Method for reprogramming T cells into induced pluripotent stem cells

Abstract

The invention discloses a method for reprogramming T cells into induced pluripotent stem cells. The method comprises the following steps: obtaining separated peripheral blood mononuclear cells; sorting the separated peripheral blood mononuclear cells, and collecting total T cells; mixing the total T cells with reprogramming plasmids, and carrying out plasmid nuclear transfection to obtain total T cells subjected to nuclear transfection; mixing the total T cells subjected to nuclear transfection with an activating factor, culturing in stages, and inducing to obtain induced pluripotent stem cells; according to the method for reprogramming the T cells into the induced pluripotent stem cells, the CD3 / CD28 and the IL-2 are adopted for activating the T cells, so that the T cells can obtain higher activity for reprogramming induction, and meanwhile, the T cells can more simply, conveniently, safely and effectively generate the iPSC in a shorter time and a higher success rate.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for reprogramming T cells into induced pluripotent stem cells. Background Art

[0002] Induced Pluripotent Stem Cells (iPSCs) are stem cells that have the characteristics of embryonic stem cells and unlimited proliferation and multidirectional differentiation potential derived from somatic cells through reprogramming. Because it does not involve ethical disputes, its unlimited proliferation and multidirectional differentiation characteristics have led to the widespread use of induced pluripotent stem cells in the fields of disease modeling, neurodegenerative disease treatment, tumor treatment, and regenerative medicine.

[0003] At present, the technology of reprogramming T cells into iPSCs still has many problems in practical applications, such as small number of T cells obtained, low vitality, poor proliferation activity, low success rate of reprogramming into iPSCs, unstable system, and long time consumption. Summary of the invention

[0004] The main purpose of the present invention is to propose a method for reprogramming T cells into induced pluripotent stem cells, aiming to solve the current problems of long time consumption, instability, low success rate and low safety performance of T cell reprogramming into induced pluripotent stem cells.

[0005] To achieve the above object, the present invention provides a method for reprogramming T cells into induced pluripotent stem cells, comprising the following steps:

[0006] Obtaining isolated peripheral blood mononuclear cells;

[0007] Sorting the separated peripheral blood mononuclear cells to collect total T cells;

[0008] The total T cells are mixed with the reprogramming plasmid, and the plasmid is nuclear transfected to obtain the total T cells after nuclear transfection;

[0009] The total T cells after nuclear transfer are mixed with activation factors and cultured in stages to induce induced pluripotent stem cells;

[0010] Wherein, the activating factors include CD3 / CD28 and IL2.

[0011] In some embodiments, the step of "mixing the total T cells after nuclear transfer with activation factors and culturing in stages to induce induced pluripotent stem cells" includes:

[0012] The total T cells that have completed the nuclear transfer are mixed with an activation factor for activation, and then mixed with a variety of culture media for staged culture to induce induced pluripotent stem cells, wherein the various culture media include Matrigel.

[0013] In some embodiments, the reprogramming plasmid comprises pCE-hOCT3 / 4, pCE-hSK, pCE-hUL and pCE-mp53DD; and / or,

[0014] The multiple culture media include T cell culture medium, induction medium N2B27 and mTeSR Plus stem cell culture medium.

[0015] In some embodiments, the step of "mixing the total T cells after nuclear transfer with an activation factor for activation, and then mixing with multiple culture media for staged culture, and inducing induced pluripotent stem cells, wherein the multiple culture media include Matrigel, the multiple culture media include T cell culture media, induction culture media N2B27 and mTeSR Plus stem cell culture media" includes:

[0016] Mixing and culturing the total T cells after nuclear transfer with a T cell culture medium to obtain a first culture medium;

[0017] The first culture medium is mixed with an activation factor and an induction medium N2B27 and cultured until early induced pluripotent stem cells appear to obtain a second culture medium;

[0018] The second culture medium is replaced with mTeSR Plus stem cell culture medium for mixed culture to obtain induced pluripotent stem cells.

[0019] In some embodiments, the step of "obtaining isolated peripheral blood mononuclear cells" comprises:

[0020] Collect human venous peripheral blood;

[0021] The human venous peripheral blood is mixed with stem cell protection solution and lymph separation solution, centrifuged, washed, purified and cultured to obtain separated peripheral blood mononuclear cells.

[0022] In some embodiments, in the step of "sorting the separated peripheral blood mononuclear cells to collect total T cells": the sorting method is magnetic bead sorting.

[0023] In some embodiments, the step of "mixing total T cells with reprogramming plasmids, performing plasmid nuclear transfection, and obtaining nuclear transfected total T cells" includes:

[0024] Resuspend the total stem cells in the total T cells to obtain a resuspended solution;

[0025] The resuspension is mixed with a T cell nuclear transfer reaction solution and a reprogramming plasmid to perform plasmid nuclear transfer, thereby obtaining total T cells that have completed nuclear transfer.

[0026] The present invention provides an induced pluripotent stem cell, wherein the induced pluripotent stem cell is obtained by any of the above-mentioned methods for reprogramming T cells into induced pluripotent stem cells.

[0027] The present invention provides a pharmaceutical composition, comprising the induced pluripotent stem cells as described above and / or active ingredients in the induced pluripotent stem cells.

[0028] The method for reprogramming T cells into induced pluripotent stem cells provided by the present invention uses CD3 / CD28 and IL-2 to activate the total T cells after nuclear transfection, which can not only enable the T cells to obtain higher activity for reprogramming induction, but also enable the T cells to effectively generate iPSCs in a shorter time, with a higher success rate, and in a simpler, safer and more efficient manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of the method for reprogramming total T cells into induced pluripotent stem cells in Example 1 of the present application;

[0030] Figure 2 This is a diagram of the morphological changes of total T cells reprogrammed into iPSCs in Example 1 of the present application;

[0031] Figure 3 It is the immunofluorescence detection of iPSC expression proteins obtained by total T cell reprogramming in Example 1 of the present application;

[0032] Figure 4 This is a schematic diagram of the detection of plasmid residues in iPSCs obtained by total T cell reprogramming in Example 1 of the present application;

[0033] Figure 5 This is a schematic diagram of detecting the differentiation potential of iPSCs obtained by total T cell reprogramming in Example 1 of the present application;

[0034] Figure 6 This is a schematic diagram of the karyotype detection of iPSCs obtained by total T cell reprogramming in Example 1 of the present application;

[0035] Figure 7 This is a schematic diagram of the detection of iPSC marker gene expression obtained by total T cell reprogramming in Example 1 of the present application. DETAILED DESCRIPTION

[0036] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0037] Induced Pluripotent Stem Cells (iPSCs) are stem cells that have the characteristics of embryonic stem cells and unlimited proliferation and multidirectional differentiation potential derived from somatic cells through reprogramming. Because it does not involve ethical disputes, its unlimited proliferation and multidirectional differentiation characteristics have led to the widespread use of induced pluripotent stem cells in the fields of disease modeling, neurodegenerative disease treatment, tumor treatment, and regenerative medicine.

[0038] At present, the technology of reprogramming T cells into iPSCs still has many problems in practical applications, such as low reproducibility, low success rate of differentiating the obtained iPSCs into T cells, and low safety performance.

[0039] In view of this, if Figure 1 As shown, the present invention provides a method for reprogramming T cells into induced pluripotent stem cells, comprising the following steps:

[0040] Obtaining isolated peripheral blood mononuclear cells;

[0041] Sorting the separated peripheral blood mononuclear cells to collect total T cells;

[0042] The total T cells are mixed with the reprogramming plasmid, and the plasmid is nuclear transfected to obtain the total T cells after nuclear transfection;

[0043] The total T cells after nuclear transfer are mixed with activation factors and cultured in stages to induce induced pluripotent stem cells;

[0044] Wherein, the activating factors include CD3 / CD28 and IL2.

[0045] In the technical scheme of the present invention, CD3 / CD28 and IL-2 are used as activation factors. The method for reprogramming T cells into induced pluripotent stem cells provided by the present invention uses CD3 / CD28 and IL-2 to activate T cells, which can not only enable T cells to obtain higher activity for reprogramming induction, but also enable T cells to effectively generate iPSCs in a shorter time, with a higher success rate, and in a simpler, safer and more efficient manner.

[0046] In any embodiment of the present invention, the step of "mixing the total T cells after nuclear transfer with activation factors for culturing in stages to induce induced pluripotent stem cells" includes: mixing the total T cells after nuclear transfer with activation factors for activation, and then mixing with multiple culture media for culturing in stages to induce induced pluripotent stem cells, wherein the multiple culture media include Matrigel gel. This is because the use of culture media and Matrigel is very important in the reprogramming stage. Multiple culture media are used for culturing at different stages, and stem cell-specific Matrigel gel is added to each culture medium to provide a cell growth environment, which replaces the use of mouse feeder cells, is safer, and more and safer iPSC clones can be obtained later.

[0047] In any embodiment of the present invention, the reprogramming plasmids include pCE-hOCT3 / 4, pCE-hSK, pCE-hUL and pCE-mp53DD, which are non-viral, non-integrating plasmids. Non-integrating plasmids do not have the risk of exogenous genes integrating into the human genome.

[0048] In any embodiment of the present invention, the multiple culture media include T cell culture medium, induction culture medium N2B27 and mTeSR Plus stem cell culture medium, wherein the T cell culture medium is an oxygen-containing organic compound, a colorless and odorless transparent liquid at room temperature, with high polarity, high boiling point, good thermal stability, non-proton, miscible with water, soluble in most organic substances such as ethanol, propanol, benzene and chloroform, stable in the air, not easy to volatilize, and the solution is easy to handle after the reaction. Induction culture medium N2B37 is a culture medium for culturing human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). It contains two supplements, N2 and B27, hence the name N2B27. This culture medium also contains a series of cytokines and small molecules for maintaining the self-renewal and pluripotency of stem cells; mTeSR Plus is a culture medium specifically designed for culturing human embryonic stem cells and induced pluripotent stem cells. It is an evolved version of mTeSR1 medium, containing more growth factors and small molecules to improve cell growth and maintain the stability of its stem cell state. mTeSR Plus can provide better cell growth performance and the stability required to maintain the stem cell state. Continuous culture of the three together can obtain more and more stable induced pluripotent stem cells.

[0049] Specifically, total T cells reprogrammed into iPSCs showed morphological changes such as Figure 2 As shown, after continuous culture for 30 days, alkaline phosphatase staining confirmed that induced pluripotent stem cells were obtained.

[0050] In any embodiment of the present invention, the step of "mixing the total T cells after nuclear transfer with an activation factor for activation, and then mixing with a plurality of culture media for culturing in stages, and inducing induced pluripotent stem cells, wherein the plurality of culture media include Matrigel gel, the plurality of culture media include T cell culture media, induction culture media N2B27 and mTeSR Plus stem cell culture media" includes:

[0051] Mixing and culturing the total T cells after nuclear transfer with a T cell culture medium to obtain a first culture medium;

[0052] The first culture medium is mixed with an activation factor and an induction medium N2B27 and cultured until early induced pluripotent stem cells appear to obtain a second culture medium;

[0053] The second culture medium is replaced with mTeSR Plus stem cell culture medium for mixed culture to obtain induced pluripotent stem cells.

[0054] The operation steps are simple, time-saving, easy to promote and use, and the use of T cell culture medium, induction medium N2B27 and mTeSR Plus stem cell culture medium for staged cultivation can enable the generation of iPSCs with a shorter time, higher success rate, and more convenient, safe and effective total T cells.

[0055] In any embodiment of the present invention, the step of "obtaining isolated peripheral blood mononuclear cells" comprises:

[0056] Collect human venous peripheral blood;

[0057] The human venous peripheral blood is mixed with stem cell protection solution and lymph separation solution, centrifuged, washed, purified and cultured to obtain separated peripheral blood mononuclear cells.

[0058] Efficient and stable somatic cell reprogramming technology is the key to obtaining iPSCs, and the selection of somatic cells also affects the subsequent application of iPSCs to a certain extent. Therefore, the use of non-invasive somatic cells from peripheral blood that have almost no impact on donors has become the choice of iPSC reprogramming, especially when establishing patient-specific iPSCs, which only requires a small amount of peripheral blood, making it easier for patients to accept. In addition, after peripheral blood-derived T cells are reprogrammed into iPSCs, they are more easily directed to differentiate into T cells, which is of great help in the subsequent treatment of immune diseases such as car-T tumors.

[0059] In any embodiment of the present invention, in the step of "sorting the separated peripheral blood mononuclear cells to collect total T cells": the sorting method is magnetic bead sorting.

[0060] This method screens out high-purity, unstimulated total T cells (PanT) from PBMC. During the sorting process, non-T cells such as monocytes, neutrophils, eosinophils, B cells, dendritic cells, and NK cells will be retained in the magnetic field. These mononuclear non-T cells can also be subsequently induced to reprogram using this protocol, thereby increasing their utilization rate.

[0061] In any embodiment of the present invention, “mixing total T cells with reprogramming plasmids, performing plasmid nuclear transfection, and obtaining nuclear transfected total T cells” includes:

[0062] Resuspend the total stem cells in the total T cells to obtain a resuspended solution;

[0063] The resuspension is mixed with a T cell nuclear transfer reaction solution and a reprogramming plasmid to perform plasmid nuclear transfer, thereby obtaining total T cells that have completed nuclear transfer.

[0064] It should be noted that the present invention uses reprogramming plasmids for nuclear transfer, and the reprogramming plasmids include pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1. It uses non-viral, non-integrating plasmids, combined with nuclear transfer technology, T cell stage technology, and stage differentiation culture technology to establish a technology with a very high success rate, simple and repeatable, safe, stable, and efficient T cell reprogramming to iPSC.

[0065] In any embodiment of the present invention, the present invention provides an induced pluripotent stem cell, which is obtained by the method of reprogramming T cells into induced pluripotent stem cells as described in any one of the above items, and therefore, also has the beneficial effects of the above-mentioned induced pluripotent stem cells, which are not described one by one here.

[0066] In any embodiment of the present invention, the present invention provides a pharmaceutical composition, which comprises the induced pluripotent stem cells as described above and / or the active ingredients in the induced pluripotent stem cells, that is, the induced pluripotent stem cells prepared by the present invention can be applied in many fields, such as the medical field, as disease modeling, neurodegenerative disease treatment, tumor treatment, regenerative medicine, etc., of course, it is not limited thereto.

[0067] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0068] Example

[0069] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0070] 1.1 Reagents and Materials:

[0071] Stem cell protection solution: weigh 2.5g BSA, add it to 500mL DPBS to fully dissolve, then add 2ml 0.5M EDTA solution, mix thoroughly, filter with a 0.22μm filter to obtain stem cell protection solution, and store at 4℃;

[0072] Lymphocyte separation fluid: human peripheral blood lymphocyte separation fluid (LTS1077) from Tianjin Haoyang / TBD Company;

[0073] Culture medium: ImmunoCult-XF T Cell Expansion Medium (10981) from stem cell company

[0074] 1.2 The separation steps include:

[0075] (1) Collect 10 mL of venous peripheral blood using a sterile EDTA anticoagulant tube and restore the lymph separation fluid and stem cell protection fluid to room temperature;

[0076] (2) Dilute the blood sample with stem cell protection solution at a ratio of 1:1 and transfer it to a 50 mL centrifuge tube;

[0077] (3) Take a 15 mL centrifuge tube, add 3 mL of lymph separation solution restored to room temperature, and then take 7 mL of diluted blood sample and slowly add it to the separation solution;

[0078] (4) Adjust the centrifuge acceleration to the minimum, centrifuge at 300 g for 20 min at room temperature, and after centrifugation, the solution can be seen to be divided into four layers from top to bottom, namely, a yellow plasma layer, a milky white lymphocyte layer, a transparent separation fluid layer, and a red blood cell layer;

[0079] (5) Carefully pipette the milky white lymphocyte layer into a 15 mL centrifuge tube using a Pasteur tube, avoiding other layers as much as possible. Add stem cell protection solution up to 12 mL, gently invert upside down to mix, centrifuge at 300 g for 10 min at room temperature, and discard the supernatant.

[0080] (6) Wash the cells again with stem cell protection solution, centrifuge at 300 g for 10 min at room temperature, and discard the supernatant;

[0081] (7) Add an appropriate amount of red blood cell lysis buffer and incubate at room temperature for 10 min to fully lyse the red blood cells;

[0082] (8) Add an appropriate amount of stem cell protection solution to terminate the reaction, gently invert to mix the cells, centrifuge at 300g for 10 min at room temperature, and discard the supernatant;

[0083] (9) Add an appropriate amount of ImmunoCult-XF T Cell Expansion Medium to resuspend the cells, transfer to a 10 cm culture dish, and culture at 37°C, 5% CO2. T cell purification is performed the next day.

[0084] 2. Isolation of T cells from PBMC

[0085] 2.1 Reagents and Materials:

[0086] T cell sorting kit: Pan T Cell Isolation Kit (Miltenyi Biotec130-096-535), obtains highly pure, unstimulated total T cells (Pan T) from PBMC by negative sorting. Sorting will retain non-T cells such as monocytes, neutrophils, eosinophils, B cells, dendritic cells, NK cells, etc. in the magnetic field. These mononuclear non-T cells can also be induced for reprogramming using this protocol.

[0087] LS Columns: Miltenyi Biotec, 130-042-401.

[0088] 2.2 Sorting steps:

[0089] (1) Transfer the PBMCs isolated and cultured overnight into a 15 mL centrifuge tube, centrifuge at 250 g for 10 min at room temperature, and remove the supernatant;

[0090] (2) Add appropriate amount of stem cell protection solution to resuspend and wash, centrifuge at 250g for 10 min at room temperature, and discard the supernatant;

[0091] (3) Resuspend the cells in 1 mL of pre-cooled stem cell protection solution, transfer to a clean 1.5 mL EP tube, and count;

[0092] (4) Based on the counting results, subsequent T cell sorting is performed with 10 million cells as one reaction;

[0093] (5) Add 40uL of pre-cooled stem cell protection solution per 10 million cells, gently resuspend the cells, incubate in a 4°C refrigerator for 5 min, then add 10uL of Pan T Cell Biotin Antibody Cocktail, mix thoroughly to avoid bubbles, and incubate in a 4°C refrigerator for 5 min;

[0094] (6) Add 30uL stem cell protection solution and 20uL Pan T Cell MicroBead Cocktail, mix gently, and incubate in a 4°C refrigerator for 10 min;

[0095] (7) During the incubation period, sterilize the MACS separator and carefully place the separation column in the separation rack, taking care not to touch the ends of the separation column. Connect a 15 mL centrifuge tube and rinse it three times with stem cell protection solution, adding 2 mL each time.

[0096] (8) After the cell incubation is completed, add an appropriate amount of stem cell protection solution to the 1.5 mL EP tube, and then immediately add the solution to the sorting column. At this time, the liquid flowing down contains T cells. When no more liquid drips, add 3 mL of stem cell protection solution to fully collect the total T cells;

[0097] (9) After sorting, add an appropriate amount of stem cell protection solution to the collected T cells, centrifuge at 250g for 10 min, and discard the supernatant; (10) Add ImmunoCult-XF T Cell Expansion Medium to resuspend the sorted T cells, transfer to a 10 cm culture dish, and culture at 37°C, 5% CO2. Induce iPSC the next day.

[0098] Reprogramming of 3T cells into iPSCs

[0099] 3.1 Reagents and Materials:

[0100] T cell culture medium: ImmunoCult-XF T Cell Expansion Medium (10981), T cell activation and expansion: ImmunoCult Human CD3 / CD28 T Cell Activator (10971), IL2;

[0101] Nuclear transfer reagents: Human T Cell Kit(VPA-1002);

[0102] Reprogramming plasmids: pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1 were purchased from Addgene;

[0103] Induction medium N2B27: composed of DMEM / F12, 1×N-2Supplement, 1×B-27Supplement, 0.1mMMEM Non-essential Amino Acids Solution, 1×GlutaMAX-I, 100μMβ-Mercaptoethanol, 100ng / mL bFGF, purchased from thermo company;

[0104] iSPC culture medium: mTeSR Plus, purchased from stem cell company;

[0105] Others: Y27632, Matrigel hESC-Qulified Matrix (354277), purchased from Thermo Corporation. Nuclear transfection instrument: Nucleofector 2D Device, purchased from Lonza Corporation.

[0106] 3.2 Steps of T cell reprogramming to iPS cells:

[0107] (1) Coat a 6-well plate with Matrigel for more than 2 hours. Remove the Matrigel before the experiment, add ImmunoCult-XF T Cell Expansion Medium, and preheat in a 37°C cell culture incubator for more than 30 minutes.

[0108] (2) Collect the above T cells into a 15 ml centrifuge tube, centrifuge at 250 g for 10 min, and discard the supernatant;

[0109] (3) Add stem cell protection solution to wash once, centrifuge and discard the supernatant;

[0110] (4) Add an appropriate amount of stem cell protection solution to resuspend the T cells, count them, transfer 5 million cells to a 1.5 ml centrifuge tube, centrifuge at 800 g for 10 min, and discard the supernatant;

[0111] (5) Prepare T cell nucleofector solution: Take 82 μl of Nucleofector Solution and 18 μl of supplement and mix them evenly to prepare the nucleofector reaction solution. Let stand at room temperature for 15 min.

[0112] (6) Add the nuclear transfer reaction solution to the resuspended cell pellet in step (4), and add the reprogramming plasmids pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1, and mix well;

[0113] (7) Transfer the cell-nucleofection solution-plasmid suspension to the bottom of the nucleofection cup and cover the cup tightly;

[0114] (8) Turn on the nuclear transfer instrument and adjust the program to V-024;

[0115] (9) inserting the nuclear transfer cup into the nuclear transfer instrument to perform nuclear transfer;

[0116] (10) When nuclear transfer is completed, the nuclear transfer cup is immediately removed, and 500 μL of pre-warmed T cell culture medium is gently added to the nuclear transfer cup. The cells are gently transferred to the culture wells coated with Matrigel using a matching Pasteur tube and cultured at 37°C and 5% CO2.

[0117] (11) 10 h after nuclear transfection, activation factors, namely antiCD3 / antiCD28 (25 μL / mL) and IL-2 (150-200 IU) were added to activate T cells;

[0118] (12) 24 h after nuclear transfection, add 1 ml of N2B27 medium containing 10 μM Y27632 (only add once). Do not aspirate the original medium.

[0119] (13) 2-6 days after nuclear transfer: Perform half-volume medium exchange, that is, discard 0.5 ml of old medium every day and add 0.5 ml of fresh N2B27 medium;

[0120] (14) 7-9 days after nuclear transfer, perform a full medium exchange, i.e., collect all old medium, centrifuge at 250 g for 5 min, discard the supernatant, add 2 mL of fresh N2B27 medium to resuspend the cells, and transfer to the culture well for continued culture;

[0121] (15) 9 days after nuclear transfection: the culture medium was replaced with mTeSR Plus stem cell culture medium containing 100 ng / ml bFGF until obvious iPSC-like cell clones appeared;

[0122] (16) After the clones have grown, pick each iPSC clone and place it in a new Matrigel-coated culture well, expand the culture, and obtain induced pluripotent stem cells.

[0123] Performance Testing

[0124] The induced pluripotent stem cells prepared in the above example were identified as follows:

[0125] 1. iPSC passaging and marker identification

[0126] (1) Coat the well plate with Matrigel in advance. When the iPSC cell density reaches 80%, add an appropriate amount of 0.5 mM EDTA digestion solution to rinse the cells once to remove the residual culture medium. Add an appropriate amount of 0.5 mM EDTA again and digest at room temperature for 5 min. Discard the EDTA digestion solution, add an appropriate amount of culture medium, gently pipette 3 to 5 times, collect the cells into a 15 ml centrifuge tube, and centrifuge at 300 rpm for 5 minutes.

[0127] (2) Discard the supernatant, add an appropriate amount of mTeSR Plus to resuspend the cell pellet, and expand and culture at a ratio of 1:6; at the same time, take a small amount of iPSC cells and inoculate them on the cell slide coated with Matrigel to perform immunofluorescence experiments to identify iPSC markers. The identification results are as follows: Figure 3 As shown, Figure 3 OTC4, SOX2, NANOG, SSEA4, TRA-1-60 and DAPI are all iPSC markers, representing indicators of pluripotency and stemness.

[0128] 2. Plasmid residue detection

[0129] The present invention uses PCR technology combined with agarose gel electrophoresis to detect plasmid residues. The experimental results are as follows Figure 4 As shown in the figure, the DL2000 DNA ladder is composed of 6 linear double-stranded DNA bands, which are used for the analysis of DNA bands in agarose gel electrophoresis. The band composition is 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, of which the 750bp band is displayed as a bright band. P2 represents 2 passages of IPSC, P5 represents 5 passages, and P10 represents 10 passages. 4 indicates that a non-viral, non-integrating episomal reprogramming plasmid is used, and the plasmid will be gradually lost as the iPSC is passaged and expanded.

[0130] 3. Karyotype identification, mRNA detection of iPSC marker gene expression, teratoma experiment

[0131] The iPSC cells obtained by T cell reprogramming were continued to be subcultured and expanded. When the cells reached 20 or more, karyotype identification, marker gene expression detection, and teratoma experiments were performed to evaluate whether the iPSCs obtained were stable and had the ability to differentiate into the three germ layers. The experimental results were as follows: Figure 5 , Figure 6 and Figure 7 :

[0132] Figure 5 This is an iPSC teratoma experiment, in which the ectoderm mainly refers to the neural rosette-like processing, the mesoderm is illustrated as muscle tissue, and the endoderm is illustrated as glands. The teratoma is a tumor composed of different types of mature tissues derived from the three germ layers (endoderm, mesoderm, and ectoderm). If iPSCs can form teratomas in vivo, it means that they have the potential to form all basic cell types of the human body, thus proving their pluripotency.

[0133] Figure 6 The karyotype is a normal diploid karyotype, indicating that the obtained iPSC genome is safe and stable.

[0134] Figure 7 To measure the results of qPCR, the vertical axis is the multiple ratio of the target genes Oct-4, Sox2 and Nanog in each group, with GAPDH as the internal reference, the horizontal axis PBMC represents peripheral blood mononuclear cells, and iPSC represents induced pluripotent stem cells. These transcription factors will activate gene expression and regulate their own expression, and the iPSC markers are detected.

[0135] In summary, the induced pluripotent stem cells obtained by the present invention have the ability to differentiate into three germ layers.

[0136] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for reprogramming T cells into induced pluripotent stem cells, characterized in that: The following steps are involved: Obtaining isolated peripheral blood mononuclear cells; Sorting the separated peripheral blood mononuclear cells to collect total T cells; The total T cells are mixed with the reprogramming plasmid, and the plasmid is nuclear transfected to obtain the total T cells after nuclear transfection; The total T cells after nuclear transfer are mixed with activation factors and cultured in stages to induce induced pluripotent stem cells; Wherein, the activating factors include CD3 / CD28 and IL2.

2. The method for reprogramming T cells into induced pluripotent stem cells according to claim 1, characterized in that: The step of "mixing the total T cells after nuclear transfer with the activation factors and culturing them in stages to induce induced pluripotent stem cells" includes: The total T cells after nuclear transfer are mixed with activation factors for activation, and then mixed with a variety of culture media for staged culture to induce induced pluripotent stem cells, wherein the various culture media include Matrigel.

3. The method for reprogramming T cells into induced pluripotent stem cells according to claim 2, characterized in that: The reprogramming plasmids include pCE-hOCT3 / 4, pCE-hSK, pCE-hUL and pCE-mp53DD; and / or, The multiple culture media include T cell culture medium, induction medium N2B27 and mTeSR Plus stem cell culture medium.

4. The method for reprogramming T cells into induced pluripotent stem cells according to claim 3, characterized in that: The step of "mixing the total T cells after nuclear transfer with an activation factor for activation, and then mixing with a plurality of culture media for culturing in stages, and inducing induced pluripotent stem cells, wherein the plurality of culture media include Matrigel gel, the plurality of culture media include T cell culture media, induction culture media N2B27 and mTeSR Plus stem cell culture media" includes: Mixing and culturing the total T cells after nuclear transfer with a T cell culture medium to obtain a first culture medium; The first culture medium is mixed with an activation factor and an induction medium N2B27 and cultured until early induced pluripotent stem cells appear to obtain a second culture medium; The second culture medium is replaced with mTeSR Plus stem cell culture medium for mixed culture to obtain induced pluripotent stem cells.

5. The method for reprogramming T cells into induced pluripotent stem cells according to claim 1, characterized in that: The step "obtaining isolated peripheral blood mononuclear cells" includes: Collect human venous peripheral blood; The human venous peripheral blood is mixed with stem cell protection solution and lymph separation solution, centrifuged, washed, purified and cultured to obtain separated peripheral blood mononuclear cells.

6. The method for reprogramming T cells into induced pluripotent stem cells according to claim 1, characterized in that: In the step "sorting the separated peripheral blood mononuclear cells to collect total T cells": the sorting method is magnetic bead sorting.

7. The method for reprogramming T cells into induced pluripotent stem cells according to claim 1, characterized in that: The step of "mixing total T cells with reprogramming plasmids, performing plasmid nuclear transfection, and obtaining nuclear transfected total T cells" includes: Resuspend the total stem cells in the total T cells to obtain a resuspended solution; The resuspension is mixed with a T cell nuclear transfer reaction solution and a reprogramming plasmid to perform plasmid nuclear transfer, thereby obtaining total T cells that have completed nuclear transfer.

8. An induced pluripotent stem cell, characterized in that The induced pluripotent stem cells are obtained by the method for reprogramming T cells into induced pluripotent stem cells according to any one of claims 1 to 7.

9. A pharmaceutical composition, characterized in that The composition comprises the induced pluripotent stem cells according to claim 8 and / or active ingredients in the induced pluripotent stem cells.

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