Cell differentiation induction method and composition for differentiation induction

Abstract

The present invention addresses the problem of providing novel technology for inducing differentiation of stem cells.　The present invention provides a cell differentiation induction method, said cell differentiation induction method comprising a culturing step for culturing stem cells in the presence of extracellular vesicles derived from a tissue specific cell, wherein the tissue specific cell and the cells obtained by differentiation induction are the same type of cells.

Description

Method for inducing cell differentiation and composition for inducing differentiation

[0001] The present invention relates to a method for inducing cell differentiation and a composition for inducing differentiation.

[0002] In regenerative medicine research, various methods are being investigated for inducing differentiation of undifferentiated stem cells into desired cells and transplanting them into the body, with the aim of restoring the function of lost biological tissue.

[0003] As a method for inducing differentiation of stem cells, for example, a method for inducing differentiation of stem cells into adipocytes in the presence of exosomes derived from stem cells differentiating into adipocytes (Patent Document 1) and a method for inducing differentiation of stem cells into chondrocytes in the presence of exosomes derived from stem cells differentiating into chondrocytes (Patent Document 2) have been proposed. Note that exosomes are a type of extracellular vesicle secreted by cells.

[0004] Special table 2017-534629 publication Special table 2018-508486 publication

[0005] However, there is a need for a technology that can more efficiently induce differentiation of stem cells.

[0006] The present invention has been made in view of the above circumstances, and an objective of the present invention is to provide a novel technique for inducing differentiation of stem cells.

[0007] The present inventors have discovered that, in inducing stem cell differentiation, the use of extracellular vesicles derived from tissue-specific cells that constitute a living body can efficiently induce differentiation of stem cells into cells of the same type as the cells from which the extracellular vesicles are derived, and have completed the present invention. More specifically, the present invention provides the following.

[0008] (1) A method for inducing cell differentiation, comprising a culture step of culturing stem cells in the presence of extracellular vesicles derived from tissue-specific cells, wherein the tissue-specific cells and the differentiation-induced cells are of the same type.

[0009] (2) The cell differentiation induction method according to (1), wherein the content of the extracellular vesicles is 0.5 μg / 500 μL or more in terms of protein relative to the culture medium used in the culture step.

[0010] (3) The method for inducing cell differentiation according to (1), wherein the average particle diameter of the extracellular vesicles is 50 nm or more and 200 nm or less.

[0011] (4) The method for inducing cell differentiation according to any one of (1) to (3), wherein the tissue-specific cells comprise at least one type of cell selected from the group consisting of lymphatic endothelial cells, vascular endothelial cells, nerve cells, fibroblasts, hepatocytes, adipocytes, osteoblasts, myoblasts, and chondrocytes.

[0012] (5) A composition for inducing differentiation, comprising extracellular vesicles primarily derived from tissue-specific cells.

[0013] According to the present invention, a novel technique for inducing differentiation of stem cells is provided.

[0014] 1 is a diagram showing the results of flow cytometry in an example. 2 is a diagram showing the proliferation ability of lymphatic endothelial cells obtained in an example. 3 is a diagram showing the results of flow cytometry in an example. 4 is a diagram showing the results of flow cytometry in an example. 5 is a diagram showing the results of flow cytometry in an example. 6 is a diagram showing the results of transplantation of lymphatic network internal tissue obtained in an example. 7 is a diagram showing the results of transplantation of lymphatic network internal tissue obtained in an example. 8 is a diagram showing the results of flow cytometry in an example.

[0015] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this.

[0016] <Method for inducing cell differentiation> The method for inducing cell differentiation of the present invention comprises a culture step of culturing stem cells in the presence of extracellular vesicles derived from tissue-specific cells. The tissue-specific cells and the differentiation-induced cells are of the same type.

[0017] As described above, there are known methods for inducing differentiation of stem cells into adipocytes in the presence of exosomes derived from stem cells differentiating into adipocytes (Patent Document 1) and for inducing differentiation of stem cells into chondrocytes in the presence of exosomes derived from stem cells differentiating into chondrocytes (Patent Document 2). According to these patent documents, "differentiated into" adipocytes or chondrocytes refers to stem cells that are "in the process of differentiating" into adipocytes or chondrocytes. Such cells are clearly distinguished from tissue-specific cells (e.g., adipocytes themselves or chondrocytes themselves).

[0018] On the other hand, the present inventors have made the unexpected discovery that the differentiation-inducing ability of extracellular vesicles (which may contain exosomes) derived from tissue-specific cells is significantly superior to the differentiation-inducing ability of exosomes derived from differentiating stem cells as described above, and that they can efficiently induce differentiation into cells of the same type as the cells from which the extracellular vesicles are derived (see "Test 1" in the Examples below).

[0019] In the present invention, "differentiation induction" includes a process of differentiating stem cells into desired cells other than stem cells. Cells that have been induced to differentiate usually lose their differentiation potential. Whether stem cells have differentiated into desired cells or whether the cells have differentiation potential can be determined by using flow cytometry or the like to confirm the presence or absence and degree of expression of markers, etc., depending on the type of cell.

[0020] The cell differentiation induction method of the present invention will be described in detail below.

[0021] (1) Stem Cells Stem cells in the present invention are the treatment targets to be induced to differentiate by the cell differentiation induction method of the present invention.

[0022] The type of stem cell is not particularly limited, and includes both tissue stem cells and pluripotent stem cells. Examples of tissue stem cells include mesenchymal stem cells and hematopoietic stem cells. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0023] As the stem cells, mesenchymal stem cells are preferred from the viewpoint that the effects of the present invention can be stably obtained. Mesenchymal stem cells can be isolated from any tissue (e.g., bone marrow, fat, dental pulp, blood (peripheral blood, umbilical cord blood, etc.), placenta, umbilical cord, etc.).

[0024] The stem cells may be cells derived from cells of the organism to which the cells obtained by the cell differentiation induction method of the present invention are to be administered (autologous cells), or may be derived from cells derived from a source other than the organism (allogeneic cells).

[0025] The living organism from which the stem cells are derived is not particularly limited, but includes humans, non-human animals (such as mammals), and the like.

[0026] (2) Extracellular vesicles derived from tissue-specific cells In the cell differentiation induction method of the present invention, stem cells are cultured in the presence of extracellular vesicles derived from tissue-specific cells. This process allows stem cell differentiation to proceed efficiently and differentiate into cells of the same species as the tissue-specific cells.

[0027] In the present invention, "extracellular vesicles derived from tissue-specific cells" refer to extracellular vesicles secreted from tissue-specific cells. In one embodiment of the present invention, extracellular vesicles derived from tissue-specific cells include extracellular vesicles derived from a cell population primarily comprising tissue-specific cells. A "cell population primarily comprising tissue-specific cells" includes a cell population in which preferably 50% or more, more preferably 70%, and even more preferably 80% or more of the cells are tissue-specific cells.

[0028] (2-1) Tissue-specific cells In the present invention, "tissue-specific cells" include cells that exist specifically in any biological tissue. In a preferred embodiment of the present invention, "tissue-specific cells" particularly refer to cells that exist specifically in fully differentiated tissue (preferably healthy tissue). Therefore, stem cells themselves and stem cells in the process of differentiation are not included in tissue-specific cells.

[0029] The tissue-specific cells may be derived from any tissue and are not particularly limited. From the viewpoint of easily achieving the effects of the present invention, examples of tissue-specific cells include at least one cell selected from the group consisting of lymphatic endothelial cells, vascular endothelial cells, nerve cells (e.g., neuroblastoma cells), fibroblasts, hepatocytes, adipocytes, osteoblasts, myoblasts, and chondrocytes. Among these, lymphatic endothelial cells are preferred from the viewpoint of easily achieving the effects of the present invention.

[0030] In a preferred embodiment of the present invention, the tissue-specific cells may be isolated from tissues or organs (for example, skin, blood vessels, liver, muscle, fat, etc.).

[0031] Whether or not a cell is a tissue-specific cell can be identified by, for example, any of the following methods: - The expression of a marker specific to tissue-specific cells is evaluated by flow cytometry or the like, and it is confirmed that the marker of interest is expressed and that markers specific to other cells (e.g., stem cells) are hardly expressed. In this embodiment, the cells expressing the marker specific to tissue-specific cells are preferably 70% or more, more preferably 80% or more, of the entire cell population. - The differentiation potential of tissue-specific cells is evaluated based on the expression of tissue-specific genes using PCR, flow cytometry, or the like, and it is confirmed that they do not have differentiation potential. - The presence of tissue-specific cells is confirmed by tissue staining.

[0032] (2-2) Extracellular vesicles "Extracellular vesicles" is a general term for vesicles with a lipid bilayer structure secreted from living cells. Extracellular vesicles include, for example, exosomes, microvesicles, and apoptotic bodies.

[0033] In a preferred embodiment of the present invention, the extracellular vesicles contain exosomes as a major component. Note that "containing exosomes as a major component" means, for example, that the content of exosomes relative to the total protein content of the extracellular vesicles is preferably 50% by mass or more, more preferably 70% by mass or more, and even more preferably 90% by mass or more.

[0034] In a preferred embodiment of the present invention, the average particle size of the extracellular vesicles is preferably 50 nm or more and 200 nm or less, more preferably 90 nm or more and 150 nm or less. When the average particle size of the extracellular vesicles is within the above range, the extracellular vesicles usually contain exosomes as a major component. In the present invention, the "average particle size" refers to a value determined by the nanotracking method.

[0035] Any conventional method (ultracentrifugation, density gradient centrifugation, immunoprecipitation using beads, size exclusion chromatography column methods, polymer precipitation methods, size exclusion methods using ultrafiltration membranes, etc.) can be used to isolate extracellular vesicles. For example, tissue-specific cells can be cultured under conditions appropriate for the cell type, and extracellular vesicles containing exosomes as a major component can be collected from the resulting culture by ultracentrifugation or the like.

[0036] In a preferred embodiment of the present invention, the method shown in the Examples can be used as a method for isolating extracellular vesicles.

[0037] To determine whether extracellular vesicles are derived from tissue-specific cells, the expression of specific markers in the extracellular vesicles can be measured using flow cytometry or the like to confirm that the marker of interest is expressed.

[0038] For example, if extracellular vesicles express the following markers, they can be determined to be extracellular vesicles derived from tissue-specific cells. These markers are not expressed on extracellular vesicles derived from differentiating stem cells. Lymphatic endothelial cell-derived extracellular vesicles: Alix, CD9, CD63, CD81, Podoplanin, LYVE-1, VEGFR-3, PROX-1 Vascular endothelial cell-derived extracellular vesicles: Alix, CD9, CD63, CD81, CD31, CD144, Tie2, vWF, ACE2, Lectin, ICAM-1 / CD54, VEGF-R2 Neuron-derived extracellular vesicles: Alix, CD9, CD63, CD81, MAP-2, NeuN, Neurofilament-H, Neurofilament-L, Neurofilament-M Fibroblast-derived extracellular vesicles: Alix, CD9, CD63, CD81, ER-TR7, D7-FIB, Prolyl-4-hydroxylase β, S100A4, HSP47 / SerpinH1, Vimentin Hepatocyte-derived extracellular vesicles: Alix, CD9, CD63, CD81, Alb, AFP, HNF-3β Adipocyte-derived extracellular vesicles: Alix, CD9, CD63, CD81, PLN1, PPARγ, UCP1, PRDM16, PGC-1α Osteoblast-derived extracellular vesicles: Alix, CD9, CD63, CD81, ALP, FGF2310, Osteocalcin Myoblast-derived extracellular vesicles: Alix, CD9, CD63, CD81, MHC, My5f, MyoD, Myogenin, MRF4, Desumin Chondrocyte-derived extracellular vesicles: Alix, CD9, CD63, CD81, Collagen Type II, Aggrecan, YKL-40

[0039] For example, if the extracellular vesicles do not express the following markers, they can be determined to be extracellular vesicles derived from tissue-specific cells. These markers are specifically expressed in extracellular vesicles derived from stem cells and stem cells undergoing differentiation. - Markers for human mesenchymal stem cells: CD73, D90, CD105 - Markers for mouse mesenchymal stem cells: CD106, Sca-1 - Markers specifically expressed in iPS cells, ES cells, and precursor cells of tissue-specific cells differentiated from them

[0040] (3) Culture Conditions Any conditions under which stem cells can grow can be adopted as culture conditions for stem cells, except that the cells are cultured in the presence of extracellular vesicles derived from tissue-specific cells.

[0041] In a preferred embodiment of the present invention, stem cells may be cultured in a culture medium supplemented with extracellular vesicles derived from tissue-specific cells. Any culture medium capable of growing stem cells (DMEM medium, RPMI medium, MEM medium, mTeSR medium, PSGro hESC / iPSC medium, etc.) can be used as the culture medium. In this embodiment, the amount of extracellular vesicles added can be adjusted appropriately depending on the differentiation efficiency to be achieved, and is preferably 0.5 μg / 500 μL or more, more preferably 1 μg / 500 μL or more, in terms of protein equivalent. Since desirable effects can be obtained even without excessive amounts of extracellular vesicles, the amount of extracellular vesicles added is typically 10 μg / 500 μL or less, in terms of protein equivalent. In a preferred embodiment of the present invention, the protein equivalent amount may correspond to the exosome equivalent amount.

[0042] The culture time for stem cells is not particularly limited as long as it is a period that allows sufficient differentiation, but is, for example, 3 to 14 days. During the culture period, the medium may be changed as necessary.

[0043] The temperature for culturing stem cells is not particularly limited as long as it allows the cells to grow appropriately, but a temperature of 35 to 37°C is preferred, for example.

[0044] According to the present invention, as described above, efficient differentiation induction can be achieved by culturing in the presence of extracellular vesicles derived from tissue-specific cells. Therefore, in a preferred embodiment of the present invention, stem cells are cultured in the absence of a differentiation-inducing medium or a differentiation inducer. Examples of differentiation-inducing media for differentiation into lymphatic endothelial cells include DMEM medium and endothelial cell growth medium MV2. Examples of differentiation inducers for differentiation into lymphatic endothelial cells include various growth factors, ascorbic acid, heparin, and the like. However, this does not exclude embodiments in which stem cells are cultured in the presence of a differentiation-inducing medium or a differentiation inducer. The differentiation-inducing medium and differentiation inducer are appropriately selected depending on the type of cells to be differentiated.

[0045] (4) Differentiation-induced cells After the culture process is completed, cells induced to differentiate from the stem cells are obtained. These cells are the same type as the cells from which the extracellular vesicles used in the culture process were derived. For example, if the extracellular vesicles are derived from "lymphatic endothelial cells," the stem cells will differentiate into "lymphatic endothelial cells."

[0046] The differentiated cells can be used for any purpose depending on the type of cell. For example, the obtained cells may be grown under conditions that allow the cells to grow, or may be made into a sheet and used for transplantation, etc.

[0047] <Composition for inducing differentiation> The present invention also encompasses a composition for inducing differentiation, which mainly contains extracellular vesicles derived from tissue-specific cells.

[0048] The differentiation-inducing composition of the present invention "mainly" comprises extracellular vesicles derived from tissue-specific cells, which means that the main component exerting the differentiation-inducing effect is extracellular vesicles derived from tissue-specific cells. The component exerting the differentiation-inducing effect in the differentiation-inducing composition of the present invention may be only extracellular vesicles derived from tissue-specific cells, or may be a combination of extracellular vesicles derived from tissue-specific cells and other components. The other components can be appropriately selected depending on the type of cells to be differentiated, and any component that does not inhibit the action of extracellular vesicles derived from tissue-specific cells can be selected. Examples of such components include various growth factors, ascorbic acid, heparin, etc.

[0049] Details regarding extracellular vesicles derived from tissue-specific cells are as described above in the cell differentiation induction method.

[0050] The content of extracellular vesicles derived from tissue-specific cells is adjusted depending on the form of the composition for inducing differentiation. For example, when the composition for inducing differentiation is liquid, the content of extracellular vesicles is preferably 0.5 μg / 500 μL or more, more preferably 1 μg / 500 μL or more, in terms of protein equivalent, relative to the composition for inducing differentiation. Since desirable effects can be obtained even without excessive amounts of extracellular vesicles, the content is usually 10 μg / 500 μL or less, in terms of protein equivalent.

[0051] Components other than extracellular vesicles derived from tissue-specific cells in the differentiation-inducing composition of the present invention are not particularly limited, and examples thereof include culture medium, various growth factors, etc. In a preferred embodiment of the present invention, the differentiation-inducing composition of the present invention comprises extracellular vesicles derived from tissue-specific cells and a culture medium.

[0052] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0053] <Test 1: Study using extracellular vesicles derived from lymphatic endothelial cells-1> Extracellular vesicles derived from lymphatic endothelial cells were collected by the following method, and their actions, properties, etc. were studied. Lymphatic endothelial cells correspond to "tissue-specific cells."

[0054] (1) Preparation of extracellular vesicles derived from tissue-specific cells Extracellular vesicles derived from lymphatic endothelial cells were collected by the following method.

[0055] (1-1) Preparation of Cells from Which Extracellular Vesicles Are Produced The following two types of cells were prepared. Cell-1: Human dermal lymphatic endothelial cells (hLEC, Cat. No. C-12216, Manufacturer: Takara) Cell-2: Human mesenchymal stem cells UE7T-13 cells (hMSC) cultured in a medium at 37°C for 10 days. The medium used was EGM-2+ supplement: EGM™-2MV Microvascular Endothelial Cell Growth Medium-2 (Cat. No. CC-3202, Manufacturer: Lonza). This medium is known to be capable of inducing differentiation of mesenchymal stem cells into lymphatic endothelial cells.

[0056] Expression markers were confirmed for each of the above "Cell-1" and "Cell-2." As a result, in "Cell-1," at least 80% of the cells in the cell group simultaneously expressed podoplanin and LYVE-1 (each a lymphatic endothelial cell-specific marker). In contrast, in "Cell-2," 20% or less of the cells in the cell group expressed podoplanin and LYVE-1 (lymphatic endothelial cell-specific markers), and 50% or more of the cells in the cell group expressed CD106 (a mesenchymal stem cell-specific marker).

[0057] (1-2) Collection of extracellular vesicles derived from lymphatic endothelial cells (ultracentrifugation) Two types of cells (1 x 10 ６ After culturing the cells (10 cm dish) in EGM-MV2 medium for 72 hours, the culture supernatant was subjected to stepwise centrifugation (300 x g, 10 minutes / 2,000 x g, 20 minutes / 10,000 x g, 30 minutes) to remove cell debris. The culture supernatant was then ultracentrifuged at 100,000 x g for 70 minutes, the supernatant was removed, and an extracellular vesicle fraction (pellet) was obtained. The extracellular vesicle fraction was washed with PBS and ultracentrifuged at 100,000 x g for 70 minutes, after which the supernatant was removed, and an extracellular vesicle fraction (pellet) was obtained. After another ultracentrifugation at 100,000 x g for 70 minutes, the resulting pellet was suspended in PBS (200 μL) and collected as lymphatic endothelial cell-derived extracellular vesicles, which were then subjected to the following tests. The above collection method is also known as a method for isolating exosomes. The obtained extracellular vesicles were stored at 4°C until they were used for testing.

[0058] Hereinafter, extracellular vesicles collected from "cell-1" will be referred to as "EVs (differentiating)", and extracellular vesicles collected from "cell-2" will be referred to as "EVs (differentiating)".

[0059] (2) Confirmation of expression markers in extracellular vesicles The expression markers of the extracellular vesicles ("EVs (differentiation)") obtained above were confirmed by Western blotting. As a result, "Alix," one of the extracellular vesicle markers, was expressed in the former.

[0060] (3) Differentiation induction using extracellular vesicles Using each of the extracellular vesicles obtained above ("EVs (differentiating)" and "EVs (differentiating)"), human mesenchymal stem cells were induced to differentiate into lymphatic endothelial cells by the following method.

[0061] (3-1) Stem Cell Culture First, human mesenchymal stem cells "UE7T-13 cells" (hMSC) were prepared. Next, hMSC (5 × 10 ４ The cells (100 cells / well) were cultured in DMEM medium (Dulbecco's modified Eagle's medium) containing extracellular vesicles at 37°C for 5 days to induce differentiation. The amount of extracellular vesicles added to the DMEM medium was 1 μg / 500 μL in terms of protein. This amount also corresponds to the amount of exosomes.

[0062] In this example, the protein equivalent amount of extracellular vesicles was determined using the "BCA Protein Assay Kit" (Cat: 23225, manufacturer: Thermofisher).

[0063] (3-2) Flow Cytometry After culturing, the expression levels of FL-1 (podoplanin) and FL-4 (LYVE-1) for the obtained cells were confirmed by flow cytometry. These are both lymphatic endothelial cell-specific markers. The results are shown in Figure 1. In Figure 1, a higher value (gMFI: Geometric Mean Fluorescence Intensity) indicates a higher expression level of the marker and a higher efficiency of differentiation into lymphatic endothelial cells.

[0064] As shown in Figure 1, human mesenchymal stem cells were induced to differentiate into lymphatic endothelial cells by culturing in the presence of extracellular vesicles ("EVs (differentiating)") derived from "Cell-1." The effect was significantly greater than when "Cell-2"-derived extracellular vesicles ("EVs (differentiating)") were used.

[0065] Furthermore, the same conclusions as above were obtained when cells after differentiation induction were immunostained and observed with a confocal laser microscope.

[0066] In this example, the differentiation induction using extracellular vesicles was performed for 5 days, but shorter or longer culture periods did not significantly affect the results. However, from the viewpoint of obtaining stable results, 5 days of culture tended to be the best.

[0067] In this example, the amount of each extracellular vesicle used in differentiation induction using extracellular vesicles was set at 1 μg / 500 μL in terms of protein amount (exosomes), but using a smaller or larger amount did not significantly affect the results. However, from the viewpoint of obtaining stable results, 1 to 2.5 μg / 500 μL tended to be the most preferable.

[0068] In this example, in the differentiation induction using extracellular vesicles, the amount of human mesenchymal stem cells to be differentiated was set to 5×10 ４ Although the concentration was set at 5 × 10 cells / well, using a smaller or larger amount did not significantly affect the results. However, from the viewpoint of obtaining stable results, a concentration of 5 × 10 cells / well was recommended. ４ cells / well tended to be the best.

[0069] (4) Proliferation Ability of Lymphatic Endothelial Cells The proliferation ability of lymphatic endothelial cells obtained by differentiation in the presence of extracellular vesicles ("EVs (differentiation)") derived from "Cell-1" in (3) above was evaluated by the following method.

[0070] Lymphatic endothelial cells (5 × 10 ３The lymphatic endothelial cells were seeded in a 96-well plate and cultured in one of the following four media. These media can culture lymphatic endothelial cells as long as they have normal characteristics.・DMEM medium containing 15% FBS ・"ECM-MV2": Endothelial Cell Growth Medium MV 2 (Ready-to-use) (Cat: C-22022, Manufacturer: Takara), a medium used for culturing human lymphatic endothelial cells (primary) ・"ECM-MV": Endothelial Cell Growth Medium MV Kit (Cat: C-22120, Manufacturer: Takara), a medium used for culturing immortalized mouse lymphatic endothelial cells ・"EGM-2+ supplement": EGM (trademark)-2MV Microvascular Endothelial Cell Growth Medium-2 (Cat: CC-3202, Manufacturer: Lonza), a medium used to induce differentiation of mesenchymal stem cells into lymphatic endothelial cells

[0071] At 24, 48, 72, and 96 hours after the start of culture, absorbance was measured with "ARVO" (measurement wavelength: 450 nm) using "Cell Counting Kit-8" (Cat: 343-07623, manufacturer: DOJINDO) (n=3). The results are shown in Figure 2.

[0072] As shown in Figure 2, lymphatic endothelial cells proliferated over time in either medium. Therefore, it was found that lymphatic endothelial cells obtained by differentiation in the presence of extracellular vesicles ("EVs (differentiation)") derived from "Cell-1" have normal proliferation ability.

[0073] (5) Various studies using extracellular vesicles As mentioned in (3) above, it was found that extracellular vesicles derived from "Cell-1" ("EVs (differentiation)") have an excellent ability to induce differentiation of mesenchymal stem cells into lymphatic endothelial cells. Therefore, their properties and effects were confirmed from various perspectives using the following methods.

[0074] (5-1) Particle size The "EVs (differentiated)" were filtered ("FILTSTAR Syringe 0.22 μm", Global Scientific) and the particle size was measured using "Nanosight (NS300)" (Malvern Panalytical) under the following conditions, and the following results were obtained. This measurement method corresponds to the nanotracking method. Average particle size: 145.4 nm Mode diameter: 106.2 nm Standard deviation: 63.7 nm

[0075] (5-2) Zeta Potential "EVs (differentiated)" were suspended in PBS, and the zeta potential was measured using "ELSZ-2000ZS" (Yamato Scientific Co., Ltd.), which was found to be "-38.6 mV."

[0076] <Test 2: Study-2 using extracellular vesicles derived from lymphatic endothelial cells> As in "Test 1" above, it was found that extracellular vesicles ("EVs (differentiation)") derived from "Cell-1" have an excellent ability to induce differentiation from human mesenchymal stem cells to lymphatic endothelial cells. Therefore, hereinafter, the differentiation-inducing effect of "EVs (differentiation)" was confirmed using mouse mesenchymal stem cells instead of human mesenchymal stem cells.

[0077] Using the "EVs (differentiated)" obtained in "Test 1" above, mouse mesenchymal stem cells were induced to differentiate into lymphatic endothelial cells by the following method. First, mouse mesenchymal stem cells "C3H10T1 / 2 cells" (mMSC) were prepared. Mouse fibroblasts "NIH3T3 cells" were also prepared as a control. Next, each cell (5 × 10 ４ The cells (1000 cells / well) were cultured in DMEM medium (Dulbecco's modified Eagle's medium) containing extracellular vesicles at 37°C for 5 days to induce differentiation. The amount of extracellular vesicles added to the DMEM medium was 1 μg / 500 μL or 2.5 μg / 500 μL in terms of protein (exosomes) (however, only 1 μg / 500 μL was used for the culture of "NIH3T3 cells").

[0078] After culturing, the expression levels of FL-1 (podoplanin) and FL-4 (LYVE-1) in the resulting cells were confirmed by flow cytometry. These are both lymphatic endothelial cell-specific markers. The results are shown in Figure 3.

[0079] As shown in Figure 3, mouse mesenchymal stem cells were induced to differentiate into lymphatic endothelial cells by culturing in the presence of "EVs (differentiation)." On the other hand, no differentiation induction was observed in NIH3T3 cells.

[0080] <Test 3: Study using extracellular vesicles derived from neuroblastoma cells> Extracellular vesicles derived from neuroblastoma cells were collected and their effects were studied using the following method. Note that neuroblastoma cells correspond to "tissue-specific cells."

[0081] (1) Preparation of extracellular vesicles derived from tissue-specific cells Extracellular vesicles derived from neuroblastoma cells were collected by the following method.

[0082] (1-1) Culture of neuroblastoma First, mouse neuroblastoma "Neuro2a" was prepared. Then, the above cells (1 × 10 ６ The cells (15 cm dish) were cultured in a medium at 37°C for 72 hours, and the culture was then collected. The medium used in this example was EMEM medium containing 10% FBS (FBS-EV Free).

[0083] (1-2) Recovery of extracellular vesicles derived from neuroblastoma cells Extracellular vesicles were recovered from the culture obtained in (1-1) above by the same method as in "(1-2) Recovery of extracellular vesicles derived from lymphatic endothelial cells" in "Test 1", to obtain extracellular vesicles derived from neuroblastoma cells.

[0084] (2) Differentiation induction using extracellular vesicles Using the extracellular vesicles obtained above, mouse mesenchymal stem cells were induced to differentiate into neuroblastoma cells by the following method. First, mouse mesenchymal stem cells "C3H10T1 / 2 cells" (mMSCs) were prepared. Next, mMSCs (5 × 10 ４The cells (1000 cells / well) were cultured in DMEM medium (Dulbecco's modified Eagle's medium) containing extracellular vesicles at 37°C for 5 days to induce differentiation. The amount of extracellular vesicles added to the DMEM medium was 2.5 μg / 500 μL in terms of protein (exosomes).

[0085] After culturing, the expression level of FL-4 (MAP-2), a neuroblastoma cell-specific marker, was confirmed by flow cytometry for the resulting cells. The results are shown in Figure 4.

[0086] As shown in Figure 4, mouse mesenchymal stem cells were induced to differentiate into neuroblastoma cells by culturing in the presence of extracellular vesicles derived from neuroblastoma cells.

[0087] <Test 4: Study using extracellular vesicles derived from vascular endothelial cells> Extracellular vesicles derived from vascular endothelial cells were collected and their effects were studied by the following method. Note that vascular endothelial cells correspond to "tissue-specific cells."

[0088] (1) Preparation of extracellular vesicles derived from tissue-specific cells Extracellular vesicles derived from vascular endothelial cells were collected by the following method.

[0089] (1-1) Culturing of Vascular Endothelial Cells First, a mouse arterial endothelial cell line "MAEC" was prepared. Then, the cells (1 x 10 ６ The cells (15 cm dish) were cultured in a medium at 37°C for 72 hours, and the culture was then collected. The medium used in this example was M199 medium containing 10% FBS (FBS-EV Free).

[0090] (1-2) Recovery of extracellular vesicles derived from vascular endothelial cells Extracellular vesicles were recovered from the culture obtained in (1-1) above by the same method as in "(1-2) Recovery of extracellular vesicles derived from lymphatic endothelial cells" in "Test 1", to obtain extracellular vesicles derived from vascular endothelial cells.

[0091] (2) Differentiation induction using extracellular vesicles Mouse mesenchymal stem cells were induced to differentiate into vascular endothelial cells using the extracellular vesicles obtained above by the following method. First, mouse mesenchymal stem cells "C3H10T1 / 2 cells" (mMSCs) were prepared. Next, mMSCs (5 × 10４ The cells (1000 cells / well) were cultured in DMEM medium (Dulbecco's modified Eagle's medium) containing extracellular vesicles at 37°C for 5 days to induce differentiation. The amount of extracellular vesicles added to the DMEM medium was 2.5 μg / 500 μL in terms of protein (exosomes).

[0092] After culturing, the expression level of FL-1 (CD31) in the obtained cells was confirmed by flow cytometry. This is a marker specific to vascular endothelial cells. The results are shown in Figure 5.

[0093] As shown in Figure 5, mouse mesenchymal stem cells were induced to differentiate into vascular endothelial cells by culturing in the presence of extracellular vesicles derived from vascular endothelial cells.

[0094] <Test 5: Preparation and transplantation of lymphatic network visceral tissue using lymphatic endothelial cells> Lymphatic network visceral tissue was prepared by the following method using lymphatic endothelial cells obtained in "Test 1" that had been induced to differentiate from human mesenchymal stem cells in the presence of "EVs (differentiation)." The obtained lymphatic network visceral tissue was then transplanted into mice.

[0095] (1) Preparation of lymphatic vessel network visceral tissue hMSC (7.5 × 10 ５ The lymphatic endothelial cells (2 x 10 cells) that had been induced to differentiate from human mesenchymal stem cells in the presence of "EVs (differentiation)" obtained in "Test 1" were seeded onto a culture plate (24-well Transwell insert, Cat: 3470, manufacturer: Corning) and centrifuged at 750 x g for 30 seconds. 18 hours after the completion of centrifugation, the cells were cultured. ５ After seeding, the culture plate was centrifuged at 750 × g for 30 seconds. After 18 hours had passed since the completion of centrifugation, hMSCs (7.5 × 10 ５ The cells were seeded onto a culture plate, and centrifuged at 750×g for 30 seconds.

[0096] Using the above method, a cell layered sheet (hMSC / lymphatic endothelial cells / hMSC) was obtained, which was further cultured in 15% FBS / DMEM medium for 5 days to form a lymphatic network tissue (three-dimensional cell structure) within the sheet.

[0097] (2) Transplantation of Lymphatic Network Tissue into Mice An incision was made in the lower limb of a BALB / c slc-nu / nu mouse, and the inguinal and popliteal lymph nodes were removed. The lymphatic network tissue obtained in (1) above was then transplanted into the site of the removed popliteal lymph node and sutured. Control mice were also prepared, which underwent lymphatic network tissue removal but were not transplanted with lymphatic network tissue. Immunostaining was performed by administering 50 μL of 1 mg / mL indocyanine green (Cat: I0535, manufacturer: MP Blomedcals) to the footpad of each mouse 1, 14, and 21 days after transplantation. The fluorescence distribution 1 hour after administration was imaged using "In-Vivo Xtreme II."

[0098] The imaging results (of the mouse lower limbs) are shown in Figure 6. The results regarding leg thickness and lymphedema size from the time of transplantation are shown in Figure 7. As these figures show, lymphatic network tissue was established at the transplant site, and lymphedema caused by the removal treatment was suppressed.

[0099] <Test 6: Study -3 Using Extracellular Vesicles Derived from Lymphatic Endothelial Cells> As in the above "Test 1," it was found that mouse mesenchymal stem cells were induced to differentiate into lymphatic endothelial cells by culturing in the presence of "EVs (differentiation)." In this example, the expression of the stem cell-specific marker FL-1 (CD44) in addition to the lymphatic endothelial cell-specific marker FL-4 (LYVE-1) was confirmed by the following method, and it was confirmed whether the differentiation induction in "Test 1" was specific to lymphatic endothelial cells.

[0100] Differentiation was induced in the same manner as in "Test 1," except that the amount of each extracellular vesicle added to the DMEM medium was set to 2 μg / 500 μL in terms of protein (exosome) amount, and flow cytometry was performed. The results are shown in Figure 8.

[0101] As shown in Figure 8, the expression level of FL-4 (LYVE-1) increased while the expression of FL-1 (CD44) was suppressed by culturing in the presence of "EVs (differentiation)." This indicates that the differentiation induction in "Test 1" is specific to lymphatic endothelial cells.

[0102] <Test 7: Study using extracellular vesicles of a different origin from that of the target cells> As shown in "Test 1" above, extracellular vesicles derived from "Cell-1," i.e., extracellular vesicles derived from lymphatic endothelial cells, were found to have an excellent effect of inducing differentiation of human mesenchymal stem cells into lymphatic endothelial cells. Furthermore, as shown in "Test 2," this effect was also found to be excellent when mouse mesenchymal stem cells were used instead of human mesenchymal stem cells. In this example, the following method was used to confirm whether induction of differentiation into lymphatic endothelial cells was also observed in such cases using extracellular vesicles derived from mouse embryonic cell-derived mesenchymal stem cells (C3H10T1 / 2 cells) instead of extracellular vesicles derived from lymphatic endothelial cells.

[0103] Extracellular vesicles were prepared using mouse embryonic cell-derived mesenchymal stem cells (C3H10T1 / 2 cells). Differentiation was induced in the same manner as in "Test 1," except that the amount of extracellular vesicles added to DMEM medium was set at 2.5 μg / 500 μL in terms of protein (exosomes). Flow cytometry was then performed. As in "Test 6," the focus was on FL-4 (LYVE-1) and FL-1 (CD44). The results are shown in Figure 9.

[0104] As shown in Figure 9, when extracellular vesicles derived from C3H10T1 / 2 cells (mMSC-EVs) were used instead of extracellular vesicles derived from lymphatic endothelial cells, no induction of differentiation into lymphatic endothelial cells was observed. This demonstrates that the differentiation-inducing effect of the present invention is achieved when the tissue-specific cells from which the extracellular vesicles are derived and the differentiation-induced cells are of the same type of cell.

Claims

1. A method for inducing cell differentiation, comprising a culture step of culturing stem cells in the presence of extracellular vesicles derived from tissue-specific cells, wherein the tissue-specific cells and the differentiation-induced cells are of the same type of cells.

2. The method for inducing cell differentiation described in claim 1, wherein the content of the extracellular vesicles is 0.5 μg / 500 μL or more in terms of protein equivalent to the culture medium used in the culture step.

3. The method for inducing cell differentiation described in claim 1, wherein the average particle diameter of the extracellular vesicles is 50 nm or more and 200 nm or less.

4. A method for inducing cell differentiation described in any one of claims 1 to 3, wherein the tissue-specific cells include at least one type of cell selected from the group consisting of lymphatic endothelial cells, vascular endothelial cells, neurons, fibroblasts, hepatocytes, adipocytes, osteoblasts, myoblasts, and chondrocytes.

5. A composition for inducing differentiation, comprising extracellular vesicles primarily derived from tissue-specific cells.

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