WO2001088100A1 - Nouvelle methode permettant de declencher une differentiation de cellules embryonnaires dans des cellules de l'ectoderme et leurs applications - Google Patents
Nouvelle methode permettant de declencher une differentiation de cellules embryonnaires dans des cellules de l'ectoderme et leurs applications Download PDFInfo
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- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
Definitions
- the present invention relates to a method for inducing functional cells from embryonic stem cells. More specifically, the present invention relates to a method for inducing differentiation of ectoderm cells or cells derived from ectoderm from embryonic stem cells, which is useful as cell therapy, the differentiated cells, and uses thereof.
- the present invention also relates to a medium used in the above method, an antibody specifically recognizing a stoma cell used in the above method, an antigen recognized by the antibody, and uses thereof.
- embryonic stem cells are all cells that can be cultured in vitro and that contain germ cells when injected into other individuals' preimplantation embryos, such as the blastocyst cavity. Means cells that can be differentiated into different types of cells, and are also called embryonic stem cells or ES cells.
- mice The relationship between early embryo development and embryonic stem cells will be described below using mice as an example.
- the mouse fertilized egg divides into 2 cells, 4 cells, and 8 cells while moving from the oviduct to the ovum, and when the 16 cell stage is reached, a compaction occurs in which the adhesion between cells increases, It reaches a stage called morula, in which the boundaries between the cells are blurred.
- a space called blastcoel is formed inside the embryo and becomes a blastocyst.
- the blastocyst is composed of the outer trophectodermal layer (trophectogerm) and the inner cell mass (ICM) force.
- the blastocyst is implanted in the uterine wall 4.5 to 5.5 days after fertilization.
- the cells on the surface facing the septum in the inner cell mass It differentiates into primitive endoderm cells. Of these, some cells migrate away from the embryo body to the middle of the trophectoderm layer to become distal endoderm cells, which secrete extracellular matrix and reichert membrane (Re 1 chert) s membrane)
- Primitive endoderm cells near the embryonic body form a cell layer called the visceral endoderm. These distal and proximal endoderm eventually provide the supporting tissue for protecting the fetal body and exchanging nutrients and waste with the mother. In the future, the cells of the inner cell mass that make up the body of the fetus will proliferate and form a cell layer called primitive ectoderm. Primitive ectoderm is also called embryonic ectoderm or epiblast. Embryos after implantation grow into a cylindrical shape as a whole, so embryos 5.5 to 7.5 days after fertilization are called egg cylinder embryos.
- the extraembryonic tissue which will form the placenta in the future, is formed from the trophectoderm in the base half of the oviduct embryo at the base of the uterus. 6.5 days after fertilization, a groove called a primitive streak appears in the primordial ectoderm layer, in which the primordial ectoderm changes into mesenchymal cells, and the primordial ectoderm layer and proximal endoderm It moves between the layers and moves left and right and back and forth from the original streak to form an embryonic mesoderm cell layer (embryonic mesoderm). This cell layer also contains cells that will become the definitive endoderm of the fetal body in the future.
- primitive ectoderm produces not only ectoderm but also three germ layers of fetal mesoderm and endoderm, and that all tissues of the fetus are derived from primitive ectoderm It is shown. It has been shown that cells of the nervous system and epidermis are formed from the ectoderm, and ectoderm that is destined to differentiate into cells of the nervous system is referred to as neural ectoderm and epidermal cells. The ectoderm that is committed to differentiation is called the non-neural ectoderm.
- the individual blastomere cells from the fertilized egg to the morula, the cells of the inner cell mass in the blastocyst, and the cells constituting the primitive ectoderm layer It is totipotent and has the properties of undifferentiated embryonic stem cells.
- the primordial ectoderm begins to differentiate into each germ layer, most of the cells lose totipotency, but some of them are primordial germ cells responsible for transferring genes to the next generation
- primordial germ cell Primordial germ cell
- Primordial germ cells are swept backwards into the embryonic mesoderm cell layer invading from the primordial streak when the primordial ectoderm differentiates into each germ layer, and the extraembryonic mesoderm at the base of the allantois (allantois) Extraembryonic mesoderm).
- Primordial germ cells eventually migrate toward the gonad primordium and form ova and sperm as the gonads are sexually differentiated.
- Embryonic stem cells can be established by transferring cells constituting the inner cell mass, which is an undifferentiated stem cell present inside a blastocyst, into culture and repeating dissociation and passage of the cell mass frequently. These cells can grow and pass almost indefinitely while maintaining a normal karyotype, and maintain pluripotency that can differentiate into all types of cells, similar to the inner cell mass. It has been known.
- embryos 1 and living stem cells When embryos 1 and living stem cells are injected into the blastocysts of another individual, they mix with the cells of the inner cell mass of the host embryo and contribute to the formation of embryos and fetuses, creating chimeric individuals. In extreme cases, individuals may be born that consist solely of embryonic stem cells that the fetal body almost injected.
- chimera individuals those in which the injected embryonic stem cells contributed to the formation of primordial germ cells that will produce eggs and sperm in the future are called germline chimeras, and embryos injected by crossing these germline chimeras Since it is possible to obtain an individual derived from an embryonic stem cell, it has been proved that the embryonic stem cell has a totipotent ability to differentiate into any cell (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereafter abbreviated as "Ma-Purating the Mouth Embryo a Laboratory” Mayua Nore "; Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) (hereinafter abbreviated as “Gene 'Targeting”); No Manual Series 8 Gene Targeting, Production of Mutant Mouse Using ES Cells, Yodosha (1995) (hereinafter “ES Cells Preparation of Mutant Mouse Used ”)).
- LIF leukaemia inhibitory factor
- Embryonic stem cells have been established. It is known that when embryonic stem cells are transplanted subcutaneously into an animal of the same strain as that of the embryonic stem cells, a teratoma mixed with various tissues is formed (manipulation of 'the' mouse embryo '). 'Laboratory' manual).
- embryoid stem cells are aggregated to form a pseudo-embryoid body, a cell mass called an embryoid body.
- EB endoderm cells
- ectoderm cells mesoderm cells
- blood cells endothelial cells
- chondrocytes skeletal muscle cells
- smooth muscle cells myocardium Cells
- glial cells nerve cells
- epithelial cells melanocytes
- keratinocytes have been reported to be able to appear (PD Rathjen et al., Reprod. Fertil. Dev., 10, 31). (1998)).
- spontaneous distribution due to the formation of cell aggregates is caused, and as a result, the appearance of target cells has been observed.
- the cell population has not yet been efficiently induced, and at the same time, the appearance of various types of tissue cells has been observed.
- Bell class III tubulin, neurofilament M subunit, GAP-43 (growth-associated protein-43), MAP-2 (microtubule-associated protein-2), which is a substrate for neurospecific calmodulin-binding kinase C, ⁇ y-Aminobutyric acid (-GABA) receptor, NMDA (N-methyl-D-aspartate) receptor, and synapsin expression are expressed at the mRNA level in neurofilament L subunit, It has been reported that expression of glutamate receptor, tyrosine hydroxylase, transcription factor Brn-3, glial fibrillary acidic protein (GFAP), and GAD (glutamic acid decarboxylase), a GABA synthase, is observed (G. Bain Biol., 168, 342 (1995); FA Michael et al., J. Neurosci., 16, 1056 (1996)).
- Brn-3 is the central nervous system (X. He et al., Nature, 340, 35 (1989)), and GAP-43 is the nerve axon (LI Benowitz and A. Routtenberg; Trends Neurosci., 20, 84 (1997)).
- MAP-2 is a neurite (LI Binder et al., Ann. NY Acad. Sci., 76, 145 (1986)) and GFAP is a glial cell (A. Bignami et al., Brain Res., 43, 429 ( 1972)),
- GABA receptor GAD is an inhibitory nerve (Y. Chang and DI Gottling; J.
- glutamate receptor NMDA receptor is excitatory Since it is known to be expressed in nerves, it has been shown that when schizophrenia is induced using retinoic acid, schizophrenia signals are simultaneously transmitted to various nervous system cells.
- GAP-43-positive cells show a glial cell-like morphology, of which about half are neurofilament-165 positive cells, but neuronal-like cells induced by retinoic acid treatment after EB formation. It has been reported that both GAP-43 and Neurofilament-165 have lower staining intensity by antibody staining than cells (WG van Inzen et al., Biochim. Biophys. Acta., 1312, 21 (1996)). Thus, it has been confirmed that cell-cell interaction due to EB formation is necessary for efficient induction of neural cell differentiation.
- the action potential was measured using the patch clamp method for cells with a glial cell-like morphology, and the potential of 5-HT (5-hydroxytryptamin), GABA, kainate, glutamic acid, dopamine, and calpachol stimulation was measured.
- 5-HT 5-hydroxytryptamin
- GABA glutamic acid
- dopamine dopamine
- calpachol stimulation was measured.
- the neuronal-like cells induced by retinoic acid treatment after EB formation which was used as a control, showed no action potential due to carbachol stimulation.
- the generation of action potentials due to noradrenaline stimulation was observed, indicating that the interaction between cells by EB formation is also important in determining the direction of neuronal differentiation (WG van Inzen et al., Biochim. Biophys. Acta., 1312, 21 (1996)).
- retinoic acid when used to induce differentiation of embryonic stem cells, it is used at a much higher concentration (10- to 100-fold) than that which is physiologically present. It is difficult to use the obtained cells for transplantation because the use of retinoic acid at a higher concentration than is physically present is a concern in terms of toxicity. Attempts have been made to induce the differentiation of embryonic stem cells into cells of the nervous system under conditions closer to physiological conditions without using retinoic acid.
- the EBs formed by suspension culture for 4 days are cultured on a tissue culture dish for 1 day and allowed to adhere, and an ITSFn medium (A. Rizzino and C. Growl containing insulin, transferrin, selenium chloride, and fibrous nectin) is attached. Natl. Acad. Sci. USA, 77, 457 (1980)) for 5 to 7 days to produce nestin-positive fatty acid binding protein positive (A.
- ITSFn medium A. Rizzino and C. Growl containing insulin, transferrin, selenium chloride, and fibrous nectin
- the mouse neuroepithelial cell-like progenitor cells induced using the ITSFn medium described above migrate and are taken up by the brain tissue.
- Astrocytes and oligodendrocytes are observed to be morphologically indistinguishable from host cells (0. Housele et al., Proc. Natl. Acad. Sci. USA, 94, 14809). (1997)).
- teratoma tumor tissue has also been observed in transplantation of neural progenitor cells derived from embryonic stem cells using retinoic acid (J. Dinsmore et al., Cell Transplant., 5, 131 (1996). T. Deacon et al., Exp. Neurol., 149, 28 (1998)).
- glial progenitor cells are derived from embryonic stem cells, and the progenitor cells are transplanted into the brain or spinal cord of rats with a myelin-deficient myelin sheath, whereby the myelin medulla is formed without teratoma formation. It has been reported that sheath repair is observed (0. Housele et al., Science, 285, 754 (1999)).
- precursor cells of further differentiated glial cells are induced from the neural epithelial cell-like precursor cells induced using the iTSFn medium and used for transplantation.
- the induced neuroepithelial cell-like progenitor cells were cultured on a polyortin-coated dish in a medium containing insulin, transfulin, progesterone, putrescine, selenium chloride, FGF2 (fibroblast growth factor 2), and laminin. Cultured for 5 days, Hanks without calcium and magnesium Cells were detached using a buffer and subcultured at a 1/5 cell density in a medium containing FGF2 and EGF (epidermal growth factor) .When the cells reached almost confluent state, the cells were again cultured at 1/5 the cell density.
- Subculture into a medium containing FGF2 and PDGF-AA (platelet-derived growth factor-M) and continued cultivation can induce differentiation of Daryah cells into progenitor cells. It has been shown that this is possible.
- the cells induced to differentiate in this way are A2B5-positive (MC Raff et al., Nature, 303, 390 (1983)), and when cultured in a medium free of FGF2 and EGF, are divided into astrocytes and oligodendrocytes. Observation at the in-vitro mouth indicates that dani is a precursor cell of Dallia cells.
- EC cells embryonic carcinoma cells
- EC cells embryonic carcinoma cells
- MJ Evans J. Erabryol. Exp. Morph., 28, 163 (1972)
- These cells express genes that serve as markers for embryonic stem cells (EG Bernstine et al., Proc. Natl. Acad. Sci. USA, 70, 3899 (1973); SB Diwan and LC Steven, J. Natl. Cancer Inst., 57, 937 (1976); D. Solter and BB Knowles ⁇ Proc. Natl. Acad. Sci. USA, 75, 5565 (1978); BA Hosier et al., Mol. Cell. Biol., 9, 5623.
- Parkinson's disease is a chronic progressive disease that is mainly caused by degeneration of the nigrostriatal dopamine neurons.
- oral therapy with a focus on L-DOPA (L-dihydroxyfuranalanine) has been performed. You will be relieved by side effects such as dyskinesia. Therefore, the search for more effective treatments has been sought, and treatments for transplanting aborted fetal brains to patients with Parkinson's disease have begun.
- Hundreds of cases of aborted fetal brain transplantation have been administered worldwide. Recently, a double-blind study of aborted fetal brain cell transplantation in 40 patients with Parkinson's disease in the United States has demonstrated its usefulness.
- a method for efficiently inducing and obtaining ectoderm-derived cells, specifically, dopaminergic neurons having a normal function, from undifferentiated stem cells can be used for patients with brain diseases such as Parkinson's disease. Important and wished from the medical point of view, but not yet developed. Disclosure of the invention
- the present invention is applicable to cell and organ transplantation medicine, a method for selectively and efficiently inducing ectoderm cells and ectoderm-derived cells from embryonic stem cells, It is intended to provide cells and their uses.
- the present invention also relates to a medium used for the method of inducing differentiation, an antibody specifically recognizing stromal cells used for the method of inducing differentiation, a method for producing the antibody, an antibody obtained thereby, an antigen recognized by the antibody
- An object of the present invention is to provide a method for obtaining the antigen, the antigen obtained thereby, and these uses.
- the present inventors have conducted intensive studies on various culture conditions that cause the differentiation of embryonic stem cells, and have found that selective and efficient We succeeded in finding a method and completed the present invention. That is, the present invention relates to the following (1) to (71).
- ectodermal cells are cells capable of differentiating into neural cells or epidermal cells.
- a method for inducing differentiation of embryonic stem cells into ectoderm-derived cells comprising a step of culturing embryonic stem cells in a non-aggregated state.
- the nervous system cell is a cell selected from the group consisting of the following (a), (b), (c) and (d).
- the nerve cell is a nerve cell selected from the group consisting of the following (a;), (b), (c) and (d).
- acetylcholinergic neuron is a motor neuron expressing islet1.
- the neural tube cell is a cell selected from the group consisting of the following (a), (b), (c) and (d).
- Neural tube ventral cells expressing HNF-3 / 3 located on the most ventral base plate of the neural tube;
- HNF_ ventral neural tube 3 i3 marker present second only to (Hepatocyte Nuclear Factor- 3 j8) Nkx2 two neural tube ventral expressing cells;
- the anticancer agent is an anticancer agent selected from the group consisting of mitomycin C, 5-fluorouracil, adriamycin, and methotrexate.
- Treatments for tissue fixation used in pathological diagnosis include microwave fixation, rapid freezing displacement fixation, daltaraldehyde fixation, paraformaldehyde fixation, formalin fixation, acetone fixation, Bouin fixation, and periodate fixation
- stromal cells are stromal cells recognized by a monoclonal antibody produced by hybridoma FERM BP-7573.
- the stromal cell is a stromal cell selected from the group consisting of the following (a), (b), (c), (d), (, (f) and (g): ) To (22).
- mice (e) mouse calvaria-derived MC3T3-G2 / PA6 cells; (f) stromal cells derived from embryonic 'I "live stem cells;
- An agent for inducing differentiation of ectodermal cells into epidermal cells comprising Bone Morphogenetic Protein 4 as an active ingredient.
- a medium comprising the culture supernatant according to (37), wherein the stromal cells are the stoma cells according to any one of (18) to (23).
- a medium comprising the culture supernatant according to (37) or (38), wherein the mucopolysaccharide is heparin.
- (41) obtaining an antibody that specifically recognizes stromal cells having an activity of inducing differentiation of embryonic stem cells into ectodermal cells and cells derived from ectoderm, using stromal cells as an immunogen how to.
- a method for obtaining an antigen recognized by the antibody which comprises using the antibody according to (43) or (44).
- a medium for cell culture comprising the antigen according to (46).
- a stromal cell-derived factor that has the activity of inducing differentiation of embryonic stem cells into ectoderm-derived cells based on the activity of inducing differentiation of embryonic stem cells into ectodermal cells and ectoderm-derived cells How to get.
- the anticancer agent is an anticancer agent selected from the group consisting of mitomycin (, 5-fluorouracil, adriamycin, methotrexate, and ara-C).
- the method described in any one of (1) to (27) above is performed in the presence of the test substance and in the absence of the test substance, and the presence of the test substance and the absence of the test substance are performed. Under the differentiation of embryonic stem cells to ectoderm or ectoderm-derived cells, characterized by comparing the differentiation process from embryonic stem cells to ectoderm or ectoderm-derived cells below Evaluation method of substances to be used. (57) The method described in any one of (1) to (27) above is performed in the presence of the test substance and in the absence of the test substance, and the presence of the test substance and the absence of the test substance are performed. Under the differentiation of embryonic stem cells to ectoderm or ectoderm-derived cells, characterized by comparing the differentiation process from embryonic stem cells to ectoderm or ectoderm-derived cells below How to screen substances.
- a medicament comprising stromal cells having an activity of inducing differentiation of ectodermal cells and cells derived from ectoderm from embryonic stem cells, or a medicament containing factors derived from the cells.
- a medicament comprising the antibody according to (43) or (44).
- Diseases based on nervous system disorders include Alzheimer's disease, Huntington's chorea, Parkinson's disease, ischemic brain disease, epilepsy, brain trauma, spinal cord injury, motor neuron disease, neurodegenerative disease, retinitis pigmentosa, Inner ear hearing loss, multiple sclerosis, amyotrophic lateral sclerosis, or a disorder caused by a neurotoxin disorder; disorders based on disorders of epidermal cells can cause burns, trauma, wound healing, rubbing on the floor, or psoriasis The medicament according to the above (68), which is
- the animals to which the present invention is applied include vertebrates, especially warm-blooded animals, and furthermore, mice, rats, monoremots, nomsters, rabbits, cats, dogs, higgins, pigs, lions, goats, monkeys, humans And other mammals.
- the lunar Pi stem cells include cells capable of culturing in the mouth of an intestine and having a pluripotency capable of differentiating into all cells constituting a living body. Examples include (a) embryonic stem cells such as mammals established by culturing early embryos before implantation, specifically, ES cells established from the inner cell mass that constitutes the early embryos Cells, EG cells established from primordial germ cells, cells isolated from a population of cells that have the diversifying potential of the preimplantation early embryo (eg, primitive ectoderm), or And cells obtained by culturing the above cells. Since EC cells established from malignant teratomas are known to exhibit the same properties as ES cells, they can be spread to Pi stem cells such as mammals established by culturing early embryos before implantation. Included in righteousness.
- embryonic stem cells such as mammals established by culturing early embryos before implantation, specifically, ES cells established from the inner cell mass that constitutes the early embryos Cells, EG cells established from primordial germ cells, cells isolated from
- the embryonic stem cells of the present invention include the embryonic stem cells of (a) above, and (b) embryonic stem cells established by culturing the early embryos produced by nuclear transfer of the nuclei of somatic cells. And (c) embryonic stem cells obtained by modifying the gene on the chromosome of the embryonic stem cells of (a) or (b) by genetic engineering techniques.
- the embryonic stem cells By culturing the embryonic stem cells as described above in a non-aggregated state by the differentiation-inducing method of the present invention, the embryonic stem cells can be induced to differentiate into ectoderm cells or cells derived from ectoderm.
- the ectoderm cells include germ cells composed of cells capable of differentiating into neural cells and epidermal cells. Examples include fetal ectoderm cells differentiated from primitive ectoderm.
- the ectoderm-derived cells include cells differentiated from ectoderm cells and functional cells constituting a living body. Specific examples include neural cells and epidermal cells. That is, by the method of the present invention, ectodermal cells can be induced into neural cells or epidermal cells.
- Neural cells include neural stem cells, neural cells, neural tube cells, neural crest cells, and the like.
- Nerve cells include neural stem cells, neural cells, neural tube cells, neural crest cells, and the like.
- a nerve cell refers to a cell having a function of transmitting a stimulus to another nerve cell, muscle, or gland cell in response to stimulation from another nerve cell or a stimulus receiving cell.
- Nerve cells are classified according to the difference in neurotransmitters produced by the neurons, and specifically, according to differences in neurotransmitters, neurotransmitter synthases, and the like.
- Neurotransmitters include both peptide and non-peptide.
- Non-peptidic neurotransmitters include dopamine, noradrenaline, adrenaline, serotonin, acetylcholine, gamma-aminobutyric acid, and glutamic acid.
- Dopamine, noradrenaline and adrenaline are called catecholamines.
- Neurons classified by these neurotransmitters include, for example, dopaminergic neurons, acetylcholinergic neurons, ⁇ -aminobutyrate nervous system cells, serotonergic neurons, noradrenergic neurons, Examples include adrenergic neurons and glutamatergic neurons. Dopaminergic neurons, noradrenergic neurons, and adrenergic neurons are collectively called catecholaminergic neurons.
- Catecholaminergic neurons commonly express tyrosine hydroxylase
- noradrenergic and adrenergic neurons commonly express dopamine-hydroxylase.
- Noradrenergic neurons express fluorethanolamine methyltransferase
- serotonergic neurons express tributophane hydroxylase
- acetylcholinergic neurons express choline acetinol transferase.
- y-aminobutyric acid-operated neurons specifically express glutamate decarboxylase. Therefore, methods for recognizing nerve cells include a method using an antibody recognizing the enzyme, a method for detecting expression of mRNA encoding the enzyme, and the like.
- Peptide neurotransmitters include corticotropin
- Substance P Neurokinin A, Neuropeptide K, Neuropeptide- ⁇ , Neurokinin B, Bombesin, Gust Gastrin-: releasing peptide; Segretin, Motilin, Glucagon, Vasoactive intestinal peptide, Growth hormone-releasing factor factor), insulin, insulin-like growtn factors, somatostatin, gastrin, cholecystokinin, neuropeptide Y ( Neuropeptide Y), Pancreatic polypeptide, Peptide YY, Corticotropin- releasing factor, Calcito Calcitonin, Calcitonin gene-related peptide Angiotensin, Bradykinin, Thyrotropin-releasing hormone, Neurotensin, Ga ⁇ nin The luteinizing hormone-releasing hormone (Luteinizing hormone-releasing hormone;) The nerve cells that produce these peptide neurotransmitters are Identification can be made by detecting the expression of an antibody that recognizes a transmitter or a neurotransmitter precursor peptide,
- Motor nerves also transmit information to skeletal muscle by secreting acetylcholine from their nerve endings, and are classified as acetylcholinergic neurons.
- a marker protein for motor neuron is islet 1 (0. Karlsson et al., Nature, 344, 879, 1990).
- the method for inducing differentiation of the present invention is suitably used for inducing differentiation into neurons, preferably dopaminergic neurons, acetylcholinergic neurons, ⁇ -aminobutyric acid-nergic cells, and serotonergic neurons.
- the dopaminergic neurons derived from embryonic stem cells by the method of the present invention express tyrosine hydroxylase that is commonly observed in catecholaminergic neurons.
- it is characterized as a cell that does not express dopamine- ⁇ -hydroxylase, whose expression is commonly observed in noradrenergic neurons and adrenergic neurons.
- Symptoms of neurodegenerative diseases such as Parkinson's disease by transplantation Have the ability to improve.
- Neural stem cells are defined as cells that have the ability to differentiate into neurons, astrocytes and oligodendrocytes, and have the ability to self-renew. Although it probably does not have the ability to differentiate into all cells like embryonic stem cells, it has the function of supplying nerve cells, astrocytes and oligodendrocytes in the brain.
- Neural stem cells having such functions can be stained with an anti-nestin antibody that recognizes the cytoskeletal protein nestin, which has been confirmed to be expressed in neural progenitor cells (R. Mckay, Science, 276, 66). (1997)). Therefore, neural stem cells can be confirmed by staining with an anti-nestin antibody.
- Nerve induction refers to the process in which the ectoderm located on the dorsal side of the early embryo differentiates into neural ectoderm upon receiving a signal from the organizer region located on the adjacent or inner part.
- the neural ectoderm formed by this nerve induction becomes a neural tube independently of the non-neural ectoderm, that is, the epidermal ectoderm, which invades the ventral side and eventually enters the neural tube ( form a neural tube).
- the ectoderm located between the neural plate and the epidermal ectoderm, forms a neural crest when invaginating. All the cells of the central nervous system develop from one layer of neuroepithelial tissue that makes up the neural tube. That is, the anterior part of the neural tube expands to form brain vesicles, which are the basis of the brain, and the posterior part of the neural tube remains in the tube and splits into the spinal cord. Neural crests are not directly involved in the differentiation of the central nervous system itself, and the cells that make up the neural crest move and move into various tissues, such as the brain, spinal ganglia, sympathetic nerves and their ganglia, adrenal medulla, or melanin pigment cells. To differentiate into such.
- Neural tube cells refer to the cells that make up the neural tube during the developmental process described above.
- Neural crest cells refer to cells constituting the neural crest in the above-mentioned developmental process.
- the method for inducing differentiation of the present invention is suitably used for inducing differentiation into neural tube cells and neural crest cells.
- Neural tube cells derived from embryonic stem cells by the method of the present invention are cells located on the ventral side in response to Sonic hedgehog (hereinafter abbreviated as “shh”), which is a ventricularizing factor of the neural tube.
- shh Sonic hedgehog
- Dorsoventral axis which has the ability to differentiate into cells that are differentiated into cells and respond to bone morphogenetic protein 4 (BMP4), a dorsal factor in the neural tube, and differentiate into cells located on the dorsal side And cells characterized as cells of the neural tube in a stage before the determination of the neural tube.
- BMP4 bone morphogenetic protein 4
- the cells differentiate and express the marker HNF-3j3 (Hepatocyte Nuclear Factor-3 ⁇ or less, abbreviated as "HNF-3”) located on the most ventral bottom plate of the neural tube.
- ventral cells of the neural tube expressing the marker Nkx2.2, which is the second present after HNF-3 / 3 from the ventral side of the neural tube, and dorsal neural tube expressing Pax-7 Cells are also included as neural tube cells derived from embryonic stem cells by the method of the present invention.
- Neural crest cells derived from embryonic stem cells by the method of the present invention include cells characterized as cells expressing AP-2 (Activator Protein 2, hereinafter abbreviated as AP-2j).
- AP-2 Activator Protein 2, hereinafter abbreviated as AP-2j.
- Shh is a secretory factor involved in early developmental morphogenesis such as the formation of the dorsal-ventral axis of the neural tube and the anterior-posterior axis of the limb bud (C. Chiang et al., Nature, 383, 407 (1996); M Bitgood et al., Curr. Biol., 6, 298 (1996)).
- ⁇ 4 is a secretory factor involved in morphogenesis in early development that acts as a dorsalizing factor, such as formation of the dorsoventral axis of the neural tube and formation of the mesoderm dorsal-ventral axis (JM Graff et al., Cell, 79 A. Suzuki et al., Proc. Natl. Acad. Sci. USA, 91, 10255 (1994)).
- HNF-3 j8 is known to be expressed in postnatal liver, small intestine, lung, and islets of Langerhans, but during development, intestinal epithelium, liver primordium, or primordium after foregutogenesis During the intestinal invagination period, it is expressed in the organs such as the dorso-oral lip, the anterior chordae, the notochord, and the ventral central part of the neural tube. Is an important factor controlling the signal for (C. Vaisse et al., Diabetes, 46, 1364 (1997); M. Levinson-Dushnik et al., Mol. Cell. Biol., 17, 3817 (1997)).
- Nkx2.2 is known to be a factor expressed on the ventral side of the neural tube during development and a factor that plays an important role in the differentiation and function of these cells (M. Price et al. Neuron, 8, 241 (1992)).
- Pax-7 is localized in the dorsal part of the neural tube (B. Jostes et al., Mech. Dev., 33> 27 (1990)), and is derived from neural crest-derived tissues in the head and central nervous system differentiation. It is a factor that plays an important role in formation (A. Mansouri et al., Development, 122, 831 (1996)).
- AP-2 is expressed in neural crest cells and their major tissues, sensory ganglia in the head, spinal ganglia, and mesenchyme of the face, in mouse embryos from embryonic days 8.5 to 12.5 and, a factor which plays an important role in the differentiation and functions of these cells (H. Schorle et, Nature, 381, 235 (1996 ), J. Zhang et al., Nature, 381, 238 (1996 ))
- 0 neural crest cells Pax_3 and twist are transcription factors other than AP2 that are expressed in and involved in cranial closure. ⁇
- neural tube and neural crest cells are known for their marker genes and factors that affect the developmental direction of these cells, such as detection of marker gene mRNA, detection of expression marker gene product itself, Alternatively, cells can be identified by examining the response to the factor.
- epidermal cells examples include epidermal cells.
- the skin is composed of the epidermis of epidermis derived from ectoderm (epidemis) and the dermis of connective tissue derived from mesoderm (dermis).
- Epidermal cells are epithelial cells that constitute epidermis. Defined.
- the epidermis basically consists of keratinized stratified squamous epithelium, stratum basale (stratum basale :), stratum spinosum (stratum) spinosum) N Granule layer (stratum granulosum), Pale light layer (stratum lucidum), Stratum corneum (stratum corneum).
- Epidermal cells are classified based on cell morphology and keratin filament expression.
- Keratins 8 and 18 are expressed early in development, they are used as markers for epithelial cells in the early fetal period (RG Oshima et al., Dev. Bio., 99, 447 (1983)). Keratin 19 is used as a marker for epithelial cells in the fetus (PC Stasiak & EB Lane, Nucleic Acids Res., 15, 10058 (1987)) 0 Keratins 5 and 14 are used as markers for epithelial cells constituting the basal layer of the epidermis (E. Fuchs & E Green, Cell, 19, 1033 (1980)).
- the keratinizing epidermal cells are called keratinocytes, and the expression of keratins 5 and 14 decreases and the expression of keratins 1 and 10 increases with keratinization (E. Fuchs & H. Green, Cell, 19, 1033 (1980); C. Bagutti et al., Dev. Biol., 179, 184 (1996)).
- Identification of epidermal cells can be performed by detecting with antibodies against each of the above keratins, antibodies against E-force doherin, a marker for non-neural ectoderm cells, or by detecting mRNA of these keratin proteins. Can be.
- the method of the present invention can suitably induce differentiation into epidermal cells in the basal lamina having a high cell division ability.
- the differentiation inducing method of the present invention includes a step of preparing a single-cell state embryonic stem cell, a step of preparing the embryonic stem cell in the presence of a stromal cell or a stromal cell-derived factor, and And a method including the step of culturing.
- stromal cells are stromal cells described in 4 below, and stromal cell-derived factors are antigens described in 8 described below, factors obtained by the method described in 5 below, and stromal cells.
- To cultivate embryonic stem cells in a non-aggregated state means to start culturing in a single cell state in which the cells are released from each other (single cell) and continue culturing.
- the single-cell state refers to a state in which individual cells that have not adhered to each other by enzymatic digestion or the like have become flat.
- the seeded cells do not aggregate and do not form an amploid body.
- the cell density is lower than that used for normal embryonic stem cell passage in embryonic stem cell culture. It can be performed by seeding and culturing at a cell density. That is, treatment such as enzyme digestion is performed on embryonic stem cells, a cell suspension in a single cell state is prepared using a culture medium, and the cell suspension is kept in a state where the cells do not come into contact with the culture system. Culture to the extent possible. This culture is fundamentally different from the conventional culture method using an amplifying body, which attempts to induce differentiation by actively aggregating cells and reproducing the state of a pseudo embryo.
- the cell density of the embryonic stem cells for seeding to such an extent that they can exist in a state where the cells do not come into contact with the culture system is preferably tens to hundreds of cells / cm 2 , and more preferably 30 to N 300 cells, which are m 2.
- Examples of a method for obtaining embryonic stem cells in a single cell state include a known enzyme digestion method used in tissue cell culture. Specifically, the medium was changed the day before, the medium was removed from the culture dish in which embryonic stem cells that had grown to several tens of percent to almost confluent state were removed, and a phosphate-buffered saline solution (hereinafter “PBS”) was used. ), Several times, preferably 2-3 times. After washing, add an appropriate enzyme digestion solution (for example, PBS containing lmM EDTA and 0.25% trypsin) to the culture dish containing embryonic stem cells, and at 37 ° C for several tens of minutes, preferably for 5 to 20 minutes Incubate.
- PBS phosphate-buffered saline solution
- the cells are suspended in the medium prepared in 2 below, centrifuged (for example, at 4 ° C, 200 X g for 5 minutes), and the embryonic stem cells are suspended in the regeneration medium to obtain a single cell.
- Embryonic stem cells in a cell state can be collected.
- Methods for culturing embryonic stem cells include manipulating the mouse, umbrio 'a' hukhovtree, 'ma-yuanore'; Methods in Enzymology, volume 225, Guide to Techniques in Mouse Development, Academic Press (1993); ES cells And a method for culturing embryonic stem cells described in the above. Serum-free culture is also possible.
- KNOCKOUT TM SR (GIBC0BRL), 2raM glutamine, 100 MEM Non-Essential Amino Acids dissolved in Dulbecco MEM medium, 50 U / ml Subculture in a medium supplemented with cylin, 50 U / ml streptomycin, 100 / iM2-mercaptoethanol, and l, 000 U / ml LIF, while maintaining the characteristics of undifferentiated embryonic stem cells. (MD Goldsborough et al., Focus, 20, 8 (1998)).
- any culture method suitable for inducing differentiation of embryonic stem cells to be used can be used.
- Examples include a monolayer culture method, a co-culture method with a supporting cell, a high-density maintenance culture method, a microcarrier culture method, a reflux culture method, and a soft agar culture method.
- a method of culturing the embryonic stem cells in the single cell state in the medium prepared in 2 described below, and the embryonic stem cells in the single cell state suspended in the medium prepared in 2 described below examples include a method of co-culturing with stromal cells prepared in advance in 4 below in a non-aggregated state for several days.
- the step of culturing embryonic stem cells in a non-aggregated state according to the present invention is preferably performed under serum-free culture conditions, but is performed under serum-free culture conditions and then cultured under serum-added culture conditions. (E.g., using a medium supplemented with preferably several tens%, more preferably 5 to 20% of mammalian serum to the basal medium described in 2 below, and 37% to several%, preferably 5% carbon dioxide can also be carried out step) that cultured in aerated C0 2 incubator scratch.
- the differentiation induction rate can be further increased by including a step of culturing the cells under the culture conditions to which the serum is added.
- the ectoderm cells or cells derived from ectoderm of the present invention can be obtained.
- embryonic stem cells are induced to differentiate into ectoderm cells and cells derived from ectoderm, and 5% or more, preferably 15% or more, of the embryonic stem cells subjected to the differentiation induction method of the present invention.
- 40% or more, and more preferably 80% or more can be induced to differentiate into ectodermal cells (ectodermal cells or cells derived from ectoderm).
- a certain ectodermal cell can be cultured by continuously culturing while appropriately changing the medium by a method including the above-described steps.
- BMP4 BMP4
- the above-mentioned steps are performed using a BMP4-free medium, and embryonic stem cells begin to differentiate into neural ectoderm (E.g., 1 to 14 days, preferably 2 to 8 days, more preferably 4 to 6 days after the start of the culture), the culture can be continued while appropriately changing the medium using a medium containing shh or BMP4. It can be carried out.
- the method for inducing differentiation of the present invention it is preferable to culture embryonic stem cells in the presence of stromal cells or factors derived from stromal cells.
- the stromal cells are as described in 4 below, and the stromal cell-derived factors include the antigens described in 8 below, the factors obtained by the method described in 5 below, culture supernatants of stromal cells, and stromal cells. Cell fragment and the like.
- the ratio of stromal cells to embryonic stem cells in a culture system is such that the embryonic stem cells can be induced to differentiate into ectoderm cells or cells derived from ectoderm. Any ratio, but 10 4 to 1 to 1 (stroma (Cell number vs. embryonic stem cell number), preferably 10 3 to 1 to 1, more preferably 10 2 to 10: 1.
- co-culture of embryonic stem cells and stroma cells is performed when the embryonic stem cells and stromal cells are in physical contact or when both cells are in the same culture system. This includes cases where the cells themselves are separated by a partition capable of coming and going and physical contact of the cells themselves is not possible.
- the pore size of the filter is preferably from 0.01 to several tens 111, more preferably from 0.02 to 12 ⁇ .
- Specific examples of such a filter include membrane cultivator insert (manufactured by Iwaki Glass), NuncTC insert (manufactured by Nunc), CO-CULTURE petri dish (manufactured by Grainer), and cell culture. Insert (made by Falcon), Chemotaxis chamber I (made by Neuro Probe Inc.) and the like. Either embryonic stem cells or stromal cells can be cultured on the filter, but stromal cells are preferably cultured on the filter.
- the recovered embryonic stem cells were prepared in 2 below.
- Medium eg, Glasgow MEM medium with 10% KNOCKOUT TM SR (GIBC0BRL), 2raM glutamine, 50U / ml penicillin, 50U / ml streptomycin, 100 MEM Non-Essential Amino Acids solution, ImM pyruvate) ⁇ 100 ⁇ medium containing 2-mercaptoethanol
- a culture vessel eg, a cell culture flask
- the stromal cells prepared in 4 described later are cultured.
- cm 2 preferably seeded N 100 cells at a cell density of m 2, 5 to 20 days During preferably 7: Number% at 37 ° C for 10 days, preferably it can be mentioned a method of culturing 5% carbon dioxide at aerated C0 2 incubator scratch.
- a medium prepared by preparing the collected embryonic stem cells in step 2 For example, 10% KNOCKOUT TM SR (GIBC0BRL), 2raM glutamine, 50U / ml penicillin, 50U / ml streptomycin, 100 ⁇ MEM Non-Essential Amino Acids solution, IraM pyruvate, 100 ⁇ L in Glasgow MEM medium ⁇ 2-mercaptoethanol and 0 ⁇ : 100 to 100 ng / ml, preferably 1 to 50 ng / ml in a medium supplemented with BMP4), and incubator in which the stromal cells prepared in 4 below are cultured.
- 10% KNOCKOUT TM SR GBC0BRL
- 2raM glutamine 50U / ml penicillin
- 50U / ml streptomycin 100 ⁇ MEM Non-Essential Amino Acids solution
- IraM pyruvate 100 ⁇ L in Glasgow MEM medium ⁇ 2-mercap
- 0P9 cells T. Nakano et al., Science, 272, 722 (1996)
- NIH / 3T3 cells in a medium for culturing embryonic stem cells.
- a medium supplemented with a culture medium of MC3T3-G2 / PA6 cells to convert embryonic stem cells to ectodermal cells.
- Cells derived from ectoderm can also be induced.
- 0P9 cells instead of co-culturing embryonic stem cells and stroma cells, 0P9 cells, NIH / 3T3 cells, MC3T3-G2 / PA6 cells (H. Kodama et al., J. Cell. Physiol., 112, 89 (1982)), ST0 cells (G. Martin, Proc. Natl. Acad. Sci.
- the step of culturing embryonic stem cells in a non-aggregated state it is preferable to culture without using retinoic acid.
- culturing without using retinoic acid means culturing without using a nonphysiological concentration of retinoic acid.
- the non-physiological concentration means a concentration that is at least 10 times the concentration that is physiologically present in a living body. Specifically, it is known that retinoic acid at a concentration of about 10-3 ⁇ 4ol / l is usually present in the blood of humans (Biochemical Dictionary 2nd edition, Tokyo Kagaku Dojin (1992)). , 10 one 7 ⁇ 1 ( ⁇ ⁇ 1 / 1 concentration range is non-live reasonable concentration.
- retinoic acid has an effect as affecting shaped state forming substance morphogenesis during development and differentiation (morphogen)
- morphogen a substance that influences the cell type.
- it is highly toxic, and there are concerns about secondary side effects when a culture system using non-physiological concentrations of retinoic acid is applied to medical treatment. Culturing without using is useful because the risk associated with the use of retinoic acid described above can be avoided.
- the differentiation of mesodermal cells is not substantially induced in the culture system.
- the mesodermal cells refer to cells constituting organs and tissues such as the muscular system, connective tissue, skeletal system, circulatory system, urinary system, and reproductive system.
- Mesoderm cells can be identified by detecting them with an antibody that specifically recognizes mesodermal cells.Detect mRNA of a protein specifically expressed in mesodermal cells The method can be carried out using a method, a method of detecting the protein using an antibody that specifically recognizes the protein, and the like.
- any incubator capable of culturing embryonic stem cells can be used, but an incubator used for cell culture is preferable.
- an incubator for cell culture include a flask, a flask for tissue culture, a dish, a petri dish, a dish for woven culture, a dish, a Normanox tissue, a manoletitette, a microplate, a microplate, and a microwellette plate. , Manoreti plate, Manorechiuenoref.
- the surface of the incubator on the side in contact with the cells can be artificially treated.
- artificially treating the surface of the incubator include collagen coat, gelatin coat, poly-L-lysine coat, fibronectin coat, laminin coat, oral teoglycan coat, glycoprotein coat, matrigel coat, and silicone. And the like. 'Also, it can be processed to have a negative charge such as a primary door (Falcon).
- the medium used in the method of the present invention for inducing differentiation of ectoderm cells and cells derived from ectoderm from embryonic stem cells can be prepared using a medium used for culturing animal cells as a basal medium.
- a basal medium BME medium (Proc. Soc. Exp. Biol. Med., 89, 362 (1965)), BGJb medium (Exp. Cell Res., 25, 41 (1961)), CMRL 1066 medium (NY Academy of Sciences, 5, 303 (1957)), Glasgow MEM medium (Virology, 16, 147 (1962)), Improved MEM Zinc Option medium (J.
- IMDM medium In Vitro, 9, 6 (1970)
- Medium 199 medium Proc. Soc. Exp. Biol. Med., 73, 1 (1950)
- Eagle MEM medium Science, 130, 432 (1959)
- Alpha MEM medium (Nature New Biology, 230, 310 (1971)
- Dulbecco MEM medium (Virology, 8, 396 (1959))
- Ham medium Exp. Cell Res., 29, 515 (1963); Proc. Natl. Acad. Sci. USA, 53, 288 (1965)
- RPMI 1640 medium JAMA, 199, 519 (1967)
- Fischer's medium Methodhods in Med.
- McCoy's medium Proc. Soc. Exp. Biol. Med., 100, 115 (1959)
- Williams E medium Exp. Cell Res., 69, 106 (1971); Exp. Cell Res., 89, 139 (1974)
- these Mixed media It can be used, so long as the medium can have use for the cultivation of animal cells.
- basal media are media containing various growth factors as serum substitutes, media supplemented with factors produced by stromal cells, etc., or protein-free media. Any of them can be used as a culture medium of the present invention. Specific examples include a serum-free medium supplemented with commercially available KNOCKOUT TM SR (MD Goldsborough et al., Focus, 20, 8 (1998)), and a serum-free medium supplemented with insulin and transferrin (eg, CH0-S- SFM II (GIBCOBRL), Hybridoma-SFM (GIBCOBRL), eRDF Dry Powdered Media (GIBCOBRL), UltraCULTURE TM (BioWhittaker), UltraDOMA TM (BioWhittaker), UltraCHO TM (BioWhittaker), UltraMDCK TM (BioWhittaker)), ITPSG medium (S.
- KNOCKOUT TM SR MD Goldsborough et al., Focus, 20, 8 (1998)
- insulin and transferrin eg, CH0-S- S
- a medium containing a factor produced by stromal cells, a medium containing an antigen component obtained in step 8 described later, a medium containing BMP4, or a protein-free medium for example, CD-CH0 (manufactured by GIBCO BRL), PFHM-II (GIBCOBRL) and UltraDOMA-PF TM (BioWhittaker).
- development is started using a method of injecting into a non-fertilized egg of the enucleated mammal, By culturing the eggs that have started to develop, eggs that have nuclei of other somatic cells and that have started to develop normally can be obtained.
- a plurality of methods are known as methods for initializing the nucleus of somatic cells.
- the following methods are known.
- the medium in which the cells providing the nucleus are cultured is removed from a medium containing 5-30%, preferably 10% fetal calf serum (eg, M2 medium) for 3-10 days, preferably 5 days.
- a medium containing 5-30%, preferably 10% fetal calf serum eg, M2 medium
- an oligotrophic medium containing 0-1%, preferably 0.5% fetal calf serum, and culturing the cells the cell cycle is induced to a quiescent state (GO phase or G1 phase).
- GO phase or G1 phase can be This method is suitable when the mammal is, for example, a sheep, a goat, or a sea lion.
- the nucleus of the cell that provides the nucleus can be initialized by injecting the nucleus of the cell that provides the nucleus into an enucleated unfertilized egg of the same mammal and incubating it for several hours, preferably about 1 to 6 hours.
- This method is suitable when the mammal is, for example, a mouse.
- the reprogrammed nuclei are able to start developing in enucleated unfertilized eggs.
- Several methods are known for initiating development of reprogrammed nuclei in enucleated unfertilized eggs.
- the cell cycle is induced to a quiescent state (GO phase or G1 phase), and the initialized nucleus is transplanted to an enucleated unfertilized egg of the same mammal by electrofusion, etc. to activate the egg and start development. Can be done.
- This method is suitable when the mammal is, for example, a sheep, a goat, or a sea lion.
- the nuclei initialized by injecting nuclei into enucleated unfertilized eggs of the same mammal are transplanted to enucleated unfertilized eggs of the same mammal again by a method using a microphone mouth manipulator, etc.
- an activator eg, strontium
- development can be initiated by treating with a cell division inhibitor (eg, cytochalasin B) to suppress the release of the second polar body.
- cytochalasin B cytochalasin B
- embryonic stem cells in which genes on chromosomes have been modified can be produced.
- the gene on the chromosome to be modified includes a histocompatibility antigen gene, a disease-related gene based on damage to neural cells or epidermal cells, and the like.
- Modification of the target gene on the chromosome can be performed using the methods described in Manipulating, The Mouse, Laboratory, Manual, Gene, Targeting, Generating Mutant Mice Using ES Cells, etc. it can.
- a genomic gene of a target gene to be modified for example, a histocompatibility antigen gene or a disease-related gene
- homologously recombine the target gene using the isolated genomic gene Is prepared.
- the resulting target vector is introduced into embryonic stem cells, and cells that have undergone homologous recombination between the target gene and the target vector are selected to produce sex stem cells with modified chromosomal genes. be able to.
- Methods for isolating the genomic gene of the target gene include Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as "Molecular 'Clawing 2nd edition')" and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter
- the genomic gene of the target gene can be isolated by using a genomic DNA library screening system (Genome Systems) or Universal GenomeWalker TM Kits (CL0NTECH).
- the target vector for homologous recombination of the target gene can be prepared according to the method described in Gene targeting; Preparation of mutant mouse using ES cells.
- As the target vector either a replacement type or an insertion type can be used.
- Methods for efficiently selecting homologous recombinants include, for example, gene targeting; methods such as positive selection, promoter selection, negative selection, and poly-A selection described in Generating mutant mice using ES cells. Can be used. Methods for selecting the desired homologous recombinant from the selected cell lines include the Southern hybridization method (molecular cloning, second edition) for genomic DNA and the PCR method (PGR Protocols, Academic Press ( 1990)).
- the stromal cells used in the method for inducing differentiation of ectoderm cells and ectoderm-derived cells from embryonic stem cells include: Any of them can be used as long as it has an inducing activity.
- the antibody of the present invention produced by the method described later in Stromal cells recognized by the monoclonal antibody produced by the hybridoma FERM BP-7573 obtained in Example 15 (5) are preferably used.
- the monoclonal antibody produced by the hybridoma FERM BP-7573 obtained in Example 15 (5) are preferably used.
- the stromal cells of the above (c), (d) and (e) are preferable, and the stromal cells of the above (e) are more preferable.
- stromal cells recognized by the monoclonal antibody produced by the hybridoma FERM BP-7573 obtained in Example 15 (5) are described in the literature (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., 1995; Enzyme Immunological assay described in Immunological Assay, 3rd Edition, Medical Shoin, 1987). Enzyme-antibody method is identified by a method such as cell separation using a flow cytometer.
- the stromal cells are preferably subcultured using the method used when they were established.
- the method of enzymology Volume 225, Guide to Techniques in Mouse Development, Academic Press (1993);
- the method for culturing feeder cells used for culturing embryonic stem cells described in Preparation etc. can also be used.
- Dulbecco MEM medium manufactured by GIBC0BRL
- Culture can be performed using a medium supplemented with 2 mM glutamine, 50 U / ml penicillin, and 50 U / ml streptomycin.
- the stromal cells grown on a suitable support such as a culture dish are left alive.
- the cells may be used, or cells that have lost the ability to proliferate due to physicochemical treatment may be used.
- Cells that have lost the ability to grow due to physicochemical treatment are cells that have lost the ability to form next-generation progeny with gene replication.
- a medium containing an anticancer drug was used. These cells are obtained by culture, irradiation with a lethal dose, or treatment for tissue fixation used for pathological diagnosis.
- the stromal cells that are still alive can be obtained, for example, by changing the culture medium the day before and washing the cells whose cell density has reached a nearly confluent state several times with PBS.
- Germ cells can be prepared by adding ectoderm cells and cells derived from ectoderm from serum stem cells. In addition, cells that have almost reached confluence are digested with an appropriate digestive enzyme (e.g., PBS containing 0.02% EDTA and 0.05 to 0.25% trypsin or actinase), and collected.
- an appropriate digestive enzyme e.g., PBS containing 0.02% EDTA and 0.05 to 0.25% trypsin or actinase
- the resulting medium of the present invention (eg, embryonic stem cells)
- the cells are suspended in a serum-free medium used for culturing the ectoderm cells and cells derived from the ectoderm from the vesicles, and then cultured in an incubator (for example, coated with 0 to:!%, Preferably 0.1% gelatin). It can also be prepared by seeding the cells on a cultured tissue culture dish) and culturing for about 1 day.
- Stromal cells that have lost their proliferative ability by culturing in a medium containing an anticancer agent can be transformed into mice by using the following methods: Mapurating The Mouse Embrio Laboratory Laboratory Gene Gene Targeting; Generating mutant mice using ES cells It can be prepared using the method described in, for example.
- cells reached approximately Konfuruento state cell density perform medium change on the day before, 1 ⁇ 100 ⁇ ⁇ / ⁇ 1, several hours in a medium preferably containing mitomycin C concentration of 10 mu g / ml, preferably Incubate for 2-3 hours, wash several times with PBS, and digest with appropriate digestive enzymes (e.g., PBS containing 0.02% EDTA and 0.05-0.25% trypsin or actinase).
- the cells are collected and suspended in the medium of the present invention obtained in the above 2 (for example, a serum-free medium used for culture of inducing differentiation of ectoderm cells and cells derived from ectoderm from embryonic stem cells). , 0-1%, preferably 0.1% gelatin).
- an anticancer drug such as 5-fluoroperacil, adoriamysin, faraC or methotrexate, is preferably used at a concentration 1/10 to 10 times the concentration described in the Japanese Pharmacopoeia for use in a living body. Can be used to prepare stromal cells that have lost the ability to grow.
- Stroma cells that have lost their proliferative ability due to lethal dose of radiation can be obtained from tissue culture technology, Asakura Shoten (1982), tissue culture technology (second edition), Asakura Shoten (1988), tissue culture It can be prepared by using the method described in, for example, the technique of the Third Edition, Asakura Shoten (1996). For example, cells whose medium density has reached a nearly confluent state the day before the medium was exchanged, were irradiated with 200 to 5,000 rads, preferably 500 to 1,000 rads of X-rays or ⁇ -rays, and the cells were counted with PBS. After washing twice, the culture medium of the present invention obtained in 6 described later is used.
- a medium for example, a serum-free medium used for culture for inducing differentiation of ectoderm cells or cells derived from ectoderm from Pi stem cells.
- a medium for example, a serum-free medium used for culture for inducing differentiation of ectoderm cells or cells derived from ectoderm from Pi stem cells.
- the irradiated cells are digested with an appropriate digestive enzyme (for example, PBS containing 0.02% EDTA and 0.05 to 0.25% trypsin or actinase) and collected, and collected as described below.
- an appropriate digestive enzyme for example, PBS containing 0.02% EDTA and 0.05 to 0.25% trypsin or actinase
- the cells After suspending in the medium of the present invention obtained in step 6 (for example, a serum-free medium used for culturing to induce differentiation of ectodermal cells or cells derived from ectoderm from embryonic stem cells), the cells are cultured in an incubator (for example, 0 to 1%, (Tissue culture dish, preferably coated with 0.1% gelatin), and cultured for about 1 day.
- an incubator for example, 0 to 1%, (Tissue culture dish, preferably coated with 0.1% gelatin
- Stroma cells that have lost their reproductive ability due to the tissue fixation procedure used for pathological diagnosis are edited by the Japan Society for Histocytochemistry and published annually, Histocytochemistry, Interdisciplinary Planning (1987 1999), Basic Techniques For example, it can be prepared using the method described in Transmission Electron Microscopy, Acad. Press (1986), Electron Microscope Chart Manual, Medical Publishing Center (1993), and the like. Concretely, by performing microphone mouth wave fixation, quick freeze displacement fixation, daltal aldehyde fixation, paraformaldehyde fixation, formalin fixation, acetone fixation, Bouin fixation, periodate fixation, methanolic fixation, or osmium acid fixation Can be prepared.
- cells that have undergone a medium change the previous day and have reached a cell density of approximately confluent at 4 ° C. can be 0.1 to 50%, preferably 1 to 10%, more preferably 3 to 5%. It can be prepared by, for example, infiltrating into a solution containing balaformaldehyde for several minutes to several hours, preferably 5 minutes to 1 hour, more preferably 30 minutes, and washing several times with PBS.
- stromal cells of the present invention From the stromal cells of the present invention, it is possible to obtain a factor having an activity of inducing the differentiation of ectoderm-derived cells from embryonic stem cells. More specifically, Rad. Cloning 2nd Edition and Ryrent. Protocols in 'Molequira I. Nobiology, Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press (1993) (hereinafter referred to as' monoclonal 'antipodes' Abbreviation), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press (1996) (hereinafter, abbreviated as “antibody 'engineering”). it can.
- a cDNA is prepared from the stromal cells of the present invention, and the cDNA is inserted downstream of a promoter of an appropriate expression vector to prepare a recombinant vector, thereby preparing a cDNA library.
- a transformant that produces a gene product produced by the stromal cell of the present invention is obtained, and cells derived from ectoderm from embryonic stem cells are obtained.
- a transformant producing a gene product having an activity of inducing differentiation is selected.
- By determining the gene sequence encoded by the cDNA introduced into the selected transformant it is possible to obtain a factor having an activity of inducing differentiation of ectoderm-derived cells from embryonic stem cells.
- CH0 cells derived from Chinese hamster ovary TT Puck et al., J. Exp. Med., 108, 945 (1985)
- MDCK cells derived from Cocka spaniel female dog kidney ⁇ R. Gaush et al., Proc. Soc. Exp. Biol. Med., 122, 931 (1966)
- DS Misfeldt et al. Proc. Natl. Acad. Sci. USA, 73, 1212 (1976)
- rat fibroblast 3 ⁇ S. Sandineyer et al. Cancer Res., 41.
- COS cells suitable for expression cloning using an SV40-based expression vector are more preferable.
- Stromal cells having an activity of inducing differentiation of ectoderm-derived cells from embryonic stem cells are preferable as cells used for cDNA production. Specific examples include fetal primary cultured fibroblasts (manipulating 'the' mouse 'embryo'a' laboratory 'manual; production of mutant mice using ES cells), ST0 cells derived from SIHM mice (G. Martin, Natl. Acad. Sci.
- NIH / 3T3 cells derived from mouse embryos JL Jainchill et al., J. Virol , 4, 549 (1969)
- 0P9 cells derived from M-CSF-deficient mouse calvaria T. Nakano et al., Science, 272, 722 (1996)
- MC3T3-G2 / PA6 cells derived from mouse calvaria H. Kodama et al., J. Cell. Physiol., 112, 89 (1982)).
- the prepared cDNA library may be used as it is, but in order to enrich the target gene, mRNA of cells that do not have the activity to induce differentiation of ectodermal cells from embryonic stem cells is used.
- a cDNA library prepared by the subtraction method (Proc. Natl. Acad. Sci. USA, 85, 5783 (1988)) can also be used.
- Examples of a method for introducing a recombinant vector, a method for obtaining a transformant, and a method for culturing the obtained transformant in a medium include the method described in the following item 8.
- a transformant producing a gene product having an activity of inducing differentiation of ectoderm-derived cells from embryonic stem cells is selected by co-culturing embryonic stem cells and transformants. can do.
- Methods for isolating the cDNA introduced into the selected transformant and for determining the gene sequence of the isolated cDNA include the method described in 8 below.
- a stromal cell-derived factor having an activity of inducing differentiation of ectoderm-derived cells from embryonic stem cells can be obtained.
- the stromal cells of the present invention were used as a starting material and added to the medium.
- Purification can be performed using the promoting effect of inducing differentiation of ectoderm-derived cells from embryonic stem cells at that time as an index. Examples of the purification method include the method described in 8 below.
- stromal cell-derived factors have the property of adsorbing to mucopolysaccharides (eg, heparin)
- embryonic stem cells can be cultured in a stromal cell culture system or in the presence of stromal cells.
- the factor in a culture system cultured in a non-aggregated state is bound to mucopolysaccharide, and then the factor can be obtained from the stromal cell-derived factor bound to mucopolysaccharide.
- stromal cell-derived factors are bound to heparin using column chromatography using heparin as a carrier, and then the bound factors are eluted, and the factors are obtained using the differentiation induction promoting effect as an index. Can be.
- the differentiation-inducing agent of the present invention may be in any form as long as it contains the above-mentioned stromal cell or a stromal cell-derived factor as an active ingredient. Specifically, there may be mentioned those containing a medium capable of culturing Pi stem cells and stromal cells and the above-mentioned stromal cells. In addition, the above-mentioned cell fragments of stromal cells and culture supernatants are also examples of the active ingredient of the differentiation inducer.
- the culture supernatant of the above-mentioned stromal cells is obtained by culturing the above-mentioned stromal cells having the activity of inducing the differentiation of embryonic stem cells into ectodermal cells and cells derived from ectoderm in a medium.
- mucopolysaccharides that have the ability to bind to factors produced by various stromal cells and that have an activity to induce the differentiation of embryonic stem cells into ectoderm cells and cells derived from ectoderm are used. Anything can be used, For example, paparin is preferred. As heparin, commercially available known heparin can be used. The above culture supernatant is prepared by culturing the above stromal cells for 5 minutes to several days using a medium containing heparin in the range of several ng / ml to several thousand ng / ml, preferably several hundred ng / ml. can do.
- the culture supernatant thus obtained is used to induce differentiation from embryonic stem cells into ectoderm cells and ectoderm-derived cells, and from embryonic stem cells to ectoderm cells and ectoderm-derived cells. It can be used effectively when obtaining an active factor that induces differentiation into cells.
- mucopolysaccharides such as paparin
- an active factor that induces differentiation of embryonic stem cells into ectoderm cells and cells derived from ectoderm.
- Evaluation and screening methods for substances involved in the regulation of the process of differentiation from embryonic stem cells to ectoderm cells or ectoderm-derived cells, or the regulation of the function of ectoderm cells or ectoderm-derived cells Evaluation method of substances to be used (1) It can be used effectively for the staring method
- BMP4 is preferably contained as an agent for inducing differentiation of ectodermal cells into epidermal cells.
- the agent for inducing differentiation of the present invention can be used as a therapeutic agent for diseases based on disorders of cells derived from ectoderm.
- Examples of the disease based on damage to cells derived from ectoderm include a disease based on damage to nervous system cells and a disease based on damage to epidermal cells.
- Diseases based on nervous system disorders include Alzheimer's disease, Huntington's chorea, Parkinson's disease, ischemic brain disease, epilepsy, brain trauma, spinal cord injury, motor neuropathy, neurodegenerative disease, retinitis pigmentosa, inner ear Deafness, multiple sclerosis, amyotrophic lateral sclerosis, diseases caused by neurotoxic damage, etc., and diseases based on damage to epidermal cells include burns, trauma, wound healing, floor rubbing, psoriasis, etc. can give.
- the medicament containing the differentiation-inducing agent of the present invention as an active ingredient can be administered alone, but usually the active ingredient is one or more pharmacologically acceptable carriers.
- a sterile solution dissolved in water or an aqueous carrier such as an aqueous solution of salt, glycine, glucose, human albumin or the like is used.
- pharmacologically acceptable additives such as buffering agents and tonicity agents for bringing the formulation solution closer to physiological conditions, for example, sodium acetate, sodium salt, sodium lactate, salt Idani potassium, sodium citrate and the like can also be added.
- it can be stored by lyophilization and dissolved in an appropriate solvent before use.
- Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
- Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
- liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, and fructose, dalicols such as polyethylene glycol and propylene glycol, and oils such as sesame oil, olive oil, and soybean oil.
- preservatives such as P-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives.
- excipients such as lactose, pudose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polybutyl It can be produced using a binder such as alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
- Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
- an injection is prepared using a carrier such as a salt solution, a pudose solution, or a mixture of both.
- Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids.
- a spray is prepared using the active ingredient itself or a carrier which does not irritate the oral or airway mucosa of the recipient and which disperses the active ingredient as fine particles to facilitate absorption.
- the carrier include lactose and glycerin.
- Formulations such as aerosols and dry powders can be made depending on the properties of the active ingredient and the carrier used. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
- stromal cells of the present invention By using the stromal cells of the present invention as an immunogen, antibodies that recognize the stromal cells of the present invention, such as polyclonal antibodies and monoclonal antibodies, can be prepared. There have been many reports of using cells as immunogens and obtaining cell surface-specific antibodies of the cells used as immunogens and antigen molecules recognized by the antibodies (N. Itoh et al., Cell, 66, 233 (1991)), many cell surface antigen molecules have been identified and are known as CD antigens.
- a polyclonal antibody can be prepared by using the stromal cells used in the present invention as an immunogen and administering them to animals.
- Animals to be administered include egrets, goats, 3-20 week old rats, mice, hamsters and the like.
- the dose of the immunogen is preferably 10 4 to 10 8 cells per animal or 0.01 to 10 mg of a cell membrane fraction prepared from the cells.
- the immunogen is administered subcutaneously or intraperitoneally together with an appropriate adjuvant (for example, Complete Freund's Adjuvant or aluminum hydroxide gel, perennial E. coli vaccine, etc.).
- an appropriate adjuvant for example, Complete Freund's Adjuvant or aluminum hydroxide gel, perennial E. coli vaccine, etc.
- the administration of the immunogen is performed 3 to 10 times every 1 to 2 weeks after the first administration.
- Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the immunogen used for immunization is determined by enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay (ELISA): published by Medical Shoin). 1976), Antibodies—A Laboratory Manual, old Spring Harbor Laboratory (1988) (hereinafter abbreviated as “Antibody's Laboratory Manual”), or a method using a flow cytometer (monoclonal. Antibody's). ) Confirm with.
- the cell membrane components of cells used as immunogens can be prepared using many known methods. For example, the method of Jones et al. (DH Jones et al. and AL Matus, Biochim. Biophys. Acta., 356, 276 (1974)), and an enzyme immunoassay can be performed using a plate coated with this cell membrane component.
- a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum. Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation (Antibodies 'Laboratories' manual), DEAE-Sepharose column, anion exchange column, There is a method in which chromatography using a protein A or G-column or a gel filtration column or the like is performed alone or in combination.
- a mouse or rat whose serum shows a sufficient antibody titer against the stromal cells of the present invention used for immunization is used as a source of antibody-producing cells.
- the spleen is removed.
- the spleen is shredded in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
- MEM medium manufactured by Nissui Pharmaceutical Co., Ltd.
- the spleen cells in the resulting precipitate fraction are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, washed three times with MEM medium, and the resulting spleen cells are used for antibody production. Use as cells.
- myeloma cells cell lines obtained from mice or rats are used.
- 8-azaguanine-resistant mouse derived from BALB / c
- myeloma cell line P3_X63Ag8-U1 (hereinafter abbreviated as “P3-U1”) (Curr. Topics. Microbiol. Immunol., 81, 1 (1978)) , Europ J. Immunol, 6, 511 (1976)), SP2 / 0- A g 14 (SP2) (Nature, 276, 269 (1978)), P3-X63 -.. Ag8653 (653) (J.
- the suspension is dispensed at 100 ⁇ l / well into a 96-well culture plate, and cultured at 37 ° C. for 7 to 14 days in a 5% CO 2 incubator.
- a part of the culture supernatant is taken and subjected to the enzyme immunoassay described in Antibody's' A 'Laboratory' Mayuaru, etc., or the flow cytometer described in Monoclonal Antibodies, etc.
- the enzyme immunoassay described in Antibody's' A 'Laboratory' Mayuaru, etc.
- the flow cytometer described in Monoclonal Antibodies etc.
- a hybridoma that specifically reacts with the stromal cells in the present invention is selected. The following method can be mentioned as a specific example of the enzyme immunoassay.
- the cell membrane fraction prepared from the stromal cells of the present invention used as the immunogen was coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below was used as the primary antibody.
- an enzyme, a chemiluminescent substance or a radioactive compound as a second antibody, the reaction according to the labeling substance is carried out.
- the cells used as the immunogen were reacted with the hybridoma supernatant or the purified antibody obtained in (d) below as the first antibody, and the second antibody was labeled with biotin or a fluorescent substance.
- the antibody was reacted and a biotin-labeled second antibody was used, it was further reacted with fluorescently-labeled avidin, and the presence or absence of staining was confirmed using a flow cytometer such as FACS.
- Those which specifically react with cells are selected as hybridomas producing the monoclonal antibody of the present invention.
- the ascites is collected from the mouse with ascites tumor, and the solid is removed by centrifugation at 3, OOO rpm for 5 minutes.
- a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
- the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
- the protein content is calculated by the Lowry method or from the absorbance at 280 nm.
- Examples of the monoclonal antibody obtained by the above method include a monoclonal antibody produced by the hybridoma FERM BP-7573 obtained in Example 15 (5).
- the antibody obtained using the stromal cells used in the present invention obtained in 4 above as an immunogen recognizes many antigen molecules present on the cell surface. Then, using the obtained antibody, an antigen molecule recognized by the antibody can be obtained.
- the expression cloning can be performed using the expression cloning method described in Molecular Cloning, 2nd Edition, Current Protocols' In 'Molecular' Biology, Monoclonal 'Antibody's, Antibody Engineering', etc. Can be.
- a cDNA is prepared from the stromal cells of the present invention, and the cDNA is inserted downstream of a promoter of an appropriate expression vector to prepare a thread-replacement vector to prepare a cDNA library.
- a transformant producing the gene product produced by the stromal cells of the present invention is obtained, and the transformant producing the gene product of the present invention is obtained.
- a transformant producing the corresponding gene product is selected.
- a host cell a phage, a bacterium, a yeast, an animal cell, an insect cell, a plant cell, and the like can be used as long as they can express a target gene.
- the expression vector those which can replicate autonomously in the above host cells or can be integrated into the chromosome, and which contain a promoter at a position where the DNA encoding the antigen molecule of the present invention can be transcribed are used.
- total RA is prepared from the stromal cells of the present invention.
- the methods include guanidine thiocyanate-cesium trifluoroacetate method (Methods in Enzymology, 154, 3 (1987)) and guanidine acid thiocyanate phenol.chloroform (AGPC) method (Analytical Biochemistry, 162, 156 (1987)). 1987), Laboratory Medicine, £ (1937 (1991)), etc.
- mRNA can be prepared by using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or Quick Prep mRNA Purification Kit (manufactured by Pharmacia). .
- Methods for preparing a cDNA library from the prepared stromal cell mRNA include the methods described in Molecular Clawing, Second Edition, Ryrent, Protocols 'in' Molecular Biology, etc., or a commercially available kit, for example, superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Life Technologies), and ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE).
- a vector for preparing a cDNA library any phage vector, plasmid vector, or the like can be used as long as it can replicate autonomously in a microorganism such as E. coli K12 and can express the introduced cDNA in a host cell. .
- a transformant into which the prepared cDNA has been introduced can be obtained, for example, by using a commercially available kit Recombinant Phage Antibody System (Pharmacia).
- the recombinant vector containing the prepared cDNA can replicate autonomously in the prokaryote, and at the same time, a promoter, a ribosome binding sequence, a cDNA gene, and a transcription termination sequence. It is preferable that the vector is composed of: It may contain a gene that controls the promoter.
- expression vectors for example, pBTrp2, pBTacl, pBTac2 (all manufactured by Beri down Gar Mannheim), pKK233-2 (Pharmacia, Inc.), P SE280, pSE380, pSE420 (Invitrogen Corp.), P AX, pMEX (MOB I TEC), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 (Agricultural Biological Chemistry, 48, 669 (1984)), pLSAl (Agric) Biol. Chem., 53, 277 (1989)), pGELl (Proc. Natl. Acad. Sci.
- Any promoter can be used as long as it can be expressed in a host cell.
- promoters such as a promoter in which two p trps are connected in series (P trp X 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.
- a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
- Host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Previpacterium, Corynepacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XLl-Blue N Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.
- Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.
- a method using calcium ions Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)
- protoplast method JP 63-248394
- yeast When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like.
- Any promoter can be used as long as it can be expressed in yeast strains.
- Examples thereof include promoters for glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.
- Examples of the host cell include microorganisms belonging to the genera Saccharomyces, Krybetia spp., Trichosporon, Schuniomyces, and the like.
- any method for introducing a recombinant vector any method can be used as long as it is a method for introducing DNA into yeast.
- Examples of the method include an elect-portion method (Methods. Enzymol., 194, 182 (1990)), and spheroplast. Natl. Acad. Sci. USA, 84 (1929 (1978)), lithium acetate method (J. Bacteriology, 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 (Proc. 1978) The methods described in this section can be improved.
- expression vectors include pcDNAI, pCDM8 (Funakoshi), pAGE107 (JP-A-3-22979; Cytotechnology, 3, 133, (1990)) and pAS3-3 (special).
- any promoter can be used as long as it can be expressed in animal cells.
- the promoter of the cytomegalovirus (CMV) IE (immediate early) gene the early promoter of SV40, the promoter of retroinoles, Examples include the meta mouth thionein promoter, the heat shock promoter, the SRa promoter, and the like.
- the enhancer of the IE gene of human CMV may be used together with the promoter.
- host cells examples include Namalwa cells, which are human cells, COS cells, which are monkey cells, CH0 cells, which are Chinese hamster cells, and HBT5637 (JP-A-63-299). it can.
- Any method for introducing a recombinant vector can be used so long as it is a method for introducing DNA into animal cells.
- the elect-portion method (Cytotechnology, 3, 133 (1990))
- the calcium phosphate method (special method) Kaihei 2-227075)
- the ribofusion method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).
- the recombinant gene transfer vector and the paculovirus are co-transfected into insect cells to obtain the recombinant virus in the culture supernatant of the insect cells, and the recombinant virus is further transmitted to the insect cells to express the protein.
- the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitorogen) and the like.
- the baculovirus for example, Autographa californica nuclear polynedrosis virus, which is a virus infecting night moth insects, can be used, such as Autographa californica, nucleus, polyhedrosis, and wi ⁇ / s. .
- Insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)) which are ovarian cells of Spodoptera frugiperda, and High 5 (manufactured by Invitrogen) which is an ovarian cell of Trichoplusiani. Etc. can be used.
- Examples of a method for co-transferring the above-mentioned recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the ribofusion method ( Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).
- expression vectors include Ti plasmid, tobacco mosaic virus vector and the like. Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the geneactin 1 promoter.
- CaMV cauliflower mosaic virus
- Examples of the host cell include plant cells of tobacco, potato, tomato, carrot, soybean, abrana, alfa alfa, rice, wheat, wheat, and the like.
- Any method for introducing a recombinant vector can be used so long as it is a method for introducing DNA into plant cells.
- the Agrobacterium method Japanese Patent Application Laid-Open Nos. 59-140885, -70080, W094 / 00977
- Electroporesis The Yon method JP-A-60-251887
- a method using a particle gun (gene gun) Japanese Patent No. 2517813
- Japanese Patent No. 2517813 Japanese Patent No. 2517813
- the gene product encoded by the introduced cDNA can be expressed by culturing the thus obtained transformant in a medium.
- the method for culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
- a culture medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture C.
- the carbon source may be any one that can be assimilated by the organism; glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or hydrolyzed starch, organic acids such as acetic acid, propionic acid, and the like. Alcohols such as ethanol and propanol can be used.
- Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, peptone, meat extract, yeast Extracts, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermenting cells and digests thereof can be used.
- inorganic salts use is made of dipotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, man sulfate, copper sulfate, calcium carbonate, etc. be able to.
- the culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
- the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
- the culture maintain the pH between 3.0 and 9.0.
- the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like. If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
- an inducer may be added to the medium, if necessary.
- an inducer may be added to the medium, if necessary.
- indoleacrylic acid or the like may be added to the medium.
- culture media for culturing the transformants obtained using animal cells as a host commonly used RPMI1640 media (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM media (Science, 122 , 501 (1952)), Dulbecco's modified MEM medium (Virology, 8, 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum etc. It is possible to use a medium or the like that has been added. Culture is carried out usually pH6 ⁇ 8, 30 ⁇ 40 ° C, 5% C0 2 present 1 to 7 days under conditions such as lower. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
- TNM_ra medium As a medium for culturing transformants obtained using insect cells as a host, generally used TNM_ra medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCe 11405 (all Jrace Biosciences), Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) and the like can be used.
- Cultivation is usually carried out under conditions of pH 6 to 7, 25 to 30 ° C, etc. for 1 to 5 days.
- an antibiotic such as gentamicin may be added to the medium during the culturing.
- a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a cell organ of a plant.
- a medium for culturing the transformant commonly used Murashige's and Sturg (MS) medium, white (White) medium, or a plant hormone such as auxin or cytokinin is added to these mediums. The added medium and the like can be used.
- the cultivation is usually performed at pH 5 to 9 and 20 to 40 ° C for 3 to 60 days.
- an antibiotic such as kanamycin or hygromycin may be added to the medium during the culture.
- a transformant expressing the gene product encoded by the cDNA can be obtained.
- Methods for selecting a transformant that produces a gene product that reacts with the antibody of the present invention include the following: Antibodies' a 'Laboratory' manual, monoclonal 'antibody', antibody'engineering, enzyme immunoassay, Enzyme immunoassay described in 3rd ed., Medical Shoin (1987) et al., Antibody's Laboratories. Maeyua Monoclonal. Antibodies, Antibody Engineering, Int. Immunol., 10 , 275 (1998), Exp.Heraatol., 25, 972 (1997), etc., or a method using a monochromator ⁇ /. Antipodis, antibody engineering, J.
- the cDNA to be introduced into the host cell is pooled into a group consisting of a plurality of types (for example, 100 to 1000 types), and a group that gives the desired transformant Can be isolated by repeating pooling into groups consisting of even fewer (eg, 10-100) cDNAs.
- the nucleotide sequence of the isolated cDNA was analyzed from the end by a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)) or ABIPRISM377 DNA
- the base sequence of the DNA can be determined by performing analysis using a base sequence analyzer such as the one (manufactured by PE Biosystems).
- an antigen molecule recognized by the antibody of the present invention can be obtained by using the expression closing method.
- cells that do not have the activity of inducing differentiation of embryonic stem cells into cells derived from ectoderm are used as host cells, and cDNA prepared from the stromal cells of the present invention is introduced and expressed.
- Expression cloning techniques selected using an antibody that recognizes cells can be mentioned.
- Cells that do not have the activity of inducing differentiation of germ stem cells from germ stem cells include CH0 cells from Chinese hamster ovary (TT Puck et al., J. Exp. Med., 108> 945 (1985) ), African green monkey kidney-derived COS cells (Y. Gluzman, Cell, 23, 175 (1981)), and Cocka spaniel female dog kidney-derived MDCK cells (CR Gaush et al., Proc. Soc. Exp. Biol. Med., 122 , 931 (1966); DS Misfeldt et al., Proc. Natl. Acad. Sci. USA, 73.1212 (1976)), rat fibroblasts. Cell 3Y1 (S.
- an antigen molecule that is recognized by the antibody can be obtained by using the antibody that recognizes the stromal cell of the present invention.
- purification can be performed using the stromal cells of the present invention as a starting material and measuring the reactivity with the antibody using the enzyme immunoassay described above as an index.
- the stromal cells of the present invention are collected by centrifugation, suspended in an aqueous buffer, and then treated with an ultrasonic crusher, French press, Mantongaulin homogenizer, Dynomill, surfactant treatment, or the like. Crush cells to obtain cell-free extract.
- a normal enzyme isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resins such as getylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), and cation exchange using resins such as S-Sepharose FF (Pharmacia) Chromatography method, hydrophobic chromatography method using resin such as ptinoresepharose, feninoresepharose, gel filtration method using molecular sieve, affinity chromatography, chromatofocusing method, isoelectric focusing It is possible to obtain an antigen-purified sample by using a technique such as the electrophoresis method alone or in combination.
- a technique such as the electrophoresis method alone or in combination.
- the culture method of the present invention for inducing differentiation of embryonic stem cells into ectodermal cells and cells derived from ectodermal cells comprises: Physiological activity in differentiation process or regulation of cell function It is useful for pharmacological evaluation and activity evaluation of substances (eg, drugs) and novel gene products with unknown functions.
- substances eg, drugs
- embryonic stem cells in which a specific gene has been modified is useful for evaluating the function of the gene in the process of differentiation of embryonic stem cells into ectodermal cells and cells derived from ectoderm. is there.
- Examples of the method of using the culture method of the present invention include the following.
- the process of differentiation of a test substance added to a medium into ectoderm cells or cells derived from ectoderm, or regulating the function of ectoderm cells or cells derived from ectoderm Can be evaluated.
- the test substance may be any substance that can be added to the culture system, such as a low molecular compound, a high molecular compound, an organic compound, an inorganic compound, a protein, a gene, a virus, and a cell. Is raised.
- the test substance other than the gene may be added directly to the culture medium.
- Methods for efficiently introducing a gene into a culture system include a method in which the gene is loaded on a viral vector such as a retrovirus, an adenovirus, an adeno-associated virus, a simple virus, a lentivirus, etc., or a ribosome.
- a viral vector such as a retrovirus, an adenovirus, an adeno-associated virus, a simple virus, a lentivirus, etc., or a ribosome.
- a method of encapsulating in a human-like vesicle structure and adding it to a culture system is exemplified.
- a specific example is a report on gene analysis using a recombinant virus vector.
- test substances can be added to the culture system in the above differentiation induction method at any time.
- the relative culture when it is desired to evaluate the effect on the process of differentiating ectoderm-derived cells from ectoderm cells at an early stage, it can be evaluated by adding a test substance relatively late in culture.
- the degree of differentiation in the culture system it can be determined by examining the expression of marker proteins of various fractionated cells resulting from differentiation from embryonic stem cells. Evaluation of test substance or screen After culturing for a predetermined period of time, for example, it can be performed by measuring a qualitative or quantitative change in the efficiency of differentiation into ectoderm cells and cells derived from ectoderm. As a method of measuring qualitative changes, specifically, there is an example in which van Inzen et al. Measured the action potential using nerve cells induced to differentiate from embryonic stem cells (Biochim. Biophys. Acta., 1312). , 21 (1996)) 0
- the stromal cells used in the present invention or the stromal cell-derived factors of the present invention, or ectodermal cells or ectoderm-derived cells obtained by inducing differentiation of embryonic stem cells with the stromal cells or the stromal cells derived from the stromal cells of the present invention, are based on the damage of the ectodermal cells. It can be used as a drug for treating, diagnosing or preventing a disease.
- Examples of the disease based on the damage of the ectoderm-derived cells include the various diseases described in 6. (2) above.
- Therapeutic agents for diseases based on the damage of ectoderm-derived cells include cells that have the same function as damaged cells, progenitor cells of damaged cells, Cells that compensate for the function of damaged cells and cells that have the function of promoting regeneration of damaged cells are used.
- the therapeutic agent of the present invention can be produced by preparing the ectoderm cells or cells derived from ectoderm derived from embryonic stem cells of the present invention and using them as an active ingredient.
- stromal cells can be produced by the above-mentioned method, and by preparing a stromal cell-derived factor by the above-mentioned method 5 and using this as an active ingredient.
- the therapeutic agent of the present invention has a function of promoting regeneration of damaged cells in a disease based on the damage of ectoderm-derived cells.
- Any known method of cell separation and purification can be used to increase the purity of the cells.
- Specific examples thereof include Antibody's Laboratory, Manila Yunore, and Monoclona. Nore Antibody, Antibody Engineering, Int. Immunol., 10, 275 (1998), Exp. Hematol., 25, 972 (1997), etc. , J. Immunol., 141, 2797 (1988), etc., and a cell utilizing the density difference of sucrose concentration described in Tissue Culture Techniques (Third Edition), Asakura Shoten (1996), etc. A fractionation method can be given.
- the method for increasing the purity of the differentiated cells of the present invention comprises the step of culturing the outer Pi leaf cells or ectoderm-derived cells obtained by inducing the differentiation of embryonic stem cells as described above in a medium containing an anticancer agent. Including.
- undifferentiated cells can be removed, and differentiated cells with higher purity can be obtained, which is more suitable as a medicine. That is, by treating with an anticancer agent, cells other than the target differentiated cells, for example, undifferentiated cells can be removed. It is feared that such undifferentiated cells may cause teratoma of the teratoma, but the danger can be avoided by treating them with anticancer drugs.
- examples of the anticancer agent include mitomycin C, 5_fluorouracil, adoriamycin, ara C, and methotrexate. These anticancer agents are preferably used at a concentration that is more cytotoxic to cells in an undifferentiated state than cells in which differentiation has been induced. Specifically, according to the method described in 4 above, cultivation using these anticancer agents can be performed to determine the optimal concentration. For example, 100% of the concentration described in the Japanese Pharmacopoeia using these anticancer agents in a living body can be determined. using a medium containing at 1-1 times the concentration of minute, with C0 2 incubator aerated with 5% carbon dioxide for several hours at 37 ° C, preferably be mentioned a method of culturing for 2 hours.
- any medium can be used as long as it can culture the differentiated cells.
- the medium described in the above item 2 and the like can be mentioned.
- these anticancer agents can be used for the cells induced to differentiate according to the method described in the above item 4.
- the therapeutic agent of the present invention contains a pharmacologically acceptable physiological saline solution, an additive, and a medium in addition to the above cells (including ectoderm cells or cells derived from ectoderm and stromal cells).
- impurities such as serum virus are not mixed.
- the therapeutic agent containing the above factors is produced by the method of 9.
- embryonic stem cells obtained by nuclear transfer of somatic cell nuclei described in 1. (2) above or 1. (3) described above This can be overcome by using embryonic stem cells in which the gene on the chromosome described in (2) has been modified.
- differentiation is induced using embryonic stem cells obtained by nuclear transfer of the nucleus of the somatic cell described in 1. (2) above, so that the ectoderm cells and the ectoderm derived from the individual who provided the somatic cell can be obtained.
- Cells can be obtained.
- the cells of such individuals are not only effective as transplantation medicines themselves, but also useful as diagnostic materials for determining whether existing drugs are effective for the individual.
- long-term cultivation of differentiated cells enables determination of susceptibility to oxidative stress and senescence, and comparison of functions and longevity with cells derived from other individuals enables the diagnosis of neurodegenerative diseases and other diseases. Individuals can be assessed for risk, and these assessments are useful in providing effective prevention of diseases diagnosed with high future incidence.
- any method can be used as long as it is suitable for the target disease, and known methods suitable for each disease are known for each disease.
- embryonic stem cells are obtained from a diseased patient, and stromal cells and stromal cell-derived factors are added thereto, and the obtained embryonic stem cells are cultured.
- the embryonic stem cell Diseases can be treated by inducing differentiation of ectodermal cells or cells derived from ectoderm from the vesicle, and then transplanting the cells to the diseased site of the patient.
- stromal cells and stromal cell-derived factors can be directly administered to the disease site of a disease patient to treat the disease.
- examples of a method for transplanting brain cells of an aborted fetus into a Parkinson's disease patient include a method described in Nature Neuroscience, 2, 1137 (1999) and the like.
- the antigen of the present invention is obtained by performing an antigen-antibody reaction using the antibody of the present invention that specifically recognizes a stromal cell having an activity of inducing differentiation of ectodermal cells or cells derived from ectodermal cells from embryonic stem cells.
- a tissue containing the antigen can be immunologically detected.
- the detection method or a drug containing the antibody can be used for diagnosis of a disease caused by a decrease or disappearance of the function of the stromal cells, such as a disease based on damage to cells derived from ectoderm.
- the detection method is also used for quantification of an antigen.
- Examples of the disease based on the damage of the cells derived from the ectoderm include the various diseases described in 6. (2) above.
- Examples of the method for immunological detection include ELISA using a microtiter plate, fluorescent antibody, Western blotting, and immunohistological staining.
- a method of immunologically quantified a sandwich ELISA method using two kinds of antibodies Epitopu different among the antibodies that react with antigens of the present invention in the liquid phase, the present invention labeled with a radioisotope such as 125 1 And an antibody recognizing the antigen of the present invention.
- a radioisotope such as 125 1
- an antibody recognizing the antigen of the present invention (4) Medicine containing antigen
- Antigen molecules specifically recognized by stromal cells having an activity of inducing differentiation of embryonic stem cells from ectodermal cells or cells derived from ectodermal cells recognized by the antibody of the present invention are: It can be used as a therapeutic drug for disorders based on disorders.
- Examples of the disease based on the damage of the cells derived from the ectoderm include the various diseases described in 6. (2) above.
- a medicament containing the medicament containing the antigen of the present invention as an active ingredient as an active ingredient can be administered alone, but usually the pharmacologically acceptable one of the active ingredient is acceptable. Alternatively, it is desirably mixed with a further carrier and provided as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
- the form, administration route, dose, use form, and the like of these pharmaceutical preparations include the form, administration route, dose, use form, and the like of the pharmaceutical preparation described in 6. (2) above.
- Fig. 1 is a micrograph showing the results of staining of colonies that appeared as a result of co-culture of ES cells EB5 and PA6 cells with antibodies against (A) NCAM, (B) tupurin, and (C) nestin. .
- FIG. 2 is a photomicrograph showing the results of staining with a NCAM antibody on a corneal knee that appeared as a result of co-culture of ES cells EB5 and MEF cells.
- FIG. 3 is a micrograph showing the results of staining the colonies that appeared as a result of co-culture of ES cells EB5 and PA6 cells with an antibody against tyrosine hydroxylase.
- FIG. 4 is a graph showing, over time, the proportion of various positive colonies among colonies that appeared as a result of co-culture of ES cells EB5 and PA6 cells.
- Fig. 5 shows, among colonies that appeared as a result of co-culture of ES cells EB5 and PA6 cells without BMP4, (A) an antibody against NCAM, (B) an antibody against nestin, (C) an antibody against E Antibody (G) Antibody against NCAM, (E)
- FIG. 4 is a micrograph showing the results of staining using an antibody against nestin, an antibody against (F) E-cadherin, and an antibody against (H, I) keratin 14.
- FIG. 4 is a micrograph showing the results of staining using an antibody against nestin, an antibody against (F) E-cadherin, and an antibody against (H, I) keratin 14.
- FIG. 4 is a graph showing the results of staining colonies that appeared with an antibody against tubulin between when the cells were cultured with (gelatin) and.
- FIG. 7 is a graph showing the results of RT-PCR analysis of the expression levels of Nurrl, Ptx3 and G3PDH in differentiated cells that appeared as a result of co-culture of ES cells EB5 and PA6 cells.
- Embryo cells in the mouse head on day 12 (denoted as Embryo), ES cells EB5 co-cultured with PA6 cells for 12 days (ES + PA6 in the figure), and ES cells EB5 cultured for 12 days as controls ( The results of RT-PCR analysis and agarose gel electrophoresis analysis using ES as the material) are shown.
- Fig. 8 shows the results of HPLC analysis of the components secreted into the medium by stimulating de-separation of the fraction cells that appeared as a result of co-culture of ES cells EB5 and PA6 cells. It is a chromatograph. As a control, the results of analysis of components secreted when similar dedifferentiation stimulus was given only to PA6 cells used as one feeder cell are shown in the upper right chromatograph.
- FIG. 9 shows the results of analyzing the reactivity of monoclonal antibody KM1306 with PA6 cells by a fluorescent antibody method using a cell sorter.
- antibody The results of the same analysis using the rat IgM monoclonal antibody KM2070 whose species and subclass were identical were shown.
- the vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity.
- FIG. 10 shows the results of analyzing the reactivity of monoclonal antibody KM1307 with PA6 cells by a fluorescent antibody method using a cell sorter.
- the results of the same analysis were performed using rat IgM monoclonal antibody KM2070, which had the same antibody subclass.
- the vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity.
- FIG. 11 shows the results of analyzing the reactivity of monoclonal antibody KM1310 with PA6 cells by a fluorescent antibody method using a cell sorter.
- the results of the same analysis were performed using rat IgM monoclonal antibody KM2070, which had the same antibody subclass.
- the vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity.
- Embryonic stem cells H. Niwa et al., Nature Genet., 24, 372, (2000); generously awarded by Dr. Hitoshi Niwa, Department of Molecular Control Medicine, Osaka University School of Medicine
- stromal cells MC3T3-G2 / PA6 cells (H. Kodama et al., J. Cell Physiol., 112, 89 (1982); hereinafter, abbreviated as " ⁇ 6 cells” or primary mouse embryonic fibroblasts (hereinafter, abbreviated as "MEF cells”) was co-cultured.
- ES cell EB5 is transfected under the undifferentiation-specific promoter (0ct3 promoter; E. Pikarsky et al., Mol. Cell. Biol., 14, 1026 (1994)) so as to express the drug f sex gene Blastocidine-R. And add Blastocidine20 / zg / ml By culturing, only undifferentiated ES cells can be selected and maintained in culture. The ES cell EB5 used in the present invention was used after confirming that it survived in a medium supplemented with Blastocidine 20 g / ml and maintained an undifferentiated state during the test period.
- ES cell EB5 was prepared by adding 10% fetal bovine serum (Fetal Bovine Serum, ES Cell-Qualified; manufactured by Litec Oriental, Inc.), 2 mM glutamine, 100 ⁇ MEM Non-Essential Amino Acids solution, 50 U / ml ⁇ in Dulbecco MEM medium.
- the cells were cultured on a dish while maintaining the undifferentiated trait according to the method described in the manual for the mouse embryo lab.
- PA6 cells were cultured in a MEM medium containing 10% fetal bovine serum (GIBC0-BRL) according to the method of Kodama et al. (H. Kodama et al., J. Cell Physiol., 112, 89 (1982)). did.
- GIBC0-BRL fetal bovine serum
- MEF cells were prepared by adding 10% fetal bovine serum (Fetal Bovine Serum, ES Cell-Qualified; manufactured by Litec Oriental Co., Ltd.), 2 mM glutamine, 50 U / ml ⁇ -sylin and 50 U / ml streptomycin to Dulbecco MEM medium. It was prepared and cultured according to the method described in the manual “Manipulating 'The Mouse' Embryo Laboratory 'Manual.
- ES cells in single cell state were co-cultured with PA6 cells or MEF cells to induce differentiation of ES cells.
- the preparation of ES cell EB5 in a single cell state was performed as follows.
- the medium was replaced, and the EB5 ES cells were grown to a 30% confluent state. After removing the medium and washing twice with PBS (-), PBS (-) containing IraM EDTA and 0.25% trypsin was added, and the cells were cultured at 37 ° C for 20 minutes.
- the culture solution was added to Glasgow MEM medium with 10% KNOCKOUT SR (GIBC0BRL), 2raM glutamine, 100 ⁇ M MEM Non-
- the cells were suspended in a medium to which Essential Amino Acids solution, ImM pyruvate, 50 U / ml penicillin, and 50 U / ml streptomycin ( ⁇ 2 -mercaptoethanol) were added (hereinafter referred to as “serum-free medium”).
- the suspension was centrifuged at 200 ⁇ g for 5 minutes at 4 ° C., and the precipitated cells were suspended again in a serum-free medium to prepare ES cells EB5 in a single cell state.
- PA6 cells or MEF cells are prepared as feeder cells by exchanging the medium in advance, washing the cells whose cell density has almost reached the confluent state, washing twice with PBS (-), and adding the serum-free medium described above. did.
- the incubator PA6 cells prepared are cultured, were seeded ES cells EB5 prepared in single cells N 10-100 cells at a cell density of m 2, 4 days, 6 days, 7 days
- the medium was exchanged using a fresh serum-free medium at 37 ° C., and the cells were cultured at 37 ° C. for 8 days in a CO 2 incubator aerated with 5% carbon dioxide.
- the above ES cells were similarly seeded in a culture vessel coated with gelatin, and cultured in the same manner.
- anti-NCAM antibody a typical nerve marker
- anti-tubulin antibody an antibody against class III jS tubulin
- a nerve-specific antibody Babco, hereinafter referred to as “anti-tubulin antibody”
- an antibody against nestin which is a neural progenitor cell-specific marker
- Anti-nestin antibody an antibody against nestin which is a neural progenitor cell-specific marker
- PA6 cells and ES cells EB5 were co-cultured for 10 days. After fixing cells in the incubator, an antibody against tyrosine hydroxylase, a marker for dopaminergic nerve (Chemicon), an antibody against VAchT, a marker for cholinergic nerve (Cheminco), GABA activation Antibody against GAD (Chemicon), a marker for nervous nerves, and Serotonin, a marker for serotonergic nerves Immunostaining was performed using an antibody (Dia Sorin) or an antibody to dopamine j8 hydroxylase, a noradrenaline neuronal marker (PR0T0S Biotech).
- a 3 cm dish for tissue culture (plastic, FALCON) was used as an incubator, 1) a dish in which PA6 cells were prepared as one feeder cell, 2) a dish in which MEF cells were prepared as one feeder cell, 3) The figure shows the results of seeding and culturing 200 ES cells EB5 on each of the dishes only coated with gelatin.
- ES cells EB5 seeded in a single cell state adhered to the surface of the feeder cells or dish without agglutinating with each other and repeated cell division to form colonies hereinafter referred to as ⁇ ES cell-derived colonies '' or simply ⁇ colony '') ).
- FIG. 1 shows the results of staining the colonies that appeared as a result of co-culture with PA6 cells with (A) an anti-NCAM antibody, (B) an anti-tubulin antibody, and (C) an anti-nestin antibody.
- Fig. 2 shows the results of staining of the appearing knee with anti-NCAM antibody as a result of co-culture with MEF cells.
- FIG. 3 shows the results of staining the colonies that appeared as a result of co-culture with PA6 cells with an antibody against tyrosine hydroxylase (hereinafter abbreviated as “anti-tyrosine hydroxylase antibody”).
- Fig. 4 shows the time-course ratio of Koney Knee of various markers positive among colonies that appeared as a result of co-culture with PA6 cells. For the ratio of kogani knee, prepare 160 plates of co-cultured dishes under each of the conditions 1), 2) and 3) above, and observe the staining intensity of all appearing kogani by microscope. It was calculated as follows.
- Colonies derived from ES cells positive for anti-tyrosine hydroxylase antibody appeared frequently in the co-culture system prepared with PA6 cells as a feeder cell under condition 1) (89%) (Fig. 3).
- nestin-positive colonies appeared three days after the start of co-culture and tubulin-positive colonies appeared four days later, as shown in Fig. 4.
- Tyrosin hydroxylase-positive colonies appeared after 5 days, and reached a peak after 10 days. During this time, immunostaining with an antibody against dopamine J3 hydroxylase, a noradrenaline neuronal marker, was negative.
- VAchT-positive colonies which are the best of cholinergic nerves, are 5 ° after 10 days.
- the colonies that are positive for GAD, a marker for GABAergic nerves, are 15 ° .
- the colonies that are positive for serotonin are 4%. Met.
- a medium was prepared by adding 0.5 nmol / l BMP4 (manufactured by R & D) to the serum-free medium described in Example 1.
- the prepared serum-free medium supplemented with BMP4 was used instead of the serum-free medium used in Example 1, and ES cells EB5 and PA6 cells were co-cultured according to the method described in Example 1.
- cell immunostaining was performed using an anti-NCAM antibody, an anti-nestin antibody, and an antibody against E-cadherin, a non-neural ectoderm cell marker (Takara Shuzo).
- co-culture was performed using a serum-free medium without BMP4. The results are shown in FIGS. 5A, B, C, D, E, and F.
- ES cells-derived cells showed a strong anti-NCAM antibody (Fig. 5A) and a strong anti-nestin antibody (Fig. 5B). Atsushi. In contrast, a small number (18%) of E-cadherin-positive colonies (Fig. 5: On the other hand, in cultures using serum-free medium supplemented with BMP4, colonies derived from ES cells were anti-NCAM antibody-negative (Fig. 5! )), Anti-nestin antibody negative (Fig. 5E), but E-cadherin positive colonies appeared frequently (98%) (Fig. 5F). No keratin 14-positive colonies appeared when using BMP4-free medium (Fig. 5A
- Co-cultured PA6 cells MEF cells, ST0 cells, NIH / 3T3 cells, 0P9 cells, CH0 cells, MDCK cells, 3Y1 cells or COS cells (hereinafter referred to as “various cells”) and ES cells EB5 .
- ST0 cells were cultured according to the method described by Evans et al. (MJ Evans et al., Nature, 292, 154 (1981)).
- NIH / 3T3 cells as described by Jainchill et al. (JL Jainchill et al., J. Virol., 4, 549 (1969)).
- 0P9 cells were cultured according to the method described by Nakano et al. (T. Nakano et al., Science, 272, 722 (1996)).
- CH0 cells were cultured according to the method described by Puck et al. (TT Puck et al., J. Exp. Med., 108, 945 (1985)).
- MDCK cells were cultured according to the method described by Misfeldt et al.
- 3Y1 cells were cultured according to the method described by Sandineyer et al. (S. Sandineyer et al., Cancer Res., 41, 830 (1981)). COS cells were cultured according to the method described by Gluzman (Cell, 23, 175 (1981)).
- Example 1 According to the method described in Example 1, the above-mentioned various cells and ES cell EB5 were co-cultured for 8 days, immunostained with an anti-NCAM antibody, and the proportion of positive ES cell-derived colonies was examined. As a result, PA6 cells, 0P9 cells, and NIH / 3T3 cells showed positive rates of 95%, 45%, and 10%, respectively, indicating that these cells have significant neural differentiation-inducing activity against ES cells. Was. On the other hand, other cells did not show significant nerve differentiation inducing activity.
- the medium was exchanged in advance, and various cells whose cell density reached almost confluent state were washed twice with PBS (-), added with a 4% baraformaldehyde solution, and fixed by standing at 4 ° C for 30 minutes.
- Various cells were prepared by washing the fixed cells several times with a PBS (-) solution.
- ES cells EB5 co-culture with ES cells EB5 was performed according to the method described in Example 1.
- ES cells were differentiated into neural cells at high rates in co-culture with PA6 cells, 0P9 cells, NIH / 3T3 cells, MEF cells, and STO cells. It was not observed in co-culture with 3Y1 cells, COS cells, MDCK cells, and CH0 cells.
- stromal cells to differentiate embryonic stem cells into neural cells In order to analyze the activity of stromal cells to differentiate embryonic stem cells into neural cells, ES cells and stromal cells are filtered through a porous filter. Co-culture with cells was performed.
- a 6-well cell culture insert having a pore size of 0.445111 (product number 3090, manufactured by FALCON) was used.
- the PA6 cells were cultured inside the cell culture insert, and the PA6 cells adhered on the filter were prepared as a single feeder cell according to the method described in Example 1.
- the EB5 ES cells suspended in the serum-free medium described in Example 1 were seeded at 400 cells / well on a gelatin-coated 6-well culture dish (manufactured by FALCON), and the PA6 cells described above were prepared as one feeder cell.
- the inserted cell culture insert was inserted into the hole and cultured. That is, ES cells EB5 seeded on a 6-well culture dish and PA6 cells prepared in a cell culture insert as a feeder cell were co-cultured through a filter membrane. 4 days after the initiation of culture, 6 days, the medium was exchanged with a new vivid serum-free medium at day 7, 5% carbon dioxide aerated C0 2 incubator one by 8 days of culture the at 37 ° C for did.
- tubulin was positive in 25% of the cells. This is about 1/3 of the efficiency when cultured without a filter (Fig. 6, PA6), but when cultured on gelatin without PA6 cells (Fig. 6, gelatin; Positive rate) (Less than 3%).
- Part 1 Analysis of brain transplantation of embryonic stem cells differentiated into dopaminergic neurons (Part 1): According to the method described in Example 1, PA6 cells were used as a feeder cell, and ES cells EB5 were serum-free without BMP4. The cells were cultured in the medium for 10 days. That is, PA6 cells grown to almost confluent on a 6 cm tissue culture dish were used as buider cells, and EB5 ES cells were seeded on the feeder cells at 2000 cells / dish.On days 4 and 6, , the medium was replaced with fresh serum-free medium on day 8, 5% carbon dioxide at 37 ° C for 10 days in culture in aerated C0 2 incubator scratch.
- the cells that had been induced to differentiate as a result of the culture were fluorescently labeled using the cell lineage tracer Dil (manufactured by Molecular Probe) according to the attached document. After labeling, use a Papain Dissociation System kit (Worthington) and perform papain enzyme treatment for 5 minutes at room temperature according to the method attached to the kit, and separate each formed ES cell-derived colony from the feeder cell as a whole. did. In order to avoid damage to the nerve cells in the colonies, each colony formed by the induction of differentiation was separated as much as possible from the single feeder cell and used for transplantation.
- Papain Dissociation System kit Wathington
- the mixture was centrifuged at 300 rpm for 5 minutes using a 15-ml centrifuge tube to collect the differentiated ES cell mass. 6cm Differentiation induced ES cell mass recovered from one sheet dish were suspended in 5 mu 1 of the New 2 added Glasgow MEM medium (Gibco Lifetech Co., Ltd.), was used for implantation below.
- 6-0HDA Dissolve “6-0HDA” in PBS at 8 mg / ml, and use a small glass tube to place one force point 4 1 on the rostral and caudal sides of the striatum on one side. Forced injection. In some mice, three days later, after confirming the extrapyramidal symptoms on the injection side, using a 26G Hamilton syringe with a flat needle tip, differentiated into neurons near the center of the striatum on the same side as described above. 2 ⁇ l of the induced ES cell mass suspension was injected over 4 minutes. Eight days after the 6-0HDA treatment, the mouse brain was perfused and fixed to prepare a tissue specimen. The tissue specimen was prepared using an antibody against tyrosine hydroxylase, a marker of dopaminergic nerve (Chemicon), and a dopamine transporter. Immunostaining was performed using an antibody (Chemicon).
- PA6 cells were used as feeder cells, and ES cells EB5 were added without BMP4.
- the cells were cultured in serum medium for 8 days. That is, PA6 cells grown to almost confluence on a 6 cm tissue culture dish were used as feeder cells, and EB5 ES cells were seeded at 2000 cells / dish on the feeder cells. On the day, the medium was replaced with a fresh serum-free medium, and the cells were cultured at 37 ° C. for 8 days in a CO 2 incubator aerated with 5% carbon dioxide.
- the cells induced to differentiate as a result of the above culture were subjected to papain enzyme treatment at room temperature for 5 minutes according to the method of the attached document.
- Each colony formed by induction of differentiation was separated as a single mass from the feeder cell in order to avoid damaging the nerve cells in the colony.
- the cells forming the colonies were reacted in a PBS solution containing 5 ig / ml CM-DiI and 4 mg / ml glucose for 20 minutes at room temperature using the cell lineage tracer Dil (Molecular Probe) according to the attached document. Then, it was fluorescently labeled. After labeling, washed with N 2 added Glasgow MEM medium, N 2 added 1 ⁇ l of Glasgow MEM medium was prepared to contain about 4 ⁇ 10 5 cells, and used for the following transplantation.
- Transplantation drug injection was performed according to the method described in 3.10 of Current Protocols in Neuroscience (John Wiley & Sons, 1999).
- a C57BL / 6 mouse anesthetized with Nemptal is fixed on a stereotaxic apparatus (Narishige), and the mouse is in accordance with The Mouse Brain in Stereotaxic Coordinates (Academic Press, 1997)! ⁇ The striatum was located.
- 6-0 HDA which destroys local dopamine nerves, is dissolved in PBS at 8 ⁇ g / Ml, and this is striated on one side according to The Mouse Brain in Streotaxic Coordinates (Academic Press, 1997) using a micro glass tube.
- mice brain 14 days after 6-0HDA treatment, the mouse brain was perfused and fixed, and immunostaining was performed using an antibody against tyrosine hydroxylase (Chemicon), a marker for dopaminergic nerve, and an antibody against dopamine transporter (Chemicon). went.
- ES cells EB5 were cultured in a serum-free medium without BMP4 for 8 days, using PA6 cells as feeder cells. That is, PA6 cells grown to almost confluent on a 3 cm tissue culture dish were used as a feeder, and EB5 ES cells were seeded at 200 cells / dish on this single feeder cell, and on days 4 and 6, , the medium was replaced with fresh serum-free medium at day 7, 5% carbon dioxide at 37 ° C for cultured for 8 days at aerated C0 2 incubator scratch.
- the cells were fixed according to the method described in Example 1, and the cells were fixed against anti-NCAM antibody, anti-class III tubulin antibody, anti-nestin antibody, and synaptophysin, a presynaptic-specific antibody.
- Antibody Sigma
- RC2 antibody Developmental Studies Hybridoma Bank
- MF20 antibody Medium germ cells
- a medium was prepared by adding 0.5 nmol / l BMP4 (R & D) to the serum-free medium described in Example 1.
- the prepared serum-free medium supplemented with BMP4 was used instead of the serum-free medium used in Example 1, and ES cells EB5 and PA6 cells were co-cultured according to the method described in Example 1.
- PA6 cells grown to almost confluent on a 3 cm tissue culture dish were used as a feeder, and ES cells were seeded at 200 cells / dish on this feeder cell.
- the medium was replaced with a fresh medium, and the cells were cultured at 37 ° C. in a CO 2 incubator to which 5% carbon dioxide was aerated for 8 days.
- Example 1 After co-culturing for 8 days, the cells were fixed according to the method described in Example 1, anti-NCAM antibody, anti-E-doherin antibody, MF20 antibody recognizing mesoderm cells (Developmental Studies Hybridoma Bank), also mesoderm cells Colonies emerged as a result of co-culture of ES cells and PA6 cells using an antibody against PDGF receptor Hyoppo Flkl (S. L Nishikawa et al., Development, 125, 1747 (1998)) was subjected to immunostaining.
- ES cells EB5 were cultured in a serum-free medium without BMP4 for 12 days using PA6 cells as feeder cells. That is, PA6 cells grown to almost confluent on a 3 cm tissue culture dish were used as a feeder, and ES cells were seeded at 200 cells / dish on this feeder cell. day 8, the medium was replaced with fresh serum-free medium at day 10, 5% carbon dioxide at 37 ° C for 12-day-old culture at aerated C0 2 incubator scratch.
- the culture of the remaining dish was continued.
- the cells were fixed according to the method described in Example 1, and the ES cells EB5 and PA6 were cultured using antibodies against class III] 3 tubulin, nestin, and tyrosine hydroxylase. Immunostaining of colonies that appeared as a result of co-culture with cells was performed. Also, the number of all cells forming a colony was counted. For the determination, nuclear staining was performed using the kit YOYO-1 from Molecular Probe.
- the percentages of class Ill iS tupurin-positive cells, nestin-positive cells, and tyrosine hydroxylase-positive cells were 52 ⁇ 9%, 47 soil 10%, and 30 ⁇ 4%, respectively.
- ES cells EB5 were cultured in a serum-free medium without BMP4 for 12 days, using PA6 cells as feeder cells. That is, PA6 cells grown to almost confluence on a 9 cm tissue culture dish were used as a feeder, and ES cells EB5 were seeded on this feeder cell at 5 ⁇ 10 4 cells / dish. day 6, day 8, the medium was replaced with fresh serum-free medium at day 10, 5% carbon dioxide at 37 ° C for 12-day-old culture at aerated C0 2 incubator scratch. Also, using the ES cell culture medium described in Example 1, ES cells EB5 were seeded at a density of 5 ⁇ 10 4 cells / dish on a 9 cm tissue culture dish. , the medium was replaced with fresh medium on day 10, 5% carbon dioxide at 37 ° C for aerated (: 0 was prepared 2 Inkyubeta one at the 12-day cultured cells as a control.
- Reaction solution (lOramol / l Tris-HCl (pH8.3), 50mraol / l KC1, 1.5mmol / l) was prepared by using a solution prepared by diluting the synthesized cDNA 50-fold with sterile water according to a standard method.
- the oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 1 and 2 were used as Nurrl-specific primers
- the oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 3 and 4 were used as Ptx3-specific primers
- SEQ ID NOS: 5 and Oligonucleotides having the nucleotide sequence represented by 6 were used as G3PDH-specific primers.
- DMS0 was added to the reaction solution to a final concentration of 5%.
- neuron-like colonies appeared as in the case of Example 1.
- Nurrl and Ptx3 expression was observed in the cell population containing the differentiated colonies, as in the positive control (Fig. 7: ES + PA6).
- Nurrl-Ptx3 expression was not detected in control ES cells (Fig. 7: ES).
- ES control ES cells
- the expression of Nurrl and Ptx3 was not detected. Therefore, it was found that the expression of Nurrl and Ptx3, markers of dopaminergic neurons in the midbrain, were increased as embryonic stem cells were induced to differentiate into neurons by co-culture with PA6 cells. Do you get it.
- ES cells # 5 were cultured in a serum-free medium without # 4 for 8 days. That is, PA6 cells grown to almost confluence on a 9 cm tissue culture dish were used as a feeder, and ES cells were seeded at 5 ⁇ 10 4 cells / dish on one feeder cell, and the cells were cultured for 4 days. Eyes, the medium was replaced with fresh serum-free medium at day 6, 5% of the secondary Sani ⁇ carbon at 37 ° C for cultured for 8 days at aerated C0 2 incubator scratch.
- Neurons differentiated from embryonic stem cells by co-culture with PA6 cells were found to release a significant amount of dopamine by stimulating dedifferentiation of 56 mmol / l KC1 (7.7 pmol / 10 6 cells ( ES cell-derived differentiated cells)).
- Significant amounts of dopamine derivatives D0PAC (3,4-dihydroxyphenylacetic acid) and riVA (homovanillic acid) were also detected (2.5 pmol / 10 6 cells (different cells derived from ES cells, respectively)).
- Vesicle) and 4.0 pmol / 10 6 cells (ES cell-derived differentiated cells)).
- ES cells Prestress epiblast force on day 6 of mouse embryo instead of EB5 ⁇ Co-culture with PA6 cells according to the method described in Example 1 or 2 using isolated cells I got it.
- mice embryo On day 6 of mouse embryo, the cells constituting the primitive ectoderm were isolated and cultured in accordance with the method described in the manual "Mu- pilating-the-mouse-emprio-laboratory".
- PA6 cells were used as a feeder cell, and the isolated embryo cells were cultured in a serum-free medium without BMP4 for 8 days. That is, PA6 cells that had grown to almost confluence on a 3 cm tissue culture dish were used as a feeder, and primitive ectoderm cells isolated on this feeder cell were seeded at 200 cells / dish, and on day 4 , day 6, the medium was replaced with fresh serum-free medium at day 7, 5% of the diacid I ⁇ oxygen at 37 ° C for cultured for 8 days at aerated C0 2 incubator scratch.
- the embryo-cells and PA6 cells were isolated according to the method described in Example 1 using a medium obtained by adding 0.5 nmol / l BMP4 (manufactured by R & D) to the serum-free medium used in Example 1. Co-culture was performed. In other words, PA6 cells grown to almost confluence on a 3 cm tissue culture dish were used as a feeder, and ES cells were seeded at 200 cells / dish on this feeder cell. the medium was replaced with fresh medium on day 7, 5% of the diacid I inhibit carbon at 37 ° C for cultured for 8 days at aerated C0 2 incubator scratch.
- Example 13 After co-culturing for 8 days, cells were fixed according to the method described in Example 1, and embryo cells and PA6 cells were isolated using anti-NCAM antibody, anti-tupurin antibody, anti-nestin antibody, and anti-E-doherin antibody. The colonies that appeared as a result of the culture were subjected to immunostaining. Even when the isolated primitive ectoderm embryo cells were used, results similar to those of Examples 1 and 2 performed using ES cells EB5 were obtained, and the appearance of neural cells and epidermal cells was observed. Was. Example 13
- PA6 cells were used as a feeder cell, and ES cells EB5 were cultured in a serum-free medium without BMP4 for 8 days.
- heparin treatment a medium containing heparin
- PA6 cells grown to almost confluence on a 3 cm tissue culture dish were cultured in a medium containing 200 ng / ml heparin for 2 days, and a dish cultured in a medium without heparin for 2 days.
- ES cells EB5 were cultured for 10 days in a serum-free medium without the addition of shh and BMP4, using PA6 cells as feeder cells. That is, PA6 cells grown to almost confluence on a 3 cm tissue culture dish were used as a feeder, and ES cells were seeded at 200 cells / dish on this feeder cell. day 6, have rows of medium exchanged with fresh serum-free medium on day 8, a 5% C0 2 at 37 ° C for 10 days in culture in the incubator one was vented.
- shh The effect of shh is determined by using the same culture method as above, and using a serum-free medium supplemented with 300 nmol / l shh (R & D) during the medium replacement on days 4, 6, and 8. evaluated.
- the effect of BMP4 is determined by using the same culture method as above, and using a serum-free medium supplemented with 0.5 nmol / l BMP4 (manufactured by R & D) during the medium replacement on the 6th and 8th days. evaluated.
- PA6 cells Induction of ES cells by co-culture with Escherichia coli, cells of the nervous system expressing HNF-3 j8, located on the most ventral baseplate of the central nervous system primordium (neural tube), Cells of the nervous system expressing the marker Nkx2.2 next to HNF-3 ⁇ from the ventral side of the neural tube (neural tube), nerve cells on the dorsal side of the neural tube expressing Pax-7, It is induced to differentiate into neural crest cells expressing AP-2 and motor neurons expressing islet1.
- shh and BMP4 which have been shown to be involved in the determination of the dorsal-ventral axis during embryonic neurogenesis, showed differentiation potential similar to that of embryonic neural progenitor cells in vivo. It was shown that co-culture of ES cells with PA6 cells induced neural tube cells at a stage before the dorsoventral axis was determined. In other words, in the cells of this neural tube, the induction of the expression of the ventral nervous system HNF-3] 3 ⁇ 1 ⁇ 1.2.2 was observed by the action of shh, a factor that abdominates the neural tube. Inhibition of expression of the lateral markers Pax-7 and AP-2 is observed.
- PA6 cells were used as immunogen. PA6 cells were cultured according to the method described in Example 1. The PA6 cells whose cell density has reached almost confluent state are washed twice with PBS (-), and then a PBS (-) solution containing lO ⁇ g / ml actinase (manufactured by Kaken Pharmaceutical) and 0.02% EDTA is added. Incubate at 37 ° C for 30 minutes and add 10% fetal bovine serum (GIBC0- A-MEM medium containing BRL) was added to stop the action of actinase. The cells were collected by centrifugation at 1000 X g for 5 minutes at C.
- PBS PBS
- the cells were administered together with the cells to three 6- to 8-week-old female SD rats. Administration than after 2 weeks, once 10 7 cells prepared in the above (1) to 1 week, was administered a total of 4 times. Blood was collected from the carotid artery of the rat, and its serum antibody titer was examined by the enzyme immunoassay shown below. The spleen was excised 3 days after the last immunization from the mouse showing a sufficient antibody titer.
- the excised spleen was minced in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, and centrifuged (250 X g, 5 minutes). Erythrocytes were removed by adding Tris-salt ammonium buffer (pH 7.6) to the resulting precipitate fraction and treating for 1-2 minutes. The obtained precipitate fraction (cell fraction) was washed three times with MEM medium and used for cell fusion.
- MEM Minimum Essential Medium
- PA6 cells were seeded on each plate of a 96-well EIA plate (Grainer) and the plate grown to a confluent state was used as an antigen plate.
- the culture supernatant of the immunized rat antiserum or monoclonal antibody was dispensed into the plate at 50 1 / well, and left at 37 ° C for 1 hour. One hour later, the added antiserum or culture supernatant was removed, PBS (-) containing 0.25% glutaraldehyde was added, and the mixture was allowed to stand at room temperature for 30 minutes.
- P3X63Ag8U.1 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P3-U1: purchased from ATCC) is cultured in a normal medium (RPI medium supplemented with 10% ⁇ fetal blood ⁇ ⁇ ), and at least 2 ⁇ 10 7 cells are fused at the time of cell fusion. Cells were obtained and used as a parent strain for cell fusion.
- mice spleen cells obtained in Example 15 (2) and the myeloma cells obtained in Example 15 (4) were mixed at a ratio of 10: 1, and centrifuged (250 ⁇ g, 5 minutes). After the cells in the precipitate fraction obtained were thoroughly loosened, a mixture of 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM medium and 0.7 ml of dimethyl sulfoxide was mixed at 37 ° C with stirring. 10 8 mouse splenocytes per 0. 5 ml was added, after MEM medium is added several times 1ml every 1-2 minutes to the suspension, the total amount was made up to 50ml by the addition of MEM medium.
- PEG-1000 polyethylene glycol-1000
- MEM medium ml of MEM medium
- dimethyl sulfoxide dimethyl sulfoxide
- the suspension was centrifuged (900 rpm, 5 minutes), and the cells of the obtained precipitate fraction were loosened gently. Then, the cells were gently sucked and aspirated with a female pipette to form a HAT medium (10% ⁇ ).
- the suspension was suspended in 100 ml of HAT Media Supplement (Boehringer-Germanheim) supplemented with RPMI medium supplemented with fetal serum. 96 ⁇ of the suspension 200 / zl / ⁇ l at a time in a 5% CO 2 incubator at 37 ° C. for 10 to 14 days.
- Example 15 After culturing, the culture supernatant is examined by the enzyme immunoassay described in Example 15 (3), and PBS (-) containing 1% BSA in response to PA6 cells (hereinafter referred to as ⁇ 1% BSA-PBS (-) A solution that did not react with the control plate coated with) was selected, and cloning by limiting dilution was repeated twice from the cells contained therein to establish an anti-PA6 monoclonal antibody-producing hybridoma. As a result, three kinds of anti-human PA6 cell antibodies KM1306, KM1307, and KM1310 were obtained using PA6 cells as antigens.
- the KM1310-producing hybridoma cell line was designated as FERM BP-7573 on April 27, 2001 by the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan 1 Central No. 6 (Postal Code 305-8566)).
- mice Eight weeks-old nude female mice (BALB) treated with pristane were intraperitoneally injected with 5 ⁇ 10 6 to 20 ⁇ 10 6 / hypridoma strains obtained in Example 15 (5). After 10 to 21 days, ascites was collected (1 to 8 ml / animal) from the mice that had accumulated ascites due to Hypuri Doma's ascites.
- the ascites was centrifuged (1200 ⁇ g, 5 minutes) to remove solids.
- Purified IgM monoclonal antibodies were obtained by purification by ammonium sulfate precipitation (Antibody 'A' Laboratory Manual).
- the subclass of the monoclonal antibody was determined to be IgM for all KM1306, KM1307, and KM1310 by ELISA using the subcluster typing kit. (7) Analysis of reactivity with PA6 cells by fluorescent antibody method (cell sorter analysis)
- PA6 cells were cultured according to the method described in Example 1. PA6 cells whose cell density has reached almost confluent state are washed twice with PBS (-), and PBS (-) containing 10 / ig / ml actinase (manufactured by Kaken Pharmaceutical) and 0.02% EDTA is used. Add the solution, incubate at 37 ° C for 30 minutes, add ⁇ -MEM medium containing 10% fetal bovine serum (GIBC0-BRL) to stop the action of actinase, and centrifuge at 4 ° C for 1000 Xg for 5 minutes. Collected by separation.
- PBS PBS
- the collected cells were suspended in ⁇ -MEM medium containing 10% fetal bovine serum (GIBCO-BRL), and 1 ⁇ 10 6 cells were dispensed into 1.5 ml tubes.
- the aliquoted cells were suspended twice in a 1% BSA-PBS (-) solution, and washed twice by centrifugation at 1000 ⁇ g for 5 minutes.
- the washed cells are suspended in 1% BSA-PBS (-) solution containing 10 / zg / ml purified antibody (or SO / g / ml ammonium sulfate precipitated antibody fraction) and incubated at 37 ° C for 30 minutes. And reacted with the antibody.
- the cells reacted with the antibody were reacted with a fluorescently labeled secondary antibody according to a conventional method, and analyzed using a cell sorter (antibody 'a' laboratory. Manual). That is, the cells reacted with the antibody are collected by centrifugation at 1000 ⁇ g for 5 minutes, suspended in a 1% BSA-PBS (-) solution containing the secondary antibody, and cultured at 37 ° C for 30 minutes. After washing twice with a% BSA-PBS (-) solution, the cells were suspended in 2 ml of a 1% BSA-PBS (-) solution, and analyzed using a cell analyzer (Coulter; EPICS XLsystem II).
- a cell analyzer Coulter; EPICS XLsystem II
- FITC-labeled anti-rat immuno Glo purine antibody (FITC-labeled catcher formic anti-rat immuno glob phosphorus (H + L); CALTAG Inc.) was diluted 30-fold 1 0 /. BSA-PBS (-) solution was used at 1001 / tube.
- thigh 2070 which is a monoclonal antibody recognizing the aging inhibitory protein of rat IgM, was reacted at 10 / ig / ml and detected similarly.
- KM2070 is an antibody produced by Hypridoma KM2070 (FERM BP-6196; W098 / 29544).
- KM2070 was used as a control antibody after confirming in advance that the expression of the antigen molecule recognized by KM2070 was not in PA6 cells.
- KM1306, KM1307, and KM1310 obtained by immunizing PA6 cells recognized PA6 cells.
- the vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity. In the figure, nega indicates the result when the antibody was not added.
- the present invention provides a method for selectively and efficiently inducing differentiation of ectoderm cells and cells derived from ectoderm from embryonic stem cells, the differentiated cells, and uses thereof. Sequence free text
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AU56767/01A AU5676701A (en) | 2000-05-16 | 2001-05-16 | Novel method of inducing the differentiation of embryonic stem cells into ectodermal cells and use thereof |
JP2001585308A JP4458222B2 (ja) | 2000-05-16 | 2001-05-16 | 胚性幹細胞の外胚葉系細胞への新規な分化誘導法及びその用途 |
CA002409703A CA2409703A1 (en) | 2000-05-16 | 2001-05-16 | Novel differentiation inducing process of embryonic stem cell to ectodermal cell and its use |
EP01930185A EP1302533A4 (en) | 2000-05-16 | 2001-05-16 | NEW METHOD FOR INDUCING DIFFERENTIATION OF EMBRYONIC STEM CELLS IN EECTODERMAL CELLS AND THEIR USE |
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EP1302533A4 (en) | 2004-11-17 |
CA2409703A1 (en) | 2001-11-22 |
AU5676701A (en) | 2001-11-26 |
EP1302533A1 (en) | 2003-04-16 |
JP4458222B2 (ja) | 2010-04-28 |
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