNA sequencing (scRNAseq) data used for the study were obtained from publicly available datasets or generated in-house. A public dataset of human primary TEC used as a control was reanalyzed from the original data from the study by Park et al. We generated our dataset from D21 organoids cultured according to our protocol. Organoids were dissociated by incubation in 0.5 mg / mL collagenase / dispase solution at 37°C for 30' and mechanical disruption, followed by incubation with TrypLE at 37°C for 5'. The obtained cell suspension was washed in cold dPBS and filtered through a 100 μm mesh. Cells were stained with 1 μg / mL of anti-EPCAM PE, anti-CD45 APC-Cy7, anti-HLA-DR PeCy5, and anti-CD205 APC antibodies and DAPI. Cells were sorted on a BD FACS ARIA using the CD45-EPCAMhi phenotype. The cell suspension was washed and loaded onto a Chromium 10X box for library generation. The count matrix data file was downloaded and preprocessed using the Seurat package in R. A quality control step was performed to ensure high quality data for downstream analysis by filtering out cells with more than 10% mitochondrial genes, less than 500, or more than 5000 counts.

[0705] Data integration and dimensionality reduction :

[0706] For the Park et al. data, multiple datasets were integrated using Seurat's integration workflow via canonical correlation analysis (CCA). This step allowed the datasets to be merged into a common space and reconciled technical variations across different experiments. After data integration, dimensionality reduction was performed via PCA by keeping the first 15 dimensions.

[0707] Identification of cell clustering and TECs :

[0708] Unsupervised clustering of cells was performed using the graph-based clustering algorithm of Seurat using Louvain parameters. The clustering results were visualized using UMAP or other visualization techniques to identify different clusters representing different cell populations. To identify TEC clusters, marker genes (EPCAM, FOXN1, PAX9) specific to known TECs were used, and differential expression analysis was performed to find genes that were highly expressed in TEC clusters compared to other clusters.

[0709] Pseudobulk generation and differential expression analysis :

[0710] Once TEC clusters were identified, a pseudobulk transcriptome for each TEC cluster was generated by summing the expression values ​​of cells within each cluster. Differential expression analysis was performed using the DESeq2 package in R to identify genes differentially expressed between TEC clusters. The pseudobulk transcriptome from each TEC cluster was 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 to 0.05) were considered differentially expressed.

[0711] Marker gene identification

[0712] Markers in the top 20% of Log2 fold change and average expression were selected. To further filter out markers that are also expressed in vivo, the expression of markers on the restricted list was manually checked against 2 public scRNAseq datasets containing TECs from the paper by Magaletta et al. and 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 association with the epithelial gene ontology was the next criterion for marker selection to reduce the risk of false positives.

Claims

1. Use of a composition comprising the following compounds: activin A, BMP4, CHIR99, EGF, FGF 8, FGF 10, IGF1, LY3, Noggin, retinoic acid and Y27, The composition is used to implement a differentiation process of induced pluripotent stem cells or iPScs into functional thymic epithelial progenitor cells or TEPs, preferably an in vitro or ex vivo differentiation process.

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

3. The use according to claim 1 or 2, wherein the iPSc expresses OCT4 and NANOG genes.

4. A method for differentiating iPSc into functional TEP in vitro or ex vivo, the method comprising the following steps: a. incubating the iPSc in a medium supplemented with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y27) compound for 24 hours to obtain Y27 medium, b. removing the Y27 medium from the cells and incubating the cells with a second medium supplemented with activin A growth factor and [[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99) compound for 24 hours to obtain the cells in ActA / CHIR99 medium, c. removing the ActA / CHIR99 medium from the cells and incubating the cells with a third medium supplemented with actinin A growth factor for 48 hours to obtain the cells in ActA medium, d. removing the ActA medium from the cells and incubating the cells in a fourth medium supplemented with actinin A growth factor and Y27 compound for 24 hours to obtain the cells in Y27 / ActA medium, e. removing the Y27 / ActA medium from the cells and incubating the cells in a fifth medium supplemented with fibroblast growth factor 8 (FGF8) growth factor and retinoic acid (RA) compound for 24 hours to obtain the cells in RA / F8 medium, f. removing the RA / F8 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-pyrazol-4-yl]-quinoline (LY3) compound for 48 hours to obtain the cells in RA / F8 / NOG / LY3 medium, g. removing the RA / F8 / NOG / LY3 medium from the cells, and incubating the cells in a seventh medium supplemented with FGF8 growth factor, retinoic acid compound, bone morphogenetic protein 4 (BMP4) growth factor, LY3 compound and CHIR99 compound for 48 hours to obtain the cells in RA / F8 / LY3 / CHIR99 / BMP medium, h. removing the RA / F8 / LY3 / CHIR99 / BMP medium from the cells and incubating 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 the cells in the first RA / F8 / CHIR99 / BMP medium, i. removing the first RA / F8 / CHIR99 / BMP medium from the cells and incubating the cells in a ninth medium supplemented with FGF8 growth factor, retinoic acid compound, bone morphogenetic protein 4 (BMP4) growth factor and CHIR99 compound for 24 hours to obtain the cells in a second RA / F8 / CHIR99 / BMP medium, j. removing the second RA / F8 / CHIR99 / BMP medium from the cells, and incubating the cells in a tenth medium supplemented with FGF8 growth factor, BMP4 growth factor, fibroblast growth factor 10 (FGF 10) growth factor, insulin-like growth factor 1 (IGF1) growth factor and epidermal growth factor (EGF) growth factor for 72 hours to obtain the cells in RA / F8 / BMP / F10 / IGF1 / EGF medium, and k. Recovering the functional TEPs from the RA / F8 / BMP / F10 / IGF1 / EGF 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 at step c) and step 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 the 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 at a concentration of 0.75 μM in step e) and step f).

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 at step j).

11. The method according to any one of claims 4 to 10, wherein the functional TEP cells express FOXN1 and PAX9 genes and are EPCAM+CD205+.

12. A functional TEP cell obtainable by or directly obtained by the method according to any one of claims 4 to 11, the cell expressing FOXN1 and PAX9 genes and being EPCAM+CD205+, wherein the cell expresses NTRK2, CDH11, FLRT3 proteins.

13. A method for obtaining thymic epithelial cells from functional TEP as defined in claim 12, the method comprising: a. incubating the functional TEPs in a culture medium supplemented with L-glutamine and a first composition and a second composition for 96 hours to obtain a first TEP differentiation culture, wherein the first composition comprises 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 factors, and the second composition comprises receptor activator of nuclear factor kappa-B ligand (RANKL), interleukin 7 (IL7), FMS-like tyrosine kinase 3 ligand (FTL3) and stem cell factor (SCF), b. removing the first TEP differentiation culture from the cells, and incubating the cells incubated with the first TEP differentiation culture with a medium supplemented with L-glutamine and a second composition comprising RANKL, IL7, FTL3 and SCF for 120 hours to obtain a second TEP differentiation culture, and c. Recovering the thymic epithelial cells from the second TEP differentiation culture.

14. A thymic epithelial cell obtainable by or directly obtained by the method according to claim 13, wherein the cell expresses AIRE, PSMB11 and HLA-DRA genes as well as FOXN1 and PAX9 genes, and is EPCAM+CD205+.

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