WO2015199127A1 - 中胚葉細胞および造血細胞の製造方法 - Google Patents
中胚葉細胞および造血細胞の製造方法 Download PDFInfo
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Definitions
- the present invention relates to a novel method for producing mesoderm cells, a mesodermal cell supported by a three-dimensional support obtained by the method, and a transplant material containing a mesoderm cell supported by the three-dimensional support. And a culture vessel for holding a plurality of three-dimensional supports supporting mesodermal cells.
- the present invention also relates to a novel method for producing hematopoietic cells, and a therapeutic agent for blood diseases containing hematopoietic cells obtained by the method.
- ES cells embryonic stem cells
- iPS induced pluripotent stem
- Non-Patent Documents 1 to 3 methods using embryoid body formation and addition of cytokines (Non-Patent Documents 1 to 3), heterologous stromal cells, The co-culture method (Non-patent Document 4) and the method using a serum-free medium (Patent Document 3) have been reported.
- Non-Patent Documents 1 to 4 methods for inducing differentiation into erythrocytes are reported in Non-Patent Documents 1 to 4, Patent Document 3, and the like, and methods for inducing differentiation into neutrophils are reported in Non-Patent Documents 3 and 5 and the like.
- Patent Documents 3 and 5 methods for inducing differentiation into neutrophils are reported in Non-Patent Documents 3 and 5 and the like.
- in order to be able to supply blood cells in large quantities and stably there is a need for further improvements in the method of inducing differentiation into blood cells, and the development of new technologies suitable for medical applications is required. .
- An object of the present invention is to provide a novel method for producing mesoderm cells, a mesoderm cell supported by a three-dimensional support obtained by the method, and a transplant containing a mesoderm cell supported by the three-dimensional support.
- An object of the present invention is to provide a culture container for holding a material for use and a plurality of three-dimensional supports supporting mesodermal cells.
- Another object of the present invention is to provide a novel method for producing hematopoietic cells and a therapeutic agent for blood diseases containing hematopoietic cells obtained by the method.
- the present inventors have induced differentiation into mesoderm cells and / or hematopoietic cells by culturing pluripotent stem cells under the condition of contact with a three-dimensional support. I succeeded in making it happen. Similarly, mesoderm cells were successfully induced to differentiate into hematopoietic cells by culturing them under contact with a three-dimensional support. Furthermore, the present inventors have found for the first time that hematopoietic cells can be stably supplied over a long period of time by culturing pluripotent stem cells and / or mesoderm cells under the condition of contact with a three-dimensional support. It was. The present invention has been completed based on such knowledge.
- the present invention provides the following matters.
- a method for producing mesoderm cells from pluripotent stem cells comprising culturing pluripotent stem cells in contact with a three-dimensional support to induce mesoderm cells.
- the mesoderm cells are KDR positive CD34 positive cells.
- the three-dimensional support is a collagen sponge.
- the collagen sponge is a collagen sponge reinforced with polyethylene terephthalate fibers.
- step of culturing pluripotent stem cells in contact with a three-dimensional support includes the following steps: (i) culturing pluripotent stem cells in a medium containing BMP4; and (ii) A step of culturing the cells obtained in step (i) in a medium containing VEGF, bFGF and SCF.
- the culture periods of the steps (i) and (ii) are 1 to 5 days and 0.5 to 3 days, respectively.
- the culture periods of the steps (i) and (ii) are 3 days and 1 day, respectively.
- the method according to (16), wherein the hematopoietic cells are myeloid cells, monocyte cells, or erythroid cells.
- the method according to (16) or (17), wherein the hematopoietic cells are myeloid cells.
- the method according to (16) or (17), wherein the hematopoietic cells are monocytic cells.
- the method according to (16) or (17), wherein the hematopoietic cells are erythroid cells.
- the step (b) comprises a step of culturing mesoderm cells retained on a three-dimensional support in a medium containing SCF, IL-3, Flt3L and thrombopoietin (TPO).
- step (b) comprises the following steps: (i) culturing mesoderm cells retained on a three-dimensional support in a medium containing SCF, IL-3, Flt3L and TPO, (ii) culturing the cells obtained in step (i) in a medium containing SCF, IL-3, Flt3L, TPO and M-CSF, and (iii) A step of culturing the cells obtained in step (ii) in a medium containing Flt3L, M-CSF and GM-CSF.
- step (b) comprises culturing mesoderm cells retained on a three-dimensional support in a medium containing erythropoietin (EPO) and SCF.
- step (4) The method according to (21) or (23), wherein the culture period in step (b) is 16 to 41 days.
- the method according to (24), wherein the culture period in step (b) is 31 days.
- the culture periods of the steps (i), (ii) and (iii) are 2 to 5 days, 2 to 5 days and 8 to 33 days, respectively.
- the method of the present invention can stably supply a large amount of hematopoietic cells.
- differentiation is induced in the form of a single cell of pluripotent stem cells, the effect can be more exerted, and various blood diseases can be treated.
- FIG. 1 shows the results of hematopoietic cell differentiation from a small mass of undifferentiated pluripotent stem cells (PSC).
- PSC pluripotent stem cells
- FIG. 2 shows the results of differentiation into a specific hematopoietic cell lineage.
- (a, b) shows the results of differentiation into myeloid cells.
- (a) shows the results of flow cytometry, and
- (b) is a photograph showing the results of Giemsa staining (May-Giemsa staining).
- the scale bar in (b) is 20 ⁇ m.
- (c, d) shows the results of differentiation into monocytic cells.
- (c) shows the results of flow cytometry
- (d) is a photograph showing the results of Giemsa staining (May-Giemsa staining).
- the scale bar in (d) is 20 ⁇ m.
- (e, f) shows the results of differentiation into erythroid cells.
- (e) shows the results of flow cytometry
- (f) is a photograph showing the results of Giemsa staining (May-Giemsa staining).
- the scale bar of (f) is 20 ⁇ m.
- (g) Shows the number of hematopoietic cells collected according to the sputum culture period. All data are representative of at least 3 independent experiments.
- FIG. 3 shows the results of hematopoietic cell differentiation from a single undifferentiated PSC.
- (a) A photograph showing a microscopic image of CS at the start of differentiation. Clear cell aggregates are not observed. The scale bar is 500 ⁇ m.
- (b-e) shows the results of flow cytometry of hematopoietic progenitor cells induced to differentiate from KhES1 on the 6th day. Dead cells and debris are excluded by DAPI staining.
- (b) shows the total number of cells
- (c) shows the percentage of CD34 positive KDR positive hematopoietic progenitor cells (HPC)
- (d) shows the number of CD34 positive KDR positive HPC
- (e) The results of flow cytometry are shown.
- (f) Flow cytometry results for myeloid cells on day 38.
- (G) Photograph showing the result of Giemsa staining of hematopoietic progenitor cells induced to differentiate from KhES1 on day 6.
- the scale bar is 20 ⁇ m.
- (h-j, q) shows the results of flow cytometry of hematopoietic progenitor cells induced to differentiate from 201B7 on the 6th day. Dead cells and debris are excluded by DAPI staining.
- (h) indicates the total number of cells
- (i) indicates the percentage of CD34 positive KDR positive HPC
- (j) ⁇ indicates the number of CD34 positive KDR positive HPC
- (q) indicates flow cytometry Results are shown.
- (k-m, r) The results of flow cytometry of hematopoietic progenitor cells induced to differentiate from 402B2 on day 6 are shown. Dead cells and debris are excluded by DAPI staining.
- (k) indicates the total number of cells
- (l) indicates the percentage of CD34 positive KDR positive HPC
- (m) ⁇ ⁇ indicates the number of CD34 positive KDR positive HPC
- (r) is the flow cytometry Results are shown.
- (n-p, s) shows the results of flow cytometry of hematopoietic progenitor cells induced to differentiate from CB-A11 on the 6th day. Dead cells and debris are excluded by DAPI staining.
- FIG. 4 is a photograph showing the result of an image obtained by a scanning electron microscope (SEM).
- SEM scanning electron microscope
- FIG. 5 shows the results of culturing using a plurality of CSs in a flask.
- (a) shows the number of hematopoietic cells recovered from the culture.
- (b, c) shows the results for myeloid cells obtained by culm culture.
- FIG. 6 shows the results of PCR on hematopoietic cells induced using CS from a small cluster of undifferentiated pluripotent stem cells (PSC).
- FIG. 7 is a photograph showing the results of CS immunostaining after differentiation induction.
- A shows the site of immunostaining in CS
- B shows an image of immunostaining with CD45 antibody at site B in (a)
- C shows C in (a).
- the present invention provides a novel method for producing hematopoietic cells and a therapeutic agent for blood diseases containing hematopoietic cells obtained by the method.
- the present invention also provides a novel method for producing mesoderm cells, and mesoderm cells supported by a three-dimensional support obtained by the method.
- the present invention provides a culture container that holds a plurality of three-dimensional supports that support mesoderm cells.
- the cell produced by the method of the present invention can be a cell induced to differentiate from a pluripotent stem cell.
- a pluripotent stem cell is not specifically limited, For example, the following are mentioned.
- a 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
- Examples include, but are not limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic reproduction Examples include cells (“EG cells”), Multilineage-differentiating Stress Enduring cells (Muse cells), and induced pluripotent stem (iPS) cells.
- ES embryonic stem
- ntES embryonic stem
- GS cells sperm stem cells
- EG cells embryonic reproduction Examples
- EG cells EG cells
- Muse cells Multilineage-differentiating Stress Enduring cells
- iPS induced pluripotent stem cells.
- Preferred pluripotent stem cells are ES cells, ntES cells and iPS cells.
- Embryonic stem 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. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod 55: 254-259; JA JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).
- ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution to which substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (basic fibroblast growth factor (bFGF)) are added. It can be carried out.
- LIF leukemia inhibitory factor
- bFGF basic fibroblast growth factor
- DMEM / F-12 culture 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 is used as the culture medium for ES cell production.
- Human ES cells can be maintained in a humid atmosphere at 37 ° C., 5% CO 2 (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932).
- 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 generally 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 alkaline phosphatase, Oct-3 / 4, Nanog
- OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).
- Human ES cell lines for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute of Regenerative Medicine, Kyoto University (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).
- 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 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination.
- the reprogramming factors include histone deacetylase (HDAC) inhibitors [for example, small molecule inhibitors such as valproate (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg Nucleic acid expression inhibitors such as HDAC1DACsiRNA Smartpool (registered trademark) (Millipore), HuSH 29mershRNA 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 BIX-01294, Suv39hl, Suv39h2, SetDBl And nucleic acid expression inhibitor
- the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), and microinjection.
- a cell membrane-permeable peptide eg, HIV-derived TAT and polyarginine
- Virus vectors include retrovirus vectors, lentivirus vectors (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).
- HAC human artificial chromosomes
- YAC yeast artificial chromosomes
- BAC bacterial artificial chromosomes
- a plasmid 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.
- GFP green fluorescent protein
- GUS ⁇ -glucuronidase
- FLAG FLAG
- 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 it may be introduced into somatic cells by techniques such as lipofection and microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine® (TriLink® Biotechnologies) is used. Yes (Warren L, (2010) Cell Stem Cell. 7: 618-630).
- a culture solution for inducing iPS cells for example, DMEM, DMEM / F12 or DME culture solution containing 10-15% FBS (in addition to these culture solutions, LIF, penicillin / streptomycin, puromycin, L- Glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc. may be included as appropriate.
- a commercially available culture medium for example, a culture medium for mouse ES cell culture (TX-WES culture medium, Thrombo X), primate ES Cell culture medium (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology).
- the somatic cell and the reprogramming factor are brought into contact with DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact of the somatic cells with the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.
- feeder cells e.g., mitomycin C-treated STO cells, SNL cells, etc.
- 10% FBS-containing DMEM culture medium including LIF, penicillin / streptomycin, etc.
- feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- 5% CO 2 at 37 ° C. can be suitably included with puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
- ES-like colonies after about 25 to about 30 days or more .
- 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).
- the culture medium is exchanged with a fresh culture medium once a day from the second day onward.
- the number of 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 culture solution containing the corresponding drug selection The established iPS cells can be selected by culturing with the culture medium.
- 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 as the transplant destination individual from the viewpoint that rejection does not occur.
- 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)
- ES cell production technology is used (Kiyaka Wakayama et al. ( 2008), Experimental Medicine, Vol.26, No.5 (extra number), pp. 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 “mesoderm cell” is a group of cells constituting the mesoderm, and the body cavity and the mesothelium, muscle, skeleton, skin dermis, connective tissue, heart / blood vessel (including vascular endothelium) that line it during the development process. ), Blood (including blood cells), lymphatic vessels and spleen, kidneys and ureters, and cells that have the ability to produce gonads (testis, uterus, gonadal epithelium).
- the “mesoderm cell” in the present invention is indicated by the expression of markers such as T (synonymous with Brachyury), VEGF receptor-2 (KDR), FOXF1, FLK1, BMP4, MOX1, SDF1, and CD34. Preferably, it is a cell that expresses KDR and CD34.
- the “mesoderm cell” in the present invention can include hematopoietic stem cells and hematopoietic progenitor cells having the ability to differentiate into hematopoietic cells, unless otherwise specified.
- hematopoietic stem cells have the ability to produce mature blood cells such as T cells, B cells, erythrocytes, platelets, eosinophils, monocytes, neutrophils, basophils, and have the ability to self-renew. It means the cell which has.
- a “hematopoietic progenitor cell” (also referred to as “HPC”) means a cell that has been differentiated more than a “hematopoietic stem cell” and whose cell differentiation direction has been determined. These cells can be detected by expression of markers such as KDR, CD34, CD90 and CD117, but the markers are not limited thereto.
- hematopoietic progenitor cells are not distinguished from “hematopoietic stem cells” unless otherwise specified.
- the mesoderm cells induced to differentiate in the present invention may be provided as a cell population containing other cell types, or may be a purified population.
- the mesoderm cells are preferably contained in 30% or more, more preferably 50% or more.
- differentiation induction into mesoderm cells is performed by culturing pluripotent stem cells in contact with a three-dimensional support.
- the “three-dimensional support” in the present invention may be any three-dimensional substance that can hold cells in a culture solution (that is, a substance that provides a scaffold for cells), for example, collagen sponge, agarose gel, gelatin , Chitosan, hyaluronic acid, proteoglycan, and biomaterials such as PGA, PLA, PLGA, and mixtures thereof, but are not limited thereto.
- a collagen sponge is used.
- the collagen sponge preferably has a reinforcing material for the purpose of supplementing its strength.
- the reinforcing material of the collagen sponge is not particularly limited, and examples thereof include glycolic acid, lactic acid, dioxanone, caprolactone, and the like, or copolymers thereof, knitted fabrics, woven fabrics, nonwoven fabrics, and other sheet-like fiber materials.
- a preferable reinforcing material is polyethylene terephthalate fiber.
- the method of reinforcing with the reinforcing material is not particularly limited.
- a reinforcing material may be laminated on one side or both sides of the collagen sponge, and when the collagen sponge is manufactured, the reinforcing material is formed in the collagen sponge. You may do it.
- the collagen sponge in the present invention can be obtained from, for example, MedGEL (# PETcol-24W).
- the three-dimensional support in the present invention has a porous structure so that cells can enter the inside thereof.
- the cells can exist on either or both of the surface and the inside of the three-dimensional support, and can be subjected to a differentiation-inducing operation at that position.
- the porous pore size can be, for example, in the range of 5 ⁇ m to 1000 ⁇ m, preferably 50 ⁇ m to 500 ⁇ m, more preferably 200 ⁇ m.
- “contact between a (pluripotent stem / mesoderm) cell and a three-dimensional support” is close by a distance until the cell and the three-dimensional support can interact with each other. Means that. Therefore, in the present specification, “(pluripotent stem / mesoderm) cells are cultured in contact with a three-dimensional support” means the distance until the cells can interact with the three-dimensional support. Means that the cells are cultured in close proximity. Examples of the “contact between the (pluripotent stem / mesoderm) cell and the three-dimensional support” include, but are not limited to, a physical form or a chemical form. Preferably, the coupling
- the method for inducing differentiation from pluripotent stem cells to mesoderm cells is not particularly limited, for example, the following method can be used.
- the medium used for inducing mesoderm cells can be prepared using a medium used for culturing animal cells as a basal medium.
- basal media include IMDM medium, MediumMedi199 medium, mTeSR1 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, Fistem's medium (Invitrogen), Mouse Embryonic fibroblast conditioned medium (MEF-CM), and mixed media thereof are included.
- mTeSR1 medium and / or StemPro34 medium is used.
- the medium may contain serum or may be serum-free.
- 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 It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
- KSR Knockout Serum Replacement
- the medium in this step may further contain a ROCK inhibitor.
- the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
- ROCK Rho kinase
- Y-27632 can be used in the present invention.
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
- the CO 2 concentration is about 2-5%, preferably 5%.
- differentiation-inducing factor into mesoderm cells examples include, but are not limited to, BMP4, VEGF, bFGF, SCF and the like, and it is known that they can be used for the differentiation-inducing step of mesoderm cells. Any factor and any factor identified that can be used in the process of inducing differentiation of mesoderm cells in the future are included.
- differentiation induction into mesoderm cells can be performed, for example, by the following steps: (i) culturing pluripotent stem cells in a medium containing BMP4 in contact with a three-dimensional support; and (ii) A step of culturing the cells obtained in step (i) in a medium containing VEGF, bFGF and SCF.
- the basal medium used in the step (i) is preferably mTeSR1 medium, and the basal medium used in the step (ii) may be preferably StemPro34 medium.
- the concentration of the differentiation-inducing factor to mesoderm cells in the medium may be any concentration as long as the target cells can be induced.
- the concentration of BMP4 in the medium of the step (i) is, for example, in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 110ng / ml, 120ng / ml, 130ng / ml, 140ng / ml, 150ng / ml, 160ng / ml, 170ng / ml, 180 ng / ml, 190 ng / ml, 200 ng / ml, but not limited thereto.
- it is 80 ng / ml.
- the concentration of VEGF in the medium of the step (ii) is, for example, in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 110ng / ml, 120ng / ml, 130ng / ml, 140ng / ml, 150ng / ml, 160ng / ml, 170ng / ml, 180 ng / ml, 190 ng / ml, 200 ng / ml, but not limited thereto.
- it is 80 ng / ml.
- the concentration of bFGF in the medium of the step (ii) is, for example, in the range of 1 ng / ml to 100 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25ng / ml, 30ng / ml, 35ng / ml, 40ng / ml, 45ng / ml, 50ng / ml, 55ng / ml, 60ng / ml, 65ng / ml, 70ng / ml, 75ng / ml, 80ng / ml, 85ng / ml, 90 ng / ml, 95 ng / ml, 100 ng / ml, but not limited thereto.
- it is 25 ng / ml in each step.
- the concentration of SCF in the medium of the step (ii) is, for example, in the range of 1 ng / ml to 250 ng / ml, such as 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 110ng / ml, 120ng / ml, 130ng / ml, 140ng / ml, 150ng / ml, 160ng / ml, 170ng / Examples include, but are not limited to, ml, 180 ng / ml, 190 ng / ml, 200 ng / ml, 210 ng / ml, 220 ng / ml, 230
- the culture time in the step (i) is, for example, 10 days or less, preferably 1 to 5 days, particularly preferably 3 days.
- the culture time in the step (ii) is, for example, culture for 5 days or less, preferably 0.5 to 3 days, and particularly preferably 1 day.
- the pluripotent stem cell in the step (i) may be in the form of a small mass in which a plurality of pluripotent stem cells are aggregated, or may be in a form dissociated into single cells. Preferably, it is a form dissociated into single cells. Therefore, the present invention can further include a step of dissociating pluripotent stem cells into a small mass or a single cell before the step (i).
- the step of dissociating pluripotent stem cells into a nodule or single cell form can be performed by, for example, a method of mechanical separation, a separation solution having protease activity and collagenase activity (for example, Accutase (TM), Accumax (TM), CTK solution), a separation solution having only collagenase activity, an enzyme-free separation solution (for example, EDTA solution) and the like.
- a separation solution having protease activity and collagenase activity for example, Accutase (TM), Accumax (TM), CTK solution
- TM Accutase
- TM Accumax
- CTK solution a separation solution having only collagenase activity
- an enzyme-free separation solution for example, EDTA solution
- a ROCK inhibitor may be included in the dissociation solution for the purpose of preventing cell death.
- the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
- ROCK Rho kinase
- the size is not particularly limited, but may be, for example, 100 ⁇ m to 1000 ⁇ m, preferably 200 ⁇ m to 800 ⁇ m, and particularly preferably 300 ⁇ m to 500 ⁇ m.
- the pluripotent stem cells small mass or single cell after dissociation are cultured for a certain period of time under non-differentiation inducing conditions in contact with a three-dimensional support, and then induced to differentiate into mesoderm cells. You may culture by. Such a pre-culture step is performed in order to achieve an appropriate adhesion state between the pluripotent stem cells and the three-dimensional support, and any culture conditions can be used as long as this purpose can be achieved. can do.
- the medium in the preculture step is, for example, IMDM medium, Medium 199 medium, mTeSR1 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 StemPro34 (Invitrogen), Mouse Embryonic fibroblast conditioned medium (MEF-CM), and mixed media thereof, but are not limited thereto.
- EMEM Eagle's Minimum Essential Medium
- DMEM Dulbecco's modified Eagle's Medium
- Ham's F12 medium RPMI 1640 medium
- Fischer's medium StemPro34 Invitrogen
- MEF-CM Mouse Embryonic fibroblast conditioned medium
- mixed media thereof but are not limited thereto.
- mTeSR1 medium is used.
- the medium in this step may further contain a ROCK inhibitor.
- Y-27632 can be used in this step.
- the culture time in the pre-culture step is, for example, 5 days or less, preferably 0.5 to 3 days, and particularly preferably 1 day.
- the culture is 6 days or less, preferably 1 to 4 days, and particularly preferably 2 days.
- the fresh medium to be replaced include, but are not limited to, mTeSR1.
- the exchange of the medium between the above steps may be performed by replacing only the medium without changing the three-dimensional support, or by moving the three-dimensional support to a container containing a new medium. May be. Preferably, it is performed by moving the three-dimensional support to the container.
- hematopoietic cell means any type of cell committed to the blood lineage.
- the “hematopoietic cell” in the present invention may be any cell committed from the mesoderm cell to the blood lineage.
- the “hematopoietic cell” may preferably be a myeloid cell, a monocytic cell or an erythroid cell.
- “hematopoietic cell” is used synonymously with “blood cell” unless otherwise specified.
- differentiation induction into hematopoietic cells is carried out by culturing mesoderm cells held on the three-dimensional support obtained by the above method in a culture vessel.
- the resulting hematopoietic cells can be recovered from the culture supernatant.
- myeloid cells means a series of cells committed to fate from mesoderm cells to the myeloid lineage.
- neutrophils Eosinophils, basophils and the like, but are not limited thereto.
- neutrophil is a kind of granulocyte having special granules stained with a neutral pigment.
- ⁇ eosinophils '' are cells having major basic protein (MBP), eosinophil maintaining protein (ECP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN) in their granules, more preferably These cells have the ability to release EDN upon stimulation with secretory immunoglobulin A (sIgA) and the ability to migrate upon stimulation with IL-5, Eotaxin and fMLP.
- the “basophil” means a cell having a large granule dyed dark purple by staining with a basic dye.
- the “myeloid cell” may preferably be a CD43-positive CD45-positive cell.
- the myeloid cell induced to differentiate in the present invention may be provided as a cell population containing other cell types, or may be a purified population.
- the myeloid cell is preferably contained in 30% or more, more preferably 50% or more.
- culture can be performed using a medium that induces differentiation of mesoderm cells into myeloid cells.
- the mesoderm cell in this step is preferably in a state where pluripotent stem cells are induced to differentiate to mesoderm cells in contact with the three-dimensional support, but the differentiation is induced by any method.
- Germ cells may be in contact with a three-dimensional support just before this step.
- a medium used for inducing myeloid cells can be prepared using a medium used for culturing animal cells as a basal medium.
- basal media include IMDM medium, MediumMedi199 medium, mTeSR1 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, Fistem's medium (Invitrogen), Mouse Embryonic fibroblast conditioned medium (MEF-CM), and mixed media thereof are included.
- StemPro34 medium is preferably used.
- the medium may contain serum or may be serum-free.
- 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 It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
- KSR Knockout Serum Replacement
- the medium in this step may further contain a ROCK inhibitor.
- the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
- ROCK Rho kinase
- Y-27632 can be used in the present invention.
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
- the CO 2 concentration is about 2-5%, preferably 5%.
- SCF stem cell factor
- TPR thrombopoietin
- Flt3 ligand proteins secreted from leukocytes and are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL.
- SCF stem cell factor
- TPR thrombopoietin
- differentiation induction into myeloid cells can be performed, for example, by the following steps: mesodermal cells held on a three-dimensional support contain SCF, IL-3, Flt3L and thrombopoietin (TPO) Culturing in a culture medium.
- mesodermal cells held on a three-dimensional support contain SCF, IL-3, Flt3L and thrombopoietin (TPO) Culturing in a culture medium.
- the basal medium used in the above step can be preferably StemPro34 medium.
- the concentration of the differentiation-inducing factor for myeloid cells in the medium is not particularly limited as long as the target cell can be induced.
- the concentration of SCF in the medium is in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, Examples include, but are not limited to, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, and 200 ng / ml. Preferably, it is 50 ng / ml.
- the concentration of IL-3 in the medium is, for example, in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 120ng / ml, 140ng / ml, 160ng / ml, 180ng / ml, 200ng / ml, but not limited to these .
- it is 50 ng / ml.
- the concentration of Flt3L in the medium is, for example, in the range of 1 ng / ml to 200 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml Examples include, but are not limited to, ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml. Preferably, it is 50 ng / ml.
- the concentration of TPO in the medium is in the range of 1 ng / ml to 20 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml , 8 ng / ml, 9 ng / ml, 10 ng / ml, 12 ng / ml, 14 ng / ml, 16 ng / ml, 18 ng / ml, 20 ng / ml, but not limited thereto.
- it is 5 ng / ml.
- the culture time in this step is, for example, 60 days or less, preferably 16 to 41 days, and particularly preferably 31 days.
- monocytic cells means a series of cells committed to fate from mesoderm cells to monocytic lineages, Examples include, but are not limited to monocytes and macrophages.
- monocytes and macrophages are both types of white blood cells, and are cells having the ability to engulf foreign substances and present antigens. These cells can be detected by expression of markers such as CD14, CD16, CD45, CD68 (the type of marker is not limited).
- the “monocyte lineage cell” may preferably be a CD14-positive CD45-positive cell.
- the monocytic cells induced to differentiate in the present invention may be provided as a cell population containing other cell types, or may be a purified population.
- the monocytic cells are preferably contained in 30% or more, more preferably 50% or more.
- culturing can be performed using a medium that induces differentiation of mesoderm cells into monocytic cells.
- the mesoderm cell in this step is preferably in a state where pluripotent stem cells are induced to differentiate to mesoderm cells in contact with the three-dimensional support, but the differentiation is induced by any method.
- Germ cells may be in contact with a three-dimensional support just before this step.
- the medium used for inducing monocytic cells can be prepared using a medium used for culturing animal cells as a basal medium.
- basal media include IMDM medium, MediumMedi199 medium, mTeSR1 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, Fistem's medium (Invitrogen), Mouse Embryonic fibroblast conditioned medium (MEF-CM), and mixed media thereof are included.
- StemPro34 medium is preferably used.
- the medium may contain serum or may be serum-free.
- 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 It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
- KSR Knockout Serum Replacement
- the medium in this step may further contain a ROCK inhibitor.
- the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
- ROCK Rho kinase
- Y-27632 can be used in the present invention.
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
- the CO 2 concentration is about 2-5%, preferably 5%.
- differentiation-inducing factors into monocyte cells include, but are not limited to, SCF, IL-3, Flt3L, TPO, M-CSF, GM-CSF, and the like. Any factor known to be usable in the differentiation induction process of the present invention and any factor identified to be used in the differentiation induction process of monocyte cells in the future are included.
- differentiation induction into monocytic cells can be performed, for example, by the following steps: (i) culturing mesoderm cells retained on a three-dimensional support in a medium containing SCF, IL-3, Flt3L and TPO, (ii) culturing the cells obtained in step (i) in a medium containing SCF, IL-3, Flt3L, TPO and M-CSF, and (iii) A step of culturing the cells obtained in step (ii) in a medium containing Flt3L, M-CSF and GM-CSF.
- the basal medium used in the steps (i) to (iii) is preferably StemPro34 medium.
- the concentration of the differentiation-inducing factor into monocyte cells in the medium is not particularly limited as long as the target cell can be induced.
- the concentration of SCF in the medium of step (i) and (ii) is in the range of 1 ng / ml to 200 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml.
- ng / ml 40ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 120ng / ml, 140ng / ml, 160ng / ml, 180ng / ml, 200
- ng / ml it is not limited to these.
- it is 50 ng / ml.
- the concentration of IL-3 in the medium of the steps (i) and (ii) is, for example, in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml.
- ml 30ng / ml, 40ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 120ng / ml, 140ng / ml, 160ng / ml, 180ng / ml, although it is 200 ng / ml, it is not limited to these. Preferably, it is 50 ng / ml.
- the concentration of Flt3L in the medium of steps (i) to (iii) is, for example, in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30ng / ml, 40ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 120ng / ml, 140ng / ml, 160ng / ml, 180ng / ml, 200ng / Although it is ml, it is not limited to these. Preferably, it is 50 ng / ml.
- the concentration of TPO in the medium in the medium of step (i) and (ii) is in the range of 1 ng / ml to 20 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml 5ng / ml /, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 12ng / ml, 14ng / ml, 16ng / ml, 18ng / ml, 20ng / ml Not.
- it is 5 ng / ml.
- the concentration of M-CSF in the medium of step (ii) and (iii) is, for example, in the range of 1 ng / ml to 200 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml.
- ml 30ng / ml, 40ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 120ng / ml, 140ng / ml, 160ng / ml, 180ng / ml, although it is 200 ng / ml, it is not limited to these. Preferably, it is 50 ng / ml.
- the concentration of GM-CSF in the medium of the step (iii) is, for example, in the range of 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml Examples include, but are not limited to, ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml. Preferably, it is 25 ng / ml in each step.
- the culture time in the step (i) is, for example, culture for 10 days or less, preferably 2 to 5 days, and particularly preferably 3 days.
- the culture time in the step (ii) is, for example, 10 days or less, preferably 2 to 5 days, and particularly preferably 3 days.
- the culture time in the step (iii) is, for example, culture for 40 days or less, preferably 8 to 33 days, and particularly preferably 23 days.
- the exchange of the medium between the steps may be performed by replacing only the medium while leaving the three-dimensional support, or by moving the three-dimensional support to a container containing a new medium. Also good. Preferably, it is performed by moving the three-dimensional support to the container.
- erythroid cells mean a series of cells committed to fate from mesoderm cells to the erythroid lineage, and examples include erythrocytes. However, it is not limited to this.
- erythrocyte means a cell rich in hemoglobin, and can be detected by expression of markers such as ⁇ -globin, ⁇ -globin, ⁇ -globin, ⁇ -globin, CD235a of hemoglobin. Preferred markers in mature erythrocytes can be ⁇ -globin and ⁇ -globin (the type of marker is not limited).
- a preferred marker for separating red blood cells by FACS may be CD235a.
- the “erythroid cell” may preferably be a CD71-positive CD235a-positive cell.
- the erythroid cells induced to differentiate may be provided as a cell population containing other cell types, or may be a purified population.
- the erythroid cell is a cell population containing other cell types, the erythroid cell is preferably contained in 30% or more, more preferably 50% or more.
- the mesoderm cells can be cultured using a medium that induces differentiation into erythroid cells.
- the mesoderm cell in this step is preferably in a state where pluripotent stem cells are induced to differentiate to mesoderm cells in contact with the three-dimensional support, but the differentiation is induced by any method.
- Germ cells may be in contact with a three-dimensional support just before this step.
- the medium used for inducing erythroid cells can be prepared using a medium used for culturing animal cells as a basal medium.
- basal media include IMDM medium, MediumMedi199 medium, mTeSR1 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, Fistem's medium (Invitrogen), Mouse Embryonic fibroblast conditioned medium (MEF-CM), and mixed media thereof are included.
- StemPro34 medium is preferably used.
- the medium may contain serum or may be serum-free.
- 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 It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
- KSR Knockout Serum Replacement
- the medium in this step may further contain a ROCK inhibitor.
- the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK).
- ROCK Rho kinase
- Y-27632 can be used in the present invention.
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
- the CO 2 concentration is about 2-5%, preferably 5%.
- factors that induce differentiation into erythroid cells include stem cell factor (Stem Cell Factor (SCF)), colony stimulating factor (Coloney-Stimulating Factor (CSF)), granulocyte colony stimulating factor (Granulocyte- (G-) CSF) ), Erythropoietin (EPO), interleukins, thrombopoietin (TPO) and Flt3 ligand.
- SCF stem cell factor
- CSF Coloney-Stimulating Factor
- G- CSF granulocyte colony stimulating factor
- EPO Erythropoietin
- interleukins are proteins secreted from leukocytes and are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL.
- interleukins are proteins secreted from leukocytes and are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7
- differentiation induction into erythroid cells can be performed, for example, by the following steps: mesoderm cells retained on a three-dimensional support are cultured in a medium containing erythropoietin (EPO) and SCF. Process.
- EPO erythropoietin
- the basal medium used in the above step can be preferably StemPro34 medium.
- the concentration of the differentiation-inducing factor into erythroid cells in the medium is not particularly limited as long as the concentration can induce the target cells.
- the concentration of EPO in the medium is, for example, in the range of 1U / ml to 20U / ml, for example, 1U / ml, 2U / ml, 3U / ml, 4U / ml, 5U / ml, 6U / ml, 7U / ml.
- concentration of EPO in the medium is, for example, in the range of 1U / ml to 20U / ml, for example, 1U / ml, 2U / ml, 3U / ml, 4U / ml, 5U / ml, 6U / ml, 7U / ml.
- the concentration of SCF in the medium is, for example, in the range of 1 ng / ml to 200 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml Examples include, but are not limited to, ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml. Preferably, it is 50 ng / ml.
- the culture time in this step is, for example, 60 days or less, preferably 16 to 41 days, and particularly preferably 31 days.
- ⁇ Method of inducing hematopoietic cells from pluripotent stem cells> the above-described ⁇ method for inducing mesoderm cells from pluripotent stem cells> and ⁇ method for inducing hematopoietic cells from mesoderm cells> may be combined to induce hematopoietic cells from pluripotent stem cells. It can.
- the differentiation induction conditions in that case include, for example, the following methods, but are not limited thereto.
- a method for inducing myeloid cells from pluripotent stem cells the following steps: (i) culturing pluripotent stem cells in a medium containing BMP4 in contact with a three-dimensional support; (ii) culturing the cells obtained in step (i) in a medium containing VEGF, bFGF and SCF; and (iii) A method including the step of culturing the cells obtained in step (ii) in a medium containing SCF, IL-3, Flt3L and thrombopoietin (TPO) is exemplified.
- TPO thrombopoietin
- a method for inducing monocytic cells from pluripotent stem cells the following steps: (i) culturing pluripotent stem cells in a medium containing BMP4 in contact with a three-dimensional support; (ii) culturing the cells obtained in step (i) in a medium containing VEGF, bFGF and SCF; (iii) culturing the cells obtained in step (ii) in a medium containing SCF, IL-3, Flt3L and TPO, (iv) culturing the cells obtained in step (iii) in a medium containing SCF, IL-3, Flt3L, TPO and M-CSF, and (v) A method comprising culturing the cells obtained in step (iv) in a medium containing Flt3L, M-CSF and GM-CSF is exemplified.
- a method for inducing erythroid cells from pluripotent stem cells the following steps: (i) culturing pluripotent stem cells in a medium containing BMP4 in contact with a three-dimensional support; (ii) culturing the cells obtained in step (i) in a medium containing VEGF, bFGF and SCF; and (iii) A method including the step of culturing the cells obtained in step (ii) in a medium containing erythropoietin (EPO) and SCF is exemplified.
- EPO erythropoietin
- any technique known in the art prior to the filing of the present application can be used.
- the present invention provides a therapeutic agent for a disease, (A) a transplant material containing a three-dimensional support containing mesodermal cells produced by the method of the present invention, or (B) produced by the method of the present invention.
- a therapeutic agent for blood diseases containing hematopoietic cells The mesodermal cells and / or hematopoietic cells in the three-dimensional support obtained by the method of the present invention may be derived from the patient who is the subject of the treatment or from other individuals. May be. Preferably, it originates from the patient who is the subject of the treatment.
- mesoderm cells and / or hematopoietic cells in the three-dimensional support are derived from other individuals, it is possible to collect somatic cells from another person with the same HLA type from the viewpoint that rejection does not occur preferable.
- the agent in the present invention may include a three-dimensional support containing mesoderm cells alone, or a buffer or antibiotic together with a three-dimensional support containing mesoderm cells, Other pharmaceutical additives may be included.
- the drug in the present invention is a scaffold material (scaffold) such as fibronectin, laminin, synthetic polymer (for example, polylactic acid) for the purpose of promoting the engraftment of cells contained in the three-dimensional support to the recipient tissue. ) May further be included.
- the drug in the present invention may contain arbitrary cell (s) other than mesoderm cells.
- the drug in the present invention may contain induced hematopoietic cells alone, or any cell other than hematopoietic cells, such as buffers, antibiotics, other pharmaceutical additives, and the like together with hematopoietic cells. (May be more than one).
- the drug in the present invention is widely effective as a therapeutic agent for blood diseases.
- the target diseases of the therapeutic agent in the present invention include congenital anemia, aplastic anemia, autoimmune anemia, myelodysplastic syndrome (MDS), granulocytopenia, lymphopenia, thrombocytopenia Decreased hematopoietic stem cells and / or hematopoietic progenitor cells associated with various cancers or tumors, decreased hematopoietic stem cells and / or hematopoietic progenitor cells associated with cancer chemotherapy or radiotherapy, acute radioactive syndrome, bone marrow / umbilical cord blood / peripheral Delayed recovery of hematopoietic stem cells and / or hematopoietic progenitor cells after blood transplantation, decreased hematopoietic stem cells and / or hematopoietic progenitor cells associated with blood transfusion, leukemia (acute myeloid leukemia (AML), acute lymph
- the route of administration of the drug to the patient is not particularly limited.
- it is preferably administered to a patient by transplantation.
- it can be performed by the same method as conventional bone marrow transplantation and cord blood transplantation.
- drug administration transplantation
- it is desirable to allow sufficient time for the desired cells to be engrafted in the tissue.
- administration forms such as intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal, intramedullary, and intracerebral can be exemplified.
- the preferred route of administration can be intravenous or intramedullary.
- the hematopoietic cells obtained by the method of the present invention can also be used in transplantation therapy. Or the same method as umbilical cord blood transplantation.
- the dose / transplant amount of the drug of the present invention to a patient varies depending on the type of disease state to be treated, symptoms and severity of disease, patient age, sex or body weight, administration method / transplant method, etc. Although it cannot be said, an appropriate dose / transplant amount can be appropriately determined by the doctor taking into consideration the above situation.
- one or a plurality of three-dimensional supports having a size capable of entering the container can be placed in one culture container and cultured.
- the three-dimensional support is used while holding pluripotent stem cells and / or mesoderm cells.
- An original support or the like can be used.
- the pluripotent stem cell from which differentiation into mesoderm cells may be in the form of a small cell or a single cell, and preferably in the form of a single cell.
- the culture vessel used in this step can be any vessel that is usually used in the art, and examples include, but are not limited to, petri dishes, flasks, culture tanks and the like.
- a large volume container capable of culturing a large amount of cells can be used.
- the medium used in the large-scale culture the same medium as that used in the above-described differentiation induction into mesoderm cells or hematopoietic cells can be used.
- a medium by any differentiation induction method known before the filing date of the present application.
- culture conditions for mass culture for example, stationary culture, shaking culture, stirring culture, and the like can be employed.
- shaking culture or stirring culture that allows cells to come into contact with the medium components efficiently is used.
- the collected hematopoietic cells can be collected by collecting a medium containing hematopoietic cells. By repeatedly collecting the medium, a large amount of hematopoietic cells can be collected.
- the collection of the culture medium may be performed manually, or may be performed using an apparatus equipped with an automatic collection device. Preferably, it is performed by an apparatus equipped with an automatic recovery device.
- Another method for collecting hematopoietic cells is a method of collecting cells adhering to a three-dimensional support. In this case, the recovered three-dimensional support is physically or enzymatically treated to dissociate the cells from the three-dimensional support and recover them. Only one or both of the hematopoietic cell recovery from the medium and the three-dimensional support may be performed.
- the collected hematopoietic cells may be used as they are, or may be used after being purified, depending on the purpose of use.
- Example 1 The following strains were used as pluripotent stem cells human ES cells (KhES1 strain) and human iPS cells (201B7 strain, 409B2 strain and CB-A11 strain).
- KhES1 strain Human ES cells were cultured by a conventional method using the KhES1 strain established by the Center for Stem Cell Medicine, Institute of Regenerative Medicine, Kyoto University (Suemori H, et al. Biochem Biophys Res Commun. 345). : 926-32, 2006). Human ES cells were used with the approval of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
- 201B7 strain 201B7 strain was prepared by the method described in Takahashi K, et al. Cell.
- epidermal vectors (pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE -hUL) was transfected by electroporation and cultured on a mitomycin-treated mouse fetal fibroblast feeder to prepare an iPS cell line. Culture was performed by conventional methods (Takahashi K, et al. Cell. 131: 861-72, 2007 and Nakagawa M, et al. Nat Biotechnol. 26: 101-6, 2008).
- iPS cell line was prepared from cord blood hematopoietic cells based on the method described in Yanagimachi, MD, et al., PLoS ONE, 8 (4): e59243, 2013. The resulting iPS cell line is maintained on mitotically inactive SNL feeder cells in primate ES Cell Medium (ReproCELL) supplemented with 5 ng / mL bFGF, or in mTeSR1 serum-free medium (STEMCELL Technologies), Maintenance culture was performed on tissue culture dishes coated with growth factor-reduced Matrigel (Becton-Dickinson). The medium was changed every day.
- ReproCELL primate ES Cell Medium
- mTeSR1 serum-free medium STMCELL Technologies
- CS collagen sponge Collagen sponge
- PET polyethylene terephthalate
- Example 2 Induction of hematopoietic cells from pluripotent stem cell nodules
- CS three-dimensional support
- each of the KhES1 strain, 201B7 strain, 409B2 strain and CB-A11 strain was treated with a CTK solution for 1 minute at room temperature, and then washed twice with phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- each cell line is peeled off from the culture plate, and after adding 1.5 mL mTeSR1 medium, each cell line is recovered in a 15 mL conical tube (Becton-Dickinson) and 300-500 ⁇ m in diameter. Dissociated by pipetting until small. The tube was left standing for 1 minute to precipitate a small mass of each cell line. Thereafter, the supernatant was removed, and a small lump of each cell line was collected. Subsequently, a small lump of each cell line was seeded on CS placed in a 24-well plate. The CS prepared by the method of Example 1 was used. After overnight incubation with a maintenance medium for undifferentiated pluripotent stem cells (mTeSR1), CS was transferred to a 12-well plate containing a differentiation medium (mTeSR1 supplemented with BMP4).
- mTeSR1 undifferentiated pluripotent stem cells
- mTeSR1 medium was supplemented with a cytokine cocktail consisting of 80 ng / mL VEGF (R & D Systems), 25 ng / mL bFGF (Wako) and 100 ng / mL SCF (R & D Systems).
- the serum medium (containing 2 mM mMglutaMAX (Invitrogen)) (Gibco) was replaced.
- CS On the 6th day from the start of differentiation, cells were collected. Briefly, to collect adherent cells on CS, CS was placed on the side wall of a conical tube (Becton-Dickinson) and centrifuged at 7000 rpm for 10 seconds to drain water from the CS. Then, 1 mL mL Accmax® (Innovative Cell Technologies, Inc.) was added to CS. After incubating at 37 ° C. for 10 minutes, 9 ⁇ mL PBS was added to the well and mixed. After squeezing CS with a spatula, it was removed from the tube, and then the tube was centrifuged at 1500 rpm for 5 minutes to obtain a cell pellet. In order to collect floating cells, CS was transferred to another well and the remaining medium was collected and centrifuged at 1500 rpm for 5 minutes to obtain a cell pellet.
- CS In order to collect floating cells, CS was transferred to another well and the remaining medium was collected and centrifuged at 1500
- KDR-positive CD34-positive HPC was obtained from all the cell lines KhES1, 201B7, 409B2, and CB-A11 (FIG. 1c).
- This differentiation induction efficiency is based on the previously reported 2D Matrigel method (Niwa, A. et al. PloS one 6, e22261, 2011 and Yanagimachi, MD et al., PLoS ONE, 8 (4): e59243, 2013). It was comparable to that used.
- each marker gene ZFP42 and Nanog as pluripotent stem cell-specific markers, and mesoderm
- FOG. 6 quantitative PCR
- medium was mixed with 50 ng / mL SCF (R & D Systems), 50 ng / mL IL-3 (R & D Systems), 5 ng / mL TPO (R & D Systems), 50 ng / mL M-CSF (R & D Systems ) And 50 ng / mL FL3 (R & D Systems) were replaced with StemPro-34 serum-free medium.
- the medium was StemPro-34 serum-free medium supplemented with 50 ng / mL FL3 (R & D Systems), 25 ng / mL GM-CSF (R & D Systems) and 50 ng / mL M-CSF (R & D Systems) was replaced.
- StemPro-34 serum-free medium supplemented with 5 IU / mL EPO (EMD Biosciences), 50 ng / mL IL-3 (R & D Systems) and 50 ng / mL SCF (R & D Systems) for erythroid cell induction
- EPO EPO
- 50 ng / mL IL-3 R & D Systems
- 50 ng / mL SCF R & D Systems
- the collected cells were subjected to flow cytometry analysis. Briefly, data for flow cytometry analysis was collected using MACS®QuantTM®Analyzer® (Miltenyi®Biotec) and analyzed by FlowJo®software® (Treestar). In addition, the morphology of the obtained cells was observed by Giemsa staining. Giemsa staining was performed by seeding cells on a glass slide with CYTOSPIN-4 (Thermo Scientific) and staining with May-Grunwald-Giemsa staining solution (MERCK) according to the manufacturer's instructions.
- Example 3 Induction of hematopoietic cells from a single pluripotent stem cell
- Induction of mesoderm cells from a single pluripotent stem cell Since the average pore size of CS is about 200 ⁇ m, PSC nodules penetrate into CS I thought it was not. Therefore, in order to utilize the three-dimensional structure of CS, whether or not CS can support hematopoietic cell differentiation of PSC dissociated into single cells was examined. Briefly, each of the KhES1 strain, 201B7 strain, 409B2 strain and CB-A11 strain was treated with a CTK solution for 1 minute at room temperature, and then washed twice with phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- the CS prepared by the method of Example 1 was used. On the next day, add 1 ml of maintenance medium (mTeSR1) for undifferentiated pluripotent stem cells, further culture for 1 day, and then transfer CS to a 12-well plate containing differentiation medium (mTeSR1 supplemented with BMP4). did. The subsequent differentiation process was performed in the same manner as in Example 2.
- maintenance medium mTeSR1 for undifferentiated pluripotent stem cells
- KDR positive CD34 positive HPC was obtained from all the cell lines of KhES1, 201B7, 409B2 and CB-A11 (FIGS. 3b-e and gs).
- the number of starting cells was examined, and in the KhES1 strain, 1 ⁇ 10 5 per CS.
- the largest production of KDR-positive CD34-positive HPC was observed with a single cell count (FIG. 3b-d), but these results differed between cell lines (FIG. 3b-d and hp).
- Example 4 Observation by scanning microscope and immunostaining method
- the method of Examples 2 and 3 was used to measure small cells and single cells, respectively.
- the hematopoietic cells induced to differentiate were observed with a scanning microscope. Briefly, CS before seeding cells, CS after differentiating PSC nodules for 41 days, CS after differentiating single PSCs for 21 days, After differentiating PSC nodules for 41 days CS, and CS after differentiating single PSC for 21 days, were fixed overnight at 4 ° C. with 4% paraformaldehyde and 2% glutaraldehyde.
- Example 5 Large-scale induction of hematopoietic cells
- PSC KhES1 nodules were induced to differentiate on multiple CSs by the method of Example 2, and on the 18th day of differentiation, 50 mL containing 25 mL medium containing myeloid cytokine cocktail. Twelve CSs were transferred together to a flask (tissue culture T75 flask (Becton-Dickinson)). Thereafter, CS was cultured under suspension culture conditions, and suspension cells were collected each time the medium was changed.
- the suspension cells were collected by suspending the flask gently to collect the medium containing the suspension cells, and then centrifuging. As a result of repeatedly collecting floating cells over 2 weeks, 1 million cells were recovered from 12 CSs (FIG. 5a). Many of the recovered cells were CD43 positive CD45 positive cells, and their morphology was consistent with immature myeloid cells (FIGS. 5b and c). That is, this suggests that the differentiation induction system by CS can be applied to enlargement in inducing hematopoietic cells from pluripotent stem cells.
- the present invention provides a method for stably supplying a large amount of hematopoietic cells. Therefore, various blood diseases can be treated.
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Abstract
Description
最近では、胚性幹細胞(ES細胞)や体細胞へ未分化細胞特異的遺伝子を導入することで得られる人工多能性幹(iPS)細胞(特許文献1および2)などの多能性を有する細胞を用いて、血球を産生する元となる造血幹細胞や造血前駆細胞、あるいはさらに成熟した赤血球や好中球などを効率的に増幅することが試みられている。
ES細胞やiPS細胞から造血幹細胞や造血前駆細胞への分化誘導法としては、これまでに、胚様体の形成とサイトカインの添加による方法(非特許文献1~3)、異種由来のストローマ細胞との共培養法(非特許文献4)、無血清培地を用いる方法(特許文献3)などが報告されている。赤血球への分化誘導法も同様に、非特許文献1~4、特許文献3などにより報告されており、好中球への分化誘導法は、非特許文献3、5などにおいて報告されている。
しかしながら、血液細胞を多量にかつ安定的に供給できるようにするためには、血液細胞への分化誘導法についてさらなる改善の必要があり、医療応用に適した新たな技術の開発が求められている。
(1)多能性幹細胞から中胚葉細胞を製造する方法であって、多能性幹細胞を三次元支持体との接触下で培養して中胚葉細胞を誘導することを含む、方法。
(2)前記中胚葉細胞が、KDR陽性CD34陽性細胞である、(1)に記載の方法。
(3)前記三次元支持体が、コラーゲンスポンジである、(1)または(2)に記載の方法。
(4)前記コラーゲンスポンジが、ポリエチレンテレフタレート繊維で補強されたコラーゲンスポンジである、(3)に記載の方法。
(5)前記多能性幹細胞と三次元支持体との接触が、三次元支持体の表面および/または内部で生じる、(1)から(4)のいずれかに記載の方法。
(6)多能性幹細胞を三次元支持体との接触下で培養する工程が、下記の工程を含む、(1)から(5)のいずれかに記載の方法:
(i) 多能性幹細胞をBMP4を含有する培地中で培養する工程、および
(ii) 工程(i)で得られた細胞をVEGF、bFGFおよびSCFを含有する培地中で培養する工程。
(7)前記工程(i)および(ii)の培養期間が、それぞれ1~5日および0.5~3日である、(6)に記載の方法。
(8)前記工程(i)および(ii)の培養期間が、それぞれ3日および1日である、(7)に記載の方法。
(9)前記工程(i)の前に、多能性幹細胞を三次元支持体と接触させて前培養を行う、(6)から(8)のいずれかに記載の方法。
(10)前記多能性幹細胞が、ヒトiPS細胞である、(1)から(9)のいずれかに記載の方法。
(11)前記多能性幹細胞が、小塊または単一細胞である、(1)から(10)のいずれかに記載の方法。
(12)前記多能性幹細胞が、単一細胞である、(11)に記載の方法。
(13)(1)から(12)のいずれかに記載の方法により製造された、三次元支持体に支持された中胚葉細胞。
(14)(13)に記載の三次元支持体に支持された中胚葉細胞を含有する、移植用材料。
(15)1またはそれ以上の三次元支持体を保持している培養容器であって、該三次元支持体に中胚葉細胞が支持されており、ここで、該中胚葉細胞が(1)から(12)のいずれかに記載の方法により製造された細胞である、培養容器。
(16)以下の工程を含む造血細胞を製造する方法;
(a)(1)から(12)のいずれかに記載の方法により中胚葉細胞を三次元支持体と接触させて製造する工程、および
(b)得られた三次元支持体に保持された中胚葉細胞を培養容器中で培養することにより造血細胞に誘導する工程。
(17)前記造血細胞が、骨髄系細胞、単球系細胞または赤血球系細胞である、(16)に記載の方法。
(18)前記造血細胞が、骨髄系細胞である、(16)または(17)に記載の方法。
(19)前記造血細胞が、単球系細胞である、(16)または(17)に記載の方法。
(20)前記造血細胞が、赤血球系細胞である、(16)または(17)に記載の方法。
(21)工程(b)が三次元支持体に保持された中胚葉細胞をSCF、IL-3、Flt3Lおよびthrombopoietin (TPO)を含有する培地中で培養する工程を含む、(18)に記載の方法。
(22)工程(b)が下記の工程を含む、(19)に記載の方法:
(i) 三次元支持体に保持された中胚葉細胞をSCF、IL-3、Flt3LおよびTPOを含有する培地中で培養する工程、
(ii) 工程(i)で得られた細胞をSCF、IL-3、Flt3L、TPOおよびM-CSFを含有する培地中で培養する工程、および
(iii) 工程(ii)で得られた細胞をFlt3L、M-CSFおよびGM-CSFを含有する培地中で培養する工程 。
(23)工程(b)が三次元支持体に保持された中胚葉細胞をerythropoietin(EPO)およびSCFを含有する培地中で培養する工程を含む、(20)に記載の方法。
(24)工程(b)の培養期間が16~41日である、(21)または(23)に記載の方法。
(25)工程(b)の培養期間が31日である、(24)に記載の方法。
(26)前記工程(i)、(ii)および(iii)の培養期間が、それぞれ2~5日、2~5日および8~33日である、(22)に記載の方法。
(27)前記工程(i)、(ii)および(iii)の培養期間が、それぞれ3日、3日および23日である、(26)に記載の方法。
(28)(16)から(27)のいずれかに記載の方法で製造された造血細胞を含有する、血液疾患治療剤。
本発明で使用可能な多能性幹細胞(「PSC」ともいう)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、以下のものに限定されないが、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」)、Multilineage-differentiating Stress Enduring cells(Muse細胞)、人工多能性幹(iPS)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。
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))を含む培養液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(竹橋正則ら(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)。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、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。
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「中胚葉細胞」は、中胚葉を構成する細胞群であり、発生の過程で体腔およびそれを裏打ちする中皮、筋肉、骨格、皮膚真皮、結合組織、心臓・血管(血管内皮も含む)、血液(血液細胞も含む)、リンパ管や脾臓、腎臓および尿管、性腺(精巣、子宮、性腺上皮)をつくる能力を有する細胞群を意味する。本発明における「中胚葉細胞」は、例えば、T(Brachyuryと同義)、VEGF receptor-2(KDR)、FOXF1、FLK1、BMP4、MOX1、SDF1およびCD34のようなマーカーの発現により示される。好ましくは、KDRおよびCD34を発現する細胞である。本発明における「中胚葉細胞」は、別段の断りがない限り、造血細胞への分化能を有する造血幹細胞や造血前駆細胞を包含し得る。
したがって、本明細書において「(多能性幹/中胚葉)細胞を三次元支持体との接触下で培養する」とは、当該細胞が三次元支持体と何らかの相互作用をし得るまでの距離で近接している状態で培養することを意味する。「(多能性幹/中胚葉)細胞と三次元支持体との接触」は、例えば、物理的な形態のもの、あるいは化学的な形態のものなどが挙げられるが、別段限定されない。好ましくは、細胞側の受容体を介した支持体との結合が挙げられる。
(i) 多能性幹細胞を三次元支持体との接触下でBMP4を含有する培地中で培養する工程、および
(ii) 工程(i)で得られた細胞をVEGF、bFGFおよびSCFを含有する培地中で培養する工程。
本発明において「造血細胞」は、血液系列にコミットしたあらゆる種類の細胞を意味する。好ましくは、本発明における「造血細胞」は、中胚葉細胞から血液系列にコミットした任意の細胞であり得る。本発明における「造血細胞」としては、例えば、骨髄系細胞、好中球、好酸球、好塩基球、赤血球系細胞、赤芽球、赤血球、単球系細胞、単球、マクロファージ、巨核球、血小板、樹状細胞などが挙げられるが、これらに限定されない。本発明において「造血細胞」は、好ましくは、骨髄系細胞、単球系細胞または赤血球系細胞であり得る。本明細書における「造血細胞」は、特に断りがない限り、「血液細胞」と同義で使用される。
本発明において「骨髄系細胞」は、中胚葉細胞から骨髄系系列への運命がコミットされた一連の細胞を意味し、例えば、好中球、好酸球、好塩基球などが挙げられるがこれに限定されない。本発明において「好中球」は、中性色素により染色される特殊顆粒を有する顆粒球の一種である。本発明において「好酸球」は、major basic protein (MBP) 、eosinophil cationic protein (ECP)、eosinophil peroxidase (EPO)、eosinophil-derived neurotoxin (EDN)をその顆粒内に有する細胞であり、より好ましくは、secretory immunoglobulin A (sIgA)の刺激によりEDNを放出する能力と、IL-5、EotaxinおよびfMLPの刺激により遊走能とを有する細胞である。本発明において「好塩基球」は、塩基性色素による染色により暗紫色に染まる大型の顆粒を有する細胞を意味する。これらの細胞は、CD43、CD45、CD19、CD13、CD33、MPOなどのマーカーの発現により検出され得る(マーカーの種類は限定されない)。本発明において「骨髄系細胞」は、好ましくは、CD43陽性CD45陽性の細胞であり得る。
本発明において「単球系細胞」は、中胚葉細胞から単球系系列への運命がコミットされた一連の細胞を意味し、例えば、単球、マクロファージなどが挙げられるがこれに限定されない。本発明において「単球」および「マクロファージ」は、いずれも白血球細胞の一種であり、異物を貪食し、抗原提示する能力を有する細胞である。これらの細胞は、CD14、CD16、CD45、CD68などのマーカーの発現により検出され得る(マーカーの種類は限定されない)。本発明において「単球系細胞」は、好ましくは、CD14陽性CD45陽性の細胞であり得る。
(i) 三次元支持体に保持された中胚葉細胞をSCF、IL-3、Flt3LおよびTPOを含有する培地中で培養する工程、
(ii) 工程(i)で得られた細胞をSCF、IL-3、Flt3L、TPOおよびM-CSFを含有する培地中で培養する工程、および
(iii) 工程(ii)で得られた細胞をFlt3L、M-CSFおよびGM-CSFを含有する培地中で培養する工程 。
ng/mlであるがこれらに限定されない。好ましくは、50ng/mlである。
本発明において「赤血球系細胞」は、中胚葉細胞から赤血球系列への運命がコミットされた一連の細胞を意味し、例えば、赤血球が挙げられるがこれに限定されない。本発明において「赤血球」は、ヘモグロビンに富んだ細胞を意味し、ヘモグロビンのα-グロビン、ε-グロビン、γ-グロビン、β-グロビン、CD235aなどのマーカーの発現により検出され得る。成熟した赤血球において好ましいマーカーは、α-グロビンおよびβ-グロビンであり得る(マーカーの種類は限定されない)。また、FACSにより赤血球を分離する場合において好ましいマーカーは、CD235aであり得る。本発明において「赤血球系細胞」は、好ましくは、CD71陽性CD235a陽性の細胞であり得る。
本発明において、上記の<多能性幹細胞から中胚葉細胞を誘導する方法>と<中胚葉細胞から造血細胞を誘導する方法>とを組み合わせて、多能性幹細胞から造血細胞を誘導することができる。その場合の分化誘導条件は、例えば、下記の方法が挙げられるがこれらに限定されない。
(i) 多能性幹細胞を三次元支持体との接触下でBMP4を含有する培地中で培養する工程、
(ii) 工程(i)で得られた細胞をVEGF、bFGFおよびSCFを含有する培地中で培養する工程、
および
(iii) 工程(ii)で得られた細胞をSCF、IL-3、Flt3Lおよびthrombopoietin (TPO)を含有する培地中で培養する工程
を含む方法が例示される。
(i) 多能性幹細胞を三次元支持体との接触下でBMP4を含有する培地中で培養する工程、
(ii) 工程(i)で得られた細胞をVEGF、bFGFおよびSCFを含有する培地中で培養する工程、
(iii) 工程(ii)で得られた細胞をSCF、IL-3、Flt3LおよびTPOを含有する培地中で培養する工程、
(iv) 工程(iii)で得られた細胞をSCF、IL-3、Flt3L、TPOおよびM-CSFを含有する培地中で培養する工程、および
(v) 工程(iv)で得られた細胞をFlt3L、M-CSFおよびGM-CSFを含有する培地中で培養する工程を含む方法が例示される。
(i) 多能性幹細胞を三次元支持体との接触下でBMP4を含有する培地中で培養する工程、
(ii) 工程(i)で得られた細胞をVEGF、bFGFおよびSCFを含有する培地中で培養する工程、
および
(iii) 工程(ii)で得られた細胞をerythropoietin(EPO)およびSCFを含有する培地中で培養する工程
を含む方法が例示される。
本発明は、疾患の治療剤として、(A)本発明の方法により製造された中胚葉細胞を含有する三次元支持体を含有する移植用材料、または(B)本発明の方法により製造された造血細胞を含有する血液疾患の治療剤を提供する。本発明の方法によって得られる三次元支持体内の中胚葉細胞および/または造血細胞は、当該治療の対象となる患者本人に由来するものであってもよいし、他の個体に由来するものであってもよい。好ましくは、当該治療の対象となる患者本人に由来するものである。三次元支持体内の中胚葉細胞および/または造血細胞が他の個体に由来するものである場合、拒絶反応が起こらないという観点から、HLAの型が同一である他人から体細胞を採取することが好ましい。
本発明において、造血細胞を大量に得るために、容器に入ることができる大きさの一つまたは複数の三次元支持体を一つの培養容器に入れて培養することができる。本工程において、三次元支持体は、多能性幹細胞および/または中胚葉細胞を保持した状態のものが使用される。例えば、上記したような、多能性幹細胞と接触させた分化誘導前の三次元支持体や多能性幹細胞を三次元支持体と接触させた条件下で中胚葉細胞まで分化させた状態の三次元支持体などが使用され得る。この場合、中胚葉細胞に分化させる元となる多能性幹細胞は、小塊または単一細胞のいずれの形態であってもよく、好ましくは、単一細胞の形態であり得る。本工程において使用される培養容器は、当分野において通常使用されるような任意の容器を用いることができ、例えば、シャーレ、フラスコ、培養タンクなどが挙げられるがこれらに限定されない。好ましくは、大量の細胞の培養が可能となる体積の大きな容器が使用され得る。大量培養において使用される培地は、上記した中胚葉細胞または造血細胞への分化誘導において使用されるものと同じ培地が使用され得る。上記の方法で得られる造血細胞をさらに成熟化した造血細胞とするために、本願出願日前に知られていた任意の分化誘導法による培地を使用することも可能である。大量培養のための培養条件としては、例えば、静置培養、振盪培養、撹拌培養などが採用され得る。好ましくは、細胞が効率良く培地成分に接し得る振盪培養または撹拌培養が使用される。
多能性幹細胞
ヒトES細胞(KhES1株)およびヒトiPS細胞(201B7株、409B2株およびCB-A11株)として以下の株を用いた。
(1)KhES1株
ヒトES細胞は、京都大学再生医科学研究所附属幹細胞医学研究センターによって樹立されたKhES1株を用い、従来の方法で培養した(Suemori H, et al. Biochem Biophys Res Commun. 345:926-32, 2006)。ヒトES細胞の使用は、文部科学省(MEXT)による承認の下に行われた。
(2)201B7株
201B7株は、Takahashi K, et al. Cell. 131: 861-72, 2007に記載の方法で作製された。
(3)409B2株
Okita. K, et al., Stem Cells. 2012 Nov 29.に記載の方法に基づき、ヒト皮膚細胞にエピソーマルベクター(pCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hUL)を電気穿孔法で遺伝子導入し、マイトマイシン処理したマウス胎仔線維芽細胞フィーダー上で培養することにより、iPS細胞株を作製した。培養は、従来の方法で行った (Takahashi K, et al. Cell. 131: 861-72, 2007およびNakagawa M, et al. Nat Biotechnol. 26: 101-6, 2008)。
(4)CB-A11株
Yanagimachi, M. D. et al., PLoS ONE, 8(4): e59243, 2013に記載の方法に基づき、臍帯血造血細胞からiPS細胞株を作製した。得られたiPS細胞株は、5 ng/mL bFGFを補充した霊長類ES Cell Medium (ReproCELL)中、分裂不活性SNLフィーダー細胞上で維持培養するか、またはmTeSR1無血清培地 (STEMCELL Technologies)中、growth factor-reduced Matrigel (Becton-Dickinson)で被覆した組織培養皿上で維持培養を行った。培地は、毎日交換した。
ポリエチレンテレフタレート(PET)繊維の組み込みにより機械的に補強したコラーゲンスポンジ(PETCol-24w、以下「CS」という)を、MedGEL44から購入した。気泡を取り除くために、多能性幹細胞用の維持培地を10 mL加えた50 mLコニカルチューブ(Becton-Dickinson)に6個のCSを入れて、10000 rpmで5分間、遠心分離した。遠心分離後、スパチュラでCSを取り出して、24ウェルプレートに入れて以下の実験に用いた。
多能性幹細胞の小塊からの造血細胞の誘導
(1)多能性幹細胞の小塊からの中胚葉細胞の誘導
三次元支持体(CS)を用いた培養条件下で多能性幹細胞の小塊から中胚葉細胞を誘導できるか否かを検討するために、下記の実験を行った。簡潔には、KhES1株、201B7株、409B2株およびCB-A11株のそれぞれを、室温で1分間、CTK溶液にて処理し、次いでphosphate-buffered saline (PBS)で2回洗浄した。次いで、スクラッパーを用いて、各細胞株を培養プレートから引き剥がし、1.5 mL mTeSR1培地を加えた後に、各細胞株を15mLコニカルチューブ(Becton-Dickinson)に回収して、直径300-500 μmの小塊になるまでピペッティングにより解離した。チューブを1分間立てた状態にしておき、各細胞株の小塊を沈殿させた。その後、上清を除去し、各細胞株の小塊を回収した。続いて、各細胞株の小塊を24ウェルプレート内に配置されたCS上に播種した。CSは、実施例1の方法で調製したものを用いた。未分化多能性幹細胞用の維持培地(mTeSR1)を用いて一晩インキュベートした後、分化培地(BMP4を補充したmTeSR1)を入れておいた12ウェルプレートにCSを移した。
さらに、得られたHPC(D6)および誘導前の多能性幹細胞株(KhES1株またはCB-A11株)において、各マーカー遺伝子(多能性幹細胞特異的マーカーとしてのZFP42およびNanog、ならびに、中胚葉前駆細胞特異的マーカーとしてのTおよびMIXL1、造血前駆細胞特異的マーカーとしてのRUNX1、ならびに内皮前駆細胞マーカーとしてのAPLNRおよびCDH5)を定量PCRで測定した(図6)。その結果、得られたHPCにおいて、ZFP42およびNanogはほとんどその発現が確認されず、T、MIXL1、RUNX1、APLNRおよびCDH5は発現が上昇することが確認された。これらの結果より、本方法で得られた細胞は、造血系中胚葉前駆細胞に誘導され、多能性幹細胞の残存はほとんどないことが確認された。
上記(1)で得られた中胚葉細胞を、特定のサイトカインの組み合わせを添加した培地中で培養することにより、特定の異なる造血細胞へ分化誘導させた。簡潔には、分化誘導開始から6日目に、培地を、異なるセットのサイトカインを含むStemPro-34無血清培地に交換し、特定の造血細胞系列へ分化させた。骨髄系細胞誘導のために、50 ng/mL SCF (R&D Systems)、50 ng/mL IL-3 (R&D Systems)、5 ng/mL TPO (R&D Systems)および50 ng/mL FL3 (R&D Systems)を添加したStemPro-34無血清培地を用いて培養し、その間4日毎に培地を交換した。単球系細胞誘導のために、6~9日目まで、50 ng/mL SCF (R&D Systems)、50 ng/mL IL-3 (R&D Systems)、5 ng/mL TPO (R&D Systems)および50 ng/mL FL3 (R&D Systems)を添加したStemPro-34無血清培地を用いて培養した。10日目に、培地を、50 ng/mL SCF (R&D Systems)、50 ng/mL IL-3 (R&D Systems)、5 ng/mL TPO (R&D Systems)、50 ng/mL M-CSF (R&D Systems)および50 ng/mL FL3 (R&D Systems) を添加したStemPro-34無血清培地に交換した。14日目に、培地を、50 ng/mL FL3 (R&D Systems)、25 ng/mL GM-CSF (R&D Systems)および50 ng/mL M-CSF (R&D Systems)を添加したStemPro-34無血清培地に交換した。赤血球系細胞誘導のために、5 IU/mL EPO (EMD Biosciences)、50 ng/mL IL-3 (R&D Systems)および50 ng/mL SCF (R&D Systems)を添加したStemPro-34無血清培地を用いて培養し、その間2日毎に培地を交換した。最初の分化誘導開始(0日目)から14日目以降は、2-5日毎に、CSを、新鮮な培地を入れたウェルに移した。CSを新たなウェルに移す度に、移した後の培地を繰り返し回収した(22日目、27日目、32日目、37日目、42日目、47日目)。
さらに、CS上の細胞を回収し、CD34+前駆細胞およびCD45+造血系細胞が実際に培養されていることを確認した(図2h)。さらに、本方法により得られる造血系細胞を評価するため、コロニー形成試験を行ったところ、CSを用いて誘導されたコロニーは37日までコロニー形成能を有していることが確認された(図2i)。得られた細胞は、骨髄性細胞への誘導傾向が見られた。
単一多能性幹細胞からの造血細胞の誘導
(1)単一多能性幹細胞からの中胚葉細胞の誘導
CSの平均ポアサイズは約200 μmであるため、PSCの小塊はCS内に浸透していないと考えられた。そこで、CSの三次元構造を利用するために、CSが単一細胞にまで解離されたPSCの造血細胞分化を支持し得るか否かについて検討を行った。簡潔には、KhES1株、201B7株、409B2株およびCB-A11株のそれぞれを、室温で1分間、CTK溶液にて処理し、次いでphosphate-buffered saline (PBS)で2回洗浄した。次いで、1 mL Accumax (Innovative Cell Technologies, Inc.)を用いて、37℃で10分間処理し、T1000ピペット先端で解離して、9 mL PBSを加えた15 mLコニカルチューブ(Becton-Dickinson)に回収した。その後、細胞を2回洗浄して、Accumax内に含まれるコラゲナーゼを除去し、1500 rpmで5分間遠心分離した。続いて、細胞ペレットを、10 mM Y27632 (abcam)を含む500-1000 μLのmTeSR1に懸濁した。それぞれの細胞株を含む50 μLの細胞懸濁物を、24ウェルプレート内に配置させておいたCSの中央に穏やかに滴下した。CSは、実施例1の方法で調製したものを用いた。翌日に、1mlの未分化多能性幹細胞用の維持培地(mTeSR1)を加えて、さらに1日間培養した後、分化培地(BMP4を補充したmTeSR1)を入れておいた12ウェルプレートにCSを移した。その後の分化工程は、実施例2と同様の方法により行った。
また、1個のCSあたりの最適な未分化多能性幹細胞の数を決定するために、いくつかの出発細胞数で検討を行ったところ、KhES1株では、1個のCSあたり1×105個の細胞数のときに、最大のKDR陽性CD34陽性 HPCの産生が観察されたが(図3b-d)、これらの結果は、細胞株間で異なっていた(図3b-dおよびh-p)。
実施例2と同様の方法により、骨髄系細胞への分化誘導を行った。その結果、多能性幹細胞の小塊を用いた場合と同様に、約20日目あたりから培養培地中に浮遊造血細胞が放出され始めた。骨髄系細胞への分化誘導においては、浮遊生細胞は、骨髄様表面マーカーおよび形態を示した(図3fおよびg)。
走査型顕微鏡および免疫染色法による観察
分化した細胞同士の相互作用や分化した細胞のスキャフォールド内での分布状況を評価するために、実施例2および3の方法によりそれぞれ小塊および単一細胞から分化誘導させた造血細胞について走査型顕微鏡による観察を行った。簡潔には、細胞を播種する前のCS、41日間PSCの小塊を分化させた後のCS、21日間単一PSCを分化させた後のCS、41日間PSCの小塊を分化させた後のCS、および21日間単一PSCを分化させた後のCSのそれぞれを、4% paraformaldehydeおよび2% glutaraldehydeを用いて、4℃で一晩、固定した。1% OsO4で3時間の後固定(post-fixation)を行った後、CSを脱水して乾燥させ、プラチナパラジウムの薄層でコートした。その後、Hitachi S-4700走査型電子顕微鏡(Hitachi, Tokyo, Japan)を用いて、標本を観察した。なお、PSCとしては、すべてKhES1が使用された。
その結果、コラーゲン繊維は、細胞内コラゲナーゼの影響で、分化の間に徐々に崩壊していた(図4a-c)。PSCの小塊を分化させたとき、球状の造血様細胞がPET繊維を取り囲むように凝集していた(図4d)。興味深いことに、これらの造血細胞はまた、PET繊維を架橋するシート様細胞に接着していた(図4e)。これらの構造はまた、単一細胞から分化させたときにおいても観察された(図4fおよびg)。全体的に、走査型顕微鏡による観察は、CS上の造血細胞ニッチが、PET繊維、造血細胞および非造血系支持細胞が緊密な位置関係を有しながら配置されていることを明らかにした。興味深いことに、このニッチを構成する要素は、以前に報告された(Leisten I, et al., Biomaterials.33(6):1736-47 (2012))インビトロでの造血微小環境と一致していた。
さらに、CS内部の細胞の特性を調べるため、CSごとに免疫染色を行ったところ、CD34+細胞およびコラーゲン繊維が嚢状(sac状)構造を形成し、CD45+細胞がCSの空洞内に取り込まれていることが確認された(図7a~c)。
造血細胞の大規模誘導
本発明によるCSを用いた培養法が大規模浮遊培養に適用できるか否かを検討するために、50 mLのフラスコを用いて実験を行った。簡潔には、実施例2の方法により、複数のCS上でPSC(KhES1)の小塊を分化誘導し、分化の18日目において、骨髄系サイトカインカクテルを含む25 mLの培地を入れた50 mLフラスコ(組織培養T75 flask (Becton-Dickinson))に12個のCSを一緒に移した。その後、CSを浮遊培養条件下で培養し、培地交換の度に浮遊細胞を回収した。浮遊細胞の回収は、フラスコを穏やかに懸濁して浮遊細胞を含む培地を回収し、次いで、遠心分離することにより行った。2週間にわたり浮遊細胞を繰り返し回収した結果、100万個の細胞が12個のCSより回収された(図5a)。回収された細胞の多くは、CD43陽性CD45陽性細胞であり、それらの形態は、未成熟な骨髄系細胞と一致していた(図5bおよびc)。すなわち、このことは、CSによる分化誘導系が、多能性幹細胞から造血細胞を誘導する際の大規模化に適用可能であることを示唆している。
Claims (28)
- 多能性幹細胞から中胚葉細胞を製造する方法であって、多能性幹細胞を三次元支持体との接触下で培養して中胚葉細胞を誘導することを含む、方法。
- 前記中胚葉細胞が、KDR陽性CD34陽性細胞である、請求項1に記載の方法。
- 前記三次元支持体が、コラーゲンスポンジである、請求項1または2に記載の方法。
- 前記コラーゲンスポンジが、ポリエチレンテレフタレート繊維で補強されたコラーゲンスポンジである、請求項3に記載の方法。
- 前記多能性幹細胞と三次元支持体との接触が、三次元支持体の表面および/または内部で生じる、請求項1から4のいずれか1項に記載の方法。
- 多能性幹細胞を三次元支持体との接触下で培養する工程が、下記の工程を含む、請求項1から5のいずれか1項に記載の方法:
(i) 多能性幹細胞をBMP4を含有する培地中で培養する工程、および
(ii) 工程(i)で得られた細胞をVEGF、bFGFおよびSCFを含有する培地中で培養する工程。 - 前記工程(i)および(ii)の培養期間が、それぞれ1~5日および0.5~3日である、請求項6に記載の方法。
- 前記工程(i)および(ii)の培養期間が、それぞれ3日および1日である、請求項7に記載の方法。
- 前記工程(i)の前に、多能性幹細胞を三次元支持体と接触させて前培養を行う、請求項6から8のいずれか1項に記載の方法。
- 前記多能性幹細胞が、ヒトiPS細胞である、請求項1から9のいずれか1項に記載の方法。
- 前記多能性幹細胞が、小塊または単一細胞である、請求項1から10のいずれか1項に記載の方法。
- 前記多能性幹細胞が、単一細胞である、請求項11に記載の方法。
- 請求項1から12のいずれか1項に記載の方法により製造された、三次元支持体に支持された中胚葉細胞。
- 請求項13に記載の三次元支持体に支持された中胚葉細胞を含有する、移植用材料。
- 1またはそれ以上の三次元支持体を保持している培養容器であって、該三次元支持体に中胚葉細胞が支持されており、ここで、該中胚葉細胞が請求項1から12のいずれか1項に記載の方法により製造された細胞である、培養容器。
- 以下の工程を含む造血細胞を製造する方法;
(a)請求項1から12のいずれか1項に記載の方法により中胚葉細胞を三次元支持体と接触させて製造する工程、および
(b)得られた三次元支持体に保持された中胚葉細胞を培養容器中で培養することにより造血細胞に誘導する工程。 - 前記造血細胞が、骨髄系細胞、単球系細胞または赤血球系細胞である、請求項16に記載の方法。
- 前記造血細胞が、骨髄系細胞である、請求項16に記載の方法。
- 前記造血細胞が、単球系細胞である、請求項16に記載の方法。
- 前記造血細胞が、赤血球系細胞である、請求項16に記載の方法。
- 工程(b)が三次元支持体に保持された中胚葉細胞をSCF、IL-3、Flt3Lおよびthrombopoietin (TPO)を含有する培地中で培養する工程を含む、請求項18に記載の方法。
- 工程(b)が下記の工程を含む、請求項19に記載の方法:
(i) 三次元支持体に保持された中胚葉細胞をSCF、IL-3、Flt3LおよびTPOを含有する培地中で培養する工程、
(ii) 工程(i)で得られた細胞をSCF、IL-3、Flt3L、TPOおよびM-CSFを含有する培地中で培養する工程、および
(iii) 工程(ii)で得られた細胞をFlt3L、M-CSFおよびGM-CSFを含有する培地中で培養する工程 。 - 工程(b)が三次元支持体に保持された中胚葉細胞をerythropoietin(EPO)およびSCFを含有する培地中で培養する工程を含む、請求項20に記載の方法。
- 工程(b)の培養期間が16~41日である、請求項21または23に記載の方法。
- 工程(b)の培養期間が31日である、請求項24に記載の方法。
- 前記工程(i)、(ii)および(iii)の培養期間が、それぞれ2~5日、2~5日および8~33日である、請求項22に記載の方法。
- 前記工程(i)、(ii)および(iii)の培養期間が、それぞれ3日、3日および23日である、請求項26に記載の方法。
- 請求項16から27のいずれか1項に記載の方法で製造された造血細胞を含有する、血液疾患治療剤。
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WO2020051453A1 (en) * | 2018-09-07 | 2020-03-12 | Wisconsin Alumni Research Foundation | Generation of hematopoietic progenitor cells from human pluripotent stem cells |
WO2021107117A1 (ja) * | 2019-11-28 | 2021-06-03 | 国立大学法人京都大学 | 多能性幹細胞からの造血細胞の製造法 |
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GB202102297D0 (en) * | 2021-02-18 | 2021-04-07 | Adaptimmune Ltd | Methods of Producing Haemogenic Endothelial Cells from Pluripotent Stem Cells |
WO2023156774A1 (en) * | 2022-02-15 | 2023-08-24 | The University Of Birmingham | Generating bone marrow organoids |
WO2023191099A1 (en) * | 2022-04-01 | 2023-10-05 | Kyoto University | Axioloid: a stem cell-based model of human axial development |
LU501820B1 (en) * | 2022-04-08 | 2023-10-10 | Care For Rare Found Stiftung Buergerlichen Rechts | Bone marrow organoids produced from induced pluripotent stem cells and uses of these organoids |
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