Method for producing hemogenic endothelial cells from pluripotent stem cells

Abstract

The present invention relates to the production of hemogenic endothelial cells (HECs). A population of induced pluripotent stem cells (iPSCs) are differentiated into mesodermal cells by sequentially culturing iPSCs in a first, second, and third mesoderm induction medium to induce differentiation into mesodermal cells. Mesodermal cells are then differentiated into HECs by sequentially culturing mesodermal cells in a first and second hemogenic endothelial (HE) induction medium to induce differentiation into HECs. HECs can be further differentiated into hematopoietic progenitor cells (HPCs) and progenitor T cells.

Description

[Technical field]

[0001] The present invention relates to the production of hemogenic endothelial cells (HECs), for use, for example, in generating T cells for immunotherapy. [Background technology]

[0002] Immunotherapeutic drugs promise long-term survival and are poised to transform the face of cancer treatment (McDermott et al., Cancer Treat Rev. 2014 Oct;40(9):1056-64). There is a clear unmet medical need for new immunomodulatory drugs to expand the scope of patient populations and tumor types. Furthermore, new agents are needed to improve the magnitude and duration of antitumor responses. The development of these agents has been made possible by an improved understanding of the fundamental principles that control T cell immunity over the past two decades (Sharma and Allison, Cell. 2015 Apr 9;161(2):205-14). This typically requires tumor-specific CD4+ and CD8+ T cells that recognize tumor-associated peptide antigens presented by MHC molecules. Various vaccination strategies and adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes have shown that in some cases tumor-specific T cells can treat advanced cancer (Rosenberg et al., Nat Med. 2004 Sep;10(9):909-15).

[0003] However, current adoptive T cell therapies are limited by a shortage of suitable patient- and tumor-specific T cells, and there is a need for therapeutically sufficient and functional antigen-specific T cells for effective use in immunotherapy. Summary of the Invention

[0004] We have developed a process for differentiating induced pluripotent stem cells (iPSCs) into hemogenic endothelial cells that results in high yields, rapid and reproducible differentiation, which may be useful, for example, for the production of clinical grade blood cells such as T cells for use in immunotherapy.

[0005] A first aspect of the invention is a method for producing a population of hemogenic endothelial cells (HECs), comprising: (a) differentiating a population of induced pluripotent stem cells (iPSCs) into mesodermal cells by sequentially culturing the iPSCs in a first, second, and third mesoderm-inducing medium to induce differentiation into mesodermal cells; and (b) Differentiating the mesodermal cells into HECs by sequentially culturing the mesodermal cells in a first and a second hemogenic endothelial (HE) induction medium to induce differentiation into HECs. The present invention provides a method comprising:

[0006] The first mesoderm induction medium stimulates SMAD1, SMAD5, and SMAD9 mediated signaling pathways. For example, the first mesoderm induction medium may include BMP4. The second mesoderm induction medium (i) stimulates SMAD1, SMAD5, and SMAD9 mediated signaling pathways, and (ii) has fibroblast growth factor (FGF) activity. For example, the second mesoderm induction medium may include BMP4 and bFGF. The third mesoderm induction medium (i) stimulates SMAD1, SMAD2, SMAD3, SMAD5, and SMAD9 mediated signaling pathways, (ii) has fibroblast growth factor (FGF) activity, and (iii) inhibits glycogen synthase kinase 3β. For example, the third mesoderm induction medium may include BMP4, bFGF, activin, and CHIR-99021.

[0007] The first HE induction medium (i) stimulates VEGFR-mediated signaling pathways and (ii) has fibroblast growth factor (FGF) activity. For example, the first HE induction medium may contain VEGF and bFGF. The second HE induction medium (i) stimulates cKIT receptor (CD117; KIT receptor tyrosine kinase)-mediated signaling pathways, (ii) stimulates VEGFR-mediated signaling pathways, and (iii) has fibroblast growth factor (FGF) activity. For example, the second HE induction medium may contain SCF, VEGF, and bFGF.

[0008] Steps (a) and (b) may be performed without purification or isolation of the cells in the population.

[0009] The method of the first aspect comprises: (c) Differentiating the HECs into HPCs by culturing a population of HECs under conditions suitable for promoting hematopoietic differentiation. It may further include.

[0010] The above steps (a), (b) and (c) may be carried out in a defined culture medium and in the absence of feeder cells, stromal cells, serum, or other undefined medium supplements.

[0011] In some preferred embodiments, HPCs produced as described herein may be used in the production of T cells.

[0012] The method of the first aspect comprises: (iv) differentiating a population of HPCs into T cell precursors; and (v) maturation of precursor T cells to produce a population of DP CD4+CD8+ T cells It may further include.

[0013] The method of the first aspect comprises: (vi) activating and expanding DP CD4+CD8+ T cells to produce a population of SP CD8+ T cells or a population of SP CD4+ T cells. It may further include.

[0014] In other embodiments, the HPCs described herein may be used for the production of NK cells. (iv) Differentiation of a population of HPCs into NK cells It may further include.

[0015] In other embodiments, the HPCs produced as described herein may be used to produce B cells. (iv) Differentiating a population of HPCs into B cells It may further include.

[0016] These and other aspects and embodiments of the invention are described in more detail below. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows a schematic diagram of an example of a six-step method for generating T cells from iPSCs. [Diagram 2] FIG. 1 shows a schematic diagram of steps 1 and 2 of a six-step method for generating T cells from iPSCs. [Diagram 3] FIG. 1 shows the yield of CD34+ cells (HECs) differentiated from manually disaggregated iPSC colonies or passaged at single cell level using Accutase solution, using the protocol of the present invention ("alternative") or the control protocol. [Figure 4] Figure 1 shows the yield of CD34+CD45+ cells (HPCs) differentiated from manually disaggregated iPSC colonies or passaged at single cells using Accutase solution, using the protocol of the present invention ("alternative") or the control protocol. [Diagram 5] FIG. 1 shows the yield of CD7+ cells (progenitor T cells) differentiated from manually disaggregated iPSC colonies or passaged at single cell level using Accutase solution, using the protocol of the present invention ("alternative") or the control protocol. [Figure 6] 1 shows expression of CD34, CD45, CD43 and CD73 in HECs differentiated from iPSCs passaged as cell clusters using the protocol of the present invention ("alternative") or a control protocol and using Versene™ solution on day 6 of differentiation. [Figure 7A] 1 shows the yield of CD34+ cells (HEC) and CD34+CD45+ cells (HPC) at day 13 of differentiation from Versene™-passaged iPSCs using the protocol of the present invention ("Alternative") or a control protocol. [Figure 7B]4 shows the fold expansion of T cell precursors obtained from CD34+ cells generated using stage 4 Versene™ passaged iPSCs using the protocol of the present invention ("alternative") or a control protocol. [Figure 8] 1 shows representative photographs of active human hemogenic endothelium (HE) in culture using the protocol of the present invention. [Figure 9] Representative differences observed in the generation of hematopoietic progenitor cells (HSPCs) in culture using the protocol of the present invention ("Alternative") or a control protocol are shown. The number of round floating cells (HSPCs) emerging from HE was increased with the Alternative protocol. [Figure 10] FIG. 1 shows improved survival after magnetic isolation of CD34+ progenitor cells using the alternative protocol compared to the control protocol in four independent experiments (**p=0.0021, two-way ANOVA). [Figure 11] Representative phenotypes of CD34+ / CD45+ cells before and after CD34+ magnetic isolation using the protocol of the invention ("alternative") or a control protocol are shown. [Figure 12] Based on three independent experiments using the protocol of the present invention ("Alternative"), a significant increase in target cell yield per area and total yield is shown compared to the control protocol (2-way ANOVA, GraphPad software, p-values ​​are indicated in the figure). [Figure 13] Figure 1 shows integrated FACS histograms of CD34+ cells isolated using the protocol of the present invention ("alternative" (light shading)) and the control protocol (dark shading). The alternative protocol generated primarily low CD34+ cells with no expression of the CD73 endothelial marker, compared to the control protocol which generated high CD34+ cells with higher levels of CD73. [Figure 14] A comparison of representative phenotypes of T cell progenitors after 3 and 5 weeks of culture of CD34+ cells generated by the protocol of the present invention ("alternative") or the control protocol is shown. Similar phenotypes were observed. Both protocols were able to generate T cells with increasing levels of TCR over time. [Figure 15]Illustrates lymphoid cell yield and fold expansion at day 21 using the protocol of the present invention ("Alternative") or the control protocol. Increased lymphoid cell growth was observed from CD34+ cells generated with the Alternative protocol. Each dot represents a different TCR+ edited cell line in culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to an efficient and reproducible process for producing hemogenic endothelial cells (HECs) from induced pluripotent stem cells (iPSCs) cultured on a support substrate in 2D tissue culture vessels.

[0019] The methods described herein provide increased yields and purity of HECs compared to previous methods. HECs produced by the methods described herein can be differentiated into HPCs with increased yields compared to HECs produced by other methods. HECs produced by the methods described herein can have low or negative CD73 expression. CD73 expression is characteristic of the endothelial lineage, which does not have hematopoietic potential, and low or negative CD73 expression can indicate HECs with the ability to generate all types of blood cells, including lymphoid cells. CD7 expression can be increased in HPCs that are progeny of HECs produced by the methods described herein. CD7 expression indicates that HPCs are committed to the thymocyte lineage (i.e., early stage thymocytes) and can further differentiate into T cells.

[0020] Preferably, the methods described herein do not employ purification steps such as cell sorting or gating, which allows for the production of HECs in a single culture vessel.

[0021] Preferably, iPSCs are differentiated into HECs in a defined culture medium in the absence of feeder cells, stromal cells such as OP9-D14 stromal cells, serum, or other undefined medium supplements. This allows for the production of clinical grade cell products, and HECs produced as described herein may be useful, for example, for the production of T cells for use in immunotherapy.

[0022] Induced pluripotent stem cells (iPSCs) are multipotent cells derived from non-pluripotent fully differentiated donor or precursor cells. iPSCs are capable of self-renewal in vitro, exhibit an undifferentiated phenotype, and have the potential to differentiate into fetal or adult cell types of any of the three germ layers (endoderm, mesoderm, and ectoderm). A population of iPSCs can be clonal, i.e., genetically identical cells that are descendants of a single common ancestral cell.

[0023] iPSCs may express one or more of the following pluripotency-associated markers: POU5f1(Oct4), Sox2, alkaline phosphatase, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF4 and c-myc, preferably one or more of POU5f1, NANOG and SOX2. iPSCs may lack markers associated with specific differentiation fates, such as Brachyury, Sox17, FoxA2, ​​αFP, Sox1, NCAM, GATA6, GATA4, Hand1 and CDX2. In particular, iPSCs may lack markers associated with endoderm fate.

[0024] Preferably, the iPSCs are human iPSCs (hiPSCs).

[0025] In some embodiments, the iPSCs may be genetically edited to inactivate or delete HLA genes or other genes associated with immunogenicity or GVHD, or may be genetically edited to include nucleic acid encoding an exogenous antigen receptor, such as a TCR, CAR, or NKCR.

[0026] IPSCs can be derived from or reprogrammed from donor cells, which can be somatic cells or other precursor cells obtained from a source such as a donor individual. The donor cells can be mammalian, preferably human, cells. Suitable donor cells include adult fibroblasts and blood cells, such as peripheral blood cells, such as HPCs, mononuclear cells, CD34+ cord blood cells, or T cells.

[0027] Donor cells suitable for reprogramming into iPSCs as described herein may be obtained from a donor individual. In some embodiments, the donor individual may be the same person as the recipient individual to whom the T cells are administered after production as described herein (autologous procedure). In other embodiments, the donor individual may be a different person than the recipient individual to whom the T cells are administered after production as described herein (allogeneic procedure). For example, the donor individual may be a healthy individual who is human leukocyte antigen (HLA)-matched (either before or after donation) with the recipient individual suffering from cancer. In other embodiments, the donor individual may not be HLA-matched with the recipient individual. Preferably, the donor individual may be a newborn (neonatal), for example, the donor cells may be obtained from a sample of umbilical cord blood.

[0028] Suitable donor individuals are preferably free of transmissible viruses (eg, HIV, HPV, CMV) and adventitious agents (eg, bacteria, mycoplasma) and free of known genetic abnormalities.

[0029] In some embodiments, a population of peripheral blood cells such as HPCs for reprogramming can be isolated from a blood sample, preferably an umbilical cord sample, obtained from a donor individual. Suitable methods for the isolation of HPCs and other peripheral blood cells are well known in the art and include, for example, magnetic activated cell sorting (see, e.g., Gaudernack et al 1986 J Immunol Methods 90 179), fluorescence activated cell sorting (FACS: see, e.g., Rheinherz et al (1979) PNAS 76 4061), and cell panning (see, e.g., Lum et al (1982) Cell Immunol 72 122). HPCs can be identified in a sample of blood cells by expression of CD34. In other embodiments, a population of fibroblasts for reprogramming can be isolated from a skin biopsy after dispersion using collagenase or trypsin and growth in appropriate cell culture conditions.

[0030] In some embodiments, IPSCs can be derived from antigen-specific T cells. For example, T cells can contain nucleic acids encoding αβTCR that bind to antigens, such as tumor antigens, that are presented in complex with class I MHC. The antigen-specific T cells used to generate iPSCs can be obtained by screening a diverse population of T cells that have peptide epitopes of target antigens presented on class I or II MHC molecules on the surface of antigen-presenting cells, such as dendritic cells, or by isolating them from tumor samples of cancer patients.

[0031] Donor cells are typically reprogrammed into iPSCs by introducing reprogramming factors, such as Oct4, Sox2 and Klf4, into the cells. The reprogramming factors may be proteins or coding nucleic acids and may be introduced into the differentiated cells by any suitable technique, including plasmids, transposons, or more preferably, viral transfection or direct protein delivery. Other reprogramming factors, such as Klf genes, such as Klf-1, Klf-2, Klf-4 and Klf-5; Myc genes, such as C-myc, L-myc and N-myc; Nanog; SV40 large T antigen; Lin28; and small hairpins (shRNAs) targeting genes such as p53, may also be introduced into the cells to increase induction efficiency. After introduction of the reprogramming factors, the donor cells may be cultured. Cells expressing pluripotency markers may be isolated and / or purified to produce a population of iPSCs. Techniques for the production of iPSCs are well known in the art (Yamanaka et al Nature 2007;448:313-7, Yamanaka 6 2007 Jun 7;1(1):39-49, Kim et al Nature.2008 Jul 31;454(7204):646-50, Takahashi Cell.2007 Nov 30;131(5):861-72. Park et al Nature.2008 Jan 10;451(7175):141-6, Kimet et al Cell Stem Cell.2009 Jun 5;4(6):472-6, Vallier,L.,et al.Stem Cells,2009.9999(999A):p. N / A, Baghbaderani et al 2016;Stem Cell Rev. 2016 Aug;12(4):394-420, Baghbaderani et al. (2015) Stem Cell Reports,5(4),647-659).

[0032] Conventional techniques can be used for the culture and maintenance of iPSCs (Vallier, L. et al Dev. Biol. 275, 403-421 (2004); Cowan, CA et al. N. Engl. J. Med. 350, 1353-1356 (2004); Joannides, A. et al. Stem Cells 24, 230-235 (2006); Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005); Ludwig, TE et al. Nat. Biotechnol. 24, 185-187 (2006)). The iPSCs used in the present method can be grown under defined conditions or on feeder cells. For example, iPSCs can be cultured in a culture dish at an appropriate density (e.g., 10 5 ~10 6 iPSCs may be conventionally cultured on a feeder cell layer such as 1000 cells / 60 mm dish, irradiated mouse embryonic fibroblasts (MEFs), or on a suitable substrate in feeder-conditioned or defined iPSC maintenance medium. iPSCs for use in the subject methods may be passaged manually as disaggregated colonies, as single cells using enzyme solutions, or as cell clusters using chelating agents. In some embodiments, iPSCs may be passaged on ECM proteins such as Matrigel™ or vitronectin in iPSC maintenance medium such as mTeSR™1 or mTeSR™plus (StemCell Technologies) or E8 flex (Life Thermo) culture medium.

[0033] iPSCs are differentiated into HECs using the two-step process described above, which includes a mesoderm stage. For example, one method includes: (a) differentiating a population of iPSCs into mesodermal cells; and (b) Differentiation of mesodermal cells into HECs may include.

[0034] Preferably, the HECs are further differentiated into HPCs. For example, a method includes: (c) Differentiation of HECs into a population of HPCs It may further include.

[0035] Steps (a), (b) and (c) can all be performed without purification of cells or subpopulations from the population of cells to produce T cell-competent HPCs. Steps (a), (b) and (c) can be performed without serum and without the use of stromal cells, such as OP9-D14 stromal cells. For example, the population of cells can remain in the same culture vessel and be subjected to only a change of culture medium to perform the differentiation steps (a), (b) and (c) described herein.

[0036] The differentiation and maturation of the cell population in the steps of the methods described herein is induced by culturing the cells in a culture medium supplemented with a set of differentiation factors. The set of differentiation factors listed for each culture medium is preferably exhaustive, and the medium may lack other differentiation factors. In a preferred embodiment, the culture medium is a chemically defined medium. For example, the culture medium may consist of a chemically defined nutrient medium supplemented with an effective amount of one or more differentiation factors, as described below. The chemically defined nutrient medium may include a basal medium supplemented with one or more serum-free culture medium supplements.

[0037] A differentiation factor is a factor that modulates, for example promotes or inhibits, a signal transduction pathway that mediates the differentiation of mammalian cells. Differentiation factors can include growth factors, cytokines, and small molecules that modulate one or more of Activin / Nodal, FGF, Wnt, or BMP signal transduction pathways. Examples of differentiation factors include Activin / Nodal, FGF, BMP, retinoic acid, vascular endothelial growth factor (VEGF), stem cell factor (SCF), TGFβ ligand, GDF, LIF, interleukin, GSK-3 inhibitor, and phosphatidylinositol 3-kinase (PI3K) inhibitor.

[0038] Differentiation factors used in one or more of the media described herein include TGFβ ligands, such as activins, fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), stem cell factors (SCFs), vascular endothelial growth factors (VEGFs), GSK-3 inhibitors (e.g., CHIR-99021), interleukins, and hormones such as IGF-1 and angiotensin II. Differentiation factors may be present in the media described herein in an amount effective to modulate signaling pathways in cells cultured in the media.

[0039] In some embodiments, the differentiation factors listed above or below in the culture medium may be replaced by factors that have the same effect (i.e., stimulation or inhibition) on the same signaling pathways. Suitable factors are known in the art and include proteins, nucleic acids, antibodies, and small molecules.

[0040] The degree of differentiation of a cell population during each step may be determined by monitoring and / or detecting the expression of one or more cell markers in the population of differentiating cells. For example, an increase in the expression of a marker characteristic of a more differentiated cell type or a decrease in the expression of a marker characteristic of a less differentiated cell type may be determined. The expression of a cell marker may be determined by any suitable technique, including immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, fluorescence activated cell sorting (FACS), and enzymatic analysis. In a preferred embodiment, a cell is said to express a marker if the marker is detectable on the cell surface. For example, a cell described herein as not expressing a marker may exhibit active transcription and intracellular expression of the marker gene, but may not have detectable levels of the marker on the cell surface.

[0041] A population of partially differentiated cells, e.g., functional non-T cell cells, e.g., mesodermal cells, HECs (i.e., HE cells), HPCs, T cell precursors, or DP T cells, produced by a step of the methods described herein can be cultured, maintained, or expanded prior to the next differentiation step. The partially differentiated cells can be expanded by any convenient technique.

[0042] Cells can be cultured in monolayers in the absence of feeder cells on a surface or substrate coated with an extracellular matrix protein, such as fibronectin, laminin, or collagen. Suitable techniques for cell culture are well known in the art (e.g., Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. US (15 Oct 2004) ISBN: 1588295451, Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., US (9 Dec 2004) ISBN: 1588292223, Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol Methods. (2006) 310: 40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430). Basic Cell Culture Protocols' by J. Pollard and See JM Walker (1997), 'Mammalian Cell Culture: Essential Techniques' by A. Doyle and JB Griffiths (1997), 'Human Embryonic Stem Cells' by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside' by A. Bongso (2005), Peterson & Loring (2012) Human Stem Cell Manual: A Laboratory Guide Academic Press, and 'Human Embryonic Stem Cell Protocols' by K. Turksen (2006). Culture media and their components may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems).The above culture steps can use standard mammalian cell culture conditions, such as 37° C., 5% or 21% oxygen, 5% carbon dioxide, etc. The medium is preferably changed every 2 days and the cells are allowed to settle by gravity.

[0043] Cells may be cultured in a culture vessel. Suitable cell culture vessels are well known in the art and include culture plates, dishes, flasks, bioreactors, and multi-well plates, such as 6-well, 12-well or 96-well plates.

[0044] The culture vessel is preferably treated for tissue culture, for example, by coating one or more surfaces of the vessel with an extracellular matrix protein, such as fibronectin, laminin, or collagen. The culture vessel may be treated for tissue culture using standard techniques, for example, by incubating with a coating solution described herein, or may be obtained pretreated from a commercial supplier.

[0045] In a first step, iPSCs may be differentiated into mesodermal cells by culturing a population of iPSCs under suitable conditions that promote mesodermal differentiation. For example, iPSC cells may be sequentially cultured in a first, second, and third mesodermal induction medium to induce differentiation into mesodermal cells.

[0046] A suitable first mesoderm induction medium may stimulate SMAD1, SMAD5 and SMAD9, and / or SMAD1, SMAD5 and SMAD9 mediated signaling pathways. For example, the first mesoderm induction medium may include BMP.

[0047] A suitable second mesoderm induction medium may (i) stimulate SMAD1, SMAD5 and SMAD9, and / or SMAD1, SMAD5 and SMAD9 mediated signaling pathways, and (ii) have fibroblast growth factor (FGF) activity. For example, the second mesoderm induction medium may contain BMP, preferably BMP4, and FGF, preferably bFGF.

[0048] A suitable third mesoderm induction medium may (i) stimulate SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9, and / or SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9 mediated signaling pathways, (ii) have fibroblast growth factor (FGF) activity, and (iii) inhibit glycogen synthase kinase 3β. For example, the third mesoderm induction medium may comprise activin, preferably activin A, BMP, preferably BMP4, FGF, preferably bFGF, and a GSK3 inhibitor, preferably CHIR99021.

[0049] The first, second, and third mesoderm induction media may lack differentiation factors other than those mentioned above.

[0050] SMAD2 and SMAD3, and / or SMAD2 and SMAD3-mediated intracellular signaling pathways can be stimulated by the third mesoderm induction medium through the presence of a first TGFβ ligand in the medium. The first TGFβ ligand can be activin. Activin (Activin A: NCBI Gene ID: 3624 nucleic acid reference sequence NM_002192.2 GI: 62953137, amino acid reference sequence NP_002183.1 GI: 4504699) is a dimeric polypeptide that exerts various cellular effects through the stimulation of the activin / Nodal pathway (Vallier et al., Cell Science 118: 4495-4509 (2005)). Activin is readily available from commercial sources (e.g. Stemgent Inc. MA USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of activin in the medium described herein may be between 0.1 and 10 ng / mL, preferably about 1 ng / mL.

[0051] The fibroblast growth factor (FGF) activity of the second and third mesoderm induction media can be provided by the presence of fibroblast growth factors (FGFs) in the media. Fibroblast growth factors (FGFs) are protein factors that stimulate cell growth, proliferation, and cell differentiation by binding to fibroblast growth factor receptors (FGFRs). Suitable fibroblast growth factors include any member of the FGF family, such as any one of FGF1 to FGF14 and FGF15 to FGF23. Preferably, the FGF is FGF2 (also known as bFGF, NCBI Gene ID: 2247, nucleic acid sequence NM_002006.3 GI: 41352694, amino acid sequence NP_001997.4 GI: 41352695), FGF7 (also known as keratinocyte growth factor (or KGF), NCBI Gene ID: 2247, nucleic acid sequence NM_002006.3 GI: 41352694, amino acid sequence NP_001997.4 GI: 41352695), or FGF10 (NCBI Gene ID: 2247, nucleic acid sequence NM_002006.3 GI: 41352694, amino acid sequence NP_001997.4 GI: 41352695). Most preferably, the fibroblast growth factor is FGF2.

[0052] Conveniently, the concentration of FGF, such as FGF2, in the medium described herein may be 0.5-50 ng / mL, preferably about 5 ng / mL. Fibroblast growth factors, such as FGF2, FGF7 and FGF10, can be produced using conventional recombinant techniques or obtained from commercial suppliers (e.g., R&D Systems, Minneapolis, MN, Stemgent Inc, USA, Miltenyi Biotec Gmbh, DE).

[0053] SMAD1, SMAD5 and SMAD9, and / or SMAD1, SMAD5 and SMAD9-mediated intracellular signaling pathways can be stimulated by the first, second, and third mesoderm induction media through the presence of a second TGFβ ligand in the media.

[0054] The second TGFβ ligand can be a bone morphogenetic protein (BMP). Bone morphogenetic protein (BMP) binds to bone morphogenetic protein receptor (BMPR) and stimulates intracellular signaling through the pathway mediated by SMAD1, SMAD5, and SMAD9. Suitable bone morphogenetic proteins include any member of the BMP family, such as BMP2, BMP3, BMP4, BMP5, BMP6, or BMP7. Preferably, the second TGFβ ligand is BMP2 (NCBI gene ID: 650, nucleic acid sequence NM_001200.2 GI: 80861484; amino acid sequence NP_001191.1 GI: 4557369) or BMP4 (NCBI gene ID: 652, nucleic acid sequence NM_001202.3 GI: 157276592; amino acid sequence NP_001193.2 GI: 157276593). Suitable BMPs include BMP4. Advantageously, the concentration of bone morphogenetic proteins such as BMP2 or BMP4 in the medium described herein can be 1-500ng / mL, preferably about 10ng / mL. BMPs can be produced using conventional recombinant techniques or obtained from commercial suppliers (e.g. R&D, Minneapolis, USA, Stemgent Inc, USA, Miltenyi Biotec Gmbh, DE).

[0055] GSK3β inhibitory activity in the third mesoderm induction medium can be provided by the presence of a GSK3β inhibitor in the medium. GSK3β inhibitors inhibit the activity of glycogen synthase kinase 3β (gene ID 2932:EC2.7.11.26). Preferred inhibitors specifically inhibit the activity of glycogen synthase kinase 3β. Suitable inhibitors include CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile; Ring DB et al., Diabetes, 52:588-595(2003)), alsterpaullone, kenpaullone, BIO (6-bromoindirubin-3'-oxime (Sato et al Nat Med.2004)) Jan;10(1):55-63), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), lithium, and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione; Coghlan et al Chem Biol. 2000 Oct;7(10):793-803). In some preferred embodiments, the GSK3β inhibitor is CHIR99021. Suitable glycogen synthase kinase 3β inhibitors can be purchased from commercial suppliers (e.g., Stemgent Inc. MA USA, Cayman Chemical Co. MI USA, Selleckchem, MA For example, the third mesoderm induction medium may contain 0.1 to 100 μM, for example, about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 95 μM, preferably 1 to 10 μM, for example, about 3 μM or about 10 μM, of a GSK3β inhibitor, for example, CHIR99021.

[0056] In a preferred embodiment, the first, second, and third mesoderm induction media are chemically defined media. For example, the first mesoderm induction medium can consist of a chemically defined nutrient medium supplemented with an effective amount of BMP, preferably BMP4, for example 10 ng / mL BMP4, the second mesoderm induction medium can consist of a chemically defined nutrient medium supplemented with an effective amount of BMP, preferably BMP4, for example 10 ng / mL BMP4, and FGF, preferably bFGF (FGF2), for example 5 ng / mL bFGF, and the third mesoderm induction medium can consist of a chemically defined nutrient medium supplemented with an effective amount of activin, preferably activin A, for example 1 ng / mL activin A, BMP, preferably BMP4, for example 10 ng / mL BMP4, FGF, preferably bFGF (FGF2), for example 5 ng / mL bFGF, and a GSK3 inhibitor, preferably CHIR-99021, for example 3 μM CHIR-99021.

[0057] A chemically defined medium (CDM) is a nutrient solution for culturing cells that contains only specific components, preferably components with known chemical structures. CDM lacks undefined components or components, including undefined components such as feeder cells, stromal cells, serum, and complex extracellular matrices such as Matrigel™. For example, CDM does not contain stromal cells such as OP9 cells that express Notch ligands such as DLL1 or DLL4.

[0058] CDM or chemically defined nutrient medium may include chemically defined basal media. Suitable chemically defined basal media include Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12, Improved Dulbecco's Modified Eagle's Medium (DMEM) (Price et al Focus (2003), 25 3-6), Williams E (Williams, GM et al Exp. Cell Research, 89, 139-142 (1974)), RPMI-1640 (Moore, GE and Woods LK, (1976) Tissue Culture Association Manual. 3, 503-508), and StemPro™-34 (ThermoFisher Scientific).

[0059] The basal medium may be supplemented with serum-free culture medium supplements and / or additional components in the medium. Suitable supplements and additional components are described above and may include L-glutamine or replacements such as GlutaMAX-1™, ascorbic acid, monothioglycerol (MTG), human serum albumin, recombinant human serum albumin such as Cellastim™ (Merck / Sigma) and Recombumin™ (albumedix.com), insulin, transferrin, and 2-mercaptoethanol. The basal medium may be supplemented with serum replacements such as Knockout Serum Replacement (KOSR; Invitrogen).

[0060] The iPSCs are cultured in a first mesoderm induction medium for 1-36 hours, e.g., any of 6, 7, 8, 9, 10, 11. 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, preferably about 24 hours, to produce a population of mesoderm cells, and then cultured in a second mesoderm induction medium for 1-36 hours, e.g., any of 6, 7, 8, 9, 10, 11. 12. The cells may be cultured for any of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, preferably for about 24 hours, and then cultured in the third mesoderm induction medium for 36 to 60 hours, for example, any of 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 hours, preferably for about 48 hours.

[0061] Mesodermal cells are partially differentiated progenitor cells committed to the mesodermal lineage and can differentiate under appropriate conditions into mesenchyme (fibroblasts), muscle, bone, fat, blood vessels, and all cell types of the hematopoietic system. Mesodermal cells may express one or more mesodermal markers. For example, mesodermal cells may express any one, two, three, four, five, six, or all seven of Brachyury, Goosecoid, Mixl1, KDR, FoxA2, ​​GATA6, and PDGFαR.

[0062] In a second step, the mesodermal cells can be differentiated into hemogenic endothelial (HE) cells by culturing the population of mesodermal cells under suitable conditions that promote HE differentiation. For example, iPSC-derived mesodermal cells can be cultured in a first HE induction medium and then cultured in a second HE induction medium.

[0063] A suitable first HE induction medium may (i) stimulate VEGFR-mediated signaling pathways and (ii) have fibroblast growth factor (FGF) activity. For example, the first HE induction medium may contain VEGF and bFGF. A suitable second HE induction medium may (i) stimulate cKIT receptor (CD117; KIT receptor tyrosine kinase)-mediated signaling pathways, (ii) stimulate VEGFR-mediated signaling pathways, and (iii) have fibroblast growth factor (FGF) activity. For example, the second HE induction medium may contain SCF, VEGF, and bFGF.

[0064] Vascular endothelial growth factor (VEGF) is a protein factor of the PDGF family that binds to the VEGFR tyrosine kinase receptor and stimulates vasculogenesis and angiogenesis. Suitable VEGFs include any member of the VEGF family, such as any one of VEGF-A to VEGF-D and PIGF. Preferably, the VEGF is VEGF-A (also known as VEGF, NCBI gene ID: 7422, nucleic acid sequence NM_001025366.2, amino acid sequence NP_001020537.2). Preferably, the VEGFR-mediated signaling pathway is the VEGFR2 (KDR / Flk-1)-mediated signaling pathway. VEGF is readily available from commercial sources (e.g., R&D Systems, USA). Advantageously, the concentration of VEGF in the first or second HE induction medium described herein may be between 1 and 100 ng / mL, such as about any of 5, 7, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45 or 50 ng / mL, preferably about 15 ng / mL.

[0065] In some examples of the first or second HE induction medium, VEGF may be replaced by a VEGF activator or agonist that stimulates VEGFR-mediated signaling pathway. Suitable VEGF activators are known in the art and include proteins such as gremlin (Mitola et al (2010) Blood 116(18)3677-3680), nucleic acids such as shRNA (e.g., Turunen et al Circ Res. 2009 Sep 11;105(6):604-9), CRISPR-based plasmids (e.g., VEGF CRISPR activating plasmid; Santa Cruz Biotech,USA), antibodies and small molecules.

[0066] Stem cell factor (SCF) is a cytokine that binds to the KIT receptor (KIT proto-oncogene, receptor tyrosine kinase) (CD117; SCFR) and is involved in hematopoiesis. SCF (also known as KITLG, NCBI gene ID: 4254) can have the reference nucleic acid sequence NM_000899.5 or NM_03994.5 and the reference amino acid sequence NP_000890.1 or NP_003985.5. SCF is readily available from commercial sources (e.g., R&D Systems, USA). Advantageously, the concentration of SCF in the HE induction medium described herein may be between 1 and 1000 ng / mL, for example about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 ng / mL, preferably about 100 ng / mL.

[0067] In a preferred embodiment, the first and second HE induction medium are chemically defined media. For example, the first HE induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of VEGF, for example 15ng / mL VEGF, and FGF, for example 5ng / mL FGF. The second HE induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of VEGF, for example 15ng / mL VEGF, FGF, for example 5ng / mL bFGF, and SCF, for example 100ng / mL SCF. Preferably, the mesodermal cells are cultured in a first HE induction medium consisting of a chemically defined nutrient medium and two differentiation factors, where the two differentiation factors are bFGF and VEGF, and in a second HE induction medium consisting of a chemically defined nutrient medium and three differentiation factors, where the three differentiation factors are bFGF, SCF, and VEGF.

[0068] Suitable chemically defined nutrient media are described above and include basal media such as StemPro™-34 (ThermoFisher Scientific), optionally supplemented with glutamine, ascorbic acid and monothioglycerol, or IMDM supplemented with albumin, insulin, selenium transferrin, and lipids, as described below.

[0069] Mesodermal cells can be cultured in HE induction medium for 2-6 days, such as any of 2, 3, 4, 5 or 6 days, preferably about 4 days, to produce a population of HECs.

[0070] Hemopoietic endothelial cells (HECs) are a transient specialized type of endothelial cells that have the ability to differentiate into hematopoietic lineages under appropriate conditions. HECs may express CD34, and in some embodiments may not express CD73 or CXCR4 (CD184). For example, in some embodiments, HECs may have the phenotype CD34+CD73-, or CD34+CD73-CXCR4-.

[0071] In a third step, hemogenic endothelial cells (HECs) can be differentiated into hematopoietic progenitor cells (HPCs) by culturing a population of HECs under suitable conditions that promote hematopoietic differentiation. For example, HECs can be cultured in hematopoietic induction medium.

[0072] A suitable hematopoietic induction medium may stimulate (i) the cKIT receptor (CD117) or the cKIT receptor (CD117)-mediated signaling pathway, and / or (ii) the VEGFR or the VEGFR-mediated signaling pathway, preferably the VEGFR2 or the VEGFR2-mediated signaling pathway. For example, the hematopoietic induction medium may include differentiation factors: VEGF-A and / or SCF.

[0073] Preferably, the hematopoietic induction medium stimulates both (i) the cKIT receptor (CD117) or the cKIT receptor (CD117)-mediated signaling pathway, and (ii) the VEGFR or the VEGFR-mediated signaling pathway, preferably the VEGFR2 or the VEGFR2-mediated signaling pathway. For example, the hematopoietic induction medium may include differentiation factors: VEGF-A and SCF.

[0074] In some embodiments, a suitable hematopoietic induction medium can (a) stimulate cKIT receptor (CD117) and / or cKIT receptor (CD117)-mediated signaling pathways, (b) stimulate VEGFR and / or VEGFR-mediated signaling pathways, (c) stimulate MPL (CD110) and / or MPL (CD110)-mediated signaling pathways, (d) stimulate FLT3 and / or FLT3-mediated signaling pathways, (e) stimulate IGF1R and / or IGF1R-mediated signaling pathways, and (f) exhibit interleukin (IL) activity. For example, the hematopoietic induction medium may include differentiation factors: VEGF, SCF, thrombopoietin (TPO), Flt3 Ligand (Flt3L), IGF-1, IL-3, and IL-6 (see Table 3), or the hematopoietic induction medium may include differentiation factors: VEGF, SCF, thrombopoietin (TPO), Flt3 Ligand (Flt3L), IGF-1, IL-3, IL-6, and IL-7 (see Table 4).

[0075] In other embodiments, a suitable hematopoietic induction medium may stimulate (i) cKIT receptor (CD117; KIT receptor tyrosine kinase) and / or cKIT receptor (CD117; KIT receptor tyrosine kinase) mediated signaling pathway, (ii) VEGFR and / or VEGFR mediated signaling pathway, preferably VEGFR2 and / or VEGFR2 mediated signaling pathway, (iii) MPL (CD110) and / or MPL (CD110) mediated signaling pathway, (iv) FLT3 and / or FLT3 mediated signaling pathway, (v) IGF1R and / or IGF1R mediated signaling pathway, (vi) SMAD1, 5 and 9 and / or SMAD1, 5 and 9 mediated signaling pathway, (vii) hedgehog and / or hedgehog signaling pathway, (viii) EpoR and / or EpoR mediated signaling pathway, and (ix) AGTR2 and / or AGTR2 mediated signaling pathway. Suitable hematopoietic induction media may also inhibit AGTR1 (angiotensin II type 1 receptor (AT1)) and / or AGTR1 (angiotensin II type 1 receptor (AT1))-mediated signaling pathways. Suitable hematopoietic induction media may also have interleukin (IL) activity and FGF activity.

[0076] For example, hematopoietic induction medium may include differentiation factors: VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMP, FGF, sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II, and angiotensin II type 1 receptor (AT1) antagonist. One example of a suitable hematopoietic induction medium is Stage 3 medium shown in Table 1 below.

[0077] Thrombopoietin (TPO) is a glycoprotein hormone that regulates the production of platelets. TPO (also known as THPO, NCBI gene ID: 7066) may have the reference nucleic acid sequence NM_000460.4 and the reference amino acid sequence NP_000451.1. TPO is readily available from commercial sources (e.g., R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of TPO in the hematopoietic induction medium described herein may be 3-300 ng / mL, preferably about 30 ng / mL.

[0078] Flt3 ligand (Fms-related tyrosine kinase 3 ligand or FLT3L) is a cytokine with hematopoietic activity that binds to the FLT3 receptor and stimulates the proliferation and differentiation of progenitor cells. Flt3 ligand (also known as FLT3LG, NCBI gene ID: 2323) can have the reference nucleic acid sequence NM_001204502.2 and the reference amino acid sequence NP_001191431.1. Flt3 is readily available from commercial sources (e.g. R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of Flt3-Ligand in the hematopoietic induction medium described herein can be between 0.25 and 250 ng / mL, such as any of about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 ng / mL, preferably about 25 ng / mL.

[0079] Interleukins (ILs) are cytokines that play an important role in immune development and function. ILs in hematopoietic induction media can include IL-3, IL-6, IL-7, and IL-11.

[0080] IL-3 (also known as IL3 or MCGF, NCBI gene ID: 3562) may have the reference nucleic acid sequence NM_000588.4 and the reference amino acid sequence NP_000579.2. IL-3 is readily available from commercial sources (e.g., R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of IL-3 in the hematopoietic induction medium described herein may be between 0.25 and 250 ng / mL, such as any of about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 ng / mL, preferably about 25 ng / mL.

[0081] IL-6 (also known as IL6 or HGF, NCBI gene ID: 3569) may have the reference nucleic acid sequence NM_000600.5 and the reference amino acid sequence NP_000591.5. IL-6 is readily available from commercial sources (e.g., R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of IL-6 in the hematopoietic induction medium described herein may be 0.1-100 ng / mL, such as about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 95 ng / mL, preferably about 10 ng / mL.

[0082] IL-7 (also known as IL7, NCBI gene ID: 3574) may have the reference nucleic acid sequence NM_000880.4 and the reference amino acid sequence NP_000871.1. IL-7 is readily available from commercial sources (e.g., R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of IL-7 in the hematopoietic induction medium described herein may be 0.1-100 ng / mL, such as about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 95 ng / mL, preferably about 10 ng / mL.

[0083] IL-11 (also known as AGIF, NCBI gene ID: 3589) may have the reference nucleic acid sequence NM_000641.4 and the reference amino acid sequence NP_000632.1. IL-11 is readily available from commercial sources (e.g. R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of IL-11 ligand in the hematopoietic induction medium described herein may be 0.5-100 ng / mL, such as about 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 95 ng / mL, preferably about 5 ng / mL.

[0084] Insulin-like growth factor 1 (IGF-1) is a hormone that binds to the tyrosine kinase IGF-1 receptor (IGF1R) and the insulin receptor, activating multiple signaling pathways. IGF-1 (also known as IGF or MGF, NCBI gene ID: 3479) can have the reference nucleic acid sequence NM_000618.5 and the reference amino acid sequence NP_000609.1. IGF-1 is readily available from commercial sources (e.g., R&D Systems, USA). Advantageously, the concentration of IGF-1 in the hematopoietic induction medium described herein may be between 0.25 and 250 ng / mL, such as any of about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 ng / mL, preferably about 25 ng / mL.

[0085] Sonic hedgehog (SHH) is a ligand of the hedgehog signaling pathway that regulates vertebrate organogenesis. SHH (also known as TPT or HHG1, NCBI gene ID: 6469) can have the reference nucleic acid sequence NM_000193.4 and the reference amino acid sequence NP_000184.1. SHH is readily available from commercial sources (e.g. R&D Systems, USA, Miltenyi Biotec Gmbh, DE). Advantageously, the concentration of SHH in the hematopoietic induction medium described herein may be between 0.25 and 250 ng / mL, such as any of about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 ng / mL, preferably about 25 ng / mL.

[0086] Erythropoietin (EPO) is a glycoprotein cytokine that binds to the erythropoietin receptor (EpoR) and stimulates red blood cell formation. EPO (also known as DBAL, NCBI gene ID: 2056) may have the reference nucleic acid sequence NM_000799.4 and the reference amino acid sequence NP_000790.2. EPO is readily available from commercial sources (e.g., R&D Systems, USA, PreproTech, USA). Advantageously, the concentration of EPO in the hematopoietic induction medium described herein may be 0.02-20 U / mL, such as about 0.025, 0.05, 0.075, 0.1, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 13, 15, 17, or 19 U / mL, preferably about 2 U / mL.

[0087] Angiotensin II is a heptapeptide hormone formed by the action of angiotensin-converting enzyme (ACE) on angiotensin I. Angiotensin II stimulates vasoconstriction. Angiotensin I and II are formed by cleavage of angiotensinogen (also known as AGT, NCBI gene ID: 183), which may have the reference nucleic acid sequence NM_000029.4 and the reference amino acid sequence NP_000020.1. Angiotensin II is readily available from commercial sources (e.g., R&D Systems, USA, Tocris, USA). Advantageously, the concentration of angiotensin II in the hematopoietic induction medium described herein may be between 0.05 and 50 ng / mL, such as any of about 0.05, 0.075, 0.1, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 ng / mL, preferably about 5 ng / mL.

[0088] Angiotensin II type 1 receptor (AT1) antagonists (ARBs) are compounds that selectively block activation of the AT1 receptor (AGTR1; gene ID 185). Suitable AT1 antagonists include losartan (2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)-4-biphenylyl]methyl}-1H-imidazol-5-yl)methanol), valsartan ((2S)-3-methyl-2-(pentanoyl{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}amino)butanoic acid), and telmisartan (4'[(1,4'-dimethyl-2'-propyl[2,6'-bi-1H-benzimidazol]-1'-yl)methyl][1,1'-biphenyl]-2-carboxylic acid). In some preferred embodiments, the AT1 antagonist is losartan. Suitable AT1 antagonists can be purchased from commercial suppliers (e.g., Tocris, USA, Cayman Chemical Co. MI). Conveniently, the concentration of the angiotensin II type 1 receptor (AT1) antagonist in the hematopoietic induction medium described herein can be 1 to 1000 μM, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 μM, preferably about 100 μM.

[0089] In some preferred embodiments, a suitable hematopoietic induction medium may lack differentiation factors that stimulate (a) SMAD1, 5 and 9 mediated signaling pathways, e.g., bone morphogenetic proteins (BMPs), e.g., BMP4; (b) Hedgehog signaling pathways, e.g., Sonic Hedgehog (SHH); (c) EpoR mediated signaling pathways, e.g., erythropoietin (EPO); and / or (d) AGTR2 mediated signaling pathways, e.g., angiotensin. A suitable hematopoietic induction medium may lack differentiation factors that inhibit AGTR1 (angiotensin II type 1 receptor (AT1))-mediated signaling pathways, such as angiotensin II type 1 receptor (AT1) antagonists (ARBs), such as losartan (2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)-4-biphenylyl]methyl}-1H-imidazol-5-yl)methanol, and differentiation factors with fibroblast growth factor (FGF) activity, such as FGFs, such as bFGF (FGF2). For example, the hematopoietic induction medium may lack BMPs, FGF.SHH, EPO, angiotensin, and losartan.

[0090] In a preferred embodiment, the hematopoietic induction medium is a chemically defined medium.

[0091] In some embodiments, the hematopoietic induction medium comprises an effective amount of VEGF, e.g., 15 ng / mL, SCF, e.g., 100 ng / mL, thrombopoietin (TPO), e.g., 30 ng / mL, Flt3 ligand (FLT3L), e.g., 25 ng. / mL, IL-3, e.g., 25 ng / mL, IL-6, e.g., 10 ng / mL, IL-7, e.g., 5 ng / mL, IL-11, e.g., 25 ng / mL, IGF-1, BMP, e.g., 10 ng / mL, BMP4, FGF, e.g., 5 ng / mL, bFGF, e.g., 25 ng / mL, sonic hedgehog (SHH), e.g., 2 u / mL, erythropoietin (EPO), e.g., 10 μg / mL, angiotensin II, ... The hematopoietic induction medium may consist of a chemically defined nutrient medium supplemented with a type 1 receptor (AT1) antagonist, for example, 100 μM losartan. In another embodiment, the hematopoietic induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of, for example, 15 ng / mL VEGF and, for example, 100 ng / mL SCF. In another embodiment, the hematopoietic induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of, for example, 15 ng / mL VEGF, for example, 100 ng / mL SCF, for example, 30 ng / mL thrombopoietin (TPO), for example, 25 ng / mL Flt3 ligand (Flt3L), for example, 25 ng / mL IL-3, for example, 10 ng / mL IL-6, for example, 25 ng / mL IGF-1, and optionally, for example, 10 ng / mL IL-7. A suitable hematopoietic induction medium may be devoid of other differentiation factors. For example, the hematopoietic induction medium can consist of a chemically defined nutrient medium supplemented with one or more differentiation factors, where the one or more differentiation factors consist of SCF and / or VEGF (i.e., the medium does not contain differentiation factors other than SCF and VEGF).Other examples of hematopoietic induction media can consist of a chemically defined nutrient medium supplemented with one or more differentiation factors, where the one or more differentiation factors consist of VEGF, SCF, thrombopoietin (TPO), Flt3 Ligand (Flt3L), IL-3, IL-6, IGF-1, and IL-7 (i.e., the medium does not contain differentiation factors other than VEGF, SCF, thrombopoietin (TPO), Flt3 Ligand (Flt3L), IL-3, IL-6, and IGF-1; or VEGF, SCF, thrombopoietin (TPO), Flt3 Ligand (Flt3L), IL-3, IL-6, IGF-1, and IL-7).

[0092] Suitable chemically defined nutrient media are described above and include basal media such as StemPro™-34 (ThermoFisher Scientific), optionally supplemented with glutamine, ascorbic acid and monothioglycerol (MTG), or IMDM supplemented with albumin, insulin, selenium transferrin, and lipids, as described below.

[0093] HECs may be cultured in hematopoietic induction medium for 8 to 35 days, preferably 12 to 28 days or 16 to 28 days, such as any of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 days, for example, about 16 days, to produce a population of HPCs.

[0094] HPCs (also known as hematopoietic stem and progenitor cells or HSPCs) are multipotent stem cells committed to the hematopoietic lineage and capable of further hematopoietic differentiation into all blood cell types, including myeloid and lymphoid lineages, including monocytes, B cells, NK cells, NKT cells, TILs, and T cells. HPCs may express CD34.

[0095] In some embodiments, HPCs may include thymocyte HPCs (tHPCs). A population of HPCs may include HPCs, thymocyte HPCs, or both HPCs and thymocyte HPCs. Thymocyte HPCs are committed to the thymocyte lineage (i.e., early stage thymocytes) and are progenitor cells capable of further differentiation into T cells. tHPCs may express CD34 and CD7, i.e., tHPCs may exhibit a CD34+CD7+ phenotype.

[0096] HPCs may co-express CD117, CD133, CD45, and FLK1 (also known as KDR or VEGFR2), and may be negative for expression of CD38 and other lineage-specific markers. For example, HPCs may exhibit one or more, preferably all, of CD34+CD133+CD45+FLK1+CD38-. Thymocyte HPCs may exhibit one or more, preferably all, of CD34+CD7+CD133+CD45+FLK1+CD38-.

[0097] HECs and HPCs can be generated from iPSCs without intermediate purification or isolation of individual cells or subsets of cells from a population of differentiating cells. For example, cells of a particular class, type, lineage or phenotype may not be separated from other cells in a population of differentiating cells. Conversely, cells not belonging to a particular class, type, lineage or phenotype in a population of differentiating cells may not be separated from cells in the population that have a particular class, type, lineage or phenotype. A purification or isolation step may increase the proportion of cells in a population that exhibit a particular class, type, lineage or phenotype. Such steps are not necessary to produce hemogenic endothelial cells in the methods described herein. Cell purification or isolation techniques are well established in the art, including cell sorting or gating techniques such as magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS) and microfluidic cell sorting.

[0098] Differentiating the population of iPSCs into HECs as described herein can be achieved in a single culture vessel by using different culture media as described herein to drive differentiation.Target cells that can differentiate into HECs may not be separated from non-target cells in the population during differentiation.For example, target cells or non-target cells may not be isolated from the cell population to increase the concentration or proportion of target cells.

[0099] After generating HPCs from HECs, HPCs expressing one or more cell surface markers, such as CD34, can be purified or isolated from other cells in the population, for example, by magnetic activated cell sorting (MACS) or other cell purification or isolation techniques, before being subjected to further differentiation. For example, a population of CD34+ HPCs can be purified. CD34+ HPCs can be purified after 8 days of culture in HE induction medium, for example, 8-10 days. CD34+ HPCs can be purified after 11 days of differentiation, for example, days 11-17 of the differentiation method, i.e., days 11, 12, 13, 14, 15, 16, and 17.

[0100] Preferably, HPCs produced as described herein are used to generate a population of T cells. The population of T cells comprises: Differentiating the population of iPSCs into HECs as described above; Differentiating a population of HECs into HPCs as described above; and Differentiation of HPCs into T cell precursors It can be produced by a method comprising:

[0101] Hematopoietic progenitor cells (HPCs) can be differentiated into progenitor T cells by culturing a population of HPCs under suitable conditions that promote lymphoid differentiation. For example, hematopoietic progenitor cells can be cultured in lymphoid expansion medium.

[0102] Hematopoietic progenitor cells (HPCs) can differentiate into precursor T cells and DP (double positive) T cells in the absence of stromal cells such as OP9-D14 stromal cells, feeder cells or serum.

[0103] Lymphoid Expansion Medium is a cell culture medium that promotes lymphoid differentiation of HPCs into precursor T cells.

[0104] A suitable lymphoid expansion medium may (i) stimulate cKIT receptor (CD117; KIT receptor tyrosine kinase) and / or cKIT receptor (CD117; KIT receptor tyrosine kinase)-mediated signaling pathways, (ii) stimulate MPL (CD110) and / or MPL (CD110)-mediated signaling pathways, (iii) stimulate FLT3 and / or FLT3-mediated signaling pathways, and (iv) have interleukin (IL) activity. For example, the lymphoid expansion medium may contain the differentiation factors SCF, FLT3L, TPO, and IL7.

[0105] In a preferred embodiment, the lymphoid expansion medium is a chemically defined medium. For example, the lymphoid expansion medium may consist of a chemically defined nutrient medium supplemented with an effective amount of the above differentiation factors. Suitable lymphoid expansion media are well known in the art and include Stemspan™ SFEM II (catalog number 9605; StemCell Technologies Inc, CA) supplemented with Stemspan™ Lymphoid Expansion Supplement (catalog number 9915; StemCell Technologies Inc, CA).

[0106] HPCs can be cultured on a surface during differentiation into progenitor T cells. For example, HPCs can be cultured on the surface of a culture vessel, beads, or other biomaterial or polymer.

[0107] Preferably, the surface can be coated with an agent that stimulates Notch signaling, such as a Notch ligand, such as delta-like 1 (DLL1) or delta-like 4 (DLL4). Suitable Notch ligands are well known in the art and are available from commercial suppliers.

[0108] The surface may be coated with an extracellular matrix protein, such as fibronectin, vitronectin, laminin or collagen, and / or one or more cell surface adhesion proteins, such as VCAM1.

[0109] In some embodiments, a surface for culturing HPCs can have a coating that includes an agent that stimulates Notch signaling, e.g., a Notch ligand such as DLL4, an extracellular matrix protein such as vitronectin, and a cell surface adhesion protein such as VCAM 1. In some embodiments, a surface for culturing HPCs can have a coating that includes an agent that stimulates Notch signaling, e.g., a Notch ligand such as DLL4, but does not include an extracellular matrix protein or a cell surface adhesion protein.

[0110] A surface can be coated with extracellular matrix proteins, factors that stimulate Notch signaling, and / or cell surface adhesion proteins by contacting the surface with a coating solution. For example, the coating solution can be incubated on the surface under conditions suitable for coating the surface. The conditions can include, for example, about 2 hours at room temperature. Coating solutions containing extracellular matrix proteins and factors that stimulate Notch signaling are available from commercial suppliers (StemSpan™ Lymphoid Differentiation Coating Material; Catalog No. 9925; Stem Cell Technologies Inc, CA).

[0111] The HPCs can be cultured in Lymphoid Expansion Medium on the substrate for a time sufficient to differentiate the HPCs into progenitor T cells, for example, the HPCs can be cultured for 2-6 weeks, or 2-4 weeks, 2-5 weeks, preferably 3 weeks.

[0112] Precursor T cells are multipotent lymphopoietic progenitor cells that can give rise to αβ T cells, γδ T cells, tissue-resident T cells, and NK T cells. Precursor T cells can commit to the αβ T cell lineage after pre-TCR selection in the thymus. Precursor T cells may be able to colonize the thymus in vivo and may commit to the αβ T cell lineage after pre-TCR selection in the thymus. Precursor T cells are cytokine-producing CD3 + They may also be able to mature into T cells.

[0113] The precursor T cells may express CD5 and CD7, i.e., the precursor T cells may have a CD5+CD7+ phenotype. The precursor T cells may co-express CD44, CD25, and CD2. For example, the precursor T cells may have a CD5+, CD7+CD44+, CD25+CD2+ phenotype. The precursor T cells may co-express CD45. The precursor T cells may lack expression, e.g., cell surface expression, of CD3, CD4, and CD8.

[0114] In a fifth step, the precursor T cells may be matured into T cells by culturing the population of precursor T cells under suitable conditions that promote T cell maturation. For example, the precursor T cells may be cultured in a T cell maturation medium.

[0115] A T cell maturation medium is a cell culture medium that promotes the maturation of precursor T cells into mature T cells. A suitable T cell maturation medium may (i) stimulate cKIT receptor (CD117; KIT receptor tyrosine kinase) and / or stimulate cKIT receptor (CD117; KIT receptor tyrosine kinase)-mediated signaling pathway, (ii) stimulate FLT3 and / or FLT3-mediated signaling pathway, and (iii) have interleukin (IL) activity. For example, a T cell maturation medium may contain differentiation factors SCF, FLT3L, and IL7.

[0116] In a preferred embodiment, the T cell maturation medium is a chemically defined medium. For example, the T cell maturation medium may consist of a chemically defined nutrient medium supplemented with an effective amount of the above differentiation factors. Suitable T cell maturation media are well known in the art and include Stemspan™ SFEM II (Cat. No. 9605; StemCell Technologies Inc, CA) supplemented with Stemspan™ T cell maturation supplement (Cat. No. 9930; StemCell Technologies Inc, CA), as well as other media suitable for the expansion of PBMCs and CD3+ cells, such as ExCellerate Human T Cell Expansion Medium (R&D Systems, USA). Other suitable T cell maturation media may include basal media such as IMDM supplemented with ITS, albumin and lipids, as described elsewhere herein, and further supplemented with an effective amount of the above differentiation factors.

[0117] The precursor T cells can be cultured on a surface. For example, the precursor T cells can be cultured on the surface of a culture vessel, beads, or other biomaterial or polymer.

[0118] Preferably, the surface may be coated with an agent that stimulates Notch signaling, e.g., a Notch ligand, such as delta-like 1 (DLL1) or delta-like 4 (DLL4). Suitable Notch ligands are well known in the art and available from commercial suppliers. The surface may be coated with an extracellular matrix protein, such as fibronectin, vitronectin, laminin or collagen, and / or one or more cell surface adhesion proteins, such as VCAM1. Suitable coatings are well known in the art and described elsewhere herein.

[0119] The precursor T cells can be cultured in a T cell maturation medium on the substrate for a time sufficient to mature the precursor T cells into T cells. For example, the precursor T cells can be cultured for 1 to 4 weeks, preferably 1 to 2 weeks.

[0120] T cells (also known as T lymphocytes) are white blood cells that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes by the presence of the T cell receptor (TCR) on the cell surface.

[0121] There are several types of T cells, each with a different function.

[0122] T helper cells (TH cells) are known as CD4+ T cells because they express the CD4 surface glycoprotein. CD4+ T cells play a key role in the adaptive immune system, aiding the activity of other immune cells by releasing T cell cytokines that help suppress or modulate the immune response. They are essential for the activation and growth of cytotoxic T cells.

[0123] Cytotoxic T cells (TC cells, CTL, killer T cells, cytolytic T cells) are known as CD8+ T cells because they express the CD8 surface glycoprotein. CD8+ T cells act to destroy virus-infected and tumor cells. Most CD8+ T cells express TCRs that can recognize specific antigens presented on the surface of infected or damaged cells by class I MHC molecules, or recognize specific antigens or peptides thereof independent of MHC presentation, and such T cells can be produced according to the methods of the present invention. Specific binding of the TCR and CD8 glycoprotein to the antigen and MHC molecule results in the destruction of the infected or damaged cells via the T cell.

[0124] The T cells produced as described herein can be mature CD3+ T cells. In some embodiments, the T cells can also express CD45 and CD28.

[0125] The T cells produced as described herein can be γδ T cells, αβ T cells, or NKT cells.

[0126] In some preferred embodiments, the T cells produced as described herein are αβ T cells. After precursor T cell maturation (stage 5), the population of T cells may be predominantly double positive (DP) CD4+CD8+ T cells.

[0127] In the sixth step, the population of T cells can be activated and / or expanded to produce or increase the proportion of single positive CD4+ T cells, or more preferably single positive CD8+ T cells. Suitable methods for activating and expanding T cells are well known in the art. For example, T cells can be exposed to a T cell receptor (TCR) agonist under suitable culture conditions. Suitable TCR agonists include ligands such as peptides presented on class I or II MHC molecules (MHC-peptide complexes) on the surface of antigen presenting cells such as beads or dendritic cells, and soluble factors such as anti-TCR antibodies, e.g., antibodies CD28 antibodies, and multimeric MHC-peptide complexes such as MHC-peptide tetramers, pentamers or dextramers.

[0128] Activation refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0129] The anti-TCR antibody may specifically bind to a component of the TCR, such as εCD3, αCD3 or αCD28. Anti-TCR antibodies suitable for TCR stimulation are well known in the art (e.g., OKT3) and available from commercial suppliers (e.g., eBioscience CO USA). In some embodiments, T cells may be activated by exposure to anti-αCD3 antibody and IL2, IL-7, or IL15. More preferably, T cells are activated by exposure to anti-αCD3 antibody and anti-αCD28 antibody. Activation may be achieved by activating CD14 +This can occur in the presence or absence of monocytes. T cells can be activated by beads coated with anti-CD3 and anti-CD28 antibodies. For example, PBMCs or CD4 + and / or CD8 + T cell subsets including cells can be activated without feeder cells (antigen presenting cells) or antigen using antibody-coated beads, e.g., magnetic beads coated with anti-CD3 and anti-CD28 antibodies, such as Dynabeads® Human T-Activator CD3 / CD28 (ThermoFisher Scientific). In other embodiments, T cells can be activated using soluble tetrameric antibody complexes that bind to CD3, CD28, and CD2 cell surface ligands, e.g., ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator or Human CD3 / CD28 T Cell Activator. In other embodiments, T cells can be activated with MHC-peptide complexes, preferably multimeric MHC-peptide complexes, optionally in combination with anti-CD28 antibodies.

[0130] T cells expressing chimeric antigen receptors can be activated using soluble antigens against the receptor. The antigens can be in multimeric form or on the surface of beads, and can optionally be used in conjunction with anti-TCR antibodies, such as anti-CD28 antibodies.

[0131] In some embodiments, double positive CD4+CD8+ T cells may be cultured in a T cell maturation medium as described herein supplemented with IL-15. The medium may be further supplemented with a T cell receptor (TCR) agonist, such as one or more anti-TCR antibodies, such as an anti-αCD3 antibody and an anti-αCD28 antibody, as described above.

[0132] The double positive CD4+CD8+ T cells can be cultured using any convenient technique to produce an expanded population. Suitable culture systems include stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, particularly hollow fiber bioreactors. The use of such systems is well known in the art.

[0133] The T cells produced as described herein can express antigen receptors that bind to target antigens.For example, the antigen receptors can specifically bind to cancer cells that express tumor antigens.The T cells can be useful, for example, in immunotherapy, as described below.

[0134] The antigen receptor can be a T cell receptor (TCR). TCRs are disulfide-linked, membrane-anchored heterodimeric proteins that typically contain highly variable alpha (α) and beta (β) chains expressed in complex with the invariant CD3 chain molecule. T cells that express these types of TCRs are called α:β (or αβ) T cells. A small number of T cells express an alternative TCR that contains variable gamma (γ) and delta (δ) chains and are called γδ T cells.

[0135] A suitable TCR specifically binds to major histocompatibility complexes (MHC) on the surface of cancer cells that present peptide fragments of tumor antigens. MHC is a set of cell surface proteins that allow the adaptive immune system to recognize "foreign" molecules. Proteins are degraded inside the cell and presented on the surface of the cell by MHC. MHC presenting "foreign" peptides, such as viruses or cancer-associated peptides, are recognized by T cells with the appropriate TCR and promote cell destruction pathways. MHC on the surface of cancer cells can present peptide fragments of tumor antigens, i.e., antigens that are present on cancer cells but not on the corresponding non-cancerous cells. T cells that recognize these peptide fragments can exert a cytotoxic effect on cancer cells.

[0136] In some embodiments, the TCR expressed by the T cell may be naturally expressed (i.e., endogenous TCR). For example, the T cell may be produced as described herein from iPSCs derived from tumor infiltrating lymphocytes (TILs). TILs, e.g., tumor-resident CD3+CD8+ cells, may be obtained from an individual with a cancer pathology using standard techniques. Alternatively, the T cell may be produced as described herein from iPSCs derived from T cells that bind to peptide fragments of a target antigen presented on class I or II MHC molecules on the surface of an antigen-presenting cell, such as a dendritic cell, or a population of T cells produced as described herein may be screened for binding to peptide fragments of a target antigen presented on class I or II MHC molecules to identify T cells that bind to the presented peptide fragments.

[0137] In other embodiments, the TCR is not naturally expressed by the cell (i.e., the TCR is exogenous or xenogeneic). A suitable xenogeneic TCR may specifically bind to a class I or II MHC molecule that presents a peptide fragment of the target antigen. For example, T cells may be engineered to express a xenogeneic αβ TCR that specifically binds to a class I or II MHC molecule that presents a peptide fragment of a tumor antigen expressed by a cancer cell of a cancer patient. Tumor antigens expressed by a cancer cell of a cancer patient may be identified using standard techniques. Preferred tumor antigens include NY-ESO1, PRAME, α-fetoprotein (AFP), MAGE A4, MAGE A1, MAGE A10 and MAGE B2, most preferably NY-ESO-1, MAGE-A4 and MAGE-A10.

[0138] Suitable TCRs may include non-conventional TCRs, e.g., non-MHC-dependent TCRs that recognize and bind non-peptide antigens presented by monomorphic antigen-presenting molecules such as CD1 and MR1, NKT cell TCRs, and intraepithelial lymphocyte (IEL) TCRs. In some embodiments, the TCR may recognize a target antigen or a peptide fragment of a target antigen on a cancer cell, independent of MHC presentation.

[0139] A heterologous TCR can be a synthetic or artificial TCR, i.e., a TCR that does not occur in nature. For example, a heterologous TCR can be engineered to increase its affinity or avidity for a tumor antigen (i.e., an affinity-improved TCR). An affinity-improved TCR can contain one or more mutations compared to a naturally occurring TCR, for example, one or more mutations in the hypervariable complementarity determining regions (CDRs) of the variable regions of the TCR α and β chains. These mutations increase the affinity of the TCR for MHC presenting peptide fragments of tumor antigens expressed by cancer cells. Suitable methods for generating affinity-improved TCRs include screening libraries of TCR mutants using phage or yeast display and are well known in the art (see, for example, Robbins et al J Immunol (2008) 180(9):6116; San Miguel et al (2015) Cancer Cell 28(3) 281-283; Schmitt et al (2013) Blood 122 348-256; Jiang et al (2015) Cancer Discovery 5 901). Preferred affinity-improved TCRs may bind to cancer cells expressing one or more of the tumor antigens NY-ESO1, PRAME, alpha-fetoprotein (AFP), MAGE A4, MAGE A1, MAGE A10 and MAGE B2.

[0140] Alternatively, the antigen receptor may be a chimeric antigen receptor (CAR). A CAR is an artificial receptor engineered to contain an immunoglobulin antigen-binding domain, such as a single chain variable fragment (scFv). A CAR may, for example, contain an scFv fused to a TCR CD3 transmembrane domain and endodomain. An scFv is a set of two distinct domains, each of which consists of a variable region (V) of an immunoglobulin heavy chain, which may be connected by a short linker peptide of approximately 10-25 amino acids. H ) and the light chain variable region (V L) fusion protein (Huston J Set al. Proc Natl Acad. Sci USA 1988;85(16):5879-5883). The linker can be glycine-rich for flexibility, serine- or threonine-rich for solubility, and V H N-terminus of V L The scFv may be connected to the C-terminus of the TCR or vice versa. The scFv may be preceded by a signal peptide to direct the protein to the endoplasmic reticulum and then to the T cell surface. In the CAR, the scFv may be fused to the TCR transmembrane and endodomains. A flexible spacer may be included between the scFv and the TCR transmembrane domain to allow variable orientation and antigen binding. The endodomain is the functional signaling domain of the receptor. The endodomain of the CAR may include, for example, the CD3 zeta chain, or the intracellular signaling domain from a receptor such as CD28, 41BB, or ICOS. The CAR may include multiple signaling domains, for example, but not limited to, CD3z-CD28-41BB or CD3z-CD28-OX40.

[0141] CAR can specifically bind to tumor-specific antigens expressed by cancer cells.For example, T cells can be modified to express CARs that specifically bind to the tumor antigens expressed by cancer cells of a particular cancer patient.The tumor antigens expressed by cancer cells of a cancer patient can be identified using standard techniques.

[0142] Alternatively, the antigen receptor may be a NK cell receptor (NKCR).

[0143] Expression of a heterologous antigen receptor, such as a heterologous TCR, NKCR or CAR, can alter the immunogenic specificity of T cells produced as described herein, such that they recognize or exhibit improved recognition of one or more target antigens, such as tumor antigens present on the surface of cancer cells in individuals with cancer. In some embodiments, T cells produced as described herein may exhibit reduced or no binding to cancer cells in the absence of a heterologous antigen receptor. For example, expression of a heterologous antigen receptor can increase the affinity and / or specificity of T cells binding to cancer cells compared to T cells that do not express the antigen receptor.

[0144] The term "heterologous" refers to a polypeptide or nucleic acid that is foreign to a particular biological system, such as a host cell, and does not naturally occur in that system. A heterologous polypeptide or nucleic acid may be introduced into a biological system using artificial means, such as recombinant techniques. For example, a heterologous nucleic acid encoding a polypeptide may be inserted into a suitable expression construct, which can then be used to transform a host cell to produce the polypeptide. A heterologous polypeptide or nucleic acid may be synthetic or artificial, or may be present in a different biological system, such as a different species or cell type. An endogenous polypeptide or nucleic acid is native to a particular biological system, such as a host cell, and naturally occurs in that system. A recombinant polypeptide is expressed from a heterologous nucleic acid that has been introduced into a cell by artificial means, such as using recombinant techniques. A recombinant polypeptide may be identical to a polypeptide that naturally occurs in the cell, or it may be different from a polypeptide that naturally occurs in the cell.

[0145] T cells can be modified to express a heterologous antigen receptor, such as a TCR or a CAR, by introducing a heterologous encoding nucleic acid into the cells at any stage in the methods described herein. For example, the heterologous encoding nucleic acid can be introduced into iPSCs, HPCs, mesodermal cells, HECs, or progenitor T cells. In some preferred embodiments, the cells can be transduced with a heterologous nucleic acid encoding an antigen receptor in lymphoid expansion medium, for example after two weeks of culture in lymphoid expansion medium as described herein (stage 4). The heterologous nucleic acid encoding an antigen receptor can encode all subunits of the receptor. For example, the nucleic acid encoding a TCR can include a nucleotide sequence encoding a TCR alpha chain and a nucleotide sequence encoding a TCR beta chain, or a nucleotide sequence encoding a TCR delta chain and a nucleotide sequence encoding a TCR gamma chain.

[0146] Nucleic acid can be introduced into cells by any convenient technique. When introducing or incorporating heterologous nucleic acid into iPSC, HPC or precursor T cell, certain considerations must be taken into account, which are well known to those skilled in the art. The nucleic acid to be inserted should be assembled in a construct or vector that contains effective regulatory elements that drive transcription in T cell. Many known techniques and protocols for manipulation and transformation of nucleic acid, for example, preparation of nucleic acid constructs, introduction of DNA into cells, and gene expression, are described in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992. In some embodiments, nucleic acid can be introduced into cells by gene editing. For example, the CRISPR / Cas9 system can induce a DNA double-stranded break (DSB) at a target site, and repair of the DSB may introduce a heterologous nucleic acid into the cellular genome at the target site, or a rAAV vector may be used to introduce the nucleic acid (AAV-mediated gene editing; Hirsch et al 2014 Methods Mol Biol 1114 291-307).

[0147] Suitable techniques for introducing an expression vector into iPSCs, HPCs or precursor T cells are well known in the art and include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, gene editing, and transduction using retroviruses such as vaccinia or lentivirus or other viruses. Preferably, the nucleic acid encoding the heterologous antigen receptor can be contained in a viral vector, most preferably a gamma retroviral vector or a lentiviral vector, such as a VSVg pseudotyped lentiviral vector. The methods described herein can include transducing a population of cells, such as iPSCs, HPCs or precursor T cells, with a viral vector to produce a transduced population of genetically modified cells. The cells can be transduced by contact with viral particles containing the nucleic acid. Viral particles for transduction can be produced according to known methods. For example, HEK293T cells can be transfected with a lentiviral vector containing a plasmid encoding viral packaging and envelope elements, as well as the coding nucleic acid. VSVg pseudotyped viral vectors may be produced in combination with the viral envelope glycoprotein G of vesicular stomatitis virus (VSVg) to produce pseudotyped viral particles. For example, solid-phase transduction can be performed without selection by culturing on tissue culture plates coated with retronectin and preloaded with the retroviral vector.

[0148] Populations of T cells, e.g., DP CD4+CD8+ cells, SP CD4+ cells or SP CD8+ cells, can be isolated and / or purified after production. Any convenient technique can be used, including fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) using antibody-coated magnetic particles.

[0149] A population of T cells, e.g., DP CD4+CD8+ cells, SP CD4+ cells or SP CD8+ cells, may be expanded and / or enriched. Optionally, a population of T cells produced as described herein may be stored, e.g., by cryopreservation, prior to use.

[0150] The population of T cells may be mixed with other reagents, such as buffers, carriers, diluents, preservatives and / or pharma- ceutically acceptable excipients. Suitable reagents are described in more detail below. The methods described herein may include mixing the population of T cells with a pharma- ceutically acceptable excipient.

[0151] Pharmaceutical compositions suitable for administration (e.g., by injection) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions that may contain antioxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. Examples of suitable isotonic vehicles used in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Suitable vehicles can be found in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0152] In some preferred embodiments, the T cells, which may be DP CD4+CD8+ T cells, SP CD4+ T cells, or preferably SP CD8+ T cells, may be formulated into a pharmaceutical composition suitable for intravenous infusion into an individual.

[0153] The term "pharmacologically acceptable" as used herein refers to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0154] Other aspects of the invention provide populations of HECs produced by the above-mentioned methods, and populations of HPCs produced by the above-mentioned methods.

[0155] Another aspect of the invention provides a population of T cells produced by the above-described methods, which can be, for example, DP CD4+CD8+ T cells, SP CD4+ T cells, or SP CD8+ T cells.

[0156] The population of T cells can be used as a medicine.For example, the population of mature T cells described herein can be used for cancer immunotherapy, such as adoptive T cell therapy.

[0157] Adoptive cell therapy or adoptive immunotherapy refers to the adoptive transfer of human T lymphocytes that express an antigen receptor specific for a target cell. For example, the human T lymphocytes may express a TCR specific for an antigen expressed on the target cell and / or a TCR specific for a peptide-MHC complex expressed on the target cell, or a chimeric antigen receptor (CAR) specific for an antigen expressed on the target cell.

[0158] This can be used to treat various diseases depending on the target selected, such as tumor-specific antigens to treat cancer. Adoptive cell therapy involves removing a portion of the donor's or patient's cells, e.g., white blood cells. These cells are then used to generate iPSCs in vitro, which are used to efficiently generate T cells specific for antigens expressed on target cells and / or T cells specific for peptide-MHC complexes on target cells, as described herein. The T cells may be expanded, washed, concentrated, and / or subsequently frozen to allow time for testing, transportation, and storage until the patient is ready to receive an infusion of the cells.

[0159] Other aspects of the invention provide use of a population of T cells described herein for the manufacture of a medicament for treating cancer, a population of T cells described herein for treating cancer, and a method of treating cancer comprising administering a population of T cells described herein to an individual in need thereof.

[0160] The population of T cells can be autologous, i.e., the T cells were originally obtained from the same individual to which they are subsequently administered (i.e., the donor and recipient individuals are the same).

[0161] The population of T cells can be allogeneic, i.e., the T cells can be originally obtained from an individual different from the individual to whom they are subsequently administered (i.e., the donor and recipient individuals are different). Allogeneic refers to a graft derived from a different animal belonging to the same species.

[0162] To avoid GVHD and other undesirable immune effects such as rejection, the HLA of the donor and recipient individuals may be matched. Alternatively, the HLA of the donor and recipient individuals may be mismatched, or the HLA genes in the cells of the donor individual may be modified, for example by gene editing, to eliminate HLA mismatches with the recipient.

[0163] A T cell population suitable for administration to a recipient individual may be produced by a method comprising providing an initial population of cells obtained from a donor individual, reprogramming the cells into iPSCs, and differentiating the iPSCs in the recipient individual into T cells that express an antigen receptor, such as αβTCR, that specifically binds to cancer cells or an antigen or peptide of an antigen on a cancer cell, optionally presented in a complex with MHC.

[0164] Following administration of the T cells, the recipient individual may mount a T cell-mediated immune response against the recipient individual's cancer cells, which may have a beneficial effect on the individual's cancer condition.

[0165] As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and refer, in either singular or plural, to cells that have undergone a malignant transformation that becomes pathological to the host organism.

[0166] Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, especially histological examination. The definition of cancer cells used herein includes not only primary cancer cells, but also any cells derived from a cancer cell ancestor. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that usually appear as solid tumors, a "clinically detectable" tumor is one that can be detected based on tumor mass, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during physical examination, and / or one that can be detected due to the expression of one or more cancer-specific antigens in a sample that can be obtained from a patient.

[0167] Cancer conditions can be characterized by the abnormal proliferation of malignant cancer cells, and can include leukemias, such as AML, CML, ALL, and CLL; lymphomas, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma; and solid cancers, such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gallbladder and bile duct, thyroid cancer, thymus cancer, bone cancer, and cerebral cancer, as well as cancer of unknown primary site (CUP).

[0168] Cancer cells in an individual may be immunologically distinct from normal somatic cells of the individual (i.e., cancerous tumors may be immunogenic). For example, cancer cells may be capable of inducing a systemic immune response in an individual against one or more antigens expressed by the cancer cells. The tumor antigens that induce the immune response may be specific to the cancer cells or may be shared by one or more normal cells in the individual.

[0169] Cancer cells of an individual suitable for the treatments described herein may express the antigen and / or may be of the correct HLA type that binds to the antigen receptor expressed by the T cell.

[0170] The individual suitable for the above-mentioned treatment can be a mammal.In a preferred embodiment, the individual is a human.In another preferred embodiment, non-human mammals can be used, particularly mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., mice, primates, pigs, dogs, or rabbits).

[0171] In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment.

[0172] An individual with cancer may show at least one distinguishable sign, symptom, or examination finding that is sufficient to diagnose cancer according to clinical criteria known in the art.Examples of such clinical criteria can be found in medical textbooks such as Harrison's Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001.In some cases, diagnosing cancer in an individual may include identifying a specific cell type (e.g., cancer cell) in a sample of body fluid or tissue obtained from the individual.

[0173] Antitumor effect is a biological effect that can be manifested by a decrease in tumor growth rate, a decrease in tumor volume, a decrease in tumor cell number, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancerous pathology. "Antitumor effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies, as well as T cells, that can be obtained according to the method of the present invention, as described herein, in preventing the development of tumors in the first place.

[0174] Treatment can be any treatment and / or therapy, whether in humans or animals (e.g., veterinary uses), that achieves some desired therapeutic effect, e.g., inhibiting or slowing the progression of a condition, including slowing the rate of progression, halting the rate of progression, ameliorating the condition, curing or remission (whether partial or complete) of the condition, preventing, slowing, alleviating or arresting one or more symptoms and / or signs of the condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0175] Treatment may be prophylactic (i.e., preventive). For example, individuals susceptible to cancer or at risk of its development or recurrence may be treated as described herein. Such treatment may prevent or delay the development or recurrence of cancer in individuals.

[0176] In particular, the treatment may include inhibition of cancer growth, including complete remission of cancer, and / or inhibition of cancer metastasis. Cancer growth generally refers to any one of several indicators that indicate a change in cancer to a more developed form. Thus, indicators for measuring inhibition of cancer growth include a reduction in cancer cell viability, a reduction in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound, or other imaging methods), slowing of tumor growth, destruction of tumor vasculature, improved performance in delayed-type skin hypersensitivity tests, increased activity of T cells, and reduced levels of tumor-specific antigens. Administration of modified T cells as described herein may improve an individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the tendency of cancer growth in the individual.

[0177] T cells or pharmaceutical compositions comprising T cells can be administered to a subject by any convenient route of administration, including but not limited to parenterally, for example, by injection, whether systemically / peripherally or at a desired site of action. Injection includes administration of T cells in a suitable composition via a needle or catheter. Typically, T cells are injected intravenously or subcutaneously, although T cells may also be injected via other parenteral routes, such as intramuscular injection and epidural routes. Suitable injection techniques are known in the art and are commonly used in therapy (see, for example, Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).

[0178] Typically, the number of cells administered is about 10 per kg of body weight. 5 pieces ~ about 10 10 For example, about 1, 2, 3, 4, 5, 6, 7, 8, or 9 x 10 cells per individual 5 , ×10 6 , ×10 7 , ×10 8 , ×10 9 , or ×10 10 Typically 2 x 10 cells per individual 8 ~2×10 10The dose is typically 30 minutes, with the treatment repeated as needed, for example at intervals of several days to several weeks. It will be understood that appropriate dosages of T cells and compositions comprising T cells may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against the risk or adverse side effects of the treatment of the present invention. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular cells, cytokine release syndrome (CRS), route of administration, time of administration, rate of cell loss or inactivation, duration of treatment, other drugs, compounds, and / or substances used in combination, as well as the patient's age, sex, weight, disease state, overall health, and medical history. The dose and route of administration of the cells are ultimately at the discretion of the physician, but generally the dosage will achieve a local concentration at the site of action that achieves the desired effect without causing substantially harmful or deleterious side effects.

[0179] T cells may be administered alone or, in some circumstances, in combination with a target antigen, APCs presenting the target antigen, CD3 / CD28 beads, IL-2, IL-7, and / or IL15 to promote in vivo expansion of the T cell population. Administration of the combination may be by separate, simultaneous, or sequential administration of the combined components.

[0180] The population of T cells may be administered in combination with one or more other therapies, for example, immuno-oncology agents including cytokines such as IL-2, cytotoxic chemotherapy, radiation, and checkpoint inhibitors such as anti-B7-H3, anti-B7-H4, anti-TIM3, anti-KIR, anti-LAG3, anti-PD-1, anti-PD-L1, and anti-CTLA4 antibodies. Administration of the combination may be by separate, simultaneous, or sequential administration of the combined components.

[0181] The one or more other therapies may be administered by any convenient means, preferably at a site remote from the site of administration of the T cells.

[0182] The T cells may be administered in one dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods for determining the most effective means and dosages of administration are well known to those skilled in the art and will vary depending on the formulation used in therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed, with the dose level and pattern being selected by the attending physician. Preferably, the T cells are administered in a dose of any of 500, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 billion T cells, for example at least 1×10 9 It is administered as a single infusion of T cells.

[0183] Other aspects of the invention provide kits and reagents for use in generating populations of HEC using the methods described above.

[0184] A kit for producing hemogenic endothelial cells may include; A first mesoderm induction medium containing BMPs, A second mesoderm induction medium comprising BMP and FGF; a third mesoderm induction medium containing activin, BMP, FGF, and a GSK3 inhibitor; A first HE induction medium containing VEGF and FGF, and A second HE induction medium containing SCF, VEGF and FGF.

[0185] The kit may further comprise one of HPC induction media as described herein, for example, (i) a hematopoietic induction medium comprising VEGF and SCF, (ii) a hematopoietic induction medium comprising VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IGF-1, IL-3, IL-6, and optionally IL-7, or (iii) a hematopoietic induction medium comprising VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMPs, FGFs, sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II, and angiotensin II type 1 receptor (AT1) antagonist.

[0186] Another aspect of the present invention also provides the use of a set of culture media or kit as described above for the production of hemogenic endothelial cells, the set of media comprising: A first mesoderm induction medium containing BMPs, A second mesoderm induction medium comprising BMP and FGF; a third mesoderm induction medium containing activin, BMP, FGF, and a GSK3 inhibitor; A first HE induction medium containing VEGF and FGF, A second HE induction medium containing SCF, VEGF and FGF, and optionally HPC induction media as described herein, for example, one of: (i) a hematopoietic induction medium comprising VEGF and SCF; (ii) a hematopoietic induction medium comprising VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IGF-1, IL-3, IL-6, and optionally IL-7; or (iii) a hematopoietic induction medium comprising VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMPs, FGFs, sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II, and angiotensin II type 1 receptor (AT1) antagonist.

[0187] Suitable media are described in more detail above.

[0188] As described elsewhere herein, the medium may be supplemented with effective amounts of the differentiation factors described above.

[0189] The one or more culture media may be formulated in deionized distilled water. The one or more media are typically sterilized prior to use, for example, by ultraviolet light, heating, irradiation or filtration to prevent contamination. The one or more media may be frozen (e.g., at -20°C or -80°C) for storage or transport.

[0190] The one or more media may be 1x formulations, or more concentrated formulations, such as 2x to 250x concentrated media formulations. In 1x formulations, each component in the media is at a concentration intended for cell culture, such as the concentrations described above. In concentrated formulations, one or more of the components are present at a higher concentration than intended for cell culture. Concentrated culture media are well known in the art. Culture media can be concentrated using known methods such as salt precipitation or selective filtration. Concentrated media can be diluted for use with water (preferably deionized and distilled) or any suitable solution, such as saline solution, aqueous buffer or culture medium.

[0191] One or more media in the kit may be contained in a hermetically sealed container.To prevent contamination, a hermetically sealed container may be preferred for transporting or storing the culture medium.The container may be any suitable container, such as a flask, a plate, a bottle, a jar, a vial, or a bag.

[0192] Other aspects and embodiments of the present invention provide for replacing the term "comprising" in the above-mentioned aspects and embodiments with the term "consisting of," as well as for replacing the term "comprising" in the above-mentioned aspects and embodiments with the term "consisting essentially of."

[0193] It is to be understood that the present application discloses any and all combinations of the above aspects and any of the above-described embodiments with each other, unless the context requires otherwise.Similarly, the present application discloses any and all combinations of preferred and / or optional features, either alone or in combination with any of the other aspects, unless the context requires otherwise.

[0194] Modifications of the above embodiments, further embodiments, and modifications thereof will be apparent to those of skill in the art upon reading this disclosure, and therefore are within the scope of the invention.

[0195] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In the practice or testing of the disclosed method, any compositions and methods similar or equivalent to those described herein may be used, but exemplary compositions and methods are described herein. Any of the disclosed aspects and embodiments described herein may be combined. For example, the subject matter of any dependent or independent claims disclosed herein may be combined in various ways (e.g., one or more recitations from each dependent claim may be combined into a single claim based on the independent claim on which they depend).

[0196] The ranges provided herein include all values ​​within the specified ranges described, as well as values ​​near the endpoints of the specified ranges. The figures and tables of this disclosure also represent ranges and individual values ​​that may constitute elements of any method disclosed herein. The concentrations described herein are determined at ambient temperature and pressure. This may be, for example, at room temperature or at the temperature and pressure in a particular part of the process stream. Preferably, the concentrations are determined at standard conditions of 20°C and 1 bar pressure. The term "about" refers to a value within 2 standard deviations of the mean of the particular measurement.

[0197] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide chain" is a reference to one or more peptide chains and includes equivalents thereof known to those skilled in the art.

[0198] All documents and sequence database entries referred to herein are incorporated by reference in their entirety for all purposes.

[0199] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, without regard to the presence or absence of the other. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. EXAMPLES

[0200] method hiPSC culture iPSCs were cultured in mTeSRplus medium (SCT) on Synthemax II-SC substrates (Corning) at 37°C in an incubator with a 5% CO2, 5% O2 atmosphere using tissue culture plasticware. iPSCs were routinely passaged in different ways, including single cell suspension with the enzyme solutions Accutase™, StemPro™, and / or generation of cell clusters, which can be generated by EDTA solution (Versene™ Gibco™) or manually using the EasyPassage tool (Invitrogen). Depending on the passaging method, iPSCs were cultured in medium containing 10uM Y27632 (R&D Systems) for 48 hours of culture or 1XClone for the first 24 hours of culture. RCells were seeded at a certain cell density on a Synthemax II-SC substrate (STC). For differentiation, hiPSCs were passaged at a low cell density on Synthemax II-SC substrate (Corning) and allowed to form one iPSC colony per field under a microscope at 5x magnification for approximately 5 days.

[0201] Differentiation of T cells from pluripotent stem cells For first and second stage differentiation, alternative and control protocols were used.

[0202] In the alternative protocol, on day 0, hiPSC maintenance medium (mTeSRplus) was removed and cells were washed twice with DMEM / F12. In wells of a 6-well plate, 2 mL of StemPro34 PLUS (StemPro34 basal medium with StemPro34 supplement, plus glutamine (2 mM; Invitrogen), ascorbic acid (50 μg / mL; Sigma Aldrich), and monothioglycerol (100 μM; Sigma Aldrich)) was further supplemented with 10 ng / mL BMP4 and incubated for 1 day. The volume depends on the surface area of ​​the culture flask, typically 1 cm. 2Add 0.2 mL of medium per day. On day 1, 1 mL of StemPro34 PLUS supplemented with mLBMP4 and mLbFGF to a final concentration of 10 ng / mL and 5 ng / mL, respectively, was added to the medium and incubated for 1 day (stage 1 medium). On day 2, the medium was replaced with 2 mL of StemPro34 PLUS supplemented with 10 ng / mL BMP4, 5 ng / mL bFGF, 1 ng / mL activin A, and 3 μM CHIR-99021 and cultured for an additional 48 hours. On day 4, the medium was removed and the cells were washed twice with DMEM / F12 to remove residual stage 1 cytokines. The medium was replaced with StemPro34 PLUS supplemented with 5 ng / mL bFGF and 15 ng / mL VEGF and incubated for 48 hours (stage 2 medium). Then, on day 6, the medium was replaced with StemPro34 PLUS supplemented with 100 ng / mL SCF, 5 ng / mL bFGF, and 15 ng / mL VEGF, and incubated for an additional 48 hours (stage 2 medium).

[0203] In the control protocol, on day 0, hiPSC maintenance medium was removed and cells were washed twice with DMEM / F12. 2 mL of StemPro34 PLUS (StemPro34 from Invitrogen; StemPro34 basal medium with supplements, plus penicillin streptomycin (1% v / v: Invitrogen) and glutamine (2 mM: Invitrogen), ascorbic acid (50 μg / mL: Sigma Aldrich) and monothioglycerol (100 μM: Sigma Aldrich), further supplemented with 50 ng / mL activin) was added and incubated for 4 hours. The volume depends on the size of the culture flask, typically at least 2 mL / 9 cm. 2 , and 20mL / 150cm 2After 4 hours, the medium was removed and the cells were washed twice with DMEM / F12 to remove residual high concentrations of Activin A. The medium was replaced with 2 mL of StemPro34 PLUS supplemented with 5 ng / mL Activin A, 10 ng / mL BMP4, and 5 ng / mL bFGF and incubated for 44 hours (Stage 1 medium). The medium was then replaced with fresh Stage 1 medium, supplemented with 10 μM CHIR-99021, and cultured for an additional 48 hours. On day 4, the medium was removed and the cells were washed twice with DMEM / F12 to remove residual Stage 1 cytokines. The medium was then replaced with StemPro34 PLUS supplemented with 100 ng / mL SCF and 15 ng / mL VEGF and incubated for 48 hours (Stage 2 medium). The medium was then replenished with fresh Stage 2 medium and the cells were cultured for an additional 48 hours.

[0204] On day 8, the medium for both the alternative and control protocols was replaced with HEM7.2 stage 3 medium as shown in Table 3 and the cells were cultured until days 11–14 (alternative protocol) or until days 16–18 (control protocol), at which point the vessels were filled with 2x medium on day 10, half-fed (1:1 (v / v)) with medium every 48 h from day 12 onwards, and the cells in suspension were collected. Typically, this involved harvesting the medium, collecting the cells in suspension by centrifugation (10 min at 300 g) and resuspending the suspended cells in fresh medium (i.e. 0.2 ml / cm ). 2 ) culture.

[0205] Approximately from day 11 to day 17, depending on the hiPSC line and the approach used, CD34+ or CD34+CD7+ cells (confirmed separately by flow cytometry before the day of harvest) were isolated from the resulting monolayer for subsequent culture. CD34+ cells were harvested by sequential incubation with Collagenase II (Invitrogen: 2 mg / mL, 30 min at 37°C) followed by Accutase (SCT: 30 min at 37°C). The cell suspension was collected and washed (2x centrifugation at 300 g for 12 min in DMEM / F12) followed by isolation of CD34+ cells by magnetic activated bead (MACS) isolation (Miltenyi: according to the manufacturer's instructions).

[0206] After MACS isolation of CD34+ cells, these were cultured at 1 × 10 6 Cells / vial were first frozen at -80°C under controlled rate freezing and then in liquid nitrogen for long term storage.

[0207] For sustained lymphoid expansion and differentiation, Stem Cell Technologies' proprietary two-stage (lymphoid expansion / T cell maturation) medium was used (according to the manufacturer's instructions).

[0208] result iPSCs passaged as clusters or single cells (manually or Accutase-passaged) showed increased yields of CD34+ cells (HECs) and CD34+CD45+ cells (HPCs) in the alternative protocol compared to the control protocol (Figures 3 and 4). The alternative protocol was also found to generate increased yields of precursor T cells at the end of stage 4 (HPC differentiation to pro-T cells) compared to the control protocol (Figure 5). By day 6, the phenotype of the CD34+ cells (HECs) in the alternative protocol was observed to be improved compared to the CD34+ cells in the control protocol, with an increased number of cells expressing CD43+, indicative of hematopoietic lineage, and a decreased number of cells expressing CD73, indicative of endothelial lineage (Figure 6).

[0209] When iPSCs were passaged as clusters using Versene™ solution, the alternative protocol was found to generate 3-fold more CD34+ cells (HECs) and 14-fold more CD34+CD45+ cells (HPCs) than the control protocol (Figure 7A). Proliferation of progenitor T cells was also found to be increased (Figure 7B).

[0210] Differentiation of eF1a-TCR+ edited iPSC cell lines was also tested using the alternative protocol. eF1a-TCR+ edited iPSC cell lines were observed to generate active human hemogenic endothelium using the alternative protocol (Figure 8). The number of round floating cells (HSPCs) emerging from HE was increased with the alternative protocol (Figure 9) compared to the control protocol. CD34+ progenitor cells generated by the alternative protocol were found to exhibit improved viability compared to the control protocol (Figure 10). A significant increase in the yield of target cells per area and total yield was observed using the alternative protocol compared to the control protocol (Figure 12). The alternative protocol was also found to generate mainly low CD34+ cells with no expression of the CD73 endothelial marker compared to the control protocol which generated high CD34+ cells with higher levels of CD73 (Figure 13). The phenotype of T progenitor cells from CD34+ generated by both protocols was similar (Figure 14). It was found that the yield of lymphoid cells was higher from CD34+ cells generated with the alternative protocol compared to CD34+ cells generated with the control protocol.

[0211] In summary, the alternative protocol was found to increase (i) the total yield and purity of CD34+ cells in stage 2, (ii) the yield of CD34+CD45+ double positive cells (DP) and CD7+ cells in stage 3, and (iii) cell viability at the end of stage 3. The alternative protocol was found to reduce CD73 expression in stages 2 and 3, indicating that the cells were more enriched for HE with the ability to generate hematopoietic cells. [Table 1]

Table 2

Table 3

Table 4

Claims

1. 1. A method for producing a population of hemogenic endothelial cells (HECs), comprising: (a) differentiating a population of induced pluripotent stem cells (iPSCs) into mesodermal cells by sequentially culturing the iPSCs in a first, second, and third mesoderm induction medium to induce differentiation into mesodermal cells; the first mesoderm induction medium stimulates SMAD1, SMAD5, and SMAD9 mediated signaling pathways; The second mesoderm induction medium (i) stimulates SMAD1, SMAD5, and SMAD9 mediated signaling pathways, and (ii) has fibroblast growth factor (FGF) activity; The third mesoderm induction medium (i) stimulates SMAD1, SMAD2, SMAD3, SMAD5, and SMAD9 mediated signaling pathways, (ii) has fibroblast growth factor (FGF) activity, and (iii) inhibits glycogen synthase kinase 3β, to differentiate the population of iPSCs; and (b) differentiating the mesodermal cells into HECs by sequentially culturing the mesodermal cells in a first and a second HE induction medium to induce differentiation into HECs; The first HE induction medium (i) stimulates a VEGFR-mediated signaling pathway, and (ii) has fibroblast growth factor (FGF) activity; The second HE induction medium (i) stimulates the cKIT receptor (CD117; KIT receptor tyrosine kinase) mediated signaling pathway, (ii) stimulates the VEGFR mediated signaling pathway, and (iii) differentiates the mesodermal cells to have fibroblast growth factor (FGF) activity. The method comprising:

2. The method of claim 1 , wherein steps (a) and (b) are carried out in the absence of stromal cells, feeder cells, or serum.

3. The first mesoderm induction medium consists of a chemically defined nutrient medium supplemented with BMP; The second mesoderm induction medium consists of a chemically defined nutrient medium supplemented with BMP and FGF; The third mesoderm induction medium consists of a chemically defined nutrient medium supplemented with activin, BMP, FGF, and a GSK3 inhibitor; The first HE induction medium consists of a chemically defined nutrient medium supplemented with FGF and VEGF; and / or 3. The method of claim 1 or 2, wherein the second HE induction medium consists of a chemically defined nutrient medium supplemented with SCF, FGF, and VEGF.

4. The method of any one of claims 1 to 3, wherein steps (a) and (b) are performed without purification or isolation of cells in said population.

5. (c) differentiating the HECs into HPCs by culturing the population of HECs in a hematopoietic induction medium to induce differentiation into HPCs. Further comprising: The hematopoietic induction medium (i) stimulates cKIT receptor (CD117) or a cKIT receptor (CD117)-mediated signaling pathway, and / or (ii) stimulates VEGFR or a VEGFR-mediated signaling pathway; The method according to any one of claims 1 to 4, wherein the hematopoietic induction medium comprises one or both of SCF and VEGF.

6. The hematopoietic induction medium comprises: (i) VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IGF-1, IL-3 and IL-6, or (ii) VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IGF-1, IL-3, IL-6, and IL-7 The method of claim 5 , comprising:

7. 7. The method of claim 5 or 6, wherein the hematopoietic induction medium lacks one or more of BMP, FGF, SHH, EPO, angiotensin II and losartan.

8. (d) differentiating the population of HPCs into progenitor T cells by a process comprising culturing the population of HPCs in Lymphoid Expansion Medium to produce progenitor T cells. The method of any one of claims 5 to 7, further comprising:

9. The method of claim 8, further comprising maturing the precursor T cells to produce a population of double positive CD8+CD4+ T cells by a process comprising culturing the population of precursor T cells in a T cell maturation medium to produce double positive CD8+CD4+ T cells.

10. 10. The method of claim 9, comprising activating and expanding the double positive CD8+CD4+ T cells to produce a population of T cells having a CD8+ single positive phenotype or a CD4+ single positive phenotype.

11. 11. The method of claim 10, wherein the T cells specifically bind to cancer cells that express a tumor antigen.

12. The method of any one of claims 1 to 11, wherein the iPSCs are derived from T cells obtained from a donor individual.

13. the T cells obtained from the donor individual are specific for a target antigen; 13. The method of claim 12, wherein the T cells obtained from the donor individual may be tumor infiltrating lymphocytes (TILs).

14. 13. The method of any one of claims 1 to 12, further comprising introducing a heterologous nucleic acid encoding an antigen receptor into said iPSCs, said HECs, said HPCs, or said progenitor T cells.

15. the antigen receptor is a TCR, The TCR may be an affinity-enhanced TCR; The method of claim 14, wherein the TCR may be a non-MHC restricted TCR.

16. The method of claim 15, wherein the TCR specifically binds to MHC presenting a peptide fragment of a tumor antigen expressed by a cancer cell, or specifically binds to a tumor antigen or a peptide fragment thereof expressed by a cancer cell independent of MHC presentation.

17. the antigen receptor is a chimeric antigen receptor (CAR) or a NKCR; The method of claim 14, wherein the CAR or the NKCR may specifically bind to a target antigen expressed by a cell.

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