WO2017183736A1 - ドーパミン産生神経前駆細胞の製造方法 - Google Patents
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- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
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Definitions
- the present invention relates to a method for producing dopaminergic neural progenitor cells and a method for producing a cell population containing Corin and / or Lrtm1 positive cells that can be differentiated into dopaminergic neural progenitor cells.
- Parkinson's disease is a neurodegenerative disease caused by the loss of midbrain substantia nigra dopamine-producing neurons, and there are currently about 4 million affected individuals worldwide.
- drug treatment with L-DOPA or a dopamine agonist, coagulation by stereotaxic surgery or deep electrical stimulation treatment, and fetal midbrain transplantation are performed.
- Fetal mesencephalic transplantation has ethical problems with the source tissue and also has a high risk of infection. Then, the treatment method using the nerve cell which induced differentiation from pluripotent stem cells, such as an embryonic stem cell (ES cell) and an induced pluripotent stem cell (iPS cell), is proposed (nonpatent literature 1). However, the possibility of forming benign tumors when transplanted with differentiation-induced neurons and dyskinesias that are thought to be caused by cells other than the desired dopamine-producing neurons have been pointed out, and are safe to take. There is a need to select and transplant cells.
- ES cell embryonic stem cell
- iPS cell induced pluripotent stem cell
- Patent Document 1 As a method for producing dopaminergic neural progenitor cells, a method including a step of selecting dopaminergic neurons or cells suitable for transplantation using a gene serving as a marker for dopaminergic neural progenitor cells has been proposed (Patent Document 1). Further improvements are required in order to reduce the effects of lot differences due to the inclusion of biological components and increase production efficiency.
- An object of the present invention is to produce dopaminergic neural progenitor cells suitable as a therapeutic agent for Parkinson's disease. Accordingly, an object of the present invention is to provide a method for producing dopaminergic neural progenitor cells and the like.
- the present inventors have found a method for efficiently producing a cell population containing cells that express cells by focusing on the cell surface membrane proteins Corin and / or Lrtm1. It was. Furthermore, the present inventors have found that dopaminergic neural progenitor cells can be obtained after cells are extracted from the cell population using Corin and / or Lrtm1 as an index and further cultured.
- a method for producing a cell population containing Corin and / or Lrtm1 positive cells comprising the following steps (1) and (2): (1) Adhesive culture of pluripotent stem cells in the presence of an extracellular matrix in an undifferentiated maintenance medium containing a Sonic hedgehog (SHH) signal stimulator and an undifferentiated maintenance factor in the absence of feeder cells; And (2) culturing the cells obtained in step (1) in a medium containing one or more differentiation-inducing factors.
- SHH Sonic hedgehog
- SHH signal stimulant is SAG (N-Methyl-N ′-(3-pyridinylbenzyl) -N ′-(3-chlorobenzo [b] thiophene-2-carbonyl) -1,4-diaminocyclohexane), shh protein Or the manufacturing method as described in [1] which is the fragment, Purmorphamine, or these combination.
- the undifferentiated maintenance medium in the step (1) is a medium further containing a TGF (Transforming growth factor) ⁇ inhibitor or a BMP (Bone Morphogenetic Protein) inhibitor.
- the differentiation inducing factor is one or more factors selected from the group consisting of BMP inhibitors, TGF ⁇ inhibitors, SHH signal stimulators, FGF8 and GSK (Glycogen synthase kinase) 3 ⁇ inhibitors, [1] The manufacturing method according to any one of to [7]. [9] The production method according to any one of [1] to [8], wherein the step (2) is a step of adhesion culture on an extracellular matrix. [10] The production method according to [9], wherein the extracellular matrix is laminin or a fragment thereof. [11] The production method according to [9], wherein the extracellular matrix is laminin 511E8.
- step (2) comprises the following steps; (A) a step of adhesion-culturing the cells obtained in step (1) in a medium containing a BMP inhibitor and a TGF ⁇ inhibitor in the presence of an extracellular matrix; (B) adhesion-culturing the cells obtained in the step (a) in a medium containing a BMP inhibitor, TGF ⁇ inhibitor, SHH signal stimulator and FGF8 in the presence of an extracellular matrix; (C) Adherent culture of the cells obtained in the step (b) in a medium containing a BMP inhibitor, TGF ⁇ inhibitor, SHH signal stimulator, FGF8 and GSK3 ⁇ inhibitor in the presence of an extracellular matrix; And (d) a step of adhesion-culturing the cells obtained in the step (c) in the presence of an extracellular matrix in a medium containing a BMP inhibitor and a GSK3 ⁇ inhibitor.
- [23] The following steps: (3) Collect Corin and / or Lrtm1 positive cells from the cell population obtained by the production method according to any one of [1] to [22] using a substance that binds to Corin and / or a substance that binds to Lrtm1 And (4) a step of subjecting Corin and / or Lrtm1 positive cells obtained in step (3) to suspension culture in a medium containing one or more neurotrophic factors,
- a method for producing dopaminergic neural progenitor cells comprising: [24] The production method according to [23], wherein the substance that binds to Corin or the substance that binds to Lrtm1 is an antibody or aptamer that binds to Corin or Lrtm1.
- step (4) is performed for at least 7 days.
- step (4) is performed for 14 to 30 days.
- step (4) is performed for 14 to 16 days.
- a cell population obtained by the production method according to any one of [1] to [22] and having a Corin and / or Lrtm1-positive cell content of 10% or more.
- a dopaminergic neural progenitor cell obtained by the production method according to any one of [23] to [32].
- a therapeutic agent for Parkinson's disease comprising dopaminergic neural progenitor cells obtained by the production method according to any one of [23] to [32].
- dopaminergic neural progenitor cells useful for Parkinson's disease therapeutic agents and the like can be obtained efficiently.
- FIG. 1 shows an example of a protocol for producing dopaminergic cells.
- “Y” indicates Y-27632.
- FIG. 2 shows the results of flow cytometry in the cells of day 12 cultured in the presence or absence of SAG (300 nM) in Example 1, that is, the Corin positive cell ratio (the ratio of Corin positive cells in the total number of cells). ).
- FIG. 3 is a diagram (photograph) showing the result of confirming whether or not the cells were differentiated into dopamine neural progenitor cells by staining FoxA2, Nurr1 (both midbrain markers) on day28 cells in Example 1. It is. DAPI-positive cells indicating the presence of nuclei were 100%, Foxa2 was 99.0%, and Nurr1 was 14.6%.
- FIG. 1 shows an example of a protocol for producing dopaminergic cells.
- “Y” indicates Y-27632.
- FIG. 2 shows the results of flow cytometry in the cells of day 12 cultured in the presence or absence of SAG (300
- FIG. 4 is a graph showing the results of flow cytometry, that is, the Corin positive cell rate, in day 12 cells cultured in Example 3 in the presence or absence of SAG (300 nM).
- FIG. 5 shows the state of the graft in Example 1 at 4 weeks after the cells of day 28 were transplanted into the brain of a rat (rat administered with 6-OHDA). It is a figure (photograph) which shows the result of having dye
- A is a transplant of cells in the presence of SAG (300 nM), and the average TH is 9.24% (individual (1) 6.1%), with HuNu positive cells indicating the presence of transplanted cells (human cell nuclei) being 100%. Individual (2) 12.37%).
- B is a graft of cells in the absence of SAG, and the average TH was 9.7% (individual (3) 6.27%, individual (4) 13.13%), with HuNu positive cells as 100%. It was.
- the pluripotent stem cell that can be used in the present invention is a stem cell that has pluripotency that can be differentiated into all cells existing in a living body and also has proliferative ability, and is not particularly limited.
- embryonic stem (ES) cells embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer
- GS cells sperm stem cells
- EG cells embryonic germ cells
- artificial pluripotency Examples include sex stem (iPS) cells, cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells).
- Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.
- ES cells are stem cells established from the inner cell mass of early embryos (for example, blastocysts) of mammals such as humans and mice, and having pluripotency and proliferation ability by self-replication.
- ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication.
- ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (JA Thomson et al.
- ES cells can be established by removing an inner cell mass from a blastocyst of a fertilized egg of a subject animal and culturing the inner cell mass on a fibroblast feeder. Cells are maintained by subculture using a medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). be able to.
- LIF leukemia inhibitory factor
- bFGF basic fibroblast growth factor
- a medium for ES cell production for example, DMEM / F-12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF, 37 Human ES cells can be maintained in a humidified atmosphere at 0 ° C. and 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224).
- ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
- ES cells can be selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index.
- gene markers such as OCT-3 / 4, NANOG, ECAD and the like can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443 -452).
- Human ES cell lines are obtained from, for example, WA01 (H1) and WA09 (H9) from the WiCell Research Institute, and KhES-1, KhES-2, and KhES-3 from Kyoto University Institute for Regenerative Medicine (Kyoto, Japan). Is possible.
- sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012).sperm stem cells are capable of self-replication in media containing glial cell line-derived neurotrophic factor (GDNF) and repeated passage under the same culture conditions as ES cells (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra), pages 41-46, Yodosha (Tokyo, Japan)).
- GDNF glial cell line-derived neurotrophic factor
- Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
- iPS Artificial pluripotent stem cells
- somatic cells in the form of DNA or protein, which is almost equivalent to ES cells
- It is an artificial stem cell derived from a somatic cell having the characteristics of, for example, differentiation pluripotency and proliferation ability by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol.
- the reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or non-coding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be constituted by a low molecular compound.
- genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, dominant negative p53, p53 gene suppressor such as shRNA, EBNA1 or Glis1, etc., and these reprogramming factors are They may be used alone or in combination.
- HDAC histone deacetylase
- VPA valproic acid
- trichostatin A sodium butyrate
- MC 1293 M344
- siRNA and shRNA against HDAC eg Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.
- MEK inhibitors eg, PD184352, PD98059, U0126, SL327 and PD0325901
- Glycogen synthase kinase- 3 inhibitors eg, Bio and CHIR99021
- DNA methyltransferase inhibitors eg, 5-azacytidine
- histone methyltransferase inhibitors eg, small molecule inhibitors such as
- Factors used for the purpose of improving establishment efficiency are also included.
- factors used for the purpose of improving establishment efficiency are not distinguished from initialization factors. . Further, one or more of these may be appropriately selected and used.
- the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, TAT and polyarginine derived from HIV), or microinjection.
- a cell membrane-permeable peptide for example, TAT and polyarginine derived from HIV
- DNA it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection.
- viral vectors examples include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like.
- artificial chromosome vectors examples include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
- a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
- the vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed.
- Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), ⁇ -glucuronidase (GUS), FLAG, etc. Can be included.
- the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.
- RNA form it may be introduced into somatic cells by techniques such as lipofection and microinjection, and RNA containing 5-methylcytidine and pseudoridine (TriLink Biotechnologies) is used to suppress degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630).
- a medium for iPS cell induction for example, DMEM, DMEM / F12 or DME medium containing 10-15% FBS (in addition to these mediums, LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential) Amino acids, 2-mercaptoethanol, etc. may be included as appropriate.
- commercially available media eg, mouse ES cell culture medium (TX-WES medium, Thrombo X), primate ES cell culture medium (primate) ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology)] and the like.
- the somatic cell and the reprogramming factor are brought into contact with DMEM or DMEM / F12 medium containing 10% FBS and cultured for about 4 to 7 days. Thereafter, the cells are repopulated on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.) and cultured in a primate ES cell culture medium containing bFGF about 10 days after contact between the somatic cells and the reprogramming factor.
- feeder cells for example, mitomycin C-treated STO cells, SNL cells, etc.
- IPS-like colonies can be generated about 30 to about 45 days or more after the contact.
- DMEM medium containing 10% FBS on feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- 5% CO 2 at 37 ° C. including LIF, penicillin / streptomycin, puro
- somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.
- iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845). During the culture, the medium is replaced with a fresh medium once a day from the second day after the start of the culture.
- somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
- iPS cells can be selected according to the shape of the formed colonies.
- a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized for example, Oct3 / 4, Nanog
- a medium containing the corresponding drug for example, Oct3 / 4, Nanog
- the marker gene is a fluorescent protein gene
- iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.
- the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell.
- Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines.
- somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells.
- somatic cells having the same or substantially the same HLA genotype of the transplant destination individual from the viewpoint of preventing rejection.
- substantially the same means that the HLA genotype matches the transplanted cells to such an extent that an immune response can be suppressed by an immunosuppressive agent.
- HLA-A, HLA-B And somatic cells having an HLA type in which 3 loci of HLA-DR or 4 loci plus HLA-C are matched.
- E Cloned embryo-derived ES cells obtained by nuclear transfer nt ES cells are cloned embryo-derived ES cells produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007) , Nature, 450: 497-502).
- an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell.
- nt ES nuclear transfer ES
- nuclear transfer technology JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52).
- Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.
- Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time, preferably 8 or 16 hours. It is a pluripotent cell obtained by suspension culture after treatment, and is positive for SSEA-3 and CD105.
- the dopaminergic neural progenitor cell may include a dopaminergic neuron or a dopaminergic neuron unless otherwise specified.
- the dopaminergic neural progenitor cell may be a cell population containing other cell types, and is preferably a cell population not containing serotonin neurons.
- the dopaminergic neural progenitor cells are preferably a cell population containing Foxa2, Nurr1, Lmx1a, Pitx3 and / or TH positive cells.
- examples of human Foxa2 include a polynucleotide represented by NCBI accession number NM_021784 or NM_153675 and proteins encoded by these.
- human Nurr1 includes a polynucleotide represented by NCBI accession number NM_006186 and a protein encoded by these.
- human TH includes a polynucleotide represented by NCBI accession number NM_000360, NM_199292 or NM_199293 and proteins encoded by these.
- human Lmx1a include polynucleotides represented by NCBI accession numbers NM_001174069 or NM_177398 and proteins encoded by these.
- Examples of human Pitx3 include a polynucleotide represented by NCBI accession number NM_005029 and a protein encoded by these.
- an SHH (Sonic hedgehog) signal stimulator means that the SHH is bound to the receptor Patched (Ptch1) and is derepressed by Smoothened (Smo) and further activated by Gli2.
- proteins belonging to the Hedgehog family specifically SHH or IHH (Indian Hedgehog), SHH receptor, SHH receptor agonist, Hh-Ag1.5 (Li, X ., et al., Nature Biotechnology, 23, 215-221 (2005).), Smoothened Agonist, SAG (N-Methyl-N '-(3-pyridinylbenzyl) -N'-(3-chlorobenzo [b] thiophene- 2-carbonyl) -1,4-diaminocyclohexane; N-methyl-N '-(3-pyridinylbenzyl) -N'-(3-chlorobenzo [b] thiophene-2-carbonyl) -1,4-diaminocyclohexane ), 20a-hydroxycholesterol, Purmorphamine (PMA; 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-
- the SHH signal stimulator used in the present invention preferably includes SHH protein (Genbank accession numbers: NM_000193, NP_000184), Purmorphamine, and SAG.
- undifferentiation maintenance factor will not be specifically limited if it is a substance which has the effect
- undifferentiated maintenance factors that are widely used by those skilled in the art include FGF signaling pathway agents, TGF ⁇ inhibitors, insulin and the like.
- FGF signal transduction pathway agent include fibroblast growth factors (for example, bFGF, FGF4, and FGF8).
- TGF ⁇ inhibitors include substances acting on TGF ⁇ signal transduction pathway and substances acting on Nodal / Activin signal transduction pathway.
- the medium in the step (1) preferably contains at least bFGF as an undifferentiation maintenance factor.
- the undifferentiation maintenance factor used in the present invention is usually a mammalian undifferentiation maintenance factor.
- mammals include rodents, ungulates, felines, primates and the like.
- Rodents include mice, rats, hamsters, guinea pigs and the like.
- Hoofed animals include pigs, cows, goats, horses, sheep and the like.
- Cat eyes include dogs, cats and the like.
- the term “primate” refers to mammals belonging to the order of primates. Examples of primates include wild monkeys such as lemurs, loris, and tuna, and monkeys such as monkeys, apes, and humans. it can.
- an undifferentiated maintenance factor can have cross-reactivity between mammalian species, any mammalian undifferentiated maintenance factor can be used as long as the undifferentiated state of the pluripotent stem cells to be cultured can be maintained.
- an undifferentiated maintenance factor of a mammal of the same species as the cell to be cultured is used.
- human undifferentiated maintenance factors eg, bFGF, FGF4, FGF8, EGF, Nodal, ActivinA, ActivinB, TGF ⁇ 1, TGF ⁇ 2, etc.
- human protein X means that protein X has the amino acid sequence of protein X that is naturally expressed in the human body.
- the undifferentiation maintenance factor used in the present invention is preferably isolated.
- isolated protein X means that an operation to remove a target component or a factor other than cells has been performed, and the state existing in nature has been removed. Therefore, “isolated protein X” does not include endogenous protein X produced from cells or tissues to be cultured and contained in the cells, tissues, or medium.
- the purity of “isolated protein X” (percentage of the weight of protein X in the total protein weight) is usually 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 99% or more, More preferably, it is 100%. Accordingly, in one aspect, the present invention includes providing an isolated undifferentiated maintenance factor.
- the process of adding the isolated undifferentiation maintenance factor exogenously (or exogenous) to the culture medium used for a process (1) is included.
- an undifferentiation maintenance factor may be added in advance to the medium used in the step (1).
- the concentration of the undifferentiated maintenance factor in the medium used in step (1) is a concentration capable of maintaining the undifferentiated state of the pluripotent stem cells to be cultured, and can be appropriately set by those skilled in the art.
- the concentration is usually about 4 ng to 500 ng / mL, preferably about 10 ng to 200 ng / mL, more preferably 30 ng to About 150 ng / mL.
- the medium used for culturing cells in the present invention can be prepared as a basal medium or a commercially available medium usually used for culturing animal cells.
- the basal medium include BME medium, BGJb medium, and CMRL. 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium , RPMI 1640 medium, Fischer's medium, Neurobasal medium or a mixed medium thereof that can be used for culturing animal cells.
- the medium used as “medium containing undifferentiated maintenance factor” (undifferentiated maintenance medium) in the present invention is a feeder-free serum-free medium. It can be prepared by adding a source or the like. Specifically, for example, it can be prepared by adding bFGF, KSR, non essential amino acid (NEAA), L-glutamine and 2-mercaptoethanol to DMEM / F12 medium. Moreover, the below-mentioned feeder free medium containing an undifferentiation maintenance factor marketed as a medium suitable for undifferentiation maintenance can also be used as an undifferentiation maintenance medium.
- the “serum-free medium” in the present invention means a medium that does not contain unconditioned or unpurified serum.
- a medium containing purified blood-derived components or animal tissue-derived components is also included in the serum-free medium unless it contains unconditioned or unpurified serum.
- the serum-free medium may contain a serum replacement.
- serum substitutes include those containing albumin, transferrin, fatty acid, collagen precursor, trace elements, 2-mercaptoethanol or 3 ′ thiolglycerol, or their equivalents as appropriate.
- Such a serum substitute can be prepared, for example, by the method described in WO98 / 30679. A commercially available product may be used as a serum substitute.
- Examples of such commercially available serum substitutes include Knockout Serum Replacement (manufactured by Life Technologies (currently ThermoFisher Scientific); hereinafter referred to as KSR), and Chemically-defined Lipid concentrated (manufactured by Life Technologies). GlutaMax (manufactured by Life Technologies), B27 (manufactured by Life Technologies), N2 supplement (manufactured by Life Technologies), and ITS supplement (manufactured by Life Technologies).
- the serum-free medium may appropriately contain fatty acids or lipids, amino acids (eg, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like. Good.
- an appropriate amount of a commercially available KSR (manufactured by Life Technologies) is used as such a serum-free medium (for example, about 0.5% to about 30%, preferably about 1% to about 20%).
- An appropriate amount (for example, about 0.1 to 5%) of an added serum-free medium for example, a medium in which about 8% KSR and Chemically-defined Lipid concentrated are added to a GMEM medium) or a commercially available B27 (manufactured by Life Technologies) in a Neurobasal medium. )
- Added serum-free medium may be used.
- the culture medium disclosed by the special table 2001-508302 as a KSR equivalent product is mentioned.
- the serum-free medium is preferably a serum-free medium containing KSR or B27, or a medium under xeno-free conditions.
- zeno-free means a condition in which a component derived from a biological species different from the biological species of the cell to be cultured is excluded.
- a feeder cell is a cell other than the stem cell that coexists when the stem cell is cultured.
- feeder cells include mouse fibroblasts (such as MEF), human fibroblasts, SNL cells, and STO cells.
- the feeder cells may be feeder cells that have been subjected to growth inhibition treatment, and examples of the growth inhibition treatment include treatment with a growth inhibitor (for example, mitomycin C), treatment with gamma irradiation or UV irradiation, and the like.
- the culture is preferably performed in the absence of feeder cells (feeder-free).
- the absence of feeder cells means culturing in the absence of feeder cells.
- the condition where no feeder cells are added as described above, or substantially no feeder cells for example, the ratio of the number of feeder cells to the total number of cells is 3% or less, The condition is preferably 0.5% or less.
- a feeder-free medium that can be used as an undifferentiated maintenance medium, many synthetic media have been developed and marketed, and examples include Essential 8 (manufactured by Life Technologies).
- Essential 8 medium was added to DMEM / F12 medium as additives: L-ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenium (14 ⁇ g / 1), insulin (19.4 mg / l), NaHCO 3 (543 mg / l), transferrin (10.7 mg / l), bFGF (100 ng / mL), and TGF ⁇ inhibitor (TGF ⁇ 1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods , 8, 424-429 (2011)).
- a feeder-free medium containing a commercially available undifferentiated maintenance factor that is, a feeder-free medium that can be used as an undifferentiated maintenance medium, is S-medium (DS Pharma Biomedical), StemPro (Life Technologies), hESF9 (Proc Natl Acad Sci US A. 2008 Sep 9; 105 (36): 13409-14), mTeSR1 (STEMCELL Technologies), mTeSR2 (STEMCELL Technologies), TeSR-E8 (STEMCELL Technologies), or StemFit (Ajinomoto) Manufactured).
- S-medium DS Pharma Biomedical
- StemPro Life Technologies
- hESF9 Proc Natl Acad Sci US A. 2008 Sep 9; 105 (36): 13409-14
- mTeSR1 STMCELL Technologies
- mTeSR2 mTeSR2
- TeSR-E8 STEMCELL Technologies
- StemFit Ajinomoto
- the extracellular matrix is a supramolecular structure existing outside the cell and may be naturally derived or an artificial product (recombinant). Examples thereof include substances such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin and laminin, or fragments thereof. These extracellular matrices may be used in combination, for example, preparations from cells such as BD Matrigel TM. Laminin or a fragment thereof is preferable.
- laminin is a protein having a heterotrimeric structure having one ⁇ chain, one ⁇ chain, and one ⁇ chain, and is an extracellular matrix protein in which isoforms having different subunit chain compositions exist. .
- Laminin has about 15 isoforms in a combination of heterotrimers of 5 ⁇ chains, 4 ⁇ chains and 3 ⁇ chains.
- the ⁇ chain is ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, or ⁇ 5, the ⁇ chain is ⁇ 1, ⁇ 2, ⁇ 3, or ⁇ 4, and the ⁇ chain is exemplified by ⁇ 1, ⁇ 2, or ⁇ 3
- the laminin used in the present invention is more preferably laminin 511 consisting of ⁇ 5, ⁇ 1 and ⁇ 1 (Nat Biotechnol 28, 611-615 (2010)).
- laminin may be a fragment and is not particularly limited as long as it has an integrin binding activity. For example, E8 fragment (EMBO J. 3: 1463-1468, 1984, J. Cell Biol., 105: 589-598, 1987).
- laminin 511E8 (preferably human laminin 511E8) described in WO2011 / 043405, preferably obtained by digesting laminin 511 with elastase, is exemplified.
- the laminin E8 fragment such as laminin 511E8 used in the present invention does not need to be an elastase digestion product of laminin, and may be a recombinant.
- Laminin 511E8 is commercially available, and can be purchased from, for example, Nippi Corporation. From the viewpoint of avoiding contamination with unidentified components, the laminin or laminin fragment used in the present invention is preferably isolated.
- the differentiation inducing factor is not particularly limited as long as it is a substance that induces differentiation from pluripotent stem cells to Corin and / or Lrtm1-positive cells or dopaminergic neural progenitor cells, but specifically, BMP inhibitor, TGF ⁇ inhibition Agents, SHH signal stimulants, FGF8 and GSK3 ⁇ inhibitors.
- the BMP inhibitor is not particularly limited as long as it is a substance that inhibits signal transduction caused by BMP, and may be any of nucleic acid, protein, and low molecular weight organic compound.
- BMP examples include BMP2, BMP4, BMP7, and GDF7.
- BMP inhibitors substances that directly act on BMP (for example, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (for example, antisense oligonucleotides, siRNA, etc.), BMP receptor (BMPR) and BMP Examples include substances that inhibit binding, and substances that inhibit physiological activity resulting from signal transduction by the BMP receptor.
- BMPR examples include ALK2 and ALK3.
- BMP signaling pathway inhibitors including proteinaceous inhibitors such as Chordin, Noggin, Follistatin, Dorsomorphin (ie, 6- [4- (2-piperidin-1- yl-ethoxy) phenyl] -3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. 2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al.
- LDN193189 is well known as a BMPR (ALK2 / 3) inhibitor (hereinafter referred to as BMPR inhibitor), and is commercially available, for example, in the form of hydrochloride.
- Dorsomorphin and LDN193189 are available from Sigma-Aldrich and Stemgent, respectively. As a BMP inhibitor, one or more of these may be appropriately selected and used.
- the BMP inhibitor used in the present invention may preferably be LDN193189.
- the concentration of LDN193189 in the medium is not particularly limited as long as it is a concentration that inhibits BMP.
- it is 100 nM.
- the TGF ⁇ inhibitor is a substance that inhibits signal transduction from binding of TGF ⁇ to the receptor of TGF ⁇ to SMAD, and is not particularly limited as long as it is a substance that inhibits the signal transduction pathway resulting from it. Any of a nucleic acid, protein, and a low molecular weight organic compound may be sufficient.
- TGF ⁇ examples include substances that directly act on TGF ⁇ (eg, proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGF ⁇ (eg, antisense oligonucleotides, siRNA, etc.), TGF ⁇ receptors and TGF ⁇
- examples include substances that inhibit binding, and substances that inhibit physiological activity resulting from signal transduction by the TGF ⁇ receptor (for example, inhibitors of TGF ⁇ receptor, inhibitors of Smad, etc.).
- TGF ⁇ receptor ALK family examples include substances that inhibit the binding to the receptor ALK family, or substances that inhibit the phosphorylation of SMAD by the ALK family.
- Lefty-1 NCBI Accession No.
- mice includes mouse: NM_010094, human: NM_020997 Exemplified), Lefty-2 (NCBI Accession No., mouse: NM_177099, human: NM_003240 and NM_001172425 are exemplified), SB431542, SB202190 (above, RKLindemann et al., Mol. Cancer, 2003, 2:20 ), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO2009 / 146408) and derivatives thereof.
- SB431542 SB202190 (above, RKLindemann et al., Mol. Cancer, 2003, 2:20 ), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY
- the TGF ⁇ inhibitor used in the present invention is preferably SB431542 (4- (5-Benzol [1,3] dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl) -benzamide. ) Or A-83-01 (3- (6-methyl-2-pyridinyl) -N-phenyl-4- (4-quinolinyl) -1H-pyrazole-1-carbothioamide), which is a TGF ⁇ receptor Known as inhibitors of the body (ALK5) and Activin receptor (ALK4 / 7). As a TGF ⁇ inhibitor, one or more of these may be appropriately selected and used.
- the TGF ⁇ inhibitor used in the present invention may more preferably be A83-01.
- the concentration of A83-01 in the medium is not particularly limited as long as it inhibits ALK5.
- it is 500 nM to 5 ⁇ M, more preferably 500 nM.
- TGF ⁇ inhibitory activity such as SB431542 and LDN193189 can be determined by methods well known to those skilled in the art, for example, by detecting phosphorylation of Smad by Western blotting (Mol Cancer Ther. (2004) 3, 737-45.) .
- the activity of SHH stimulants such as Purmorphamine and SAG can be determined by methods well known to those skilled in the art, for example, a reporter gene assay focusing on the expression of the Gli1 gene (Oncogene (2007) 26, 5163-5168).
- the GSK3 ⁇ inhibitor is defined as a substance that inhibits the kinase activity of GSK3 ⁇ protein (for example, phosphorylation ability for ⁇ -catenin), and many of them are already known.
- an indirubin derivative is used.
- BIO also known as GSK3 ⁇ inhibitor IX; 6-bromoindirubin 3′-oxime
- a maleimide derivative SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) ) -1H-pyrrole-2,5-dione
- GSK3 ⁇ inhibitor VII (4-dibromoacetophenone
- a phenyl ⁇ bromomethyl ketone compound also known as GSK3 ⁇ peptide inhibition
- a cell membrane permeation type phosphorylated peptide Agent Myr-N-GKEAPPAPPQpSP-NH 2 (SEQ ID NO: 1)
- CHIR99021 (6- [2- [4- (2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazole) having high selectivity -2-yl) pyrimidin-2-ylamino] ethylamino] pyridine-3-carbonitrile
- GSK3 ⁇ inhibitor As a GSK3 ⁇ inhibitor, one or more of these may be appropriately selected and used. These compounds are commercially available from, for example, Calbiochem and Biomol, and can be easily used, but may be obtained from other sources or may be prepared by themselves.
- the GSK3 ⁇ inhibitor used in the present invention may preferably be CHIR99021.
- the concentration of CHIR99021 in the medium is, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, although it is 40 ⁇ M and 50 ⁇ M, it is not limited to these. Preferably, it is 1 ⁇ M.
- FGF8 is not particularly limited, but in the case of human FGF8, four splicing forms of FGF8a, FGF8b, FGF8e, or FGF8f are exemplified, and FGF8b is more preferable.
- FGF8 is commercially available from, for example, Wako, R & D systems, etc., and can be easily used, but may be obtained by forced expression in cells by methods known to those skilled in the art.
- the concentration of FGF8 in the medium is, for example, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, although it is 2000 ng / mL and 5000 ng / mL, it is not limited to these. Preferably, it is 100 ng / mL.
- Corin positive cells and / or Lrtm1 positive cells are cells in which the amount of Corin protein and / or Lrtm1 protein expressed by an anti-Corin antibody or anti-Lrtm1 antibody can be recognized. That is, a cell expressing an amount of Corin protein or Ltrm1 protein capable of recognizing cells by the following “method for selecting cells” on the cell surface can be mentioned.
- a substance that specifically binds to Corin or Lrtm1 in order to select Corin positive cells and / or Lrtm1 positive cells from the cell population, a substance that specifically binds to Corin or Lrtm1 can be used.
- an antibody or an aptamer can be used, and preferably an antibody or an antigen-binding fragment thereof.
- the antibody may be a polyclonal or monoclonal antibody.
- the antibody when the antibody is a polyclonal antibody, a protein encoded by Corin or Lrtm1 expressed in E. coli or a mammalian cell line, etc. and purified according to a conventional method, an oligopeptide or glycolipid having a partial amino acid sequence is purified. It is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, it can be obtained from hybridoma cells prepared by cell fusion of spleen cells and myeloma cells obtained from the immunized non-human animal described above (Current protocols in Molecular Biology).
- antigen-binding fragment of an antibody examples include a part of an antibody (eg, a Fab fragment) or a synthetic antibody fragment (eg, a single chain Fv fragment “ScFv”).
- Antibody fragments such as Fab and F (ab) 2 fragments can also be prepared by methods well known in genetic engineering. For example, antibodies against Corin can be obtained by the production methods described in WO2004 / 065599 and WO2006 / 009241, and antibodies against Lrtm1 can be obtained by the production methods described in WO2013 / 015457.
- the sequence of human Corin can be obtained by NCBI accession number NM_006587.
- the sequence of human Lrtm1 can be obtained by NM_020678.
- the substance to be bound is, for example, a detectable substance such as fluorescent label, radioactive label, chemiluminescent label, enzyme, biotin or streptavidin or protein A Alternatively, it may be bound or conjugated to a substance allowing isolation and extraction, such as protein G, beads or magnetic beads.
- the binding substance may also be indirectly labeled.
- a method for detecting dopaminergic neural progenitor cells includes using a flow cytometer or a protein chip.
- Methods for extracting dopaminergic neural progenitor cells include a method in which particles are joined to the binding substance and precipitated, a method in which cells are magnetically sorted using magnetic beads (e.g., MACS), and a cell sorter using a fluorescent label. Examples thereof include a method of using, or a method of using a carrier (eg, a cell concentration column) on which an antibody or the like is immobilized.
- aptamers that specifically bind to Corin or Lrtm1 can be prepared using techniques well known to those skilled in the art (SELEX (systematic evolution of ligand by exponential enrichment) method: Ellington, AD & Szostak). , JW (1990) Nature, 346, 818-822., Tuerk, C. & Gold, L. (1990) Science, 249, 505-510.).
- a neurotrophic factor is a ligand to a membrane receptor that plays an important role in survival and function maintenance of motor neurons, such as Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF) , Neurotrophin 3 (NT-3), Neurotrophin 4/5 (NT-4 / 5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor ( HGF), Insulin, Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF- ⁇ 2, TGF- ⁇ 3, Interleukin 6 (IL-6 ), Ciliary Neurotrophic Factor (CNTF) and LIF.
- NGF Nerve Growth Factor
- BDNF Brain-derived Neurotrophic Factor
- NT-3 Neurotrophin 3
- Neurotrophin 4/5 NT-4 / 5
- Neurotrophin 6 Neurotrophin 6
- a preferred neurotrophic factor in the present invention is a factor selected from the group consisting of GDNF and BDNF.
- the neurotrophic factor is commercially available from, for example, Wako and R & D systems, and can be easily used. However, it may be obtained by forced expression in cells by methods known to those skilled in the art.
- the concentration of GDNF1 in the medium is, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / Examples include, but are not limited to, mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL. Preferably, it is 10 ng / mL.
- the concentration of BDNF1 in the medium is, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / Examples include, but are not limited to, mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL. Preferably, it is 20 ng / mL.
- the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
- ROCK Rho kinase
- Y-27632 for example, Ishizaki et al., Mol. Pharmacol. 57, 976- 983 (2000); see Narumiya et al., Methods Enzymol. 325,273-284 (2000)
- Fasudil / HA1077 see, eg, Uenata et al., Nature 389: 990-994 (1997)
- H-1152 eg, Sasaki et al., Pharmacol. Ther.
- ROCK inhibitors may be used.
- the ROCK inhibitor used in the present invention may preferably be Y-27632.
- the concentration of Y-27632 is, for example, 100 nM, 500 nM, 750 nM, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M. It is not limited to. Preferably, it is 10 ⁇ M.
- medium X-containing medium and “in the presence of substance X” mean a medium to which exogenous substance X is added or a medium containing exogenous substance X, or foreign Means the presence of sex substance X. That is, when cells or tissues present in the medium express, secrete or produce the substance X endogenously, the endogenous substance X is distinguished from the foreign substance X, and the foreign substance It is understood that a medium not containing X does not fall under the category of “medium containing substance X” even if it contains endogenous substance X.
- the present invention provides a method for producing a cell population containing Corin and / or Lrtm1 positive cells, comprising the following steps (1) and (2): (1) Adhesive culture of pluripotent stem cells in an undifferentiated maintenance medium containing an SHH signal stimulator and an undifferentiated maintenance factor in the absence of feeder cells, and (2) obtained in step (1) Culturing the cell population in a medium containing one or more differentiation-inducing factors.
- Preferred pluripotent stem cells in the step (1) are induced pluripotent stem cells or embryonic stem cells (ES cells, iPS cells), more preferably human induced pluripotent stem cells or human embryonic stem cells (ES cells, iPS cells). ).
- the method for producing induced pluripotent stem cells is not particularly limited, and as described above, it can be produced by a method well known to those skilled in the art. It is desirable that the step of establishing pluripotent stem cells is also performed feeder-free.
- the method for obtaining embryonic stem cells (ES cells, iPS cells) is not particularly limited, and can be produced by methods well known to those skilled in the art as described above.
- Embryonic stem cells (ES cells, iPS cells) It is desirable to perform the manufacturing process in a feeder free manner.
- the maintenance / expansion culture of pluripotent stem cells can be carried out by adhesion culture or suspension culture, but is preferably carried out by adhesion culture.
- Maintenance culture / expansion culture of pluripotent stem cells may be performed in the presence of a feeder or feeder-free, but is preferably performed feeder-free.
- feeder-free in the absence of feeder cells (feeder-free) in maintenance culture and expansion culture of pluripotent stem cells is a condition that does not substantially contain feeder cells (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less) Means.
- maintenance culture and expansion culture of pluripotent stem cells are performed under conditions that do not include feeder cells.
- it can be performed in the presence of a ROCK inhibitor.
- the undifferentiated maintenance medium used in step (1) is not particularly limited as long as it is a medium in which an undifferentiated maintenance factor is added to the basal medium and the pluripotent stem cells can survive while maintaining pluripotency.
- the differentiation maintenance factor and the basal medium are as described above.
- the undifferentiated maintenance medium used in step (1) specifically, a commercially available pluripotent stem cell medium to which an undifferentiated maintenance factor has already been added can be used. Alternatively, TGF ⁇ and the like can be mentioned.
- an undifferentiated maintenance medium for example, Essential 8 (manufactured by Life Technologies), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Life Technologies), hESF9 (Proc Natl Acad Sci US A. 2008 Sep 9 105 (36): 13409-14), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), StemFit (manufactured by Ajinomoto Co., Inc.), preferably Examples include Essential 8 or StemFit. Further, the above-described ROCK inhibitor can be appropriately added to an undifferentiated maintenance medium for use.
- the absence of feeder cells means a condition that substantially does not contain feeder cells (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less).
- step (1) is performed under conditions that do not include feeder cells.
- the pluripotent stem cell culture in the step (1) may be performed under any conditions of suspension culture and adhesion culture, but is preferably performed by adhesion culture.
- the incubator used for the adhesion culture is not particularly limited as long as it can perform “adhesion culture”, but a cell adhesion incubator is preferable.
- Examples of the cell-adhesive incubator include an incubator in which the surface of the incubator is artificially treated for the purpose of improving the adhesion to cells. Specifically, the inside described above is coated with a coating agent. Incubator.
- laminin 511 examples include laminin ⁇ 5 ⁇ 1 ⁇ 1 (hereinafter referred to as laminin 511), laminin ⁇ 1 ⁇ 1 ⁇ 1 (hereinafter referred to as laminin 111), and laminin fragments (such as laminin 511E8), entactin, collagen, gelatin, vitronectin (Vitronectin), Examples include an extracellular matrix such as Synthemax (Corning) and Matrigel, or a polymer such as polylysine and polyornithine.
- a culture vessel having a surface processed such as a positive charge treatment can also be used.
- laminin is mentioned, More preferably, laminin 511E-8 is mentioned.
- Laminin 511E-8 can be purchased commercially (eg, iMatrix-511, Nippi).
- the medium used in step (1) contains a SHH stimulant.
- Corrin-expressing cells can be produced with high efficiency by treating pluripotent stem cells with an SHH stimulator in step (1) and then subjecting them to adhesion culture in the presence of a differentiation-inducing factor in step (2).
- the concentration of the SHH stimulant can be appropriately set as long as the above-described effect can be achieved.
- Shh protein is usually used at a concentration of 20 to 1000 ng / ml, preferably 50 to 300 ng / ml.
- SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM, more preferably 30 to 600 nM, and even more preferably 30 to 300 nM.
- PMA is usually used at a concentration of 0.002 to 20 ⁇ M, preferably 0.02 to 2 ⁇ M.
- the SHH stimulant can be appropriately used in an amount having a SHH stimulating action equivalent to that of SAG at the above concentration.
- the medium used in step (1) may be a serum medium or a serum-free medium, but is preferably a serum-free medium from the viewpoint of avoiding contamination with chemically undecided components.
- the medium used in the step (1) may be a medium in which the components are chemically determined from the viewpoint of avoiding contamination of chemically undecided components.
- an appropriate matrix may be used as the scaffold in order to provide a scaffold instead of feeder cells for the pluripotent stem cells.
- the pluripotent stem cells are adhered and cultured in a cell container whose surface is coated with a matrix as a scaffold.
- Matrix that can be used as a scaffold includes laminin (Nat Biotechnol 28, 611-615 (2010)), laminin fragment (Nat Commun 3, 1236 (2012)), basement membrane preparation (Nat Biotechnol 19, 971-974 ( 2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, vitronectin and the like.
- the surface is coated with isolated laminin 511 or an E8 fragment of laminin 511 (more preferably, an E8 fragment of laminin 511).
- pluripotent stem cells are cultured for adhesion.
- the culture time of pluripotent stem cells in step (1) is not particularly limited as long as the effect of improving the quality of the cell population formed in step (2) can be achieved, but usually 0.5 to 48 hours. It is.
- the culture time of pluripotent stem cells in the step (1) is preferably 1 hour or more, 2 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, or 24 hours or more.
- the culture time of pluripotent stem cells in step (1) is preferably within 36 hours or within 28 hours.
- the range of pluripotent stem cell culture time in step (1) is preferably 2 to 48 hours, more preferably 6 to 48 hours, still more preferably 12 to 36 hours, and still more preferably 18 to 28. Time (eg, 24 hours).
- the first step is started 0.5 to 48 hours (preferably 18 to 28 hours) before the start of the step (2), and the step (2) is continued after the completion of the step (1).
- the range of culture time of pluripotent stem cells in step (1) is preferably 18 to 48 hours, 18 to 36 hours, 18 to 28 hours, or 24 to 28 hours.
- the respective treatment times can be within the above-described culture time range.
- step (1) is performed without adding a ROCK inhibitor.
- Culture conditions such as culture temperature and CO 2 concentration in step (1) can be set as appropriate.
- the culture temperature is, for example, about 30 ° C. to about 40 ° C., preferably about 37 ° C.
- the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
- human pluripotent stem cells are adherently cultured in a serum-free medium containing bFGF and an SHH signal stimulator in the absence of feeder cells.
- the adhesion culture is preferably performed in a cell container whose surface is coated with laminin 511, an E8 fragment of laminin 511 or vitronectin.
- the adhesion culture is preferably performed using Essential 8, TeSR medium, mTeSR medium, mTeSR-E8 medium, or StemFit medium, more preferably Essential 8 or StemFit medium as a feeder-free medium.
- human pluripotent stem cells eg, human iPS cells
- a serum-free medium containing bFGF and an SHH signal stimulator in the absence of feeder cells.
- human pluripotent stem cells may form aggregates of human pluripotent stem cells.
- the cell obtained by step (1) is a cell in which the pluripotent-like state is maintained, and the pluripotent-like property is maintained through step (1).
- a pluripotent-like property means a state in which at least a part of a trait peculiar to pluripotent stem cells common to pluripotent stem cells, including pluripotency, is maintained. The pluripotency-like nature does not require strict pluripotency. Specifically, a state in which all or part of a marker serving as an indicator of a pluripotent state is expressed as “pluripotent-like property”. Markers of pluripotency-like properties include Oct3 / 4 positive, alkaline phosphatase positive and the like.
- cells that retain pluripotent-like properties are Oct3 / 4 positive. Even when the expression level of Nanog is lower than that of ES cells or iPS cells, it falls under “cells exhibiting pluripotency-like properties”.
- the cells obtained by step (1) are Corin and / or Lrtm1 positive cells, dopaminergic neural progenitor cells, or stem cells having the ability to differentiate into dopaminergic neurons.
- the cells obtained by step (1) are Oct3 / 4 positive stem cells having the ability to differentiate into at least Corin and / or Lrtm1 positive cells, dopaminergic neural progenitor cells, or dopaminergic neurons.
- human pluripotent stem cells are adherently cultured in a serum-free medium containing a SHH signal stimulator and bFGF in the absence of feeder cells.
- Said adhesion culture is preferably carried out in a cell container whose surface is coated with laminin 511 or E8 fragment of laminin 511.
- the SHH signal stimulator is preferably Shh protein, SAG or Purmorphamine (PMA), more preferably SAG.
- a combination of a TGF ⁇ inhibitor eg, Lefty, SB431542, A-83-01
- a BMP inhibitor eg, LDN193189
- SHH signal stimulator eg, Shh protein, SAG, PMA
- Specific examples include a combination of SAG and A-83-01, SAG and LDN193189.
- the culture time is 0.5 to 48 hours (preferably 18 to 48 hours, 18 to 36 hours, 18 to 28 hours, or 24 to 28 hours).
- pluripotent stem cells are maintained and cultured in a medium containing an undifferentiated maintenance factor (undifferentiated maintenance medium) in the absence of feeder cells, and SHH signal stimulation is performed in this medium. It is carried out by adding an agent or changing the medium to an undifferentiated maintenance medium containing an SHH signal stimulator and continuing the culture.
- human pluripotent stem cells eg, human iPS cells
- the maintenance culture is preferably performed by adhesion culture.
- the adhesion culture is preferably performed in a cell container whose surface is coated with vitronectin, laminin 511 or E8 fragment of laminin 511.
- an SHH signal stimulator is added to the medium, and the culture is continued.
- the SHH signal stimulator is preferably Shh protein, SAG or PMA.
- TGF ⁇ inhibitors eg, Lefty, SB431542, A-83-01
- BMP inhibitors eg, LDN193189
- the culture is continued for 0.5 to 48 hours (preferably 18 to 48 hours, 18 to 28 hours, or about 24 hours).
- the step (2) can be performed using a medium containing a differentiation-inducing factor that can induce differentiation of pluripotent stem cells into Corin and / or Lrtm1-positive cells.
- a step of adhesion-culturing pluripotent stem cells on an extracellular matrix in a medium containing a BMP inhibitor and a TGF ⁇ inhibitor a step of adhering and culturing the cells obtained in the step (a) on an extracellular matrix in a medium containing a BMP inhibitor, a TGF ⁇ inhibitor, an SHH signal stimulator, and FGF8;
- C Adherent culture of the cells obtained in the step (b) on an extracellular matrix in a medium containing a BMP inhibitor, a TGF ⁇ inhibitor, an SHH signal stimulator, FGF8 and a GSK3 ⁇ inhibitor
- D A step of adhesion-culturing the cells obtained in the step (c) on an extracellular matrix in a medium containing a BMP inhibitor, a TGF ⁇ inhibitor, an SHH
- the medium used in the step (2) is a medium used for culturing animal cells, and specifically used for differentiating cells from undifferentiated cells such as pluripotent stem cells.
- the medium can be prepared as a basal medium.
- As the basal medium for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 Medium, Fischer's medium, Neurobasal Medium (manufactured by Life Technologies), mixed media thereof and the like are included.
- GMEM Glasgow's Minimal Essential Medium
- IMDM Medium IMDM medium
- Medium 199 medium medium
- EMEM Eagle's Minimum Essential Medium
- DMEM Dulbecco's modified Eagle's Medium
- Ham's F12 medium RPMI 1640 Medium
- Fischer's medium Neurobasal Medium (manufacture
- the medium may contain serum or may be serum-free. If necessary, the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen precursor, trace element, 2 - May contain one or more serum replacements such as mercaptoethanol, 3'-thiol glycerol, lipids, amino acids, L-glutamine, GlutaMax (Life Technologies), non-essential amino acids, vitamins, growth factors, low It may also contain one or more substances such as molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
- KSR Knockout Serum Replacement
- a preferred medium is a GMEM medium containing KSR, 2-mercaptoethanol, non-essential amino acids and pyruvate.
- step (2) for example, when the step (2) is performed by the steps (a), (b), (c) and (d) above, in the step (b) and the step (c) in the step (2)
- SHH signal stimulating agent any one of the above-mentioned SHH signal stimulating agents may be appropriately selected or used, but preferably, Purmorphamine is used.
- the concentration of Purmorphamine in the medium is not particularly limited as long as it activates Gli2, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, Examples include, but are not limited to, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 40 ⁇ M, and 50 ⁇ M. Preferably, it is 2 ⁇ M.
- step (2) adhesion culture in the presence of an extracellular matrix, preferably on the extracellular matrix, can be performed by culturing using a culture vessel coated with the extracellular matrix.
- the coating treatment can be performed by placing a solution containing an extracellular matrix in a culture container and then removing the solution as appropriate.
- step (a) is performed in a medium further containing a ROCK inhibitor. That is, the step (a) may be a “step of adherent culture of pluripotent stem cells on a extracellular matrix in a medium containing a ROCK inhibitor, a BMP inhibitor and a TGF ⁇ inhibitor”.
- the culture temperature is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C.
- the culture is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%.
- the culture period is not particularly limited as long as Corin and / or Lrtm1 positive cells appear, but the ratio of Corin and / or Lrtm1 positive cells contained in the cell population obtained after completion of step (2) is Culturing is preferably performed so as to be 10% or more, and step (2) is desirably performed for at least 10 days. More preferably, it is 12 days to 21 days, and more preferably 12 days to 14 days.
- the number of days is 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more.
- the number of days is 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more.
- the number of days is 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more.
- 4 days Preferably, 4 days.
- the number of days is 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more.
- it is 5 days.
- Pluripotent stem cells may be used after dissociating the cells.
- the method for dissociating the cells include a method of dynamically dissociating, a dissociation solution having protease activity and collagenase activity (for example, Accutase (trademark) and And a dissociation method using a dissociation solution having only collagenase activity.
- a method of dissociating human pluripotent stem cells using trypsin or an alternative thereof is used.
- Step (3)> The step of collecting Corin and / or Lrtm1-positive cells in step (3) can be performed based on the above-described ⁇ Method for selecting cells>.
- the medium used in step (4) can be prepared using a medium used for culturing animal cells as a basal medium.
- a basal medium for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 Medium, Fischer's medium, Neurobasal Medium (manufactured by Life Technologies), mixed media thereof and the like are included.
- GMEM Glasgow's Minimal Essential Medium
- IMDM Medium IMDM medium
- Medium 199 medium for example, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 Medium, Fischer's medium, Neurobasal Medium (manufactured by Life Technologies), mixed media thereof and the like are included.
- DMEM
- the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMax (Life Technologies), non-essential amino acids It may also contain one or more substances such as vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, nucleic acids (eg, Dibutyryl cyclic AMP (dbcAMP)).
- KSR Knockout Serum Replacement
- dbcAMP Dibutyryl cyclic AMP
- a preferred medium is Neurobasal Medium containing B27 supplement, ascorbic acid and dbcAMP.
- a neurotrophic factor can be appropriately added to this medium and cultured.
- the suspension culture in the step (4) is culturing cells in a non-adherent state in a culture vessel, and is not particularly limited, but artificially treated for the purpose of improving adhesion with cells (for example, Culture containers not coated with extracellular matrix, etc., or artificially suppress adhesion (eg, polyhydroxyethyl methacrylic acid (poly-HEMA), nonionic surfactant polyol (Pluronic F-127) Etc.) or a phospholipid-like structure (for example, a water-soluble polymer (Lipidure) containing 2-methacryloyloxyethyl phosphorylcholine as a structural unit) can be used.
- poly-HEMA polyhydroxyethyl methacrylic acid
- Pluronic F-127 nonionic surfactant polyol
- the culture temperature is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C.
- the culture is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%.
- the culture period is not particularly limited as long as Nurr1 or Foxa2 positive cells appear, but step (iii) is preferably performed for at least 7 days. More preferably, it is 7 days to 30 days, more preferably 14 days to 21 days, or 14 days to 16 days, and most preferably 16 days.
- a ROCK inhibitor When performing the step (4) following the step (3), it is desirable to add a ROCK inhibitor appropriately and culture. When a ROCK inhibitor is added, it may be added and cultured for at least 1 day, more preferably 1 day.
- the dopaminergic neural progenitor cells obtained in the present invention can be administered as a preparation to Parkinson's disease patients.
- the obtained dopaminergic neural progenitor cells are suspended in physiological saline or the like and transplanted to the striatal region of the patient.
- the present invention provides a therapeutic agent for Parkinson's disease comprising dopaminergic neural progenitor cells obtained from pluripotent stem cells by the above method.
- the number of dopaminergic neural progenitor cells contained in the therapeutic agent for Parkinson's disease is not particularly limited as long as the graft can be engrafted after administration, but may be contained, for example, 15 ⁇ 10 4 or more.
- Transplantation of dopaminergic neural progenitor cells into a diseased site can be performed by a technique as described in, for example, Nature Neuroscience, 2,1137 (1999) or N Engl J Med.; 344: 710-9 (2001). it can.
- kits for producing dopaminergic neural progenitor cells from pluripotent stem cells includes a medium, an additive, a culture container, or the like used in each step of producing the above-described dopaminergic neural progenitor cells.
- SHH signal stimulator eg, SAG
- BMP inhibitor eg, LDN193189
- TGF ⁇ inhibitor used in step (2)
- SHH signal stimulators eg, Purmorphamine
- FGF8 GSK3 ⁇ inhibitors eg, CHIR99021
- extracellular matrix eg, laminin 511E8
- neurotrophic factors eg, BDNF and GDNF
- Kits may further include a document or instructions describing the procedure of the manufacturing process.
- Example 1 The protocol for producing cells and cultured dopaminergic cells is shown in FIG.
- Human iPS cells, QHJ-I01 are dominant negatives of Oct3 / 4, Sox2, Klf4, L-MYC, LIN28 and p53 (Okita, K., et al. Stem Cells 31, 458-66 (2013); WO2013 / 176233) was received from Professor Yamanaka of Kyoto University as cells obtained by introducing episomal vectors into human PBMC.
- iPS cells were cultured by the method described in Miyazaki T et al., Nat Commun. 3: 1236, 2012.
- the cell population containing the iPS cells after culturing for 24 hours in the presence of SAG was dissociated using TrypLE CTS (Life Technologies), and 1 well was added to a 6-well plate coated with Laminin511E8 (iMatrix-511, Nippon) prepared separately. 5 ⁇ 10 6 cells per cell, and the medium was differentiated into basic medium A [8% KSR (Invitrogen) containing differentiation medium [10 ⁇ M Y-27632 (WAKO), 0.1 ⁇ M LDN193189 (STEMGENT) and 0.5 ⁇ M A83-01 (WAKO)].
- the medium was changed to a basic medium A containing 0.1 ⁇ M LDN193189, 0.5 ⁇ M A83-01, 2 ⁇ M Purmorphamine (WAKO) and 100 ng / mL FGF8 (WAKO).
- the medium was changed to a basic medium A containing 0.1 ⁇ M LDN193189, 0.5 ⁇ M A83-01, 2 ⁇ M Purmorphamine, 100 ng / mL FGF8 and 3 ⁇ M CHIR99021 (WAKO).
- the medium was changed to basal medium A containing 0.1 ⁇ M LDN193189 and 3 ⁇ M CHIR99021.
- the medium was changed to basal medium A containing 0.1 ⁇ M LDN193189 and 3 ⁇ M CHIR99021.
- the medium was changed once a day.
- flow cytometry analysis using an anti-Corin antibody was performed (FIG. 1).
- the anti-Corin antibody was prepared by the following method. Of the cynomolgus monkey Corin gene, a gene sequence encoding a part of the extracellular region (79-453 amino acids) was introduced into 293E cells, and the extracellular region fragment of Corin protein was expressed and recovered. After immunizing mice with the recovered protein, lymphocyte cells were taken out and fused with myeloma cells. Clones reactive to Corin were selected from the fused cell population. The culture supernatant of this clone was used after applying a fluorescent label as an anti-Corin monoclonal antibody.
- the result of measuring the Corin positive rate is shown in FIG.
- the left bar graph shows the target cells in the case of undifferentiated maintenance culture (preconditioning) in the absence of SAG
- the right bar graph shows the Corin positive rate in the case of undifferentiated maintenance culture (preconditioning) in the presence of 300 nM SAG. Indicates.
- the ratio of Corin positive cells was improved by treatment with SAG in an undifferentiated maintenance medium before differentiation induction.
- the collected Corin positive cells are transferred to PrimeSurface 96U plate (Sumitomo Bakelite) at 20000 cells / well, and basic medium B (B27 Supplement without vitamin A (Invitrogen), 20 ng / mL BDNF, 10 ng / mL GDNF, 200 mM Ascorbic acid and 0.4 Suspension culture was carried out using Neurobasal medium (Invitrogen) supplemented with mM dbcAMP (Sigma). At this time, a medium supplemented with 30 ⁇ M Y-27632 was used as the first medium. However, when half the amount was changed once every three days, a medium without Y-27632 was used.
- Suspension culture was carried out 16 days after sorting (day 28), and FoxA2 and Nurr1 (both midbrain markers) were stained to confirm whether they differentiated into dopamine neural progenitor cells. As a result, as shown in FIG. 3, the obtained cells were found to express FoxA2 and Nurr1. At this time, DAPI positive cells indicating the presence of nuclei were taken as 100%, Foxa2 was 99.0%, and Nurr1 was 14.6%.
- Example 2 In the same manner as in Example 1, experiments in which the concentration of SAG to be added was changed and experiments in which A83-01 or LDN193189 was added simultaneously with SAG were performed, and the Corin positive rate was measured. Table 1 shows the examination conditions and the Corin positive rate under each condition. From the above results, it was found that when A83-1 or LDN193189 is added in addition to SAG or SAG, the Corin positive rate is improved depending on the addition concentration of SAG as compared with the control. On the other hand, when only A83-1 and / or LDN193189 was added, no effect was shown.
- Example 3 Manufactured by iPS cells prepared by another method LPF11, which is a human iPS cell, introduced Oct3 / 4, Sox2, Klf4, L-MYC into human PBMC by Sendai virus vector (Cytotune 2.0L, manufactured by ID Pharma) StemFit medium according to the method described in the Kyoto University public protocol (Establishment and maintenance culture of human iPS cells, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyoto-u.ac.jp/j/research/protocol.html) (Ajinomoto Co., Inc.) and Laminin511-E8 (Nippi) were used.
- Example 1 Using these iPS cells, culture and differentiation induction were performed in the same manner as in Example 1. On day 12 (day 12), the ratio of Corin positive cells to the total number of cells was measured using an anti-Corin antibody. The result of measuring the Corin positive rate is shown in FIG. The left bar graph shows the Corin positive rate in the case of undifferentiated maintenance culture (preconditioning) in the absence of SAG, and the right bar graph shows the Corin positive in the case of undifferentiated maintenance culture (preconditioning) in the presence of 300 nM SAG. Indicates the rate. As a result, as in Example 1, it was found that the ratio of Corin positive cells was improved by treatment with SAG in an undifferentiated maintenance medium before differentiation induction, regardless of the type of iPS cells. .
- Example 4 Cell transplantation to animals It was examined whether the cells (corresponding to day 28) prepared in Example 1 were suitable for transplantation. At this time, the medium was exchanged by half every 3 days. When the cells of Day28 were immunostained for TH (dopaminergic neuron marker), Foxa2 and Nurr1 (all of which are midbrain markers), the obtained cells expressed FoxA2 and Nurr1 as shown in FIG. I found out. At this time, DAPI positive cells indicating the presence of nuclei were taken as 100%, Foxa2 was 99.0%, and Nurr1 was 14.6%.
- TH dopaminergic neuron marker
- Foxa2 and Nurr1 all of which are midbrain markers
- Example 1 (day 28) can be confirmed to be graft engraftment and maturation into dopaminergic cells both with and without SAG treatment, and are suitable as cells used for the treatment of Parkinson's disease. It was suggested that
- the present invention is useful for regenerative medicine, particularly for the treatment of Parkinson's disease.
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Abstract
Description
[1] 下記工程(1)および(2)を含む、Corinおよび/またはLrtm1陽性細胞を含む細胞集団の製造方法;
(1)多能性幹細胞を、フィーダー細胞非存在下で、Sonic hedgehog (SHH)シグナル刺激剤及び未分化維持因子を含む未分化維持培地中、細胞外基質の存在下で、接着培養する工程、および
(2)工程(1)で得られた細胞を、1以上の分化誘導因子を含む培地中で培養する工程。
[2] SHHシグナル刺激剤が、SAG(N-Methyl-N’-(3-pyridinylbenzyl)-N’-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane)、shhタンパク質もしくはそのフラグメント、Purmorphamine又はこれらの組合せである、[1]に記載の製造方法。
[3] 工程(1)における未分化維持培地が、更にTGF(Transforming growth factor)β阻害剤又はBMP(Bone Morphogenetic Protein)阻害剤を含む培地である、[1]又は[2]に記載の製造方法。
[4] TGFβ阻害剤が、A83-01であり、BMP阻害剤がLDN193189である、[3]に記載の製造方法。
[5] 未分化維持因子が、少なくともFGF(Fibroblast growth factor)シグナル伝達経路作用物質を含む、[1]~[4]のいずれかに記載の製造方法。
[6] FGFシグナル伝達経路作用物質が、bFGFである、[5]に記載の製造方法。
[7] 前記工程(1)において、多能性幹細胞を、48時間を超えない期間培養する、[1]~[6]のいずれかに記載の製造方法。
[8] 分化誘導因子が、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK(Glycogen synthase kinase)3β阻害剤から成る群より選択される1又は複数の因子である、[1]~[7]のいずれかに記載の製造方法。
[9] 工程(2)が、細胞外基質上にて接着培養する工程である、[1]~[8]のいずれかに記載の製造方法。
[10] 細胞外基質が、ラミニンまたはその断片である、[9]に記載の製造方法。
[11] 細胞外基質が、ラミニン511E8である、[9]に記載の製造方法。
[12] 工程(2)が次の工程を含む、[1]~[11]のいずれかに記載の方法;
(a)工程(1)で得られる細胞を、細胞外基質の存在下で、BMP阻害剤およびTGFβ阻害剤を含む培地中で接着培養する工程、
(b)前記工程(a)で得られた細胞を、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤およびFGF8を含む培地中で、細胞外基質存在下で接着培養する工程、
(c)前記工程(b)で得られた細胞を、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK3β阻害剤を含む培地中で、細胞外基質存在下で接着培養する工程、および(d)前記工程(c)で得られた細胞を、BMP阻害剤およびGSK3β阻害剤を含む培地中で細胞外基質存在下で接着培養する工程。
[13] 工程(a)における培地が、更にROCK阻害剤を含む、[12]に記載の製造方法。
[14] ROCK阻害剤がY-27632である、[13]に記載の製造方法。
[15] 工程(2)におけるBMP阻害剤がLDN193189である、[12]~[14]のいずれかに記載の製造方法。
[16] 工程(2)におけるTGFβ阻害剤がA83-01である、[12]~[15]のいずれかに記載の製造方法。
[17] 工程(2)におけるSHHシグナル刺激剤がPurmorphamineである、[12]~[16]のいずれかに記載の製造方法。
[18] GSK3β阻害剤がCHIR99021である、[12]~[17]のいずれかに記載の製造方法。
[19] 工程(2)が少なくとも10日間行われる、[1]~[18]のいずれかに記載の製造方法。
[20] 工程(2)が、12日間~21日間行われる、[19]に記載の製造方法。
[21] 工程(2)が、12日間~14日間行われる、[19]に記載の製造方法。
[22] 工程(2)で得られる細胞集団中に含まれるCorinおよび/またはLrtm1陽性細胞の割合が、10%以上である[1]~[21]のいずれかに記載の製造方法。
[23] 以下の工程:
(3)[1]~[22]のいずれかに記載の製造方法により得られる細胞集団から、Corinに結合する物質および/またはLrtm1に結合する物質を用いてCorinおよび/またはLrtm1陽性細胞を収集する工程、及び
(4)工程(3)で得られるCorinおよび/またはLrtm1陽性細胞を、1又は複数の神経栄養因子を含む培地中で浮遊培養する工程、
を含む、ドーパミン産生神経前駆細胞の製造方法。
[24] Corinに結合する物質またはLrtm1に結合する物質が、CorinまたはLrtm1に結合する抗体またはアプタマーである、[23]に記載の製造方法。
[25] 神経栄養因子が、BDNFおよびGDNFである、[23]または[24]に記載の製造方法。
[26] 神経栄養因子を含む培地が、B27サプリメント、アスコルビン酸およびcAMPアナログをさらに含む、[23]~[25]のいずれかに記載の製造方法。
[27] cAMPアナログが、Dibutyryl cyclic AMPである、[26]に記載の製造方法。
[28] 工程(4)における培地が、更にROCK阻害剤を含む、[23]~[27]のいずれかに記載の製造方法。
[29] ROCK阻害剤がY-27632である、[28]に記載の製造方法。
[30] 工程(4)が、少なくとも7日間行われる、[23]~[29]のいずれかに記載の製造方法。
[31] 工程(4)が、14日間~30日間行われる、[30]に記載の製造方法。
[32] 工程(4)が、14日間~16日間行われる、[30]に記載の製造方法。
[33] [1]~[22]のいずれかに記載の製造方法により得られるCorinおよび/またはLrtm1陽性細胞の含有率が10%以上である細胞集団。
[34] [23]~[32]のいずれかに記載の製造方法により得られるドーパミン産生神経前駆細胞。
[35] [23]~[32]のいずれかに記載の製造方法により得られるドーパミン産生神経前駆細胞を含むパーキンソン病治療剤。
<多能性幹細胞>
本発明で使用可能な多能性幹細胞は、生体に存在するすべての細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞であり、それには、特に限定されないが、例えば胚性幹(ES)細胞、核移植により得られるクローン胚由来の胚性幹(ntES)細胞、精子幹細胞(「GS細胞」)、胚性生殖細胞(「EG細胞」)、人工多能性幹(iPS)細胞、培養線維芽細胞や骨髄幹細胞由来の多能性細胞(Muse細胞)などが含まれる。好ましい多能性幹細胞は、ES細胞、ntES細胞、およびiPS細胞である。
ES細胞は、ヒトやマウスなどの哺乳動物の初期胚(例えば胚盤胞)の内部細胞塊から樹立された、多能性と自己複製による増殖能を有する幹細胞である。
ES細胞は、受精卵の8細胞期、桑実胚後の胚である胚盤胞の内部細胞塊に由来する胚由来の幹細胞であり、成体を構成するあらゆる細胞に分化する能力、いわゆる分化多能性と、自己複製による増殖能とを有している。ES細胞は、マウスで1981年に発見され(M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156)、その後、ヒト、サルなどの霊長類でもES細胞株が樹立された (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848;J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165)。
ES細胞作製のための培地として、例えば0.1mM 2-メルカプトエタノール、0.1mM 非必須アミノ酸、2mM L-グルタミン酸、20% KSRおよび4ng/ml bFGFを補充したDMEM/F-12培地を使用し、37℃、2% CO2/98% 空気の湿潤雰囲気下でヒトES細胞を維持することができる(O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224)。また、ES細胞は、3~4日おきに継代する必要があり、このとき、継代は、例えば1mM CaCl2および20% KSRを含有するPBS中の0.25% トリプシンおよび0.1mg/mlコラゲナーゼIVを用いて行うことができる。
ES細胞の選択は、一般に、アルカリホスファターゼ、Oct-3/4、Nanogなどの遺伝子マーカーの発現を指標にしてReal-Time PCR法で行うことができる。特に、ヒトES細胞の選択では、OCT-3/4、NANOG、ECADなどの遺伝子マーカーの発現を指標とすることができる(E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452)。
ヒトES細胞株は、例えばWA01(H1)およびWA09(H9)は、WiCell Reserch Instituteから、KhES-1、KhES-2およびKhES-3は、京都大学再生医科学研究所(京都、日本)から入手可能である。
精子幹細胞は、精巣由来の多能性幹細胞であり、精子形成のための起源となる細胞である。この細胞は、ES細胞と同様に、種々の系列の細胞に分化誘導可能であり、例えばマウス胚盤胞に移植するとキメラマウスを作出できるなどの性質をもつ(M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012)。神経膠細胞系由来神経栄養因子(glial cell line-derived neurotrophic factor (GDNF))を含む培地で自己複製可能であるし、またES細胞と同様の培養条件下で継代を繰り返すことによって、精子幹細胞を得ることができる(竹林正則ら(2008),実験医学,26巻,5号(増刊),41~46頁,羊土社(東京、日本))。
胚性生殖細胞は、胎生期の始原生殖細胞から樹立される、ES細胞と同様な多能性をもつ細胞であり、LIF、bFGF、幹細胞因子(stem cell factor)などの物質の存在下で始原生殖細胞を培養することによって樹立しうる(Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551)。
人工多能性幹(iPS)細胞は、特定の初期化因子を、DNA又はタンパク質の形態で体細胞に導入することによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能、を有する体細胞由来の人工の幹細胞である(K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M.ら,Nat. Biotechnol. 26:101-106 (2008);国際公開WO 2007/069666;Okita, K., et al.Stem Cells 31, 458-66 (2013))。初期化因子は、ES細胞に特異的に発現している遺伝子、その遺伝子産物もしくはnon-coding RNAまたはES細胞の未分化維持に重要な役割を果たす遺伝子、その遺伝子産物もしくはnon-coding RNA、あるいは低分子化合物によって構成されてもよい。初期化因子に含まれる遺伝子として、例えば、Oct3/4、Sox2、Sox1、Sox3、Sox15、Sox17、Klf4、Klf2、c-Myc、N-Myc、L-Myc、Nanog、Lin28、Fbx15、ERas、ECAT15-2、Tcl1、beta-catenin、Lin28b、Sall1、Sall4、Esrrb、Nr5a2、Tbx3、ドミナントネガティブ体p53, shRNA等のp53遺伝子の抑制因子、EBNA1またはGlis1等が例示され、これらの初期化因子は、単独で用いても良く、組み合わせて用いても良い。初期化因子の組み合わせとしては、WO2007/069666、WO2008/118820、WO2009/007852、WO2009/032194、WO2009/058413、WO2009/057831、WO2009/075119、WO2009/079007、WO2009/091659、WO2009/101084、WO2009/101407、WO2009/102983、WO2009/114949、WO2009/117439、WO2009/126250、WO2009/126251、WO2009/126655、WO2009/157593、WO2010/009015、WO2010/033906、WO2010/033920、WO2010/042800、WO2010/050626、WO 2010/056831、WO2010/068955、WO2010/098419、WO2010/102267、WO 2010/111409、WO 2010/111422、WO2010/115050、WO2010/124290、WO2010/147395、WO2010/147612、WO2011/16588、WO2013/176233、Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797、Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528、Eminli S, et al. (2008), Stem Cells. 26:2467-2474、Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275、Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574、Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479、Marson A, (2008), Cell Stem Cell, 3, 132-135、Feng B, et al. (2009), Nat Cell Biol. 11:197-203、R.L. Judson et al., (2009), Nat. Biotech., 27:459-461、Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917、Kim JB, et al. (2009), Nature. 461:649-643、Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503、Heng JC, et al. (2010), Cell Stem Cell. 6:167-74、Han J, et al. (2010), Nature. 463:1096-100、Mali P, et al. (2010), Stem Cells. 28:713-720、Maekawa M, et al. (2011), Nature. 474:225-9.に記載の組み合わせが例示される。
上記初期化因子には、ヒストンデアセチラーゼ(HDAC)阻害剤[例えば、バルプロ酸 (VPA)、トリコスタチンA、酪酸ナトリウム、MC 1293、M344等の低分子阻害剤、HDACに対するsiRNAおよびshRNA(例、HDAC1 siRNA Smartpool (Millipore)、HuSH 29mer shRNA Constructs against HDAC1 (OriGene)等)等の核酸性発現阻害剤など]、MEK阻害剤(例えば、PD184352、PD98059、U0126、SL327およびPD0325901)、Glycogen synthase kinase-3阻害剤(例えば、BioおよびCHIR99021)、DNAメチルトランスフェラーゼ阻害剤(例えば、5-azacytidine)、ヒストンメチルトランスフェラーゼ阻害剤(例えば、BIX-01294 等の低分子阻害剤、Suv39hl、Suv39h2、SetDBlおよびG9aに対するsiRNAおよびshRNA等の核酸性発現阻害剤など)、L-channel calcium agonist (例えばBayk8644)、酪酸、TGFβ阻害剤またはALK5阻害剤(例えば、LY364947、SB431542、616453およびA83-01)、p53阻害剤(例えばp53に対するドミナントネガティブ体、siRNAおよびshRNA)、ARID3A阻害剤(例えば、ARID3Aに対するsiRNAおよびshRNA)、miR-291-3p、miR-294、miR-295およびmir-302などのmiRNA、Wnt Signaling(例えばsoluble Wnt3a)、神経ペプチドY、プロスタグランジン類(例えば、プロスタグランジンE2およびプロスタグランジンJ2)、hTERT、SV40LT、UTF1、IRX6、GLISl、PITX2、DMRTBl等の樹立効率を高めることを目的として用いられる因子も含まれており、本明細書においては、これらの樹立効率の改善目的にて用いられた因子についても初期化因子と別段の区別をしないものとする。また、これら一種または二種以上を適宜選択して使用してもよい。
初期化因子は、タンパク質の形態の場合、例えばリポフェクション、細胞膜透過性ペプチド(例えば、HIV由来のTATおよびポリアルギニン)との融合、マイクロインジェクションなどの手法によって体細胞内に導入してもよい。
一方、DNAの形態の場合、例えば、ウイルス、プラスミド、人工染色体などのベクター、リポフェクション、リポソーム、マイクロインジェクションなどの手法によって体細胞内に導入することができる。ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター(以上、Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007)、アデノウイルスベクター(Science, 322, 945-949, 2008)、アデノ随伴ウイルスベクター、センダイウイルスベクター(WO 2010/008054)などが例示される。また、人工染色体ベクターとしては、例えばヒト人工染色体(HAC)、酵母人工染色体(YAC)、細菌人工染色体(BAC、PAC)などが含まれる。プラスミドとしては、哺乳動物細胞用プラスミドを使用しうる(Science, 322:949-953, 2008)。ベクターには、核初期化物質が発現可能なように、プロモーター、エンハンサー、リボゾーム結合配列、ターミネーター、ポリアデニル化サイトなどの制御配列を含むことができるし、さらに、必要に応じて、薬剤耐性遺伝子(例えばカナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子など)、チミジンキナーゼ遺伝子、ジフテリアトキシン遺伝子などの選択マーカー配列、緑色蛍光タンパク質(GFP)、βグルクロニダーゼ(GUS)、FLAGなどのレポーター遺伝子配列などを含むことができる。また、上記ベクターには、体細胞への導入後、初期化因子をコードする遺伝子もしくはプロモーターとそれに結合する初期化因子をコードする遺伝子を共に切除するために、それらの前後にLoxP配列を有してもよい。
また、RNAの形態の場合、例えばリポフェクション、マイクロインジェクションなどの手法によって体細胞内に導入しても良く、分解を抑制するため、5-メチルシチジンおよびpseudouridine (TriLink Biotechnologies)を取り込ませたRNAを用いても良い(Warren L, (2010) Cell Stem Cell. 7:618-630)。
iPS細胞誘導のための培地としては、例えば、10~15%FBSを含有するDMEM、DMEM/F12又はDME培地(これらの培地にはさらに、LIF、penicillin/streptomycin、puromycin、L-グルタミン、非必須アミノ酸類、2-メルカプトエタノールなどを適宜含むことができる。)または市販の培地[例えば、マウスES細胞培養用培地(TX-WES培地、トロンボX社)、霊長類ES細胞培養用培地(霊長類ES/iPS細胞用培地、リプロセル社)、無血清培地(mTeSR、Stemcell Technology社)]などが含まれる。
培養法の例としては、たとえば、37℃、5%CO2存在下にて、10%FBS含有DMEM又はDMEM/F12培地上で体細胞と初期化因子とを接触させ約4~7日間培養し、その後、細胞をフィーダー細胞(たとえば、マイトマイシンC処理STO細胞、SNL細胞等)上にまきなおし、体細胞と初期化因子の接触から約10日後からbFGF含有霊長類ES細胞培養用培地で培養し、該接触から約30~約45日又はそれ以上ののちにiPS様コロニーを生じさせることができる。
あるいは、37℃、5% CO2存在下にて、フィーダー細胞(たとえば、マイトマイシンC処理STO細胞、SNL細胞等)上で10%FBS含有DMEM培地(これにはさらに、LIF、ペニシリン/ストレプトマイシン、ピューロマイシン、L-グルタミン、非必須アミノ酸類、2-メルカプトエタノールなどを適宜含むことができる。)で培養し、約25~約30日又はそれ以上ののちにES様コロニーを生じさせることができる。望ましくは、フィーダー細胞の代わりに、初期化される体細胞そのものを用いる(Takahashi K, et al. (2009), PLoS One. 4:e8067またはWO2010/137746)、もしくは細胞外基質(例えば、Laminin-5(WO2009/123349)およびマトリゲル(BD社))を用いる方法が例示される。
この他にも、血清を含有しない培地を用いて培養する方法も例示される(Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106:15720-15725)。さらに、樹立効率を上げるため、低酸素条件(0.1%以上、15%以下の酸素濃度)によりiPS細胞を樹立しても良い(Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241またはWO2010/013845)。
上記培養の間には、培養開始2日目以降から毎日1回新鮮な培地と培地交換を行う。また、核初期化に使用する体細胞の細胞数は、限定されないが、培養ディッシュ100cm2あたり約5×103~約5×106細胞の範囲である。
iPS細胞は、形成したコロニーの形状により選択することが可能である。一方、体細胞が初期化された場合に発現する遺伝子(例えば、Oct3/4、Nanog)と連動して発現する薬剤耐性遺伝子をマーカー遺伝子として導入した場合は、対応する薬剤を含む培地(選択培地)で培養を行うことにより樹立したiPS細胞を選択することができる。また、マーカー遺伝子が蛍光タンパク質遺伝子の場合は蛍光顕微鏡で観察することによって、発光酵素遺伝子の場合は発光基質を加えることによって、また発色酵素遺伝子の場合は発色基質を加えることによって、iPS細胞を選択することができる。
また、iPS細胞を移植用細胞の材料として用いる場合、拒絶反応が起こらないという観点から、移植先の個体のHLA遺伝子型が同一もしくは実質的に同一である体細胞を用いることが望ましい。ここで、「実質的に同一」とは、移植した細胞に対して免疫抑制剤により免疫反応が抑制できる程度にHLA遺伝子型が一致していることであり、例えば、HLA-A、HLA-BおよびHLA-DRの3遺伝子座あるいはHLA-Cを加えた4遺伝子座が一致するHLA型を有する体細胞である。
nt ES細胞は、核移植技術によって作製されたクローン胚由来のES細胞であり、受精卵由来のES細胞とほぼ同じ特性を有している(T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502)。すなわち、未受精卵の核を体細胞の核と置換することによって得られたクローン胚由来の胚盤胞の内部細胞塊から樹立されたES細胞がnt ES(nuclear transfer ES)細胞である。nt ES細胞の作製のためには、核移植技術(J.B. Cibelli et al. (1998), Nature Biotechnol., 16:642-646)とES細胞作製技術(上記)との組み合わせが利用される(若山清香ら(2008),実験医学,26巻,5号(増刊), 47~52頁)。核移植においては、哺乳動物の除核した未受精卵に、体細胞の核を注入し、数時間培養することで初期化することができる。
Muse細胞は、WO2011/007900に記載された方法にて製造された多能性幹細胞であり、詳細には、線維芽細胞または骨髄間質細胞を長時間トリプシン処理、好ましくは8時間または16時間トリプシン処理した後、浮遊培養することで得られる多能性を有した細胞であり、SSEA-3およびCD105が陽性である。
本発明において、ドーパミン産生神経前駆細胞とは、特に断りがなければ、ドーパミン産生神経細胞またはドーパミン作動性ニューロンなどを含んでもよいものとする。また、ドーパミン産生神経前駆細胞は、他の細胞種が含まれている細胞集団であってもよく、好ましくはセロトニン神経細胞を含まない細胞集団である。ドーパミン産生神経前駆細胞は、Foxa2、Nurr1、Lmx1a、Pitx3および/またはTH陽性細胞を含有する細胞集団であることが望ましい。本発明において、ヒトFoxa2としては、NCBIアクセッション番号NM_021784またはNM_153675で示されるポリヌクレオチドおよびこれらがコードするタンパク質が挙げられる。本発明において、ヒトNurr1としては、NCBIアクセッション番号NM_006186で示されるポリヌクレオチドおよびこれらがコードするタンパク質が挙げられる。本発明において、ヒトTHとしては、NCBIアクセッション番号NM_000360、NM_199292またはNM_199293で示されるポリヌクレオチドおよびこれらがコードするタンパク質が挙げられる。ヒトLmx1aとしては、NCBIアクセッション番号NM_001174069またはNM_177398で示されるポリヌクレオチドおよびこれらがコードするタンパク質が挙げられる。ヒトPitx3としては、NCBIアクセッション番号NM_005029で示されるポリヌクレオチドおよびこれらがコードするタンパク質が挙げられる。
本発明において、SHH(Sonic hedgehog;ソニック・ヘッジホッグ)シグナル刺激剤とは、SHHが受容体であるPatched (Ptch1)に結合して引き起こされるSmoothened (Smo)の脱抑制およびさらに続くGli2の活性化を引き起こす物質として定義され、例えば、Hedgehogファミリーに属するタンパク質、具体的にはSHHもしくはIHH(Indian Hedgehog;インディアン・ヘッジホッグ)、SHH受容体、SHH受容体アゴニスト、Hh-Ag1.5 (Li, X., et al., Nature Biotechnology, 23, 215~ 221 (2005).)、Smoothened Agonist, SAG (N-Methyl-N’-(3-pyridinylbenzyl)-N’-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane;N-メチル-N'-(3-ピリジニルベンジル)-N'-(3-クロロベンゾ[b]チオフェン-2-カルボニル)-1,4-ジアミノシクロヘキサン)、20a-hydroxycholesterol、Purmorphamine(PMA;9-シクロヘキシル-N-[4-(4-モルホリニル)フェニル]-2-(1-ナグタレニルオキシ)-9H-プリン-6-アミン)およびこれらの誘導体などが例示される(Stanton BZ, Peng LF., Mol Biosyst. 6:44-54, 2010)。SHHシグナル刺激剤として、これら一種または二種以上を適宜選択して使用してもよい。
本発明で使用されるSHHシグナル刺激剤として、好ましくは、SHHタンパク質(Genbankアクセッション番号:NM_000193、NP_000184)、Purmorphamine、SAGが挙げられる。
未分化維持因子は、多能性幹細胞の分化を抑制する作用を有する物質であれば特に限定はない。当業者に汎用されている未分化維持因子としては、プライムド多能性幹細胞(Primed pluripotent stem cells)(例えば、ヒトES細胞やヒトiPS細胞)の場合、FGFシグナル伝達経路作用物質、TGFβ阻害剤、insulin等を挙げることができる。FGFシグナル伝達経路作用物質として具体的には、線維芽細胞増殖因子(例えば、bFGF、FGF4やFGF8)が挙げられる。また、TGFβ阻害剤としては、TGFβシグナル伝達経路作用物質、Nodal/Activinシグナル伝達経路作用物質が挙げられる。TGFβシグナル伝達経路作用物質としては、例えばTGFβ1、TGFβ2が挙げられる。Nodal/Activinシグナル伝達経路作用物質としては、例えばNodal、ActivinA、ActivinBが挙げられる。未分化維持因子として、これら一種または二種以上を適宜選択して使用してもよい。ヒト多能性幹細胞(ヒトES細胞、ヒトiPS細胞)を培養する場合、前記工程(1)における培地は、好ましくは未分化維持因子として、少なくともbFGFを含む。
本明細書において、哺乳動物としては、げっ歯類、有蹄類、ネコ目、霊長類等が包含される。げっ歯類には、マウス、ラット、ハムスター、モルモット等が包含される。有蹄類には、ブタ、ウシ、ヤギ、ウマ、ヒツジ等が包含される。ネコ目には、イヌ、ネコ等が包含される。本発明における「霊長類」とは、霊長目に属するほ乳類動物をいい、霊長類としては、キツネザルやロリス、ツバイなどの原猿亜目と、サル、類人猿、ヒトなどの真猿亜目を挙げることができる。
未分化維持因子は、哺乳動物の種間で交差反応性を有し得るので、培養対象の多能性幹細胞の未分化状態を維持可能な限り、いずれの哺乳動物の未分化維持因子を用いてもよいが、好適には培養する細胞と同一種の哺乳動物の未分化維持因子が用いられる。例えば、ヒト多能性幹細胞の培養には、ヒト未分化維持因子(例、bFGF、FGF4、FGF8、EGF、Nodal、ActivinA、ActivinB、TGFβ1、TGFβ2等)が用いられる。ここで「ヒトタンパク質X」と言う場合には、タンパク質Xが、ヒト生体内で天然に発現するタンパク質Xのアミノ酸配列を有することを意味する。
本発明に用いる未分化維持因子は、好ましくは単離されている。「単離」とは、目的とする成分や細胞以外の因子を除去する操作がなされ、天然に存在する状態を脱していることを意味する。従って、「単離されたタンパク質X」には、培養対象の細胞や組織から産生され細胞や組織及び培地中に含まれている内在性のタンパク質Xは包含されない。「単離されたタンパク質X」の純度(総タンパク質重量に占めるタンパク質Xの重量の百分率)は、通常70%以上、好ましくは80%以上、より好ましくは90%以上、更に好ましくは99%以上、更に好ましくは100%である。従って、一態様において、本発明は、単離された未分化維持因子を提供する工程を含む。また、一態様において、工程(1)に用いる培地中へ、単離された未分化維持因子を外来性(又は外因性)に添加する工程を含む。あるいは、前記工程(1)に用いる培地に予め未分化維持因子が添加されていてもよい。
工程(1)において用いられる培地中の未分化維持因子の濃度は、培養する多能性幹細胞の未分化状態を維持可能な濃度であり、当業者であれば、適宜設定することができる。例えば、具体的には、未分化維持因子として、フィーダー細胞非存在下でbFGFを用いる場合、その濃度は、通常4ng~500ng/mL程度、好ましくは10ng~200ng/mL程度、より好ましくは30ng~150ng/mL程度である。
本発明において細胞の培養に用いられる培地は、動物細胞の培養に通常用いられる培地を基礎培地として調製又は市販品として購入することができ、基礎培地としては、例えば、BME培地、BGJb培地、CMRL 1066培地、Glasgow MEM (GMEM)培地、Improved MEM Zinc Option培地、IMDM培地、Medium 199培地、Eagle MEM培地、αMEM培地、DMEM培地、F-12培地、DMEM/F12培地、IMDM/F12培地、ハム培地、RPMI 1640培地、Fischer’s培地、Neurobasal培地又はこれらの混合培地など、動物細胞の培養に用いることのできる培地を挙げることができる。これらの基礎培地から、本発明の製造方法の各工程において使用される培地を調製することができる。
本発明において「未分化維持因子を含む培地」(未分化維持培地)として用いられる培地は、フィーダーフリーの無血清であり、例えば、基礎培地に未分化維持因子及び後述の血清代替物及び適宜栄養源等を添加することにより、調製することができる。具体的には、例えばDMEM/F12培地にbFGF、KSR、non essential amino acid (NEAA)、L-グルタミン及び2-メルカプトエタノールを添加することにより、調製することができる。
また、未分化維持に適する培地として市販されている、後述の、未分化維持因子を含むフィーダーフリー培地を未分化維持培地として使用することもできる。
本発明における「無血清培地」とは、無調整又は未精製の血清を含まない培地を意味する。本発明では、精製された血液由来成分や動物組織由来成分(例えば、増殖因子)が混入している培地も、無調整又は未精製の血清を含まない限り無血清培地に含まれる。
無血清培地は、血清代替物を含有していてもよい。血清代替物としては、例えば、アルブミン、トランスフェリン、脂肪酸、コラーゲン前駆体、微量元素、2-メルカプトエタノール又は3’チオールグリセロール、あるいはこれらの均等物などを適宜含有するものを挙げることができる。かかる血清代替物は、例えば、WO98/30679に記載の方法により調製することができる。血清代替物としては市販品を利用してもよい。かかる市販の血清代替物としては、例えば、Knockout Serum Replacement(ライフテクノロジー(Life Technologies、現ThermoFisher Scientific)社製;以下、KSRと記すこともある。)、Chemically-defined Lipid concentrated(Life Technologies社製)、GlutaMax(Life Technologies社製)、B27(Life Technologies社製)、N2サプリメント(Life Technologies社製)、ITSサプリメント(Life Technologies社製)が挙げられる。
無血清培地は、適宜、脂肪酸又は脂質、アミノ酸(例えば、非必須アミノ酸)、ビタミン、増殖因子、サイトカイン、抗酸化剤、2-メルカプトエタノール、ピルビン酸、緩衝剤、無機塩類等を含有してもよい。
調製の煩雑さを回避するために、かかる無血清培地として、市販のKSR(Life Technologies社製)を適量(例えば、約0.5%から約30%、好ましくは約1%から約20%)添加した無血清培地(例えば、GMEM培地に約8% KSR、Chemically-defined Lipid concentratedを添加した培地)、又はNeurobasal培地に市販のB27(Life Technologies社製)を適量(例えば、約0.1~5%)添加した無血清培地を使用してもよい。また、KSR同等品として特表2001-508302に開示された培地が挙げられる。
未分化維持培地として使用可能なフィーダーフリー培地として、多くの合成培地が開発・市販されており、例えばEssential 8(Life Technologies社製)が挙げられる。Essential 8培地は、DMEM/F12培地に、添加剤として、L-ascorbic acid-2-phosphate magnesium (64 mg/l), sodium selenium(14 μg/1), insulin(19.4mg /l), NaHCO3(543 mg/l), transferrin (10.7 mg/l), bFGF (100 ng/mL)、及び、TGFβ阻害剤(TGFβ1(2 ng/mL) または Nodal (100 ng/mL))を含む(Nature Methods, 8, 424-429 (2011))。市販の未分化維持因子を含むフィーダーフリー培地、すなわち未分化維持培地として使用可能なフィーダーフリー培地として、S-medium(DSファーマバイオメディカル社製)、StemPro(Life Technologies社製)、hESF9(Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13409-14)、mTeSR1 (STEMCELL Technologies社製)、mTeSR2 (STEMCELL Technologies社製)、TeSR-E8(STEMCELL Technologies社製)、又はStemFit(味の素社製)が挙げられる。上記工程(1)ではこれらを用いることにより、簡便に本発明を実施することが出来る。
本発明において、細胞外基質とは、細胞の外に存在する超分子構造体であり、天然由来であっても、人工物(組換え体)であってもよい。例えば、コラーゲン、プロテオグリカン、フィブロネクチン、ヒアルロン酸、テネイシン、エンタクチン、エラスチン、フィブリリンおよびラミニンといった物質またはこれらの断片が挙げられる。これらの細胞外基質は、組み合わせて用いられてもよく、例えば、BD Matrigel(商標)などの細胞からの調製物であってもよい。好ましくは、ラミニンまたはその断片である。本発明においてラミニンとは、α鎖、β鎖、γ鎖をそれぞれ1本ずつ持つヘテロ三量体構造を有するタンパク質であり、サブユニット鎖の組成が異なるアイソフォームが存在する細胞外マトリックスタンパク質である。ラミニンは、5種のα鎖、4種のβ鎖および3種のγ鎖のヘテロ三量体の組合せで約15種類のアイソフォームを有する。特に限定されないが、例えば、α鎖は、α1、α2、α3、α4またはα5であり、β鎖は、β1、β2、β3またはβ4であり、ならびにγ鎖は、γ1、γ2またはγ3が例示される。本発明において用いられるラミニンは、より好ましくは、α5、β1およびγ1からなるラミニン511である(Nat Biotechnol 28, 611-615 (2010))。
本発明では、ラミニンは断片であってもよく、インテグリン結合活性を有している断片であれば、特に限定されないが、例えば、エラスターゼにて消化して得られる断片であるE8フラグメント(EMBO J., 3:1463-1468, 1984、J. Cell Biol., 105:589-598, 1987)であってもよい。従って、本発明では、好ましくは、ラミニン511をエラスターゼで消化して得られる、WO2011/043405に記載されたラミニン511E8(好ましくはヒトラミニン511E8)が例示される。尚、本発明で用いられるラミニン511E8等のラミニンE8フラグメントは、ラミニンのエラスターゼ消化産物であることを要するものではなく、組換え体であってもよい。またラミニン511E8は市販されており、例えばニッピ株式会社等から購入可能である。
未同定成分の混入を回避する観点から、本発明において用いられるラミニン又はラミニン断片は、好ましくは単離されている。
分化誘導因子としては、多能性幹細胞からCorinおよび/またはLrtm1陽性細胞、またはドーパミン産生神経前駆細胞へ分化誘導する物質であれば特に限定はないが、具体的には、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK3β阻害剤を例示することができる。
本発明において、BMP阻害剤とは、BMPに起因するシグナル伝達を阻害する物質であれば特に限定は無く、核酸、タンパク質、低分子有機化合物のいずれであってもよい。ここでBMPとしては、BMP2、BMP4、BMP7及びGDF7が挙げられる。BMP阻害剤として、BMPに直接作用する物質(例えば抗体、アプタマー等)、BMPをコードする遺伝子の発現を抑制する物質(例えばアンチセンスオリゴヌクレオチド、siRNA等)、BMP受容体(BMPR)とBMPの結合を阻害する物質、BMP受容体によるシグナル伝達に起因する生理活性を阻害する物質を挙げることができる。BMPRとして、ALK2又はALK3を挙げることができる。BMPシグナル伝達経路阻害物質として、当業者に周知の化合物を使用することができ、Chordin、Noggin、Follistatin、などのタンパク質性阻害剤、Dorsomorphin (すなわち、6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine)、その誘導体 (P. B. Yu et al. (2007), Circulation, 116:II_60; P.B. Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904)およびLDN193189(すなわち、4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline;4-[6-(4-ピペラジン-1-イルフェニル)ピラゾロ[1,5-a]ピリミジン-3-イル]キノリン)が例示される。ここでLDN193189は、BMPR(ALK2/3)阻害剤(以下、BMPR阻害剤)として周知であり、例えば塩酸塩の形態で市販されている。DorsomorphinおよびLDN193189は、それぞれSigma-Aldrich社およびStemgent社から入手可能である。BMP阻害剤として、これら一種または二種以上を適宜選択して使用してもよい。本発明で使用されるBMP阻害剤は、好ましくは、LDN193189であり得る。
培地中におけるLDN193189の濃度は、BMPを阻害する濃度であれば特に限定されないが、例えば、1nM、10nM、50nM、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、15μM、20μM、25μM、30μM、40μM、50μMであるがこれらに限定されない。好ましくは、100nMである。
本発明において、TGFβ阻害剤とは、TGFβのTGFβの受容体への結合からSMADへと続くシグナル伝達を阻害する物質であり、起因するシグナル伝達経路を阻害する物質であれば特に限定は無く、核酸、タンパク質、低分子有機化合物のいずれであってもよい。当該物質として例えば、TGFβに直接作用する物質(例えば、タンパク質、抗体、アプタマー等)、TGFβをコードする遺伝子の発現を抑制する物質(例えばアンチセンスオリゴヌクレオチド、siRNA等)、TGFβ受容体とTGFβの結合を阻害する物質、TGFβ受容体によるシグナル伝達に起因する生理活性を阻害する物質(例えば、TGFβ受容体の阻害剤、Smadの阻害剤等)を挙げることができる。受容体であるALKファミリーへの結合を阻害する物質、またはALKファミリーによるSMADのリン酸化を阻害する物質が挙げられ、例えば、Lefty-1(NCBI Accession No.として、マウス:NM_010094、ヒト:NM_020997が例示される)、Lefty-2(NCBI Accession No.として、マウス:NM_177099、ヒト:NM_003240およびNM_001172425が例示される)、SB431542、SB202190(以上、R.K.Lindemann et al., Mol. Cancer, 2003, 2:20)、SB505124 (GlaxoSmithKline)、NPC30345、SD093、SD908、SD208 (Scios)、LY2109761、LY364947、 LY580276 (Lilly Research Laboratories)、A83-01(WO2009/146408) およびこれらの誘導体などが例示される。本発明で使用されるTGFβ阻害剤として、好ましくはSB431542(4-(5-ベンゾール[1,3]ジオキソール-5-イル-4-ピリジン-2-イル-1H-イミダゾール-2-イル)-ベンズアミド)又はA-83-01(3-(6-メチル-2-ピリジニル)-N-フェニル-4-(4-キノリニル)-1H-ピラゾール-1-カルボチオアミド)が挙げられ、これらは、TGFβ受容体(ALK5)及びActivin受容体(ALK4/7)の阻害剤として公知である。TGFβ阻害剤として、これら一種または二種以上を適宜選択して使用してもよい。本発明で使用されるTGFβ阻害剤は、更に好ましくは、A83-01であり得る。
培地中におけるA83-01の濃度は、ALK5を阻害する濃度であれば特に限定されないが、例えば、1nM、10nM、50nM、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、15μM、20μM、25μM、30μM、40μM、50μMであるがこれらに限定されない。好ましくは、500nMから5μMであり、より好ましくは、500nMである。
Purmorphamine、SAG等のSHH刺激剤の活性は、当業者に周知の方法、例えばGli1遺伝子の発現に着目したレポータージーンアッセイにて決定することができる(Oncogene (2007) 26, 5163-5168)。
本発明において、GSK3β阻害剤とは、GSK3βタンパク質のキナーゼ活性(例えば、βカテニンに対するリン酸化能)を阻害する物質として定義され、既に多数のものが知られているが、例えば、インジルビン誘導体であるBIO(別名、GSK3β阻害剤IX;6-ブロモインジルビン3'-オキシム)、マレイミド誘導体であるSB216763(3-(2,4-ジクロロフェニル)-4-(1-メチル-1H-インドール-3-イル)-1H-ピロール-2,5-ジオン)、フェニルαブロモメチルケトン化合物であるGSK3β阻害剤VII(4-ジブロモアセトフェノン)、細胞膜透過型のリン酸化ペプチドであるL803-mts(別名、GSK3βペプチド阻害剤;Myr-N-GKEAPPAPPQpSP-NH2(配列番号1))および高い選択性を有するCHIR99021(6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile)が挙げられる。GSK3β阻害剤として、これら一種または二種以上を適宜選択して使用してもよい。これらの化合物は、例えばCalbiochem社やBiomol社等から市販されており容易に利用することが可能であるが、他の入手先から入手してもよく、あるいはまた自ら作製してもよい。本発明で使用されるGSK3β阻害剤は、好ましくは、CHIR99021であり得る。
培地中におけるCHIR99021の濃度は、例えば、1nM、10nM、50nM、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、15μM、20μM、25μM、30μM、40μM、50μMであるがこれらに限定されない。好ましくは、1μMである。
本発明において、FGF8とは、特に限定されないが、ヒトFGF8の場合、FGF8a、FGF8b、FGF8eまたはFGF8fの4つのスプライシングフォームが例示され、より好ましくは、FGF8bである。FGF8は、例えばWako社やR&D systems社等から市販されており容易に利用することが可能であるが、当業者に公知の方法によって細胞へ強制発現させることによって得てもよい。
培地中におけるFGF8の濃度は、例えば、1ng/mL、5ng/mL、10ng/mL、50ng/mL、100ng/mL、150ng/mL、200ng/mL、250ng/mL、500ng/mL、1000ng/mL、2000ng/mL、5000ng/mLであるがこれらに限定されない。好ましくは、100ng/mLである。
Corin陽性細胞および/またはLrtm1陽性細胞とは、Corinタンパク質および/またはLrtm1タンパク質が、抗Corin抗体または抗Lrtm1抗体により認識できる量発現している細胞である。すなわち、下記の「細胞を選択する方法」で細胞を認識可能な量のCorinタンパク質、又はLtrm1タンパク質を細胞表面上に発現している細胞が挙げられる。
本発明において、細胞集団よりCorin陽性細胞および/またはLrtm1陽性細胞を選択するために、CorinまたはLrtm1に特異的に結合する物質を用いて行うことができる。CorinまたはLrtm1に結合する物質は、抗体、アプタマーを用いることができ、好ましくは、抗体もしくはその抗原結合断片である。
本発明において、抗体はポリクローナルまたはモノクローナル抗体であってよい。これらの抗体は、当業者に周知の技術を用いて作成することが可能である(Current protocols in Molecular Biology edit.Ausubel et al.(1987) Publish.John Wiley and Sons.Section 11.12-11.13)。具体的には、抗体がポリクローナル抗体の場合には、常法に従って大腸菌または哺乳類細胞株等で発現し精製したCorinまたはLrtm1がコードするタンパク質、部分アミノ酸配列を有するオリゴペプチドあるいは糖脂質を精製して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、上述の免疫された非ヒト動物から得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞の中から得ることができる(Current protocols in Molecular Biology edit.Ausubel et al.(1987) Publish.John Wiley and Sons.Section 11.4-11.11)。抗体の抗原結合断片としては、抗体の一部(たとえばFab断片)または合成抗体断片(たとえば、一本鎖Fv断片「ScFv」)が例示される。FabおよびF(ab)2断片などの抗体の断片もまた、遺伝子工学的に周知の方法によって作製することができる。例えば、Corinに対する抗体はWO2004/065599、WO2006/009241に記載の作製法により、Lrtm1に対する抗体はWO2013/015457に記載の作製法により得ることができる。
ヒトCorinは、NCBIのアクセッション番号NM_006587によりその配列を得ることができる。同様に、ヒトLrtm1は、NM_020678によりその配列を得ることができる。
CorinまたはLrtm1を発現する細胞を認識または分離することを目的として、当該結合する物質は、例えば、蛍光標識、放射性標識、化学発光標識、酵素、ビオチンまたはストレプトアビジン等の検出可能な物質またはプロテインA、プロテインG、ビーズまたは磁気ビーズ等の単離抽出を可能とさせる物質と結合または接合されていてもよい。
当該結合する物質はまた、間接的に標識してもよい。当業者に公知の様々な方法を使用して行い得るが、例えば、当該抗体に特異的に結合する予め標識された抗体(二次抗体)を用いる方法が挙げられる。
ドーパミン産生神経前駆細胞を検出する方法として、フローサイトメーターまたはプロテインチップ等を用いることが含まれる。
ドーパミン産生神経前駆細胞を抽出する方法には、当該結合する物質へ粒子を接合させ沈降させる方法、磁気ビーズを用いて磁性により細胞を選別する方法(例えば、MACS)、蛍光標識を用いてセルソーターを用いる方法、または抗体等が固定化された担体(例えば、細胞濃縮カラム)を用いる方法等が例示される。
本発明において、CorinまたはLrtm1に特異的に結合するアプタマーは、当業者に周知の技術を用いて作成することが可能である(SELEX(systematic evolution of ligand by exponential enrichment)法:Ellington, A.D. & Szostak, J.W.(1990)Nature,346,818-822., Tuerk, C. & Gold, L.(1990)Science,249,505-510.)。
本発明において、神経栄養因子とは、運動ニューロンの生存と機能維持に重要な役割を果たしている膜受容体へのリガンドであり、例えば、Nerve Growth Factor (NGF)、Brain-derived Neurotrophic Factor (BDNF)、Neurotrophin 3 (NT-3)、Neurotrophin 4/5 (NT-4/5)、Neurotrophin 6 (NT-6)、basic FGF、acidic FGF、FGF-5、Epidermal Growth Factor (EGF)、Hepatocyte Growth Factor (HGF)、Insulin、Insulin Like Growth Factor 1 (IGF 1)、Insulin Like Growth Factor 2 (IGF 2)、Glia cell line-derived Neurotrophic Factor (GDNF)、TGF-β2、TGF-β3、Interleukin 6 (IL-6)、Ciliary Neurotrophic Factor (CNTF)およびLIFなどが挙げられる。また、これらの一種又は二種以上を適宜選択して用いてもよい。本発明において好ましい神経栄養因子は、GDNF、およびBDNFから成るグループより選択される因子である。神経栄養因子は、例えばWako社やR&D systems社等から市販されており容易に利用することが可能であるが、当業者に公知の方法によって細胞へ強制発現させることによって得てもよい。
培地中におけるGDNF1の濃度は、例えば、0.1ng/mL、0.5ng/mL、1ng/mL、5ng/mL、10ng/mL、15ng/mL、20ng/mL、25ng/mL、30ng/mL、40ng/mL、50ng/mL、100ng/mL、200ng/mL、500ng/mLであるがこれらに限定されない。好ましくは、10ng/mLである。培地中におけるBDNF1の濃度は、例えば、0.1ng/mL、0.5ng/mL、1ng/mL、5ng/mL、10ng/mL、15ng/mL、20ng/mL、25ng/mL、30ng/mL、40ng/mL、50ng/mL、100ng/mL、200ng/mL、500ng/mLであるがこれらに限定されない。好ましくは、20ng/mLである。
本発明において、ROCK阻害剤とは、Rhoキナーゼ(ROCK)の機能を抑制できるものである限り特に限定されず、例えば、Y-27632(例えば、Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000);Narumiya et al., Methods Enzymol. 325,273-284 (2000)参照)、Fasudil/HA1077(例えば、Uenata et al., Nature 389: 990-994 (1997)参照)、H-1152(例えば、Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)参照)、Wf-536(例えば、Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)参照)およびそれらの誘導体、ならびにROCKに対するアンチセンス核酸、RNA干渉誘導性核酸(例えば、siRNA)、ドミナントネガティブ変異体、及びそれらの発現ベクターが挙げられる。また、ROCK阻害剤としては他の低分子化合物も知られているので、本発明においてはこのような化合物またはそれらの誘導体も使用できる(例えば、米国特許出願公開第20050209261号、同第20050192304号、同第20040014755号、同第20040002508号、同第20040002507号、同第20030125344号、同第20030087919号、及び国際公開第2003/062227号、同第2003/059913号、同第2003/062225号、同第2002/076976号、同第2004/039796号参照)。本発明では、1種または2種以上のROCK阻害剤が使用され得る。本発明で使用されるROCK阻害剤は、好ましくは、Y-27632であり得る。
Y-27632の濃度は、例えば、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、15μM、20μM、25μM、30μM、40μM、50μMであるがこれらに限定されない。好ましくは、10μMである。
本発明は、下記工程(1)および(2)を含む、Corinおよび/またはLrtm1陽性細胞を含む細胞集団の製造方法を提供する;
(1)多能性幹細胞を、フィーダー細胞非存在下で、SHHシグナル刺激剤、及び未分化維持因子を含む未分化維持培地中、接着培養する工程、および
(2)工程(1)で得られた細胞集団を、1以上の分化誘導因子を含む培地中で培養する工程。
前記工程(1)における好ましい多能性幹細胞として、人工多能性幹細胞又は胚性幹細胞(ES細胞、iPS細胞)、より好ましくはヒト人工多能性幹細胞又はヒト胚性幹細胞(ES細胞、iPS細胞)が挙げられる。ここで人工多能性幹細胞の製造方法には特に限定はなく、上述のとおり当業者に周知の方法で製造することができるが、人工多能性幹細胞の作製工程(すなわち、体細胞を初期化し多能性幹細胞を樹立する工程)もフィーダーフリーで行うことが望ましい。ここで胚性幹細胞(ES細胞、iPS細胞)を取得する方法には特に限定はなく、上述のとおり当業者に周知の方法で製造することができるが、胚性幹細胞(ES細胞、iPS細胞)の作製工程もフィーダーフリーで行うことが望ましい。
工程(1)で用いられる未分化維持培地は、基礎培地に未分化維持因子が添加され、多能性幹細胞が多能性を維持したまま生存可能な培地であれば特に限定は無く、当該未分化維持因子及び基礎培地については前述のとおりである。工程(1)で用いられる未分化維持培地として、具体的には、既に未分化維持因子が添加された市販の多能性幹細胞用培地を用いることができ、未分化維持因子として、好ましくはbFGF又はTGFβ等が挙げられる。未分化維持培地としては、例えば、Essential 8(Life Technologies社製)、S-medium(DSファーマバイオメディカル社製)、StemPro(Life Technologies社製)、hESF9(Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13409-14)、mTeSR1 (STEMCELL Technologies社製)、mTeSR2 (STEMCELL Technologies社製)、TeSR-E8(STEMCELL Technologies社製)、StemFit(味の素社製)が挙げられ、好ましくは、Essential 8又はStemFitが挙げられる。また、未分化維持培地に、適宜前述のROCK阻害剤を添加して用いることができる。
接着培養を行う際に用いられる培養器は、「接着培養する」ことが可能なものであれば特に限定されないが、細胞接着性の培養器が好ましい。細胞接着性の培養器としては、培養器の表面が、細胞との接着性を向上させる目的で人工的に処理された培養器が挙げられ、具体的には前述した内部がコーティング剤で被覆された培養器が挙げられる。コーティング剤としては、例えば、ラミニン[ラミニンα5β1γ1(以下、ラミニン511)、ラミニンα1β1γ1(以下、ラミニン111)等及びラミニン断片(ラミニン511E8等)を含む]、エンタクチン、コラーゲン、ゼラチン、ビトロネクチン(Vitronectin)、シンセマックス(コーニング社)、マトリゲル等の細胞外マトリクス等、又は、ポリリジン、ポリオルニチン等の高分子等が挙げられる。また、正電荷処理等の表面加工された培養容器を使用することもできる。好ましくは、ラミニンが挙げられ、より好ましくは、ラミニン511E-8が挙げられる。ラミニン511E-8は、市販品を購入する事ができる(例:iMatrix-511、ニッピ)。
SHH刺激剤の濃度は、上述の効果を達成可能な範囲で適宜設定することが可能である。例えば、Shhタンパク質は、通常20~1000 ng/ml、好ましくは50~300 ng/mlの濃度で使用される。SAGは、通常、1~2000nM、好ましくは10~700nM、より好ましくは30~600nM、更に好ましくは30~300nMの濃度で使用される。PMAは、通常0.002~20μM、好ましくは0.02~2μMの濃度で使用される。また、一態様において、SHH刺激剤は、前記濃度のSAGと同等のSHH刺激作用を有する量で適宜使用することができる。
工程(1)において用いられる培地は、化学的に未決定な成分の混入を回避する観点から、含有成分が化学的に決定された培地であってもよい。
足場として用いることのできるマトリクスとしては、ラミニン(Nat Biotechnol 28, 611-615 (2010))、ラミニン断片(Nat Commun 3, 1236 (2012))、基底膜標品 (Nat Biotechnol 19, 971-974 (2001))、ゼラチン、コラーゲン、ヘパラン硫酸プロテオグリカン、エンタクチン、ビトロネクチン(Vitronectin)等が挙げられる。
好ましくは、工程(1)におけるフィーダーフリー条件での多能性幹細胞の培養においては、単離されたラミニン511又はラミニン511のE8フラグメント(更に好ましくは、ラミニン511のE8フラグメント)により、表面をコーティングした細胞容器中で、多能性幹細胞を接着培養する。
一態様において、工程(1)により得られる細胞は、Corinおよび/またはLrtm1陽性細胞、ドーパミン産生神経前駆細胞、又はドーパミン神経細胞へ分化する能力を有する幹細胞である。一態様において、工程(1)により得られる細胞は、少なくともCorinおよび/またはLrtm1陽性細胞、ドーパミン産生神経前駆細胞、又はドーパミン神経細胞へ分化する能力を有する、Oct3/4陽性の幹細胞である。
上記の当該接着培養は、好ましくは、ラミニン511又はラミニン511のE8フラグメントで表面をコーティングした細胞容器中で実施される。SHHシグナル刺激剤は、好ましくはShhタンパク質、SAG又はPurmorphamine(PMA)、より好ましくはSAGである。TGFβ阻害剤(例、Lefty、SB431542、A-83-01)もしくはBMP阻害剤(例、LDN193189)と、SHHシグナル刺激剤(例、Shhタンパク質、SAG、PMA)とを組み合わせて用いてもよい。具体的にはSAG及びA-83-01、SAG及びLDN193189の組合せが挙げられる。培養時間は、0.5~48時間(好ましくは、18~48時間、18~36時間、18~28時間、又は24~28時間)である。
例えば、ヒト多能性幹細胞(例、ヒトiPS細胞)を、フィーダー細胞非存在下で、bFGFを含む無血清培地中で維持培養する。当該維持培養は、好ましくは接着培養により行われる。当該接着培養は、好ましくは、ビトロネクチン、ラミニン511又はラミニン511のE8フラグメントで表面をコーティングした細胞容器中で実施される。そして、工程(1)において、この培地中へSHHシグナル刺激剤を添加し、培養を継続する。SHHシグナル刺激剤は、好ましくはShhタンパク質、SAG又はPMAである。ここで、TGFβ阻害剤(例、Lefty、SB431542、A-83-01)もしくはBMP阻害剤(例、LDN193189)を、SHHシグナル刺激剤(例、Shhタンパク質、SAG、PMA)と組み合わせて用いてもよい。添加後、0.5~48時間(好ましくは、18~48時間、18~28時間、又は約24時間)培養を継続する。
本発明において、前記工程(2)は多能性幹細胞をCorinおよび/またはLrtm1陽性細胞に分化誘導することのできる分化誘導因子を含む培地を用いて行うことができるが、次の多段階の工程によって行われることが望ましい;
(a)多能性幹細胞を細胞外基質上でBMP阻害剤およびTGFβ阻害剤を含む培地中で接着培養する工程、
(b)前記工程(a)で得られた細胞をBMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤およびFGF8を含む培地中で細胞外基質上にて接着培養する工程、
(c)前記工程(b)で得られた細胞をBMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK3β阻害剤を含む培地中で細胞外基質上にて接着培養する工程、
(d)前記工程(c)で得られた細胞をBMP阻害剤およびGSK3β阻害剤を含む培地中で細胞外基質上にて接着培養する工程。
Corinおよび/またはLrtm1陽性細胞を含む細胞集団へ分化させるために、これらの培地へ適宜上述のBMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK3β阻害剤から成る群より選択される試薬を加えて培養することができる。
工程(2)、例えば工程(2)が上記(a)、(b)、(c)及び(d)の工程によって行われる場合においては工程(2)中工程(b)及び工程(c)において用いられるSHHシグナル刺激剤としては、上述したいずれのSHHシグナル刺激剤を、一種または二種以上適宜選択して用いてもよいが、好ましくはPurmorphamineが用いられる。
培地中におけるPurmorphamineの濃度は、Gli2を活性化する濃度であれば特に限定されないが、例えば、1nM、10nM、50nM、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、15μM、20μM、25μM、30μM、40μM、50μMであるがこれらに限定されない。好ましくは、2μMである。
通常は、工程(2)中工程(a)は更にROCK阻害剤を含む培地で行われる。すなわち、工程(a)は、「多能性幹細胞を細胞外基質上でROCK阻害剤、BMP阻害剤およびTGFβ阻害剤を含む培地中で接着培養する工程」であってもよい。
培養条件について、培養温度は、特に限定されないが、約30~40℃、好ましくは約37℃であり、CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2~5%である。
培養期間は、Corinおよび/またはLrtm1陽性細胞が出現する期間であれば、特に限定されないが、工程(2)の終了後に得られる細胞集団中に含まれるCorinおよび/またはLrtm1陽性細胞の割合が、10%以上となるように培養を行うことが好ましく、工程(2)は、少なくとも10日間行われることが望ましい。より好ましくは、12日間から21日間であり、さらに好ましくは、12日間から14日間である。
また工程(2)内の工程(a)では、1日以上、2日以上、3日以上、4日以上、5日以上、6日以上、7日以上、またはそれ以上の日数が挙げられる。好ましくは、1日である。同様に、工程(b)では、1日以上、2日以上、3日以上、4日以上、5日以上、6日以上、7日以上、またはそれ以上の日数が挙げられる。好ましくは、2日である。同様に、工程(c)では、1日以上、2日以上、3日以上、4日以上、5日以上、6日以上、7日以上、またはそれ以上の日数が挙げられる。好ましくは、4日である。同様に、工程(d)では、1日以上、2日以上、3日以上、4日以上、5日以上、6日以上、7日以上、またはそれ以上の日数が挙げられる。好ましくは、5日である。
多能性幹細胞は、細胞を解離させて用いてもよく、細胞を解離させる方法としては、例えば、力学的に解離する方法、プロテアーゼ活性とコラゲナーゼ活性を有する解離溶液(例えば、Accutase(商標)およびAccumax(商標)など)またはコラゲナーゼ活性のみを有する解離溶液を用いた解離方法が挙げられる。好ましくは、トリプシンまたはその代替物(TrypLE CTS(Life Technologies)が例示される)を用いてヒト多能性幹細胞を解離する方法が用いられる。細胞を解離させた場合、ROCK阻害剤を適宜、解離後に添加して培養することが望ましい。ROCK阻害剤を添加する場合、少なくとも1日間添加して培養すればよく、より好ましくは1日間である。
<工程(3)>
工程(3)のCorinおよび/またはLrtm1陽性細胞を収集する工程は、上述した<細胞を選択する方法>に基づいて行うことができる。
工程(4)で用いる培地は、動物細胞の培養に用いられる培地を基礎培地として調製することができる。基礎培地としては、例えば、Glasgow's Minimal Essential Medium(GMEM)培地、IMDM培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium(Life Technologies社製)およびこれらの混合培地などが包含される。好ましくは、Neurobasal Mediumである。培地には、血清が含有されていてもよいし、あるいは無血清でもよい。必要に応じて、培地は、例えば、アルブミン、トランスフェリン、Knockout Serum Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2サプリメント(Invitrogen)、B27サプリメント(Invitrogen)、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、3'-チオールグリセロールなどの1つ以上の血清代替物を含んでもよいし、脂質、アミノ酸、L-グルタミン、GlutaMax(Life Technologies社製)、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類、核酸(例えば、Dibutyryl cyclic AMP(dbcAMP))などの1つ以上の物質も含有し得る。好ましい培地は、B27サプリメント、アスコルビン酸およびdbcAMPを含有するNeurobasal Mediumである。この培地へ適宜神経栄養因子を加えて培養することができる。
工程(4)における、浮遊培養とは、細胞を培養容器へ非接着の状態で培養することであり、特に限定はされないが、細胞との接着性を向上させる目的で人工的に処理(例えば、細胞外マトリックス等によるコーティング処理)されていない培養容器、若しくは、人工的に接着を抑制する処理(例えば、ポリヒドロキシエチルメタクリル酸(poly-HEMA)、非イオン性の界面活性ポリオール(Pluronic F-127等)またはリン脂質類似構造物(例えば、2-メタクリロイルオキシエチルホスホリルコリンを構成単位とする水溶性ポリマー(Lipidure))によるコーティング処理した培養容器を使用して行うことができる。
培養条件について、培養温度は、特に限定されないが、約30~40℃、好ましくは約37℃であり、CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2~5%である。
培養期間は、Nurr1またはFoxa2陽性細胞が出現する期間であれば、特に限定されないが、工程(iii)は、少なくとも7日間行われることが望ましい。より好ましくは、7日間から30日間であり、さらに好ましくは、14日間から21日間、または14日間から16日間であり、最も好ましくは、16日間である。
工程(3)に続いて工程(4)を行う場合、ROCK阻害剤を適宜添加して培養することが望ましい。ROCK阻害剤を添加する場合、少なくとも1日間添加して培養すればよく、より好ましくは1日間である。
<パーキンソン病治療剤>
本発明で得られたドーパミン産生神経前駆細胞は、製剤としてパーキンソン病患者に投与することができる。得られたドーパミン産生神経前駆細胞を生理食塩水等に懸濁させ、患者の線条体領域に移植することによって行われる。従って、本発明では、上記の方法で多能性幹細胞より得られたドーパミン産生神経前駆細胞を含むパーキンソン病治療剤を提供する。
本発明において、パーキンソン病治療剤に含まれるドーパミン産生神経前駆細胞の細胞数は、移植片が投与後に生着できれば特に限定されないが、例えば、15×104個以上含まれ得る。また、症状や体躯の大きさに合わせて適宜増減して調製されてもよい。
ドーパミン産生神経前駆細胞の疾患部位への移植は、例えば、Nature Neuroscience,2,1137(1999)もしくはN Engl J Med. ;344:710-9(2001)に記載されるような手法によって行うことができる。
本発明での他の実施態様において、多能性幹細胞からドーパミン産生神経前駆細胞を作製するキットが含まれる。当該キットには、上述したドーパミン産生神経前駆細胞を作製する各工程に使用する培地、添加剤または培養容器等が含まれる。例えば、抗Corin抗体及び/又は抗Lrtm1抗体、工程(1)に用いられるSHHシグナル刺激剤(例えば、SAG)、工程(2)に用いられる、BMP阻害剤(例えば、LDN193189)、TGFβ阻害剤(例えば、A83-01)、SHHシグナル刺激剤(例えば、Purmorphamine)、FGF8、GSK3β阻害剤(例えば、CHIR99021)、細胞外基質(例えばラミニン511E8)、および神経栄養因子(例えば、BDNF及びGDNF)を含むキットが挙げられる。本キットには、さらに製造工程の手順を記載した書面や説明書を含んでもよい。
細胞および培養
ドーパミン産生細胞製造のプロトコールを図1に示した。
ヒトiPS細胞であるQHJ-I01は、Oct3/4、Sox2、Klf4、L-MYC、LIN28およびp53のドミナントネガティブ体(Okita, K., et al.Stem Cells 31, 458-66 (2013);WO2013/176233)をヒトPBMCにエピソーマルベクターにより導入して得られた細胞として京都大学の山中教授らより受領した。
iPS細胞は、Miyazaki T et al., Nat Commun.3:1236, 2012に記載に準拠した方法で培養した。すなわち簡潔には、Laminin511E8でコーティングされた6 well plate上にてFGF2(bFGF)を含む未分化維持培地により維持培養した。
分化誘導開始、すなわち分化培地への交換の24時間前に、SHHシグナル刺激剤であるSAG(Enzo Life Sciences,Inc, 300nM)を含む未分化維持培地、StemFit:AK-03N(Ajinomoto)へと交換し、iPS細胞を含む細胞集団を得た。
SAG存在下で24時間培養した後の前記iPS細胞を含む細胞集団を、TrypLE CTS(Life Technologies)を用いて解離し、別途用意したLaminin511E8(iMatrix-511、Nippi)でコーティングした6 well plateに1wellあたり5×106個で播種し、培地を分化培地〔10μM Y-27632(WAKO)、0.1μM LDN193189(STEMGENT)および0.5μM A83-01(WAKO)を含有する基本培地A〔8%KSR(Invitrogen)、1mM Sodium pyruvate(Invitrogen)、0.1mM MEM non essential amino acid(Invitrogen)および0.1mM 2-Mercaptoethanol(WAKO)を含有するGMEM(Invitrogen)〕に交換した。翌日(day1)、0.1μM LDN193189、0.5μM A83-01、2μM Purmorphamine(WAKO)および100ng/mL FGF8(WAKO)を含有する基本培地Aへ培地を交換した。2日後(day3)、0.1μM LDN193189、0.5μM A83-01、2μM Purmorphamine、100ng/mL FGF8および3μM CHIR99021(WAKO)を含有する基本培地Aへ培地を交換した。4日後(day7)、0.1μM LDN193189および3μM CHIR99021を含有する基本培地Aへ培地を交換した。0.1μM LDN193189および3μM CHIR99021を含有する基本培地Aへ培地を交換した。これらの期間、培地交換は1日に一度行った。分化誘導開始後12日目(day12)に、抗Corin抗体を用いたフローサイトメトリー解析を実施した(図1)。
尚、抗Corin抗体は以下の方法で作製した。カニクイザルCorin遺伝子のうち、細胞外領域の一部(79-453アミノ酸)をコードする遺伝子配列を293E細胞に導入して、Corinタンパク質の細胞外領域断片を発現させて回収した。回収したタンパク質をマウスに免疫したのち、リンパ球細胞を取り出してミエローマ細胞とフュージョンさせた。フュージョンさせた細胞集団より、Corinに反応性を持つクローンを選択した。このクローンの培養上清を抗Corinモノクローナル抗体として蛍光ラベルを施した後に用いた。
0.1μM LDN193189および3μM CHIR99021を含有する基本培地Aでの培養から5日後、すなわち分化誘導開始後12日目(day12)、TrypLE CTSを用いて細胞を解離し、2%FBS、10μM Y-27632(WAKO)、20 mM D-glucoseおよび50μg/ml Penicillin/Streptomycinを含有するCa2+Mg2+-free HBSS(Invitrogen)へ懸濁させた。上記の抗Corin抗体を添加し、4℃で20分間インキュベートし、FACS(BD)を用いてソーティングを行いCorin陽性細胞を回収し、細胞数を計測した。Corin陽性率を測定した結果を図2に示す。左側の棒グラフはSAG非存在下で未分化維持培養(プレコンディショニング)した場合の対象細胞を示し、右側の棒グラフは、300nMのSAG存在下で未分化維持培養(プレコンディショニング)した場合のCorin陽性率を示す。
その結果、分化誘導前に未分化維持培地中でSAGによる処理を行うことによってCorin陽性細胞割合が向上することが見出された。
回収したCorin陽性細胞をPrimeSurface 96Uプレート(住友ベークライト)に20000個/wellにて移し、基本培地B(B27 Supplement without vitamin A(Invitrogen)、20ng/mL BDNF、 10ng/mL GDNF、200mM Ascorbic acidおよび0.4mM dbcAMP(Sigma)を添加したNeurobasal medium(Invitrogen))を用いて浮遊培養した。この時、最初の培地は、30μMのY-27632を添加した培地を用いたが、3日に一度、半量ずつ交換した際には、Y-27632を添加しない培地を用いた。ソーティングから16日後(day28)まで浮遊培養を実施してFoxA2, Nurr1(何れも中脳マーカー)の染色を行い、ドパミン神経前駆細胞へ分化しているかどうかを確認した。その結果、図3に示すとおり、得られた細胞はFoxA2及びNurr1を発現していることがわかった。この際、核の存在を示すDAPI陽性細胞を100%として、Foxa2は99.0%、Nurr1は14.6%であった。
実施例1と同様に、添加するSAGの濃度を変更した実験、及びSAGと同時にA83-01もしくはLDN193189を添加した実験を行い、Corin陽性率を測定した。検討条件及びそれぞれの条件下でのCorin陽性率を表1に示した。
別方法で調製したiPS細胞による製造
ヒトiPS細胞であるLPF11は、Oct3/4, Sox2, Klf4,L-MYCをヒトPBMC にセンダイウイルスベクター(Cytotune2.0L、ID Pharma社製)により導入して、京都大学の公開プロトコル(ヒトiPS細胞の樹立・維持培養、CiRA_Ff-iPSC_protocol_JP_v140310、http://www.cira.kyoto-u.ac.jp/j/research/protocol.html)記載の方法に従って、StemFit培地 (味の素社製)、Laminin511-E8(ニッピ社製)を用いて樹立した。
このiPS細胞を使用して、実施例1と同じ方法で、培養及び分化誘導を行い、12日目(day12)に、抗Corin抗体を用いてCorin陽性細胞の全細胞数に対する割合を測定した。Corin陽性率を測定した結果を図4に示す。左側の棒グラフはSAG非存在下で未分化維持培養(プレコンディショニング)した場合のCorin陽性率を示し、右側の棒グラフは、300nMのSAG存在下で未分化維持培養(プレコンディショニング)した場合のCorin陽性率を示す。
その結果、実施例1の場合と同様、iPS細胞の種類によらず、分化誘導前に未分化維持培地中でSAGによる処理を行うことによってCorin陽性細胞の割合が向上することが見出された。
動物への細胞移植
実施例1で調製した、細胞(day28に相当)が移植に適しているか検討をおこなった。この時、培地は、3日に一度、半量ずつ交換した。
Day28の細胞を、TH(ドーパミン産生神経細胞マーカー)、Foxa2およびNurr1(いずれも中脳マーカー)に対して免疫染色を行ったところ、図3に示すとおり、得られた細胞はFoxA2及びNurr1を発現していることがわかった。この際、核の存在を示すDAPI陽性細胞を100%として、Foxa2は99.0%、Nurr1は14.6%であった。
得られた細胞4×105個の細胞を含む複数の細胞塊を2μl(生理食塩水)に懸濁させ、パーキンソン病モデルラット(6-OHDA片側投与ラット)の線条体に22ゲージの注射針を用いて投与し、4週後の移植片の様子を観察した(図5)。移植片の解析では、TH陽性の線維、および細胞体が確認できた(図5AおよびB)。このとき、SAG処理、非処理の細胞移植片において、HuNu陽性細胞数(ヒト細胞核、移植細胞の生着数を示す)あたりのTH陽性細胞数の割合に差が見られず、同等の染色像を示した(図5AおよびB)。
以上の結果より、実施例1で製造した細胞(day28)はSAG処理、非処理ともに移植片の生着およびドーパミン産生細胞への成熟を確認でき、パーキンソン病の治療のために使用する細胞として適していることが示唆された。
Claims (23)
- 下記工程(1)および(2)を含む、Corinおよび/またはLrtm1陽性細胞を含む細胞集団の製造方法;
(1)多能性幹細胞を、フィーダー細胞非存在下で、Sonic hedgehog (SHH)シグナル刺激剤及び未分化維持因子を含む未分化維持培地中、細胞外基質の存在下で、接着培養する工程、および
(2)工程(1)で得られた細胞を、1以上の分化誘導因子を含む培地中で培養する工程。 - SHHシグナル刺激剤が、SAG(N-Methyl-N’-(3-pyridinylbenzyl)-N’-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane)、shhタンパク質もしくはそのフラグメント、Purmorphamine又はこれらの組合せである、請求項1に記載の製造方法。
- 工程(1)における未分化維持培地が、更にTGFβ阻害剤又はBMP阻害剤を含む培地である、請求項1又は2に記載の製造方法。
- TGFβ阻害剤が、A83-01であり、BMP阻害剤がLDN193189である、請求項3に記載の製造方法。
- 未分化維持因子が、少なくともFGFシグナル伝達経路作用物質を含む、請求項1~4のいずれか一項に記載の製造方法。
- FGFシグナル伝達経路作用物質が、bFGFである、請求項5に記載の製造方法。
- 前記工程(1)において、多能性幹細胞を、48時間を超えない期間培養する、請求項1~6のいずれか一項に記載の製造方法。
- 分化誘導因子が、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK3β阻害剤から成る群より選択される1又は複数の因子である、請求項1~7のいずれか一項に記載の製造方法。
- 工程(2)が、細胞外基質上にて接着培養する工程である、請求項1~8のいずれか一項に記載の製造方法。
- 細胞外基質が、ラミニンまたはその断片である、請求項9に記載の製造方法。
- 細胞外基質が、ラミニン511E8である、請求項10に記載の製造方法。
- 工程(2)が次の工程を含む、請求項1~11のいずれか一項に記載の方法;
(a)工程(1)で得られる細胞を、細胞外基質の存在下で、BMP阻害剤およびTGFβ阻害剤を含む培地中で接着培養する工程、
(b)前記工程(a)で得られた細胞を、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤およびFGF8を含む培地中で、細胞外基質存在下で接着培養する工程、
(c)前記工程(b)で得られた細胞を、BMP阻害剤、TGFβ阻害剤、SHHシグナル刺激剤、FGF8およびGSK3β阻害剤を含む培地中で、細胞外基質存在下で接着培養する工程、および
(d)前記工程(c)で得られた細胞を、BMP阻害剤およびGSK3β阻害剤を含む培地中で細胞外基質存在下で接着培養する工程。 - 工程(a)における培地が、更にROCK阻害剤を含む、請求項12に記載の製造方法。
- 工程(2)で得られる細胞集団中に含まれるCorinおよび/またはLrtm1陽性細胞の割合が、10%以上である請求項1~13のいずれか一項に記載の製造方法。
- 以下の工程:
(3)請求項1~14のいずれか一項に記載の製造方法により得られる細胞集団から、Corinに結合する物質および/またはLrtm1に結合する物質を用いてCorinおよび/またはLrtm1陽性細胞を収集する工程、及び
(4)工程(3)で得られるCorinおよび/またはLrtm1陽性細胞を、1又は複数の神経栄養因子を含む培地中で浮遊培養する工程、
を含む、ドーパミン産生神経前駆細胞の製造方法。 - Corinに結合する物質またはLrtm1に結合する物質が、CorinまたはLrtm1に結合する抗体またはアプタマーである、請求項15に記載の製造方法。
- 神経栄養因子が、BDNFおよびGDNFである、請求項15または16に記載の製造方法。
- 神経栄養因子を含む培地が、B27サプリメント、アスコルビン酸およびcAMPアナログをさらに含む、請求項15~17のいずれか一項に記載の製造方法。
- cAMPアナログが、Dibutyryl cyclic AMPである、請求項18に記載の製造方法。
- 神経栄養因子を含む培地中での浮遊培養が、少なくとも7日間行われる、請求項15~19のいずれか一項に記載の製造方法。
- 請求項1~14のいずれか一項に記載の製造方法により得られるCorinおよび/またはLrtm1陽性細胞の含有率が10%以上である細胞集団。
- 請求項15~20のいずれか一項に記載の製造方法により得られるドーパミン産生神経前駆細胞。
- 請求項15~20のいずれか一項に記載の製造方法により得られるドーパミン産生神経前駆細胞を含むパーキンソン病治療剤。
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