Method for producing t cell progenitors from human pluripotent stem cells

Abstract

The present invention relates to a method for generating T cell progenitors from pluripotent stem cells, and to the therapeutic use of the generated T cell progenitors.

Description

METHOD FOR PRODUCING T CELL PROGENITORS FROM HUMANPLURIPOTENT STEM CELLSFIELD OF INVENTION

[0001] The present invention relates to in vitro methods for producing T cell progenitors with therapeutic potential. In particular, the present invention relates to a method for producing T cell progenitors from pluripotent stem cells.BACKGROUND OF INVENTION

[0002] T cells are a crucial component of the immune system, and present a promising therapeutic potential for preventing or treating various diseases. In particular, the administration of T cells has been described or suggested in the art for treating diseases, such as for example autoimmune diseases, infections, inflammatory diseases or cancer, in particular to reconstitute and / or complement the immune system of the diseased subject.

[0003] It has also been described that progenitor of T lymphocytes (called proT cells or T cells progenitor) transplantation can accelerate T-cell compartment reconstitution after Progenitor and Hematopoietic Stem / Progenitor Cell (HSPC) transplantation. HSPC transplantation is nowadays considered as a referent therapeutic option for many severe hereditary immune deficiencies, as well as for many malignant or nonmalignant hematopoietic diseases, and for some solid tumors and autoimmune diseases. T cell progenitors could in particular reduce the risk linked to the persistence of T immunodeficiency in these subjects (including, for example, infections or relapses). In view of the therapeutic potential of T cells, and thus also of T cell progenitors that may be administered to differentiate in vivo into T cells, there is a need to develop a method to produce T cell progenitors in large numbers for clinical applications.

[0004] T cell progenitors are characterized by the expression of CD7, by their ability to home to the patient's thymus once injected, and to give rise to a large number of functional T cells. T cell progenitors can be produced from Hematopoietic Stem and progenitor cells, sorted from healthy donors (cord blood, bone marrow or blood after mobilization). However, all these sources can be used only a limited number of times (e.g., only once with cord blood) and allow the generation of a limited number of T cell progenitors, which hamper their broad use and their industrial production. There is thus still a need for an improved source of T cell progenitors, that avoids these drawbacks.

[0005] In the present invention, the Applicants aimed at using Pluripotent Stem Cells (PSCs) as a source of T cell progenitors. Because of their unlimited self-renewal property (capacity of the cells to divide indefinitely with maintenance of an undifferentiated state), PSCs may provide a renewable cell source for unlimited supply of ready to be use “off- the-shelf’ allogeneic T cell progenitors for clinical applications. However, the production of clinically applicable T cell progenitors in large numbers from PSCs is still significantly challenging. Most of the currently existing methods to produce T cells from PSCs use mouse derived stromal feeder cells that restricts their clinical use. Feeder cell-free methods are described in the art. However, these methods allow generation of T cell progenitors with only limited yield or with low purity.

[0006] Therefore, there is still a need to provide a method for generating T cell progenitors from PSC, with a high purity and with an increased yield as compared to the methods of the prior art.

[0007] In the present invention, Applicants define a novel method of producing T cell progenitors from PSCs, resulting on a highly pure T cell progenitor population with a strikingly high yield.SUMMARY

[0008] The present invention relates to an in vitro method for generating T cell progenitors (proT cells) comprising the steps of:a) obtaining embryoid bodies (EBs) from pluripotent stem cells (PSCs), b) isolating CD34+CD144+CD31+ cells from said embryoid bodies obtained in step a), c) culturing the population of CD34+ CD144+CD31+ cells obtained in step b) in a culture medium comprising a notch ligand, fibronectin or a fragment thereof, and an antagonist of the Aryl hydrocarbon / Dioxin receptor, thereby obtaining a population of HPCs, and d) culturing the population of HPCs obtained in step c) in a cytokine comprising medium, thereby obtaining a population of proT cells.

[0009] In some embodiments, at step a), the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0010] In some embodiments, at step a) the pluripotent stem cells are cultured in a medium comprising Ascorbic acid, 1 -thioglycerol, Transferrin, BMP4 (bone morphogenetic protein 4), Rock inhibitor, bFGF (basic fibroblast growth factor), an inhibitor of the activin receptor-like kinase (ALK) receptors, GSK3 inhibitor, VEGF (vascular endothelial growth factor), IL-6, IL-11, EPO (erythropoietin), IGF-1, and SCF (stem cell factor).

[0011] In some embodiments, the EBs obtained at step a) are dissociated to obtain a single cell suspension, preferably using an enzymatic treatment.

[0012] In some embodiments, at step c), the notch ligand is an immobilized Notch Ligand, preferably an immobilized Delta-like-4 ligand or a fragment thereof, and wherein the fibronectin or the fragment thereof is an immobilized fibronectin or fragment thereof, preferably an immobilized CH-296 (Retronectin®).

[0013] In some embodiments, the antagonist of the Aryl hydrocarbon / Dioxin receptor is StemRegenin 1 (SRI).

[0014] In some embodiments, at step c), the cells are cultured in presence of an immobilized Notch Ligand, an immobilized fibronectin or fragment thereof and anantagonist of the Aryl hydrocarb on / Dioxin receptor for more than 5 days and less than 12 days, preferably for about 9 days.

[0015] In some embodiments, at step d), the cells are cultured in the cytokine comprising medium for more than 5 days and less than 9 days, preferably for about 7 days and wherein said cytokine comprising medium comprises TNF-alpha, interleukin-7 (IL-7), Stem Cell Factor (SCF), thrombopoietin (TPO) and Flt3 ligand (FLT3L).

[0016] In some embodiments, at step d) the cells are also cultured in presence of an immobilized Notch Ligand, preferably an immobilized Delta-like-4 ligand or a fragment thereof, and an immobilized fibronectin or fragment thereof, preferably an immobilized CH-296 (Retronectin®), and optionally with Stromal cell-derived factor la (SDFla) and / or a p38 inhibitor.

[0017] In some embodiments, the in vitro method comprises an additional step of transducing or transfecting the cells with a vector encoding a transgene, preferably wherein said transgene encodes a Chimeric Antigen Receptor (CAR) or a T-cell receptor (TCR), or wherein said transgene is a safety switch gene, or encodes a potentiator of T- cell killing, wherein said additional step is performed at the beginning of step c).

[0018] In some embodiments, the in vitro method comprises an additional step of modifying the cells such as for example with a system of gene editing to express or delete expression of a specific gene, wherein said additional step is performed at the beginning of step c) of the method.

[0019] The present invention further relates to a population of T cell progenitors obtained by the in vitro method according to the invention, wherein at least 70%, preferably at least 80% of the cells are CD7+ and wherein at least 15%, preferably at least 20% of CD7+ cells are CD5+.

[0020] In some embodiments, the population of T cell progenitors obtained by the in vitro method according to the invention express CD161 and CD56, and does not express at least one of CD3, CD 19 or CD la.

[0021] In some embodiments, the population of T cell progenitors obtained by the in vitro method according to the invention is for use for increasing the number of T cell population in a subject in need thereof.

[0022] In some embodiments, the population of T cell progenitors obtained by the in vitro method according to the invention is for use for increasing the number of T cell population in a subject in need thereof, wherein the subject in need thereof has a medical condition causing or resulting in lymphopenia, preferably wherein the lymphopenia results from cancer, infection (e,g., HIV), partial thymectomy, autoimmune disease and / or organ transplantation.DEFINITIONS

[0023] In the present invention, the following terms have the following meanings:

[0024] The term "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0025] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclose method.

[0026] The term "administration", or a variant thereof (e.g. "administering"), means providing the active agent or active ingredient, alone or as part of a pharmaceutically acceptable composition, to the subject in whom / which the condition, symptom, or disease is to be treated or prevented.

[0027] The term “chimeric antigen receptor or CAR” refers to one polypeptide or to a set of polypeptides, typically two in the simplest embodiments, which when in an immune cell, provides the cell with specificity for a target ligand and with intracellular signal generation. Thus, a CAR usually comprises an extracellular ligand binding domain and a signaling domain. In some embodiments, the set of polypeptides are contiguous with each other. In some embodiments, the CAR is a chimeric fusion protein comprising the set of polypeptides. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple a ligand binding domain to an intracellular signaling domain. In some embodiments, the CAR comprises an optional leader sequence at the aminoterminus (N-ter). In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular ligand binding domain, wherein the leader sequence is optionally cleaved from the ligand binding domain during cellular processing and localization of the chimeric receptor to the cellular membrane.

[0028] The term “embryoid body (EB)” refers here to an in vitro differentiation model for pluripotent stem cells. EBs are characterized as three-dimensional aggregates of pluripotent stem cells. Embryoid bodies (EBs) can be obtained from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Pluripotent stem cells are differentiated into embryoid bodies comprising the cells to become ectoderm, mesoderm or endoderm.

[0029] The term “embryonic stem cells (ESCs)” refers here to a type of pluripotent stem cell derived from the blastocyst stage of early mammalian embryos and that have the ability to differentiate into an unlimited number of distinct cell types (z.e., all somatic cell types in the embryo). In some embodiments, the method of the invention does not comprise a step of destruction of human embryo to obtain ESCs.

[0030] The terms “expressing”, “positive”, or “+” and “not expressing”, “negative”, or are well known in the art and refer to the expression level of a cell marker of interest, in that the expression level of the cell marker corresponding to “+” is high or intermediate or low (i.e., the cell marker is expressed or present at the cell surface), and the expression level of the cell marker corresponding to is null (i.e., the cell marker is not expressed, or is absent, at the cell surface).

[0031] The term “hematopoietic progenitor cells (HPCs)” refers here to multipotent cells that can both self-renew and differentiate into all types of blood cells, including myeloid- lineage and lymphoid-lineage.

[0032] The term ‘hemogenic endothelial cells” (all call hemogenic endothelium) refers here to a subset of endothelial cells that can differentiate into multilineage hematopoietic stem and progenitor cells.

[0033] The term “induced pluripotent stem cells (iPSCs)” refers here to a type of pluripotent stem cell that can be obtained from animal or human differentiated cells using reprogramming approaches, said reprogramming approaches being well known in the art and including, without limitation, introduction of stem cell-associated genes (e.g., Oct4 (Pou5fl), Sox2, Klf4 or cMyc) into the cells.

[0034] The term “pharmaceutically acceptable excipient” (that may also be referred to as “pharmaceutically acceptable carrier”) refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the regulatory offices such as the FDA or EMA.

[0035] The term “pluripotent stem cells (PSCs)” refers here to cells that have the ability to undergo self-renewal, i.e., that have the capacity to divide indefinitely with maintenance of the undifferentiated state. PSCs have the capacity to differentiate to all the somatic cells / tissues. “PSCs” refers thus to an unlimited source of any type of animal or human cell needed for therapeutic purposes, e.g. dopaminergic neurons, insulinsecreting beta-cells, cardiomyocytes, immune cells for adoptive transfer, blood cells for transfusion, etc.

[0036] The term “subject” refers to a mammal, preferably a human. In some embodiments, a subject may be a “patient”, i.e. a warm-blooded animal, more preferablya human, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease. In some embodiments, the subject is an adult (for example a subject above the age of 18). In another embodiment, the subject is a child (for example a subject below the age of 18). In some embodiments, the subject is a male. In another embodiment, the subject is a female.

[0037] The terms “therapeutically effective amount” refers to an amount of the cells or of the composition as described herein, effective to achieve a particular biological result. Thus, the terms “therapeutically effective amount” mean a level or amount of a composition or a number of cells that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of the targeted disease or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the targeted disease or condition; (3) bringing about ameliorations of the symptoms of the targeted disease or condition; (4) reducing the severity or incidence of the targeted disease or condition; or (5) curing the targeted disease or condition. A therapeutically effective amount may be administered prior to the onset of the targeted disease or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the targeted disease or condition, for a therapeutic action.

[0038] The term “transfection” or “transduction” refers to a process by which an exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid, and includes the primary subject cell and its progeny.

[0039] The term “transgene” refers to a gene transferred or introduced into the host cell by different techniques well known in the art. A transgene encodes such as for example a Chimeric Antigen Receptor (CAR) and / or a T-cell receptor (TCR).

[0040] The term “treatment” or “treating” refers to therapeutic treatment, or prophylactic or preventative measures or to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted disease orcondition. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented. A subject is successfully “treated” for a disease or condition if, after receiving a therapeutic amount of cells or compositions as described herein, the subject shows observable and / or measurable improvement in one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; relief to some extent of one or more of the symptoms associated with the specific condition; reduced morbidity and mortality, and / or improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the condition are readily measurable by routine procedures familiar to a physician.DETAILED DESCRIPTION

[0041] The present invention relates to an in vitro method for generating T cell progenitors (proT cells) comprising the steps of: a. obtaining embryoid bodies (EBs) from pluripotent stem cells (PSCs), b. isolating CD34+CD144+CD31+ cells from said embryoid bodies obtained at step a), c. culturing the population of CD34+ CD144+CD31+ cells obtained at step b) in a culture medium comprising a Notch ligand (or a fragment thereof), fibronectin or a fragment thereof, and an antagonist of the Aryl hydrocarbon / Dioxin receptor, thereby obtaining a population of HPCs, and d. culturing the population of HPCs obtained in step c) in a cytokine comprising medium, thereby obtaining a population of proT cells.

[0042] At each culture step of the methods, cells may be cultured in one or more culture vessels. Said culture vessel may in particular be a conventional culture vessel selected from the group comprising, but not limited to, culture plates from 6 to 96 wells, petri dishes, flasks, stirrer bottles, bioreactor, culture chambers (e.g. Coming® CellSTACK® Culture Chambers), micro titer plates, test tubes, hollow fiber devices, cell foam and bags. The skilled artisan would now that the quantity of cells seeded for each culture step may be adapted according to the culture vessel used.

[0043] In some embodiments, the culture media used in step a) to step b) of the present invention are feeder cell-free. In particular, in some embodiments, the culture media used at step a) to step b) are free of OP-9 feeder cells or any other mouse derived stromal feeder cells.

[0044] In some embodiments, the total duration of the method described herein ranges from 20 to 30 days, preferably is about 24 days.

[0045] In some embodiments, pluripotent stem cells (PSCs) are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Examples of commercial cells that may be used in the method of the invention include, but are not limited to ATCC-DYR0100, WiCell iPS(IMR90)-4, iPS DF19-9-11T.H and Merck SCC271.

[0046] At step a) of the method of the present invention, pluripotent stem cells (PSCs) are cultured to obtain embryoid bodies (EBs).

[0047] In some embodiments, at step a), the cells are cultured for more than 5 days and less than 12 days, preferably for about 8 days. For example, PSCs may be cultured at step a) during about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days.

[0048] In some embodiments, the PSCs are seeded at a concentration ranging from about 5xl04to about 5xl05cells / ml of culture medium, preferably at a concentration ranging from 1x105to about 2.5xl05cells / ml. In some embodiments, the PSCs are seeded at a concentration ranging from about 10.000 to about 100.000 cells / cm2.of culture medium, preferably at a concentration ranging from about 30.000 to about 50.000 cells / cm2

[0049] In some embodiments, the PSCs are cultured at step a) in a hypoxia environment. For example, the PSCs may be cultured at step a) in a 5% CO2 / 5% 02 / 90% N2 environment. In some embodiments, the pluripotent stem cells (PSCs) are cultured at step a) at 37°C.

[0050] At step a), cells may be cultured in any culture medium adapted for the culture of PSCs. Examples of culture medium adapted for culture of PSCs comprise, but are not limited to, StemPro™-34 SFM, Essential 8™, SFM hESC StemPro™, StemFlex™, StemScale™ PSC, Ham’s F12, IMDM (Gibco™ , ThermoFisher Scientific).

[0051] For example, PSCs may be cultured at step a) in a mixture of media adapted for the culture of pluripotent stem cells. In another example, the PSCs may be cultured at step a) in a mixture of IMDM medium and Ham’s F-12 medium. Still in another example, the PSCs may be cultured at step a) in StemPro™-34 SFM medium (Gibco™, Thermo Fisher Scientific).

[0052] In some embodiments, the culture medium used at step a) comprises at least one of component selected from the group comprising or consisting of Ascorbic acid (AscH2, vitamin C), 1 -thioglycerol, Transferrin, BMP4 (bone morphogenetic protein 4), Rock inhibitor, bFGF, an inhibitor of the activin receptor-like kinase (ALK) receptors, a GSK3 inhibitor, VEGF (vascular endothelial growth factor), IL-6, IL-11, EPO (erythropoietin), IGF-1, SCF (stem cell factor). Ascorbic acid (AscH2, vitamin C), 1 -thioglycerol, Transferrin, BMP4 (bone morphogenetic protein 4), Rock inhibitor, bFGF, an inhibitor of the activin receptor-like kinase (ALK) receptors, a GSK3 inhibitor, VEGF (vascular endothelial growth factor), IL-6, IL-11, EPO (erythropoietin), IGF-1, SCF (stem cell factor) may be human components.

[0053] In some embodiments, the culture medium used at step a) is a serum-free medium. In some embodiments, the culture medium used at step a) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of fetal bovine serum (FBS) or fetal calf serum (FCS). In some embodiments, the culture medium used at step a) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of KnockOut™ serum replacement (GibcoTM,ThermoFisher Scientific). In some embodiments, the culture medium used at step a) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of serum -free alternative component such as for example (but not limited) UltraGRO™ cell culture supplement (HELIOS®, AventaCell BioMedical Corp.) or XerumFree™ cell culture supplement (TNCBIO).

[0054] In some embodiments, the culture medium used at step a) is supplemented with bovine serum albumin (BSA, Sigma-Aldrich). In some embodiments, the culture medium used at step a) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of BSA. In some embodiments, the culture medium used at step a) is supplemented with human serum albumin (HSA, Sigma-Aldrich). In some embodiments, the culture medium used at step a) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v HSA.

[0055] In some embodiments, the culture medium used at step a) is supplemented with at least one amino acid. In some embodiments, the culture medium used at step a) is supplemented with L-Glutamine (IX, GibcoTM , ThermoFisher Scientific) or an alternative such as for example GlutaMAX™ (GibcoTM , ThermoFisher Scientific).

[0056] The composition of the culture medium used at step a) may be the same during all this step, or may be different depending on the day of culture.

[0057] In some embodiments, at step a), the culture medium comprises Ascorbic acid (AA). Ascorbic Acid is a compound showing antioxidant activity, particularly against reactive oxygen species. Ascorbic Acid chemical formula is C6H8O6. This component is sold in particular by the companies MERCK (Sigma-Aldrich) or STEMCELL™ Technologies.

[0058] AA may be used at step a) at a concentration ranging from about 1 to about 500pg / ml, preferably from about 50 to about 500pg / ml, such as for example of at least about 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500pg / ml. For example, Ascorbic acid may be used at a concentration of 176 pg / ml.

[0059] AA may be present in the culture medium from day 0 of step a) to the end of step a), such as, for example, from day 0 of step a) and for about 1, 2, 3, 4, 5, 6, 7 or 8 days.

[0060] In some embodiments, at step a), the culture medium comprises transferrin. Transferrin is a glycoprotein with approximate molecular weights of 78 to 80 Kd, that provides iron to cells in culture. This component is sold in particular by the companies MERCK (Sigma-Aldrich) or Lonza.

[0061] Transferrin may be used at step a) at a concentration ranging from about 1 to about lOOOpg / ml, preferably from about 1 to about 200pg / ml, such as for example of at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200pg / ml. For example, Transferrin may be used at a concentration of 150pg / ml.

[0062] Transferrin may be present in the culture medium from day 0 of step a) to the end of step a), such as, for example, from day 0 of step a) and for about 1, 2, 3, 4, 5, 6, 7 or 8 days.

[0063] In some embodiments, at step a), the culture medium comprises 1 -thioglycerol (also call a-Monothioglycerol, a- Thioglycerol, 3 -Mercapto- 1,2-propanediol or Monothioglycerol or MTG). MTG is a compound showing inhibitor of glycerol kinase activity. 1 -thioglycerol chemical formula is HSCH2CH(OH)CH2OH. This component is sold in particular by the companies MERCK (Sigma-Aldrich) or Santa Cruz BioTechnology.

[0064] MTG may be used at step a) at a concentration ranging from about 50 to about lOOOpM, preferably from about 100 to about 600pM, such as for example of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 pM. For example, 1 -thioglycerol may be used at a concentration of 400pM.

[0065] MTG may be present in the culture medium from day 0 of step a) to the end of step a), such as, for example, from day 0 and for about 1, 2, 3, 4, 5, 6, 7 or 8 days.

[0066] In some embodiments, at step a), the culture medium comprises BMP4 (bone morphogenetic protein 4). BMP4 (corresponding for example to the Human UniProtaccession Q53XC5) is a polypeptide of the transforming growth factor P (TGF-P) superfamily that plays essential roles in many developmental processes, including neurogenesis, angiogenesis or osteogenesis. In some embodiments, BMP4 comprises or consists for example of amino acids 1-408 of SEQ ID NO: 1. This component is sold in particular by the companies MERCK (Sigma- Aldrich), STEMCELL Technologies or Miltenyi Biotec.

[0067] SEQ ID NO : 1MIPGNRMLMVVLLCQVLLGGASHASLIPETGKKKVAEIQGHAGGRRSGQSHEL LRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPE RPASRANTVRSFHHEEHLENIPGTSENSAFRFLFNLSSIPENEVISSAELRLFREQV DQGPDWERGFHRINIYEVMKPPAEVVPGHLITRLLDTRLVHHNVTRWETFDVS PAVLRWTREKQPNYGLAIEVTHLHQTRTHQGQHVRISRSLPQGSGNWAQLRPL LVTFGHDGRGHALTRRRRAKRSPKHHSQRARKKNKNCRRHSLYVDFSDVGW NDWIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSSIPKACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR

[0068] BMP4 may be used at step a) at a concentration ranging from about 1 to about 50ng / ml preferably from about 1 to about 15ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ng / ml. For example, BMP4 may be used at a concentration of 10 ng / ml.

[0069] BMP4 (bone morphogenetic protein 4) may be present in the culture medium from day 0 of step a) to the end of step a), such as, for example, from day 0 and for about 1, 2, 3, or 4, days.

[0070] In some embodiments, at step a), the culture medium comprises Rho-kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor). Examples of Rho- kinase inhibitor include, but are not limited to, AT-13148, BA-210, [3-Elemene, Belumosudil, Chroman 1, GSK-576371, GSK429286A, Y-27632, Y-30141, Y-33075 or Y-39983. For example, at step a), the culture medium may comprise Y-27632.

[0071] Y-27632 is a compound showing inhibitor of Rho-associated kinase (ROCK) activity. Y-27632 is known to improve cells viability. Y-27632 chemical formula is C14H21N3O. This component is sold in particular by the companies MERCK (Sigma- Aldrich), STEMCELL Technologies or Miltenyi Biotec.

[0072] The ROCK inhibitor may be used at step a) at a concentration ranging from about 1 to about 50pM, preferably from about 1 to about lOpM, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, lOpM. For example, the ROCK inhibitor may be used at a concentration of 5pM.

[0073] The Rock inhibitor may be present in the culture medium from day 0 of step a) to the end of step a), such as, for example, from day 0 and during about 1 day.

[0074] In some embodiments, at step a), the culture medium comprises bFGF (basic fibroblast growth factor also known as Fibroblast growth factor 2). bFGF (e.g., corresponding to the Human Uniprot Accession P09038) is a growth factor involved in several biological processes, such as, for example, cell growth or cell survival. In some embodiments, bFGF comprises or consists for example of amino acids 1-288 of SEQ ID NO: 2. In some embodiments, bFGF comprises or consists for example of any described isoform of SEQ ID NO: 2. This component is sold in particular by the companies MERCK (Sigma-Aldrich), STEMCELL Technologies or R&D Systems.

[0075] SEQ ID NO: 2MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPS VNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERV GGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGH FKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCA NRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTG QYKLGSKTGPGQKAILFLPMSAKS

[0076] bFGF may be used at step a) at a concentration ranging from about 1 to about 50 ng / ml, preferably from about 1 to about lOng / ml, such as for example of at least about 1,2, 3, 4, 5, 6, 7, 8, 9, lOng / ml. For example, bFGF may be used at a concentration of 5 ng / ml.

[0077] bFGF may be present in the culture medium from day 0 of step a) to the end of step a). bFGF may also be present in the culture medium from day 1 of step a) to the end of step a), such as, for example, from day 1 and during about 1, 2, 3, 4, 5, 6 or 7 days.

[0078] In some embodiments, at step a), the culture medium comprises an inhibitor of the activin receptor-like kinase (ALK) receptors. Examples of inhibitors of the activin receptor-like kinase (ALK) receptors include, but are not limited to, SB-431542, A 83- 01, SB-505124, Nodal. For example, at step a), the culture medium may comprise SB- 431542.

[0079] SB-431542 (4-[4-(2H-l,3-Benzodioxol-5-yniportyridin-2-yl)-lH-imidazol-2- yl]benzamide) is an inhibitor of the activin receptor-like kinase (ALK) receptors, ALK4, ALK5 and ALK7, developed by GlaxoSmithKline (GSK). SB-431542 chemical formula is C22H16N4O3. This component is sold in particular by the companies MERCK (Sigma- Aldrich), or STEMCELL Technologies.

[0080] The inhibitor of the activin receptor-like kinase (ALK) receptors, such as, for example, SB-431542 may be used at step a) at a concentration ranging from about 1 to about 50pM, preferably from about 1 to about lOpM, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, lOpM. For example, the inhibitor of the activin receptor-like kinase (ALK) receptors may be used at a concentration of 6pM.

[0081] The inhibitor of the activin receptor-like kinase (ALK) receptors may be present in the culture medium from day 0 to the end of step a). The inhibitor of the activin receptor-like kinase (ALK) receptors may also be present in the culture medium from day 2 of step a) to day 3 of step a).

[0082] In some embodiments, at step a), the culture medium comprises a GSK3 inhibitor (glycogen synthase kinase) or an activator of Wnt signaling. Examples of inhibitor of the enzyme GSK-3 (glycogen synthase kinase) include, but are not limited to, metal cations (e.g. Copper, lithium, mercury ...), and ATP-competitive agents (e.g. CHIR99021,CHIR98014, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR- A014418...), non-ATP competitive agents (e.g. Manzamine A, TDZD-8, NP00111, NP031115, L803-mts, L807-mts). In some embodiments, at step a), the culture medium comprises CHIR-99021.

[0083] CHIR-99021 (6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-lH-imidazol-2- yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile) is a compound showing GSK-3 inhibitor activity and Wnt signaling pathway regulator activity. This component is sold in particular by the companies MERCK (Sigma- Aldrich), or Bio-Techne.

[0084] The GSK3 inhibitor may be used at step a) at a concentration ranging from about 1 to about 50pM, preferably from about 1 to about lOpM, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, lOpM. For example, the GSK3 inhibitor may be used at a concentration of about 3pM.

[0085] The GSK3 inhibitor may be present from day 0 to the end of step a). The GSK3 inhibitor may also be present in the culture medium from day 2 of step a) to day 3 of step a).

[0086] In some embodiments, at step a), the culture medium comprises VEGF (vascular endothelial growth factor). VEGF (e.g., corresponding to the Human Uniprot accession Pl 5692) is a growth factor having a role in angiogenesis and endothelial cell growth. VEGF is also known to endothelial cell proliferation or promotes cell migration. In some embodiments, VEGF comprises or consists for example of amino acids 1-395 of SEQ ID NO: 3. In some embodiments, VEGF comprises or consists of any described isoform of SEQ ID NO: 3. This component is sold in particular by the companies MERCK (Sigma- Aldrich), ThermoFisher Scientific or R&D Systems.

[0087] SEQ ID NO: 3MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVAL KLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERG PQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEES GPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRC GGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRA RQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCSC KNTDSRCKARQLELNERTCRCDKPRR

[0088] VEGF may be used at step a) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. For example, VEGF may be used at a concentration of 15ng / ml.

[0089] VEGF may be present in the culture medium during all step a), or from day 4 of step a) to the end of step a). For example, VEGF may be present in the culture medium from day 4 and during at least about 1, 2, 3 or 4 days.

[0090] In some embodiments, at step a), the culture medium comprises Interleukin -6 (IL-6). IL-6 (e.g., corresponding to the Human UniProt Accession P05231) is a pro- inflammatory cytokine having a wide variety of biological functions in immunity and metabolism. In some embodiments, IL-6 comprises or consists for example of amino acids 1-212 of SEQ ID NO: 4. This component is sold in particular by the companies MERCK (Sigma-Aldrich), GenScript, STEMCELL™ Technologies or Miltenyi Biotec.

[0091] SEQ ID NO: 4MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDK QIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE TCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAI TTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

[0092] IL-6 may be used at step a) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. For example, IL- 6 is used at a concentration of about lOng / ml.

[0093] IL-6 may be present in the culture medium for all step a), or from day 4 of step a) to the end of step a). For example, IL-6 may be present in the culture medium at step a) since day 4 and during at least about 1, 2, 3 or 4 days.

[0094] In some embodiments, at step a), the culture medium comprises Interleukin- 11 (IL-11). IL-11 (e.g., corresponding to the Human UniProt Accession P20809) is a cytokine having wide variety of biological functions such as for example in adipogenesis or platelet maturation. In some embodiments, IL-11 comprises or consists for example of amino acids 1-199 of SEQ ID NO: 5. In some embodiments, IL-11 comprises or consists for example of amino acids 80-199 of SEQ ID NO: 5. This component is sold in particular by the companies MERCK (Sigma-Aldrich) or GenScript.

[0095] SEQ ID NO: 5MNCVCRLVLVVLSLWPDTAVAPGPPPGPPRVSPDPRAELDSTVLLTRSLLADTR QLAAQLRDKFPADGDHNLDSLPTLAMSAGALGALQLPGVLTRLRADLLSYLRH VQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLALPQPPPDPPAPPL APPSSAWGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL

[0096] IL- 11 may be used at step a) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. For example, IL- 11 may be used at a concentration of 5ng / ml.

[0097] IL-11 may be present in the culture medium for all step a), or from day 4 of step a) to the end of step a). For example, IL-11 may be present in the culture medium at step a) since day 4 and during at least about 1, 2, 3 or 4 days.

[0098] In some embodiments, at step a), the culture medium comprises erythropoietin (EPO). EPO (e.g., corresponding to the Human Uniprot accession P01588) is a glycoprotein hormone involved in the regulation of erythrocyte proliferation and differentiation. In some embodiments, EPO comprises or consists for example of amino acids 1-193 of SEQ ID NO: 6. This component is sold in particular by the companies MERCK (Sigma-Aldrich), Bio-Techne or R&D Systems.

[0099] SEQ ID NO: 6MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITT GCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQ ALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

[0100] EPO may be used at step a) at a concentration ranging from about 1 to about 50U / ml, preferably from about 1 to about lOU / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or lOU / ml. For example, EPO may be used at a concentration of 2 U / ml.

[0101] EPO may be present in the culture medium for all step a), or from day 6 of step a) to the end of step a). For example, EPO may be present in the culture medium at step a) from day 6 and during about 1 or 2 days.

[0102] In some embodiments, at step a), the culture medium comprises insulin-like growth factor-1 (IGF-1). IGF-l(e.g., corresponding to the Human Uniprot accession P05019) is a peptide hormone in particular involved in mediating growth and development. In some embodiments, IGF-1 comprises or consists for example of amino acids 1-195 of SEQ ID NO: 7. In some embodiments, IGF-1 comprises or consists for example of any described isoform of SEQ ID NO: 7. This component is sold in particular by the companies STEMCELL Technologies or Miltenyi Biotec.

[0103] SEQ ID NO: 7MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLC GAELVD ALQF VCGDRGF YFNKPTGYGS S SRRAPQTGIVDECCFRSCDLRRLEM YCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPGG EQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK

[0104] IGF-1 may be used at step a) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 30ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30ng / ml. For example, IGF-1 may be used at a concentration of 25ng / ml.

[0105] IGF-1 may be present in the culture medium during all step a), or from day 6 of step a) to the end of step a). For example, IGF-1 (insulin-like growth factor-1) may be present in the culture medium at step a) from day 6 and for about 1 or 2 days.

[0106] In some embodiments, at step a), the culture medium comprises stem cell factor (SCF, also known as kit-ligand). SCF (stem cell factor, e.g., corresponding Human Uniprot accession: P21583) have an essential role in the regulation of cell survival and proliferation or hematopoiesis. In some embodiments, SCF comprises or consists for example of amino acids 1-273 of SEQ ID NO: 8. In some embodiments, SCF comprises or consists for example of any described isoform of SEQ ID NO: 8.

[0107] SEQ ID NO: 8MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMI TLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIV DDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCV VS STLSPEKD SRVS VTKPFMLPP VAAS SLRND S S S SNRKAKNPPGD S SLHW AAM ALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV

[0108] SCF may be used at step a) at a concentration ranging from about 1 to about 500ng / ml, preferably from about 10 to about lOOng / ml, such as for example of at least about 10, ,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or lOOng / ml. For example, SCF is used at a concentration of 50ng / ml.

[0109] SCF may be present in the culture medium during all step a), or from day 6 of step a) to the end of step a). For example, SCF may be present in the culture medium at step a) since day 6 and during at least about 1 or 2 days.

[0110] In some embodiments, at step a), the culture medium does not comprise a solubilized basement membrane matrix, i.e., Matrigel®, or an alternative product.

[0111] In some embodiments, step a) comprises or consists of the following steps: i. at TO, PSCs are seeded in a serum-free medium comprising ascorbic acid, Transferrin, MTG, BMP4, and ROCK inhibitor,ii. at day 2, cells are collected and resuspended in serum-free media comprising ascorbic acid, Transferrin, MTG, inhibitor of the activin receptor-like kinase (ALK) receptors, GSK3 inhibitor, bFGF, and BMP4, iii. at day 4, the medium is replaced by human serum albumin (HSA)-based media comprising ascorbic acid, Transferrin, MTG, VEGF, IL-6, IL-11 and bFGF, iv. at day 6, the medium is replaced by a human serum albumin (HSA)-based media comprising ascorbic acid, Transferrin, MTG, bFGF, VEGF, IL-6, IGF-1, IL-11, EPO and SCF, and v. optionally, after 8 days of culture, embryoid bodies (EB) are dissociated.

[0112] In some embodiments, at the end of the step a), the method comprises an additional step of recovering and dissociating embryoid bodies, thereby obtaining a cell suspension, preferably a single cell suspension.

[0113] As used herein, the term “dissociated” refers to a process / method in which aggregated cells or tissues are broken down / separated into a single cell suspension. Methods of dissociation of aggregated cells or tissues are well known in the art and comprise, but are not limited to, mechanical methods (e.g., cutting, crushing, or scraping), chemical methods (e.g., GTA, or egtazic acid) or enzymatic methods (e.g., trypsin, collagenase or protease).

[0114] At the end of the step a), EBs may be recovered and dissociated with an enzymatic method, thereby obtaining a single cell suspension of embryoid body cells. Said recovering and dissociation step may be performed using at least one enzyme, such as, for example, one or two enzymes. When two enzymes are used, they may be used simultaneously or sequentially. Examples of enzymes include, but are not limited to, trypsin, dissolution solutions, and collagenase. Non-limitative examples of dissociation solutions include, but are not limited to, Gibco™ Enzyme (IX) TrypLE™ Express or Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent.

[0115] EBs may be recovered and dissociated using trypsin and / or a dissociation solution. At the end of the step a), EBs may also be recovered and dissociated using first trypsin or an alternative dissociation solution and second using a collagenase.

[0116] Thus, in some embodiments, a single cell suspension of embryoid body cells is obtained at the end of step a).

[0117] According to the method of the present invention, at step b), CD34+CD144+CD31+ cells are isolated from embryoid bodies (or from single cell suspension) obtained at step a).

[0118] The isolation of CD34+CD144+CD31+ cells may be based on the isolation of CD34+ cells. Indeed, all CD34+ cells present in the embryoid bodies also express CD144 and CD31. Alternatively, the isolation of CD34+CD144+CD31+ cells may be based on the isolation of CD31+ cells. Indeed, all CD31+ cells present in the embryoid bodies also express CD144 and CD34. Alternatively, the isolation of CD34+CD144+CD31+ cells may be based on the isolation of CD144+ cells. Indeed, all CD144+ cells present in the embryoid bodies also express CD34 and CD31.

[0119] In some embodiments, at step b), CD34+CD144+ CD31+ cells are isolated from a single cell suspension of embryoid body cells obtained at step a).

[0120] In some embodiments, the CD34+CD144+CD31+ cells consist of hemogenic endothelial cells. In some embodiments, the CD34+ CD144+CD31+ cells comprise hemogenic endothelial cells. As used herein, “hemogenic endothelial cells” (or EH cells) are cells having the following phenotype: CD34+CD144+CD31+CD43-CD73-CD184-.

[0121] In some embodiments, the method comprises a step of isolating hemogenic endothelial cells.

[0122] Methods for isolating cells from a larger population of cells are well-known in the art. Cell isolation may be done by positively selecting the cells of interest or by removing the cells with no interest. Examples of conventional methods for isolating cells include, but are not limited to, Fluorescence Activated Cell Sorting (FACS) (e.g., for CD34, CD31 or CD144) or Magnetic-activated cell sorting (e.g., targeting CD34, CD144 or CD31, and using kits, such as, for example, the kit Indirect CD34 MicroBead Kit, human; Miltenyi Biotech), microfluidics, cell centrifugation (such as, for example,density gradient centrifugation), limiting dilution, manual cell picking, specific adhesion procedures.

[0123] In some embodiments, the step c) comprises or consists of culturing the CD34+CD144+CD31+ cells or the EH cells in a culture medium promoting the induction hematopoietic progenitor cells (HPCs). Said step may thus be referred to as an endothelial to hematopoietic transition (EHT) step.

[0124] In practice, the culture medium used at step c) is adapted for the culture of CD34+ CD144+CD31+ cells, HE cells and HPCs. Examples of culture media adapted for culture of CD34+ CD144+CD31+ cells, HE cells and HPCs comprise, but are not limited to, StemPro™-34 SFM, Essential 8™, SFM hESC StemPro™, Stem Fl ex™, StemScale™ PSC, IMDM, or Ham’s F12 (Gibco™ , ThermoFisher Scientific). In some embodiments, the HPCs are cultured at step c) in StemPro™-34 SFM medium (Gibco™, Thermo Fisher Scientific).

[0125] In some embodiments, the culture medium used at step c) is a serum-free medium. In some embodiments, the culture medium used at step c) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of fetal bovine serum (FBS) or fetal calf serum (FCS). In some embodiments, the culture medium used at step c) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of KnockOut™ serum replacement (GibcoTM, ThermoFisher Scientific). In some embodiments, the culture medium used at step c) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of serum -free alternative component such as for example (but not limited) UltraGRO™ cell culture supplement (HELIOS®, AventaCell BioMedical Corp.) or XerumFree™ cell culture supplement (TNCBIO).

[0126] In some embodiments, the culture medium used at step c) is supplemented with bovine serum albumin (BSA, Sigma-Aldrich). In some embodiments, the culture medium used at step c) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of BSA. In some embodiments, the culture medium used at step c) is supplemented with human serum albumin (HSA, Sigma-Aldrich). Insome embodiments, the culture medium used at step c) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of HSA.

[0127] In some embodiments, the culture medium used at step c) is supplemented with at least one amino acid such as for example L-Glutamine (that may be provided, for example, by GibcoTM , ThermoFisher Scientific) or an alternative of L-Glutamine, such as for example GlutaMAX™ (GibcoTM , ThermoFisher Scientific).

[0128] In some embodiments, at the beginning of step c), the cells isolated at step b) of the method are seeded at a concentration ranging from about 0.1 million to about 0.5 million cells / ml of culture medium, preferably at a concentration from about O. lmillion to about 0.2 million cells / ml.

[0129] In some embodiments, at the beginning of step c), the cells isolated at step b) are seeded at a concentration ranging from about 0.03 million to about 0.15 million cells / cm2of culture medium preferably at a concentration ranging from about 0.03 million to about 0.06 million cells / cm2.

[0130] Notch proteins are transmembrane receptors that regulate the cellular response to a large number of environmental signals. In mammals, four Notch receptors (Notch 1-4) and five ligands (Delta-like- 1, Delta-like-3, Delta-like-4, Jagged-1 and Jagged-2) have been described (Weinmaster Curr Opin Genet Dev 2000: 10: 363-369).

[0131] In some embodiments, the culture medium of step c) comprises a Notch ligand or fragment thereof, wherein the Notch ligand is Delta-like-4, and more preferably human Delta-like-4 (also known as DL-4, Uniprot accession number: Q9NR61). Delta-like-4 may for example comprise or consist for example of amino acids 1-685 of SEQ ID NO: 12.

[0132] SEQ ID NO: 12MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEP GCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLP FNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQT STLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTP WQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGV DCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFN GGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRM CRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVR TSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPWVAVS LGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQL KNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKS LGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVIATEV

[0133] In some embodiments, the Notch ligand or fragment thereof comprises the soluble domain of at least one Notch ligand. In some embodiments, the Notch ligand or fragment thereof (preferably the soluble domain of the Notch ligand) is fused to a Fc region of an IgG protein, such as, for example, a human IgG protein, preferably a human IgG2 protein.

[0134] In some embodiments, the Notch ligand or fragment thereof is used at a concentration ranging from about 1 to 50pg / ml, preferably from about 1 to about 20pg / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20pg / ml. In some embodiments, the Notch ligand is used at a concentration of 5pg / ml.

[0135] The Notch ligand or fragment thereof may be immobilized on the culture vessel or on beads.

[0136] A method to coat a culture vessel or beads with a Notch ligand is disclosed in WO2016 / 055396, which is incorporated herein by reference. About 75% of the Notch ligand, in particular DL-4, may adhere to a culture vessel surface or to bead surface when 5 pg / ml is used. Thus, the composition used for coating a culture vessel or beads with a Notch ligand or fragment thereof may comprise a concentration of a Notch ligand or fragment thereof higher or equal to 1.25 pg / ml and preferably ranging from 2.5 and 5 pg / ml.

[0137] In some embodiments, the Notch ligand is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, at step c), cells are cultured at in presence of a Notch ligand or a fragment thereof for more than 5 days and less than 12 days, preferably for about 9 days.

[0138] Fibronectin corresponds to a protein having the human uniprot accession number: P02751. Fibronectin is a protein, which in its natural form is a v-shaped large dimer of 100 nm long and 460 kDa. The two monomers are connected by two disulfide bridges at their C-terminus. The term "fibronectin" or “fibronectin fragment” is understood to mean the natural fibronectin protein (i.e., any isoform produced by alternative splicing), but also a monomer of this protein, or a fragment of this protein (containing, when specified, the RGDS motif, CS-1 motif and heparin binding site).

[0139] In some embodiments, the fibronectin fragment comprises or consists of an RGDS motif, a connecting segment 1 (CS-1) motif and / or a heparin binding domain; preferably, the fibronectin fragment comprises or consists of an RGDS motif, a CS-1 motif and a heparin binding domain.

[0140] In some embodiments, fibronectin or the fragment thereof is Retronectin®. This protein corresponds to a fragment of a human fibronectin (CH-296 fragment, Kimizuka et al., J Biochem., 1991 Aug. 110 (2):284-91, Chono et al., J Biochem 2001 Sep 130 (3):331-4) and contains the cell-binding C domain (comprising the RGDS motif, the heparin-binding domain and the CS-1 motif). This protein is sold in particular by the companies Takara Bio Inc. (Shiga, Japan), Clinisciences (Nanterre, France, also called NovoNectin®) and Fisher scientific (Hampton, United-States).

[0141] In some embodiments, fibronectin or the fragment thereof is immobilized on the culture vessel or on beads. In some embodiments, the Notch ligand or the fragment thereof and fibronectin or the fragment thereof are immobilized on the same beads. In other embodiments, the Notch ligand or the fragment thereof and fibronectin or the fragment thereof are immobilized on distinct beads.

[0142] In some embodiments, fibronectin or the fragment thereof is used at a concentration ranging from about 1 to lOOpg / ml, preferably from about 10 to about50pg / ml, such as for example of at least about 10, 15, 20, 25, 30, 35, 40, 45 or 50pg / ml. In some embodiments, the fibronectin or fragment thereof is used at a concentration of 25pg / ml.

[0143] A method to coat a culture vessel or beads with fibronectin or a fragment thereof is disclosed in WO2016 / 055396, which is incorporated herein by reference. In some embodiments, the composition used for coating a culture vessel or beads with fibronectin or a fragment thereof comprises a concentration of fibronectin or a fragment thereof ranging from 10 and 100 pg / ml, preferably of about 25 pg / ml.

[0144] In some embodiments, fibronectin or the fragment thereof is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, the cells are cultured at step c) in presence of fibronectin or the fragment thereof for more than 5 days and less than 12 days, preferably for about 9 days.

[0145] Examples of antagonist of the Aryl hydrocarbon / Dioxin receptor include, but are not limited to, StemRegenin, resveratrol, omeprazole, Luteolin, alpha-naphthoflavone, mexiletine, tranilast, 6,2',4'-Trimethoxyflavone, CH 223191 (l-Methyl-N-[2-methyl-4- [2-(2-methylphenyl)diazenyl]phenyl-lH-pyrazole-5-carboxamide, CAS 301326-22-7).

[0146] In some embodiments, the antagonist of the Aryl hydrocarbon / Dioxin receptor is StemRegenin 1 (also known as SRI). This component is sold in particular by the companies StemCell Technologies or Bio-techne.

[0147] In some embodiments, the antagonist of the Aryl hydrocarbon / Dioxin receptor is used at a concentration ranging from about 200 to lOOOng / ml, preferably from about 500 to about lOOOng / ml, such as for example of at least about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or lOOOng / ml. In some embodiments, the Aryl hydrocarbon / Dioxin receptor is used at a concentration of 800ng / ml.

[0148] In some embodiments, the antagonist of the Aryl hydrocarbon / Dioxin receptor is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, cells are cultured at step c) in presence of an antagonist of the Arylhydrocarbon / Dioxin receptor for more than 5 days and less than 12 days, preferably for about 9 days.

[0149] In some embodiments, the culture medium used at step c) comprises an immobilized Notch ligand; an immobilized fibronectin or fragment thereof; and an antagonist of the Aryl hydrocarbon / Dioxin receptor.

[0150] In some embodiments, the culture medium used at step c) further comprises at least one component selected from the group comprising or consisting of Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt3 ligand (Flt-3L), IL-3 and BMP4. Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt3 ligand (Flt-3L), IL-3 or BMP4. Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt3 ligand (Flt-3L), IL-3 or BMP4 may be human components.

[0151] In some embodiments, at step c), the culture medium comprises Ascorbic acid (AA).

[0152] In some embodiments, AA is used at step c) at a concentration ranging from about 1 to about 500pg / ml, preferably from about 1 to about lOOpg / ml, such as for example of at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or lOOpg / ml. In some embodiments, Ascorbic acid is used at a concentration of 50pg / ml.

[0153] In some embodiments, AA is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, Ascorbic acid is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0154] In some embodiments, at step c), the culture medium comprises transferrin.

[0155] In some embodiments, Transferrin is used at step c) at a concentration ranging from about 1 to about lOOOpg / ml, preferably from about 1 to about 200pg / ml, such as for example of at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200pg / ml. In some embodiments, Transferrin is used at a concentration of 150pg / ml.

[0156] In some embodiments, Transferrin is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, Transferrin is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0157] In some embodiments, at step c), the culture medium comprises 1 -thioglycerol (also call a-Monothioglycerol, a- Thioglycerol, 3 -Mercapto- 1,2-propanediol or Monothioglycerol or MTG).

[0158] In some embodiments, MTG is used at step c) at a concentration ranging from about 1 to about lOOOpM, preferably from about 100 to about 600pM, such as for example of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 pM. In some embodiments, 1 -thioglycerol is used at a concentration of 400pM.

[0159] In some embodiments, MTG is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, 1 -thioglycerol is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0160] In some embodiments, at step c), the culture medium comprises BMP4 (bone morphogenetic protein 4).

[0161] In some embodiments, BMP4 is used at step c) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. In some embodiments, BMP4 is used at a concentration of lOng / ml.

[0162] In some embodiments, BMP4 is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, BM4 is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0163] In some embodiments, at step c), the culture medium comprises bFGF (basic fibroblast growth factor also known as Fibroblast growth factor 2).

[0164] In some embodiments, bFGF is used at step c) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for exampleof at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. In some embodiments, bFGF is used at a concentration of 5 ng / ml.

[0165] In some embodiments, bFGF is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, bFGF is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0166] In some embodiments, at step c), the culture medium comprises VEGF (vascular endothelial growth factor).

[0167] In some embodiments, VEGF is used at step c) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. In some embodiments, VEGF is used at a concentration of 5ng / ml.

[0168] In some embodiments, VEGF is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, VEGF is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0169] In some embodiments, at step c), the culture medium comprises Interleukin -6 (IL-6).

[0170] In some embodiments, IL-6 is used at step c) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. In some embodiments, IL-6 is used at a concentration of lOng / ml.

[0171] In some embodiments, IL-6 is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, IL-6 is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0172] In some embodiments, at step c), the culture medium comprises Interleukin- 11 (IL-H).

[0173] In some embodiments, IL-11 is used at step c) at a concentration ranging from about 1 to about lOOng / ml, preferably from about 1 to about 20ng / ml, such as for example of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 ng / ml. In some embodiments, IL-11 is used at a concentration of 5ng / ml.

[0174] In some embodiments, IL-11 is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, IL-11 is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0175] In some embodiments, at step c), the culture medium comprises insulin-like growth factor- 1 (IGF-1).

[0176] In some embodiments, IGF-1 is used at a concentration ranging from about 1 to about lOOng / ml, preferably from about 10 to about 50ng / ml, such as for example of at least about 10, 15, 20, 25, 30, 35, 40, 45 or 50ng / ml. In some embodiments, IGF-1 is used at a concentration of 25 ng / ml.

[0177] In some embodiments, IGF-1 is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, IGF-1 (insulin-like growth factor-1), is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0178] In some embodiments, at step c), the culture medium comprises stem cell factor (SCF, also known as kit-ligand).

[0179] In some embodiments, SCF is used at step c) at a concentration ranging from about 1 to 500ng / ml, preferably from about 50 to about 200ng / ml, such as for example of at least about 50, 60, 70, 80, 90, 100, 110, 150, 160, 170, 180, 190, 200ng / ml. In some embodiments, SCF is used at a concentration of lOOng / ml.

[0180] In some embodiments, SCF is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, SCF is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0181] In some embodiments, at step c), the culture medium comprises Thrombopoietin (TPO). TPO (Thrombopoietin, e.g., corresponding to the uniprot accession number P40225) is known to promote the survival, self-renewal and expansion of megakaryocytes. In some embodiments, TPO comprises or consists for example of amino acids 1-353 of SEQ ID NO: 9. In some embodiments, TPO comprises or consists for example of any described isoform of SEQ ID NO: 9. This component is sold in particular by the companies R&D systems or MERCK (Sigma-Aldrich).

[0182] SEQ ID NO: 9MELTELLLVVMLLLTARLTLSSPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVH PLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCL SSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVR FLMLVGGSTLCVRRAPPTTAVPSRTSLVLTLNELPNRTSGLLETNFTASARTTGS GLLKWQQGFRAKIPGLLNQTSRSLDQIPGYLNRIHELLNGTRGLFPGPSRRTLGA PDISSGTSDTGSLPPNLQPGYSPSPTHPPTGQYTLFPLPPTLPTPVVQLHPLLPDPS APTPTPTSPLLNTSYTHSQNLSQEG

[0183] In some embodiments, TPO is used at step c) at a concentration ranging from about 1 to 500ng / ml, preferably from about 5 to about lOOng / ml, such as for example of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / ml. In some embodiments, TPO is used at a concentration of 30ng / ml.

[0184] In some embodiments, TPO is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, TPO is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0185] In some embodiments, at step c), the culture medium comprises Flt3-L (Fms- related tyrosine kinase 3 ligand). Flt3-L (Flt3 ligand, e.g., corresponding to the human uniprot accession number: P49771) is a hematopoietic cytokine. In some embodiments, Flt3-L comprises or consists for example of amino acids 1-235 of SEQ ID NO: 10. In some embodiments, Flt3-L comprises or consists for example of any described isoform of SEQ ID NO: 10. This component is sold in particular by the companies R&D systems or MERCK (Sigma-Aldrich).

[0186] SEQ ID NO: 10MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQD YPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHF VTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDS STLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPR PGEQVPPVPSPQDLLLVEH

[0187] In some embodiments, Flt3-L is used at step c) at a concentration ranging from about 1 to 200 ng / ml, preferably from about 5 to 100 ng / ml, more preferably from about 10 to 20 ng / ml. In practice, a concentration of Flt3-L of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ng / ml may be used. In some embodiments, Flt3-L is used at a concentration of lOng / ml.

[0188] In some embodiments, Flt3-L (Fms-related tyrosine kinase 3 ligand) is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, Flt3-L is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0189] In some embodiments, at step c), the culture medium comprises Interleukin-3 (il- 3). IL-3 (interleukin-3, e.g. corresponding to the human uniprot accession number P08700) is a cytokine also called Hematopoietic Growth Factor. In some embodiments, IL-3 comprises or consists for example of amino acids 1-152 of SEQ ID NO: 11. This component is sold in particular by the companies R&D systems or MERCK (Sigma- Aldrich).

[0190] SEQ ID NO: 11MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLD FNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATA APTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF

[0191] In some embodiments, IL-3 is used at step c) at a concentration ranging from about 1 to 500ng / ml, preferably from about 10 to about lOOng / ml, such as for example ofat least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or lOOng / ml. In some embodiments, IL-3 is used at a concentration of 30ng / ml.

[0192] In some embodiments, IL-3 (interleukin-3) is present in the culture medium from day 0 of step c) to the end of step c). In some embodiments, IL-3 is present in the culture medium at step c) from day 0 and for about 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0193] In some embodiments, at step c), the culture medium does not comprise Vascular cell adhesion protein 1 (VCAM-1).

[0194] In some embodiments, at step c), the culture medium does not comprise a solubilized basement membrane matrix, i.e., Matrigel®, or an alternative product.

[0195] In some embodiments, the cells are cultured at step c) in a medium comprising at least one of Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt-3L, IL-3 and BMP4, in presence of a Notch ligand; fibronectin or fragment thereof and an antagonist of the Aryl hydrocarbon / Dioxin receptor.

[0196] In some embodiments, the cells are cultured at step c) in a medium comprising Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt-3L, IL-3 and BMP4, in presence of a Notch ligand; fibronectin or fragment thereof and an antagonist of the Aryl hydrocarbon / Dioxin receptor.

[0197] In some embodiments, the cells are cultured at step c) in a medium comprising at least one of Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt-3L, IL-3 and BMP4, in presence of an immobilized Notch ligand or fragment thereof, an immobilized fibronectin or fragment thereof and an antagonist of the Aryl hydrocarbon / Dioxin receptor.

[0198] In some embodiments, the cells are cultured at step c) in a medium comprising Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt-3L, IL-3 and BMP4, in presence of an immobilized Notch ligand or fragment thereof, an immobilized fibronectin or fragment thereof and an antagonist of the Aryl hydrocarbon / Dioxin receptor.

[0199] According to the invention, at the end of the step c) a population of hematopoietic progenitor cells (HPCs) is obtained.

[0200] Optionally, at the end of the step c), HPCs are recovered / harvested.

[0201] In some embodiments, step d) comprises or consists of culturing the population of HPCs obtained at step c) in a culture medium promoting the induction of T cell progenitors from said HPCs.

[0202] Methods to obtain a population of T cell progenitors from HPC are disclosed in WO2016 / 055396 and WO2018 / 146297 which are incorporated herein by reference.

[0203] In some embodiments, the culture medium used at step d) is adapted for the culture of HPCs and T cell progenitors. Examples of culture medium adapted for culture of HPCs and T cell progenitors comprise, but are not limited a-MEM, DMEM, RPMI 1640, IMDM, BME, McCoy's 5A, SFII (StemCell Technologies) media, Fischer's medium and X-VIVOTM medium (Lonza, Basel, Switzerland). In some embodiments, hematopoietic progenitor cells (HPCs) are cultured in a-MEM medium (Thermo Fischer Scientic).

[0204] In some embodiments, the culture medium used at step d) is a serum-free medium. In some embodiments, the culture medium used at step d) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of fetal bovine serum (FBS) or fetal calf serum (FCS). In some embodiments, the culture medium used at step d) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of KnockOut™ serum replacement (GibcoTM , ThermoFisher Scientific). In some embodiments, the culture medium used at step d) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of serum -free alternative component such as for example (but not limited) UltraGRO™ cell culture supplement (HELIOS®, AventaCell BioMedical Corp.) or XerumFree™ cell culture supplement (TNCBIO). In some embodiments, the culture medium used at step d) is supplemented with 20% FBS Hyclone (Cytiva).

[0205] In some embodiments, the culture medium used at step d) is supplemented with bovine serum albumin solution (BSA, Sigma-Aldrich). In some embodiments, the culture medium used at step d) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of BSA. In some embodiments, the culture medium used at step d) is supplemented with human serum albumin solution (HSA, Sigma-Aldrich). In some embodiments, the culture medium used at step d) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v of HSA.

[0206] In some embodiments, at the beginning of step d), HPCs obtained at step c) are seeded on a culture vessel.

[0207] In some embodiments, HPCs obtained at step c) are cultured at step d) of the method of the invention for more than 5 days and less than 9 days, preferably for about 7 days.

[0208] In some embodiments, the culture medium used at step d) comprises TNF-alpha (TNF-a) and at least one compound selected from the group comprising or consisting of interleukin-7 (IL-7), Stem Cell Factor (SCF), thrombopoietin (TPO) and Flt3 ligand (FLT3L). In some embodiments, the culture medium used at step d) comprises TNF-a, IL-7, SCF, TPO and FLT3L.

[0209] In some embodiments, the culture medium used at step d) comprises human TNF- alpha (TNF-a), human interleukin-7 (hIL-7), human Stem Cell Factor (hSCF), human thrombopoietin (hTPO) and human Flt3 ligand (hFLT3L).

[0210] In some embodiments, the culture medium used at step d) comprises human TNF- a and at least one compound selected from the group comprising or consisting of human IL-7, human SCF, human TPO and human FLT3L. In some embodiments, the culture medium used at step d) comprises human TNF-a, human IL-7, human SCF, human TPO and human FLT3L.

[0211] In some embodiments, at step d), the culture medium comprises TNF-alpha (TNF-a). TNF-a a cytokine with pro-inflammatory properties and immuno-regulatoryfunctions. In some embodiments, TNF-a (Tumor Necrosis Factor, e.g. corresponding to the human Uniprot accession number: P01375) comprises or consists for example of the amino acid 1 to 233 of SEQ ID NO: 13.

[0212] SEQ ID NO: 13MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGV IGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRA NALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSY QTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY LDFAESGQVYFGIIAL

[0213] In some embodiments, the concentration of TNF-a (preferably human TNF-a) ranges from about Ito about 200 ng / ml, preferably from about 5 to about 100 ng / ml and more preferably is of about 10 ng / ml.

[0214] In some embodiments, TNF-a (preferably human TNF-a) is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs are cultured at step d) in presence of TNF-a (preferably human TNF-a) for more than 5 days and less than 9 days, preferably for about 7 days.

[0215] In some embodiments, at step d), the culture medium comprises stem cell factor (SCF, also known as kit-ligand).

[0216] In some embodiments, the concentration of SCF (preferably hSCF) used at step d) ranges from about 2 to about 300 ng / ml, preferably from about 40 to about 200 ng / ml and more preferably is of about 100 ng / ml.

[0217] In some embodiments, SCF (preferably hSCF) is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs obtained at step c) are cultured at step d) in presence of SCF (preferably hSCF) for more than 5 days and less than 9 days, preferably for about 7 days.

[0218] In some embodiments, at step d), the culture medium comprises Flt3-L (Fms- related tyrosine kinase 3 ligand).

[0219] In some embodiments, the concentration of Flt3-L (preferably hFlt3-L) at step d) ranges from about 2 to about 300 ng / ml, preferably from about 40 to about 200 ng / ml and more preferably is of about 100 ng / ml.

[0220] In some embodiments, Flt3-L (preferably hFlt3-L) is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs are cultured at step d) in presence of Flt3-L (preferably hFlt3-L) for more than 5 days and less than 9 days, preferably for about 7 days.

[0221] In some embodiments, at step d), the culture medium comprises Thrombopoietin (TPO).

[0222] In some embodiments, the concentration of TPO (preferably hTPO) at step d) ranges from about 2 to about 300 ng / ml, preferably from about 40 to about 200 ng / ml and more preferably is of about 100 ng / ml.

[0223] In some embodiments, TPO (preferably hTPO) is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs are cultured at step d) in presence of TPO (preferably hTPO) for more than 5 days and less than 9 days, preferably for about 7 days.

[0224] In some embodiments, at step d), the culture medium comprises Interleukin-7 (IL-7). IL-7 (Interleukin- 7, e.g. corresponding to the Human Uniprot Pl 3232) is a cytokine that play an essential role in the development and expansion of some immune cells. In some embodiments, IL-7 comprises or consists of for example the amino acid 1 to 177 of SEQ ID NO: 14.

[0225] SEQ ID NO: 14MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEI GSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVS EGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQE IKTCWNKILMTGKEH

[0226] In some embodiments, the concentration of IL-7 (preferably hIL-7) ranges from about 2 to about 300 ng / ml, preferably from about 40 to about 200 ng / ml and more preferably is of about 100 ng / ml.

[0227] In some embodiments, IL-7 (preferably hIL-7) is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs are cultured at step d) in presence of IL-7 (preferably hIL-7) for more than 5 days and less than 9 days, preferably for about 7 days.

[0228] In some embodiments embodiment, the culture medium at step d) does not comprise IL-3.

[0229] In some embodiments embodiment, the culture medium at step d) does not comprise an antagonist of the Aryl hydrocarbon / Dioxin receptor. In some embodiments, the culture medium at step d) does not comprise SRI.

[0230] In some embodiments, the culture medium used at step d) further comprises a Notch Ligand or fragment thereof and a fibronectin or fragment thereof, and optionally Stromal cell-derived factor la (SDFla) or a p38 inhibitor.

[0231] In some embodiments, at step d), the culture medium comprises a Notch ligand or fragment thereof as described herein.

[0232] In some embodiments, at step d), the culture medium comprises fibronectin or fragment thereof as described herein.

[0233] In some embodiments, at step d), the culture medium comprises Stromal cell- derived factor la (preferably human SDFla). Stromal cell-derived factor la (also known as CXCL12, e.g. corresponding to the human Uniprot accession P48061) is a chemokine protein that plays a critical role in diverse biological functions, including embryogenesis, immune surveillance, inflammatory response or tissue homeostasis. In some embodiments, SDFla comprises or consists of the amino acid 1 to 93 of SEQ ID NO: 15. This component is sold in particular by the companies R&D systems or MERCK (Sigma- Aldrich).

[0234] SEQ ID NO: 15MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTP NCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRFKM

[0235] In some embodiments, SDFla is used at a concentration range from about 50 to about 1000 ng / ml, preferably from about 40 to about 200 ng / ml and more preferably is about 100 ng / ml.

[0236] In some embodiments, SDFla is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs are cultured at step d) in presence of SDF 1 a thereof for more than 5 days and less than 9 days, preferably for about 7 days.

[0237] In some embodiments embodiment, at step d), the culture medium comprises a p38 inhibitor. Examples of p38 inhibitors include, but are not limited to, SB203580, pamapimod, PH-797804, BIRB 796, VX-702, SB239063, SB202190, SCIO 469, and BMS 582949. In some embodiments, at step d), the culture medium comprises SB203580.

[0238] SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4-pyridyl)-lH- imidazole) is a specific inhibitor of p38a and p38[3 MAP kinase. This component is sold in particular by the companies STEMCELL Technologies, MERCK (Sigma-Aldrich) or InvivoGen.

[0239] In some embodiments, the p38 inhibitor is used at a concentration ranging from about 1 to about 20 pM, preferably from about 5 to about 15pM and more preferably is about 15pM.

[0240] In some embodiments, the p38 inhibitor is present in the culture medium from day 0 of step d) to the end of step d). In some embodiments, HPCs are cultures at step d) in presence of a p38 inhibitor for more than 5 days and less than 9 days, preferably for about 7 days.

[0241] In some embodiments, the culture medium used at step d) further comprises a Notch Ligand or fragment thereof, fibronectin or fragment thereof and Stromal cell- derived factor la.

[0242] In some embodiments, the culture medium used at step d) further comprises a Notch Ligand or fragment thereof, fibronectin or fragment thereof and a p38 inhibitor.

[0243] In some embodiments, the culture medium used at step d) further comprises a Notch Ligand or fragment thereof, fibronectin or fragment thereof, Stromal cell-derived factor la and a p38 inhibitor.

[0244] In some embodiments, the culture medium used at step d) further comprises an immobilized Notch Ligand and an immobilized fibronectin or fragment thereof, and optionally Stromal cell-derived factor la (SDFla) and / or p38 inhibitor.

[0245] In some embodiments, the culture medium used at step d) comprises TNF-alpha, IL-7, SCF, TPO, FLT3L, Notch Ligand or fragment thereof, fibronectin or fragment thereof, and optionally Stromal cell-derived factor la (SDFla) and / or p38 inhibitor.

[0246] In some embodiments, HPSc are cultured at step d) in presence of TNF-alpha (preferably human TNF-a), IL-7 (preferably hIL-7), SCF (preferably hSCF), TPO (preferably hTPO), FLT3L (preferably hFLT3L), Notch Ligand or fragment thereof, fibronectin or fragment thereof and SDFla.

[0247] In some embodiments, HPSc are cultured at step d) in presence of TNF-alpha (preferably human TNF-a), IL-7 (preferably hIL-7), SCF (preferably hSCF), TPO (preferably hTPO), FLT3L (preferably hFLT3L), Notch Ligand or fragment thereof, fibronectin or fragment thereof and a p38 inhibitor.

[0248] In some embodiments, HPSc are cultured at step d) in presence of TNF-alpha (preferably human TNF-a), IL-7 (preferably hIL-7), SCF (preferably hSCF), TPO (preferably hTPO), FLT3L (preferably hFLT3L), Notch Ligand or fragment thereof, fibronectin or fragment thereof, SDFla and a p38 inhibitor.

[0249] According to the invention, at the end of the step d), a population of T cell progenitors is obtained.

[0250] At the end of the step d), the method may further comprise a step of recovering or harvesting the T cell progenitors. Methods for recovering or harvesting cells are well known in the art.

[0251] In addition, the method of the invention may further comprise a step of freezing the T cell progenitors.

[0252] In some embodiments, the method of the invention comprises one or more additional step of genetic modification.

[0253] Genetic modification step(s) may be carried out at the beginning of step c), i.e., the population of cells obtained at step b) may by genetically modified.

[0254] Genetic modification step(s) may correspond(s) to a gene disruption step, a gene correction step or a gene addition step. In some embodiments, the genetic modification step(s) is / are carried out by a method selected from the group comprising, but not limited to, transfection, transduction or gene editing.

[0255] Examples of methods of gene editing that may be used in the present invention include, but are not limited to, methods based on engineered nucleases, methods based on recombinant Adeno-Associated Virus (or AAV), methods based on Transposons (e.g., Sleeping Beauty transposon system), methods based on homologous recombination, conditional targeting using site-specific recombinases (e.g., Cre-LoxP and Flp-FRT systems), and Multiplex Automated Genomic Engineering (MAGE). Other examples of methods of gene editing that may be used in the present invention include, but are not limited to, methods based on nickases.

[0256] Non-limiting examples of engineered nucleases include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR) transcriptionactivator like effector nuclease (TALEN), zinc finger endonuclease (ZFN), meganuclease (mn, also known as homing endonuclease), or megaTAL (combining a TAL effector with a mn cleavage domain). Other non-limitative examples of engineered nucleases are baseeditors or prime editors. Such systems may use natural or synthetic transposon systems e.g., the Sleeping Beauty (SB) transposon system, based on transposase activity.

[0257] Genetic modification (such as, for example, the transduction of transfection of cells) may be performed one day after the seeding of the population of cells obtained at step b).

[0258] In some embodiments, for the genetic modification step, the population of cells obtained at step b), is seeded at a concentration ranging from about 0.1 million to about 1 million cells / ml of culture medium, more preferably ranging from about 0.2 million to about 0.5 million cells / ml.

[0259] In some embodiments, for the transduction or transfection step, the population of cells obtained at step b) is seeded at a concentration ranging from about 0.1 million to about 1 million cells / ml of culture medium, more preferably ranging from about 0.2 million to about 0.5 million cells / ml.

[0260] In some embodiments, the medium used for the genetic modification step is a medium comprising Ascorbic acid, Transferrin, Monothio glycerol, VEGF, bFGF, SCF, IL-6, IL-11, IGF-1, TPO, Flt3 ligand (Flt-3L), IL-3 or BMP4, as described herein.

[0261] In some embodiments, the medium used for the genetic modification step further comprises a Notch ligand or fragment thereof and / or fibronectin or fragment thereof, as described herein. In some embodiments, the medium used the genetic modification step further comprises an immobilized Notch ligand or fragment thereof and / or an immobilized fibronectin or fragment thereof, as described herein.

[0262] The additional step of transducing or transfecting cells may correspond to the introduction into the cells a vector, a plasmid or a nucleic acid sequence encoding a transgene. Examples of transgenes include, but are not limited to, a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR), a T-cell receptor (TCR), a safety switch gene, a suicide gene and a potentiator of T-cell killing. Examples of potentiators of T-cell killing include, but are not limited to, dominant negative TGFRb (thereby rendering the cell TGFb resistant), IL-7 (that may, for example, increase endogenous immuneresponse), Interleukin- 15 (IL- 15) and its receptor subunit (IL-15Ralpha, IL-2 and IL-21), CCL-19 (Chemokine (C-C motif) ligand 19) and CCL21 Chemokine (C-C motif) ligand 21) (that may, for example, improve tumor infiltration), dominant negative FAS (thereby rendering the cell FAS-L resistant, thereby limiting or avoiding T cell apoptosis). Examples of suicide genes include, but are not limited to, tEGFR (truncated human epidermal growth factor receptor), iCasp9 (Inducible Caspase9), HSV-TK (Herpes Simplex Virus Thymidine Kinase) or an antigen-specific induction system such as synNotch receptors.

[0263] The genetic modification step(s) may correspond to a gene disruption step, aiming at decreasing or abolishing expression of specific genes. Examples of genetic modifications include, but are not limited to, genetic modification for correcting a genetic defect, genetic modifications for modulating cytokine signaling (for example via gene overexpression or gene disruption) for downstream T-cell activation and expansion in order to bolster antitumor activity and counter the immunosuppressive effect of tumor microenvironment ; deletion or downregulation of genes encoding an immune checkpoint inhibitor such as, for example PDCD1 (z.e., the gene that encodes PD1), LAG3 encoding gene, CTLA-4 encoding gene, DGKa (Diacylglycerol kinase alpha) encoding gene or DGK^ (Diacylglycerol kinase zeta ) encoding gene, to increase T cell function; deletion or downregulation of the DNMT3A and TET2 genes or overexpression of CARD 11- PIK3R3 (overexpression of this gene is hypothesized to increase the therapeutic efficacy by enhancing T-cell persistence), deletion or downregulation of RAG1 or RAG2 (to avoid further rearrangement in iPSC); and genetic modification aiming at disrupting the gene encoding an endogenous TCR and / or aiming at disrupting the gene encoding human leucocyte antigen (HLA) system, thereby eliminating or reducing GvHD concerns and / or avoiding alloreactivity. The genetic modification step(s) may also correspond to a gene addition step, aiming at increasing the expression of a specific gene or aiming at expressing a nucleic acid sequence not naturally expressed by the cell. For example, the genetic modification step may correspond to the induction of the expression of a transgenic TCR or CAR (such as, for example, a CAR with low tonic signal) thereby redirecting the engineered T cells derived from the T cell progenitors of the invention tospecific tumor antigens or induction of the expression of engineered dominant negative inhibitory receptors such as PD1 or TGFpR thereby enhancing T cell function.

[0264] The genetic modification step may correspond to the induction of the expression of a CAR with an additional 2B4. Without willing to be link to any theory, the expression of a CAR with an additional 2B4 may decrease the tonic signal during T cell differentiation from iPSC, thereby avoiding the bias towards CD8aa T cells and favor CD8aP T cell production.

[0265] In some embodiments, the transduction or transfection step is performed during at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, preferably during at least 6 to 24 hours, more preferably during at least 16 hours.

[0266] The present invention also relates to a population of T cell progenitors obtained or susceptible to be obtained by the method as described herein.

[0267] In some embodiments, said T cell progenitors are or comprise CD34-.

[0268] In some embodiments, said T cell progenitors are or comprise CD7+. In some embodiments, said T cell progenitors are or comprise CD34- CD7+. In some embodiments, said T cell progenitors are or comprise CD7+ CD5+ cells. In some embodiments, said T cell progenitors are or comprise CD34- CD7+ CD5+ cells.

[0269] In some embodiments, the population of T cell progenitors of the invention is a population wherein at least 70%, preferably 80 %, and more preferably at least 90 % of the cells express the CD7 marker. In some embodiments, the population of T cell progenitors of the invention is a population wherein at least 15 %, preferably 25% and more preferably at least 30 % of the CD7+ cells express the CD5 marker.

[0270] In some embodiments, the population of T cell progenitors of the invention comprises at least about 70, 71, 72, 73, 74, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95% or more CD7+ cells, preferably at least about 80% of CD7+ cells and more preferably at least about 90% of CD7+.

[0271] In some embodiments, the population of T cell progenitors of the invention comprises at least about 10, 15, 20, 25, 30, 35, 40, 45 or 50% of CD7+ cells that are CD5+, preferably at least about 15% of CD7+ cells that are CD5+.

[0272] In some embodiments, the population of T cell progenitors of the invention comprises or consists of cells that express the marker CD 161 and / or the marker CD56. In some embodiments, population comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21,22, 23,24, 25, 26, 27, 28, 29 or 30 % cells that express the marker CD 161 and / or the marker CD56.

[0273] In some embodiments, the population of T cell progenitors of the invention comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21,22, 23,24, 25, 26, 27, 28, 29 or 30 % cells that express the marker CD161.

[0274] In some embodiments, the population of T cell progenitors of the invention comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 % cells that express the marker CD56.

[0275] In some embodiments, the population of T cell progenitors of the invention comprises or consists of cells that does not express at least one of the markers comprising CD3, CD19 or CDla. In some embodiments, the population of T cell progenitors of the invention comprises less than about 1, 2, or 3 % of cells that express CD3, CD19 or CDla.

[0276] In some embodiments, the population of T cell progenitors of the invention comprises less than about 1, 2, or 3 % of cells that express CD3.

[0277] In some embodiments, the population of T cell progenitors of the invention comprises less than about 1, 2, or 3 % of cells that express CD 19.

[0278] In some embodiments, the population of T cell progenitors of the invention comprises less than about 1, 2, or 3 % of cells that express CDla.

[0279] In some embodiments, the T cell progenitors of the invention are capable of differentiating into T cells. In some embodiment, the T cell progenitors of the invention are capable of differentiating into NK cells.

[0280] The present invention further relates to a composition comprising T cell progenitors as described herein. In some embodiments, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient. The pharmaceutical may consist essentially of T cell progenitors as described herein.

[0281] Pharmaceutically acceptable excipients that may be used in the pharmaceutical composition of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as, for example, human serum albumin, buffer substances such as, for example, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as, for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0282] The present invention further relates to T cell progenitors obtained by the method as described herein for increasing the number of T cells in a subject in need thereof.

[0283] The present invention further relates to the use of T cell progenitors obtained by the method as described herein for increasing the number of T cells in a subject in need thereof, or for the manufacture of a medicament for increasing the number of T cells in a subject in need thereof.

[0284] In some embodiments, T cell progenitors obtained by the method as described herein are, or are for use as, a medicament, preferably for increasing the number of T cells in a subject in need thereof.

[0285] In some embodiments, the medicament comprises, consists essentially of or consists of T cell progenitors as described herein.

[0286] As used herein, “consists essentially of’ means that the T cell progenitors are the only therapeutic ingredients in the medicament or pharmaceutical composition.

[0287] The present invention further relates to a method for increasing the number of T cells in a subject, comprising administering to the subject T cell progenitors obtained by a method disclosed herein.

[0288] In some embodiments, a therapeutically effective amount of T cell progenitors is administered, or is to be administered, to the subject. In some embodiments, the therapeutically effective amount ranges from about 0. IxlO6to about 5xl07cells / kg body weight.

[0289] In some embodiments, the T cell progenitors are used for treating immunodeficiency.

[0290] In some embodiments, the T cell progenitors are used for complementing the T cell compartment.

[0291] In some embodiments, the T cell progenitors are used for treating a disease selected from autoimmune diseases, infections, inflammatory diseases and cancers.

[0292] In some embodiments, the T cell progenitors are used in adoptive therapies for cancer, infections, lymphoid reconstitution, inflammatory diseases or autoimmune diseases.

[0293] Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, diabetes type I, chronic hepatitis, psoriasis, celiac disease, myasthenia gravis, graves’ disease, Sjogren’s syndrome; inflammatory bowel disease, multiple sclerosis and systemic lupus erythematosus.

[0294] Examples of cancers include, but are not limited to hematological malignancies such as leukemias, lymphomas and myelomas, and solid tumors such as liver cancer, lung cancer, breast cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, kidney cancer, head and neck cancer, colon and rectal cancer and melanoma

[0295] In some embodiments, the number of T cell progenitors that may be administered to a subject ranges from about IxlO6to about 5xl07cells / kg body weight.

[0296] T cell progenitors may be administrated in a subject with an autoimmune disease (such as, for example, to eliminate autoreactive B cells) or to a subject with a defect in the T cell compartment (such as, for example, to replenishing exhaustive T cells or to improve the capacity of the immune system to fight a disease, an infection or a cancer).

[0297] In some embodiments, T cell progenitors are administered in a subject just prior to, just after or concomitantly with other treatments and / or therapeutics agents.

[0298] Non-limiting examples of therapeutics agents include, but are not limited to, Progenitor and Hematopoietic Stem / Progenitor Cell (HSPC) transplant, cytokines, chemokines, growth factors, antibodies, chemotherapy molecules, hormones, sex streroids, metformine, immunosuppressive drugs, cell populations or recombinant proteins.

[0299] In some embodiments, the T cell progenitors, the population of T cell progenitors, the composition, the pharmaceutical composition or the medicament of the invention is administered (or is to be administered or is for administration) by intravesical administration, intravaginal administration, intraosseous administration, intraperitoneal administration, intrauterine administration, intraocular administration, intradermal administration, intraarterial administration, intracerebral administration, intranasal administration, enteral administration, buccal administration, intranasal administration, oral administration, rectal administration, or by inhalation.

[0300] In some embodiments, the T cell progenitors, the population of T cell progenitors, the composition, the pharmaceutical composition or the medicament of the invention is administered (or is to be administered or is for administration) by injection, including, without limitation, subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intra-sternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0301] Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like.

[0302] In some embodiments, the subject is a human. In some embodiments, the subject is a patient.

[0303] In some embodiments, the subject is an immunosuppressed patient. The reasons for the deficiency may be multiple: hereditary immune deficiency, genetic immunodeficiency, chemotherapy for leukemia, conditioning, graft containing only stem cells, post-graft treatment for prophylaxis of GVHD (graft-versus-host disease), age of patient, and complications such as infections.

[0304] In some embodiments, the patient can be immunosuppressed due to the depletion of its immune cells following therapy before hematopoietic stem cells transplantation.

[0305] In some embodiments, the subject has a medical condition causing or resulting in lymphopenia. In some embodiments, the lymphopenia results from cancer, infection (e,g., HIV), partial thymectomy, autoimmune disease and / or organ transplantation. In some embodiments, the subject has a cancer, an infection (e,g., HIV), a partial thymectomy, an autoimmune disease and / or an organ transplantation.

[0306] The present invention further relates to a kit for performing the method as described herein, comprising or consisting of: a) a medium comprising Ascorbic acid, MTG, Transferrin, BMP4, Rock inhibitor, bFGF, an inhibitor of the activin receptor-like kinase (ALK) receptors, an GSK3 inhibitor, VEGF, IL-6, IL-11, EPO, IGF-1, SCF; b) a medium comprising a Notch Ligand or a fragment thereof, fibronectin or a fragment thereof, an antagonist of the Aryl hydrocarbon / Dioxin receptor, Ascorbic acid, Transferrin, MTG, VEGF, bFGF, SCF, IL-6, IL- 11, IGF-1, TPO, Flt-3L, IL-3 and BMP4; and c) a medium comprising TNF-alpha, interleukin-7 (IL-7), Stem Cell Factor (SCF), thrombopoietin (TPO) and Flt3 ligand (FLT3L); a Notch Ligand or a fragment thereof, fibronectin or a fragment thereof, and optionally with Stromal cell-derived factor la (SDFla) and / or a p38 inhibitor.

[0307] In some embodiments, the method of the present invention presents the following advantages over the methods previously described in the art: increased purity (e.g., more than 80%, preferably more than 90% of the cells obtained at the end of the method are T cell progenitors) and / or an increased yield. For example, using the method of the present invention, about one T cell progenitor may be produced from 1 to 10 PSC seeded, for example from two PSC seeded.BRIEF DESCRIPTION OF THE DRAWINGS

[0308] Figures 1A-1C are a combination of schematic representation and Flow cytometry plots showing the differentiation of PSC-derived EB cells into CD7+ progenitors after 7 days of EHT culture on Matrigel followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture) in presence or absence of Ascorbic acid, Transferrin and Insulin growth factor 1. Fig. 1A is a schematic representation of differentiation of PSC-derived EB cells into CD7+ progenitors. Fig. IB is a combination of dot plots representative of the phenotype CD34 and CD45 of cells obtained after culture. Fig. 1C is a combination of dot plots showing the frequencies of CD7+ progenitors obtained after culture.

[0309] Figures 2A-2C are a combination of schematic representation and Flow cytometry plots showing the differentiation of PSC-derived EB cells into CD7+ progenitors after 9 days of EHT culture (either on Matrigel or DLL4 and RetroNectin in presence or absence of SRI) followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture). Fig. 2A is a schematic representation of differentiation of PSC-derived EB cells into CD7+ progenitors. Fig. 2B is a combination of dot plots representative of the phenotype CD34 and CD45 of cells obtained after culture. Fig. 2C is a combination of dot plots showing the frequencies of CD7+ progenitors obtained after culture.

[0310] Figures 3A-3F are a combination of schematic representation, Flow cytometry plots and graph showing the differentiation of PSC-derived EB cells into CD7+ progenitors after 9 days of EHT culture on DLL4 and RetroNectin in presence or absence of SRI followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture) inpresence or absence of either SDFla or SB203580 alone or both. Fig. 3A is a schematic representation of differentiation of PSC-derived EB cells into CD7+ progenitors. Fig. 3B is a combination of dot plots representative of the phenotype CD34 and CD45 of cells obtained after 9 days of EHT culture on DLL4 and RetroNectin in presence or absence of SRI. Fig. 3C is a combination of dot plots showing the frequencies of CD7+ progenitors after 9 days of EHT culture on DLL4 and RetroNectin in presence or absence of SRI followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture) in presence or absence of either SDFla or SB203580 alone or both. Fig. 3D is a graph showing the CD7+ progenitor cell number obtained per CD34+ cell after 9 days of EHT culture on DLL4 and RetroNectin in presence or absence of SRI followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture) in presence or absence of either SDFla or SB203580 alone or both. Fig. 3E is a combination of dot plots showing the frequencies of CD7+CD5+ progenitors after 9 days of EHT culture on DLL4 and RetroNectin in presence or absence of SRI followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture) in presence or absence of either SDFla or SB203580 alone or both. Fig. 3F is a graph showing the CD7+CD5+ progenitor cell number obtained per CD34+ cell after 9 days of EHT and 7 days of DLL4 culture.

[0311] Figures 4A-4D are a combination of schematic representation and Flow cytometry plots showing the generation of transduced CD7+ progenitors from PSC- derived EB cells after EHT on DLL4 and RetroNectin with SRI. Fig. 4A is a schematic representation of differentiation of PSC-derived EB cells into CD7+ progenitors comprising the addition of transduction step. Fig. 4B and 4C are a combination of dot plots representative of the phenotype of non-transduced (mock) and transduced cells after 9 days of EHT on DLL4 and RetroNectin with SRI . Fig. 4D is a combination of dot plots showing the frequencies of non-transduced (mock) and transduced CD7+ progenitors after 9 days of EHT on DLL4 and RetroNectin with SRI followed by 7 days of culture on DLL4 and RetroNectin (DLL4 culture) in presence or absence of either SDFla or SB203580 alone or both.

[0312] Figures 5A-5D are a combination of Flow cytometry plots showing the phenotype of non-transduced (mock) and transduced CD7+ progenitors from PSC-derived EB cells obtained after 9 days of EHT on DLL4 and RetroNectin with SRI followed by 7 days of DLL4 culture in presence or absence of either SDFla or SB203580 alone or both. Fig. 5A is a combination of dot plots showing the expressions of CD7 and CD161. Fig. 5B is a combination of dot plots showing the expressions of CD56 and CD161. Fig. 5C is a combination of dot plots showing the expressions ofCD3 and CD 19. Fig. 5D is a combination of dot plots showing the expressions of CD la.

[0313] Figures 6A-6D are a combination of Flow cytometry plots showing the in vitro T cell differentiation potential of the non-transduced (mock) and transduced CD7+ progenitors generated from PSCs and obtained after 9 days of EHT on DLL4 and RetroNectin with SRI followed by 7 days of DLL4 culture in presence or absence of either SDFla or SB203580 alone or both. Fig. 6A is a combination of dot plots showing the differentiation of CD4+CD8+ cells from non-transduced (mock) progenitors (left) and from transduced progenitors (right) obtained after 1, 2 and 3 weeks of co-culture of PSC-derived CD7+ progenitors with OP9-hDLLl stromal cells. Fig. 6B is a combination of dot plots showing the differentiation of TCRyS or TCR a[3 expressing CD3+ cells from non-transduced (mock) progenitors obtained after 1, 2 and 3 weeks of co-culture of PSC- derived CD7+ progenitors with OP9-hDLLl stromal cells. Fig. 6C is a combination of dot plots showing the differentiation of TCR y5 or TCR a[3 expressing CD3+ cells from transduced progenitors obtained after 1, 2 and 3 weeks of co-culture of PSC-derived CD7+ progenitors with OP9-hDLLl stromal cells. Fig. 6D is a combination of dot plots showing stable transduction of cells during co-culture of PSC-derived CD7+ progenitors with OP9-hDLLl stromal cells for 3 weeks.

[0314] Figure 7 is a combination of Flow cytometry plots showing the differentiation of PSC-derived non-transduced (mock) and transduced CD7+ progenitors obtained from different DLL4 culture conditions (in presence or absence of either SDFla or SB203580 alone or both) into CD56+ NK cells after 1 week of feeder cell-free NK differentiation culture.EXAMPLES

[0315] The present invention is further illustrated by the following example.Example 1 :Materials and MethodsEmbryoid body (EB)

[0316] Embryoid body (EB) are obtained from the culture of pluripotent human embryoid stem cell line H9 in a 5% CO2 / 5% 02 / 90% N2 environment. At TO, H9 cells were seeded at a density of l-2.5xlOA5 cell / ml in a serum-free medium supplemented with L-glutamine (lx) penicillin / streptomycin (lx), ascorbic acid (176pg / ml), Transferrin (150pg / ml), mono-thioglycerol (MTG, 400 pM), BMP4 (10 ng / ml), and Y-27632 (5 pM). On day 1, bFGF (5ng / ml) was added to the medium. On day 2, cells were collected and resuspended in serum-free media supplemented with L-glutamine (lx) penicillin / streptomycin (lx), ascorbic acid (50pg / ml), Transferrin (150pg / ml), monothioglycerol (MTG, 400 pM), SB431542 (6 pM), CHIR99021 (3 pM), bFGF (5 ng / ml), and BMP4 (10 ng / ml). On day 4, medium was replaced by human serum albumin (HSA)- based media supplemented with L-glutamine (lx) penicillin / streptomycin (lx), ascorbic acid (50pg / ml), Transferrin (150pg / ml), mono-thioglycerol (MTG, 400 pM), VEGF (15 ng / ml), IL-6 (lOng / ml), IL-11 (5ng / ml) and bFGF (5 ng / ml). At day 6, medium was replaced by human serum albumin (HSA)-based media supplemented with L-glutamine (lx), penicillin / streptomycin (lx), ascorbic acid (50pg / ml), Transferrin (150pg / ml), mono-thioglycerol (MTG, 400 pM), bFGF (5 ng / ml), VEGF (15 ng / ml), IL-6 (10 ng / ml), IGF-1 (25 ng / ml), EPO (2U / ml), IL-11 (5 ng / ml), and SCF (50ng / ml). After 8 days of culture, embryoid body (EB) cells were dissociated via a first incubation with trypsin followed by digestion with Collagenase Type II.Isolation ofhemogenic endothelial (HE) (CD34+ CD 43 -CD 73 -CD 184-) and CD34+ cells from EB

[0317] Hemogenic endothelial (HE) (CD34+ CD43-CD73-CD184-) and CD34+ cells were isolated from dissociated EB using respectively Fluorescence Activated Cell Sorting(FACS) or Magnetic-activated cell sorting. For FACS sorting, dissociated EB cells were stained with antibodies against human CD34, CD43, CD73 and CD184 in 10 million (M) cells / ml of Iscove’s Modification of DMEM (IMDM) (Corning, NY, US) containing 5% Knock-out serum replacement (KOSR) (Gibco, Life Technologies, Carlsbad, CA) (FACS buffer) for 30min in ice. The stained cells were then washed with FACS buffer and then resuspended in 5M cells / ml of FACS buffer. The hemogenic endothelial (HE) (CD34+CD43-CD73-CD184-) cells were sorted in FACS Aria II (Becton Dickinson). For Magnetic-activated cell sorting, the CD34+ cells from EB were magnetically isolated using Indirect CD34 MicroBead Kit, human (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturers’ instructions.Transition of hemogenic endothelial (HE) cells to hematopoietic progenitor steps

[0318] The transition of HE cells to hematopoietic progenitors (HET step) was performed using either FACS sorted CD34+CD43-CD73-CD184- cells or magnetically sorted CD34+ cells. The transition from CD34+CD43-CD73-CD184- cells was performed either on Matrigel (Corning)- or on DLL4-Fc fusion protein (DLL4) (GTP Technology ) / RetroNectin (Takara Bio) - coated conditions, whereas the transition from CD34+ cells were performed on DLL4 / RetroNectin coated conditions.

[0319] For transition on Matrigel: The sorted CD34+CD43-CD73-CD184- cells were washed and resuspended in 0.2M cells / ml of HE medium (Table 1). Then 50pl of cell suspension was added into wells of 96 well ultra-low attachment microplate (Corning) to let them reaggregate overnight inside CO2 incubator. The reaggregated cells were then transferred into Growth Factor Reduced (GFR) Matrigel (Corning) coated 24 well plates. The cells were incubated inside CO2 incubator for 6h. After 6h, fresh 1ml of HE was added into the wells and cultured the HE cells for a total of 7 or 9 days to induce them to produce hematopoietic progenitors.

[0320] For transition on DLL4 / RetroNectin: The sorted CD34+CD43-CD73-CD184- cells were washed and resuspended in 0.4M cells / ml of HE medium (Table 1). Then 50 pl of cell suspension was seeded into DLL4 (5 g / ml) (GTP Technology) and RetroNectin (25pg / ml) (Takara Bio) coated wells of 24 well plate in 1ml of HE medium with orwithout SRI (800ng / ml) (Stem Cell Technologies, STEMCELL Technologies, Vancouver, Canada) and cultured for 9 days to induce them to produce hematopoietic progenitors.

[0321] For transition from CD34+ cells on DLL4 / RetroNectin: The magnetically isolated CD34+ cells were washed and resuspended in 0.2M cells / ml of HE medium(Table 1) without SRI or HE medium supplemented with SRI. Then lOOpl of cell suspensions were seeded into DLL4 and RetroNectin coated wells of 96 well plate. lOOpl of the respective culture media (HE with or without SRI) were added the next day and cultured for 9 days to induce the cells to produce hematopoietic progenitors.

[0322] Table 1: HE mediumTransduction of cells

[0323] The transduction was performed during HE to hematopoietic transition culture. One day after the seeding of CD34+ cells on DLL4 and RetroNectin coated wells at a cell concentration of 0.27M / ml, they were transduced with a lentiviral vector encoding human LNGFR at multiplicity of infection (MOI) of 20 at the same cell concentration overnight.After overnight transduction, the transduction was neutralized by adding fresh HEmedium (with or without SRI) to make 0.1M cells / ml of culture medium. The cells were cultured for a total of 9 days to produce transduced hematopoietic progenitors.Culture of hematopoietic progenitors

[0324] For hematopoietic progenitors produced from FACS sorted CD34+CD43-CD73-CD184 cells The hematopoietic progenitors were harvested after 7 or 9 days of HE to hematopoietic transition. The harvested cells were seeded into DLL4 (5pg / ml) (GTP Technology) and RetroNectin (25pg / ml) (Takara Bio) coated 48 well plates. The cells harvested from one well of 24 well plates were seeded into one well of 48 well plates. The cells were cultured in alpha minimal essential medium (a-MEM) (Gibco, Life Technologies, Carlsbad, CA) containing 20% FBS Hyclone (Cytiva, MA, US) and supplemented with lOOng / ml Stem cell factor (SCF), lOOng / ml Thrombopoietin (TPO), lOOng / ml FMS-like tyrosine kinase 3 ligand (Flt3-L), lOOng / ml Interleukin 7 (IL-7); all from Peprotech Inc., Rocky Hill, NJ, lOng / ml Tumor necrosis factor alpha (TNF-a) (R&D Systems) and with or without Ascorbic acid (AA) (50ug / ml) (Merck), Transferrin (150ug / ml) (Roche) and Insulin growth factor (IGF) 1 (25ng / ml) (R&D Systems, Minneapolis, MN) for 7 days. After 7 days of culture, the cells were harvested and analyzed by flow cytometry.

[0325] For hematopoietic progenitors produced from CD34+ cells: The hematopoietic progenitors were harvested after 9 days of HE to hematopoietic transition. The harvested cells were seeded into DLL4 (5pg / ml) and RetroNectin (25pg / ml) (Takara Bio) coated 48 well plates. The cells harvested from one well of 96 well plates were seeded into one well of 48 well plates. The cells were cultured in a-MEM (Gibco) containing 20% FBS Hyclone (Cytiva) and supplemented with lOOng / ml Stem cell factor (SCF), lOOng / ml Thrombopoietin (TPO), lOOng / ml FMS-like tyrosine kinase 3 ligand (Flt3-L), lOOng / ml Interleukin 7 (IL-7) (Peprotech), lOng / ml Tumor necrosis factor alpha (TNF-a) (R&D Systems) and in the presence or absence of either Stromal cell-derived factor 1 a (SDFla) (lOOng / ml) (Peprotech) or SB203580 (p38 inhibitor) (15pM) (R&D Systems) alone or both. After 7 days of culture, the cells were harvested and analyzed by flow cytometry.In vitro T-cell differentiation assay

[0326] The in vitro T-lymphoid potential of PSC-derived non-transduced (mock) or transduced CD7+ progenitors was assessed by using an OP9-hDLLl co-culture system. CD7+ progenitors (transduced or non-transduced) generated from PSC were co-cultured on OP9-hDLLl stromal cells for 4 weeks in a-MEM medium (Gibco) containing 20% FBS Hyclone (Cytiva) and supplemented with SCF (lOng / ml), Flt3-L (5ng / ml), TPO (lOng / ml) and IL-7 (2ng / ml) (Peprotech). Every week, a quarter of the cultured cells were analyzed (using FACS) for the presence of CD4+CD8+ and TCRa[3- or TCRyS- expressing CD3+ T cells; three-quarters of the cells were reseeded on fresh OP9-hDLLl cells.In vitro NK cell differentiation Assay

[0327] The in vitro NK cell differentiation potential of PSC-derived non-transduced or transduced CD7+ progenitors was assessed by using a feeder cell-free NK differentiation method. The CD7+ progenitors were cultured in RPMI medium (Gibco) containing 10% FBS Hyclone (Cytiva) and supplemented with SCF (50ng / ml), Flt3-L (50ng / ml), IL-15 (20ng / ml), IL-7 (20ng / ml) (Peprotech) and IL-2 (500IU / ml) (Novartis, Bale, Switzerland) for one week.Flow Cytometry

[0328] Anti-human CD4-PE (clone REA623), CD8-APCVio770 (clone REA734), CD34-APC (clone AC136), CD45-APCVio770 (clone REA747), LNGFR-Vioblue (clone REA844), CD56-PEVio770 (clone REA196), CD56-APCVio770 (clone REA196), CD161-APC (clone 191B8) and 7-aminoactinomycin D (7-AAD) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany). Anti-human CD7-PE (clone MT701), CD5-APCCy7 (clone UCHT2), CD43-PE (clone 1G10), CD73-PECy7 (clone AD2), CD14-PECy7 (clone McpP9) and CD15-PECy7 (clone HI98) were purchased from BD Biosciences (San Jose, CA). Anti-human CD3-BV421 (clone UCHT1), CD3-BV510 (clone UCHT1), CD3-PECy7 (clone UCHT1) and CD45-BV510 (clone HI30) antibodies were purchased from Sony Biotechnology (San Jose, CA). Antihuman CD19-APCCy7 (HIB19), TCRa[3-APC (IP26A) and CD184-BV421 (clone12G5), antibodies were purchased from BioLegend (San Diego, CA). Anti-human TCRyS-FITC (IMMU510) was purchased from Beckman Coulter (Brea, CA).

[0329] For surface staining, cells were incubated with the appropriate antibodies for 15 min on ice, washed and then resuspended in MACS buffer (0.5% bovine serum albumin (BSA) in IX PBS with 2.5mM EDTA).

[0330] All flow cytometry data were acquired with either a Gallios flow cytometer (Beckman Coulter, Krefeld, Germany) or Novocyte flow cytometer (Agilent, CA, US) and then analyzed using FlowJo software (version 10.2, TreeStar, Ashland, OR). During FACS analyses, all gatings were performed on live cells (determined by exclusion of the dye 7-AAD).ResultsFailure of differentiation of PSC-derived EB cells into CD7+ progenitors using Matrigel for Transition of hemogenic endothelial (HE) cells to hematopoietic progenitors

[0331] In a first attempt to generate human T cell progenitors from pluripotent stem cells (PSCs), hemogenic endothelial (HE) (CD34+CD43-CD73-CD184-) cells were FACS sorted from Embryoid body (EB) differentiated from pluripotent human embryonic stem cell line H9 and differentiated into hematopoietic progenitor cells (HPCs) (Endothelial to hematopoietic transition, EHT) on Matrigel for 7 days (Figure 1A). After 7 days, the HPCs obtained were cultured on DLL4 and RetroNectin in the presence of a cytokine cocktail with or without Ascorbic acid (AA), Transferrin and Insulin growth factor (IGF) 1 for 7 days to produce T cell progenitors (Figure 1A). As shown in Figures IB and 1C, although CD45+ hematopoietic cells were observed after a total of 14 days of culture (7 days of EHT and 7 days of DLL4 culture), no significant population of CD7+ T cell progenitors were observed in both the culture conditions with and without Ascorbic acid (AA), Transferrin and Insulin growth factor (IGF) 1.Addition of StemReginin 1 during EHT leads to the generation of high numbers of CD7+ progenitors from PSCs

[0332] Then, the differentiation of FACS sorted CD34+CD43-CD73-CD184- cells into HPCs was performed on either Matrigel or DLL4 and RetroNectin (with or without StemReginin 1, SRI) for 9 days to explore if we could produce T cell progenitors upon these modifications during EHT step (Figure 2A). Interestingly, after 7 days of DLL4 culture of the HPCs obtained, the EHT on DLL4 and RetroNectin increased the frequencies of CD45+ progenitors (Figure 2B). However, CD7+ progenitor cells were observed only in the condition of EHT culture on DLL4 and RetroNectin in presence of SRI (Figure 2C). Although the pluripotent stem cells could give rise to CD7+ progenitors using this method, their proportion was very low.

[0333] Considering the very low proportion of CD7+ progenitors obtained, next experimentations were performed to optimize the method with the aim of producing a pure population of CD7+ progenitors in high numbers. From this perspective, CD34+cells were used instead of FACS sorted CD34+CD43-CD73-CD184- cells to avoid the step of FACS cell sorting and to have more HE cells for their differentiation into HPCs. For this, CD34+ cells were magnetically isolated from EB and subjected to differentiation into HPCs on DLL4 and RetroNectin in presence or absence of SRI for 9 days (Figure 3A). Interestingly, the addition of SRI increased the frequency of CD45+CD34+ hematopoietic progenitors (Figure 3B). The HPCs obtained were then cultured on DLL4 and RetroNectin in presence of either Stromal cell-derived factor la (SDFla) or SB203580 (p38 inhibitor) alone or both for 7 days (Figure 3A). After 7 days of DLL4 culture, all the culture conditions gave rise to CD7+ progenitors (Figure 3C). Essentially, the addition of SRI during the EHT step increased the frequencies of CD7+ progenitors (reaching up to >90%) (Figure 3C) with a striking increase in their yield (up to >80 progenitor per CD34+ cell (Figure 3D) in all the corresponding conditions. Notably, the addition of SRI during EHT step also increased the proportion of CD5+CD7+ progenitors (SR1+: between 16 and 25%; SRI- : between 4,7 and 20% depending of the presence or not of SDFla or SB203580) (Figure 3E) and their yield (up to 20 progenitors per CD34+) (Figure 3F). Taken together, these results suggest that the addition of SRI during EHT culture on DLL4 and RetroNectin increases the frequency and yield of CD7+ progenitors obtained after DLL4 culture of the resulted HPCs.Generation of transduced CD7+ progenitors from PSCs

[0334] Next, the method described hereinabove was combined with transduction to check if transduced CD7+ progenitors can be generated. For this, CD34+ cells were transduced with a lentiviral vector encoding human LNGFR during their differentiation into HPCs on DLL4 and RetroNectin (Figure 4A). Analysis of the cells after 9 days of differentiation showed an efficient transduction of progenitors with >90% transduction efficiency (Figure 4B). However, the frequency of CD45+CD34+ cells obtained after 9 days of culture is lower in the transduced condition than in non-transduced mock condition (Figure 4C). Moreover, when hematopoietic progenitors obtained from 9 days of EHT were cultured on DLL4 and RetroNectin in presence or absence of either SDFla or SB203580 or both (Figure 4A), transduced LNGFR+CD7+ progenitors were generated under all culture conditions with comparable frequencies to those of their corresponding mock non-transduced culture conditions (Figure 4D).

[0335] Further phenotypic analyses of the progenitors obtained after 9 days of EHT followed by 7 days of DLL4 culture showed the presence of CD161+CD7+ and CD161+CD56+ progenitors where the frequencies of CD161+CD7+ progenitors were decreased in the DLL4 culture conditions performed in presence of SB203580 (alone or with SDFla) (Figures 5A and 5B). Importantly, no significant population of CD3+, CD 19+ or CDla+ cells were observed in all the culture conditions (Figures 5C and 5D).In vitro T cell differentiation potential of PSC-derived CD7+ progenitors

[0336] Finally, the in vitro T cell differentiation potential of the PSC-derived nontransduced and transduced CD7+ progenitors was tested using OP9-hDLLl (murine bone marrow stromal cell line expressing human DLL1) co-culture system. Weekly analysis during co-culture with OP9-hDLLl stromal cells showed the appearance of CD4+CD8+ (double positive, DP) and TCRyS expressing CD3+ cells since week 1 for both nontransduced and transduced progenitors (Figures 6A, 6B, 6C and 6D). The TCRaP expressing CD3+ cells appeared since week 2 of co-culture (Figures 6C and 6D). The frequencies of DP cells, TCRyS or a[3 expressing CD3+ cells were higher in conditions of DLL4 culture performed in presence of SB203580 regardless of the presence orabsence of SDFla than those conditions without SB203580 (Figures 6A, 6B, 6C and 6D). Notably, the transduction was stable during in vitro T cell differentiation with >80% of transduction after 3 weeks of differentiation (Figure 6D).

[0337] These results demonstrate the in vitro T cell differentiation potential of the non- transduced or transduced CD7+ progenitors produced from PSCs.In vitro NK cell differentiation potential of PSC-derived CD7+ progenitors

[0338] The in vitro NK differentiation potential of PSC-derived CD7+ progenitors were also explored using a feeder cell-free culture method. The PSC-derived non-transduced (mock) and transduced CD7+ progenitors obtained from different DLL4 culture conditions (in presence or absence of either SDFla or SB203580 or both) were subjected to feeder cell-free NK differentiation culture for one week. Analysis of the cultures after one week of differentiation showed that both non-transduced and transduced CD7+ progenitors obtained from all DLL4 culture conditions could give rise to CD3-CD56+ NK cells (Figure 7) demonstrating the in vitro NK differentiation potential of PSC- derived CD7+ progenitors.

Claims

CLAIMS1. An in vitro method for generating T cell progenitors (proT cells) comprising the steps of: a) obtaining embryoid bodies (EBs) from pluripotent stem cells (PSCs), b) isolating CD34+CD144+CD31+ cells from said embryoid bodies obtained in step a), c) culturing the population of CD34+ CD144+CD31+ cells obtained in step b) in a culture medium comprising a Notch ligand, fibronectin or a fragment thereof, and an antagonist of the Aryl hydrocarbon / Dioxin receptor, thereby obtaining a population of HPCs, and d) culturing the population of HPCs obtained in step c) in a cytokine comprising medium, thereby obtaining a population of proT cells.

2. The in vitro method according to claim 1, wherein at step a), the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

3. The in vitro method according to claim 1 or 2; wherein at step a) the pluripotent stem cells are cultured in a medium comprising Ascorbic acid, 1 -thioglycerol, Transferrin, BMP4 (bone morphogenetic protein 4), Rock inhibitor, bFGF (basic fibroblast growth factor), an inhibitor of the activin receptor-like kinase (ALK) receptors, GSK3 inhibitor, VEGF (vascular endothelial growth factor), IL-6, IL-11, EPO (erythropoietin), IGF-1, and SCF (stem cell factor).

4. The in vitro method according to any one of claims 1 to 3, wherein the EBs obtained at step a) are dissociated to obtain a single cell suspension, preferably using an enzymatic treatment.

5. The in vitro method according to any one of claim 1 to 4, wherein at step c), the Notch ligand is an immobilized Notch Ligand, preferably an immobilized Delta- like-4 ligand or a fragment thereof, and wherein the fibronectin or the fragmentthereof is an immobilized fibronectin or fragment thereof, preferably an immobilized CH-296 (Retronectin®)6. The in vitro method according to any one of claim 1 to 5, wherein the antagonist of the Aryl hydrocarbon / Dioxin receptor is StemRegenin 1 (SRI).

7. The in vitro method according to any one of claim 1 to 6, wherein at step c), the cells are cultured in presence of an immobilized Notch Ligand, an immobilized fibronectin or fragment thereof and an antagonist of the Aryl hydrocarbon / Dioxin receptor for more than 5 days and less than 12 days, preferably for about 9 days.

8. The in vitro method according to any one of claims 1 to 7, wherein at step d), the cells are cultured in the cytokine comprising medium for more than 5 days and less than 9 days, preferably for about 7 days and wherein said cytokine comprising medium comprises TNF-alpha, interleukin-7 (IL-7), Stem Cell Factor (SCF), thrombopoietin (TPO) and Flt3 ligand (FLT3L).

9. The in vitro method according to claim 8, wherein at step d), the cells are also cultured in presence of an immobilized Notch Ligand, preferably a immobilized Delta-like-4 ligand or a fragment thereof, and an immobilized fibronectin or fragment thereof, preferably an immobilized CH-296 (Retronectin®), and optionally with Stromal cell-derived factor la (SDFla) and / or a p38 inhibitor.

10. The in vitro method according to any one of claims 1 to 9, comprising an additional step of transducing or transfecting the cells with a vector encoding a transgene, preferably wherein said transgene encodes a Chimeric Antigen Receptor (CAR) or a T-cell receptor (TCR), or wherein said transgene is a safety switch gene, or encodes a potentiator of T-cell killing, wherein said additional step is performed at the beginning of step c).

11. The in vitro method according to any one of claims 1 to 10, comprising an additional step of modifying the cells such as for example with a system of gene editing to express or delete expression of a specific gene, wherein said additional step is performed at the beginning of step c) of the method.

12. A population of T cell progenitors obtained by the in vitro method according to any one of claims 1 to 11, wherein at least 70%, preferably at least 80% of the cells are CD7+ and wherein at least 15%, preferably at least 20% of CD7+ cells are CD5+.

13. The population of T cell progenitors obtained by the in vitro method according to claim 12, wherein the cells express CD161 and CD56, and does not express at least one of CD3, CD19 or CDla.

14. The population of T cell progenitors obtained by the in vitro method according to claim 12 or claim 13, for use for increasing the number of T cell population in a subject in need thereof.

15. The population of T cell progenitors obtained by the in vitro method according to claim 14, for use for increasing the number of T cell population in a subject in need thereof, wherein the subject in need thereof has a medical condition causing or resulting in lymphopenia, preferably wherein the lymphopenia results from cancer, infection (e,g., HIV), partial thymectomy, autoimmune disease and / or organ transplantation.

Images