WO2017150294A1 - Procédé de production de sphéroïde de type cellule souche pluripotente, et sphéroïde de type cellule souche pluripotente - Google Patents

Procédé de production de sphéroïde de type cellule souche pluripotente, et sphéroïde de type cellule souche pluripotente Download PDF

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WO2017150294A1
WO2017150294A1 PCT/JP2017/006546 JP2017006546W WO2017150294A1 WO 2017150294 A1 WO2017150294 A1 WO 2017150294A1 JP 2017006546 W JP2017006546 W JP 2017006546W WO 2017150294 A1 WO2017150294 A1 WO 2017150294A1
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cells
cell
pluripotent stem
stem cell
culture
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良考 山口
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良考 山口
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

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  • the present invention relates to a method for producing pluripotent stem cell-like spheroids and pluripotent stem cell-like spheroids.
  • Patent Document 1 prepares a cell suspension derived from heart tissue and sorts a cell group including stem cells by a density gradient method.
  • the sorted cell group is suspended in a medium containing fibroblast growth factor and epidermal growth factor at a cell concentration of 1 ⁇ 10 4 to 2 ⁇ 10 4 cells / ml at the start of culture, and suspended spheres Are selected and separated to obtain pluripotent stem cells.
  • This pluripotent stem cell is said to have the ability to differentiate into cardiomyocytes and smooth muscle cells.
  • the floating sphere is formed by repeated cell division of pluripotent stem cells (Patent Document 1, paragraph “0034”). Stem cells proliferated from single cells are selectively separated, and thus the stem cells themselves are highly uniform and clinically useful (Patent Document 1, paragraph “0015”).
  • Patent Document 2 There is also a method for efficiently inducing differentiation of stem cells into cerebral cortex progenitor cells (Patent Document 2).
  • a method of differentiation culture of stem cells comprising a step of forming uniform aggregates of stem cells in a serum-free medium, and proliferation of Nodal signal promoter, Wnt signal promoter, FGF signal promoter, BMP signal promoter, retinoic acid, etc. Differentiation is induced by culturing pluripotent stem cells as floating aggregates in a serum-free medium substantially free of factors and insulins, and progenitor cells of hypothalamic neurons can be isolated from the culture (patent) Reference 2, paragraph "0020").
  • Patent Document 3 there is a method of selecting or proliferating pluripotent stem cells derived from human skin fibroblasts (Patent Document 3).
  • a characteristic cell cluster is formed at a very low frequency from an untreated human bone marrow mesenchymal cell fraction, and a low-sleep tissue stem cell is activated by stress stimulation (Patent Document 3, paragraph “ 0024)), cells that are stressed by various methods and survived when culturing mesenchymal cells or mesodermal cells such as bone marrow mesenchymal cell fraction and skin fibroblast fraction Is to collect.
  • This stem cell is a pluripotent stem cell characterized by SSEA-3 positive and CD105 positive, self-renewal ability, etc., and is named Muse cell. Muse cells exist in mesodermal or mesenchymal tissues of living organisms. Cells or cell fractions present in these tissues are separated (Patent Document 3, paragraph “0040”), non-stem cells are removed by stress stimulation, and remaining stem cells are enriched (Patent Document 3, Paragraph "0167").
  • Muse cells differ from iPS cells and ES cells in that they can be obtained directly from living bodies and tissues. Further, it has a feature that it can be obtained without requiring reprogramming or induction of dedifferentiation (Patent Document 3, paragraph “0036”).
  • human skin fibroblast fraction was used as mesenchymal cells, and the cells were incubated for 16 hours in trypsin (0.25% trypsin-HBSS) as a stress stimulus, and the living cells were collected to contain methylcellulose. Cultured in a medium at a density of 8,000 cells / ml for 7 days (Patent Document 3, paragraph “0138”).
  • a cell population formed from the human fibroblast fraction subjected to the 16-hour trypsin incubation described above is referred to as a “rich Muse cell fraction”, and a single cell obtained from the population is cultured in suspension to obtain a rich Muse cell fraction. Since cell mass formation is observed in 9 to 10% of the cells, about 9 to 10% of Muse cells are contained in the rich Muse cell fraction (Patent Document 3, paragraph “0139”).
  • Patent Documents 1 to 3 are techniques for selecting and proliferating stem cells contained in living tissue, there are also methods for generating pluripotent cells from differentiated cells by reprogramming (Patent Document 4).
  • CD45-positive lymphocytes derived from Oct4-GFP mice were used, and when exposed to a low pH solution, spherical colonies were observed within 3 days of exposure, and embryonic stem cell markers were expressed (Patent Document 4, paragraph) “0149”).
  • CD45 negative cells or CD45 negative / Oct4 positive cells are shown, and on the 7th day, CD45 expression disappeared and Oct4 expressing cells were observed (Patent Document 4, paragraph “0158”). Evaluating the appearance of early embryogenesis markers on day 7 cells to confirm reprogramming demonstrated that pluripotent cell markers such as Oct4, Nanog, Sox2 were expressed and reprogrammed by external stimuli. (Patent Document 4, paragraph “0159”).
  • Non-patent Document 1 there is an introduced protein that brings cells into a pluripotent stem cell-like state (Non-patent Document 1). It is called reprotin, and it is said that fibroblasts form spheroids through an epithelial-mesenchymal transition state in a short time, and reprogramming / initialization is remarkably enhanced. When fibroblasts are initialized using this reprotin, pluripotent stem cell colony-like spheroids can be produced with an efficiency of 10% or more in about 1 to 3 days.
  • Spheroids were strongly positive by alkaline phosphatase staining, and pluripotency markers SSEA-3 and TRA-1-81 were also positive at the same time, and the cells constituting the spheroids are considered to have changed to a pluripotent state.
  • the structure of reprotin is unknown.
  • the neural stem cell marker Nestin becomes positive, and pluripotent stem cells and neural stem cells can be easily prepared from fibroblasts.
  • Reprotech gene-free pluripotent stem cell creation technology
  • neural stem cell marker Shin Nakabayashi, Gene Try, Inc, Reprotech website, http://reprotech.jimdo.com/reprotech%E3%81%A8%E3%81%AF /, Search on August 29, 2016
  • Patent Document 1 described above is a method for selectively sorting pluripotent stem cells having excellent differentiation ability from heart tissue into cardiomyocytes and the like.
  • the cells obtained are limited to pluripotent stem cells that are excellent in the ability to differentiate into cardiomyocytes.
  • a cell suspension derived from heart tissue is used as a raw material, a cell population containing the target stem cells is separated by a density gradient method, and the separated cell population is separated in a suspension medium containing fibroblast growth factor and epidermal growth factor.
  • the process of culturing and selectively separating spheres of floating pluripotent stem cells from the cell group contained in the culture is necessary, and the operation is complicated.
  • the stem cells are aggregated in a serum-free medium to form a qualitatively uniform stem cell group, and the aggregated stem cells are suspended in the serum-free medium.
  • a network is formed in a test tube.
  • This method is not a technique for creating stem cells, but a method for synchronizing existing stem cell groups with the same properties.
  • Patent Document 1 and Patent Document 2 both cultivate stem cells that exist slightly in advance, and are not techniques for preparing pluripotent stem cells from differentiated mature cells.
  • the Muse cell described in Patent Document 3 is also used for culturing preexisting stem cells as in Patent Document 1 and the like.
  • Very rare pluripotent stem cells contained in human dermal fibroblasts are sorted and proliferated using SSEA-3 marker as an indicator. It is excellent in that human skin fibroblasts that can be easily obtained can be used as a raw material, but most cells are killed by an overload of stress, and Muse cells in surviving cells are selected. Recovery rate of Muse cells Is extremely low. Further, it is necessary to use an expensive instrument for separating the target cells, and the operation is complicated because a special technique is used.
  • Patent Document 3 describes that Muse cells can be proliferated by a series of cycles of Muse cell-derived embryoid somatic cell mass and clonal expansion, whereby a large amount of Muse cells can be obtained from a mesenchymal cell population.
  • Patent Document 3, paragraph “0142” the size of the cell cluster has a maximum diameter of about 150 ⁇ m
  • Patent Document 3, paragraph “0167” the size of the cell cluster has a maximum diameter of about 150 ⁇ m
  • Muse cells have multiple security systems to prevent explosive growth and can avoid abnormal growth (Patent Document 3, paragraph “0167”), large stem cells can be operated with simpler operations. Can be used for stem cell research, regenerative medicine, treatment, etc. without performing a proliferation operation, and is excellent in versatility.
  • Patent Document 4 generates pluripotent cells from differentiated cells by exposure to a low pH solution.
  • CD45-positive cells collected from the spleen of Oct4-GFP (GOF) mice were exposed to an acidic solution at pH 5.4 to 5.6, GFP-expressing cells were sorted and cultured in B27-LIF medium. The number of dead cells increases on the seventh day of culture.
  • Patent Document 4 describes that the cell characteristics were gradually changed by stress treatment and specific culture, and successfully changed cells expressing Oct4 could be selected (Patent Document 4, paragraph “0158”), but for 7 days.
  • a cell mass having a diameter of 70 ⁇ m is obtained only by the growth over a wide range. Therefore, development of a method for producing spheroids composed of large-sized pluripotent stem cells and a production technique for converting all used somatic cells into stem cells are desired.
  • Non-Patent Document 1 prepares pluripotent-like stem cells by culturing using an introduced protein called Reprotin. Although gene transfer is not necessary, the structure of reprotin is not clear, and safety and the like are insufficient for use in regenerative medicine, stem cell therapy, and the like. Therefore, development of a method capable of preparing pluripotent stem cells without adding an extracellular compound is desired.
  • an object of the present invention is to provide a method for producing a pluripotent stem cell-like spheroid expressing a pluripotent stem cell marker from differentiated somatic cells by a simple method.
  • Another object of the present invention is to provide a polyclonal pluripotent stem cell-like spheroid having a diameter of 0.1 to 4 mm.
  • the present inventor prepared a cell suspension containing spheroidized fibroblasts obtained by enzymatic treatment of differentiated fibroblasts and individually separating and suspending the cells, and the spheroidized cells were separated from the cell culture container.
  • the cells are statically cultured at a high density or in a close contact state, the cells start to aggregate, and the aggregates increase in size with the passage of time to form spheroids, and the cells constituting the spheroids are Oct3. / 4 and SSEA-3 were found to express proteins characteristic of pluripotent stem cells, and the present invention was completed.
  • pluripotent stem cells do not require the introduction of artificial compounds, vectors / genes, exposure to strong stress environments (low pH, culture temperature lower or higher than 37 ° C, low oxygen conditions, etc.), hormones, proliferation Spheroids exhibiting the properties of pluripotent stem cells can be produced from mature somatic cells without using stimulating factors such as factors, pluripotency-inducing proteins, and other pluripotent cell-inducing factors.
  • the inventor named this pluripotent stem cell as a natural stem cell.
  • the present invention separates the living cells by enzymatic treatment of the differentiated living cells, and the separated living cells are 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml.
  • the present invention provides a method for producing a pluripotent stem cell-like spheroid, characterized by performing static culture in a cell non-adherent cell culture vessel at a density.
  • the present invention also provides the method for producing the pluripotent stem cell-like spheroid, wherein the enzyme treatment comprises treating the differentiated living cells with a solution having a trypsin concentration of 0.1 to 1% for 1 to 60 minutes. Is.
  • the present invention also provides a method for producing the pluripotent stem cell-like spheroid, wherein the differentiated living cells are fibroblasts.
  • the pluripotent stem cell-like spheroid is at least one pluripotent stem cell marker selected from the group consisting of OCT3 / 4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1
  • the separated living cells are statically cultured in a cell non-adherent cell culture vessel at a cell density of 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml, and the cell number is accommodated.
  • the present invention provides a method for producing the pluripotent stem cell-like spheroid, characterized by forming a spheroid having a diameter of 0.1 to 4 mm.
  • the present invention also relates to at least one pluripotent stem cell marker selected from the group consisting of OCT3 / 4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA- Providing a clonal pluripotent stem cell-like spheroid having a diameter of 0.1 to 4 mm and composed of cells expressing at least one pluripotent stem cell marker selected from the group consisting of 4 and ALP It is.
  • at least one pluripotent stem cell marker selected from the group consisting of OCT3 / 4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA- Providing a clonal pluripotent stem cell-like spheroid having a diameter of 0.1 to 4 mm and composed of cells expressing at least one pluripotent stem cell marker selected from the group consisting of 4 and ALP It is.
  • the differentiated living cells are treated with enzymes to separate the living cells individually, and the separated living cells are separated by 1 ⁇ 10 5 cells / cm 2 to 7 ⁇ 10 6 cells / cm 2 per medium area.
  • the present invention provides a method for producing a pluripotent stem cell-like spheroid, characterized by performing static culture in a flat-bottom non-adherent cell culture vessel at a cell density of 5%.
  • the present invention also provides an enzyme treatment of differentiated living cells to separate the living cells individually, and the separated living cells are separated by 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml.
  • the present invention provides a method for expressing a pluripotent stem cell marker, characterized by forming a spheroid by stationary culture in a cell non-adherent cell culture vessel at a cell density.
  • living cells individually separated and spheroidized are statically cultured in a cell non-adherent cell culture vessel in a high density state, thereby comprising cells that express a pluripotent stem cell marker.
  • Spheroids can be formed.
  • Example 1 It is a figure which shows the result of Example 1, and is a differential interference microscope image of 0 hours after culture
  • Example 2 It is a figure which shows the result of Example 2, and is a figure which shows the result of the expression of PAR4 which is a pluripotent stem cell marker. It is a figure which shows the result of Example 2, and is a figure which shows the result of the expression of SSEA-3 which is a pluripotent stem cell marker. It is a figure which shows the result of Example 2, and is a figure which shows the result of the expression of SSEA-4 which is a pluripotent stem cell marker. It is a figure which shows the result of Example 2, and is a figure which shows the result of the expression of SOX2 which is a pluripotent stem cell marker.
  • Example 2 It is a figure which shows the result of Example 2, and is a figure which shows the result of the expression of TRA-1-60 which is a pluripotent stem cell marker. It is a figure which shows the result of Example 2, and is a figure which shows the result of the expression of TRA-1-81 which is a pluripotent stem cell marker. It is a figure which shows the result of (4) of Example 3.
  • the spheroids obtained in the step (1) of Example 3 were dropped and adhered to a normal form of human skin fibroblasts that were adhered and alive on an adhesive cell culture vessel, and co-cultured. Spheroids migrated (transformed) into normal forms of human skin fibroblasts.
  • the figure shows differential interference microscopic images at the start of co-culture, 2 hours after co-culture, 4 hours after co-culture, and 26 hours after co-culture. It is a figure which shows the result of (5) of Example 3.
  • the spheroids obtained by static culture for 6 hours in the step (1) of Example 3 were cultured using the cell culture vessel for adherent cells and without adding the normal form of adherent human skin fibroblasts. It is a differential interference microscope image of.
  • FIG. 4 It is a figure which shows the result of Example 4, and is the microscope image of the centrifuge tube bottom part when the cell isolate
  • the left shows innumerable spheroidized living cell cultures that have been sunk at the bottom of the centrifuge tube before the start of culture, and the right shows spheroids formed by culture.
  • Example 5 It is a figure which shows the result of Example 5, and it is the stereoscopic microscope image of the several droplet of a cell at the time of the start of culture
  • the image of one droplet is the result of observing the same droplet over time.
  • the image of one droplet is the result of observing the same droplet over time.
  • FIG. 6 shows the results of Example 7, which is a stereomicroscopic image of a spheroid having a diameter of about 0.1 to 0.3 mm. It is a figure which shows the result of Example 7, and is a figure which shows the result of the expression of the obtained spheroid DNA dyeing
  • the differentiated living cells are treated with enzymes to separate the living cells individually, and the separated living cells are separated from 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml.
  • This is a method for producing a pluripotent stem cell-like spheroid, characterized by performing static culture in a cell non-adherent cell culture vessel at a cell density of 5%.
  • biological cells can broadly target cells that constitute mammalian tissues and organs. Examples of such mammals include primates such as humans and monkeys, rodents such as mice and rats, lepidoptera such as rabbits, cloven-hoofed animals such as cows, equine eyes such as horses, cetaceans such as pigs, Meats such as dogs and cats can be exemplified.
  • the living cells used in this specification are limited to differentiated cells.
  • a “differentiated living cell” means a cell specialized from the time of development of the living body, and includes both a cell that has been differentiated and a cell that has been specialized before the final differentiation but has been developed. . Since tissue stem cells can be differentiated into final tissue cells, they are included in differentiated living cells. On the other hand, embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) are not included in “differentiated living cells”.
  • ES cells embryonic stem cells
  • EG cells embryonic germ stem cells
  • differentiated biological cells include muscle tissue, connective tissue, circulatory tissue, excretory tissue, reproductive tissue, bone, cartilage, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon.
  • the differentiated living cell may be a commercially available product, and a living tissue is separated into individual cells constituting a physical or chemical treatment, and a cell containing the target cell is separated by a density gradient method or the like. It may be stored in
  • Differentiated living cells are usually cultured in a medium or culture vessel most suitable for the cells.
  • a medium or culture vessel examples include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, D-MEM medium, Ham medium, RPMI 1640 medium, Fisher medium, and the like.
  • culture vessels for adherent cells include plastic or glass culture vessels coated with extracellular substrates such as various collagens, fibronectin, laminin, gelatin, elastin, proteoglycan, vitronectin, and various poly-lysines.
  • the cell non-adhesive cell culture container include a plastic or glass culture container that is not coated with a cell-adhesive extracellular matrix. It can select suitably according to the biological cell to be used.
  • the differentiated living cells are individually separated by enzyme treatment.
  • the separated living cells spheroidize and float in the culture medium.
  • fibroblasts are adherent cells that adhere to a culture container during culture or cells adhere to each other.
  • enzyme treatment is an operation for obtaining a cell suspension in which living cells are individually separated.
  • enzymes used include serine proteases such as trypsin and chymotrypsin, aspartic proteases such as pepsin, cysteine proteases such as papain and chymopapain, metalloproteases such as thermolysin, glutamate protease, N-terminal threonine protease, collagenase, and despase proteases. There is. In this method, trypsin is preferred.
  • the condition of the enzyme treatment is a concentration used for peeling off the living cells adhered to the culture vessel when handling the adherent cells.
  • the concentration is 0.1 to 1% by mass, more preferably 0.1 to 0%. 3% by mass.
  • the temperature is 30 to 40 ° C, more preferably 33 to 38 ° C.
  • the treatment time is 1 to 60 minutes, preferably 1 to 30 minutes, more preferably 2 to 5 minutes.
  • collagenase is used, the content is 0.1 to 0.6% by mass, preferably 0.2 to 0.4% by mass.
  • the temperature is 25 to 40 ° C, more preferably 30 to 40 ° C.
  • the processing time is 30 minutes to 24 hours.
  • an enzyme inhibitor used in the enzyme treatment may be contained in the culture medium or storage medium of “differentiated living cells”. In that case, before the enzyme treatment, the inhibitor is removed by washing the cells with a culture solution not containing the enzyme inhibitor, PBS or other buffer solution, and then the enzyme treatment is performed.
  • the individually separated living cells contained in the cell suspension are spherical. Centrifugation is suitable for recovering the spheroidized living cells. Usually, centrifugation is performed at 500 to 1,500 rpm for 2 to 5 minutes, the supernatant is removed, and the precipitated cells are collected.
  • the collected spheroidized living cells have a cell density of 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml, preferably 4 ⁇ 10 5 cells / ml to 7 ⁇ 10 7 cells / ml, More preferably, static culture is performed at 5 ⁇ 10 5 cells / ml to 3 ⁇ 10 7 cells / ml, particularly preferably 1 ⁇ 10 6 cells / ml to 2 ⁇ 10 7 cells / ml. Generally, cells are roughly classified into adhesion culture cells and suspension culture cells.
  • adherent culture cells are seeded in a culture container coated with an adhesive substance such as poly-L-lysine, and cultured and propagated while attached to the container.
  • suspension culture cells are cultured in a state of being suspended in a cell non-adhesive cell culture vessel.
  • stationary culture is performed regardless of whether the differentiated living cells are adhesion culture systems or suspension culture systems.
  • the “stationary culture” means culturing the culture solution without stirring or swirling.
  • poly-D-lysine poly-L-lysine
  • 2-methacryloyloxyethyl Phosphoryl Choline poly (2-hydroxyethyl methacrylate) (Poly (2-hydroxyethyl methacrylate))
  • a so-called non-coated container that is not coated with collagen or the like is suitable.
  • feeder cells are not necessary.
  • the container for stationary culture for example, flasks, tissue culture flasks, dishes, petri dishes, cell culture dishes, multi dishes, micro plates, micro well plates, multi plates, multi well plates, Chamber slides, petri dishes, test tubes, centrifuge tubes, trays, culture bags, roller bottles, plastic plates, plastic microtubes, and the like can be used.
  • a flat-bottomed floating cell culture flask may be used, and a deep container with a curved bottom surface such as a test tube or centrifuge tube may be used.
  • Static culture may be performed.
  • a preferable cell density when culturing in a flat bottom is preferably about 1 ⁇ 10 5 cells / cm 2 to 7 ⁇ 10 6 cells / cm 2 per medium area.
  • the cell density per medium area in the case of a deep container is higher than this, but if static culture is performed at a cell density of 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml, the medium There is no limitation on the number of cells per area.
  • the cells When the individually separated cells are statically cultured at the above cell density, the cells naturally fall in the culture solution with the passage of time, and other cells are stacked on top of the cells dropped on the bottom of the container. Under the above-mentioned cell density conditions, a number of cell layers stacked on the bottom layer of cells that have been sunk on the bottom of the container are formed, and the cells may contact and adhere within the culture solution.
  • the feature of this method is that, when statically cultured living cells are individually cultured, the cell density is adjusted to a cell density of 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml to start the culture.
  • a cell culture container As a basic property of mammalian cells, when a cell culture container is full, that is, confluent, adherent cells naturally detach and die, and floating cells also die by apoptosis.
  • a culture environment such as a culture vessel or a nutrient source is prepared, if a high-density state in which the cells are in close contact with each other continues, the cells cannot survive for a long time.
  • a general high-density state including normal cells, cell lines, and cancer cells is 8 ⁇ 10 3 to 2 ⁇ 10 4 cells / cm 2 with respect to the area of the culture vessel. Since normal cell culture is performed at a lower cell density than this, the cells hardly come into contact with each other, and the cell proliferation ability and the risk of cell death can be avoided. The cell morphology and properties peculiar to the cell type can be maintained. In this method, static culture is performed at a high density, and in the case of a flat bottom container, the cell density is 1 ⁇ 10 5 cells / cm 2 to 7 ⁇ 10 6 cells / cm 2 , or the cell density per solution Is 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml.
  • the number of seeded cells is too large, the distance between the cells is close or close, and the cell becomes confluent in a short period of time. For this reason, there may be a risk of reduced proliferation ability, cell-specific morphology failure, and cell death.
  • individual spheroidized living cells are allowed to stand in a non-adherent cell culture vessel and allowed to stand at the above cell density, the suspended cells in the culture solution will naturally fall over time and the bottom of the vessel It was found that cells contacted each other and formed spheroids in 5 to 8 hours. Surprisingly, the cells that make up the spheroids express embryonic and pluripotent stem cell markers.
  • this method can be said to be a method for forming spheroids from differentiated living cells and a method for expressing pluripotent stem cell markers in spheroids derived from differentiated living cells.
  • the pluripotent stem cell markers include NANOG, SOX2, Oct-3 / 4, Klf4, Lin28, TRA-1-60, SSEA-1, SSEA-4, c-Myc, PAR4, TRA-1-81, SSEA- 3, ALP, etc.
  • NANOG, SOX2, Oct-3 / 4 are essential markers for pluripotent stem cells.
  • the spheroids formed by the above are at least one pluripotent stem cell marker selected from the group consisting of OCT3 / 4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3 Expressing at least one pluripotent stem cell marker selected from the group consisting of SSEA-4 and ALP.
  • the spheroids formed by the above operations express NANOG, SOX2 and Oct-4, and PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4 and ALP was expressed.
  • Expression of a pluripotent stem cell marker indicates that the cell has changed to a pluripotent state.
  • the “pluripotent stem cell-like spheroid” is an aggregate of cells, and the cells constituting the aggregate are at least one kind selected from the group consisting of OCT3 / 4, SOX2 and NANOG. Expressing a pluripotent stem cell marker and at least one pluripotent stem cell marker selected from the group consisting of PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4 and ALP And
  • Pluripotent stem cells express markers of different cell types with the progress of their differentiation. For example, when they differentiate into hepatocytes, albumin is expressed. Ultimately differentiated hepatocytes express albumin and the expression of pluripotent stem cell markers disappears. The level of expression of markers in different cell types and the level of disappearance of pluripotent stem cell markers vary depending on the degree of differentiation, etc., but the expression of embryonic stem cells and pluripotent stem cell markers are differentiated in both. Means there is no initialization state.
  • the spheroids prepared as described above are pluripotent stem cell spheroids in an initialized state because they express embryonic stem cells and pluripotent stem cell markers. Therefore, this method can be said to be a method for initializing differentiated living cells.
  • the method for producing a pluripotent stem cell-like spheroid of this method is free from stress caused by long-term enzyme treatment, and is an artificial nucleic acid such as OCT3 / 4, SOX2, KLF4, c-Myc, or reprotin (Non-patent Document) It is not necessary to use an introduced protein such as 1).
  • spheroidized fibroblasts individually separated by enzyme treatment are adjusted to 3 ⁇ 10 5 cells / ml to 7 ⁇ 10 8 cells / ml, and D containing 10% fetal calf serum is used.
  • the proportion of cells expressing the pluripotent stem cell marker is 95 to 100%. Moreover, since there is no stress load in the operation process, almost no dead cells are observed. The number of dead cells in static culture is as low as 0 to 5%. This means that it was possible to convert the differentiated living cells into cells expressing the pluripotent stem cell marker at a conversion rate of 90-100%, more preferably 95-98%.
  • the conversion rate from differentiated living cells to cells that express pluripotent stem cell markers is high, it is possible to adjust the diameter of the spheroids according to the number of cells by preparing the number of cells when stationary culture is performed it can.
  • the cells gather over time in one place.
  • 30 ⁇ l of a cell suspension with a cell density of 1.25 ⁇ 10 7 cells can be cultured to form a spheroid with a diameter of about 1 mm, and 500 ⁇ l can be cultured to form a spheroid with a diameter of about 4 mm. can do.
  • pluripotent stem cell-like spheroids having a diameter of 0.1 to 4 mm can be produced according to the number of cultured cells. For this reason, the process of concentration operation using the antibody of a pluripotent stem cell, the process of enlarging spheroid or enlarging by carrying out suspension culture after isolate
  • the spheroids formed by this method are formed by aggregation of neighboring cells during stationary culture, and are not formed by the growth of one cell. Therefore, it can be said to be a “multiple clone” in which different cells, that is, different clones are assembled. Moreover, the cell aggregation occurs with time and without exception, and the spheroid size increases with the progress of aggregation.
  • pluripotent stem cell-like spheroids are prepared in a short time of 6 hours. That is, if spheroidized fibroblasts are prepared, pluripotent stem cell-like spheroids can be prepared in an extremely large amount in a short time by one step of stationary culture.
  • the Muse cell described in Patent Document 3 is obtained by culturing and selecting human skin fibroblasts by applying a stress stimulus of incubation treatment with trypsin for 16 hours, and culturing and sorting the resulting single cells.
  • a cell mass formed in the Muse cell fraction It can be said to be a “monoclone” because it proliferates in culture.
  • the preparation of the rich Muse cell fraction is not easy, and the number of Muse cells is increased by subjecting a single cell obtained from this fraction to a plurality of suspension cultures and adhesion cultures. A multi-stage cell growth operation is required, and it takes a long time to obtain the desired number of cells.
  • a large amount of pluripotent stem cell-like spheroids can be prepared in a short time.
  • spheroids with a diameter of 0.5 mm can be visually observed and can be recovered without requiring an expensive centrifuge because they settle by their own weight. It is easy to prepare as much as you need for research and treatment, saving time and money. Since the operation is simple, there is little stress on the cells, and unnecessary operation errors can be avoided.
  • Pluripotent stem cell-like spheroids prepared by this method can be applied to regenerative medicine.
  • a technique has been developed in which iPS cells derived from a subject are injected into a blastocyst of a pig that cannot produce a specific organ, and the organ composed of the iPS cell-derived cells is regenerated into a pig. If pluripotent stem cell-like spheroids prepared by this method from differentiated living cells of a subject are used instead of iPS cells, problems such as canceration can be avoided and organs can be regenerated.
  • stem cells derived from subjects there are stem cells obtained from autologous bone marrow, placenta, umbilical cord blood, umbilical cord, amniotic membrane, etc., and there is also a “stem cell bank” in which these are stored at a low temperature using liquid nitrogen or the like.
  • pluripotent stem cell-like spheroids can be prepared from living cells such as fibroblasts of a subject, there is no need for cryopreservation, and there are problems such as a decrease in the number of living cells upon lysis. There is no need to do.
  • the second of the present invention is at least one pluripotent stem cell marker selected from the group consisting of OCT3 / 4, SOX2 and NANOG, and PAR4, TRA-1-60, TRA-1-81, SSEA-3, A clonal pluripotent stem cell-like spheroid having a diameter of 0.1 to 4 mm and composed of cells expressing at least one pluripotent stem cell marker selected from the group consisting of SSEA-4 and ALP .
  • This pluripotent stem cell-like spheroid can be produced by the first of the present invention using differentiated living cells as a raw material, but is not limited thereto. Conventionally, monoclonal spheroids obtained by proliferating stem cells exist.
  • Muse cells have a diameter of about 150 ⁇ m. Muse cells have multiple security systems to prevent explosive growth. For this reason, it is not easy to prepare a large spheroid.
  • the pluripotent stem cell-like spheroid of the present invention has a diameter of about 0.1 to 4 mm, more preferably 0.5 to 3 mm. Since it is larger than conventional spheroids, it does not need to be enlarged by cell proliferation.
  • the pluripotent stem cell-like spheroid of the present invention expresses a pluripotent stem cell marker, functions as a pluripotent stem cell, and can differentiate into any tissue / cell type. It can be used for regenerative medicine.
  • the pluripotent stem cell-like spheroid of the present invention can also be used for transplantation, etc.
  • a cell suspension of pluripotent stem cell-like spheroid is prepared, attached to the tissue and proliferated, and used for various organ cell types it can.
  • the transplanted tissue includes skin, cerebral spinal cord, liver, muscle and the like.
  • pluripotent stem cell-like spheroid of the present invention By administering the pluripotent stem cell-like spheroid of the present invention directly or nearby to a damaged or damaged tissue, organ, etc., cells expressing a pluripotent stem cell marker enter the tissue, organ, It can differentiate into cells peculiar to tissues and regenerate tissues and organs.
  • Administration can be performed by subcutaneous injection, intravenous or arterial injection, intramuscular injection, intraperitoneal injection, parenteral administration such as direct injection into a damaged or defective organ tissue, intrauterine injection into an embryo, or the like.
  • the dose can be appropriately determined depending on the type and desired size of the organ or tissue in the organ to be regenerated and the degree of regeneration.
  • the pluripotent stem cell-like spheroid of the present invention can be induced to differentiate in advance, and the differentiated cells can be used for regenerative medicine. As shown in Example 6 to be described later, when the size after spheroid culture was evaluated over time, it reached its maximum at 67 hours after the culture, and then maintained the size at the time of transformation until 94 hours after the culture.
  • the pluripotent stem cell-like spheroid of the present invention was not prepared by introducing a tumorigenic gene or the like, and no tumorigenicity estimated by size increase after culture was observed. The possibility of canceration is extremely low.
  • the pluripotent stem cell-like spheroid of the present invention can be used for basic research such as elucidation of disease mechanisms, therapeutic drug development, screening for drug effects and toxicity, drug evaluation, and the like.
  • embryonic body-like cell clusters made from pluripotent stem cell-like spheroids, pluripotent stem cell-like spheroids, and cells, tissues, and organs obtained by differentiation from the embryoid body-like cell clusters are used for drug evaluation and drug screening. It can be used as a material.
  • the pluripotent stem cell-like spheroid of the present invention has basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), dimethyl sulfoxide (DMSO) and isoproterenol, or fibroblast as a differentiation inducing factor.
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • DMSO dimethyl sulfoxide
  • isoproterenol or fibroblast as a differentiation inducing factor.
  • FGF4 vascular endothelial growth factor
  • DMSO dimethyl sulfoxide
  • isoproterenol or fibroblast as a differentiation inducing factor.
  • FGF4 vascular endothelial growth factor
  • DMSO dimethyl sulfoxide
  • isoproterenol fibroblast as a differentiation inducing factor.
  • FGF4 vascular endothelial growth factor
  • HGF hepatocyte growth
  • the present invention it is possible to obtain a spheroid expressing a pluripotent stem cell marker from a differentiated living cell without introducing a foreign gene, transcript, protein, nuclear component, cytoplasm, or other foreign reprogramming factors. it can.
  • the reason for expressing pluripotent stem cell markers is unknown. If this phenomenon is reprogramming, the only difference from the conventional cell culture is that the cells are cultured in a high density or contact state under the cell culture conditions. Although high-density contact culture can be considered as a kind of stress load, since cells in a living body are in contact and survive in the first place, it is difficult to think of stress, and even in the examples, there are almost no dead cells in culture. Even if the above phenomenon is interpreted as reprogramming, the reprogramming expression mechanism in the pluripotent stem cell-like spheroid of the present invention is unknown at present.
  • Example 1 Cell culture petri dish for adherent cells (Sumitomo Bakelite Co., Ltd., trade name “Petri dish for cell culture 90 ⁇ (deep type)”), 1.5 ⁇ 10 6 human skin fibroblasts (HDF, Purchased from Kurabo Industries Co., Ltd.), D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., 10% fetal bovine serum, 1% penicillin / streptomycin contained) 10 ml, 37 ° C., 5% CO 2 , 80% confluent The culture was continued for 48 hours until.
  • FIG. 1 shows differential interference microscopic images at the start of culture, after 5 hours of culture, after 14 hours of culture, and after 26 hours of culture. Little dead cells were observed.
  • Example 2 (1) Cell culture petri dish for adherent cells (Sumitomo Bakelite Co., Ltd., trade name “Petri dish for cell culture 90 ⁇ (deep type)”) and cell culture petri dish for adherent cells (Sumitomo Bakelite Co., Ltd., trade name “Cell” 2 ⁇ 10 6 cells and 0.6 ⁇ 10 6 human skin fibroblasts (HDF, purchased from Kurabo Industries), respectively, were used in a D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd. 10% fetal bovine serum, 1% penicillin / streptomycin-containing 10 ml and 3 ml were cultured for 48 hours under conditions of 37 ° C. and 5% CO 2 .
  • D-MEM medium High Glucose, Wako Pure Chemical Industries, Ltd. 10% fetal bovine serum, 1% penicillin / streptomycin-containing 10 ml and 3 ml were cultured for 48 hours under conditions of 37 ° C. and 5%
  • the cell density was 3.7 ⁇ 10 6 cells / ml, about 1.2 ⁇ 10 6 cells / cm 2 .
  • Numerous spheroids were formed from almost all of the human skin fibroblasts in the upper lid.
  • the upper left is a differential interference microscope image (DIC image)
  • the lower left is stained with 4 ′, 6-diamidino-2-phenylindole, and an image observed with a fluorescence microscope (DAPI image)
  • DAPI image an image obtained by staining the marker protein of each pluripotent stem cell with a fluorescent dye (Alexa 488) and observed with a fluorescent microscope
  • the upper right is an image obtained by superimposing the DAPI image and the fluorescent dye image.
  • HDF human dermal fibroblasts
  • Example 3 (1) (1) to (3) of Example 1 using four cell culture dishes for adherent cells (manufactured by Sumitomo Bakelite Co., Ltd., trade name “Petri dish for cell culture 90 ⁇ (deep type)”) The same operation was performed to obtain 1.1 ⁇ 10 7 individually isolated human skin fibroblasts. To the obtained human skin fibroblasts, 10 ml of D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., 10% fetal bovine serum, 1% penicillin / streptomycin) was added to prepare a cell suspension.
  • D-MEM medium High Glucose, Wako Pure Chemical Industries, Ltd., 10% fetal bovine serum, 1% penicillin / streptomycin
  • suspension culture multiplate manufactured by Sumitomo Bakelite Co., Ltd., trade name “with suspension culture plate 12F (independent well) with lid”, non-coated, flat bottom, 1 well bottom area: 3 6 cm 2
  • static culture was performed at 37 ° C. and 5% CO 2 for 9 hours.
  • the cell density is 1.1 ⁇ 10 6 cells / ml and 1.5 ⁇ 10 5 cells / cm 2 .
  • spheroids were formed from human dermal fibroblasts.
  • step (2) Transfer 200 ⁇ l of the cell suspension prepared in step (1) to a chamber slide (manufactured by AGC Techno Glass Co., Ltd., IWAKI, trade name “Chamber Slide II”, collagen coating, 1 well bottom area: 9 cm 2 ). 1 ml of D-MEM medium was added and static culture was performed at 37 ° C. and 5% CO 2 for 9 hours. The cell density is 1.8 ⁇ 10 5 cells / ml, 2.4 ⁇ 10 4 cells / cm 2 . The cultured human skin fibroblasts took the form of normal human skin fibroblasts adhered to a slide glass.
  • Example 3 The spheroids obtained in step (1) of Example 3 were collected in a 1.5 ml microtube and centrifuged at 6,200 rpm for 10 seconds to obtain spheroid pellets.
  • step (3) of Example 3 To the normal human skin fibroblasts obtained in step (2) of Example 3, the spheroid pellet recovered in step (3) of Example 3 was added and co-cultured for 26 hours.
  • spheroids in contact with normal forms of human skin fibroblasts changed to normal human skin fibroblasts over time.
  • FIG. 11 is a differential interference microscope image after 2 hours, 4 hours, and 26 hours at the start of co-culture.
  • Spheroids were prepared by static culture for 6 hours in the step (1) of Example 3.
  • the spheroids were collected and D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., 10% bovine) in a chamber slide (manufactured by IWAKI, AGC Techno Glass, trade name “Chamber Slide II”, collagen coating, 1 well).
  • Fetal serum, containing 1% penicillin / streptomycin was cultured in an environment free of normal forms of human skin fibroblasts. As a result, the spheroid maintained the shape of the cell aggregate. The result is shown in FIG. FIG.
  • Example 4 (1) In the same manner as in Example 2, 1.25 ⁇ 10 7 individually isolated human skin fibroblasts were obtained. This was suspended in 1 ml of D-MEM medium. (2) 30 ⁇ l (3.8 ⁇ 10 5 cells), 60 ⁇ l (7.5 ⁇ 10 5 cells), 125 ⁇ l (1.6 ⁇ 10 6 cells), 250 ⁇ l (1.5 ⁇ l centrifuge tube) 3.1 ⁇ 10 6 ) and 500 ⁇ l (6.3 ⁇ 10 6 ) were transferred, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was removed. D-MEM medium was added to each test tube so that the final volume was 90 ⁇ l, followed by static culture at 37 ° C. and 5% CO 2 for 15 hours. In each test tube, one spheroid was formed, and the diameters thereof were 1 mm, 1.4 mm, 1.8 mm, 2.8 mm, and 4 mm, respectively. The results are shown in FIGS.
  • Example 5 By operating in the same manner as in Example 1, 3 ⁇ 10 6 individually isolated human skin fibroblasts were obtained. This was suspended in 1 ml of D-MEM medium to prepare a cell suspension. (2) The lid for 96-well plate (manufactured by Greiner bio-one, trade name “L ID FOR MICROPLATE, non-coated”), 1 ⁇ l, 2 ⁇ l, 3 ⁇ l, 5 ⁇ l (1.5 ⁇ 10 4 pieces), 8 ⁇ l (2.4 ⁇ 10 4 pieces), 10 ⁇ l (3 ⁇ 10 4 pieces) were dropped, and stationary culture was performed in the form of droplets under conditions of 37 ° C. and 5% CO 2 .
  • Greiner bio-one trade name “L ID FOR MICROPLATE, non-coated”
  • 15 and 16 show the microscopic images of each droplet of cells at the start of culturing of each droplet, 15 hours after culturing, and 24 hours after culturing, and one droplet.
  • the image of one droplet is the result of observing the same droplet over time, and the drawings of 1 to 5 ⁇ l of each well plate in Fig. 16 are 1 ⁇ l, 2 ⁇ l, It is the figure which carried out stationary culture of 3 microliters and 5 microliters of droplets, and the image of one droplet is It is the result of ⁇ l droplet.
  • Example 6 In the same manner as in Example 1, 7.5 ⁇ 10 6 individually separated human skin fibroblast cell suspensions were obtained. To this, 15 ml of D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., containing 10% fetal bovine serum, 1% penicillin / streptomycin) was added to obtain a cell suspension of 5 ⁇ 10 5 cells / ml. .
  • D-MEM medium High Glucose, Wako Pure Chemical Industries, Ltd., containing 10% fetal bovine serum, 1% penicillin / streptomycin
  • the culture was allowed to stand for 17 hours, 36 hours, 67 hours, and 94 hours under the conditions.
  • the result of the same well image is shown in FIG.
  • a plurality of spheres were formed over time from the start of culture, and a large fused spheroid was formed 67 hours later. However, the spheroid size after 94 hours was equivalent to that at 67 hours.
  • Example 7 The same operation as in Example 1 was performed except that human normal dermal fibroblasts (CCD-1079SK) were used instead of human dermal fibroblasts (HDF, purchased from Kurabo Industries). The formation of spheroids was confirmed after 9 hours of culture. The results are shown in FIG. FIG. 18 is a stereomicroscopic image of a spheroid having a maximum diameter of about 0.1 to 0.3 mm.
  • step (3) of Example 2 was carried out by immunocytochemical staining using an antibody specific for the marker protein of pluripotent stem cells, and OCT3 / 4, TRA-1-81 and The expression of NANOG protein was confirmed.
  • the results are shown in FIG. Spheroids obtained from human normal skin fibroblasts (CCD-1079SK) also expressed OCT3 / 4, TRA-1-81 and NANOG and were considered pluripotent stem cell-like spheroids.
  • D-MEM medium High Glucose, Wako Pure Chemical Industries, Ltd., 10% fetal bovine serum, 1 ⁇ 7 ⁇ 10 6 individually isolated human skin fibroblasts obtained in step (3) of Example 1 15 ml of penicillin / streptomycin was added to adjust the cell density to 2.5 ⁇ 10 5 / ml, and 1.1 ml of the slide chamber (AGC Techno Glass, Type I collagen coating, 1 well area 8.36 cm 2 ) And static culture at 37 ° C. and 5% CO 2 . The cell density per culture area is 3.3 ⁇ 10 4 / cm 2 . Spheroids were not formed even after 15 hours of culture. The results are shown in FIG. FIG. 20 is an optical microscope image.
  • D-MEM medium High Glucose, Wako Pure Chemical Industries, Ltd., 10% fetal calf serum, 1 ⁇ 10 7 individually isolated human skin fibroblasts obtained in step (3) of Example 1 10 ml of penicillin / streptomycin was added to make the cell density 1.2 ⁇ 10 6 / ml, and 20 ⁇ l thereof and 1 ml of the D-MED medium were added to a glass bottom dish (manufactured by FPI, uncoated, hole diameter 1 4 mm, culture area 9.6 cm 2 ), and static culture was performed under conditions of 37 ° C. and 5% CO 2 . The cell density per culture area is 2.5 ⁇ 10 3 / cm 2 . When cultured for 19 hours, although it was weak, it survived adhesion, but no spheroids were formed.
  • FIG. 21 shows differential interference microscopic images after starting culture and 19 hours later.
  • Example 1 and FIG. 1 when human dermal fibroblasts isolated individually were cultured at high density in a floating state, cells in the vicinity aggregated over time, and spheroids were formed. Formed. The formed spheroids were not formed by the growth of one cell, but were derived from the aggregation of proximal cells. Therefore, it is a polyclonal spheroid formed by aggregation of a plurality of cells. In addition, as shown in Example 7, spheroids were similarly formed in human normal skin fibroblasts of the same cell type and other cell lines. (2) As shown in Example 2 and FIGS. 2 to 10, cells constituting spheroids expressed pluripotent stem cell markers.
  • the cells constituting the spheroids express pluripotent stem cell markers, and living cells are transformed into pluripotent-like cells at a very high rate.
  • Example 6 From Example 6, it was shown that the pluripotent stem cell-like spheroid of the present invention has no cell proliferation ability. This can be said to indicate that the pluripotent stem cell-like spheroid of the present invention does not have a carcinogenic effect.
  • the doubling time is about 24 hours, so that the doubling time is doubled after 24 hours of culture and 16 times of culture for 4 days. The proliferative and cancerous cells found in cancerous cells were not detected even after 4 days of culture.
  • Example 5 As shown in Example 5, when a cell suspension having the same cell density is used and the amount of droplets is changed and the culture is statically cultured in a floating state, droplets are obtained as shown in FIGS.
  • the amount of spheroids increased corresponding to the amount, that is, the amount of cells. It has been shown that a large number of spheroids can be prepared by increasing the number of cells. Moreover, the reproducibility was confirmed from the fact that spheroids were formed in all droplets 24 hours after the start of culture. (6) As shown in Example 4, it was found that a large-diameter spheroid was formed by using a culture vessel with a shape such as a centrifuge tube that easily collects cells when statically cultured in a floating state. did. When combined with Example 5, the number and size of spheroids can be adjusted by selecting the number of cultured cells and the shape of the culture vessel.

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Abstract

L'invention concerne un procédé de production d'un sphéroïde de type cellule souche pluripotente et un sphéroïde de type cellule souche pluripotente. Des cellules vivantes différenciées sont traitées avec une enzyme pour donner des cellules sphériques. Ensuite, les cellules sphériques sont cultivées de manière constante à une densité cellulaire de 3×105-7×108/ml en utilisant un contenant de culture de cellules sans adhérence cellulaire. Ainsi, les cellules forment une masse d'agrégation cellulaire dont la taille correspond à la numération cellulaire. La masse d'agrégation cellulaire obtenue (un sphéroïde) exprime un marqueur de cellules souches pluripotentes et se transforme en cellules de type cellules souches pluripotentes.
PCT/JP2017/006546 2016-03-04 2017-02-22 Procédé de production de sphéroïde de type cellule souche pluripotente, et sphéroïde de type cellule souche pluripotente WO2017150294A1 (fr)

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