WO2011129446A1 - 人工多能性幹細胞の製造方法 - Google Patents
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Definitions
- the present invention relates to a method for producing induced pluripotent stem cells.
- iPS cells Artificial pluripotent stem cells
- iPS cells are useful in transplantation therapy for various diseases, and are expected to be applied to regenerative medicine.
- somatic cells such as fibroblasts and hepatocytes and expressed. It has been reported that cells can be produced (for example, WO 2007/069666 International Publication, Takahashi K, Yamanaka S. (2006). “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”.
- an object is to provide a method for producing iPS cells with low invasiveness and high efficiency.
- the method for producing induced pluripotent stem cells (iPS cells) according to the present invention is characterized by using a mononuclear cell population derived from peripheral blood as a material.
- the above-mentioned iPS cell production method comprises the steps of (a) culturing a mononuclear cell population derived from peripheral blood for 3 to 14 days in the presence of anti-CD3 antibody and interleukin 2, and (b) the mononuclear cells after culturing. And a step of performing a dedifferentiation treatment on the population.
- step (b) it is more preferable to introduce a dedifferentiation factor into the mononuclear cell population.
- a dedifferentiation factor an operation of introducing a recombinant expression vector that expresses the dedifferentiation factor may be performed.
- the dedifferentiation factor is preferably Sox2, Oct3 / 4, Klf4 and c-Myc, and the recombinant expression vector is more preferably a Sendai virus vector.
- the method for producing iPS cells according to the present invention preferably further includes (c) a step of culturing the mononuclear cell population subjected to the dedifferentiation treatment for 14 to 25 days in the presence of a growth factor.
- peripheral blood is derived from a human.
- alkaline phosphatase-positive stem cells obtained by adding a virus at a MOI of 1 to 20 to a medium of a mononuclear cell population cultured in the presence of a CD3 antibody to dedifferentiate the mononuclear cell population. It is the graph which showed the colony rate of.
- it is a microscope picture of the iPS cell manufactured from the peripheral blood origin mononuclear cell dye
- Example of this invention it is a photograph of the immunohistochemical dyeing
- the mononuclear cell population when the mononuclear cell population is cultured in the presence of anti-CD3 antibody and interleukin 2 to produce iPS cells (anti-CD3 + IL2 + group), the mononuclear cell population is divided into anti-CD3 antibody and interleukin. It is a graph which shows iPS establishment efficiency in the case of culturing in the absence of leukin 2 to produce iPS cells (anti-CD3-IL2-group).
- Peripheral blood is preferably derived from mammals and may be derived from animals such as mice, rats, hamsters, guinea pigs, rabbits, sheep, horses, pigs, cats, dogs, monkeys, etc. Is more preferable.
- the growth stage of these animals may be any of adults, pups, fetuses and embryos, and is not limited as long as peripheral blood containing mononuclear cells is obtained.
- the blood collection method may be appropriately selected from well-known methods by those skilled in the art in consideration of the size of the animal and the amount of blood collected, and is not particularly limited. However, in order to reduce the burden on the animal, it is preferable to collect blood with a syringe.
- the peripheral blood is preferably derived from the same animal as the patient or animal. And more preferably derived from the patient itself.
- the mononuclear cell population may be mixed with peripheral blood-derived cells and components other than mononuclear cells or may contain only mononuclear cells. However, considering the production efficiency of iPS cells, It is preferable that it is contained in a high ratio.
- a method for preparing a mononuclear cell population from peripheral blood can be appropriately selected by those skilled in the art from well-known methods. For example, density gradient centrifugation, lymphoquick method (One ⁇ Lambda), immunomagnetic bead method, etc. It may be used.
- a solution of a water-soluble copolymer of sucrose and epichlorohydrin such as sucrose solution or ficoll solution, or ficoll-conlay, ficoll-hypac, or the like, or Those skilled in the art may appropriately select from well-known solutions such as a solution of colloidal silica coated with polyvinylpyrrolidone such as Percoll.
- the peripheral blood is preferably fresh, but may be refrigerated or frozen.
- the culture conditions may be appropriately selected by those skilled in the art.
- the culture may be performed at 35 to 40 ° C, preferably 37 ° C in the presence of 5% CO 2 .
- the medium can be appropriately selected by those skilled in the art from the medium normally used for mononuclear cell culture.
- KBM502, DMEM / F12, DMEM, KBM530, KBM540, KBM560, RPM1640 may be used, and KBM502 is preferable. .
- the anti-CD3 antibody may be fixed to a culture dish or a culture tube, or may be suspended in a liquid medium.
- the anti-CD3 antibody may be immobilized, for example, it may be immobilized on a plastic or the like constituting the culture dish or culture tube via a covalent bond or a non-covalent bond such as electrostatic interaction.
- the immobilization method is not particularly limited, and can be appropriately selected from methods well known to those skilled in the art.
- a culture dish in which an anti-CD3 antibody is immobilized can be purchased from BD BioCoat.
- the concentration of the anti-CD3 antibody in the liquid medium may be determined by those skilled in the art, but is preferably 1 to 100 ⁇ g / ml in view of the final iPS cell yield.
- the anti-CD3 antibody may be any antibody as long as it can specifically stimulate the CD3 antigen of mononuclear cells as a material and promote the proliferation of CD3 antigen-positive mononuclear cells. Or an antibody obtained from any animal, and it is not particularly limited.
- the antibody may be a part of an antibody including an antigen binding site including a variable region, and may be, for example, a Fab fragment, an F (ab ′) 2 fragment, or the like.
- the interleukin may be any known interleukin, and examples include interleukin 1, interleukin 2, interleukin 4, interleukin 6, interleukin 8, interleukin 11, and interleukin 12. Considering the ease of handling and preparation, it is preferable to use a commercially available interleukin 2.
- one or more kinds of substances usually added to mononuclear cell culture may be contained in the medium.
- substances usually added to mononuclear cell culture include growth factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), FBS, knockoutknserum replacement (Invitrogen), L-glutamine, non-essential amino acids, penicillin, streptomycin, etc.
- FGF fibroblast growth factor
- EGF epidermal growth factor
- Invitrogen knockoutknserum replacement
- L-glutamine L-glutamine
- non-essential amino acids penicillin, streptomycin, etc.
- Antibiotics, mercaptoethanol, and the like but are not limited thereto.
- the dedifferentiation treatment of the mononuclear cell population can be performed by a method well known to those skilled in the art, and is not limited as long as desired iPS cells can be produced.
- a dedifferentiation factor may be used, or a well-known agent that promotes mononuclear cell dedifferentiation may be administered.
- a dedifferentiation factor (initialization factor) that is usually used when producing iPS cells can be used.
- the initialization method described in Takahashi et al.'S paper (Cell 2007 vol.131: 861-872) can be used, and is incorporated herein by reference.
- genes belonging to the Oct gene group include each gene such as Oct3 / 4, Oct1A, and Oct6.
- genes belonging to the Klf gene group include Klf1, Klf2, Klf4, Klf5 and the like.
- genes belonging to the Sox gene group include Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, and Sox18.
- genes belonging to the Myc gene group include genes such as c-Myc, N-Myc, and L-Myc.
- cytokines and compounds may be added to the medium as cofactors.
- dedifferentiation factors examples include combinations including the gene product of Oct gene group, the gene product of Sox gene group, the gene product of Nanog gene, the gene product of lin-28 gene, and the like. Further, in addition to the combination of these dedifferentiation factors, for example, SV40 LargeT antigen gene product, TERT gene product, immortalization inducing factor and the like may be used.
- the dedifferentiation factor in the mononuclear cells that are dedifferentiated by the dedifferentiation factor, when one or more of the above dedifferentiation factors are already expressed, the dedifferentiation factor can be omitted.
- the gene product of the Myc gene group may be replaced with a cytokine or a compound, and examples of the cytokine in this case include SCF and bFGF.
- examples of compounds that can replace the c-Myc gene and the Klf4 gene include valproic acid.
- genes encoding dedifferentiation factors are all highly conserved genes in vertebrates, and unless otherwise indicated in the present specification, the genes including homologs are expressed.
- gene products having mutations, including gene polymorphisms have a function equivalent to that of the wild type gene product, for example, 1 to 10, preferably 1 to 6, of the wild type gene product, More preferably, mutant gene products in which 1 to 4, more preferably 1 to 3, particularly preferably 1 to 2 amino acids are substituted, inserted, or deleted are also included.
- the method of dedifferentiation of the mononuclear cell population using the dedifferentiation factor as described above is not particularly limited.
- the dedifferentiation treatment is performed by introducing the dedifferentiation factor into the mononuclear cell population.
- a complex of a cationic lipid reagent such as SAINT-PhD (Cosmo Bio Inc.) or Cellvader (GEderHealthcare) and a dedifferentiation factor, or a peptide called Protein ⁇ ⁇ Transduction Domain (PTD)
- SAINT-PhD Cosmo Bio Inc.
- GEderHealthcare Cellvader
- PTD Protein ⁇ ⁇ Transduction Domain
- a dedifferentiation factor can be introduced into the mononuclear cells by adding these complexes to the culture medium of the mononuclear cell population and bringing them into contact with the mononuclear cell population.
- the mononuclear cells thus introduced with the dedifferentiation factor are dedifferentiated and acquire pluripotency.
- the amount of dedifferentiation factor to be added can be appropriately determined by those skilled in the art.
- the mononuclear cell population may be dedifferentiated by introducing an expression vector capable of expressing the dedifferentiation factor into the mononuclear cell population.
- an expression vector capable of expressing the dedifferentiation factor into the mononuclear cell population.
- a gene encoding a dedifferentiation factor (dedifferentiation factor gene) is introduced downstream of an appropriate promoter for expression in mononuclear cells to prepare a recombinant expression vector.
- a dedifferentiation factor gene is expressed using a virus-derived promoter.
- an RNA encoding the dedifferentiation factor is used.
- a recombinant virus vector in the genome is prepared.
- the expression vector to be used is not particularly limited as long as it has a desired dedifferentiation-inducing function, and is not limited to any of wild type, mutant type, natural type, artificially modified type, etc. It is preferable that For example, it may be a viral vector such as Sendai virus-derived, retrovirus-derived, adenovirus-derived, adeno-associated virus-derived, or box virus-derived, but it is derived from Sendai virus having the feature of not causing fusion due to transfer to the host chromosome. It is preferable that it is a vector.
- Such a Sendai virus vector preferably has a gene encoding a protein necessary for genome replication, such as N protein, P protein, and L protein, in order to perform a function of inducing dedifferentiation in mononuclear cells.
- the artificially modified Sendai virus vector may be a Sendai virus vector having a mutation regarding cytotoxicity or temperature sensitivity.
- a Sendai virus vector having a mutation or deletion in a gene encoding a viral envelope protein or outer shell protein such as F gene, H gene, HN gene, or G gene derived from a minus-strand RNA virus Good (see WO00 / 70055, WO00 / 70070, Li, H.-O. et al., J. Virol. 74 (14) 6564-6569 (2000)).
- Such Sendai virus vectors are highly safe because they can replicate the genome in mononuclear cells but cannot form infectious virus particles.
- the recombinant expression vector and virus particles thus prepared are introduced into mononuclear cells.
- the gene of interest can be introduced into mononuclear cells by adding a recombinant expression vector such as a plasmid or virus particles to the medium.
- a recombinant expression vector such as a plasmid
- the recombinant expression vector can be introduced into mononuclear cells by treatment according to known gene transfer methods such as the calcium phosphate method, lipofection method, DEAE dextran method, electroporation method, etc.
- virus particles such as recombinant Sendai virus are added, the target gene is introduced into the mononuclear cells by infecting the mononuclear cells with the virus.
- a mononuclear cell into which a recombinant expression vector has been introduced expresses a dedifferentiation factor
- the mononuclear cell is dedifferentiated by the expressed dedifferentiation factor.
- the amount of the recombinant expression vector to be added can be appropriately determined by those skilled in the art.
- the cells are cultured in a serum-free medium for 1 to 5 days, preferably 2 days in the presence of 5% CO 2 at 35 ° C. to 40 ° C., preferably 37 ° C. .
- the serum-free medium may be DMEM / F12, VP-SFM, DMEM, KBM530, KBM540, KBM560, ROM1640, or KBM502, preferably KBM502.
- MEF mouse embryonic fibroblast
- SNL strained mouse embryonic fibroblast
- the mononuclear cell population subjected to the dedifferentiation treatment as described above is cultured under general conditions for culturing iPS cells for 10 to 30 days, preferably 14 to 25 days, more preferably 20 days.
- growth factor-containing DMEM / F12 may be used and cultured at 35 ° C. to 40 ° C., preferably 37 ° C. in the presence of 5% CO 2 .
- a medium such as DMEM may be used, and feeder cells prepared with MEF or SNL may be used.
- the growth factor is not particularly limited and can be appropriately selected by those skilled in the art from known growth factors.
- FGF fibroblast growth factor
- EGF epidermal growth factor
- one or more kinds of substances that are usually added to the culture of mononuclear cells may be contained in the medium.
- examples of such substances include, but are not limited to, serum such as FBS, knockout serum replacement (Invitrogen), L-glutamine, non-essential amino acids, antibiotics such as penicillin and streptomycin, and mercaptoethanol.
- This example shows that iPS cells can be efficiently produced from a peripheral blood-derived mononuclear cell population by the iPS cell production method according to the present invention. All cultures were performed at 37 ° C. and 5% CO 2 .
- Sendai virus was prepared by introducing the dedifferentiation factor genes (1) to (4) into a SeV18 + / TS ⁇ F vector (WO2010 / 008054) according to a known method (WO2010 / 008054).
- RNA viral genomic RNA minus strand
- plasmid vector expressing a recombinant viral genome of cDNA encoding the complementary strand (plus strand) and a protein (F, N, P, L, T7 RNA polymerase) necessary for virus self-replication.
- the cells were introduced into 293T / 17 cells, and further cultured by overlaying LLC-MK2 / F / A cells expressing F protein, and the culture supernatant containing the produced virus was collected.
- Anti-CD3 antibody (BD Biosciences) was prepared to 10 ⁇ g / ml with PBS. The bottom of the 6-well dish was covered with this antibody dilution, and incubated at 37 ° C. for 30 minutes to 3 hours. Immediately before use, the antibody dilution was removed and washed with PBS to give an anti-CD3 antibody-binding dish.
- Peripheral blood-derived mononuclear cell populations are seeded in anti-CD3 antibody-binding dishes at a density of 1 ⁇ 10 5 to 1 ⁇ 10 6 cells / ml and contain KBM502 medium (containing 20 U / ml recombinant interleukin 2) Cultured in 10 ml for 5 days.
- KBM502 medium containing 20 U / ml recombinant interleukin 2
- Dedifferentiation treatment day 2 Cells were peeled off with a cell scraper, and the medium containing the cells was collected in a tube for each well. After centrifuging at 20 ° C. and 800 to 1000 rpm for 5 minutes, 2 ml of KBM502 medium was added to the pellet, and pipetting was performed several times. The pellet was broken to such an extent that it would not become a single cell and suspended. This suspension was returned to the well of the original dish and cultured in KBM502 medium for 24 hours.
- the cells were peeled off with a cell scraper, and the medium containing the cells was collected in a tube for each well.
- the cells were made into single cells by pipetting, and the number of cells was counted. After centrifugation at 20 ° C. and 800-1000 rpm for 5 minutes, an appropriate amount of KBM502 medium was added to the pellet.
- the pellet is made into a single cell by pipetting, and mononuclear cells are seeded at a density of 5 ⁇ 10 4 , 5 ⁇ 10 5 , 5 ⁇ 10 6 / dish on feeder cells (SNL) prepared in a 10 cm dish, and KBM502 The cells were cultured in the medium for 24 hours.
- SNL feeder cells
- the iPS cell culture medium is DMEM / F12 (Invitrogen), 20% knockout serum replacement (Invitrogen), 2 mM L-glutamine (Invitrogen), 1 ⁇ 10 ⁇ 4 M non-essential amino acid (Invitrogen), 1 ⁇ 10 ⁇ 4 M2-mercaptoethanol (Invitrogen), 0.5% penicillin-streptomycin (Wako Pure Chemical Industries).
- colonies of clones obtained by the dedifferentiation treatment were fixed with 10% neutral buffered formalin solution (Wako Pure Chemical Industries), then anti-Nanog antibody (Reprocell, diluted 1000 times), anti-Oct3 / 4 antibody (Santa Cruz, diluted 100-fold), anti-SSEA3 antibody (Millipore, diluted 200-fold), anti-SSEA4 antibody (Millipore, diluted 200-fold), anti-Tral60 antibody (Millipore, diluted 200-fold), anti-Tral81 antibody ( Millipore (200-fold dilution).
- anti-rabbit IgG antibody, anti-mouse IgG antibody or anti-mouse IgM antibody labeled with Alexa 488 or Alexa 568 was appropriately used as a secondary antibody (all from Molecular Probes).
- Alexa 488 or Alexa 568 was appropriately used as a secondary antibody (all from Molecular Probes).
- the stained cells were observed under a fluorescence microscope (IX70, Olympus Corporation), as shown in FIG. 3, all the examined stem cell marker proteins were detected in all three clones.
- Nanog-F CAGCCCCGATTCTTCCACCAGTCCC (SEQ ID NO: 1)
- Nanog-R CGGAAGATTCCCAGTCGGGTTCACC (SEQ ID NO: 2)
- Oct 3 / 4-F GACAGGGGGAGGGGAGGAGCTAGG (SEQ ID NO: 3)
- Oct 3 / 4-R CTTCCCTCCAACCAGTTGCCCCAAAC (SEQ ID NO: 4)
- Sox 2-F GGGAAATGGGAGGGGTGCAAAAGAGG
- Sox 2-R TTGCGTGAGTGTGGATGGGATTGGTG (SEQ ID NO: 6)
- Klf 4-F ACGATCGTGGCCCCGGAAAAGGACC (SEQ ID NO: 7)
- Klf 4-R TGATTGTAGTGCTTTCTGGCTGGGCTCC (SEQ ID NO: 8)
- cMyc-F GCGTCCTGGGAAGGGAGATCCGGAGC (SEQ ID NO: 9)
- cMyc-R TTGAGGGG
- anti-CD3-positive T cells are not isolated from the peripheral blood-derived mononuclear cell population using FACS or the like, and the peripheral blood-derived mononuclear cell population is cultured in the presence of the anti-CD3 antibody and then subjected to dedifferentiation treatment.
- iPS cells can be produced with high efficiency.
- T cells are selected from the peripheral blood mononuclear cell fraction by FACS to produce iPS cells.
- CD3-positive T cells were selected by FACS from the same amount of peripheral blood-derived mononuclear cell fraction used in the examples. By this selection, about 30 to 40% of the peripheral blood-derived mononuclear cell fraction was obtained as CD3-positive T cells.
- the selected T cells were seeded on an anti-CD3 antibody-binding dish and cultured for 5 days in 10 ml of KBM502 medium containing recombinant interleukin 2 (20 U / ml).
- the cultured T cells were prepared at a density of 7.5 ⁇ 10 5 cells / ml, and recombinant Sendai virus was added at MOI 3 or 20 in the same manner as in Example to introduce a recombinant vector, and CD3 positive T cells The dedifferentiation process was performed.
- the colony cells obtained by the dedifferentiation treatment were subjected to alkaline phosphatase staining and crystal violet staining.
- FIG. 5 shows a graph comparing the number of colonies when the dedifferentiation process is performed with MOI 20 in this comparative example with the number of colonies when the dedifferentiation process is performed with MOI 20 in the example.
- the colonies obtained in the comparative examples were several colonies including alkaline phosphatase positive and negative.
- the method of the example showed that the efficiency of iPS cell establishment was about 100 times higher than the method of the comparative example.
- Comparative Example 2 This comparative example shows that when producing iPS cells from the mononuclear cell population described in the Examples, a step of culturing the mononuclear cell population in the presence of anti-CD3 antibody and interleukin 2 is necessary.
- a mononuclear cell population obtained from peripheral blood was seeded in an anti-CD3 antibody-binding dish, and cells cultured for 5 days in the presence of interleukin 2 were dedifferentiated to establish iPS cells (anti-antibody).
- CD3 + IL2 + group On the other hand, a mononuclear cell population obtained from peripheral blood was seeded in a dish not bound with anti-CD3 antibody, and the cells cultured for 5 days using KBM502 medium not containing interleukin 2 were dedifferentiated to obtain iPS cells. Established (anti-CD3-IL2-group).
- the mononuclear cell dedifferentiation treatment was performed by adding Sendai virus expressing the reprogramming factor at MOI 3 to the mononuclear cell population.
- the ratio (%) of the number of alkaline phosphatase-positive colonies to the number of mononuclear cells added with Sendai virus was calculated as the iPS cell establishment efficiency. As shown in FIG. It was 0% in the CD3-IL2-group, but 0.016% in the anti-CD3 + IL2 + group.
- a culture step in the presence of anti-CD3 antibody and interleukin 2 is required.
- iPS cells with high efficiency from mononuclear cells derived from peripheral blood.
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Abstract
Description
本出願は、2010年4月16日付で出願した日本国特許出願2010-95404に基づく優先権を主張するものであり、当該基礎出願を引用することにより、本明細書に含めるものとする。
まず、末梢血から調製した単核球集団を、抗CD3抗体およびインターロイキンの存在下で3~14日間、好ましくは3~7日間培養する。この培養により、単核球のうち、CD3陽性T細胞の増殖が特異的に促進されると考えられる。
次に、単核球集団に対して脱分化処理を行う。
以上のようにして脱分化処理した単核球集団を10~30日、好ましくは14~25日、より好ましくは20日間、iPS細胞を培養する一般的な条件下で培養する。例えば、増殖因子含有DMEM/F12を用い、5%CO2存在下、35℃~40℃、好ましくは37℃で培養すればよい。あるいは、DMEM等の培地を用いてもよく、MEFやSNL等で作製したフィーダー細胞を用いてもよい。また、増殖因子は特に制限されず、周知の増殖因子から当業者が適宜選択できるが、例えば、繊維芽細胞増殖因子(FGF)や上皮成長因子(EGF)であってもよい。
慶應大学病院倫理委員会で承認されたプロトコールに従って、インフォームドコンセントを行った各健常人ボランティア(11歳~66歳、男女、計5名)から、1~20mlの末梢血を採血した。フィコール・ハイパック(GE Healthcare 社)を比重液とし、遠心分離(30分間、400×g)を行い、末梢血から単核球分画を単離した。
以下(1)~(4)の脱分化因子遺伝子を公知の方法(WO2010/008054)に従って、SeV18+/TSΔFベクター(WO2010/008054)に導入したセンダイウイルスの調製を行った。
(1)Oct3/4遺伝子
(2)Klf4遺伝子
(3)c-Myc遺伝子
(4)Sox2遺伝子
具体的には、(1)~(4)の遺伝子を含む、マイナス鎖RNAウイルスゲノムRNA(マイナス鎖)またはその相補鎖(プラス鎖)をコードするcDNAの組み換えウイルスゲノムを発現するプラスミドベクター、及びウイルスの自己複製に必要な蛋白質(F、N、P、L、T7RNAポリメラーゼ)を発現するプラスミドベクターを293T/17細胞に導入し、さらにFタンパク質を発現するLLC-MK2/F/A細胞を重層して培養を行い、生成したウイルスを含む培養上清を回収することにより製造した。
(株化マウス胚性繊維芽細胞、SNL)
ディッシュに0.1%ゼラチンを加え、37℃で約1時間静置し、ディッシュをコーティングした。SNL(EGACC社)を1.5×105細胞/mlの密度に調製し、1ディッシュ(直径10cm)あたり10mlを加え、一晩培養し、フィーダー細胞を調製した。
抗CD3抗体(BD Biosciences 社)をPBSで10μg/mlに調製した。この抗体希釈液で6ウェルディッシュの底面を覆い、37℃で30分間~3時間インキュベートした。使用直前に抗体希釈液を取り除き、PBSで洗浄して抗CD3抗体結合ディッシュとした。
(脱分化処理1日目)
抗CD3抗体を用いて培養した単核球集団を7.5×105細胞/mlの密度に調製し、ここにMOI 1、3、5、10、あるいは20でセンダイウイルスを添加することによって、組み換えベクターを導入した後、KBM502培地中で24時間培養した。
セルスクレーパーで細胞を剥がし取り、細胞を含む培地を、ウェル毎にチューブに回収した。20℃、800~1000rpmで5分間遠心した後、ペレットにKBM502培地2mlを加え、数回ピペッティングし、ペレットをシングルセルにならない程度に破壊し、懸濁した。この懸濁液を、元のディッシュのウェルに戻し、KBM502培地中で24時間培養した。
セルスクレーパーで細胞を剥がし取り、細胞を含む培地をウェル毎にチューブに回収した。ピペッティングによって、細胞をシングルセルにし、細胞数を計数した。20℃、800~1000rpmで5分間遠心した後、ペレットに適量のKBM502培地を加えた。ピペッティングによって、ペレットをシングルセルにし、10cmディッシュに調製したフィーダー細胞(SNL)上に、5×104、5×105、5×106/ディッシュの密度で単核球を播種し、KBM502培地中で24時間培養した。
上記のように脱分化処理した細胞を、フィーダー細胞(SNL)上に播種し、培地を10ng/mlヒト塩基性繊維芽細胞増殖因子(bFGF、和光純薬工業)添加iPS細胞培地(10ml/10cmディッシュ)に交換した。その後、48時間毎に培地を交換し、20日間培養を続けた。なお、iPS細胞培地は、DMEM/F12(Invitrogen 社)、20%knockout serum replacement (Invitrogen 社)、2mM L-グルタミン、(Invitrogen 社)、1×10-4M 非必須アミノ酸(Invitrogen 社)、1×10-4M2-メルカプトエタノール(Invitrogen 社)、0.5%ペニシリン-ストレプトマイシン(和光純薬工業)から成る。
脱分化処理により得られたコロニーに対し、アルカリフォスファターゼ染色、および、クリスタルバイオレット染色を行った。まず、コロニーを10%中性緩衝ホルマリン液(和光純薬工業)で固定した後、1-Step NBT/BCIP(Pierce 社)で染色した。さらに、クリスタルバイオレットをメタノールに溶解して4%クリスタルバイオレット溶液を調製し、細胞に添加して30分間染色した。なお、アルカリフォスファターゼは幹細胞で発現することが知られており、幹細胞のマーカーとして用いられている(例えば、Riekstina U. et al., Stem Cell Rev. 2009 Dec 5(4): 378-386 参照)。また、クリスタルバイオレット染色により生細胞のみが染色される。さらに、DAPI(Molecular Probes 社)を用いて適宜核の対比染色を行った。
上記アルカリフォスファターゼ陽性細胞のコロニーのうち、3コロニーをランダムにクローニングし、これらの細胞がiPS細胞であることを確認するため、DAPI染色およびアルカリフォスファターゼ染色を行い形態学的観察を行ったところ、3つのクローンとも、胚性幹細胞あるいはiPS細胞に典型的な形態を有し、アルカリフォスファターゼ陽性であった。
さらに、各クローンの細胞について各種幹細胞マーカーのタンパク質および遺伝子発現を、免疫組織学的染色および逆転写ポリメラーゼ連鎖反応(RT-PCR)法により解析した。RT-PCR法では、脱分化処理前の単核球、および、脱分化処理後の単核球について同様に解析を行い、陽性コントロール細胞としてヒト胚性幹細胞を用いた(KhES-2、京都大学より入手)。
プライマー:
Nanog-F: CAGCCCCGATTCTTCCACCAGTCCC(配列番号1)
Nanog-R: CGGAAGATTCCCAGTCGGGTTCACC(配列番号2)
Oct 3/4-F: GACAGGGGGAGGGGAGGAGCTAGG(配列番号3)
Oct 3/4-R: CTTCCCTCCAACCAGTTGCCCCAAAC(配列番号4)
Sox 2-F: GGGAAATGGGAGGGGTGCAAAAGAGG(配列番号5)
Sox 2-R: TTGCGTGAGTGTGGATGGGATTGGTG(配列番号6)
Klf 4-F: ACGATCGTGGCCCCGGAAAAGGACC(配列番号7)
Klf 4-R: TGATTGTAGTGCTTTCTGGCTGGGCTCC(配列番号8)
cMyc-F: GCGTCCTGGGAAGGGAGATCCGGAGC(配列番号9)
cMyc-R: TTGAGGGGCATCGTCGCGGGAGGCTG(配列番号10)
GDF 3-F: CTTATGCTACGTAAAGGAGCTGGG(配列番号11)
GDF 3-R: GTGCCAACCCAGGTCCCGGAAGTT(配列番号12)
Rex 1-F: CAGATCCTAAACAGCTCGCAGAAT(配列番号13)
Rex 1-R: GCGTACGCAAATTAAAGTCCAGA(配列番号14)
DPPA 4-F: GGAGCCGCCTGCCCTGGAAAATTC(配列番号15)
DPPA 4-R: TTTTTCCTGATATTCTATTCCCAT(配列番号16)
DPPA 2-F: CCGTCCCCGCAATCTCCTTCCATC(配列番号17)
DPPA 2-R: ATGATGCCAACATGGCTCCCGGTG(配列番号18)
GAPDH-F: CAGAACATCATCCCTGCCTCTAG(配列番号19)
GAPDH-R: TTGAAGTCAGAGGAGACCACCTG(配列番号20)
比較例では、FACSにより末梢血単核球分画からT細胞を選別し、iPS細胞を製造する。
本比較例では、実施例に記載の単核球集団からiPS細胞を作製する際、抗CD3抗体およびインターロイキン2の存在下で単核球集団を培養する工程が必要であることを示す。
Claims (8)
- 人工多能性幹細胞の製造方法であって、
末梢血由来の単核球集団を材料とすることを特徴とする方法。 - 請求項1に記載の人工多能性幹細胞の製造方法であって、
(イ)末梢血由来の単核球集団を抗CD3抗体およびインターロイキン2の存在下で3~14日間培養する工程と、
(ロ)培養後の前記単核球集団に対して脱分化処理を行う工程と
を含むことを特徴とする方法。 - 請求項2に記載の人工多能性幹細胞の製造方法であって、
前記工程(ロ)において、前記単核球集団に脱分化因子の導入操作を行うことを特徴とする方法。 - 請求項3に記載の人工多能性幹細胞の製造方法であって、
前記脱分化因子の導入操作において、前記脱分化因子を発現する組み換え発現ベクターの導入操作を行うことを特徴とする方法。 - 請求項3または4に記載の人工多能性幹細胞の製造方法であって、
前記工程(ロ)において、前記脱分化因子がSox2、Oct3/4、Klf4およびc-Mycであることを特徴とする方法。 - 請求項4または5に記載の人工多能性幹細胞の製造方法であって、
前記組み換え発現ベクターがセンダイウイルスベクターであることを特徴とする方法。 - 請求項2~6のいずれかに記載の人工多能性幹細胞の製造方法であって、
(ハ)脱分化処理を行った前記単核球集団を、増殖因子の存在下で14~25日間培養する工程をさらに含むことを特徴とする方法。 - 請求項1~7のいずれかに記載の人工多能性幹細胞の製造方法であって、
前記末梢血がヒト由来であることを特徴とする方法。
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US13/641,370 US9447432B2 (en) | 2010-04-16 | 2011-04-15 | Method for producing induced pluripotent stem cells |
SG2012076873A SG184892A1 (en) | 2010-04-16 | 2011-04-15 | Method for producing induced pluripotent stem cells |
JP2012510720A JP5856949B2 (ja) | 2010-04-16 | 2011-04-15 | 人工多能性幹細胞の製造方法 |
EP11768967.9A EP2559757B1 (en) | 2010-04-16 | 2011-04-15 | Method for producing induced pluripotent stem cells |
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US11572544B2 (en) | 2017-06-14 | 2023-02-07 | The Children's Medical Center Corporation | Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells by episomal plasmid gene transfer |
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EP2559757B1 (en) | 2017-12-06 |
JPWO2011129446A1 (ja) | 2013-07-18 |
JP5856949B2 (ja) | 2016-02-10 |
EP2559757A4 (en) | 2014-01-22 |
CA2796599A1 (en) | 2011-10-20 |
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US20130189786A1 (en) | 2013-07-25 |
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