Method for producing induced pluripotent stem cell, nucleic acid introduction carrier and kit

Abstract

To provide a method for producing an induced pluripotent stem cell, a nucleic acid introduction carrier and a kit.SOLUTION: According to one embodiment, a method for producing an induced pluripotent stem cell includes bringing a first lipid particle into contact at least once with a fibroblast, followed by bringing a second lipid particle into contact at least once with the fibroblast. The first lipid particle and the second lipid particle each contain a reprogramming factor. The lipid component composition ratios of the first lipid particle and the second lipid particle are different from each other.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Embodiments of the present invention relate to a method for producing induced pluripotent stem cells, a nucleic acid introduction carrier, and a kit.

Background Art

[0002] Induced pluripotent stem cells, generally called iPS cells, are produced by expressing reprogramming factors in cells using a viral vector. When using a viral vector, there is a risk that the vector remains in the infected cells and continues to express reprogramming factors. In addition, the residual virus in the final product can also pose a further risk. On the other hand, there is also a method using lipofection, but when using this method, it is not easy to produce iPS cells stably or homogeneously.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The problem to be solved by the present invention is to provide a method for producing induced pluripotent stem cells, a nucleic acid introduction carrier, and a kit.

Means for Solving the Problems

[0004] The method according to the embodiment is a method for producing induced pluripotent stem cells. The method includes contacting a first lipid particle with fibroblasts one or more times, and subsequently contacting a second lipid particle with the fibroblasts one or more times. The first lipid particle and the second lipid particle each encapsulate a reprogramming factor. The lipid component composition ratio of the first lipid particle and the lipid component composition ratio of the second lipid particle are different from each other.

Brief Description of the Drawings

[0005]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0006] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially the same constituent parts are denoted by the same reference numerals, and the description thereof may be partially omitted. The drawings are schematic, and the relationship between the thickness and the planar dimensions of each part, the ratio of the thicknesses of each part, etc. may be different from the actual ones.

[0007] (First Embodiment) The method according to the embodiment is a method for producing induced pluripotent stem cells using lipid particles, for example, liposomes. Hereinafter, induced pluripotent stem cells are referred to as iPS cells. First, with reference to FIG. 1, the lipid particles used in the method of the embodiment will be described. The introduction carrier set 2001 shown in FIG. 1 includes a first introduction carrier 101a and a second introduction carrier 201b. The first introduction carrier 101a includes a hollow lipid particle 11a (for example, liposome 11a) and a reprogramming factor 12a encapsulated therein. The second introduction carrier 201b includes a hollow lipid particle 11b (for example, liposome 11b) and a reprogramming factor 12a encapsulated therein. The lipid particles 11a and 11b are hollow bodies formed of a lipid membrane, and for example, it is a liposome. The lipid component composition ratio of the lipid particles of the second introduction carrier 201b is different from the lipid component composition ratio of the lipid particles of the first introduction carrier 101a. In other words, the first introduction carrier 101a is the first lipid particle encapsulating the reprogramming factor, and the second introduction carrier 101a is the second lipid particle encapsulating the reprogramming factor.

[0008] As shown in FIG. 2, the method includes contacting first lipid particles with fibroblasts one or more times (S21), subsequently contacting second lipid particles with the fibroblasts one or more times (S22), and obtaining induced pluripotent stem (iPS) cells (S23). Here, the first lipid particles and the second lipid particles each encapsulate reprogramming factors. Also, the lipid component composition ratios of the first lipid particles and the second lipid particles are different from each other. By such an introduction carrier set 2001, that is, by repeatedly introducing reprogramming factors into fibroblasts using two types of lipid particles composed of different lipid components, it becomes possible to prevent the reprogramming setback that may occur in the first half of reprogramming. Thereby, it becomes possible to maintain and / or enhance the effect of the reprogramming factors. And thereby, it becomes possible to efficiently produce iPS cells. Also, in such a method, since iPS cells are produced using a non-viral material, unlike the case of using a viral material, there is no risk of infection or virus origin. Since stable production is possible, it becomes possible to produce iPS cells homogeneously. Here, obtaining iPS cells may be such that, by such contact, the fibroblasts substantially migrate to the iPS cells, and may or may not be accompanied by physical recovery of the iPS cells.

[0009] The concept of the method will be further described with reference to FIG. 3. FIG. 3 shows an image of the procedure along the progress of the method and also an image showing the change in the state of a single fibroblast over time. A first introduction carrier 101a is administered to the fibroblast 20a as the starting material (FIG. 3(a)). The first introduction carrier 101a contacts the fibroblast 20a which is the starting material and releases the initialization factor 12a encapsulated in the lipid particle 11a into its cytoplasm (FIG. 3(a)). The released initialization factor 12a forms an initialization substance a in the cytoplasm (FIG. 3(a)). Due to the initialization substance a, the information of the nucleus 21a of the starting material fibroblast 20a is gradually reprogrammed, and the state of the cell shifts to the first half of initialization (FIG. 3(a)→(b)). In the first half of this initialization, a second introduction carrier 201b is administered (FIG. 3(b)). The administered second introduction carrier 201b contacts the fibroblast 20b in the state of the first half of initialization and releases the initialization factor 12a encapsulated in the lipid particle 11b into its cytoplasm (FIG. 3(b)). The released initialization factor 12a forms a further initialization substance a in the cytoplasm (FIG. 3(b)). Due to the initialization substance a, the information of the nucleus 21b of the fibroblast 20b in the first half of initialization is gradually reprogrammed, and the state of the cell shifts to the second half of initialization (FIG. 3(b)→(c)). In the second half of this initialization, the second introduction carrier 201b is readministered (FIG. 3(c)). The administered second introduction carrier 201b contacts the fibroblast 20c in the state of the second half of initialization and releases the initialization factor 12a encapsulated in the lipid particle 11b into its cytoplasm (FIG. 3(c)). The released initialization factor 12a forms a further initialization substance a in the cytoplasm (FIG. 3(c)). Due to the initialization substance a, the information of the nucleus 21c of the fibroblast 20c in the second half of initialization is gradually reprogrammed, and the state of the cell shifts to the state where initialization is completed (FIG. 3(c)→(d)). Thus, the initialization is completed, and an initialized cell 20d, that is, an iPS cell is obtained.

[0010] As described above, when the initialization substance a is provided to the cytoplasm, the information of the nucleus 21a of the starting material fibroblast 20a is gradually reprogrammed, and the state of the cell changes from the starting material fibroblast 20a, to the fibroblast 20b in the first half of initialization, to the fibroblast 20c in the second half of initialization, and to the initialized cell 20d (iPS cell) (i.e., transitions) (Figs. 3(a), (b), (c), (d)). The initialization factor 12a is, for example, RNA. When the factors necessary for initialization are introduced as RNA, the RNA remains in the cytoplasm and is translated into protein. The protein to be translated is, for example, a transcription factor. The gene information in the nucleus is reprogrammed by the transcription factor. By the way, the introduced RNA is degraded in the cytoplasm. The degradation of RNA occurs 24 to 48 hours after introduction. The cell changes that occur approximately 24 to approximately 48 hours after introduction correspond to the first half of initialization. Including the second half of initialization, the time until the fibroblast as the starting material completes initialization and becomes an initialized cell (i.e., an initialized cell (iPS cell)) is approximately 96 hours (approximately 4 days) after introduction. Particularly in the first half of initialization, a phenomenon called regression of initialization is likely to occur.

[0011] The steps from the start of the method to the first half of initialization are shown in Figs. 3(a) to (b), and the detailed steps therein are as follows; administering a first introduction carrier 101a to the starting material fibroblast 20a, the first introduction carrier 101a contacting the fibroblast 20a, the first introduction carrier 101a releasing the initialization factor 12a encapsulated in the lipid particle 11a into its cytoplasm, the released initialization factor 12a forming the initialization substance a in the cytoplasm, the information of the nucleus 21a of the starting material fibroblast 20a being started to be reprogrammed by the initialization substance a, and the state of the fibroblast 20a transitioning to the first half of the initialization period. Resetting initialization cancels the reaction that has progressed to the first half of initialization, that is, the reaction by maintaining constancy. As a result, the reprogramming induced by the first introduction carrier 101a is regarded as not having occurred, and the state of the cell returns to the state of the starting material.

[0012] The method according to the embodiment is derived from the inventors' attention to the fact that such resetting occurs even if the administration of the first introduction carrier 101a is continued. That is, in the first half of initialization, by further introducing the initialization factor 12a using the second lipid particle having a lipid component composition ratio different from that of the lipid component composition ratio of the first lipid particle, initialization can be efficiently completed, and the target cell can be obtained.

[0013] The first lipid particle is configured to have a lipid component composition ratio having an appropriate affinity for the state of the starting material. The second lipid particle is configured to have a lipid component composition ratio having an appropriate affinity for the cell in which initialization has been started. By combining these first lipid particles and second lipid particles and sequentially administering them, it becomes possible to efficiently perform initialization. Thereby, it becomes possible to stably obtain initialized cells. Also, thereby, it becomes possible to obtain uniform initialized cells.

[0014] The one or more contacts between the first lipid particles and fibroblasts as the initial material may be, for example, 1, 2, 3, 4, or one or more, two or more, three or more, four or more times. The one or more contacts between the second lipid particles and fibroblasts in the first half of the initialization may be, for example, 1, 2, 3, 4, or one or more, two or more, three or more, four or more times. The contact between the first lipid particles and fibroblasts as the initial material is also referred to as the first contact. The contact between the second lipid particles and fibroblasts in the first half of the initialization is also referred to as the second contact. Basically, the first contact is performed as a previous step prior to the second contact. Alternatively, the first contact may be performed before the second contact. For example, after the first contact of the first contact, the first contact of the second contact may be performed. For example, any contact after the second contact of the first contact may be simultaneous with the first contact of the second contact. For example, any contact after the second contact of the first contact may be before, simultaneous with, or after the first contact of the second contact. For example, it is also possible to produce iPS cells by adding the first lipid particles (for example, liposome A) on Day0 and adding the second lipid particles (for example, liposome B) on Day2 and Day4. Although not limited thereto, for example, the composition of liposome A includes FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and its mol% ratio is 58:0:0:16:24:2, and the lipid composition of liposome B includes FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and its mol% ratio may be 0:32:5:9:51:3.

[0015] The first contact and the second contact can be performed, for example, in a medium, a physiological aqueous solution, etc. in an environment suitable for maintaining fibroblasts as the starting material, fibroblasts at the start of initialization, and cells that have completed the initialization of fibroblasts. If necessary, the medium or physiological aqueous solution may be exchanged. During or after any contact, or until a subsequent further contact, the cells are maintained in an environment suitable for them. For example, it may be incubation. The incubation conditions may be general conditions suitable for fibroblasts, for example, conditions of 35°C to 38°C.

[0016] Lipid particles 11a and lipid particles 11b are designed to have an affinity for their respective target cells. In other words, lipid particles 11a have an appropriate affinity for target cells in the first state, and lipid particles 11b have an appropriate affinity for target cells in the second state. The target cells in the first state are fibroblasts in the state of the starting material. The target cells in the second state are fibroblasts that have contacted lipid particles 11a, fibroblasts in which initialization has started, or fibroblasts in which reprogramming has started, and are fibroblasts in a state that has shifted (changed) after lipid particles 11a have contacted the target cells in the first state. For example, the target cells in the second state may be fibroblasts at any time after contacting lipid particles 11a. It can be a cell that has contacted lipid particles 11a and has not completed the reversion. For example, the target cells in the second state may be cells within 24 hours, within 36 hours, or within 48 hours after contacting lipid particles 11a. For example, the second contact may be performed multiple times during these periods, for example, 1 or more times, 2 or more times, 3 or more times, 4 or more times, 5 or more times. Also, for example, the first contact may be performed using the first lipid particles 11a as a further contact within 1 hour, within 2 hours, within 3 hours, within 6 hours, within 12 hours, or within 24 hours after the first contact.

[0017] Here, "appropriate affinity" means, for example, when referring to target cells in the first state, having a higher affinity compared to similar introduction carriers by general design under normal and / or general contact conditions with the target cells in the first state. Such appropriate affinity is achieved, as will be described in detail later, by adjusting the lipid composition of the lipid particles.

[0018] Here, the "target cell" refers to a cell to which an initialization factor should act, and it can be any common fibroblast. The organ from which the fibroblast is derived is not particularly limited. Examples of fibroblasts include, but are not limited to, human fibroblasts, rat fibroblasts, cat fibroblasts, dog fibroblasts, etc. They may be selected according to the purpose of use. For example, for use in treatment, fibroblasts previously collected from a patient to be treated may be used. Fibroblasts are cells that can be obtained relatively easily. Therefore, compared with other cells, the technology of reprogramming fibroblasts to produce induced pluripotent stem cells or iPS cells is extremely useful in various fields such as medicine, beauty, and basic research.

[0019] Here, the "initialization factor" may be any factor that can initialize and reprogram the DNA information in the nucleus when encapsulated in lipid particles and introduced into cells. For example, the initialization factor is mRNA. In that case, the initialization factor is introduced into the cytoplasm of the cell, remains in the cytoplasm, and is translated into a transcription factor that is a protein as an initialization substance to reprogram the DNA in the nucleus. The type of initialization factor may be one type or two or more types. For the required initialization factors, they may be introduced using the first lipid particles or the second lipid particles according to the state of the cells and the administration time. Examples of initialization factors include, but are not limited to, messenger RNAs of Oct3 / 4, Sox2, Klf4, cMyc, Nanog, Lin28).

[0020] As described above, the lipid particles can be liposomes. It can be a particle of a lipid membrane encapsulating an aqueous core, for example, a particle of a lipid bilayer membrane. Any known liposome may be used for the liposome itself. For example, the lipid composition forming the liposome may contain, as its components, the first lipid (FFT-10) of formula (I) and / or the second lipid (FFT-20) of formula (II). These lipids are biodegradable lipids. By adjusting the lipid composition of the lipid particles using these lipids, appropriate affinity can be achieved.

Chemical formula

[0021] In addition to the above-mentioned first lipid and second lipid, the lipid particles may contain additional lipids. Among the compositions of the lipid molecular materials constituting the lipid particles, the fraction consisting of the first lipid and the second lipid is hereinafter referred to as the "first fraction". Also, the fraction consisting of lipid molecular materials other than the first lipid and the second lipid is hereinafter referred to as the "second fraction". The lipids contained in the second fraction are collectively hereinafter also referred to as the "third lipid".

[0022] The terms "first fraction" and "second fraction" represent the composition of the components of the lipid particles, and do not indicate the physical positions of the lipids contained therein. For example, the components of the first fraction and the second fraction do not necessarily form one aggregate in the lipid particles, and the lipids contained in the first fraction and the lipids contained in the second fraction may be mixed and present. The blending ratio of the first fraction with respect to the entire lipid material constituting the lipid particles can be, for example, 30% to 70%, 30% to 65%, etc.

[0023] In other words, the total content of FFT-10 and / or FFT-20 can be, for example, 30% to 70%, or 30% to 65%, etc. as the blending ratio of the lipid particles. Here, the percentage is % mol / % mol unless otherwise specified.

[0024] The average particle diameter of the lipid particles can be changed according to the use, but can be adjusted, for example, to about 50 nm to about 300 nm. For example, it can be about 70 nm to about 100 nm.

[0025] The types of the third lipid contained in the second fraction of the lipid particles are not limited. For example, the second fraction contains a base lipid. As the base lipid, for example, lipids that are the main components of biological membranes can be used. The base lipid can be a phospholipid or a sphingolipid, such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin or cerebroside, or a combination thereof, etc.

[0026] For example, as the base lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or a combination thereof is preferably used. As the above base lipid, it is particularly preferable to use a cationic lipid or a neutral lipid, and the acid dissociation constant of the lipid particles can be adjusted by its content. It is preferable to use DOTAP as the cationic lipid and DOPE as the neutral lipid.

[0027] The second fraction preferably also contains a lipid that prevents aggregation of lipid particles. For example, the lipid that prevents aggregation may further contain a PEG-modified lipid, such as polyethylene glycol (PEG) dimyristoyl glycerol (DMG-PEG), a polyamide oligomer derived from an omega-amino (oligoethylene glycol) alkanoic acid monomer (U.S. Patent No. 6,320,017), or monosialoganglioside, etc.

[0028] The second fraction may further contain lipids such as a relatively low-toxicity lipid for adjusting toxicity; a lipid having a functional group for binding a ligand to the lipid particle; a lipid for suppressing leakage of inclusions such as sterols, for example, cholesterol. In particular, it is preferable to include cholesterol.

[0029] The type and composition of the lipid used for the second fraction may be appropriately selected in consideration of the acid dissociation constant (pKa) of the target lipid particle or the particle diameter of the lipid particle, the type of the included active agent, or the stability in cells, etc.

[0030] For example, when the second fraction contains DOPE, DOTAP, cholesterol, and DMG-PEG, it is preferable because the delivery efficiency of the active agent is particularly excellent.

[0031] In addition to the active agent, further components may be included in the lipid particle as needed. The further components are, for example, a pH adjuster, an osmotic pressure adjuster, a gene activator, etc. The pH adjuster is, for example, an organic acid such as citric acid and its salts, etc. The osmotic pressure adjuster is sugar, an amino acid, etc. Here, the gene activator can be any substance that promotes or supports the activity of the active agent when the active agent is a gene.

[0032] Lipid particles encapsulating an active agent and, if necessary, other substances can be produced using, for example, known methods used when encapsulating small molecules in lipid particles, such as the Bangham method, organic solvent extraction method, surfactant removal method, or freeze-thaw method. For example, a lipid mixture obtained by dissolving the materials of the lipid particles in an organic solvent such as alcohol at a desired ratio and an aqueous buffer containing components to be encapsulated such as an activator are prepared, and the aqueous buffer is added to the lipid mixture. By stirring and suspending the resulting mixture, lipid particles encapsulating an activator or the like are formed. The lipid particles thus obtained are an example of liposomes.

[0033] For example, the lipid composition of the first lipid particle includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof may be in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 50 to 65:0:5 to 25:15 to 35:1 to 15. Alternatively, for example, the lipid composition of the first lipid particle includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof may be in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 53 to 63:0:9 to 21:19 to 29:1 to 7. Also, for example, the lipid composition of the second lipid particle includes FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof may be in the range of FFT-20:DOPE:DOTAP:cholesterol:DMG-PEG = 20 to 40:1 to 15:1 to 15:40 to 65:1 to 15. Alternatively, for example, the lipid composition of the second lipid particle includes FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof may be in the range of FFT-20:DOPE:DOTAP:cholesterol:DMG-PEG = 26 to 37:1 to 9:4 to 14:46 to 61:1 to 8. These ratios are such that in the lipid composition of the first lipid particle or the second lipid particle, each value is selected so that the total is 100. Alternatively, for example, the lipid composition of the first lipid particle may include FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar ratio thereof may be 58:0:0:16:24:2. The lipid composition of the first lipid particle may be a lipid particle having a lipid component composition ratio of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 58.3:16.5:23.6:1.6 in terms of molar percentage. For example, the lipid composition of the second lipid particle may include FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar ratio thereof may be 0:32:5:9:51:3.The lipid composition of the second lipid particles includes FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG, and may be lipid particles having a lipid component composition ratio with a molar percentage of FFT-20:DOPE:DOTAP:cholesterol:DMG-PEG = 31.7:4.5:9.0:51.4:3.4%.

[0034] Thus, according to the method according to the embodiment, it is possible to change the composition of the introduction liposome and continuously introduce the initialization factor along with the progress of the initialization process of the target cells. Further, by changing the composition of the liposome, it is possible to maintain the amount of the initialization factor in the target cells at a certain amount or more at any time in the first half and the second half of the initialization. When producing iPS cells by introducing an initialization factor into fibroblasts as the starting material, it is possible to maintain a sufficient amount of the initialization factor in the cells for a certain period (for example, 4 days or more) in order to prevent the initialization from reverting.

[0035] (Second Embodiment) The introduction carrier set according to the second embodiment will be described with reference to FIG. 1. The introduction carrier set 2001 includes a first introduction carrier 101a and a second introduction carrier 201b. The introduction carrier 101a includes lipid particles 11a (for example, liposomes 11a) and an initialization factor 12a encapsulated therein. The lipid particles 11a have an appropriate affinity for fibroblasts as the starting material. The introduction carrier 201b includes lipid particles 11b (for example, liposomes 11b) and an initialization factor 12a encapsulated therein. The lipid particles 11b have an appropriate affinity for fibroblasts in a state where initialization has been started. The introduction carrier set 2001 can maintain and / or enhance the initialization by repeatedly introducing the initialization factor using two types of lipid particles having different component ratios. Further, it is thereby possible to efficiently perform the initialization and obtain homogeneous initialized cells and iPS cells.

[0036] (Third Embodiment) As described above, as an introduction carrier set according to the second embodiment, it may be provided in a state immediately usable for a desired target cell, or may be provided in a form as a material so that the user of the above-described introduction carrier set can adjust it at the time of use. The third embodiment is an introduction carrier production kit provided in such a form.

[0037] The kit may include, for example, a material for the first lipid particle for encapsulating an initialization factor, having a lipid composition designed to exhibit an appropriate affinity for fibroblasts as a starting material, and a material for the second lipid particle, which has a lipid composition different from that of the first lipid particle and is designed to exhibit an appropriate affinity for fibroblasts in a state where initialization has been started, and for encapsulating a second activator an initialization factor, and may include.

[0038] These materials for the first and second lipid particles, and the initialization factor may be provided in a container in an appropriate state so that each is stably provided as a substance. Here, the initialization factor is an initialization factor to be encapsulated in the materials for the first and second lipid particles, respectively. Furthermore, the kit may include instructions regarding the production of the introduction carrier, for example, a production manual, for the user to appropriately perform the production.

[0039] By repeatedly introducing the initialization factor using two types of lipid particles having different component ratios using the introduction carrier production kit, it is possible to maintain and / or enhance the initialization. Also, it becomes possible to efficiently perform the initialization, and it becomes possible to obtain homogeneous initialized cells, iPS cells.

[0040] [Example] Hereinafter, an example of producing and using the lipid particles of the embodiment as lipid nanoparticles will be described.

[0041] Experiment 1. Production of Lipid Nanoparticles Encapsulating an Initialization Factor Group As the nucleic acid encapsulated in the lipid nanoparticles, messenger RNAs of six types of reprogramming factors (Oct3 / 4, Sox2, Klf4, cMyc, Nanog, Lin28) were used. The nucleic acid was suspended in 10 mM HEPES (pH 7.3) to obtain a nucleic acid solution.

[0042] FFT-10, FFT-20, DOPE, DOTAP, cholesterol and DMG-PEG were dissolved in ethanol at the charged lipid composition ratios shown in Table 1 to obtain a lipid solution. The lipid solution and the above nucleic acid solution were mixed using a microfluidic chip and a syringe pump. The mixed solution was diluted 10-fold with 10 mM HEPES (pH 7.3) and then concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain lipid nanoparticles. The obtained lipid nanoparticles were to be used in subsequent experiments with Example 1 as the first lipid particles and Example 2 as the second lipid particles.

[0043]

Table 1

[0044] Experiment 2. Measurement of Encapsulated Nucleic Acid Concentration The encapsulated nucleic acid concentrations of the respective lipid nanoparticles prepared in Experiment 1 were measured using the QuantiFluor RNA system (manufactured by Promega). Triton X-100 (0.1 v / v%, manufactured by Eastman Kodak Company) was added to the lipid nanoparticle solution and mixed with a vortex mixer, and then the mixture was allowed to stand at room temperature for 30 minutes. An equal volume of QuantiFluor solution was added and the mixture was allowed to stand at room temperature for 10 minutes, and then the fluorescence intensity was measured using a single-tube fluorescence measuring device, Quantus Fluormeter (manufactured by Promega). The concentration of the nucleic acid encapsulated in the lipid nanoparticles was calculated by subtracting the fluorescence intensity of the sample without Triton X-100 from the fluorescence intensity of the sample with Triton X-100. Table 2 shows the encapsulated nucleic acid concentrations of the respective lipid nanoparticles.

[0045]

Table 2

[0046] Experiment 3. Cell Reprogramming Test Human fibroblasts (Fibroblast, manufactured by KURABO, Lot.06444) cultured in Dulbecco's Modified Eagle Medium (DMEM, manufactured by Gibco) were centrifuged and collected. After that, they were seeded in a 24-well culture plate at a density of 1×104 cells / 300 μL of DMEM medium (supplemented with 10% fetal bovine serum) per well, and the lipid nanoparticles prepared in Experiment 1 were added under the conditions shown in Table 3. The culture plates were placed in an incubator, and the cells were cultured in an atmosphere of 37°C and 5% CO2. Cell culture was continued while adding AK03N medium (supplemented with Rock Inhibitor Y27632) every 3 - 4 days, and it was observed under an optical microscope whether iPS cell colonies were formed. Figure 4 shows the image diagrams of the lipid compositions of the liposomes of Example 1 and Example 2 used (Figure 4(a) and (b)), and the cell images observed under an optical microscope after sequentially adding the lipid nanoparticles of Example 1 and Example 2 under Condition 3 to the target cells and culturing for 4 weeks (Figure 4(c) and (d)). None of the observed cell colonies showed the characteristics of fibroblasts, such as elongated and multi-directionally extended protrusions. Instead, the characteristics of iPS cell colonies arranged in a round paving stone shape were observed. From the morphological characteristics, it was confirmed that iPS cells were produced from fibroblasts under Condition 3.

[0047] As a comparative experiment, messenger RNAs of six types of reprogramming factors (Oct3 / 4, Sox2, Klf4, cMyc, Nanog, Lin28) were introduced using a lipofection reagent (Lipofectamine RNAiMAX, manufactured by Thermo Fisher Scientific), and microscopic observation was performed.

[0048] Figure 5 shows the results of the comparative experiment conducted by the lipofection method for Example 1 and 2, and the comparative example. Table 3 summarizes the conditions used and the experimental results.

[0049]

Table 3

[0050] Among the three conditions carried out by changing the type of lipid nanoparticles to be added and the timing of addition, Condition 3, that is, the one in which the lipid nanoparticles of Example 1 were added on Day 0 and the lipid nanoparticles of Example 2 were added on Day 2 and Day 4, showed that colonies of iPS cells were formed after about 4 weeks. The morphology of the same colonies under the conditions according to the embodiment (Figs. 5(c) and (d)) was observed to be equal to or better than that of the comparative experiment conducted as a comparative example, that is, the colonies of iPS cells formed when the lipofection reagent was administered continuously 4 times from Day 0 to Day 3 (Figs. 5(a) and (b)). In the case of Condition 1 (adding the lipid nanoparticles of Example 1 on Day 0) and Condition 2 (administering the lipid nanoparticles of Example 1 continuously 4 times from Day 0 to Day 3) corresponding to the condition of using only the first lipid particles, sufficient formation of the target iPS cell colonies was not observed.

[0051] Experiment 4. Examination of the addition timing of reprogramming factors Similar to Experiments 1 to 3, experiments were conducted by changing the addition timing of reprogramming factors using lipid nanoparticles, and the formation of iPS cell colonies was confirmed. The results are shown in Table 4.

[0052]

Table 4

[0053] The results were described with the addition of the lipid nanoparticles of Example 1 as A and the addition of the lipid nanoparticles of Example 2 as B. For the addition, in Condition 1, A was added once on Day 0, in Condition 2, A was added 4 times respectively from Day 0 to Day 3, and in Condition 3, a total of 3 additions were made, that is, A was added once on Day 0, B was added once on Day 2, and B was added once on Day 4. In Condition 3, colonies of iPS cells were observed well. In contrast, in Conditions 1 and 2, formation of iPS cell colonies was not observed.

[0054] From the above results, it was confirmed that fibroblasts can be reprogrammed by the lipid particles and each method according to the embodiments, thereby enabling the production of iPS cells.

[0055] Examples of further embodiments are described below. [1] Contacting a first lipid particle encapsulating a reprogramming factor with fibroblasts one or more times, and after at least one of the above contacts, contacting the fibroblasts one or more times with a second lipid particle encapsulating the reprogramming factor and having a lipid component composition ratio different from that of the first lipid particle; obtaining induced pluripotent stem cells A method for producing induced pluripotent stem cells comprising the above steps. [2] The method according to [1], wherein the first and second lipid particles are lipid particles composed of a lipid component containing biodegradable lipids FFT-10 and / or FFT-20. [3] The method according to [1] or [2], wherein in the first and second lipid particles, the proportion of FFT-10 and / or FFT-20 is 30% or more. [4] The method according to any one of [1] to [3], wherein the lipid component of the first lipid particle has a higher content of FFT-10 than the second lipid particle. [5] The lipid composition of the first lipid particle contains FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 50 - 65:0:5 - 25:15 - 35:1 - 15, and each value is selected so that the total is 100, the lipid composition of the second lipid particle contains FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 20 - 40:1 - 15:1 - 15:40 - 65:1 - 15, and each value is selected so that the total is 100 The method according to any one of [1] to [5]. [6] The lipid composition of the first lipid particle includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 53 to 63:0:9 to 21:19 to 29:1 to 7, and each value is selected so that the total is 100. The lipid composition of the second lipid particle includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 26 to 37:1 to 9:4 to 14:46 to 61:1 to 8, and each value is selected so that the total is 100. [1]~[5] The method according to any one of items [1] to [5]. [7] The lipid composition of the first lipid particle includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is a lipid particle composed of a lipid component composition ratio of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 58.3:16.5:23.6:1.61. The lipid composition of the second lipid particle includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is a lipid particle composed of a lipid component composition ratio of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 31.7:4.5:9.0:51.4:3.4. [1]~[5] The method according to any one of items [1] to [5]. [8] An introduction carrier set for use in the method according to any one of items [1] to [7], A first lipid particle having a lipid composition that encapsulates an initialization factor and is designed to exhibit an appropriate affinity for fibroblasts in the state of the starting material. A second lipid particle having a lipid composition that encapsulates the initialization factor, has a different lipid composition from the first lipid particle, and is designed to exhibit an appropriate affinity for fibroblasts in the state where initialization has started. And a carrier set comprising the same. [9] A kit for manufacturing an introduction carrier set used in the method according to any one of [1] to [7], comprising: A first lipid particle material having a lipid composition designed to exhibit an appropriate affinity for fibroblasts in the state of the starting material, A second lipid particle material having a lipid composition different from that of the first lipid particle and designed to exhibit an appropriate affinity for fibroblasts in the initialized state, An initialization factor to be encapsulated in the first lipid particle and the second lipid particle, and a kit comprising the same.

[10] The kit according to [9], further comprising a production manual.

[0056] Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are included in the invention described in the claims and its equivalent scope.

Explanation of Reference Numerals

[0057] 11a, 11b... lipid particles, 12a... initialization factor, 20a, 20b, 20c, 20d... target cells, 21a, 21b, 21c, 21d... nuclei of target cells, 101a... first introduction carrier, 201b... second introduction carrier, 2001... introduction carrier set, a... initialization substance

Claims

**Claim 1** contacting fibroblast cells with a first lipid particle encapsulating an initialization factor one or more times; and after at least one said contact, contacting said fibroblast cells with a second lipid particle encapsulating said initialization factor and having a lipid component composition ratio different from that of said first lipid particle one or more times; obtaining induced pluripotent stem cells A method for producing induced pluripotent stem cells, comprising the above steps. **Claim 2** The method according to claim 1, wherein said first and second lipid particles are lipid particles composed of a lipid component containing biodegradable lipids FFT-10 and / or FFT-20. **Claim 3** The method according to claim 1, wherein in said first and second lipid particles, the proportion of said FFT-10 and / or FFT-20 is 30% or more. **Claim 4** The method according to claim 1, wherein the lipid component of said first lipid particle has a higher content of FFT-10 than that of said second lipid particle. **Claim 5** The lipid composition of said first lipid particle contains FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof is in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 50-65:0:5-25:15-35:1-15, and each value is selected so that the total is 100. The lipid composition of said second lipid particle contains FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof is in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 20-40:1-15:1-15:40-65:1-15, and each value is selected so that the total is 100. The method according to claim 1. **Claim 6** The lipid composition of said first lipid particle contains FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof is in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 53-63:0:9-21:19-29:1-7, and each value is selected so that the total is 100. The lipid composition of said second lipid particle contains FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio thereof is in the range of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 26-37:1-9:4-14:46-61:1-8, and each value is selected so that the total is 100. The method according to claim 1.

7. The lipid composition of the first lipid particles includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is a lipid particle having a lipid component composition ratio of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 58.3:16.5:23.6:1.

6. The lipid composition of the second lipid particles includes FFT-10, DOPE, DOTAP, cholesterol, and DMG-PEG, and the molar percentage ratio is a lipid particle having a lipid component composition ratio of FFT-10:DOPE:DOTAP:cholesterol:DMG-PEG = 31.7:4.5:9.0:51.4:3.

4. The method according to claim 1.

8. An introduction carrier set for use in the method according to claim 1, A first lipid particle having a lipid composition designed to encapsulate an initialization factor and exhibit appropriate affinity for fibroblasts in the state of the starting material, A second lipid particle having a lipid composition designed to encapsulate the initialization factor, having a different lipid composition from the first lipid particle, and exhibiting appropriate affinity for fibroblasts in the state where initialization has started A carrier set comprising.

9. A kit for manufacturing an introduction carrier set for use in the method according to claim 1, A first lipid particle material having a lipid composition designed to exhibit appropriate affinity for fibroblasts in the state of the starting material, A second lipid particle material having a lipid composition designed to exhibit appropriate affinity for fibroblasts in the state where initialization has started, having a different lipid composition from the first lipid particle And an initialization factor to be encapsulated in the first lipid particle and the second lipid particle A kit comprising.

10. The kit according to claim 9, further comprising a production manual.

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