WO2014189144A1 - 骨髄異形成症候群等の治療/予防薬のスクリーニング方法 - Google Patents
骨髄異形成症候群等の治療/予防薬のスクリーニング方法 Download PDFInfo
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Definitions
- the present invention relates to a method for screening a therapeutic and / or prophylactic agent for myelodysplastic syndrome (MDS), which is acute myeloid leukemia or its related disease.
- MDS myelodysplastic syndrome
- the present invention also relates to a therapeutic agent for myelodysplastic syndrome (MDS), comprising hematopoietic progenitor cells derived from normal iPS cells.
- Myelodysplastic syndrome is a clonal acquired hematopoietic disorder with a poor prognosis associated with chronic treatment-resistant anemia, cytopenias (refractory anemia), and pre-leukemia that is likely to shift to acute myeloid leukemia.
- Bone marrow disease It is characterized by a relatively large number of elderly people, and the probability of onset (secondary MDS) after chemotherapy or radiation therapy for malignant tumors increases several to several tens of times. Coupled with the spread of chemotherapy, MDS cases continue to increase.
- iPS cells mouse and human induced pluripotent stem cells
- Yamanaka et al. Succeeded in establishing iPS cells by introducing 4 genes of Oct3 / 4, Sox2, Klf4 and c-Myc into human skin-derived fibroblasts (Patent Document 1 and Non-Patent Document 1).
- the iPS cells obtained in this way can be differentiated into cells of each tissue after being prepared using cells derived from the patient to be treated, it is possible to reproduce the disease state in vitro It is considered.
- no example of successful production of iPS cells using somatic cells derived from MDS patients has been reported.
- a method for screening for a therapeutic or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome comprising the following steps: (A) Hematopoietic progenitor cells derived from induced pluripotent stem (iPS) cells produced from non-T cells in blood mononuclear cells isolated from patients with myelodysplastic syndromes in the presence and absence of the test substance A step of forming colonies, and (b) when the number of colonies in the presence of the test substance is increased as compared to the number of colonies in the absence of the test substance, Selecting as a candidate for the treatment or prevention of acute myeloid leukemia or myelodysplastic syndrome, Including the method.
- iPS induced pluripotent stem
- a method for screening for a therapeutic or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome comprising the following steps: (A) a step of inducing iPS cells produced from non-T cells in blood mononuclear cells isolated from a patient with myelodysplastic syndrome to hematopoietic progenitor cells, (B) inducing the hematopoietic progenitor cells into blood cells in the presence and absence of the test substance, and (c) in the presence of the test substance, the number of blood cells is higher than in the absence. If increased, selecting the test substance as a candidate for the treatment or prevention of acute myeloid leukemia or myelodysplastic syndrome, Including the method.
- step (b) includes the following steps: (I) culturing hematopoietic progenitor cells in a medium containing VEGF, IL-6, IL-3, IL-11, SCF, FLT3L, erythropoietin (EPO) and thrombopoietin (TPO); (Ii) culturing the cells obtained in step (i) in a medium containing IL-3, SCF and EPO, and (iii) containing the cells obtained in step (ii) containing SCF and EPO Culturing in a medium.
- step (b) comprises culturing hematopoietic progenitor cells in a medium containing GM-CSF and / or G-CSF.
- step (b) includes a step of culturing hematopoietic progenitor cells in a medium containing TPO and SCF.
- hematopoietic progenitor cells are hematopoietic progenitor cells induced by a method comprising a step of co-culturing iPS cells with feeder cells.
- the feeder cell is an OP9 cell line.
- a therapeutic agent for myelodysplastic syndrome comprising hematopoietic progenitor cells produced by inducing differentiation of iPS cells produced from T cells of a patient with myelodysplastic syndrome.
- hematopoietic progenitor cells are hematopoietic progenitor cells induced by a method comprising a step of co-culturing iPS cells with feeder cells.
- the feeder cell is an OP9 cell line.
- a method for screening for a therapeutic or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome comprising the following steps: (A) Hematopoietic progenitor cells (non-T cell-derived hematopoietic progenitor cells) derived from induced pluripotent stem (iPS) cells produced from non-T cells in blood mononuclear cells isolated from patients with myelodysplastic syndrome And a step of bringing a hematopoietic progenitor cell (control hematopoietic progenitor cell) derived from a control iPS cell into contact with a test substance, and (b) the number of non-T cell-derived hematopoietic progenitor cells after contact with the test substance, A step of selecting the test substance as a candidate for the treatment or prevention of acute myeloid leukemia or myelodysplastic syndrome when the number of control hematopoietic progenitor cells after contact with the test substance
- control hematopoietic progenitor cells are hematopoietic progenitor cells derived from iPS cells produced from T cells in blood mononuclear cells isolated from the same patient as non-T cells.
- the present invention enables screening for therapeutic and / or preventive drugs for acute myeloid leukemia or myelodysplastic syndrome (MDS) using a novel tool.
- MDS myelodysplastic syndrome
- cells derived from the same patient and derived from the same blood mononuclear cells can be used as a control, the conditions of cells other than the presence or absence of a disease state can be aligned, and a more accurate screening system can be obtained.
- FIG. 1 shows a stained image of blood in an MDS patient with established iPS cells.
- A shows blasts, bone marrow cells and giant platelets in KM3 (MDS patient)
- B shows blasts, bone marrow cells, erythroblasts and megakaryocytes in KM5 (MDS patient).
- FIG. 2 shows the measurement results of the copy number of the chromosome 18p11.3 gene (USP14, NDC810 and MYL12A) in iPS cells derived from KM3 blood (non-T cells and T cells).
- FIG. 3 shows the results of karyotype and CGH analysis of MDS-iPS cells.
- FIG. 15 shows the results of colony forming unit (CFU) assay of hematopoietic progenitor cells (HPC) induced to differentiate from abnormal MDS-iPSC (MDS or MD) and Normal ⁇ iPSC (Normal or N) derived from KM5, KM15 and KM16. Show.
- CFU colony forming unit
- FIG. 17 shows the results of flow cytometry when abnormal MDS-iPSC ⁇ (MDS or MD) and Normal iPSC (Normal or N) derived from KM3, KM5, KM15 and KM16 were differentiated into neutrophils.
- MDS or MD abnormal MDS-iPSC ⁇
- Normal iPSC Normal or N
- the vertical axis represents the ratio of neutrophils (CD66b +) in CD11b + bone marrow cells.
- 5M-B1 is an iPS cell line that exhibits a normal karyotype.
- FIG. 18 shows the results of flow cytometry when differentiated KM3-derived mutant iPS cells (KM3-A4) and normal iPS cells (KM3-A3) into megakaryocytes.
- mutant MDS-iPS cells As used herein, the terms “mutant MDS-iPS cells”, “mutant MDS-iPSCs” and “MDS-iPSC” are the following “normal MDS-iPS cells”, “normal MDS-iPSCs” and “Normal”. In contrast to “iPSC”, it can also be read as “abnormal MDS-iPS cells”.
- normal MDS-iPS cells normal MDS-iPS cells
- normal MDS-iPSCs normal iPSC or an equivalent term is an iPS cell produced from a somatic cell of a patient with myelodysplastic syndrome. It means iPS cells that do not have the intracellular pathology characteristic of myelodysplastic syndrome.
- IPS cells produced from T cells in blood mononuclear cells isolated from patients with myelodysplastic syndromes did not contain intracellular pathologies such as chromosomal abnormalities unique to myelodysplastic syndromes, and were derived from these iPS cells.
- Hematopoietic progenitor cells are preferably iPS cells produced from T cells because they have normal colony forming ability and ability to differentiate into hematopoietic cells.
- an iPS cell introduces a specific nuclear reprogramming substance into a somatic cell in the form of a protein or a nucleic acid encoding the same, or the nuclear reprogramming substance endogenous by a drug.
- Artificial stem cells derived from somatic cells that have almost the same properties as ES cells, such as pluripotency of differentiation and ability to proliferate through 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 M. Nakagawa et al. (2008) Nat.
- the nuclear reprogramming substance is not particularly limited as long as it is a gene specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof.
- nucleotide sequences of mouse and human cDNA of each nuclear reprogramming substance and amino acid sequence information of the protein encoded by the cDNA refer to NCBI accession numbers described in WO 2007/069666, and L-Myc, Lin28 , Lin28b, Esrrb, Esrrg and Glis1 mouse and human cDNA sequences and amino acid sequence information can be obtained by referring to the following NCBI accession numbers, respectively.
- a person skilled in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.
- nuclear reprogramming substances may be introduced into somatic cells in the form of proteins, for example, by lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA, for example, It can be introduced into somatic cells by techniques such as viruses, plasmids, artificial chromosomes, vectors, lipofection, liposomes, and microinjection.
- 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 (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) and the like.
- artificial chromosome vectors 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, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that a nuclear reprogramming substance can be expressed.
- promoter used examples include EF1 ⁇ promoter, CAG promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV- A TK (herpes simplex virus thymidine kinase) promoter or the like is used.
- EF1 ⁇ promoter, CAG promoter, MoMuLV LTR, CMV promoter, SR ⁇ promoter and the like can be mentioned.
- drug resistance genes for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.
- thymidine kinase gene diphtheria toxin gene and other selectable marker sequences
- green fluorescent protein (GFP) green fluorescent protein
- GUS ⁇ -glucuronidase
- reporter gene sequences such as FLAG, and the like.
- the above vector contains a LoxP sequence before and after the gene or promoter encoding the nuclear reprogramming substance and the gene encoding the nuclear reprogramming substance that binds to it. You may have.
- the vector replicates without chromosomal integration and is episomal, so that the origin and replication of lymphotropic herpesvirus (lymphotrophic herpes virus), BK virus and bovine papillomavirus
- lymphotropic herpesvirus lymphotropic herpesvirus
- BK virus BK virus
- sequence which concerns on may be included. Examples include EBNA-1 and oriP or LargeLT and SV40ori sequences (WO 2009/115295, WO 2009/157201 and WO 2009/149233).
- an expression vector for polycistronic expression may be used.
- the gene coding sequence may be linked by an IRES or foot-and-mouth disease virus (FMDV) 2A coding region (Science, 322: 949-953, 2008 and WO 2009 / 092042, WO 2009/152529).
- FMDV foot-and-mouth disease virus
- HDAC histone deacetylase
- VPA valproic acid
- MC 1293 sodium butyrate
- M344 small molecule inhibitors
- siRNA and shRNA against HDAC e.g., HDAC1 siRNA (Smartpool® (Millipore), HuSH 29mer shRNA Nucleic acid expression inhibitors such as Constructs against HDAC1 (OriGene) etc.
- DNA methyltransferase inhibitors eg 5'-azacytidine
- G9a Histone methyltransferase inhibitors eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology) Etc.), etc.], L-channel calcium agonist (for example, Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (eg siRNA and shRNA against p53) (CellpStem Cell, 3, 475-479 (2008)), Wnt Signaling activator (eg soluble Wnt3a ) (Cell Stem Cell, 3, 132-135 (2008)), growth factors such as LIF or bFGF, ALK5 inhibitors (eg, SB431542) (Nat.
- small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (
- Examples of the drug in the method for increasing the expression of the endogenous protein of the nuclear reprogramming substance by the drug include 6-bromoindirubin-3'-oxime, indirubin-5-nitro-3'-oxime, valproic acid, 2- (3- (6-methylpyridin-2-yl) -lH-pyrazol-4-yl) -1,5-naphthyridine, 1- (4-methylphenyl) -2- (4,5,6,7-tetrahydro-2-imino- 3 (2H) -benzothiazolyl) ethanone HBr (pifithrin-alpha), prostaglandin J2, and prostaglandin E2 are exemplified (WO 2010/068955).
- DMEM DMEM / F12 or DME medium containing 10-15% FBS (in addition to LIF, penicillin / streptomycin, puromycin, L- Glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc. may be included as appropriate.
- ES cell culture medium containing bFGF or SCF for example, mouse ES cell culture medium (for example, TX-WES medium, Thrombo X) or primate ES cell culture medium (eg, primate (human & monkey) ES cell culture medium (Reprocell, Kyoto, Japan), mTeSR-1).
- a somatic cell and a nuclear reprogramming substance are brought into contact with each other in DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO 2 at 37 ° C.
- Culture for ⁇ 7 days then re-spread cells onto feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.), and bFGF-containing primate ES cells approximately 10 days after contact between somatic cells and nuclear reprogramming substance
- the cells can be cultured in a culture medium and ES cell-like colonies can be generated about 30 to about 45 days or more after the contact.
- the cells may be cultured under conditions of an oxygen concentration as low as 5-10%.
- 10% FBS-containing DMEM medium for example, LIF, penicillin / streptomycin, puromycin, L-glutamine, mitomycin C-treated STO cells, SNL cells, etc.
- Non-essential amino acids, ⁇ -mercaptoethanol, etc. can be included as appropriate.
- ES-like colonies after about 25 to about 30 days or more.
- the medium is replaced with a fresh medium once a day from the second day after the start of the culture.
- 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.
- a marker gene-expressing cell When a gene containing a drug resistance gene is used as a marker gene, a marker gene-expressing cell can be selected by culturing in a medium (selective medium) containing the corresponding drug.
- the marker gene is a fluorescent protein gene
- the marker gene-expressing cells can be obtained by observing with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, and by adding a chromogenic substrate in the case of a chromogenic enzyme gene. Can be detected.
- a “somatic cell” may be any cell other than a germ cell derived from a mammal (eg, human, mouse, monkey, pig, rat, etc.), for example, keratinized epithelial cell (Eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface), exocrine glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medullary cells), cells for metabolism and storage (Eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells (eg, vascular endothelial cells) in the inner chain, and ciliated cells that are capable of transporting (Eg, airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (e
- undifferentiated progenitor cells including somatic stem cells
- terminally differentiated mature cells It can be used as the source of somatic cells in the invention.
- undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
- tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
- non-T cells in blood mononuclear cells in order to produce iPS cells having an intracellular pathology characteristic of myelodysplastic syndrome.
- non-T cells mean cells other than T cells among mononuclear cells in blood, and are characterized by, for example, that all markers of CD3, CD4, CD8 and CD9 are negative.
- Non-T cells are a group of cells that are concentrated by culturing mononuclear cells in blood in a culture solution containing IL-3, IL-6, G-CSF and GM-CSF. good.
- the T cell is characterized by, for example, that at least one of markers selected from CD3, CD4, CD8 and CD9 is positive.
- the T cells may be a group of cells that are enriched by culturing mononuclear cells in blood in a culture solution containing IL-2, CD3 antibody, and CD28 antibody.
- the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human.
- somatic cells are collected from patients with known myelodysplastic syndromes. It is desirable to produce it. In this case, it is desirable that the iPS cells have a chromosomal abnormality peculiar to myelodysplastic syndrome.
- Chromosomal abnormalities in myelodysplastic syndrome include partial chromosome abnormalities and numerical chromosome abnormalities. Chromosome partial abnormalities include, but are not limited to, duplication, deletion, translocation, and the like.
- Examples of the numerical abnormality of the chromosome include “monosomy” that is one, “trisomy” that is three, “tetrasomy” that is four, and “pentasomy” that is five.
- Examples of chromosomal abnormalities in myelodysplastic syndromes include, for example, deletion of part or all of the long arm of chromosome 4 (4q) (ie, a decrease in copy number), deletion of part or all of the long arm of chromosome 5 (5q) (Ie, a decrease in copy number), a loss of part or all of chromosome 7 long arm (7q) (ie, a decrease in copy number), a partial or complete overlap of chromosome 9 short arm (9p) (ie , Increase in copy number), deletion of part or all of chromosome 11 long arm (11q) (ie, decrease in copy number), deletion of part or all of chromosome 17 short arm (17p) (ie, copy) Increase or decrease), deletion of part or all of chro
- iPS cells used in a method for screening a therapeutic and / or prophylactic agent for myelodysplastic syndrome are produced from, for example, non-T cells collected from patients with known myelodysplastic syndromes. IPS cells.
- iPS cells produced from non-T cells collected from patients with myelodysplastic syndromes even when they do not have the aforementioned chromosomal abnormalities, the hematopoietic progenitor cells induced to differentiate from them are bone marrow It may be possible to develop a pathology of dysplasia syndrome.
- Hematopoietic progenitor cells differentiated from iPS cells as a control required for this screening method may be derived from normal somatic cells collected from patients with known myelodysplastic syndrome, or It may be derived from a somatic cell derived from a normal individual.
- IPS cells prepared from normal somatic cells collected from patients with myelodysplastic syndromes used to produce iPS cells with chromosomal abnormalities characteristic of myelodysplastic syndromes, from the standpoint of other conditions Is preferably used as a control iPS cell. More preferably, iPS cells produced from T cells of patients with myelodysplastic syndrome are used as a control for the production of hematopoietic progenitor cells.
- Induction of differentiation into hematopoietic progenitor cells As a method for inducing differentiation of hematopoietic progenitor cells from pluripotent stem cells, for example, a method by formation of embryoid bodies and addition of cytokines (Chadwick et al. Blood 2003, 102: 906-15 , Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62 and Saeki et al. Stem Cells 2009, 27: 59-67), co-culture with heterologous stromal cells (Niwa A et al. J Cell Physiol 2009 Nov; 221 (2): 367-77.), A method using a serum-free medium (WO2011 / 115308) and the like, but are not limited thereto.
- hematopoietic progenitor cell means a cell that has been differentiated from 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. Preferred are CD43 positive, CD34 positive, and CD38 negative cells.
- 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-replicate Means a cell. Unless otherwise specified, “hematopoietic progenitor cells” in the present specification include “hematopoietic stem cells”.
- the hematopoietic progenitor 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.
- pluripotent stem cells such as ES cells and iPS cells may be isolated by any method and induced by suspension culture, or adhesion culture using a coated culture dish. You may guide by.
- a separation solution having protease activity and collagenase activity for example, Accutase (TM) and Accumax (TM), etc.
- a separation solution having protease activity and collagenase activity for example, Accutase (TM) and Accumax (TM), etc.
- TM Accutase
- TM Accutase
- TM Accutase
- TM Accutase
- TM Accutase
- TM Accutase
- the human pluripotent stem cell used is a colony cultured until it becomes 80% confluent with respect to the used dish.
- a separation method using 0.25% trypsin / EDTA can be mentioned.
- the suspension culture means that an embryoid body is formed by culturing cells in a non-adherent state on a culture dish, and is not particularly limited, but is artificial for the purpose of improving adhesion to cells.
- Culture dishes that have not been treated (eg, coated with an extracellular matrix) or artificially suppressed adhesion (eg, coated with polyhydroxyethyl methacrylic acid (poly-HEMA)) Can be done using.
- the feeder cell means another cell that plays an auxiliary role used for adjusting the culture condition of the target cell.
- a cell obtained from the AGM region of a mammalian fetus for example, AGM -S3 cell line: JP 2001-37471
- mouse mesenchymal cells for example, C3H10T1 / 2 cell line: available from RIKEN BioResource Center
- stromal cells derived from bone marrow for example, OP9 Cell lines
- the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof.
- a medium for inducing hematopoietic progenitor cells can be prepared using a medium used for culturing animal cells as a basal medium.
- the basal medium include IMDM medium, MediumMedi199 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, Vischer's medium , And mixed media thereof.
- 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 for differentiation into hematopoietic progenitor cells preferably contains vascular endothelial growth factor (VEGF).
- VEGF vascular endothelial growth factor
- concentration of VEGF in the medium is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 1 ⁇ g / ml, but not limited thereto.
- it is 20 ng / ml.
- a more preferable medium is an ⁇ MEM medium (also referred to as HPC differentiation medium) containing 10% FBS, VEGF, transferrin, L-glutamine, ⁇ -monothioglycerol (MTG) and ascorbic acid. obtain.
- ⁇ MEM medium also referred to as HPC differentiation medium
- the culture period is, for example, 20 days or less, preferably 12 days or more and 14 days or less, particularly preferably 13 days.
- a step of removing feeder cells and concentrating / purifying hematopoietic progenitor cells can be included. This step can be achieved by collecting and removing only the feeder cells after the hematopoietic progenitor cells are peeled from the culture dish together with the feeder cells.
- Examples of the method of peeling off from the culture dish include, but are not limited to, a method of separating mechanically, a separation method using a separation solution having protease activity and collagenase activity, or a separation solution having only collagenase activity.
- a method using collagenase Type IV and / or Trypsin / EDTA is used.
- 0.05% trypsin / EDTA is preferably used.
- differentiation induction into hematopoietic progenitor cells may be performed, for example, by the following steps.
- (i) Process of forming EB ii) Process of forming primitive streak / mesoderm
- a medium in which an arbitrary substance necessary for the basic medium is added to induce a target cell or to achieve the object in each step can be used.
- a medium supplemented with the following substances is used in each step.
- the basal medium used in the steps (i) to (iii) is preferably StemPro-34 supplemented with L-glutamic acid, thioglycerol and ascorbic acid.
- the concentration of BMP-4 in the medium of the step (i) is not particularly limited as long as it can form EB.
- the concentration of bFGF in the medium of step (ii) is, for example, 100 pg / ml, 250 pg / ml, 500 pg / ml, 750 pg / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml. It is not limited to. Preferably, it is 1 ng / ml.
- the concentration of VEGF in the medium of step (iii) is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml. However, it is not limited to these. Preferably, it is 10 ng / ml.
- the concentration of IL-6 in the medium of the step (iii) is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng. / ml, but not limited to. Preferably, it is 10 ng / ml.
- the concentration of IL-3 in the medium of the step (iii) is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng. / ml, but not limited to. Preferably, it is 40 ng / ml.
- the concentration of IL-11 in the medium of the step (iii) is, for example, 500 pg / ml, 750 pg / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng. / ml, but not limited to. Preferably, it is 5 ng / ml.
- the concentration of SCF in the medium of step (iii) is, for example, 1 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml. However, it is not limited to these. Preferably, it is 100 ng / ml.
- the concentration of FLT3L in the medium of the step (iii) is, for example, 1 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml. However, it is not limited to these. Preferably, it is 100 ng / ml.
- the culture time in the step (i) is, for example, 5 days or less, preferably 1 to 3 days, particularly preferably 1 day.
- the culture time in the step (ii) is, for example, 10 days or less, preferably 1 to 5 days, particularly preferably 3 days.
- the culture time for the step (iii) is, for example, 10 days or less, preferably 2 to 6 days, and particularly preferably 4 days.
- a step of purifying and purifying mesoderm cells after completion of the production step (ii) may be included.
- mesoderm cells any method that can separate mesoderm cells from a population of cells including mesoderm cells with high purity can be used.
- purification and purification by FACS Purification may be mentioned.
- cells can be selected using SSEA-1-negative (ie, SSEA-1-) as an index so that undifferentiated cells are not included in the purified and purified mesoderm population.
- the step of selecting Flk1-positive (ie, Flk1 +) and SSEA-1 ⁇ cells may be performed simultaneously or as separate steps. For example, selection of cells that are Flk1 + / SSEA-1- can be done simultaneously using FACS.
- 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%.
- the culture medium may further contain a ROCK inhibitor.
- a 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.
- Induction of differentiation into erythrocytes As a method for inducing differentiation of pluripotent stem cells into blood cells, particularly erythrocytes, for example, a method by formation of embryoid bodies and addition of cytokines (Chadwick et al. Blood 2003, 102: 906- 15, Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62 and Saeki et al. Stem Cells 2009, 27: 59-67), co-culture with heterologous stromal cells (Niwa A et al. J Cell) Physiol. 2009 Nov; 221 (2): 367-77), a method using a serum-free medium (WO 2011/115308), and the like, but are not limited thereto.
- cytokines Chadwick et al. Blood 2003, 102: 906- 15, Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62 and Saeki et al. Stem Cells 2009,
- erythrocyte means a cell rich in hemoglobin.
- erythrocytes can be detected by expression of markers such as ⁇ -globin, ⁇ -globin, ⁇ -globin, ⁇ -globin and CD235a of hemoglobin, but the marker is not limited thereto.
- Preferred markers in mature erythrocytes may be ⁇ -globin and ⁇ -globin.
- a preferred marker when separating red blood cells by FACS may be CD235a.
- Erythrocytes 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 above-described hematopoietic progenitor cells may be separated by any method and cultured by suspension culture, or may be adherently cultured using a coated culture dish.
- suspension culture is preferably employed.
- hematopoietic progenitor cells may be separated and used, or the state obtained by the above method may be used as it is.
- Suspension culture means culturing cells in a non-adherent state on a culture dish.
- artificial treatment for example, coating with an extracellular matrix or the like
- it can be performed using a culture dish that has not been treated, or a culture dish that has been artificially treated to suppress adhesion (for example, coating treatment with polyhydroxyethyl methacrylic acid (poly-HEMA)).
- poly-HEMA polyhydroxyethyl methacrylic acid
- the cells are cultured in an arbitrary medium on feeder cells or in a coated culture dish.
- the feeder cells mean other cells that play an auxiliary role used to adjust the culture conditions of the target cells.
- the feeder cells in this step are, for example, stromal cells (stromal cells) derived from bone marrow, and preferably OP9 cells can be used.
- the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof.
- the medium in this step can be prepared using a medium used for animal cell culture as a basal medium.
- basal media include 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, StemPro-34 medium. , And mixed media thereof.
- EMEM Eagle's Minimum Essential Medium
- DMEM Dulbecco's modified Eagle's Medium
- Ham's F12 medium RPMI 1640 medium
- Fischer's medium StemPro-34 medium.
- StemPro-34 medium The medium may contain serum or may be serum-free.
- the medium may be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), EasyDiff TM Erythroid Supplement ( Lonza), may contain one or more serum replacements such as fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen ), One or more substances such as non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
- KSR Knockout Serum Replacement
- N2 supplement Invitrogen
- B27 supplement Invitrogen
- EasyDiff TM Erythroid Supplement Lonza
- serum replacements such as fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol (2
- the medium in this step may further contain a ROCK inhibitor.
- this step includes a step of dispersing human pluripotent stem cells into single cells, it is preferable that the medium contains 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%.
- Examples of the differentiation-inducing factor into erythrocytes include stem cell factor (Stem ⁇ ⁇ ⁇ ⁇ Cell Factor (SCF)), colony stimulating factor (Coloney-Stimulating Factor (CSF)), granulocyte colony stimulating factor (Granulocyte- (G-) CSF), There are erythropoietin (EPO), interleukins, thrombopoietin (TPO) and Flt3 ligand.
- 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, IL-8, and IL.
- interleukins are proteins secreted from leukocytes and are IL-1, IL-2
- differentiation induction from hematopoietic progenitor cells to erythrocytes may be performed, for example, by the following steps: (i) culturing hematopoietic progenitor cells in a medium containing VEGF, IL-6, IL-3, IL-11, SCF, FLT3L, EPO and TPO; (ii) culturing the cells obtained in step (i) in a medium containing IL-3, SCF and EPO, and (iii) A step of culturing the cells obtained in step (ii) in a medium containing SCF and EPO.
- the concentration of the differentiation-inducing factor for erythrocytes in the medium is not particularly limited as long as the concentration can induce the target cells.
- the concentration of VEGF in the medium is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, but not limited thereto Not.
- it is 10 ng / ml.
- the concentration of IL-6 in the medium is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml. It is not limited to. Preferably, it is 10 ng / ml.
- the concentration of IL-3 in the medium is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml. It is not limited to. Preferably, it is 40 ng / ml.
- the concentration of IL-11 in the medium is, for example, 500 pg / ml, 750 pg / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml. It is not limited to. Preferably, it is 5 ng / ml.
- the concentration of SCF in the medium is, for example, 1 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, but not limited thereto Not. Preferably, it is 100 ng / ml.
- the concentration of FLT3L in the medium is, for example, 1 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, but is not limited thereto Not. Preferably, it is 100 ng / ml.
- the concentration of EPO in the medium is, for example, 1U / ml, 2U / ml, 3U / ml, 4U / ml, 5U / ml, 6U / ml, 7U / ml, 8U / ml, 9U / ml, 10U / ml
- it is not limited to these.
- it is 4U / ml.
- the concentration of TPO in the medium is, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, but not limited thereto Not. Preferably, it is 50 ng / ml.
- the culture time in the step (i) is, for example, 10 days or less, preferably 2 to 6 days, and particularly preferably 4 days.
- the culture time of the step (ii) is, for example, 20 days or less, preferably 5 to 15 days, particularly preferably 10 days.
- the culture time in the step (iii) is, for example, 20 days or less, preferably 12 to 16 days, and particularly preferably 14 days.
- the medium can be replaced with a medium containing SCF and EPO during the culture.
- the culture time is, for example, 12 days or less before the medium exchange, preferably 7-9 days, particularly preferably 8 days, and after the medium exchange, for example, 8 days or less. It is preferably 5 to 7 days, particularly preferably 6 days.
- Differentiation induction method to neutrophils include, for example, Saeki et al., Stem Cells 27: 59-67 and Morishima et al., J Cell Physiol. 2011 May ; 226 (5): 1283-91, but is not limited thereto.
- neutrophil is a type of white blood cell and means a cell that controls an immune reaction in bacterial infection or the like.
- “neutrophils” can be detected by expression of markers such as Ly6g, CD11b, and CD66b, but the markers are not limited thereto.
- a preferred marker for neutrophils can be CD66b.
- the neutrophils 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 hematopoietic progenitor cells may be cultured by suspension culture, or may be cultured by adhesion using a coated culture dish.
- Adhesion culture is preferably employed as the culture method in the present invention.
- cells are cultured in a non-adherent state on a culture dish, and are not particularly limited, but artificially treated (for example, coating with an extracellular matrix) for the purpose of improving adhesion to cells.
- Culture dishes that have not been treated, or artificially treated to suppress adhesion for example, coating treatment with polyhydroxyethyl methacrylic acid (poly-HEMA)).
- the cells are cultured in an arbitrary medium on feeder cells or in a coated culture dish.
- the feeder cells mean other cells that play an auxiliary role used to adjust the culture conditions of the target cells.
- the feeder cells in this step are, for example, stromal cells (stromal cells) derived from bone marrow, and preferably OP9 cells can be used.
- the coating agent include matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof.
- the medium in this step can be prepared using a medium used for animal cell culture as a basal medium.
- the basal medium include 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, and mixtures thereof. Media and the like are included. ⁇ MEM medium or IMDM medium is preferable.
- the medium may contain serum or may be serum-free.
- the medium for differentiation into neutrophils preferably contains GM-CSF and / or G-CSF.
- concentration of GM-CSF in the medium is, for example, 1 ng / ml, 10 ng / ml, 50 ng / ml, 100 ng / ml, 150 ng / ml, 175 ng / ml, 200 ng / ml, 225 ng / ml, 250 ng / ml, 300 ng / ml 400 ng / ml, 500 ng / ml, and 1 ⁇ g / ml, but not limited thereto.
- it is 200 ng / ml.
- the concentration of G-CSF in the medium is, for example, 1 ng / ml, 10 ng / ml, 25 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 110 ng / ml 120 ng / ml, 130 ng / ml, 140 ng / ml, 150 ng / ml, 175 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 1 ⁇ g / ml, but not limited thereto.
- it is 100 ng / ml.
- differentiation induction from hematopoietic progenitor cells to neutrophils is performed, for example, by the following steps. (i) Inducing myeloid progenitor cells from hematopoietic progenitor cells (ii) Inducing neutrophils from myeloid progenitor cells
- a preferable medium in step (i) contains, for example, 10% FBS, 5.5 mg / ml human transferrin, 2 mM L-glutamine, 0.5 mM ⁇ -monothioglycerol, 50 ⁇ g / ml ascorbic acid, and 200ng / ml GM-CSF It can be an ⁇ -MEM medium (also called a medium for proliferation of pluripotent myeloid progenitor cells).
- the megakaryocytes 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 hematopoietic progenitor cells may be cultured by suspension culture, or may be cultured by adhesion using a coated culture dish.
- Adhesion culture is preferably employed as the culture method in the present invention.
- cells are cultured in a non-adherent state on a culture dish, and are not particularly limited, but artificially treated (for example, coating with an extracellular matrix) for the purpose of improving adhesion to cells.
- Culture dishes that have not been treated, or artificially treated to suppress adhesion for example, coating treatment with polyhydroxyethyl methacrylic acid (poly-HEMA)).
- the medium in this step can be prepared using a medium used for animal cell culture as a basal medium.
- the basal medium include 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, and mixtures thereof. Media and the like are included. ⁇ MEM medium is preferable.
- the medium may contain serum or may be serum-free.
- the medium for differentiation into megakaryocytes preferably contains TPO and / or SCF.
- concentration of TPO in the medium is, for example, 1 ng / ml, 10 ng / ml, 25 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 110 ng / ml, 120 ng / ml, 130 ng / ml, 140 ng / ml, 150 ng / ml, 175 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 1 ⁇ g / ml, but are not limited thereto.
- the concentration of SCF in the medium is, for example, 1 ng / ml, 10 ng / ml, 25 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 110 ng / ml, 120 ng / ml, 130 ng / ml, 140 ng / ml, 150 ng / ml, 175 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 1 ⁇ g / ml, but are not limited thereto.
- it is 100 ng / ml.
- a preferable medium in the present invention is, for example, an ⁇ -MEM medium containing 10% FBS, 5.5 mg / ml human transferrin, 2mMmL-glutamine, 0.5 mM ⁇ -monothioglycerol, 50 ⁇ g / ml ascorbic acid and 200ng / ml GM-CSF. (Also referred to as megakaryocyte differentiation medium).
- 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%.
- the number of culture days is, for example, 20 days or less, preferably 5-15 days, particularly preferably 10 days. It is also possible to add an additional medium appropriately during the culture period, or to exchange a part or all of the medium.
- the present invention relates to a method for screening a therapeutic and / or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome (MDS) .
- the present invention comprises contacting a test substance with an iPS cell-derived hematopoietic progenitor cell obtained as described above.
- a method for screening a test substance for a therapeutic and / or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome (MDS) using the indicator is provided.
- iPS cells produced from non-T cells in blood mononuclear cells isolated from patients with myelodysplastic syndrome are preferably derived from somatic cells of patients with myelodysplastic syndrome having chromosomal abnormalities Cells.
- the chromosomal abnormality is a chromosomal abnormality of the above-mentioned myelodysplastic syndrome.
- chromosome 20 long arm (20q) Decrease in copy number of chromosome 20 long arm (20q), decrease in copy number of chromosome 5 long arm (5q), decrease in copy number of chromosome 7 long arm (7q), chromosome 11 long arm (11q) And at least one chromosomal abnormality selected from the group consisting of a translocation of the long arm of chromosome 3 and the long arm of chromosome 21.
- the non-T cell-derived hematopoietic progenitor cells used here are hematopoietic progenitor cells produced by inducing differentiation of normal MDS-iPS cells produced from somatic cells of patients with myelodysplastic syndrome when colony formation assay is performed. Compared with, it shows low colony forming ability.
- the colonies showing a low colony-forming ability are, for example, granulocyte / macrophage colony (GM), macrophage colony (G) and granulocyte colony (M), but are not limited thereto.
- the control iPS cells used here are collected from patients with myelodysplastic syndromes used to produce iPS cells with chromosomal abnormalities that are characteristic of myelodysplastic syndromes, in terms of other conditions. It is desirable to use iPS cells prepared from normal somatic cells. More preferably, iPS cells produced from T cells of patients with myelodysplastic syndrome are used. In this screening method, more preferably, the change in the number of control hematopoietic progenitor cells before and after contact with the test substance is measured, and the decrease in the number of control hematopoietic progenitor cells is suppressed.
- the test substance can be selected by using as a further indicator that the value of the test substance is not decreased.
- form colony means that hematopoietic progenitor cells are separated and cultured in a semi-solid medium such as methylcellulose or soft agar in the presence of cytokines to form a cell population (ie, colonies).
- a semi-solid medium such as methylcellulose or soft agar in the presence of cytokines to form a cell population (ie, colonies).
- the cytokine used in colony formation is exemplified by one or more selected from the group consisting of SCF, G-CSF, GM-CSF, IL-3, IL-6 and EPO. Formation of a colony can be performed using a commercially available kit, and examples thereof include Cell Biolab's CytoSelect.
- the number of colonies may be counted visually or may be mechanically counted using an in-cell analyzer.
- the obtained colonies may be dissolved and counted using Cyquant GR Dye from Cell Biolabs.
- a method including the following steps is exemplified: (A) a step of inducing iPS cells produced from non-T cells in blood mononuclear cells isolated from a patient with myelodysplastic syndrome to hematopoietic progenitor cells, (B) inducing the hematopoietic progenitor cells into blood cells in the presence and absence of the test substance, and (c) in the presence of the test substance, the number of blood cells is higher than in the absence.
- the hematopoietic cell is selected from the group consisting of neutrophils, eosinophils, basophils, erythrocytes and megakaryocytes.
- a therapeutic or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome can be screened by a method comprising the following steps: (A) a step of inducing iPS cells produced from non-T cells in blood mononuclear cells isolated from a patient with myelodysplastic syndrome to hematopoietic progenitor cells, (B) a step of inducing the hematopoietic progenitor cells into red blood cells in the presence and absence of the test substance; and (c) in the presence of the test substance, The step of selecting the test substance as a candidate for the treatment or prevention of acute myeloid leukemia or myelodysplastic syndrome.
- the blood cell is a neutrophil
- Screening for treatment or prevention of acute myeloid leukemia or myelodysplastic syndrome by a method comprising the following steps: (A) a step of inducing iPS cells produced from non-T cells in blood mononuclear cells isolated from a patient with myelodysplastic syndrome to hematopoietic progenitor cells, (B) Inducing the hematopoietic progenitor cells into neutrophils in the presence and absence of the test substance, and (c) In the presence of the test substance, the number of neutrophils increased compared to the absence.
- a step of selecting the test substance as a candidate for a therapeutic or prophylactic agent for acute myeloid leukemia or myelodysplastic syndrome a method comprising the following steps: (A) a step of inducing iPS cells produced from non-T cells in blood mononuclear cells isolated from a patient with myelodysplastic syndrome to hematopoietic progen
- Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67).
- Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al.
- the target diseases of the drug selected in the screening method of the present invention are 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 ⁇ Delayed recovery of hematopoietic stem cells and / or hematopoietic progenitor cells after umbilical cord blood / peripheral blood transplantation, decreased hematopoietic stem cells and / or hematopoietic progenitor cells associated with blood transfusion, leukemia (acute myeloid leukemia (AML), acute lymphoblastic leukemia) (ALL), chronic myelog
- the present invention provides a therapeutic agent for myelodysplastic syndrome comprising hematopoietic progenitor cells produced by inducing differentiation of iPS cells produced from somatic cells of a patient with myelodysplastic syndrome.
- the somatic cell of a myelodysplastic syndrome patient used in the present invention is preferably a patient-derived cell and has no chromosomal abnormality.
- the somatic cell of a patient with myelodysplastic syndrome used in the present invention is, for example, a T cell, but is not limited thereto.
- the drug in the present invention is not limited to acute myeloid leukemia or myelodysplastic syndrome (MDS), for example, a disease with a decrease in the number of red blood cells, a disease with a decrease in the number of neutrophils, a decrease in hematopoietic progenitor cells and It is also effective as a therapeutic agent for diseases associated with decreased hematopoietic function.
- MDS myelodysplastic syndrome
- 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 lymphoblastic leukemia ALL (ALL), chronic Myelogenous leukemia
- the route of administration of a drug containing hematopoietic progenitor cells to a patient is not particularly limited.
- 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.
- iPS cells serving as a normal control were obtained from the same patient in KM15 (FIG. 14A right figure, KM15-I2).
- SNP-CGH array analysis was performed on iPS cells established from blood cells derived from MDS patients (KM16)
- the copy number of chromosome 7 long arm (7q) decreased in C1 strain (KM16-C1).
- the long arm of chromosome 3 and the long arm of chromosome 21 were translocated (FIG. 14B).
- a similar mutation was also confirmed in the karyotype analysis by the G-band method at this time (left figure in FIG. 14B). It was confirmed that iPS cells serving as a normal control can be obtained from the same patient in KM16 (FIG.
- pluripotency markers were confirmed by RT-PCR for the resulting KM3 and KM5-derived iPS cells, pluripotency markers were found in the same manner as existing ES cells (KhES3 or H1) and iPS cells (692D2 or 585A1). It was confirmed that it was expressed (FIG. 5). Moreover, it was confirmed that the pluripotent surface marker is also expressed similarly (FIG. 6). Furthermore, it confirmed that it had the teratoma formation ability (FIG. 7).
- iPS cells differentiated into hematopoietic progenitor cells MDS-iPSCs and Normal iPSCs
- HPCs hematopoietic progenitor cells
- HPC differentiation medium 10% FBS, 5.5 mg / ml human transferrin, 2 mM L-glutamine, 0.5 mM ⁇ -monothioglycerol, 50 ⁇ g / mL ascorbic acid, and 20 ng / ml vascular endothelial ⁇ -MEM supplemented with growth factor (VEGF) was used to seed small clusters ( ⁇ 100 cells) of iPS cells in 10 cm dishes previously coated with gelatin and OP9 cultured overconfluent. On the next day, the medium was replaced with 20 ml of fresh HPC differentiation medium, and thereafter, the medium was replaced with HPC differentiation medium every three days.
- HPC differentiation medium 10% FBS, 5.5 mg / ml human transferrin, 2 mM L-glutamine, 0.5 mM ⁇ -monothioglycerol, 50 ⁇ g / mL ascorbic acid, and 20 ng / ml vascular endothelial ⁇ -MEM
- iPS cells without feeder cells (10-20 cells) in an aggregation medium consisting of StemPro-34 supplemented with 2 mM glutamine, penicillin / streptomycin, 0.4 mM ⁇ -monothioglycerol and 50 ⁇ g / mL ascorbic acid Embryoid bodies (EBs) were prepared by culturing for a time.
- BMP-4 human bone morphogenetic protein-4
- VEGF vascular endothelial growth factor
- bFGF interleukin-6
- IL-6 10 ng / mL
- StemPro-34 supplemented with IL-3 (40 ng / mL)
- IL-11 5 ng / mL
- SCF stem cell factor
- FLT3L human FLT3 ligand
- MDS-iPSCs MDS-iPSCs
- T-iPS fibroblast growth factor
- Embryo-like cells are cultured for 24 hours by culturing small nodules (10-20 cells) of iPS cells without feeder cells in an aggregation medium consisting of StemPro-34 supplemented with 0.4 mM ⁇ -monothioglycerol and 50 ⁇ g / mL ascorbic acid. Body (EBs) was prepared.
- VEGF 10 ng / mL
- bFGF 1 ng / mL
- IL-6 10 ng / mL
- IL-3 40 ng / mL
- IL-11 5 ng / mL
- SCF 100 ng / mL
- FLT3L 100 ng / ml
- the cells were dissociated to a single cell suspension with a 1000 ⁇ l pipette to obtain hematopoietic progenitor cells.
- the above hematopoietic progenitor cells were seeded at a density of 10 6 cells / mL and supplemented with SCF (100 ng / ml), IL-3 (5 ng / ml) and EPO (4 U / mL).
- SCF 100 ng / ml
- IL-3 5 ng / ml
- EPO 4 U / mL
- the obtained cells were cultured in fresh StemPro-34 supplemented with SCF and EPO, and the percentage of CD235a positive cells was determined by flow cytometry on days 30-33.
- MDS-iPSCs showed a lower ability to induce differentiation into erythrocytes than normal iPSCs (FIGS. 12 and 16).
- MDS-iPSCs and Normal iPSCs into megakaryocyte-induced iPS cells (MDS-iPSCs and Normal iPSCs) into hematopoietic progenitor cells (HPCs)
- HPCs hematopoietic progenitor cells
- IPS cells were cultured. On days 12-14, colonies were treated with 5 ml collagenase Type IV (1 mg / ml) for 30 minutes and dissociated with 0.05% Trypsin-EDTA for 20 minutes at 37 ° C.
- iPS cell-derived differentiated cells derived from MDS patients are useful in screening for therapeutic agents for MDS and acute myeloid leukemia that has migrated from it.
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Abstract
Description
[1] 急性骨髄性白血病または骨髄異形成症候群の治療または予防薬をスクリーニングする方法であって、以下の工程:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造した人工多能性幹(iPS)細胞から誘導された造血前駆細胞を被検物質の存在下および非存在下で、コロニーを形成させる工程、および
(b)被検物質の存在下でのコロニー数が、被検物質の非存在下でのコロニー数と比較して、増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程、
を含む、方法。
[2] 前記骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞が、第9染色体短腕(9p)のコピー数の増加、第18染色体短腕(18p)のコピー数の減少、第20染色体長腕(20q)のコピー数の減少、第5染色体長腕(5q)のコピー数の減少、第7染色体長腕(7q)のコピー数の減少、第11染色体長腕(11q)のコピー数の減少、および第3染色体長腕と第21染色体長腕との転座から成る群より選択される少なくとも一つの染色体異常を有している、[1]または[2]に記載の方法。
[3] 急性骨髄性白血病または骨髄異形成症候群の治療または予防薬をスクリーニングする方法であって、以下の工程:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞を造血前駆細胞へ誘導する工程、
(b)前記造血前駆細胞を被検物質の存在下、および非存在下で、血球系細胞へ誘導する工程、および
(c)被検物質の存在下において、非存在下より血球系細胞数が増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程、
を含む、方法。
[4]前記骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞が、第9染色体短腕(9p)のコピー数の増加、第18染色体短腕(18p)のコピー数の減少、第20染色体長腕(20q)のコピー数の減少、第5染色体長腕(5q)のコピー数の減少、第7染色体長腕(7q)のコピー数の減少、第11染色体長腕(11q)のコピー数の減少、および第3染色体長腕と第21染色体長腕との転座から成る群より選択される少なくとも一つの染色体異常を有している、[3]に記載の方法。
[5]前記工程(b)で誘導する血球系細胞が、赤血球である、[3]または[4]に記載の方法。
[6]前記工程(b)で誘導する血球系細胞が、好中球である、[3]または[4]に記載の方法。
[7]前記工程(b)で誘導する血球系細胞が、巨核球である、[3]または[4]に記載の方法。
[8] 前記工程(b)が次の工程を含む、[5]に記載の方法:
(i)造血前駆細胞をVEGF、IL-6、IL-3、IL-11、SCF、FLT3L、erythropoietin(EPO)およびthrombopoietin (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IL-3、SCFおよびEPO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およびEPOを含有する培地中で培養する工程。
[9]前記工程(b)が造血前駆細胞をGM-CSFおよび/またはG-CSFを含有する培地中で培養する工程を含む、[6]に記載の方法。
[10]前記工程(b)が造血前駆細胞をTPOおよびSCFを含有する培地中で培養する工程を含む、[7]に記載の方法。
[11] 前記工程(a)が次の工程から成る、[3]から[10]のいずれかに記載の方法:
(1)BMP-4を含有する培地中でiPS細胞から胚様体を形成させる工程、
(2)前記胚様体をbFGFおよびBMP-4を含有する培地中で培養する工程、および
(3)工程(2)で得られた細胞をbFGF、VEGF、IL-6、IL-3、IL-11、stem cell factor (SCF)およびFLT3Lを含有する培地中で培養する工程。
[12] 前記造血前駆細胞が、iPS細胞をフィーダー細胞と共培養する工程を含む方法により誘導された造血前駆細胞である、[3]から[10]のいずれかに記載の方法。
[13] 前記フィーダー細胞がOP9細胞株である、[12]に記載の方法。
[14] 骨髄異形成症候群患者のT細胞から製造したiPS細胞を分化誘導して作製した造血前駆細胞を含む、骨髄異形成症候群の治療剤。
[15] 前記iPS細胞から造血前駆細胞への分化誘導が次の工程から成る、[14]に記載の剤:
(1)BMP-4を含有する培地中でiPS細胞から胚様体を形成させる工程、
(2)前記胚様体をbFGFおよびBMP-4を含有する培地中で培養する工程、および
(3)工程(2)で得られた細胞をbFGF、VEGF、IL-6、IL-3、IL-11、stem cell factor (SCF)およびFLT3Lを含有する培地中で培養する工程。
[16] 前記造血前駆細胞が、iPS細胞をフィーダー細胞と共培養する工程を含む方法により誘導された造血前駆細胞である、[14]に記載の剤。
[17] 前記フィーダー細胞がOP9細胞株である、[16]に記載の剤。
[18] 急性骨髄性白血病または骨髄異形成症候群の治療または予防薬をスクリーニングする方法であって、以下の工程:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造した人工多能性幹(iPS)細胞から誘導された造血前駆細胞(非T細胞由来の造血前駆細胞)および対照iPS細胞から誘導された造血前駆細胞(対照造血前駆細胞)を被検物質と接触させる工程、および
(b)当該被検物質接触後の非T細胞由来の造血前駆細胞数が、当該被検物質接触後の対照造血前駆細胞数と比較して、減少した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程、
を含む、方法。
[19] 前記対照造血前駆細胞が、非T細胞と同一患者から単離した血液単核球中のT細胞から製造したiPS細胞から誘導された造血前駆細胞である、[18]に記載の方法。
[20] 前記骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞が、第9染色体短腕(9p)のコピー数の増加、第18染色体短腕(18p)のコピー数の減少、第20染色体長腕(20q)のコピー数の減少、第5染色体長腕(5q)のコピー数の減少、第7染色体長腕(7q)のコピー数の減少、第11染色体長腕(11q)のコピー数の減少、および第3染色体長腕と第21染色体長腕との転座から成る群より選択される少なくとも一つの染色体異常を有している、[18]または[19]に記載の方法。
[21] 前記造血前駆細胞が以下の工程を含む方法によりiPS細胞から誘導された造血前駆細胞である、[18]から[20]のいずれかに記載の方法:
(1)iPS細胞をBMP-4を含有する培地中で培養し、胚様体を形成させる工程、
(2)前記胚様体をbFGFおよびBMP-4を含有する培地中で培養する工程、および
(3)工程(2)で得られた細胞をbFGF、VEGF、IL-6、IL-3、IL-11、stem cell factor (SCF)およびFLT3Lを含有する培地中で培養する工程。
[22]前記造血前駆細胞が、iPS細胞をフィーダー細胞と共培養する工程を含む方法により誘導された造血前駆細胞である、[18]から[20]のいずれかに記載の方法。
[23] 前記フィーダー細胞がOP9細胞株である、[22]に記載の方法。
本発明において、iPS細胞は、ある特定の核初期化物質を、タンパク質またはそれをコードする核酸の形態で体細胞に導入すること、または薬剤によって内在性の当該核初期化物質のmRNAおよびタンパク質の発現を上昇させ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、M. Nakagawa et al. (2008) Nat. Biotechnol., 26: 101-106、WO 2007/069666およびWO 2010/068955)。核初期化物質は、ES細胞に特異的に発現している遺伝子またはES細胞の未分化維持に重要な役割を果たす遺伝子もしくはその遺伝子産物であればよく、特に限定されないが、例えば、Oct3/4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, EsrrgまたはGlis1が例示される。これらの初期化物質は、iPS細胞樹立の際には、組み合わされて使用されてもよい。例えば、上記初期化物質を、少なくとも1つ、2つもしくは3つ含む組み合わせであり、好ましくは4つを含む組み合わせである。
遺伝子名 マウス ヒト
L-Myc NM_008506 NM_001033081
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM_001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438
Glis1 NM_147221 NM_147193
多能性幹細胞から造血前駆細胞を分化誘導する方法としては、例えば、胚様体の形成とサイトカインの添加による方法(Chadwick et al. Blood 2003, 102:906-15、Vijayaragavan et al. Cell Stem Cell 2009, 4:248-62およびSaeki et al. Stem Cells 2009, 27:59-67)、異種由来のストローマ細胞との共培養法(Niwa A et al. J Cell Physiol. 2009 Nov;221(2):367-77.)、無血清培地を用いる方法(WO2011/115308)などが挙げられるがこれらに限定されない。
いられ得る。コーティング剤としては、例えば、マトリゲル(BD)、コラーゲン、ゼラチン、ラミニン、ヘパラン硫酸プロテオグリカン、またはエンタクチン、およびこれらの組み合わせが挙げられる。
(i) EBを形成する工程
(ii) 原条(primitive streak)/中胚葉を形成する工程
(iii) 造血前駆細胞へ特化させる工程
(i) BMP-4
(ii) bFGF
(iii) VEGF、bFGF、IL-6、IL-3、IL-11、SCFおよびFLT3L
多能性幹細胞から血球系細胞、特に赤血球を分化誘導する方法としては、例えば、胚様体の形成とサイトカインの添加による方法(Chadwick et al. Blood 2003, 102:906-15、Vijayaragavan et al. Cell Stem Cell 2009, 4:248-62およびSaeki et al. Stem Cells 2009, 27:59-67)、異種由来のストローマ細胞との共培養法(Niwa A et al. J Cell Physiol. 2009 Nov;221(2):367-77)、無血清培地を用いる方法(WO 2011/115308)などが挙げられるがこれらに限定されない。
(i) 造血前駆細胞をVEGF、IL-6、IL-3、IL-11、SCF、FLT3L、EPO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IL-3、SCFおよびEPO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およびEPOを含有する培地中で培養する工程。
多能性幹細胞から好中球を分化誘導する方法としては、例えば、Saeki et al., Stem Cells 27: 59-67やMorishima et al., J Cell Physiol. 2011 May;226(5):1283-91などが挙げられるがこれらに限定されない。
(i) 造血前駆細胞から骨髄系前駆細胞を誘導する工程
(ii) 骨髄系前駆細胞から好中球を誘導する工程
(i) GM-CSF
(ii) G-CSF
多能性幹細胞から巨核球を分化誘導する方法としては、例えば、WO2008/041370、WO2009/122747、Takayama et al.,Blood,111:5298-5306 2008などが挙げられるがこれらに限定されない。
本発明は、前述のように得られたiPS細胞由来の造血前駆細胞と被検物質とを接触させ、各指標を用いて、急性骨髄性白血病または骨髄異形成症候群(MDS)の治療および/または予防薬の被検物質をスクリーニングする方法を提供する。
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞から誘導された造血前駆細胞(非T細胞由来の造血前駆細胞)および対照iPS細胞から誘導された造血前駆細胞(対照造血前駆細胞)を被検物質と接触させる工程、および
(b)当該被検物質接触後の非T細胞由来の造血前駆細胞数が、当該被検物質接触後の対照造血前駆細胞数と比較して、減少した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程。
本スクリーニング法において、より好ましくは、被検物質との接触前後での対照造血前駆細胞数の変化を測定し、対照造血前駆細胞数の減少が抑制された、望ましくは実質的に対照造血細胞数を減少させないことをさらなる指標として、被検物質を選択することができる。このようにして選出された薬剤を、後述の、骨髄異形成症候群患者のT細胞由来iPS細胞から分化誘導された造血前駆細胞を有効成分とする治療剤と併用すれば、異常な造血細胞の増幅を抑制しつつ、正常な造血細胞の増幅を実質的に妨げないので、より有効な治療が可能となる。
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞から誘導された造血前駆細胞を被検物質の存在下および非存在下で、コロニーを形成させる工程、および
(b)被検物質の存在下でのコロニー数が、被検物質の非存在下でのコロニー数と比較して、増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程。
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞を造血前駆細胞へ誘導する工程、
(b)前記造血前駆細胞を被検物質の存在下、および非存在下で、血球系細胞へ誘導する工程、および
(c)被検物質の存在下において、非存在下より血球系細胞数が増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程。
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞を造血前駆細胞へ誘導する工程、
(b)前記造血前駆細胞を被検物質の存在下および非存在下で赤血球へ誘導する工程、および
(c)被検物質の存在下において、非存在下より赤血球数が増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程。
次の工程を含む方法によって急性骨髄性白血病または骨髄異形成症候群(MDS)の治療または予防薬をスクリーニングすることができる:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞を造血前駆細胞へ誘導する工程、
(b)前記造血前駆細胞を被検物質の存在下および非存在下で好中球へ誘導する工程、および
(c)被検物質の存在下において、非存在下より好中球数が増加した場合、前記被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程。
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞を造血前駆細胞へ誘導する工程、
(b)前記造血前駆細胞を被検物質の存在下および非存在下で巨核球へ誘導する工程、および
(c)被検物質の存在下において、非存在下より巨核球数が増加した場合、前記被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程。
本発明は、骨髄異形成症候群患者の体細胞から製造したiPS細胞を分化誘導して作製した造血前駆細胞を含む、骨髄異形成症候群の治療剤を提供する。本発明において使用される骨髄異形成症候群患者の体細胞は、患者由来の細胞であり、かつ、染色体異常を有していないことが好ましい。本発明において使用される骨髄異形成症候群患者の体細胞は、例えばT細胞であるが、これに限定されない。
芽球の出現、骨髄・末梢血における血球異形成像が確認された4名の骨髄異形成症候群(MDS)患者(KM3、KM5、KM15およびKM16; KM3およびKM5由来の血液の染色像を図1に示す)の末梢血から、Okita. K, et al., Stem Cells. 2012 Nov 29.に記載の方法を用いてiPS細胞を作製した。詳細は次の通りである。京都大学病院においてこれらのMDS患者から同意を得た後採血し、採取した血液から、Ficoll-paque Plus (GE Healthcare)またはBD Vacutainer CPT (BD)を用いた密度勾配遠心法にて、末梢血単核球(PMNC)を回収した。Nucleofector 2b Device(Lonza)およびAmaxa(R) Human T Cell Nucleofector(R) Kitを用いて、3 μgの発現プラスミド混合物を3から5×106個のPMNCに導入した。この時、発現プラスミドは、pCXLE-hOCT3/4-shp53-F、pCXLE-hSKおよびpCXLE-hUL、およびpCXWB-EBNA1の組み合わせを用いた。発現プラスミドを導入した3 × 104から1 ×106個の細胞を、あらかじめMEFフィーダー細胞(マイトマイシンC処理済)を播種しておいた6 well培養プレート (Falcon) へ移し、37℃、5% CO2の条件下で培養した。この時T細胞由来のiPS細胞を樹立するためには、30 U/ml IL-2 (PeproTech)および 5 μl/well Dynabeads Human T-activator CD3/CD28を添加したX-vivo10培地(Lonza)を用いた。一方、T細胞以外の血球(non-T細胞)由来のiPS細胞を樹立するためには10% FCS, 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml G-CSFおよび10 ng/ml GM-CSFを添加したαMEM培地の培地を用いた。プラスミド導入2日目に、bFGFおよび10 μM Y27632を添加した等量の霊長類ES細胞用培地(リプロセル)を各ウェルに加えた。次いで、プラスミド導入4日目に、培養培地を、bFGFおよび10 μM Y27632を添加した霊長類ES細胞用培地(リプロセル)に交換した。プラスミド導入20~25日目 にES様コロニー(iPS細胞コロニー)をピックアップした。このようにしてT細胞由来iPS細胞とnon-T細胞由来iPS細胞を作製した。
続いて、MDS患者(KM3)由来の血液より作製した各iPS細胞株のUSP14、NDC80およびMYL12Aのコピー数を測定したところ、A4株(KM3-A4:non-T細胞由来iPS細胞)においてコピー数が半数以下であることが確認された(図2)。さらに、同KM3-A4株においてCGH(Comparative genomic hybridization)アレイ解析を行ったところ、第9染色体短腕(9p)のコピー数が増加していること、および第18染色体短腕(18p)のコピー数が減少していることが確認された(図3A右図)。この時のGバンド法による核型解析においても同様の変異が確認された(図3A左図)。一方、同じMDS患者(KM3)のT細胞から作製したiPS細胞株(G1、H1、H3、H4、H5、H7、I1、J1)はいずれも染色体異常は認められなかった(図2)。
以上より、MDS患者(KM3)のnon-T細胞から樹立されたKM3-A4株は染色体変異を持った細胞由来のiPS細胞であり、T細胞由来のiPS細胞はMDS患者由来であってもすべて正常の染色体を有するiPS細胞であることが確認された。このことは、同一患者のT細胞由来のiPS細胞は、正常対照として用いることができることを意味する。
同様に、MDS患者(KM5)由来の血液細胞から樹立した9株のiPS細胞についてSNP-CGHアレイ解析を行ったところ、A1、A2、A3、B1、B2、B3およびB4株(それぞれ、KM5-A1、KM5-A2、KM5-A3、KM5-B1、KM5-B2、KM5-B3およびKM5-B4)において第20染色体長腕(20q)のコピー数が減少していることが確認された(図3B右図)。この時のGバンド法による核型解析においても同様の変異が確認された(図3B左図)。KM5においても同一の患者から正常対照となるiPS細胞が得られることが確認された(図3B右図、KM5-D2、KM5-E1)。さらに、FISH法により20q部の数を確認したところ、変異iPS細胞では、20q12-13は1対しか確認できなかった(図4)。
同様に、MDS患者(KM15)由来の血液細胞から樹立したiPS細胞についてSNP-CGHアレイ解析を行ったところ、I7株(KM15-I7)において第5染色体長腕(5q)および第11染色体長腕(11q)のコピー数が減少していることが確認された(図14A)。この時のGバンド法による核型解析においても同様の変異が確認された(図14A左図)。KM15においても同一の患者から正常対照となるiPS細胞が得られることが確認された(図14A右図、KM15-I2)。
同様に、MDS患者(KM16)由来の血液細胞から樹立したiPS細胞についてSNP-CGHアレイ解析を行ったところ、C1株(KM16-C1)において第7染色体長腕(7q)のコピー数が減少し、第3染色体長腕と第21染色体長腕とが転座していることが確認された(図14B)。この時のGバンド法による核型解析においても同様の変異が確認された(図14B左図)。KM16においても同一の患者から正常対照となるiPS細胞が得られることが確認された(図14B右図、KM16-D1)。
得られたKM3およびKM5由来のiPS細胞について多能性マーカーをRT-PCRにて確認したところ、既存のES細胞(KhES3またはH1)やiPS細胞(692D2または585A1)と同様に多能性マーカーが発現していることが確認された(図5)。また、多能性の表面マーカーも同様に発現していることが確認された(図6)。さらに、奇形腫形成能をもつことを確認した(図7)。以上より、変異iPS細胞(MDS-iPSCs)と正常iPS細胞(Normal iPSCsまたはT-iPSCs)は、多能性の機能において違いが見られないことが確認された。
一方、マイクロアレイ法によりES細胞、既存のiPS細胞およびMDS患者由来のiPS細胞における遺伝子発現を解析し、階層クラスタリングしたところ、同一人由来のiPS細胞であっても変異iPS細胞が正常iPS細胞と異なる遺伝子発現プロファイルを有する傾向があることが確認された(図8)。
iPS細胞(MDS-iPSCsおよびNormal iPSCs)を造血前駆細胞 (HPCs) に分化させるために、OP9ストローマ細胞共培養系およびEB法のそれぞれを用いてiPS細胞を培養した。
OP9ストローマ細胞共培養系においては、10mlのHPC分化培地 (10% FBS, 5.5 mg/ml human transferrin, 2 mM L-glutamine, 0.5 mM α-monothioglycerol, 50μg/mL ascorbic acid, および20ng/ml vascular endothelial growth factor (VEGF)を補充したα-MEM) を用いて、前もってゼラチンで被覆しOP9をオーバーコンフルエントに培養した10cmディッシュへiPS細胞の小塊(<100細胞) を播種した。翌日、20 mlの新鮮なHPC分化培地に培地交換し、その後、3日ごとにHPC分化培地で培地交換した。12-14日目に、コロニーを5 mlのcollagenase Type IV (1mg/ml) で30分間処理し、0.05% Trypsin-EDTAを用いて37℃で20分間解離させた。ストローマ細胞を除去するために、解離させた細胞に5倍のOP9培地を加えて再懸濁し、37℃で45分間培養容器上で培養した後、浮遊細胞を回収した。さらにストローマ細胞および凝集細胞を除去するために、回収した細胞を100 μmフィルターに通過させて、分化誘導した細胞をFACSにより評価した。
一方、EB法においては、下記の手順により造血前駆細胞へ分化誘導させた。
(day0-day1)6ウェル低接着プレートを用いて、37℃、5% CO2、5% O2および90% N2の環境で、10 ng/mL human bone morphogenetic protein-4 (BMP-4)、2mM glutamine、penicillin/streptomycin、0.4mM α-monothioglycerolおよび50 μg/mL ascorbic acidを補充したStemPro-34からなる凝集培地中、フィーダー細胞を含まないiPS細胞の小塊 (10-20細胞) を24時間培養することにより、胚様体(EBs)を作製した。
(day1-day4)得られたEBsを回収、洗浄し、5 ng/mL bFGF、 10 ng/mL BMP-4、2mM glutamine、penicillin/streptomycin、0.4mM α-monothioglycerolおよび50 μg/mL ascorbic acidを補充したStemPro-34中でさらに3日間培養して、原条(primitive streak)/中胚葉形成を誘導した。
(day4-day8)4日目に、EBsを再び回収し、vascular endothelial growth factor (VEGF; 10 ng/mL)、bFGF (1 ng/mL)、interleukin-6 (IL-6; 10 ng/mL)、IL-3 (40 ng/mL)、IL-11 (5 ng/mL)、stem cell factor (SCF; 100 ng/mL)およびhuman FLT3 ligand (FLT3L; 100ng/ml)を補充したStemPro-34中で、造血前駆細胞への特化および発生のために4日間再培養した。
(day8-day15)8日目に、EBsを5% CO2/air環境に移し、VEGF (10 ng/mL)、erythropoietin (EPO; 4 U/mL)、thrombopoietin (TPO; 50 ng/mL)、SCF、FLT3L、IL-6、IL-11およびIL-3を補充したStemPro-34中に移し、造血前駆細胞成熟(赤芽球および巨核芽球系前駆細胞への成熟)および増殖のためさらに7日間培養した。0.25% Trypsin-EDTAを用いて37℃で5-10分間のインキュベーション後、1000 μlピペットにより細胞を単一細胞懸濁液にまで解離させ、次いで、解離させた細胞を70μmフィルターに通過させて、分化誘導した細胞をFACSにより評価した。
以上の結果、OP9ストローマ細胞共培養系、EB法のいずれの方法を用いた場合においても、MDS-iPSCsの造血前駆細胞への分化能は、Normal iPSCsと同程度であった(図9および図11)。
35 mm培養皿を用いて、SCF、G-CSF、GM-CSF、IL-3、IL-6およびEPO (MethoCult H4435)を含む2 mlのメチルセルロース培地中に、実施例2の方法によりiPS細胞(異常MDS-iPSCs (NonT-iPS)およびNormal iPSCs (T-iPS))から分化誘導し、フローサイトメーターにより抽出したCD43+CD34+CD38-画分の細胞を2500個播種した。15日後、顕微鏡下でコロニーの数を計測した。さらに、サイトスピン後、ライト染色し、顕微鏡観察により、コロニー型の確認を行った。
その結果、MDS-iPSCs由来の造血前駆細胞はNormal iPSCsと比較して、有意にコロニー形成能が低いことが確認された(図10および15)。また、コロニーアッセイday15の有核細胞中に占める好中球(分葉核球及び桿状核球)の割合は、Normal iPSC由来造血前駆細胞では40%であったのに対し、MDS-iPSC由来造血前駆細胞では2%と低値であった。ここで、5M-B1は、核型に変異のないMDS-iPSCsであることから、nonT細胞由来のiPS細胞は核型の変異に依拠しないMDSの特徴を有していると考えられる。
分化誘導前に、線維芽細胞成長因子(bFGF)を補充したマウス胚性線維芽細胞条件培地中、Matrigelで被覆したプレート上で72~96時間培養することで、iPS細胞(MDS-iPSCs (MDS-iPS) およびNormal iPSCs (T-iPS))からフィーダー細胞の混入を除去した。
(day0-day1)6ウェル低接着プレートを用いて、37℃、5% CO2、 5% O2および90% N2の環境で、10 ng/mL BMP-4、2mM glutamine、penicillin/streptomycin、 0.4mM α-monothioglycerol および50 μg/mL ascorbic acidを補充したStemPro-34からなる凝集培地中、フィーダー細胞を含まないiPS細胞の小塊 (10-20細胞) を24時間培養することにより、胚様体(EBs)を作製した。
(day1-day4)得られたEBsを回収、洗浄し、5 ng/mL bFGF、10 ng/mL BMP-4、2mM glutamine、penicillin/streptomycin、0.4 mM α-monothioglycerolおよび50 μg/mL ascorbic acidを補充したStemPro-34でさらに3日間培養して、原条(primitive streak)/中胚葉形成を誘導した。
(day4-day8)4日目に、EBsを再び回収し、VEGF (10 ng/mL)、bFGF (1 ng/mL)、IL-6(10 ng/mL)、IL-3 (40 ng/mL)、IL-11 (5 ng/mL)SCF(100 ng/mL) およびFLT3L(100 ng/ml)を補充したStemPro-34中で、造血前駆細胞への特化および発生のために4日間再培養した。
(day8-day18)8日目に、EBsを5% CO2/air環境に移し、VEGF (10 ng/mL) 、EPO(4 U/mL) 、TPO(50 ng/mL)、SCF、FLT3L(100 ng/ml)、IL-6(10 ng/mL)、IL-11(5 ng/mL)およびIL-3(40 ng/mL)を補充したStemPro-34中に移し、造血前駆細胞成熟(赤芽球および巨核芽球系前駆細胞への成熟)および増殖のためにさらに10日間培養した。0.25% Trypsin-EDTAを用いて37℃で5-10分間のインキュベーション後、1000 μlピペットにより細胞を単一細胞懸濁液にまで解離させ、造血前駆細胞を得た。
(day18~day26)上記の造血前駆細胞を106細胞/mLの密度で播種し、SCF (100 ng/ml)、IL-3 (5 ng/ml)およびEPO (4 U/mL)を補充したStemPro-34中で8日間培養した。
(day26~day33)得られた細胞を、SCFおよびEPOを補充した新鮮なStemPro-34中で培養し、30~33日目に、フローサイトメトリーによりCD235a陽性細胞の割合を決定した。
その結果、MDS-iPSCsは、Normal iPSCsと比較して、赤血球への低い分化誘導能を示した(図12および16)。
iPS細胞(異常MDS-iPSCs (NonT-iPS)およびNormal iPSCs (T-iPS))を造血前駆細胞 (HPCs) に分化させるために、OP9ストローマ細胞共培養系を用いてiPS細胞を培養した。簡潔には、10mlのHPC分化培地を用いて、前もってゼラチンで被覆しOP9をオーバーコンフルエントに培養した10cmディッシュへiPS細胞の小塊(<100細胞)を播種した。翌日、20 mlの新鮮なHPC分化培地と培地交換し、その後、3日ごとにHPC分化培地を交換した。11-12日目に、コロニーを5mlのcollagenase Type IV (1mg/ml) で30分間処理し、0.05% Trypsin-EDTAを用いて37℃で20分間解離させた。解離させた細胞に多能性骨髄系前駆細胞増殖用培地 (10% FBS, 5.5 mg/ml human transferrin, 2 mM L-glutamine, 0.5 mM α-monothioglycerol, 50 μg/ml ascorbic acid, および20 ng/ml GM-CSFを補充したα-MEM) を加えて再懸濁した。2日後に骨髄系培養物を回収し、凝集細胞を除去するために、細胞を70 μmフィルターに通過させた。フローサイトメトリーによりLin-CD34+CD43+CD45+前駆体細胞を抽出し、2.5 mlの好中球分化培地 (20% FBSおよび100 ng/ml G-CSFを補充したIMDM) 中、OP9をセミコンフルエントに培養した6ウェルプレート上に播種した。3日目に、2.5 mlの好中球分化培地を加えて、6日目に、好中球分化培地の半分を交換した。9-10日後に、フローサイトメトリーにより、CD11b陽性骨髄細胞中のCD66b陽性好中球細胞の割合を決定した。
その結果、MDS-iPSCsは、Normal iPSCsと比較して、好中球への低い分化誘導能を示した(図13および17)。
iPS細胞(MDS-iPSCsおよびNormal iPSCs)を造血前駆細胞 (HPCs) に分化させるために、実施例5に記載のOP9ストローマ細胞共培養系を用いてiPS細胞を培養した。12-14日目に、コロニーを5 mlのcollagenase Type IV (1mg/ml) で30分間処理し、0.05% Trypsin-EDTAを用いて37℃で20分間解離させた。骨髄間質細胞を除去するために、5倍のOP9培地を加えて解離させた細胞を再懸濁して、培養容器上に播種し、37℃で45分間インキュベートした後、浮遊細胞を回収した。さらに、骨髄間質細胞および凝集細胞を除去するために、細胞を100 μmフィルターに通過させて、2 mlの巨核球分化培地 (10% FBS, 5.5 mg/ml human transferrin, 2 mM L-glutamine, 0.5 mM α-monothioglycerol, 50 μg/ml ascorbic acid, 100 ng/ml TPOおよび100 ng/ml SCFを補充したα-MEM)を補充したOP9セミコンフルエント6ウェルプレートに播種した。2日後に浮遊細胞を回収し、2 mlの巨核球分化培地を補充した新しいOP9セミコンフルエント6ウェルプレートに播種した。4日目に、2 mlの巨核球分化培地を加えた。6日目および8日目に、培地の半分を交換した。10日目に、フローサイトメトリーにより、CD45陽性細胞中、CD41a/CD42b二重陽性細胞の割合を決定した。
その結果、MDS-iPSCsは、Normal iPSCsと比較して、巨核球への低い分化誘導能を示した(図18)。
MDS-iPS細胞から誘導された造血前駆細胞における低下したコロニー形成能が、p38阻害剤の効果により回復するかを検討するために、p38阻害剤を加えることを除いては実施例3と同様の方法によりコロニー形成アッセイを行った。簡潔には、35 mm培養皿を用いて、SCF、G-CSF、GM-CSF、IL-3、IL-6、EPOおよび10 μM SB203580(p38阻害剤)を含む1.1 mlのメチルセルロース培地中に、実施例2の方法によりiPS細胞(MDS-iPSCs (NonT-iPS))およびNormal iPSCs (T-iPS))から分化誘導し、フローサイトメーターにより抽出したCD43+CD34+CD38-画分の細胞を2500個播種した。14または15日後、顕微鏡下でコロニーの数を計測した。さらに、サイトスピン後ライト染色し、顕微鏡観察により、コロニー型の確認を行った。
Claims (23)
- 急性骨髄性白血病または骨髄異形成症候群の治療または予防薬をスクリーニングする方法であって、以下の工程:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造した人工多能性幹(iPS)細胞から誘導された造血前駆細胞を被検物質の存在下および非存在下で、コロニーを形成させる工程、および
(b)被検物質の存在下でのコロニー数が、被検物質の非存在下でのコロニー数と比較して、増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程、
を含む、方法。 - 前記骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞が、第9染色体短腕(9p)のコピー数の増加、第18染色体短腕(18p)のコピー数の減少、第20染色体長腕(20q)のコピー数の減少、第5染色体長腕(5q)のコピー数の減少、第7染色体長腕(7q)のコピー数の減少、第11染色体長腕(11q)のコピー数の減少、および第3染色体長腕と第21染色体長腕との転座から成る群より選択される少なくとも一つの染色体異常を有している、請求項1または2に記載の方法。
- 急性骨髄性白血病または骨髄異形成症候群の治療または予防薬をスクリーニングする方法であって、以下の工程:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞を造血前駆細胞へ誘導する工程、
(b)前記造血前駆細胞を被検物質の存在下、および非存在下で、血球系細胞へ誘導する工程、および
(c)被検物質の存在下において、非存在下より血球系細胞数が増加した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程、
を含む、方法。 - 前記骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞が、第9染色体短腕(9p)のコピー数の増加、第18染色体短腕(18p)のコピー数の減少、第20染色体長腕(20q)のコピー数の減少、第5染色体長腕(5q)のコピー数の減少、第7染色体長腕(7q)のコピー数の減少、第11染色体長腕(11q)のコピー数の減少、および第3染色体長腕と第21染色体長腕との転座から成る群より選択される少なくとも一つの染色体異常を有している、請求項3に記載の方法。
- 前記工程(b)で誘導する血球系細胞が、赤血球である、請求項3または4に記載の方法。
- 前記工程(b)で誘導する血球系細胞が、好中球である、請求項3または4に記載の方法。
- 前記工程(b)で誘導する血球系細胞が、巨核球である、請求項3または4に記載の方法。
- 前記工程(b)が次の工程を含む、請求項5に記載の方法:
(i)造血前駆細胞をVEGF、IL-6、IL-3、IL-11、SCF、FLT3L、erythropoietin(EPO)およびthrombopoietin (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IL-3、SCFおよびEPO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およびEPOを含有する培地中で培養する工程。 - 前記工程(b)が造血前駆細胞をGM-CSFおよび/またはG-CSFを含有する培地中で培養する工程を含む、請求項6に記載の方法。
- 前記工程(b)が造血前駆細胞をTPO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7に記載の方法。
- 前記工程(a)が次の工程から成る、請求項3から10のいずれか1項に記載の方法:
(1)BMP-4を含有する培地中でiPS細胞から胚様体を形成させる工程、
(2)前記胚様体をbFGFおよびBMP-4を含有する培地中で培養する工程、および
(3)工程(2)で得られた細胞をbFGF、VEGF、IL-6、IL-3、IL-11、stem cell factor (SCF)およびFLT3Lを含有する培地中で培養する工程。 - 前記造血前駆細胞が、iPS細胞をフィーダー細胞と共培養する工程を含む方法により誘導された造血前駆細胞である、請求項3から10のいずれか1項に記載の方法。
- 前記フィーダー細胞がOP9細胞株である、請求項12に記載の方法。
- 骨髄異形成症候群患者のT細胞から製造したiPS細胞を分化誘導して作製した造血前駆細胞を含む、骨髄異形成症候群の治療剤。
- 前記iPS細胞から造血前駆細胞への分化誘導が次の工程から成る、請求項14に記載の剤:
(1)BMP-4を含有する培地中でiPS細胞から胚様体を形成させる工程、
(2)前記胚様体をbFGFおよびBMP-4を含有する培地中で培養する工程、および
(3)工程(2)で得られた細胞をbFGF、VEGF、IL-6、IL-3、IL-11、stem cell factor (SCF)およびFLT3Lを含有する培地中で培養する工程。 - 前記造血前駆細胞が、iPS細胞をフィーダー細胞と共培養する工程を含む方法により誘導された造血前駆細胞である、請求項14に記載の剤。
- 前記フィーダー細胞がOP9細胞株である、請求項16に記載の剤。
- 急性骨髄性白血病または骨髄異形成症候群の治療または予防薬をスクリーニングする方法であって、以下の工程:
(a)骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造した人工多能性幹(iPS)細胞から誘導された造血前駆細胞(非T細胞由来の造血前駆細胞)および対照iPS細胞から誘導された造血前駆細胞(対照造血前駆細胞)を被検物質と接触させる工程、および
(b)当該被検物質接触後の非T細胞由来の造血前駆細胞数が、当該被検物質接触後の対照造血前駆細胞数と比較して、減少した場合、当該被検物質を急性骨髄性白血病または骨髄異形成症候群の治療または予防薬の候補として選出する工程、
を含む、方法。 - 前記対照造血前駆細胞が、非T細胞と同一患者から単離した血液単核球中のT細胞から製造したiPS細胞から誘導された造血前駆細胞である、請求項18に記載の方法。
- 前記骨髄異形成症候群患者から単離した血液単核球中の非T細胞から製造したiPS細胞が、第9染色体短腕(9p)のコピー数の増加、第18染色体短腕(18p)のコピー数の減少、第20染色体長腕(20q)のコピー数の減少、第5染色体長腕(5q)のコピー数の減少、第7染色体長腕(7q)のコピー数の減少、第11染色体長腕(11q)のコピー数の減少、および第3染色体長腕と第21染色体長腕との転座から成る群より選択される少なくとも一つの染色体異常を有している、請求項18または19に記載の方法。
- 前記造血前駆細胞が以下の工程を含む方法によりiPS細胞から誘導された造血前駆細胞である、請求項18から20のいずれか1項に記載の方法:
(1)iPS細胞をBMP-4を含有する培地中で培養し、胚様体を形成させる工程、
(2)前記胚様体をbFGFおよびBMP-4を含有する培地中で培養する工程、および
(3)工程(2)で得られた細胞をbFGF、VEGF、IL-6、IL-3、IL-11、stem cell factor (SCF)およびFLT3Lを含有する培地中で培養する工程。 - 前記造血前駆細胞が、iPS細胞をフィーダー細胞と共培養する工程を含む方法により誘導された造血前駆細胞である、請求項18から20のいずれか1項に記載の方法。
- 前記フィーダー細胞がOP9細胞株である、請求項22に記載の方法。
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