CN117305241B - Method for inducing and differentiating hiPSCs into NK cells - Google Patents
Method for inducing and differentiating hiPSCs into NK cells Download PDFInfo
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- CN117305241B CN117305241B CN202311595331.XA CN202311595331A CN117305241B CN 117305241 B CN117305241 B CN 117305241B CN 202311595331 A CN202311595331 A CN 202311595331A CN 117305241 B CN117305241 B CN 117305241B
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Abstract
The invention provides a method for inducing and differentiating hiPSCs into NK cells, which comprises the steps of inducing and differentiating hiPSCs into embryoid body cells, inducing and differentiating embryoid body cells into mesodermal cells, and inducing and differentiating mesodermal cells into CD34 + Hematopoietic stem cells, CD34 + Inducing and differentiating the hematopoietic stem cells into NK cells and performing amplification culture on the NK cells; in the step of inducing and differentiating the hiPSCs into embryoid body cells, a culture medium is DMEM/F-12 culture medium, and Y-27632 with a final concentration of 10 mu M, SB431542 with a final concentration of 10 mu M, VEGF with a final concentration of 50ng/mL, IGF1 with a final concentration of 10ng/mL and L-ascorbic acid with a final concentration of 100ng/mL are added. CD34 of the invention + The induction rate of hematopoietic stem cells and NK cells is higher, and the NK cells obtained by the invention have higher in vitro killing rate on target cells.
Description
Technical Field
The invention relates to a method for inducing and differentiating hiPSCs into NK cells, belonging to the technical field of genetic engineering.
Background
NK cells are an important component of the innate immune system, and are capable of killing malignant and virally infected cells without MHC restriction and prior insensitivity. The killing activity of NK cells against tumor cells or infected cells is mediated by activating or inhibiting cell surface receptors, including Killer Immunoglobulin Receptors (KIRs), natural Cytotoxic Receptors (NCRs) and fcγ receptor (fcγriiia) CD16a that mediate Antibody Dependent Cellular Cytotoxicity (ADCC), important features enabling NK cells to act as alloeffector cells for the treatment of refractory cancers and chronic infectious diseases.
At present, the majority of adoptive immunotherapy clinical trials based on NK cells use PB-NK cells derived from peripheral blood of a donor and UCB-NK cells derived from umbilical cord blood. These NK cells are of limited origin, vary in yield and quality, and are usually mixtures of NK cells and immune cells. Studies show that NK cells from iPSC have strong anti-tumor and antiviral activities.
CN102822332a relates to a process of inducing induced pluripotent stem cells and human embryonic stem cells into vascular cells and then inducing NK cells, single cells obtained by EB digestion of the induced pluripotent stem cells and human embryonic stem cells are inoculated into a methylellulose culture system to induce vascular cell populations, which is unfavorable for clinical scale production, and the final induction efficiency is relatively low.
CN114774365a has at least some of the following problems: (1) Expensive commercial culture media with undefined components (such as Stempro 34) are used in the whole process, so that the cost of the product is too high; (2) CD34 expression rate was low, about 75%.
CN115896019A is induced to CD34 by iPSCs cells + Direct mutagenesis in hematopoietic Stem cellsThe purity of the guided cells is 65.5%, the purity reaches 95.8% after sorting, the cost in the NK cell production process is increased by using magnetic bead sorting, and the killing power level of the NK cells obtained by the patent is limited.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for inducing and differentiating hiPSCs into NK cells, which realizes the following aims: CD34 enhancement + The induction rate of hematopoietic stem cells and NK cells improves the killing power of NK cells.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for inducing differentiation of hiPSCs into NK cells, which comprises inducing differentiation of hiPSCs into embryoid body cells, inducing differentiation of embryoid body cells into mesodermal cells, and inducing differentiation of mesodermal cells into CD34 + Hematopoietic stem cells, CD34 + Inducing and differentiating the hematopoietic stem cells into NK cells and performing amplification culture on the NK cells; in the step of inducing and differentiating the hiPSCs into embryoid body cells, a culture medium is DMEM/F-12 culture medium, and Y-27632 with a final concentration of 10 mu M, SB431542 with a final concentration of 10 mu M, VEGF with a final concentration of 50ng/mL, IGF1 with a final concentration of 10ng/mL and L-ascorbic acid with a final concentration of 100ng/mL are added.
In the step of inducing and differentiating embryoid body cells into mesodermal cells, a serum-free differentiation medium is adopted, and BMP4, 50ng/mL VEGF, 50ng/mL FGF2, 10 mu M CHIR99021 and 10 mu M SB431542 with the final concentration of 25ng/mL are added into the serum-free differentiation medium.
The mesoderm cells induce differentiation into CD34 + In the hematopoietic stem cell procedure, the medium used was serum-free differentiation medium supplemented with BMP4 at a final concentration of 25ng/mL, SCF at 30ng/mL, TPO at 50ng/mL, FLT-3L at 20ng/mL, IL-6 at 20ng/mL, IL-7 at 20ng/mL, thiazolidine at 25. Mu.M, LIF at 50 ng/mL.
The CD34 + In the step of inducing and differentiating hematopoietic stem cells into NK cells, a culture medium is adopted, and SCF with a final concentration of 50ng/mL, TPO with a final concentration of 30ng/mL, FLT-3L with a final concentration of 50ng/mL, IL-2 with a final concentration of 30ng/mL, IL-6 with a final concentration of 20ng/mL and IL-15 with a final concentration of 20ng/mL are added into the NK cell differentiation culture medium.
In the NK cell amplification culture step, the adopted culture medium is that IL-2, IL-6, IL-12 and IL-18 factors with the final concentration of 20ng/mL are added into an NK cell amplification culture medium, and in addition, the chickpea alkaloid with the final concentration of 5 mu M is added.
The serum-free differentiation medium is DMEM/F-12 medium, and is added with polyvinyl alcohol with a final concentration of 100 mu g/mL, thioglycerol with a final concentration of 100 mu M, ethanolamine with a final concentration of 20 mu M, r-HSA with a final concentration of 100 mu g/mL, L-ascorbic acid with a final concentration of 125 mu M, linoleic acid with a final concentration of 0.25 mu M, IGF1 with a final concentration of 10ng/mL and heparin with a final concentration of 100 ng/mL.
Compared with the prior art, the invention has the following beneficial effects:
in the process of differentiating hiPSCs into NK cells, CD34 + The expression level of hematopoietic stem cells was 91.8%; CD3 - CD56 + The rate of NK cells was 84.7%, significantly higher than control NK cells;
the killing capacity of the NK cells induced by the invention to target cells is consistent with or even stronger than that of the NK cells derived from cord blood;
compared with the control NK cells, the NK cells induced by the method have higher in-vitro killing rate on target cells, and have stronger cytotoxicity effect when the effective target ratio gradient dependence is higher, and the killing rate of the NK cells on the target cells is up to 94.25% when the effective target ratio is 8:1, which is obviously higher than that of the control NK cells.
Drawings
FIG. 1 is a flow chart showing the experimental procedure for obtaining NK cells by inducing the differentiation of hiPSCs according to the present invention;
FIG. 2 is a microscopic view of EB cells induced by example 2;
FIG. 3 is CD34 obtained in example 3 + Flow charts of hematopoietic stem cells;
FIG. 4 is a microscopic image of NK cells and a flow chart of cell markers obtained in example 5;
wherein FIG. 4A is a microscopic image of NK cells obtained after 2 weeks of expansion of example 5; FIG. 4B is a flow chart of cell markers CD3 and CD 56;
FIG. 5 is a graph showing comparison of killing ability of NK cells, control NK cells and cord blood-derived NK cells prepared by the present invention against tumor cell HCC-827.
Detailed Description
EXAMPLE 1 resuscitation and passaging of hiPSCs
hiPSCs, vitronectin (human vitronectin), ROCK inhibitor Blebbistatin, EDTA digest, nuwacell, ncEpic hPSC Medium suit were all purchased from the company of biosciences, inc.
(1) T75 bottle coating
Thawing the coated protein Vitronectin at room temperature, subpackaging into 120 mu L/tube, adding 9mL of DMEM/F12 culture medium into 120 mu L of Vitronectin, gently mixing and diluting, adding into a T75 bottle, gently shaking and mixing to obtain a T75 bottle coated with Vitronectin coating liquid, standing at room temperature for 2h, and using, and sucking the coating liquid with a pipette.
(2) Cell resuscitation
The water bath was preheated to 40℃and 1 frozen hiPSCs (1 mL) were removed, placed in a 40℃water bath, gently shaken by hand, thawed within 1 min, and removed when the ice crystals in the cell suspension were about to completely disappear, as observed with the naked eye, to give a cell suspension. Wiping the surface of the freezing and storing tube with 75% alcohol dust-free paper, and transferring into an ultra-clean bench; transferring the cell suspension into a 50mL centrifuge tube prepared in advance, then dropwise adding 10mL DMEM/F12 culture medium, gently shaking and uniformly mixing the cells in the process, and centrifuging for 5min at a speed of 850 rpm; the supernatant was aspirated off, 10mL of ncEpic complete medium (pre-warmed to room temperature in advance) containing Blebb istatin (final concentration 2.5. Mu.M) was added, the cells were mixed, blowing was avoided as much as possible, and a cell culture solution was obtained.
Absorbing and removing the coating liquid of the Vitronectin from the T75 bottle coated in the step (1), inoculating the uniformly mixed cell culture liquid into the T75 bottle according to 10 mL/bottle, wherein the cell density is 2 multiplied by 10 5 Shaking horizontally for three times at 37 deg.C with 5% CO 2 The culture was carried out in a concentration incubator for 24 hours, and then replaced with new ncEpic complete medium (10 mL was added per bottle), followed by daily replacement of ncEpic complete medium.
ncEpic complete medium: 4mL of the ncEpic additive (ncEpic Supplement) was added to 496mL of ncEpic basal medium (ncEpic Basal Medium) and mixed to obtain the final product.
(3) Passage of cells
Passaging was performed at a cell confluence of about 85% according to 1:5 passaging was carried out and transferred to the cells of passage 2, and when the cell confluency was 80% or more, the cells were used for the following test.
Example 2 Induction of differentiation of hiPSCs into embryoid body cells (EBs)
(1) Taking out hiPSCs in incubator, digesting with EDTA digestive juice for 7min, observing cell digestion condition under microscope, sucking digestive juice after most cells become round to obtain digested single hiPSCs, blowing off cells with induction medium, inoculating into T75 bottle, and inoculating with cell density of 1×10 6 The amount of the first induction medium added to each cell/mL was 10mL, designated day0, and the cells/mL were placed at 37℃in 5% CO 2 Culturing in an incubator.
(2) The EB cells formed were collected at day1 (see FIG. 2), 100g, and centrifuged for 5min for subsequent differentiation.
Wherein the induction medium I was DMEM/F-12 medium (available from Gibco corporation), 10. Mu.M Y-27632, 10. Mu.M SB431542, 50ng/mL VEGF, 10ng/mL IGF1, 100ng/mL L-ascorbic acid (all available from MCE) were added.
Example 3 EB cell to CD34 + Hematopoietic stem cell differentiation
(1) The centrifuged EB cells were cultured in 10mL of serum-free differentiation medium containing cytokines, and after 2 days of culture, fresh serum-free differentiation medium containing cytokines was replaced, and the induction was continued for 2 days to obtain mesodermal cells, designated day5.
The serum-free differentiation medium containing cytokines is prepared by adding BMP4, VEGF, FGF2, CHIR99021 and SB431542 (all available from MCE) at the final concentration of 25ng/mL, 50ng/mL, 10 mu M and the final concentration of 10 mu M to the serum-free differentiation medium.
(2) Collecting the induced mesoderm cells into a 50mL centrifuge tube, centrifuging 400g for 5min, discarding the supernatant, adding 10mL of serum-free differentiation medium containing cytokines and chemical small molecules, and culturing for 3 days to obtain CD34 + Hematopoietic stem cells, analysis of CD34 using flow cytometry + The expression level, as shown in FIG. 3, was CD34 + The expression level of (2) was 91.8%.
The serum-free differentiation medium containing cytokines and chemical small molecules is prepared by adding 25ng/mL of BMP4, 30ng/mL of SCF, 50ng/mL of TPO, 20ng/mL of FLT-3L, 20ng/mL of IL-6, 20ng/mL of IL-7, 25 mu M of thiazolidine and 50ng/mL of LIF into the serum-free differentiation medium.
Wherein the serum-free differentiation medium is DMEM/F-12 medium (available from Gibco corporation) containing polyvinyl alcohol (available from Merck) at a final concentration of 100. Mu.g/mL, 100. Mu.M thioglycerol (available from Sigma), 20. Mu.M ethanolamine (available from Sigma), 100. Mu.g/mL r-HSA, 125. Mu.M L-ascorbic acid, 0.25. Mu.M linoleic acid, 10ng/mL IGF1, 100ng/mL heparin (all available from MCE).
Example 4 Induction of CD34 + Differentiation of hematopoietic Stem cells into NK cells
Cell culture flasks were first coated with DLL4-Fc fusion protein. Collection of induced CD34 + Hematopoietic stem cells at 5000/cm 2 Is used for the inoculation of the cell density of the strain. Before inoculation, washing the coated cell culture flask with basic medium (namely NK cell differentiation medium), adding 10mL of NK cell differentiation medium containing factors, and placing at 37deg.C and 5% CO 2 Culturing in an incubator for 2 weeks, half-changing NK cell differentiation medium containing factors every three days, and collecting cells after differentiation to obtain total differentiated cells.
The NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, TPO with the final concentration of 30ng/mL, FLT-3L with the final concentration of 50ng/mL, IL-2 with the final concentration of 30ng/mL, IL-6 with the final concentration of 20ng/mL and IL-15 with the final concentration of 20ng/mL into the NK cell differentiation medium;
wherein NK cell differentiation medium was 50% Vol IMEM and 50% F-12 medium (all from Gibco) were mixed and contained 1% Vol glutamine, 250. Mu.M ascorbic acid, 2mM nicotinamide.
Example 5 expansion culture of NK cells
Centrifuging the collected NK cells to remove NK cell differentiation medium at 5×10 5 The cells/mL are inoculated into NK cell expansion culture medium containing additives,placing at 37deg.C and 5% CO 2 Culturing in incubator for 2 weeks (as shown in FIG. 4), half-changing NK cell amplification medium containing additives every three days, collecting amplified NK cells, and detecting cell markers CD3 and CD56 with flow antibody, and the result is shown in FIG. 4, CD3 - CD56 + The NK cell ratio was 84.7%.
The NK cell expansion medium containing the additive is obtained by adding IL-2, IL-6, IL-12 and IL-18 factors with the final concentration of 20ng/mL into the NK cell expansion medium, and adding fagomine (purchased from Sigma) with the final concentration of 5 mu M.
The NK cell expansion medium was a mixture of 50% Vol IMEM and 50% F-12 medium (both available from Gibco) containing 1% Vol glutamine, 2mM nicotinamide, 100 μg/mL heparin.
EXAMPLE 6 preparation of control NK cells
The experimental procedure of examples 2 and 3 above was followed using the hiPSCs of example 1 above as the inducer cells, with the following modifications: adopting the E3 culture medium of CN115896019A to replace the induction culture medium I; adopting the SFDM culture medium containing the cytokines to replace the serum-free differentiation culture medium containing the cytokines; the SFDM culture medium containing hematopoietic supporting factors is adopted to replace the serum-free differentiation culture medium containing cytokines and chemical small molecules; obtaining CD34 + Hematopoietic stem cells were examined for the cell marker CD34 using a flow antibody, resulting in a CD34 expression rate of 62.4%.
Then separating and purifying CD34 according to CN115896019A + Hematopoietic stem cells are sorted, and sorted CD34 + The expression rate was 96.1%.
For CD34 after sorting + Hematopoietic stem cells were induced to give NK cells according to the methods of examples 4 and 5 of the present invention, except that:
the types and concentrations of cytokines added to the NK cell differentiation medium and additives added to the NK cell expansion medium are all as disclosed in CN115896019A, the rest methods are all the operations of examples 4 and 5 of the invention, control NK cells are obtained by induction, detection of cell markers CD3 and CD56 is carried out by using a flow antibody, and the ratio of CD3-CD16+56+NK cells is 78.9%.
It can be seen that the expression rate of CD34 and the ratio of CD3-CD16+56+NK cells in example 6 were significantly lower than those of the present invention.
EXAMPLE 7 NK cell killing Activity assay
First, HCC-827 cells (human non-small cell lung cancer cells) with luciferase markers are placed in a 96-well plate for adherence culture, 10000 cells are inoculated in each well, NK cells induced by the method, control NK cells and NK cells from cord blood (obtained by culturing according to a conventional method) are taken as effector cells, and effector NK cells are added according to different target ratios of 8:1,4:1,2:1,1:1 and 1:2. Each group is provided with 3 compound holes, and the average value of the 3 compound holes is taken. The detection time is 6 hours after the cells are mixed, the fluorescence intensity is measured by using an enzyme-labeled instrument, and the killing efficiency is analyzed.
As shown in fig. 5 and table 1, the NK from cord blood and the NK prepared by the invention have consistent killing effect on target cells when the effective target ratio is greater than 1:1, and have stronger killing ability on tumor than the NK cells from cord blood when the effective target ratio is 1:1, which indicates that the killing ability of the NK cells prepared by the invention on target cells is consistent with or even stronger than the NK cells from cord blood.
Compared with the control NK cells, the NK cells induced by the method have higher in-vitro killing rate on target cells, and have stronger cytotoxicity effect when the effective target ratio gradient dependence is higher, and the killing rate of the NK cells on the target cells is up to 94.25% when the effective target ratio is 8:1, which is obviously higher than that of the control NK cells.
TABLE 1 comparison of cell killing rates for two groups of different target ratios
。
Claims (1)
1. A method for the induced differentiation of hiPSCs into NK cells, characterized by: the method comprises the steps of inducing and differentiating hiPSCs into embryoid body cells, inducing and differentiating embryoid body cells into mesodermal cells and mesodermal fineCell induced differentiation to CD34 + Hematopoietic stem cells, CD34 + Inducing and differentiating the hematopoietic stem cells into NK cells and performing amplification culture on the NK cells;
the method for inducing and differentiating the hiPSCs into embryoid cells comprises the steps of blowing off and inoculating the hiPSCs into an induction medium I, marking the hiPSCs as day0, culturing, and collecting the embryoid cells formed in day 1;
the first induction medium is DMEM/F-12 medium, and Y-27632 with the final concentration of 10 mu M, SB431542 with the final concentration of 10 mu M, VEGF with the final concentration of 50ng/mL, IGF1 with the final concentration of 10ng/mL and L-ascorbic acid with the final concentration of 100ng/mL are added into the first induction medium;
the method for inducing and differentiating the embryoid-like cells into mesoderm cells comprises the steps of adding the centrifuged embryoid-like cells into a serum-free differentiation medium containing cytokines for culture, replacing fresh serum-free differentiation medium containing cytokines after 2 days of culture, and continuously inducing for 2 days to obtain mesoderm cells;
the serum-free differentiation medium containing the cytokines is prepared by adding BMP4, VEGF, FGF2, CHIR99021 and SB431542 into a serum-free differentiation medium, wherein the final concentration of the BMP4, the VEGF and the CHIR99021 is 25ng/mL, 50ng/mL, 10 mu M and 10 mu M respectively;
the mesoderm cells induce differentiation into CD34 + The hematopoietic stem cells are obtained by centrifuging the induced mesodermal cells, removing the supernatant, adding serum-free differentiation medium containing cytokines and chemical small molecules, and culturing for 3 days to obtain CD34 + Hematopoietic stem cells;
the serum-free differentiation medium containing cytokines and chemical small molecules is prepared by adding BMP4, SCF, TPO, FLT-3L, IL-6, IL-7, thiazolidine and LIF into serum-free differentiation medium, wherein the final concentration of the thiazolidine, the thiazolidine and LIF is 25ng/mL, 30ng/mL, 50ng/mL, 20ng/mL, and IL-6, 20 ng/mL;
the CD34 + The method for inducing and differentiating hematopoietic stem cells into NK cells comprises collecting CD34 obtained by induction + Hematopoietic stem cells are inoculated, added with NK cell differentiation medium containing factors, cultured for 2 weeks, half-replaced every three days, and the cells are collected after differentiation to obtain total differentiated cells, namely NK cells;
the NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, TPO with the final concentration of 30ng/mL, FLT-3L with the final concentration of 50ng/mL, IL-2 with the final concentration of 30ng/mL, IL-6 with the final concentration of 20ng/mL and IL-15 with the final concentration of 20ng/mL into the NK cell differentiation medium;
the NK cell amplification culture method comprises the steps of centrifugally removing an NK cell differentiation culture medium from collected NK cells, inoculating the NK cell differentiation culture medium into an NK cell amplification culture medium containing additives, culturing for 2 weeks, performing half-quantity replacement of the NK cell amplification culture medium containing the additives every three days, and collecting amplified NK cells;
the NK cell expansion medium containing the additive is prepared by adding IL-2, IL-6, IL-12 and IL-18 factors with the final concentration of 20ng/mL into the NK cell expansion medium, and adding falcone with the final concentration of 5 mu M;
the serum-free differentiation medium is a DMEM/F-12 medium, and polyvinyl alcohol with a final concentration of 100 mu g/mL, thioglycerol with a final concentration of 100 mu M, ethanolamine with a final concentration of 20 mu M, r-HSA with a final concentration of 100 mu g/mL, L-ascorbic acid with a final concentration of 125 mu M, linoleic acid with a final concentration of 0.25 mu M, IGF1 with a final concentration of 10ng/mL and heparin with a final concentration of 100ng/mL are added;
the NK cell differentiation medium is formed by mixing 50% of Vol IMEM and 50% of F-12 medium, and contains 1% of Vol glutamine, 250 mu M of ascorbic acid and 2mM of nicotinamide;
the NK cell expansion medium is formed by mixing 50% of Vol IMEM and 50% of F-12 medium, and contains 1% of Vol glutamine, 2mM of nicotinamide and 100 mug/mL of heparin.
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