WO2018025975A1 - インビトロで多能性幹細胞を分化誘導する方法 - Google Patents
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Definitions
- the present invention relates to a method for inducing differentiation of pluripotent stem cells in vitro. More specifically, the present invention relates to a pluripotent stem cell or a cell fraction enriched with the pluripotent stem cell as a damaged cell and / or a dead cell (including a part thereof) derived from an information-presenting cell. The present invention relates to a method for inducing differentiation of pluripotent stem cells into cells having the same phenotype and function as information-presenting cells in vitro, including co-culture.
- pluripotent stem cells collected from one's own or another family, pluripotent stem cells stored in banks, or cells induced to differentiate from these pluripotent stem cells are transplanted into the target disease to treat the disease. It has been tried to do.
- pluripotent stem cells derived from healthy subjects and target cells derived from disease-specific pluripotent stem cells obtained from patients are used for analysis and screening, and new drugs for these diseases are used. Is expected to develop. Furthermore, in the drug safety test, it is expected that differentiation-induced cells are used for screening of the toxicity of the drug being developed or developed.
- Non-patent Document 1 a method of inducing cardiomyocyte differentiation without using cytokines has also been proposed (Non-patent Document 1).
- this proposal after feeder-less culture, the cells are isolated into single cells and then seeded at a high density to perform adhesion culture.
- the cells can be adapted to feeder-less culture.
- SSEA-3 Serial-Specific present in the mesenchymal cell fraction found by Mr. Dezawa, one of the present inventors.
- a typical example is Muse (Multilineage-differentiating Stress Ending cells) expressing Embryonic Antigen-3) as a surface antigen (Patent Document 3; Non-Patent Document 2).
- Muse cells are different from other pluripotent stem cells known as induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), neural stem / progenitor cells (NSPC), cord blood stem cells (UCBC), It is easy to procure cells, does not require gene transfer for procuring such as iPS cells, and does not have tumorigenicity.
- iPS cells induced pluripotent stem cells
- ES cells embryonic stem cells
- NSPC neural stem / progenitor cells
- UCBC cord blood stem cells
- Muse cells have been widely known for their contribution to regenerative medicine, and can be found in the treatment of myocardial infarction, brain tumor, cerebral infarction, renal disorder, etc.
- Patent Document 4 For example, in the treatment of myocardial infarction, it has been found that the treatment of myocardial infarction is achieved by injecting Muse cells from veins, the Muse cells reach the infarcted site, and differentiate into myocardial cells.
- the mechanism by which Muse cells administered intravenously migrate to a diseased site is gradually known, and, for example, a migration factor (injury signal) such as SIP1 that is considered to be released from the diseased site is involved.
- a migration factor injury signal
- SIP1 a migration factor
- the treatment of diseases using these cells may involve cell injury and inflammatory reaction associated with it as in acute diseases such as myocardial infarction that emits injury signals from the injury site, but the damaged cells.
- acute diseases such as myocardial infarction that emits injury signals from the injury site, but the damaged cells.
- Muse cell administration a series of Muse cell administration, migration, and differentiation while some of them are still in place, that is, "field logic" is alive. It can be said.
- the release of injury signals and the “field logic” has already disappeared.
- the aforementioned Muse cell blood vessels In many cases, it cannot be achieved only by internal administration.
- pluripotent stem cells such as Muse cells in large quantities, stably, easily and at low cost, and desired cells can be obtained, regenerative medicine, drug discovery research, drug safety Further application to various uses such as sex testing becomes possible.
- an object of the present invention is to provide a method for easily and efficiently preparing various differentiated cells having uniform and high functions.
- Muse cells have phagocytic properties in vitro and differentiate into these cells by phagocytosing damaged or dead cells (including some of them). It came to complete.
- the present invention is as follows.
- [1] In vitro, including co-culture of pluripotent stem cells or a cell fraction enriched with the pluripotent stem cells with damaged cells, dead cells, and / or parts thereof derived from information-presenting cells
- [2] The method according to [1] above, wherein the pluripotent stem cells phagocytose damaged cells derived from information-presenting cells, dead cells, and / or a part thereof.
- the pluripotent stem cells are SSEA-3-positive cells isolated from mesenchymal tissue or cultured mesenchymal cells in a living body.
- the pluripotent stem cell is CD105 positive.
- the pluripotent stem cells are CD117 negative and CD146 negative.
- the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative, and CD271 negative.
- the pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative.
- the pluripotent stem cell is a pluripotent stem cell having all of the following properties: (I) low or no telomerase activity; (Ii) has the ability to differentiate into cells of any germ layer of the three germ layers; (Iii) no neoplastic growth; and (iv) self-renewal ability.
- the information-presenting cell is an ectoderm-derived cell.
- the ectoderm-derived cells are selected from the group consisting of nerve cells, glial cells, pigment cells, skin cells, inner ear cells, retinal cells, corneal cells, and hair root cells.
- the information-presenting cell is a mesoderm-derived cell.
- the mesoderm-derived cells are selected from the group consisting of cardiomyocytes, skeletal muscle cells, smooth muscle cells, bone cells, chondrocytes, germline cells, and hematopoietic cells. .
- the information-presenting cell is derived from endoderm.
- the endoderm-derived cell is selected from the group consisting of pancreatic ⁇ cells, hepatocytes, bile duct cells, respiratory epithelial cells, gastrointestinal epithelial cells, vascular endothelial cells, renal constituent cells, bladder epithelial cells, and pancreatic exocrine cells.
- [17] The method according to any one of [1] to [10] above, wherein the information-presenting cell is a rare cell.
- the rare cells are motor neurons, dopamine neurons, interneurons, glutamatergic neurons, pituitary cells, thyroid cells, adrenal cortex, adrenal medulla, carotid pressure sensor cells, cardiac stimulation transmission system, choroid plexus
- a method for confirming that cells contained in a cell preparation in vitro are viable pluripotent stem cells, (A) co-culturing cells in a cell preparation with damaged cells, dead cells, and / or parts thereof; and (b) cells in the cell preparation are damaged cells, dead cells, and / or thereof When differentiated into cells having the same phenotype and function as ectoderm cells, mesodermal cells, and / or endoderm cells derived from a part, pluripotent stem cells survive in the cell preparation.
- the method comprising determining to be.
- pluripotent stem cells are pluripotent by coculturing pluripotent stem cells with injured cells, dead cells, and / or parts thereof derived from information-presenting cells. It can be easily differentiated from sex stem cells. The differentiated cells thus obtained can be applied to various uses such as regenerative medicine, drug discovery research, and drug safety testing.
- FIGS. 1A and 1B show the results of detection of mRNA expression of various markers when Muse cells were added to culture supernatants or exosomes obtained from normal mouse cardiomyocytes, respectively.
- FIG. 1C shows the results of examination of mRNA expression when normal mouse cardiomyocytes were brought into direct contact with Muse cells.
- FIG. 1D shows the results of examining the presence or absence of expression of GATA-4 by immunostaining after 3 days and 7 days of culture by direct contact and observing under a fluorescence microscope. The result of having verified about the differentiation induction using an injured mouse
- FIG. 7A is a fluorescence microscope image showing that Muse cells (green) express myogenin (red), which is a marker of skeletal muscle cells.
- FIG. 7B shows that Muse cells differentiated into skeletal muscle cells using MyoD as an index. It is a result which shows the differentiation induction to a neural progenitor cell.
- Muse cells differentiated into nerve cells using Neuro2A as an index It is a result which shows the differentiation induction to (beta) cell. It shows that Muse cells have differentiated into ⁇ cells using the precursor cell markers (Nkx2.2 and NeuroD1) and the mature ⁇ cell marker (Islet1) as indices. It is a result which shows the differentiation induction to a hepatocyte. It shows that Muse cells differentiated into hepatocytes using AFP as an index.
- the present invention is a method for inducing differentiation of pluripotent stem cells in vitro, and by coculturing pluripotent stem cells with damaged cells or dead cells, the same phenotype as the cells that are the origin of damaged cells or dead cells And a method for obtaining a cell having a function.
- the present invention will be described below.
- the present invention relates to an in vitro method comprising co-culturing a pluripotent stem cell or a cell fraction enriched with the pluripotent stem cell with a damaged cell, a dead cell, and / or a part thereof derived from an information-presenting cell.
- the present invention relates to a method for inducing differentiation of pluripotent stem cells into information-presenting cells.
- the pluripotent stem cell has the same phenotype as the original information-presenting cell from which the damaged cell or the like is derived by phagocytosing the damaged cell, dead cell, and / or part thereof in co-culture. Differentiate into functional cells.
- Muse cells As for the phagocytosis of pluripotent stem cells, particularly Muse cells, Muse cells have been known to phagocytose ferrite (magnetic material) (JP 2016-028614), but Muse cells are used as information recognition cells. It is not known to differentiate by phagocytosing damaged cells, which are information-presenting cells. As described above, the present invention is based on the first discovery that Muse cells can differentiate into original information-presenting cells that are the origin of damaged cells and the like by phagocytosing damaged cells and the like. In vitro differentiation induction using the phagocytosis of such pluripotent stem cells can be achieved by simply co-culturing pluripotent stem cells with damaged cells or the like. The method to be performed can be easily performed based on a method commonly used by those skilled in the art, and is not limited.
- the present invention provides: (A) culturing information-presenting cells; (B) a step of injuring or killing information-presenting cells in the culture system; and (c) adding pluripotent stem cells to the culture system of step (b) and culturing the pluripotent stem cells in vitro.
- a method of inducing differentiation may be used.
- the present invention provides: (A ′) culturing pluripotent stem cells; and (b ′) injuring or killing information-presenting cells to obtain injured cells, dead cells, and / or parts thereof; and (c ′) step Pluripotent stem cells may be induced to differentiate in vitro, including the step of adding the damaged cells, dead cells, and / or part thereof obtained in step (b) to the culture system of (a) and culturing them. .
- Pluripotent stem cell Typically, the pluripotent stem cell used in the differentiation induction method of the present invention was found by Mr. Dezawa, one of the present inventors, in human body, Muse (Multilineage-differentiating Stress Ending) cell ”. Muse cells are obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)) and dermal connective tissue. Can also be scattered in the connective tissue of each organ. In addition, this cell is a cell having the properties of both pluripotent stem cells and mesenchymal stem cells.
- Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, these antigen markers as indicators.
- Muse cells are stress-resistant and can be concentrated from mesenchymal tissues or cultured mesenchymal cells by various stress stimuli.
- a cell fraction in which Muse cells are concentrated by stress stimulation can also be used. Details such as a method for separating Muse cells, an identification method, and characteristics are disclosed in International Publication No. WO2011 / 007900. Also, as reported by Wakao et al.
- Muse cells when separating Muse cells from living mesenchymal tissue or cultured mesenchymal stem cells, Muse cells can be purified and used simply with SSEA-3 as an antigen marker.
- pluripotent stem cells Muse cells isolated from living mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker, which can be used in the differentiation induction method of the present invention.
- SSEA-3 positive cells a cell population containing Muse cells.
- “non-Muse cells” refer to cells other than “SSEA-3-positive cells”, which are cells contained in a mesenchymal tissue or cultured mesenchymal tissue in a living body.
- Muse cells or cell populations containing Muse cells can be obtained from living tissue (eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively) , Mesenchymal tissue).
- living tissue eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively
- Mesenchymal tissue e.g., Mesenchymal tissue.
- “living body” means a living body of a mammal. In the present invention, the living body does not include embryos whose developmental stage is earlier than the fertilized egg or blastocyst stage, but includes embryos in the developmental stage after the blastocyst stage including the fetus and blastocyst.
- Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. It is done.
- Muse cells used in the differentiation induction method of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are separated from living tissues using markers directly.
- ES cells embryonic stem cells
- iPS cells embryonic stem cells
- “Mesenchymal tissue” refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, umbilical cord blood, and tissues present in various organs.
- Muse cells can be obtained from bone marrow, skin, or adipose tissue.
- Muse cells may be separated from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the separation means.
- the Muse cell used may be autologous to the recipient or may be autologous.
- a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using, for example, SSEA-3 positive and SSEA-3 and CD105 double positive as an index.
- SSEA-3 positive and SSEA-3 and CD105 double positive are known to include various types of stem cells and progenitor cells.
- Muse cells are not the same as these cells.
- Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblast (MB), perivascular cells (PC), endothelial progenitor cells (EP), adipose-derived stem cells (ADSC). ).
- Muse cells can be isolated using “non-expression” of a marker unique to these cells as an index.
- Muse cells are CD34 (EP and ADSC markers), CD117 (c-kit) (MB markers), CD146 (PC and ADSC markers), CD271 (NGFR) (NCSC markers), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and At least one of 11 markers selected from the group consisting of Dct (MB marker), for example 2, 3, 4, 5, 6, 7, 8, 9, 10 The non-expression of individual or eleven markers can be separated into indicators.
- non-expression of CD117 and CD146 can be separated as an index
- non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index
- the non-expression of 11 markers can be separated as an index.
- the Muse cell having the above characteristics used in the differentiation induction method of the present invention is as follows: (I) low or no telomerase activity; (Ii) has the ability to differentiate into cells of any germ layer of the three germ layers; It may have at least one property selected from the group consisting of (iii) showing no neoplastic growth; and (iv) having a self-renewal capability.
- the Muse cell used in the differentiation induction method of the present invention has all the above properties.
- telomerase activity is low or absent means that, for example, when telomerase activity is detected using TRAPEZE XL telomerase detection kit (Millipore), it is low or cannot be detected.
- telomerase activity means, for example, telomerase having a telomerase activity comparable to that of somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less compared to Hela cells. It means having activity.
- the Muse cell has the ability to differentiate into three germ layers (endoderm, mesodermal, and ectoderm) in vitro and in vivo, for example, induction culture in vitro Can be differentiated into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. In addition, when transplanted to the testis in vivo, it may show the ability to differentiate into three germ layers.
- Muse cells grow at a growth rate of about 1.3 days in suspension culture, but grow from 1 cell in suspension culture to form an embryoid body-like cell mass and stop growing in about 14 days. However, when these embryoid body-like cell masses are brought into an adhesion culture, cell proliferation is started again, and the proliferated cells spread from the cell masses. Furthermore, when transplanted to the testis, it has the property of not becoming cancerous for at least half a year.
- a Muse cell has self-renewal (self-replication) ability.
- self-renewal means that differentiation from cells contained in embryoid body-like cell clusters obtained by culturing in suspension culture from one Muse cell to trioderm cells can be confirmed, Bring the cells of embryoid body-like cell mass to suspension culture with one cell again to form the next generation embryoid body-like cell mass, from which again embryos in trioderm differentiation and suspension culture This means that a clot-like cell mass can be confirmed.
- the self-renewal may be repeated once or multiple times.
- the cell fraction containing Muse cells used in the differentiation induction method of the present invention provides external stress stimulation to living mesenchymal tissue or cultured mesenchymal cells and recovers stress-resistant cells. It may be a cell fraction enriched in SSEA-3 positive and CD105 positive pluripotent stem cells having at least one, preferably all, of the following properties obtained by the method comprising: (I) SSEA-3 positive; (Ii) CD105 positive; (Iii) low or no telomerase activity; (Iv) has the ability to differentiate into three germ layers; (V) exhibits no neoplastic growth; and (vi) has the ability to self-renew.
- the above external stress includes protease treatment, culture at low oxygen concentration, culture under low phosphate conditions, culture at low serum concentration, culture under low nutrient conditions, culture under exposure to heat shock, low temperature Incubation in freezing, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment or physical impact Any one or a plurality of combinations may be used.
- the protease treatment time is preferably 0.5 to 36 hours in total in order to give external stress to the cells.
- the protease concentration may be a concentration used when peeling cells adhered to the culture vessel, separating the cell mass into single cells, or collecting single cells from the tissue.
- the protease is preferably a serine protease, aspartic protease, cysteine protease, metalloprotease, glutamic acid protease or N-terminal threonine protease. Further, the protease is preferably trypsin, collagenase or dispase.
- Information-presenting cell used in the in vitro differentiation-inducing method of the present invention has the same phenotype and functional characteristics as a cell finally differentiated from a pluripotent stem cell, It means a cell before differentiation induction, which is a source of damaged cells or dead cells.
- the information-presenting cells used in the present invention may have any origin as long as desired differentiated cells can be obtained from pluripotent stem cells.
- the origin of the cell may originate from various mammals, depending on its purpose, and is not limited to humans, chimpanzees, other primates, livestock animals such as dogs, cats, livestock animals such as cows, pigs, Derived from horses, sheep, goats, laboratory animals such as rabbits, rats, mice, guinea pigs and the like.
- the damaged cells or dead cells used for differentiating pluripotent stem cells into desired cells can originate from the information-presenting cells.
- the term “damaged cell” refers to, for example, proliferation, even when cultured under suitable culture conditions, for example, because it is damaged by artificial or environmental stress. Although difficult, the metabolic activity of the cell is reduced compared to a living cell, but it refers to a cell in a state that is significantly more active than a dead cell.
- the term “dead cell” refers to a cell that is incapable of growing even when cultured under suitable culture conditions and does not exhibit metabolic activity.
- the “dead cell” may be a dead cell due to necrosis or a cell death due to apoptosis.
- the phagocytosis of pluripotent stem cells is not limited to the above-mentioned damaged cells and dead cells, but some of them (for example, cytoplasm, cell membrane, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, cytoplasm) Can also be shown for cytoskeleton, enzyme, protein, RNA, DNA) or cell debris.
- the method of obtaining damaged cells, dead cells, and / or a part thereof from information-presenting cells is not limited, and can be performed according to a commonly used method.
- a drug that inhibits cell growth as known in the art can be used.
- agents include, but are not limited to, those that significantly reduce the percentage of cells in S phase, and typically agents that block the progression of the cell division cycle, such as G1 arrest and M phase arrest.
- antimetabolite / anticancer agents eg, azathioprine, 6-mercaptopurine, 6-thioguanine, fludarabine, pentostatin, cladribine, 5-fluorouracil ( 5FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), methotrexate, trimethoprim, pyrimethamine, pemetrexed, capecitabine, gemcitabine, cytarabine); alkylating agents (eg, cyclophosphamide, mechlorethamine, uramustine, melphalan) , Chlorambucil, thiotepa / chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, dibromomannitol, cisplatin, carboplatin, nedaplatin
- antimetabolite / anticancer agents eg, azathi
- the concentration of the reagent, the method of use, further the culture conditions, etc. are as usual, and those skilled in the art can appropriately determine them.
- the concentration suitable for obtaining can be changed according to the type of reagent used, but 1 to 100 ⁇ M is preferable.
- the information-presenting cells may be subjected to UV irradiation and / or X-ray treatment.
- UV irradiation and X-ray treatment can be performed by appropriately selecting conditions known in the art. Note that UV irradiation and X-ray treatment may be performed in combination with the above reagents.
- Reagents that can be used to make dead cells include those that can be used to make the above-mentioned damaged cells, for example to increase their use concentration or achieve their purpose by long-term use. Can do. Further, when performing UV irradiation or X-ray processing, for example, the intensity of UV or X-rays may be increased by extending the time of irradiation or the like.
- means for producing damaged or dead cells include, but are not limited to, heat, cold, acid or alkali treatment, ultrasound or vortex treatment, freeze thawing,
- Examples of the method include low oxygen treatment, low nutrient treatment, and active oxygen treatment. Regarding the use of these means, those skilled in the art can select and use conditions as appropriate.
- the method of the present invention may include means for confirming whether damaged cells and dead cells obtained by the above method have been prepared.
- the confirmation means include, but are not limited to, observation under a microscope, use of a commercially available kit for measuring cell damage, and physiological or immunological measurement of contents secreted from cells.
- dead cells dead cells resulting from apoptosis can be determined based on observation of unique morphological abnormalities such as cell surface curvature, nuclear chromatin condensation, and chromosomal DNA fragmentation.
- a method of staining dead cells with trypan blue, or colorimetric analysis (MTT assay) with tetrazolium can be used.
- the present invention relates to a method for inducing differentiation of pluripotent stem cells in vitro by co-culturing pluripotent stem cells with damaged cells, dead cells, and / or parts thereof.
- co-culture means culturing two or more types of cells (or a part) in the same environment, and pluripotent stem cells are placed in the same culture vessel or petri dish. It refers to culturing with a predetermined number of damaged cells.
- the ratio of the number of pluripotent stem cells to the number of injured or dead cells derived from information presenting cells is 1: 10,000 to 10,000: 1, for example 1: 10,000, 1: 7,000. 1: 5,000, 1: 3,000, 1: 2,000, 1: 1,000, 1: 700, 1: 500, 1: 300, 1: 200, 1: 100, 1:70, 1 : 50, 1:30, 1:20, 1:10, 1: 7, 1: 5, 1: 3, 1: 2, 1: 1, 2: 1, 3: 1, 5: 1, 7: 1 10: 1, 20: 1, 30: 1, 50: 1, 70: 1, 100: 1, 200: 1, 300: 1, 500: 1, 700: 1, 1,000: 1, 2,000 1: 3,000: 1, 5,000: 1, 7,000: 1, 10,000: 1.
- the culture solution for culturing the cells may be a medium that matches the cells to be actually cultured, and generally DMEM, RPMI-1640, Ham's F12, etc. can be used. Ordinarily used antibiotics, fetal bovine serum, antibiotics for selecting transgenic cells, etc. may be added at a concentration of 0.1 to 10%.
- Culture conditions such as culture temperature are in accordance with normal cell culture conditions, for example, 36 to 38 ° C., preferably 37 ° C., and 4 to 6% CO 2 , preferably 5% CO 2 atmosphere. obtain.
- the co-culture time is approximately 2 to 10 days, although it depends on the cell type that is induced and finally obtained.
- Differentiation induction means that a pluripotent stem cell is led to a cell constituting a specific organ, organ or tissue or a progenitor cell thereof by some means. Includes differentiation into a group of cells including many types of cells such as endoderm cells, mesoderm cells, and ectoderm cells. According to the present invention, differentiation induction from pluripotent stem cells to specific cells is achieved by simply co-culturing pluripotent stem cells with damaged cells and the like, and reagents other than damaged cells and UV irradiation are required. It is characterized by not.
- Such differentiation induction is caused by phagocytosis of pluripotent stem cells against damaged cells, etc., and any humoral factor or exosome secreted from the corresponding information-presenting cell before injury or death, or information-presenting cell It was suggested that this was not due to direct contact between cells (see Example 1).
- Confirmation of differentiation-induced cells can be performed by a technique performed in the technical field, and is typically performed by examining the presence or absence of gene expression or protein expression of a predetermined differentiation marker in cells. it can. Examples of such methods include RT-PCR method, Northern blot method, flow cytometry method, Western blot method, ELISA method and the like. For example, whether or not pluripotent stem cells have been differentiated into cardiomyocytes is determined by Nkx2.5 (NK-2 transcription factor related, locus 5), GATA-4, troponin-T, ANP (atrial) It can be confirmed at the mRNA level using the RT-PCR method that the gene expression of natriuretic peptide) is observed in differentiation-induced cells (Example 3).
- (6) Use Cells that contain pluripotent stem cells by utilizing the property that pluripotent stem cells differentiate by phagocytosing damaged cells, dead cells, and / or parts thereof into cells derived therefrom. It can be applied to quality control of pharmaceutical preparations.
- a method for confirming that cells contained in a cell preparation in vitro are viable pluripotent stem cells, (A) co-culturing cells in a cell preparation with damaged cells, dead cells, and / or parts thereof; and (b) cells in the cell preparations being damaged cells, dead cells, and / or parts thereof.
- pluripotent stem cells When pluripotent stem cells are alive in the cell preparation when differentiated into cells having the same phenotype and function as ectoderm cells, mesoderm cells, and / or endoderm cells derived from A method is provided that includes determining.
- the method for confirming whether pluripotent cells for example, Muse cells
- three germ layer cells ectodermal cells, mesodermal cells, and endoderm cells
- the method comprises co-culturing a pluripotent stem cell or a cell fraction enriched with the pluripotent stem cell with a damaged cell, a dead cell, and / or a part thereof derived from an information-presenting cell,
- a method for producing cells having the same phenotype and function as information-presenting cells from pluripotent stem cells in vitro can be provided.
- Example 1 Analysis of differentiation induction mechanism of Muse cells Muse cells migrate to an injured site by an injury signal (eg, S1P) emitted from the injured site in vivo, and after differentiation, differentiate according to "field logic". Although induced, the mechanism by which Muse cells differentiate is not known in detail. In this example, the mechanism by which differentiation induction of Muse cells is performed was verified in vitro. Elucidating how Muse cells receive “field logic” information, that is, what factors are thought to be related to differentiation induction, disappears from “field logic”. It becomes the basis for the application to regeneration of rare cells and tissues and organs that are difficult to reach through chronic diseases and blood vessels.
- an injury signal eg, S1P
- Muse cells As information of “field logic” that is considered to be received by Muse cells, humoral factors and exosomes secreted from information-presenting cells, or direct contact with information-presenting cells without being mediated by such factors, Alternatively, it is assumed that Muse cells phagocytose damaged information-presenting cells. Therefore, the possibility of inducing differentiation of Muse cells by humoral factors, exosomes, and direct contact was examined using normal mouse cardiomyocytes and damaged mouse cardiomyocytes.
- human Muse cells were obtained by the method described in International Publication No. WO2011 / 007900. Muse cells were seeded on a plate (Nunc multidisch 6 unclon delta SI, Thermo, # 140675) at 1 ⁇ 10 4 cells / well and cultured for 24 hours. Subsequently, the culture supernatant or exosome derived from normal mouse cardiomyocytes obtained above was added, and the culture was continued. The cultured Muse cells were collected 3 days, 1 week, 2 weeks, and 3 weeks after the addition, and it was examined whether the Muse cells were differentiated into the same cardiomyocytes as the information-presenting cells.
- Nkx2.5 and GATA-4 were selected as early differentiation markers expressing myocardial progenitor cells, ANP as an undifferentiated cardiomyocyte marker, and troponin-T as a mature myocardial differentiation marker.
- Muse cells express cardiomyocyte markers when using either humoral factors (culture supernatant) (FIG. 1A) or exosomes (FIG. 1B) obtained from normal mouse cardiomyocytes. And differentiation was not induced in cardiomyocytes.
- the primers used for various markers as detection targets in RT-PCR are Nkx2.5 (NM 004387; SEQ ID NO: 1), GATA-4 (NM 002052; SEQ ID NO: 4), troponin-T (NM 000364; SEQ ID NO: 7), and ANP (NM Designed and synthesized based on the nucleotide sequence of 006172; SEQ ID NO: 10). Specifically, it is as follows.
- Nkx2.5 Forward primer 5′-CAGGACCAGACTCTGGAGCTG-3 ′ (SEQ ID NO: 2) (corresponding to 821-841 nt of SEQ ID NO: 1)
- Reverse primer 5'-CGCCGAAGTTCACGAAGTTGT-3 '(SEQ ID NO: 3) (corresponding to 1118-1098nt of SEQ ID NO: 1)
- GATA-4 Forward primer 5′-AGCAGCTCCGTGTCCCACACGTT-3 ′ (SEQ ID NO: 5) (corresponding to 1683-1705 nt of SEQ ID NO: 4)
- Reverse primer 5′-CCAACTCACAGGGAGATGGCAGTGTG-3 ′ (SEQ ID NO: 6) (corresponding to 2139-2114nt of SEQ ID NO: 4)
- Troponin-T Forward primer 5′-CTCAAAAGACAGGATCGAGAG-3 ′ (SEQ ID NO: 8) (corresponding to 489-508nt of SEQ ID NO: 7)
- Reverse primer 5′-GATCTTCATCA
- Muse cells could be induced to differentiate by direct contact with normal mouse cardiomyocytes.
- gene transfer was performed on normal mouse cardiomyocytes so as to express green fluorescent protein (GFP).
- GFP-labeled cardiomyocytes were seeded at 3 ⁇ 10 4 cells / well on a plate (Nunc multidissimilar 6 nunclon delta SI, Thermo, # 140675) and cultured. Thereafter, human Muse cells whose nuclei were previously labeled with DAPI were added at 1 ⁇ 10 4 cells / well, and the culture was continued.
- FIG. 1C After 3 days, 1 week, 2 weeks, and 3 weeks after the start of culture, the presence or absence of human-specific mRNA expression of a myocardial differentiation marker was examined by RT-PCR, as described above (FIG. 1C). Similar to the culture supernatant and exosome results described above, no cardiomyocyte marker expression was observed. Also, after 3 days and 7 days in culture, the presence or absence of GATA-4 expression was examined by immunostaining, and the results of observation under a fluorescence microscope are shown in FIG. 1D. In Muse cells whose nuclei were labeled in blue, GATA-4 expression was not observed. From the above results, it was found that Muse cells were not induced to differentiate by cell contact with normal mouse cardiomyocytes.
- Injured mouse cardiomyocytes were prepared using etoposide that induces apoptosis. Briefly, GFP-labeled mouse cardiomyocytes were seeded at 3 ⁇ 10 4 cells / well, and 50 ⁇ M etoposide (SIGMA, # E1383) was added after 2 days of culture. One day after the culture, the culture supernatant of apoptosis-inducing mouse cardiomyocytes was collected, and human Muse cells seeded at 1 ⁇ 10 4 cells / well were cultured.
- Example 2 Verification of phagocytosis of Muse cells Muse cells have been reported to phagocytose ferrite, but it has not been investigated whether damaged cells, dead cells, or parts thereof can also phagocytose. Therefore, the phagocytosis of Muse cells was re-verified using PKH26PCL beads.
- PKH26PCL (1 ⁇ 10 ⁇ 3 M stock in ethanol; SIGMA, # PKH26PCL) is a dye that can selectively label phagocytic cells such as macrophages, neutrophils, and microglia. Widely used in the field.
- Example 3 Differentiation induction by co-culture of Muse cells and damaged cells The possibility that Muse cells directly receive information on "field logic" and differentiate by phagocytosing damaged cells was examined.
- Injured cardiomyocytes were prepared using etoposide which is a topoisomerase inhibitor.
- etoposide which is a topoisomerase inhibitor.
- the GFP-labeled mouse-derived cardiomyocyte prepared in Example 1 was used as a host cardiomyocyte.
- GPR mouse cardiomyocytes were seeded on a plate at a cell density of 3 ⁇ 10 4 cells / well, and 50 ⁇ M etoposide was added to each well in a culture system cultured for 1 day. Thereafter, the cells were further cultured for 1 day. Next, labeled human Muse cells were added at 1 ⁇ 10 4 cells / well, and co-culture was performed.
- annexin V is added to the culture system to suppress the phagocytic ability of the cells. Therefore, it was examined whether differentiation induction was inhibited.
- etoposide 50 ⁇ M
- 1 ⁇ g / mL Annexin V was added and cultured for 8 hours in the same culture system as the above co-culture.
- Muse cells (1 ⁇ 10 4 cells / well) were added to this system, and the culture was continued.
- Total RNA was extracted from Muse cells 3 days, 1 week, 2 weeks, and 3 weeks after the addition of Annexin V, and the presence or absence of gene expression of each marker was confirmed by RT-PCR (FIG. 6).
- Nkx2.5 and GATA4 are differentiation markers that are early in the differentiation process and are expressed in younger cardiomyocytes.
- Muse cells In the samples without annexin V added after 3 days and 1 week, Muse cells However, expression of these differentiation markers was not observed in the sample to which Annexin V was added. Similarly, even in ANP and troponin T (TNT) whose expression is observed at a later stage, the expression of differentiation markers was not observed in Muse cells due to annexin V. In this way, inhibition of Muse cell phagocytosis inhibited differentiation into cardiomyocytes, so Muse cells phagocytose dead cells or dying cells (damaged cells) to obtain “field logic” information. Obtained and shown to be induced to differentiate.
- FIGS. 7A and 7B The results of observation under a fluorescence microscope and the results of RP-PCR of MyoD expression are shown in FIGS. 7A and 7B, respectively.
- Muse cells green
- myogenin red
- myoD mRNA expression one week after the start of co-culture.
- C2C12 C2C12 that induced injury It was shown to differentiate into skeletal muscle cells, which are mesoderm cells.
- the primers used for various markers as detection targets in RT-PCR are MyoD (NM 002478; designed and synthesized based on the nucleotide sequence of SEQ ID NO: 13). Specifically, it is as follows. Forward primer: 5′-GAGCAATCCAAAACCAGCGGTTG-3 ′ (SEQ ID NO: 14) (corresponding to 631-652nt of SEQ ID NO: 13) Reverse primer: 5′-TAGTAGGCGCCTTCGTAGCAG-3 ′ (SEQ ID NO: 15) (corresponding to 912-892nt of SEQ ID NO: 13)
- ectoderm cells Differentiation into ectoderm cells (neuronal cells) Induction of differentiation of Muse cells into ectoderm cells was examined using injured nerve cells (Neuro2A, ATCC, # CCL-131). In the same manner as described above, nerves in which injury was induced by etoposide treatment were prepared and co-cultured with Muse cells. Total RNA was extracted from Muse cells 3 days and 1 week after the start of co-culture and verified for differentiation induction. More specifically, NeuN mRNA expression, which is known as a neuronal cell marker, was examined by RT-PCR. There was no NeuN expression in mouse embryos (15-day culture) and human MSC-Muse cells (L26).
- ⁇ cells ⁇ cells in which injury was induced by etoposide treatment were prepared and co-cultured with Muse cells. Three days after the start of co-culture and 1-3 weeks later, total RNA was extracted from Muse cells and verified for differentiation induction. More specifically, the mRNA expression of Nkx2.2 and NeuroD1 known as ⁇ cell progenitor cell markers and Islet1 known as mature ⁇ cell markers was examined by RT-PCR. As shown in FIG.
- Muse cells differentiate from progenitor cells to mature ⁇ cells over time of co-culture Muse cells were shown to be differentiated into ⁇ cells that are endoderm cells by co-culture with injured ⁇ cells.
- the primers used for various markers as detection targets in RT-PCR are Nkx2.2 (NM 002509; SEQ ID NO: 16), NeuroD1 (NM 002500; SEQ ID NO: 19), Islet1 (NM Designed and synthesized based on the nucleotide sequence of SEQ ID NO: 22). Specifically, it is as follows.
- hepatocytes Differentiation into endoderm cells (hepatocytes) Using hepatocytes (mouse hepatoma cell line: Hepa1-6) (ATCC, # CRL-1830) in which injury was induced, to endoderm cells of Muse cells Differentiation induction was examined. In the same manner as described above, hepatocytes in which injury was induced by etoposide treatment were prepared and co-cultured with Muse cells. Total RNA was extracted from Muse cells 3 days after the start of co-culture and 1-2 weeks later, and differentiation induction was verified. More specifically, ⁇ -fetoprotein (AFP) mRNA expression observed in the early stage of hepatocyte development was examined by RP-PCR. As shown in FIG. 10, AFP expression is observed one week after the start of co-culture, and Muse cells can be differentiated into hepatocytes that are endoderm cells by co-culture with damaged hepatocytes. Indicated.
- AFP ⁇ -fetoprotein
- the primers used for various markers as detection targets in RT-PCR are AFP (NM Designed and synthesized based on the nucleotide sequence of 001134; SEQ ID NO: 25). Specifically, it is as follows. Forward primer: 5′-CCGAACTTTCCAAGCCATAACTG-3 ′ (SEQ ID NO: 26) (corresponding to 740-762nt of SEQ ID NO: 25) Reverse primer: 5′-CACTTCTCCAAATAACTCCCTGGTATC-3 ′ (SEQ ID NO: 27) (corresponding to 1195-1171nt of SEQ ID NO: 25)
- the method for inducing differentiation of pluripotent stem cells in vitro may induce differentiation into endoderm cells, mesoderm cells, and ectoderm cells using a common system from pluripotent stem cells, particularly Muse cells. it can. Furthermore, the present invention can be applied to the regeneration of a chronic disease in which the “field logic” has already disappeared, a tissue or an organ that is difficult to reach through blood vessels, or a rare cell.
- the differentiation induction method of the present invention can induce differentiated cells in a large amount from pluripotent stem cells such as Muse cells in a stable, easy and low-cost manner. Unlike cells in which a foreign gene has been introduced and differentiation-induced, there is no possibility of becoming a tumor and it can be used more safely.
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Abstract
Description
[1]多能性幹細胞又は該多能性幹細胞が濃縮された細胞画分を、情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部と共培養することを含む、インビトロで多能性幹細胞を情報提示細胞と同じ表現型及び機能を有する細胞に分化誘導する方法。
[2]多能性幹細胞が情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部を貪食することを特徴とする、上記[1]に記載の方法。
[3]前記多能性幹細胞が、生体の間葉系組織又は培養間葉系細胞から分離されたSSEA-3陽性細胞である、上記[1]又は[2]に記載の方法。
[4]前記多能性幹細胞が、CD105陽性である、上記[1]~[3]のいずれかに記載の方法。
[5]前記多能性幹細胞が、CD117陰性及びCD146陰性である、上記[1]~[4]のいずれかに記載の方法。
[6]前記多能性幹細胞が、CD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性、及びCD271陰性である、上記[1]~[5]のいずれかに記載の方法。
[7]前記多能性幹細胞が、CD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snai1陰性、Slug陰性、Tyrp1陰性、及びDct陰性である、上記[1]~[6]のいずれかに記載の方法。
[8]前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、上記[1]~[7]のいずれかに記載の方法:
(i)テロメラーゼ活性が低いか又は無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;及び
(iv)セルフリニューアル能を持つ。
[9]多能性幹細胞の数と情報提示細胞由来の傷害細胞又は死細胞の数の比が1:10,000~10,000:1である、上記[1]~[8]のいずれかに記載の方法。
[10]情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部が、アポトーシス誘導剤、代謝拮抗薬、アルキル化剤、アントラサイクリン、抗生物質、有糸分裂阻害剤、トポイソメラーゼ阻害剤、プロテアソーム阻害剤、抗がん剤、熱、低温、酸、アルカリ、超音波、及びボルテックスなどによる物理的破砕からなる群から選択される薬剤の添加によって得られたものである、上記[1]~[9]のいずれかに記載の方法。
[11]情報提示細胞が、外胚葉由来細胞である、上記[1]~[10]のいずれかに記載の方法。
[12]外胚葉由来細胞が、神経細胞、グリア細胞、色素細胞、皮膚細胞、内耳細胞、網膜細胞、角膜細胞、及び毛根細胞からなる群から選択される、上記[11]に記載の方法。
[13]情報提示細胞が、中胚葉由来細胞である、上記[1]~[10]のいずれかに記載の方法。
[14]中胚葉由来細胞が、心筋細胞、骨格筋細胞、平滑筋細胞、骨細胞、軟骨細胞、生殖系細胞、及び造血系細胞からなる群から選択される、上記[13]に記載の方法。
[15]情報提示細胞が、内胚葉由来である、上記[1]~[10]のいずれかに記載の方法。
[16]内胚葉由来細胞が、膵β細胞、肝細胞、胆管細胞、呼吸器系上皮細胞、消化管上皮細胞、血管内皮細胞、腎構成細胞、膀胱上皮細胞、及び膵外分泌細胞からなる群から選択される、上記[15]に記載の方法。
[17]情報提示細胞が、希少細胞である、上記[1]~[10]のいずれかに記載の方法。
[18]前記希少細胞が、運動ニューロン、ドーパミン神経、介在ニューロン、グルタミン作動性ニューロン、脳下垂体細胞、甲状腺細胞、副腎皮質、副腎髄質、頸動脈の圧センサー細胞、心臓刺激伝達系、脈絡叢上皮細胞、膵-alpha細胞、膵-delta細胞、及び肺クララ細胞からなる群から選択される、上記[17]に記載の方法。
[19]インビトロにおいて細胞製剤中に含有される細胞が、生存する多能性幹細胞であることを確認するための方法であって、
(a)細胞製剤中の細胞と、傷害細胞、死細胞、及び/又はその一部とを共培養し;及び
(b)該細胞製剤中の細胞が、傷害細胞、死細胞、及び/又はその一部に由来する外胚葉系細胞、中胚葉系細胞、及び/又は内胚葉系細胞と同じ表現型及び機能を有する細胞に分化した場合に、該細胞製剤中に多能性幹細胞が生存していると判断する
ことを含む上記方法。
[20]多能性幹細胞又は該多能性幹細胞が濃縮された細胞画分を、情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部と共培養することを含む、インビトロで多能性幹細胞から情報提示細胞と同じ表現型及び機能を有する細胞を製造する方法。
(a)情報提示細胞を培養する工程;
(b)上記培養系で情報提示細胞を傷害又は死滅させる工程;及び
(c)多能性幹細胞を工程(b)の培養系に添加し、培養する工程
を含む、多能性幹細胞をインビトロで分化誘導する方法であってもよい。
(a’)多能性幹細胞を培養する工程;及び
(b’)情報提示細胞を傷害又は死滅させて、傷害細胞、死細胞、及び/又はその一部を得る工程;及び
(c’)工程(a)の培養系に工程(b)で得られた傷害細胞、死細胞、及び/又はその一部を添加し、培養する工程
を含む、多能性幹細胞インビトロで分化誘導であってもよい。
本発明の分化誘導法に使用される多能性幹細胞は、典型的には、本発明者らの一人である出澤氏が、ヒト生体内にその存在を見出し、「Muse(Multilineage-differentiating Stress Enduring)細胞」と命名した細胞である。Muse細胞は、骨髄液、脂肪組織(Ogura,F.,et al.,Stem Cells Dev.,Nov 20,2013(Epub)(published on Jan 17,2014))や真皮結合組織等の皮膚組織から得ることができ、各臓器の結合組織にも散在する。また、この細胞は、多能性幹細胞と間葉系幹細胞の両方の性質を有する細胞であり、例えば、それぞれの細胞表面マーカーである「SSEA-3(Stage-specific embryonic antigen-3)」と「CD105」のダブル陽性として同定される。したがって、Muse細胞又はMuse細胞を含む細胞集団は、例えば、これらの抗原マーカーを指標として生体組織から分離することができる。また、Muse細胞はストレス耐性であり、間葉系組織又は培養間葉系細胞から種々のストレス刺激により濃縮することができる。本発明の分化誘導法には、ストレス刺激によりMuse細胞が濃縮された細胞画分を用いることもできる。Muse細胞の分離法、同定法、及び特徴などの詳細は、国際公開第WO2011/007900号に開示されている。また、Wakaoら(2011、上述)によって報告されているように、骨髄、皮膚などから間葉系細胞を培養し、それをMuse細胞の母集団として用いる場合、SSEA-3陽性細胞の全てがCD105陽性細胞であることが分かっている。したがって、本発明によれば、生体の間葉系組織又は培養間葉系幹細胞からMuse細胞を分離する場合は、単にSSEA-3を抗原マーカーとしてMuse細胞を精製し、使用することができる。なお、本明細書においては、本発明の分化誘導法において使用され得る、SSEA-3を抗原マーカーとして、生体の間葉系組織又は培養間葉系組織から分離された多能性幹細胞(Muse細胞)又はMuse細胞を含む細胞集団を単に「SSEA-3陽性細胞」と記載することがある。また、本明細書においては、「非Muse細胞」とは、生体の間葉系組織又は培養間葉系組織に含まれる細胞であって、「SSEA-3陽性細胞」以外の細胞を指す。
(i)テロメラーゼ活性が低いか又は無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;及び
(iv)セルフリニューアル能を持つ
からなる群から選択される少なくとも1つの性質を有してもよい。本発明の一局面では、本発明の分化誘導法に使用されるMuse細胞は、上記性質を全て有する。ここで、上記(i)について、「テロメラーゼ活性が低いか又は無い」とは、例えば、TRAPEZE XL telomerase detection kit(Millipore社)を用いてテロメラーゼ活性を検出した場合に、低いか又は検出できないことをいう。テロメラーゼ活性が「低い」とは、例えば、体細胞であるヒト線維芽細胞と同程度のテロメラーゼ活性を有しているか、又はHela細胞に比べて1/5以下、好ましくは1/10以下のテロメラーゼ活性を有していることをいう。上記(ii)について、Muse細胞は、in vitro及びin vivoにおいて、三胚葉(内胚葉系、中胚葉系、及び外胚葉系)に分化する能力を有し、例えば、in vitroで誘導培養することにより、肝細胞、神経細胞、骨格筋細胞、平滑筋細胞、骨細胞、脂肪細胞等に分化し得る。また、in vivoで精巣に移植した場合にも三胚葉に分化する能力を示す場合がある。さらに、静注により生体に移植することで損傷を受けた臓器(心臓、皮膚、脊髄、肝、筋肉等)に遊走及び生着し、組織に応じた細胞に分化する能力を有する。上記(iii)について、Muse細胞は、浮遊培養では増殖速度約1.3日で増殖するが、浮遊培養では1細胞から増殖し、胚様体様細胞塊を作り14日間程度で増殖が止まる、という性質を有するが、これらの胚様体様細胞塊を接着培養に持っていくと、再び細胞増殖が開始され、細胞塊から増殖した細胞が広がっていく。さらに精巣に移植した場合、少なくとも半年間は癌化しないという性質を有する。また、上記(iv)について、Muse細胞は、セルフリニューアル(自己複製)能を有する。ここで、「セルフリニューアル」とは、1個のMuse細胞から浮遊培養で培養することにより得られる胚様体様細胞塊に含まれる細胞から3胚葉性の細胞への分化が確認できると同時に、胚様体様細胞塊の細胞を再び1細胞で浮遊培養に持っていくことにより、次の世代の胚様体様細胞塊を形成させ、そこから再び3胚葉性の分化と浮遊培養での胚様体様細胞塊が確認できることをいう。セルフリニューアルは1回又は複数回のサイクルを繰り返せばよい。
(i)SSEA-3陽性;
(ii)CD105陽性;
(iii)テロメラーゼ活性が低いか又は無い;
(iv)三胚葉に分化する能力を持つ;
(v)腫瘍性増殖を示さない;及び
(vi)セルフリニューアル能を持つ。
本発明のインビトロ分化誘導法において使用される「情報提示細胞」とは、多能性幹細胞から最終的に分化した状態にある細胞と同じ表現型及び機能特性を有し、分化誘導される前の細胞であって、傷害細胞又は死細胞の起源となる細胞を意味する。本発明において使用される情報提示細胞は、多能性幹細胞から所望の分化細胞を得ることができれば、その起源はいずれであってもよい。例えば、細胞の起源は、その目的に応じ、様々な哺乳動物を起源としてよく、限定されないが、ヒト、チンパンジー、その他の霊長類、家畜動物、例えばイヌ、ネコ、畜産動物、例えばウシ、ブタ、ウマ、ヒツジ、ヤギ、実験用動物、例えばウサギ、ラット、マウス、モルモットなどに由来する。
本発明によれば、多能性幹細胞を所望の細胞に分化させるために使用される傷害細胞又は死細胞は、上記の情報提示細胞を起源とすることができる。本明細書で使用するとき、用語「傷害細胞」とは、例えば、人為的ストレス又は環境的ストレスにより傷害を受けているために、一般に好適な培養条件によって培養した場合であっても、増殖は困難であるが、その細胞が有する代謝活性は、生細胞と比較すると低下しているが、死細胞と比較すると有意に活性を有する状態の細胞をいう。本明細書で使用するとき、用語「死細胞」とは、好適な培養条件によって培養した場合であっても増殖は不可能であって、代謝活性を示さない状態の細胞をいう。「死細胞」は、ネクローシスによる死細胞又はアポトーシスによる細胞死のいずれであってもよい。なお、本発明によれば、多能性幹細胞の貪食性は、上記の傷害細胞や死細胞に限らず、それらの一部(例えば、細胞質、細胞膜、核、ミトコンドリア、ゴルジ体、小胞体、細胞質に含まれる細胞骨格、酵素、蛋白、RNA、DNA)又は細胞の破砕物に対しても示され得る。
本発明は、多能性幹細胞と傷害細胞、死細胞、及び/又はその一部を共培養することによって、インビトロで多能性幹細胞を分化誘導する方法に関する。本明細書で使用するとき、「共培養」とは、同一環境下において2種類以上の細胞(又は一部)を培養することを意味し、同一の培養容器又はシャーレ内に多能性幹細胞と傷害細胞等をそれぞれ所定数入れて培養することを指す。共培養の態様としては、(i)培養中の多能性幹細胞に傷害細胞等を添加して共培養する系、(ii)情報提示細胞から傷害細胞等を作製後、多能性幹細胞を添加して共培養する系、(iii)培養容器内に多能性幹細胞と傷害細胞等を所定する混在させて共培養する系などが挙げられる。
本発明で使用するとき、「分化誘導」とは、何らかの手段によって、多能性幹細胞から特定の臓器、器官若しくは組織を構成する細胞又はその前駆細胞へと導くことを意味するが、内胚葉細胞、中胚葉細胞及び外胚葉細胞などの多種類の細胞を含む細胞群に分化させることも含む。本発明によれば、多能性幹細胞から特定の細胞への分化誘導は、多能性幹細胞を傷害細胞等と単に共培養することによって達成され、傷害細胞等以外の試薬やUV照射などを必要としないことを特徴とする。このような分化誘導は、多能性幹細胞の傷害細胞等に対する貪食性に起因するものであり、傷害又は死滅前の対応する情報提示細胞から分泌される何らかの液性因子やエキソソーム、又は情報提示細胞との細胞間の直接的接触によるものでないことが示唆された(実施例1参照)。
多能性幹細胞が、傷害細胞、死細胞、及び又はその一部を貪食することにより、これらに由来する細胞に分化するという性質を利用して、多能性幹細胞を含有する細胞製剤の品質管理に応用することができる。本発明によれば、インビトロにおいて細胞製剤中に含有される細胞が、生存する多能性幹細胞であることを確認するための方法であって、
(a)細胞製剤中の細胞と、傷害細胞、死細胞、及び又はその一部とを共培養し;及び
(b)該細胞製剤中の細胞が、傷害細胞、死細胞、及び又はその一部に由来する外胚葉系細胞、中胚葉系細胞、及び/又は内胚葉系細胞と同じ表現型及び機能を有する細胞に分化した場合に、該細胞製剤中に多能性幹細胞が生存していると判断する
ことを含む上記方法が提供される。ここで、多能性細胞(例えば、Muse細胞)が、三胚葉系細胞(外胚葉系細胞、中胚葉系細胞、及び内胚葉系細胞)に分化したかどうかを確認する方法は、上述の通り、各種細胞マーカーの発現の有無を調べることによって可能である。
Muse細胞は、生体内で傷害部位から発せられる傷害シグナル(例えば、S1P)により、傷害部位に遊走し、生着後、「場の論理」に従って分化誘導されるが、Muse細胞が分化するメカニズムについて詳細には分かっていない。本実施例では、Muse細胞の分化誘導がどのようなメカニズムで行われているのかについて、インビトロにおいて検証した。Muse細胞が、「場の論理」の情報をどのように受け取っているか、すなわち、分化誘導に関係すると考えられる要因がどのようなものかを解明することは、「場の論理」が消失している慢性疾患や血管を介した到達が困難な組織や臓器、又は希少細胞の再生への応用の基礎となる。
日本エスエルシー(株)から購入したC57BL/6―TG (CAG-EGFP)から生まれた生後1日目のマウス心臓より樹立した正常マウス心筋細胞を約10日間培養後、細胞の培養上清を回収し、これを液性因子による分化誘導の可能性を検討するための材料とした。次に、無血清培地で3日間培養した上記心筋細胞の培養上清を回収し、該培養上清をExoQuick Exosome Precipitation Solution(System Biosciences)と混合後、遠心分離をすることによって、心筋細胞より分泌されたエキソソームを得た(Douglas,D.,et al.,Molecular Biology,728(4),235~246(2011)参照)。エキソソームが得られたことの確認は、HSP70を指標にウェスタンブロットによって行った。
(1)Nkx2.5
フォワードプライマー:5’-CAGGACCAGACTCTGGAGCTG-3’(配列番号2)(配列番号1の821-841ntに対応)
リバースプライマー:5’-CGCCGAAGTTCACGAAGTTGT-3’(配列番号3)(配列番号1の1118-1098ntに対応)
(2)GATA-4
フォワードプライマー:5’-AGCAGCTCCGTGTCCCAGACGTT-3’(配列番号5)(配列番号4の1683-1705ntに対応)
リバースプライマー:5’-CCAACTCACAGGAGAGATGCAGTGTG-3’(配列番号6)(配列番号4の2139-2114ntに対応)
(3)トロポニン-T
フォワードプライマー:5’-CTCAAAGACAGGATCGAGAG-3’(配列番号8)(配列番号7の489-508ntに対応)
リバースプライマー:5’-GATCTTCATTCAGGTGGTCA-3’ 配列番号9)(配列番号7の795-771ntに対応)
(4)ANP
フォワードプライマー:5’-TCCAGCTCCTAGGTCAGACC-3’(配列番号11)(配列番号10の149-168ntに対応)
リバースプライマー:5’-TCTGGGCTCCAATCCTGTCC-3’(配列番号12)(配列番号10の523-504ntに対応)
傷害マウス心筋細胞を用いた場合において、Muse細胞が分化され得るかどうかを検討した。傷害マウス心筋細胞の作製は、アポトーシスを誘導するエトポシドを用いて行った。簡単には、GFP標識したマウス心筋細胞を3×104細胞/ウェルで播種し、培養2日後に50μMのエトポシド(SIGMA,#E1383)を添加した。培養1日後、アポトーシス誘導マウス心筋細胞の培養上清を回収し、1×104細胞/ウェルで播種したヒトMuse細胞を培養した。正常マウス心筋細胞の場合と同様に、培養開始から3日後、1週間後、2週間後、及び3週間後に心筋分化マーカーのヒト特異的なmRNA発現の有無をRT-PCRによって検討した(図2A)。しかしながら、いずれのマーカーも確認できなかったことから、傷害細胞の培養上清に含まれる因子だけでは、Muse細胞の心筋細胞への分化は起こらないことが判明した。
Muse細胞は、フェライトを貪食することは報告されているが、傷害細胞、死細胞、又はその一部もまた貪食し得るのかについては検討されていない。そこで、Muse細胞の貪食性について、PKH26PCLビーズを用いて再検証した。
Muse細胞が、傷害細胞を貪食することにより、直接「場の論理」の情報を受け取り、分化するという可能性を検証した。
トポイソメラーゼ阻害剤であるエトポシドを用いて、傷害心筋細胞を調製した。宿主となる心筋細胞として、実施例1で調製したGFP標識したマウス由来の心筋細胞を用いた。GPRマウス心筋細胞を3×104細胞/ウェルの細胞密度でプレートに播種し、1日間培養した培養系に、50μMのエトポシドを各ウェルに添加した。その後、さらに1日間培養した。次に、標識したヒトMuse細胞を1×104細胞/ウェルで添加し、共培養を行った。共培養を開始してから3日後に、Muse細胞による傷害心筋細胞の貪食能を蛍光顕微鏡下で検証した。図4に示されるように、アポトーシスを起こしたGFPマウス心筋細胞と共培養したMuse細胞は、壊れた心筋細胞の一部(緑色)をMuse細胞内(赤色)に取り込んでいるため、Muse細胞の貪食能が確認された。
マウスC2C12(横紋筋肉腫由来細胞株)(ATCC,#CRL-1772)を用いて、Muse細胞の中胚葉系細胞への分化誘導について検討した。エトポシド処理したC2C12細胞とGFP標識したMuse細胞を共培養し、分化誘導を試みた。具体的には、5×104細胞/ウェルの細胞濃度でC2C12細胞を播種し、エトポシドで処理後、同細胞濃度のMuse細胞を播種し、共培養を行った。共培養を開始してから14日後に、ミオゲニン(骨格筋細胞の分化マーカー)の発現の有無を免疫染色によって検討した。蛍光顕微鏡下で観察した結果及びMyoD発現のRP-PCRの結果をそれぞれ図7A及びBに示す。Muse細胞(緑色)は、ミオゲニン(赤色)を発現し、多核であり、さらにMyoDのmRNA発現(共培養開始から1週間後)を確認できたことから、傷害を誘導したC2C12との共培養によって中胚葉系細胞である骨格筋細胞に分化されることが示された。
フォワードプライマー:5’-GAGCAATCCAAACCAGCGGTTG-3’(配列番号14)(配列番号13の631-652ntに対応)
リバースプライマー:5’-TAGTAGGCGCCTTCGTAGCAG-3’(配列番号15)(配列番号13の912-892ntに対応)
傷害を誘導した神経細胞(Neuro2A,ATCC,#CCL-131)を用いて、Muse細胞の外胚葉系細胞への分化誘導について検討した。上記と同様にエトポシド処理で傷害を誘導した神経を作製し、Muse細胞と共培養した。共培養の開始から3日後と1週間後に、Muse細胞から総RNAを抽出し、分化誘導について検証した。より具体的には、神経細胞マーカーとして知られているNeuNのmRNA発現をRT-PCRによって調べた。マウス胚(15日培養)及びヒトMSC-Muse細胞(L26)では、NeuNの発現はなかった。一方、傷害を誘導したNeuro2Aとの共培養の系では、共培養開始後1週間でMuse細胞においてNeuNの発現が確認された。これにより、Muse細胞は、傷害神経細胞との共培養により、外胚葉系細胞である神経細胞に分化されることが示された。
傷害を誘導したマウスβ細胞(NIT-1)(ATCC,#CRL-2055)を用いて、Muse細胞の内胚葉系細胞への分化誘導について検討した。上記と同様にエトポシド処理で傷害を誘導したβ細胞を作製し、Muse細胞と共培養した。共培養の開始から3日後と1~3週間後に、Muse細胞から総RNAを抽出し、分化誘導について検証した。より具体的には、β細胞の前駆細胞マーカーとして知られているNkx2.2とNeuroD1、並びに成熟β細胞マーカーとして知られているIslet1のmRNA発現をRT-PCRによって調べた。図9に示されるように、各種マーカーの経時的な発現の変化から、Muse細胞は、共培養の時間経過とともに前駆細胞から成熟β細胞へと分化していくことが確認された。Muse細胞は、傷害β細胞との共培養により、内胚葉系細胞であるβ細胞に分化されることが示された。
1)Nkx2.2
フォワードプライマー:5’-GACATAAATTTTGGGGTCT-3’(配列番号17)(配列番号16の25-43ntに対応)
リバースプライマー:5’-GGTTCTGGAACCAGATCTT-3’(配列番号18)(配列番号16の584-566ntに対応)
2)NeuroD1
フォワードプライマー:5’-GCACAATTTGAGCAATTCAT-3’(配列番号20)(配列番号19の1998-2017ntに対応)
リバースプライマー:5’-CAAGCTTGTGCAAGTAATGTG-3’(配列番号21)(配列番号19の2144-2124ntに対応)
3)Islet1
フォワードプライマー:5’-GGCTGTTCACCAACTGTA-3’(配列番号23)(配列番号22の490-507ntに対応)
リバースプライマー:5’-ACTCGATGTGATACACCTTG-3’(配列番号24)(配列番号22の858-839ntに対応
傷害を誘導した肝細胞(マウス肝癌細胞株:Hepa1-6)(ATCC,#CRL-1830)を用いて、Muse細胞の内胚葉系細胞への分化誘導について検討した。上記と同様にエトポシド処理で傷害を誘導した肝細胞を作製し、Muse細胞と共培養した。共培養の開始から3日後と1~2週間後に、Muse細胞から総RNAを抽出し、分化誘導について検証した。より具体的には、肝細胞の発生初期に見られるα-フェトプロテイン(AFP)のmRNA発現をRP-PCRによって調べた。図10に示されるように、AFPの発現は、共培養開始後1週間で観察され、Muse細胞は、傷害肝細胞との共培養により、内胚葉系細胞である肝細胞に分化されることが示された。
フォワードプライマー:5’-CCGAACTTTCCAAGCCATAACTG-3’(配列番号26)(配列番号25の740-762ntに対応)
リバースプライマー:5’-CACTTCTCCAATAACTCCTGGTATC-3’(配列番号27)(配列番号25の1195-1171ntに対応)
Claims (20)
- 多能性幹細胞又は該多能性幹細胞が濃縮された細胞画分を、情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部と共培養することを含む、インビトロで多能性幹細胞を情報提示細胞と同じ表現型及び機能を有する細胞に分化誘導する方法。
- 多能性幹細胞が情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部を貪食することを特徴とする、請求項1に記載の方法。
- 前記多能性幹細胞が、生体の間葉系組織又は培養間葉系細胞から分離されたSSEA-3陽性細胞である、請求項1又は2に記載の方法。
- 前記多能性幹細胞が、CD105陽性である、請求項1~3のいずれか1項に記載の方法。
- 前記多能性幹細胞が、CD117陰性及びCD146陰性である、請求項1~4のいずれか1項に記載の方法。
- 前記多能性幹細胞が、CD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性、及びCD271陰性である、請求項1~5のいずれか1項に記載の方法。
- 前記多能性幹細胞が、CD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snai1陰性、Slug陰性、Tyrp1陰性、及びDct陰性である、請求項1~6のいずれか1項に記載の方法。
- 前記多能性幹細胞が、以下の性質の全てを有する多能性幹細胞である、請求項1~7のいずれか1項に記載の方法:
(i)テロメラーゼ活性が低いか又は無い;
(ii)三胚葉のいずれの胚葉の細胞に分化する能力を持つ;
(iii)腫瘍性増殖を示さない;及び
(iv)セルフリニューアル能を持つ。 - 多能性幹細胞の数と情報提示細胞由来の傷害細胞又は死細胞の数の比が1:10,000~10,000:1である、請求項1~8のいずれか1項に記載の方法。
- 情報提示細胞由来の傷害細胞、死細胞、及び・又はその一部が、アポトーシス誘導剤、代謝拮抗薬、アルキル化剤、アントラサイクリン、抗生物質、有糸分裂阻害剤、トポイソメラーゼ阻害剤、プロテアソーム阻害剤、抗がん剤、熱、低温、酸、アルカリ、超音波、及びボルテックスなどによる物理的破砕からなる群から選択される薬剤の添加によって得られたものである、請求項1~9のいずれか1項に記載の方法。
- 情報提示細胞が、外胚葉由来細胞である、請求項1~10のいずれか1項に記載の方法。
- 外胚葉由来細胞が、神経細胞、グリア細胞、色素細胞、皮膚細胞、内耳細胞、網膜細胞、角膜細胞、及び毛根細胞からなる群から選択される、請求項11に記載の方法。
- 情報提示細胞が、中胚葉由来細胞である、請求項1~10のいずれか1項に記載の方法。
- 中胚葉由来細胞が、心筋細胞、骨格筋細胞、平滑筋細胞、骨細胞、軟骨細胞、生殖系細胞、及び造血系細胞からなる群から選択される、請求項13に記載の方法。
- 情報提示細胞が、内胚葉由来細胞である、請求項1~10のいずれか1項に記載の方法。
- 内胚葉由来細胞が、膵β細胞、肝細胞、胆管細胞、呼吸器系上皮細胞、消化管上皮細胞、血管内皮細胞、腎構成細胞、膀胱上皮細胞、及び膵外分泌細胞からなる群から選択される、請求項15に記載の方法。
- 情報提示細胞が、希少細胞である、請求項1~10のいずれか1項に記載の方法。
- 前記希少細胞が、運動ニューロン、ドーパミン神経、介在ニューロン、グルタミン作動性ニューロン、脳下垂体細胞、甲状腺細胞、副腎皮質、副腎髄質、頸動脈の圧センサー細胞、心臓刺激伝達系、脈絡叢上皮細胞、膵-alpha細胞、膵-delta細胞、及び肺クララ細胞からなる群から選択される、請求項17に記載の方法。
- インビトロにおいて細胞製剤中に含有される細胞が、生存する多能性幹細胞であることを確認するための方法であって、
(a)細胞製剤中の細胞と、傷害細胞若しくは死細胞又はその一部とを共培養し;及び
(b)該細胞製剤中の細胞が、傷害細胞若しくは死細胞又はその一部に由来する外胚葉系細胞、中胚葉系細胞、及び/又は内胚葉系細胞と同じ表現型及び機能を有する細胞に分化した場合に、該細胞製剤中に多能性幹細胞が生存していると判断する
ことを含む上記方法。 - 多能性幹細胞又は該多能性幹細胞が濃縮された細胞画分を、情報提示細胞由来の傷害細胞、死細胞、及び/又はその一部と共培養することを含む、インビトロで多能性幹細胞から情報提示細胞と同じ表現型及び機能を有する細胞を製造する方法。
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