Method for improving efficiency of differentiating iPSC into CD34 + cells and iPSC-NK cells

Abstract

The invention relates to the technical field of cell engineering, in particular to a method for improving the efficiency of differentiating induced pluripotent stem cells (iPSC) into CD34 + cells and induced pluripotent stem cell-derived natural killer (iPSC-NK) cells and novel application of long-chain non-coding RNA (lncRNAs). According to the technical scheme, the lncRNAs capable of regulating and controlling proliferation, survival, activation and functions of the natural killer cells (NK) are systematically screened out, then the method for improving the differentiation efficiency of the iPSC to the NK cells is provided, a large number of NK cells are efficiently obtained, uniform and stable cell products are produced on a large scale, and wider clinical application of the iPSC-NK cells is promoted. According to the technical scheme, the technical problems of low in-vitro amplification efficiency, difficulty in activity maintenance and easiness in function depletion in a tumor microenvironment of the iPSC-NK cells in the prior art can be solved, and the iPSC-NK cells have ideal application and popularization prospects.

Description

Technical Field

[0001] The present invention relates to the field of cell engineering technology, and in particular to a method for enhancing the differentiation of iPSC into CD34 + A method for evaluating the efficiency of iPSC-NK cells and iPSC-NK cells and a new use of lncRNA. Background Art

[0002] Natural killer cells (NK cells) are key members of the human immune system. Activated NK cells participate in immune regulation through cytotoxicity or secretion of cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). The activation and function of NK cells are comprehensively regulated by a series of activating / inhibiting surface receptors or downstream cascade reactions such as cytokines IL-2, IL-12 and IL-15. For example, NK cells recognize the Fc segment of IgG on target cells with the help of their activating receptors CD16 and CD32, triggering antibody-dependent cell-mediated cytotoxicity (ADCC); cytokines can activate downstream signaling pathways such as JAK-STAT by binding to NK cell receptors, promoting NK cell development, proliferation, survival, activation and function.

[0003] Due to its unique target cell recognition mechanism, NK cells can be used for allogeneic transplantation for tumor immunotherapy. Some clinical trials have confirmed that NK cells derived from NK-92 cell lines, peripheral blood (PB-NK), induced pluripotent stem cells (iPSCs) and umbilical cord blood (UCB-NK), whether or not they express chimeric antigen receptors (CAR), can be used for allogeneic transplantation to treat tumors, and will not cause the common serious side effects in T cell immunotherapy, such as cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD), and therefore have received widespread attention in the industry.

[0004] iPSC-derived NK cells have been tested in clinical trials as a standardized "off-the-shelf" (OTS) product for the treatment of hematological malignancies and solid tumors, and early studies have demonstrated their effectiveness and safety. The single-cell cluster embryoid body (Spin-EBs, also known as EBs) technology is used to promote the formation of hematopoietic stem / progenitor cells (HSPC) from iPSCs, and then differentiate to obtain iPSC-derived NK cells (iPSC-NK). The iPSC-NK cell acquisition system effectively avoids the problems of difficult gene editing and cell senescence in existing PB-NK cell expansion strategies by performing gene editing at the iPSC level and large-scale expansion of HSPCs. However, the low in vitro expansion efficiency, difficulty in maintaining activity, and easy functional exhaustion of iPSC-NK cells in the tumor microenvironment (TME) are still the key factors limiting their therapeutic efficacy. Therefore, in-depth understanding of the regulatory network of NK cell development, proliferation, survival, activation and function, and the use of engineered CAR or other methods to improve the proliferation, survival and anti-tumor activity of iPSC-NK cells provide new ideas and new targets for breaking through the bottleneck problems encountered by NK cells in clinical treatment. It has important scientific value and clinical significance and is a major scientific problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a method for enhancing the differentiation of iPSC into CD34 + The invention relates to a method for improving the efficiency of induced pluripotent stem cell-derived natural killer (iPSC-NK) cells and iPSC-NK cells to solve the technical problems of low in vitro expansion efficiency, difficulty in maintaining activity, and easy exhaustion of function in the tumor microenvironment of the prior art induced pluripotent stem cell-derived natural killer (iPSC-NK) cells.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method to enhance the differentiation of iPSC into CD34 + The method for increasing the efficiency of iPSC-NK cells and iPSC-NK cells comprises step S1: obtaining induced pluripotent stem cells overexpressing MiR155HG.

[0008] Furthermore, MiR155HG overexpressing induced pluripotent stem cells were obtained by the following method:

[0009] The induced pluripotent stem cells were cultured using PGM1 medium containing Y-27632; the induced pluripotent stem cells were then infected with lentivirus, and after screening, induced pluripotent stem cells with stable miR155HG overexpression were obtained.

[0010] Furthermore, the lentivirus is a lentivirus packaged with an expression plasmid, wherein the expression plasmid is formed by integrating a gene fragment of miR155HG as shown in SEQID NO.1 into a vector GV513; the vector GV513 contains a green fluorescent protein reporter gene, and the positive rate of green fluorescent protein in induced pluripotent stem cells with stable miR155HG overexpression is >90%.

[0011] Furthermore, the method also includes step S2: inducing the differentiation of induced pluripotent stem cells overexpressing MiR155HG into hematopoietic stem / progenitor cells expressing CD34 on the surface, and step S3: inducing the differentiation of hematopoietic stem / progenitor cells expressing CD34 on the surface into induced pluripotent stem cell-derived natural killer cells.

[0012] Further, in S2, induced pluripotent stem cells overexpressing MiR155HG are induced and cultured using EBs differentiation medium to form embryoid bodies containing hematopoietic stem / progenitor cells expressing CD34 on their surface; preferably, the proportion of hematopoietic stem / progenitor cells expressing CD34 on their surface is >30%.

[0013] Furthermore, in S3, embryoid bodies containing hematopoietic stem / progenitor cells expressing CD34 on their surface are cultured adherently using NK differentiation medium; the embryoid bodies first grow adherently, then form suspended cells of hematopoietic stem / progenitor cells, and finally develop into induced pluripotent stem cell-derived natural killer cells.

[0014] Furthermore, the culture time of step S3 is 21-28 days, and the positive rate of induced pluripotent stem cell-derived natural killer cells positive for surface markers CD45 and CD56 is greater than 90%.

[0015] The technical solution also provides the use of long-chain non-coding RNA MiR155HG in hematopoietic stem / progenitor cells and natural killer cells obtained by induced culture of induced pluripotent stem cells. The gene sequence of long-chain non-coding RNA MiR155HG is shown in SEQID NO.1.

[0016] Furthermore, long noncoding RNA MiR155HG was used to increase CD34 + and CD34 + CD43 + The proportion of hematopoietic stem / progenitor cells; used to increase CD45 + CD56 + Increase the number of natural killer cells, CD56 + NKP46 + 、CD56 + CD16 + and Ki67 +The ratio of natural killer cells; used to enhance the expression of IFN-γ in natural killer cells.

[0017] Furthermore, the use of long non-coding RNA MiR155HG in obtaining hematopoietic stem / progenitor cells and natural killer cells by inducing culture of induced pluripotent stem cells comprises the following steps performed in sequence:

[0018] S1: Obtain MiR155HG overexpressing induced pluripotent stem cells;

[0019] S2: Inducing the differentiation of induced pluripotent stem cells overexpressing MiR155HG into hematopoietic stem / progenitor cells expressing CD34 on their surface:

[0020] S3: A step of inducing the differentiation of hematopoietic stem / progenitor cells expressing CD34 on their surface into induced pluripotent stem cell-derived natural killer cells.

[0021] The technical principle and beneficial effects of this technical solution are:

[0022] As a new class of regulatory molecules, long noncoding RNAs (lncRNAs) have attracted widespread attention in the field of biomedical research in recent years. Although these molecules do not encode proteins, they play a vital role in gene expression regulation, cell cycle control and differentiation. Despite this, research on how lncRNAs specifically affect the differentiation, development, proliferation, activation and function of natural killer (NK) cells is still in its early stages. As an important innate immune cell, NK cells play a key role in tumor immune surveillance and antiviral infection. Therefore, in-depth exploration of the regulatory mechanism of lncRNAs on NK cell function will not only help deepen our understanding of NK cell biology, but also provide the possibility for the development of new immunotherapy strategies.

[0023] This technical solution uses NCBI GEO public database resources, combined with advanced bioinformatics analysis methods and experimental research methods, to systematically screen out lncRNAs that have important regulatory effects on NK cell proliferation, survival, activation and its effector functions. Through this study, a type of lncRNA was discovered that can effectively improve the efficiency of induced pluripotent stem cells (iPSC) differentiation into NK cells, thereby achieving large-scale production of high-quality, uniform and stable NK cell products. This will greatly promote the use of iPSC-derived NK cells in a wider range of clinical applications, including but not limited to cancer immunotherapy, viral disease treatment, and regenerative medicine.

[0024] The lncRNAs screened by this technical solution is specifically miR155HG. Through a series of bioinformatics analysis and experimental verification, it was confirmed that miR155HG has a significant effect in regulating the proliferation, activation and effector function of NK cells. Based on this discovery, we constructed an iPSC model with overexpression of miR155HG and successfully induced it to differentiate into hematopoietic stem and progenitor cells (HSPCs), and then induced it to become NK cells (iPSC-NK cells). The results showed that the overexpression of miR155HG can not only promote the differentiation of iPSC into CD34 + CD34 + CD43 + The transformation of cells can also significantly increase the total number of iPSC-NK cells produced, especially specific subsets such as CD45 + CD56 + 、CD56 + NKP46 + and CD56 + CD16 + ratio and increase Ki67 + The results showed that overexpression of miR155HG in iPSC-NK cells could enhance the expression of IFN-γ, indicating that miR155HG plays an important role in promoting the differentiation of iPSC into NK cells.

[0025] In summary, miR155HG is not only an effective target for optimizing the iPSC-NK cell acquisition system, but its unique regulatory mechanism also opens up new avenues for basic research and clinical application of NK cells. In the future, as the understanding of lncRNAs and their regulatory networks continues to deepen, more innovative therapies based on iPSC-NK cells are expected to be developed to meet the current challenges in the medical and health field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the map of the vector GV513 of Example 1.

[0027] Figure 2 This is the map of the vector GV644 of Example 2.

[0028] Figure 3 These are the experimental study results of Example 1 showing that MiR155HG promotes hematopoiesis and the generation of iPSC-NK cells.

[0029] Figure 4 This is the experimental result of screening lncRNAs related to NK cell activation in Example 2. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

[0031] Example 1: Acquisition of induced pluripotent stem cell-derived natural killer (iPSC-NK) cells

[0032] (1) Acquisition and culture of iPSCs

[0033] In the experiments of this protocol, the specific cell line used for induced pluripotent stem cells (iPSC) is hiPSC-B1, which is purchased from Cellapy. iPSC is cultured and maintained in a conventional manner in the prior art, for example: iPSC is inoculated in a culture dish containing specific feeder layer cells (such as mouse embryonic fibroblasts), and cultured using iPSC-specific culture medium to maintain its pluripotency. The culture and maintenance of iPSC is a conventional method in the prior art, which will not be described in detail here. This protocol mainly uses iPSC to induce natural killer cells (NK cells).

[0034] (2) iPSC recovery and passaging

[0035] Cell recovery

[0036] 1×Matrigel (Corning, catalog number: 354277) was prepared in advance. After the Matrigel was melted, the Matrigel was diluted with DMEM / F12 medium according to the dilution multiple in the instructions provided by the manufacturer. 1 mL of the diluted Matrigel was added to a 6-well plate (3 wells) and allowed to stand at 37°C for 1 hour. Y-27632 (BioGems, BG0787SKU129, 1293823-10MG) was added to the PGM1 complete medium (human pluripotent stem cell medium, Pluripotency Growth Master 1, Cellapy) equilibrated at room temperature to obtain a PGM1 medium containing Y-27632. Among them, the final concentration of Y-27632 in the PGM1 medium containing Y-27632 was 10 μM. Remove iPSC cells from liquid nitrogen, thaw quickly in a 37°C water bath, transfer the cell suspension to a 15mL centrifuge tube with 4mL PGM1 complete medium, centrifuge at 300×g for 5 minutes, remove the supernatant and resuspend the cells with 1mL PGM1 medium containing Y-27632. Add 1mL PGM1 medium (containing 10μM Y-27632, i.e.: PGM1 medium containing Y-27632) to each well of a 6-well plate, transfer 1mL of cell suspension (at a ratio of 1:1, 1:2, 1:4) to three culture wells, shake the culture plate in a cross method to mix the cells, and observe the cell state under a microscope. Culture at 37°C and 5% CO2.

[0037] The above culture method lasts for less than 24 hours, then the medium is changed and the cells are cultured using PGM1 complete medium without Y-27632. The specific operation is: take out the cells from the incubator, observe under a microscope and take pictures. The culture supernatant is aspirated, 2mL DPBS buffer (Seville) is added along the wall of the well to rinse the cells and aspirate. Then, 2mL PGM1 complete medium is added and the cells are returned to the cell culture incubator for continued culture. The cells are changed every day until the growth confluence is 70-80%.

[0038] Cell passaging

[0039] Prepare PGM1 medium, PGM1 medium containing Y-27632, DPBS solution, and TrypLE in advance. TMExpressEnzyme (1×) digestion solution (Gibco). Take out iPSC cells with a confluence of 70-80% from the incubator, remove the culture medium, add 2 mL of DPBS to rinse, remove the DPBS, add 1 mL of Tryple digestion solution, digest for 3 minutes, and add 2 mL of PGM1 complete medium to terminate the digestion. Add 1 mL of PGM1 medium containing Y-27632 to a 6-well plate pre-coated with Matrigel (coating method is the same as before), and pass the cells at a ratio of 1:10-1:15. Remove the old culture medium (less than 24 hours) on the second day, replace with new PGM1 complete medium (without Y-27632), and pass the cells 1-2 times.

[0040] (3) Viral infection of iPSCs

[0041] Referring to the above method, a 6-well plate was pre-coated with matrigel, and 1 mL of PGM1 medium containing Y-27632 was added to each well of the 6-well plate. iPSC cells with a confluence of 70-80% were taken out from the incubator, the medium was removed, 2 mL of DPBS was added for rinsing, 1 mL of Tryple digestion solution was added, digestion was performed for 3 minutes, 2 mL of PGM1 complete medium was added to terminate the digestion, and the cell pellet was retained after centrifugation at 300 × g for 5 minutes. 1 mL of PGM1 medium containing Y-27632 was added to the cells, the cells were resuspended and counted, and 150,000 to 300,000 cells were inoculated in a 12-well plate, and the virus infection was performed after 18-24 hours of culture (no more than 24 hours).

[0042] 18-24 hours after cell inoculation, the cells are in a monolayer growth state. Take out the cells from the incubator, remove the old culture medium, add 500μL PGM1 complete culture medium, add the infection reagent (virus infection enhancement reagent, GeneCare Gene) according to the recommended ratio of the reagent, put the cells back into the incubator, culture for 15 minutes, select MOI = 10, 15, 20, add the virus solution, mix well, and put it back into the incubator.

[0043] 10-12 hours after adding the virus solution, take out the cells from the incubator, remove the virus-containing culture medium, add 1 mL of PGM1 complete culture medium, continue to culture for 2-3 days, and observe the GFP fluorescence expression of the cells under a fluorescence microscope. It was found that the fluorescence intensity of the cells with MOI=10 was lower than that of MOI=15 or 20, and the fluorescence intensity of the cells with MOI=15 and MOI=20 was similar. Therefore, the best MOI for infecting iPSC cells is 15.

[0044] The lentivirus (virus solution) used to infect cells was purchased from GeneCare Gene. The lentivirus was specifically a lentivirus packaged with an expression plasmid. The expression plasmid was composed of a full-length gene fragment of miR155HG (NCBI gene ID No. 114614, MIR155 hostgene, SEQ ID NO. 1) integrated into the vector GV513 (GeneCare Company, sequence map see Figure 1 ), the full-length sequence of MiR155HG is inserted into the multiple cloning site BamHI / NheI of the GV513 vector. The vector GV513 is a conventional lentiviral expression vector in the prior art, which integrates resistance genes and reporter genes for conventional screening of iPSC cells after viral transfection to ensure that iPSC cells transfected with expression plasmids are obtained and used for the subsequent process of inducing differentiation into NK cells. The resistance gene is specifically the puromycin resistance gene (Puro r ), which encodes a puromycin N-acetyltransferase that can resist the toxicity of puromycin in the screening medium by acetylation of puromycin. The reporter gene is specifically the green fluorescent protein gene (EGFP). By detecting the GFP signal in the cells by flow cytometry, the percentage of cells transfected with the expression plasmid in multiple cells can be detected. Among them, the sequence information of SEQ ID NO.1 is specifically (NR_001458.3Homo sapiens MIR155 host gene (MIR155HG), long non-coding RNA):

[0045]

[0046] The construction of expression plasmid and packaging of lentivirus can be obtained by conventional technical means of the prior art. The general process is: integrate the full-length gene fragment of miR155HG into the empty vector GV513 (insert into the GV644 multiple cloning sites AgeI and EcoRI) to obtain the expression plasmid GV513-miR155HG; use the expression plasmid and packaging plasmid to co-transfect the packaging cell line (such as HEK293T), collect the virus supernatant after transfection, and obtain the lentivirus. The lentivirus provided by Jikai Gene is obtained by the above conventional method. This scheme mainly uses the lentivirus to transfer the expression plasmid (GV513 vector integrated with the full-length gene of miR155HG) into iPSC, which overexpresses miR155HG, and then regulates iPSC, that is, iPSC cells overexpressing miR155HG can be obtained by the above method.

[0047] In contrast, the present technical solution also uses the shRNA method to reduce the expression of miR155HG in iPSC. The construction method of iPSC with reduced expression of miR155HG is similar to that of iPSC with overexpression of miR155HG. The gene fragment of shmiR155HG (SEQ ID NO.2: CTGGGATGTTCAACCTTAA) was integrated into the empty vector GV644 (GeneCare Company, sequence map see Figure 2 ), obtain the expression plasmid GV644-shmiR155HG; co-transfect the packaging cell line (such as HEK293T) with the expression plasmid and the packaging plasmid, collect the virus supernatant after transfection, and obtain the lentivirus. The lentivirus provided by GeneCare is obtained by the above conventional method. Then use the above lentivirus to transfect iPSC according to the conventional method to obtain iPSC cells with reduced miR155HG expression.

[0048] (4) Subculture, resistance screening, and seed preservation

[0049] After the optimal MOI virus transfection is completed, the virus-containing medium is removed, and PGM1 complete medium is added to continue culturing the cells until the confluence is 70-80%, and subculture, resistance screening, and seed preservation are performed according to the conventional methods of the prior art. The positive cell screening process is as follows: the cells of the first subculture (1:10) after virus infection are screened and cultured with 0.5-1ng / mL puromycin until the cell confluence reaches 70-80%, and the cells are digested for the second subculture, and the GFP expression level is detected by flow cytometry. If the GFP positive rate reaches 50%, 2-3ng / mL puromycin is added to the PGM1 medium containing Y-27632 on the first day of subculture for screening and culture, and the cells are continuously cultured, subcultured, and resistance screened until the cell expression reporter gene GFP positive rate is greater than 90% (that is, the percentage of iPSC cells transfected with the expression plasmid GV513-miR155HG or GV644-shmiR155HG in multiple iPSC cells is greater than 90%). If the GFP positive rate does not reach 50%, continue to screen for resistance with 0.5-1ng / mL puromycin until the GFP positive rate reaches 50%, and then perform screening culture with 2-3ng / mL puromycin. According to the above method, a stable iPSC strain transfected with a miR155HG expression plasmid (i.e., a stable iPSC strain overexpressing miR155HG, with a GFP positive rate greater than 90%) was obtained. Cells with a GFP positive rate greater than 90% were collected, and RNA was extracted to detect the RNA expression level of miR155HG. It was found that the expression level of miR155HG RNA (its gene sequence is shown in SEQ ID NO.1) was significantly reduced (iPSC cells with reduced miR155HG expression) or increased (iPSC cells overexpressing miR155HG), indicating that the stable strain was successfully constructed.

[0050] (5) Induction of hematopoietic stem / progenitor cells (HSPC)

[0051] It generally includes the following steps:

[0052] (5.1) Preparation before differentiation of induced pluripotent stem cells (iPSC): iPSC stable lines overexpressing miR155HG were pre-passaged twice to allow the cell confluence to reach 70-80%.

[0053] (5.2) Formation of embryoid bodies (EBs): iPSC single-cell suspensions are prepared and EBs are generated by in vitro centrifugation through the method of single-cell aggregation. At the same time, EBs are cultured in an induction medium containing specific cytokines (such as VEGF, SCF, BMP4, Flt3-L, etc.) to promote the differentiation of iPSCs into HSPCs. During the induction process, iPSCs will spontaneously aggregate to form EBs, which is an important step in simulating the embryonic development process in vivo.

[0054] (5.3) HSPC generation: After a period of induction and culture, HSPCs are generated from EBs. At this time, HSPCs have the potential to further differentiate into various blood cells.

[0055] More specifically, EBs were induced and cultured using EBs differentiation medium, including EBs differentiation medium 1, EBs differentiation medium 2, and EBs differentiation medium 3. When the growth confluence of the iPSC stable strain overexpressing miR155HG reached 70-80%, Tryple digestion solution was added and digested at 37°C for 2-3 minutes; the cell suspension was transferred to a 15 ml centrifuge tube, centrifuged at 1000 rpm for 4 minutes, the supernatant was discarded, and the cells were resuspended with an appropriate volume of EBs differentiation medium 1. EBs differentiation medium 1 is an APEL2 medium (Stem cell, catalog number: 05275) containing a first specific cytokine, and the first specific cytokine includes SCF with a final concentration of 40 ng / mL, VEGF 165 with a final concentration of 50 ng / mL, BMP4 with a final concentration of 20 ng / mL, FGF-basic with a final concentration of 20 ng / mL, Activin A with a final concentration of 10 ng / mL, and Y-27632 with a final concentration of 10 μM. The resuspended cells were counted and the cell density was adjusted to 80,000 cells / mL. 100 μL of cell suspension was added to the middle 60 wells of the 96-well plate, and 150 μL of sterile water was added to each of the 36 wells at the edge to reduce the volatilization volume of the culture medium in the cell suspension.

[0056] On the 4th day of EBs culture, the medium was changed to EBs differentiation medium 2. EBs differentiation medium 2 is APEL2 medium (Stem cell, catalog number: 05275) containing a second specific cytokine, which includes SCF at a final concentration of 50 ng / mL, BMP4 at a final concentration of 20 ng / mL, FGF-basic at a final concentration of 20 ng / mL, VEGF165 at a final concentration of 100 ng / mL, IGF-II at a final concentration of 100 ng / mL, and SB 431542 at a final concentration of 3 μM).

[0057] On the 8th day of EBs culture, the culture medium was replaced with EBs differentiation medium 3 until the end of EBs differentiation culture (CD34 +Cell ratio>30%), and EBs differentiation medium 3 was used for culture. EBs differentiation medium 3 is APEL2 medium containing the third specific cytokine, and the third specific cytokine includes SCF with a final concentration of 50 ng / mL, FGF-basic with a final concentration of 20 ng / mL, VEGF 165 with a final concentration of 200 ng / mL, IGF-II with a final concentration of 20 ng / mL, Flt3-Ligand with a final concentration of 25 ng / mL, TPO with a final concentration of 25 ng / mL, IL-3 with a final concentration of 50 ng / mL, EPO with a final concentration of 3 U / mL, and IL-6 with a final concentration of 25 ng / mL.

[0058] The above-mentioned cytokines or reagents are described in detail. These cytokines or reagents are conventional substances in the prior art and can be obtained by commercial means. SCF is Stem Cell Factor; VEGF 165 is Vascular Endothelial Growth Factor 165; BMP4 is Bone Morphogenetic Protein 4; FGF-basic is Basic Fibroblast Growth Factor; Activin A is a member of the TGF-β superfamily and is a cytokine involved in regulating cell proliferation, differentiation and inflammatory response; IGF-II is Insulin-like Growth Factor II. II); Flt3-Ligand is Fms-related tyrosine kinase 3 ligand; TPO is thrombopoietin; IL-3 is interleukin-3; EPO is erythropoietin; IL-6 is interleukin-6; Y-27632 is an inhibitor of Rho-associated coiled-coil protein kinase (ROCK); SB 431542 is a small molecule inhibitor of ALK5 / TGFBR1 (transforming growth factor β receptor type I).

[0059] On the 8th and 12th days of EBs culture, 15-20 wells of EBs were collected and flow cytometry was used to detect the expression of CD34 (endothelial cells or endothelial progenitor cells) in embryoid body cells. +When the cell ratio was >30%, EBs were transferred to NK differentiation conditions for adherent culture. On the 7th day of EBs adherent culture (for details of adherent culture, see the description in (6) below), the HSPC surface markers CD34, CD43, CD45, etc. in the generated suspension cells were detected.

[0060] (6) Differentiation of natural killer cells (NK cells)

[0061] In the process of natural killer cell differentiation, embryoid body EBs are first cultured on the wall. During the culture process, a large number of suspension cells are produced under the induction of specific cytokines, and the suspension cells are further differentiated into NK cells.

[0062] More specifically, the culture medium used in the culture process is NK differentiation medium, including NK differentiation medium 1 and NK differentiation medium 2. Carefully transfer EBs from the 96-well plate to a 10 cm culture dish using a 10 mL serological pipette, add DMEM / F12 and gently blow down the EBs, discard the medium, and add 2 mL of NK differentiation medium 1. Transfer 14-16 EBs to each well of a 6-well plate coated with 2% gelatin. The base medium of NK differentiation medium 1 is GlutaMAX TM F12 medium (Thermo, 31765035) and GlutaMAX TM DMEM medium (Thermo, 10569010) was mixed at a ratio of 2:1 to form a basal medium, and a fourth specific cytokine was added, including: IL-3 at a final concentration of 5 ng / ml, Flt3 ligand at a final concentration of 10 ng / ml, IL-15 (Interleukin-15) at a final concentration of 10 ng / ml, SCF at a final concentration of 20 ng / ml, and IL-7 (Interleukin-7) at a final concentration of 20 ng / ml. NK differentiation medium 1 was used to culture at 37°C, 5% CO2 to induce the differentiation of cells into NK cells. In the second week of differentiation, NK differentiation medium 1 was replaced with NK differentiation medium 2. The only difference between the two culture media is that NK differentiation medium 2 does not add IL-3 at a final concentration of 5 ng / ml, and the rest is exactly the same.

[0063] During this stage, EBs first adhere to the wall, then suspension cells gradually appear, and finally develop into NK cells after induction differentiation. During the NK cell differentiation process, the culture medium is changed half a week. After 21-28 days of EBs transfer to NK differentiation culture, >90% CD45 + CD56 + NK cells. CD45 + CD56 +NK cell surface markers such as CD16 and NKp46 were detected.

[0064] (7) Culture and functional detection of NK cells:

[0065] Culture: After embryoid bodies were transferred to NK differentiation culture for 21-28 days, >90% CD45+CD56+NK cells were obtained and then cultured in NK differentiation medium (GlutaMAX TM F12 medium (Thermo, 31765035) and GlutaMAX TM DMEM medium (Thermo, 10569010) was mixed at a ratio of 2:1 and supplemented with specific cytokines such as IL-7 and IL-15), and the medium was replaced regularly to provide adequate nutrients and growth factors.

[0066] Functional testing: Detect the functions of NK cells by flow cytometry, cytotoxicity assays, etc., such as NK cell-specific markers (such as CD56, CD16, etc.), cytotoxicity, degranulation, and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0067] This technical solution intervenes in the expression of miR155HG in iPSCs, thereby inducing NK cells (iPSC-NK cells, induced pluripotent stem cell-derived natural killer cells). On the 12th day of EBs formation, miR155HG can promote CD34 + Cells and CD34 + CD43 + The proportion of cells in EBs adherent culture was significantly increased, and the effect was more obvious in cells on the 7th day of EBs adherent culture. On the 28th day of NK cell differentiation, miR155HG was found to increase the proliferation of iPSC-NK cells (CD45 + CD56 + ) (increased nearly 3 times), CD56 + NKP46 + iPSC-NK cells and CD56 + CD16 + iPSC-NK cells and Ki67 + The proportion of iPSC-NK cells was increased, and the proportion of early and late apoptotic cells in iPSC-NK cells was reduced. Functional experiments found that overexpression of miR155HG in iPSC-NK cells increased the expression of IFN-γ. The above results suggest that miR155HG can be used as an effective target for optimizing the iPSC-NK cell acquisition system.

[0068] Specifically, in Figure 3In the figure, AB represents the expression of surface antigens (CD34, CD43 and CD45) of hematopoietic progenitor cells of iPSC-shCtrl / shmiR155HG (A) or iPSC-Ctrl / miR155HG (B) on day 8 and day 12 of embryonic body (EB) formation and day 7 of adherent culture differentiation by flow cytometry analysis. iPSC-shCtrl specifically refers to iPSC cells transfected with empty expression plasmid GV644 by lentivirus. iPSC-shmiR155HG specifically refers to iPSC cells transfected with expression plasmid GV644-shmiR155HG by lentivirus (iPSC cells with reduced expression of miR155HG). iPSC-Ctrl specifically refers to iPSC cells transfected with empty expression plasmid GV513 by lentivirus. iPSC-miR155HG specifically refers to iPSC cells transfected with the expression plasmid GV513-miR155HG by lentivirus (iPSC cells overexpressing miR155HG). CG is the total number of suspended cells detected by cell counting experiment (C), and the CD56 expression of iPSC-miR155HG and iPSC-Ctrl cells differentiated into NK cells on day 28 of iPSC-NK cell differentiation by flow cytometry. + NKP46 + iPSC-NK cells (D), CD56 + CD16 + iPSC-NK cells (E), Ki67 + The percentage of iPSC-NK cells (F) and early apoptosis (Annexin V+7-AAD-) and late apoptosis (Annexin V+7-AAD+) (G). H is the expression level of IFN-γ in iPSC-NK-shCtrl / shmiR155HG after 24 hours of IL-15 stimulation by flow cytometry.

[0069] iPSCs were infected with lentivirus containing expression plasmids expressing full-length miR155HG or shmiR155HG and their control plasmids (Ctrl or shCtrl) and conventionally differentiated into hematopoietic stem / progenitor cells (HSPCs) and NK cells. To investigate the role of miR155HG in hematopoietic development, we analyzed the expression of surface antigens (CD34, CD43, and CD45) by hematopoietic progenitor cells generated on days 8 and 12 of EB formation and on day 7 of adherent culture of EBs under NK differentiation conditions. Knockdown of miR155HG reduced CD34 on day 12 of EB formation. + Cells and CD34 + CD43 +The proportion of HSPC cells was significantly increased in iPSCs, while overexpression of miR155HG in iPSCs increased the percentage of these cells, and the effect was more obvious in cells on day 7 of adherent culture ( Figure 3 AB). Among them, CD34 + Cells are cells that express the CD34 marker on their surface. CD34 is a glycosylated transmembrane protein that is often used as a marker for HSPCs. These cells have multi-lineage differentiation potential, meaning they can differentiate into all types of blood cells, including red blood cells, white blood cells, and platelets. CD43 (also known as the leukocyte adhesion molecule sialophorin) is also a large molecular weight transmembrane glycoprotein that is widely expressed on many types of blood cells. CD34 + CD43 + The cells are cells that express both markers at the same time. Compared with cells that only express CD34, they further develop along the differentiation path toward NK cells. They are progenitor cells that are in the early development stage and have the potential to become NK cells (precursor cells that have the potential to develop into NK cells). Therefore, the experimental data show that the overexpression of miR155HG promotes the differentiation of iPSCs into NK cells.

[0070] On day 28 of NK cell differentiation, overexpression of miR155HG increased the expression of iPSC-NK cells (CD45 + CD56 + ) (increased nearly 3 times) Figure 3 C), CD56 + NKP46 + iPSC-NK cells ( Figure 3 D) and CD56 + CD16 + iPSC-NK cells ( Figure 3 E). CD45 is called leukocyte common antigen (LCA), which is a marker that is widely present on all types of white blood cells, except mature red blood cells and platelets. It plays an important role in regulating cell signaling and is an important marker for identifying white blood cells in the blood system. CD56 is a neural cell adhesion molecule (NCAM), which is used as an important marker for identifying natural killer cells (NK cells). CD45 + CD56 +The cells specifically refer to natural killer cells (NK cells). NKP46 (also known as NCR1, natural cytotoxicity receptor 1) is an activating receptor that is almost exclusively expressed on the surface of all NK cells. NKP46 is involved in the process of NK cells recognizing and killing target cells. It triggers the activation and cytotoxic effects of NK cells by recognizing ligands on virus-infected or transformed cells. CD16 (FcγRIIIa) is a low-affinity immunoglobulin G Fc region receptor that is mainly expressed on NK cells, macrophages and neutrophils. For NK cells, the presence of CD16 is particularly associated with antibody-dependent cell-mediated cytotoxicity (ADCC). By binding to the surface of target cells that have bound antibodies, CD16 triggers NK cells to attack these target cells. CD56 + NKP46 + Cells and CD56 + CD16 + CD45 + CD56 + A subtype of NK cells, which to some extent reflects the immune killing function of NK cells. From the above data, it can be seen that overexpression of miR155HG in iPSC cells can increase the total amount of NK cells (CD45 + CD56 + iPSC-NK cells) and to a certain extent increase the number of functional (immune) NK cells (CD56 + NKP46 + iPSC-NK cells and CD56 + CD16 + iPSC-NK cells).

[0071] At day 28 after embryoid bodies were transferred to NK cells for differentiation, it was found that overexpression of miR155HG increased Ki67 + The proportion of iPSC-NK cells ( Figure 3 F), and reduced the proportion of early and late apoptotic cells in iPSC-NK cells ( Figure 3 G). Ki67 is a nuclear protein whose expression is closely related to cell proliferation. Ki67 antigen is expressed in the G1, S, G2, and M phases of the cell cycle, but not in cells in the quiescent phase (G0 phase). Therefore, Ki67 is often used as a marker to assess cell proliferation activity and is widely used in tumor biology, pathology research, and immune cell dynamics research. Ki67 +iPSC-NK cells refer to natural killer cells (NK cells) that are undergoing active division and proliferation. From the above data, it can be seen that overexpression of miR155HG in iPSC cells can enhance the proliferation activity of NK cells through conventional induction culture using existing technologies. Expression of miR155HG can reduce the proportion of early and late apoptotic cells in iPSC-NK cells, which means that overexpression of miR155HG can effectively promote the survival rate of iPSC-NK cells and reduce cell loss caused by programmed cell death (i.e., apoptosis). Specifically, early apoptosis refers to cells that have begun to initiate the apoptosis program but have not completely lost their function, while late apoptosis involves cells that have irreversibly entered the cell death pathway. Overexpression of miR155HG helps maintain the health of iPSC-NK cells and prevents them from entering the apoptotic pathway, thereby increasing the quantity and quality of the functional NK cells ultimately obtained.

[0072] Functional experiments found that overexpression of miR155HG in iPSC-NK cells increased the expression of IFN-γ ( Figure 3 H). IFN-γ, or interferon-gamma, is an important cytokine that plays a key role in the immune system. It is mainly produced by activated T lymphocytes (especially helper T cell type 1, Th1 cells), natural killer cells (NK cells) and partially activated macrophages. By overexpressing miR155HG in iPSC cells, the NK cells obtained by conventional induction culture of existing technologies have not only been increased in number, but also further enhanced in efficacy.

[0073] In summary, miR155HG can promote the differentiation, proliferation, survival and effector function of NK cells.

[0074] Example 2: Screening of long noncoding RNA (lncRNA) associated with NK cell activation

[0075] To screen lncRNAs associated with NK cell activation, the GEO database (GSE110446) was used for screening. The screening criteria were: (1) up-regulated more than 2-fold in activated NK cells compared with the resting state; (2) located in the intergenic region of the genome; (3) co-expressed with NK cell effector molecules (NCR1, EOMES, TBX21, CD69, DNAM1, KLRK1, PERF, IFNG or GZMB) in tumors. Among them, NCR1 (Natural Cytotoxicity Triggering Receptor 1), also known as NKp46, is an activating receptor on the surface of natural killer cells (NK cells) that is involved in the recognition and killing of tumor cells and virus-infected cells. EOMES (Eomesodermin) is a transcription factor that is essential for the functional maturation of T cells and natural killer cells (NK cells), especially in enhancing cytotoxic function. TBX21 encodes T-bet protein, which is also a transcription factor. It is essential for Th1 cell differentiation and plays an important role in the development and function of natural killer cells (NK cells), CD8+T cells, etc. CD69 is a C-type lectin, which is expressed on the surface of activated lymphocytes, natural killer cells, etc. as an early activation marker and participates in cell activation signal transduction. DNAM1 (DNAX auxiliary molecule 1) is CD226, which is a co-stimulatory molecule expressed on activated T cells and natural killer cells, promoting cell-mediated cytotoxicity and cytokine production. KLRK1, also known as NKG2D, is an activation receptor on natural killer cells and several other immune cells, used to recognize cells after stress or infection. PERF is perforin, a protein released by cytotoxic T lymphocytes and natural killer cells that can form holes in the target cell membrane, leading to cell death. IFN-γ (Interferon Gamma) encoded by IFNG is an important cytokine, mainly produced by natural killer cells, Th1 cells, CD8+T cells, etc., with antiviral, immunomodulatory and anti-tumor properties. Granzyme B, a protein encoded by GZMB, is a serine protease stored in cytotoxic granules and released into target cells by cytotoxic T lymphocytes and natural killer cells to induce apoptosis of target cells.

[0076] The results showed that (see Figure 4), in the GEO database (GSE110446), compared with resting NK cells, a total of 141 lncRNAs were expressed at least under one activation stimulus, 11 lncRNAs were expressed under ADCC and cytokine stimulus, 17 lncRNAs were expressed under ADCC and K562 co-culture, 14 lncRNAs were expressed under K562 co-culture and cytokine stimulus, and 8 lncRNAs were expressed under all three activation stimulus conditions. After experimental verification and co-expression analysis with NK cell effector molecules, miR155HG had the most prominent effect, and was used as a follow-up research object to explore its role in NK cell proliferation, activation and effector function.

[0077] Specifically, in Figure 4 In Figure 1, A is the screening process of lncRNAs related to NK cell activation. Cytokines are interleukin (IL)-12 and IL-18; ADCC is antibody-dependent cellular cytotoxicity; K562 is a tumor cell line sensitive to NK cells. B is a Venn diagram showing the upregulated lncRNAs in NK cells activated under three different stimulation conditions. C is a histogram showing the upregulated expression levels of 8 lncRNAs in NK cells under three different activation stimulation conditions. D is the expression level changes of the 8 lncRNAs in C after NK92 cells were stimulated with IL-15 alone or with IL-2 plus IL-12 for 12 hours. E is the correlation between miR155HG and RP11-63K6.7 TPM (transcripts per kilobase of exon model per million mapped reads) and NK effector function molecule TPM (NCR1, EOMES, TBX21, CD69, DNAM1, KLRK1, PERF, IFNG and GZMB) in 33 different cancer types in TCGA dataset (http: / / gepia2.cancer-pku.cn / #index). There is a significantly higher correlation between miR155HG and NK effector function molecules.

[0078] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method to enhance the differentiation of iPSC into CD34 + The method for improving the efficiency of cells and iPSC-NK cells is characterized by: The method comprises step S1: obtaining induced pluripotent stem cells overexpressing MiR155HG.

2. A method for enhancing the differentiation of iPSC into CD34 + The method for improving the efficiency of cells and iPSC-NK cells is characterized by: MiR155HG overexpressing induced pluripotent stem cells were obtained by the following method: The induced pluripotent stem cells were cultured using PGM1 medium containing Y-27632; the induced pluripotent stem cells were then infected with lentivirus, and after screening, induced pluripotent stem cells with stable miR155HG overexpression were obtained.

3. A method for enhancing the differentiation of iPSC into CD34 + The method for improving the efficiency of cells and iPSC-NK cells is characterized by: The lentivirus is a lentivirus packaged with an expression plasmid, wherein the expression plasmid is formed by integrating a gene fragment of miR155HG shown in SEQ ID NO.1 into a vector GV513; the vector GV513 contains a green fluorescent protein reporter gene, and the positive rate of green fluorescent protein in induced pluripotent stem cells stably overexpressing miR155HG is greater than 90%.

4. A method for improving the efficiency of iPSC differentiation into CD34+ cells and iPSC-NK cells according to any one of claims 1 to 3, characterized in that: The method further comprises step S2: inducing the differentiation of induced pluripotent stem cells overexpressing MiR155HG into hematopoietic stem / progenitor cells expressing CD34 on their surface, and step S3: inducing the differentiation of hematopoietic stem / progenitor cells expressing CD34 on their surface into induced pluripotent stem cell-derived natural killer cells.

5. A method for enhancing the differentiation of iPSC into CD34 + The method for improving the efficiency of cells and iPSC-NK cells is characterized by: In S2, induced pluripotent stem cells overexpressing MiR155HG are induced and cultured using EBs differentiation medium to form embryoid bodies containing hematopoietic stem / progenitor cells expressing CD34 on their surface; preferably, the proportion of hematopoietic stem / progenitor cells expressing CD34 on their surface is >30%.

6. A method for enhancing the differentiation of iPSC into CD34 + The method for improving the efficiency of cells and iPSC-NK cells is characterized by: In S3, embryoid bodies containing hematopoietic stem / progenitor cells expressing CD34 on their surface are cultured adherently using NK differentiation medium; the embryoid bodies first grow adherently, then form suspended cells of hematopoietic stem / progenitor cells, and finally develop into induced pluripotent stem cell-derived natural killer cells.

7. A method for enhancing the differentiation of iPSC into CD34 + The method for improving the efficiency of cells and iPSC-NK cells is characterized by: The culture time of step S3 is 21-28 days, and the positive rate of induced pluripotent stem cell-derived natural killer cells positive for surface markers CD45 and CD56 is greater than 90%.

8. Use of long non-coding RNA MiR155HG in induced pluripotent stem cell culture to obtain hematopoietic stem / progenitor cells and natural killer cells, characterized in that: The gene sequence of long non-coding RNA MiR155HG is shown in SEQ ID NO.

1.

9. The use of the long non-coding RNA MiR155HG according to claim 8 in obtaining hematopoietic stem / progenitor cells and natural killer cells by inducing pluripotent stem cells, characterized in that: It is used to increase CD34 + and CD34 + CD43 + The proportion of hematopoietic stem / progenitor cells; used to increase CD45 + CD56 + Increase the number of natural killer cells, CD56 + NKP46 + 、CD56 + CD16 + and Ki67 + The ratio of natural killer cells; used to enhance the expression of IFN-γ in natural killer cells.

10. The use of the long non-coding RNA MiR155HG according to claim 8 in obtaining hematopoietic stem / progenitor cells and natural killer cells by inducing pluripotent stem cells for culture, characterized in that: The method includes the following steps in sequence: S1: Obtain MiR155HG overexpressing induced pluripotent stem cells; S2: Inducing the differentiation of induced pluripotent stem cells overexpressing MiR155HG into hematopoietic stem / progenitor cells expressing CD34 on their surface: S3: A step of inducing the differentiation of hematopoietic stem / progenitor cells expressing CD34 on their surface into induced pluripotent stem cell-derived natural killer cells.

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