A method of directing differentiation of iPSCs to iNKs

Abstract

The present application relates to the technical field of cell differentiation, in particular to a method for differentiating iPSC into iNK. Specifically, the present application relates to a method for preparing CD34+ hematopoietic stem / progenitor cells and iNK on a shaker, which comprises the following steps: forming embryoid bodies (EB) of iPSC on a shaker, and then obtaining iNK by collecting single-cell supernatant. By adjusting the concentration of factors and the method of collecting supernatant, the present application can obtain a high proportion of CD34+ hematopoietic stem / progenitor cells and further induce the differentiation into iNK. The iNK obtained by batch harvesting can reach more than 100 iNK before the iPSC is expanded.

Description

Technical Field

[0001] This invention relates to the field of cell differentiation technology, and more specifically to a method for directing the differentiation of iPSCs into iNK cells. Background Technology

[0002] Natural killer (NK) cells, as core effector cells of the innate immune system, recognize and kill tumor cells through non-MHC-restricted mechanisms. They possess the ability to directly kill tumor cells and virus-infected cells and are less likely 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 cell source that can be infinitely expanded and has high homogeneity, gene editing flexibility, and genetic stability, are expected to overcome the donor limitations and batch heterogeneity bottlenecks of traditional PBMC / UCB sources, becoming the core source of "off-the-shelf" NK cell therapy. Future efforts should focus on optimizing differentiation mechanisms and improving safety to accelerate its clinical application translation. Existing iPSC-to-iNK differentiation protocols suffer from problems such as long cycles (>30 days) and insufficient functional maturity.

[0003] Therefore, how to provide a method that can significantly improve the efficiency of iPSC to iNK directional differentiation has become a hot topic and pain point in this industry. Summary of the Invention

[0004] To address the technical problems mentioned in the background art, this invention provides a method for directional iPSC differentiation into iNK, the technical solution of which is as follows:

[0005] A method and a dedicated culture medium system for efficiently and stably differentiating human induced pluripotent stem cells (iPSCs) into induced natural killer cells (iNKs) by optimizing the culture medium combination and differentiation sequence.

[0006] A method for targeted iPSC differentiation into iNK includes the following steps:

[0007] Phase 1: iPSC-induced differentiation to form embryoid bodies between day 1 and day 0;

[0008] The second stage: between 0 and 2 days, embryoid bodies are induced to differentiate into mesodermal cells;

[0009] The third stage: between 1 and 6 days, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells;

[0010] Phase 4: Between 4 and 9 days, CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells;

[0011] Phase 5: iNK is formed between 8 and 28 days.

[0012] Preferably, in the first stage, on the first day, cells are digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, resuspended in mTeSR, and then diluted with 1×10⁻⁶ water. 5 ~1×10 6 Cells were seeded at a density of cell number / mL into low-adsorption six-well plates, 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.

[0013] Preferably, in the second stage, at day 0, the basal medium is EB differentiation medium, with 6~10μM CHIR99021 added. The cells are centrifuged and the medium is changed. The cells are placed in a 5% CO2, 37℃ constant temperature incubator and cultured on a shaker at 80rpm for 24~48 hours.

[0014] The EB differentiation medium consists of the following components: 47% IMDM medium, 47% F12 medium, 1% ITS liquid medium supplement, 1% lipid concentrate, 2 mM L-glutamine, and 6 g / L human serum albumin.

[0015] Preferably, in the third stage, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells between 1 and 3 days.

[0016] On day 1, add 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF to the EB differentiation medium. Tilt the plate at 45° and wait for the cells to settle. Remove the supernatant and replace the medium with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 48 hours. Then replace the medium completely and culture for another 24 hours.

[0017] The fourth stage: CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells between 4 and 7 days;

[0018] On day 4, the EB differentiation medium was prepared by adding 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. The plate was tilted at 45° and the cells were allowed to settle. The supernatant was removed, and the cells were replaced with the EB differentiation medium. The plate was then placed in a 5% CO2 incubator at 37°C and cultured on a shaker at 80 rpm for 96 hours. The medium was replaced every 48 hours.

[0019] Preferably, the concentration of CHIR99021 is 10 μM.

[0020] Preferably, in the third stage, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells between 2 and 6 days.

[0021] 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 at 45° and wait for the cells to settle. Remove the supernatant and replace the cells with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 120 hours. Replace the medium completely every 48 hours.

[0022] The fourth stage: CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells between 7 and 9 days;

[0023] On day 7, using the EB differentiation medium as described above, add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. Tilt the plate at 45° and after the cells settle, remove the supernatant, replace the cells with this medium, and place it in a 5% CO2, 37°C constant temperature incubator. Culture the cells on a shaker at 80 rpm for 72 hours, replacing the medium every 48 hours.

[0024] Preferably, in the first stage, the cell seeding density is 1×10⁻⁶. 5 Cell count / mL.

[0025] Preferably, in the third stage, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells between 2 and 4 days.

[0026] 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 at 45° and wait for the cells to settle. Remove the supernatant and replace the cells with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 72 hours. Replace the medium completely every 48 hours.

[0027] The fourth stage: CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells between 5 and 7 days;

[0028] On day 5, using the EB differentiation medium as described above, add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. Tilt the plate at 45° and after the cells settle, remove the supernatant, replace the cells with this medium, and place it in a 5% CO2, 37°C constant temperature incubator. Culture the cells on a shaker at 80 rpm for 72 hours, replacing the medium every 48 hours.

[0029] Preferably, the fifth stage specifically includes the following steps:

[0030] At 8 days, the EB spheres harvested in the fourth stage were resuspended in iNK differentiation medium 1 and placed in a 5% CO2, 37°C constant temperature incubator. The cells were cultured on a shaker at 80 rpm for 48 hours.

[0031] The iNK differentiation medium 1 is based on OptiVitro NK cell expansion serum-free medium PO1, 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 (volume ratio).

[0032] At day 10, add iNK differentiation medium 1 and continue culturing for another 96 hours;

[0033] At 14 days, the iNK differentiation medium was changed halfway every 72 hours and cultured in a constant temperature incubator of 37°C with 2.5% CO2 until 28 days were completed.

[0034] The iNK differentiation medium 2 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, and 10% human AB serum by volume.

[0035] Preferably, before the first stage, a preparatory stage is further included, which specifically includes the following steps:

[0036] Four days prior, the matrix gel was added to the TC six-well plate and placed in a 5% CO2, 37°C constant temperature incubator for more than one hour to coat the plate.

[0037] Select iPSCs that have been passaged three times or more after resuscitation, and when the confluence reaches more than 50%, passage them. Add a mild cell digestion enzyme and digest at 37°C for 5-10 minutes. After removing the digestion solution, blow the cells off with mTeSR, seed them into plates, and incubate them in a 5% CO2, 37°C constant temperature incubator. Change the medium daily.

[0038] The main advantages of this invention are: the batch harvesting of iNK cells can yield more than 100 iNK cells from one iPSC. Through a combination of stage-specific factors and a dynamic culture strategy, this invention can obtain iNK cells with high purity (>80%) within 14 days (the time from hematopoietic stem differentiation to iNK) and high cytotoxicity marker expression (>95%) within 21 days, significantly enhancing its clinical translational potential. Attached Figure Description

[0039] Figure 1 A flowchart for differentiating iNK from iPSC;

[0040] Figure 2 Morphological image of iPSC cultured under a 4x optical microscope (1 day prior).

[0041] Figure 3 This is a diagram showing the morphology of EB in an orifice plate;

[0042] Figure 4 The iNK morphology image under a 20x optical microscope after 28 days;

[0043] Figure 5 The flow cytometry results for CD34 and CD45 under condition 7 are shown in the figure.

[0044] Figure 6 Flow cytometry results of CD34 in hematopoietic stem / progenitor cells differentiated under different conditions;

[0045] Figure 7 Flow cytometry results of CD56, CD3, and CD16 detected by iNK assay after 28 days;

[0046] Figure 8 The results of NKp30, NKp44, NKp46, NKG2A, NKG2D, and FasL were obtained from iNK testing over 28 days.

[0047] Figure 9 A graph showing the amplification fold results of iNK amplification after 14 days;

[0048] Figure 10 A graph showing the kill data of iNK 14 days after amplification;

[0049] Figure 11 The image shows the flow cytometry results of CD56, CD3, and CD16 detected by iNK over 21 days. Detailed Implementation

[0050] The technical solutions of the present invention will be further described below with reference to specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] As used in this article, the term "iPSC" refers to induced pluripotent stem cells, which are cells obtained by reprogramming human cells, possessing the ability to differentiate into all three germ layers and into various human cells and organs, and capable of unlimited replication and self-renewal. The cell line used in this article is iPSC derived from blood reprogramming.

[0052] As used herein, embryoid bodies (EBs) obtained from directed differentiation of iPSCs are referred to as "iEBs" or induced embryoid cells. The preparation of iEBs from iPSCs is a crucial step in stem cell research, widely applied in organoids, cell differentiation, drug screening, and many other fields. An embryoid body formed from iPSCs is a three-dimensional structure that spontaneously forms from iPSCs under specific culture conditions. This structure resembles the tissue of an early embryo and can exhibit the differentiation potential of the ectoderm, mesoderm, and endoderm. Embryoid body formation typically involves suspending single-cell or clump-shaped iPSCs in a feeder-free medium, allowing them to aggregate into spherical structures in the absence of adherent tissue. Many methods are known in the art for preparing embryoid bodies. EB spheres are formed in microplates or low-adsorption plates, shake flasks, or bioreactors. Shaking methods, hanging drop methods, or spin aggregation methods are commonly used to promote aggregation. The addition of ROCK inhibitors, such as Y27632, has proven to be a good and important small molecule for spherical aggregation, resulting in the formation of uniformly sized EBs within one day.

[0053] As used in this article, HSPCs obtained through directed differentiation of iPSCs are referred to as "iHSPCs" or induced hematopoietic stem cells and progenitor cells. Hematopoietic stem cells and progenitor cells are important cell types in the blood system, playing a crucial role in maintaining blood system stability and generating various blood cells. Hematopoietic stem cells are the original cells that generate various blood cells, also known as pluripotent hematopoietic stem cells. They possess high self-renewal capacity and multi-lineage differentiation potential, capable of differentiating into erythrocytes, leukocytes, platelets, and lymphocytes, etc. Hematopoietic progenitor cells are cells differentiated from hematopoietic stem cells and can only proliferate and differentiate into one or a few blood cell lines; they are also called directed 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.

[0054] As used in this article, NK cells obtained from iPSC-directed differentiation are referred to as "iNK" or induced natural killer cells.

[0055] Natural killer (NK) cells are key effector cells of the innate immune system, capable of directly killing tumor cells and virus-infected cells. Their functional characteristics can be verified through various experimental methods, including cell surface marker detection, cytotoxicity analysis, and cytokine secretion assays. The identification and functional analysis of NK cells rely on the expression of their specific surface markers. Based on maturity and functional status, NK cells can express the following key molecules: panleukocyte markers such as CD45 (a common marker of all hematopoietic cells, used to distinguish immune cells from other cell types); and CD56 (NCAM) (the main marker of NK cells, usually classified according to expression levels: CD56 bright (high expression): strong cytokine secretion capacity (such as IFN-γ), but low cytotoxicity). dim (low expression): High expression of CD16 (FcγRIII), exhibiting strong antibody-dependent cell-mediated cytotoxicity (ADCC) ability. Activating and inhibitory receptors include CD16 (FcγRIII) (mediates ADCC, triggering NK cell killing of target cells after binding to IgG antibodies); NKG2D (recognizes stress cell surface molecules (such as MICA / MICB), promoting NK cell activation); KIR (killer cell immunoglobulin-like receptor) (recognizes MHC-I molecules, transmitting inhibitory signals to prevent self-attack); NKG2A (CD94 / NKG2A) (inhibitory receptor, binding to HLA-E to inhibit NK cell activity); natural cytotoxic receptors (NCRs) (NKp30, NKp44, NKp46, recognize virus or tumor-associated ligands, enhancing NK cell killing ability); while CD3: negative expression (distinguishing it from T cells). The cytotoxic mechanism of NK cells involves multiple signaling pathways and molecules, including the death receptor pathway, cytokine pathway, and antibody-dependent cytotoxicity (ADCC) pathway. NK cells primarily mediate cytotoxicity through two mechanisms: one is the release of cleavage particles containing perforin and granzymes, and the other is the induction of apoptosis in target cells through the expression of death ligands such as TRAIL and FasL. These death ligands activate apoptosis signaling pathways by binding to corresponding receptors on target cells, ultimately leading to cell death. For example, FasL binds to the Fas receptor on target cells, triggering apoptosis signaling; while TRAIL induces apoptosis by binding to DR4 and DR5 receptors. The use of TRAIL and FasL differs in NK cells at different maturity stages. Immature CD161+ / CD56- NK cells mainly rely on TRAIL-mediated cytotoxicity, rather than FasL or particle release-dependent mechanisms; while mature CD56+ NK cells use both FasL and particle release-dependent mechanisms. Furthermore, TRAIL plays an important role in inhibiting tumor metastasis in IFN-γ-dependent NK cells.NK cells also regulate immune responses and the tumor microenvironment by secreting various cytokines (such as IFN-γ and TNF-α) and chemokines (such as CCL3, CCL4, and CCL5). These cytokines not only promote the activation of other immune cells but also directly induce tumor cell apoptosis. Therefore, the maturity and functional characteristics of NK cells, including their cytotoxicity and tumor-killing ability, can be assessed by detecting the expression levels of NK cells such as Fas ligand (FasL), tumor necrosis factor (TNF) α, TNF-associated apoptosis-inducing ligand (TRAIL), or other cytokines and chemokines. This article used flow cytometry to detect CD56, CD16, CD3, NKp30, NKp44, NKp46, NKG2A, NKG2D, and FasL.

[0056] The first stage involves iPSC induction and differentiation into embryoid bodies between day 1 and day 0. The second stage involves embryoid bodies induction and differentiation into mesodermal cells between day 0 and day 1. The third stage involves mesodermal cells induction and differentiation into CD34+ hematopoietic endothelial cells between day 1 and day 4. The fourth stage involves CD34+ hematopoietic endothelial cells induction and differentiation into CD34+ / CD45+ cells between day 4 and day 8. The fifth stage involves iNK cell formation between day 8 and day 28.

[0057] Examples 1 and 2: Preparation of HSPC from iPSC (Conditions #6 and #7)

[0058] 1. Experimental Materials

[0059] The experimental materials involved in the embodiments of this invention are shown in Table 1.

[0060] Table 1 Experimental Materials

[0061]

[0062] 2. iPSC culture

[0063] 1) The iPSCs were obtained from Zhongyuan Union Cell & Gene Engineering Co., Ltd., and were reprogrammed from CBMC. After resuscitation, they were passaged to the third or higher level as seed cells.

[0064] For the first 4 days, add 1 mL of matrix gel to a TC six-well plate and place it in a 5% CO2, 37℃ constant temperature incubator for more than one hour to coat it.

[0065] 2) Select wells with a cell confluence of more than 50% for passage. After discarding the culture medium, add 1 mL of mild cell digestion enzyme and digest at 37°C for 5-10 min. After removing the digestion solution, blow off the cells with 1 mL of mTeSR and seed them into a plate. Place the plate in a 5% CO2 incubator at 37°C and culture the cells. Change the medium daily.

[0066] 3) One day prior, cells were digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, resuspended in mTeSR, and diluted with 1×10⁻⁶ mol / L. 5 Cells were seeded at a density of 3 mL / mL into a low-adsorption six-well plate, and 15 μM MY27632 was added. The plate was then placed in a 5% CO2, 37°C incubator and cultured on a shaker at 80 rpm for 24 hours to form embryoid bodies.

[0067] 3. Mesodermal differentiation

[0068] Table 2 EB Differentiation Culture Medium

[0069]

[0070] On day 0, prepare the mesodermal differentiation medium: the base is EB differentiation medium, add 6 μM CHIR99021 in condition 6, add 10 μM CHIR99021 in condition 7, centrifuge the cells and change the medium, place them in a 5% CO2, 37℃ constant temperature incubator, and culture the cells at 80 rpm for 24 hours on a shaker.

[0071] 4. Hematopoietic endothelial differentiation:

[0072] Add factor BMP4 (5ng / mL), VEGF (50ng / mL), and bFGF (10ng / mL) to EB differentiation medium on day 1. Tilt the plate at 45° and wait for the cells to settle. Remove the supernatant and replace the cells with this medium. Place the plate in a 5% CO2, 37°C constant temperature incubator and culture the cells on a shaker at 80 rpm for 48 hours. Then completely replace the medium and culture for another 24 hours.

[0073] 5. Hematopoietic stem / progenitor differentiation:

[0074] 4-day preparation of hematopoietic stem / progenitor differentiation medium: Using EB differentiation medium as the base, add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. Tilt the plate at 45° and allow cells to settle. Remove the supernatant and replace the medium with this new medium. Incubate in a 5% CO2, 37°C incubator at 80 rpm on a shaker for 96 hours, changing the medium every 48 hours. Subsequently, the ratio of CD34 to CD45 in the cells is measured.

[0075] Example 3: Preparation of HSPC from iPSC (Condition #1)

[0076] The day before, cells were digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, resuspended in mTeSR, and then diluted with 1×10⁻⁶ water. 5Cells were seeded at a density of 3 mL per cell number in a low-adsorption six-well plate, and 15 μM MY27632 was added. The plate was then placed in a 5% CO2, 37°C incubator and cultured on a shaker at 80 rpm for 24 hours.

[0077] Preparation of mesodermal differentiation medium on day 0: The base is EB differentiation medium, with 6 μM CHIR99021 added. After centrifuging and changing the medium, the cells are placed in a 5% CO2, 37℃ constant temperature incubator and cultured on a shaker at 80 rpm for 48 hours.

[0078] Two days later, add 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF to the EB differentiation medium. Tilt the plate at 45° and wait for the cells to settle. Remove the supernatant and replace the cells with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 120 hours. Replace the medium completely every 48 hours.

[0079] 7-day preparation of hematopoietic stem / progenitor differentiation medium: Using EB differentiation medium as the base, add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. Tilt the plate at 45° and allow cells to settle. Remove the supernatant and replace the medium with this new medium. Incubate in a 5% CO2, 37°C incubator at 80 rpm on a shaker for 72 hours, changing the medium every 48 hours. Subsequently, the ratio of CD34 to CD45 in the cells is measured.

[0080] Examples 4 and 5: Preparation of HSPC from iPSC (Conditions #2 and #3)

[0081] The culture methods for Examples 4 and 5 (conditions #2 and #3) were basically the same as those for Example 3, except that the cell seeding density was different on the first day. The density for condition 2 was 5 × 10⁻⁶ cells / day. 5 Cell count / mL, 3mL; density in condition 3 is 1×10⁻⁶. 6 Cell count / mL, 3mL.

[0082] Examples 6 and 7: Preparation of HSPC from iPSC (Conditions #4 and #5)

[0083] The day before, cells were digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, resuspended in mTeSR, and then diluted with 1×10⁻⁶ water. 5 Cells were seeded at a density of 3 mL per cell number in a low-adsorption six-well plate, and 15 μM MY27632 was added. The plate was then placed in a 5% CO2, 37°C incubator and cultured on a shaker at 80 rpm for 24 hours.

[0084] On day 0, prepare the mesodermal differentiation medium: the base is EB differentiation medium, add 6 μM CHIR99021 in condition 4, add 10 μM CHIR99021 in condition 5, centrifuge the cells and change the medium, place them in a 5% CO2, 37℃ constant temperature incubator, and culture the cells at 80 rpm for 48 hours on a shaker.

[0085] Two days later, add 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF to the EB differentiation medium. Tilt the plate at 45° and wait for the cells to settle. Remove the supernatant and replace the cells with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 72 hours. Replace the medium completely every 48 hours.

[0086] Prepare hematopoietic stem / progenitor differentiation medium for 5 days: Add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF to the basal EB differentiation medium. Tilt the plate at 45° and allow cells to settle. Remove the supernatant and replace the medium with this new medium. Incubate in a 5% CO2, 37°C incubator at 80 rpm on a shaker for 72 hours, changing the medium every 48 hours. Subsequently, measure the ratio of CD34 to CD45 within the cells.

[0087] Example 8: Preparation of iNK cells by HSPC

[0088] Table 3 iNK differentiation medium 1

[0089]

[0090] 8-day iNK differentiation medium 1 preparation: Add the components in the table to PO1 medium (OptiVitro NK cell expansion serum-free medium PO1), mix well, resuspend the EB globules harvested in Examples 1-7 in iNK differentiation medium 1, place in a 5% CO2, 37℃ constant temperature incubator, and culture the cells on a shaker at 80 rpm for 48 hours.

[0091] After 10 days, supplement with 1.2 mL of iNK differentiation medium and continue culturing for another 96 hours.

[0092] Table 4 iNK differentiation medium 2

[0093]

[0094] Prepare iNK differentiation medium 2 for 14 days: Add the ingredients in the table to P01 medium and mix well.

[0095] From day 14 to 28, the medium was changed halfway every 72 hours, maintaining a total volume of 4 mL after each change. The method involved tilting the six-well plate at a 45° angle, collecting the supernatant, centrifuging at 1000 rpm for 5 minutes, discarding half of the old medium, adding half of fresh iNK differentiation medium 2, and finally incubating in a 5% CO2 incubator at 37°C. On day 28, iNK cell counting and flow cytometry were performed. The results showed that more than 100 iNK cells could be harvested from one iPSC before amplification. Existing literature (Advances in NK cell production, Fang F, Xie S. 2022. PMCID: PMC8975878.) indicates that eight weeks of differentiation are needed to achieve the experimental results of four weeks of differentiation in this application, meaning that more than 100 high-purity iNK cells can be harvested from one iPSC.

[0096] Example 9: iNK amplification

[0097] 1000 IU / mL IL-2, 100 ng / mL IL-12, 50 ng / mL IL-15, 25 ng / mL IL-18, 50 ng / mL IL-21, and 10% AB serum were added to PO1 medium. iNK cells were collected and combined after 28 days. Cells were then cultured at a rate of 2 × 10⁻⁶ cells / mL. 5 Cell count / mLiNK and 4×10 5 Cell count / mL: K562-IL-21 cells were co-cultured in 6-well plates, 2 mL per well, marked as day 0. 2 mL of culture medium was added on day 3, and cell counts were performed on day 5, followed by a 5 × 10⁻⁶ cell count. 5 Cells were expanded by adding fresh medium at a density of 1:1, and transferred to multiple T75 cells. Cell counts were performed after 7 days, and feeder cells (K562-IL-21) were added at a 1:1 ratio. Cells were then introduced at a concentration of 5 × 10⁻⁶ cells / mL. 5 Cells / mL iNK density were added to fresh culture medium for amplification. After 10 days, the cells were transferred to shake flasks and cultured at 5 × 10⁻⁶ cells / mL. 5 Amplification was carried out by adding fresh culture medium at a cell count / mL density, and fresh culture medium was added every 12 days at a rate of 5 × 10⁶ cells / mL. 5 Cells were seeded at a density of 100 cells / mL in multiple shake flasks, counted after 14 days, and the final cells were collected.

[0098] Example 10: iNK lethality

[0099] (1) Target cell staining

[0100] Target cells were 1×10 5To determine the baseline unit cell for the effector-to-target ratio, calculate the target cell suspension volume required for the killing assay. Collect cells by centrifugation at 300g for 5 min. Dilute CFSE staining solution to the working concentration (200 nM) with PBS preheated to 37℃. Resuspend the target cells in 500 μL of the staining working solution and incubate for staining at 37℃ with 5% CO2 for 20 min. After staining, wash the stained target cells twice with PO1 medium at 300g for 5 min, and resuspend to a volume of 2×10⁻⁶ cells / mL. 5 Cell count / mL.

[0101] Simultaneously, some target cells were prepared without CFSE staining, serving as samples for voltage adjustment and compensation in FACS detection.

[0102] (2) iNK cell count

[0103] Take iNK cells expanded to D14, centrifuge, resuspend in PO1 complete medium, count, and take the required number of NK cells. Centrifuge again, resuspend in PO1 complete medium to a suitable density (4 × 10⁻⁶). 6 Cell count / mL, 4×10 5 Cell count / mL).

[0104] (3) Incubation of target cells

[0105] A gradient of effector-to-target ratio was set up, with 1 mL seeding volume per well in a 12-well plate. Effector-to-target cell killing incubation was performed using P01 complete medium. The cells were incubated at 37°C in a 5% CO2 incubator for 4 hours.

[0106] (4) PI staining incubation

[0107] A portion of NC K562 cells and CFSE-stained K562 cells were used for PI staining, followed by FACS compensation and regulation preparation. The remaining effector-target mixed incubation cells were collected by centrifugation (500g, 5min), and the cells were resuspended in PI staining solution at 500X with PBS (300μL / sample) and incubated at room temperature in the dark for 30min.

[0108] (5) FACS detection

[0109] After incubation and staining, the target cells were washed twice with 500 μL PBS, resuspended in 500 μL PBS, and detected by flow cytometry.

[0110] Example 11: Flow cytometry detection of EB spheroids and suspended single cells

[0111] Specific testing steps for EB balls

[0112] 1) Take about 6 EB spheres into a 1.5mL centrifuge tube, let them settle naturally, and then remove the cell supernatant;

[0113] 2) Wash once with 1 mL of PBS, allow to settle naturally, and then remove the supernatant;

[0114] 3) Add 0.5 mL of cell dissociation reagent and incubate at 37°C for 30 min;

[0115] 4) Add 0.5 mL of DMF12 to blow EB spheres into single cells, and filter through a 40 μm filter membrane;

[0116] 5) Transfer to a flow cytometer, 300g, 5min, remove supernatant;

[0117] 6) Add 1 mL of PBS and wash once, 300 g, 5 min, then remove the supernatant;

[0118] 7) Add 100 μL PBS and 1 μL CD34 and CD45 antibodies, and incubate at room temperature in the dark for 10 min;

[0119] 8) Add 1 mL of PBS and wash once, 300 g, 5 min, then remove the supernatant;

[0120] 9) After resuspending the cells in 300 μL of PBS, perform the analysis.

[0121] Specific steps for detecting suspended single cells:

[0122] Take an appropriate amount of supernatant into a flow cytometer, add 1 mL of PBS, incubate at 300 g for 5 min, and then remove the supernatant.

[0123] 2) Add 100 μL of PBS, and then add 1 μL of CD56-PE, CD3-APC, CD16-FITC, NKP44-APC, NKP46-FITC, NKP30-APC, NKG2D-FITC, NKG2A-FITC, and FasL-APC-Cy7 antibodies respectively. Incubate at room temperature in the dark for 10 min.

[0124] 3) Add 1 mL of PBS and wash once, 300 g, 5 min, then remove the supernatant;

[0125] 4) After resuspending the cells in 300 μL of PBS, perform the analysis.

[0126] 8. Experimental Results

[0127] The overall flowchart of the iPSC differentiation iNK of this invention is shown below. Figure 1 The optimized conditions for iPSC differentiation into hematopoietic stem / progenitor cells are detailed in Table 5.

[0128] Table 5

[0129]

[0130] Morphological images of iPSC culture under a 4x optical microscope (first day) are shown below. Figure 2 The cells have neat edges, smooth and flat surfaces, and dense colonies, which is a normal morphology of iPSC.

[0131] The morphology diagram of EB in the orifice plate is shown below. Figure 3 (The first 7 images are morphological images under a 4x optical microscope, and the last image is morphological images under a 10x optical microscope.) In the early stage, EB spheres are regular spheres. They begin to sprout on the seventh day and enter the later differentiation stage. Single cells begin to overflow from the EB spheres and differentiate into iNK cells.

[0132] See the iNK morphology image under a 20x optical microscope after 28 days. Figure 4 The differentiated iNK cells are spindle-shaped. The flow cytometry results for CD34 and CD45 under condition 7 are shown in the figure. Figure 5 The flow cytometry results of CD34 in hematopoietic stem / progenitor cells differentiated under different conditions are shown in the figure. Figure 6 Comparing conditions 1, 2, and 3, the optimal differentiation density is found to be 1 × 10⁻⁶. 5 Cell count / mL. Comparing conditions 1 and 4, the optimal number of days for the second stage of differentiation is 3 days. Comparing conditions 4, 5 or 6, 7, the optimal concentration of CHIR99021 is 10 μM.

[0133] The flow cytometry results for CD56, CD3, and CD16 detected by iNK over 21 days are shown below. Figure 11 The proportion of CD56 reached over 80%. Flow cytometry results for CD56, CD3, and CD16 detected by iNK over 28 days are shown below. Figure 7 The iNK purity is relatively high. See the results for NKp30, NKp44, NKp46, NKG2A, NKG2D, and FasL obtained from 28-day iNK testing. Figure 8 The remaining phenotypes of iNK are relatively complete. The fold increase results of iNK amplification after 14 days are shown in [link to data]. Figure 9 iNK cells exhibit good amplification capabilities. Killer data 14 days after iNK amplification can be found in... Figure 10 The amplified iNK has lethal capabilities.

[0134] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for directional differentiation of iPSCs into iNKs, characterized in that, Includes the following steps: Phase 1: iPSC-induced differentiation to form embryoid bodies between day 1 and day 0; The second stage: between 0 and 2 days, embryoid bodies are induced to differentiate into mesodermal cells; The third stage: between 1 and 6 days, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells; Phase 4: Between 4 and 9 days, CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells; Phase 5: iNK is formed between 8 and 28 days; In the first stage, on the first day, cells were digested into single cells using a cell dissociation reagent, centrifuged at 1200 rpm for 5 min, resuspended in mTeSR, and then diluted with 1×10⁻⁶ mol / L. 5 Cells were seeded at a density of cell number / mL into low-adsorption six-well plates, and 15 μM MY27632 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. In the second stage, at day 0, the basal medium was EB differentiation medium, with 10 μM CHIR99021 added. The cells were centrifuged and the medium was changed. 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% IMDM medium, 47% F12 medium, 1% ITS liquid medium supplement, 1% lipid concentrate, 2 mM L-glutamine, and 6 g / L human serum albumin. In the third stage, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells between 1 and 3 days. On day 1, add 5 ng / mL BMP4, 50 ng / mL VEGF, and 10 ng / mL bFGF to the EB differentiation medium. Tilt the plate at 45° and wait for the cells to settle. Remove the supernatant and replace the medium with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 48 hours. Then replace the medium completely and culture for another 24 hours. The fourth stage: CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells between 4 and 7 days; On day 4, the EB differentiation medium was prepared by adding 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. The plate was tilted at 45° and the cells were allowed to settle. The supernatant was removed and the cells were replaced with the EB differentiation medium. The plate was placed in a 5% CO2 incubator at 37°C and cultured on a shaker at 80 rpm for 96 hours. The medium was replaced every 48 hours. Alternatively, in the third stage, mesodermal cells are induced to differentiate into CD34+ hematopoietic endothelial cells between 2 and 4 days. 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 at 45° and wait for the cells to settle. Remove the supernatant and replace the cells with this medium. Place the plate in a 5% CO2, 37°C incubator and culture the cells on a shaker at 80 rpm for 72 hours. Replace the medium completely every 48 hours. The fourth stage: CD34+ hematopoietic endothelial cells are induced to differentiate into CD34+ / CD45+ cells between 5 and 7 days; On day 5, using the EB differentiation medium as described above, add 50 ng / mL VEGF, 10 μM SB431542, and 10 ng / mL bFGF. Tilt the plate at 45° and after the cells settle, remove the supernatant, replace the cells with this medium, and place it in a 5% CO2, 37°C constant temperature incubator. Culture the cells on a shaker at 80 rpm for 72 hours, replacing the medium every 48 hours. The fifth stage specifically includes the following steps: On day 8, the EB spheres harvested in the fourth stage were suspended in iNK differentiation medium 1 and placed in a 5% CO2, 37°C constant temperature incubator. The cells were 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 PO1, 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 (volume ratio). At day 10, add iNK differentiation medium 1 and continue culturing for another 96 hours; At 14 days, the medium was changed halfway every 72 hours. The iNK differentiation medium was 2.5% CO2 and cultured in a constant temperature incubator at 37°C until 28 days were completed. The iNK differentiation medium 2 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, and 10% human AB serum (by volume). Before the first stage, there is a preparatory stage, which specifically includes the following steps: Four days prior, the matrix gel was added to the TC six-well plate and placed in a 5% CO2, 37°C constant temperature incubator for more than one hour to coat the plate. Select iPSCs that have been passaged three times or more after resuscitation, and when the confluence reaches more than 50%, passage them. Add a mild cell digestion enzyme and digest at 37°C for 5-10 minutes. After removing the digestion solution, blow the cells off with mTeSR, seed them into plates, and place them in a 5% CO2, 37°C constant temperature incubator for cell culture. Change the medium daily.

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