[go: up one dir, main page]

WO2017121386A1 - Procédé de préparation de cellules souches pluripotentes de type petit arn exogène et son utilisation - Google Patents

Procédé de préparation de cellules souches pluripotentes de type petit arn exogène et son utilisation Download PDF

Info

Publication number
WO2017121386A1
WO2017121386A1 PCT/CN2017/071140 CN2017071140W WO2017121386A1 WO 2017121386 A1 WO2017121386 A1 WO 2017121386A1 CN 2017071140 W CN2017071140 W CN 2017071140W WO 2017121386 A1 WO2017121386 A1 WO 2017121386A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
stem cells
cells
group
small rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/071140
Other languages
English (en)
Chinese (zh)
Inventor
张辰宇
曾科
张峻峰
陈熹
梁宏伟
周桢
付正
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Micromedmark Biotech Co Ltd
Original Assignee
Jiangsu Micromedmark Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Micromedmark Biotech Co Ltd filed Critical Jiangsu Micromedmark Biotech Co Ltd
Publication of WO2017121386A1 publication Critical patent/WO2017121386A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/65—MicroRNA
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/07—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from endocrine cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
    • C12N2506/098—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells from cells of secretory glands, e.g. parotid gland, salivary glands, sweat glands, lacrymal glands
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1315—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from cardiomyocytes
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1323—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from skeletal muscle cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1338—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from smooth muscle cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/14—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from hepatocytes
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/25—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from renal cells, from cells of the urinary tract

Definitions

  • the present invention relates to the field of molecular biology, and in particular to a method and use of exogenous small RNA for preparing pluripotent stem cells.
  • the pluripotent stem cell has the potential to differentiate into a variety of cellular tissues, which can differentiate into all cells in the body, thereby forming all tissues and organs of the body. Therefore, the study of pluripotent stem cells not only has important theoretical significance, but also has great application value in organ regeneration, repair and disease treatment.
  • pluripotent stem cells can only be obtained from human embryos.
  • KLF4, OCT4, SOX2 and C-MYC four genes can be converted from normal somatic cells into pluripotent stem cells.
  • the pluripotent stem cells induced by this gene are called Induced Pluripotent Stem Cells (iPs).
  • iPs Induced Pluripotent Stem Cells
  • other somatic cells can also produce iPs.
  • iPs have been successfully cultured and differentiated into various somatic cells and different tissues such as myocardium, nerves, pancreas and bone. iPS technology is a major breakthrough in the field of stem cell research.
  • induced pluripotent stem cells must use retroviral vectors for genome integration. Due to the random nature of genomic integration, mutations can occur and can even cause cancer and genetic diseases. Finding an efficient way to promote the transformation of somatic cells into pluripotent stem cells is an urgent problem to be solved.
  • Pluripotent stem cells have also become a hot spot and focus of current stem cell research.
  • Japanese scientists exposed cells isolated from newborn mice to a weakly acidic environment to restore the cells to an undifferentiated state and have the potential to differentiate into any cell type, but this pure physical stimulation enables differentiated cells. The discovery of the phenomenon of returning to the pluripotent state has been greatly questioned.
  • a first aspect of the invention provides the use of an exogenous small RNA molecule for the preparation of a reagent or kit for promoting the conversion of somatic or multipotent stem cells into pluripotent stem cells.
  • the reagent comprises a transfection reagent.
  • the somatic cells include: epithelial cells, nerve cells, red blood cells, white fine Cells, platelets, phagocytic cells (phagocytic neutrophils, basophils, phagocytic granulocytes, etc.), B lymphocytes, effector B cells, memory B cells, T lymphocytes, memory T cells, effector T cells, Cardiomyocytes, smooth muscle cells, skeletal muscle cells, cardiomyocytes, osteoblasts, glial cells, hepatocytes, kidney cells, gland cells, endocrine cells (thyroid cells, thymocytes, islet B cells, islet cells, etc.).
  • epithelial cells nerve cells, red blood cells, white fine Cells, platelets, phagocytic cells (phagocytic neutrophils, basophils, phagocytic granulocytes, etc.)
  • B lymphocytes effector B cells
  • memory B cells T lymphocytes
  • memory T cells memory T cells
  • effector T cells Cardiomyocyte
  • the pluripotent stem cells include: hematopoietic stem cells, embryonic stem cells, bone marrow mesenchymal stem cells, neural stem cells, muscle stem cells, osteogenic stem cells, endoderm stem cells, retinal stem cells, pancreatic stem cells.
  • the small RNA molecule is selected from the group consisting of miRNA, siRNA- or a combination thereof.
  • the small RNA molecule comprises at least two or all of the miRNAs selected from the group consisting of (A): miR-432, miR-320, miR-27b, miR-103.
  • the small RNA molecule further comprises at least one or all of the miRNAs selected from the group consisting of: miR-423, miR-185, miR-378, miR-130b.
  • the small RNA molecule further comprises at least one or all of the miRNAs selected from the group (C): let-7g, miR-107.
  • the small RNA molecule comprises ⁇ 4 species, preferably ⁇ 6 species, more preferably ⁇ 7 species, selected from the group (A), the group (B), and/or the group (C). Optimally ⁇ 8 or all miRNAs.
  • the small RNA molecule comprises at least three or all of the miRNAs selected from the group (A). .
  • the small RNA molecule comprises at least four or all of the miRNAs selected from the group consisting of: hsa-miR-432-5p, hsa-miR-320a, hsa-miR-27b-3p, hsa-miR-103a-3p, hsa-miR-423-5p, hsa-miR-185-5p, hsa-miR-378a-3p, hsa-let-7g-5p, hsa-miR-130b-3p, or combination.
  • the small RNA molecule comprises at least 4 or all of the miRNAs selected from the group consisting of: bta-miR-107, bta-miR-432, bta-miR-320a, bta-miR- 27b, bta-miR-103, bta-miR-423-3p, bta-miR-423-5p, bta-miR-185, bta-miR-378, bta-let-7g, bta-miR-130b, or combination.
  • the miRNAs selected from the group consisting of: bta-miR-107, bta-miR-432, bta-miR-320a, bta-miR- 27b, bta-miR-103, bta-miR-423-3p, bta-miR-423-5p, bt
  • the small RNA molecule comprises at least 4 or all of the miRNAs selected from the group consisting of: mmu-miR-320-3p, mmu-miR-27b-3p, mmu-miR-103- 3p, mmu-miR-423-5p, mmu-miR-185-5p, mmu-let-7g-5p, mmu-miR-130b-3p, or a combination thereof.
  • the small RNA molecule comprises at least one or all of the miRNAs selected from the group consisting of: miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369- 3p, miR-369-5p, miR-21, miR-214, or a combination thereof.
  • the agent is also used to upregulate expression of a transcription factor in the cell.
  • the transcription factor is selected from the group consisting of Oct4, Sox2, c-Myc, n-Myc, Klf4, Nanog, Lin28, Zeb2, Mecp2, p21, wisp1, mbd2, p53, Jun, or combination.
  • the transcription factor is selected from the group consisting of Oct4, Sox2, c-Myc, n-Myc, Klf4, Nanog, Lin28, or a combination thereof.
  • the transcription factor is selected from the group consisting of Oct4, Sox2, c-Myc, Klf4, or a combination thereof.
  • the transcription factor is selected from the group consisting of Zeb2, Mecp2, p21, wisp1, mbd2, p53, Jun, or a combination thereof.
  • the transcription factor is selected from the group consisting of mbd2, p53, Jun, or a combination thereof.
  • the small RNA molecule is synthetic, recombinant, or naturally occurring.
  • the small RNA molecule is derived from humans, cows, pigs, sheep, rodents.
  • the small RNA molecule has a size of from 12 to 80 nt, preferably from 14 to 60 nt, more preferably from 15 to 30 nt, most preferably from 18 to 22 nt.
  • the mammal comprises a human or a non-human mammal.
  • the non-human mammal comprises a rodent such as a mouse or a rat.
  • the transfection reagent comprises or is a pH acidic regulator.
  • the transfection reagent comprises or is a microparticle preparation.
  • the microparticle formulation comprises microparticles and the small RNA molecule encapsulated in the microparticles.
  • a second aspect of the invention provides a method for promoting the transformation of somatic or multipotent stem cells into pluripotent stem cells in vitro, comprising the steps of:
  • a transfection system comprising: (a) a buffer and/or a culture; (b) somatic or multipotent stem cells; and (c) small RNA;
  • the transfection system is an aqueous system.
  • the cell is a mammalian cell.
  • the transfection conditions comprise an acidic pH treatment.
  • the acidic pH treatment refers to treatment at a pH of 2.5 to 6.0, preferably 2.8 to 5.5, more preferably 2.9 to 4.5, most preferably 3.0 to 4.0.
  • the acidic pH treatment is carried out for a period of from 0.5 minutes to 24 hours.
  • the treatment time of step (ii) is from 0.5 minutes to 24 hours, preferably from 1 minute to 6 hours, more preferably from 5 minutes to 2 hours, most preferably from 0.25 to 1 hour.
  • the temperature is 4 to 50 ° C, preferably 15 to 45 ° C, more preferably 25 to 40 ° C, most preferably 35 to 39 ° C. .
  • step (iii) comprising: transfecting the somatic cells obtained in the previous step Or a competent stem cell is transferred into a somatic cell of the exogenous nucleic acid, and induced to obtain a pluripotent stem cell.
  • the induced culture comprises the following conditions: plasmid vector mediated, calcium phosphate coprecipitation, electroporation, DEAE-dextran and polybrene, mechanical method ( Microinjection and gene gun), cationic liposome reagent transfection.
  • the method is non-therapeutic and non-diagnostic.
  • a third aspect of the invention provides a composition or combination for promoting the transformation of somatic or multipotent stem cells into pluripotent stem cells, comprising:
  • a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.
  • the small RNA molecule comprises at least two or all of the miRNAs selected from the group consisting of (A): miR-432, miR-320, miR-27b, miR-103.
  • a fourth aspect of the invention provides the use of a composition according to the third aspect of the invention for the preparation of a kit for promoting the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.
  • the kit is further for regulating pluripotent stem cell transcription factor expression.
  • a fifth aspect of the invention provides a kit, the kit comprising:
  • kits (ii) a label or instructions indicating that the kit is for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells.
  • the kit further comprises (iii) a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.
  • the pH adjusting agent is an acid
  • the small RNA molecule comprises from 3 to 100, preferably from 4 to 50, more preferably from 5 to 20.
  • the small RNA molecules are placed separately or in combination.
  • Figure 1 shows the relative content of osa-156a in wild type 293T cells treated with different pH.
  • Figure 2 shows a schematic view of Lipofectamine TM 2000 and acid-induced fluorescent FAM siRNA transfection results.
  • RNA can be more efficiently entered into eukaryotic cells.
  • small RNA can regulate a variety of pluripotent stem cell transcription factors and promote the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.
  • the method of the invention is convenient and efficient to operate, does not need to add an exogenous vector, has low toxicity to cells, high transfection rate, and the introduced nucleic acid molecule is more stable. On the basis of this, the present invention has been completed.
  • Small RNA small RNA
  • small ribonucleic acid refers to a small fragment of RNA of a length of twenty-five nucleotides; according to the widely accepted classification method proposed by Steven Buckingham in May 2003, small RNA (small RNAs) are non-coding RNAs other than transcribed RNA (including ribosomal RNA and transfer RNA), including microRNAs, short interfering RNAs (siRNA), small nucleolar RNA ( snoRNA) and small nuclear RNA (snRNA).
  • small RNA small ribonucleic acid
  • small RNA are non-coding RNAs other than transcribed RNA (including ribosomal RNA and transfer RNA), including microRNAs, short interfering RNAs (siRNA), small nucleolar RNA ( snoRNA) and small nuclear RNA (snRNA).
  • microRNAs are a class of single-stranded small RNA molecules of about 19-23 nucleotides in length, located in the non-coding region of the genome, which are highly conserved in evolution and can inhibit the translation process of target genes.
  • Gene expression is regulated and closely related to many normal physiological activities of animals, such as biological individual development, tissue differentiation, apoptosis and energy metabolism, and is also closely related to the occurrence and development of many diseases.
  • Existing studies have also confirmed that plant miRNAs can also enter the animal through food intake and participate in regulatory activities.
  • RNA interference is a way of post-transcriptional regulation of genes.
  • siRNA can specifically recognize its target gene and recruit a protein complex called RNA induced silencing complex (RISC).
  • RISC contains ribonuclease and the like, and can specifically and efficiently inhibit gene expression by targeting cleavage of homologous mRNA. Since RNA interference technology can specifically eliminate or turn off the expression of specific genes, this technology has been widely used in biomedical experimental research and treatment of various diseases.
  • cell microparticles In the present invention, the terms “cell microparticles”, “microparticles”, and “microvesicles” are used interchangeably.
  • Cell microparticles are membrane corpuscles with diameters between 30 and 1000 nm that are secreted by cells in the body under normal and pathological conditions. Natural small particles, including extracellular bodies, containing cell contents, surrounded by cell membrane-like membrane structures. (exosome) and shedding vesicle (shedding vesicle). Both in vivo and in vitro experiments have demonstrated that cell microparticles can be secreted by a variety of cells such as red blood cells, B cells, T cells, dendritic cells, mast cells, epithelial cells, and tumor cells. The cells encapsulate specific biologically active molecules such as proteins, mRNAs, etc.
  • this cell-microparticle-mediated intercellular communication plays a very important role in some physiological and pathological processes.
  • the microparticles of the present invention include natural biovesicles having a lipid bilayer membrane secreted by cells, each having a size between 10 and 500 nm, including exosome, shedding vesicle, and A special name for shedding vesicles secreted by various cells.
  • the present invention provides a kit for promoting the transformation of somatic cells and/or primary competent stem cells into pluripotent stem cells, the kit comprising:
  • RNA molecule preferably in the form of a microparticle preparation or a first transfection reagent located in the container;
  • kits (ii) a label or instructions indicating that the kit is for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells.
  • the kit further comprises (iii) a pH acidity regulator (second transfection reagent) for providing an acidic pH having a pH of from 2.5 to 6.0.
  • a pH acidity regulator second transfection reagent
  • the pH adjusting agent is an acid
  • the small RNA molecule comprises from 3 to 100, preferably from 4 to 50, more preferably from 5 to 20.
  • the small RNA molecules are placed separately or in combination.
  • the first transfection reagent and the second transfection reagent are separate reagents or two-in-one or the same transfection reagent.
  • the method of the present invention increases the efficiency of small RNA transfected cells by adjusting the cell culture environment to be acidic.
  • the method of the present invention has low toxicity, high transfection rate, and high stability of introduced small RNA molecules.
  • RNA molecules can regulate at least three pluripotent stem cell transcription factors and promote the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.
  • RNA molecules can regulate at least three pluripotent stem cell transcription factors and promote the transformation of somatic cells into pluripotent stem cells under the guidance of cell microparticles.
  • the experimental method is as follows:
  • the container and operating equipment were sterilized, and polylysine was purchased from sigma.
  • the 12-well plate was taken out and the polylysine was aspirated with a pipette. Change the straw, add PBS, 3-4 drops per well, rinse three times. Repeat the above steps three times.
  • the 12-well plate was placed in the incubator for use.
  • DMEM medium (10% FBS, 1% double antibody), cultured at 37 ° C, 5% CO 2 for 24 h.
  • the DMEM culture solution (2% FBS, 1% double antibody) used in the experiment was prepared, and the pH was determined to be 7.65, and then the culture solution was adjusted to a pH of 3.15 with hydrochloric acid for use.
  • the transferred nucleic acid was selected from the exogenously synthesized rice single-stranded microRNA, osa-miR156a (synthesized by Gima, sequence UGACAGAAGAGAGUGAGCAC (SEQ ID NO.: 12)), and the rice osa-miR156a differed greatly from the transferred human 293T cells. It can effectively avoid the interference of own microRNA in animal cells and reduce the possibility of error in the experimental results.
  • osa-miR156a was added to a final concentration of 40 pmol/ml.
  • the 12-well plate with 293T cells was taken out from the incubator, the original culture solution was removed, and the above two pH-valued culture solutions supplemented with exogenous miRNA were added to the two 12-well plates, and incubated at 37 ° C for 0.5 h.
  • RNase digest was prepared: RNase A (purchased from Thermo) was added to DMEM medium (2% FBS, 1% double antibody) in a volume ratio of 2 ⁇ l/ml, ie >100 u/ml.
  • Real-time fluorescent quantitative PCR technology is to add a fluorescent group in the reaction system of PCR, and accumulate the fluorescent signal to monitor the whole PCR process in real time.
  • a gene-specific reverse primer containing the same stem-loop structure was designed for each miRNA, and the specific cDNA was reversed, and finally PCR reaction was carried out.
  • RNA and other reagents required for the reaction are formulated into a reverse transcription reaction system, and the reaction is carried out under the following conditions, and the reverse transcription reaction conditions are as follows:
  • Step 1 16 ° C 30 min
  • Step 2 42 ° C 30 min
  • Step 3 85 ° C 5 min
  • Step 4 Place at 4 ° C
  • Step 1 95 ° C 5 min
  • Step 2 95 ° C 15s
  • Step 3 60 ° C 1 min
  • Step 2 - Step 3 50 cycles
  • the processing of the data uses a relative comparison method and is also considered to be the ⁇ Ct method.
  • the expression level of the acid-treated exogenous microRNA relative to the control wild-type plant can be expressed by Equation 2 - ⁇ CT .
  • Figure 1 shows the relative values of two pH media transferred into the cells osa-miR156a, pH 7.65 control group The relative value of 1 showed that the exogenous microRNA content in the cells after pH3.15 transformation was significantly higher than that in the pH7.65 control group.
  • the experimental method is as follows:
  • Lipofectamine TM 2000 available from Invitrogen, FAM siRNA available from Zimmer, FAM siRNA with a fluorophore, after transfection by fluorescence microscopy, confocal microscopy or flow cytometry for detection.
  • the operation steps are carried out according to the instructions. The approximate steps are as follows.
  • the cells were inoculated to a 12-well plate one day before transfection, and 400 ⁇ l of DMEM medium containing 10% calf serum (containing no double antibody) was added to each well, and the cell density should be 90% when transfected.
  • the diluted Lipofectamine TM 2000 siRNA and mixed gently and incubated at room temperature for 20 minutes to form a complex.
  • FAM is a green fluorescent group excited by blue light with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.
  • Lipofectamine TM 2000 can be detected after 6 hours of transfection, and the cell treatment process before detection needs to be protected from light.
  • the light path can be aligned with the hole that is not transfected with FMA siRNA, and the focal length is adjusted to turn on the excitation light. The observation time should not be too long to avoid the fluorescence being quenched.
  • FAM green fluorescence which is dispersed in the cytoplasm.
  • Figure 2 is a schematic diagram showing the results of fluorescence induction of Lipofectamine TM 2000 and acid-induced transfection of FAM siRNA, showing that the transfection efficiency is comparable, indicating that the acid-treated group also has the ability to efficiently transfect siRNA.
  • Example 3 fetal bovine serum is rich in miRNA
  • solexa sequencing The expression profile of fetal bovine serum was detected by solexa sequencing.
  • the specific steps of solexa sequencing include:
  • the purified DNA was directly used for cluster generation and subjected to sequencing analysis using an Illumina Genome Analyzer.
  • Bioinformatics methods are used to predict miRNAs that regulate pluripotent stem cells.
  • microRNAs may be involved in the regulation of transcription factors of pluripotent stem cells.
  • miRNAs that promote iPSC include: miR-291-3p, miR-294, miR-295, miR-302b, miR-372, miR-200c, miR-302s, miR-369s, miR-302a-3p, miR- 302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214; miRNAs that promote Wisp1 include: miR-486; miRNAs that promote p21 include: miR-423- 5p; miRNAs that promote AOF1 include: miR-320a, miR-130; miRNAs that promote MECP2-1 include: miR-185, miR-432, miR-107, miR-103, miR-378, miR-27b, miR- 423-3p, miR-877, let-7g.
  • the transcription factors regulating iPSC include: Oct4, MET, Sox2, c-Myc, n-Myc, Klf4, Nanog, Lin28, AOF1, AOF2, MECP1-p66, MECP2, MBD2, p21, p53, Wisp1, ZEB2, Jun.
  • miRNAs rich in fetal bovine serum can inhibit genes such as p21 and p53 that can prevent somatic cells from becoming pluripotent stem cells, suggesting that miRNAs rich in fetal bovine serum may induce somatic cells to become Multifunctional stem cells.
  • the pluripotent stem cells are efficiently prepared by stimulating exogenous small RNA to induce the transformation of somatic cells into pluripotent stem cells by acid-mediated, cell microparticle-mediated, plasmid-mediated, and the like.
  • acid such as phosphoric acid, sulfuric acid, or acetic acid
  • Another part of the cells was colonized in a Petri dish without cell adhesive, and cultured in a DMEM/F12 medium supplemented with 1000 U of Leukemia Inhibitor Factor (LIF) and 2% B27, and some cells were separated every day for 1-10 days.
  • LIF Leukemia Inhibitor Factor
  • the expression of pluripotent stem cell-specific genes such as OCT4 was examined, and the induction rate of pluripotent stem cells was observed and counted.
  • CD45 + hematopoietic stem cells were isolated from mice by flow cytometry, and 10 6 CD45 + hematopoietic stem cells were transferred to DMEM/F12 medium containing 1000 U of Leukemia Inhibitor Factor (LIF) and 2% B27, followed by utilization.
  • LIF Leukemia Inhibitor Factor
  • Liposomes are loaded into cells by plasmids that are capable of overexpressing miRNAs in fetal calf serum that may induce somatic cells to become pluripotent stem cells, followed by isolation of some cells such as OCT4 every day for 1-10 days. The expression of functional stem cell-specific genes was observed and the induction rate of pluripotent stem cells was observed and counted.
  • the plasmid expression vector of the miRNA of pluripotent stem cells is not particularly limited, and includes an expression vector which is commercially available or prepared by a conventional method.
  • Representative examples include, but are not limited to, pcDNATM6.2-GW/miR, pcDNA3, pMIR-REPORT miRNA, pAdTrack-CMV, pCAMBIA3101+pUC-35S, pCMVp-NEO-BAN, pBI121, pBin438, pCAMBIA1301, pSV2 , CMV4 expression vectors, pmiR-RB-Report TM, pshOK-basic, mmu-mir 300-399miRNASelect TM, pshRNA-copGFP Lentivector, GV317, GV309, GV253, GV250, GV249, GV234, GV233, GV232, GV201, GV159 , or other GV series expression vector.
  • plasmid-mediated exogenous small RNA can regulate the expression of pluripotent stem cell-specific genes and significantly increase the induction rate of pluripotent stem cells.
  • Microvesicles (MV) in fetal bovine serum were separated by ultracentrifugation (100000 g, 70 min).
  • the miRNA in the MV was then detected using qRT-PCR technology.
  • the specific steps of qRT-PCR include:
  • the primary cells were cultured to a suitable density, and microvesicles (MV) in fetal bovine serum were added to the medium, incubated for 10-30 min under cell culture conditions, and cultured continuously and observed until stem cells were confirmed to be present.
  • MV microvesicles
  • the cells were isolated to detect the expression of pluripotent stem cell-specific genes such as OCT4, and the induction rate of pluripotent stem cells was observed and counted.
  • Cellular microparticles mediated stimulation of fetal bovine serum miRNA promotes the transformation of somatic cells into pluripotent stem cells.
  • the invention also increases the expression level of exogenous small RNA or stimulates small RNA to promote the transformation of somatic cells into pluripotent stem cells, and further comprises calcium phosphate coprecipitation, electroporation, DEAE-dextran and polybrene, mechanical method (microinjection) And gene gun), cationic liposome reagent transfection method, and the like.
  • Acid-stimulated exogenous siRNA is introduced into somatic cells to efficiently prepare pluripotent stem cells
  • Exogenous siRNA was selected for subsequent experimental studies.
  • the sequence information is shown in Table 2. The first sequence of each gene was selected for the experiment.
  • Transfection is used as a transfection group. RNAiMAX transfection.
  • Lipofectamine TM RNAiMAX were purchased from Invitrogen, FAM siRNA available from Zimmer, FAM siRNA with a fluorophore, after transfection by fluorescence microscopy, confocal microscopy or flow cytometry for detection. The operation steps are carried out in accordance with the instructions, and the steps are as described in Embodiment 2.
  • Group T (initial cell density 2 ⁇ 10 4 ) See Table 4.
  • OKS represents a positive control group, and normal induction is not treated
  • DR represents a negative control group, only infected with DsRed virus for estimating infection efficiency, and does not induce positive clones;
  • T-3si represents the siRNA concentration of jun, mbd2 and p53 genes transfected to 10nM/25nM
  • T+Jun-si represents a siRNA concentration of 10nM/25Nm for transfection-induced jun gene
  • Group A was an acid-treated group (the cells were relatively dying due to acid treatment, and the number of cells was relatively high when plating), the acid pH was 3.5, and the acid treatment time was 15 min.
  • Group A (Initial cell density 5.5 ⁇ 10 4 ) See Table 5.
  • NA-3si represents acid-free treatment, only the above three siRNAs are added;
  • A-3si represents acid-induced jun, mbd2, p53 three genes siRNA concentration of 10nM/25nM;
  • A+Jun-si represents the acid-induced jun gene siRNA concentration of 10nM/25nM
  • the sequence information is the same as the T group.
  • Both group T and group A were treated every two days from the next day to the eighth day. Positive clones were counted on the ninth day, and the number of induced stem cell clones that emitted green fluorescence was manually counted under a fluorescence microscope.
  • the number of positive clones was significantly increased by siRNA targeting jun, mbd2, and p53, and the number of positive clones transfected with siRNA specifically inhibiting jun gene. increase.
  • the number of positive clones of siRNA, mbd2, p53, and the number of positive clones increased; the number of positive clones of acid-induced jun gene increased. It is indicated that by directly adjusting the pH of the medium to pH 3.5, siRNAs of jun, mbd2 and p53 genes can be induced to enter cells, thereby inhibiting the expression of these three genes and improving the efficiency of inducing stem cells; The medium pH is temporarily adjusted to pH 3.5 for induction, which can promote the jun gene siRNA to enter the cell, exert the effect of inhibiting the expression of the jun gene, and improve the efficiency of inducing stem cells.
  • the T group was the transfection group
  • the group A was the acid treatment group
  • the acid treatment time was 15 min;
  • OKS positive control group, normal induction is not treated
  • P53-si siRNA 10nM/25nM transfected/acid-induced p53 gene
  • the number of positive clones that induced siRNA that specifically inhibits the p53 gene was increased as compared with the control group (OKS). It is indicated that siRNA transfected with p53 gene by liposome can inhibit the expression of p53 gene and promote the efficiency of inducing stem cells.
  • the number of positive clones of siRNA-induced p53 gene siRNA increased compared with the control group (OKSA, NA+3si), indicating that induction by temporarily adjusting the pH of the medium to pH 3.5 can promote The siRNA of the p53 gene enters the cell, exerts an effect of inhibiting the expression of the p53 gene, and increases the efficiency of inducing stem cells.
  • siRNAs include: Zeb2 siRNA, Mecp2 siRNA, p21 siRNA, and wisp1 siRNA.
  • Table 10 The sequence information of other siRNAs is shown in Table 10.
  • Acid-stimulated exogenous miRNAs are introduced into somatic cells to efficiently prepare pluripotent stem cells
  • miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214 were selected for group experiments.
  • Each miRNA was transfected with liposome and acid-treated, and the concentration of each miRNA was 10 nM/25 nM.
  • the sequence information of miRNAs is shown in Table 11.
  • the T group was the transfection group
  • the group A was the acid treatment group
  • the acid treatment time was 15 min;
  • Both group T and group A were treated every two days from the next day to the eighth day. Positive clone counts were performed on the ninth day.
  • transfection-inducible specific miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR- were compared with the control group (OKS).
  • the number of positive clones of 369-5p, miR-21 and miR-214 increased.
  • the use of liposome to transfect miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214 for the induction of stem cells Efficiency has a positive effect.
  • kits for promoting transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells comprising:
  • a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.
  • the small RNA molecule comprises from 3 to 100, preferably from 4 to 50, more preferably from 5 to 20, preferably one or more combinations of the miRNAs shown in Table 1.
  • the exogenous small RNA molecules are placed separately or in combination.
  • the above kit can be used to regulate the expression of pluripotent stem cell transcription factors; and/or to promote the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Transplantation (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de préparation de cellules souches pluripotentes de type petit ARN exogène. Une utilisation du petit ARN exogène pour préparer un kit de réactifs destiné à faciliter la conversion de cellules somatiques ou de cellules souches spécialisées en cellules souches pluripotentes est en outre décrite.
PCT/CN2017/071140 2016-01-15 2017-01-13 Procédé de préparation de cellules souches pluripotentes de type petit arn exogène et son utilisation Ceased WO2017121386A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610028091.9A CN106978398A (zh) 2016-01-15 2016-01-15 一种外源性小rna制备多能干细胞的方法及其用途
CN201610028091.9 2016-01-15

Publications (1)

Publication Number Publication Date
WO2017121386A1 true WO2017121386A1 (fr) 2017-07-20

Family

ID=59310809

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/071140 Ceased WO2017121386A1 (fr) 2016-01-15 2017-01-13 Procédé de préparation de cellules souches pluripotentes de type petit arn exogène et son utilisation

Country Status (2)

Country Link
CN (1) CN106978398A (fr)
WO (1) WO2017121386A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102712904A (zh) * 2009-11-11 2012-10-03 桑福德—伯恩哈姆医学研究协会 用于产生和调节iPS细胞的方法及其组合物
WO2013188679A1 (fr) * 2012-06-13 2013-12-19 Stemgent, Inc. Méthodes de préparation de cellules souches pluripotentes
WO2015073625A2 (fr) * 2013-11-15 2015-05-21 The Mclean Hospital Corporation Reprogrammation synergique non intégrative du génome par des microarn et des facteurs de transcription

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102712904A (zh) * 2009-11-11 2012-10-03 桑福德—伯恩哈姆医学研究协会 用于产生和调节iPS细胞的方法及其组合物
WO2013188679A1 (fr) * 2012-06-13 2013-12-19 Stemgent, Inc. Méthodes de préparation de cellules souches pluripotentes
WO2015073625A2 (fr) * 2013-11-15 2015-05-21 The Mclean Hospital Corporation Reprogrammation synergique non intégrative du génome par des microarn et des facteurs de transcription

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MORRISEY EE: "Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency", CELL STEM CELL, vol. 8, no. 4, 8 April 2011 (2011-04-08), pages 376 - 388, XP055547506, ISSN: 1934-5909, DOI: doi:10.1016/j.stem.2011.03.001 *
WANG, ET AL.: "Role of microRNA in induced pluripotent stem cell", HEREDITAS, vol. 34, no. 12, 31 December 2012 (2012-12-31), pages 1545 - 1550, ISSN: 0253-9772 *

Also Published As

Publication number Publication date
CN106978398A (zh) 2017-07-25

Similar Documents

Publication Publication Date Title
US9353352B2 (en) Embryonic stem cell specific microRNAs promote induced pluripotency
JP5840119B2 (ja) 誘導多能性幹細胞の製造方法
CN107002031B (zh) 选择分化细胞的方法
Bem et al. Transient microRNA expression enhances myogenic potential of mouse embryonic stem cells
Lei et al. Cultured pluripotent planarian stem cells retain potency and express proteins from exogenously introduced mRNAs
CN114807135A (zh) 一种牛骨骼肌发育相关环状RNA circMYH8的生物学鉴定方法及应用
US9163234B2 (en) Culture method
US11624067B2 (en) In-vitro induction of adult stem cell expansion and derivation
WO2017121386A1 (fr) Procédé de préparation de cellules souches pluripotentes de type petit arn exogène et son utilisation
US20230348938A1 (en) Methods and apparatuses for biomolecule delivery to primary human hematopoietic stem cells using nanostraws
US20210171949A1 (en) Targeting Mirnas for Exosomal Delivery or Cellular Retention
US20130273655A1 (en) ENGRAFTMENT POTENTIAL OF EMBRYONIC STEM CELLS AND PLURIPOTENT STEM CELLS USING MICRO RNAs
CN119120480B (zh) 一种用于华西牛育种调节的环状rna及其应用
CN120249284B (zh) 一种lncRNA及在山羊卵巢颗粒细胞中的应用
US20190256830A1 (en) Method for cell-specifically controlling nuclease
CN115806982B (zh) 一种抑制牛CD44基因表达的shRNA序列及应用
CN112210554A (zh) 源于鹿茸由CNBP介导的microRNA及其应用
Kumari Understanding the Internalization of Follicular Fluid Extracellular MiRNAs in Buffalo Granulosa Cells
Mikolajczyk IDENTIFYING THE FUNCTIONS OF SOLUTE CARRIER PROTEINS
Krems et al. Marlene Geyer
Stuart Drosha Expression and Microprocessor Activity in Maturing Bovine Oocytes and Cultured Granulosa Cells
CN106978444B (zh) 一种向细胞中导入核酸的方法
CN115058419A (zh) 与猪骨骼肌卫星细胞增殖相关的circRNA及其应用
Barros A long non-coding RNA regulating cellular reprogramming
CN111334507A (zh) 绵羊Lrh-1短发夹RNA及其干扰载体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17738199

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17738199

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 01/07/2019)

122 Ep: pct application non-entry in european phase

Ref document number: 17738199

Country of ref document: EP

Kind code of ref document: A1