JP6235795B2 - Composition for cell reprogramming - Google Patents

Composition for cell reprogramming Download PDF

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JP6235795B2
JP6235795B2 JP2013113297A JP2013113297A JP6235795B2 JP 6235795 B2 JP6235795 B2 JP 6235795B2 JP 2013113297 A JP2013113297 A JP 2013113297A JP 2013113297 A JP2013113297 A JP 2013113297A JP 6235795 B2 JP6235795 B2 JP 6235795B2
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reprogramming
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JP2014230521A (en
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育男 森田
育男 森田
基浩 小牧
基浩 小牧
剣吾 岩崎
剣吾 岩崎
尚毅 横山
尚毅 横山
弘人 菖蒲
弘人 菖蒲
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国立大学法人 東京医科歯科大学
国立大学法人 東京医科歯科大学
大日本印刷株式会社
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  The present invention relates to differentiation induction and production of therapeutic cells / tissues by enhancing expression of transcription factors involved in a plurality of reprogramming in the field of cell regenerative medicine. More specifically, the present invention relates to exosomes capable of enhancing the expression of transcription factors involved in a plurality of reprogramming, and cells having somatic cell reprogramming and pluripotency (such as iPS cells) using the exosomes. Regarding induction.

  Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can be cultured while maintaining pluripotency capable of differentiating into all cells present in the living body. Utilizing this property, pluripotent stem cells are expected as a means of cell transplantation for many diseases such as Parkinson's disease, juvenile diabetes and leukemia. However, ES cells have a problem of causing rejection similar to organ transplantation. There are also many disagreements from the ethical point of view regarding the use of ES cells established by destroying embryos.

  iPS cells are expected to be usable as ideal pluripotent cells without rejection and ethical problems. The following Patent Document 1 is known as a method for establishing iPS cells. Patent Document 1 describes a process of reprogramming by introducing a plurality of specific factors (four factors of Oct3 / 4 gene, Sox-2 gene, Klf4 gene and c-Myc gene) into a somatic cell. Since a retrovirus or the like is introduced into a somatic cell for gene introduction, there is a concern about the risk of tumor development in iPS cells or cells obtained by inducing differentiation of iPS cells.

  In recent years, studies have been made to try to dedifferentiate cells by administering small lipid vesicles called exosomes or microvesicles to cells. For example, in Non-Patent Document 1, when an ES cell-derived microvesicle containing a large amount of Oct3 / 4 gene and Nanog gene is added to a mouse bone marrow-derived mononuclear cell culture system, the Oct4 gene is expressed in the cell. It has been reported. In Non-Patent Document 2, when the Oct4 gene, Sox-2 gene, and miRNA are introduced into Muller cells of the retina via ES cell microvesicles, the expression of Oct4 gene and Sox-2 gene is enhanced. It has been reported.

Pamphlet of International Publication WO2007 / 069666

Leukemia (2006), 20, 847-856, Embryonic Stem Cell-Derived Microvesicles Preprogram Hematopoietic Progenitors: Evidence for Horizontal Transfer of mRNA and Protein Delivery PLOS ONE (2012), 7, e50417, Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Muller Cells of the Retina

  In Non-Patent Documents 1 and 2, since ES cells are used, there is an ethical problem with the acquisition method. Furthermore, Non-Patent Documents 1 and 2 increase the expression level of Oct4 gene and Sox-2 gene, but do not increase the expression level of Nanog gene involved in stem cell nature.

  The present invention has been made in view of the above circumstances, and is a means for enhancing the expression of a plurality of transcription factors involved in reprogramming in somatic cells without using ES cells. The aim is to provide a low means.

  The inventors of the present invention focused on the fact that the use of placenta-derived cells for the production of exosomes can solve at least the ethical problems that arise when using ES cells. In addition, since placenta-derived cells have extremely low or no tumorigenicity or tumor proliferative potential, the inventors have also paid attention to the fact that the risk of tumor development can be reduced like conventional iPS cells. Based on these findings, the present inventors have further researched and used the placenta-derived cells to enhance not only the Oct3 / 4 gene and the Sox-2 gene but also the ability to enhance the expression of the Nanog gene. It was found that exosomes possessed can be extracted.

That is, the present invention includes the following.
(1) A composition for enhancing the expression of a plurality of transcription factors involved in reprogramming in a target cell,
The said composition containing the exosome extracted from the placenta origin cell colonized by culture | cultivation.
(2) The composition according to (1), wherein the placenta-derived cells comprise placenta-derived stem cells.
(3) The composition according to (1) or (2), which is a composition for enhancing the expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene as a transcription factor involved in reprogramming.
(4) The composition according to any one of (1) to (3), wherein the exosome does not substantially contain an Oct3 / 4 gene and a Sox-2 gene.
(5) The composition according to any one of (1) to (4), which is a composition for reprogramming target cells.
(6) A method for producing a composition for enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell,
a) culturing the placenta-derived cells to form colonies;
b) obtaining colony-formed placenta-derived cells and further culturing;
c) a step of extracting exosomes from the culture supernatant obtained in step b);
Said method.
(7) The method according to (6), wherein step a) is a step of seeding and culturing placenta-derived cells at a density of 14 cells / cm 2 or less to form colonies.
(8) The method according to (6) or (7), wherein the composition is a composition for enhancing expression of Nanog gene, Oct3 / 4 gene and Sox-2 gene as transcription factors involved in reprogramming.
(9) The method according to any one of (6) to (8), wherein the placenta-derived cells include placenta-derived stem cells.
(10) A method for enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell,
a) culturing the placenta-derived cells to form colonies;
b) obtaining colony-formed placenta-derived cells and further culturing;
c) a step of extracting exosomes from the culture supernatant obtained in step b);
d) contacting the exosome with the target cell;
Said method.
(11) The method according to (10), wherein step a) is a step of seeding and culturing placenta-derived cells at a density of 14 cells / cm 2 or less to form colonies.
(12) The method according to (10) or (11), wherein expression of Nanog gene, Oct3 / 4 gene and Sox-2 gene is enhanced as a transcription factor involved in reprogramming.
(13) The method according to any one of (10) to (12), wherein the placenta-derived cells include placenta-derived stem cells.
(14) The method according to any one of (10) to (13), wherein the target cell is reprogrammed by enhancing expression of a plurality of transcription factors involved in reprogramming.
(15) The method according to any one of (10) to (13), which is a method for producing a multipotent cell by enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell.
(16) A placenta-derived cell colonized by culturing, wherein the cell produces an exosome that enhances expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene of a somatic cell.

  According to the present invention, it is possible to provide a means for enhancing the expression of a plurality of transcription factors involved in reprogramming in somatic cells without using ES cells and having a low risk of tumor development.

For exosomes extracted from colonized human placenta-derived cells, real-time PCR is used to perform melting curve analysis using periodontal ligament-derived stem cells as a positive control to quantify Oct3 / 4, Sox-2, and Nanog genes. The results are shown. The exosome real-time PCR product extracted from colonized human placenta-derived cells was subjected to electrophoresis using an agarose gel, stained with ethidium bromide, and the size of the real-time PCR product was qualitatively examined. FIG. 3a shows the results of comparing the expression level of Oct3 / 4 gene in human dermal fibroblasts contacted with exosomes extracted from colonized human placenta-derived cells, compared to the control. FIG. 3b shows the result of comparing the expression level of Oct3 / 4 gene in human dermal fibroblasts contacted with exosomes extracted from human placenta-derived cells that have not formed colonies, compared to the control. FIG. 3c shows the results of comparing the expression level of Oct3 / 4 gene in human skin fibroblasts contacted with exosomes extracted from non-colonized human calvarial osteoblasts, compared to the control. The control shows the expression level of Oct3 / 4 gene in human dermal fibroblasts not contacted with exosomes. FIG. 4a shows the result of comparing the expression level of Sox-2 gene in human skin fibroblasts contacted with exosomes extracted from colonized human placenta-derived cells, compared to the control. FIG. 4 b shows the result of comparing the expression level of Sox-2 gene in human skin fibroblasts contacted with exosomes extracted from human placenta-derived cells that have not formed colonies, compared to the control. FIG. 4 c shows the result of comparing the expression level of the Sox-2 gene in human skin fibroblasts contacted with exosomes extracted from non-colonized human calvarial osteoblasts, compared to the control. The control shows the expression level of the Sox-2 gene in human dermal fibroblasts not contacted with exosomes. FIG. 5a shows the result of comparison of the expression level of Nanog gene in human dermal fibroblasts contacted with exosomes extracted from colonized human placenta-derived cells, with a control. FIG. 5b shows the result of comparison of the expression level of Nanog gene in human dermal fibroblasts contacted with exosomes extracted from human placenta-derived cells that were not colonized, with the control. FIG. 5 c shows the result of comparison of the expression level of Nanog gene in human skin fibroblasts contacted with exosomes extracted from non-colonized human calvarial osteoblasts, compared to the control. The control shows the expression level of Nanog gene in human skin fibroblasts not contacted with exosomes.

  The present inventors use exosomes extracted from placenta-derived cells colonized by culture (hereinafter sometimes referred to as exosomes of the present invention) to enhance the expression of multiple transcription factors involved in reprogramming in target cells. I found out that I can do it.

  An exosome is a small lipid vesicle having a diameter of 30 to 100 nm derived from a late endosomal compartment and containing not only lipid but also protein and nucleic acid (Nature Reviews Immunology 9, 581-593, 2009). Exosomes are membrane vesicles surrounded by lipid bilayers secreted by cells, and it has become clear that communication between cells and regulation of biological phenomena are carried out by taking in other cells. ing. An exosome is a biological material produced by a cell, is a stable membrane particle that is not easily decomposed even in blood or the like, and has a property that a nucleic acid inside the exosome functions in other cells into which the exosome has been incorporated.

  The exosome used in the present invention is extracted from placenta-derived cells. It is not particularly limited as long as it is derived from an animal having a placenta. For example, those derived from mammals (eg, human, mouse, rat, monkey, dog, cat, cow, horse, etc.) can be used. Use an animal derived from the same species as the target cell.

  In the present invention, exosomes are extracted from placenta-derived cells colonized by culture. Placenta-derived cells also include amnion-derived cells and umbilical cord-derived cells. Preferably, exosomes are extracted from those containing placenta-derived stem cells. Placenta-derived stem cells are derived from the fetus, have low immunogenicity, and have the ability to differentiate into three germ layers. Placenta-derived stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, placental pluripotent cells and embryonic-like stem cells.

  Methods for preparing placenta-derived cells are not particularly limited, and methods known in the art can be used. For example, an outgrowth method using a crushed tissue or a method using a digestive enzyme can be used. Digestive enzymes used include, but are not limited to, matrix metalloproteinases (eg, collagenase), trypsin, chymotrypsin, papain, pepsin, proteinase K, dispase, carboxypeptidase, calpain, and subtilisin.

  Methods for disrupting tissue are known in the art and include, for example, mincing, mixing, and the like. Breaking the placental tissue results in a tissue mixture of floating placental cells and placental tissue. By applying the tissue mixture to one or more sieves, eg strainers of various pore sizes ranging from 50 μm to 500 μm, it is possible to separate the tissue from floating placental cells. For example, the placental tissue mixture can be repeatedly passed through a small pore size mesh to finally remove almost all of the tissue and obtain a placental cell suspension (eg, a single cell suspension).

  In addition to placenta-derived stem cells, the placental cell suspension may contain one or more other placenta-derived cell types, such as epithelial cells (eg, embryonic ciliary cells). Placenta-derived cells other than placenta-derived stem cells can be separated using established methods such as one or more washes, centrifugation, density gradient centrifugation, elution, positive selection and negative selection.

The method for culturing placenta-derived cells to form colonies is not particularly limited, and methods known in the art can be used. For example, the placenta-derived cells obtained as described above can be colonized by seeding and culturing on the culture surface of the culture vessel at a low density. The placenta-derived cells, preferably 5,000cells / cm 2 or less in density, more preferably 500cells / cm 2 or less in density, more preferably a density of 50cells / cm 2 or less, even more preferably 25cells / cm 2 or less of Colony formation by seeding and culturing at a density of preferably 0.5 cells / cm 2 or more, more preferably 1.5 cells / cm 2 or more, and even more preferably 5 cells / cm 2 or more Can be made.

  Colonies are formed by seeding and culturing cells at low density under conditions that do not cause confluence, and only cells having colony-forming ability can be selected. Culture conditions are not particularly limited, and methods known in the art can be used. As the medium, a known medium used for culture of placenta-derived cells can be used. For example, MSCGM medium, 199 medium, BME medium (BME-Earle, BME-Hanks), Ames' medium, Ham's F10 medium, Ham's F12 medium, HAT medium, L-15 medium, McCoy5a medium, RPMI1629 medium, RPMI1640 medium, Alpha-MEM medium, MEM medium (MEM-Earle, MEM-Hanks), D-MEM medium (low glucose (1 0.0 g / L glucose), high glucose (4.5 g / L glucose)), CD medium (Chemically-Defind) and the like can be used. The culture is performed at a temperature of 36 to 38 ° C. for 12 to 16 days while changing the medium.

  Colony formation is effective for concentrating and culturing cells having a reprogramming effect on target cells from primary cultured cells containing various cells. Since cells having a reprogramming effect have a high proliferation ability, the ratio can be increased among many types of cells by performing colony formation. As specific methods for forming colonies, methods known in the art can be used (STEM CELLS (2001), 19, 219-225, Rat Marrow Stromal Cells are More Sensitive to Plating Density and Expand More Rapidly from Single- Cell-Derived Colonies than Human Marrow Stromal Cells).

  The placenta-derived cells colonized in this way are cells having excellent proliferation ability, and exosomes extracted from these placenta-derived cells are more effective than exosomes extracted from confluent placenta-derived cells. It is excellent in the ability to enhance the expression of transcription factors involved in reprogramming, and excellent in the ability to reprogram cells and to induce multipotent cells.

It is preferable to obtain only colony-forming placenta-derived cells (hereinafter sometimes referred to as colony-forming cells), proliferate by further culturing, and extract exosomes from the proliferated placenta-derived cells. For example, colony-forming cells are seeded in another culture vessel and cultured. When 60-80% confluent, the cells are washed with PBS and the medium is changed under conditions of 37 ° C., 5% CO 2 and saturated steam. Incubate, collect the culture supernatant, centrifuge, and filter. By using a serum-free medium as the medium, contamination of exosomes derived from other cell types can be prevented. Examples of the serum-free medium include 199 medium, BME medium (BME-Earle, BME-Hanks), Ames' medium, Ham's F10 medium, Ham's F12 medium, HAT medium, L-15 medium, McCoy5a medium, RPMI1629 medium, RPMI1640 medium, Alpha-MEM medium, MEM medium (MEM-Earle, MEM-Hanks), D-MEM medium (low glucose (1.0 g / L glucose), high glucose (4.5 g / L glucose)) ) And CD medium (Chemically-Defind). If there is no problem even if exosomes derived from other cells are mixed, the medium is not particularly limited to a serum-free medium.

At this time, the density for seeding the cells is not particularly limited, but is preferably a density of 500 cells / cm 2 or less, more preferably a density of 50 cells / cm 2 or less, preferably a density of 1.5 cells / cm 2 or more, more preferably Is a density of 5 cells / cm 2 or more.

  Exosomes can be prepared from the cell culture supernatant of colony forming cells using any suitable technique (see, for example, the method described in Current Protocols in Cell Biology (2006) 3.22.1-3.22.29). ). Specifically, first, the obtained culture supernatant is centrifuged at a speed of 5000 to 20000 g, preferably 8000 to 12000 g for 15 to 60 minutes, preferably 20 to 40 minutes. Next, the obtained supernatant is further centrifuged at a speed of 50,000 to 150,000 g, preferably 90000 to 110000 g for 50 to 100 minutes, preferably 60 to 80 minutes. Subsequently, the obtained pellet is suspended in PBS and centrifuged at a speed of 50,000 to 150,000 g, preferably 90000 to 110000 g, for 50 to 100 minutes, preferably 60 to 80 minutes. The pellet thus obtained can be obtained as an exosome.

  Another preferred method for preparing the exosome fraction includes a method using centrifugation and a gel filtration column. Specifically, first, the obtained culture supernatant is centrifuged for the first time under the condition that the exosome fraction does not precipitate. For example, centrifugation is performed at 1 to 30 ° C. and a speed of 500 to 4000 g for 5 to 30 minutes. An optimal example is centrifugation at room temperature, 2000 g for 15 minutes. Further, as the second centrifugation, the centrifugation is performed at a speed higher than that of the first centrifugation and under the condition that the exosome fraction does not precipitate. Examples of the second centrifugation can include centrifugation at 1 to 30 ° C. and 5000 to 25000 g for 5 to 120 minutes, and most preferred examples include centrifugation at room temperature and 12000 g for 35 minutes. it can. The obtained supernatant fraction is applied to a gel filtration column used for general biochemical experiments, the absorbance at 260 nm of the eluate is measured, and the fraction with high absorbance is used as the exosome fraction. As the gel filtration column, a carrier may be purchased and produced by itself, or a commercially available product may be used. Here, Sephacryl S-400 HR (GE Healthcare Bioscience) can be cited as a suitable example. In addition, as the fraction having a high absorbance, the upper 10 fractions of the absorbance value may be used. Of course, the fraction exhibiting the highest value can be used as the exosome fraction.

  Another preferred method for preparing the exosome fraction is, for example, differential centrifugation. As a preferred specific example, Raposo et. al. J. et al. Exp. Med. 183: 1161-1172 (1996) is described below. The cell culture is centrifuged at 300 g for 10 minutes. The obtained supernatant is centrifuged twice at 300 g for 10 minutes, once at 10000 g for 30 minutes, and once at 70,000 g for 60 minutes. The resulting precipitate is dissolved in a solution containing 2.5 M sucrose, 20 mM Hepes / NaOH, pH 7.2 and overlaid with a sucrose solution having a density gradient of 2 to 0.25 M. After centrifugation at 100000 × g for 15 hours, the obtained residue is dissolved in PBS, and ultracentrifugation is further performed at 200000 × g for 1 hour.

  The exosome of the present invention can enhance the expression of a plurality of transcription factors involved in reprogramming in a target cell, but preferably all genes encoding transcription factors involved in reprogramming that can be enhanced, for example, those that can be enhanced It does not include all mRNA and DNA encoding transcription factors involved in reprogramming. The exosome of the present invention is preferably substantially free of Oct3 / 4 gene and Sox-2 gene, that is, mRNA and DNA encoding them. Here, “substantially free of a gene” means that it does not contain within a detectable range. For example, in quantitative PCR of cDNA obtained through total RNA extraction from exosomes and reverse transcription, Oct3 / 4 The gene and the Sox-2 gene are not detected. It is surprising that the exosomes of the present invention can enhance the expression of genes encoding them in target cells, even though they do not contain Oct3 / 4 and Sox-2 genes.

  By bringing the exosome of the present invention into contact with a target cell, expression of a plurality of transcription factors involved in reprogramming can be enhanced in the target cell. The exosome of the present invention enhances the expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene in the target cell, in particular, it can reprogram the target cell and has multipotency from the somatic cell. Can be induced.

  The ability to reprogram cells by enhancing the expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene is, for example, Differentiation (2010), 80, 123-129 (Rapid and efficient reprogramming of human amnion-derived cells into pluripotency by three factors OCT4 / SOX2 / NANOG).

  “Reprogramming” or “reprogramming” refers to the reverse process of cell differentiation, that is, regaining the undifferentiated state that the differentiated cell once held. This process is called “nuclear reprogramming” or “reprogramming”, especially when the cell has acquired pluripotency with trigermogenesis and ontogeny by reprogramming. Such cells are referred to as “iPS cells”. This “nuclear reprogramming” or “initialization” typically refers to the return of the somatic nucleus to the fertilized egg state.

  A transcription factor involved in reprogramming refers to a nuclear protein that can be used to reprogram a cell when expressed independently or in combination with a target cell. Transcription factors involved in reprogramming include an Oct family gene (eg, Oct3 / 4 gene), a Sox family gene (eg, Sox-2 gene), a Nanog family gene (eg, Nanog gene), a Klf family gene (eg, Klf4 gene), and Each gene of the Myc family gene (for example, c-Myc gene) is known (WO2007 / 069666). The exosome of the present invention is characterized by enhancing the expression of a plurality of transcription factors involved in reprogramming in a target cell, and at least enhances the expression of the Nanog gene, and preferably further comprises a Sox-2 gene and an Oct3. / 4 gene expression is enhanced.

  Oct3 / 4 is known to play a critical role in maintaining pluripotency. The absence of Oct3 / 4 in Oct3 / 4 + cells such as blastomeres and embryonic stem cells leads to spontaneous differentiation of the trophoblast, and thus the presence of Oct3 / 4 is associated with the pluripotency and differentiation potential of embryonic stem cells. It is considered to be the original. Examples of Oct3 / 4 proteins include those encoded by the mouse Oct3 / 4 gene (Genbank accession number NM_013633) and the human Oct3 / 4 gene (Genbank accession number NM_002701).

  Sox family genes are associated with maintenance of pluripotency similar to Oct3 / 4, but are associated with pluripotent and pluripotent stem cells as opposed to Oct3 / 4 expressed only in pluripotent stem cells To do. Sox-2 was the first gene used for induction (Takahashi et al, Cell, 2006, 126: 663-76; Takahashi et al. Cell, 2007, 131-861-72; Yu et al. al, Science 2007, 318: 1917), other genes of the Sox family have been found to act in the induction process as well. Examples of Sox-2 proteins include proteins encoded by the mouse Sox-2 gene (Genbank accession number NM — 014443) and the human Sox-2 gene (Genbank accession number NM — 003106).

  Nanog is a homeobox transcription factor. When a gene is knocked out, ES cells lose pluripotency and differentiate into primitive endoderm cells. In addition, it has been reported that when the Nanog gene is expressed several times normal, mouse ES cells can maintain pluripotency in the absence of LIF / STAT3 signal. Therefore, Nanog is considered to be a necessary and sufficient factor for differentiation pluripotency of ES cells (Yamanaka et al, Cell, 2003, 113: 631-642). Examples of Nanog proteins include those encoded by the mouse Nanog gene (Genbank accession number NM — 028016) and the human Nanog gene (Genbank accession number NM — 024865).

  Increasing (increasing) expression of transcription factors involved in reprogramming in target cells includes overexpression of transcription factors involved in reprogramming, increasing the amount of transcription factor proteins involved in reprogramming in target cells. And increasing the amount of mRNA encoding a transcription factor involved in reprogramming in the target cell. In addition, the transcription factor genes involved in reprogramming exemplified above include orthologs and homologs of each gene. In the present invention, the gene includes nucleic acids including DNA and RNA, RNA includes mRNA, and DNA includes genomic DNA and cDNA.

  In the target cell contacted with the exosome of the present invention, expression of a transcription factor involved in reprogramming is enhanced, while the expression level of a gene encoding telomere reverse transcriptase (TERT) does not change. TERT has been reported to be associated with tumor growth and teratoma formation, and the expression level of TERT gene is used as an indicator of tumor growth or tumor growth. Therefore, it is considered that the exosome of the present invention does not promote tumorigenesis or canceration of the contacted target cells, and has low or no tumorigenicity or tumor growth.

  In the present invention, the target cells to be reprogrammed are preferably differentiated somatic cells excluding stem cells. For example, fibroblasts (eg skin fibroblasts), epithelial cells (eg gastric epithelial cells, liver epithelial cells), endothelial cells (eg blood vessels, lymphatic vessels), nerve cells (eg neurons, glial cells), pancreas Cells, blood cells, bone marrow cells, muscle cells (eg, skeletal muscle cells, smooth muscle cells, cardiomyocytes), liver parenchymal cells, non-hepatic parenchymal cells, fat cells, osteoblasts, cells constituting periodontal tissues (eg, , Periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), cells constituting the kidney, eye and ear.

  According to a preferred embodiment of the invention, the cells targeted for reprogramming are fibroblasts, in particular skin fibroblasts. In the present invention, the target cell for reprogramming may be derived from any animal, but is preferably derived from a mammal (eg, human, mouse, rat, monkey, dog, cat, cow, horse, etc.). Cells, more preferably cells derived from mice or humans.

  As a use of the cell reprogrammed as described above, it can first be used as a therapeutic cell. Examples of such therapeutic cells include cells reprogrammed to differentiated states of tissue stem cells and their progenitor cells. That is, reprogrammed cells can be used for treatment of diseases, and specifically, various diseases can be treated using cells reprogrammed to differentiated states of tissue stem cells and their precursor cells. For example, Takahashi et al., Experimental Medicine, vol. 26, no. 5 (Special Issue), 2008 (Yodosha); Regenerative Medicine, Vol. 7, No. 3, 2008 (Medical Review); and Ayumi of Medicine, vol. 229, no. 9, 2009 (Medical and Dental Publishing Co., Ltd.).

  For example, when hematopoietic stem cells are obtained by reprogramming blood cells, they can be used for hematopoietic stem cell transplantation. Specifically, by transplanting the obtained hematopoietic stem cells using an infusion or a catheter or the like, congenital metabolic disorders including leukemia, lymphoma, autoimmune disease, lysosomal disease and peroxisomal disease (for example, mucopolysaccharidosis) , Gaucher disease, Fabry disease, Pompe disease, adrenoleukodystrophy, I-cell disease, metachromatic leukodystrophy, GM1 gangliodosis, and the like. When myeloid or lymphoid progenitor cells are obtained, these cells themselves, or further to erythrocytes, neutrophils, basophils, eosinophils, platelets, dendritic cells or regulatory T cells. After induction of differentiation, by direct injection or transplantation by infusion, immune recovery after hematopoietic stem cell transplantation or anticancer chemotherapy, anticancer cell therapy, autoimmune disease cell therapy, rheumatism therapy, multiple sclerosis therapy, It can be used to treat anemia such as aplastic anemia, thrombocytopenia, and trauma. In addition, when mesenchymal stem cells are obtained, they can be used for the treatment of graft-versus-host disease (GvHD) or the treatment of joint disorders by transplanting them using an infusion or a catheter.

  Similarly, in the nervous system, for example, when dopamine-producing cells are obtained by reprogramming from somatic cells, they can be used for treatment of Parkinson's disease by transplanting them with a catheter or the like. For example, when oligodendrocyte progenitor cells are obtained, they can be used for treatment of spinal cord injury by direct injection into the affected area or transplantation using a catheter or the like. Further, for example, when neuron precursor cells or astrocyte precursor cells are obtained, they can be used for the treatment of motor neuropathy by directly injecting them into the affected area or transplanting them using a catheter or the like. For example, when neural stem cells or neural progenitor cells that can be identified by the expression of differentiation markers such as Nestin and Musashi are obtained, they can be directly injected into the affected area or transplanted using a catheter to treat cerebral infarction or cerebral hemorrhage. Available.

  Similarly, blood vessels such as arteries and veins / lymphatic vessels, muscle tissue systems such as heart muscle / smooth muscle / skeletal muscle / oval cells, digestive systems such as pancreas, liver, stomach, intestine, and skin such as epidermis / dermis / hair It is also possible to obtain therapeutic cells in the visual system such as the cornea, the cornea, and the auditory system such as the inner ear. For example, in the intestinal system, when intestinal progenitor cells such as crypt cells are obtained, they can be used for the treatment of ulcerative colitis, Crohn's disease, short bowel disease, etc. by transplanting them with a catheter or the like. In the visual system, for example, when retinal progenitor cells, retinal pigment epithelial cells and photoreceptor cells are obtained by reprogramming from somatic cells, they can be directly injected or transplanted into a cell sheet for transplantation to age-related macular degeneration or retinal pigments. Can be used to treat degeneration. In the pancreatic system, for example, when β cells or insulin-producing cells are obtained, they can be used for the treatment of diabetes by encapsulating them with a catheter or the like or encapsulating them in an immune blocking device. In the liver system, for example, when albumin-producing cells are obtained, they can be used for the treatment of trauma with massive bleeding by encapsulating them in a catheter or the like or encapsulating them in an immune blocking device. In addition, for example, when blood coagulation factor-producing cells are obtained, they can be encapsulated in a catheter or an immune blocking device and transplanted to treat congenital genetic diseases such as trauma treatment with hemorrhage and hemophilia Available to.

  Cells reprogrammed in accordance with the present invention can also be used as therapeutic regenerative tissues, organs, organ materials. For example, myocardial cells, or myocardial progenitor cells that express the islet-1 gene, etc., or cells that express myocardial differentiation-related marker factors such as the nkx-2.5 gene and the gata-4 gene are induced and directly induced by a catheter or the like. In addition to transplantation, the induced cardiomyocytes and the like can be applied in the form of a sheet / stacked and transplanted to a heart disease such as myocardial infarction or dilated cardiomyopathy. Cell sheeting / stacking can be performed using, for example, a temperature-sensitive culture dish. Also, by mixing vascular endothelial cells in addition to the induced cardiomyocytes to form a cell sheet / laminate, a cell laminate sheet in which the vascular network is reconstructed can be produced.

  Similarly, when mesenchymal stem cells are obtained from somatic cells such as fat cells, bone marrow cells, and blood cells, for example, chondrocytes are induced and transplanted directly with a catheter or the like, and the induced chondrocytes are polymerized. By transplanting with a support carrier, cartilage and bone tissue can be applied to reconstruct and treat joint disorders. Similarly, culture of therapeutic tissues in digestive systems such as pancreas, liver, stomach and intestines, skin systems such as epidermis / dermis / hair, and auditory systems such as inner ear using a polymer support carrier. Can also be obtained. For example, when obtained by inducing liver tissue, it can be used for the treatment of liver cancer, cirrhosis, acute liver failure, and liver metabolic disorders such as hemochromatosis. When obtained by inducing a muscular tissue system such as smooth muscle / skeletal muscle, it can be used for muscular dystrophy treatment by direct injection or transplantation using a catheter or the like. When lung cell tissue is obtained, it can be used for treatment of pulmonary respiratory diseases such as cystic fibrosis and asthma by transplanting it to the affected area. In the kidney system, when a tissue containing mesangial cells, tubular epithelial cells, glomerular cells, etc. is obtained, it can be directly transplanted to be used for treatment of renal failure or nephritis, dialysis treatment, and the like. In blood vessels / lymphatic vessels such as arteriovenous, blood vessels are constructed using cell sheets or polymer support carriers, etc., and then directly transplanted to treat heart diseases such as coronary artery aortic bypass grafts, lower limb ischemic diseases It can be used for treatment, ischemic heart disease treatment and the like.

  Furthermore, the cells reprogrammed according to the present invention can be used as a drug discovery research tool. For example, when human somatic cells are reprogrammed to obtain human tissue stem cells, their progenitor cells, or iPS cells, these cells are used to induce cells of the desired differentiation state, or tissues or organs. Can be used for evaluating the efficacy / toxicity of test substances, elucidating the mechanism of action, or analyzing the mechanism of biological phenomena.

  Further, the present invention can be applied to cells for producing a gene recombinant preparation. That is, when cells that produce hormones, metabolic enzymes, and the like are obtained by the reprogramming method of the present invention, they can be used as cells for producing therapeutic proteins. For example, by obtaining cells that can metabolize the liver, albumin-producing cells, blood coagulation factor-producing cells, α1-antitrypsin and other metabolic enzyme-producing cells are produced, and the produced metabolic enzymes are directly injected or infused. By administering it, it can be used to treat deficiencies of these proteins. For example, insulin-producing cells can be prepared by obtaining pancreatic cells that can metabolize substances such as β cells, and the produced insulin can be directly injected to be used for the treatment of type I diabetes. Furthermore, by introducing a desired foreign gene into the obtained cells, it is possible to obtain cells for producing a therapeutic protein preparation to which a gene recombinant is added. Similarly, by introducing into a cell a functional gene that adds a function to the produced protein or enhances the secretion ability, the substance production ability of the cell can be enhanced.

  The composition containing the exosome of the present invention can be formulated as a liquid as it is, or as a pharmaceutical composition of an appropriate dosage form together with a pharmacologically acceptable carrier, diluent or excipient. Here, as a pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, solvents (dispersing agents), solubilizing agents, suspending agents, and stabilizing agents are used. It is added as an agent, isotonic agent, buffer, pH adjuster, soothing agent and the like. If necessary, formulation additives such as preservatives and antioxidants can also be used.

  Examples of the dosage form of the pharmaceutical composition include injectable preparations such as injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, intraarterial injections, etc.) and infusions. Can be mentioned.

  The pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia. The content of exosomes in the pharmaceutical composition varies depending on the dosage form, dosage, etc., but is, for example, about 0.01 to about 50% by weight. Administration is oral administration such as breast milk, nasal administration, parenteral administration such as intravenous, intraperitoneal, intramuscular, subcutaneous and transdermal administration.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the scope of the examples.

(1) Placenta-derived cell collection Human placental tissue was obtained by cesarean delivery. The placental tissue was washed 3 times with phosphate buffered saline (PBS). The outer membrane was removed from the placenta tissue, and the tissue was separated into pieces using a scalpel or a razor. Enzyme treatment was performed at 37 ° C. for 60 minutes with a solution containing 40 ml of 3 mg / ml collagenase and 4 mg / ml dispase per 10 g of placental tissue. Thereafter, the mixture was filtered with a strainer having a pore diameter of 100 μm, centrifuged at 600 g for 5 minutes with a centrifugal separator, and the supernatant was aspirated. Next, hemolysis was performed with 35 ml of ACK buffer per 10 g of placental tissue for 5 minutes, and the hemolysis reaction was stopped with 35 ml of 3% fetal bovine serum (FBS) and 5 U / ml heparin (Heparin) per 10 g of placental tissue. Further, the mixture was centrifuged at 600 g for 15 minutes with a centrifuge, and the supernatant was aspirated. The pellet was resuspended in MSCGM medium (Lonza) and the number of cells was counted.

(2) Colony formation 800 placenta-derived cells obtained as described above were seeded in a φ10 cm dish containing MSCGM medium (14 cells / cm 2 ). The cells were cultured for 2 weeks while changing the medium every 3 days. The proliferated cells were designated as CFC (colony forming cells).

(3) Preparation of culture supernatant According to a conventional method, 2 × 10 6 CFCs were seeded in a φ15 cm dish (13,000 cells / cm 2 ). When it became 70% confluent, the medium was replaced with serum-free DMEM after washing 3 times with PBS. Thereafter, the cells were incubated for 48 hours at 37 ° C., 5% CO 2 and saturated water vapor conditions. After 48 hours, the culture supernatant was collected, centrifuged at 2000 g for 20 minutes with a centrifuge, and the supernatant was collected and filtered with a filter having a pore size of 0.2 μm to remove cell components. .

(4) Preparation of exosomes The above culture supernatant was centrifuged at 10,000 g for 30 minutes with a centrifuge, and then the supernatant was further centrifuged at 100,000 g for 70 minutes. This pellet was suspended in PBS and further centrifuged at 100,000 g for 70 minutes. The pellet was suspended in 100 μl PBS per 20 ml culture supernatant. Exosome 1 was produced as described above.

  In the same method, the exosome extracted from the human placenta-derived cells not colonized in the step (2) is called exosome 2, and the exosome extracted from the human calvarial osteoblasts not colonized is Prepared as exosome 3.

  The exosome 1 was subjected to melting curve analysis using the LightCycler Real-Time PCR System (Roche Applied Science) with periodontal ligament-derived stem cells as a positive control, and the Oct3 / 4 gene, Sox-2 gene, and Nanog gene were quantified. Tried. It was suggested that the exosome 1 does not substantially contain Oct3 / 4 gene and Sox-2 gene, and that Nanog gene may be contained (FIG. 1). In addition, the real-time PCR product was subjected to electrophoresis using an agarose gel and stained with ethidium bromide, and the size of the real-time PCR product was qualitatively examined (FIG. 2). From the size of the real-time PCR product, it was suggested that the exosome 1 does not substantially contain the Oct3 / 4 gene and the Sox-2 gene and may contain the Nanog gene.

(5) Addition experiment to human dermal fibroblasts A 6-well plate (AGC Asahi Glass) for tissue culture was prepared, and 1 × 10 5 human dermal fibroblasts (obtained from Lonza) were seeded on this dish. After 24 hours, 100 μl of each of exosomes 1 to 3 was added to the dish (this time is defined as day 0). At the timing of day 1 and day 3, total RNA was recovered using ISOGEN II (NIPPON GENE).

(6) Quantification of mRNA in human skin fibroblasts by real-time RT-PCR From the above RNA, using the LightCycler Real-Time PCR System (Roche Applied Science), Oct3 / 4 gene, Sox-2 gene, Nanog gene, TERT Gene mRNA was quantified.

(7) The results are shown in FIGS. A vertical axis | shaft shows the relative expression level of mRNA of each gene.

  It was confirmed that the expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene was enhanced by bringing exosomes extracted from colonized placenta-derived cells into contact with dermal fibroblasts. On the other hand, even when exosomes extracted from human placenta-derived cells and human calvarial osteoblasts that were not colonized were brought into contact with dermal fibroblasts, the increased expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene was Not observed. Moreover, no change was observed in the expression level of the TERT gene in dermal fibroblasts contacted with exosomes extracted from colonized placenta-derived cells.

Claims (17)

  1. A composition for enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell,
    The said composition containing the exosome extracted from the placenta origin cell colonized by culture | cultivation.
  2.   The composition of claim 1, wherein the placenta-derived cells comprise placenta-derived stem cells.
  3.   The composition according to claim 1 or 2, which is a composition for enhancing the expression of Oct3 / 4 gene, Nanog gene and Sox-2 gene as transcription factors involved in reprogramming.
  4.   The composition according to any one of claims 1 to 3, wherein the exosome is substantially free of Oct3 / 4 gene and Sox-2 gene.
  5.   The composition of any one of Claims 1-4 which is a composition for reprogramming a target cell.
  6. A method for producing a composition for enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell,
    a) culturing the placenta-derived cells to form colonies;
    b) obtaining colony-formed placenta-derived cells and further culturing;
    c) a step of extracting exosomes from the culture supernatant obtained in step b);
    Said method.
  7. The method according to claim 6, wherein step a) is a step of seeding and culturing placenta-derived cells at a density of 14 cells / cm 2 or less to form colonies.
  8.   The method according to claim 6 or 7, wherein step b) is a step of further culturing in a serum-free medium.
  9.   The method according to any one of claims 6 to 8, wherein the composition is a composition for enhancing the expression of Nanog gene, Oct3 / 4 gene and Sox-2 gene as a transcription factor involved in reprogramming.
  10.   The method according to any one of claims 6 to 9, wherein the placenta-derived cells comprise placenta-derived stem cells.
  11. A method for enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell,
    a) culturing the placenta-derived cells to form colonies;
    b) obtaining colony-formed placenta-derived cells and further culturing;
    c) a step of extracting exosomes from the culture supernatant obtained in step b);
    d) contacting the exosome with the target cell;
    Said method.
  12. The method according to claim 11, wherein step a) is a step of seeding and culturing placenta-derived cells at a density of 14 cells / cm 2 or less to form colonies.
  13.   The method according to claim 11 or 12, wherein step b) is a step of further culturing in a serum-free medium.
  14.   The method according to any one of claims 11 to 13, wherein expression of Nanog gene, Oct3 / 4 gene and Sox-2 gene is enhanced as a transcription factor involved in reprogramming.
  15.   The method according to any one of claims 11 to 14, wherein the placenta-derived cells comprise placenta-derived stem cells.
  16.   The method according to any one of claims 11 to 15, which is a method for reprogramming a target cell by enhancing expression of a plurality of transcription factors involved in reprogramming in the target cell.
  17.   The method according to any one of claims 11 to 15, which is a method for producing a multipotent cell by enhancing expression of a plurality of transcription factors involved in reprogramming in a target cell.
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