WO2021201175A1 - 下垂体ホルモン産生細胞及びその前駆細胞の分離法 - Google Patents
下垂体ホルモン産生細胞及びその前駆細胞の分離法 Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0613—Cells from endocrine organs
- C12N5/0616—Pituitary gland
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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
- C12M45/00—Means for pre-treatment of biological substances
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Definitions
- the present invention relates to a method for separating and purifying pituitary hormone-producing cells and their progenitor cells induced to differentiate from pluripotent stem cells in vitro.
- the pituitary gland is a small endocrine organ that exists in contact with the lower part of the diencephalon and plays a major role as a control center for various hormones. For example, it produces a variety of pituitary hormones, including adrenocorticotropic hormone (ACTH), which promotes the production of adrenocorticotropic hormone, which is essential for life support, and growth hormone, which promotes the growth of children. Therefore, pituitary dysfunction causes serious systemic illness.
- ACTH adrenocorticotropic hormone
- Patent Document 1 non-patent.
- Documents 1 and 2 In these methods, three-dimensional culture forms a hypothalamic-pituitary complex within a single cell mass.
- the glandular pituitary gland and its precursor tissue are mainly located on the surface layer of the cell mass, and by exfoliating this and transplanting it under the renal capsule of the pituitary gland-excised mouse, the activity, survival, and body weight Therapeutic effects such as improvement of reduction have been confirmed (Patent Document 1, Non-Patent Document 1).
- Patent Document 1 Non-Patent Document 1
- no cell surface marker for separating the glandular pituitary gland and its precursor tissue from the above complex tissue is known, and the identification of the exfoliating glandular pituitary gland and its precursor tissue is based on the morphology of the experimenter. It relied on subjective judgment.
- An object of the present invention is to provide an objective and reliable method for separating target cells using a cell surface antigen for separating pituitary hormone-producing cells and their progenitor cells.
- the present inventors first screened from 332 types of human cell surface antigen panels in order to obtain surface antigens specific to the cell lineage of the anterior pituitary gland. As a result, several types of pituitary hormone-producing cells including epithelial cell adhesion molecule (hereinafter abbreviated as EpCAM) and / or surface antigens having high specificity for their progenitor cells were identified.
- EpCAM epithelial cell adhesion molecule
- the identified surface antigen, EpCAM can withstand treatment with enzymes and the like when dispersing cell clusters of pluripotent stem cells, and by sorting with an anti-EpCAM antibody.
- EpCAM the identified surface antigen
- pituitary hormone-producing cells and their progenitor cells can be effectively isolated and purified from the cell mass of pluripotent stem cells.
- the separated and purified cells can be reaggregated and maintained-cultured, and even after the maintenance-culture, they show excellent pituitary hormone secretory ability by physiological stimulation of pituitary hormone secretion and are functional.
- the present invention is as follows.
- a method for separating pituitary hormone-producing cells and / or progenitor cells thereof which comprises a step of separating cells expressing EpCAM from a cell aggregate containing an anterior pituitary gland and / or its progenitor tissue.
- the cell aggregate comprises the hypothalamic neuroepithelial tissue.
- the cell aggregate containing the glandular pituitary gland and / or its precursor tissue is shredded, or the glandular pituitary gland and / or its precursor tissue is contained.
- Any one of [1] to [4], wherein the cell aggregate containing the anterior pituitary gland and / or its precursor tissue is a cell aggregate obtained by inducing differentiation of pluripotent stem cells.
- the method described in. [6] The method according to [5], wherein the pluripotent stem cell is a human induced pluripotent stem cell.
- a method for producing pituitary hormone-producing cells and / or their progenitor cells which comprises a step of separating cells expressing EpCAM from a cell aggregate containing an anterior pituitary gland and / or its progenitor tissue.
- the cell aggregate containing the glandular pituitary and / or its precursor tissue is shredded, or the glandular pituitary and / or its precursor is shredded.
- the method according to [9] or [10] which comprises the step of physically making a notch in a cell aggregate containing tissue.
- the pituitary hormone-producing cells are growth hormone (GH) -producing cells, prolactin (PRL) -producing cells, corticostimulating hormone (ACTH) -producing cells, thyroid-stimulating hormone (TSH) -producing cells, and follicular-stimulating hormone (FSH).
- GH growth hormone
- PRL prolactin
- ACTH corticostimulating hormone
- TSH thyroid-stimulating hormone
- FSH follicular-stimulating hormone
- functional pituitary hormone-producing cells and / or progenitor cells thereof can be efficiently separated and purified from pluripotent stem cell-derived differentiated tissues by using EpCAM.
- the separated and purified pituitary hormone-producing cells show excellent pituitary hormone secretory ability by physiological stimulation of pituitary hormone secretion, the cells can be used to treat diseases related to the pituitary gland. can.
- FIG. 1 shows the expression of EpCAM in pituitary progenitor cells derived from human iPS cells.
- the figure on the left shows an example in which human iPS cell clusters on day 44 of induction of differentiation were dispersed and then fluorescently immunostained with Cytokeratin (pituitary progenitor cell marker) and EpCAM (A).
- the figure on the right shows the results of quantifying the expression rates of Cytokeratin (CK) and EpCAM in all cells on days 44 to 51 of induction of differentiation (B). In the results, the numbers show the average of the four experiments (B).
- FIG. 2 shows immunohistochemical analysis of EpCAM in differentiated tissues derived from human iPS cells.
- the figure above shows immunohistochemical staining of human iPS cell clusters on day 48 of induction of differentiation (A).
- EpCAM is expressed in Cytokeratin-positive pituitary progenitor cells (A).
- the central figure shows immunohistochemical staining of human iPS cell clusters on day 51 of induction of differentiation (B).
- EpCAM is expressed in Pitx1-positive pituitary progenitor cells (B).
- the figure below shows immunohistochemical staining of human iPS cell clusters on day 147 of induction of differentiation (C).
- EpCAM is expressed in ACTH-positive ACTH-producing cells and Lhx3-positive pituitary progenitor cells (C).
- FIG 3 shows purification of ACTH-producing cells and pituitary progenitor cells by MACS using EpCAM as a marker.
- the figure above shows the procedure of MACS (A).
- the lower left figure is a bright-field image (left) of a cell mass reaggregated after MACS and an immunostaining image of ACTH / Lhx3 (right) (B).
- the lower right figure shows the amount of ACTH secreted from the cell mass that reaggregated after MACS (C).
- C the ACTH concentration in the culture supernatant before and after the addition of CRH was measured
- Pluripotent stem cells are daughter cells that have the ability to differentiate into all the cells that make up the living body (pluripotency) and the same differentiation ability as self through cell division. A cell that also has the ability to produce (self-renewal ability).
- Differentiation pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing for the presence or absence of teratomy including each of the three germ layers (ectoderm, mesoderm, endoderm). can.
- pluripotent stem cells examples include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), and the like.
- the cell is not limited to this as long as it is a cell having both pluripotency and self-renewal ability.
- ES cells or iPS cells are preferably used.
- the pluripotent stem cell is an ES cell or an arbitrary cell derived from a human embryo, the cell is a cell produced by destroying an embryo, even if the cell is produced by destroying the embryo. It may be, but preferably cells produced without destroying the embryo.
- ES cells can be established, for example, by culturing an early embryo before implantation, an inner cell mass constituting the early embryo, a single blastomere, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994), Thomson, J. A. et al., Science, 282, 1145-1147 (1998)).
- an early embryo prepared by nuclear transfer of a somatic cell nucleus may be used (Wilmut et al., (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280,). 1256 (1998)), Akira Iriya et al.
- Participatory embryos may be used as early embryos (Kim et al., (Science, 315, 482-486 (2007)), Nakajima et al., (Stem Cells, 25,983-985 (2007)), Kim. et al., (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al., (Cloning Stem Cells, 9, 432-449 (2007)), Revazova et al., (Cloning Stem Cells, 10) , 11-24 (2008))).
- Fusion ES cells obtained by cell fusion of ES cells and somatic cells are also included in the ES cells used in the method of the present invention.
- ES cells can be obtained from a designated institution, and commercial products can also be purchased.
- human ES cells KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University.
- EG cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, SCF, etc. (Matsui et al., Cell, 70, 841-847 (1992), Shamblott et al., Proc. Natl. Acad. Sci. USA, 95 (23), 13726-13731 (1998), Turnpenny et al., Stem Cells, 21 (5), 598-609, (2003)).
- IPS cells are cells that have artificially acquired pluripotency and self-renewal ability by contacting somatic cells (for example, fibroblasts, skin cells, lymphocytes, etc.) with nuclear reprogramming factors.
- somatic cells for example, fibroblasts, skin cells, lymphocytes, etc.
- nuclear reprogramming factors consisting of Oct3 / 4, Sox2, Klf4 and c-Myc into somatic cells (eg fibroblasts, skin cells, etc.) (Cell, 126: p). . 663-676, 2006). Since then, many researchers have made various improvements in the combination of reprogramming factors and the method of introducing the factors, and various methods for producing iPS cells have been reported.
- the nuclear reprogramming factor is a substance (group) capable of inducing cells having pluripotency and self-renewal ability from somatic cells such as fibroblasts, it is a proteinaceous factor or a nucleic acid (vector) encoding the same. It may be composed of any substance (including forms incorporated in) or low molecular weight compounds.
- the nuclear reprogramming factor is a proteinaceous factor or a nucleic acid encoding the same, the following combinations are preferably exemplified (in the following, only the name of the proteinaceous factor will be described).
- c-Myc can be replaced with T58A (active mutant), N-Myc, L-Myc.) (2) Oct3 / 4, Klf4, Sox2 (3) Oct3 / 4, Klf4, c-Myc (4) Oct3 / 4, Sox2, Nanog, Lin28 (5) Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Lin28 (6) Oct3 / 4, Klf4, Sox2, bFGF (7) Oct3 / 4, Klf4, Sox2, SCF (8) Oct3 / 4, Klf4, c-Myc, Sox2, bFGF (9) Oct3 / 4, Klf4, c-Myc, Sox2, SCF
- the combination of the three factors Oct3 / 4, Sox2 and Klf4 is preferable when considering the use of the obtained iPS cells for therapeutic purposes.
- the four factors Oct3 / 4, Klf4, Sox2 and c-Myc, or c-Myc, or Five factors with the addition of Lin28 or Nanog are preferred.
- IPS cells are preferably used for autologous transplantation.
- Pluripotent stem cells in which genes on chromosomes are modified by using known genetic engineering techniques can also be used in the present invention.
- a pluripotent stem cell uses a known method to knock in a labeled gene (for example, a fluorescent protein such as GFP) in-frame to a gene encoding a differentiation marker, thereby corresponding to the differentiation stage using the expression of the labeled gene as an index. It may be a cell that can identify that it has reached.
- a labeled gene for example, a fluorescent protein such as GFP
- pluripotent stem cell for example, a warm-blooded animal, preferably a mammalian pluripotent stem cell can be used. Mammals include, for example, rodents such as mice, rats, hamsters and guinea pigs, experimental animals such as rabbits, domestic animals such as pigs, cows, goats, horses and sheep, pets such as dogs and cats, humans and monkeys. Primates such as orchid dogs and chimpanzees can be mentioned. Pluripotent stem cells are preferably pluripotent stem cells of rodents (mouse, rat, etc.) or primates (human, etc.), and most preferably human pluripotent stem cells.
- Pluripotent stem cells can be maintained and cultured by a method known per se.
- pluripotent stem cells are preferably maintained by serum-free culture using a serum substitute such as Knockout TM Serum Replacement (KSR) or feeder cell culture.
- KSR Knockout TM Serum Replacement
- the pluripotent stem cells used in the present invention are preferably isolated. "Isolation” means that an operation has been performed to remove factors other than the target cells and components, and the naturally occurring state has been removed.
- the purity of "isolated human pluripotent stem cells” is usually 70% or more, preferably 80% or more, more preferably 90% or more, still more preferable. Is 99% or more, most preferably 100%.
- pluripotent stem cell aggregates are obtained by culturing dispersed pluripotent stem cells in an incubator under non-adherent conditions (ie, suspension culture) and multiple pluripotents. It can be obtained by assembling sex stem cells to form aggregates.
- the incubator used for forming the agglomerates is not particularly limited, and for example, a flask, a tissue culture flask, a dish, a petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a micropore, and a multi-plate. , Multi-well plates, flask slides, petri dishes, tubes, trays, culture bags, roller bottles.
- the incubator is preferably cell non-adhesive to allow culturing under non-adhesive conditions.
- Non-cell-adhesive incubators include those in which the surface of the incubator is artificially treated so as to be non-cell-adhesive, or those in which the surface of the incubator is artificially treated for the purpose of improving adhesion to cells (for example). , Those that have not been coated with an extracellular matrix or the like) can be used.
- the medium used for the formation of agglomerates can be prepared using the medium used for culturing mammalian cells as the basal medium.
- the medium is not particularly limited as long as it can be used for culture.
- a mixed medium of IMDM medium and Ham's F-12 medium is used.
- the medium used for culturing may be a serum-containing medium or a serum-free medium.
- the serum-free medium means a medium containing no prepared or unpurified serum, and a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors) corresponds to a serum-free medium. It shall be. From the viewpoint of avoiding contamination of chemically undetermined components, a serum-free medium is preferably used in the present invention.
- the medium used for the formation of agglomerates may contain a serum substitute.
- Serum substitutes may optionally contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'thiolglycerol, or equivalents thereof.
- serum substitutes can be prepared, for example, by the method described in WO98 / 30679.
- a commercially available serum substitute can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), and Glutamax (manufactured by Gibco).
- the medium used for the formation of aggregates can contain other additives as long as it does not adversely affect the induction of differentiation of pluripotent stem cells into pituitary gland or its partial tissues or its precursor tissues.
- Additives include, for example, insulin, iron sources (eg, transferrin, etc.), minerals (eg, sodium selenate, etc.), sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), serum proteins (eg, albumin, etc.).
- Amino acids eg L-glutamine, etc.
- reducing agents eg 2-mercaptoethanol, etc.
- vitamins eg, ascorbic acid, d-biotin, etc.
- antibiotics eg, streptomycin, penicillin, gentamicin, etc.
- buffers eg, streptomycin, penicillin, gentamicin, etc.
- HEPES HEPES, etc.
- the medium used for forming the agglomerates may be the medium used in the first culture step, which will be described later.
- the pluripotent stem cells are collected from the subculture and dispersed into a single cell or a state close to this. Dispersion of pluripotent stem cells is performed using a suitable cell dissociation solution.
- a suitable cell dissociation solution for example, EDTA; proteolytic enzymes such as trypsin, collagenase IV, and metalloprotease can be used alone or in combination as appropriate. Among them, those having less cytotoxicity are preferable, and commercially available products such as Dispase (EIDIA), TrypLE (Invitrogen) and Accutase (MILLIPORE) are available as such cell dissociation solutions.
- the dispersed pluripotent stem cells are suspended in the above medium.
- ROCK Rho-associated coiled-coil kinase
- the concentration of the ROCK inhibitor used in the suspension culture is a concentration capable of suppressing cell death of pluripotent stem cells induced by dispersion.
- concentrations are typically about 0.1-200 ⁇ M, preferably about 2-50 ⁇ M.
- concentration of the ROCK inhibitor may be varied within the period of addition, for example the concentration can be halved in the second half of the period.
- a plurality of pluripotent stem cells are sown in the above-mentioned incubator, and the dispersed pluripotent stem cells are cultured in the incubator under non-adhesive conditions. Pluripotent stem cells assemble to form aggregates. At this time, by seeding the dispersed pluripotent stem cells in a relatively large incubator such as a 10 cm dish, even if agglomerates of a plurality of pluripotent stem cells are simultaneously formed in one culture compartment. This is good, but this causes a large variation in the size of each agglomerate and the number of pluripotent stem cells contained therein, and this variation causes the pluripotent stem cells to pituitary or its partial tissue, or a precursor thereof.
- dispersed pluripotent stem cells Differences occur between aggregates in the degree of tissue differentiation, resulting in reduced efficiency of differentiation induction. Therefore, it is preferable to rapidly aggregate dispersed pluripotent stem cells to form one aggregate in one culture compartment.
- a method for rapidly aggregating such dispersed pluripotent stem cells include the following methods: 1) A method in which dispersed pluripotent stem cells are confined in a culture compartment having a relatively small volume (for example, 1 ml or less, 500 ⁇ l or less, 200 ⁇ l or less, 100 ⁇ l or less), and one aggregate is formed in the compartment. The culture compartment is allowed to stand after confining preferably dispersed pluripotent stem cells.
- Examples of the culture compartment include multi-well plates (384 wells, 192 wells, 96 wells, 48 wells, 24 wells, etc.), wells in micropores, chamber slides, etc., tubes, droplets of medium in the hanging drop method, etc. However, it is not limited to these.
- the dispersed pluripotent stem cells confined in the compartment are urged by gravity to settle in one place, or the cells adhere to each other to form one agglomerate per culture compartment.
- the shape of the bottom of the multi-well plate, micropore, chamber slide, tube, etc. is preferably U-bottom or V-bottom so that dispersed pluripotent stem cells can be easily precipitated in one place.
- a method in which dispersed pluripotent stem cells are placed in a centrifuge tube, and the pluripotent stem cells are centrifuged to precipitate pluripotent stem cells in one place to form one agglomerate in the tube.
- the number of pluripotent stem cells seeded in one culture compartment is such that one agglomerate is formed per one culture compartment, and according to the method of the present invention, in the agglomerate, from the pluripotent stem cell, the pituitary gland or its pituitary gland or its agglomerate.
- it is not particularly limited, but is usually about 1 ⁇ 10 3 to about 5 ⁇ 10 4 per culture compartment, preferably about 1 ⁇ 10 3 to. Seed about 2 ⁇ 10 4 cells, more preferably about 2 ⁇ 10 3 to about 1.2 ⁇ 10 4 pluripotent stem cells.
- pluripotent stem cells usually about 1 ⁇ 10 3 to about 5 ⁇ 10 4 cells, preferably about 1 ⁇ 10 3 to about 2 ⁇ 10 4 cells, and more per culture compartment.
- pluripotent stem cells usually about 1 ⁇ 10 3 to about 5 ⁇ 10 4 cells, preferably about 1 ⁇ 10 3 to about 2 ⁇ 10 4 cells, and more per culture compartment.
- one cell aggregate consisting of about 2 ⁇ 10 3 to about 1.2 ⁇ 10 4 pluripotent stem cells is formed.
- the time to agglomeration is such that one agglomerate is formed per compartment, and in the agglomerate, from pluripotent stem cells to the pituitary gland or its partial tissue, or its precursor tissue, according to the method of the present invention.
- efficient differentiation induction into the target pituitary gland, its partial tissue, or its precursor tissue can be expected.
- the time until the formation of agglomerates can be appropriately adjusted by those skilled in the art by adjusting a tool for agglutinating cells, centrifugation conditions, and the like.
- culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C, preferably about 37 ° C.
- the CO 2 concentration is, for example, about 1 to 10%, preferably about 5%.
- pluripotent stem cell aggregates By preparing multiple culture compartments under the same culture conditions and forming a single pluripotent stem cell aggregate in each culture compartment, a qualitatively uniform population of pluripotent stem cell aggregates can be obtained. Obtainable.
- the qualitative homogeneity of pluripotent stem cell aggregates means aggregate size and cell number, macroscopic morphology, micromorphology and its homogeneity by tissue staining analysis, expression of differentiated and undifferentiated markers and It can be evaluated based on its homogeneity, the regulation of expression of differentiation markers and their synchrony, and the reproducibility of differentiation efficiency between agglomerates.
- the population of pluripotent stem cell aggregates used in the method of the invention has a uniform number of pluripotent stem cells contained in the aggregate.
- a "uniform" population of pluripotent stem cell aggregates means that more than 90% of the population of pluripotent stem cells has an average value of ⁇ 10 for that parameter in the population of aggregates. It means that it is within the range of%, preferably within the range of the average value ⁇ 5%.
- the cell aggregate containing the glandular pituitary and / or its precursor tissue transmits the aggregate of pluripotent stem cells to bone-forming factor signaling. It can be obtained by methods including suspension culture in a medium containing a pathway activator and a sonic hedgehog (Shh) signal pathway activator.
- the glandular pituitary refers to a tissue containing at least one type of anterior or middle pituitary pituitary hormone-producing cells.
- the pituitary hormone-producing cells include growth hormone (GH) -producing cells, prolactin (PRL) -producing cells, adrenal cortex stimulating hormone (ACTH) -producing cells, thyroid stimulating hormone (TSH) -producing cells, and follicle-stimulating hormone (FSH) -producing cells.
- GH growth hormone
- PRL prolactin
- ACTH adrenal cortex stimulating hormone
- TSH thyroid stimulating hormone
- FSH follicle-stimulating hormone
- pituitary hormone-producing cells can express EpCAM as a marker in addition to the expression of pituitary hormone specific to each cell type.
- the glandular pituitary gland is at least one, preferably two or more, selected from the group consisting of GH-producing cells, PRL-producing cells, ACTH-producing cells, TSH-producing cells, FSH-producing cells, and LH-producing cells. Includes (2, 3, 4, 5 or 6) pituitary hormone-producing cells.
- the glandular pituitary gland is at least one, preferably two, and more preferably three pituitary hormone-producing cells selected from the group consisting of GH-producing cells, PRL-producing cells, and ACTH-producing cells. including.
- a tissue refers to a structure of a cell population having a structure in which a plurality of types of cells having different morphologies and properties are three-dimensionally arranged in a certain pattern.
- Examples of the precursor tissue of the glandular pituitary gland include pituitary placode, Rathke's pouch and the like.
- the pituitary placode is a thickened structure formed in the epithelial ectoderm region (oral ectoderm) during embryogenesis, and expresses at least EpCAM, which is a pituitary progenitor cell marker, and Lhx3 or Pitx1. Obtained, preferably one capable of expressing all of EpCAM, Lhx3, and Pitx1.
- Rathke's pouch refers to a sac-like structure formed by the invagination of the pituitary placode.
- Rathke's pouch can express at least the pituitary progenitor cell markers EpCAM and Lhx3 or Pitx1, preferably all of EpCAM, Lhx3, and Pitx1.
- EpCAM and Lhx3 or Pitx1 preferably all of EpCAM, Lhx3, and Pitx1.
- the expression of Isl1 / 2, Cytokeratin, etc. may be further confirmed as the pituitary progenitor cell marker.
- cells capable of expressing at least EpCAM, which is a pituitary progenitor cell marker, and Lhx3 or Pitx1 are referred to as pituitary progenitor cells, and the progenitor cells preferably express all of EpCAM, Lhx3, and Pitx1. obtain.
- the pituitary progenitor cell is also referred to as a progenitor cell of a pituitary hormone-producing cell.
- a progenitor cell of a pituitary hormone-producing cell for example, a cell aggregate positive for at least one marker selected from LHX3, NKX2.1, PITX1 and ACTH.
- examples of the "cell agglutinin containing the glandular pituitary gland and / or its precursor tissue” include LHX3, NKX2.1, PITX1 and ACTH-positive cell agglutinin.
- the cell aggregate containing the glandular pituitary gland and / or its precursor tissue used in the separation method and the production method of the present invention can be produced by the following method. Specifically, the method involves suspension-culturing agglomerates of pluripotent stem cells in a medium containing an osteogenic factor signaling pathway activator and a Shh signaling pathway activator to create hypothalamic neuroepithelial tissue and.
- epidermal ectoderm including oral ectoderm
- hypothalamic neuroepithelial tissue By further suspension-culturing the cell agglomerates containing epidermal ectoderm (including oral ectoderm) in a medium containing an osteogenic factor signal transduction pathway activator and a Shh signal pathway activator, 1) hypothalamic neuroepithelium Includes tissue and 2) obtaining cell agglomerates containing pitoderm placode and / or rattake sac (second culture step).
- the first culture step induces differentiation of pluripotent stem cells into hypothalamic neuroepithelial tissue and epidermal ectoderm (including oral ectoderm), and the obtained hypothalamic neuroepithelial tissue and epidermal ectoderm (oral cavity).
- submission of cell aggregates containing (including ectoderm) to the second culture step induces further differentiation from the epidermal ectoderm region (oral ectoderm) into pituitary placodes and / or ratoke sac. ..
- the cell aggregate containing the glandular pituitary and / or its precursor tissue may be the cell aggregate obtained by the second culture step.
- Floating culture of pluripotent stem cell aggregates means culturing pluripotent stem cell aggregates in a medium under non-adherent conditions to an incubator. This enables efficient induction of the glandular pituitary gland or its precursor tissue, which has been difficult in the past.
- the medium used for suspension culture contains a bone-forming factor signaling pathway activator and a Shh signaling pathway activator.
- the action of osteogenic factor signaling pathway activators and Shh signaling pathway activators induces the differentiation of pluripotent stem cells into hypothalamic neuroepithelial tissue and epidermal ectoderm.
- the bone-forming factor signal transduction pathway activating substance means any substance that activates the pathway in which a signal is transmitted by the binding between the bone-forming factor and the receptor.
- bone-forming factor signaling pathway activators include BMP2, BMP4, BMP7, GDF5 and the like.
- the bone-forming factor signaling pathway activator is BMP4.
- BMP4 will be mainly described, but the bone-forming factor signal transduction pathway activator used in the present invention is not limited to BMP4.
- BMP4 is a known cytokine, and its amino acid sequence is also known.
- the BMP4 used in the present invention is a mammalian BMP4.
- Mammals include, for example, rodents such as mice, rats, hamsters and guinea pigs, experimental animals such as rabbits, domestic animals such as pigs, cows, goats, horses and sheep, pets such as dogs and cats, humans and monkeys. Primates such as orchid dogs and chimpanzees can be mentioned.
- BMP4 is preferably rodent (mouse, rat, etc.) or primate (human, etc.) BMP4, and most preferably human BMP4.
- Human BMP4 means that BMP4 has an amino acid sequence of BMP4 that is naturally expressed in humans in vivo.
- Typical amino acid sequences of human BMP4 are NCBI accession numbers, NP_001193.2 (updated June 15, 2013), NP_570911.2 (updated June 15, 2013), NP_570912.2 (2013). (Updated on June 15), an amino acid sequence (mature human BMP4 amino acid sequence) in which the N-terminal signal sequence (1-24) is removed from each of these amino acid sequences can be exemplified.
- the Shh signal pathway agent is not particularly limited as long as it can enhance signal transduction mediated by Shh.
- the Shh signal pathway agonist include proteins belonging to the Hedgehog family or fragments thereof (for example, Shh, Ihh, Shh (C24II) N-Terminus, Shh (C25II) N-Terminus), Shh receptor, and Shh receptor agonist.
- Purmorphamine, Smoothened Agonist (SAG) (3-Chloro-N- [trans-4- (methylamino) cyclohexyl] -N-[[3- (4-pyridinyl) phenyl] methyl]-benzo [b] thiophene-2- Carboxamide), but not limited to these. Of these, SAG is preferable.
- bone-forming factor signaling pathway activator and Shh signaling pathway-acting substance is BMP4 and SAG.
- the concentration of the bone-forming factor signaling pathway activator in the medium can be appropriately set within a range that can induce the differentiation of pluripotent stem cells into the hypothalamic neuroepithelial tissue and epidermal ectoderm in the cell aggregate.
- BMP4 is used as the bone-forming factor signal transduction pathway activator, its concentration is usually 0.01 nM or more, preferably 0.1 nM or more, and more preferably 1 nM or more.
- the BMP4 concentration in the medium is usually 0.01 to 1000 nM, preferably 0.1 to 100 nM, more preferably 1 to 10 nM (eg, 5 nM).
- Exogenous bone-forming factor signaling pathway activators in particular, 1) actively form epidermal ectoderm, and 2) induce differentiation of neuroepithelial tissue in the hypothalamus rather than the cerebral agglomerates into cell aggregates. It is contained in the medium at a concentration that can achieve these effects, as it contributes to that.
- the bone-forming factor signaling pathway activator does not have to be contained in the medium for the entire period of the first culture step. For example, no bone-forming factor signaling pathway activator was added to the medium for 2-4 days (eg, 3 days) from the start of suspension culture of pluripotent stem cell aggregates, followed by the bone-forming factor signaling pathway. The activator may be added to the medium.
- the concentration of the Shh signal pathway agent in the medium can be appropriately set as long as it can induce the differentiation of pluripotent stem cells into the hypothalamic neuroepithelial tissue and the epidermal ectoderm in the cell aggregate, although it can be appropriately set.
- SAG is used as a signal pathway agent
- its concentration is usually 1 nM or more, preferably 10 nM or more, and more preferably 100 nM or more.
- the SAG concentration in the medium is usually 1 nM to 1000 ⁇ M, preferably 10 nM to 100 ⁇ M, more preferably 100 nM to 10 ⁇ M (eg, 2 ⁇ M).
- This effect can be achieved because extrinsic Shh signaling pathway agents play a role in inducing differentiation of the neuroepithelial tissue of the hypothalamus (preferably the ventral hypothalamus), rather than the nerve retina, into cell aggregates. Included in the medium in concentration.
- the Shh signaling pathway agent does not have to be contained in the medium for the entire period of the first culture step. For example, no Shh signal pathway agonist was added to the medium for 5-7 days (eg, 6 days) from the start of suspension culture of pluripotent stem cell aggregates, after which the Shh signal pathway agonist was added to the medium. You may.
- pluripotent stem cell aggregates are suspended in culture medium free of osteogenic factor signaling pathway activators and Shh signaling pathway activators for 2-4 days, and the resulting aggregates are then subjected to suspension.
- Shh signal pathway activator and Shh signal pathway activator and Shh signal pathway activator and Shh signal pathway activator and Shh signal pathway activator and Shh signal pathway Incubate in medium containing the agent until the hypothalamic neuroepithelial tissue and epidermal ectoderm are induced.
- Bone-forming factor signaling pathway activators such as BMP4 used in the present invention are preferably isolated.
- isolated protein X means that an operation has been performed to remove a target component or a factor other than a cell, and the state of being naturally present has been removed. Therefore, the "isolated protein X" does not include the endogenous protein X produced from the cells or tissues to be cultured.
- the purity of "isolated protein X" (percentage of the weight of protein X in total protein weight) is usually 70% or higher, preferably 80% or higher, more preferably 90% or higher, still more preferably 99% or higher. Most preferably 100%.
- the isolated bone-forming factor signaling pathway activator contained in the medium used for suspension culture is extrinsically added to the medium.
- the invention comprises the step of exogenously adding an isolated bone-forming factor signaling pathway activator into the medium used in the first culture step.
- the medium used in the first culture step is an inhibitor of Rho-associated coiled-coil kinase (ROCK) in order to suppress cell death of pluripotent stem cells (particularly human pluripotent stem cells) induced by dispersion.
- ROCK Rho-associated coiled-coil kinase
- the ROCK inhibitor is added, for example, within 15 days, preferably within 10 days, more preferably within 6 days from the start of culture.
- Examples of the ROCK inhibitor include Y-27632 ((+)-(R) -trans-4- (1-aminoethyl) -N- (4-pyridyl) cyclohexanecarboxamide dihydrochloride).
- the concentration of the ROCK inhibitor used in the suspension culture is a concentration capable of suppressing cell death of pluripotent stem cells induced by dispersion.
- concentrations are typically about 0.1-200 ⁇ M, preferably about 2-50 ⁇ M.
- concentration of the ROCK inhibitor may be varied within the period of addition, for example the concentration can be halved in the second half of the period.
- the medium used for suspension culture of cell aggregates can be prepared using the medium used for culture of mammalian cells as the basal medium.
- the medium is not particularly limited as long as it can be used for culture.
- a mixed medium of IMDM medium and Ham's F-12 medium is used.
- the medium used for culturing may be a serum-containing medium or a serum-free medium.
- the medium used for suspension culture of cell aggregates is preferably a serum-free medium.
- the medium used for suspension culture of cell aggregates may contain a serum substitute.
- Serum substitutes may optionally contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'thiolglycerol, or equivalents thereof.
- Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679.
- a commercially available serum substitute can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), and Glutamax (manufactured by Gibco).
- the medium used for suspension culture of cell aggregates can contain other additives as long as it does not adversely affect the induction of differentiation of pluripotent stem cells into hypothalamic neuroepithelial tissue and epidermal ectoderm.
- Additives include, for example, insulin, iron sources (eg, transferrin, etc.), minerals (eg, sodium selenate, etc.), sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), serum proteins (eg, albumin, etc.).
- Amino acids eg L-glutamine, etc.
- reducing agents eg 2-mercaptoethanol, etc.
- vitamins eg, ascorbic acid, d-biotin, etc.
- antibiotics eg, streptomycin, penicillin, gentamicin, etc.
- buffers eg, streptomycin, penicillin, gentamicin, etc.
- HEPES HEPES, etc.
- the medium used for suspension culture of cell aggregates is included in the medium from the viewpoint of not adversely affecting the induction of differentiation into the hypothalamic neuroepithelial tissue and epidermal epithelial tissue.
- a serum substitute KSR, etc.
- KSR serum substitute
- gfCDM chemically synthesized medium
- the "growth factor” as used herein includes Fgf; BMP; pattern-forming factors such as Wnt, Nodal, Notch, and Shh; insulin and Lipid-rich albumin.
- Examples of the chemically synthesized medium containing no growth factor include gfCDM disclosed in Wataya et al, Proc Natl Acad Sci USA, 105 (33): 11796-11801, 2008.
- culture temperature is, for example, about 30-40 ° C, preferably about 37 ° C.
- the CO 2 concentration is, for example, about 1-10%, preferably about 5%.
- the O 2 concentration is, for example, about 20%.
- a qualitatively homogeneous population of pluripotent stem cell aggregates is suspended in culture medium containing an osteogenic factor signaling pathway activator and a Shh signaling pathway activator.
- a qualitatively uniform population of pluripotent stem cell aggregates the difference between the aggregates in the degree of differentiation into the glandular pituitary or its precursor tissue is minimized and the purpose is It is possible to improve the efficiency of differentiation induction.
- Suspension culture of a qualitatively homogeneous population of pluripotent stem cell agglomerates includes the following aspects: 1) Prepare multiple culture compartments and sow a qualitatively uniform population of pluripotent stem cell aggregates so that one culture compartment contains one pluripotent stem cell aggregate.
- one pluripotent stem cell aggregate is placed in each well of a 96-well plate.
- one pluripotent stem cell aggregate activates the osteogenic factor signaling pathway.
- put agglomerates of multiple pluripotent stem cells in a 10 cm dish. Then, in the compartment, agglutinates of multiple pluripotent stem cells are subjected to osteogenic factor signaling pathway activator and Shh signaling pathway action.
- any of the above aspects 1) and 2) may be adopted, and the aspect may be changed during culturing from the aspect ⁇ 1) to the aspect 2), or 2).
- ) To 1)> In one aspect, the aspect 1) is adopted in the first culturing step, and the aspect 2) is adopted in the second culturing step.
- the first culture step is carried out for a period sufficient to induce differentiation of pluripotent stem cells into hypothalamic neuroepithelial tissue and epidermal ectoderm.
- Differentiation into hypothalamic neuroepithelial tissue and epidermal ectoderm can be detected by, for example, RT-PCR or immunohistochemistry using marker-specific antibodies of hypothalamic neuroepithelial tissue or epidermal ectoderm.
- 10% or more, preferably 30% or more, more preferably 50% or more of the cell aggregates in culture are carried out until the hypothalamic neuroepithelial tissue and epidermal ectoderm are contained.
- the culture period can vary depending on the animal species of pluripotent stem cells and the types of osteogenic factor signaling pathway activators and Shh signaling pathway activators, and therefore cannot be unequivocally specified, but for example, When using human pluripotent stem cells, the first culture step is usually 15 to 20 days (eg, 18 days).
- a cell aggregate containing hypothalamic neuroepithelial tissue and epidermal ectoderm can be obtained.
- the hypothalamic neuroepithelial tissue is a neuroepithelial tissue that expresses a hypothalamic marker, and is a hypothalamus that can differentiate into cells constituting the hypothalamus (for example, neurons, glial cells, etc.) and has self-renewal ability. Tissue containing progenitor cells and neurons, glial cells, etc. that have lost their self-renewal ability due to differentiation.
- the proportion of hypothalamic progenitor cells and their differentiated cells in the hypothalamic neuroepithelial tissue varies depending on the degree of differentiation. Has been confirmed to differentiate into hypothalamic neurons (Cell Reports, 30, 18-24, January 7, 2020).
- the hypothalamus includes the ventral hypothalamus and the dorsal hypothalamus.
- Examples of the hypothalamus marker include NKx2.1 (ventral hypothalamus marker) and Pax6 (dorsal hypothalamus marker).
- the ventral hypothalamic neuroepithelial tissue is Rx-positive, Chx10-negative, Pax6-negative, and Nkx2.1-positive neuroepithelial tissue.
- the dorsal hypothalamic neuroepithelial tissue is Rx-positive, Chx10-negative, Nkx2.1-negative, and Pax6-positive neuroepithelial tissue.
- the hypothalamic neuroepithelial tissue contained in the cell aggregate obtained in the first culture step is preferably the ventral hypothalamic neuroepithelial tissue.
- EpCAM is not expressed in any of the above hypothalamic neuroepithelial tissues, which means that all cells constituting the hypothalamic neuroepithelial tissue (not only hypothalamic progenitor cells but also differentiate and self-renewal ability). It is shown that EpCAM is not expressed in lost neurons, glial cells, etc.).
- the epidermal ectoderm is an ectoderm cell layer formed on the surface layer of an embryo during embryogenesis.
- Examples of epidermal ectoderm markers include pan-cytokeratin.
- the epidermal ectoderm can generally differentiate into anterior pituitary gland, skin, oral epithelium, tooth enamel, skin glands and the like.
- the epidermal ectoderm is an E-cadherin-positive and pan-cytokeratin-positive cell layer.
- the hypothalamic neuroepithelial tissue occupies the inside of the cell aggregate, and the monolayer epidermal epithelial cells are the surface of the cell aggregate.
- the epidermal ectoderm may contain a thickened epidermal placode as a part thereof.
- (3.2) Second culture step In the second culture step, cell aggregates containing the hypothalamic neuroepithelial tissue and epidermal ectoderm (including oral ectoderm) obtained in the first culture step are sown. Cells containing 1) hypothalamic neuroepithelial tissue and 2) pitoderm placode and / or rattake sac by further suspension culture in medium containing osteogenic factor signaling pathway activators and Shh signaling pathway activators. Obtain agglomerates. The action of osteogenic factor signaling pathway activators and Shh signaling pathway activators induces differentiation from epidermal ectoderm into pituitary placodes and / or Rathke's pouch.
- the definitions of the bone-forming factor signal transduction pathway activator and the Shh signal pathway activator are as described in the description of the first culture step.
- the bone-forming factor signaling pathway activator used in the second culture step is BMP4 as in the first culture step.
- the Shh signaling pathway agent used in the second culture step is SAG as in the first culture step.
- bone-forming factor signaling pathway activator and Shh signaling pathway-acting substance is BMP4 and SAG.
- the concentration of the bone-forming factor signaling pathway activator in the medium can be appropriately set within a range capable of inducing differentiation of epidermal ectoderm into pituitary placode and / or Rathke's pouch in the cell aggregate.
- BMP4 is used as the bone-forming factor signal transduction pathway activator
- its concentration is usually 0.01 nM or more, preferably 0.1 nM or more, and more preferably 1 nM or more.
- the BMP4 concentration in the medium is usually 0.01 to 1000 nM, preferably 0.1 to 100 nM, more preferably 1 to 10 nM (eg, 5 nM).
- the concentration of the bone-forming factor signaling pathway activator may be varied within the period of addition, eg, at the start of the second culture step, at the above concentration, in stages at a rate of half every 2-4 days. The concentration can be reduced.
- the concentration of the Shh signal pathway agent in the medium can be appropriately set within a range capable of inducing differentiation of the epidermal ectoderm into the pituitary placode and / or Rathke's pouch in the cell aggregate, although the Shh signal can be appropriately set.
- SAG SAG
- its concentration is usually 1 nM or more, preferably 10 nM or more, and more preferably 100 nM or more.
- concentration is usually 1000 ⁇ M or less, preferably 100 ⁇ M or less, more preferably 10 ⁇ M or less.
- the SAG concentration in the medium is usually 1 nM to 1000 ⁇ M, preferably 10 nM to 100 ⁇ M, more preferably 100 nM to 10 ⁇ M (eg, 2 ⁇ M).
- the medium used in the second culture step contains FGF2.
- FGF2 promotes differentiation from epidermal ectoderm into pituitary placode.
- FGF2 is a known cytokine also called basic fibroblast growth factor (bFGF), and its amino acid sequence is also known.
- the FGF2 used in the present invention is usually a mammalian FGF2. Examples of mammals include the above. Since FGF2 has cross-reactivity between many mammalian species, any mammalian FGF2 may be used as long as the object of the present invention can be achieved, but preferably the same as the cells to be cultured. A type of mammalian FGF2 is used. For example, rodents (mouse, rat, etc.) or primates (humans, etc.) FGF2 are used.
- mouse FGF2 means that FGF2 has an amino acid sequence of FGF2 that is naturally expressed in a mouse in vivo.
- a typical amino acid sequence of mouse FGF2 is NCBI accession number, NP_032032.1 (updated February 18, 2014), and the amino acid sequence obtained by removing the N-terminal signal sequence (1-9) from this amino acid sequence (updated on February 18, 2014).
- Mature mouse FGF2 amino acid sequence) and the like can be exemplified.
- NP_001997.5 updated on February 18, 2014
- NCBI accession number of NCBI.
- the concentration of FGF2 in the medium is not particularly limited as long as it can promote the differentiation of epidermal ectoderm into pituitary placode, but is usually 1 ng / ml or more, preferably 10 ng / ml or more. Is. There is no particular upper limit of the FGF2 concentration unless it adversely affects the differentiation into pituitary placode and / or Rathke's pouch, but from the viewpoint of culture cost, it is usually 1000 ng / ml or less, preferably 500 ng / ml or less. .. In one embodiment, the FGF2 concentration in the medium is usually 1 to 1000 ng / ml, preferably 10 to 100 ng / ml.
- the bone-forming factor signaling pathway activator such as BMP4 and FGF2 used in the present invention are preferably isolated.
- the isolated bone-forming factor signaling pathway activator and the isolated FGF2 contained in the medium used in the second culture step were extrinsically added to the medium. Therefore, in one embodiment, the present invention extrinsically adds an isolated bone-forming factor signaling pathway activator (and optionally isolated FGF2) into the medium used in the second culture step.
- the medium used for culturing mammalian cells can be prepared as the basal medium in the same manner as the medium used in the first culturing step.
- the medium is not particularly limited as long as it can be used for culture.
- a mixed medium of IMDM medium and Ham's F-12 medium is used.
- the medium used for culturing may be a serum-containing medium or a serum-free medium.
- the medium used for suspension culture of cell aggregates is preferably a serum-free medium.
- the medium used for suspension culture of cell aggregates may contain a serum substitute.
- Serum substitutes may optionally contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'thiolglycerol, or equivalents thereof.
- Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679.
- a commercially available serum substitute can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), and Glutamax (manufactured by Gibco).
- the medium used for suspension culture of cell aggregates can contain other additives as long as it does not adversely affect the induction of differentiation from epidermal ectoderm to pituitary placode and / or Rathke's pouch.
- Additives include, for example, insulin, iron sources (eg, transferrin, etc.), minerals (eg, sodium selenate, etc.), sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), serum proteins (eg, albumin, etc.).
- Amino acids eg L-glutamine, etc.
- reducing agents eg 2-mercaptoethanol, etc.
- vitamins eg, ascorbic acid, d-biotin, etc.
- antibiotics eg, streptomycin, penicillin, gentamicin, etc.
- buffers eg, streptomycin, penicillin, gentamicin, etc.
- HEPES HEPES, etc.
- the medium used for suspension culture of cell aggregates is included in the medium from the viewpoint of not adversely affecting the induction of differentiation into pituitary placode and / or Rathke's pouch.
- a serum substitute KSR, etc.
- KSR serum substitute
- gfCDM chemically synthesized medium
- the "growth factor” as used herein includes Fgf; BMP; pattern-forming factors such as Wnt, Nodal, Notch, and Shh; insulin and Lipid-rich albumin.
- Examples of the chemically synthesized medium containing no growth factor include gfCDM disclosed in Wataya et al, Proc Natl Acad Sci USA, 105 (33): 11796-11801, 2008.
- the suspension culture in the second culture step is preferably performed under high oxygen partial pressure conditions.
- oxygen can reach the inside of the cell aggregate and the cell aggregate can be maintained and cultured for a long period of time. Achieved, allowing efficient induction of differentiation into pituitary placode and / or Rathke's pouch.
- the high oxygen partial pressure condition means an oxygen partial pressure condition that exceeds the oxygen partial pressure in air (20%).
- the oxygen partial pressure in the second culture step is, for example, 30 to 60%, preferably 35 to 60%, and more preferably 38 to 60%.
- culture temperature is, for example, about 30-40 ° C, preferably about 37 ° C.
- the CO 2 concentration is, for example, about 1-10%, preferably about 5%.
- the second culture step is carried out for a period sufficient to induce differentiation from epidermal ectoderm into pituitary placode and / or Rathke's pouch.
- a pituitary placode is formed in the epidermal ectoderm (more specifically, in the oral ectoderm). Also, some or all of the pituitary placode can invade the interior of the cell aggregate (ie, the adjacent hypothalamic nerve epithelium) and form Rathke's pouch.
- the interaction between the epithelial epithelial tissue and the hypothalamic neuroepithelial tissue is essential for the differentiation of epidermal epithelial to pituitary placode and / or ratoke sac.
- the hypothalamic neuroepithelial tissue and epithelial epithelial tissue are simultaneously formed in the cell aggregate by the first culture step, and in a preferred embodiment, the hypothalamic neuroepithelial tissue occupies the inside of the cell aggregate.
- Monolayer epithelial epithelial cells constitute the surface of the cell aggregate.
- the second culture step is performed until 10% or more, preferably 30% or more, more preferably 50% or more of the cell aggregates in culture contain pituitary placode and / or Rathke's pouch. Will be implemented. Since the culture period can vary depending on the animal species of pluripotent stem cells and the types of osteogenic factor signaling pathway activators and Shh signaling pathway activators, it cannot be unequivocally specified, but for example, When using human pluripotent stem cells, the second culture step is usually 6 days or longer, for example 6-12 days.
- a cell aggregate containing 1) hypothalamic neuroepithelial tissue and 2) pituitary placode and / or Rathke's pouch can be obtained.
- the cell aggregate containing the glandular pituitary and / or its precursor tissue may be the cell aggregate obtained by the second culture step.
- Shh signal pathway agent The definition of the Shh signal pathway agent is as described in the description of the first culture step.
- the Shh signal pathway agent used in the third culture step is SAG as in the first and second culture steps.
- the concentration of the Shh signal pathway agent in the medium can be appropriately set as long as it can induce the differentiation of pituitary placode and / or Rathke's pouch into pituitary hormone-producing cells in the cell aggregate.
- SAG SAG
- its concentration is usually 1 nM or more, preferably 10 nM or more, and more preferably 100 nM or more.
- the SAG concentration in the medium is usually 1 nM to 1000 ⁇ M, preferably 10 nM to 100 ⁇ M, more preferably 100 nM to 10 ⁇ M (eg, 2 ⁇ M).
- the medium used in the third culture step contains FGF2.
- FGF2 promotes differentiation of pituitary placode and / or Rathke's pouch into pituitary hormone-producing cells.
- FGF2 The definition of FGF2 is as described in the explanation of the second culture step.
- the concentration of FGF2 in the medium is not particularly limited as long as it can promote the differentiation of pituitary placode and / or Rathke's pouch into pituitary hormone-producing cells, but is usually 1 ng / ml or more. Preferably, it is 10 ng / ml or more. There is no particular upper limit of the FGF2 concentration unless it adversely affects the differentiation into pituitary hormone-producing cells, but from the viewpoint of culture cost, it is usually 1000 ng / ml or less, preferably 500 ng / ml or less. In one embodiment, the FGF2 concentration in the medium is usually 1 to 1000 ng / ml, preferably 10 to 100 ng / ml.
- the medium used in the third culture step contains a Notch signal inhibitor.
- Notch signaling inhibitors promote the differentiation of pituitary placodes and / or Rathke's pouch into pituitary hormone-producing cells (particularly ACTH-producing cells).
- Notch signaling inhibitors increase the expression of the transcription factor Tbx19, which regulates ACTH production upstream.
- the Notch signal inhibitor is not particularly limited as long as it can suppress Notch-mediated signal transduction.
- Examples of Notch signal inhibitors include gamma secretase inhibitors such as DAPT (N- [N- (3,5-difluorophenacetyl) -l-alanyl] -S-phenylglycine t-butyl ester), DBZ, and MDL28170.
- DAPT N- [N- (3,5-difluorophenacetyl) -l-alanyl] -S-phenylglycine t-butyl ester
- DBZ DBZ
- MDL28170 MDL28170
- the concentration of the Notch signal inhibitor in the medium is not particularly limited as long as it can promote the differentiation of pituitary placode and / or Rathke's pouch into pituitary hormone-producing cells (particularly ACTH-producing cells).
- DAPT for example, it is usually 0.1 ⁇ M or more, preferably 1 ⁇ M or more.
- the DAPT concentration in the medium is usually 0.1 to 1000 ⁇ M, preferably 1 to 100 ⁇ M (eg, 10 ⁇ M).
- the medium used in the third culture step is free of osteogenic factor signaling pathway activators.
- the FGF2 used in the present invention is preferably isolated.
- the isolated FGF2 contained in the medium used in the third culture step is extrinsically added to the medium. Therefore, in one aspect, the present invention comprises the step of extrinsically adding the isolated FGF2 into the medium used for the second culture step.
- cell aggregates may be treated with corticosteroids by adding corticosteroids to the medium.
- corticohormones pituitary hormone-producing cells other than ACTH-producing cells (that is, GH-producing cells, PRL-producing cells, TSH-producing cells, LH-producing cells, FSH-producing cells, etc.) from the pituitary placode and / or Rathke's pouch, etc. ) Is promoted.
- corticosteroids examples include natural glucocorticoids such as hydrocortisone, cortisone acetate, and fludrocortisone acetate; artificially synthesized glucocorticoids such as dexamesaone, betamethasone, prednisolone, methylprednisolone, and triamcinolone. Yes, but not limited to these.
- the concentration of adrenocorticotropic hormones in the medium is not particularly limited as long as it can promote the differentiation of pituitary placode and / or ratoke sac into pituitary hormone-producing cells (excluding ACTH-producing cells). Further, it can be appropriately set depending on the type of adrenocorticotropic hormones. For example, in the case of hydrocortisone, it is usually 100 ng / ml or more, preferably 1 ⁇ g / ml or more.
- hydrocortisone concentration there is no particular upper limit of the hydrocortisone concentration unless it adversely affects the differentiation into pituitary hormone-producing cells (excluding ACTH-producing cells), but from the viewpoint of culture cost, it is usually 1000 ⁇ g / ml or less, preferably 100 ⁇ g. It is less than / ml.
- the concentration of hydrocortisone in the medium is usually 100 ng / ml to 1000 ⁇ g / ml, preferably 1 to 100 ⁇ g / ml.
- dexamethasone is used as an adrenocortical hormone, its concentration in the medium can be about 1/25 of that of hydrocortisone.
- the timing of adding corticosteroids to the medium promotes the differentiation of pituitary hormones and / or ratoke sac into pituitary hormone-producing cells (excluding ACTH-producing cells).
- corticosteroids may be added to the medium from the start of the third culture step, or after the start of the third culture step, the culture is carried out for a certain period of time in a medium to which no corticosteroids are added. Later, corticosteroids may be added to the medium.
- corticosteroids may be added to the medium after starting the third culture step, corticosteroids are added to the medium when the appearance of ACTH-producing cells is confirmed in the cell aggregate.
- the cell aggregates are cultured in a medium to which no corticohormones are added, and after the appearance of ACTH-producing cells is confirmed, the adrenal gland Continue the third culture step in medium containing cortical hormones.
- the appearance of ACTH-producing cells can be confirmed by immunohistological staining using an antibody against ACTH.
- human pluripotent stem cells in general, the appearance of ACTH-producing cells can be expected 37 days after the start of the third culture step. Therefore, in one embodiment, 37 from the start of the third culture step. After the day, add corticosteroids to the medium.
- the period of treatment of cell aggregates with adrenocorticotropic hormones is not particularly limited as long as it can promote the differentiation of pituitary hormones and / or ratoke sac into pituitary hormone-producing cells (excluding ACTH-producing cells).
- cells are usually found to promote differentiation into pituitary hormone-producing cells (excluding ACTH-producing cells) in the adrenocorticotropic hormone-treated group as compared to the adrenocorticotropic hormone-untreated group. Treat aggregates with adrenocorticotropic hormones.
- the treatment period is usually 7 days or longer, preferably 12 days or longer.
- the upper limit of the treatment period is not particularly limited, but the differentiation into pituitary hormone-producing cells (excluding ACTH-producing cells) in the corticosteroid-treated group is compared with that in the corticosteroid-untreated group. Corticosteroids may be removed from the medium when promotion is confirmed.
- adrenocorticotropic hormones acts suppressively on the induction of differentiation of ACTH-producing cells by inhibiting feedback.
- the medium used for culturing mammalian cells can be prepared as the basal medium in the same manner as the medium used in the first and second culture steps.
- the medium is not particularly limited as long as it can be used for culture.
- a mixed medium of IMDM medium and Ham's F-12 medium is used.
- the medium used for culturing may be a serum-containing medium or a serum-free medium.
- the medium used for suspension culture of cell aggregates is preferably a serum-free medium.
- the medium used for suspension culture of cell aggregates may contain a serum substitute.
- Serum substitutes may optionally contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'thiolglycerol, or equivalents thereof.
- Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679.
- a commercially available serum substitute can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), and Glutamax (manufactured by Gibco).
- the medium used for suspension culture of cell aggregates is added with other mediums to the extent that it does not adversely affect the induction of differentiation from pluripotent stem cells into pituitary placode and / or Rathke's pouch and pituitary hormone-producing cells.
- Additives include, for example, insulin, iron sources (eg, transferrin, etc.), minerals (eg, sodium selenate, etc.), sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), serum proteins (eg, albumin, etc.).
- Amino acids eg L-glutamine, etc.
- reducing agents eg 2-mercaptoethanol, etc.
- vitamins eg, ascorbic acid, d-biotin, etc.
- antibiotics eg, streptomycin, penicillin, gentamicin, etc.
- buffers eg, streptomycin, penicillin, gentamicin, etc.
- HEPES HEPES, etc.
- the medium used for suspension culture of cell aggregates is other than those specifically described in the present specification from the viewpoint of not adversely affecting the induction of differentiation into pituitary hormone-producing cells.
- a serum substitute KSR, etc.
- KSR serum substitute
- gfCDM Chemically Defined Medium
- the "growth factor” as used herein includes Fgf; BMP; pattern-forming factors such as Wnt, Nodal, Notch, and Shh; insulin and Lipid-rich albumin.
- Examples of the chemically synthesized medium containing no growth factor include gfCDM disclosed in Wataya et al, Proc Natl Acad Sci USA, 105 (33): 11796-11801, 2008.
- the suspension culture in the third culture step is preferably performed under high oxygen partial pressure conditions. Under high oxygen partial pressure conditions, 1) the hypothalamic neuroepithelial tissue, and 2) the arrival of oxygen inside the cell aggregate by further suspension culture of the cell aggregate containing the pituitary placode and / or Rathke's pouch. And long-term maintenance culture of cell aggregates is achieved, and efficient induction of differentiation into pituitary hormone-producing cells becomes possible.
- the high oxygen partial pressure condition means an oxygen partial pressure condition that exceeds the oxygen partial pressure in air (20%).
- the oxygen partial pressure in the third culture step is, for example, 30 to 60%, preferably 35 to 60%, and more preferably 38 to 60%.
- culture temperature is, for example, about 30-40 ° C, preferably about 37 ° C.
- the CO 2 concentration is, for example, about 1-10%, preferably about 5%.
- the third culture step is carried out for a period sufficient to induce differentiation of the pituitary placode and / or Rathke's pouch into pituitary hormone-producing cells.
- differentiation of the pituitary placode and / or Rathke's pouch into pituitary hormone-producing cells is induced, and the pituitary hormone-producing cells in the pituitary placode and / or Rathke's pouch.
- pituitary hormone-producing cells derived from pituitary placode and / or Rathke's pouch
- examples of pituitary hormone-producing cells derived from pituitary placode and / or Rathke's pouch include growth hormone (GH) -producing cells, prolactin (PRL) -producing cells, and adrenocorticotropic hormone (ACTH) -producing cells.
- GH growth hormone
- PRL prolactin
- ACTH adrenocorticotropic hormone
- the adrenocorticotropic hormone (ACTH) -producing cells secrete ACTH in response to CRH stimulation, and the ACTH secretion is feedback-suppressed by glucocorticoids.
- GH growth hormone
- PRL prolactin
- ACTH adrenal cortex stimulating hormone
- a glandular pituitary gland containing at least one, preferably two, and more preferably three pituitary hormone-producing cells selected from the group is formed.
- GH growth hormone
- PRL prolactin
- ACTH adrenocorticotropic hormone
- TSH thyroid-stimulating hormone
- FSH stimulating hormone
- LH luteinizing hormone
- MSH melanin cell stimulating hormone
- the glandular pituitary formed by the third culture step is at least selected from the group consisting of growth hormone (GH) -producing cells, prolactin (PRL) -producing cells, and adrenocorticotropic hormone (ACTH) -producing cells.
- GH growth hormone
- PRL prolactin
- ACTH adrenocorticotropic hormone
- TSH adrenocorticotropic hormone
- FSH follicle-stimulating hormone
- LH luteinizing hormone
- MSH melanin cell-stimulating hormone
- Other pituitary hormone-producing cells such as producing cells may be included.
- Differentiation into pituitary hormone-producing cells can be confirmed, for example, by detecting pituitary hormone-positive cells by immunohistochemistry using a specific antibody against pituitary hormone. For example, it is carried out until 10% or more, preferably 30% or more, more preferably 50% or more of the cell aggregates in the culture contain pituitary hormone-producing cells. Since the culture period can vary depending on the animal species of pluripotent stem cells and the type of Shh signal pathway activator, it cannot be unconditionally specified, but for example, when human pluripotent stem cells are used, the first The culture step of 3 is usually 37 days or more, for example 37 to 70 days.
- a cell aggregate containing a glandular pituitary gland can be obtained.
- suspension culture of aggregates is performed under either the presence or absence of feeder cells.
- the incubator used for suspension culture of cell aggregates is not particularly limited, but for example, a flask, a tissue culture flask, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, and the like. Examples include microwell plates, micropores, multiplates, multiwell plates, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles.
- the incubator is preferably cell non-adhesive to allow culturing under non-adhesive conditions.
- Non-cell-adhesive incubators include those in which the surface of the incubator is artificially treated so as to be non-cell-adhesive, or those in which the surface of the incubator is artificially treated for the purpose of improving adhesion to cells (for example). , Those that have not been coated with an extracellular matrix or the like) can be used.
- an oxygen permeable one may be used as the incubator used for suspension culture of cell aggregates.
- an oxygen permeable incubator By using an oxygen permeable incubator, the supply of oxygen to the cell agglomerates can be improved, which can contribute to long-term maintenance culture of the cell agglomerates.
- the agglutinin may be statically cultured as long as the non-adherent state of the agglutinin to the incubator can be maintained, or the agglutinin may be consciously moved by swirling culture or shaking culture.
- the static culture is a culture method in which agglomerates are cultivated without being consciously moved.
- the medium may convect with a local change in the medium temperature, and the agglomerates may move due to the flow, but the agglomerates are not consciously moved.
- the static culture may be carried out throughout the whole period of the suspension culture, or the static culture may be carried out only for a part of the period.
- static culture is performed throughout the suspension culture.
- the static culture does not require a device, is expected to cause less damage to the cell mass, and is advantageous in that the amount of the culture solution can be reduced.
- the present invention comprises a step of separating EpCAM-expressing cells from a cell aggregate containing a glandular pituitary gland and / or its progenitor tissue.
- EpCAM can be expressed on the surface of pituitary hormone-producing cells and their progenitor cells, but not on the surface of hypothalamic neuroepithelial tissue. Therefore, using EpCAM as a marker, pituitary hormone-producing cells and their progenitor cells can be separated from the hypothalamic neuroepithelial tissue.
- the cell agglutinin containing the glandular pituitary gland and / or its precursor tissue used in the separation method of the present invention is either a cell agglutinin obtained by the second culture step or a cell agglutinin obtained by the third culture step.
- Cell aggregates may be used.
- the specific number of days after the initiation of differentiation induction from the pluripotent stem cells to the pituitary gland or its partial tissue or its precursor tissue is, for example, 30 to 600 days, preferably 90 to 500 days. More preferably, it is 150 to 400 days, and even more preferably 200 to 350 days.
- the separation method of the present invention includes the following steps.
- a pituitary hormone-producing cell and / or a precursor thereof which comprises a step of separating EpCAM-expressing cells from a cell aggregate containing the glandular pituitary gland and / or its precursor tissue of the present invention.
- the method of separating cells is as follows: (B) Before the step of separating cells (population) expressing EpCAM, (A) a cell aggregate containing a glandular pituitary gland and / or its progenitor tissue is dispersed to simply disperse the cell aggregate. It may include the step of obtaining a population of one cell.
- the cell aggregate containing the glandular pituitary gland and / or its precursor tissue is subjected to enzymatic treatment. It may include a step of dispersing (described later (4.2)). Prior to the step of dispersing by the enzymatic treatment, (a2) shredded cell agglomerates containing the glandular pituitary gland and / or its precursor tissue, or cell aggregation containing the glandular pituitary gland and / or its precursor tissue. It may include a step of physically making a notch in the mass (described later (4.2)).
- the cell aggregate containing the glandular pituitary gland and / or its precursor tissue is optionally treated with a ROCK inhibitor. It may include a step of processing. When the step is carried out, it is preferable to carry out the step at the beginning of the step of obtaining a population of single cells (described later (4.1)).
- the step of dispersing by enzymatic treatment (a2)
- the step of separating the cells (population) expressing EpCAM the step of separating the cells (population) expressing EpCAM and the cells (population) not expressing EpCAM). ((4.3) described later).
- an optional step (C) of re-aggregating the obtained cells (population) may be performed (described later (4.4)).
- ) Specifically, (c1) the obtained cells (population) are seeded in an incubator and adherently cultured, and / or (c2) the obtained cells (population) are seeded in an incubator. This can be done by suspension culture.
- (4.1) Pretreatment Step with ROCK Inhibitor In the separation method of the present invention, the obtained cell agglutinin can be pretreated with a ROCK inhibitor.
- a ROCK inhibitor is added before the start of the pretreatment in order to suppress cell death of pluripotent stem cells (particularly human pluripotent stem cells) induced by the subsequent dispersion of cell aggregates. It is preferable to do so.
- the ROCK inhibitor is added, for example, at least 24 hours, at least 12 hours, at least 6 hours, at least 3 hours, at least 2 hours, and at least 1 hour before the start of the treatment.
- Examples of the ROCK inhibitor include Y-27632 ((+)-(R) -trans-4- (1-aminoethyl) -N- (4-pyridyl) cyclohexanecarboxamide dihydrochloride).
- the concentration of the ROCK inhibitor used in the treatment is a concentration capable of suppressing cell death of pluripotent stem cells induced by subsequent dispersion of cell aggregates.
- concentrations are typically about 0.1-200 ⁇ M, preferably about 2-50 ⁇ M.
- concentration of the ROCK inhibitor may be varied within the period of addition, for example the concentration can be halved in the second half of the period. In any of the steps (4.2) to (4.4) described later, it is preferable to use a solution containing a ROCK inhibitor at the same concentration as the solution to be brought into contact with the cells.
- the medium used for the pretreatment is the cell aggregate obtained in the second culture step
- the medium used in the second culture step is the medium used in the third culture step
- the cell aggregate obtained in the third culture step is used.
- the medium used in the third culture step can be used. If the ROCK inhibitor has already been added to the medium at a desired concentration, it is not necessary to perform the pretreatment step.
- the enzyme for dispersion is not particularly limited as long as the cells can be dispersed, and examples thereof include enzymes such as EDTA; trypsin, collagenase (collagenase types I to VII), metalloprotease, hyaluronidase, elastase, dispase, deoxyribonuclease, and the like.
- the mixture of. Preferred enzymes include collagenase, more preferably collagenase type I.
- the conditions for enzyme treatment can be appropriately set depending on the enzyme to be used and the like.
- the cell aggregate is physically shredded (eg, female, scissors, etc.) or physically (eg, female, scissors, etc.) before the treatment. ) May be performed.
- the enzyme treatment can also be carried out with the above-mentioned enzymes and the like, and preferred enzymes include EDTA; trypsin, and more preferably EDTA; trypsin and deoxyribonuclease. Further, instead of EDTA; trypsin, a commercially available product such as TrypLE (Invitrogen) may be used.
- the conditions for enzyme treatment (temperature, time, etc.) can be appropriately set depending on the enzyme to be used and the like.
- a single cell suspension can be prepared by the above series of enzymatic treatments. Further, when preparing the single cell suspension, dead cells may be removed by a method known per se.
- EpCAM-positive cells As a method for separating desired cells expressing EpCAM from the cell population contained in the above-prepared single cell suspension, a method using flow cytometry or mass cytometry, or magnetism Examples include cell separation methods, and these methods can be performed using methods known per se.
- cells expressing EpCAM can be separated by a method including a step of contacting the cells with a substance (eg, antibody or the like) that specifically binds to the EpCAM molecule.
- the substances include those with a detectable label (eg, GFP, PE) attached to themselves, and those without a label attached to themselves.
- the separation is possible by further using a substance with a detectable label that directly or indirectly recognizes the substance.
- a substance with a detectable label that directly or indirectly recognizes the substance.
- the substance is an antibody
- a fluorescent dye, metal isotope or beads eg, magnetic beads
- the antibody used at this time may be only one type or two or more types of antibodies.
- the separated cell population is seeded in an incubator and adherently cultured, and may be used as a cell sheet, for example. It may be allowed to form a cell aggregate.
- the cell population may be maintained and cultured as it is until use, or may be further induced to differentiate into desired cells according to the differentiation state of the separated cells.
- the method described in the above-mentioned third culture step may be carried out as it is, or may be appropriately modified by a method known per se as necessary.
- the adhesive culture when the adhesive culture is also carried out, the adhesive culture itself can be carried out by a method known per se, and the contents described in the above-mentioned third culture step (eg, medium composition, etc.) may be used as appropriate. .. Further, in the case of adhesive culture, it is preferable to coat the incubator with an extracellular matrix or the like (eg, laminin, collagen, etc.).
- an extracellular matrix or the like eg, laminin, collagen, etc.
- the pituitary hormone-producing cells and their progenitor cells obtained by the separation method or production method of the present invention can be used for transplantation medicine. ..
- the relevant damaged part is supplemented.
- pituitary hormone-producing cells and / or precursor cells thereof obtained by the method of the present invention can be used.
- the anterior pituitary gland Disorder-based disorders or injured conditions of the anterior pituitary gland can be treated.
- the site of transplantation is not particularly limited as long as the transplanted pituitary hormone-producing cells and / or progenitor cells thereof can function as a substitute for the impaired glandular pituitary gland, and examples thereof include subcapsular gland.
- glandular pituitary disorders include panhypopituitarism, pituitary dwarfism, hypopituitarism, partial hypopituitarism, and anterior pituitary hormone deficiency alone. Be done. Further, the injured state of these glandular pituitaries includes patients after glandular pituitary removal, patients after irradiation of pituitary tumors, and trauma.
- pluripotent stem cells eg, iPS cells
- the recipient in the method of the present invention, by using pluripotent stem cells (eg, iPS cells) established from the somatic cells of the recipient as the pluripotent stem cells, the recipient is immunologically self-sufficient. It produces glandular pituitary gland or its precursor tissue, or pituitary hormone-producing cells, which are transplanted into the recipient.
- the pituitary hormone-producing cells and their progenitor cells obtained by the method of the present invention can be used for drug screening and evaluation. Specifically, it can be applied to, for example, screening of substances that suppress or promote the production of pituitary hormones, side effect / toxicity tests of pharmaceutical products, and the like.
- the above screening, test, etc. include, for example, a step of culturing a cell population containing pituitary hormone-producing cells and / or progenitor cells thereof in the presence or absence of a test substance (negative control), and treatment with the test substance. It may include a step of comparing the production amount of the target hormone in the cell population with that of the negative control, and a step of selecting a test substance that suppresses or promotes the production of pituitary hormone as a candidate substance.
- the present invention is a step of separating cells expressing EpCAM from cell aggregates containing glandular pituitary gland and / or progenitor cells thereof.
- the pituitary hormone-producing cell and / or its precursor cell may be a pituitary hormone-producing cell and / or its precursor cell, and a population of pituitary hormone-producing cells (cell population) and / or its precursor cell.
- EpCAM can be expressed on the surface of pituitary hormone-producing cells and their progenitor cells, but not on the surface of hypothalamic neuroepithelial tissue. Therefore, using EpCAM as a marker, pituitary hormone-producing cells and their progenitor cells can be separated from the hypothalamic neuroepithelial tissue.
- a cell population containing pituitary hormone-producing cells and / or progenitor cells thereof, or pituitary hormone-producing cells and / or progenitor cells thereof with high purity eg, 80% or more of the cell population, preferably Is 85% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 99% or more, most preferably 100%.
- the step of separating the cells expressing EpCAM from the cell aggregate containing the anterior pituitary gland and / or its precursor tissue included in the production method of the present invention is described in the above-mentioned "Summary of the separation step of the present invention". All the contents of the described steps and the like can be incorporated. Further, for each process, all the contents of (4,1) to (4.4) described above can be incorporated.
- the pituitary hormone-producing cells (population) and / or their progenitor cells (population) produced by the method of the present invention may be maintained and cultured as they are until use as described in (4.1) above, or isolated cells.
- culturing eg, a third culturing step
- the pituitary hormone-producing cells (population) and / or their progenitor cells (population) produced by the method of the present invention are directly transplanted into a subject requiring treatment or the like by the cell population. Alternatively, it may be transplanted to the subject after further increasing (purifying) the purity by a method known per se.
- the purified cells may be seeded in an incubator and adherently cultured as described in (4.4) above, and may be shaped like a cell sheet, and seeded in the incubator. It may be reaggregated by suspension culture. A cell sheet or cell agglutination obtained through the adhesive culture or suspension culture may be transplanted to the above-mentioned subject. Further, the adhesive culture or suspension culture may be carried out for a short period of time (eg, 3 to 6 days) or for a long period of time (eg, 7 to 30 days). In the production method of the present invention, all of the contents relating to "(5) Use of isolated pituitary hormone-producing cells and progenitor cells thereof" from "(1) pluripotent stem cells" can be incorporated.
- Example 1 Screening of cell surface antigens Human iPS cells (201B7 strain) are glandularized by a method known per se as described in International Publication No. 2016/013669 or Cell Reports, 30, 18-24, January 7, 2020. Differentiation was induced into a cell aggregate containing the pituitary gland or its precursor tissue. Cell clusters on day 47 after induction of differentiation from human iPS cells (201B7 strain) were treated with Accumax (Innovative Cell Technologies, # AM105) at 37 ° C. for 5 to 10 minutes to disperse, and a single cell suspension was prepared.
- Accumax Innovative Cell Technologies, # AM105
- EpCAM may be able to identify pituitary progenitor cells.
- Example 2 Quantification of EpCAM and Cytokeratin expression rates After preparing a single cell suspension in the same manner as for cell surface antigen screening, PE-labeled anti-EpCAM antibody (Miltenyi, # 130-098-115), FITC-labeled anti-Cytokeratin antibody. , Hoechst 33342 and images were acquired with a fluorescence microscope (Leica DMI6000B). Using the Cell counter tool of the image analysis software Image J, EpCAM + / Cytokeratin + cells, EpCAM + / Cytokeratin- cells, EpCAM- / Cytokeratin + cells, and EpCAM- / Cytokeratin- cells were counted, and the respective ratios in all cells were calculated.
- EpCAM Epithelial cell adhesion molecule
- Example 3 Purification of EpCAM-positive cells by Magnetic-Activated Cell Sorting (MACS) 90-500 days after the start of differentiation induction, cell clusters were enzymatically dispersed and used for MACS. The procedure is shown below.
- Y27632 In order to suppress cell death due to dispersion treatment, Y27632 (Wako, # 034-24024) was added to the medium at a final concentration of 20 ⁇ M at least 1 hour before the start of work to prepare cell clumps. Preprocessed. In subsequent operations, 20 ⁇ M Y27632 was added to all solutions to which the cells were exposed (except PBS).
- the supernatant containing floating cells was transferred to a new 15 ml tube, and the remaining cell mass was washed with PBS (the washing solution was also collected in the same tube as the supernatant).
- Floating cells were pelleted by centrifuging the 15 ml tube from which the supernatant was collected at 4 ° C. and 1000 rpm for 5 minutes.
- the cell mass was loosened by pipetting about 20 times with a P1000 micropipette together with the pellets of. After suspending in 10 ml of gfCDM + 20% KSR (cell mass differentiation and maintenance medium) to neutralize trypsin, the cells were centrifuged at 4 ° C. and 1000 rpm for 5 minutes. Add 1 ml of gf CDM + 20% KSR + 10 ⁇ g / ml DNase I to the cell pellet after centrifugation, pipette vigorously about 30 times with a P1000 micropipette to disperse the cell mass, and pass the aggregate through a 70 ⁇ m cell strainer. Removed. A single cell suspension was prepared by the above operation.
- EpCAM-positive cells were suspended in MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) and stained with PE-labeled anti-EpCAM antibody (10 minutes refrigeration). EpCAM-positive cells were subsequently labeled with magnetic beads by treatment with anti-PE-microbeads (Miltenyi, # 130-105-639). Next, the cells were passed through a magnetic column (LS column; Miltenyi, # 130-042-401) to separate and collect magnetic bead-labeled cells (EpCAM positive cells) and unlabeled cells (EpCAM negative cells).
- Example 4 Reaggregation culture of purified cells Each purified cell population (EpCAM positive cells, negative cells) was seeded on a low-adhesion V-bottom 96-well plate (PrimeSurface plate 96V; Sumitomo Bakelite, # MS-9096V). It was reaggregated. Each well was seeded with 15,000 cells in 200 ⁇ l medium (gfCDM + 20% KSR + 30 ⁇ M Y27632). After that, half the amount was replaced with a medium containing no Y27632 every 3 days.
- Example 5 Immunohistochemistry A cell mass cultured for 48 days or more after the start of differentiation induction and a cell mass cultured for 6 days or more after reaggregation after MACS purification were used. After fixing the cell mass with a 4% paraformaldehyde solution, the cell mass was gradually immersed in a 10, 20, 30% sucrose solution for sucrose substitution. Subsequently, it was embedded with OCT compound, and frozen sections having a thickness of 4 to 10 ⁇ m were prepared with a cryostat and attached to an anti-peeling coated slide glass (PLATINUM PRO; Matsunami Glass).
- the sections were washed with PBS and then blocked with a blocking solution (5% normal donkey serum + 0.1% TritonX-100 in PBS) at room temperature for 30 to 60 minutes. Next, the reaction was carried out overnight at 4 ° C. with a primary antibody diluted with a blocking solution. Subsequently, the reaction was carried out with a fluorescently labeled secondary antibody and DAPI (nuclear stain) at room temperature for 1 hour.
- the stained sections were encapsulated with Fluoromount (Diagnostic BioSystems, # K024) and then fluorescently observed with a confocal laser scanning microscope LSM-710 (Zeiss). As a result, EpCAM-positive thickened epithelial tissue was observed on the surface of the aggregate.
- This EpCAM-positive epithelial formula was vertically thicker than 3 cells. It was found that this EpCAM positive was co-positive with Cytokeratin (Fig. 2A). Furthermore, it was found that this EpCAM positive was co-positive with Pitx1 (Fig. 2B).
- Example 6 ACTH secretion test (CRH stimulation test) Cell clusters that were reaggregated after MACS purification and cultured for 6 days or longer were used. Six cell clusters were transferred to a 1.5 ml microtube and kept warm in 250 ⁇ l of HBSS (+) (Wako, # 084-08965) at 37 ° C. for 10 minutes, and then HBSS was recovered. Subsequently, HBSS was recovered after keeping warm at 37 ° C. for 10 minutes in 250 ⁇ l of HBSS containing 1 ⁇ g / ml CRH (Peptide Institute, # 4136-s). The ACTH concentration in the recovered HBSS was measured by the ECLIA method used in clinical tests (testing outsourced to SRL Co., Ltd.).
- the glandular pituitary gland and its precursors express Cytokeratin as a marker.
- Cytokeratin is a cytoskeletal protein that can be detected with high sensitivity in a short time by immunostaining. Therefore, cell clusters induced to differentiate from human iPS cells were dispersed, and surface antigen screening was performed using a commercially available antibody panel targeting Cytokeratin-positive cells.
- As the differentiation time for screening we selected about 50 days of culture in which pituitary progenitor cells were already induced and tissue dispersion was possible with gentle enzyme treatment. As a result, it was possible to prevent the degradation of surface antigens and secure a sufficient amount of cells for screening.
- EpCAM Epithelial cell adhesion molecule
- Fig. 1A EpCAM was expressed in about 80% of Cytokeratin-positive cells, and about 95% of EpCAM-positive cells were Cytokeratin-positive (Fig. 1B).
- Immunohistochemical analysis of cell clusters revealed that EpCAM was expressed in Cytokeratin, Pitx1, or Lhx3-positive pituitary progenitor cells (FIGS. 2A, B, C).
- EpCAM expression was maintained in pituitary hormone-producing cells (ACTH-positive cells) differentiated from the cells (Fig. 2C).
- EpCAM-negative cell clusters no increase in ACTH secretion was observed due to CRH addition, and the amount of secretion before CRH addition was less than half on average compared to EpCAM-positive cell clusters (Fig. 3C). .. From the above results, it is understood that functional pituitary hormone-producing cells and their progenitor cells can be purified from differentiated tissues derived from human pluripotent stem cells by using EpCAM as a marker.
- the separation method and production method of the present invention efficiently separate and purify functional pituitary hormone-producing cells and / or progenitor cells thereof from differentiated tissues derived from pluripotent stem cells by using EpCAM as a marker. Especially available.
- separated and purified pituitary hormone-producing cells and the like show excellent pituitary hormone secretory ability by physiological stimulation of pituitary hormone secretion, and therefore can be used for treatment of diseases related to the pituitary gland using the cells.
- This application is based on Japanese Patent Application No. 2020-065346 (Filing date: March 31, 2nd year of Reiwa), the entire contents of which are incorporated herein by reference.
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Abstract
Description
[1]腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞を分離する方法。
[2]細胞凝集塊が、視床下部神経上皮組織を含む、[1]に記載の方法。
[3]EpCAMを発現する細胞を分離する工程の前に、細胞凝集塊を分散し、単一細胞の集団を得る工程を含む、[1]又は[2]に記載の方法。
[4]EpCAMを発現する細胞を分離する工程の前に、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を細断するか、又は腺性下垂体及び/又はその前駆組織を含む細胞凝集塊に物理的に切れ込みを入れる工程を含む、[1]~[3]のいずれか1つ記載の方法。
[5]腺性下垂体及び/又はその前駆組織を含む細胞凝集塊が、多能性幹細胞を分化誘導することにより得られる細胞凝集塊である、[1]~[4]のいずれか1つに記載の方法。
[6]前記多能性幹細胞がヒト人工多能性幹細胞である、[5]に記載の方法。
[7]前記前駆組織が、下垂体プラコード及び/又はラトケ嚢である、[1]~[6]のいずれか1つに記載の方法。
[8]腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞の製造方法。
[9]以下の工程:
(A)腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を分散し、単一細胞の集団を得る工程、
(B)前記集団から、EpCAMを発現する細胞の集団を分離する工程、及び
(C)前記(B)で得られる集団を、再凝集させる工程
を含む、[8]に記載の製造方法。
[10]下垂体ホルモン産生細胞及び/又はその前駆細胞が、細胞凝集塊又は細胞シートである、[8]又は[9]に記載の製造方法。
[11]EpCAMを発現する細胞を分離する工程(B)の前に、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を細断するか、又は腺性下垂体及び/又はその前駆組織を含む細胞凝集塊に物理的に切れ込みを入れる工程を含む、[9]又は[10]に記載の方法。
[12]前記下垂体ホルモン産生細胞が、成長ホルモン(GH)産生細胞、プロラクチン(PRL)産生細胞、副腎皮質刺激ホルモン(ACTH)産生細胞、甲状腺刺激ホルモン(TSH)産生細胞、卵胞刺激ホルモン(FSH)産生細胞、黄体化ホルモン(LH)産生細胞からなる群から選択される少なくとも1種である、[8]~[11]のいずれか1つに記載の方法。
「多能性幹細胞」とは、生体を構成するすべての細胞に分化しうる能力(分化多能性)と、細胞分裂を経て自己と同一の分化能を有する娘細胞を生み出す能力(自己複製能)とを併せ持つ細胞をいう。
(1) Oct3/4, Klf4, Sox2, c-Myc(ここで、Sox2はSox1, Sox3, Sox15, Sox17又はSox18で置換可能である。また、Klf4はKlf1, Klf2又はKlf5で置換可能である。さらに、c-MycはT58A(活性型変異体), N-Myc, L-Mycで置換可能である。)
(2) Oct3/4, Klf4, Sox2
(3) Oct3/4, Klf4, c-Myc
(4) Oct3/4, Sox2, Nanog, Lin28
(5) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28
(6) Oct3/4, Klf4, Sox2, bFGF
(7) Oct3/4, Klf4, Sox2, SCF
(8) Oct3/4, Klf4, c-Myc, Sox2, bFGF
(9) Oct3/4, Klf4, c-Myc, Sox2, SCF
多能性幹細胞の凝集塊を調製する方法に特に限定はなく、分散させた多能性幹細胞を、培養容器に対して、非接着性の条件で培養(即ち浮遊培養)してもよいし、接着性の条件下で培養(即ち、接着培養)してもよい。
好ましい一態様において、多能性幹細胞の凝集塊は、分散させた多能性幹細胞を、培養器に対して、非接着性の条件下で培養し(即ち、浮遊培養し)、複数の多能性幹細胞を集合させて凝集塊を形成させることにより、得ることができる。
1)比較的小さな体積(例えば、1ml以下、500μl以下、200μl以下、100μl以下)の培養コンパートメント中に、分散した多能性幹細胞を閉じ込め、該コンパートメント中に1個の凝集塊を形成する方法。好ましくは分散した多能性幹細胞を閉じ込めた後、培養コンパートメントを静置する。培養コンパートメントとしては、マルチウェルプレート(384ウェル、192ウェル、96ウェル、48ウェル、24ウェル等)、マイクロポア、チャンバースライド等におけるウェルや、チューブ、ハンギングドロップ法における培地の液滴等を挙げることができるが、これらに限定されない。該コンパートメントに閉じ込められた分散した多能性幹細胞が、重力にうながされて1箇所に沈殿し、或いは細胞同士が接着することにより、1つの培養コンパートメントにつき、1つの凝集塊が形成される。マルチウェルプレート、マイクロポア、チャンバースライド、チューブ等の底の形状は、分散した多能性幹細胞が1箇所へ沈殿するのが容易となるように、U底又はV底とすることが好ましい。
2)遠心チューブに分散した多能性幹細胞を入れ、これを遠心し、1箇所に多能性幹細胞を沈殿させることにより、該チューブ中に1個の凝集塊を形成する方法。
本発明において、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊は、多能性幹細胞の凝集塊を、骨形成因子シグナル伝達経路活性化物質及びソニック・ヘッジホッグ(Shh)シグナル経路作用物質を含む培地中で浮遊培養することを含む方法にて取得することができる。
本明細書における「腺性下垂体及び/又はその前駆組織を含む細胞凝集塊」として、例えば、LHX3、NKX2.1、PITX1及びACTHから選択される少なくとも一つのマーカーが陽性である細胞凝集体が挙げられる。
また、「腺性下垂体及び/又はその前駆組織を含む細胞凝集塊」として、例えば、LHX3、NKX2.1、PITX1及びACTH陽性である細胞凝集塊が挙げられる。
本発明の分離方法及び製造方法において、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊とは、第2の培養工程により得られる細胞凝集塊であってもよい。
第1の培養工程においては、多能性幹細胞の凝集塊を、骨形成因子シグナル伝達経路活性化物質及びShhシグナル経路作用物質を含む培地中で浮遊培養する。
1)複数の培養コンパートメントを用意し、1つの培養コンパートメントに1つの多能性幹細胞の凝集塊が含まれるように、質的に均一な、多能性幹細胞の凝集塊の集団を播く。(例えば、96ウェルプレートの各ウェルに1つずつ、多能性幹細胞の凝集塊を入れる。)そして、各培養コンパートメントにおいて、1つの多能性幹細胞の凝集塊を骨形成因子シグナル伝達経路活性化物質及びShhシグナル経路作用物質を含む培地中で浮遊培養する。
2)1つの培養コンパートメントに複数の多能性幹細胞の凝集塊が含まれるように、質的に均一な、多能性幹細胞の凝集塊の集団を1つの培養コンパートメントに播く。(例えば、10cmディッシュに、複数の多能性幹細胞の凝集塊を入れる。)そして、該コンパートメントにおいて、複数の多能性幹細胞の凝集塊を骨形成因子シグナル伝達経路活性化物質及びShhシグナル経路作用物質を含む培地中で浮遊培養する。
第2の培養工程においては、第1の培養工程で得られた視床下部神経上皮組織及び表皮外胚葉(口腔外胚葉を含む)を含む細胞凝集塊を、骨形成因子シグナル伝達経路活性化物質及びShhシグナル経路作用物質を含む培地中で更に浮遊培養することにより、1)視床下部神経上皮組織、及び2)下垂体プラコード及び/又はラトケ嚢を含む細胞凝集塊を得る。骨形成因子シグナル伝達経路活性化物質及びShhシグナル経路作用物質の作用により、表皮外胚葉から下垂体プラコード及び/又はラトケ嚢への分化が誘導される。
第2の培養工程で得られた1)視床下部神経上皮組織、及び2)下垂体プラコード及び/又はラトケ嚢を含む細胞凝集塊を、Shhシグナル経路作用物質を含む培地中で更に浮遊培養することにより、腺性下垂体を含む細胞凝集塊を得ることができる(第3の培養工程)。第3の培養工程により、下垂体プラコード及び/又はラトケ嚢から、下垂体ホルモン産生細胞への分化が誘導され、下垂体プラコード及び/又はラトケ嚢中に下垂体ホルモン産生細胞が生じ、腺性下垂体が形成される。
本発明の分離方法及び製造方法において、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊とは、第3の培養工程により得られる細胞凝集塊であってもよい。
本発明は、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞を分離する方法を提供する。下垂体ホルモン産生細胞及びその前駆細胞の表面にはEpCAMが発現し得るが、視床下部神経上皮組織の表面には、EpCAMが発現し得ない。従って、EpCAMをマーカーとして、下垂体ホルモン産生細胞及びその前駆細胞と、視床下部神経上皮組織とを分離することができる。
本発明の腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞を分離する方法は、(B)EpCAMを発現する細胞(集団)を分離する工程の前に、(A)腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を分散させて、単一細胞の集団を得る工程を含み得る。
当該細胞凝集塊を分散させて、単一細胞の集団を得る工程(A)は、具体的には、(a1)腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を、酵素処理により分散させる工程を含み得る(後述する(4.2))。該酵素処理により分散させる工程の前に、(a2)腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を細断するか、又は腺性下垂体及び/又はその前駆組織を含む細胞凝集塊に物理的に切れ込みを入れる工程を含んでもよい(後述する(4.2))。
当該細胞凝集塊を分散させて、単一細胞の集団を得る工程(A)は、任意により、(a-1)腺性下垂体及び/又はその前駆組織を含む細胞凝集塊をROCK阻害剤で処理する工程を含んでもよい。該工程を行う場合、単一細胞の集団を得る工程の最初に行なうことが好ましい(後述する(4.1))。
酵素処理により分散させる工程(a2)後、(B)EpCAMを発現する細胞(集団)を分離する工程(EpCAMを発現する細胞(集団)と、EpCAMを発現しない細胞(集団)を分離する工程)を行う(後述する(4.3))。
上記細胞(集団)の分離工程(B)後、任意により、(C)得られた細胞(集団)を、再凝集させる工程を行ってもよい(後述する(4.4))当該再凝集工程(C)は、具体的には、(c1)得られた細胞(集団)を、培養器に播種して接着培養すること、及び/又は(c2)得られた細胞(集団)を、培養器に播種し浮遊培養することにより行い得る。
(4.1)ROCK阻害剤による前処理工程
本発明の分離方法では、まず得られた細胞凝集塊のROCK阻害剤による前処理を行い得る。当該前処理は、以降の細胞凝集塊の分散により誘導される多能性幹細胞(特に、ヒト多能性幹細胞)の細胞死を抑制するために、ROCK阻害剤を、当該前処理開始前から添加することが好ましい。ROCK阻害剤は、例えば、当該処理開始の少なくとも24時間前、少なくとも12時間前、少なくとも6時間前、少なくとも3時間前、少なくとも2時間前、少なくとも1時間前に添加する。ROCK阻害剤としては、Y-27632((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride)等を挙げることができる。当該処理に際して用いられるROCK阻害剤の濃度は、以降の細胞凝集塊の分散により誘導される多能性幹細胞の細胞死を抑制し得る濃度である。例えば、Y-27632について、このような濃度は、通常約0.1~200μM、好ましくは約2~50μMである。ROCK阻害剤の濃度を添加する期間内で変動させてもよく、例えば期間の後半で濃度を半減させることができる。後述の(4.2)~(4.4)のいずれの工程においても、細胞と接触させる溶液については、ROCK阻害剤を同様の濃度で含む溶液を用いることが好ましい。
当該前処理を行うための培地は、第2の培養工程により得られる細胞凝集塊を用いる場合、第2の培養工程において用いる培地を、第3の培養工程により得られる細胞凝集塊を用いる場合、第3の培養工程において用いる培地を用いることができる。なお、上記培地中に既に所望する濃度でROCK阻害剤が添加されている場合は、当該前処理工程を行う必要はない。
次に、上記前処理を行った細胞凝集塊を、酵素処理により分散させる。具体的には、まず、上記前処理を行った細胞凝集塊を、第2あるいは第3の培養工程に記載の培地(例:DMEM/F-12培地(DMEM培地:Ham's F-12培地=1:1の混合培地)等)を含む培養器(例:チューブ等)に移し、同培地で洗浄する。分散のための酵素としては、細胞を分散し得る限り特に限定されないが、例えば、EDTA;トリプシン、コラゲナーゼ(コラゲナーゼ タイプI~VII)、メタロプロテアーゼ、ヒアルロニダーゼ、エラスターゼ、ディスパーゼ、デオキシリボヌクレアーゼ等の酵素やこれらの混合物が挙げらる。好ましい酵素としては、コラゲナーゼが挙げられ、より好ましくはコラゲナーゼ タイプIである。酵素処理の条件(温度、時間等)は、用いる酵素等により適宜設定し得る。また、当該酵素処理を促進させるために、当該処理前に、細胞凝集塊を物理的(例:メス、ハサミ等)に細断するか、或いは細胞凝集塊に物理的(例:メス、ハサミ等)に切れ込みを入れる工程を行ってもよい。
上記調製した単細胞懸濁液に含まれる細胞集団から、EpCAMを発現する所望の細胞を分離する方法としては、フローサイトメトリーやマスサイトメトリーを用いた方法、磁気細胞分離法などが挙げられ、これらの方法は、自体公知の方法を用いて行うことができる。例えば、EpCAMを発現する細胞は、当該細胞とEpCAM分子に特異的に結合する物質(例:抗体等)とを接触させる工程を含む方法により分離することができる。上記物質には、それ自体に検出可能な標識(例:GFP、PE)が付加されているもの、およびそれ自体には標識が付加されていないものも含まれる。上記物質が、それ自体に標識が付加されていないものである場合、当該物質を直接または間接的に認識する検出可能な標識が付加された物質をさらに使用することで前記分離が可能となる。例えば、前記物質が抗体である場合には、蛍光色素、金属同位体又はビーズ(例:磁気ビーズ)を当該抗体に直接的又は間接的に担持させ、それにより細胞表面のマーカーを標識することが可能であり、当該標識に基づいて細胞を分離することが可能である。この際用いる抗体は、1種類のみ、又は2種類以上の抗体であってもよい。
分離した細胞集団は、培養器に播種して接着培養して、例えば、細胞シートとしてもよく、培養器に播種し浮遊培養することで、再凝集させて、細胞凝集塊としてもよい。該細胞集団は、そのまま使用まで維持培養してもよく、分離した細胞の分化状態に合わせて、所望する細胞へと更に分化誘導させてもよい。該維持培養や更なる分化誘導を行う場合、上述の第3の培養工程に記載の方法をそのまま行ってもよく、必要に応じて、適宜、自体公知の方法により修正して行ってもよい。また、該接着培養を行う場合も、接着培養自体は、自体公知の方法により行うことができ、適宜、上述の第3の培養工程に記載の内容(例:培地組成等)を用いてもよい。また、接着培養に際しては、培養器を細胞外マトリックス等(例:ラミニン、コラーゲン等)でコーティングすることが好ましい。
本発明の分離方法又は製造方法により得られた下垂体ホルモン産生細胞及びその前駆細胞は、移植医療のために使用することができる。例えば、腺性下垂体(前葉又は中葉、好ましくは前葉)の障害に基づく疾患の治療薬として、或いは腺性下垂体(前葉又は中葉、好ましくは前葉)の損傷状態において、該当する損傷部分を補充するために、本発明の方法により得られた下垂体ホルモン産生細胞及び/又はその前駆細胞を用いることができる。腺性下垂体の障害に基づく疾患、又は腺性下垂体の損傷状態の患者に、本発明により得られた下垂体ホルモン産生細胞及び/又はその前駆細胞を移植することにより、腺性下垂体の障害に基づく疾患、又は腺性下垂体の損傷状態を治療することができる。移植部位は、移植した下垂体ホルモン産生細胞及び/又はその前駆細胞が、障害された腺性下垂体の代替として機能し得る限り特に限定されないが、例えば、腎被膜下等を挙げることができる。腺性下垂体の障害に基づく疾患としては、汎下垂体機能低下症、下垂体性小人症、副腎皮質機能低下症、部分的下垂体機能低下症、下垂体前葉ホルモン単独欠損症などが挙げられる。さらに、これら腺性下垂体の損傷状態としては、腺性下垂体摘出後の患者、下垂体腫瘍への放射線照射後の患者、外傷が挙げられる。
本発明は、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞を製造する方法を提供する。本発明において、下垂体ホルモン産生細胞及び/又はその前駆細胞とは、下垂体ホルモン産生細胞及び/又はその前駆細胞でもよく、下垂体ホルモン産生細胞の集団(細胞集団)及び/又はその前駆細胞の集団(細胞集団)でもよく、あるいは、下垂体ホルモン産生細胞及び/又はその前駆細胞を高い純度で含む細胞集団であってもよい。下垂体ホルモン産生細胞及びその前駆細胞の表面にはEpCAMが発現し得るが、視床下部神経上皮組織の表面には、EpCAMが発現し得ない。従って、EpCAMをマーカーとして、下垂体ホルモン産生細胞及びその前駆細胞と、視床下部神経上皮組織とを分離することができる。該分離工程を用いることで、下垂体ホルモン産生細胞及び/又はその前駆細胞、あるいは下垂体ホルモン産生細胞及び/又はその前駆細胞を高い純度で含む細胞集団(例:細胞集団の80%以上、好ましくは85%以上、より好ましくは90%、更に好ましくは95%以上、なお一層好ましくは99%以上、最も好ましくは100%)を製造することができる。
本発明の製造方法に含まれる、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程は、上述の「・本発明の分離工程の概要」に記載の工程等の内容を全て援用することができる。また、各工程に関しても、上述の(4,1)~(4.4)の内容を全て援用することができる。
ヒトiPS細胞(201B7株)を、国際公開第2016/013669又はCell Reports, 30, 18-24, January 7, 2020に記載の自体公知の方法により、腺性下垂体又はその前駆組織を含む細胞凝集塊に分化誘導した。
ヒトiPS細胞(201B7株)から分化誘導して47日目の細胞塊をAccumax (Innovative Cell Technologies, #AM105)で37℃、5~10分処理して分散し、単細胞懸濁液を調製した。BioLegend社製のヒト表面抗原用PE標識抗体パネル(LEGENDScreen Human PE Kit, #700007)で細胞を染色後、IntraStain (Dako, #K2311)を用いて細胞の固定および膜透過処理を行った。続いてFITC標識抗Cytokeratin抗体(Miltenyi, #130-080-101)による細胞内Cytokeratinの染色、Hoechst 33342による核染色、HCS CellMask Deep Red Stain (Invitrogen, #H32721)による細胞質の染色を行った。染色後の細胞について、ParkinElmer社製のイメージングアナライザー(Opera Phenix)で画像取得・解析を行い、Cytokeratin陽性細胞に共存率の高い表面抗原を探索した。その結果、EpCAMで下垂体前駆細胞を識別できる可能性が示唆された。
細胞表面抗原のスクリーニングと同様に単細胞懸濁液を調製後、PE標識抗EpCAM抗体(Miltenyi, #130-098-115)、FITC標識抗Cytokeratin抗体、Hoechst 33342で染色を行い、蛍光顕微鏡(Leica DMI6000B)で画像を取得した。画像解析ソフトImage JのCell counterツールを用いて、EpCAM+/Cytokeratin+細胞、EpCAM+/Cytokeratin-細胞、EpCAM-/Cytokeratin+細胞、EpCAM-/Cytokeratin-細胞をカウントし、全細胞におけるそれぞれの割合を計算した。その結果、Cytokeratin陽性細胞に特異性の高い表面抗原として、Epithelial cell adhesion molecule (EpCAM; 別名CD326)を同定した(図1A)。該EpCAMはCytokeratin陽性細胞の約80%で発現し、EpCAM陽性細胞の約95%がCytokeratin陽性であった(図1B)。
分化誘導開始後90~500日の細胞塊を酵素的に分散し、MACSに使用した。以下に手順を示す。
(1)Y27632による前処理
分散処理による細胞死を抑制するために、作業開始の1時間以上前にY27632 (Wako, #034-24024)を終濃度20 μMで培地に添加して、細胞塊を前処理した。以降の操作では、細胞が曝されるすべての溶液(PBSは除く)に20 μM Y27632を添加した。
(2)細胞塊の分散
酵素処理による分散を促進するために、細胞塊をメスで細断または切込みを入れた。次に細胞塊を50 mlチューブに移して、DMEM/F12 (Wako, #042-30555)で洗浄した。次にコラゲナーゼ溶液1~3 mlを加えて、37℃で40分旋回振盪(140-150 rpm)した。コラゲナーゼ溶液の組成は、上記のDMEM/F12に0.2%コラゲナーゼtype I (Wako, #031-17601)および0.1% BSA (Sigma, #A9418)を添加したものである。
コラゲナーゼ処理終了後、浮遊細胞を含む上清を新しい15 mlチューブに移し、残りの細胞塊をPBSで洗浄した(洗浄液も上清と同じチューブに回収)。上清を回収した15 mlチューブを4℃、1000 rpm, 5分遠心して浮遊細胞をペレットにした。50 mlチューブの細胞塊に0.25% Trypsin/EDTA (Gibco, #25200072) + 0.2 mg/ml DNase I (Roche, #11284932001)を1 ml加えて、37℃で5~10分保温後、15 mlチューブのペレットと合わせてP1000マイクロピペットで約20回ピペッティングして細胞塊をほぐした。gfCDM + 20%KSR(細胞塊の分化および維持用培地)10 mlで懸濁してトリプシンを中和後、4℃、1000 rpm, 5分遠心した。遠心後の細胞ペレットにgfCDM + 20%KSR + 10 μg/ml DNase Iを1 ml加えて、P1000マイクロピペットで約30回激しくピペッティングして細胞塊を分散し、70 μmセルストレイナーを通して凝集塊を取り除いた。以上の操作により、単細胞懸濁液を調製した。
分散した細胞を死細胞除去キット(Miltenyi, #130-090-101)で処理することにより、死細胞およびアポトーシスを起こした細胞を除去した。
回収した生細胞をMACS buffer (PBS + 0.5% BSA + 2 mM EDTA)に懸濁し、PE標識抗EpCAM抗体で染色した(冷蔵10分)。続いて、抗PE-マイクロビーズ(Miltenyi, #130-105-639)で処理することにより、EpCAM陽性細胞を磁気ビーズで標識した。次に細胞を磁気カラム(LSカラム; Miltenyi, #130-042-401)に通すことで、磁気ビーズ標識細胞(EpCAM陽性細胞)と未標識細胞(EpCAM陰性細胞)を分離・回収した。
精製したそれぞれの細胞集団(EpCAM陽性細胞、陰性細胞)を低接着V底96ウェルプレート(PrimeSurface plate 96V; Sumitomo Bakelite, #MS-9096V)に播種して再凝集させた。1ウェル当たり、15,000細胞 in 200 μl培地(gfCDM + 20% KSR + 30 μM Y27632)で播種した。以降は3日ごとにY27632を含まない培地で半量交換した。
分化誘導開始後48日以上培養した細胞塊及びMACS精製後に再凝集させて6日以上培養した細胞塊を使用した。細胞塊を4%パラホルムアルデヒド溶液で固定後、10, 20, 30%スクロース溶液に段階的に浸してスクロース置換を行った。続いてOCTコンパウンドで包埋し、クリオスタットで4~10 μm厚の凍結切片を作製して、剥離防止コートスライドグラス(PLATINUM PRO; Matsunami Glass)に貼り付けた。切片をPBSで洗浄後、ブロッキング溶液(5%正常ロバ血清+0.1% TritonX-100 in PBS)で室温30~60分ブロッキング処理を行った。次に、ブロッキング溶液で希釈した一次抗体で4℃、一晩反応を行った。続いて、蛍光標識二次抗体およびDAPI(核染色剤)で室温1時間反応を行った。染色後の切片は、Fluoromount (Diagnostic BioSystems, #K024)で封入後、共焦点レーザー顕微鏡LSM-710 (Zeiss)で蛍光観察を行った。
その結果、凝集体の表面にEpCAM陽性の肥厚した上皮組織が観察された。このEpCAM陽性の上皮式は、垂直方向に3細胞以上の厚さがあった。そしてこのEpCAM陽性がCytokeratinと共陽性であることが分かった(図2A)。さらに、このEpCAM陽性がPitx1と共陽性であることが分かった(図2B)。
MACS精製後に再凝集させて6日以上培養した細胞塊を使用した。細胞塊6個を1.5 mlマイクロチューブに移し、HBSS(+) (Wako, # 084-08965) 250 μl中で37℃, 10分保温後、HBSSを回収した。続いて、1 μg/ml CRH (Peptide Institute, #4136-s)を含むHBSS 250 μl中で37℃, 10分保温後、HBSSを回収した。回収したHBSS中のACTH濃度を臨床検査で使われているECLIA法によって測定した(株式会社SRLへ検査委託)。
腺性下垂体及びその前駆組織はCytokeratin(サイトケラチン)をマーカーとして発現する。Cytokeratinは細胞骨格タンパク質であり、免疫染色により短時間で高感度に検出することが可能である。そこでヒトiPS細胞から分化誘導した細胞塊を分散し、Cytokeratin陽性細胞をターゲットとして、市販の抗体パネルにより表面抗原スクリーニングを実施した。スクリーニングを行う分化時期としては、すでに下垂体前駆細胞が誘導され、なおかつ穏やかな酵素処理で組織分散が可能な培養50日頃を選択した。これにより、表面抗原の分解を防ぐとともに、スクリーニングに足る細胞量を確保することができた。332種類のヒト表面抗原をスクリーニングした結果、Cytokeratin陽性細胞に特異性の高い表面抗原として、Epithelial cell adhesion molecule (EpCAM; 別名CD326)を同定した(図1A)。EpCAMはCytokeratin陽性細胞の約80%で発現し、EpCAM陽性細胞の約95%がCytokeratin陽性であった(図1B)。
細胞塊の免疫組織化学的解析により、EpCAMはCytokeratin、Pitx1、又はLhx3陽性の下垂体前駆細胞において発現が認められた(図2A、B、C)。また、そこから分化した下垂体ホルモン産生細胞(ACTH陽性細胞)においてもEpCAMの発現が維持されていた(図2C)。
90日以上分化誘導した細胞塊を用いて、MACSによりEpCAM陽性細胞と陰性細胞を分離して再凝集を試みた(図3A)。EpCAM陽性と陰性のいずれの細胞集団も凝集塊を形成したが、EpCAM陽性の細胞塊にはACTH陽性細胞およびLhx3陽性細胞が含まれるのに対し、EpCAM陰性の細胞塊には、ACTH陽性細胞及びLhx3陽性細胞が含まれないことを確認した(図3B)。ACTH分泌試験を行った結果、EpCAM陽性の細胞塊では、生理的な分泌刺激因子であるCRHの添加によってACTH分泌量が平均で2.1倍に増加した(図3C)。一方、EpCAM陰性の細胞塊では、CRH添加によるACTH分泌の増加は認められず、CRH添加前の分泌量もEpCAM陽性の細胞塊と比べて平均で2分の1未満であった(図3C)。
以上の結果から、EpCAMをマーカーとすることで、ヒト多能性幹細胞由来の分化組織から機能的な下垂体ホルモン産生細胞及びその前駆細胞を精製することが可能であることが理解される。
Claims (12)
- 腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞を分離する方法。
- 細胞凝集塊が、視床下部神経上皮組織を含む、請求項1に記載の方法。
- EpCAMを発現する細胞を分離する工程の前に、細胞凝集塊を分散し、単一細胞の集団を得る工程を含む、請求項1又は2に記載の方法。
- EpCAMを発現する細胞を分離する工程の前に、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を細断するか、又は腺性下垂体及び/又はその前駆組織を含む細胞凝集塊に物理的に切れ込みを入れる工程を含む、請求項1~3のいずれか一項に記載の方法。
- 腺性下垂体及び/又はその前駆組織を含む細胞凝集塊が、多能性幹細胞を分化誘導することにより得られる細胞凝集塊である、請求項1~4のいずれか一項に記載の方法。
- 前記多能性幹細胞がヒト人工多能性幹細胞である、請求項5に記載の方法。
- 前記前駆組織が、下垂体プラコード及び/又はラトケ嚢である、請求項1~6のいずれか一項に記載の方法。
- 腺性下垂体及び/又はその前駆組織を含む細胞凝集塊から、EpCAMを発現する細胞を分離する工程を含む、下垂体ホルモン産生細胞及び/又はその前駆細胞の製造方法。
- 以下の工程:
(A)腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を分散し、単一細胞の集団を得る工程、
(B)前記集団から、EpCAMを発現する細胞の集団を分離する工程、及び
(C)前記(B)で得られる集団を、再凝集させる工程
を含む、請求項8に記載の製造方法。 - 下垂体ホルモン産生細胞及び/又はその前駆細胞が、細胞凝集塊又は細胞シートである、請求項8又は9に記載の製造方法。
- EpCAMを発現する細胞を分離する工程(B)の前に、腺性下垂体及び/又はその前駆組織を含む細胞凝集塊を細断するか、又は腺性下垂体及び/又はその前駆組織を含む細胞凝集塊に物理的に切れ込みを入れる工程を含む、請求項9又は10に記載の方法。
- 前記下垂体ホルモン産生細胞が、成長ホルモン(GH)産生細胞、プロラクチン(PRL)産生細胞、副腎皮質刺激ホルモン(ACTH)産生細胞、甲状腺刺激ホルモン(TSH)産生細胞、卵胞刺激ホルモン(FSH)産生細胞、黄体化ホルモン(LH)産生細胞からなる群から選択される少なくとも1種である、請求項8~11のいずれか一項に記載の方法。
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