PRDM1 overexpression embryoid body as well as preparation method and application of PRDM1 overexpression embryoid body

Abstract

The invention discloses a preparation method of an embryoid body for overexpressing PRDM1, which accelerates the formation and maturation process of the embryoid body and shortens the amplification period of the embryoid body through a lentivirus-mediated PRDM1 overexpression system, reveals the regulation mechanism of the embryoid body in combination with a genomics technology, and provides a new technical strategy and theoretical basis for optimizing the differentiation efficiency of stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to an embryoid body overexpressing PRDM1, a preparation method thereof, and an application thereof. Background Art

[0002] PRDM1, namely B lymphocyte-induced maturation protein (Blimp-1), is an important transcription factor. PRDM1 plays a key role in the immune system. Especially in liver type 1 innate lymphoid cells (Type 1 ILCs), PRDM1 exhibits its unique regulatory function. The deletion of PRDM1 will lead to changes in the ratio of cNK cells to ILC1s in the liver, tending to increase the number of cNK cells, and at the same time affecting the maturation and function of these cells. In the field of stem cell research, PRDM1 is considered an important factor for human primordial germ cells because it can regulate the differentiation of the endoderm of human primordial germ cells and promote the differentiation of human pluripotent stem cells into human primordial germ cells without the action of cytokines such as BMP4.

[0003] An embryoid body (EB) is a cell aggregate spontaneously formed by embryonic stem cells or induced pluripotent stem cells under specific suspension culture or defined attachment conditions in vitro. Its core feature is the ability to mimic the early embryonic development process and gradually form a multi-lineage cell structure containing endoderm, mesoderm, and ectoderm through non-directed differentiation. In the initial stage of in vitro culture, stem cells form loose aggregates through cell-cell adhesion. At this time, the diameter of the embryoid body is usually between dozens and hundreds of micrometers. As the culture time extends, the embryoid body gradually increases to hundreds to thousands of micrometers through division and proliferation and extracellular matrix secretion. The increase in the embryoid body is accompanied by a change in the aggregate morphology, with the outer layer of cells tending to epithelial-like transformation and the inner layer of cells maintaining an undifferentiated or progenitor cell state. The embryoid body technology has been widely used in in vitro organoid construction, disease model simulation, and regenerative medicine research. However, the randomness of its differentiation direction and the differences between batches are still key issues to be optimized.

[0004] The directed differentiation process of traditional pluripotent stem cells usually needs to go through the embryoid body formation stage, the embryoid body amplification stage, the directed induction differentiation stage, and the target cell / organoid maturation stage. The overall differentiation cycle is about four weeks. Among them, the embryoid body amplification stage involves the dynamic proliferation and morphological remodeling of cell populations. Its time consumption accounts for about 30-40% of the total process and is easily affected by multiple factors such as the culture microenvironment, process parameter instability, or insufficient regulation of key signaling pathways. As a result, the embryoid body amplification process is the bottleneck restricting the improvement of stem cell differentiation efficiency. Therefore, it is urgent to adjust the culture strategy in the embryoid body amplification stage, shorten the amplification cycle, achieve the efficient transformation of embryoid bodies into target lineages, and optimize the differentiation efficiency of pluripotent stem cells. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art. To solve the problem of the too long directed differentiation cycle of stem cells, through a lentivirus-mediated PRDM1 overexpression system, the formation and maturation process of embryoid bodies is accelerated, the amplification cycle of embryoid bodies is shortened, and its regulatory mechanism is revealed by combining genomics technology, providing a new technical strategy and theoretical basis for optimizing the differentiation efficiency of stem cells.

[0006] The present invention is achieved by the following technical solutions:

[0007] On the one hand, the present invention provides a method for preparing embryoid bodies overexpressing PRDM1, comprising the following steps:

[0008] S1, pluripotent stem cell construction: adding lentiviral particles overexpressing PRDM1 to pluripotent stem cells to be transfected, and obtaining pluripotent stem cells with low-purity overexpression of PRDM1 after transfection;

[0009] S2, purification: subjecting the pluripotent stem cells with low-purity overexpression of PRDM1 to Puro drug screening to obtain pluripotent stem cells with high-purity overexpression of PRDM1;

[0010] S3, differentiation: differentiating the pluripotent stem cells with high-purity overexpression of PRDM1 to obtain embryoid bodies overexpressing PRDM1.

[0011] In the above technical solution, the nucleotide sequence of PRDM1 is as shown in SEQ ID NO.1.

[0012] In the above technical solution, the pluripotent stem cells to be transfected are H9.

[0013] On the other hand, the present invention provides a method for preparing embryoid bodies overexpressing PRDM1, comprising the following steps:

[0014] Step 1, preparation of a lentiviral construct overexpressing PRDM1:

[0015] Select the full-length cDNA sequence of PRDM1, and clone it into a lentiviral expression vector carrying the RSV promoter; obtain a recombinant lentiviral expression vector;

[0016] For the above recombinant lentiviral expression vector with correct sequencing, use a plasmid large-scale extraction kit to extract and purify to obtain a high-quality recombinant plasmid, that is, a lentiviral construct overexpressing PRDM1, for lentiviral packaging and transfection of target cells;

[0017] Step 2, preparation of lentiviral particles overexpressing PRDM1:

[0018] Transfect the cells to be transfected with the packaged overexpressed PRDM1 lentiviral construct, and after culturing, collect the cell culture supernatant to obtain the overexpressed PRDM1 lentiviral particles;

[0019] Step 3, Preparation of pluripotent stem cells with high-purity overexpression of PRDM1:

[0020] Infect the pluripotent stem cells to be transfected with the overexpressed PRDM1 lentiviral particles, and screen through Puro (Puromycin) to obtain pluripotent stem cells with high-purity overexpression of PRDM1;

[0021] Step 4, Differentiate to generate embryoid bodies overexpressing PRDM1:

[0022] Digest the pluripotent stem cells with high-purity overexpression of PRDM1 into single cells, and further perform initial culture and amplification culture to obtain embryoid bodies overexpressing PRDM1.

[0023] In the above technical solution, the nucleotide sequence of PRDM1 is as shown in SEQ ID NO.1.

[0024] In the above technical solution, in step 2, the cells to be transfected are 293FT.

[0025] In the above technical solution, in step 2, the culturing process is completed in a constant temperature environment of 37°C containing 5% CO2.

[0026] In the above technical solution, in step 2, the process of packaging the overexpressed PRDM1 lentiviral construct is as follows:

[0027] Add the overexpressed PRDM1 lentiviral construct, packaging helper plasmid PAX2, and packaging helper plasmid VSVG to Opti-MEM; the mass ratio of the overexpressed PRDM1 lentiviral construct, packaging helper plasmid PAX2, and packaging helper plasmid VSVG is 2:1:1; add 16 micrograms of the overexpressed PRDM1 lentiviral construct to every 0.92 milliliters of Opti-MEM; obtain the first mixture;

[0028] Add PEI transfection reagent to the obtained first mixture and mix again, and incubate at room temperature for 15 minutes to obtain the second mixture, that is, obtain the packaged overexpressed PRDM1 lentiviral construct; wherein, add 80-100 microliters of PEI transfection reagent to every 0.8-1 milliliter of Opti-MEM.

[0029] In the above technical solution, in step 2, after culturing, collect the cell culture supernatant, and obtain the PRDM1 overexpressed lentiviral particles through the ultracentrifugation concentration process.

[0030] In the above technical solution, in step 3, the pluripotent stem cells to be transfected are H9.

[0031] In the above technical solution, in step 3, the initial concentration of the Puro screening process is Puro at a concentration of 0.5 μg / ml, and the final concentration is Puro at 2.5 μg / ml.

[0032] In the above technical solution, in step 4, the medium used for the initial culture is the embryoid body formation medium, and the components are: bone morphogenetic protein 4: 15 - 25 μg / L, stem cell factor: 35 - 45 μg / L, vascular endothelial growth factor: 15 - 25 μg / L, ROCK inhibitor: 8 - 12 μmol / L, and the balance is the basic medium for stem cell differentiation;

[0033] The components of the embryoid body amplification medium used for amplification culture are: bone morphogenetic protein 4: 15 - 25 μg / L, stem cell factor: 35 - 45 μg / L, vascular endothelial growth factor: 15 - 25 μg / L, and the balance is the basic medium for stem cell differentiation;

[0034] On the other hand, the present invention provides an embryoid body overexpressing PRDM1, which is prepared by the method described in any one of the above.

[0035] The embryoid body overexpressing PRDM1 provided by the present invention has the characteristics of accelerating the formation and maturation of embryoid bodies, and is an ideal cell material for studying the specific functions and regulatory mechanisms of PRDM1 in embryoid bodies, etc.

[0036] On the other hand, the present invention provides the application of the embryoid body overexpressing PRDM1 as described above in studying the function of the PRDM1 gene or its regulatory mechanism, or in optimizing the efficiency of stem cell differentiation.

[0037] It should be noted that the applications provided by the present invention are not limited to the above-mentioned research on the function of the PRDM1 gene and its regulatory mechanism and drug screening, etc. Using the embryoid body overexpressing PRDM1 provided by the present invention for research or use in other fields also belongs to the protection scope of the present invention.

[0038] Optionally, in some embodiments of the present invention, the above research is for non-diagnostic or therapeutic purposes of diseases.

[0039] The advantages and beneficial effects of the present invention are:

[0040] Overexpression of PRDM1 reduces the expression of genes related to DNA-binding transcription activator (sequence-specific DNA binding), ATP-dependent plus-end-directed microtubule motor activity, cell adhesion molecule binding, calcium ion binding, calmodulin binding, etc. At the same time, the expression of genes related to chemokine activity, cytokine activity, CCR chemokine receptor binding, extracellular matrix structural constituent conferring tensile strength, platelet-derived growth factor binding, etc. increases.

[0041] It is suggested that overexpression of PRDM1 synergistically leads to an increase in the diameter of embryoid bodies through a multi-dimensional molecular regulation network, specifically manifested as follows: ① inhibiting pluripotency maintenance genes, promoting differentiation, and increasing the number of cells in embryoid bodies; ② weakening cell-cell junctions and enhancing extracellular matrix deposition, providing physical space and signal support for the expansion of embryoid bodies; ③ the chemokine / cytokine network drives cell migration and proliferation, further expanding the volume of embryoid bodies. This mechanism is highly consistent with the phenotypes of "significantly increased diameter of embryoid bodies" and "significantly shortened duration required for the amplification stage" observed in the experiment, providing more possibilities for revealing the related functions of PRDM1 and optimizing the efficiency of stem cell differentiation.

[0042] In addition, overexpression of PRDM1 enhances the directional migration and aggregation effects between cells by upregulating the expression levels of chemokines and related inflammation regulatory genes, promotes the establishment of local microenvironment homeostasis by cells through autocrine / paracrine signals, and reduces the perturbation of cell behavior by external physical or chemical interference, thereby enhancing the stability of differentiation. Brief Description of the Drawings

[0043] Figure 1 a is the flow cytometry histogram of pluripotent stem cells overexpressing PRDM1 in the present invention.

[0044] Figure 1b is a bar graph of the fluorescence intensity of the pluripotent stem cells overexpressing PRDM1 of the present invention;

[0045] Figure 2 a is a microscope picture of the embryoid body overexpressing PRDM1 of the present invention;

[0046] Figure 2 b is a bar graph of the diameter of the embryoid body overexpressing PRDM1 of the present invention;

[0047] Figure 3a is the GO_f enrichment bubble chart of the up-regulated genes by gene sequencing of the present invention;

[0048] Figure 3b is the GO_f enrichment bubble chart of the down-regulated genes by gene sequencing of the present invention;

[0049] Figure 4 is the schematic diagram of the lentiviral vector structure in Example 1 of the present invention.

[0050] For those of ordinary skill in the art, other related drawings can be obtained based on the above drawings without creative efforts. Detailed implementation manners

[0051] In order to enable the personnel in the technical field to better understand the solution of the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0052] Example 1

[0053] A method for preparing an embryoid body overexpressing PRDM1 includes the following steps:

[0054] S1, preparation of the overexpressing PRDM1 lentiviral construction system,

[0055] Select the full-length cDNA sequence of PRDM1 and clone it into the lentiviral expression vector carrying the RSV promoter. At the same time, insert a reporter gene (such as green fluorescent protein) into the vector for screening and tracking expression; obtain the recombinant lentiviral expression vector;

[0056] For the above-mentioned recombinant lentiviral expression vector with correct sequencing, use PureLink TM HiPure Plasmid Maxi Kit to extract and purify to obtain a high-quality recombinant plasmid, that is, the overexpressing PRDM1 lentiviral construction body, for lentiviral packaging and transfection of target cells;

[0057] The viral plasmid was designed by Genewiz, as Figure 4 shown;

[0058] The full-length cDNA sequence of the said PRDM1 is as shown in SEQ ID NO.1:

[0059] SEQ ID NO.1

[0060]

[0061] The amino acid sequence encoded by the full-length cDNA sequence of PRDM1 is shown in SEQ ID NO.2:

[0062] SEQ ID NO.2

[0063] MLDICLEKRVGTTLAAPKCNSSTVRFQGLAEGTKGTMKMDMEDADMTLWTEAEFEEKCTYIVNDHPWDSGADGGTSVQAEASLPRNLLFKYATNSEEVIGVMSKEYIPKGTRFGPLIGEIYTNDTVPKNANRKYFWRIYSRGELHHFIDGFNEEKSNWMRYVNPAHSPREQNLAACQNGMNIYFYTIKPIPANQELLVWYCRDFAERLHYPYPGELTMMNLTQTQSSLKQPSTEKNELCPKNVPKREYSVKEILKLDSNPSKGKDLYRSNISPLTSEKDLDDFRRRGSPEMPFYPRVVYPIRAPLPEDFLKASLAYGIERPTYITRSPIPSSTTPSPSARSSPDQSLKSSSPHSSPGNTVSPVGPGSQEHRDSYAYLNASYGTEGLGSYPGYAPLPHLPPAFIPSYNAHYPKFLLPPYGMNCNGLSAVSSMNGINNFGLFPRLCPVYSNLLGGGSLPHPMLNPTSLPSSLPSDGARRLLQPEHPREVLVPAPHSAFSFTGAAASMKDKACSPTSGSPTAGTAATAEHVVQPKATSAAMAAPSSDEAMNLIKNKRNMTGYKTLPYPLKKQNGKIKYECNVCAKTFGQLSNLKVHLRVHSGERPFKCQTCNKGFTQLAHLQKHYLVHTGEKPHECQVCHKRFSSTSNLKTHLRLHSGEKPYQCKVCPAKFTQFVHLKLHKRLHTRERPHKCSQCHKNYIHLCSLKVHLKGNCAAAPAPGLPLEDLTRINEEIEKFDISDNADRLEDVEDDISVISVVEKEILAVVRKEKEETGLKVSLQRNMGNGLLSSGCSLYESSDLPLMKLPPSNPLPLVPVKVKQETVEPMDP*

[0064] S2. Preparation of lentiviral particles overexpressing PRDM1

[0065] (1) Cell culture preparation:

[0066] Use 293FT cells, which are a fast-growing and highly transfectable cell line derived from human embryonic kidney cells and transformed with SV40 large T antigen, and are very suitable as a production host for lentivirus. When the cell density approaches 90%, digest the cells with trypsin preheated to 37°C to separate the cells.

[0067] After digestion, resuspend the cells evenly by centrifugation, and then seed them in a 10-cm culture dish.

[0068] Place the culture dish in an incubator at 37°C with 5% CO2 for 24 hours until the cells adhere and the coverage area reaches more than 50% of the total area of the culture dish. At this time, start the preparation for virus packaging.

[0069] (2) Virus packaging:

[0070] Prepare two sterile 50-ml centrifuge tubes. In one centrifuge tube (Tube B), add 920 μl of Opti-MEM, and add the overexpressed PRDM1 lentiviral construct prepared in S1, 8 μg of packaging plasmid PAX2, and 8 μg of packaging plasmid VSVG. The mass ratio here is overexpressed PRDM1 lentiviral construct:PAX2:VSVG = 2:1:1.

[0071] In another centrifuge tube (Tube A), add 92 μl of PEI transfection reagent.

[0072] After standing Tubes A and B for 5 minutes, slowly add the PEI mixture in Tube A to Tube B, mix well, and stand for 15 minutes to obtain the packaged overexpressed PRDM1 lentiviral construct, which is beneficial to improving the transfection efficiency.

[0073] (3) Transfect 29 cells with the packaging plasmid:

[0074] Dropwise add the packaged overexpressed PRDM1 lentiviral construct evenly into the 293FT cell culture dish prepared in step S2(1), and gently shake the culture dish to ensure that the mixture is evenly distributed.

[0075] Return the culture dish to the incubator at 37°C with 5% CO2 and continue to incubate.

[0076] (4) Change the culture medium:

[0077] After 11 - 12 hours, aspirate the old culture medium, add fresh, preheated to 37°C complete culture medium, and continue to incubate the cells.

[0078] (5) Virus collection:

[0079] At about 48 hours after transfection, the cell culture supernatant containing lentiviral particles was collected, and cells and cell debris were removed by centrifugation to obtain a clarified virus suspension.

[0080] (6) Ultracentrifugation to concentrate the virus

[0081] The clarified virus suspension was concentrated by ultracentrifugation. The supernatant was carefully discarded, and the pellet was gently resuspended with sterile PBS. The virus suspension was aliquoted into sterile cryotubes, 1 mL per tube, and repeated freeze-thaw cycles were avoided. Finally, the virus titer was determined, and the qualified standard was not less than 1×10 5 TU / mL. After the above steps were completed, lentiviral particles overexpressing PRDM1 were obtained.

[0082] The main operation of step (5) was to collect the virus. The centrifugation in step (5) was a routine low-speed centrifugation in the laboratory, aiming to remove impurities. The ultracentrifugation in step (6) was to concentrate the virus so that it met the qualified titer standard.

[0083] Preparation of pluripotent stem cells with high purity overexpressing PRDM1

[0084] The pluripotent stem cells H9 to be transfected were plated on a 24-well plate. After 12 - 24 h, the medium was changed, and the optimal density was 30% - 50%. The lentiviral particles overexpressing PRDM1 (obtained in step S2(6)) were added for culture. Re-infection with the virus solution could be carried out according to the situation. After the cells grew confluent, the expression of the recombinant plasmid was detected.

[0085] At 48 - 72 hours after infection, cells with fluorescence were observed under a fluorescence microscope to preliminarily determine the infection effect, and then the transfection rate was analyzed by flow cytometry. The FITC channel was selected for the detection of pUC-RSV-hPRDM1-Puro-EGFP.

[0086] The preliminary detection showed that the transfection rate was about 15%. At this time, they were pluripotent stem cells with low purity overexpressing PRDM1. Positive clones were screened by Puro. The cells could tolerate 0.5 μg / ml concentration of Puro in about two weeks. After staining with PRDM1 antibody and detecting again by flow cytometry, it was found that the protein expression level of PRDM1 increased significantly. Subsequently, the concentration of the Puro drug was gradually increased until the cells could tolerate 2.5 μg / ml of Puro and the protein expression level of PRDM1 no longer increased, thus obtaining pluripotent stem cells with high purity overexpressing PRDM1. The cells were collected into an ep tube for further verification experiments.

[0087] S4. Differentiate to generate embryoid bodies overexpressing PRDM1

[0088] The pluripotent stem cells with high-purity overexpression of PRDM1 are differentiated into embryoid bodies overexpressing PRDM1.

[0089] (1) Embryoid body formation stage (Day 1)

[0090] a Prepare embryoid body formation medium and embryoid body amplification medium:

[0091] The embryoid body formation medium contains: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, vascular endothelial growth factor at 20 μg / L, and ROCK inhibitor at 10 μmol / L, dissolved in the basic stem cell differentiation medium. The embryoid body amplification medium contains: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, and vascular endothelial growth factor at 20 μg / L, dissolved in the basic stem cell differentiation medium;

[0092] b For the pluripotent stem cells with high-purity overexpression of PRDM1 obtained in S3: First, use a pipette to aspirate the old medium, wash the cells with 1 mL of 1×PBS solution, aspirate the PBS, and then add 1 mL of Accutase enzyme. Incubate the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to detach the cells, add 10 ml of DF12, transfer to a centrifuge tube, and centrifuge at 1000 g for 5 minutes.

[0093] c Resuspend the centrifuged precipitate with the embryoid body formation medium, add 5000 cells per well with a volume of 100 μl to a V-bottom 96-well plate, centrifuge at 500 g for 3 minutes, and culture the embryoid bodies in an incubator.

[0094] (2) Embryoid body amplification stage (Day 4)

[0095] It can be seen under a microscope that the embryoid bodies have become significantly larger, with a diameter mostly around 400 μm. At this time, aspirate the original medium, add 100 μl of embryoid body amplification medium, observe 7 embryoid bodies under the microscope and take pictures, and record the diameter of the embryoid bodies. When the embryoid bodies have clearly differentiated into the inner, middle, and outer germ layers, it indicates that the embryoid bodies are mature. At this time, embryoid bodies overexpressing PRDM1 are obtained. The time required from adding the embryoid body amplification medium until the embryoid bodies are mature is recorded as the duration of the amplification stage.

[0096] Comparative Example 1

[0097] The preparation of embryoid bodies from ordinary pluripotent stem cells includes the following steps:

[0098] S1 Embryoid body formation stage (Day 1)

[0099] a Prepare embryoid body formation medium and embryoid body amplification medium:

[0100] Embryoid body formation medium, with the following components: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, vascular endothelial growth factor at 20 μg / L, and ROCK inhibitor at 10 μmol / L, dissolved in the basic medium for stem cell differentiation. The embryoid body amplification medium has the following components: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, and vascular endothelial growth factor at 20 μg / L, dissolved in the basic medium for stem cell differentiation;

[0101] b Digest ordinary pluripotent stem cells into single cells: First, use a pipette to aspirate the old medium, rinse the cells with 1 mL of 1×PBS solution, aspirate the PBS, and then add 1 mL of Accutase enzyme. Incubate the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to make the cells detach, add 10 mL of DF12, transfer to a centrifuge tube, and centrifuge at 1000 g for 5 minutes.

[0102] c Resuspend the precipitate obtained by centrifugation with the embryoid body formation medium, add 5000 cells per well with a volume of 100 μL to a V-bottom 96-well plate, centrifuge at 500 g for 3 minutes, and culture the obtained embryoid bodies in an incubator.

[0103] S2 Embryoid body amplification stage (Day 4)

[0104] It can be observed under a microscope that the embryoid bodies are significantly enlarged, with a diameter mostly around 200 μm. At this time, aspirate the original medium, add 100 μL of embryoid body amplification medium, observe 7 embryoid bodies under the microscope and take pictures, and record the diameter of the embryoid bodies. When the embryoid bodies differentiate into obvious endoderm, mesoderm, and ectoderm, it indicates the maturation of the embryoid bodies. At this time, embryoid bodies derived from ordinary pluripotent stem cells are obtained. The time required for the amplification stage is recorded from the start of adding the embryoid body amplification medium until the embryoid bodies mature.

[0105] Example 2

[0106] Verification during the preparation of embryoid bodies overexpressing PRDM1, including:

[0107] Cell culture preparation: Prepare ordinary pluripotent stem cells H9, and place the culture dish in a 37°C incubator containing 5% CO2 for daily culture for subsequent verification experiments.

[0108] 1. Verification of the expression level of the target gene of highly purified pluripotent stem cells overexpressing PRDM1

[0109] Flow cytometry detection was performed on the highly purified pluripotent stem cells overexpressing PRDM1 (labeled as H9-PRDM1-Stemcell) obtained in S3 of Experimental Example 1 and the ordinary pluripotent stem cells in Comparative Example 1 (labeled as H9-Stem cell) after staining with the antibody PRDM1. (See Appendix Figure 1 )

[0110] Attached Figure 1 a is a flow cytometry histogram. The vertical axis represents the number of cells, the horizontal axis represents the fluorescence signal intensity of PRDM1, and the fluorescence channel is PE. Different colors represent different groups. Gray represents the comparative example H9 - Stem cell, and red represents the example H9 - PRDM1 - Stem cell. Attached Figure 1 b is a bar graph of fluorescence intensity. The vertical axis represents the average fluorescence signal intensity of PRDM1, and the horizontal axis represents the groups. It can be seen that compared with the comparative example H9 - Stem cell, the protein expression level of PRDM1 in the example H9 - PRDM1 - Stem cell is significantly increased.

[0111] 2. Microscope pictures and amplification stage duration of embryoid bodies overexpressing PRDM1

[0112] The embryoid bodies overexpressing PRDM1 obtained in S4 of Experimental Example 1 (labeled as H9 - PRDM1 - EB) and the embryoid bodies derived from ordinary pluripotent stem cells obtained in Comparative Example 1 (labeled as H9 - EB) were placed under a microscope for observation and photographed. To exclude accidental results, 7 samples were randomly selected from each group. The photographing parameters were set as: objective magnification ×10, constant exposure time (200 ms), and unified white balance calibration. (See Attached Figure 2 )

[0113] Attached Figure 2 a is a microscope picture of embryoid bodies, in which the diameters of H9 - PRDM1 - EB and H9 - EB are marked; Attached Figure 2 b is a bar graph of diameters. The vertical axis represents the diameter of embryoid bodies, and the horizontal axis represents the groups. There are 7 pairs of data in total, and paired t - test was used for significance test. It can be seen that the diameter of H9 - PRDM1 - EB is about 400 microns, the diameter of H9 - EB is about 200 microns, and the diameter of H9 - PRDM1 - EB is about twice that of H9 - EB, with significant differences. It is suggested that overexpression of PRDM1 promotes the amplification and maturation of embryoid bodies.

[0114] In addition, the duration required for the amplification stage of H9 - PRDM1 - EB is mostly 4 days, and the duration required for the amplification stage of H9 - EB is mostly 7 days. It can be seen that overexpression of PRDM1 shortens the amplification cycle of embryoid bodies.

[0115] Example 3 Mechanism exploration of embryoid bodies overexpressing PRDM1

[0116] RNA sequencing was performed on the embryoid bodies overexpressing PRDM1 obtained in Experimental Example 1 (labeled as H9-PRDM1-EB) and the embryoid bodies derived from ordinary pluripotent stem cells obtained in Comparative Example 1 (labeled as H9-EB). GO functional enrichment analysis was carried out on the "differentially expressed genes between H9-PRDM1-EB and H9-EB", with a focus on the enrichment results of gene molecular functions (go_f). When visualizing, a bubble chart was used to present the association between the enrichment degree and gene count. (See Appendix Figure 3a and 3b )

[0117] The results showed that the expression of genes related to DNA-binding transcription activator activity (DNA-binding transcription activator, sequence-specific DNA binding), ATP-dependent plus-end-directed microtubule motor activity, cell adhesion molecule binding activity, calcium ion binding activity, calmodulin binding activity, etc. decreased in the embryoid bodies overexpressing PRDM1. At the same time, the expression of genes related to chemokine activity, cytokine activity, CCR chemokine receptor binding, extracellular matrix structural constituent conferring tensile strength, platelet-derived growth factor binding, etc. increased.

[0118] It is suggested that overexpression of PRDM1 synergistically leads to an increase in the diameter of embryoid bodies through a multi-dimensional molecular regulatory network, specifically manifested as: ① inhibiting pluripotency-maintaining genes, promoting differentiation, and increasing the number of cells in the embryoid bodies; ② weakening cell-cell junctions and enhancing extracellular matrix deposition to provide physical space and signal support for the expansion of embryoid bodies; ③ the chemokine / cytokine network driving cell migration and proliferation to further enlarge the volume of embryoid bodies. This mechanism is highly consistent with the phenotype of "significantly increased diameter of embryoid bodies" observed in the experiment, providing more possibilities for revealing the related functions of PRDM1 and optimizing the efficiency of stem cell differentiation.

[0119] The specific implementation steps not described in the above embodiments are generally common steps. The following are some examples:

[0120] Steps for large-scale plasmid extraction

[0121] Before starting:

[0122] RNase A needs to be added to the resuspension buffer (R3), mixed well, and marked as added. Store at 4°C.

[0123] If the lysis buffer (L7) contains salt precipitation, heat it in a 37°C water bath until clear. Do not shake L7

[0124] (1) Obtain enriched E. coli precipitate. Centrifuge at 4,000 g for 10 minutes. Divide into 6 tubes, 50 ml of enrichment each time.

[0125] (2) Add 10 ml of RNase A buffer (R3) to the precipitate and resuspend by pipetting up and down.

[0126] (3) Add 10 ml of lysis buffer (L7) and invert the tube 5 times up and down. Let it stand at room temperature for 5 minutes.

[0127] During the waiting period, place the column on a conical flask, add 30 ml of equilibration buffer (EQ1) to the column, and let it flow out naturally by gravity.

[0128] (4) Add 10 ml of precipitation buffer (N3). Immediately invert the tube to mix well. Centrifuge at 7,000 g for 10 minutes at 4°C.

[0129] (5) Transfer the supernatant obtained by centrifugation to the column. (Try not to aspirate the white material)

[0130] (6) Add 60 ml of W8 until it has all flowed out.

[0131] (7) Transfer the column to a new 50-ml centrifuge tube, add 15 ml of elution buffer (E4) to the column, and let it flow out naturally. (The centrifuge tube now contains the purified plasmid)

[0132] (8) Add 10.5 ml of isopropanol to the centrifuge tube and mix well. Centrifuge at 7,000 g for 2 h at 4°C. White precipitate can be seen on the side walls of the centrifuge tube at this time

[0133] (9) After aspirating the supernatant with a pipette, enter the cell culture room, add 1 ml of 70% ethanol to dissolve and mix well, transfer to a screw-cap centrifuge tube, and centrifuge at 12,000 rpm for 1 minute

[0134] (10) After centrifugation, aspirate the supernatant (try to aspirate it as clean as possible) and let it air-dry naturally (ethanol is volatile)

[0135] (11) Add 300 - 600 μL of sterile ultrapure water and mix well.

[0136] (12) Use nanodrop to detect the plasmid concentration.

[0137] Daily culture of pluripotent stem cells:

[0138] (1) Cell medium change:

[0139] Pluripotent stem cells are cultured daily in a T25 flask. During the culture process, ensure that the cells form colonies and avoid dissociating them into single cells. Change the culture medium every 24 hours.

[0140] (2) Cell passage:

[0141] Pre - coat a new T25 flask with Matrix gel (2.5 ml of basal medium + 30 μl of Matrix concentrate) overnight for the next cell passage.

[0142] First, use a pipette to aspirate the old culture medium, wash the cells with 1 mL of 1×PBS buffer, aspirate the PBS, then add 1 mL of ReLeSR to wash the cells and aspirate the ReLeSR. Digest the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to detach the cells, and add 1 mL of basal medium to terminate the digestion. Aspirate 1 ml of the cell suspension and drop 8 drops into the pre - coated T25 flask, and check under the microscope whether the cells in the T25 flask are evenly distributed.

[0143] (3) Cell cryopreservation:

[0144] Pre - warm the cell cryopreservation box to room temperature, prepare the cryopreservation solution according to the ratio of serum - free medium: DMSO = 9:1. After obtaining the cell suspension according to the cell passage method, centrifuge at 1000 rpm for 5 minutes, resuspend the cell pellet with the cryopreservation solution, add 1 - 1.5 mL of the mixture to each cryopreservation tube, place it in the cryopreservation box, store at - 80°C, and transfer to a liquid nitrogen tank for storage after the cells are completely frozen.

[0145] (4) Cell thawing:

[0146] Pre - coat a new T25 flask with Matrix gel (2.5 ml of basal medium + 30 μl of Matrix concentrate) overnight for the next cell thawing.

[0147] Adjust the water bath temperature to 37°C. Remove cryovials from the liquid nitrogen tank or -80°C freezer according to the records. Carefully grasp the cryovials with tweezers and gently shake them in a water bath until the solids inside the tubes are completely melted. Add approximately 10 mL of stem cell culture medium to a 15 mL centrifuge tube to dilute the DMSO concentration in the cryopreservative. Add the cell suspension to the 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. During this time, prepare cell recovery medium (4 mL stem cell culture medium + 4 μL YZ7632). After centrifugation, discard the supernatant and carefully pipette the cell pellet with cell recovery medium until it forms a clumping mass (be careful not to break up the cells during this step). Transfer the pellet to a coated T25 culture flask and culture in a 37°C incubator.

[0148] Flow cytometry staining steps for PRDM1

[0149] PRDM1 is mainly present in the cytoplasm. Therefore, before staining with PRDM1 antibody, it is necessary to use BD Cytofix / Cytoperm Plus to permeabilize the membrane, then stain with PRDM1 primary antibody, and finally stain with PRDM1 secondary antibody. The specific steps are as follows:

[0150] (1) Wash the digested cells twice with PBS and resuspend them in 200 μl of Fixation / Permeabilization solution. Fix at 4°C for 20 minutes. (During this time, prepare 1× Perm / Wash solution, i.e., 4.5 ml ultrapure water + 0.5 ml 10× Perm / Wash solution.)

[0151] (2) Wash the fixed cells twice with 250ul 1×Perm / Wash solution

[0152] (3) Resuspend with 50ul 1×Pem / Wash buffer, add 1ul PRDM1 primary antibody, and incubate at room temperature in the dark for 20 minutes

[0153] (4) Wash the cells twice with 250ul 1×Perm / Wash solution to remove the primary antibody.

[0154] (5) Resuspend with 50ul 1×Pem / Wash solution, add 1ul PRDM1 secondary antibody, and incubate at room temperature in the dark for 20 minutes

[0155] (6) After staining, wash once with 250ul 1× Perm / Wash solution, then wash once more with PBS, resuspend in PBS to 200ul, filter with gauze, and analyze on the instrument. (The detection channel of this PRDM1 antibody is PE)

[0156] List of main reagents

[0157] Reagent Name Catalog Number Company <![CDATA[PureLink TM HiPure Plasmid Maxiprep Kit]]> K210006 Thermo Fisher Opti-MEM 31985062 Gibco PEI 40816ES02 Yeasen Accutase 07922 07920 Stem Cell mTeSR Plus 100-0276 100-1130 Stem Cell DMEM-F12 Medium KGM12500NH-500 KeyGen Biotech ReLeSR 100-0483 Stem Cell DMSO D806645 MACKLIN BD Cytofix / Cytoperm Plus 554715 BD

[0158] Main self-prepared reagents

[0159]

[0160]

[0161]

[0162] The above makes an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification or equivalent replacement that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. A method for preparing embryoid bodies overexpressing PRDM1, characterized in that, It includes the following steps: S1. Pluripotent stem cell construction: Add lentiviral particles overexpressing PRDM1 to the pluripotent stem cells to be transfected. After transfection, pluripotent stem cells with low purity overexpressing PRDM1 are obtained. S2. Purification: Subject the pluripotent stem cells with low purity overexpressing PRDM1 to Puro drug screening to obtain pluripotent stem cells with high purity overexpressing PRDM1. S3. Differentiation: Differentiate the pluripotent stem cells with high purity overexpressing PRDM1 to obtain embryoid bodies overexpressing PRDM1.

2. The method for preparing embryoid bodies overexpressing PRDM1 according to claim 1, characterized in that, The nucleotide sequence of the PRDM1 is shown as SEQ ID NO.1; preferably, the pluripotent stem cells to be transfected are H9.

3. A method for preparing embryoid bodies overexpressing PRDM1, characterized in that, It includes the following steps: Step 1. Preparation of the lentiviral construct overexpressing PRDM1: Select the full-length cDNA sequence of PRDM1 and clone it into a lentiviral expression vector carrying the RSV promoter; a recombinant lentiviral expression vector is obtained. For the above recombinant lentiviral expression vector with correct sequencing, use a plasmid large-scale extraction kit to extract and purify to obtain a high-quality recombinant plasmid, namely the lentiviral construct overexpressing PRDM1, for lentiviral packaging and transfection of target cells. Step 2. Preparation of the lentiviral particles overexpressing PRDM1: Transfect the lentiviral construct overexpressing PRDM1 after packaging into the cells to be transfected. After culture, collect the cell culture supernatant to obtain the lentiviral particles overexpressing PRDM1. Step 3. Preparation of pluripotent stem cells with high purity overexpressing PRDM1: The lentiviral particles overexpressing PRDM1 infect the pluripotent stem cells to be transfected, and pluripotent stem cells with high purity overexpressing PRDM1 are obtained through Puro (Puromycin) screening. Step 4. Differentiate to generate embryoid bodies overexpressing PRDM1: Digest the pluripotent stem cells with high purity overexpressing PRDM1 into single cells, and perform initial culture and amplification culture to obtain embryoid bodies overexpressing PRDM1.

4. The method for preparing embryoid bodies overexpressing PRDM1 according to claim 3, characterized in that, The nucleotide sequence of the PRDM1 is shown as SEQ ID NO.

1.

5. The method for preparing embryoid bodies overexpressing PRDM1 according to claim 3, wherein, In Step 2, the cells to be transfected are 293FT; Preferably, the culture process is completed in a constant temperature environment of 37°C with 5% CO2. Preferably, after culture, collect the cell culture supernatant, and obtain the lentiviral particles overexpressing PRDM1 through an ultracentrifugation concentration process.

6. The method for preparing embryoid bodies overexpressing PRDM1 according to claim 3, wherein In Step 2, the process of packaging the lentiviral construct overexpressing PRDM1 is: Add the lentiviral construct overexpressing PRDM1, the packaging auxiliary plasmid PAX2, and the packaging auxiliary plasmid VSVG to Opti-MEM; the mass ratio of the lentiviral construct overexpressing PRDM1, the packaging auxiliary plasmid PAX2, and the packaging auxiliary plasmid VSVG is 2:1:1; add 16 micrograms of the lentiviral construct overexpressing PRDM1 to every 0.92 milliliters of Opti-MEM; a first mixture is obtained. Add the PEI transfection reagent to the obtained first mixture and mix again, and incubate at room temperature for 15 minutes to obtain a second mixture, that is, the overexpressed PRDM1 lentiviral construct after packaging; wherein, 80-100 μL of the PEI transfection reagent is added to every 0.8-1 mL of Opti-MEM.

7. The method for preparing embryoid bodies overexpressing PRDM1 according to claim 3, characterized in that, In step 3, the pluripotent stem cells to be transfected are H9; Preferably, the initial concentration of the Puro screening process is Puro at a concentration of 0.5 μg / ml, and the final concentration is Puro at 2.5 μg / ml.

8. The method for preparing embryoid bodies overexpressing PRDM1 according to claim 3, characterized in that, In step 4, the medium used for the initial culture is an embryoid body formation medium, and the components are: bone morphogenetic protein 4: 15-25 μg / L, stem cell factor: 35-45 μg / L, vascular endothelial growth factor: 15-25 μg / L, ROCK inhibitor: 8-12 μmol / L, and the balance is a basic medium for stem cell differentiation; The components of the embryoid body amplification medium used for amplification culture are: bone morphogenetic protein 4: 15-25 μg / L, stem cell factor: 35-45 μg / L, vascular endothelial growth factor: 15-25 μg / L, and the balance is a basic medium for stem cell differentiation.

9. An embryoid body overexpressing PRDM1, characterized in that, It is prepared by the method according to any one of claims 1 to 8.

10. An overexpressed PRDM1 embryoid body as described in claim 9 is used in the study of the function of the PRDM1 gene or its regulatory mechanism, or in the application of optimizing the efficiency of stem cell differentiation.

Images