A method for preparing reprogrammed induced pluripotent stem cells from PBMCs and a kit

Abstract

The present invention provides a method and a kit for preparing reprogrammed induced pluripotent stem cells from PBMCs. Specifically, it provides an mRNA-LNP-based reprogramming of PBMCs. More specifically, it relates to a method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells. The mRNA mixture of multiple reprogramming factors is encapsulated by the cationic lipid SM102 to prepare a nano-liposomal mRNA cocktail-LNP. The peripheral blood mononuclear cells are reprogrammed with the nano-liposomal mRNA-LNP to obtain reprogrammed induced pluripotent stem cells, realizing a safer iPSCs reprogramming technology with low cost, low toxicity, high efficiency, stability and repeatability. The present invention also provides a reprogramming kit and a method for preparing induced pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to the fields of regenerative medicine and cells, and particularly to a method for preparing reprogrammed induced pluripotent stem cells from PBMCs and a kit. Background Art

[0002] The fields of regenerative medicine and cell therapy have always been the focus of scientific research, because they have great potential to solve numerous diseases and health problems. In recent years, stem cell research has gradually deepened. The ability of stem cells to self-renew and differentiate into multiple cell types makes them an important resource in the field of regenerative medicine. The reprogramming method of human adult somatic cells into induced pluripotent stem cells (iPSCs) is an important technical means to obtain stem cells. In 2006, the Yamanaka team (Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663 - 676, 2006.) invented a mixture composed of four genes, Oct4, Sox2, Klf4, and c-Myc, and successfully reprogrammed terminally differentiated skin fibroblasts into induced pluripotent stem cells (iPSCs) by viral infection. One year later, James Thomson (Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920, 2007.) successfully reprogrammed human fibroblasts into iPSCs by episomal plasmid transfection using a different combination of four factors (Oct4, Sox2, Nanog, and Lin28 combination). This technology can overcome ethical problems and expand treatment possibilities. However, the clinical application of iPSCs faces many challenges, among which the integration of exogenous genes into the genome poses a huge obstacle to the therapeutic application of induced pluripotent stem cells. Therefore, it is imperative to seek a simple and reproducible method to generate integration-free clinical-grade iPSCs. <> <>

[0003] Currently, the preparation of iPSCs mainly relies on sources such as skin fibroblasts or CD34+ hematopoietic stem cells. However, the acquisition process of these cells requires invasive operations (such as skin or tissue biopsies and long-term in vitro primary cell culture), which limits the feasibility of clinical applications. In contrast, peripheral blood mononuclear cells (PBMCs), as a common component in peripheral blood, can be obtained almost non-invasively by routine blood drawing, without additional surgical risks and pain. The acceptance rate of donors (especially patients) is relatively high, and they can be cryopreserved for long-term use, showing significant advantages in convenience and repeatability. Therefore, PBMCs are particularly suitable as a source of samples for clinical-grade cell banks. However, as terminally differentiated cells (such as lymphocytes and monocytes), PBMCs have a highly closed epigenetic state, resulting in low efficiency of traditional reprogramming techniques. Existing methods also heavily rely on viral vectors (such as retroviruses and lentiviruses) or electroporation delivery systems. Although viral vectors can efficiently introduce reprogramming genes, they carry the risk of insertional mutations caused by random genomic integration, which may lead to genomic instability of iPSCs or the obtained iPSCs being impure, containing exogenous gene sequences, and thus difficult to meet strict clinical safety requirements. Electroporation technology requires multiple transfections and causes significant cell damage, resulting in an apoptosis rate of more than 50% in PBMCs, severely restricting its practicality.

[0004] In recent years, the RNA-lipid nanoparticle (RNA-LNP) system has demonstrated dual advantages of efficient delivery and low toxicity in mRNA vaccines (such as COVID-19 vaccines). Moreover, since mRNA degrades rapidly in vivo and does not persist or integrate into the genome for a long time, RNA-based technologies can obtain safer and purer iPSCs. Such safer and purer iPSC cell products are particularly valuable for clinical applications in the field of cell gene therapy and some applications in the protection of endangered animals. In the patent CN118109518A, we proposed reprogramming human fibroblasts into iPSCs using mRNA-LNP. On this basis, we attempted to reprogram PBMCs but failed to successfully induce them into iPSCs. There are still many difficulties to be solved in directly using the RNA-LNP-based delivery system for reprogramming cells such as PBMCs. Large-scale iPSC cell bank construction requires the starting cell source to be as simple and easily obtainable as possible. Therefore, developing a safe and pure reprogramming method and kit that can directly and successfully reprogram PBMC cells remains a technical challenge in the industry.

[0005] CN118109518A is incorporated herein by reference in its entirety. Summary of the Invention

[0006] Based on the patent CN118109518A, the present invention realizes for the first time the non-viral reprogramming of PBMC based on RNA-LNP.

[0007] The present invention provides a method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells, comprising the following steps:

[0008] S1: Prepare nano-liposome mRNA-LNP by encapsulating a mixture of mRNA of reprogramming factors with cationic lipid SM102.

[0009] The nano-liposome is mainly composed of one or more of the following parts in structure:

[0010] (1) Cationic lipid: Cationic lipid is the most critical component in the LNP delivery system. A cationic lipid molecule usually contains one or more positively charged head groups, a hydrophobic tail (usually a long-chain hydrocarbon group or a saturated / unsaturated fatty acid chain), and a linking part (such as a glycerol backbone) that connects the head group and the hydrophobic chain of the tail.

[0011] (2) Co-lipid: Co-lipids are usually uncharged lipids, such as phosphatidylcholine. They also consist of a head group, a hydrophobic tail, and a structure connecting the two. Co-lipids help to stabilize the structure of the liposome, reduce the positive charge density on the surface of the cationic liposome, alleviate possible toxicity, and can adjust the fluidity and size of the liposome to make it more suitable for drug delivery.

[0012] (3) Cholesterol: Cholesterol is a sterol lipid, containing a polycyclic structure and a hydrophobic tail. Cholesterol is embedded in the bilayer of the liposome. It can enhance the stability of the liposome, reduce drug leakage, adjust the fluidity of the liposome, and may contribute to the fusion process of the liposome with the cell membrane.

[0013] (4) Targeting ligand: The targeting ligand can be an antibody, protein, polypeptide, or small molecule, which can specifically bind to receptors or other molecules on the cell surface. The targeting ligand helps to direct the liposome to specific types of cells, improving the selectivity of the drug and the intracellular delivery efficiency.

[0014] (5) Polyethylene glycol (PEG) modification: PEG is a widely used polymer, which has a chain structure composed of repeating ethylene glycol units.

[0015] In some embodiments, the cationic lipid is selected from one or more of SM102 (CAS: 2089251-47-6), MC3 (1224606-06-7), ALC0315 (CAS: 2036272-55-4), and ALC0159 (CAS: 1849616-42-7). In some embodiments, the cationic lipid is selected from SM102.

[0016] The LNP may comprise one or more cationic lipids, non-cationic lipids, and / or PEGylated lipids. In some embodiments, the LNP may comprise at least one of the following cationic lipids: SM102, MC3, ALC0315, or ALC0159. In some embodiments, the LNP further comprises cholesterol and / or a PEGylated lipid.

[0017] The traditional reprogramming four factors are Oct4, Sox2, Klf4, and c-Myc, namely OSKM. The method provided by the present invention comprises a mixture of more than five reprogramming factor mRNAs. Specifically, the mixture of reprogramming factor mRNAs is a mixture of 5 or more factors among mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, mRNA-hLin28, mRNA-hNanog, and mRNA-hGlis.

[0018] In some embodiments, the mRNA mixture of reprogramming factors is a mixture of mRNAs of five reprogramming factors. Specifically, it can be one of the four factors mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf, and mRNA-hcMyc and one of the three factors mRNA-hLin28, mRNA-hNanog, and mRNA-hGlis. In some embodiments, the mRNA mixture of reprogramming factors is a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, and mRNA-hGlis, namely the five-factor OSKMG. In some embodiments, the mRNA mixture of reprogramming factors is a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, and mRNA-hLin28, namely the five-factor OSKML.

[0019] In some embodiments, the mRNA is linear RNA. In some embodiments, the mRNA is circular RNA.

[0020] S2: Induce the reprogramming of PBMCs with nano-liposome mRNA-LNP to obtain reprogrammed induced pluripotent stem cells.

[0021] Peripheral blood mononuclear cells (PBMCs) are a group of mononuclear cell populations isolated from peripheral blood, mainly including lymphocytes (T cells, B cells, NK cells) and monocytes. They are an important part of the immune system. Since peripheral blood samples are relatively easy to obtain and cause less trauma, they have significant advantages in clinical applications.

[0022] In some embodiments, peripheral blood mononuclear cells are cultured in PBMC medium and then contacted with mRNA-LNP. The PBMC medium does not contain serum and is a serum-free medium. In some embodiments, the components of the PBMC medium include PBMC complete medium and B18R protein. In some embodiments, the PBMC medium is PBMC complete medium supplemented with or containing B18R protein. In some embodiments, the PBMC medium further contains an auxiliary protein, and the auxiliary protein is selected from bovine serum albumin or apolipoprotein. In some embodiments, the auxiliary protein is apolipoprotein. In some embodiments, the PBMC medium is a serum-free medium containing PBMC complete medium, B18R protein, and an auxiliary protein. In some embodiments, the PBMC medium is a serum-free medium containing PBMC complete medium, B18R protein, and apolipoprotein.

[0023] In some embodiments, the reprogramming of induced pluripotent stem cells is carried out in a reprogramming medium, and the reprogramming medium does not contain serum and is a serum-free medium. Specifically, the components of the reprogramming medium include reprogramming complete medium and B18R protein. In some embodiments, the reprogramming medium is reprogramming complete medium supplemented with or containing B18R protein. In some embodiments, the reprogramming medium further contains an auxiliary protein, and the auxiliary protein is selected from bovine serum albumin or apolipoprotein. In some embodiments, the auxiliary protein is apolipoprotein. In some embodiments, the reprogramming medium is a serum-free medium containing reprogramming complete medium, B18R protein, and an auxiliary protein. In some embodiments, the reprogramming medium is a serum-free medium containing reprogramming complete medium, B18R protein, and apolipoprotein.

[0024] B18R protein is an immunomodulatory protein derived from Vaccinia Virus and belongs to the soluble receptor of type I interferon (IFN-α / β). It can bind and neutralize type I interferon with high affinity, thereby inhibiting the host's innate immune response, and plays an important role in viral evasion of the immune system and biotechnological applications.

[0025] Serum (such as fetal bovine serum, FBS) is a core component in traditional cell culture, with complex and diverse functions to meet the growth requirements of cells. Although serum-free media have low cell adaptability and survival rate and limited application scenarios, their advantages lie in reducing batch differences, lowering the risk of pathogen contamination, and being suitable for standardized production and the culture of specific cell types. The method provided by the present invention still maintains good cell adaptability and survival rate under the condition of not using serum throughout the process.

[0026] PBMC (peripheral blood mononuclear cell) complete medium is a special medium used for isolating, culturing, and studying mononuclear cells (such as lymphocytes, monocytes, etc.) in peripheral blood. Its core components are a basal medium (such as RPMI-1640, SFEMII, or DMEM) combined with necessary additives, such as serum, antibiotics, etc., to provide the nutrients, growth factors, and suitable environment required for cell growth.

[0027] Reprogramming Complete Medium is a medium specifically used for inducing somatic cells (such as fibroblasts, peripheral blood mononuclear cells, etc.) into induced pluripotent stem cells (iPSCs, induced Pluripotent Stem Cells). Such media usually have the characteristics of defined chemical composition, serum-free, and xeno-free to improve the reprogramming efficiency and reduce batch differences and the risk of immune rejection.

[0028] Bovine Serum Albumin (BSA) is a spherical water-soluble protein isolated from bovine serum and is the most abundant protein in bovine serum (accounting for about 50% of the total serum protein).

[0029] Apolipoprotein (Apo) is the protein component in lipoprotein. According to structure and function, it can be divided into multiple families. In some embodiments, the apolipoprotein is selected from one or more of ApoA, ApoB, ApoC, and ApoE.

[0030] Furthermore, the peripheral blood mononuclear cells in the method for preparing induced pluripotent stem cells by reprogramming peripheral blood mononuclear cells are derived from warm-blooded animals. In some embodiments, the somatic cells are derived from mammals or birds. Specifically, the mammal can be a human or other mammals such as monkeys, cows, horses, sheep, or mice.

[0031] The present invention also provides a reprogramming kit, which can reprogram peripheral blood mononuclear cells by any of the above methods for preparing induced pluripotent stem cells by reprogramming peripheral blood mononuclear cells.

[0032] The present invention also provides a method for preparing induced pluripotent stem cells, which includes reprogramming peripheral blood mononuclear cells with the above reprogramming kit, and then obtaining the induced pluripotent stem cells.

[0033] The present invention provides a method for preparing induced pluripotent stem cells by reprogramming peripheral blood mononuclear cells. First, a mixture of mRNAs encoding reprogramming factors is encapsulated with the cationic lipid SM102 to prepare nano-liposomal mRNA-LNP. Then, the nano-liposomal mRNA-LNP is used to induce the reprogramming of peripheral blood mononuclear cells, as Figure 1 shown, to obtain reprogrammed induced pluripotent stem cells. This technology successfully delivers reprogramming factors to PBMC cells via LNP and enables the successful reprogramming of PBMC cells into induced pluripotent stem cells. PBMC cells are easier to obtain compared to other adult somatic cells, providing new strategies and tools for research in the field of cell reprogramming. In addition, the method provided by the present invention can be carried out smoothly under serum-free conditions, which is beneficial for reducing batch differences and the risk of pathogen contamination. In the future, this technology is expected to be widely applied in the biomedical field, bringing revolutionary changes to areas such as disease treatment, tissue regeneration, and personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To better understand the present invention and more clearly show how to implement the present invention, the features of the embodiments according to the present invention are described by way of example and with reference to the accompanying drawings, wherein:

[0035] Figure 1 : Process flow chart of PBMC reprogramming technology based on mRNA-LNP.

[0036] Figure 2 : Changes in cell morphology after PBMC transfection tested with a commercial RNA reprogramming kit.

[0037] Figure 3 : Graph of changes in cell morphology at different times during PBMC reprogramming.

[0038] Figure 4 : Detection of marker expression in iPSCs by immunofluorescence staining.

[0039] Figure 5 : Detection of marker expression in iPSCs by flow cytometry.

[0040] Figure 6 : Detection of in vitro self-differentiation of iPSCs into three germ layer cells by immunofluorescence staining.

[0041] Figure 7 : Karyotype analysis of iPSCs derived from PBMC cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] Definition: To provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0043] When used in a claim and / or the specification in conjunction with the term "comprising", the use of the word "a" can mean "one", but it is also known to have the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" can mean at least a second or a plurality.

[0044] As used in this specification and the claims, the words "comprising" (and any form thereof, such as "comprises" and "comprising"), "having" (and any form of having, "has", "comprises", and "contains") are inclusive and open-ended and do not exclude additional unlisted elements or process steps.

[0045] Examples: The present invention will be more readily understood by reference to the following examples, which are used to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way.

[0046] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0047] As used herein, the terms "induced pluripotent stem cell" and "iPSC" refer to pluripotent cells generated from various differentiated (i.e., pluripotent or non-pluripotent) somatic cells. iPSCs are genetically substantially identical to their respective differentiated origin somatic cells and exhibit characteristics similar to more potent cells such as embryonic stem (ES) cells, which include the ability to self-renew indefinitely in culture and the ability to differentiate into other cell types.

[0048] As used herein, the term "reprogramming" refers to the process of changing the differentiation state of a cell (such as a somatic cell, a unipotent cell, or a progenitor cell). In some embodiments, reprogramming a cell can include converting the cell from a first cell type to a second cell type. In some embodiments, reprogramming can include changing the phenotype of a differentiated cell to a pluripotent phenotype. In some embodiments, reprogramming can refer to the process of "induced differentiation" or "transcription factor-mediated differentiation", in which iPSCs are converted into differentiated cells.

[0049] As used herein, the term "reprogramming factor" refers to any factor or combination of factors that promotes cellular reprogramming. A reprogramming factor can be, for example, a transcription factor. Exemplary reprogramming factors for generating iPSCs from differentiated cells include Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc.

[0050] As used herein, the terms "lipid nanoparticle" and "LNP" describe lipid-based particles in the submicron range. LNPs can have the structural characteristics of liposomes and / or can have alternative non-bilayer types of structures. LNPs can be conjugated to nucleic acids (such as DNA or RNA molecules) and used to deliver nucleic acids to cells.

[0051] The present invention provides a method for inducing PBMCs into iPSCs based on mRNA-LNP. The illustrated embodiments are not intended to limit the scope of the claims of the present invention, but merely to exemplify certain embodiments. Any variations contemplated by those skilled in the art in the exemplary methods will fall within the scope of the present invention. Other embodiments that can be obtained according to the principles of the present invention are all within the scope defined by the claims of the present invention.

[0052] The experimental methods not specifically described in the present invention are all carried out according to the specific methods in "Molecular Cloning: A Laboratory Manual" (Fourth Edition) by J. Sambrook (Joseph.Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition)), or in accordance with the relevant product specifications. When used herein, unless otherwise specified, all terms in the present invention should be understood in their ordinary meanings as known in the art. The biological reagents used in the present invention, unless otherwise specified, can be obtained commercially.

[0053] Example 1: Preparation of mRNA cocktail-LNP

[0054] 1) mRNA was obtained by in vitro transcription.

[0055] Obtain the plasmid vector containing reprogramming factors through gene synthesis method (General Biology (Anhui) Co., Ltd.). Referring to CN118109518A, the reprogramming factors are hOct3 / 4, hKlf4, hSox2, hcMyc, and hLin28 respectively. Then use restriction endonuclease to digest the plasmid to obtain a linearized plasmid DNA template. In this example, NsiI restriction endonuclease is preferably used. Use the T7 High Yield RNA Synthesis Kit (Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.) to synthesize mRNA by in vitro transcription and capping from the linear plasmid DNA template. After in vitro transcription, treat the reaction product with DNase I (Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.) for 30 minutes to remove the DNA template. After DNase I treatment, purify the mRNA. The purification methods can include column purification, ethanol precipitation, lithium chloride precipitation, or HPLC purification, etc. Lithium chloride precipitation is preferably used in this example.

[0056] 2) Prepare mRNA cocktail-LNP based on a microfluidic chip using SM102 cationic lipid.

[0057] Use SM102 as the cationic lipid to prepare mRNA cocktail-LNP. Among them, the formula of the mRNA cocktail is mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, mRNA-hLin28, that is, OSKML.

[0058] The specific method for preparing LNP is as follows: Inject raw materials such as liposomes, single nucleic acid, or multiple nucleic acids into the microfluidic mixing chip through a microfluidic device. Then, by adjusting parameters such as pressure, flow rate, and temperature, make the raw materials fully mixed and emulsified in the chip to form stable LNP. Prepare mRNA cocktail-LNP through the outlet of the microfluidic device.

[0059] Example 2: Reprogram PBMC using a commercial RNA reprogramming kit

[0060] In this example, try to reprogram PBMC into iPSC using a commercial RNA reprogramming kit, and select StemRNA™ 3rd Gen Reprogramming Kit (Reprocell, 00-0076). The specific implementation process is as follows:

[0061] 1) Day 0: Inoculate PBMC cells and perform RNA transfection.

[0062] Coat the culture plate with Matrigel. In this example, the coating is preferably LN521, and the culture plate is preferably a 6-well plate. Inoculate PBMC into the 6-well plate at a cell density of 5x105 cells / well, and culture with the prepared PBMC medium. The PBMC medium consists of SFEM II basal medium, supplemented with IL3, IL6, TPO, Flt3L and SCF. Transfect the reprogramming cocktail into PBMC using RNAiMAX transfection reagent according to the instructions.

[0063] 2) Day 1 - 7: Transfect every day.

[0064] Collect the cell suspension every day. After low-speed centrifugation, discard the supernatant, resuspend the cells with the reprogramming complete medium, and transfer them to the original coated wells. Transfect the reprogramming cocktail reagent with the same formulation as in Example 1 into PBMC using RNAiMAX transfection reagent according to the instructions.

[0065] Repeat the transfection 8 times in total, and continuously observe the morphological changes of the cells. The results are shown in Figure 2 . As shown by Figure 2 , as the number of transfection times increases, the cell state gradually deteriorates. Dead cells can be observed. After 8 times of transfection, no adherent cells or deformed cells are observed. Detect the cell viability of the supernatant on the 8th day, and the viability is 0%, indicating that the cells are basically dead. This result shows that it is difficult to induce PBMC into iPSC based on RNA.

[0066] Example 3: Reprogramming of PBMC into iPSC by mRNA cocktail-LNP

[0067] Deliver the mRNA cocktail-LNP obtained from the lipid formulation in Example 1 into PBMC cells for reprogramming to generate iPSCs. The materials used include: PBMC isolated from blood extraction; prepared LN-521 coated plate (BioLamina); PBMC complete medium (STEMCELL Technologies, #09655); reprogramming complete medium (STEMCELL Technologies., #05926); B18R protein; accessory protein; sterile pipette tips (1000 μl, 200 μl, 10 μl).

[0068] Seven groups of experiments were set up. The auxiliary protein in group I was BSA; the auxiliary protein in group II was apolipoprotein A; the auxiliary protein in group III was apolipoprotein B; the auxiliary protein in group IV was apolipoprotein C1; the auxiliary protein in group V was apolipoprotein C2; the auxiliary protein in group VI was apolipoprotein H; and the auxiliary protein in group VII was apolipoprotein E. The auxiliary protein dosage was 0.1-2 μg / mL, preferably 1 μg / mL in this embodiment.

[0069] The specific implementation process is:

[0070] 1) Day 0: PBMC cells were seeded and mRNA cocktail-LNP was added dropwise.

[0071] PBMCs were plated at 5x10 5 Cells were seeded at a density of 1 μg / well in a 12-well plate and cultured in PBMC medium containing 400 ng / mL B18R protein. After 4-6 hours of culture, mRNA cocktail-LNPs were added dropwise to the cells. In this example, the preferred amount of LNP was 1 μg of total mRNA / well.

[0072] 2) Day 2: Transfer cells to the coated plate and add mRNA cocktail-LNP.

[0073] Coat the culture plate with Matrigel. In this example, LN521 is the preferred coating medium, and a 6-well plate is preferred. After coating, transfer the cells treated on Day 0 to the coated 6-well plate and add reprogramming medium containing 400 ng / mL B18R protein at a 1:1 volume ratio. Subsequently, add the mRNA cocktail-LNP dropwise to the cells. In this example, the preferred amount of LNP is 1 μg of total mRNA per well.

[0074] 3) Days 4, 6, and 8: Change the medium every other day and add mRNA cocktail-LNP dropwise.

[0075] Every other day, the cell suspension was collected, centrifuged at low speed, and the supernatant was discarded. The cells were resuspended in reprogramming medium containing 400 ng / mL B18R protein and transferred to the original coated wells. The corresponding auxiliary protein was added to each group. Subsequently, mRNA cocktail-LNP was added dropwise to the cells. In this example, the dosage of mRNA cocktail-LNP was preferably 1 μg total mRNA / well. Cell morphological changes were continuously observed. Among them, colonies with typical iPSC morphology were observed in Group VII experiments, such as Figure 3As shown, adherent cells gradually appeared in the PBMCs treated with mRNA cocktail-LNP as the reprogramming time increased, and obvious morphological changes occurred in the adherent cells over time. The cells gradually proliferated and expanded during the culture process, and colonies with typical iPSCs morphology could be observed on the 15th day of reprogramming.

[0076] Example 4: Detection of the pluripotency and differentiation ability of iPSCs obtained by reprogramming

[0077] The iPSCs induced by mRNA cocktail-LNP in Example 3 were picked out for expansion culture, and the iPSCs were identified. The identification methods include but are not limited to the detection of stem cell pluripotency markers, AP staining, in vitro differentiation, karyotype experiments, etc. In this example, the detection of the stemness, differentiation ability and karyotype of iPSCs is preferably carried out.

[0078] 1) Immunofluorescence staining was used to detect the expression of markers in iPSCs.

[0079] In this example, immunofluorescence staining was preferably used to detect pluripotent genes, and the stemness markers were preferably Oct3 / 4, SSEA4 and TRA-1-60, etc. When the iPSCs derived from PBMCs induced by mRNA cocktail-LNP grew to a sufficient size, they were fixed with 4% paraformaldehyde, and immunostaining was performed using anti-OCT3 / 4 antibody (Santa Cruz, catalog number SC-5279), anti-SSEA4 antibody (Santa Cruz, catalog number SC-21704) and anti-TRA-1-60 antibody (Santa Cruz, catalog number SC-21705). At the same time, Hoechst 33342 was used to stain the cell nuclei, and the observation was carried out under a fluorescence microscope. Figure 4 The results showed that the stemness markers in the iPSCs obtained by reprogramming PBMCs were all positive. [[ID=!6]]

[0080] 2) Flow cytometry was used to detect the expression of markers in iPSCs.

[0081] The detection methods for the stemness of iPSCs include RT-qPCR method, immunofluorescence staining method and flow cytometry method, etc. The expression of pluripotent genes in multiple cells was detected. In this example, flow cytometry was preferably used to detect pluripotent genes, and the stemness markers were preferably SSEA4 and Nanog, etc.

[0082] EDTA (Thermofisher) was used to dissociate the iPSCs into single-cell suspensions. Flow antibodies such as SSEA4 and Nanog were used to stain the iPSCs. Subsequently, the stained cells were analyzed using a BD flow cytometer, and the flow results are shown in Figure 5 and Table 1.

[0083] Figure 5 As shown in Figure 1 and Table 1, all the stemness markers in the iPSCs obtained by reprogramming were positive. Compared with the control group, the positive cells of SSEA4 and Nanog in the iPSCs were 94.79% and 94.9% respectively. This result indicates that the pluripotent genes in the iPSCs obtained by reprogramming are normally expressed and have good stemness.

[0084] Table 1 Detection results of different markers by flow cytometry

[0085]

[0086] 3) Detect the in vitro self-differentiation ability of iPSCs by immunofluorescence staining.

[0087] Stem cells generally have the ability of self-differentiation and can form embryoid bodies (EBs) and self-differentiate into the three germ layers. The iPSCs obtained by reprogramming were digested into small clumps by EDTA and inoculated in the differentiation medium to form EB balls by self-aggregation. The preferred differentiation medium in this example is DMEM / F12 medium containing 20% KOSR, 1% NEAA, 1% Glutamax-1, and 100 μM β-mercaptoethanol. After the EB balls grew for 7 days, the EB balls were inoculated into a 12-well plate coated with 0.1% Gelatin and continued to be cultured for 7 days with the differentiation medium containing 5% fetal bovine serum. Then, the cells were fixed with 4% paraformaldehyde and stained with antibodies against the three germ layer differentiation markers. The preferred antibodies in this example are endoderm: FoxA2; mesoderm: SMA; ectoderm: Nestin. After washing the secondary antibody staining, observe and take pictures with a fluorescence microscope. The staining results are shown in Figure 6 . Figure 6 The results showed that the iPSCs obtained by reprogramming could form EB balls in vitro and successfully differentiated into cells of the three germ layers, indicating that the cells obtained by reprogramming meet the identification criteria of iPSCs and have the ability of self-differentiation.

[0088] 4) Karyotype analysis of iPSCs

[0089] The karyotype analysis of iPSCs derived from PBMC cells was entrusted to Weifang People's Hospital. The results showed that they all had 46 chromosomes and normal karyotypes ( Figure 7 ).

[0090] In summary, the reprogramming method of induced pluripotent stem cells provided by the present invention successfully delivered reprogramming factors to PBMC cells through LNP and successfully reprogrammed PBMC cells into induced pluripotent stem cells. The method provided by the present invention can be carried out smoothly under serum-free conditions, which is beneficial to reducing batch differences and the risk of pathogen contamination.

[0091] The present invention provides a simple and effective reprogramming method in the fields of regenerative medicine and cells, which is expected to bring new breakthroughs in disease treatment and tissue regeneration. Further research and optimization will lay a solid foundation for clinical applications, bringing better quality of life and hope to patients.

[0092] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided for illustration rather than limitation of the present invention. Other embodiments that can be obtained according to the principles of the present invention fall within the scope defined by the claims of the present invention.

Claims

1. A method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells, characterized in that, Comprising the following steps: S1: Prepare a nano-liposomal mRNA-LNP by encapsulating a mixture of mRNAs of reprogramming factors with the cationic lipid SM102, wherein the nano-liposomal mRNA-LNP is composed of the cationic lipid SM102 and the mRNAs of the reprogramming factors; S2: Induce the reprogramming of the peripheral blood mononuclear cells with the nano-liposomal mRNA-LNP to obtain reprogrammed induced pluripotent stem cells, wherein the induction of the reprogramming of the peripheral blood mononuclear cells is carried out in a reprogramming medium, the reprogramming medium is a serum-free medium, and the reprogramming medium is supplemented with apolipoprotein E, B18R protein and bovine serum albumin.

2. The method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells according to claim 1, wherein The peripheral blood mononuclear cells are cultured in a PBMC medium and then contacted with mRNA-LNP, and the PBMC medium is a complete PBMC medium supplemented with or containing B18R protein.

3. The method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells according to claim 2, wherein The PBMC medium is a serum-free medium and also contains an auxiliary protein, and the auxiliary protein is selected from bovine serum albumin or apolipoprotein.

4. The method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells according to any one of claims 1 to 3, characterized in that, The peripheral blood mononuclear cells are derived from a homeothermic animal.

5. The method for preparing reprogrammed induced pluripotent stem cells from peripheral blood mononuclear cells according to claim 4, characterized in that, The homeothermic animal is a mammal or a bird, and the mammal is a human, a monkey, a cow, a horse, a sheep or a mouse.

6. A reprogramming kit, characterized in that, The reprogramming kit reprograms the peripheral blood mononuclear cells by the method for preparing induced pluripotent stem cells by reprogramming the peripheral blood mononuclear cells according to any one of claims 1 to 5.

7. A method for preparing induced pluripotent stem cells, characterized in that, The method includes reprogramming the peripheral blood mononuclear cells with the reprogramming kit according to claim 6 to obtain the induced pluripotent stem cells.

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