WO2022244670A1 - Procédé de production de cellules souches pluripotentes - Google Patents

Procédé de production de cellules souches pluripotentes Download PDF

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WO2022244670A1
WO2022244670A1 PCT/JP2022/020037 JP2022020037W WO2022244670A1 WO 2022244670 A1 WO2022244670 A1 WO 2022244670A1 JP 2022020037 W JP2022020037 W JP 2022020037W WO 2022244670 A1 WO2022244670 A1 WO 2022244670A1
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laminin
cells
mrna
fragment
reprogramming
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誠人 中川
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国立大学法人京都大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to a method for producing pluripotent stem cells.
  • iPS cells Induced pluripotent stem cells
  • iPS cells Human pluripotent stem cells
  • plasmid vector is inserted into the iPS cell genomic DNA. Therefore, it is necessary to conduct a test to confirm that there is no plasmid vector insertion before shipment.
  • Sendai virus it is necessary to confirm the survival of the virus before shipment, as with plasmid vectors.
  • the Sendai virus must be handled at biosafety level 2, which imposes restrictions on manufacturing sites.
  • the method of producing iPS cells by introducing the mRNA of the reprogramming factor gene into somatic cells and expressing the reprogramming factors for reprogramming has the great advantage that there is no concern about residual reprogramming factors.
  • the conversion efficiency is also high (Non-Patent Document 1).
  • the production of iPS cells by introducing mRNA of reprogramming factor genes has not yet been achieved.
  • fibroblasts collected from the skin are mainly used for iPS cell production, but blood cells, which are less invasive, are one of the useful somatic cell sources for iPS cell production.
  • Patent Document 1 discloses a method for establishing iPS cells, which includes the step of contacting somatic cells with a nuclear reprogramming substance in the presence of modified laminin.
  • the modified laminin used in this method is a composite molecule having a structure in which a cell growth regulatory molecule is bound to laminin or a laminin fragment forming a heterotrimer.
  • a reprogramming gene was introduced into adult skin-derived fibroblasts using an episomal plasmid vector in the presence of modified laminin to establish human iPS cells.
  • An object 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.
  • the present invention includes the following inventions.
  • a method for producing pluripotent stem cells comprising step 1 of introducing mRNA of a reprogramming factor gene into a somatic cell and step 2 of culturing the somatic cell into which the mRNA has been introduced, wherein in step 1, A production method comprising introducing mRNA of a reprogramming factor gene into a somatic cell in the presence of laminin having an ⁇ 5 chain or a fragment thereof having integrin-binding activity.
  • the production method according to [1] or [2] above, wherein the somatic cells are planktonic cells.
  • the planktonic cells are peripheral blood mononuclear cells.
  • the above step 1 is carried out by culturing the somatic cells in a medium containing laminin having an ⁇ 5 chain or a fragment thereof having integrin-binding activity, a transfection reagent, and mRNA of a reprogramming factor gene.
  • the manufacturing method according to any one of [1] to [4].
  • the present invention can provide a method for producing pluripotent stem cells by introducing mRNA of reprogramming factor genes into blood cells.
  • Example 1 of the production method of the present invention reprogramming factor gene mRNA (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, E3, K3 and B18R (EKB)) was introduced four times, and phase-contrast microscope images of cells 9 days after the first introduction.
  • 4 mRNAs of reprogramming factor genes OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB
  • PBMC peripheral blood mononuclear cells
  • FIG. 10 shows the results of comparing the number of iPS cell-like colonies formed in each group of cells after 9 days from the first introduction after the first introduction.
  • FIG. 2 shows the results of seeding human peripheral blood mononuclear cells (PBMC) together with a laminin 511E8 fragment and measuring the number of viable cells adhering to the plate the next day.
  • PBMC peripheral blood mononuclear cells
  • Example 2 is a diagram showing the results of comparing the number of iPS cell-like colonies generated from human peripheral blood mononuclear cells (PBMC) by conducting the same experiment as in Example 1 using a mouse EHS sarcoma-derived laminin solution.
  • the reprogramming factor mRNA OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB
  • MDM4 mouse double Minute 4
  • FIG. 4 is a diagram showing an image
  • the reprogramming factor mRNA (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB) used in Example 1 has a dominant negative mutation of p53 as a p53 inhibitor.
  • the same experiment as in Example 1 was performed by adding each mRNA of p53DN, MDM2 (mouse double minute 2), and MDM4, respectively, and comparing the number of iPS cell-like colonies generated from human peripheral blood mononuclear cells (PBMC).
  • FIG. 10 is a diagram showing the results of the experiment.
  • FIG. 10 is a diagram showing the results of the experiment.
  • FIG. 7 is a diagram showing the results of comparing the number of iPS cell-like colonies produced by conducting the same experiment as the experiment in FIG. 6 using human dermal fibroblasts (HDF).
  • the reprogramming factor mRNAs OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB
  • NANOG and/or LIN28 are not used.
  • FIG. 10 is a diagram showing the results of comparing the number of iPS cell-like colonies generated from human peripheral blood mononuclear cells (PBMC) after conducting the same experiment as in Example 1 using human dermal fibroblasts (HDF). .
  • PBMC peripheral blood mononuclear cells
  • 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 a somatic cell, and step 2 of culturing the somatic cell into which the mRNA has been introduced. Any method may be used as long as it introduces reprogramming factor gene mRNA into somatic cells in the presence of a fragment having integrin-binding activity.
  • Pluripotent stem cells mean stem cells that have pluripotency capable of differentiating into all cells existing in the body and that also have the ability to proliferate.
  • pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), cloned embryo-derived embryonic stem cells (ntES cells) obtained by nuclear transfer, and spermatogonial stem cells (GS cells). , embryonic germ cells (EG cells), cultured fibroblasts, and pluripotent cells (Muse cells) derived from bone marrow stem cells.
  • Pluripotent stem cells produced by the production method of the present invention are iPS cells produced by reprogramming somatic cells.
  • Laminin is a major cell adhesion molecule in the basement membrane, and is a heterotrimer consisting of three subunit chains, ⁇ , ⁇ , and ⁇ chains, and is a large glycoprotein with a molecular weight of approximately 800,000.
  • the three subunit chains associate at their C-terminal sides into a coiled-coil structure to form a heterotrimeric molecule stabilized by disulfide bonds.
  • Five types of ⁇ chain, ⁇ 1 to ⁇ 5, three types of ⁇ chain, ⁇ 1 to ⁇ 3, and three types of ⁇ chain, ⁇ 1 to ⁇ 3, are known, and at least 12 isoforms exist by combining them ( (see Table 1).
  • laminin isoforms are referred to as laminin-111, for example. Further, in the present specification, the term “laminin” simply means “full-length laminin”. Laminin having an ⁇ 5 chain used in the production method of the present invention includes laminin-511 and laminin-521.
  • a laminin fragment with integrin-binding activity is a fragment containing the laminin ⁇ -chain C-terminal LG1-3 and the ⁇ -chain C-terminal region, which form a heterotrimer with the ⁇ -chain C-terminal region of laminin. Fragments are preferred.
  • a laminin E8 fragment (hereinafter sometimes referred to as “laminin E8” or “E8”) can be suitably used as a laminin fragment having integrin-binding activity. 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 fragment forming a heterotrimer obtained by digesting mouse laminin 111 with elastase, and was identified as a fragment with strong cell adhesion activity (Edgar D et al., J. Cell Biol., 105:589-598, 1987). It is presumed that laminins other than mouse laminin 111 also have a fragment corresponding to mouse laminin 111E8 when digested with elastase. . Therefore, the laminin E8 having an ⁇ 5 chain used in the production method of the present invention does not have to be an elastase digestion product of laminin having an ⁇ 5 chain, and a laminin fragment having an ⁇ 5 chain having a similar structure good.
  • Laminin having an ⁇ 5 chain or a fragment thereof having integrin-binding activity may be laminin of any organism, but mammalian laminin is preferred. Mammals include, but are not limited to, humans, mice, rats, cows, pigs, and the like. Among them, humans are preferred.
  • Laminin and laminin fragments can be produced as recombinant proteins by appropriately using known gene recombination techniques.
  • the nucleotide sequence information of the genes encoding the ⁇ -, ⁇ -, and ⁇ -chains that constitute laminin of major mammals and the amino acid sequence information of each chain can be obtained from known databases (NCBI, etc.).
  • commercially available products such as human recombinant full-length laminin 511 (BioLamina), human recombinant full-length laminin 521 (BioLamina), human recombinant laminin 511E8 fragment (Nippi, trade name: iMatrix-511) can be used.
  • Somatic cells mean all animal cells except germline cells such as eggs, oocytes, ES cells, and totipotent cells. Somatic cells include fetal somatic cells, neonatal somatic cells, mature healthy or diseased somatic cells. Also included are both primary cultured cells and established cell lines.
  • somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue progenitor cells, (3) lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosa cells, enterocytes, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells , differentiated cells such as adipocytes, and the like.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells
  • tissue progenitor cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosa cells, enterocytes
  • the somatic cells used in the production method of the present invention may be adherent cells or suspension cells.
  • Adherent cells include, for example, dermal fibroblasts, mesenchymal stem cells, epithelial cells, and the like.
  • Floating cells include, for example, peripheral blood mononuclear cells (PBMC), blood cells such as bone marrow-derived cells and cord blood-derived cells. Suspended cells are preferred, blood cells are more preferred, and peripheral blood mononuclear cells are even more preferred.
  • PBMC peripheral blood mononuclear cells
  • Suspended cells are preferred, blood cells are more preferred, and peripheral blood mononuclear cells are even more preferred.
  • the somatic cells used in the production method of the present invention are preferably mammalian somatic cells, and mammals include, for example, humans, mice, rats, cows, and pigs. Of these, humans or mice are more preferred, and humans are most preferred.
  • 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, Glis1 and the like.
  • a single initialization factor may be used, or a plurality thereof may be used in combination.
  • Combinations of initialization factors are not particularly limited, but for example, WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009659/0 ⁇ WO2009/101084 ⁇ WO2009/101407 ⁇ WO2009/102983 ⁇ WO2009/114949 ⁇ WO2009/117439 ⁇ WO2009/126250 ⁇ WO2009/126251 ⁇ WO2009/126655 ⁇ 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.
  • the combination of reprogramming factors may be a combination of OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and microRNA302/367.
  • microRNA302/367 are highly expressed in pluripotent stem cells and are important microRNAs for maintaining an undifferentiated state, and may be replaced with microRNAs having similar expression characteristics and functions.
  • HDAC histone deacetylase
  • small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNAs and shRNAs against HDACs (e.g. , HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.
  • MEK inhibitors e.g., PD184352, PD98059, U0126, SL327 and PD0325901
  • Glycogen synthase kinase- 3 inhibitors e.g.
  • DNA methyltransferase inhibitors e.g. 5-azacytidine
  • histone methyltransferase inhibitors e.g. small molecule inhibitors such as BIX-01294, against Suv39hl, Suv39h2, SetDBl and G9a nucleic acid expression inhibitors such as siRNA and shRNA
  • L-channel calcium agonists e.g. Bayk8644
  • butyric acid TGF ⁇ inhibitors or ALK5 inhibitors
  • ALK5 inhibitors e.g.
  • p53 inhibition Agents e.g., siRNA and shRNA against p53, dominant-negative mutants 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 Wnt3a
  • neuropeptide Y e.g.
  • prostaglandin E2 and prostaglandin J2 hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl and other factors used for the purpose of increasing reprogramming efficiency (iPS cell establishment efficiency). Factors used for the purpose of improving the efficiency of initialization shall not be distinguished from initialization factors.
  • Combinations of reprogramming factors used in the production method of the present invention include, 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 MDM4", " OCT4, SOX2, KLF4, c-MYC, NANOG, microRNA302/367
  • Step 1 of the production method of the present invention includes an operation of introducing reprogramming factor gene mRNA into somatic cells in the presence of laminin having an ⁇ 5 chain or a fragment thereof having integrin-binding activity.
  • laminin or a laminin fragment may be dissolved in a medium, It may exist in a coated state in the culture vessel. Preferably, it is dissolved in a medium.
  • the concentration is, for example, 0.1 ⁇ g/mL or more, 0.2 ⁇ g/mL or more, 0.3 ⁇ g/mL or more, 0.4 ⁇ g/mL or more, 0.5 ⁇ g/mL or more, 0.6 ⁇ g/mL or more. It may be 0.7 ⁇ g/mL or more, 2.0 ⁇ g/mL or less, 1.9 ⁇ g/mL or less, 1.8 ⁇ g/mL, 1.7 ⁇ g/mL or less, or 1.6 ⁇ g/mL or less.
  • the coating concentration is, for example, 0.05 ⁇ g/cm 2 or more, 0.1 ⁇ g/cm 2 or more, 0.15 ⁇ g/cm 2 or more, 0.2 ⁇ g/cm 2 or more, 0.25 ⁇ g/cm 2 or more. cm 2 or more, 0.3 ⁇ g/cm 2 or more, 0.35 ⁇ g/cm 2 or more, 1.0 ⁇ g/cm 2 or less, 0.95 ⁇ g/cm 2 or less, 0.9 ⁇ g/cm 2 or less, 0.85 ⁇ g/cm 2 or less , may be 0.8 ⁇ g/cm 2 or less.
  • Coating of laminin or laminin fragments can be achieved by exposing laminin or laminin fragments to neutral pH in a suitable solvent such as PBS, saline, trishydroxymethylaminomethane or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. It can be carried out by diluting with diluted physiological saline or the like, adding this solution to a culture vessel, and allowing it to stand at about room temperature to about 37° C. for about 1 to about 12 hours.
  • a suitable solvent such as PBS, saline, trishydroxymethylaminomethane or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. It can be carried out by diluting with diluted physiological saline or the like, adding this solution to a culture vessel, and allowing it to stand at about 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 in vitro transcription from the expression vector.
  • In vitro transcription can be performed using, for example, commercially available reagents (eg, trade name: MEGAscript T7 Transcription Kit (Thermo Fisher Scientific), etc.).
  • Base sequence information of DNAs encoding reprogramming factors can be obtained from known databases (NCBI, etc.).
  • the method for introducing the mRNA of the reprogramming factor gene into somatic cells is not particularly limited, and is appropriately selected from known methods such as the liposome method, lipofection method, microinjection method, calcium phosphate method, electroporation method, and DEAE dextran method.
  • a commercially available transfection reagent for introducing RNA eg, trade name: Lipofectamine MessengerMAX (Thermo Fisher Scientific), etc. may be used.
  • a method for introducing reprogramming factor gene mRNA into somatic cells comprises suspending somatic cells in a medium containing laminin having an ⁇ 5 chain or a fragment thereof having integrin-binding activity, a transfection reagent, and reprogramming factor gene mRNA.
  • it may be a method of introducing the mRNA of the reprogramming factor gene into the somatic cells by seeding them in a culture vessel and culturing them.
  • a method of introducing mRNA of a reprogramming factor gene into a somatic cell comprises suspending somatic cells in a medium containing a transfection reagent and mRNA of the reprogramming factor gene, and obtaining laminin having an ⁇ 5 chain or its integrin-binding activity.
  • a method of introducing the mRNA of the reprogramming factor gene into the somatic cells by seeding and culturing the culture vessel coated with the fragment may also be used.
  • the somatic cells When using adherent somatic cells, the somatic cells are adherently cultured in a culture vessel pre-coated with laminin having an ⁇ 5 chain or a fragment thereof having integrin-binding activity, and containing a transfection reagent and reprogramming factor gene mRNA.
  • a method of introducing mRNA of a reprogramming factor gene into somatic cells by exchanging the medium and culturing the somatic cells may also be used.
  • somatic cells are adherently cultured in a culture vessel not coated with laminin or laminin fragments, and placed in a medium containing laminin having an ⁇ 5 chain or its integrin-binding fragment, transfection reagent, and reprogramming factor gene mRNA.
  • a method of introducing mRNA of a reprogramming factor gene into somatic cells by culturing after exchanging the medium may be used.
  • the cell number of the cell suspension may be between 2 ⁇ 10 5 /ml and 4 ⁇ 10 5 /ml, preferably between 2.5 ⁇ 10 5 /ml and 3.5 ⁇ 10 5 /ml. Concentrations of laminin or laminin fragments are as described above.
  • the amount of reprogramming factor gene mRNA to be added may be 300 ng/mL to 700 ng/mL, preferably 400 ng/mL to 600 ng/mL. Depending on the transfection reagent used, the number of cells, and the amount of mRNA to be added, the concentration recommended by the instruction manual may be adopted as the concentration of the transfection reagent.
  • the culture vessel to be used is not particularly limited as long as it can be used for culturing pluripotent stem cells.
  • the number of seeded cells can be appropriately changed according to the culture vessel to be used. For example, when using a 24-well plate, 0.5 mL to 1.5 mL of the above cell concentration suspension may be seeded per well.
  • the culture time may be 16-32 hours, preferably 18-30 hours, more preferably 22-26 hours.
  • the medium used in step 2 can be replaced and proceed to step 2.
  • introduction of reprogramming factor gene mRNA into somatic cells may be repeated without proceeding to step 2.
  • the number of times the reprogramming factor gene mRNA is introduced into somatic cells is not particularly limited, and may be two times, three times, four times, or five times or more. good. For example, the number of times that achieves the highest efficiency of reprogramming into pluripotent stem cells may be set based on a preliminary study.
  • 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 had undergone the first mRNA transfer were collected, and the collected somatic cells were suspended in a new medium containing the transfection reagent and the mRNA of the reprogramming factor gene, but not containing laminin or laminin fragments, and then resuspended. It may be sown and cultured. If a culture vessel coated with laminin or a laminin fragment is used in the first mRNA introduction, a culture vessel not coated with laminin or a laminin fragment may be used for the second and subsequent mRNA introductions.
  • the mRNA of the reprogramming factor gene to be introduced into the somatic cells from the second time onward may be the same as or different from the already introduced mRNA of the reprogramming factor gene. Preferably, it is the same as the mRNA of the reprogramming factor gene that has already been introduced.
  • the somatic cells into which the mRNA of the reprogramming factor gene has been introduced a predetermined number of times can be replaced with the medium used in step 2 and proceed to step 2.
  • step 2 of the production method of the present invention somatic cells into which the mRNA of the reprogramming factor gene has been introduced in step 1 are cultured.
  • the medium used in step 2 for example, DMEM, DMEM/F12 or DME culture medium containing 10 to 15% FBS (these culture mediums further include LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential Amino acids, ⁇ -mercaptoethanol, etc. can be appropriately included.) can be used.
  • ReproMed iPSC Medium Reprocell
  • mTeSR1 Stemcell Technology
  • Essential 8 Thermo Fisher Scientific
  • StemFit AK03N StemFit AK03N
  • the culture method is, for example, at 37° C. in the presence of 5% CO 2 , on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.) with 10% FBS-containing DMEM culture medium (this further includes LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.), and after about 25 to about 30 days or more, generate iPS cell-like colonies. be able to.
  • somatic cells themselves that are reprogrammed instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010/137746) method, extracellular matrix (e.g., laminin 511E8 (Nippi), Matrigel (BD Biosciences, etc.) may also be used.
  • iPS cells can be selected according to the shape of the formed colonies. Specifically, colonies that exhibit a single-layer flattened epithelial cell-like shape can be selected as iPS cell-like colonies. Alternatively, iPS cell-like colonies or iPS cells can be selected by confirming the expression of genes that are expressed when somatic cells are reprogrammed (e.g., Oct3/4, Nanog, TRA-1-60). .
  • the method of the present application can include a step of expanding somatic cells prior to step 1.
  • the culture conditions may be appropriately determined by those skilled in the art, and are not particularly limited.
  • the somatic cells are peripheral blood mononuclear cells (PBMC)
  • PBMC peripheral blood mononuclear cells
  • Known media for CD34-positive cells can be appropriately used, and examples thereof include media containing cytokines represented by IL-6, SCF, TPO, Flt-3L, IL-3 and G-CSF.
  • the period of 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 changing the medium as appropriate.
  • continuous medium exchange perfusion culture
  • Example 1 Production of iPS cells from peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • S6F StemSpan ACF
  • reprogramming factor gene mRNA OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB attached to StemRNA-3rd Gen Reprogramming Kit (Reprocell)
  • transfection reagent MessengerMAX, Thermo Fisher Scientific
  • laminin 511E8 fragment iMatrix-511, Nippi
  • the amount of laminin 511E8 fragment was set to 0.3125 ⁇ g/well, 0.625 ⁇ g/well or 1.25 ⁇ g/well, and a control group containing no laminin 511E8 fragment was provided. Twenty-four hours later (Day 5), the second mRNA transfer was performed by replacing with SS6F containing freshly prepared reprogramming factor gene mRNA and transfection reagent but not containing laminin 511E8 fragment. Twenty-four hours later (Day 6), the third mRNA transduction was performed by replacing with SS6F containing freshly prepared reprogramming factor gene mRNA and transfection reagent but not containing laminin 511E8 fragment.
  • the 4th mRNA transduction was carried out by replacing with SS6F containing freshly prepared reprogramming factor gene mRNA and transfection reagent but not containing laminin 511E8 fragment.
  • the medium was replaced with a 1:1 mixture of SS6F and StemFit AK03N (bFGF+).
  • the medium was replaced with StemFit AK03N (bFGF+) only, and culture was continued. Thereafter, the medium was replaced with StemFit AK03N (bFGF+) every 2 days.
  • FIG. 1 shows a phase-contrast microscope image 9 days after introduction of the reprogramming factor gene mRNA (Day 13). No iPS cell-like colonies were observed in the group not using the laminin 511E8 fragment. On the other hand, multiple iPS cell-like colonies were observed in all groups using the laminin 511E8 fragment.
  • the production method of the present invention reveals that the mRNA method can be used to reprogram blood cells to produce iPS cells. became.
  • the above-mentioned reprogramming efficiency is a numerical value that is sufficiently practical at the iPS cell manufacturing site.
  • the reprogramming efficiency when generating iPS cells from human dermal fibroblasts by the mRNA method (without using laminin 511E8 fragment) was about 0.5%.
  • the reprogramming efficiency when producing iPS cells by the viral method (SeV method) was about 0.45%.
  • Example 2 Examination of extracellular matrices other than laminin-511
  • laminin 511E8 iMatrix-511, Nippi
  • Geltrex trade name, Thermo Fisher Scientific
  • vitronectin Fujifilm Wako Pure Chemical Industries
  • type I collagen Nippi
  • fibronectin Fujifilm Wako Pure Chemical Industries
  • laminin solution derived from mouse EHS sarcoma (Fujifilm Wako Pure Chemical Industries) used.
  • Example 3 Examination of reprogramming effect by p53 inhibitor (1)
  • Suppression of p53 function is known to improve reprogramming efficiency. Therefore, MDM4, a p53 inhibitor, was added to the eight reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB) used in Example 1, The effect on reprogramming of MDM4 was evaluated.
  • Example 4 Examination of reprogramming effect by p53 inhibitor (2)
  • p53DN a dominant-negative mutant of p53
  • MDM2 a dominant-negative mutant of p53
  • the amount of laminin 511E8 (iMatrix-511) used was 1.25 ⁇ g/well. Eleven days after the first mRNA introduction (Day 15), the number of iPS cell-like colonies in each group was counted under a phase-contrast microscope.
  • FIG. (A) is a diagram comparing the number of iPS cell-like colonies per well in each group, and (B) is 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. It is the figure which calculated and evaluated the ratio of.
  • the +p53DN group tended to have higher reprogramming efficiency than the +mCherry group.
  • the +MDM4 group significantly improved the reprogramming efficiency compared to the +mCherry group, and the improvement in reprogramming efficiency was more than doubled compared to the +p53DN group.
  • no improvement in reprogramming efficiency was observed in the +MDM2 group.
  • Example 5 Examination of reprogramming effect by p53 inhibitor (3)
  • HDF human dermal fibroblasts
  • the amount of laminin 511E8 (iMatrix-511) used was 0.625 ⁇ g/well. Eleven days after the first mRNA introduction (Day 15), the number of iPS cell-like colonies in each group was counted under a phase-contrast microscope.
  • Example 6 Examination of types of initialization factors
  • OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, microRNA302/367, EKB the reprogramming efficiency was examined when NANOG and/or LIN28 were not used.
  • 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-N: OCT4, SOX2, KLF4, c-MYC, LIN28, microRNA302/367, EKB

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Abstract

L'invention concerne un procédé de production de cellules souches pluripotentes, le procédé étant caractérisé en ce qu'il comprend une étape 1 d'introduction d'ARNm d'un gène de facteur de reprogrammation dans des cellules somatiques, et une étape 2 de culture des cellules somatiques dans lesquelles l'ARNm a été introduit, et étant également caractérisé en ce que, à l'étape 1, l'ARNm du gène du facteur de reprogrammation est introduit dans les cellules somatiques en présence de laminine ayant une chaîne α5 ou un fragment de celle-ci ayant une activité de liaison à l'intégrine.
PCT/JP2022/020037 2021-05-18 2022-05-12 Procédé de production de cellules souches pluripotentes WO2022244670A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012137970A1 (fr) * 2011-04-08 2012-10-11 国立大学法人大阪大学 Laminine modifiée et utilisation de celle-ci
WO2017040548A1 (fr) * 2015-08-31 2017-03-09 I Peace, Inc. Système de production de cellules souches pluripotentes, et procédé de production de cellules souches pluripotentes induites
WO2018155595A1 (fr) * 2017-02-24 2018-08-30 剛士 田邊 Procédé de production de cellules souches pluripotentes artificielles

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012137970A1 (fr) * 2011-04-08 2012-10-11 国立大学法人大阪大学 Laminine modifiée et utilisation de celle-ci
WO2017040548A1 (fr) * 2015-08-31 2017-03-09 I Peace, Inc. Système de production de cellules souches pluripotentes, et procédé de production de cellules souches pluripotentes induites
WO2018155595A1 (fr) * 2017-02-24 2018-08-30 剛士 田邊 Procédé de production de cellules souches pluripotentes artificielles

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MASATO NAKAGAWA, YUKIMASA TANIGUCHI, SHO SENDA, NANAKO TAKIZAWA, TOMOKO ICHISAKA, KANAKO ASANO, ASUKA MORIZANE, DAISUKE DOI, JUN T: "A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells", SCIENTIFIC REPORTS, vol. 4, 8 January 2014 (2014-01-08), US , pages 1 - 7, XP055132358, ISSN: 2045-2322, DOI: 10.1038/srep03594 *
TERUHISA KAWAMURA, JOTARO SUZUKI, YUNYUAN V. WANG, SERGIO MENENDEZ, LAURA BATLLE MORERA, ANGEL RAYA, GEOFFREY M. WAHL, JUAN CARLOS: "Linking the p53 tumour suppressor pathway to somatic cell reprogramming", NATURE, vol. 460, no. 7259, 1 August 2009 (2009-08-01), London, pages 1140 - 1144, XP002632653, ISSN: 0028-0836, DOI: 10.1038/NATURE08311 *

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