Method for directionally differentiating iPSC into iNK

Abstract

The invention relates to the technical field of cell differentiation, in particular to a method for directionally differentiating iPSC into iNK. Specifically, the method for preparing the CD34 + hematopoietic stem / progenitor cell and the iNK on the shaking table comprises the following steps: forming an embryoid (EB) by the iPSC on the shaking table, and then collecting a single cell supernatant to obtain the iNK. According to the method, high-proportion CD34 < + > hematopoietic stem / progenitor cells are obtained by adjusting the factor concentration and collecting the supernatant and are further induced and differentiated into the iNK, and more than 100 iNKs can be harvested before amplification of one iPSC according to the iNK harvested in batches.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell differentiation, and in particular to a method for differentiating iPSC into iNK. BACKGROUND

[0002] Natural killer (NK) cells, as the core effector cells of the innate immune system, can recognize and kill tumor cells through non-MHC restricted mechanism, and have the ability to directly kill tumor cells and virus infected cells, and are not prone to induce graft-versus-host disease (GVHD), thus showing great potential in cancer immunotherapy. Traditional sources of NK cells include peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB) and NK cell lines, but these sources have certain limitations, such as donor dependence, cell heterogeneity and low expansion efficiency. In recent years, induced pluripotent stem cells (iPSCs) as a kind of cell source that can be infinitely expanded and has high homogeneity, genetic editing flexibility and genetic stability, are expected to break through the donor limitation and batch heterogeneity bottleneck of traditional PBMC / UCB sources, and become the core source of "spot-type" NK cell therapy. In the future, we need to focus on the optimization of differentiation mechanism and the improvement of safety to accelerate the clinical application transformation. The existing differentiation scheme of iPSC to iNK has problems such as long cycle (more than 30 days) and insufficient functional maturity.

[0003] Therefore, how to provide a method for significantly improving the directional differentiation efficiency of iPSC to iNK has become a hot and painful point in the industry. SUMMARY

[0004] In order to solve the technical problems mentioned in the background art, the present application provides a method for differentiating iPSC into iNK, and the technical scheme is as follows: A method for efficiently and stably differentiating human induced pluripotent stem cells (iPSCs) into induced natural killer cells (iNKs) by optimizing the medium combination and differentiation timing, and a special medium system.

[0005] A method for differentiating iPSC into iNK, comprising the following steps: First stage: iPSCs are induced to differentiate into embryoid bodies between day -1 and day 0; Second stage: embryoid bodies are induced to differentiate into mesoderm cells between day 0 and day 2; Third stage: mesoderm cells are induced to differentiate into CD34+hemogenic endothelial cells between day 1 and day 6; Fourth stage: CD34+hemogenic endothelial cells are induced to differentiate into CD34+ / CD45+cells between day 4 and day 9; Fifth stage: iNK is formed between day 8 and day 28.

[0006] Preferably, the first stage, day 1, cells are digested into single cells with cell dissociation reagent, resuspended with mTeSR after 5 min centrifugation at 1200 rpm and seeded at a density of 1 x 10 5 ~1×10 6 Cells are seeded in low attachment six-well plates at a density of 1 x 10

[0007] Preferably, the second stage, day 0, the base is EB differentiation medium, 6-10 μΜ CHIR99021 is added, cells are centrifuged and replaced with fresh medium, and cells are cultured in a 5% CO2, 37°C incubator on a shaker at 80 rpm for 24-48 hours. The EB differentiation medium is composed of the following components: 47% (by volume) IMDM medium, 47% (by volume) F12 medium, 1% (by volume) ITS liquid medium supplement, 1% (by volume) lipid concentrate, 2 mM L-glutamine, and 6 g / L human serum albumin.

[0008] Preferably, the third stage, mesodermal cells are induced to differentiate into CD34+hemogenic endothelial cells between day 1 and day 3. On day 1, 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF are added to the EB differentiation medium, the plate is tilted at 45°, after the cells settle, the supernatant is removed, the cells are replaced with this medium, and the cells are cultured in a 5% CO2, 37°C incubator on a shaker at 80 rpm for 48 hours, and then the medium is completely replaced with this medium for another 24 hours. The fourth stage: CD34+hemogenic endothelial cells are induced to differentiate into CD34+ / CD45+cells between day 4 and day 7. On day 4, the base is the EB differentiation medium, 50 ng / mL VEGF, 10 μΜ SB431542, and 10 ng / mL bFGF are added, the plate is tilted at 45°, after the cells settle, the supernatant is removed, the cells are replaced with this medium, and the cells are cultured in a 5% CO2, 37°C incubator on a shaker at 80 rpm for 96 hours, and the medium is replaced every 48 hours.

[0009] Preferably, the concentration of CHIR99021 is 10 μΜ.

[0010] Preferably, the third stage, mesodermal cells are induced to differentiate into CD34+hemogenic endothelial cells between day 2 and day 6. 2 days, the EB differentiation medium is added with factors 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF, the plate is inclined at 45°, after the cells are settled, the supernatant is removed, the cells are replaced with the medium, and the cells are cultured in a 5% CO2, 37°C constant-temperature incubator on a shaking table at 80 rpm for 120 hours, and the medium is completely replaced every 48 hours; The fourth stage is CD34+hemogenic endothelial cell induction and differentiation to form CD34+ / CD45+cells between 7 days and 9 days; 7 days, the EB differentiation medium is added with factors 50 ng / mL VEGF and 10 ng / mL bFGF, the plate is inclined at 45°, after the cells are settled, the supernatant is removed, the cells are replaced with the medium, and the cells are cultured in a 5% CO2, 37°C constant-temperature incubator on a shaking table at 80 rpm for 72 hours, and the medium is replaced every 48 hours.

[0011] Preferably, in the first stage, the cell inoculation density is 1×10 5 cells / mL.

[0012] Preferably, in the third stage, mesoderm cell induction and differentiation form CD34+hemogenic endothelial cells between 2 days and 4 days; 2 days, the EB differentiation medium is added with factors 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF, the plate is inclined at 45°, after the cells are settled, the supernatant is removed, the cells are replaced with the medium, and the cells are cultured in a 5% CO2, 37°C constant-temperature incubator on a shaking table at 80 rpm for 120 hours, and the medium is completely replaced every 48 hours; The fourth stage is CD34+hemogenic endothelial cell induction and differentiation to form CD34+ / CD45+cells between 5 days and 7 days; 7 days, the EB differentiation medium is added with factors 50 ng / mL VEGF and 10 ng / mL bFGF, the plate is inclined at 45°, after the cells are settled, the supernatant is removed, the cells are replaced with the medium, and the cells are cultured in a 5% CO2, 37°C constant-temperature incubator on a shaking table at 80 rpm for 72 hours, and the medium is replaced every 48 hours.

[0013] Preferably, the fifth stage specifically comprises the following steps: 8 days, the EB spheres harvested in the fourth stage are resuspended in the iNK differentiation medium 1, and the cells are cultured in a 5% CO2, 37°C constant-temperature incubator on a shaking table at 80 rpm for 48 hours; The iNK differentiation medium 1 is: based on OptiVitro NK cell expansion serum-free medium P01, and the following ingredients are added: 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL Flt3, 10 ng / mL IL-15, 5 ng / mL IL-3, and 10% by volume human AB serum; At 10 days, the iNK differentiation medium 1 is supplemented, and the culture is continued for 96 hours; At 14 days, the iNK differentiation medium 2 is half-replaced every 72 hours, and the culture is continued in a 5% CO2, 37℃ constant temperature incubator until 28 days; The iNK differentiation medium 2 is: based on OptiVitro NK cell expansion serum-free medium P01, and the following ingredients are added: 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL Flt3, 10 ng / mL IL-15, and 10% by volume human AB serum.

[0014] Preferably, before the first stage, a pre-stage is further included, and the pre-stage specifically includes the following steps: At the first 4 days, Matrigel is added in a TC six-well plate, and the plate is placed in a 5% CO2, 37℃ constant temperature incubator for more than one hour for coating; The iPSCs that have been passed for more than three generations after resuscitation are selected for passaging when the confluence reaches more than 50%, cell gentle digestion enzyme is added, and the cells are digested at 37° for 5-10 min; after the digestion solution is removed, the cells are blown down with mTeSR and inoculated into a plate, which is placed in a 5% CO2, 37℃ constant temperature incubator for cell culture, and the liquid is changed daily.

[0015] The beneficial effects of the present application mainly lie in that the iNKs harvested in batches can reach more than 100 iNKs per iPSC. The present application obtains iNK cells with high purity (>80%) within 14 days (the time for hematopoietic stem cell differentiation into iNK) and high expression of cytotoxicity markers (>95%) within 21 days through the combination of stage-specific factors and dynamic culture strategies, which significantly improves the clinical conversion potential. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The overall flow chart for iPSC differentiation of iNKs; Figure 2 The morphology chart of iPSC culture under a 4-fold optical microscope (the first day); Figure 3 The morphology chart of EBs in a well plate; Figure 4 The morphology chart of iNKs under a 20-fold optical microscope at 28 days; Figure 5 Figure for flow cytometry detection of CD34 and CD45 under condition 7; Figure 6 Figure for flow cytometry detection of CD34 of hematopoietic stem / progenitor cells differentiated under different conditions; Figure 7 Figure for flow cytometry detection of CD56, CD3 and CD16 of iNK detected at 28 days; Figure 8 Figure for flow cytometry detection of NKp30, NKp44, NKp46, NKG2A, NKG2D and FasL of iNK detected at 28 days; Figure 9 Figure for amplification fold of iNK amplified for 14 days; Figure 10 Figure for killing data of iNK amplified for 14 days; Figure 11 Figure for flow cytometry detection of CD56, CD3 and CD16 of iNK detected at 21 days. DETAILED DESCRIPTION

[0017] The technical solutions of the present application will be further described below in combination with specific embodiments. The technical solutions of the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0018] As used herein, the term "iPSC" refers to induced pluripotent stem cells, which are cells reprogrammed from human somatic cells, have the ability to differentiate into three germ layers, and can differentiate into various cells and organs of the human body, can replicate indefinitely, and can self-renew. The cell line used herein is an iPSC derived from blood reprogramming.

[0019] As used herein, an embryoid body (EB) obtained from the directed differentiation of iPSCs is referred to as an "iEB" or induced embryoid cell. The preparation of iPSCs to iEBs is a key step in stem cell research and is widely used in multiple fields such as organoids, cell differentiation, drug screening, etc. The embryoid body formed by iPSCs is a three-dimensional structure formed spontaneously by iPSCs under specific culture conditions. This structure is similar to the organization of early embryos and can exhibit the differentiation potential of ectoderm, mesoderm and endoderm. The formation of embryoid bodies is usually achieved by suspending iPSCs in single cell or clump form in feeder-free culture medium, allowing them to aggregate into spherical structures under conditions lacking adhesion. Many methods are known in the art for preparing embryoid bodies. EB spheres are formed in microwell plates or low-attachment plates, in shake flasks or bioreactors. Methods using a shaker, or hanging drop methods, or spin aggregation methods are commonly used to promote aggregation. The addition of ROCK inhibitors, such as Y27632, has been shown to be a good and important small molecule for aggregation into spheres, which can form uniform-sized EBs in one day.

[0020] As used herein, HSPCs obtained from the directed differentiation of iPSCs are referred to as "iHSPC" or induced hematopoietic stem and progenitor cells. Hematopoietic stem cells and hematopoietic progenitor cells are important cell types in the blood system, which play a key role in maintaining the stability of the blood system and generating various blood cells. Hematopoietic stem cells are primitive cells that generate various blood cells, also known as multipotent hematopoietic stem cells. They have a high self-renewal capacity and multi-directional differentiation potential, and can differentiate into red blood cells, white blood cells, platelets and lymphocytes, etc. Hematopoietic progenitor cells are cells differentiated from hematopoietic stem cells, which can only proliferate and differentiate into one or several blood cell lines, also known as committed stem cells. They are more mature than hematopoietic stem cells and have a shorter self-renewal capacity. They have the potential to differentiate into lymphoid and myeloid cells.

[0021] As used herein, NK cells obtained from the directed differentiation of iPSCs are referred to as "iNK" or induced natural killer cells.

[0022] Natural killer (NK) cells are key effector cells of the innate immune system, with the ability to directly kill tumor cells and virus-infected cells. Their functional properties can be verified by various experimental methods, including cell surface marker detection, cytotoxicity analysis, and cytokine secretion determination. The identification and functional analysis of NK cells rely on the expression of specific surface markers. According to maturity and functional status, NK cells can express the following key molecules: pan-leukocyte markers such as CD45 (a common marker for all hematopoietic cells, used to distinguish immune cells from other cell types). CD56 (NCAM) (a major marker for NK cells, usually divided into: CD56 bright (high expression): with strong cytokine secretion capacity (such as IFN-γ), but lower cytotoxicity. CD56 dim (low expression): high expression of CD16 (FcγRIII), with strong antibody-dependent cell-mediated cytotoxicity (ADCC) capacity) Activation and inhibitory receptors such as CD16 (FcγRIII) (mediates ADCC, binds to IgG antibodies to trigger NK cell killing of target cells); NKG2D (recognizes stress cell surface molecules (such as MICA / MICB), promotes NK cell activation); KIR (killer cell immunoglobulin-like receptor) (recognizes MHC-I molecules, transmits inhibitory signals to prevent self-attack); NKG2A (CD94 / NKG2A) (inhibitory receptor, binds to HLA-E to inhibit NK cell activity); natural cytotoxicity receptors (NCRs) (NKp30, NKp44, NKp46, recognize virus or tumor-associated ligands, enhance NK cell killing capacity); and CD3: negative expression (distinguishes from T cells). The cytotoxic mechanism of NK cells involves multiple signaling pathways and molecules, including death receptor pathways, cytokine pathways, and antibody-dependent cellular cytotoxicity (ADCC) pathways. NK cells mainly mediate cytotoxicity through two mechanisms: one is the release of lytic granules containing perforin and granzymes, and the other is the induction of target cell apoptosis by expressing death ligands such as TRAIL and FasL. These death ligands bind to the corresponding receptors on target cells, activate the apoptosis signaling pathway, and ultimately lead to cell death. For example, FasL binds to the Fas receptor on target cells, triggering the apoptosis signal; while TRAIL induces apoptosis by binding to DR4 and DR5 receptors. There are differences in the use of TRAIL and FasL in NK cells at different maturity stages. Immature CD161+ / CD56-NK cells mainly rely on TRAIL-mediated cytotoxicity, not FasL or granule release-dependent; while mature CD56+NK cells use both FasL and granule release-dependent mechanisms. In addition, TRAIL plays an important role in inhibiting tumor metastasis in IFN-γ-dependent NK cells.NK cells also modulate immune responses and tumor microenvironments by secreting various cytokines (e.g., IFN-γ, TNF-α) and chemokines (e.g., CCL3, CCL4, CCL5). These cytokines not only promote the activation of other immune cells, but also directly induce tumor cell apoptosis. Thus, the maturity and functional properties of NK cells, including their cytotoxicity and tumor-killing capacity, can be assessed by detecting the expression levels of such as Fas ligand (FasL), tumor necrosis factor (TNF) alpha, TNF-related apoptosis-inducing ligand (TRAIL), or other cytokines and chemokines. In this paper, CD56, CD16, CD3, NKp30, NKp44, NKp46, NKG2A, NKG2D, FasL were detected by flow cytometry.

[0023] The first stage is iPSC induced differentiation to form a blastula between 1 day before and 0 day, the second stage is blastula induced differentiation to form mesoderm cells between 0 day and 1 day, the third stage is mesoderm cell induced differentiation to form CD34+hemogenic endothelial cells between 1 day and 4 days, the fourth stage is CD34+hemogenic endothelial cell induced differentiation to form CD34+ / CD45+cells between 4 days and 8 days. The fifth stage is to form iNK between 8 days and 28 days.

[0024] Examples 1, 2: iPSC preparation of HSPC (condition #6, #7) 1. Experimental materials

[0025] The experimental materials involved in the embodiments of the present application are shown in Table 1.

[0026] Table 1 Experimental materials 2. iPSC culture

[0027] 1) iPSCs were derived from Zhongxuan Union Company and obtained by CBMC reprogramming. After resuscitation, three generations or more were transmitted as inoculated cells; Add 1 mL Matrigel to the TC six-well plate for the first 4 days, and place it in a 5% CO2, 37°C constant temperature incubator for more than one hour; 2) Choose the well with a confluence of more than 50% to subculture. After discarding the culture medium, add 1 mL of cell gentle digestion enzyme and digest at 37°C for 5-10 min. After removing the digestion solution, blow the cells down with 1 mL of mTeSR and inoculate them into the plate. Place it in a 5% CO2, 37°C constant temperature incubator to culture the cells, and change the liquid every day; 3) On the first day, digest the cells into single cells with cell dissociation reagent, centrifuge at 1200 rpm for 5 min, resuspend with mTeSR at 1x10 5Cells were seeded at a density of 1 x 105cells / mL in a volume of 3 mL in low-attachment six-well plates and 15 μM Y27632 was added. Cells were then incubated in a 5% C02, 37 °C incubator on a shaker at 80 rpm for 24 hours to form embryoid bodies.

[0028] 3. Mesoderm differentiation Table 2. EB differentiation media table

[0029] 0 days Mesoderm differentiation media was prepared: EB differentiation media was the base, condition 6 was supplemented with 6 μΜ CHIR99021 and condition 7 was supplemented with 10 μΜ CHIR99021. Cells were centrifuged and resuspended in the media and incubated in a 5% C02, 37 °C incubator on a shaker at 80 rpm for 24 hours.

[0030] 4. Hemogenic endothelium differentiation: 1 day Factor BMP4 (5 ng / mL), VEGF (50 ng / mL), bFGF (10 ng / mL) were added to the EB differentiation media. Plates were tilted at 45° and after the cells had settled, the supernatant was removed and the cells were resuspended in the media. Cells were incubated in a 5% C02, 37 °C incubator on a shaker at 80 rpm for 48 hours and then the media was changed and the cells were incubated for a further 24 hours.

[0031] 5. Hematopoietic stem / progenitor differentiation: 4 days Hematopoietic stem / progenitor differentiation media was prepared: EB differentiation media was the base, supplemented with 50 ng / mL VEGF, 10 μΜ SB431542, 10 ng / mL bFGF. Plates were tilted at 45° and after the cells had settled, the supernatant was removed and the cells were resuspended in the media. Cells were incubated in a 5% C02, 37 °C incubator on a shaker at 80 rpm for 96 hours and the media was changed every 48 hours. The ratio of CD34+to CD45+cells within the spheres was then determined.

[0032] Example 3: iPSCs to HSPCs (condition #1) At day 1, cells were dissociated into single cells using cell dissociation reagent and centrifuged at 1200 rpm for 5 min. The cells were resuspended in mTeSR at 1 x 105cells / mL and seeded in a volume of 3 mL in low-attachment six-well plates and 15 μM Y27632 was added. Cells were then incubated in a 5% C02, 37 °C incubator on a shaker at 80 rpm for 24 hours. 5 Cells were seeded at a density of 1 x 105cells / mL in a volume of 3 mL in low-attachment six-well plates and 15 μM Y27632 was added. Cells were then incubated in a 5% C02, 37 °C incubator on a shaker at 80 rpm for 24 hours.

[0033] On day 0, prepare mesoderm differentiation medium: use EB differentiation medium as the base, add 6 μM CHIR99021, centrifuge the cells and change the medium, place them in a 5% CO2, 37°C constant temperature incubator, and culture the cells on a shaker at 80 rpm for 48 hours.

[0034] On day 2, add 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF to the EB differentiation medium. Tilt the plate 45°. After the cells settle, remove the supernatant and replace the cells with the medium. Place the cells in a 5% CO2, 37°C constant temperature incubator and culture the cells on a shaker at 80 rpm for 120 hours. Completely replace the medium every 48 hours.

[0035] On day 7, prepare hematopoietic stem / progenitor differentiation medium: Add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF to the basal EB differentiation medium. Tilt the plate 45°. After the cells settle, remove the supernatant and replace the culture medium with the culture medium. Incubate the cells in a 5% CO2, 37°C incubator on a shaker at 80 rpm for 72 hours, replacing the culture medium every 48 hours. The ratio of CD34 and CD45 in the spheres is then measured.

[0036] Examples 4, 5: Generation of HSPCs from iPSCs (Conditions #2, #3) The culture methods of Examples 4 and 5 (Conditions #2 and #3) were basically the same as those of Example 3, except that the cell seeding density on the first day was different. The density of Condition 2 was 5×10 5 Cell number / mL, 3mL; density of condition 3 is 1×10 6 Cells / mL, 3mL.

[0037] Examples 6, 7: Generation of HSPCs from iPSCs (Conditions #4, #5) One day before, cells were digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, and then resuspended in mTeSR and plated at 1×10 5 The cells were seeded in a low-adhesion six-well plate at a density of 10 cells / mL and a volume of 3 mL. 15 μM Y27632 was added and then placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 24 h.

[0038] On day 0, prepare mesoderm differentiation medium: the base medium is EB differentiation medium, and 6 μM CHIR99021 is added for condition 4 and 10 μM CHIR99021 is added for condition 5. Centrifuge the cells and replace the medium. Place the cells in a 5% CO2, 37°C constant temperature incubator and culture them on a shaker at 80 rpm for 48 hours.

[0039] 2 days Add factors 5 ng / mL BMP4, 50 ng / mL VEGF, 10 ng / mL bFGF to EB differentiation medium, tilt plate at 45°, remove supernatant after cells settle, replace cells with this medium, place in 5% CO2, 37°C incubator, culture cells on shaker at 80 rpm for 72 hours, replace medium completely every 48 hours.

[0040] 5 days Configure hematopoietic stem / progenitor differentiation medium: in EB differentiation medium, add factors 50 ng / mL VEGF, 10 mM SB431542, 10 ng / mL bFGF, tilt plate at 45°, remove supernatant after cells settle, replace cells with this medium, place in 5% CO2, 37°C incubator, culture cells on shaker at 80 rpm for 72 hours, replace medium every 48 hours. Then detect the ratio of CD34 and CD45 in the sphere.

[0041] Example 8: Preparation of iNK from HSPC Table 3 iNK differentiation medium 1

[0042] 8 days Configure iNK differentiation medium 1: add ingredients in the table to P01 medium (OptiVitro NK Cell Expansion Serum-Free Medium P01), mix well, resuspend the EB spheres harvested in examples 1-7 in iNK differentiation medium 1, place in 5% CO2, 37°C incubator, culture cells on shaker at 80 rpm for 48 hours.

[0043] 10 days, supplement iNK differentiation medium 1, 2 mL, continue to culture for 96 hours.

[0044] Table 4 iNK differentiation medium 2

[0045] 14 days Configure iNK differentiation medium 2: add ingredients in the table to P01 medium, mix well.

[0046] 14 days-28 days every 72h, half of the liquid was replaced once, and the total volume was maintained at 4mL after the replacement. The specific method was to tilt the six-hole plate at 45°, collect the supernatant culture medium, centrifuge at 1000rpm for 5min, discard half of the old culture medium, add half of the fresh iNK differentiation culture medium 2, and finally put it in a 5% CO2, 37°C constant temperature incubator for culture. At the 28th day, iNK cell counting and flow detection were performed, and the experimental results were: one iPSC could harvest more than 100 iNK before expansion. Prior art documents (Advances in NK cell production (NK cell production progress), Fang F, Xie S. 2022. PMCID: PMC8975878.) recorded that eight weeks of differentiation could achieve the experimental effect of four weeks of differentiation in the present application, that is, one iPSC could harvest more than 100 high-purity iNK after expansion.

[0047] Example 9: iNK expansion 1000IU / mL IL-2, 100ng / mL IL-12, 50ng / mL IL-15, 25ng / mL IL-18, 50ng / mL IL-21, 10% AB serum were added to P01 culture medium, and the iNK collected and combined at 28 days was 2×10 5 The number of cells / mL iNK and 4×10 5 The number of cells / mL K562-IL-21 co-cultured in a 6-well plate was 2mL per well, counted as 0 days, 2mL of culture medium was supplemented at 3 days, counted at 5 days, and the density of 5×10 5 The number of cells / mL of the new culture medium was supplemented at 5×10 5 The number of cells / mL iNK was supplemented with new culture medium for expansion, and the cells were transferred to multiple T75 at 10 days, and the density of 5×10 5 The number of cells / mL of the new culture medium was supplemented at 5×10 5 The number of cells / mL of the new culture medium was supplemented at 5×10

[0048] Example 10: iNK killing (1) Target cell staining The target cells were stained at a density of 1×10 5For the basis of unit cell, calculate the volume of target cell suspension required for killing test, 300g, 5min centrifugal collection of cells. Take the preheated to 37℃ PBS dilution CFSE staining solution to the working concentration (200nM), 500μL staining working solution resuspended target cells, 37℃ 5% CO2 incubator incubation staining 20min. After staining, wash the stained target cells twice with P01 medium, 300g, 5min, resuspend to 2×10 5 Cells / mL.

[0049] At the same time, part of the target cells are not stained with CFSE, which are used as FACS detection to adjust voltage and compensate samples.

[0050] (2) iNK cell counting Take the iNK cells expanded to D14, resuspend and count after centrifugation with P01 complete medium, take the required number of NK cells, resuspend to the appropriate density (4×10 6 Cells / mL, 4×10 5 Cells / mL) after centrifugation with P01 complete medium.

[0051] (3) Effector-target cell incubation Set the effector-target ratio gradient, 1mL per well in a 12-well plate as the inoculum volume, and perform effector-target cell killing incubation. The total incubation medium is P01 complete medium. Killing incubation at 37℃ 5% CO2 incubator for 4h.

[0052] (4) PI staining incubation Take part of the NK562 cells and CFSE single-stained K562 cells for PI staining, and then prepare for FACS compensation adjustment. The rest of the effector-target mixed incubation cells are centrifuged (500g, 5min), resuspended with 500X diluted PI staining solution (300μL / sample) with PBS, and incubated at room temperature for 30min.

[0053] (5) FACS detection After incubation and staining, wash the effector-target cells twice with 500μL PBS, resuspend with 500μL PBS, and detect by flow cytometry.

[0054] Example 11: Flow cytometry detection of EB spheres and suspended single cells EB sphere detection steps 1) Take 6 EB spheres in a 1.5mL centrifuge tube, and remove the supernatant after natural settlement; 2) Wash once with 1mL PBS, and remove the supernatant after natural settlement; 3) Add 0.5mL cell dissociation reagent, and place in a 37℃ incubator for 30min; 4) Add 0.5 mL DMF12 to blow the EBs into single cells, and pass through a 40-micron filter; 5) Transfer into a flow tube, 300g, 5 min, remove supernatant; 6) Add 1 mL PBS to wash once, 300g, 5 min, remove supernatant; 7) Add 100 μL PBS, 1 μL CD34, CD45 antibody, incubate at room temperature for 10 min in the dark; 8) Add 1 mL PBS to wash once, 300g, 5 min, remove supernatant; 9) Add 300 μL PBS to resuspend the cells before machine detection.

[0055] Suspension of single cells specific detection steps: Take an appropriate amount of supernatant in a flow tube, add 1 mL PBS, 300g, 5 min, remove supernatant; 2) Add 100 μL PBS, respectively add 1 μL CD56-PE, CD3-APC, CD16-FITC, NKP44-APC, NKP46-FITC, NKP30-APC, NKG2D-FITC, NKG2A-FITC, FasL-APC-Cy7 antibody, incubate at room temperature for 10 min in the dark; 3) Add 1 mL PBS to wash once, 300g, 5 min, remove supernatant; 4) Add 300 μL PBS to resuspend the cells before machine detection.

[0056] 8. Experimental results The overall flow chart of iPSC differentiation of iNK is shown in Figure 1 , and the details of the optimized conditions for iPSC differentiation of hematopoietic stem / progenitor cells are shown in Table 5.

[0057] Table 5

[0058] The morphology of iPSC culture under a 4x optical microscope (the first day) is shown in Figure 2 , the edges of the cells are neat, the surface is smooth and flat, and the colonies are tight, which are normal morphology iPSCs.

[0059] The morphology of EBs in the well plate is shown in Figure 3 (the first 7 are under a 4x optical microscope, and the last one is under a 10x optical microscope) The early stage of EBs is a regular spherical ball, starts to bud on the seventh day, enters the late stage of differentiation, and the single cells in the EBs start to overflow and differentiate into iNK.

[0060] The morphology of iNK under a 20x optical microscope at 28 days is shown in Figure 4, differentiated iNKs are spindle-like. The flow cytometry results of CD34 and CD45 of the hematopoietic stem / progenitor cells differentiated under different conditions are shown in Figure 5 , the hematopoietic stem / progenitor cells differentiated under different conditions, the flow cytometry results of CD34 are shown in Figure 6 , comparing conditions 1, 2 and 3, it can be obtained that the optimal differentiation density is 1x10 5 cells / mL, comparing conditions 1 and 4, it can be obtained that the optimal number of days for the second-stage differentiation is 3 days, and comparing conditions 4, 5 or 6 and 7, it can be obtained that the optimal concentration of CHIR99021 is 10 μM.

[0061] The flow cytometry results of CD56, CD3 and CD16 of the iNKs detected at 21 days are shown in Figure 11 , the proportion of CD56 reaches more than 80%, the flow cytometry results of CD56, CD3 and CD16 of the iNKs detected at 28 days are shown in Figure 7 , the purity of the iNKs is high. The flow cytometry results of NKp30, NKp44, NKp46, NKG2A, NKG2D and FasL of the iNKs detected at 28 days are shown in Figure 8 , the remaining phenotypes of the iNKs are complete. The amplification fold of the iNKs amplified for 14 days is shown in Figure 9 , the iNKs have good amplification capacity. The killing data of the iNKs amplified for 14 days are shown in Figure 10 , the amplified iNKs have killing capacity.

[0062] The above-described embodiments only express several embodiments of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for directing the differentiation of iPSCs into iNK cells, characterized in that: The following steps are involved: Stage 1: Between day 1 and day 0, iPSCs are induced to differentiate into embryoid bodies; The second stage: between 0 and 2 days, embryoid bodies are induced to differentiate into mesoderm cells; The third stage: between 1 and 6 days, mesodermal cells are induced to differentiate into CD34+ hemogenic endothelial cells; Stage 4: Between days 4 and 9, CD34+ hemogenic endothelial cells are induced to differentiate into CD34+ / CD45+ cells; The fifth stage: iNK is formed between 8 and 28 days.

2. The method for directing iPSC differentiation into iNK cells according to claim 1, wherein: In the first stage, one day before, cells were digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, resuspended in mTeSR, and concentrated at 1×10 5 ~1×10 6 The cells were seeded in a low-adhesion six-well plate at a density of 100 cells / mL, and 15 μM Y27632 was added. The plates were then placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 24 hours to form embryoid bodies.

3. The method for directing iPSC differentiation into iNK cells according to claim 2, wherein: In the second stage, on day 0, the basal medium was EB differentiation medium, 6-10 μM CHIR99021 was added, the cells were centrifuged and the medium was changed, and the cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 24-48 hours; The EB differentiation medium consists of the following components: 47% by volume of IMDM medium, 47% by volume of F12 medium, 1% by volume of ITS liquid medium supplement, 1% by volume of lipid concentrate, 2 mM L-glutamine, and 6 g / L of human serum albumin.

4. The method for directing iPSC differentiation into iNK cells according to claim 3, wherein: In the third stage, mesodermal cells are induced to differentiate into CD34+ hemogenic endothelial cells between 1 day and 3 days; On day 1, 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF were added to the EB differentiation medium. The plate was tilted 45°, and after the cells settled, the supernatant was removed and the cells were replaced with the medium. The cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 48 hours. The medium was then completely replaced with the medium and cultured for another 24 hours. The fourth stage: between 4 and 7 days, CD34+ hemogenic endothelial cells are induced to differentiate into CD34+ / CD45+ cells; On day 4, the basal medium was the EB differentiation medium, with the addition of 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. The plate was tilted 45°, and after the cells settled, the supernatant was removed and the cells were replaced with the medium. The cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 96 hours, with new medium replaced every 48 hours.

5. The method for directing iPSC differentiation into iNK cells according to claim 3, wherein: The concentration of CHIR99021 was 10 μM.

6. The method for directing iPSC differentiation into iNK cells according to claim 3, wherein: In the third stage, between 2 and 6 days, mesodermal cells are induced to differentiate into CD34+ hemogenic endothelial cells; On day 2, 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF were added to the EB differentiation medium. The plate was tilted 45°, and after the cells settled, the supernatant was removed and the cells were replaced with the medium. The cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 120 hours. The medium was completely replaced every 48 hours. The fourth stage: between 7 and 9 days, CD34+ hemogenic endothelial cells are induced to differentiate into CD34+ / CD45+ cells; On day 7, the basal EB differentiation medium was used, with the addition of 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. The plate was tilted 45°, and after the cells settled, the supernatant was removed and the cells were replaced with the medium. The cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 72 hours, with new medium replaced every 48 hours.

7. The method for directing iPSC differentiation into iNK cells according to claim 2, wherein: In the first stage, the cell seeding density was: 1×10 5 Cell number / mL.

8. The method for directing the differentiation of iPSCs into iNK cells according to claim 3, wherein: In the third stage, between 2 and 4 days, mesodermal cells are induced to differentiate into CD34+ hemogenic endothelial cells; On day 2, 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF were added to the EB differentiation medium. The plate was tilted 45°, and after the cells settled, the supernatant was removed and the cells were replaced with the medium. The cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 72 hours. The medium was completely replaced every 48 hours. The fourth stage: between 5 and 7 days, CD34+ hemogenic endothelial cells are induced to differentiate into CD34+ / CD45+ cells; On day 5, the basal EB differentiation medium was used, with the addition of 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. The plate was tilted 45°, and after the cells settled, the supernatant was removed and the cells were replaced with the medium. The cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured on a shaker at 80 rpm for 72 hours, with new medium replaced every 48 hours.

9. The method for directing the differentiation of iPSCs into iNK cells according to claim 1, wherein: The fifth stage specifically includes the following steps: On day 8, the EB spheres harvested from the fourth stage were resuspended in iNK differentiation medium 1, placed in a 5% CO 2 , 37° C. constant temperature incubator, and cultured on a shaker at 80 rpm for 48 hours; The iNK differentiation medium 1 is based on OptiVitro NK cell expansion serum-free medium P01, with the following components added: 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL Flt3, 10 ng / mL IL-15, 5 ng / mL IL-3, and 10% human AB serum by volume; On day 10, iNK differentiation medium 1 was added and culture was continued for 96 h; On day 14, the medium was half-changed every 72 h in iNK differentiation medium 2, 5% CO , and cultured in a 37°C constant temperature incubator until day 28; The iNK differentiation medium 2 is based on OptiVitro NK cell expansion serum-free medium P01, and the following components are added: 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL Flt3, 10 ng / mL IL-15, and 10% human AB serum by volume.

10. The method for directing iPSC differentiation into iNK cells according to claim 1, wherein: Before the first stage, there is also a pre-stage, which specifically includes the following steps: On the first 4 days, Matrigel was added to the TC six-well plate and placed in a 5% CO2, 37°C constant temperature incubator for more than one hour; Select iPSCs that have been passaged for three generations or more after recovery, and then subculture them when the confluence reaches more than 50%. Add cell-gentle digestion enzymes and digest at 37° for 5-10 minutes. After removing the digestion solution, blow off the cells with mTeSR, inoculate them into plates, and place them in a 5% CO2, 37°C constant temperature incubator for culture, changing the medium daily.

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