JP7664656B2 - Method for producing pluripotent stem cells - Google Patents

Description

The present invention relates to a method for producing pluripotent stem cells.

Human pluripotent stem cells have attracted worldwide attention for their application in regenerative medicine. Currently, induced pluripotent stem cells (hereinafter referred to as "iPS cells") used in clinical applications are created by introducing reprogramming factors into cells in the blood using a plasmid vector. The problem with this method is that the plasmid vector is inserted into the genomic DNA of the iPS cells. Therefore, a test is required before shipping to confirm that the plasmid vector has not been inserted. It is also possible to introduce reprogramming factors using the Sendai virus, but as with plasmid vectors, it is necessary to check for residual virus before shipping. In addition, the Sendai virus must be handled at biosafety level 2, which imposes restrictions on manufacturing sites.

The method of producing iPS cells by introducing mRNA of a reprogramming factor gene into somatic cells and expressing the reprogramming factor for reprogramming has the great advantage of being free of concerns about residual reprogramming factors, and generally has high reprogramming efficiency (Non-Patent Document 1). However, production of iPS cells by introducing mRNA of a reprogramming factor gene into blood cells has not yet been achieved. Traditionally, fibroblasts collected from the skin have been mainly used to produce iPS cells, but blood cells, which are less invasive, are one of the useful somatic cell sources for producing iPS cells, so there is a demand for the establishment of a method of efficiently producing iPS cells using the mRNA introduction method, regardless of the type of somatic cell.

Patent Document 1 discloses a method for establishing iPS cells, which includes a step of contacting a nuclear reprogramming substance with somatic cells in the presence of modified laminin. The modified laminin used in this method is a composite molecule having a structure in which a cell proliferation control molecule is bound to laminin or a laminin fragment forming a heterotrimer. Specifically, it discloses that a reprogramming gene was introduced into adult skin-derived fibroblasts using an episomal plasmid vector in the presence of modified laminin, and human iPS cells were established.

WO2012/137970

Kogut et al., Nature Communications, 2018 Feb 21;9(1):745.

The objective of the present invention is to provide a method for producing pluripotent stem cells by introducing mRNA of a reprogramming factor gene into blood cells.

In order to solve the above problems, the present invention includes the following inventions.
[1] A method for producing pluripotent stem cells, comprising step 1 of introducing mRNA of a reprogramming factor gene into somatic cells, and step 2 of culturing the somatic cells into which the mRNA has been introduced, wherein in step 1, the mRNA of the reprogramming factor gene is introduced into somatic cells in the presence of laminin having an α5 chain or a fragment thereof having integrin-binding activity.
[2] The method of production described in [1] above, wherein the reprogramming factor gene includes the MDM4 gene.
[3] The method of production described in [1] or [2] above, wherein the somatic cells are floating cells.
[4] The method for producing the present invention described in [3], wherein the non-adherent cells are peripheral blood mononuclear cells.
[5] The method for production according to any one of [1] to [4] above, wherein step 1 is carried out by culturing the somatic cell in a medium containing laminin having an α5 chain or a fragment thereof having integrin-binding activity, a transfection reagent, and mRNA of a reprogramming factor gene.
[6] The method according to any one of [1] to [5] above, wherein the laminin having an α5 chain is laminin α5β1γ1 or laminin α5β2γ1.
[7] The method for producing according to any one of [1] to [6] above, wherein the fragment having integrin-binding activity is a laminin E8 fragment.
[8] The method according to any one of [1] to [7] above, wherein step 1 involves repeating the introduction of mRNA of the reprogramming factor gene into somatic cells two or more times.
[9] The method for producing the present invention described in [8] above, wherein the second or subsequent mRNA introduction is carried out in the absence of laminin having an α5 chain or a fragment thereof having integrin-binding activity.

The present invention provides a method for producing pluripotent stem cells by introducing mRNA of a reprogramming factor gene into blood cells.

FIG. 1 shows phase-contrast microscopy images of human peripheral blood mononuclear cells (PBMCs) 9 days after the first transfection of mRNAs of reprogramming factor genes (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, E3, K3 and B18R (EKB)) introduced four times in Example 1 of the production method of the present invention. FIG. 1 shows results of Example 1 of the production method of the present invention, in which mRNAs of reprogramming factor genes (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB) were introduced into human peripheral blood mononuclear cells (PBMCs) four times, and the number of iPS cell-like colonies formed in cells of each group 9 days after the first introduction was compared. FIG. 1 shows the results of seeding human peripheral blood mononuclear cells (PBMCs) together with laminin 511E8 fragments and measuring the number of viable cells attached to the plate the following day. FIG. 1 shows the results of comparing the numbers of iPS cell-like colonies generated from human peripheral blood mononuclear cells (PBMCs) in an experiment similar to that in Example 1, using iMatrix-511 (trade name), iMatrix-221 (trade name), iMatrix-411 (trade name), Geltrex (trade name), vitronectin, type I collagen, fibronectin, and mouse EHS sarcoma-derived laminin solution as extracellular matrices in the production method of the present invention. FIG. 1 shows phase contrast microscopy images and immunostained images with anti-TRA-1-60 antibody of cells (colonies) taken 14 days after the first mRNA introduction, in a production method of the present invention, in which an experiment similar to that of Example 1 was performed by adding mRNA of MDM4 (mouse double minute 4), a p53 inhibitor, to the mRNA of the reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB) used in Example 1. FIG. 1 shows the results of comparing the numbers of iPS cell-like colonies generated from human peripheral blood mononuclear cells (PBMCs) in an experiment similar to that of Example 1, in which the mRNAs of the reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB) used in Example 1 were added as p53 inhibitors, respectively, to the mRNAs of the reprogramming factors used in Example 1. FIG. 7 shows the results of a comparison of the numbers of iPS cell-like colonies formed when the same experiment as in FIG. 6 was performed using human dermal fibroblasts (HDF). FIG. 1 shows the results of a comparison of the numbers of iPS cell-like colonies generated from human peripheral blood mononuclear cells (PBMCs) in an experiment similar to that of Example 1 conducted in the production method of the present invention using human dermal fibroblasts (HDFs) in a case in which NANOG and/or LIN28 was not used among the mRNAs of the reprogramming factors used in Example 1 (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB).

The present invention provides a method for producing pluripotent stem cells (hereinafter referred to as the "production method of the present invention"). The production method of the present invention comprises step 1 of introducing mRNA of a reprogramming factor gene into somatic cells, and step 2 of culturing the somatic cells into which the mRNA has been introduced, and in step 1, it is sufficient that the mRNA of the reprogramming factor gene is introduced into somatic cells in the presence of laminin having an α5 chain or a fragment thereof having integrin-binding activity.

Pluripotent stem cells refer to stem cells that have the pluripotency to differentiate into all cells present in a living organism and also have the ability to proliferate. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), and pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. The pluripotent stem cells produced by the production method of the present invention are iPS cells produced by reprogramming somatic cells.

Laminin is the main cell adhesion molecule in the basement membrane. It is a heterotrimer consisting of three subunit chains, α, β, and γ chains, and is a gigantic glycoprotein with a molecular weight of about 800,000. The three subunit chains associate at the C-terminus to form a coiled-coil structure, forming a heterotrimeric molecule stabilized by disulfide bonds. There are five known types of α chains, α1 to α5, three known types of β chains, β1 to β3, and three known types of γ chains, γ1 to γ3, and there are at least 12 or more isoforms that are combinations of these (see Table 1). In this specification, laminin isoforms are expressed as, for example, laminin 111. In addition, when simply expressed as "laminin" in this specification, it means "full-length laminin". The laminins having an α5 chain used in the production method of the present invention include laminin 511 and laminin 521.

It is known that at least three globular domains (LG1-3) on the C-terminal side of the α chain and the C-terminal region of the γ chain are involved in the expression of integrin-binding activity of laminin. Therefore, it is preferable that the laminin fragment having integrin-binding activity is a fragment including LG1-3 on the C-terminal side of the α chain and the C-terminal region of the γ chain, which form a heterotrimer together with the C-terminal region of the β chain. As a laminin fragment having integrin-binding activity, a laminin E8 fragment (hereinafter sometimes referred to as "laminin E8" or "E8") can be preferably used. Therefore, the laminin fragment used in the production method of the present invention may be a laminin 511E8 fragment (laminin 511E8) or a laminin 521E8 fragment (laminin 521E8).

Laminin E8 is a heterotrimer-forming fragment obtained by digesting mouse laminin 111 with elastase, and has been identified as a fragment with strong cell adhesion activity (Edgar D et al., J. Cell Biol., 105:589-598, 1987). It is presumed that a fragment equivalent to mouse laminin 111E8 exists when laminins other than mouse laminin 111 are digested with elastase, but there have been no reports of isolating and identifying E8 by digesting laminins other than mouse laminin 111 with elastase. Therefore, the laminin E8 having an α5 chain used in the production method of the present invention does not necessarily have to be an elastase digestion product of laminin having an α5 chain, but may be a fragment of laminin having an α5 chain with a similar structure.

The laminin having an α5 chain or a fragment thereof having integrin-binding activity may be laminin from any organism, but is preferably from a mammal. Examples of mammals include, but are not limited to, humans, mice, rats, cows, pigs, etc. Among these, humans are preferred.

Laminin and laminin fragments can be produced as recombinant proteins by appropriately using known gene recombination techniques. The base sequence information of the genes encoding the α-chain, β-chain, and γ-chain that constitute the laminins of major mammals and the amino acid sequence information of each chain can be obtained from known databases (NCBI, etc.). In addition, commercially available products such as human recombinant full-length laminin 511 (BioLamina), human recombinant full-length laminin 521 (BioLamina), and human recombinant laminin 511 E8 fragment (Nippi, product name: iMatrix-511) can be used.

Somatic cells refer to any animal cell except germline cells such as eggs, oocytes, and ES cells, and totipotent cells. Somatic cells include fetal somatic cells, neonatal somatic cells, and mature healthy or diseased somatic cells. They also include both primary cultured cells and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

The somatic cells used in the production method of the present invention may be adhesive cells or floating cells. Examples of adhesive cells include skin fibroblasts, mesenchymal stem cells, and epithelial cells. Examples of floating cells include blood cells such as peripheral blood mononuclear cells (PBMCs), bone marrow-derived cells, and umbilical cord blood-derived cells. Preferred are floating cells, more preferably blood cells, and even more preferably peripheral blood mononuclear cells. In addition, the somatic cells used in the production method of the present invention are preferably somatic cells of mammals, and examples of mammals include humans, mice, rats, cows, and pigs. Of these, humans or mice are more preferred, and humans are most preferred.

Examples of reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. Reprogramming factors may be used alone or in combination. The combination of reprogramming factors is not particularly limited, but may be any of those described in, for example, WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/101084, WO2009/ 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/1266 55, WO2009/157593, WO2010/009015, WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO 2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO 2010/111409, WO 2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9. The combination of reprogramming factors may be a combination of OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and microRNA302/367. MicroRNA302/367 is a microRNA that is highly expressed in pluripotent stem cells and is important for maintaining the undifferentiated state, and may be replaced with a microRNA that has similar expression characteristics and functions.

The above-mentioned reprogramming factors include histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, etc., and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene))], MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., BIX-01294, small molecule inhibitors such as Suv39hl, Suv39h2, SetDBl and nucleic acid expression inhibitors such as siRNA and shRNA against G9a), L-channel calcium agonists (e.g. Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g. LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (e.g. siRNA and shRNA against p53, dominant negative mutant of p53 (p53DN), MDM2 (mouse double minute 2), MDM4 (mouse double minute 4) etc.), ARID3A inhibitors (e.g. siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling (e.g. soluble These factors used for the purpose of improving reprogramming efficiency (iPS cell establishment efficiency), such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, and DMRTBl, are also included, and in this specification, factors used for the purpose of improving reprogramming efficiency will not be distinguished from reprogramming factors.

The combination of reprogramming factors used in the production method of the present invention is, for example, "OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, and EKB", "OCT4, SOX2, KLF4, c-MYC, LIN28, microRNA302/367, and EKB", "OCT4, SOX2, KLF4, c-MYC, NANOG, microRNA302/367, and EKB", "OCT4, SOX2, KLF4, c-MYC, microRNA302/367, and EKB", "OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB, and MDM4", "OCT4, SOX2, KLF4, c-MYC, LIN28, microRNA302/367, EKB, and and MDM4", "OCT4, SOX2, KLF4, c-MYC, NANOG, microRNA302/367, EKB, and MDM4", "OCT4, SOX2, KLF4, c-MYC, microRNA302/367, EKB, and MDM4", "OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB, and p53DN", "OCT4, SOX2, KLF4, c-MYC, LIN28, microRNA302/367, EKB, and p53DN", "OCT4, SOX2, KLF4, c-MYC, NANOG, microRNA302/367, EKB, and p53DN", "OCT4, SOX2, KLF4, c-MYC, microRNA302/367, EKB, and p53DN". However, the present invention is not limited to these. EKB is known to have the effect of suppressing inflammatory reactions induced in cells by the introduction of RNA, and may be substituted with other factors that can exert a similar effect.

Step 1 of the production method of the present invention includes an operation of introducing mRNA of a reprogramming factor gene into somatic cells in the presence of laminin having an α5 chain or a fragment thereof having integrin binding activity. In the operation of introducing mRNA of a reprogramming factor gene into somatic cells in the presence of laminin having an α5 chain or a fragment thereof having integrin binding activity, the laminin or laminin fragment may be present in a state dissolved in a medium, or may be present in a state coated on a culture vessel. It is preferably in a state dissolved in a medium. When laminin or laminin fragment is dissolved in a medium, its concentration may be, for example, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.4 μg/mL or more, 0.5 μg/mL or more, 0.6 μg/mL or more, 0.7 μg/mL or more, or 2.0 μg/mL or less, 1.9 μg/mL or less, 1.8 μg/mL, 1.7 μg/mL or less, or 1.6 μg/mL or less.

When laminin or laminin fragments are coated onto a culture vessel, the coating concentration may be, for example, 0.05 μg/cm2 or ^more , 0.1 μg/cm2 or ^more , 0.15 μg/ ^cm2 or more, 0.2 μg/ ^cm2 or ^more , 0.25 μg/cm2 or more, 0.3 μg/ ^cm2 or more, 0.35 μg/ ^cm2 or more, or 1.0 μg/cm2 or ^less , 0.95 μg/cm2 or ^less , 0.9 μg/ ^cm2 or less, 0.85 μg/cm2 or ^less , or 0.8 μg/ ^cm2 or less. Coating with laminin or laminin fragments can be carried out by diluting laminin or laminin fragments with an appropriate solvent, such as PBS, saline, or saline adjusted to a neutral pH with trishydroxymethylaminomethane or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, adding this solution to the culture vessel, and allowing it to stand at room temperature to about 37°C for about 1 to about 12 hours.

The mRNA of the reprogramming factor gene can be produced, for example, by constructing an expression vector into which DNA encoding the reprogramming factor is inserted and performing in vitro transcription from the expression vector. In vitro transcription can be performed, for example, using a commercially available reagent (e.g., MEGAscript T7 Transcription Kit (Thermo Fisher Scientific)). The base sequence information of the DNA encoding the reprogramming factor can be obtained from a publicly known database (e.g., NCBI).

The method for introducing the mRNA of the reprogramming factor gene into somatic cells is not particularly limited, and may be appropriately selected from known methods such as the liposome method, lipofection method, microinjection method, calcium phosphate method, electroporation method, and DEAE-dextran method. Commercially available transfection reagents for RNA introduction (e.g., trade name: Lipofectamine MessengerMAX (Thermo Fisher Scientific), etc.) may also be used.

The method for introducing the mRNA of the reprogramming factor gene into somatic cells may be a method for introducing the mRNA of the reprogramming factor gene into somatic cells by suspending the somatic cells in a medium containing laminin having an α5 chain or a fragment thereof having integrin-binding activity, a transfection reagent, and the mRNA of the reprogramming factor gene, and seeding and culturing the somatic cells in a culture vessel. The method for introducing the mRNA of the reprogramming factor gene into somatic cells may be a method for introducing the mRNA of the reprogramming factor gene into somatic cells by suspending the somatic cells in a medium containing a transfection reagent and the mRNA of the reprogramming factor gene, and seeding and culturing the somatic cells in a culture vessel coated with laminin having an α5 chain or a fragment thereof having integrin-binding activity.

When adhesive somatic cells are used, a method may be used in which somatic cells are cultured as adherent cells in a culture vessel that has been coated in advance with laminin having an α5 chain or a fragment thereof having integrin-binding activity, and the medium is replaced with a medium containing a transfection reagent and the mRNA of the reprogramming factor gene, followed by culture, to introduce the mRNA of the reprogramming factor gene into the somatic cells. Alternatively, a method may be used in which somatic cells are cultured as adherent cells in a culture vessel that has not been coated with laminin or a laminin fragment, and the medium is replaced with a medium containing laminin having an α5 chain or a fragment thereof having integrin-binding activity, a transfection reagent, and the mRNA of the reprogramming factor gene, followed by culture, to introduce the mRNA of the reprogramming factor gene into the somatic cells.

The number of cells in the cell suspension may be 2×10 ⁵ cells/mL to 4×10 ⁵ cells/mL, preferably 2.5×10 ⁵ cells/mL to 3.5× ^{10 5} cells/mL. The concentration of laminin or laminin fragment is as described above. The amount of mRNA of the reprogramming factor gene added may be 300 ng/mL to 700 ng/mL, preferably 400 ng/mL to 600 ng/mL. The concentration of the transfection reagent may be the concentration recommended in the instruction manual depending on the transfection reagent used, the number of cells, and the amount of mRNA added. The culture vessel used is not particularly limited as long as it can be used for culturing pluripotent stem cells, and for example, a glass or plastic petri dish, flask, multi-well plate, etc. can be used. The number of cells to be seeded can be appropriately changed depending on the culture vessel used. For example, when a 24-well plate is used, 0.5 mL to 1.5 mL of the suspension having the above cell concentration may be seeded per well. The culture time may be 16 to 32 hours, preferably 18 to 30 hours, and more preferably 22 to 26 hours.

After the specified culture time has elapsed, the medium can be replaced with the medium used in step 2, and the process can proceed to step 2. Alternatively, the introduction of the mRNA of the reprogramming factor gene into the somatic cells can be repeated without proceeding to step 2. The number of times that the mRNA of the reprogramming factor gene is introduced into the somatic cells is not particularly limited, and may be two, three, four, five or more times. For example, the number of times that results in the highest efficiency of reprogramming into pluripotent stem cells can be set based on preliminary studies.

The culture system for the second and subsequent mRNA introductions may or may not contain laminin or laminin fragments. That is, the somatic cells that have undergone the first mRNA introduction may be collected, suspended in a new medium containing a transfection reagent and the mRNA of the reprogramming factor gene, but not containing laminin or laminin fragments, and then reseeded and cultured. If a culture vessel coated with laminin or laminin fragments is used for the first mRNA introduction, a culture vessel not coated with laminin or laminin fragments may be used for the second and subsequent mRNA introductions. In addition, the mRNA of the reprogramming factor gene introduced into the somatic cells from the second time onwards may be the same as or different from the mRNA of the reprogramming factor gene already introduced. Preferably, it is the same as the mRNA of the reprogramming factor gene already introduced. The somatic cells into which the mRNA of the reprogramming factor gene has been introduced a predetermined number of times may be replaced with the medium used in step 2 and proceed to step 2.

In step 2 of the production method of the present invention, somatic cells into which mRNA of the reprogramming factor gene has been introduced in step 1 are cultured. The medium used in step 2 may be, for example, DMEM, DMEM/F12 or DME culture medium containing 10-15% FBS (these culture medium may further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate). In addition, commercially available media for ES/iPS cell culture may be used, such as ReproMed iPSC Medium (ReproCell), mTeSR1 (Stemcell Technology), Essential 8 (Thermo Fisher Scientific), StemFit AK03N (AJINOMOTO), etc.

For example, the culture method may involve _culturing on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM culture medium (which may further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate) at 37°C in the presence of 5% CO2, and after about 25 to about 30 days or more, iPS cell-like colonies can be generated. Methods using the somatic cells themselves to be reprogrammed instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010/137746), or methods using extracellular matrices (e.g., laminin 511E8 (Nippi), Matrigel (BD Biosciences), etc.) may also be used.

iPS cells can be selected based on the shape of the colonies formed; specifically, colonies that exhibit a monolayer, flat epithelial cell-like shape can be selected as iPS cell-like colonies. In addition, iPS cell-like colonies or iPS cells can be selected by confirming the expression of genes (e.g., Oct3/4, Nanog, TRA-1-60) that are expressed when somatic cells are reprogrammed.

In one embodiment, the method of the present application may include a step of expanding somatic cells prior to step 1. The culture conditions may be appropriately determined by a person skilled in the art and are not particularly limited. For example, when the somatic cells are peripheral blood mononuclear cells (PBMCs), the CD34-positive cells may be increased by culturing them in a medium for CD34-positive cells. As the medium for CD34-positive cells, a known medium may be appropriately used, for example, a medium containing cytokines such as IL-6, SCF, TPO, Flt-3L, IL-3, and G-CSF. The period of the expansion culture may be appropriately determined by a person skilled in the art and is not particularly limited, but may be 1 to 14 days, 3 to 10 days, for example, 4 to 7 days, while exchanging the medium appropriately. In addition, continuous medium exchange (perfusion culture) may be performed by using an automated culture device or the like.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. In the following examples and reference examples, uncoated culture vessels were used unless otherwise specified.

Example 1: Production of iPS cells from peripheral blood mononuclear cells
1. Experimental method Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and culture was initiated (Day 0) using StemSpan ACF (STEMCELL Technologies, hereafter referred to as "SS6F"; in other examples, an equivalent product, StemSpan AOF, was also used). On day 4, PBMCs were suspended in SS6F containing mRNAs of reprogramming factor genes (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB included in StemRNA-3rd Gen Reprogramming Kit (ReproCell)), transfection reagent (MessengerMAX, Thermo Fisher Scientific), and laminin 511E8 fragment (iMatrix-511, Nippi), and plated at 2.5 × 10 ⁵ cells/800 μL/well in a 24-well plate, and the mRNAs of the reprogramming factor genes were transfected into the PBMCs. The amount of mRNA of the reprogramming factor genes was 400 ng/well. The amount of laminin 511E8 fragment was 0.3125 μg/well, 0.625 μg/well, or 1.25 μg/well, and a control group without laminin 511E8 fragment was set up.
After 24 hours (Day5), the medium was replaced with SS6F containing freshly prepared mRNA of the reprogramming factor gene and transfection reagent but not containing the laminin 511E8 fragment for the second mRNA introduction. After 24 hours (Day6), the medium was replaced with SS6F containing freshly prepared mRNA of the reprogramming factor gene and transfection reagent but not containing the laminin 511E8 fragment for the third mRNA introduction. After 24 hours (Day7), the medium was replaced with SS6F containing freshly prepared mRNA of the reprogramming factor gene and transfection reagent but not containing the laminin 511E8 fragment for the fourth mRNA introduction. After 24 hours (Day8), the medium was replaced with a 1:1 mixture of SS6F and StemFit AK03N (bFGF+). After 3 days (Day11), the medium was replaced with StemFit AK03N (bFGF+) only and the culture was continued. Thereafter, the medium was replaced with StemFit AK03N (bFGF+) every two days.

2. Results Figure 1 shows phase contrast microscopy images taken 9 days (Day 13) after introduction of the mRNA of the reprogramming factor gene. No iPS cell-like colonies were observed in the group in which the laminin 511E8 fragment was not used. On the other hand, multiple iPS cell-like colonies were observed in all groups in which the laminin 511E8 fragment was used. Until now, it was believed that blood cells could not be reprogrammed using the mRNA method, but it has been revealed that the production method of the present invention makes it possible to reprogram blood cells using the mRNA method to produce iPS cells.

The results of comparing the number of iPS cell-like colonies per well in each group are shown in Figure 2. It was revealed that the number of iPS cell-like colonies increased depending on the concentration of laminin 511E8 fragment, with a peak at around 0.625 μg. As shown in Figure 2, when laminin 511E8 fragment was used at 0.625 μg/well, about 50 iPS cell-like colonies were obtained from 2.5 × 10 ⁵ PBMCs (reprogramming efficiency = 0.02%). If iPS cells are produced from PBMCs using the production method of the present invention, there is no need to check for residual vectors before shipping the iPS cells, and there is no need to handle them at biosafety level 2, which can greatly reduce the burden on the production site. In addition, the reprogramming efficiency mentioned above is a value that is fully practical for use at iPS cell production sites.
For reference, in the inventor's laboratory, the reprogramming efficiency when producing iPS cells from human skin fibroblasts by the mRNA method (without using laminin 511E8 fragment) was approximately 0.5%, and the reprogramming efficiency when producing iPS cells from PBMCs by the Sendai virus method (SeV method) was approximately 0.45%.

Reference Example 1: Effect of laminin 511E8 fragment on adhesiveness of peripheral blood mononuclear cells
As mentioned above, the mRNA method can achieve very high reprogramming efficiency in adherent HDFs. Therefore, in Example 1, we examined the possibility that the laminin 511E8 fragment improved the adhesiveness of PBMCs and thus enabled the generation of iPS cell-like colonies.
1. Experimental method Peripheral blood mononuclear cells (PBMCs) were cultured in medium containing laminin 511E8 fragment, and the number of viable cells attached to the plate was measured the next day by measuring the absorbance at 450 nm using Cell Counting Kit-8 (DOJINDO).

The results are shown in Figure 3. Contrary to expectations, the absorbance tended to decrease upon addition of the laminin 511E8 fragment. Furthermore, it was also revealed that the absorbance did not change significantly depending on the concentration of the laminin 511E8 fragment, and at least did not correlate with the result of the number of iPS cell-like colonies obtained in Example 1 (Figure 2).
This indicates that the adhesion of PBMCs to culture vessels is not improved by the laminin 511E8 fragment in the medium, at least at the fragment concentration range where PBMCs can be sufficiently reprogrammed by the mRNA method. This result indicates that the addition of the laminin 511E8 fragment to the medium does not increase the reprogramming efficiency by forcing PBMCs to adhere.

Example 2: Examination of extracellular matrices other than laminin 511
In Example 1, laminin 511E8 (iMatrix-511, Nippi) was used as the extracellular matrix, whereas in Example 2, in addition to laminin 511E8, laminin 221E8 (iMatrix-221, Nippi), laminin 411E8 (iMatrix-411), Geltrex (trade name, Thermo Fisher Scientific), vitronectin (Fujifilm Wako Pure Chemical Industries), type I collagen (Nippi), fibronectin (Fujifilm Wako Pure Chemical Industries), laminin solution, and mouse EHS sarcoma-derived (Fujifilm Wako Pure Chemical Industries) were used.

1. Experimental Method Frozen PBMCs (different lot from Example 1) were thawed, and culture was started using SS6F in the same manner as in Example 1 (=Day 0). The above eight types of extracellular matrices were used at a concentration of 1.25 μg/well, and the introduction of mRNA of reprogramming factor genes (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB included in StemRNA-3rd Gen Reprogramming Kit (ReproCell)) was repeated four times in the same manner as in Example 1. The experiment was performed in one well of a 24-well plate (n=1 for each group) for each extracellular matrix. Nine days after the first mRNA introduction (Day 13), the number of iPS cell-like colonies was counted using a phase contrast microscope.

2. Results The results are shown in FIG. 4. iPS cell-like colonies were obtained in the wells using laminin 511E8, but no iPS cell-like colonies appeared in extracellular matrices other than laminin 511E8. Therefore, it was revealed that extracellular matrices other than laminin having an α5 chain or a fragment thereof having integrin binding activity could not promote the reprogramming efficiency by the mRNA method. The present inventors have experienced a large variation in the number of iPS cell-like colonies that appeared depending on the lot of PBMC used.

Example 3: Examination of the reprogramming-promoting effect of p53 inhibitors (1)
It is known that suppressing p53 function improves reprogramming efficiency. Therefore, we added MDM4, a p53 inhibitor, to the eight reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, and EKB) used in Example 1, and evaluated the effect of MDM4 on reprogramming.

1. Experimental method
MDM4 was added as a p53 inhibitor to carry out the experiment. Frozen and stored PBMC (different lot from those in Examples 1 and 2) were thawed, and culture was started using SS6F in the same manner as in Example 1 (=Day 0). A group using the mRNA of the eight types of reprogramming factors used in Example 1 (control group) and a group to which the mRNA of the MDM4 gene was further added (+MDM4 group) were set up, and mRNA introduction was repeated four times in the same manner as in Example 1. The amount of laminin 511E8 (iMatrix-511) used was 1.25 μg/well. 14 days after the first mRNA introduction (Day 18), phase contrast microscopic images of the colonies were obtained. Furthermore, immunostaining was performed using anti-TRA-1-60 antibody, and fluorescent microscopic images of the colonies were obtained.

2. Results Representative images of each group are shown in Figure 5. The left is the control group, the right is the +MDM4 group, the top row is a phase contrast microscope image, and the bottom row is a fluorescent microscope image after immunostaining. As shown in Figure 5, many TRA-1-60 positive iPS cell-like colonies were observed in the +MDM4 group. This shows that the addition of MDM4 mRNA significantly improves reprogramming efficiency.

Example 4: Examination of the reprogramming-promoting effect of p53 inhibitors (2)
1. Experimental method
As p53 inhibitors, p53 dominant negative mutant (p53DN) and MDM2 were used in addition to MDM4. Frozen PBMCs (different lots from those in Examples 1, 2, and 3) were thawed, and culture was started using SS6F in the same manner as in Example 1 (=Day 0). Four groups were set up (n=4 for each group): a group in which mCherry (red fluorescent protein) gene mRNA was added to the mRNA of the eight types of reprogramming factors used in Example 1 (+mCherry group, control), a group in which p53DN gene mRNA was added (+p53DN group), a group in which MDM2 gene mRNA was added (+MDM2 group), and a group in which MDM4 gene mRNA was added (+MDM4 group), and mRNA introduction was repeated four times in the same manner as in Example 1. The amount of laminin 511E8 (iMatrix-511) used was 1.25 μg/well. Eleven days after the first mRNA transfection (Day 15), the number of iPS cell-like colonies in each group was counted using a phase-contrast microscope.

2. Results The results are shown in Figure 6. (A) is a diagram comparing the number of iPS cell-like colonies per well in each group, and (B) is a diagram evaluating the ratio of the average number of iPS cell-like colonies in each group, when the average number of colonies in the +mCherry group is set to 1. The +p53DN group showed a tendency for improved reprogramming efficiency compared to the +mCherry group. The +MDM4 group showed a marked improvement in reprogramming efficiency compared to the +mCherry group, and was more than twice as efficient as the +p53DN group. Surprisingly, however, no improvement in reprogramming efficiency was observed in the +MDM2 group.

Example 5: Examination of the reprogramming-promoting effect of p53 inhibitors (3)
1. Experimental method
The experiment was carried out using human dermal fibroblasts (HDF) instead of PBMC. Four groups were set up (n=3 for each group): a group in which EGFP (green fluorescent protein) gene mRNA was added to the mRNA of the eight types of reprogramming factors used in Example 1 (+EGFP group, control), a group in which p53DN gene mRNA was added (+p53DN group), a group in which MDM2 gene mRNA was added (+MDM4 group), and a group in which MDM4 gene mRNA was added (+MDM4 group), and mRNA introduction was repeated four times in the same manner as in Example 1. The amount of laminin 511E8 (iMatrix-511) used was 0.625 μg/well. 11 days after the first mRNA introduction (Day 15), the number of iPS cell-like colonies in each group was counted using a phase contrast microscope.

Furthermore, the same experiment was repeated in three groups (n=5 in each group): a group in which EGFP (green fluorescent protein) gene mRNA was added to the mRNA of the eight types of reprogramming factors used in Example 1 (+EGFP group, control), a group in which p53DN gene mRNA was added (+p53DN group), and a group in which MDM4 gene mRNA was added (+MDM4 group).

2. Results The results are shown in Figure 7. (A) shows the results of the first experiment, and (B) shows the results of the second experiment. In HDFs, the reprogramming efficiency was significantly improved in the +p53DN and +MDM4 groups compared to the +EGFP group, while the reprogramming efficiency of the +p53DN and +MDM4 groups was equivalent. In addition, the reprogramming efficiency of the +MDM2 group tended to improve, although it was much smaller than that of the +p53DN group.

Therefore, the results of Examples 3 to 5 demonstrated that in a method of reprogramming somatic cells by introducing mRNA of a reprogramming factor gene in the presence of laminin having an α5 chain or a fragment thereof having integrin-binding activity, the addition of MDM4 as a reprogramming factor provides a remarkable reprogramming-promoting effect equivalent to or greater than that obtained when a dominant-negative mutant of p53 is added. It was also shown that this promoting effect is particularly remarkable in the reprogramming of blood cell lineage cells.

Example 6: Examination of types of reprogramming factors
Among the reprogramming factors used in Example 1 (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB), the reprogramming efficiency was examined when NANOG and/or LIN28 was not used.

1. Experimental Method HDFs were used as cells. mRNA transfection of reprogramming factor genes was repeated four times according to the same schedule as in Example 1. The amount of laminin 511E8 (iMatrix-511) used was 0.625 μg/well. Nine days after the first mRNA transfection (Day 13), the number of iPS cell-like colonies was counted using a phase contrast microscope. The same experiment was performed twice.
The combinations of reprogramming factors for each group (n=5) are shown below.
NK: OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB
NK-N: OCT4, SOX2, KLF4, c-MYC, LIN28, microRNA302/367, EKB
NK-L: OCT4, SOX2, KLF4, c-MYC, NANOG, microRNA302/367, EKB
NK-NL: OCT4, SOX2, KLF4, c-MYC, microRNA302/367, EKB
NK+M4: OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB, MDM4
NK+M4-N: OCT4, SOX2, KLF4, c-MYC, LIN28, microRNA302/367, EKB, MDM4
NK+M4-L: OCT4, SOX2, KLF4, c-MYC, NANOG, microRNA302/367, EKB, MDM4
NK+M4-NL: OCT4, SOX2, KLF4, c-MYC, microRNA302/367, EKB, MDM4

2. Results The results are shown in Figure 8. (A) shows the results of the first experiment, and (B) shows the results of the second experiment.
Since iPS cell-like colonies were observed in both groups, it was shown that reprogramming is possible even if NANOG or LIN28 is omitted from the eight types of reprogramming factors used in Example 1 (NK group) or the nine types of reprogramming factors obtained by adding MDM4 to the eight types of reprogramming factors (NK+M4 group), or even if NANOG and LIN28 are omitted. Furthermore, although the results are omitted, a large number of iPS-like colonies were obtained even with the seven types of reprogramming factors obtained by removing KLF4 from the eight types, and it was confirmed that the number of iPS-like colonies increases significantly with the addition of MDM4. Therefore, it has become clear that various combinations of reprogramming factor genes can be used in the reprogramming method according to the present invention.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. In addition, all academic and patent documents described in this specification are incorporated herein by reference.

Claims (7)

A method for producing pluripotent stem cells, comprising step 1 of introducing mRNA of a reprogramming factor gene into blood cells, and step 2 of culturing the blood cells into which the mRNA has been introduced, wherein step 1 is performed by culturing the blood cells in a medium containing laminin having an α5 chain or a fragment thereof having integrin-binding activity, a transfection reagent, and the mRNA of the reprogramming factor gene. The method of claim 1, wherein the reprogramming factor gene includes the MDM4 gene. The method according to claim 1, wherein the blood cells are peripheral blood mononuclear cells. The method according to claim 1, wherein the laminin having an α5 chain is laminin α5β1γ1 or laminin α5β2γ1. The method according to claim 1, wherein the fragment having integrin binding activity is a laminin E8 fragment. The method according to any one of claims 1 to 5, wherein the step 1 comprises repeating the introduction of mRNA of the reprogramming factor gene into blood cells two or more times. The method according to claim 6, wherein the second and subsequent mRNA introductions are performed in the absence of laminin having an α5 chain or a fragment thereof having integrin-binding activity.