Method for reversing ips cells through peripheral blood mononuclear cells and application

Abstract

The invention relates to the technical field of biological materials, in particular to a method for reversing ips cells through peripheral blood mononuclear cells and application, and the method comprises the following steps: separating the peripheral blood mononuclear cells through a density gradient centrifugation method, and pretreating the peripheral blood mononuclear cells; meanwhile, a reprogramming factor mRNA is encapsulated in an LNP carrier, and the reprogramming factor comprises Oct4, Sox2, Klf4, Glis1, Nanog and Lin28, so that an LNP-reprogramming factor compound is obtained; and introducing the LNP-reprogramming factor compound into the pretreated mononuclear cells through an electroporation method, and carrying out induced culture on the mononuclear cells until the IPS cells are obtained. The method provides a reliable technical scheme for efficient and high-quality preparation of the peripheral blood-derived iPS cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a method and application of reversing ips cells by peripheral blood mononuclear cells. Background Art

[0002] The discovery of induced pluripotent stem cells (iPS cells) marked a milestone in stem cell research. In 2006, Takahashi K and Yamanaka S published a study in the journal Cell demonstrating the successful reprogramming of mouse fibroblasts into iPS cells using a retroviral vector to introduce four transcription factors—Oct4, Sox2, c-Myc, and Klf4—into them. This study pioneered the reprogramming of adult cells. The following year, Takahashi K et al. also demonstrated in Cell that the same four transcription factors could also be used to reprogram adult human fibroblasts into iPS cells using a retroviral vector, extending this technology to human cells. However, viral vector-mediated reprogramming carries the risk of exogenous gene integration, which can increase the tumorigenicity of cells and limit its clinical application. Since then, researchers have continued to explore safer reprogramming methods. In 2009, Yusa K et al. published a study in Nature Methods, using the piggyBac transposon system to reprogram mouse fibroblasts into iPS cells. This method can obtain iPS cells without integrating exogenous genes, reducing the risk of tumorigenicity. At the same time, non-viral vector-mediated reprogramming technologies have also continued to advance. In 2009, Chang CW et al. published in Stem Cells a method for reprogramming using polycistronic lentiviral vectors. This method allows for the efficient expression of multiple reprogramming factors in a short period of time, enabling the efficient reprogramming of fibroblasts into iPS cells. That same year, Zhou H et al. published in Cell Stem Cell using recombinant proteins to reprogram fibroblasts into iPS cells, avoiding the issues of genomic integration and genetic material transfer. In 2010, Jia F et al. published in Nature Methods a method for introducing reprogramming factors into human fibroblasts using a non-viral small plasmid vector, successfully generating iPS cells and avoiding the genomic integration risks associated with viral vectors.

[0003] In addition, other technologies are being explored to improve the safety and efficiency of iPS cell generation. In 2014, Ichida JK et al. reported in Nature Chemical Biology that inhibiting the Notch signaling pathway can achieve oncogene-independent iPS cell generation, providing a new approach to improving reprogramming safety. In 2016, Iseki H et al. reported in Stem Cells that combined overexpression of four genes, JARID2, PRDM14, ESRRB, and SALL4A, significantly increased the efficiency and speed of fibroblast-to-iPS cell reprogramming. Although existing technologies have made significant progress in reprogramming fibroblasts or adult cells into iPS cells, research on reversing the conversion of peripheral blood mononuclear cells into iPS cells remains in its infancy.

[0004] Chinese invention patent application CN103097521A discloses a method for producing artificial pluripotent stem cells. This method uses a mononuclear cell population from peripheral blood to be cultured in an environment of anti-CD3 antibodies and interleukin 2, and introduces factors such as Sox2, Oct4, Klf4 and c-Myc. This solves the problems of the burden of tissue collection and low efficiency in existing iPS cell production methods, and achieves efficient and low-invasive iPS cell production. This technology uses Sendai virus vectors as recombinant expression vectors. The disadvantage of this type of method is that random insertion of the genome may bring potential risks and high costs. Based on this, in May 2013, Professor Zhang Xiaobing successfully reprogrammed human peripheral blood mononuclear cells into iPS cells using modified EVs expressing Oct4 / Sox2 / Klf4 / c-Myc. Meanwhile, Chinese invention patent No. CN103756970B further optimized the episomal technology for establishing human iPS. By optimizing culture conditions and technical means, it improved the reprogramming efficiency of episomal technology on human peripheral blood cells, solved the problem of low efficiency in the prior art, and achieved efficient and safe iPS cell establishment and expansion. Similarly, Chinese invention patent No. CN117487759A further disclosed an induced pluripotent stem cell obtained by reprogramming peripheral blood mononuclear cells, a method, and its use. In this method, induced pluripotent stem cells are obtained by direct electroporation of peripheral blood mononuclear cells through reprogramming, and the cell culture conditions and electroporation conditions in this process are optimized. The reprogramming efficiency is high, the iPSC cells obtained are of high purity, and the entire reprogramming cycle is short, thereby improving efficiency.

[0005] In preliminary research, it was found that the existing technology generally uses fico11 gradient centrifugation technology to separate peripheral blood mononuclear cells. The initial state of the separated peripheral blood mononuclear cells is crucial for subsequent reprogramming. Good pretreatment can stimulate cell potential and resist damage, which meets the needs of improving the success rate of reprogramming. However, the existing technology has little research on this. Summary of the Invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a method and application of reversing ips cells with peripheral blood mononuclear cells. This method effectively improves the feasibility of reversing ips cells with peripheral blood mononuclear cells by special pretreatment of peripheral blood mononuclear cells, thereby solving the problems of the prior art.

[0007] In a first aspect, the present invention aims to provide a method for reversing ips cells by peripheral blood mononuclear cells, which specifically comprises the following steps: S1: Peripheral blood mononuclear cells were isolated by density gradient centrifugation; S2: incubate the peripheral blood mononuclear cells in the pretreatment solution, wash them with PBS and set aside to obtain pretreated mononuclear cells; S3: Encapsulating reprogramming factor mRNA in LNP vector, wherein the reprogramming factor comprises Oct4, Sox2, Klf4, Glis1, Nanog and Lin28, thereby obtaining LNP-reprogramming factor complex; S4: The LNP-reprogramming factor complex is introduced into the pretreated monocytes by electroporation, and then induced and cultured until IPS cells are obtained.

[0008] Preferably, the centrifugation step in step S1 includes: initial centrifugation at 400–500 g for 20–30 minutes; collecting the PBMC layer, diluting it with PBS, and then washing it by centrifugation at 300–400 g for 8–10 minutes; repeating the centrifugation and washing steps once to obtain peripheral blood mononuclear cells.

[0009] Preferably, the pretreatment solution in step S2 contains M-CSF 20–100 ng / mL, GM-CSF 10–50 ng / mL, and NAC 4–10 mM; More preferably, the concentration of M-CSF in the pretreatment solution is 50 ng / mL, the concentration of GM-CSF is 25 ng / mL, and the concentration of NAC is 8 mM.

[0010] Preferably, the incubation conditions are: incubation at 37° C., 5% CO 2 in an incubator with a shaker at 100 rpm for 1-3 hours, followed by washing 2-3 times with calcium- and magnesium-free PBS.

[0011] Furthermore, the preparation method of the LNP-reprogramming factor complex comprises the following steps: (a) preparing a lipid solution, wherein the lipid solution is prepared using anhydrous ethanol as a solvent and having a total lipid concentration of 8-10 mg / mL; (b) Dissolve 15 μg each of Oct4 and Sox2, 10 μg each of Klf4 and Glis1, and 3 μg each of Nanog and Lin28 in 0.1 M citric acid buffer (pH 4.0) to obtain mRNA solution; (c) The lipid solution and the mRNA solution were mixed in a volume ratio of 1:3, and the lipid solution was rapidly injected into the mRNA solution, and vortexed at 1500-2000 rpm for 10 seconds; then, mixing was performed using a microfluidic device, with the flow rate ratio controlled at 3:1 during the mixing process and a total flow rate of 12 mL / min. The mixture was sterilized by low-pressure filtration (≤0.5 bar) through a 0.22 μm PES filter membrane to obtain the LNP-reprogramming factor complex.

[0012] Preferably, in the lipid solution, the molar ratio of Lin-MC3-DMA, DOPE, cholesterol and DMG-PEG2000 is 50:10:38.5:1.5.

[0013] Preferably, the electroporation method in step S4 is: Take 1.0×10 5 The pretreated cells were incubated with 2 μg of LNP complexes at room temperature for 20 minutes and electroporated with a single pulse of 90-120 V and 5-10 ms.

[0014] Preferably, the induction culture in step S4 includes three stages, specifically including: In the first stage, 0-3 days after electroporation, mTeSR™ Plus was used as the basal medium, supplemented with Y-27632 (10 μM), ascorbic acid (100 μg / mL), and bFGF (20 ng / mL), with full medium replacement every 24 hours. In the second stage, 3-10 days after electroporation, mTeSR™ Plus was used as the basal medium, and PD0325901 (50 nM), CHIR99021 3 μM, VPA 0.3 mM, and L-ascorbic acid 0.2 mM were added to it, and half of the medium was replaced every 48 hours; In the third stage, 11-25 days after electroporation, mTeSR™ Plus was used as the basal culture medium, and bFGF (20 ng / mL) and L-ascorbic acid 0.2 mM were added to it, with half the medium replaced every 48 hours.

[0015] Furthermore, the induction culture conditions in step S4 are: 37° C., 5% O 2 , 5% CO 2 .

[0016] In a second aspect, the present invention further provides the use of iPS cells obtained by the method provided in the first aspect in constructing disease models or drug screening, wherein the use is for non-therapeutic purposes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for reversing the conversion of peripheral blood mononuclear cells into iPS cells by systematically optimizing the key steps of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells (iPS cells), significantly improving the practicality and reliability of the technology. The method includes: first, the improvement of the pretreatment step of the present invention effectively regulates the oxidative stress state of the cells, providing a healthier starting cell foundation for subsequent reprogramming and reducing cell dysfunction caused by environmental damage; second, the phased culture medium design precisely matches the requirements of different reprogramming stages, from anti-apoptosis to epigenetic regulation to pluripotency maintenance, greatly improving the conversion efficiency of pluripotent stem cells; in addition, through the coordinated optimization of centrifugation, delivery vector preparation and electroporation conditions, not only the operational stability is improved, but also the quality of the final iPS cells is guaranteed - the cell pluripotency genes are more fully activated, the epigenetic state is more stable, and the risk of karyotype abnormalities is reduced. Overall, the present invention provides a scientific and feasible technical solution for the efficient preparation of peripheral blood-derived iPS cells, reduces the difficulty of experimental operation, improves the reproducibility of results, and lays a more solid foundation for its application in regenerative medicine, disease model construction and other fields. DETAILED DESCRIPTION

[0018] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are therefore only used as examples, and cannot be used to limit the scope of protection of the present invention. It should be understood by those skilled in the art that without certain specific details, the present invention can be implemented equally. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Unless otherwise specified, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention are all understood to include inevitable systematic errors in industrial production.

[0019] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used without indicating the manufacturer are all reagents and materials that can be obtained from commercial channels. If no specific conditions are specified in the examples, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in this technical field.

[0020] Example 1 This embodiment provides a method for reversing ips cells by peripheral blood mononuclear cells, which specifically includes the following steps: Step S1: Isolation of peripheral blood mononuclear cells Fresh peripheral blood samples were collected and separated using density gradient centrifugation. Initial centrifugation was performed at 400g for 20 minutes. After centrifugation, the PBMC layer was carefully collected, diluted with PBS, and then washed by centrifugation at 300g for 8 minutes. This centrifugation and washing step was repeated once to obtain peripheral blood mononuclear cells. Step S2: Pretreatment of monocytes Prepare a pretreatment solution containing 20 ng / mL M-CSF, 10 ng / mL GM-CSF, and 4 mM NAC. Place the peripheral blood mononuclear cells in this pretreatment solution and incubate in a 37°C, 5% CO2 incubator at 100 rpm for 1 hour. After incubation, wash the cells twice with calcium- and magnesium-free PBS and set aside to obtain the pretreated mononuclear cells. Step S3: preparing LNP-reprogramming factor complex, comprising the steps of: (a) Lipid solution was prepared using anhydrous ethanol as the solvent. The total lipid concentration was controlled at 8 mg / mL. The molar ratio of Lin-MC3-DMA, DOPE, cholesterol, and DMG-PEG2000 in the lipid solution was 50:10:38.5:1.5. (b) Dissolve 15 μg each of Oct4 and Sox2, 10 μg each of Klf4 and Glis1, and 3 μg each of Nanog and Lin28 in 0.1 M citrate buffer (pH 4.0) to obtain an mRNA solution. (c) The lipid solution and mRNA solution were mixed at a 1:3 volume ratio. The lipid solution was rapidly injected into the mRNA solution and vortexed at 1500 rpm for 10 seconds. Mixing was then performed using a microfluidic device, with a flow rate ratio of 3:1 and a total flow rate of 12 mL / min. Finally, the solution was sterilized by low-pressure filtration (≤0.5 bar) through a 0.22 μm PES filter to obtain the LNP-reprogramming factor complex. Step S4: Introduce the complex and induce culture Take 1.0×10 5 The pretreated cells were incubated with 2 μg of LNP complex at room temperature for 20 minutes, and electroporated into the cells using a single pulse of 90 V and 5 ms duration. The cells were then cultured under the following conditions: 0-3 days after electroporation, use mTeSR™ Plus as the basal medium and supplement it with Y-27632 (10 μM), ascorbic acid (100 μg / mL), and bFGF (20 ng / mL). The medium is replaced every 24 hours. 3-10 days after electroporation, use mTeSR™ Plus as the basal medium and add PD0325901 (50 nM), CHIR99021 3 μM, VPA 0.3 mM, and L-ascorbic acid 0.2 mM. Change half the medium every 48 hours. Eleven to 25 days after electroporation, use mTeSR™ Plus as the basal medium and supplement it with bFGF (20 ng / mL) and 0.2 mM L-ascorbic acid. Change the medium halfway every 48 hours. Induce the culture at 37°C, 5% O₂, 5% CO₂ until IPS cells are obtained. Example 2 This embodiment provides a method for reversing ips cells by peripheral blood mononuclear cells, which specifically includes the following steps: Step S1: Isolation of peripheral blood mononuclear cells Peripheral blood samples were obtained and separated by density gradient centrifugation. Initial centrifugation was performed at 450g for 25 minutes. The PBMC layer was collected and diluted with PBS. Peripheral blood mononuclear cells were then obtained by centrifugation at 350g for 9 minutes. Step S2: Pretreatment of monocytes Prepare a pretreatment solution containing 50 ng / mL M-CSF, 25 ng / mL GM-CSF, and 8 mM NAC. Place peripheral blood mononuclear cells in the pretreatment solution and incubate in a 37°C, 5% CO2 incubator with a shaker at 100 rpm for 2 hours. Wash three times with calcium- and magnesium-free PBS to obtain pretreated monocytes. Step S3: Preparation of LNP-reprogramming factor complexes (a) Lipid solution was prepared using anhydrous ethanol as the solvent. The total lipid concentration was 9 mg / mL and the molar ratio of the lipid components Lin-MC3-DMA, DOPE, cholesterol, and DMG-PEG2000 was maintained at 50:10:38.5:1.5. (b) 15 μg each of Oct4 and Sox2, 10 μg each of Klf4 and Glis1, and 3 μg each of Nanog and Lin28 were dissolved in 0.1 M citrate buffer (pH 4.0) to prepare mRNA solution. (c) The lipid solution and mRNA solution were mixed in a 1:3 volume ratio. The lipid solution was rapidly injected into the mRNA solution and vortexed at 1800 rpm for 10 s. The mixture was mixed using a microfluidic device with a flow rate ratio of 3:1 and a total flow rate of 12 mL / min. The mixture was sterilized by low-pressure filtration (≤0.5 bar) through a 0.22 μm PES filter to obtain the LNP-reprogramming factor complex. Step S4: Introduce the complex and induce culture Take 1.0×10 5 The pretreated cells were incubated with 2 μg of LNP complex at room temperature for 20 minutes, and electroporated with a single pulse of 100 V and 8 ms duration. The cells were then cultured under the following conditions: 0-3 days after electroporation, use mTeSR™ Plus as the basal medium, supplemented with Y-27632 (10 μM), ascorbic acid (100 μg / mL), and bFGF (20 ng / mL), and replace the medium every 24 hours. 3-10 days after electroporation, use mTeSR™ Plus basal medium supplemented with PD0325901 (50 nM), CHIR99021 3 μM, VPA 0.3 mM, and L-ascorbic acid 0.2 mM, and change half the medium every 48 hours; Eleven to 25 days after electroporation, culture the cells in mTeSR™ Plus basal medium supplemented with bFGF (20 ng / mL) and 0.2 mM L-ascorbic acid. Change the medium halfway every 48 hours. Induce the cells at 37°C, 5% O₂, 5% CO₂ until IPS cells are obtained. Example 3 This embodiment provides a method for reversing ips cells by peripheral blood mononuclear cells, which specifically includes the following steps: Step S1: Isolation of peripheral blood mononuclear cells Peripheral blood was collected and separated by density gradient centrifugation. Initial centrifugation parameters were set at 500g for 30 minutes. The PBMC layer was collected and diluted with PBS. The cells were then washed by centrifugation at 400g for 10 minutes. This was repeated once to obtain peripheral blood mononuclear cells. Step S2: Pretreatment of monocytes Prepare a pretreatment solution containing 100 ng / mL M-CSF, 50 ng / mL GM-CSF, and 10 mM NAC. Place peripheral blood mononuclear cells in the pretreatment solution and incubate in a 37°C, 5% CO2 incubator with a shaker at 100 rpm for 3 hours. Wash three times with calcium- and magnesium-free PBS to obtain pretreated monocytes. Step S3: Preparation of LNP-reprogramming factor complexes (a) Lipid solution was prepared using anhydrous ethanol as the solvent. The total lipid concentration was 10 mg / mL and the molar ratio of Lin-MC3-DMA, DOPE, cholesterol, and DMG-PEG2000 in the lipids was 50:10:38.5:1.5. (b) Dissolve 15 μg each of Oct4 and Sox2, 10 μg each of Klf4 and Glis1, and 3 μg each of Nanog and Lin28 in 0.1 M citrate buffer (pH 4.0) to obtain an mRNA solution. (c) The lipid solution and mRNA solution were mixed in a 1:3 volume ratio. The lipid solution was rapidly injected into the mRNA solution and vortexed at 2000 rpm for 10 s. The mixture was mixed using a microfluidic device with a flow rate ratio of 3:1 and a total flow rate of 12 mL / min. The mixture was sterilized by low-pressure filtration (≤0.5 bar) through a 0.22 μm PES filter to obtain the LNP-reprogramming factor complex. Step S4: Introduce the complex and induce culture Take 1.0×10 5The pretreated cells were incubated with 2 μg of LNP complex at room temperature for 20 minutes, and electroporated into the cells using a single pulse of 120 V and 10 ms duration. The cells were then cultured under the following conditions: 0-3 days after electroporation, use mTeSR™ Plus as the basal medium, supplemented with Y-27632 (10 μM), ascorbic acid (100 μg / mL), and bFGF (20 ng / mL), and replace the medium every 24 hours. 3-10 days after electroporation, use mTeSR™ Plus basal medium supplemented with PD0325901 (50 nM), CHIR99021 3 μM, VPA 0.3 mM, and L-ascorbic acid 0.2 mM, and change half the medium every 48 hours; Eleven to 25 days after electroporation, use mTeSR™ Plus basal medium supplemented with bFGF (20 ng / mL) and 0.2 mM L-ascorbic acid, with a half-volume medium change every 48 hours. Induce and culture at 37°C, 5% O2, 5% CO2 until IPS cells are obtained.

[0021] Comparative Example 1 The purpose of this comparative example is to provide multiple comparative examples with respect to Example 2 to verify the effect of the pretreatment liquid NAC. This comparative example includes: (1) Comparative Example 1-1: In this group, NAC was removed from the pretreatment solution, and other parameters and treatment methods were the same as those in Example 2 (M-CSF 50 ng / mL, GM-CSF 25 ng / mL).

[0022] (2) Comparative Example 1-2: The composition of this group of pretreatment solutions is as follows: M-CSF concentration is 50 ng / mL, GM-CSF concentration is 25 ng / mL, and NAC concentration is 2 mM; other parameters and treatment methods are the same as those in Example 2.

[0023] (3) Comparative Examples 1-3: The composition of the pretreatment solution in this group was as follows: M-CSF concentration was 50 ng / mL, GM-CSF concentration was 25 ng / mL, and NAC concentration was 12 mM; other parameters and treatment methods were the same as those in Example 2.

[0024] Comparative Example 2 The purpose of this comparative example is to provide multiple comparative examples with respect to Example 2 to verify the necessity of the stage induction culture of this technical solution. This comparative example specifically includes: (1) Comparative Example 2-1: This group of steps S1 to S3 are the same as those in Example 2, and only step S4 is different from Example 2. Specifically, step S4 of this comparative example is as follows: Step S4: Introduce the complex and induce culture Take 1.0×10 5 The pretreated cells were incubated with 2 μg of LNP complex at room temperature for 20 minutes, and electroporated with a single pulse of 100 V and 8 ms duration. The cells were then cultured under the following conditions: The same culture medium was used for culture from 0 to 25 days after electroporation. Specifically, mTeSR™ Plus was used as the basal medium, supplemented with Y-27632 (10 μM), ascorbic acid (100 μg / mL), and bFGF (20 ng / mL). From 0 to 3 days, the medium was fully replaced every 24 hours; after 3 days, the medium was half replaced every 48 hours.

[0025] (2) Comparative Example 2-2: This group differs from Example 2 only in step S4. Specifically, step S4 of this comparative example is as follows: The electroporation conditions were the same, and the culture conditions after electroporation were as follows: The first stage: 0-3 days after electroporation, same as Example 2; Stage 2: 3-10 days after electroporation, use mTeSR™ Plus basal medium supplemented with PD0325901 (50 nM), CHIR99021 3 μM, and L-ascorbic acid 0.2 mM, and change the medium halfway every 48 hours. The third stage: the same as in Example 2; (3) Comparative Example 2-3: This group differs from Example 2 only in step S4. Specifically, step S4 of this comparative example is as follows: The electroporation conditions were the same, and the culture conditions after electroporation were as follows: The first stage: 0-3 days after electroporation, same as Example 2; Phase II: 3-10 days after electroporation, use mTeSR™ Plus basal medium supplemented with PD0325901 (50 nM), CHIR99021 3 μM, VPA 0.5 mM, and L-ascorbic acid 0.2 mM, with a half-volume medium change every 48 hours. The third stage: the same as in Example 2; (3) Comparative Examples 2-4: This group differs from Example 2 only in step S4. Specifically, step S4 of this comparative example is as follows: The electroporation conditions were the same, and the culture conditions after electroporation were as follows: Phase 1: 0-5 days after electroporation, use mTeSR™ Plus as the basal medium, supplemented with Y-27632 (10 μM), ascorbic acid (100 μg / mL), and bFGF (20 ng / mL), and replace the medium every 24 hours. Phase II: 5-10 days after electroporation, use mTeSR™ Plus basal medium supplemented with PD0325901 (50 nM), CHIR99021 3 μM, VPA 0.3 mM, and L-ascorbic acid 0.2 mM, and change the medium halfway every 48 hours. The third stage: the same as in Example 2; Test example This test example compares the methods of the above embodiments and comparative examples, including: (1) Detect ROS levels 1 hour after pretreatment: Fluorescence probe method was used for detection (DCFH-DA 10 μM loading for 30 minutes, flow cytometry detection of FL1 channel fluorescence intensity, excitation 488 nm / emission 530 nm), and relative fluorescence intensity was calculated to evaluate the oxidative stress status and the antioxidant efficacy of NAC. The experimental results are shown in Table 1.

[0026] Table 1 Differences in ROS accumulation levels after cell pretreatment in various examples and comparative examples (2) Detect cell apoptosis rate (%) 72 hours (3 days) after electroporation: Flow cytometry (Annexin V-FITC / PI double staining, BD Biosciences kit, gating to exclude debris and counting early apoptotic cells) was used to quantify the cytotoxicity of electroporation / LNP introduction to optimize delivery conditions. The experimental results are shown in Table 2.

[0027] Table 2 Cell apoptosis rate of each example and comparative example 3 days (72 hours) after electroporation (3) AP detection on the 20th day of induction + Number of clones Alkaline phosphatase staining (Stemgent® AP staining kit, microscopic counting of positive colonies of ≥50 cells) was used as a core efficiency indicator to reflect the number of successfully transformed pluripotent stem cells. The experimental results are shown in Table 3.

[0028] Table 3 AP of cells in various examples and comparative examples 20 days after electroporation + Difference in clone number (4) On the 25th day of induction, the expression level of OCT4, the proportion of cells with normal karyotype (%), and the methylation rate of OCT4 promoter (%) were detected. OCT4 expression: Quantitative PCR was used to detect OCT4 expression, characterizing the degree of activation of core pluripotency genes and assessing the transcriptome completeness of reprogramming; The proportion of cells with normal karyotype: analyzed by G-banding karyotype analysis (collect cells using the colchicine block method, stain with Giemsa, and scan and analyze ≥50 metaphases using the Metafer system); OCT4 promoter methylation rate: Bisulfite sequencing (DNA treated with the EZ DNA Methylation Kit, promoter region -500 to +200 bp amplified by PCR, ≥50 clones analyzed on the Illumina MiSeq platform) was used to quantify the degree of epigenetic silencing relief at the pluripotency locus to predict cell quality stability. The experimental results are shown in Table 4: Table 4 Differences in cells electroporated 25 days after each embodiment and comparative example First, the above experimental results show that: (1) The addition of NAC significantly affected the oxidative stress level (ROS), cell apoptosis rate, and reprogramming efficiency (AP+ clone number), specifically: The ROS level in Comparative Example 1-1 (no NAC) (282.4 ± 13.1) was significantly higher than that in Example 2 (105.5 ± 4.1), indicating that NAC can effectively reduce oxidative stress. The ROS levels in Comparative Examples 1-2 (2 mM NAC) and 1-3 (12 mM NAC) were still higher than those in Example 2 (8 mM NAC), indicating that 8 mM is the optimal concentration of NAC.

[0029] The apoptosis rate of Comparative Example 1-1 (no NAC) was significantly higher than that of Example 2, and the apoptosis rate of Comparative Example 1-2 (2 mM NAC) was even higher than that of 1-1. This may be because low-concentration NAC cannot effectively prevent oxidation and instead aggravates damage due to changes in osmotic pressure. The apoptosis rate of Comparative Example 1-3 (12 mM NAC) (28.1 ± 3.5%), although lower than that of 1-1, was still higher than that of Example 2, indicating that high-concentration NAC may be toxic.

[0030] The number of clones in Comparative Example 1-1 (without NAC) (6.2 ± 0.6) was significantly lower than that in Example 2 (34.7 ± 3.2). Comparative Examples 1-2 (15.1 ± 2.4) and 1-3 (11.3 ± 2.1) were also much lower than that in Example 2, further verifying that NAC (8 mM) significantly improved reprogramming efficiency by reducing oxidative stress and apoptosis.

[0031] The OCT4 expression level and normal karyotype ratio of Comparative Example 1-1 (without NAC) were significantly lower than those of Example 2, and the methylation rate was significantly higher than that of Example 2, indicating that NAC promoted pluripotency gene activation (OCT4) and epigenetic reprogramming (reduced methylation rate) by reducing oxidative stress.

[0032] (2) Culturing cells in stages after electroporation and adjusting the culture medium components in stages are the keys to successful reprogramming. Specifically: The same culture medium was used throughout the process (Comparative Example 2-1): The number of AP+ clones (12.3 ± 2.5) was only 35% of that in Example 2 (34.7 ± 3.2), the OCT4 expression level and the normal karyotype ratio were significantly lower than those in Example 2, and the methylation rate was significantly higher than that in Example 2, indicating that the use of early culture medium throughout the process cannot meet the reprogramming requirements at different stages.

[0033] In the second stage without VPA (Comparative Example 2-2), the number of AP+ clones was lower than that in Example 2, the OCT4 expression level and the normal karyotype ratio were also significantly reduced, and the methylation rate was higher than that in Example 2, indicating that VPA (histone deacetylation inhibitor) promoted epigenetic reprogramming in the second stage.

[0034] In the second stage, VPA was at high concentration (Comparative Examples 2-3): The number of AP+ clones was extremely low, the OCT4 expression level and the normal karyotype ratio were significantly reduced, and the methylation rate was extremely high, indicating that 0.5 mM VPA may inhibit cell survival and reprogramming due to toxicity.

[0035] The first stage culture time was extended (Comparative Example 2-4): the number of AP+ clones was close to that of Comparative Example 2-2, the OCT4 expression level and the normal karyotype ratio were better than those of Comparative Example 2-2 but still lower than those of Example 2, and the methylation rate was lower than that of Comparative Example 2-2, indicating that extending the first stage to 5 days may partially compensate for the shortcomings of the second stage, but it is not the optimal solution.

[0036] In summary, the inventors believe that: Adding 8mM NAC to the pretreatment solution significantly reduced oxidative stress (ROS) and apoptosis, thereby improving reprogramming efficiency and pluripotency quality. Adjusting the culture medium in stages (0-3 days, 3-10 days, and 11-25 days) is essential for successful reprogramming, with the addition of 0.3mM VPA in the second stage being a key optimization point. Among Examples 1-3, the parameter combination of Example 2 was the optimal solution, achieving significantly better overall performance than the other groups.

[0037] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that the technical solutions of the present invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, and such modifications or equivalents shall be encompassed by the claims of the present invention. Any techniques, shapes, and structures not described in detail herein are well known.

Claims

1. A method for reversing ips cells by using peripheral blood mononuclear cells, characterized in that: The following steps are involved: S1: Peripheral blood mononuclear cells were isolated by density gradient centrifugation; S2: incubate the peripheral blood mononuclear cells in the pretreatment solution, wash them with PBS and set aside to obtain pretreated mononuclear cells; S3: Encapsulating reprogramming factor mRNA in LNP vector, wherein the reprogramming factor comprises Oct4, Sox2, Klf4, Glis1, Nanog and Lin28, thereby obtaining LNP-reprogramming factor complex; S4: The LNP-reprogramming factor complex is introduced into the pretreated monocytes by electroporation, and then induced and cultured until IPS cells are obtained.

2. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The centrifugation step in step S1 includes: initial centrifugation at 400–500 g for 20–30 minutes; collecting the PBMC layer, diluting it with PBS, and then washing it by centrifugation at 300–400 g for 8–10 minutes; repeating the centrifugation and washing steps once to obtain peripheral blood mononuclear cells.

3. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The pretreatment solution in step S2 contains M-CSF 20–100 ng / mL, GM-CSF 10–50 ng / mL, and NAC 4–10 mM.

4. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The incubation conditions are as follows: incubate at 37° C., 5% CO 2 in a 100 rpm incubator for 1-3 hours, and then wash 2-3 times with calcium- and magnesium-free PBS.

5. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The preparation method of the LNP-reprogramming factor complex comprises the following steps: (a) preparing a lipid solution, wherein the lipid solution is prepared using anhydrous ethanol as a solvent and having a total lipid concentration of 8-10 mg / mL; (b) Dissolve 15 μg each of Oct4 and Sox2, 10 μg each of Klf4 and Glis1, and 3 μg each of Nanog and Lin28 in 0.1 M citric acid buffer to obtain mRNA solution; (c) The lipid solution and the mRNA solution were mixed in a volume ratio of 1:3, and the lipid solution was rapidly injected into the mRNA solution, and vortexed at 1500-2000 rpm for 10 seconds; then, mixing was performed using a microfluidic device, with the flow rate ratio controlled at 3:1 during the mixing process and a total flow rate of 12 mL / min. The mixture was sterilized by low-pressure filtration through a 0.22 μm PES filter membrane to obtain the LNP-reprogramming factor complex.

6. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 5, wherein: In the lipid solution, the molar ratio of Lin-MC3-DMA, DOPE, cholesterol and DMG-PEG2000 is 50:10:38.5:1.

5.

7. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The electroporation method in step S4 is: Take 1.0×10 5 The pretreated cells were incubated with 2 μg of LNP complexes at room temperature for 20 minutes and electroporated with a single pulse of 90-120 V and 5-10 ms.

8. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The induction culture in step S4 specifically includes: 0-3 days after electroporation, use mTeSR™ Plus as the basal medium and supplement it with Y-27632, ascorbic acid, and bFGF. Change the medium completely every 24 hours. 3-10 days after electroporation, use mTeSR™ Plus as the basal medium and supplement it with PD0325901, CHIR99021, VPA, and L-ascorbic acid. Change half the medium every 48 hours. 11-25 days after electroporation, use mTeSR™ Plus as the basal medium, supplement it with bFGF and L-ascorbic acid, and change half the medium every 48 hours.

9. The method for reversing ips cells from peripheral blood mononuclear cells according to claim 1, wherein: The induction culture conditions in step S4 are: 37° C., 5% O 2 , 5% CO 2 .

10. Use of iPS cells obtained by the method according to any one of claims 1 to 9 in constructing disease models or drug screening, characterized in that: The use is for non-therapeutic purposes.

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