Method for differentiating lymphoid lineage cells from pluripotent stem cell-derived hemogenic endothelial cells

Abstract

To provide a method of preparing a lymphoid lineage cell from a pluripotent stem cell.SOLUTION: Provided is a method of preparing a lymphoid lineage cell from a pluripotent stem cell, the method comprising the steps of: differentiating a pluripotent stem cell into a mesodermal cell by culturing in a first medium containing a medium composition containing bFGF, VEGF, and SCF; differentiating the mesodermal cell into an early hemogenic endothelial cell by culturing in a second medium containing a medium composition containing TPO, EPO, and IGF-1; and differentiating the EHE cell into a late hemogenic endothelial cell by culturing in a third medium containing a medium composition containing IL-5, IL-7, and DLL.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2021-0016845, filed on February 5, 2021, the entire specification of which is incorporated herein by reference. The present invention relates to a method for differentiating lymphoid cells from hematopoietic endothelial cells derived from pluripotent stem cells. [Background technology]

[0002] The development and maintenance of all blood cells relies on the extremely small number of hematopoietic stem cells present in adult bone marrow. Transplantation of hematopoietic stem cells derived from bone marrow, mobilized peripheral blood, or umbilical cord blood (UCB) is used as the standard of care for a variety of genetic and malignant diseases. However, the limited availability of human leukocyte antigen (HLA)-matched donors remains a major challenge. Despite antigenic incompatibility, the relatively small amount of hematopoietic stem cells in umbilical cord blood can cause various problems, such as delayed engraftment or poor post-transplant engraftment.

[0003] Meanwhile, Japanese Patent No. 5995237 discloses a method for differentiating animal AGM (aorta-gonad-mesonephros)-derived vascular stem cells into human blood cells, and U.S. Patent Publication No. 2004-0235160 discloses a method for effectively differentiating animal hematopoietic stem cells. These findings highlight the importance of generating vascular stem cells and developing methods for differentiating them into blood cells. However, there have been few reports on vascular cell differentiation and culture techniques for directly differentiating human vascular stem cells into blood cells, and no methods for inducing them to lymphoid cells. Therefore, it is necessary to establish an optimized long-term culture method for vascular stem cells that can generate blood cells. Summary of the Invention [Problem to be solved by the invention]

[0004] One aspect provides a method for producing lymphoid lineage blood cells from pluripotent stem cells, comprising the steps of: culturing pluripotent stem cells in a first medium containing bFGF, VEGF, and SCF to differentiate them into mesodermal cells; culturing the mesodermal cells in a second medium containing TPO, EPO, and IGF-1 to differentiate them into early hemogenic endothelial cells (EHE); and culturing the EHE in a third medium containing IL-5, IL-7, and DLL to differentiate them into late hemogenic endothelial cells (LHE). [Means for solving the problem]

[0005] One aspect provides a method for producing lymphoid lineage blood cells from pluripotent stem cells, comprising: culturing pluripotent stem cells in a first medium comprising a first medium composition comprising bFGF, VEGF, and SCF to differentiate them into mesodermal cells; culturing the mesodermal cells in a second medium comprising a second medium composition comprising TPO, EPO, and IGF-1 to differentiate them into early hemogenic endothelial cells (EHE); and culturing the EHE in a third medium comprising a third medium composition comprising IL-5, IL-7, and DLL to differentiate them into late hemogenic endothelial cells (LHE).

[0006] As used herein, the term "pluripotent stem cells" refers to stem cells that have the pluripotent or totipotent properties to differentiate into all tissue cells of an individual, and have the ability to self-renew. Examples of pluripotent stem cells include embryonic stem cells, induced pluripotent stem cells (iPSCs), and somatic cell nuclear transfer-derived embryonic stem cells.

[0007] As used herein, the term "hemogenic endothelial cells" refers to rare differentiated vascular endothelial cells that can differentiate into hematopoietic cells (hematoblasts) during embryonic development. In the embryo, hematopoietic cell development proceeds sequentially from mesoderm through vascular stem cells, to vascular endothelium, and to hematopoietic precursor cells.

[0008] As used herein, the term "angioblast (or vasoformative cell)" refers to a type of endothelial precursor cell derived from bone marrow, and is one of the mesenchymal cells that can develop into the endothelium of blood vessels.

[0009] As used herein, the term "lymphoid lineage blood cells" refers to lineage cells differentiated from hematopoietic stem cells and are primarily involved in the adaptive and innate immune systems. Examples of lymphoid lineage cells include natural killer cells (NK cells), T lymphocytes, and B lymphocytes. Therefore, a method according to one embodiment allows efficient differentiation of pluripotent stem cells into lymphoid lineage cells, such as NK cells, T lymphocytes, or B lymphocytes, in vitro.

[0010] In one embodiment, the method comprises differentiating pluripotent stem cells into mesodermal cells by culturing them in a first medium comprising a first medium composition containing bFGF, VEGFA, and SCF, which is also contained in conventional cell culture media. Examples of the cell culture medium include DMEM (Dulbecco's Modified Eagle's Medium, GIBCO, USA), MEM (Minimal Essential Medium, GIBCO, USA), BME (Basal Medium Eagle, GIBCO, USA), RPMI 1640 (GIBCO, USA), DMEM / F10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10, GIBCO, USA), DMEM / F12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12, GIBCO, USA), α-MEM (α-Minimal Essential Medium, GIBCO, USA), G-MEM (Glasgow's Minimal Essential Medium, GIBCO, USA), IMDM (Isocove's Modified Dulbecco's Medium, GIBCO, USA), Apel II (STEMdiff APEL 2 Medium), EGM-2 (EGM Endothelial Cell Growth Medium).

[0011] The bFGF may be contained at a concentration of 1 to 25 ng / ml. For example, the bFGF may be contained at a concentration of 1 to 25 ng / ml, 1 to 20 ng / ml, 1 to 15 ng / ml, 5 to 25 ng / ml, 5 to 20 ng / ml, 5 to 15 ng / ml, or 7 to 12 ng / ml. If the bFGF content exceeds the above range, the differentiation rate of pluripotent stem cells into mesodermal cells may be slow or they may take on the tendencies of embryonic stem cells.

[0012] The VEGFA may be contained at a concentration of 5 to 40 ng / ml. For example, the VEGFA may be contained at a concentration of 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 5 to 20 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 10 to 30 ng / ml, or 15 to 25 ng / ml. If the VEGFA content is below this range, cell proliferation may be impaired, whereas if it exceeds this range, pluripotent stem cells may be differentiated into vascular endothelial cells.

[0013] The SCF may also be contained at a concentration of 20 to 70 ng / ml, and the SGF may also be contained at a concentration of, for example, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, or 40 to 60 ng / ml.

[0014] In one embodiment, the first composition further comprises ascorbic acid, BMP4, CHIR, or a mixture thereof. The ascorbic acid may be present at a concentration of 50 to 250 ng / ml. For example, the ascorbic acid may be present at a concentration of 50 to 250 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 70 to 250 ng / ml, 70 to 230 ng / ml, 70 to 150 ng / ml, or 80 to 150 ng / ml.

[0015] The BMP4 may also be contained at a concentration of 10 to 50 ng / ml, e.g., 10 to 50 ng / ml, 10 to 45 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 10 to 30 ng / ml, 15 to 50 ng / ml, 15 to 45 ng / ml, 15 to 40 ng / ml, or 20 to 35 ng / ml.

[0016] The CHIR may also be contained at a concentration of 0.5 to 10 nM. For example, the CHIR may be contained at a concentration of 0.5 to 10 nM, 1 to 10 nM, 1 to 8 nM, 1 to 6 nM, 1 to 4 nM, 2 to 9 nM, 2 to 7 nM, 2 to 5 nM, or 3 to 6 nM. If the CHIR content is below this range, the pluripotent stem cells may not differentiate smoothly into mesoderm, whereas if it exceeds this range, the pluripotent stem cells may differentiate into hepatocytes, etc.

[0017] In one embodiment, the above steps may be performed for 1 to 3 days. For example, the above steps may be performed for 1 to 3 days, 1 to 2 days, or 2 to 3 days. If the culture period is shorter than this range, the pluripotent stem cells may not be sufficiently differentiated into mesodermal cells. If the culture period is longer than this range, the pluripotent stem cells may not be differentiated from mesodermal cells into hematopoietic endothelial cells, but may be differentiated into cells of other lineages.

[0018] In one embodiment, the method includes culturing the mesodermal cells in a second medium comprising a second medium composition comprising a basal medium plus TPO, EPO, and IGF-1, to differentiate the mesodermal cells into early hemogenic endothelial cells (EHE).

[0019] The TPO may be contained at a concentration of 50 to 250 ng / ml. For example, the TPO may be contained at a concentration of 50 to 250 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 70 to 250 ng / ml, 70 to 230 ng / ml, 70 to 150 ng / ml, or 80 to 150 ng / ml. If the TPO content is below this range, the definitive hematopoiesis process may not proceed smoothly, while if it is above this range, the TPO may be concentratedly differentiated into megakaryocytes.

[0020] The EPO may also be contained at a concentration of 5 to 40 ng / ml. For example, the EPO may be contained at a concentration of 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 5 to 20 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 10 to 30 ng / ml, or 15 to 25 ng / ml. If the EPO content is below this range, a problem occurs in that it takes a long time for erythropoiesis.

[0021] The IGF-1 may be contained at a concentration of 20 to 70 ng / ml. For example, the IGF-1 may be contained at a concentration of 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, or 40 to 60 ng / ml. If the IGF-1 content is below this range, the hematopoietic commitment stage may not proceed smoothly. If the IGF-1 content is above this range, the hematopoietic commitment stage may be limited to the myeloid lineage. In one embodiment, the second composition further comprises bFGF, VEGFA, SCF, FLT3L, GCSF, and IL-6, or a mixture thereof.

[0022] The bFGF may be contained at a concentration of 1 to 25 ng / ml, for example, 1 to 25 ng / ml, 1 to 20 ng / ml, 1 to 15 ng / ml, 5 to 25 ng / ml, 5 to 20 ng / ml, 5 to 15 ng / ml, or 7 to 12 ng / ml.

[0023] The VEGFA may also be contained at a concentration of 50 to 250 ng / ml, e.g., 50 to 250 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 70 to 250 ng / ml, 70 to 230 ng / ml, 70 to 150 ng / ml, or 80 to 150 ng / ml.

[0024] The SCF may also be contained at a concentration of 100 to 500 ng / ml, e.g., 100 to 500 ng / ml, 100 to 450 ng / ml, 100 to 400 ng / ml, 100 to 350 ng / ml, 150 to 500 ng / ml, 150 to 450 ng / ml, 150 to 350 ng / ml, 200 to 500 ng / ml, 250 to 400 ng / ml, 170 to 330 ng / ml, or 220 to 300 ng / ml.

[0025] The FLT3L may also be contained at a concentration of 100 to 400 ng / ml. For example, the FLT3L may be contained at a concentration of 100 to 400 ng / ml, 100 to 350 ng / ml, 100 to 320 ng / ml, 100 to 300 ng / ml, 100 to 280 ng / ml, 100 to 260 ng / ml, 100 to 240 ng / ml, 100 to 220 ng / ml, 150 to 400 ng / ml, 150 to 300 ng / ml, or 150 to 250 ng / ml. If the FLT3L concentration is below the above range, the degree of differentiation of early hematopoietic endothelial cells into blood cells may be reduced.

[0026] The GCSF may be contained at a concentration of 5 to 50 ng / ml. For example, the GCSF may be contained at a concentration of 5 to 50 ng / ml, 5 to 45 ng / ml, 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 10 to 50 ng / ml, 10 to 45 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 15 to 30 ng / ml, or 15 to 25 ng / ml. If the GCSF content is below the above range, the differentiation of early hematopoietic endothelial cells into myeloid cells is not effectively stimulated.

[0027] The IL-6 may also be contained at a concentration of 10 to 70 ng / ml. For example, the IL-6 may be contained at a concentration of 10 to 70 ng / ml, 10 to 65 ng / ml, 10 to 60 ng / ml, 10 to 55 ng / ml, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 30 to 60 ng / ml, or 45 to 55 ng / ml. If the IL-6 content is below this range, definitive lymphopoiesis is not achieved well.

[0028] In one embodiment, this step may be performed for 6 to 8 days. If the culture period is shorter than this range, the growth and expansion of hemogenic endothelial cells may be reduced, whereas if the culture period is longer than this range, the differentiation of hemogenic endothelial cells in the mesoderm may not be sufficiently initiated, resulting in a low frequency of hemogenic endothelial cells.

[0029] The early hemogenic endothelial cells also have one or more characteristics selected from the following (a) to (d): (a) Having rod-type morphological characteristics: (b) Having a bright yellow light: (c) a doubling time of 15 to 35 hours; and (d) Having surface antigen characteristics of CD31+, Tie-2+, CD44+, CD34+, or a combination thereof.

[0030] The method includes culturing the EHE in a third medium comprising a third medium composition containing IL-5, IL-7, and DLL to differentiate the EHE into late hemogenic endothelial cells (LHE).

[0031] The IL-5 may be contained at a concentration of 20 to 70 ng / ml. For example, the IL-5 may be contained at a concentration of 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 30 to 60 ng / ml, or 45 to 55 ng / ml. If the IL-5 content is below this range, the number of lymphoid cells may decrease, while if it exceeds this range, the number of red blood cells may increase.

[0032] The IL-7 may also be contained at a concentration of 5 to 40 ng / ml. For example, the IL-7 may be contained at a concentration of 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 10 to 30 ng / ml, or 15 to 35 ng / ml. If the IL-7 content is below this range, differentiation into NK cells will not proceed smoothly.

[0033] The DDL1 may be contained at a concentration of 5 to 50 ng / ml. The DDL1 may be contained at a concentration of, for example, 5 to 50 ng / ml, 5 to 45 ng / ml, 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 10 to 50 ng / ml, 10 to 45 ng / ml, 10 to 40 ng / ml, 10 to 30 ng / ml, 15 to 30 ng / ml, or 20 to 30 ng / ml. If the DDL1 content is below the above range, problems may occur, such as a slow proliferation rate of lymphoid cells within blood cells or poor separation of lymphoid cells from vascular stem cells. If the DDL1 content is above the above range, differentiation into T cells is more likely than that into NK cells.

[0034] In one embodiment, the third medium further comprises bFGF, VEGFA, SCF, FLT3L, GCSF, and IL-6, IGF-1, IL-15, or a mixture thereof.

[0035] The bFGF may be contained at a concentration of 1 to 25 ng / ml, for example, 1 to 25 ng / ml, 1 to 20 ng / ml, 1 to 15 ng / ml, 5 to 25 ng / ml, 5 to 20 ng / ml, 5 to 15 ng / ml, or 7 to 12 ng / ml.

[0036] The VEGFA may be contained at a concentration of 5 to 250 ng / ml, for example, 50 to 250 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 70 to 250 ng / ml, 70 to 230 ng / ml, 70 to 150 ng / ml, or 80 to 150 ng / ml.

[0037] The SCF may also be contained at a concentration of 100 to 500 ng / ml, e.g., 100 to 500 ng / ml, 100 to 450 ng / ml, 100 to 350 ng / ml, 100 to 250 ng / ml, 100 to 150 ng / ml, 150 to 500 ng / ml, 150 to 400 ng / ml, 150 to 300 ng / ml, 170 to 250 ng / ml, 200 to 250 ng / ml, or 300 to 350 ng / ml.

[0038] The FLT3L may also be contained at a concentration of 100 to 400 ng / ml, e.g., 100 to 400 ng / ml, 100 to 350 ng / ml, 100 to 320 ng / ml, 100 to 300 ng / ml, 100 to 280 ng / ml, 100 to 260 ng / ml, 100 to 240 ng / ml, 100 to 220 ng / ml, 150 to 400 ng / ml, 150 to 300 ng / ml, or 150 to 250 ng / ml.

[0039] The GCSF may also be contained at a concentration of 5 to 50 ng / ml, e.g., 5 to 50 ng / ml, 5 to 45 ng / ml, 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 10 to 50 ng / ml, 10 to 45 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 15 to 30 ng / ml, or 15 to 25 ng / ml.

[0040] The IL-6 may also be contained at a concentration of 10 to 70 ng / ml, e.g., 10 to 70 ng / ml, 10 to 65 ng / ml, 10 to 60 ng / ml, 10 to 55 ng / ml, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 30 to 60 ng / ml, or 45 to 55 ng / ml.

[0041] The IGF-1 may also be contained at a concentration of 20 to 70 ng / ml, e.g., 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, or 40 to 60 ng / ml.

[0042] The IL-15 may be contained at a concentration of 5 to 40 ng / ml. For example, the IL-15 may be contained at a concentration of 5 to 40 ng / ml, 5 to 35 ng / ml, 5 to 30 ng / ml, 5 to 25 ng / ml, 5 to 20 ng / ml, 10 to 40 ng / ml, 10 to 35 ng / ml, 10 to 30 ng / ml, or 15 to 25 ng / ml. If the IL-15 content is below this range, differentiation into and maintenance of NK cells may be difficult. If the IL-15 content exceeds this range, encouragement to differentiate into other cells, such as T cells, may also occur, making it difficult to target only NK cells or selectively differentiate into only one lymphoid cell type.

[0043] In one embodiment, the above steps may be performed for 12 to 13 days. If the differentiation period is shorter than this range, the primary hematopoiesis process may not occur sufficiently, resulting in a problem of low cell production and frequency in the definitive hematopoiesis process.

[0044] The late hemogenic endothelial cells also have one or more characteristics selected from the following (a) or (b): (a) having morphological characteristics of a cobble stone-like type; and (b) Having surface antigen characteristics of CD31+, CD34+, CD144+, Flk-1+, CD144+CD31+, CD144+CD34+, CD31+CD34+, Flk-1+CD34+, or a combination thereof.

[0045] The method may further include the step of further culturing the LHE for 13 to 28 days to differentiate them into lymphoid cells. For example, the LHE may be further cultured for 13 to 28 days, 13 to 27 days, 13 to 26 days, 13 to 25 days, 13 to 24 days, 13 to 22 days, 13 to 20 days, 15 to 28 days, 15 to 25 days, or 20 to 28 days. If the culture period is shorter than this range, the hemogenic endothelial cells may not be sufficiently differentiated into lymphoid cells. If the culture period is longer than this range, the promotion of lymphoid cells may be delayed, resulting in insufficient lymphoid cells.

[0046] The step of differentiating into lymphoid cells also involves culturing LHE in an EGM-2 basal medium containing a medium composition further comprising one or more selected from the group consisting of bFGF, VEGFA, SCF, FLT3L, GCSF, IL-6, IGF-1, IL-7, IL-15, IL-5, and DLL1.

[0047] That is, in one embodiment, the method optimizes the culture medium conditions suited to the differentiation suitability of lymphoid cells, and by applying the culture medium according to the differentiation stage, long-term culture of hemogenic endothelial cells becomes possible.

[0048] Another aspect provides lymphoid cells produced by the above method.

[0049] In yet another aspect, there is provided a pharmaceutical composition for preventing or treating cancer, comprising the lymphoid cells as an active ingredient. In yet another aspect, there is provided a cell therapy agent, comprising the lymphoid cells as an active ingredient. There is also provided a method for preventing or treating cancer, comprising administering the lymphoid cells, a cell population thereof, or a culture medium thereof as the active ingredient to an individual in need thereof.

[0050] The lymphoid cells produced by the method have immunological activity and can be used as an immunological anti-cancer agent. [Effects of the Invention]

[0051] In one embodiment, the method allows for long-term culture of hemogenic endothelial cells, which can be induced to develop into lymphoid cells, but are also susceptible to inducing the production of immune cells such as T cells, B cells, and NK cells. [Brief explanation of the drawings]

[0052] [Figure 1] 1 shows photographs showing the properties of early and late hemogenic endothelial cells. [Figure 2A] 1 shows photographs illustrating cell morphology according to culture time of late stage hemogenic endothelial cells, and a graph showing proliferation of single cells. [Figure 2B] Photographs confirming the expression of marker proteins for late stage hemogenic endothelial cells. [Figure 3A] HE was cultured in Apel II basal medium, and the photographs show the cell shape on days 1, 3, and 7, as well as thereafter. [Figure 3B]These are photographs showing the cell morphology on days 1 and 3 after culturing HE in EGM-2 basal medium. [Figure 3C] HE was cultured in DMEM / F12 basal medium, and the photographs show the cell shape on days 1, 3, and 7, as well as thereafter. [Figure 4A] This is the result of confirming marker proteins in late hemogenic endothelial cells. [Figure 4B] This is a photograph confirming Tie-2 enrichment in CD31 cells. [Figure 5A] These results confirm the generation of lymphoid cells in hemogenic endothelial cells in the mouse liver. [Figure 5B] These results confirm the generation of lymphoid cells in hemogenic endothelial cells within the mouse AGM. [Figure 5C] The properties and colony formation of hemogenic endothelial cells cultured in each basal medium, and the degree of production of lymphoid cells and myeloid cells were plotted, and the morphology of the hemogenic endothelial cells was confirmed. [Figure 5D] 1 is a graph showing the results of analyzing the expression levels of CD45, CD4, CD8, and NK1.1 using differentiated cells in AGM. [Figure 6A] The results show the cell characteristics and expression markers at each stage of lymphoid cell generation from hematopoietic endothelial cells derived from human pluripotent stem cells. [Figure 6B] This is a graph confirming whether differentiation into lymphoid cells occurs in spots treated with IL-5 and DDL1, based on the expression of lymphoid transcription factors. [Figure 7A] This is the result of confirming the surface markers of γδT cells, which are T lymphocyte precursor cells, by FACS on day 31 of culture of late-stage hematopoietic endothelial cells derived from human pluripotent stem cells. [Figure 7B] This is a graph showing the quantification of surface markers of γδ T cells, which are T lymphocyte precursor cells, on day 31 of culture of late-stage hematopoietic endothelial cells derived from human allopotent stem cells. [Figure 7C]This is the result of confirming the cell morphology of γδT cells, which are T lymphocyte precursor cells, on day 31 of culture of late-stage hematopoietic endothelial cells derived from human pluripotent stem cells. DETAILED DESCRIPTION OF THE INVENTION

[0053] In order to aid in understanding the present invention, preferred examples are presented below. However, the following examples are provided only to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0054] [Manufacturing example] Production Example 1. Establishment of culture conditions for differentiation and maintenance of stem cells into hemogenic endothelial cells The differentiation of stem cells into hemogenic endothelial cells and the culture conditions were confirmed. Specifically, 2.0 x 10 stem cells (hereinafter referred to as "CHA52 cells") obtained from CHA Biotech Co., Ltd. were cultured. 5The cells were then cultured for 2 days in a mesoderm-specific conditioned medium (MSC) containing 7 ng / ml bFGF, 1 nM CHIR, 60 ng / ml ascorbic acid, 15 ng / ml VEGFA, 35 ng / ml SCF, and 18 ng / ml BMP4 in basal medium (Stemline II medium). The cells were then cultured for 3 days in an optimized medium containing 7 ng / ml bFGF, 15 ng / ml VEGFA, 180 ng / ml SCF, 150 ng / ml FLT3L, 18 ng / ml GCSF, 80 ng / ml TPO, 17 ng / ml EPO, 25 ng / ml IL-6, and 30 ng / ml IGF-1 in basal medium (Apel II medium). The cells were then cultured for 6 days in an optimized medium containing 7 ng / ml bFGF, 15 ng / ml VEGFA, 180 ng / ml SCF, 150 ng / ml FLT3L, 18 ng / ml GCSF, 80 ng / ml TPO, 17 ng / ml EPO, 25 ng / ml IL-6, and 30 ng / ml IGF-1 in basal medium (Apel II medium). The period up to day 6 was designated pro-hemogenic endothelial cells (pro HE), and on day 6, hemogenic endothelial cells were subcultured and isolated to a high degree of purity. The culture medium was treated with 0.25% trypsin / EDTA for 3 minutes, and the non-hemogenic endothelial cells distributed in a single layer were observed to fall off first. The medium was then washed with DPBS, and further treated with 0.25% trypsin / EDTA to isolate hemogenic endothelial cell clusters as single cells. The isolated single cells were filtered through a 0.44 μm filter, and the cells from one well were divided into two wells. They were subcultured for 21 days in conditioned medium (Apel II medium) containing 11 ng / ml bFGF, 50 ng / ml VEGFA, 170 ng / ml SCF, 150 ng / ml FLT3L, 18 ng / ml GCSF, 35 ng / ml IL-6, 25 ng / ml IGF-1, 15 ng / ml IL-7, 10 ng / ml IL-15, 25 ng / ml IL-5, and 7 ng / ml DLL1 (hereafter referred to as "Phase III"). The medium was replaced every 3-4 days until day 21.

[0055] Manufacturing Examples 2-4 The same method as in Preparation Example 1 was performed, except that the components and contents in each step were as shown in Tables 1 to 3 below.

[0056] [Table 1]

[0057] [Table 2]

[0058] [Table 3]

[0059] [Example] Example 1. Characterization of hemogenic endothelial cells The hemogenic endothelial cells cultured in Step III of Preparation Example 1 were divided into early hemogenic endothelial cells (EHE) and late hemogenic endothelial cells (LHE) on day 13 of culture, and the properties of each cell type were confirmed using a microscope (x100, x200, and x400).

[0060] FIG. 1 is a photograph showing the properties of early and late hemogenic endothelial cells.

[0061] As a result, as shown in Figure 1, primary hemogenic endothelial cells were observed to form clusters, with cell aggregates distributed sparsely, and other cells were observed to be distributed outside the primary hemogenic endothelial cells. At this time, expression of hemogenic endothelial cell marker proteins such as CD31 and VE-cadherin was also observed.

[0062] Furthermore, after day 13 of culture, late hemogenic endothelial cells were observed to aggregate more cells than early hemogenic endothelial cells, and the blood cells that emerged from these aggregates resembled definitive blood. As the culture progressed, the cytoplasm of these late hemogenic endothelial cells became thinner and thinner, and they were observed to reach the end of their lifespan. Furthermore, FLK-1+ megakaryocyte and erythrocyte progenitors were observed to emerge from these late hemogenic endothelial cells. From a single late hemogenic endothelial cell, several megakaryocyte and erythrocyte progenitors were generated. Clonal expansion of these cells confirmed that the hemogenic endothelial cells primarily exhibited rod-like morphology, distinct from the typical vascular endothelial cell morphology of cobblestone-like cells and mesenchymal stromal cells. The blood cells formed by these late hemopoietic endothelial cells were primarily primitive blood cells (mainly myeloid lineage) at an early stage, while cells derived from late hemopoietic endothelial cells contained definitive blood cells (including both myeloid and lymphoid lineages), and it was confirmed that tube formation occurred in the generated vascular endothelial cells. Inferred from the above results, hemopoietic endothelial cells are thought to possess the properties of both vascular endothelial cells and blood cells.

[0063] FIG. 2A shows photographs illustrating the cell morphology of late hemogenic endothelial cells over time in culture, and a graph showing the proliferation of single cells.

[0064] As shown in Figure 2A, on day 1 of culture of late stage hemogenic endothelial cells, a bright yellow light (indicated by the white arrow) was observed under the microscope, presumably due to 5-aminolevulinate synthase, a precursor of porpyrin. However, after all erythroblasts were generated, the bright light disappeared in the hemogenic endothelial cells (D9, after day 10). Furthermore, on day 5, erythroblast budding was observed in the hemogenic endothelial cells, and after day 7, erythroblast formation was observed. The doubling day of the hemogenic endothelial cells was approximately 1.2 days, and the average doubling time was approximately 29.5 hours.

[0065] FIG. 2B is a photograph confirming the expression of marker proteins of late hemogenic endothelial cells.

[0066] As a result, as shown in Figure 2B, after day 9, marker proteins such as CD31, Tie-2, and CD34 were observed to be expressed in clusters of hemogenic endothelial cells, and mesodermal markers such as RUNX1 and Brachyury remained in the nuclei of some cells. Furthermore, when blood cells emerged from the hemogenic endothelial cells, FLK-1 protein was observed to be expressed intensively in the blood cells.

[0067] Example 2. Growth and differentiation of late hemogenic endothelial cells depending on culture conditions The late hemogenic endothelial cells cultured in Example 1 were frozen for one month and then thawed in a 37°C water bath. To identify media suitable for the proliferation and differentiation of late hemogenic endothelial cells, three basal media (Apel II, EGM-2, and DMEM / F12) were used. Apel II and DMEM / F12 media were supplemented with 7 ng / ml bFGF, 15 ng / ml VEGFA, 180 ng / ml SCF, 150 ng / ml FLT3L, 18 ng / ml GCSF, 80 ng / ml TPO, 17 ng / ml EPO, 25 ng / ml IL-6, 30 ng / ml IGF-1, 45 ng / ml IL-3, and 1 nM CHIR. EGM-2 medium was further supplemented with the same types and amounts of cytokines as in Step III of Preparation Example 1. The frozen and thawed late hemogenic endothelial cells were then cultured in each of the three media, with half-changes of all media maintained every 3–4 days. Because the late hemogenic endothelial cells were frozen and thawed vascular endothelial cells, they were expected to attach to the culture dish and proliferate.

[0068] FIG. 3A is a set of photographs showing the cell morphology of hemogenic endothelial cells cultured in Apel II basal medium on days 1, 3, and 7, and thereafter.

[0069] As shown in Figure 3A, most hemogenic endothelial cells in Apel II basal medium failed to attach to the culture dish and died rapidly. However, for hemogenic endothelial cells that survived beyond day 7, once their endothelial function stabilized, they were observed to produce WBC-like cells and form erythroid colonies. However, the amount of blood cells produced was significantly lower than that of hemogenic endothelial cells cultured in BGM-2 basal medium and DMEM / F12 basal medium.

[0070] FIG. 3B is a photograph showing the cell morphology on days 1 and 3 after culturing hemogenic endothelial cells in EGM-2 basal medium.

[0071] As shown in Figure 3B, hemogenic endothelial cells cultured in EGM-2 basal medium adhered to the culture dish and proliferated stably, with a faster and more stable proliferation rate than those cultured in other basal media. In particular, hemogenic endothelial cells grown for more than 7 days were separated into a population of endothelial progenitor cells with a typical cobblestone morphology and a population of cells with atypical endothelial cell morphology. In contrast, cells cultured in Apel II basal medium that did not adhere were cultured in EGM-2 medium for 3 days to induce adherent cell proliferation, and then further cultured in Apel II medium. Normal proliferation of hemogenic endothelial cells was observed. Thus, EGM-2 basal medium is positive for the proliferation and engraftment of hemogenic endothelial cells, and the stable adhesion and proliferation of hemogenic endothelial cells in the culture dish may affect blood cell production.

[0072] FIG. 3C is a photograph showing the cell shape of hemogenic endothelial cells cultured in DMEM / F12 basal medium on days 1, 3, and 7, and thereafter.

[0073] As a result, as shown in Figure 3C, hemogenic endothelial cells cultured in DMEM / F12 basal medium were observed to adhere to the culture dish and proliferate. Although the proliferation rate was delayed by about 4-5 days compared to hemogenic endothelial cells cultured in EGM-2 basal medium, numerous hemogenic endothelial cell colonies were formed, and from day 8 onwards, a large number of blood cells were stably produced.

[0074] Example 3. Identification of protein markers expressed in hemogenic endothelial cells The expression of marker proteins expressed in hemogenic endothelial cells was compared using the basal medium of Example 2. As a result, there was no significant difference in the expression of marker proteins depending on the basal medium, and it was confirmed that typical marker proteins were expressed in hemogenic endothelial cells.

[0075] FIG. 4A shows the results of confirming marker proteins in hemogenic endothelial cells cultured in EGM-2 basal medium for 3 days.

[0076] Figure 4B is a photograph confirming Tie-2 enrichment in hemogenic endothelial cells cultured in EGM-2 basal medium for 14 days.

[0077] As shown in Figure 4A, in the hemogenic endothelial cell group cultured in EGM-2 basal medium, the protein markers expressed were CD31 (27.9%), CD34 (8.2%), CD144 (29.9%), and Flk-1 (4.5%), while the protein markers expressed were CD144+CD31+ (11.3%), CD144+CD34+ (8.5%), CD31+CD34+ (10.2%), and Flk-1+CD34+ (4.9%). Furthermore, as shown in Figure 4B, in the group sorted by MACS using CD31, Tie-2 expression was enriched by 24.9% in the CD31+ cell group and 1.8% in the CD31- cell group. These results suggest the commitment of CD31+ cells to hemogenic endothelial cells. We also confirmed that hemogenic endothelial cell markers were expressed in the following order: Tie2 > CD144 > CD31 > CD34 > FLK1.

[0078] Example 4. Identification of culture conditions for hemogenic endothelial cells to induce lymphoid cell generation Based on the results of Example 2, the culture conditions for hemogenic endothelial cells to induce lymphocyte generation were further confirmed. First, aorta gonad mesonephros (AGM) and liver tissue were isolated from 10.5-11.5 day old mouse fetuses. The tissues were then minced using the inside of a 1.5 ml Eppendorf tube lid, washed with DPBS, and cultured in the same three media as in Example 2 until day 20, the day of birth of the mice. Hematoxylin and eosin staining was performed to confirm the morphology of hemogenic endothelial cells cultured in each medium.

[0079] Figure 5A shows the results of lymphoid cell generation in hemogenic endothelial cells in the mouse liver, Figure 5B shows the results of lymphoid cell generation in hemogenic endothelial cells in the mouse AGM, and Figure 5C shows the results of examining the morphology of hemogenic endothelial cells cultured in each basal medium, graphically illustrating the properties and colony formation of hemogenic endothelial cells, and the extent of lymphoid and myeloid cell generation.

[0080] Although liver tissue-derived blood cells rarely yielded hemogenic endothelial cells, the process of infiltration and maturation of blood cells derived from these hemogenic endothelial cells into the liver showed different trends depending on the medium used. Specifically, as shown in Figure 5A, in EGM-2 basal medium, all hemogenic endothelial cells died. In contrast, in Apel II basal medium, cells were rapidly induced to develop into megakaryocytes, erythroid cells, pronormoblasts, and orthochromic normoblasts within 3 days, and the cells disappeared within 6 days. Furthermore, in DMEM / F12 basal medium, differentiation into megakaryocytes, erythroid-derived megakaryocytes, and megakaryocytes (MK-II) was more vigorous than erythroid differentiation, and the formation of proplatelet processes and silia was clearly observed. In particular, it was confirmed that more white blood cells were produced than red blood cells with Heme.

[0081] As shown in Figure 5B, the extent of culture of hemogenic endothelial cells present in AGM varied depending on the medium used, up to day 9. Specifically, the disappearance of blood cells was significantly increased in EGM-2 basal medium. This indicates that, unlike human endothelial cells derived from full differentiation potential, mouse hemogenic endothelial cells do not survive for long periods. However, the enrichment of erythroid cells was rapid and the proliferation of leukocytes was significantly increased in Apel II basal medium. Furthermore, the differentiation of megakaryocytes and leukocytes was significantly higher in DMEM / F12 basal medium than in erythroid cells. As a result, as shown in Figure 5C, liver- and AGM-derived cells exhibited more active adhesion and proliferation in DMEM / F12 basal medium and Apel II basal medium than in EGM-2 basal medium, indicating that long-term differentiation into hemogenic endothelial cells and blood cells is possible.

[0082] FIG. 5D is a graph showing the results of analyzing the expression levels of CD45, CD4, CD8, and NK1.1 using differentiated cells in the AGM.

[0083] As a result, as shown in Figure 5D, in Apel II basal medium, the induction of spontaneously killing cells in suspension cells was highest on day 10, while in DMEM / F12 basal medium, the induction of megakaryocytes as well as CD4 and CD8 cells was rapidly promoted and maintained on day 6. This indicates that AGM-derived blood cells can be maintained in both of these basal media.

[0084] Example 5. Confirmation of differentiation of human pluripotent stem cells into hemogenic endothelial cells and blood cells Based on the findings in Example 4 that hemogenic endothelial cells derived from mouse fetuses differentiate into blood cells, the late-stage hemogenic endothelial cells cultured in Example 1 were differentiated and proliferated in the medium of Example 2 to differentiate into blood cells, and their surface markers were then identified. Specifically, the hemogenic endothelial cells cultured for 21 days through Steps I to III in Preparation Example 1 were further cultured until day 34. Since hemogenic endothelial cells began to shed and their frequency of cell death increased after day 28 of culture, the functional window of hemogenic endothelial cells was determined to be days 21 to 28. Only the hemogenic endothelial cells cultured until day 34 were further treated with 0.25% trypsin / EDTA to dissociate cell clusters into single cells. Subsequently, a CFU assay was performed to determine whether myeloid cell colonies formed.

[0085] FIG. 6A shows the cell characteristics and expression markers at each stage of lymphoid cell generation from human pluripotent stem cell-derived hematopoietic endothelial cells.

[0086] FIG. 6B is a graph showing whether or not differentiation into lymphoid cells occurs in spots treated with IL-5 and DDL1, as confirmed by the expression of lymphoid transcription factors.

[0087] As a result, as shown in Figure 6A, it was confirmed that hemogenic endothelial cells cultured from day 13 to day 34 generated colonies of myeloid cells and differentiated. Furthermore, as shown in Figure 6B, it was confirmed that lymphoid transcription factors were expressed in large numbers in spots treated with IL-5 and DLL1. This indicates that the medium containing the IL-5 medium is capable of long-term culture of human pluripotent stem cells and differentiation into hemogenic endothelial cells and vascular cells (lymphoid cells).

[0088] Example 6. Confirmation of differentiation of human pluripotent stem cells into T lymphocyte precursor cells Based on the findings in Example 4 that hemogenic endothelial cells derived from mouse fetuses differentiate into blood cells, the late hemogenic endothelial cells cultured in Example 1 were differentiated and proliferated in the medium of Example 2 to differentiate into T lymphocyte precursor cells, i.e., γδT cells, which are precursors of αβT cells, and then surface markers were identified. Specifically, the hemogenic endothelial cells cultured for 21 days through steps I to III in Preparation Example 1 were further cultured until day 31. Since lymphoid cells are generated through the definitive hematopoietic generation process on day 31 of culture, the functional window of hemogenic endothelial cells was determined to be between days 21 and 31. On day 31 of culture, FACS was performed to analyze cell surface markers.

[0089] FIG. 7A shows the results of FACS analysis of surface markers of γδT cells, which are T lymphocyte precursor cells, on day 31 of culture of late-stage hematopoietic endothelial cells derived from human allopotent stem cells.

[0090] FIG. 7B is a graph showing the quantification of surface markers of γδ T cells, which are T lymphocyte precursor cells, on day 31 of culture of late-stage hematopoietic endothelial cells derived from human allopotent stem cells.

[0091] FIG. 7C shows the results of examining the cell morphology of γδ T cells, which are T lymphocyte precursor cells, on day 31 of culture of late hemogenic endothelial cells derived from human pluripotent stem cells.

[0092] As shown in Figures 7A and 7B, late hemogenic endothelial cells began to express CD3 on day 22 of culture as they progressed to the definitive hematopoietic cell stage. Furthermore, on day 31 of culture, the number of CD3-positive cells was significantly higher in suspension cells than in adherent cells, with a frequency of approximately 40%. Furthermore, TCR receptor subtypes were identified in CD3-positive cells, and TCR Vδ2 expression was significantly higher in suspension cells than in adherent cells.

[0093] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting. The present invention also relates to the following: [Item 1] Culturing pluripotent stem cells in a first medium containing a medium composition including bFGF, VEGF, and SCF to differentiate the cells into mesodermal cells; Culturing the mesodermal cells in a second medium containing a medium composition including TPO, EPO, and IGF-1 to differentiate them into early hemogenic endothelial cells (EHE); and culturing the EHE in a third medium containing a medium composition including IL-5, IL-7, and DLL to differentiate the EHE into late hemogenic endothelial cells (LHE). [Item 2] Item 2. The method according to item 1, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer derived stem cells, and adult-derived mesenchymal stem cells. [Item 3] 2. The method of claim 1, wherein the first culture medium further comprises ascorbic acid, BMP4, or a mixture thereof. [Item 4] 2. The method according to item 1, wherein the second culture medium further comprises bFGF, VEGFA, SCF, FLT3L, GCSF and IL-6, or a mixture thereof. [Item 5] Item 1, wherein the EHE has one or more characteristics selected from the following (a) to (d): (a) having rod-type morphological characteristics; (b) having a bright yellow light; (c) a doubling time of 15 to 35 hours; and (d) Having surface antigen characteristics of CD31+, Tie-2+, CD144+, CD34+, or a combination thereof. [Item 6] 2. The method of claim 1, wherein the third culture medium further comprises bFGF, VEGFA, SCF, FLT3L, GCSF, IL-6, IGF-1, IL15, or a mixture thereof. [Item 7] The method according to item 1, wherein the LHE has one or more properties selected from the following (a) to (b): (a) have morphological characteristics of the cobblestone-like type; and (b) Having surface antigen characteristics of CD31+, CD34+, CD144+, Flk-1+, CD144+CD31+, CD144+CD34+, CD31+CD34+, Flk-1+CD34+, or a combination thereof. [Item 8] 2. The method according to item 1, wherein the step of differentiating the pluripotent stem cells into mesodermal cells is carried out for 1 to 3 days. [Item 9] 2. The method according to item 1, wherein the step of differentiating the mesodermal cells into EHE is carried out for 6 to 8 days. [Item 10] 2. The method according to item 1, wherein the step of differentiating the EHE into the LHE is carried out for 12 to 13 days. [Item 11] 2. The method according to item 1, further comprising the step of culturing the LHE for 13 to 28 days to differentiate them into lymphoid cells. [Item 12] Item 12. The method of item 11, wherein the LHE is cultured in an EGM-2 basal medium containing a medium composition further comprising any one or more selected from the group consisting of bFGF, VEGFA, SCF, FLT3L, GCSF, IL-6, IGF-1, IL-7, IL-15, IL-5, and DLL1.

Claims

1. Culturing pluripotent stem cells in a first medium containing a medium composition including bFGF, VEGF, and SCF to differentiate the cells into mesodermal cells; Culturing the mesodermal cells in a second medium containing a medium composition including TPO, EPO, and IGF-1 to differentiate them into early hemogenic endothelial cells (EHE); and culturing the EHE in a third medium containing a medium composition including IL-5, IL-7, and DLL to differentiate the EHE into late hemogenic endothelial cells (LHE).

2. 2. The method of claim 1, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer derived stem cells, and adult-derived mesenchymal stem cells.

3. 10. The method of claim 1, wherein the first culture medium further comprises ascorbic acid, BMP4, or a mixture thereof.

4. 2. The method of claim 1, wherein the second culture medium further comprises bFGF, VEGFA, SCF, FLT3L, GCSF, and IL-6, or a mixture thereof.

5. 10. The method of claim 1, wherein the EHE has one or more characteristics selected from the following (a) to (d): (a) having rod-type morphological characteristics; (b) having a bright yellow light; (c) a doubling time of 15 to 35 hours; and (d) having surface antigen characteristics of CD31+, Tie-2+, CD144+, CD34+, or a combination thereof;

6. 2. The method of claim 1, wherein the third culture medium further comprises bFGF, VEGFA, SCF, FLT3L, GCSF, IL-6, IGF-1, IL15, or a mixture thereof.

7. 10. The method of claim 1, wherein the LHE has one or more properties selected from the following (a) or (b): (a) having morphological characteristics of a cobblestone-like type; and (b) having surface antigen characteristics of CD31+, CD34+, CD144+, Flk-1+, CD144+CD31+, CD144+CD34+, CD31+CD34+, Flk-1+CD34+, or a combination thereof;

8. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into mesodermal cells is carried out for 1 to 3 days.

9. The method of claim 1, wherein the step of differentiating the mesodermal cells into EHE is carried out for 6 to 8 days.

10. The method of claim 1, wherein the step of differentiating the EHE into the LHE is carried out for 12 to 13 days.

11. The method of claim 1, further comprising the step of culturing the LHE for an additional 13 to 28 days to differentiate into lymphoid cells.

12. The method of claim 11, wherein the LHE is cultured in an EGM-2 basal medium containing a medium composition further comprising any one or more selected from the group consisting of bFGF, VEGFA, SCF, FLT3L, GCSF, IL-6, IGF-1, IL-7, IL-15, IL-5, and DLL1.

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