Initialization agent and kit for initializing blood cells derived from peripheral blood, and initialization method

Abstract

To provide a technology for homogeneously producing induced pluripotent stem cells.SOLUTION: According to one embodiment, an initialization agent for initializing blood cells derived from peripheral blood includes an initialization factor group for producing iPS cells by initializing a group of blood cells including mononuclear cells derived from peripheral blood, and a group of lipid nanoparticles that encapsulate the initialization factor group. At least 40% of the components of the lipid nanoparticles are FFT-10 and FFT-20. FFT-10 and FFT-20 are included in equal amounts.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Embodiments of the present invention relate to an initialization agent and kit for initializing blood cells derived from peripheral blood, and an initialization method.

Background Art

[0002] Induced pluripotent stem cells, generally called iPS cells, are produced by expressing an initialization factor 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 the initialization factor. In addition, the remaining virus in the final product can also be 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 technique for producing induced pluripotent stem cells homogeneously.

Means for Solving the Problems

[0004] The initialization agent according to the embodiment is for initializing blood cells derived from peripheral blood. The initialization agent includes a group of initialization factors for initializing a blood cell group containing mononuclear cells derived from peripheral blood to produce iPS cells, and a group of lipid nanoparticles encapsulating the group of initialization factors. At least 40% or more of the components of the lipid nanoparticles are FFT-10 and FFT-20. FFT-10 and FFT-20 are included in equal amounts.

Brief Description of the Drawings

[0005]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

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 initialization agent according to the embodiment is for initializing blood cells derived from peripheral blood. The initialization agent includes an initialization factor group for initializing a blood cell group including mononuclear cells derived from peripheral blood to produce iPS cells, and a group of lipid nanoparticles encapsulating the initialization factor group. At least 40% or more of the components of the lipid nanoparticles are FFT-10 and FFT-20. FFT-10 and FFT-20 are included in equal amounts. An example of its structure is shown in FIG. 1. The initialization agent may include, for example, a nucleic acid introduction carrier. The nucleic acid introduction carrier 101 includes a hollow lipid nanoparticle 11 and an initialization factor group 12 encapsulated therein. The initialization factor group 12 is an initialization factor group for initializing a blood cell group including mononuclear cells derived from peripheral blood to produce iPS cells.

[0008] The initialization factor group 12 is a factor that initializes mononuclear cells to produce iPS cells when the nucleic acid introduction carrier 101 is added to a blood cell group containing mononuclear cells derived from peripheral blood. For example, the initialization factor group 12 may be any factor that can initialize and reprogram the DNA information in the nucleus. 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, and reprograms the DAN in the nucleus. The number of types of initialization factors may be one or two or more. Regarding the necessary initialization factors, they may be introduced by the nucleic acid introduction carrier 101 according to the state of the cells and the administration timing. Examples of the initialization factors include, but are not limited to, messenger RNAs of Oct3 / 4, Sox2, Klf4, cMyc, Nanog, Lin28. For example, the initialization factor group may include Oct3 / 4, Sox2, Klf4, cMyc. Further, the initialization factor group may include Nanog, Lin28, and may further include these in addition to Oct3 / 4, Sox2, Klf4, cMyc. The initialization factor group encapsulated in the lipid nanoparticle may be included, for example, in the form of DNA, RNA, and / or protein.

[0009] For example, as shown in FIG. 2, the lipid nanoparticle may include the initialization factor group to be introduced by dividing it into two parts. In that case, it may be provided as a nucleic acid introduction carrier set 1001 including a first nucleic acid introduction carrier 101a encapsulating a first initialization factor group 12a and a second nucleic acid introduction carrier 101b encapsulating a second initialization factor group 12b in the lipid nanoparticle 11. Alternatively, as shown in FIG. 3, as a nucleic acid introduction carrier set 2001, each of the n types of initialization factors included in the initialization factor group to be introduced may be encapsulated in the first nucleic acid introduction carrier 101a to the nth nucleic acid introduction carrier 101n. Here, n is an integer of 2 or more. Alternatively, it may be divided and included in a desired number of nucleic acid introduction carriers for each desired type of initialization factor.

[0010] The lipid nanoparticles can be liposomes. It can be a particle with a lipid membrane encapsulating an aqueous core, for example, a particle with a lipid bilayer membrane. The lipid composition forming the liposome contains, as its components, a first lipid (FFT-10) of formula (I) and / or a second lipid (FFT-20) of formula (II). These lipids are biodegradable lipids. The lipid nanoparticles have at least 40% or more of their components as FFT-10 and FFT-20, and FFT-10 and FFT-20 are contained in equal amounts. By using lipid particles with such a lipid composition, it becomes possible to prime blood cells derived from peripheral blood.

Chemical formula

[0011] In addition to containing FFT-10 and FFT-20 as the first lipid and the second lipid, the lipid nanoparticles may further contain additional lipids. In the composition of the lipid molecular materials constituting the lipid nanoparticles, 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 also hereinafter referred to as the "third lipid".

[0012] The terms "first fraction" and "second fraction" represent the composition of the components of the lipid nanoparticles 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 aggregation within the lipid nanoparticles, and the lipids contained in the first fraction and the lipids contained in the second fraction may exist in a mixed state. The blending ratio of the first fraction with respect to the entire lipid material constituting the lipid nanoparticles can be, for example, 40% or more, for example, 40% - 45%, 40% - 50%, etc. FFT-10 and FFT-20 are contained in equal amounts, for example, 17% - 23% each. Here, the percentages are expressed in mol% unless otherwise specified.

[0013] The types of the third lipid contained in the second fraction of the lipid nanoparticles are not limited. For example, the second fraction contains a base lipid. As the base lipid, for example, a lipid that is a main component of a biological membrane can be used. The base lipid can be a phospholipid or a sphingolipid, such as dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin or cerebroside, or a combination thereof, etc.

[0014] 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), It is preferable to use these or any combination thereof. As the 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.

[0015] The second fraction preferably also contains a lipid that prevents aggregation of the lipid nanoparticles. 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.

[0016] The second fraction may further contain a lipid that is relatively less toxic for adjusting toxicity; a lipid having a functional group for binding a ligand to the lipid particles; a lipid for suppressing leakage of inclusions such as sterols, for example, cholesterol. In particular, it is preferable to include cholesterol.

[0017] In addition to the active agent, additional components may be encapsulated in the lipid nanoparticles as needed. The additional 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. The osmotic pressure adjuster is sugar or 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. Furthermore, the lipid nanoparticles may contain additional factors other than the initialization factor group. For example, such additional factors can be factors that suppress the response of interferon, miRNAs that improve the iPS cell establishment efficiency, etc.

[0018] Lipid nanoparticles encapsulating an active agent and, if necessary, other substances can be produced using, for example, known methods used when encapsulating small molecules into lipid nanoparticles, 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 nanoparticles 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 nanoparticles encapsulating an activator or the like are formed. The lipid nanoparticles thus obtained are an example of liposomes.

[0019] For example, in the lipid nanoparticles, 60% or less of the components can be neutral lipids, cationic lipids, cholesterol, or PEG-modified lipids. For example, the lipid nanoparticles can contain more cationic lipids than neutral lipids. Also, the PEG-modified lipid in the lipid nanoparticles may be a PEG-modified lipid having a maleimide-modified functional group. For example, the lipid composition ratio of the lipid nanoparticles can be FFT-10:FFT-20:cationic lipid:cholesterol:PEG-modified lipid = 17 - 23:17 - 23:7 - 13:45 - 51:1.5 - 2.5 (mol%, total 100 mol%). With such a configuration, it becomes possible to more efficiently initialize the blood cell population.

[0020] (Second Embodiment) Such an initialization agent may be provided in a state immediately usable for a desired target cell as an introduction carrier set according to the first embodiment described above, or may be provided in the form of a material so that the user of the above introduction carrier set can adjust it at the time of use. The second embodiment can be an introduction carrier production kit or an initialization agent production kit provided in such a form.

[0021] The kit is for reprogramming blood cells derived from peripheral blood. The kit comprises a nucleic acid delivery carrier material and a group of reprogramming factors for reprogramming a blood cell population containing mononuclear cells derived from peripheral blood to produce iPS cells. The nucleic acid delivery carrier material can be a lipid nanoparticle material for encapsulating the group of reprogramming factors. At least 40% or more of the components of the lipid nanoparticle material are FFT-10 and FFT-20, and FFT-10 and FFT-20 are included in equal amounts.

[0022] The kit can be provided with the lipid nanoparticle material and the group of reprogramming factors contained in a container in an appropriate state so that each is stably provided as a substance. Here, the group of reprogramming factors may be provided as a group of reprogramming factors or may be provided in containers for each type. Prior to use, the kit is constructed by known means itself into a group of lipid nanoparticles encapsulating the group of initial factors and is used for use. Furthermore, the kit may include instructions for producing an introduction carrier for the user to appropriately perform production, for example, a production manual.

[0023] It is possible to reprogram blood cells derived from peripheral blood using such an introduction carrier production kit. Thereby, it becomes possible to obtain homogeneous reprogrammed cells, iPS cells.

[0024] (Third Embodiment) The third embodiment is a method for reprogramming a blood cell population containing mononuclear cells derived from peripheral blood to produce iPS cells. As shown in FIG. 4, this method includes contacting the lipid nanoparticles encapsulating the above-described group of reprogramming factors with the blood cell population at least once (S21) and obtaining iPS cells from the blood cell population (S22).

[0025] The contact between the lipid nanoparticles encapsulating the group of reprogramming factors and the blood cell population only needs to be at least once, and may be, for example, once, twice, three times, four times, five times, or one or more times, two or more times, three or more times, four or more times, or, for example, one to three times, one to four times, one to five times, etc.

[0026] When performing such contact multiple times, the interval between each contact may be, for example, every 12 hours, every 24 hours, every 36 hours, every 48 hours, or a combination thereof.

[0027] Obtaining iPS cells from a blood cell group can be achieved by performing incubation after contact between a blood cell containing mononuclear cells derived from peripheral blood, which is the starting material cell, and the lipid nanoparticles. "Obtaining iPS cells" here may mean that iPS cells are formed from the starting material cells.

[0028] The group of reprogramming factors to be introduced may be added to the blood cell group simultaneously, or the reprogramming factors encapsulated in lipid nanoparticles and divided into desired groups may be added to the blood cell group simultaneously or in sequence.

[0029] For example, it may be incubation. Incubation conditions may be general conditions suitable for blood cells, for example, conditions of 35°C to 38°C.

[0030] Here, the "blood cell group containing mononuclear cells derived from peripheral blood" is the cell on which the reprogramming factors should act. The blood cell group can be used without separating peripheral blood collected by a general method into individual blood cells. For example, it is possible to use it as a starting material cell group by obtaining a blood cell component from blood collected from a peripheral blood vessel. Substantially, mononuclear cells contained in such a starting material cell group contact the lipid nanoparticles, and iPS cells are formed by the action of the reprogramming factors encapsulated therein. Also, before such a starting material cell group contacts the lipid nanoparticles, it may be cultured for a certain period under culture conditions in which hematopoietic stem cells contained in the starting material cell group are likely to proliferate.

[0031] The inventors are conducting research on forming iPS cells by reprogramming various cells. Among such research, as described in the above embodiments, it has been discovered that a blood cell group containing mononuclear cells derived from peripheral blood can be used as a starting material cell group for more efficient reprogramming. The collection of peripheral blood is also beneficial because it is less invasive and imposes less burden on the subject being collected.

[0032] [Example] Hereinafter, an example of producing and using the lipid particles of the embodiment as lipid nanoparticles will be described.

[0033] Experiment 1. Preparation of Lipid Nanoparticles Encapsulating GFP Gene As the nucleic acid encapsulated in the lipid nanoparticles, messenger RNA of the green fluorescent protein (GFP) gene was used. The nucleic acid was suspended in 10 mM HEPES (pH 7.3) to obtain a nucleic acid solution. FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG were each dissolved in ethanol at a molar ratio of 0:25.8:4.9:9.8:55.8:3.7 to obtain a lipid solution. The lipid solution and the above nucleic acid solution were mixed using a microfluidic chip and a syringe pump. After diluting the mixed solution 10-fold with 10 mM HEPES (pH 7.3), it was concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain the lipid nanoparticles of Examples 1 to 4.

[0034]

Table 1

[0035] Experiment 2. Lipid Quantification of Lipid Nanoparticles by LC / MS To compare the charged lipid composition ratio during the preparation of lipid nanoparticles and the composition ratio of the lipids contained in the prepared lipid nanoparticles, and to evaluate the error range of the lipid composition ratio, lipid quantification was performed by liquid chromatography-mass spectrometry (LC / MS). Each lipid nanoparticle prepared in Experiment 1 was diluted with methanol, and FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG contained in each lipid nanoparticle were quantified under the conditions shown in Table 2 using an LC / MS apparatus (ACQUITY UPLC / QToF system, Waters).

[0036]

Table 2

[0037]

Table 3

[0038]

Table 4

[0039] As a result of calculating the error amount with the charged lipid composition ratio as the theoretical value and the lipid composition ratio by LC / MS analysis as the measured value, it was found that the error range in the above lipid nanoparticle preparation method is 0.1 mol% to 4 mol%. When describing the lipid composition ratio here, an error of 0.1 mol% to 4 mol% may be included as a possibility.

[0040] Experiment 3. Preparation of Lipid Nanoparticles Encapsulating GFP Gene As the nucleic acid encapsulated in the lipid nanoparticles, messenger RNA of the GFP gene was used. The nucleic acid was suspended in 10 mM HEPES (pH 7.3) to obtain a nucleic acid solution. FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG were dissolved in ethanol at the charged lipid composition ratios shown in Table 5 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 the lipid nanoparticles of Examples 5 to 9.

[0041]

Table 5

[0042] Experiment 4. Measurement of gene expression intensity Human peripheral blood mononuclear cells (PBMC, Precision For Medicine) cultured in StemSpan SFEMII medium (Stemcell technologies) containing 6 types of cytokines (IL-6, SCF, TPO, Flt-3L, IL-3, G-CSF; all from Wako) were centrifuged and collected. Then, they were seeded in a 96-well culture plate at 4 × 104 cells / 100 μL of StemFit AK03N medium (supplemented with Rock Inhibitor Y27632), and each lipid nanoparticle prepared in Experiment 3 was added at 1.3 μL / well. The culture plate was placed in an incubator, and the cells were cultured at 37°C in a 5% CO2 atmosphere.

[0043] From the day after the addition of the lipid nanoparticles to the 6th day, the fluorescence intensity of the GFP protein expressed from the GFP gene was photographed with a fluorescence microscope (KEYENCE BZ-X810), and the average luminance in each well was measured using image processing software (ImageJ). The measurement was performed according to the manual attached to the fluorescence microscope. Figure 5 shows the measurement results of the relative fluorescence intensity of the GFP protein.

[0044] Among the five types of lipid nanoparticles with different charged lipid composition ratios (Examples 5 to 9), only the lipid nanoparticles with FFT-10:FFT-20:DOPE:DOTAP:cholesterol:DMG-PEG = 20:20:0:10:48:2 (mol%) (Example 5) showed a relative fluorescence intensity of about 2.5 exceeding the reference value of 1.5 on the first day of measurement (D1) and maintained a relative fluorescence intensity of 2.5 or more until the sixth day of measurement (D6). None of the other lipid nanoparticles (Examples 6 to 9) showed a fluorescence intensity exceeding that of Example 5. From the above results, it was shown that the lipid nanoparticles composed of the lipid composition ratio of Example 5 can efficiently deliver nucleic acids to PBMC cultured under the above conditions.

[0045] Experiment 5. Preparation of Lipid Nanoparticles Encapsulating a Group of Reprogramming Factors 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. FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG were dissolved in ethanol at a molar ratio of 20:20:0:10:48:2, respectively, 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 the lipid nanoparticles of Example 10.

[0046] Experiment 6. Cell Reprogramming Test Human peripheral blood mononuclear cells (PBMC, Precision For Medicine) cultured in StemSpan SFEMII medium (Stemcell technologies) containing six types of cytokines (IL-6, SCF, TPO, Flt-3L, IL-3, G-CSF; all from Wako) were centrifuged and collected. After that, they were seeded in 24-well culture plates at a density of 2×105 cells / 600 μL / well of StemFit AK03N medium (supplemented with Rock Inhibitor Y27632), and the lipid nanoparticles prepared in Experiment 5 were added under the conditions shown in Table 6.

[0047]

Table 6

[0048] The culture plates were placed in an incubator, and the cells were cultured at 37°C in a 5% CO2 atmosphere. Cell culture was continued while adding fresh medium every 3 - 4 days, and the formation of iPS cell colonies was observed under an optical microscope. Figures 6 - 8 show the cell images observed under an optical microscope before passage, after the first passage, and after the second passage for Conditions 1 - 3, respectively. In addition, the cells under Condition 3 were collected, Alexa647-labeled TRA-1-60 antibody (R&D systems) was added, and the expression of the pluripotent stem cell marker TRA-1-60 was measured using a flow cytometer (Sony SA3800). The measurement was performed according to the manual attached to the flow cytometer. Table 7 shows the viable cell rate and TRA-1-60 expression rate measured by the flow cytometer.

[0049]

Table 7

[0050] Three conditions were carried out by changing the timing and amount of addition of lipid nanoparticles. In all cases, the formation of iPS cell colonies could be observed before passage (about 10 days after adding lipid nanoparticles). Also, the formation of iPS cell colonies could be observed at the second passage (about 24 days later). The iPS cells with colony formation were collected, and the expression of TRA-1-60, an initialization marker, was measured by flow cytometry. As a result, condition 3 (cells cultured with lipid nanoparticles added once on the first day) showed a TRA-1-60 expression rate of 96%.

[0051] From the above results, it was confirmed that the technology for producing iPS cells by initializing a blood cell group containing mononuclear cells derived from peripheral blood was provided by the lipid nanoparticles and each method according to the embodiment.

[0052] Examples of further embodiments are described below. [1] A medicament for initializing a blood cell group containing mononuclear cells derived from peripheral blood to produce iPS cells, comprising an initialization factor group for initializing the blood cell group and a group of lipid nanoparticles encapsulating the initialization factor group, wherein at least 40% or more of the lipid components of the lipid nanoparticles are FFT-10 and FFT-20, and FFT-10 and FFT-20 are contained in equal amounts. [2] The medicament according to [1], wherein 60% or less of the components of the lipid nanoparticles are neutral lipids, cationic lipids, cholesterol, and PEG-modified lipids. [3] The medicament according to [1] or [2], wherein the lipid nanoparticles contain more cationic lipids than neutral lipids. [4] The medicament according to any one of [1] to [3], wherein the PEG-modified lipid of the lipid nanoparticles is a PEG-modified lipid having a maleimide-modified functional group. [5] The medicament according to any one of [1] to [4], wherein the lipid composition ratio of the lipid nanoparticles is, in mol%, FFT-10:FFT-20:cationic lipid:cholesterol:PEG-modified lipid = 17-23:17-23:7-13:45-51:1.5-2.5, and the values are selected so that the total of each component is 100 mol%. [6] The agent according to any one of [1] to [5], wherein the group of reprogramming factors encapsulated in the lipid nanoparticles includes Oct3 / 4, Sox2, Klf4, and cMyc. [7] The agent according to any one of [1] to [6], wherein the group of reprogramming factors encapsulated in the lipid nanoparticles further includes Nanog and Lin28. [8] The agent according to any one of [1] to [7], wherein the group of reprogramming factors encapsulated in the lipid nanoparticles is DNA, RNA, and / or protein. [9] The agent according to any one of [1] to [8], wherein the lipid nanoparticles further contain additional factors other than the group of reprogramming factors.

[10] The agent according to any one of [1] to [9], wherein the lipid nanoparticles contain, as an inclusion other than the group of reprogramming factors, a factor that suppresses the interferon response.

[11] The agent according to any one of [1] to

[10] , wherein the lipid nanoparticles contain, as an inclusion other than the group of reprogramming factors, miRNA that improves the efficiency of iPS cell establishment.

[12] The agent according to any one of [1] to

[11] , wherein the encapsulation of the group of reprogramming factors into the lipid nanoparticles is such that all the reprogramming factors are encapsulated together per particle, or the group of reprogramming factors is divided and encapsulated into a plurality of the lipid nanoparticles.

[13] A kit for preparing an agent for reprogramming a blood cell group containing mononuclear cells derived from peripheral blood to produce iPS cells, the kit including a group of reprogramming factors and a lipid nanoparticle material encapsulating the group of reprogramming factors, wherein at least 40% or more of the lipid components of the lipid nanoparticles are FFT-10 and FFT-20, and FFT-10 and FFT-20 are included in equal amounts.

[14] The kit according to

[13] , wherein 60% or less of the components of the lipid nanoparticles are neutral lipids, cationic lipids, cholesterol, and PEG-modified lipids.

[15] The kit according to

[13] or

[14] , wherein the lipid nanoparticles contain more cationic lipids than neutral lipids.

[16] The kit according to any one of

[13] to

[15] , wherein the lipid nanoparticles are PEG-modified lipids having a maleimide-modified functional group.

[17] The kit according to any one of

[13] to

[16] , wherein the lipid composition ratio of the lipid nanoparticles is, as mol%, FFT-10:FFT-20:cationic lipid:cholesterol:PEG-modified lipid = 17 to 23:17 to 23:7 to 13:45 to 51:1.5 to 2.5, and the values of each component are selected so that the total is 100 mol%.

[18] The kit according to any one of

[13] to

[17] , wherein the group of reprogramming factors encapsulated in the lipid nanoparticles includes Oct3 / 4, Sox2, Klf4, and cMyc.

[19] The kit according to

[18] , wherein the group of reprogramming factors encapsulated in the lipid nanoparticles further includes Nanog and Lin28.

[20] The kit according to any one of

[13] to

[19] , wherein the group of reprogramming factors encapsulated in the lipid nanoparticles is DNA, RNA, and / or protein.

[21] The kit according to any one of

[13] to

[20] , wherein the lipid nanoparticles further include additional factors other than the group of reprogramming factors.

[22] The kit according to any one of

[13] to

[21] , wherein the lipid nanoparticles further include a factor that suppresses the interferon response as an inclusion other than the group of reprogramming factors.

[23] The kit according to any one of

[13] to

[22] , wherein the lipid nanoparticles further include miRNA that improves the efficiency of iPS cell establishment as an inclusion other than the group of reprogramming factors.

[24] The kit according to any one of

[13] to

[23] , wherein the encapsulation of the group of reprogramming factors into the lipid nanoparticles is such that all the reprogramming factors are encapsulated together per particle, or the group of reprogramming factors is divided and encapsulated into a plurality of the lipid nanoparticles.

[25] The kit according to any one of

[13] to

[24] , further comprising a production manual. A method for producing induced pluripotent stem cells (iPS cells) from a blood cell group, comprising contacting the initialization agent according to any one of [1] to

[12] with a blood cell group containing blood-derived mononuclear cells, and obtaining iPS cells from the blood cell group.

[0053] 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 the equivalent scope thereof.

Explanation of Signs

[0054] 11…lipid particle, 12, 12a, 12b, 12n…initialization factor or group of initialization factors, 101a…first introduction carrier, 101b…second introduction carrier, 101n…nth introduction carrier, 1001, 2001…introduction carrier set

Claims

1. An initialization factor group for producing iPS cells by initializing a blood cell group containing mononuclear cells derived from peripheral blood, and a lipid nanoparticle group encapsulating the initialization factor group, wherein at least 40% or more of the lipid components of the lipid nanoparticles are FFT-10 and FFT-20, and FFT-10 and FFT-20 are contained in equal amounts, a drug for initializing a blood cell group.

2. The drug according to claim 1, wherein 60% or less of the components of the lipid nanoparticles are neutral lipids, cationic lipids, cholesterol and PEG-modified lipids.

3. The drug according to claim 1, wherein the lipid nanoparticles contain more cationic lipids than neutral lipids.

4. The drug according to claim 2, wherein the PEG-modified lipid of the lipid nanoparticles is a PEG-modified lipid having a maleimide-modified functional group.

5. The drug according to claim 1, wherein the lipid composition ratio of the lipid nanoparticles is, as mol%, FFT-10:FFT-20:cationic lipid:cholesterol:PEG-modified lipid = 17-23:17-23:7-13:45-51:1.5-2.5, and the values of each component are selected so that the total is 100 mol%.

6. The drug according to claim 1, wherein the initialization factor group encapsulated in the lipid nanoparticles includes Oct3 / 4, Sox2, Klf4, and cMyc.

7. The drug according to claim 6, wherein the initialization factor group encapsulated in the lipid nanoparticles further includes Nanog and Lin28.

8. The drug according to claim 1, wherein the initialization factor group encapsulated in the lipid nanoparticles is in the form of DNA, RNA and / or protein.

9. The drug according to claim 1, wherein the lipid nanoparticles further contain additional factors other than the initialization factor group.

10. The drug according to claim 1, wherein the lipid nanoparticles further contain a factor that suppresses the response of interferon as an inclusion other than the initialization factor group.

11. The drug according to claim 1, wherein the lipid nanoparticles further contain miRNA that improves the efficiency of iPS cell establishment as an inclusion other than the initialization factor group.

12. The drug according to claim 1, wherein all of the initialization factor groups are encapsulated together per particle, or the initialization factor groups are divided and encapsulated in a plurality of the lipid nanoparticles.

13. An initialization factor group for initializing a blood cell group containing mononuclear cells derived from peripheral blood to produce iPS cells, and a lipid nanoparticle material encapsulating the initialization factor group, wherein at least 40% or more of the lipid components of the lipid nanoparticles are FFT-10 and FFT-20, and FFT-10 and FFT-20 are contained in equal amounts, a production kit for a drug for initializing a blood cell group.

14. The kit according to claim 13, wherein 60% or less of the components of the lipid nanoparticles are neutral lipids, cationic lipids, cholesterol, and PEG-modified lipids.

15. The kit according to claim 13, wherein the lipid nanoparticles contain more cationic lipids than neutral lipids.

16. The kit according to claim 14, wherein the PEG-modified lipid of the lipid nanoparticles is a PEG-modified lipid having a maleimide-modified functional group.

17. The kit according to claim 13, wherein the lipid composition ratio of the lipid nanoparticles is, in mol%, FFT-10:FFT-20:cationic lipid:cholesterol:PEG-modified lipid = 17-23:17-23:7-13:45-51:1.5-2.5, and the values of each component are selected so that the total is 100 mol%.

18. The kit according to claim 13, wherein the initialization factor group encapsulated in the lipid nanoparticles includes Oct3 / 4, Sox2, Klf4, and cMyc.

19. The kit according to claim 18, wherein the initialization factor group encapsulated in the lipid nanoparticles further includes Nanog and Lin28.

20. A method for producing iPS cells from a blood cell group, comprising contacting the initialization drug according to claim 1 with a blood cell group containing mononuclear cells derived from blood, and obtaining iPS cells from the blood cell group.

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