Mrna-LNP-based reprogramming method for induced pluripotent stem cells

Abstract

An mRNA-LNP-based reprogramming method for induced pluripotent stem cells. Specifically, a cationic lipid is used to encapsulate an mRNA mixture of a plurality of reprogramming factors to prepare a nano-liposome containing the reprogramming factors; a certain amount of the nano-liposome containing the reprogramming factors is dropwise added into adult cells in a feeder-free system to obtain reprogrammed induced pluripotent stem cells, thereby achieving a low-cost, low-toxicity, efficient, stable, repeatable, and safer iPSCs reprogramming technology.

Description

Method for reprogramming of induced pluripotent stem cells based on mRNA-LNP TECHNICAL FIELD

[0001] The present invention relates to the field of regenerative medicine and cells, in particular to a method for reprogramming human somatic cells into human induced pluripotent stem cells (iPSCs) using mRNA-LNP. BACKGROUND

[0002] The field of regenerative medicine and cell therapy has been the focus of researchers due to its great potential to solve many diseases and health problems. In recent years, stem cell research has gradually deepened, and stem cells have the ability of self-renewal and differentiation into various cell types, making them an important resource in the field of regenerative medicine. The reprogramming method of human somatic cells to induced pluripotent stem cells (iPSCs) is an important technical means to obtain stem cells. In 2006, Yamanaka's 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 of Oct4, Sox2, Klf4 and c-Myc, and successfully reprogrammed terminally differentiated skin fibroblasts into induced pluripotent stem cells (iPSCs) by viral infection. A 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.) used a different combination of four factors (Oct4, Sox2, Nanog and Lin28 combination) by episomal plasmid transfection to successfully reprogram human fibroblasts into iPSCs. 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 great obstacle to the therapeutic application of induced pluripotent stem cells. Therefore, it is imperative to seek a simple and repeatable method to generate integration-free clinical-grade iPSCs.

[0003] Current non-integrating reprogramming techniques mainly include the use of excisable lentivirus and transposon vectors, episomal and adenovirus vectors to obtain iPSCs. In addition, iPSCs can also be obtained by DNA-free methods, such as using recombinant proteins containing cell-penetrating peptide fragments for sequence protein transduction, or using Sendai virus for transgene delivery. However, these methods inevitably introduce non-human special functional nucleic acids to a greater or lesser extent, and still have potential risks and uncertainties. The strategies designed so far for non-integrating iPSCs still have limitations, such as the risk of gene recombination or insertion mutation for DNA transfection methods, the challenge of recombinant protein generation and removal, the strict control of Sendai virus use, and the current low induction efficiency.

[0004] In recent years, it has been found that non-integrating iPSCs can also be obtained by repeated transfection of mRNA. However, the current mRNA transfection technology is costly, relying on expensive transfection reagents such as Lipofectamine RNAiMAX. In addition, the transfection reagent has high toxicity, and the efficiency of transfection mRNA is low, usually requiring the addition of multiple microRNAs to improve the reprogramming efficiency, thereby increasing the cost and potential other risks and uncertainties. Therefore, it is crucial to seek a simple and effective reprogramming method.

[0005] The mRNA nanoliposome (mRNA-LNP) delivery system has wide application prospects in the field of biomedicine, especially in the fields of gene therapy and vaccine development. However, there is no systematic research reported in the field of cell reprogramming, and it is still a challenge to establish a highly efficient and repeatable reprogramming technology using nanoliposomes.

[0006] SUMMARY

[0007] The inventors of the present application found that using the nanoliposome (Lipid Nanoparticle, LNP) delivery system to deliver the mRNA cocktail mixed with multiple reprogramming genes (mRNA-LNP) not only avoids the risk of gene integration and gene contamination, but also has simpler components, does not require the addition of microRNAs, and does not require the use of expensive transfection reagents, thereby achieving reprogramming. The mRNA cocktail-LNP prepared in the present application not only successfully reprograms somatic cells (such as human dermal fibroblasts) to prepare iPSCs, but also has a very high reprogramming efficiency.

[0008] The present application provides a reprogramming method for inducing pluripotent stem cells, comprising the following steps:

[0009] S1: encapsulating the mRNA cocktail of reprogramming factors with cationic lipids to prepare nanoliposomes;

[0010] The main method for preparing nanoliposomes is to mix the material used for encapsulating nucleic acids, such as cationic lipids, with the buffer containing nucleic acids. Current mixing methods suitable for small-volume LNP production in the laboratory include, but are not limited to, microfluidic control mixing devices or chips, T- or y-shaped mixers, ethanol injection, and manual mixing. In some embodiments, the preferred method of preparation is a microfluidic chip.

[0011] Nanoliposomes are mainly composed of the following parts in structure:

[0012] (1) Cationic Lipid: Cationic lipids are the most critical component in LNP delivery systems. Cationic lipid molecules usually contain 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) connecting the head group and the hydrophobic tail.

[0013] (2) Helper Lipid: Helper lipids are usually uncharged lipids, such as phosphatidylcholine. They are also composed of head groups, hydrophobic tails, and structures connecting the two. Helper lipids help stabilize the structure of the liposome, reduce the positive charge density on the surface of the cationic liposome, reduce possible toxicity, and can adjust the fluidity and size of the liposome to make it more suitable for drug delivery.

[0014] (3) Cholesterol (if any): Cholesterol is a sterol lipid containing a polycyclic structure and a hydrophobic tail. Cholesterol is embedded in the bilayer of the liposome, which can enhance the stability of the liposome, reduce drug leakage, adjust the fluidity of the liposome, and may help the fusion process of the liposome with the cell membrane.

[0015] (4) Targeting Ligand (if any): Targeting ligands can be antibodies, proteins, polypeptides, or small molecules that can specifically bind to receptors or other molecules on the surface of cells. Targeting ligands help direct the liposome to specific types of cells, improving drug selectivity and intracellular delivery efficiency.

[0016] (5) PEG Modification (if any): PEG is a widely used polymer composed of a chain-like structure of repeating ethylene glycol units.

[0017] In some embodiments, the cationic lipid is selected from one or more of SM102 (CAS: 2089251-47-6), MC3 (CAS: 1224606-06-7), ALC0315 (CAS: 2036272-55-4), and ALC0159 (CAS: 1849616-42-7). In some embodiments, the cationic lipid is selected from SM102.

[0018] The LNP can comprise one or more cationic lipids, non-cationic lipids, and / or PEG-modified lipids. In some embodiments, the LNP can 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 PEG-modified lipid.

[0019] The traditional reprogramming four factors are Oct4, Sox2, Klf4 and c-Myc, i.e. OSKM. The method provided in the present application comprises a mixture of five or more reprogramming factor mRNAs, specifically, the mixture of reprogramming factor mRNAs is a mixture of five or more factors selected from the group consisting of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, mRNA-hLin28, mRNA-hNanog and mRNA-hGlis.

[0020] In some embodiments, the mixture of reprogramming factor mRNAs is a mixture of five reprogramming factors, specifically, it can be a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc and one of mRNA-hLin28, mRNA-hNanog and mRNA-hGlis. In some embodiments, the mixture of reprogramming factor mRNAs is a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc and mRNA-hGlis, i.e. OSKMG five factors. In some embodiments, the mixture of reprogramming factor mRNAs is a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc and mRNA-hLin28, i.e. OSKML five factors.

[0021] S2: inducing reprogramming of the somatic cells using the nano-liposomes to obtain reprogrammed induced pluripotent stem cells.

[0022] In the feeder-free system, a certain amount of nano-liposomes containing reprogramming factors are added dropwise to the somatic cells to obtain reprogrammed human induced pluripotent stem cells.

[0023] Somatic cells refer to cells that have differentiated and have specific functions, as opposed to stem cells. They make up various tissues and organs of the human body and play an important role in maintaining life and function. Somatic cells generally cannot be differentiated into other types of cells.

[0024] In some embodiments, the somatic cells are selected from skin fibroblasts, oral epithelial cells, liver cells, stomach cells, keratinocytes, adipocytes, muscle cells or blood cells.

[0025] In some embodiments, the somatic cells are derived from a mammal. Specifically, the mammal can be a human, a monkey, a cow, a horse, a sheep, or other mammals such as a mouse. Specifically, the somatic cells are human dermal fibroblasts (HDFs).

[0026] The culture system of iPSCs can be divided into feeder-dependent and feeder-independent systems according to the presence or absence of feeder cells. Feeder cells are composed of a layer of non-dividing cells, so they are also called feeder layer cells. They provide extracellular secretory substances to help the proliferation of other cells. The feeder-dependent culture system is the traditional stem cell culture system, but the feeder cells also have the risk of heterologous contamination while maintaining the stem cell state; while the feeder-independent culture system is a culture system that has gradually emerged in recent years for the application of stem cells in clinical practice, which can avoid the contamination of heterologous cells. In some embodiments, the feeder-independent system is selected from a basement membrane matrix with animal-derived components, a vitronectin adhesion matrix without animal-derived components, and a recombinant human laminin matrix without animal-derived components.

[0027] The present application also provides a reprogrammed human induced pluripotent stem cell prepared by the reprogramming method described in any one of the above. In some embodiments, the somatic cells for reprogramming are human dermal fibroblasts. Human dermal fibroblasts are a common type of somatic cells, mainly existing in the dermis of the human body, and are one of the main cell types in the dermal tissue. The dermis is the deep layer of the skin, containing elastic fibers and collagen fibers, and plays a supporting, protective and nutritional role.

[0028] The present application provides a reprogramming method of human induced pluripotent stem cells, which uses cationic liposomes to encapsulate the mRNA cocktail of reprogramming factors to prepare nanoliposomal mRNA cocktail-LNP; and then adds a certain amount of nanoliposomes containing reprogramming factors to human somatic cells in a feeder-independent system to obtain reprogrammed human induced pluripotent stem cells. This technology successfully reprograms human somatic cells to prepare high-quality and high-safety iPSCs, providing new strategies and tools for the field of cell reprogramming. In the future, this technology is expected to be widely used in the field of biomedicine, bringing revolutionary changes to the fields of disease treatment, tissue regeneration, and personalized medicine. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to better understand the present application and more clearly show how to implement the present application, the features of the embodiments according to the present application are described by way of example and with reference to the accompanying drawings, in which:

[0030] Figure 1: Process flow chart of mRNA-LNP based reprogramming technology, wherein: A is a schematic diagram of mRNA cocktail-LNP preparation based on microfluidic chip; B is a schematic diagram of morphological changes of human dermal fibroblasts reprogrammed into iPSCs;

[0031] Figure 2: Characterization diagram of RNA cocktail-LNP induced reprogramming of different cationic lipid formulations, wherein: A is a diagram of morphological changes of cells during RNA cocktail-LNP induced reprogramming of three different cationic lipids, the arrow points to a typical iPSCs colony; B is an AP staining identification diagram, wherein AP positive is an iPSCs clone, and the calculation formula of reprogramming efficiency is: reprogramming efficiency = AP positive clone number / initial inoculation cell number x 100%;

[0032] Figure 3: Characterization diagram of iPSCs stemness identification obtained by reprogramming, wherein: A is a flow cytometry detection of pluripotency gene expression of iPSCs; B is an immunofluorescence staining identification of self-differentiation ability of iPSCs;

[0033] Figure 4: Characterization diagram of RNA cocktail-LNP induced reprogramming of different reprogramming factor formulations, wherein: A is a flow chart of LNP induced reprogramming; B is a diagram of morphological changes of cells during two five-factor mRNA cocktail-LNP induced reprogramming, the arrow points to a typical iPSCs colony;

[0034] Figure 5: Diagram of morphological changes of iPSCs reprogramming by different Matrigel based on mRNA-LNP;

[0035] Figure 6: Characterization diagram of iPSCs reprogramming by transfection method, wherein: A is a flow chart of reprogramming by transfection method; B is a diagram of morphological changes of cells during reprogramming induced by different RNA cocktails, the arrow points to a typical iPSCs colony. DETAILED DESCRIPTION

[0036] Definitions: In order to provide a clear and consistent understanding of the terms used in the specification of the present application, some definitions are provided in the following. In addition, all technical and scientific terms used in the present application have the same meaning as generally understood by those of ordinary skill in the art to which the present application belongs, unless otherwise specified.

[0037] When used in the claims 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 mean "one or more", "at least one", and "one or more than one".

[0038] The words "comprise" (and any form of comprise, such as 'comprises' and 'comprising'), "have" (and any form of have, including 'has' and 'having'), "include" (and any form of include, including 'includes' and 'including') and "contain" (and any form of contain, including 'contains' and 'containing') are to be construed inclusively and open-ended, unless otherwise indicated to the contrary.

[0039] Embodiments: The present application will be more readily understood by reference to the following examples, which are offered by way of illustration of the present application and are not intended to limit the scope of the application in any way.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be apparent to those skilled in the art that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application.

[0041] The present application provides methods for inducing human dermal fibroblasts into iPSCs based on mRNA-LNP. The present application is exemplified with an mRNA cocktail comprising at least 5 reprogramming factors, and further particularized. The examples are not intended to limit the scope of the patent claims of the present application, but to exemplify certain embodiments. Any variations to the exemplary methods that will occur to those skilled in the art will fall within the scope of the present application. Other embodiments that can be derived from the principles of the present application fall within the scope of the claims of the present application.

[0042] Experimental methods not specifically described in the present application are performed according to the methods described in the book "Molecular Cloning: A Laboratory Manual (Fourth Edition)" by J. Sambrook, or according to the instructions of the relevant products. When used in the present text, all terms in the present application should be understood according to their ordinary meanings known in the art, unless otherwise specified. Biological reagents used in the present application, unless otherwise specified, can be obtained from commercial sources.

[0043] Example 1: Preparation of mRNA cocktail-LNP with different lipid formulations

[0044] 1) Obtain mRNA by in vitro transcription

[0045] The plasmid vector containing the reprogramming factor sequence is obtained by gene synthesis method (General Biotech (Anhui) Co., Ltd.), and the reprogramming factors are hOct3 / 4 (SEQ ID NO: 1 / 7), hKlf4 (SEQ ID NO: 2 / 8), hSox2 (SEQ ID NO: 3 / 9), hcMyc (SEQ ID NO: 4 / 10) and hLin28 (SEQ ID NO: 5 / 11). Then the plasmid is digested with restriction endonuclease to obtain linearized plasmid DNA template, and Nsil restriction endonuclease is preferred in this embodiment. The mRNA is synthesized by in vitro transcription and capping from the linear plasmid DNA template using T7 high yield RNA synthesis kit (Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.). After in vitro transcription, the reaction product is treated with DNase I (Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.) for 30 minutes to remove the DNA template. After DNase I treatment, the mRNA is purified, and the purification method can be selected from column purification, ethanol precipitation, lithium chloride precipitation or HPLC purification, etc., and lithium chloride precipitation is preferred in this embodiment.

[0046] 2) Preparation of mRNA-LNP of different cationic lipids based on microfluidic chip

[0047] Three groups of formulations A, B and C are set, wherein the formulations of five-factor mRNA cocktail are all mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc and mRNA-hLin28, that is, OSKML, the formulation A uses SM102 as the cationic lipid to prepare mRNA cocktail-SM102-LNP, the formulation B uses MC3 as the cationic lipid to prepare mRNA cocktail-MC3-LNP, and the formulation C uses ALC0315 as the cationic lipid to prepare mRNA cocktail-ALC0315-LNP.

[0048] The flowchart of generating iPSCs based on mRNA-LNP is shown in FIG. 1. The raw materials such as lipids, single nucleic acid or multiple nucleic acids are injected into the microfluidic mixing chip through the microfluidic device. Then, by adjusting the parameters such as pressure, flow rate and temperature, the raw materials are fully mixed and emulsified in the chip to form stable LNP. Through the outlet of the microfluidic device, the prepared LNP is collected, and then subjected to ultrafiltration concentration treatment to obtain the final mRNA cocktail-SM102-LNP corresponding to the formulation A, mRNA cocktail-MC3-LNP corresponding to the formulation B, and mRNA cocktail-ALC0315-LNP corresponding to the formulation C.

[0049] Example 2: mRNA cocktail-LNP with different lipid formulations induce reprogramming of iPSCs

[0050] LNP with different lipid formulations from Example 1 were delivered to HDF cells for reprogramming to generate iPSCs. The materials used include: primary cultured HDF cells; LN-521 coated plates (BioLamina); reprogramming induction medium (StemCell); sterile pipette tips (1000 μL, 200 μL, 10 μL). The detailed implementation process is as follows:

[0051] 1) Day 0: HDF cell seeding

[0052] HDF cells were seeded in LN-521 coated 12-well plates at a cell density of 1000-5000 cells per well. The cells were cultured with DMEM high glucose medium containing 10% fetal bovine serum and 1% Glutamax overnight.

[0053] 2) Day 1-8: LNP drop

[0054] The reprogramming induction medium containing 200 ng / mL of B18R recombinant protein was replaced. LNP was added to the cells, and the amount of LNP used in this example was preferably 0.5-2 μg total mRNA per well, and the process was repeated for 6-8 days.

[0055] The morphological changes of the cells were continuously observed. The results are shown in Figure 2A.

[0056] The results show that during the reprogramming process of LNP containing MC3 and ALC0315, there is no obvious change in cell morphology, and no colony with iPSC-like morphology is observed after 18 days of culture. The cells treated with SM102-LNP show obvious morphological changes in the early stage of reprogramming, and there is no obvious cell death during the culture process, and colonies with typical iPSC morphology can be observed on the 13th day of reprogramming. The iPSC clone formation rate is high, indicating that the mRNA cocktail-SM102-LNP containing reprogramming factors provided by the present application can efficiently induce iPSCs.

[0057] 3) Alkaline phosphatase (AP) staining

[0058] In order to characterize the iPSCs and calculate the reprogramming efficiency, the present application uses an alkaline phosphatase staining kit (Yixing Biotech (Shanghai) Co., Ltd.) to perform AP staining on the 18th day of reprogramming samples, and the specific operation is performed according to the instructions.

[0059] AP activity is mainly expressed in embryonic stem cells and some adult stem cells, and is not expressed in normal human cells. After AP transfection, the cells forming iPSCs clones have AP activity, while the cells not forming iPSCs clones are negative after staining, and the staining results are shown in B of FIG. 2.

[0060] The results show that no AP positive cells are found in mRNA cocktail-MC3-LNP and mRNA cocktail-ALC0315-LNP, while more AP positive cells are found in mRNA cocktail-SM102-LNP, and the reprogramming efficiency reaches 10.6% (AP positive clone number / initial cell density x 100%). This result further proves that the mRNA cocktail-SM102-LNP containing reprogramming factors provided by the present application can induce iPSCs, and the reprogramming efficiency is high, which can reach 10%. In addition, the LNP provided by the present application has no obvious toxicity and simple composition.

[0061] Example 3: Identification of the stemness of iPSCs obtained by reprogramming

[0062] When the iPSCs induced by mRNA cocktail-SM102-LNP grow to be large enough, the clones are picked out for expansion culture, and the iPSCs are identified. The identification methods include but are not limited to detection of stem cell pluripotency markers, AP staining, in vitro differentiation experiment, etc. In this embodiment, the stemness and differentiation ability of iPSCs are preferably detected.

[0063] 1) Flow cytometry detection of pluripotency gene expression

[0064] The stemness detection methods of iPSCs include RT-qPCR method, immunofluorescence staining method and flow cytometry method, etc. for detecting the expression of pluripotency genes in cells. In this embodiment, the flow cytometry method is preferably used to detect pluripotency genes, and the stemness markers are preferably SSEA4, TRA-1-18, Sox2 and Nanog, etc.

[0065] iPSCs are dissociated into single cell suspension using EDTA (Thermofisher). Flow cytometry antibodies SSEA4, TRA-1-18, Sox2 and Nanog, etc. are used to stain iPSCs, and HDF cells are stained as negative controls. Then, the stained cells are analyzed using a BD flow cytometer, and the flow cytometry results are shown in A of FIG. 3 and Table 1.

[0066] The results show that the iPSCs obtained by reprogramming are positive for the stemness markers. Compared with the control group, the positive cells for SSEA4, TRA-1-18, Sox2 and Nanog in the iPSCs are 99.99%, 96.41%, 96.9% and 99.8%, respectively. This result indicates that the pluripotency genes in the iPSCs obtained by reprogramming are normally expressed, and the stemness is good.

[0067] Table 1 Flow cytometry detection results of different markers

[0068] 2) Detection of in vitro self-differentiation ability of iPSCs by immunofluorescence staining

[0069] Stem cells generally have self-differentiation ability, can form embryoid bodies (EBs) and self-differentiate into three germ layers. The iPSCs obtained by reprogramming are digested into small clumps by EDTA and inoculated in a differentiation medium to form EB balls. The preferred differentiation medium in this embodiment is DMEM / F12 medium containing 20% KOSR, 1% NEAA, 1% Glutamax-1 and 100 μM β-mercaptoethanol. After the EB balls are formed and grow for 7 days, the EB balls are inoculated in a 0.1% gelatin-coated 12-well plate and cultured in a differentiation medium containing 5% fetal bovine serum for 7 days. Then, the cells are fixed with 4% paraformaldehyde and stained with antibodies for three germ layer differentiation markers. The preferred antibodies in this embodiment are endoderm: FoxA2; mesoderm: SMA; ectoderm: MAP2. After staining and washing, the cells are observed and photographed under a fluorescence microscope. The staining results are shown in B of FIG. 3. The results show that the iPSCs obtained by reprogramming can form EB balls in vitro and successfully differentiate into three germ layer cells, indicating that the cells obtained by reprogramming meet the identification standards of iPSCs and have self-differentiation ability.

[0070] Example 4: iPSCs reprogramming by mRNA cocktail-LNP of different reprogramming factor formulations

[0071] This example utilizes LNP to deliver different reprogramming mRNA cocktail formulations to HDF cells for reprogramming to iPSCs. Two five-factor mRNA cocktail formulations were selected, mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, mRNA-hGlis (SEQ ID NO: 6 / 12) (OSKMG) and mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc, mRNA-hLin28 (OSKML). The preferred cationic lipid formulation for LNP preparation in this example is SM102. The preferred reprogramming flowchart in this example is shown in FIG. 4A, and the detailed procedure is as follows:

[0072] 1) Day 0: HDF cell seeding

[0073] HDF cells were seeded at a density of 1000-5000 cells per well in a 6-well plate coated with matrigel. The cells were cultured overnight with DMEM high glucose medium containing 10% fetal bovine serum and 1% Glutamax.

[0074] 2) Day 1-8: LNP addition

[0075] The reprogramming induction medium containing 200 ng / mL of B18R recombinant protein was replaced. LNP was added to the cells, and the amount of LNP used in this example was preferably 0.5-2 μg total mRNA per well, and the addition was repeated for 6 days.

[0076] The morphological changes of the cells were continuously observed, and the results are shown in FIG. 4B. The results showed that obvious iPSC clones appeared on day 16 of reprogramming, indicating that both mRNA-LNP formulations of OSKMG and OSKML could reprogram HDF cells into iPSCs.

[0077] Example 5: iPSCs reprogramming based on mRNA-LNP with different matrigels

[0078] This example describes a method for iPSCs reprogramming based on mRNA-LNP with different matrigels. The matrigels used in this example are preferably conventional Matrigel with animal-derived components, Vitronectin adhesion substrate without animal-derived components, and Laminin-521, a recombinant human laminin without animal-derived components. The mRNA-LNP used in this example is mRNA cocktail-LNP prepared using SM102. The detailed procedure is as follows:

[0079] 1) Day 0: HDF cell seeding

[0080] HDFs were seeded at a density of 1000-5000 cells per well in 6-well plates coated with Matrigel. Cells were cultured overnight in DMEM high glucose medium containing 10% fetal bovine serum, 1% Glutamax.

[0081] 2) Day 1-8: LNP was added dropwise

[0082] The reprogramming induction medium was replaced with the one containing 200 ng / mL of B18R recombinant protein. LNP was added dropwise to the cells, and the amount of LNP used in this example was preferably 0.5-2 μg total mRNA per well, and the process was repeated for 6-8 days.

[0083] The morphological changes of the cells were continuously observed, and the results are shown in Figure 5. The results show that human dermal fibroblasts (HDFs) can be successfully reprogrammed by mRNA-LNP based on three different Matrigels, and the cells undergo obvious morphological changes and can form typical iPSCs clones. The typical iPSCs colonies are indicated by arrows.

[0084] Example 6: iPSCs reprogramming by transfection

[0085] As a comparison, a commonly used transfection reagent method for mRNA reprogramming was tested. The transfection reagent can be selected, for example, liposome transfection, calcium transfection, or PEI transfection, etc. In this example, Lipofectamine RNAiMAX transfection reagent (Invitrogen) was preferred.

[0086] This example describes a method for reprogramming human dermal fibroblasts (HDFs) to generate iPSCs by transfection of mRNA into the cells. The materials used include: HDF cells (donated by another laboratory); Lipofectamine RNAiMAX transfection reagent (Invitrogen); Opti-MEM (Gibco); LN-521 coated plates prepared; reprogramming induction medium; sterile 1.5 mL Eppendorf tubes; sterile pipette tips (1000 μL, 200 μL, 10 μL). The reprogramming mRNA and microRNA used include: (1) mRNA-hOCT3 / 4; (2) mRNA-hKlf4; (3) mRNA-hSox2; (4) mRNA-hcMyc; (5) mRNA-hLin28; (6) mRNA-hNanog (SEQ ID NO: 13 / 14); (7) mRNA-hGlis; (8) microRNA302 / 367.

[0087] The schematic diagram of transfection reprogramming is shown in Figure 6A, and the specific implementation process is as follows:

[0088] 1) Day 0: HDF cells seeding

[0089] HDF cells were seeded in LN-521 coated 6-well plates at a cell density of 50000 cells per well.

[0090] 2) Day 1-5: Lipofectamine RNAiMAX transfection

[0091] The reprogramming induction medium was replaced with a reprogramming induction medium containing 200 ng / mL of B18R recombinant protein. After medium replacement, mRNA cocktails were prepared. In this example, two mRNA cocktails were prepared. mRNA cocktail 1 did not contain microRNA and mainly included (1) mRNA-hOCT3 / 4; (2) mRNA-hKlf4; (3) mRNA-hSox2; (4) mRNA-hcMyc; (5) mRNA-hLin28; (6) mRNA-hNanog; and (7) mRNA-hGlis. mRNA cocktail 2 was prepared based on mRNA cocktail 1 and contained microRNA. In this example, microRNA 302 / 367 was preferentially selected. Subsequently, transfection was performed using RNAiMax liposome reagent according to the manufacturer's instructions.

[0092] Additional four transfections were performed on days 2-5 of reprogramming (see the schematic diagram of FIG. 6A).

[0093] 3) Day 6-18: Reprogramming induction medium replacement

[0094] Cell morphological changes were continuously observed. The results are shown in FIG. 6B. The results showed that mRNA cocktail 1 without microRNA had a large number of cell death in the late stage of reprogramming, and no iPSC-like colony was found. mRNA cocktail 2 with microRNA could induce iPSCs, but there was also a large number of cell death, and the number of iPSC clones formed was small, and the reprogramming efficiency was calculated to be 0.033% (iPSC colony number / initial seeding cell density x 100%). This result further proved that the commonly used transfection reagent method has various limitations, including high cytotoxicity of transfection reagent, low reprogramming efficiency, complex composition, and the need for the presence of microRNA.

[0095] In summary, the reprogramming method of human induced pluripotent stem cells provided by the application not only utilizes LNP technology to deliver mRNA, but also has good compatibility, can be cultured in a feeder-free system, avoids the risk of gene integration and gene pollution. The transcription factor components used are simple, and microRNA does not need to be added during the reprogramming process, expensive transfection reagents are not needed, and the cost is lower. The application also finds that the nano-liposome of the reprogramming factor prepared by using the cationic lipid SM102 has a significantly improved reprogramming efficiency compared to the method of traditional transfection reagents, and a simpler reprogramming gene formula is used.

[0096] The application provides a simple and effective reprogramming method in the field 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 application and bring better life quality and hope to patients.

[0097] Although the application is described in detail with reference to the embodiments of the application, these embodiments are provided for illustration rather than limitation of the application. Other embodiments that can be obtained according to the principles of the application all belong to the scope defined by the claims of the application.

[0098] The sequences involved in the application are as follows:

[0099] Human Oct3 / 4 amino acid sequence (SEQ ID NO: 1)

[0100] Human Klf4 amino acid sequence (SEQ ID NO: 2)

[0101] Human Sox2 amino acid sequence (SEQ ID NO: 3)

[0102] Human cMyc amino acid sequence (SEQ ID NO: 4)

[0103] Human Lin28 amino acid sequence (SEQ ID NO: 5)

[0104] Human Glis amino acid sequence (SEQ ID NO: 6)

[0105] Human Oct3 / 4 mRNA sequence (SEQ ID NO: 7)

[0106] Human Klf4 mRNA sequence (SEQ ID NO: 8)

[0107] mRNA sequence of human Sox2 (SEQ ID NO: 9)

[0108] mRNA sequence of human cMyc (SEQ ID NO: 10)

[0109] mRNA sequence of human Lin28 (SEQ ID NO: 11)

[0110] mRNA sequence of human Glis (SEQ ID NO: 12)

[0111] Amino acid sequence of human Nanog (SEQ ID NO: 13)

[0112] mRNA sequence of human Nanog (SEQ ID NO: 14)

Claims

1. A method of inducing reprogramming of a pluripotent stem cell, characterized by, The method comprises the following steps: S1: preparing nano-liposome mRNA-LNP by encapsulating a mixture of reprogramming factor mRNAs with cationic lipids; S2: inducing reprogramming of somatic cells with the nano-liposome mRNA-LNP to obtain reprogrammed induced pluripotent stem cells.

2. The reprogramming method of claim 1, wherein, The encapsulation of the nano-liposome mRNA-LNP further comprises other lipids or non-lipids in addition to the cationic lipids.

3. The reprogramming method as described in claim 1, characterized in that, The cationic lipids are SM102.

4. The reprogramming method of claim 1, wherein, The reprogramming of the induced pluripotent stem cells is cultured in a feeder-free system.

5. The reprogramming method as described in claim 4, characterized in that, The Matrigel of the feeder-free system is selected from one or more of animal-derived basal membrane matrix, animal-free vitronectin adhesion matrix and animal-free recombinant human laminin matrix.

6. The reprogramming method of claim 1, wherein, The mixture of reprogramming factor mRNAs comprises: 1) mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4 and mRNA-hcMyc; and 2) one or more of mRNA-hLin28, mRNA-hNanog and mRNA-hGlis.

7. The reprogramming method of claim 6, wherein, The mixture of reprogramming factor mRNAs is a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc and mRNA-hGlis.

8. The reprogramming method as described in claim 7, characterized in that, The mRNA-hOCT3 / 4 comprises the sequence shown in SEQ ID NO: 7, the mRNA-hSox2 comprises the sequence shown in SEQ ID NO: 9, the mRNA-hKlf4 comprises the sequence shown in SEQ ID NO: 8, the mRNA-hcMyc comprises the sequence shown in SEQ ID NO: 10, and the mRNA-hGlis comprises the sequence shown in SEQ ID NO:

12.

9. The reprogramming method of claim 6, wherein, The mixture of reprogramming factor mRNAs is a mixture of mRNA-hOCT3 / 4, mRNA-hSox2, mRNA-hKlf4, mRNA-hcMyc and mRNA-hLin28.

10. The reprogramming method as described in claim 9, characterized in that, The mRNA-hOCT3 / 4 comprises the sequence shown in SEQ ID NO: 7, the mRNA-hSox2 comprises the sequence shown in SEQ ID NO: 9, the mRNA-hKlf4 comprises the sequence shown in SEQ ID NO: 8, the mRNA-hcMyc comprises the sequence shown in SEQ ID NO: 10, and the mRNA-hLin28 comprises the sequence shown in SEQ ID NO:

11.

11. The reprogramming method of any one of claims 1 to 10, wherein the reprogramming method is performed in the presence of a small molecule. The somatic cells are selected from skin fibroblasts, oral epithelial cells, liver cells, stomach cells, keratinocytes, adipocytes, muscle cells or blood cells.

12. The reprogramming method of claim 11, wherein, The somatic cells are derived from mammals.

13. The reprogramming method as described in claim 12, characterized in that, The somatic cells are derived from monkeys, cows, horses, sheep or mice.

14. The reprogramming method as described in claim 12, characterized in that, The somatic cells are derived from humans.

15. The reprogramming method as described in claim 14, characterized in that, The somatic cells are human dermal fibroblasts.

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