WO2015178496A1 - Procédé de production de cellules précurseurs des poumons - Google Patents

Procédé de production de cellules précurseurs des poumons Download PDF

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WO2015178496A1
WO2015178496A1 PCT/JP2015/064838 JP2015064838W WO2015178496A1 WO 2015178496 A1 WO2015178496 A1 WO 2015178496A1 JP 2015064838 W JP2015064838 W JP 2015064838W WO 2015178496 A1 WO2015178496 A1 WO 2015178496A1
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cells
family
lung
transcription factors
sox21
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直登 二宮
俊喜 横須賀
浅島　誠
晃 栗崎
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国立研究開発法人産業技術総合研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N5/0688—Cells from the lungs or the respiratory tract
    • C12N5/0689—Stem cells; Progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • 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
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • the present invention relates to a method for specifically producing lung progenitor cells.
  • tissue cells or precursor cells In order to perform regenerative medicine safely and reliably, it is necessary to stably prepare tissue cells or precursor cells to be treated.
  • chronic obstructive pulmonary disease which is closely related to smoking in lung tissue, is a chronic disease in which the alveoli are irreversibly destroyed.
  • symptoms using bronchodilators, expectorants, antitussives, etc. Although it is possible to slow the progression of lung cancer, there is no way to restore lung function at the tissue level.
  • pluripotent stem cells such as iPS cells has progressed, but only the cells of the target tissue are selectively differentiated and suitable for transplantation sites. Development of technology to prepare cell populations is ongoing.
  • Non-patent Documents 10 and 12 a method for differentiating lung progenitor cells.
  • a technique for differentiating stem cells into endoderm cells and further differentiating into tissue cells constituting the lung is well known.
  • a method of culturing ES cells in vitro and aggregating them to form a cell mass called an embryoid body, or a method of inducing differentiation by planar culture to form endoderm cells has been widely used (non-contained).
  • Patent Document 9 and Patent Document 3 a method for efficiently producing endoderm cells by adding a high concentration of activin or the like is used (Non-patent Documents 10 and 12).
  • Non-patent Document 1 a production method for controlling differentiation based on endoderm cells differentiated from iPS cells has been studied. Furthermore, in order to induce differentiation of endoderm cells into anterior endoderm, a BMP signal inhibitor such as Dorsomorphin and SB431542 and a TGF-beta / activin signal inhibitor are added and cultured in a medium containing BMP and FGF. It is also possible to produce lung progenitor cells by the culture method (Non-patent Documents 11 and 16). However, the differentiation method using ES / iPS cells has not yet established a method for eliminating tumorigenesis such as teratoma, and many steps have been taken after producing very undifferentiated iPS cells.
  • Non-Patent Document 2 In order to re-differentiate cells into target tissue cells under strict control, it takes a long time and labor to prepare cells, and there are still a number of problems such as contamination of cells other than the target cells. Therefore, clinical application using cells differentiated from iPS cells requires time especially for verification of safety, reliability of differentiated cells, stability of differentiation efficiency, and the like. On the other hand, cell therapy methods that do not use ES / iPS cells are being studied for the purpose of avoiding the risk of tumorigenesis. For example, it has been reported that transplanting mesenchymal stem cells promotes normalization of tissues by suppressing excessive inflammatory reactions and promoting angiogenesis mainly by paracrine action via secretory factors. (Non-Patent Document 2).
  • Non-Patent Document 3 A practical method has not been established yet. Further, in recent years, new methods have been developed as methods for producing target cells without using ES / iPS cells.
  • An iPS cell is a method in which undifferentiated pluripotent stem cells very similar to ES cells are transformed from differentiated fibroblasts by introducing four genes, Oct4, Sox2, Klf4, and cMyc.
  • Non-Patent Document 4 Although it was made (Non-Patent Document 4), by selecting and introducing an appropriate gene for each target differentiated cell, the nerve is directly transmitted from the fibroblast without going through the iPS cell (Non-Patent Document 5). ), Liver (Non-Patent Documents 6 and 7), cartilage (Non-Patent Document 8) and the like have been reported to be prepared, and these new differentiation methods are called “direct reprogramming methods”. In this method, since iPS cell-like pluripotent stem cells do not pass through the process of introducing a gene to change the fate of cells, the cells are safe without risk of canceration such as teratomas. Expected. However, there has never been such a direct differentiation method for lung tissue cells. In order to increase the QOL of patients with chronic obstructive pulmonary disease and patients after lung resection, a safe method for producing lung cells is desired, and development of a direct differentiation method for lung tissue cells has been urgently required.
  • the present invention intends to provide a “direct reprogramming method” for lung cells, in which lung precursor cells are directly produced from somatic cells, as a new method for producing lung precursor cells. .
  • the present inventors focused on transcription factors that are specifically expressed in the developmental process of the lung, and literature information on genes that are specifically expressed at that time in the fetal progenitor tissue, where lung tissue begins to form. And database information were comprehensively examined.
  • genes that are highly expressed in the progenitor cells that are the source of lung cells that will differentiate into various cells that will form lung tissue in the future are selected from among 2000 or more types of transcription factors, particularly Foxa2, Gata6, Four types of transcription factors, Sox21 and NMyc, and five types of transcription factors including Irx3 were selected.
  • fibroblasts were successfully transformed into lung progenitor cells by expressing the 4 transcription factor or 5 transcription factor in fibroblasts.
  • Lung progenitor cells directly differentiated with these five transcription factors plus Irx3 transcription factor have the ability to differentiate into lung tissue cells, including alveolar epithelial type I cells, by in vitro culture. It could be confirmed. Based on these findings, the present invention relating to a method for producing lung progenitor cells capable of differentiating into alveolar epithelial cells and the like, which has never been achieved, has been completed. Furthermore, in the case of human cells, it was found that by adding Sox9 in addition to the above five factors, it can be efficiently differentiated directly into lung progenitor cells. That is, the present invention is as follows.
  • (a) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd8, Fox3, Foxd8 , Foxf1, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox1, Fox3
  • a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6
  • (C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21
  • [6] The reagent according to any one of [1] to [5], which contains at least Foxa2, Gata6, Sox21, NMyc and Irx3 transcription factors.
  • (c) The reagent according to any one of [1] to [6] above, which comprises two or more transcription factors including Sox9 from the Sox family.
  • [8] The reagent according to [6] or [7] above, which contains at least the transcription factors of Foxa2, Gata6, Sox21, NMyc, Irx3 and Sox9.
  • a method for producing lung progenitor cells expressing Nkx2.1 from somatic cells which comprises the following steps (1) to (3): (1) a step of selecting one or more transcription factors from the following transcription factor families (a) to (d): (A) Fox family, (B) Gata family, (C) Sox family, and (d) Myc family, (2) a step of causing a group of transcription factors including the four types of transcription factors to act in a target somatic cell; (3) A step of confirming that the Nkx2.1 gene is expressed in the cell after the action of the transcription factor. [10-1] The method according to [9] above, wherein the step (2) includes a step of introducing a nucleic acid encoding the transcription factor into a target somatic cell.
  • a transcriptional gene can be introduced into a somatic cell isolated by a viral vector or directly into a somatic cell of a lung tissue using a viral vector or a gene introduction agent (Laube BL, Transl Respir Med). .2014 2, 3).
  • the transcription factor gene is not limited to DNA, and mRNA of the transcription factor can be directly introduced into human lung tissue using an RNA introduction reagent or an RNA virus vector such as Sendai virus.
  • the step (2) is administered in the step of adding the transcription factor to the target somatic cell culture medium, or in the lung tissue containing the target somatic cell alone or with a pharmacologically acceptable carrier
  • (a) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd3, Foxd8 , Foxe3, Foxf1, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn5, Foxn1, Fox3, Foxn3 Including some transcription factors, (B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6; (C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18
  • the method further includes (e) selecting one or more transcription factors from transcription factors in the transcription factor family belonging to the Irx family, and the transcription factor is also included in the step (2).
  • the transcription factor selected from (e) Irx family comprises any one of Irx1, Irx2, Irx3, Irx4, Irx5, and Irx6.
  • the somatic cell is a human-derived somatic cell, and in step (1), the method includes (c) selecting two or more transcription factors including Sox9 from the Sox family, and in step (2), the transcription factor The method according to any one of [14] to [16] above, wherein a group of transcription factors including 6 types of transcription factors including the above is allowed to act.
  • [19] Applying the method according to any one of [11] to [18] to isolated somatic cells to produce lung progenitor cells expressing Nkx2.1, and then the obtained lung A method for producing differentiated lung tissue cells, comprising culturing precursor cells in a medium for inducing differentiation into desired lung tissue cells.
  • the differentiation-inducing medium is a medium obtained by adding a lung tissue cell differentiation-inducing factor to a low-serum or serum-free medium for epithelial cells.
  • a screening method for an agent for inducing differentiation from a lung progenitor cell expressing Nkx2.1 into a desired lung tissue cell comprising the following steps (1) to (4): Method; (1) A step of producing lung progenitor cells expressing Nkx2.1 by applying the method according to any of [11] to [18] to isolated somatic cells, (2) culturing a part of the obtained lung progenitor cells in a medium for inducing differentiation into desired lung tissue cells, (3) Another part of lung progenitor cells is cultured in a medium obtained by adding a test substance to the differentiation-inducing medium used in step (2), or after introducing the test substance into lung progenitor cells Culturing in the differentiation-inducing medium used in (2), (4) When the expression level of a desired lung tissue cell-specific marker of the lung progenitor cells used in step (2) and step (3) is measured after a certain period of time and compared, and the latter expression level is high, A step of evaluating and selecting a test substance as a candidate substance for
  • a screening kit for an agent for promoting differentiation from a lung progenitor cell expressing Nkx2.1 into a desired lung tissue cell which is prepared by the method according to any one of [11] to [18] A kit comprising lung progenitor cells.
  • a composition for use in a method of supplementing lung progenitor cells or lung tissue cells expressing Nkx2.1 comprises one or more transcription factors selected from the following transcription factor families (a) to (d): Said method induces direct differentiation of somatic cells into lung progenitor cells expressing Nkx2.1; (A) Fox family, (B) Gata family, (C) Sox family, and (d) Myc family.
  • the transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd8, Fox3, Foxd8, Fox3 , Foxf1, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox1, Fox3 Including transcription factors, (B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6; (C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox18, Sox
  • lung progenitor cells can be produced directly from somatic cells. Since lung progenitor cells produced by this method have the ability to differentiate into various cells of lung tissue such as alveolar epithelial cells, alveolar epithelial cells differentiated in vitro can be used for toxicity tests on lung tissue. It can be applied to cell kits. It can also be used as a screening cell for a differentiation agent when differentiating lung progenitor cells into more mature lung tissue cells. Furthermore, by producing lung progenitor cells from cells retaining a causative gene of a disease associated with a gene mutation such as lung cancer, it can also be used as a cell kit for screening therapeutic drug candidates for diseases associated with a gene mutation such as lung cancer.
  • f Expression of a lung progenitor cell-specific transcription factor Nkx2.1 gene when narrowing down essential factors for producing lung progenitor cells by combining transcription factors obtained by removing one factor from thirteen transcription factors.
  • f Expression of E-cadherin in sample e. g. From the 6 factors narrowed down by ef, the essential factors were again identified using the expression of the Nkx2.1 gene as an index.
  • h Expression of E-cadherin in g samples.
  • i The essential factors were further identified from the 5 factors narrowed down by the above gh, and the expression of Nkx2.1 gene was used as an index.
  • j Expression of E-cadherin in i samples.
  • lung progenitor cells made directly from mouse embryonic dorsal epidermis fibroblasts are lung-specific progenitor cells.
  • Dorsal skin dorsal epidermis fibroblasts of mouse fetus
  • Limb skin mouse embryo limb-derived fibroblasts
  • MDF adult mouse head epidermis fibroblasts.
  • lung progenitor cells directly produced from mouse embryonic limb-derived fibroblasts are lung-specific progenitor cells.
  • d Specificity of expression of lung progenitor cell specific marker protein.
  • the lung progenitor cells directly induced to differentiate for 15 days were further cultured for up to 51 days, and then the lung cell differentiation ability was verified by immunofluorescence staining.
  • Mucus secreting cell marker Moc5AC
  • alveolar epithelial type I cells AQP5
  • Lung progenitor cells prepared from mouse fibroblasts spontaneously form a three-dimensional structure.
  • Specific Transcription Factor for Producing Lung Progenitor Cells of the Present Invention include at least a Fox family transcription factor, a Gata family transcription factor, a Sox family transcription factor, and a Myc family. It includes each transcription factor gene belonging to the transcription factor or a protein as its expression product, and preferably further contains an Irx family transcription factor or a protein as its expression product. In particular, when producing human lung progenitor cells, it is preferable that two types of transcription factors or their expression products are included from the Sox family.
  • the Fox family transcription factors include Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd2, Fox1, Fox1, Fox1, Fox1, Fox1 , Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Foxo1, Foxo3a, Foxo4, Foxp1, Fox3, Foxp2, Foxp1, Fox3
  • the domain is a highly conserved Foxa1, Foxa2, Foxa3 transcription factor.
  • the amino acid sequences of these transcription factors and the base sequences of the genes thereof can be obtained from public databases.
  • GenBank (NCBI) human-Foxa2 (Protein: NP — 068556) and mouse-Foxa2 (Protein: NP — 034576) can be used.
  • Gata family transcription factor include Gata1, Gata2, Gata3, Gata4, Gata5, and Gata6, and preferably Gata6, Gata5, Gata4 transcription factors whose functional domains are highly conserved, such as human-Gata6.
  • NP_005248 mouse-Gata6
  • NP_034388 can be used.
  • the functional domains of which are highly conserved Sox21, Sox3, Sox1, Sox15, Sox2 and Sox9 transcription factors such as human-Sox21 (Protein: NP_009015), mouse-Sox21 (Protein: NP_808421) and human- Sox9 (Protein: NP_000337) can be used.
  • Examples of the Myc family transcription factor include cMyc, LMyc, and NMyc whose functional domains are highly conserved, and are preferably NMyc transcription factors such as humanNMycgene (mRNA: NM_005378), mouseNMyc gene (mRNA: NM_008709). Can be used. In particular, when producing human lung progenitor cells, it is preferable to have two types of transcription factors including Sox9.
  • examples of the Irx family transcription factor include Irx1, Irx2, Irx3, Irx4, Irx5 and Irx6, preferably Irx3, Irx1, Irx2, Irx4 and Irx6 transcription factors whose functional domains are highly conserved.
  • human-Irx3 (Protein: NP — 077312) and mouse-Irx3 (Protein: NP — 032419) can be used.
  • These transcription factors ie, Fox family transcription factors, Gata family transcription factors, Sox family transcription factors, and Myc family transcription factors, as well as transcription factors comprising Irx family transcription factors, preferably Foxa2, Gata6, Sox21 and NMyc transcription Somatic cells such as fibroblasts by gene transfer of factors, more preferably Foxa2, Gata6, Irx3, Sox21 and NMyc transcription factors, particularly preferably Transcription factors including Foxa2, Gata6, Irx3, Sox21, NMyc and Sox9 transcription factors By expressing in (tissue cells) and restructuring the network, somatic cells can be directly differentiated into lung progenitor cells.
  • each gene is typically expressed simultaneously by introducing each gene into a somatic cell such as a fibroblast with or without a viral or non-viral vector.
  • a somatic cell such as a fibroblast with or without a viral or non-viral vector.
  • any one or more of these 4, 5 or 6 types of transcription factors may be introduced into a somatic cell as a protein of a gene expression product instead of gene introduction.
  • Expression-inducing activity of low-molecular-weight compounds having an activity to induce expression for example, melatonin having an expression-inducing activity of Foxa2 (Non-patent Document 13)
  • miRNA and inhibitors thereof for example, anti-miR124a2 having an activity of inducing expression of Foxa2 and NMyc
  • anti-miR-34a can be replaced by acting on somatic cells.
  • the Foxa2 transcription factor or the gene encoding it can be obtained from the public database (such as GenBank (NCBI)) as described above.
  • the Foxa2 amino acid sequence is human-Foxa2 (Protein: NP — 068556) and mouse-Foxa2 (Protein: NP — 034576), and the Foxa2 gene can use a base sequence encoding the amino acid sequence.
  • the activity as a Foxa2 transcription factor is maintained, it is not necessary to use the full length, and even if it is a partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Foxa2 transcription factor.
  • human Foxa1 is about 53%
  • human Foxa2 and human Foxa3 are about 42%
  • human Foxa2 and human Foxb1 are about 48%
  • human Foxa2 and human Foxb2 are about 43%.
  • human Foxa1, human Foxa2, human Foxa3, human Foxb1 and human Foxb2 have conserved consensus sequences, and any amino acid sequence is 40% or more, preferably 50% or more, more preferably 80% or more, and further Preferably, an amino acid sequence having 90% or more homology (identity) can be used similarly to Foxa2.
  • the Gata6 transcription factor or the gene encoding the same For example, Gata6 consisting of an amino acid sequence of human-Gata6 (Protein: NP_005248) or mouse-Gata6 (Protein: NP — 034388) obtained from GenBank (NCBI) or the like. A Gata6 gene encoding the amino acid sequence can be used.
  • Gata6 transcription factor it is not necessary to use the full length, and if the activity as a Gata6 transcription factor is maintained, even if it is the partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Gata6 transcription factor.
  • the homology at the amino acid sequence level of human Gata6 and mouse Gata6 is about 88%, whereas Gata4 and Gata5 having properties very similar to Gata6 in the same Gata family as Gata6 are seen as human Gata6.
  • human Gata4 is about 50%, and human Gata6 and human Gata5 are about 44%. Therefore, for example, the common sequences of human Gata6, human Gata4 and human Gata5 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more with any amino acid sequence.
  • An amino acid sequence having homology (identity) can also be used in the same manner as Gata6. The same applies to the Sox21 transcription factor or a gene encoding the same.
  • human-Sox21 obtained from GenBank (NCBI) or the like, Sox21 consisting of an amino acid sequence of mouse-Sox21 (Protein: NP_808421) or A Sox21 gene encoding the amino acid sequence can be used.
  • Sox21 transcription factor if the activity as a Sox21 transcription factor is maintained, it is not necessary to use the full length, and even if its partial length is about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Sox21 transcription factor.
  • human Sox21 SEQ ID NO: 7
  • mouse Sox21 SEQ ID NO: 8
  • Sox21 and human Sox14 are about 58%
  • human Sox21 and human Sox3 are about 48%
  • human Sox21 and human Sox1 are about 47%
  • human Sox21 and human Sox15 are approximately 44%
  • human Sox21 and human Sox2 are approximately 43%.
  • the consensus sequences of human Sox21, human Sox14 and human Sox2 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, more preferably 90% or more with any amino acid sequence.
  • An amino acid sequence having homology (identity) can also be used in the same manner as Sox21.
  • the same applies to the Sox9 transcription factor or the gene encoding it for example, human-Sox9 (Protein: NP_000337) obtained from GenBank (NCBI) or the like, Sox9 consisting of the amino acid sequence of mouse-Sox9 (Protein: NP_0357578) or A Sox9 gene encoding the amino acid sequence can be used.
  • Sox9 transcription factor it is not necessary to use the full length, and if the activity as a Sox9 transcription factor is maintained, even if it is a partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Sox9 transcription factor.
  • the homology at the amino acid sequence level of human Sox9 (SEQ ID NO: NP — 000337) and mouse Sox9 (SEQ ID NO: NP — 03557) is about 97%, while it has properties very similar to Sox9 within the same Sox family as Sox9.
  • the human Sox9 and human Sox10 are about 58%, and the human Sox9 and human Sox8 are about 48%.
  • the consensus sequences of human Sox9, human Sox8 and human Sox10 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, more preferably 90% or more with any amino acid sequence.
  • An amino acid sequence having homology (identity) can also be used in the same manner as Sox9. The same applies to the NMyc transcription factor or the gene encoding the same.
  • the human NMycgene (mRNA: NM_005378) obtained from GenBank (NCBI) or the like, the NMyc gene consisting of the base sequence of mouse NMyc gene (mRNA: NM_008709) or the base NMyc encoded by the sequence can be used.
  • NMyc transcription factor it is not necessary to use the full length, and if the activity as an NMyc transcription factor is maintained, even if it is the partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as an NMyc transcription factor.
  • the homology at the amino acid sequence level of human NMyc and mouse NMyc is about 85%, whereas LMyc and cMyc having properties very similar to NMyc within the same Myc family as NMyc Human LMyc is about 37%, human NMyc and human cMyc are about 32%. Therefore, for example, the common sequences of human NMyc, human LMyc, and human cMyc are conserved, and any amino acid sequence is 40% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more. An amino acid sequence having homology (identity) can also be used similarly to NMyc. The same applies to the Irx3 transcription factor or a gene encoding the same.
  • Irx3 consisting of an amino acid sequence of human-Irx3 (Protein: NP — 077312), mouse-Irx3 (Protein: NP — 032419) obtained from GenBank (NCBI) or the like
  • the Irx3 gene encoding the amino acid sequence can be used.
  • the activity as an Irx3 transcription factor is maintained without using the full length, even if it is the partial length, about 1 to several at the amino acid level (note that several are 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as an Irx3 transcription factor.
  • human Irx3 and mouse Irx3 are about 86%
  • Irx1 and Irx2 having properties very similar to Irx3 in the same Irx family as Irx3
  • human Irx3 And human Irx1 is about 45%
  • human Irx3 and human Irx2 are about 38%. Therefore, for example, the common sequences of human Irx3, human Irx1 and human Irx2 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more with any amino acid sequence.
  • An amino acid sequence having homology (identity) can also be used in the same manner as Irx3. 2.
  • lung progenitor cell refers to a progenitor cell that has pluripotency into various tissue cells constituting the lung, It refers to cells that are positive for Nkx2.1, a progenitor cell marker.
  • the expected lung region Fetal lung progenitor cells appearing in (2-2) Method for Differentiating into Lung Progenitor Cells
  • a method for producing lung progenitor cells from somatic cells (tissue cells) such as fibroblasts in one step was developed.
  • a transcription factor group including a Fox family transcription factor, a Gata family transcription factor, a Sox family transcription factor, a Myc family transcription factor, and an Irx family transcription factor is introduced into fibroblasts and the like.
  • transcription factors of Foxa2, Gata6, Sox21 and NMyc preferably Foxa2, Gata6, Irx3, Sox21 and NMyc, five transcription factors, especially in the case of humans, Sox9 in Foxa2, Gata6, Irx3, Sox21 and NMyc.
  • Genes of transcription factors including the added 6 transcription factors are introduced into somatic cells such as fibroblasts, or adipocyte-derived fibroblasts, and cultured for 2 to 3 weeks to facilitate pulmonary progenitor cell marker positive cells Can be produced.
  • Somatic cells can be directly differentiated into lung progenitor cells by expressing foreign genes in somatic cells such as fibroblasts by gene transfer, etc. to create and act on a lung progenitor cell-specific transcription factor network.
  • “somatic cells” are somatic cells derived from mammals including humans and mice, and include cases of somatic cells derived from fetuses, somatic cells derived from adults, and somatic cells derived from patients.
  • it is a fibroblast derived from a mammal such as a human or a mouse.
  • epithelial tissues are covered with stromal tissues in living tissues, but fibroblasts are present in various stromal tissues of such adults and can be collected.
  • epidermis-derived fibroblasts or adipose tissue-derived fibroblasts can be preferably used as human adult-derived fibroblasts.
  • the somatic cells of the present invention include not only isolated somatic cells but also somatic cells in lung tissues including humans.
  • any virus such as lentivirus, adenovirus, adeno-associated virus, and Sendai virus can be used in addition to retrovirus.
  • various methods such as lipofection, electroporation, and hydrodynamic methods can be used as gene transfer methods.
  • a transcription gene can be directly introduced into somatic cells of lung tissue using a viral vector or a gene introduction agent (see Laube BL, Transl Respir Med. 2014 2, 3.).
  • RNA introduction reagent When using mRNA of a transcription factor, it can be directly introduced into human lung tissue by using an RNA introduction reagent or an RNA virus vector such as Sendai virus.
  • RNA virus vector such as Sendai virus.
  • the somatic cells after gene introduction can be easily produced as a lung progenitor cell marker positive cell by culturing for 2 to 3 weeks.
  • introducing a drug selection marker gene at the time of gene introduction it is possible to easily select only cells into which the 4 or 5 types of transcription factor genes have been introduced.
  • the medium used in the present invention is preferably a medium suitable for culturing somatic cells into which genes are introduced.
  • a medium suitable for culturing somatic cells into which genes are introduced for example, in the case of fibroblasts, a DMEM medium containing 10% fetal bovine serum or a commercially available fibroblast culture medium is suitable.
  • the lung progenitor cells produced in this way have a certain degree of proliferative ability and can be used after being proliferated as much as necessary. (2-4) Method for detecting lung progenitor cells
  • somatic cells that have undergone the culture process after introduction of the four or five types of transcription factor genes are directly transmitted to the lung progenitors without going through iPS cell-like undifferentiated cells.
  • the differentiation into cells means that the expression of marker genes specific to the original somatic cells, such as Thy1 in the case of fibroblasts, and various markers specific to ES / iPS cells, such as Oct4, Nanog and Klf4, etc. What is necessary is just to confirm that Nkx2.1 which is a marker peculiar to a lung progenitor cell is expressing, without expressing. Furthermore, it is preferable to confirm the expression of E-cadherin and the like in addition to SPC, which is a lung tissue specific marker, and more preferably to confirm that other tissue specific markers are not expressed. In order to confirm the expression of each marker, each antibody, for example, Nkx2.1 or E-cadherin specific antibody can be used.
  • the lung progenitor cells of the present invention have pluripotency into various tissue cells constituting the lung. Therefore, it is possible to differentiate into desired lung tissue cells by applying a known differentiation induction method to each lung tissue cell. For example, when pulmonary epithelial cells are to be obtained, the lung progenitor cells obtained in the present invention are 0.2% fetal bovine serum known as a low serum medium among typical endoderm-derived epithelial cell culture media.
  • Non-patent Documents 1 and 11 are also reports of differentiation into ciliated cells, basal cells, Clara cells, and the like by differentiation induction by adding various cell growth factors to the medium. These differentiation induction processes can be differentiated into various lung tissue cells not only in vitro but also in vivo conditions using experimental animals such as mice (Non-patent Document 11).
  • the lung progenitor cells can be differentiated into alveolar epithelial cells or the like by transplanting under a renal capsule where capillary vessels develop and the engraftment efficiency of the transplanted cells is high.
  • a low serum medium or a serum-free medium among typical epithelial cell culture media as differentiation induction media, differentiation into various lung mature cells occurs, and growth factors (for example, Wnt3a, FGF10, KGF Addition of the mixture) further increases the efficiency of differentiation into mature lung cells such as mucus-secreting cells, alveolar epithelial type I cells, alveolar epithelial type II cells, and Clara cells.
  • a growth factor for example, Wnt3a, FGF10, KGF
  • a low serum RPMI1640 medium for example, an Advanced RPMI1640 medium containing 0.1 to 0.5% fetal bovine serum
  • growth factors for example, Wnt3a, FGF10, KGF
  • DMEM + KSR medium not only mucus-secreting cells but also alveolar epithelial type I cells, alveolar epithelial type II cells, and Clara. It shows high differentiation ability for all types of mature lung cells such as cells. 4).
  • the lung progenitor cells of the present invention can also be used as screening cells for differentiation drugs used in the process of differentiation into mature lung tissue cells.
  • the test drug can be evaluated by adding a test substance and a test drug in the desired differentiation process into lung tissue cells and observing the differentiation state.
  • the test substance is a nucleic acid such as a test gene
  • it is preferably introduced into the cell by applying a known nucleic acid introduction method such as using an expression vector in lung progenitor cells.
  • test substances and test drugs such as compounds, proteins, peptides, and sugars, they are added to the differentiation induction medium.
  • the lung progenitor cells of the present invention can be used for transplantation treatment of lung tissue cells to damaged sites of lung tissue by applying a normal cell transplantation technique.
  • lung tissue cells such as alveolar epithelial cells obtained by differentiating lung progenitor cells of the present invention in vitro can be applied to cell kits for toxicity tests on lung tissues. For example, a multi-sample sample in which these cells are seeded in a multi-well dish can be used for drug toxicity tests and safety tests.
  • lung progenitor cells from fibroblasts introduced with various causative genes for lung cancer by the method for producing lung progenitor cells of the present invention, the obtained lung progenitor cells have the effect of various anticancer agents. It can be used as an evaluation system, that is, as a cell kit for screening a lung cancer therapeutic drug candidate. Similarly, by producing lung progenitor cells from fibroblasts into which another causative gene for lung disease has been introduced, it can also be used as a cell kit for screening lung disease therapeutic drug candidates.
  • EXAMPLES EXAMPLES
  • this invention is demonstrated in more detail based on an Example, the technical scope of this invention is not restrict
  • Example 1 Direct differentiation method from fibroblasts to lung progenitor cells using a combination of transcription factors 13 types of transcription factors that are specifically expressed in the lung formation stage of the initial lung are selected from known literatures and databases, After inserting the cDNA into the pMYs retrovirus vector, a retrovirus is prepared using PlatE cells, which are cells for retrovirus production, and mouse fetal fibroblasts (mouse fetal dorsal epidermis-derived fibroblasts: also called Dorsal skin MEF or simply MEF) .) One week later, the culture was continued by changing to a medium in which 0.2% fetal calf serum was added to Advanced RPMI1640, which is a general medium for endoderm-derived epithelial cells.
  • PlatE cells which are cells for retrovirus production
  • mouse fetal fibroblasts mouse fetal dorsal epidermis-derived fibroblasts: also called Dorsal skin MEF or simply MEF
  • Direct differentiation into lung progenitor cells was evaluated using as an index the appearance of colonies that were positive for the expression of lung progenitor cell-specific marker gene Nkx2.1 and one of epithelial markers, E-cadherin (FIG. 1a).
  • Nkx2.1 epithelial markers
  • E-cadherin epithelial markers
  • the expression of Nkx2.1 was increased in particular, and 7 transcription factors showing a suppressive action were discarded, and the other 6 transcription factors were selected (FIGS. 1e-f).
  • the expression of Nkx2.1 was not decreased even if Sox9 was removed, so it was narrowed down to 5 factors of Foxa2, Gata6, Irx3, Sox21 and NMyc. (Fig. 1g-h).
  • the essential factors were searched by subtracting one factor from five factors, the expression of Nkx2.1 almost disappeared in the case of Foxa2, Gata6, Sox21 and NMyc factors even when any of these factors was removed.
  • Nkx2.1-positive lung progenitor cells it was concluded that it is preferable to use 5 factors obtained by adding Irx3 to the above 4 factors.
  • the combination of 4 factors of Foxa2, Gata6, Sox21 and NMyc is the minimum combination of necessary genes.
  • Irx3 factor is added to these 4 factors. Nkx2.1 positive lung progenitor cells using a combination containing 5 factors were examined.
  • Example 2 Verification of specificity of lung progenitor cells prepared by gene transfer of transcription factors (2-1) Lung progenitor cells differentiated from mouse fetal fibroblasts (MEF) 5 selected in (Example 1) above The lung tissue specificity of lung progenitor cells prepared with two transcription factors was verified by immunofluorescence staining.
  • 5 transcription factors are transfected into the same mouse embryo-derived fibroblasts as in Example 1 and cultured in a general endoderm-derived epithelial cell culture medium (Advanced RPMI 1640 + 0.2% fetal bovine serum), 2 to 3 Colonies consisting of epithelial-like cells began to appear after a week (FIG.
  • Nkx2.1 is known to be expressed not only in pulmonary progenitor cells but also in thyroid tissue and brain diencephalon that develop from the anterior endoderm during embryonic period (FIG. 2c). Therefore, with regard to lung tissue specificity of lung progenitor cells prepared with transcription factors, immunofluorescence staining is performed using Pax8 specifically expressed in thyroid tissue, Tuj1 expressed in diencephalon, E-cadherin expressed in epithelial cells, etc. as indicators.
  • Nkx2.1 is positive and E-cadherin (+), Tuj1 (-), Pax8 (-) and not thyroid gland or diencephalon cells but lung progenitor cells was suggested (FIGS. 2d-e).
  • (2-2) Preparation of lung progenitor cells from other tissue-derived fibroblasts
  • mouse fetal dorsal epidermis fibroblasts Dorsal skin MEF
  • mouse fetal limb fibrocytes (Libb MEF)
  • MDF adult mouse epidermis-derived fibroblasts
  • the differentiated cells from the mouse fetal fibroblasts obtained in (2-1) above were Nkx2.1 positive, the epithelial cell markers E-cadherin and EpCAM were positive, and the thyroid tissue markers Pax8 and It was confirmed that Tuj1 as a diencephalic tissue marker was negative, and that Sox2 expressed together with Nkx2.1 in lung progenitor cells was also positive (FIGS. 2 and 3). As a result, it was confirmed that mouse embryo fibroblasts were directly differentiated into lung progenitor cells by the above five transcription factors.
  • the Nkx2.1-positive differentiated cells from the adult mouse-derived fibroblasts of (2-2) above are obtained by using 10% fetal bovine serum-containing DMEM from fibroblasts excised from the epidermis behind the ears of adult (12-week-old) mice. Prepared by culturing in medium. Five transcription factors were introduced into the adult mouse-derived fibroblasts in the same manner as in Example 1, and cultured in the same differentiation medium for 2 to 3 weeks. As described in (2-2) above, , Nkx2.1 positive colonies were generated (FIG. 2f). Results of verification by immunofluorescence staining of Nkx2.1-positive differentiated cells from the adult mouse-derived fibroblasts using the various tissue markers used in (2-1) and (3-1) above as indices.
  • fibroblasts derived from mouse embryos are Nkx2.1 positive and E-cadherin (+), Tuj1 ( ⁇ ), Pax8 ( ⁇ ), EpCAM (+), and Sox2 (+) It was confirmed that the cells differentiated into lung progenitor cells (FIGS. 5a to 5e). That is, it was confirmed that adult mouse-derived fibroblasts were also directly differentiated into lung progenitor cells by the above five transcription factors. (3-4) Verification of conversion from fibroblasts to Nkx2.1-positive epithelial cells Obtained from Nkx2.1-positive mouse fibroblasts (mesenchymal cells) in (2-1) above.
  • E-cadherin epithelial cell marker
  • Thy1, Fsp1 mesenchymal cell marker
  • Example 4 Gene expression analysis by microarray of lung progenitor cells prepared by introducing five transcription factors (4-1) Comprehensive gene analysis by microarray-1
  • lungs derived from the three types of mouse fibroblasts (Dorsal skin MEF, Limb MEF, MDF) obtained in Example 2 were used.
  • Exhaustive gene analysis was performed on the progenitor cells using a microarray.
  • the gene expression of lung progenitor cells prepared from mouse fibroblasts showed a gene expression pattern similar to that of mouse fetal lung tissue cells compared to mouse fibroblasts before differentiation. (FIGS. 7a-b).
  • the prepared lung progenitor cells were compared with other tissues such as adult bronchi, lung, stomach, intestine, liver, heart, kidney, hypothalamus, skeletal muscle, and fat. Results The gene expression pattern of the prepared lung progenitor cells was found to be most similar to the lung derived from E11.5 fetus, and not similar to adult bronchi, lung and other tissues (FIG. 8a). ). (4-2) Comprehensive gene analysis by microarray-2 Furthermore, using three new types of mouse fibroblasts (Dorsal skin MEF, Limb MEF, MDF), five transcription factors were introduced in the same manner as in Example (2-1) to establish lung progenitor cells. Later, the number of samples was increased for further microarray analysis.
  • mucus-secreting cell markers When differentiated in 4 media, the mucus-secreting cell markers (Muc5AC, Muc5b) are highly expressed, so it is considered that they differentiated mainly into mucus-secreting lung cells. From this, it was confirmed that mucus-secreting cells and alveolar epithelial type I cells were differentiated by using RPMI1640 medium containing low serum as suggested in the above Example (5-1). Addition of (for example, Wnt3a, FGF10, KGF) was shown to increase the specificity to mucus-secreting cells. On the other hand, No. which is a typical serum-free medium for animal cells. No. 5 medium (DMEM + 10% KSR) with growth factors added.
  • a representative serum-free medium as a differentiation-inducing medium and adding growth factors (eg, Wnt3a, FGF10, KGF), not only mucus-secreting cells but also alveolar epithelial type I cells and It has been shown to efficiently differentiate into mature lung cells such as alveolar epithelial type II cells and Clara cells. From the above results, it is important to select a serum-free or low-serum medium as a differentiation induction medium for lung cells. When growth factors (for example, Wnt3a, FGF10, KGF) are added, differentiation induction efficiency is further improved. To do. That is, it can be said that these growth factors function as “pulmonary tissue cell differentiation inducing factors”.
  • growth factors for example, Wnt3a, FGF10, KGF
  • a low serum-containing RPMI1640 medium or a medium prepared by adding a lung tissue cell differentiation inducing factor to serum-free DMEM + KSR medium is preferable.
  • Preparation of human lung progenitor cells from human fibroblasts (8-1) Preparation of human lung progenitor cells from human neonatal skin-derived fibroblasts Five factors of Foxa2, Gata6, Irx3, Sox21 and NMyc Using a retrovirus expression vector inserted into the pMYs retrovirus vector, a virus was prepared with PlatGP cells, which are pseudolentivirus-producing cells, and infected with human neonatal skin-derived fibroblasts (obtained from ATCC cell bank).

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Abstract

L'invention fournit un procédé destiné à produire directement des cellules précurseurs des poumons ainsi que des cellules de poumons mûres à partir de cellules somatiques, et fournit aussi des cellules précurseurs des poumons ou des cellules de poumons mûres produites selon ce procédé. Concrètement, un facteur 4 constitué des facteurs de transcription Foxa2, Gata6, Irx3, Sox 21 et NMyc, de préférence, un composé à faible masse moléculaire ou un microARN qui présente une activité d'induction d'expression d'un gène de facteur 5 auquel est ajouté le facteur de transcription Irx3, une protéine et ces facteurs, est introduit dans une cellule somatique, et une cellule précurseur des poumons directe est ainsi produite. En outre, dans le cas d'une cellule somatique humaine, une différenciation directe en cellule précurseur des poumons de manière efficace, est possible par ajout du facteur 5, et par ajout de Sox9. Les cellules précurseurs des poumons ainsi produites, et des cellules de poumons mûres de différentes sortes telles que des cellules de l'épithélium des poumons différenciées des cellules précurseurs des poumons, peuvent être mises en œuvre en tant que kit ou matériau de cellule pour évaluation in vitro et in vivo se révélant excellents pour évaluer la différenciation en cellule de poumons fonctionnelle ou effectuer une évaluation de toxicité.
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CN112877364B (zh) * 2019-11-29 2023-07-28 中国医学科学院药物研究所 软骨下骨细胞向关节软骨细胞直接转化的重编程诱导方案
CN112608879A (zh) * 2021-01-12 2021-04-06 北京大学 一种从胚胎干细胞分化获得肺上皮细胞的方法及其使用的培养基

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