JP7033318B2 - Method for producing pluripotent stem cells - Google Patents
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Description
本発明は、多能性幹細胞の製造方法に関する。 The present invention relates to a method for producing pluripotent stem cells.
胚性幹細胞（ＥＳ細胞）は、胚盤胞期の内部細胞塊から作られる細胞であり、様々な細胞に分化できる多能性幹細胞である。１９９８年にカリフォルニア大学のジェームズ・トムソンらが、ヒトのＥＳ細胞の作製に成功したことをきっかけに、ＥＳ細胞を様々な細胞に分化させ、再生医療に利用することが期待されてきた。しかし、ヒトのＥＳ細胞を作製するにはヒトの受精卵を用いる必要があり、医療への利用は生命倫理上問題がある。そのため、現在ではＥＳ細胞を再生医療に利用することは見送られている。 Embryonic stem cells (ES cells) are cells made from the inner cell mass of the blastocyst stage and are pluripotent stem cells that can differentiate into various cells. In 1998, James Thomson et al. Of the University of California succeeded in producing human ES cells, and it has been expected that ES cells will be differentiated into various cells and used for regenerative medicine. However, in order to produce human ES cells, it is necessary to use fertilized human eggs, and their use in medical treatment poses a bioethical problem. Therefore, the use of ES cells for regenerative medicine is currently postponed.
これに対して、山中らは乳腺由来の体細胞から、ＥＳ細胞と同等の機能を有する多能性幹細胞（ｉＰＳ細胞）の作製にマウスで成功し、その後、ヒトのｉＰＳ細胞の作製にも成功している（非特許文献１：Cell、３０巻、８６１頁－８７２頁、２００７年）。ヒトのｉＰＳ細胞は、４つの遺伝子（ＳＯＸ２、ＯＣＴ３／４、ｃ－Ｍｙｃ、Ｋｌｆ－４遺伝子）を、遺伝子組み換えにより同時に体細胞に導入して発現させることにより得られる。ｉＰＳ細胞はＥＳ細胞と同様の性質を有して、様々な細胞や臓器に分化できる上に、ヒト胚を使用しないことから、再生医療への利用が期待されている。しかし、ｉＰＳ細胞は、遺伝子操作により得るため、体細胞あたりのｉＰＳ細胞の変換率は非常に低い。そのため、現在までｉＰＳ細胞の作製方法と選別方法に様々な改良が加えられてきた（特許文献１：特開２００８－２８３９７２号公報、特許文献２：特開２００９－１６５４８０号公報、特許文献３：特開２０１４－００００８３号公報、特許文献４：国際公開第２０１３／１６３２９６号）。しかしながら、依然として体細胞からｉＰＳ細胞への変換率は低く、取得までに時間がかかることがｉＰＳ細胞の問題点の１つである。 On the other hand, Yamanaka et al. Succeeded in producing pluripotent stem cells (iPS cells) having the same function as ES cells from somatic cells derived from breast glands in mice, and subsequently succeeded in producing human iPS cells. (Non-Patent Document 1: Cell, Vol. 30, pp. 861-872, 2007). Human iPS cells are obtained by simultaneously introducing and expressing four genes (SOX2, OCT3 / 4, c-Myc, Klf-4 genes) into somatic cells by genetic recombination. Since iPS cells have the same properties as ES cells and can differentiate into various cells and organs and do not use human embryos, they are expected to be used for regenerative medicine. However, since iPS cells are obtained by genetic engineering, the conversion rate of iPS cells per somatic cell is very low. Therefore, various improvements have been made to the method for producing and selecting iPS cells (Patent Document 1: Japanese Patent Application Laid-Open No. 2008-283972, Patent Document 2: Japanese Patent Application Laid-Open No. 2009-165480, Patent Document 3: Japanese Unexamined Patent Publication No. 2014-000083, Patent Document 4: International Publication No. 2013/163296). However, the conversion rate from somatic cells to iPS cells is still low, and it takes time to obtain them, which is one of the problems of iPS cells.
さらに、細胞は細胞表面に提示したＨＬＡ抗原により、自己と他人を区別している。ｉＰＳ細胞で作製した細胞組織や臓器を再生医療に利用するためには、このＨＬＡ抗原の型を一致させる必要がある。言い換えれば、ｉＰＳ細胞による再生医療では、すべてのＨＬＡ型を有するｉＰＳ細胞群を準備し、それをすべての細胞や臓器に分化したものを前もって準備しておく必要がある。しかし、ＨＬＡ抗原の型は非常に多岐にわたっており、ＨＬＡ抗原の型が一致する確率は千人から１万人に一人にすぎない。ＨＬＡ型を有するｉＰＳ細胞群とそこから分化した細胞群を準備することは不可能ではないが、現実的には極めて困難である。さらに、細胞の自己と他人の区別は、ＨＬＡ抗原以外でも行われているため、ＨＬＡ抗原型の一致したｉＰＳ細胞により、再生医療を行ったとしても、患者は自己免疫反応が生じ、免疫抑制剤を投与し続ける必要がある。このことは、患者にとって大きい負担を強いることになる。 Furthermore, cells distinguish themselves from others by the HLA antigen presented on the cell surface. In order to utilize cell tissues and organs prepared from iPS cells for regenerative medicine, it is necessary to match the types of this HLA antigen. In other words, in regenerative medicine using iPS cells, it is necessary to prepare a group of iPS cells having all HLA types, and to prepare in advance those differentiated into all cells and organs. However, the types of HLA antigens are very diverse, and the probability of matching HLA antigen types is only 1 in 1,000 to 10,000. It is not impossible to prepare an iPS cell group having an HLA type and a cell group differentiated from the iPS cell group, but it is extremely difficult in reality. Furthermore, since the distinction between the cell self and others is also performed by other than the HLA antigen, even if regenerative medicine is performed with iPS cells having the same HLA antigen type, the patient will have an autoimmune reaction and the immunosuppressant. Need to be continued. This imposes a heavy burden on the patient.
上記の２つの問題は、「患者から直接細胞を取り出し、それを再生医療に使う」ことにより解決できる。しかし、ｉＰＳ細胞では、（１）４つの遺伝子を遺伝子操作により導入し選別するため、作製の時間と手間がかかる、（２）ｉＰＳ細胞を得る確率は低く再生医療に十分な細胞を確保するまでに時間がかかる、（３）遺伝子を導入するために得られたｉＰＳ細胞の安全確認に時間がかかる、（４）ひとり当たりに膨大な労力が必要になり高額の医療費が発生するなどの理由により、患者自身の細胞を再生医療に利用することは極めて困難である。 The above two problems can be solved by "taking cells directly from the patient and using them for regenerative medicine". However, in iPS cells, (1) four genes are introduced and selected by genetic manipulation, so it takes time and effort to prepare them. (2) The probability of obtaining iPS cells is low and until sufficient cells for regenerative medicine are secured. (3) It takes time to confirm the safety of the iPS cells obtained for introducing the gene, (4) It requires a huge amount of labor per person and a large amount of medical expenses are incurred. Therefore, it is extremely difficult to utilize the patient's own cells for regenerative medicine.
一方、遺伝子組換えを用いることなく、短時間、高効率で多能性幹細胞を作製する方法を開発できれば、患者自身の細胞から、多能性幹細胞を作製することが可能になり、再生医療の実現性は非常に高まる。このような細胞の作製法に関する報告は、現在２つしか報告されていない。 On the other hand, if a method for producing pluripotent stem cells in a short time and with high efficiency can be developed without using genetic recombination, it will be possible to produce pluripotent stem cells from the patient's own cells, which will lead to regenerative medicine. The feasibility is very high. Currently, only two reports have been made on methods for producing such cells.
その１つは、ＳＴＡＰ細胞である（非特許文献２：Nature、５０５巻、６４１頁－６４７頁、２０１４年、特許文献４）。理化学研究所の小保方らのグループは、紅茶の成分を使うことでｉＰＳ細胞と同等の細胞であるＳＴＡＰ細胞を作製できることを発見した。しかし、その後に数多くの検証がなされ、ＳＴＡＰ細胞は、ＥＳ細胞自身を用いた可能性が高く、ＳＴＡＰ細胞の作製方法を提示した論文そのものが、捏造または誤りであることが立証された。 One of them is STAP cells (Non-Patent Document 2: Nature, Vol. 505, pp. 641-647, 2014, Patent Document 4). Obokata et al.'S group at RIKEN discovered that STAP cells, which are cells equivalent to iPS cells, can be produced by using black tea components. However, many verifications have been made since then, and it is highly possible that the STAP cells used the ES cells themselves, and it was proved that the paper itself, which presented the method for producing the STAP cells, was forged or erroneous.
もう１つは、２０１６年にＫｉｍらにより報告された研究（非特許文献３：Biochemical and Biophysical Research Communications、４７２巻、５８９頁－５９１頁、２０１６年）であり、これが最新の研究である。彼らは癌細胞に刺激を与え、生き残った細胞がｉＰＳ細胞に近い細胞であることを発見した。この論文は、「ＳＴＡＰ細胞を作製できた」という見出しで、一部の報道が誤って報道したが、当該論文で得られた細胞は、ｉＰＳ細胞マーカーを発現しておらず、多能性幹細胞ではない。論文の著者自身の結論も「多能性幹細胞は得られなかった」と結論している。 The other is a study reported by Kim et al. In 2016 (Non-Patent Document 3: Biochemical and Biophysical Research Communications, Vol. 472, pp. 589-591, 2016), which is the latest study. They stimulated cancer cells and found that the surviving cells were similar to iPS cells. In this paper, under the heading "STAP cells could be produced", some reports mistakenly reported, but the cells obtained in this paper did not express iPS cell markers and were pluripotent stem cells. is not it. The author's own conclusion of the paper also concludes that "pluripotent stem cells were not obtained."
すなわち、２０１６年現在においても、化合物で刺激することにより多能性幹細胞を誘導する方法は知られていない。 That is, even as of 2016, there is no known method for inducing pluripotent stem cells by stimulating with a compound.
遺伝子組換えを用いることなく、短時間、高効率でｉＰＳ細胞と同等の機能を有する多能性幹細胞を製造できれば、患者自身の細胞を使って、多能性幹細胞を製造することも可能であり、再生医療の実現性は非常に高まる。しかしながら、上述のとおり、そのような多能性幹細胞の製造方法は、これまで全く見出されていない。 If pluripotent stem cells having the same function as iPS cells can be produced in a short time and with high efficiency without using genetic recombination, it is also possible to produce pluripotent stem cells using the patient's own cells. , The feasibility of regenerative medicine is greatly enhanced. However, as described above, no method for producing such pluripotent stem cells has been found so far.
本発明は、上記の課題に鑑みてなされたものであり、ｉＰＳ細胞と同等の機能を有する多能性幹細胞を得ることのできる、多能性幹細胞の製造方法を提供することを目的とする。 The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for producing pluripotent stem cells, which can obtain pluripotent stem cells having a function equivalent to that of iPS cells.
本発明は、以下のとおりである。
［１］ ポリアミン、または、ＤＮＡメチル化阻害物質を含む培地中で、動物細胞を培養することにより、多能性幹細胞を取得する、多能性幹細胞の製造方法。
［２］ 前記ポリアミンは、スペルミン、スペルミジン、プトレシン、および、それらのアセチル化体、並びに、それらポリアミンの２つ以上の重合体、からなる群から選択される少なくとも１種である、［１］に記載の製造方法。
［３］ 前記ＤＮＡメチル化阻害物質は、ＤＮＡメチル化酵素をコードする遺伝子の発現を阻害するｓｉＲＮＡ、５－アザ－２’－デオキシシチジン、シネフンギン、ゼブラリン、エチオニン、アセチルメチオニン、および、セレノメチオニンからなる群から選択される少なくとも１種である、［１］または［２］に記載の製造方法。The present invention is as follows.
[1] A method for producing pluripotent stem cells, which obtains pluripotent stem cells by culturing animal cells in a medium containing polyamine or a DNA methylation inhibitor.
[2] The polyamine is at least one selected from the group consisting of spermine, spermidine, putrescine, and acetylated products thereof, and two or more polymers of the polyamines, [1]. The manufacturing method described.
[3] The DNA methylation inhibitor is derived from siRNA, 5-aza-2'-deoxycytidine, cinefungin, zebularine, ethionine, acetylmethionine, and selenomethionine, which inhibit the expression of genes encoding DNA methylase. The production method according to [1] or [2], which is at least one selected from the group.
本発明者は、細胞を初期化する働きのある物質について、数多くの物質を用いて探索し、得られた細胞の性質を調べた。本発明者の鋭意努力の結果、ポリアミンに、細胞の初期化に関する遺伝子群の発現を誘導する働きがあることを発見した。さらに、本発明者は、ポリアミンが結果的にＤＮＡのメチル化を阻害し、それが細胞を初期化していることを発見し、ＤＮＡメチル化阻害物質を用いても同様の細胞を得られることを見出した。また、ポリアミン、または、ＤＮＡメチル化阻害剤による細胞の分化誘導を進めると、ＥＳ細胞またはｉＰＳ細胞のマーカータンパク質を有する細胞に変化し、当該細胞が多能性幹細胞になることを発見した。 The present inventor searched for substances having a function of reprogramming cells using a large number of substances, and investigated the properties of the obtained cells. As a result of the diligent efforts of the present inventor, it has been discovered that polyamines have a function of inducing the expression of genes related to cell reprogramming. Furthermore, the present inventor has discovered that polyamines result in inhibition of DNA methylation, which reprograms cells, and that similar cells can be obtained using DNA methylation inhibitors. I found it. It was also discovered that when the differentiation of cells is induced by polyamine or DNA methylation inhibitor, the cells are transformed into cells having marker proteins of ES cells or iPS cells, and the cells become pluripotent stem cells.
それだけでなく、当該多能性幹細胞は、多能性を保持した状態で培養可能であり、胚様体に分化させる培地で、内胚葉、中胚葉、外胚葉などの様々な体細胞に再分化することも確認でき、本発明を完成するに至った。 Not only that, the pluripotent stem cells can be cultured in a state of maintaining pluripotency, and are redifferentiated into various somatic cells such as endoderm, mesoderm, and ectoderm in a medium that differentiates into embryoid bodies. It was also confirmed that the present invention was completed.
以下、本明細書において、ポリアミンを用いて得られた多能性幹細胞をＰＩＳ細胞（Polyamine-Induced Stem cells）と呼び、ＤＮＡメチル化阻害剤を用いて得られた多能性幹細胞をＤＩＳ細胞（Demethylation-Induced Stem Cells）と呼ぶ場合がある。 Hereinafter, in the present specification, pluripotent stem cells obtained by using polyamine are referred to as PIS cells (Polyamine-Induced Stem cells), and pluripotent stem cells obtained by using a DNA methylation inhibitor are referred to as DIS cells (DIS cells). Demethylation-Induced Stem Cells).
本発明によれば、ｉＰＳ細胞と同等の機能を有する多能性幹細胞を得ることのできる、多能性幹細胞の製造方法を提供することができる。なお、本発明の製造方法は、全く新しい方法であるだけでなく、遺伝子組換えを用いることなく、動物細胞から多能性幹細胞を得た点において、本発明の有用性は極めて高い。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for producing pluripotent stem cells, which can obtain pluripotent stem cells having a function equivalent to that of iPS cells. The production method of the present invention is not only a completely new method, but also the usefulness of the present invention is extremely high in that pluripotent stem cells are obtained from animal cells without using gene recombination.
さらに、ＰＩＳ細胞およびＤＩＳ細胞では、１～３日で得られる上に、当該細胞への転換効率も非常に高く、細胞の分離も容易という優れた特徴がある。ｉＰＳ細胞は、時間と手間がかかるだけでなく、ｉＰＳ細胞を得る確率は低いことから、ＰＩＳ細胞およびＤＩＳ細胞はｉＰＳ細胞の作製方法と比較して、進歩性が極めて高い。 Further, PIS cells and DIS cells are excellent in that they can be obtained in 1 to 3 days, the efficiency of conversion to the cells is very high, and the cells can be easily separated. Since iPS cells are not only time-consuming and laborious, but also have a low probability of obtaining iPS cells, PIS cells and DIS cells are extremely advanced as compared with the method for producing iPS cells.
また、患者から取得した細胞をｉＰＳ細胞に分化して再生医療に利用することは、既に記載の理由により困難であり、そのことが再生医療の実用化における大きな障害となっている。本発明のＰＩＳ細胞またはＤＩＳ細胞を作製する方法では、さまざまな細胞を短時間且つ高効率でＰＩＳ細胞またはＤＩＳ細胞に変換できる。このため、患者の一部の組織または脂肪組織等を採取すれば、再生医療に十分なＰＩＳ細胞またはＤＩＳ細胞の細胞量を短期間で確保することができる。即ち、ＰＩＳ細胞またはＤＩＳ細胞を作製する方法を利用すれば、患者自身の細胞を用いて短期間で再生医療を行うことが可能になり、治療後に免疫抑制剤を使用する必要がなく、理想的な再生医療を行うことが可能である。ｉＰＳ細胞と比較した場合、この点において本発明の進歩性および有効性は極めて高い。 In addition, it is difficult to differentiate cells obtained from patients into iPS cells and use them for regenerative medicine for the reasons already described, which is a major obstacle to the practical application of regenerative medicine. In the method for producing PIS cells or DIS cells of the present invention, various cells can be converted into PIS cells or DIS cells in a short time and with high efficiency. Therefore, if a part of the tissue or adipose tissue of the patient is collected, it is possible to secure a sufficient cell amount of PIS cells or DIS cells for regenerative medicine in a short period of time. That is, if a method for producing PIS cells or DIS cells is used, regenerative medicine can be performed in a short period of time using the patient's own cells, and it is not necessary to use an immunosuppressive agent after treatment, which is ideal. It is possible to perform various regenerative medicine. The inventive step and effectiveness of the present invention are extremely high in this respect when compared with iPS cells.
本発明は、培地に、ポリアミン、または、ＤＮＡメチル化阻害物質を添加し、動物細胞を（ＥＳ細胞またはｉＰＳ細胞と類似の形態を有するまで）培養することにより、多能性幹細胞を取得する、多能性幹細胞の製造方法である。 The present invention obtains pluripotent stem cells by adding polyamines or DNA methylation inhibitors to the medium and culturing animal cells (until they have a morphology similar to ES cells or iPS cells). It is a method for producing pluripotent stem cells.
なお、動物細胞とは、培養により増殖可能で正常な動物の細胞であればよい。ここで、動物はヒトを含み、好ましくは哺乳動物である。ヒト以外の哺乳動物としては、特に限定されないが、例えば、マウス、ラット、ウサギ、ヤギなどが挙げられる。哺乳動物以外の動物としては、例えば、ショウジョウバエなどの昆虫類などが挙げられる。 The animal cell may be a normal animal cell that can be proliferated by culturing. Here, the animals include humans, preferably mammals. Mammals other than humans are not particularly limited, and examples thereof include mice, rats, rabbits, and goats. Examples of animals other than mammals include insects such as Drosophila.
動物細胞としては、例えば、線維芽細胞、表皮細胞、乳腺細胞、脂肪細胞、筋芽細胞、骨芽細胞、肝細胞、血管内皮細胞、または、それらの前駆細胞などが挙げられる。他の動物細胞としては、例えば、血球系幹細胞、間葉系幹細胞、神経幹細胞などの幹細胞が挙げられる。 Examples of animal cells include fibroblasts, epidermal cells, mammary gland cells, fat cells, myoblasts, osteoblasts, hepatocytes, vascular endothelial cells, or precursor cells thereof. Examples of other animal cells include stem cells such as blood cell stem cells, mesenchymal stem cells, and neural stem cells.
例えば、ヒトの細胞は、ヒト正常細胞の分譲を行っている保存機関（ＡＴＣＣ、ＪＣＲＢなど）から分譲を受けるか、ヒト正常細胞の販売を行う市販のメーカーから購入するか、または、ヒトから細胞を取り出すことにより取得できる。例えば、ヒトなどの動物の組織から細胞を取り出して用いる場合は、組織検査の装置を用いた組織の一部のサンプリング、手術等による組織の部分的切除、脂肪吸引等の方法により、組織を得た後、トリプシン等の酵素により細胞を分離し、動物細胞用の培地で培養する。 For example, human cells are sold from storage institutions (ATCC, JCRB, etc.) that sell normal human cells, purchased from commercial manufacturers that sell normal human cells, or cells from humans. Can be obtained by taking out. For example, when cells are taken out from the tissue of an animal such as a human and used, the tissue is obtained by a method such as sampling a part of the tissue using a tissue inspection device, partial excision of the tissue by surgery, or aspiration of fat. After that, the cells are separated by an enzyme such as trypsin and cultured in a medium for animal cells.
（ポリアミン）
本発明に用いられるポリアミン（polyamine）は、直鎖脂肪族炭化水素に第一級アミノ基あるいは第二級アミノ基が合計で２つ以上結合した化合物を意味する。(Polyamine)
The polyamine used in the present invention means a compound in which two or more primary amino groups or secondary amino groups are bonded to a linear aliphatic hydrocarbon in total.
ポリアミンとしては、例えば、スペルミン（CAS登録番号71-44-3）、スペルミジン（CAS登録番号124-20-9）、プトレシン（CAS登録番号110-60-1）、および、それらのアセチル化体、並びに、それらポリアミンの２つ以上の重合体が挙げられる。 Examples of polyamines include spermine (CAS Registry Number 71-44-3), spermidine (CAS Registry Number 124-20-9), putrescine (CAS Registry Number 110-60-1), and their acetylated forms. Also mentioned are two or more polymers of these polyamines.
ポリアミンのアセチル化体（アセチル化ポリアミン）としては、例えば、Ｎ－アセチルプトレシン、Ｎ－アセチルスペルミジン、Ｎ－アセチルスペルミン、ジアセチルプトレシン、ジアセチルスペルミジン、ジアセチルスペルミンが挙げられる。なお、ポリアミンの２つ以上の重合体は、化学合成により得ることができる。 Examples of the acetylated form of polyamine (acetylated polyamine) include N-acetylputrescine, N-acetylspermidine, N-acetylspermine, diacetylputrescine, diacetylspermidine, and diacetylspermine. Two or more polymers of polyamines can be obtained by chemical synthesis.
ポリアミンは、炭素鎖３～４の間隔でアミノ基（－ＮＨ２基または－ＮＨ－基）が存在することで、ヒストンまたは染色体に作用し、細胞を初期化する遺伝子群の発現を誘導する特徴がある。したがって、上記以外のポリアミン（生体内に存在する他のポリアミン、化学合成により得られた他のポリアミンなど）のうち、スペルミン、スペルミジン、プトレシン等と類似の構造を有する化合物も、本発明に用いることができる。なお、ポリアミンの遺伝子発現を誘導する能力は、スペルミン、スペルミジン、プトレシンの順に強く、直鎖分子の長さが長いほど、モル濃度当たりの遺伝子の発現を誘導する効果は大きい。Polyamines are characterized by the presence of amino groups (-NH 2 or -NH- groups) at intervals of 3 to 4 carbon chains, which act on histones or chromosomes and induce the expression of genes that reprogram cells. There is. Therefore, among polyamines other than the above (other polyamines existing in the living body, other polyamines obtained by chemical synthesis, etc.), compounds having a structure similar to that of spermine, spermidine, putrescine, etc. are also used in the present invention. Can be done. The ability to induce gene expression of polyamines is stronger in the order of spermine, spermidine, and putrescine, and the longer the length of the linear molecule, the greater the effect of inducing gene expression per molar concentration.
なお、ポリアミンは水溶性のため、１０～１００ｍＭ程度の濃度のポリアミン水溶液を作製し、０．２μｍの濾過フィルターにより滅菌処理した後、適切な濃度になるように培地に添加して使用することができる。 Since polyamines are water-soluble, an aqueous polyamine solution having a concentration of about 10 to 100 mM can be prepared, sterilized with a 0.2 μm filtration filter, and then added to the medium to an appropriate concentration before use. can.
（ＤＮＡメチル化阻害物質）
本発明に用いられるＤＮＡメチル化阻害物質としては、例えば、以下の２種類が挙げられる。(DNA methylation inhibitor)
Examples of the DNA methylation inhibitor used in the present invention include the following two types.
ＤＮＡメチル化阻害物質の１種としては、ＤＮＡメチル化酵素をコードする遺伝子（ＤＮＭＴ）の発現を阻害するｓｉＲＮＡが挙げられる。ＤＮＡメチル化酵素とは、細胞を複製する際に親細胞から娘細胞にＤＮＡのメチル化情報を正確に伝える酵素（ＤＮＭＴ１）、娘細胞のＤＮＡをメチル化する酵素（ＤＮＭＴ３）などである。ｓｉＲＮＡとは、特定の遺伝子のｍＲＮＡと相補する短いＲＮＡであり、該特定の遺伝子の発現を阻害することができる。ヒトやマウスでは、ＤＮＭＴ１コード遺伝子またはＤＮＭＴ３コード遺伝子が、ＤＮＭＴをコードする遺伝子に該当し、例えば、それらの発現を阻害するｓｉＲＮＡとして、キアゲン社のｓｉＲＮＡをマニュアルに従って添加することにより、ＤＮＡのメチル化を効率よく阻害することができる。 One of the DNA methylation inhibitors includes siRNA that inhibits the expression of the gene encoding the DNA methyltransferase (DNMT). The DNA methylase is an enzyme (DNMT1) that accurately conveys DNA methylation information from a parent cell to a daughter cell when replicating a cell, an enzyme that methylates the DNA of a daughter cell (DNMT3), and the like. The siRNA is a short RNA that complements the mRNA of a specific gene and can inhibit the expression of the specific gene. In humans and mice, the DNMT1 coding gene or the DNMT3 coding gene corresponds to the gene encoding DNMT, for example, DNA methylation by adding Chiagen's siRNA as a siRNA that inhibits their expression according to the manual. Can be efficiently inhibited.
ＤＮＡメチル化阻害物質の他の１種としては、メチオニン類似物質が挙げられる。メチオニン類似物質は、メチル化反応の反応基質であるアデノシルメチオニンの合成を阻害することにより、メチル化反応を阻害する。 Another type of DNA methylation inhibitor includes methionine-like substances. Methionine-like substances inhibit the methylation reaction by inhibiting the synthesis of adenosylmethionine, which is a reaction substrate for the methylation reaction.
メチオニン類似物質としては、例えば、５－アザ－２’－デオキシシチジン（5-Aza-dc，CAS登録番号2353-33-5）、シネフンギン（CAS登録番号58944-73-3）、ゼブラリン（CAS登録番号360-10-6）、エチオニン（CAS登録番号13073-35-3）、アセチルメチオニン（CAS登録番号567-82-7）、セレノメチオニン（CAS登録番号3211-76-5）が挙げられる。これらのメチオニン類似物質は、水溶性であるため、１０－１００ｍＭ程度の濃度の水溶液を調製し、該水溶液を０．２μｍの濾過フィルターにより滅菌処理した後、適切な濃度になるように培地に添加して使用することができる。 Examples of methionine-like substances include 5-Aza-2'-deoxycitidine (5-Aza-dc, CAS Registry Number 2353-33-5), Cinefungin (CAS Registry Number 58944-73-3), and Zebularine (CAS Registry Number). Numbers 360-10-6), ethionine (CAS Registry Number 13073-35-3), acetylmethionine (CAS Registry Number 567-82-7), zebularine (CAS Registry Number 3211-76-5). Since these methionine-like substances are water-soluble, an aqueous solution having a concentration of about 10-100 mM is prepared, the aqueous solution is sterilized with a 0.2 μm filtration filter, and then added to the medium to an appropriate concentration. Can be used.
ポリアミンによる多能性幹細胞（ＰＩＳ細胞）、または、ＤＮＡメチル化阻害物質による多能性幹細胞（ＤＩＳ細胞）への誘導は、動物細胞をポリアミンまたはＤＮＡメチル化阻害物質の在下で培養することにより実施できる。細胞培養用のフラスコ、デッィシュあるはプレートに、細胞の増殖で使用される通常の培地を添加し、細胞を播種し、通常の培養温度（３７℃程度）のＣＯ２インキュベーターで培養することにより、細胞をフラスコ底面で増殖している状態にする。ヒト細胞の増殖で使用される通常の培地として、例えば、最少培地（Minimum Essential medium：ＭＥＭ）やｉＰＳ細胞用の培地などを利用することができ、２５ｃｍ２の培養フラスコの場合、１０万～１００万個程度の細胞を播種することが好ましい。Induction of pluripotent stem cells (PIS cells) by polyamines or pluripotent stem cells (DIS cells) by DNA methylation inhibitors is carried out by culturing animal cells in the presence of polyamines or DNA methylation inhibitors. can. By adding a normal medium used for cell growth to a flask, dish or plate for cell culture, seeding the cells, and culturing in a CO 2 incubator at a normal culture temperature (about 37 ° C.), Brings the cells to the bottom of the flask in a proliferating state. As a normal medium used for the growth of human cells, for example, a minimum medium (MEM) or a medium for iPS cells can be used, and in the case of a 25 cm 2 culture flask, 100,000 to 100 It is preferable to seed about 10,000 cells.
フラスコ底面に細胞が付着して細胞の増殖が開始した後、適切な濃度のポリアミン水溶液、または、ＤＮＡメチル化阻害物質の水溶液を添加し、さらに通常の培養温度（３７℃程度）のＣＯ２インキュベーターで１～４日間程度培養する。当該操作により、細胞内には無数の小さな球体が生じ、フラスコ底面に付着していた細胞が浮遊し、小さな球体の入った球状の細胞となって、ＥＳ細胞またはｉＰＳ細胞と類似の形態に変化する。この細胞が、多能性幹細胞のＰＩＳ細胞またはＤＩＳ細胞である。After the cells adhere to the bottom of the flask and the cells start to grow, an aqueous solution of polyamine or an aqueous solution of DNA methylation inhibitor at an appropriate concentration is added, and a CO 2 incubator at a normal culture temperature (about 37 ° C) is added. Incubate for 1 to 4 days. By this operation, innumerable small spheres are generated in the cells, the cells attached to the bottom of the flask float, become spherical cells containing small spheres, and change to a morphology similar to ES cells or iPS cells. do. These cells are pluripotent stem cell PIS cells or DIS cells.
ポリアミンまたはＤＮＡメチル化阻害物質の添加量が少ない場合、ＰＩＳ細胞またはＤＩＳ細胞への変化が起こらず、添加量が多い場合、細胞のアポトーシス（細胞死）が誘導される。そのため、ポリアミンまたはＤＮＡメチル化阻害物質は、ＰＩＳ細胞またはＤＩＳ細胞への変化が起こる最低濃度で添加し、可能な限りアポトーシスを起こさないようにする必要がある。ポリアミンまたはＤＮＡメチル化阻害物質の添加濃度と培養時間は、細胞種と細胞数に依存する。そのため、ポリアミンまたはＤＮＡメチル化阻害物質の添加濃度は、光学顕微鏡観察により、ＰＩＳ細胞またはＤＩＳ細胞に変化することを確認できる最少濃度を、予備実験により確認することが望ましい。 When the amount of polyamine or DNA methylation inhibitor added is small, no change to PIS cells or DIS cells occurs, and when the amount added is large, cell apoptosis (cell death) is induced. Therefore, polyamines or DNA methylation inhibitors should be added at the lowest concentrations that cause changes to PIS cells or DIS cells to prevent apoptosis as much as possible. The concentration of polyamine or DNA methylation inhibitor added and the culture time depend on the cell type and cell number. Therefore, it is desirable to confirm by preliminary experiments that the concentration of the polyamine or DNA methylation inhibitor added can be confirmed to change to PIS cells or DIS cells by observation with an optical microscope.
例えば、１０～１００万個の正常な線維芽細胞、脂肪細胞の場合、スペルミンを１０μＭ～１００μＭ程度添加した培地で１～２日間培養すれば、それらの細胞をＰＩＳ細胞に変化させることができる。さらに、上記の繊維芽細胞等に対しては、ＤＮＭＴをコードする遺伝子の発現を阻害するｓｉＲＮＡを５～１０ｎＭ程度添加した培地、５－Ａｚａ－ｄｃを２０～８０μＭ程度添加した培地、または、エチオニンを１～１０ｍＭ程度添加した培地で、１～３日間培養すれば、それらの細胞をＤＩＳ細胞に変化させることができる。 For example, in the case of 100 to 1 million normal fibroblasts and adipocytes, those cells can be transformed into PIS cells by culturing in a medium containing about 10 μM to 100 μM of spermine for 1 to 2 days. Further, for the above-mentioned fibroblasts and the like, a medium containing about 5 to 10 nM of siRNA that inhibits the expression of the gene encoding DNMT, a medium containing about 20 to 80 μM of 5-Aza-dc, or ethionine. The cells can be transformed into DIS cells by culturing the cells in a medium supplemented with about 1 to 10 mM for 1 to 3 days.
上記ポリアミンまたは上記ｓｉＲＮＡを含有する培地で培養することにより得られたＰＩＳ細胞またはＤＩＳ細胞は、上記ポリアミンまたは上記ｓｉＲＮＡを含有する培地で１～４日間程度培養できる。しかし、アポトーシスにより、次第に生存率が低下するため、光学顕微鏡によりＰＩＳ細胞への変化を確認すると、０～１日以内に、浮遊したＰＩＳ細胞またはＤＩＳ細胞を含む培養液を回収し、通常の速度（８００～２０００ｒｐｍで３分間程度）で遠心分離を行うことにより、ＰＩＳ細胞を集めることが好ましい。 The PIS cells or DIS cells obtained by culturing in the medium containing the polyamine or the siRNA can be cultured in the medium containing the polyamine or the siRNA for about 1 to 4 days. However, since the survival rate gradually decreases due to apoptosis, when the change to PIS cells is confirmed by an optical microscope, the culture medium containing the suspended PIS cells or DIS cells is collected within 0 to 1 day, and the normal rate is obtained. It is preferable to collect PIS cells by centrifuging at (800 to 2000 rpm for about 3 minutes).
また、培地には、培養の際に、ＰＩＳ細胞の生存率の低下を防ぐための阻害剤を添加してもよい。生存率の低下を防ぐための阻害剤としては、例えば、Ｒｏｃｋ阻害剤およびアポトーシス阻害剤が使用できる。Ｒｏｃｋ阻害剤としては、例えば、Ｙ－２７６３２、チアゾビビン、ＳＢ４３１５４２、ＰＤ０３２５９０１、アポトーシス阻害剤としては、例えば、ｐ５３阻害剤、Ｂａｘ阻害剤、カスパーゼ阻害剤が挙げられる。 In addition, an inhibitor for preventing a decrease in the viability of PIS cells may be added to the medium during culturing. As the inhibitor for preventing the decrease in survival rate, for example, a Rock inhibitor and an apoptosis inhibitor can be used. Examples of the Rock inhibitor include Y-27632, thiazobibin, SB431542, PD0325901, and examples of the apoptosis inhibitor include p53 inhibitor, Bax inhibitor, and caspase inhibitor.
一方、ＤＮＡメチル化阻害物質として、５－アザ－２’－デオキシシチジン、シネフンギン、ゼブラリン、エチオニン、アセチルメチオニン、セレノメチオニンを用いた場合、ＤＮＡのメチル化だけでなく様々なメチル化を阻害し、強い細胞毒性を有する。したがって、細胞の浮遊が起こるとすぐに、浮遊したＤＩＳ細胞を含む培養液を回収し、通常の速度（８００～２０００ｒｐｍで３分間程度）で遠心分離を行うことによりＤＩＳ細胞を集めることが好ましい。 On the other hand, when 5-aza-2'-deoxycytidine, cinefungin, zebularine, ethionine, acetylmethionine, and selenomethionine were used as DNA methylation inhibitors, they inhibited not only DNA methylation but also various methylation. Has strong cytotoxicity. Therefore, it is preferable to collect the DIS cells by collecting the culture medium containing the suspended DIS cells and centrifuging at a normal speed (about 3 minutes at 800 to 2000 rpm) as soon as the cells float.
遠心分離により回収したＰＩＳ細胞またはＤＩＳ細胞は、細胞からの分化が非常に高い割合で起こるため、胚様体（ＥＢ）を経由した内胚葉、外胚葉、中胚葉への再分化を直接行うことができる。しかしながら、再生医療に使う場合、「ＥＳ細胞またはｉＰＳ細胞と同等の形質を安定して発現させ、且つＰＩＳ細胞またはＤＩＳ細胞のみを選別する」必要がある。この場合は、ＥＳ細胞またはｉＰＳ細胞様の多能性の形質を獲得し且つ保持するための培地に添加することが好ましい。ＰＩＳ細胞またはＤＩＳ細胞の多能性の形質を獲得し且つ保持するための培地として、例えば、ヒトの細胞の場合、Ｃｅｌｌａｒｔｉｓ（登録商標） ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ Ｓｙｓｔｅｍ（タカラバイオ株式会社）、ｍＴｅＳＲ１（ベリタス社）など多くの市販の培地を用いることができる。また、当該培地での培養において、ＰＩＳ細胞の生存率の低下を防ぐためには、Ｒｏｃｋ阻害剤Ｙ－２７６３２、チアゾビビン、ＳＢ４３１５４２、ＰＤ０３２５９０１を添加することも可能である。 Since PIS cells or DIS cells recovered by centrifugation occur at a very high rate of cell differentiation, direct redifferentiation into endoderm, ectoderm, and mesoderm via the embryoid body (EB) should be performed. Can be done. However, when used in regenerative medicine, it is necessary to "stablely express traits equivalent to ES cells or iPS cells and select only PIS cells or DIS cells". In this case, it is preferable to add it to the medium for acquiring and retaining the pluripotent traits such as ES cells or iPS cells. As a medium for acquiring and retaining pluripotent traits of PIS cells or DIS cells, for example, in the case of human cells, Cellartis® DEF-CS 500 Culture System (Takara Bio Inc.), mTeSR1 ( Many commercially available media such as Veritas) can be used. In addition, in order to prevent a decrease in the viability of PIS cells in culturing in the medium, it is also possible to add a Rock inhibitor Y-27632, thiazobibin, SB431542, PD0325901.
これらの培地でＰＩＳ細胞またはＤＩＳ細胞の集合体を培養すると、弱く付着（または浮遊）する。ピペッティング（ピペットにより培養液を吸引、排出を繰りかえして懸濁する操作）により当該細胞を浮遊させ、培地と細胞を回収し、遠心分離により細胞を集めた後、新しい培地に移す操作を行う。ＰＩＳ細胞またはＤＩＳ細胞に分化していないもとの体細胞は、強固に付着するため、ピペッティングでは剥がれず、ＰＩＳ細胞の集合体またはＤＩＳ細胞の集合体のみを回収できる。 Culturing PIS cells or aggregates of DIS cells in these media causes weak attachment (or suspension). The cells are suspended by pipetting (the operation of sucking the culture medium with a pipette and suspending the cells repeatedly), the medium and the cells are collected, the cells are collected by centrifugation, and then transferred to a new medium. Since the original somatic cells that have not differentiated into PIS cells or DIS cells adhere firmly, they cannot be peeled off by pipetting, and only the aggregates of PIS cells or DIS cells can be recovered.
さらに、ＰＩＳ細胞またはＤＩＳ細胞の集合体を、Ｔｒｙｐ ＬＥ Ｓｅｌｅｃｔ（Ｇｉｂｃｏ）、０．２５％トリプシン溶液（Ｇｉｂｃｏ）、Ａｃｃｕｔａｓｅ－Ｓｏｌｕｔｉｏｎ（フナコシ社）等によって、細胞が分散した状態にし、Ｃｅｌｌａｒｔｉｓ（登録商標） ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ Ｓｙｓｔｅｍ（タカラバイオ株式会社）で培養した場合、個々のＰＩＳ細胞を培養器の底面に弱く付着した状態で増殖させることができる。 Further, the cells are dispersed by using Trip LE Select (Gibco), 0.25% trypsin solution (Gibco), Incubate-Solution (Funakoshi), or the like to disperse the cells of PIS cells or aggregates of DIS cells, and Cellartis (registered trademark). ) When cultured in DEF-CS 500 Culture System (Takara Bio Inc.), individual PIS cells can be grown in a state of being weakly attached to the bottom surface of the incubator.
Ｒｏｃｋ阻害剤およびアポトーシス阻害剤を添加して培養した場合、細胞にわずかな収縮が見られるが、細胞の浮遊は起こらない。この場合、Ｔｒｙｐ ＬＥ Ｓｅｌｅｃｔ、０．２５％トリプシン、Ａｃｃｕｔａｓｅ－Ｓｏｌｕｔｉｏｎ等により細胞を剥離させ、細胞をＰＢＳ等で洗浄後、ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ Ｓｙｔｅｍ等のｉＰＳ細胞用の培地で１－３日間培養すればよい。こうして得られたＰＩＳ細胞は、ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ ＳｙｔｅｍあるいはＳｔｅｍＳｕｒｅ（登録商標） ゼラチン溶液（和光純薬（株））等のゼラチン溶液でプレートをコートすることで、付着培養することができる。これにより、細胞の障害が修復され、アポトーシスを起こさず、且つ、高効率でＰＩＳ細胞を得ることができる。 When cultured with the addition of Rock inhibitor and apoptosis inhibitor, cells show slight contraction, but cell suspension does not occur. In this case, the cells are detached with Tryp LE Select, 0.25% trypsin, Accutase-Solution, etc., the cells are washed with PBS, etc., and then cultured in a medium for iPS cells such as DEF-CS 500 Culture System for 1-3 days. do it. The PIS cells thus obtained can be adherently cultured by coating the plate with a gelatin solution such as DEF-CS 500 Culture System or StemSure (registered trademark) gelatin solution (Wako Pure Chemical Industries, Ltd.). As a result, cell damage can be repaired, apoptosis does not occur, and PIS cells can be obtained with high efficiency.
ＰＩＳ細胞またはＤＩＳ細胞がｉＰＳ細胞と同等の形質を有することは、ｉＰＳ細胞に特異的なＳＯＸ２、Ｏｃｔ３／４、ｃ－Ｍｙｃなどの遺伝子マーカーの発現、アルカリホスファターゼ活性の発現、または、ＳＳＥＡ－４、ＴＲＡ１－６０、ＴＲＡ１－８１などの表面抗原マーカーの発現等を調べることにより確認でき、遺伝子の発現上昇の確認にはＲＴ－ＰＣＲまたはリアルタイムＰＣＲを、ｉＰＳ細胞に特異的な酵素または表面抗原の確認には、抗体を用いた免疫染色、ウエスタンブロッティングまたはＦＡＣＳを利用すればよい。 The fact that PIS cells or DIS cells have the same traits as iPS cells means that iPS cell-specific gene markers such as SOX2, Oct3 / 4, c-Myc, etc. are expressed, alkaline phosphatase activity is expressed, or SSEA-4. , TRA1-60, TRA1-81, etc. can be confirmed by examining the expression, etc., and RT-PCR or real-time PCR is used to confirm the increase in gene expression, and iPS cell-specific enzymes or surface antigens are used. For confirmation, immunostaining with an antibody, Western blotting or FACS may be used.
ＰＩＳ細胞またはＤＩＳ細胞から様々な細胞への分化誘導は、ｉＰＳ細胞で使用される誘導方法と同様の方法により行うことができる。 Induction of differentiation from PIS cells or DIS cells into various cells can be performed by the same method as the induction method used for iPS cells.
ＰＩＳ細胞またはＤＩＳ細胞は、ｉＰＳ細胞と同様に、Ｅｍｂｒｙｏｉｄ ｂｏｄｙ（ＥＢ）を経て、内胚葉、中胚葉、外胚葉などに分化し、その後、心筋細胞、脂肪細胞、神経細胞など様々な体細胞に分化できる。細胞の分化において、ｉＰＳ細胞と同様に、均一なＥＢを形成することで、ＰＩＳ細胞またはＤＩＳ細胞の分化効率が高まる。 Similar to iPS cells, PIS cells or DIS cells differentiate into endoderm, mesoderm, ectoderm, etc. via Embryoid body (EB), and then into various somatic cells such as myocardial cells, fat cells, and nerve cells. Can be differentiated. In cell differentiation, similar to iPS cells, the formation of uniform EB enhances the differentiation efficiency of PIS cells or DIS cells.
均一なＥＢの形成には、数多くの市販のプレートが存在し、それら利用することも有効な手段である。例えば、胚様体形成プレートＡｇｒｏｗｅｌｌ（ベリタス社）、リピジュア（登録商標）－コートプレート（日油株式会社）、ＥＺＳＰＨＥＲＥ（ＡＧＣテクノグラス）などを用いることで均一なＥＢを得ることができる。 There are many commercially available plates for the formation of uniform EB, and it is also an effective means to utilize them. For example, a uniform EB can be obtained by using an embryoid body forming plate Agrowell (Veritas), Lipidure (registered trademark) -coat plate (NOF Corporation), EZSPHERE (AGC technoglass) and the like.
また、様々な細胞への分化もｉＰＳ細胞の場合と全く同じ培地で行うことができる。様々な細胞への分化誘導を行える市販の試薬が数多く存在し、それら利用することも有効な手段である。例えば、胚体内胚葉（ＥＤ）への分化はＣｅｌｌａｒｔｉｓ（登録商標） Definitive Endoderm ChiPSC18（タカラバイオ株式会社）、心筋細胞や肝細胞への分化はPSdif-Cardio Cardiomyocyte Differentiation Kit（フナコシ株式会社）、Ｃｅｌｌａｒｔｉｓ（登録商標） iPS Cell to Hepatocyte Differentiation System（タカラバイオ株式会社）、神経細胞への分化には、SETMdiff Neural Induced Medium（ベリタス社）を用いることにより、ＰＩＳ細胞またはＤＩＳ細胞から効率よく細胞を分化することができる。 In addition, differentiation into various cells can be performed in exactly the same medium as in the case of iPS cells. There are many commercially available reagents that can induce differentiation into various cells, and it is also an effective means to utilize them. For example, differentiation into intraembryonic embryo (ED) is Cellartis (registered trademark) Definitive Endoderm ChiPSC18 (Takara Bio Co., Ltd.), differentiation into myocardial cells and hepatocytes is PSdif-Cardio Cardiomyocyte Differentiation Kit (Funakoshi Co., Ltd.), Cellartis ( Registered trademark) iPS Cell to Hepatocyte Differentiation System (Takara Bio Co., Ltd.), Efficiently differentiate cells from PIS cells or DIS cells by using SETMdiff Neural Induced Medium (Veritas) for differentiation into nerve cells. Can be done.
本発明を以下の実施例によりさらに詳細に説明する。しかしながら、以下の実施例は本発明の例示であって、本発明を限定することを意図するものではない。 The present invention will be described in more detail with reference to the following examples. However, the following examples are examples of the present invention and are not intended to limit the present invention.
実施例１：ポリアミンに細胞を初期化する能力があることの証明 Example 1: Proof that polyamines have the ability to reprogram cells
この実験は、ポリアミンが、初期化する能力があることを証明するために実施した。 This experiment was performed to prove that polyamines have the ability to reprogram.
ヒト肺由来の線維芽細胞ＴＩＧ－１－２０をＪＣＲＢ細胞バンク（ＪＣＲＢ０５０１株）より入手した。ヒト線維芽細胞ＴＩＧ－１－２０（分裂回数３３回、１０万個）を、６ｍＬの最少培地（ＭＥＭ）を含む２５ｃｍ２の培養フラスコで、１日間培養し、細胞を付着させた。１０ｍＭのプトレシン、２００μＭのスペルミジン、または４０μＭのスペルミンを添加したものと、それらを添加していないもの（コントロール）とを、３７℃で１日間ＣＯ２インキュベーター中で培養した。Human lung-derived fibroblasts TIG-1-20 were obtained from the JCRB cell bank (JCRB0501 strain). Human fibroblasts TIG-1-20 (33 divisions, 100,000) were cultured for 1 day in a 25 cm 2 culture flask containing 6 mL of minimal medium (MEM) and adhered to the cells. Those supplemented with 10 mM putrescine, 200 μM spermidine, or 40 μM spermine and those without them (control) were cultured at 37 ° C. for 1 day in a CO 2 incubator.
スペルミンを添加して培養したときの結果を、図１に示した。繊維芽細胞ＴＩＧ－１－２０（図１Ａ）の細胞内に６時間後には細かな粒子上のものが出現し（図１Ｂ）、その後培養を続けると、細胞がフラスコ底部からはがれてＥＳ細胞に近い細胞（図１Ｃ）に変化した。 The results of culturing with spermine added are shown in FIG. After 6 hours, fine particles appeared in the cells of fibroblast TIG-1-20 (Fig. 1A) (Fig. 1B), and when the culture was continued thereafter, the cells peeled off from the bottom of the flask and became ES cells. It changed to a close cell (Fig. 1C).
細胞が剥がれてから１２時間後、培地中に懸濁した当該細胞（ＰＩＳ細胞）を遠心分離（８００ｒｐｍ、３分間）により回収し、Ｃｅｌｌａｒｔｉｓ（登録商標） ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて培養した。培養は、２４穴のプレートにＤＥＦ－ＣＳ ＣＯＡＴ－１のＰＢＳ（＋）希釈液を１時間処理した後、ＰＩＳ細胞を６ｍＬの培地添加剤を添加したＤＥＦ－ＣＳ Ｂａｓａｌ Ｍｅｄｉｕｍに懸濁し、２ｍＬずつ３ウェルに分注して、３７℃で２日間培養した。細胞をピペッティングにより剥がし、遠心分離（８００ｒｐｍ、３分間）によりＰＩＳ細胞を回収した。得られた細胞は、RNeasy Lipid Tissue Mini KitとQuantiTech Reverse Transcription Kit （キアゲン社）を用いて、ｍＲＮＡの抽出とｃＤＮＡの合成を行い、リアルタイムＰＣＲ用のプライマーとリアルタイムＰＣＲ（キアゲン社）を用いて、ＴＥＲＴ、ＤＭＮＴ１、ＳＯＸ２、ＯＣＴ３／４のｍＲＮＡの発現量を調べた。 Twelve hours after the cells were detached, the cells (PIS cells) suspended in the medium were collected by centrifugation (800 rpm, 3 minutes), and Cellartis® DEF-CS 500 Culture System (Takara Bio Inc.) ) Was used for culturing. For culture, a 24-well plate was treated with a PBS (+) diluted solution of DEF-CS COAT-1 for 1 hour, and then PIS cells were suspended in DEF-CS Basic Medium supplemented with 6 mL of medium additive, 2 mL each. The cells were dispensed into 3 wells and cultured at 37 ° C. for 2 days. The cells were peeled off by pipetting and the PIS cells were collected by centrifugation (800 rpm, 3 minutes). The obtained cells were subjected to mRNA extraction and cDNA synthesis using the RNeasy Lipid Tissue Mini Kit and QuantiTech Reverse Transcription Kit (Qiagen), and were used with primers for real-time PCR and real-time PCR (Qiagen). The expression levels of TRT, DMNT1, SOX2, and OCT3 / 4 mRNAs were examined.
リアルタイムＰＣＲの結果を図２に示した。ＤＮＭＴ１遺伝子は、ＤＮＡ複製の際に、親細胞ＤＮＡのメチル化情報を娘細胞に伝える役割のメチル化転移酵素をコードする遺伝子である。図２に示すように、ＰＩＳ細胞では、ＤＮＭＴ１の発現が完全に抑制されており、ＤＮＡのメチル化の初期化が起こることがわかった。また、テロメアを伸長するテロメラーゼをコードする遺伝子（ＴＥＲＴ遺伝子）の発現が著しく増加した。つまり、老化により短くなったテロメア伸長（初期化）が起こっていた。さらに、ＥＳ細胞のマーカー遺伝子であり細胞の初期化を誘導するＳＯＸ２とＯＣＴ３／４遺伝子の発現量が増加していた。以上のように、ポリアミンは、細胞の初期化を誘導することが立証された。 The results of real-time PCR are shown in FIG. The DNMT1 gene is a gene encoding a methyltransferase that plays a role in transmitting methylation information of parent cell DNA to daughter cells during DNA replication. As shown in FIG. 2, it was found that the expression of DNMT1 was completely suppressed in PIS cells, and the reprogramming of DNA methylation occurred. In addition, the expression of the gene encoding telomerase that extends telomeres (TERT gene) was significantly increased. In other words, telomere elongation (initialization) shortened due to aging occurred. Furthermore, the expression levels of SOX2 and OCT3 / 4 genes, which are marker genes for ES cells and induce cell reprogramming, were increased. As described above, it was proved that polyamines induce cell reprogramming.
実施例２：ＰＩＳ細胞がｉＰＳ細胞と同等の形質を有していることの証明 Example 2: Proof that PIS cells have the same traits as iPS cells
この実験は、ＰＩＳ細胞が、ｉＰＳ細胞に特異的なタンパク質を発現していることを証明するために行った。 This experiment was performed to prove that PIS cells express proteins specific to iPS cells.
実施例１で用いた方法と全く同じ方法によりＰＩＳ細胞を、Ｃｅｌｌａｒｔｉｓ（登録商標） ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて培養し、形質の安定したＰＩＳ細胞を得た。当該培地中で、弱く付着したＰＩＳ細胞をピペティングにより懸濁した後、１，５００ｒｐｍで４分間遠心分離して細胞を集めた。 PIS cells were cultured using Cellartis (registered trademark) DEF-CS 500 Culture System (Takara Bio Inc.) by the same method as that used in Example 1 to obtain PIS cells having stable traits. Weakly attached PIS cells were suspended in the medium by pipetting and then centrifuged at 1,500 rpm for 4 minutes to collect the cells.
得られたＰＩＳ細胞は、Human ES/iPS Cell Characterization Kit （Applied Stem Cell社）により、アルカリホスファターゼ活性と３つの表面抗原（ＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１）の確認を行った。また、ｉＰＳ細胞は、アルカリホスファターゼを発現する特徴がある。さらに、ＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１は、ｉＰＳ細胞のマーカー遺伝子であり、ｉＰＳ細胞に特異的に発現するタンパク質である。 The obtained PIS cells were confirmed by the Human ES / iPS Cell characterization Kit (Applied Stem Cell) for alkaline phosphatase activity and three surface antigens (SSEA-4, TRA-1-60, TRA-1-81). went. In addition, iPS cells are characterized by expressing alkaline phosphatase. Furthermore, SSEA-4, TRA-1-60, and TRA-1-81 are marker genes for iPS cells and are proteins specifically expressed in iPS cells.
ＰＩＳ細胞を０．２ｍＬ用のサンプルチューブに入れ、懸濁細胞の状態で固定化処理、透過処理、ブロッキングをした後、１次抗体および２次抗体を結合させた。２次抗体で標識した細胞は、マウント液と１：１で混合してスライドグラスを作製し、蛍光顕微鏡（ＥＶＯＳ社、ＥＶＯＳ ＦＬ Ａｕｔｏ）により蛍光を確認した。結果を図３に示した。 PIS cells were placed in a 0.2 mL sample tube, immobilized, permeabilized, and blocked in the state of suspended cells, and then the primary antibody and the secondary antibody were bound. The cells labeled with the secondary antibody were mixed 1: 1 with the mount solution to prepare a slide glass, and fluorescence was confirmed by a fluorescence microscope (EVOS, EVOS FL Auto). The results are shown in FIG.
図３は、染色体を蛍光染色した図であり、蛍光物質により、活性染色または免疫染色の図である。蛍光がある場合、図３の写真では灰色に示される。図３から明らかなように、アルカリホスファターゼ活性の強い活性およびＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１抗原の強い発現を確認した。以上のように、ＰＩＳ細胞はｉＰＳ細胞に特異的なタンパク質を発現していることを証明できた。 FIG. 3 is a diagram of fluorescent staining of chromosomes, and is a diagram of active staining or immunostaining with a fluorescent substance. If there is fluorescence, it is shown in gray in the photo of FIG. As is clear from FIG. 3, strong activity of alkaline phosphatase activity and strong expression of SSEA-4, TRA-1-60, and TRA-1-81 antigens were confirmed. As described above, it was possible to prove that PIS cells express proteins specific to iPS cells.
実施例３：ＰＩＳ細胞が様々な細胞に分化する能力を保持していることの確認 Example 3: Confirmation that PIS cells retain the ability to differentiate into various cells
この実験は、ＰＩＳ細胞が、胚様体を形成し、様々な細胞に再分化できる能力があることを証明するために行った。 This experiment was performed to prove that PIS cells have the ability to form embryoid bodies and redifferentiate into various cells.
実施例２において得られたＰＩＳ細胞を用いて、心筋細胞に分化させる実験を行った。PSdif-Cardio Cardiomyocyte Differentiation Kit（フナコシ株式会社）を用い、当該キットが提供するマニュアルに従って、培養を行った。心筋細胞に分化誘導した結果を図４に示した。ＰＩＳ細胞は、ＥＢを形成し（図４Ａ）、前駆心筋細胞（図４Ｂ）に変化し、最終的には微細な振動をする心筋細胞（図４Ｃ）に変化した。以上のように、ＰＩＳ細胞は、胚葉体および体細胞に分化する能力を有する多能性幹細胞であることを証明できた。 Using the PIS cells obtained in Example 2, an experiment for differentiating into cardiomyocytes was performed. Culturing was performed using the PSdif-Cardio Cardiomyocyte Differentiation Kit (Funakoshi Co., Ltd.) according to the manual provided by the kit. The results of inducing differentiation into cardiomyocytes are shown in FIG. PIS cells formed EBs (FIG. 4A), transformed into prodromal cardiomyocytes (FIG. 4B), and finally transformed into microvibrating cardiomyocytes (FIG. 4C). As described above, it was proved that PIS cells are pluripotent stem cells having the ability to differentiate into germ layer and somatic cells.
実施例４：ヒト線維芽細胞以外の細胞でもＰＩＳ細胞を作製できることの確認 Example 4: Confirmation that PIS cells can be produced using cells other than human fibroblasts
この実験は、ヒト線維芽細胞以外の細胞から、ＰＩＳ細胞を作製できることを確認するために行った。 This experiment was performed to confirm that PIS cells can be produced from cells other than human fibroblasts.
マウス前駆脂肪細胞株３Ｔ３－Ｌ１（図５Ａ）をＥＤＣＣより入手した。３Ｔ３－Ｌ１株を６ｍＬのＤＭＥＭ（Dulbecco's modified Eagle Medium）を含む２５ｃｍ２の培養フラスコで１日間培養し、細胞を付着させた。１ｍＭのスペルミンを添加したものを、３７℃で３日間ＣＯ２インキュベーター中で培養した。その結果、実施例１と同様に、ＥＳ細胞様の細胞（ＰＩＳ－Ｌ１細胞）を得ることができ、図５のＢに示すようにＰＩＳ－Ｌ１細胞は、ＥＢを形成した。以上のように、ＰＩＳ細胞の作製方法は、ヒト線維芽細胞だけでなく、マウス脂肪細胞など様々な種の様々な細胞に適用できる方法であることを証明できた。Mouse precursor adipocyte line 3T3-L1 (FIG. 5A) was obtained from EDCC. The 3T3-L1 strain was cultured in a 25 cm 2 culture flask containing 6 mL DMEM (Dulbecco's modified Eagle Medium) for 1 day, and cells were attached. Those supplemented with 1 mM spermine were cultured at 37 ° C. for 3 days in a CO 2 incubator. As a result, ES cell-like cells (PIS-L1 cells) could be obtained as in Example 1, and as shown in FIG. 5B, the PIS-L1 cells formed EB. As described above, it was proved that the method for producing PIS cells can be applied not only to human fibroblasts but also to various cells of various species such as mouse adipocytes.
実施例５：ＤＮＡメチル化阻害物質に細胞を初期化する能力があり、ＤＩＳ細胞に変化することの証明 Example 5: Proof that DNA methylation inhibitors have the ability to reprogram cells and transform into DIS cells
この実験は、ＤＮＡメチル化阻害剤に、細胞をＥＳ細胞の状態まで初期化する能力があり、ＤＩＳ細胞を作製できることを証明するために実施した。 This experiment was performed to prove that DNA methylation inhibitors have the ability to reprogram cells to the state of ES cells and can generate DIS cells.
キアゲン社のＤＮＡメチル化酵素（ＤＮＭＴ１、ＤＮＭＴ３ＡおよびＤＮＭＴ３Ｂ）をコードする遺伝子の発現を阻害するｓｉＲＮＡ（カタログ番号SI000189910、SI00982338およびSI00165382）を入手した。また、マウス脂肪細胞由来株Ｃ３Ｈ１０Ｔ１／２（EC90110523-F0）をＥＣＡＣＣより入手した。２４穴のプレートを用いて、ＤＭＥＭ中で該細胞を１日間培養し、細胞をプレートの各ウェルの底面に付着させた。ｓｉＲＮＡによる遺伝子のサイレンシングは、RNAi Human/Mouse Starter Kit（キアゲン社）を用いて行った。これら３つのｓｉＲＮＡ（５ｎＭ）を含む２００μＬのＤＭＥＭ（血清を含まない）に、６μＬのHyperfect Transfer Reagentを加えてｓｉＲＮＡの沈澱物を形成した後、１日間培養した。その後、２００μＬのＬＩＦ（Ｗａｋｏ（株） カタログ番号129-05601）を添加したＥＳ細胞用無血清培地（ＤＳファーマバイオ社）と交換しさらに３日間培養した。その結果、図６のＡに示すように、マウス脂肪細胞由来株Ｃ３Ｈ１０Ｔ１／２は、ＤＩＳ細胞（ＤＩＳ－ＳＩ細胞）に変化したが、変化しないマウス脂肪細胞由来株Ｃ３Ｈ１０Ｔ１／２は細胞底面に付着したままであり、ＤＩＳ－ＳＩ細胞のみを培地から回収できた。 We obtained siRNAs (catalog numbers SI000189910, SI00982338 and SI00165382) that inhibit the expression of genes encoding DNA methylases (DNMT1, DNMT3A and DNMT3B) from Qiagen. In addition, a mouse adipocyte-derived strain C3H10T1 / 2 (EC90110523-F0) was obtained from ECACC. Using a 24-hole plate, the cells were cultured in DMEM for 1 day and the cells were attached to the bottom of each well of the plate. Gene silencing by siRNA was performed using the RNAi Human / Mouse Starter Kit (Qiagen). To 200 μL of DMEM (without serum) containing these three siRNAs (5 nM), 6 μL of Hyperfect Transfer Reagent was added to form a siRNA precipitate, which was then cultured for 1 day. Then, it was replaced with a serum-free medium for ES cells (DS Pharmabio) supplemented with 200 μL of LIF (Wako Co., Ltd. Catalog No. 129-05601) and cultured for another 3 days. As a result, as shown in A of FIG. 6, the mouse adipocyte-derived strain C3H10T1 / 2 changed to DIS cells (DIS-SI cells), but the unchanged mouse adipocyte-derived strain C3H10T1 / 2 adhered to the cell bottom. Only DIS-SI cells could be recovered from the medium.
５－Ａｚａ－ｄｃについても同様の実験を行った。Ｃ３Ｈ１０Ｔ１／２株を５００ｍＬのＤＭＥＭを含む２５ｃｍ２の培養フラスコで１日間培養し、細胞を付着させた４０μＭの５－Ａｚａ－ｄｃを添加したＤＭＥＭで培養した後、６ｍＬのＬＩＦ（Ｗａｋｏ（株） カタログ番号129-05601）と４０μＭの５－Ａｚａ－ｄｃを添加したＥＳ細胞用無血清培地（ＤＳファーマバイオ社）と交換しさらに３日間培養した。その結果、図６のＢに示すように、前駆脂肪細胞の３Ｔ３－Ｌ１は、ＤＩＳ細胞（ＤＩＳ－Ａｚａ細胞と呼ぶ）に変化したが３Ｔ３－Ｌ１細胞は細胞底面に付着したままであり、ＤＩＳ－Ａｚａ細胞のみを培地から回収できた。A similar experiment was performed for 5-Aza-dc. The C3H10T1 / 2 strain was cultured in a 25 cm 2 culture flask containing 500 mL of DMEM for 1 day, cultured in DMEM supplemented with 40 μM 5-Aza-dc to which cells were attached, and then 6 mL of LIF (Wako Co., Ltd.). It was replaced with a serum-free medium for ES cells (DS Pharmabio) supplemented with Catalog No. 129-05601) and 40 μM 5-Aza-dc, and cultured for another 3 days. As a result, as shown in B of FIG. 6, the precursor adipocyte 3T3-L1 was transformed into DIS cells (referred to as DIS-Aza cells), but the 3T3-L1 cells remained attached to the cell bottom and DIS. -Only Aza cells could be recovered from the medium.
さらに、得られたＤＩＳ－Ａｚａ細胞をマウスＥＳ細胞から造血系細胞への分化培地であるES-Cut Hematopoietic Differentiation Kit with Cytokine（ベリタス社、カタログ番号ST-03160）を用いて、初期分化を行った。その結果、図６のＣに示すように血球系細胞への初期分化を確認した。また、神経前駆細胞への分化培地（STEMdiff Neural Induction Medium、ベリタス社）を用いて、ＤＩＳ－Ａｚａ細胞を神経細胞に分化させた。図６のＤに示すように、ＤＩＳ－Ａｚａ細胞は、神経幹細胞および神経様細胞に分化した。以上のように、ＤＮＡメチル化阻害物質を用いてもポリアミンと同様に、細胞を多能性幹細胞に初期化する能力があり、様々な細胞に分化するＤＩＳ細胞を作製できることを証明できた。 Furthermore, the obtained DIS-Aza cells were initially differentiated using ES-Cut Hematopoietic Differentiation Kit with Cytokine (Veritas, Catalog No. ST-03160), which is a medium for differentiating mouse ES cells into hematopoietic cells. .. As a result, as shown in C of FIG. 6, early differentiation into blood cell lineage cells was confirmed. In addition, DIS-Aza cells were differentiated into nerve cells using a differentiation medium for neural progenitor cells (STEMdiff Neural Induction Medium, Veritas). As shown in D of FIG. 6, DIS-Aza cells differentiated into neural stem cells and neural-like cells. As described above, it was proved that even if a DNA methylation inhibitor is used, it has the ability to reprogram cells into pluripotent stem cells and can produce DIS cells that differentiate into various cells, similar to polyamines.
実施例６：高効率でＰＩＳ細胞を作製できることの確認 Example 6: Confirmation that PIS cells can be produced with high efficiency
この実験は、（１）体細胞を高効率でＰＩＳ細胞にでき、（２）得られたＰＩＳ細胞を高効率で外胚葉、中胚葉または内胚葉に分化することが可能であり、（３）高効率で様々な細胞に分化できることを証明するために実施した。 In this experiment, (1) somatic cells can be converted into PIS cells with high efficiency, and (2) the obtained PIS cells can be differentiated into ectodermal, mesodermal or endoderm with high efficiency, and (3). It was carried out to prove that it can differentiate into various cells with high efficiency.
ヒト肺由来の線維芽細胞ＴＩＧ－１－２０（分裂回数３０－３５回）を、６ｍLの最少培地（ＭＥＭ）を用いて、２５ｃｍ２の培養フラスコ中で1日間培養し、フラスコの底面に付着させた。培養フラスコに、Ｒｏｃｋ阻害剤である２０μＭのＹ－２７６３２および５μＭのチアゾビビンと、アポトーシス阻害剤である６０μＭのｐ５３阻害剤（Ｃｙｃｌｉｃ ｐｉｆｔｈｒｉｎ―α―ｈｙｄｒｏｂｔｏｍｉｄｅ）および３０μＭのＢａｘ阻害剤（Ｂａｘ Ｉｎｈｉｂｉｔｏｒ Ｐｅｐｔｉｄｅ Ｖ５）とを添加して８時間培養し、この４種類の阻害剤を十分に細胞内に取り込ませた。その後、１００ｍＭのスペルミン溶液を添加して、２４時間培養した。培養した細胞は、阻害剤の影響によりＲｏｃｋの活性化とアポトーシスとが抑制されるためにやや縮んだ状態になるが、それ以外には変化が見られなかった。また、Ｈｏｅｃｈｓｔ ３３３４２で核染色した場合も、アポトーシス時に起こる染色体の断片化は見られなかった。Human lung-derived fibroblasts TIG-1-20 (30-35 divisions) were cultured in a 25 cm 2 culture flask for 1 day using 6 mL of minimal medium (MEM) and attached to the bottom of the flask. I let you. In a culture flask, 20 μM Y-27632 and 5 μM thiazobibin, which are Rock inhibitors, 60 μM p53 inhibitor (Cyclic Pifthrin-α-hydroxytomide), which is an apoptosis inhibitor, and 30 μM Bax Inhibitor Pet. And was added and cultured for 8 hours, and these four kinds of inhibitors were sufficiently incorporated into the cells. Then, 100 mM spermine solution was added and the cells were cultured for 24 hours. The cultured cells became slightly shrunk due to the suppression of Rock activation and apoptosis due to the influence of the inhibitor, but no other changes were observed. In addition, nuclear staining with Hoechst 33342 did not show chromosomal fragmentation that occurs during apoptosis.
培養フラスコに、Ｔｒｙｐ－ＬＥ Ｓｅｌｅｃｔ溶液（Ｇｉｂｃｏ）０．８ｍＬを添加して細胞をはがし、ＰＢＳ溶液１０ｍＬに入れた後、遠心分離（８００ｒｐｍ、３分間）により細胞を回収した。上澄みを取り除いた後、ＰＢＳ溶液１０ｍＬを用いて同様の操作を行った。得られた細胞を、ＤＥＦ－ＣＳ ５００ Ｃｕｌｔｕｒｅ Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて２日間培養し、細胞の修復を行うと同時に、安定したＰＩＳ細胞を得た。その後、このＰＩＳ細胞を、Ｓｔｅｍ ＸＶｉｖｏ Ｅｃｔｏｄｅｒｍ Ｋｉｔ、Ｓｔｅｍ ＸＶｉｖｏ Ｍｅｓｏｄｅｒｍ Ｋｉｔ、Ｓｔｅｍ ＸＶｉｖｏ Ｅｎｄｏｄｅｒｍ Ｋｉｔ （Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社）を用いて、外胚葉、中胚葉または内胚葉に分化させた。分化した細胞は、これらのキットに含まれている抗ヒトＯｃｔ２ヤギ抗体、抗ヒトＢｒａｃｈｙｕｒｙヤギ抗体および抗ヒトＳＯＸ１７ヤギ抗体を一次抗体として用い、Ａｌｅｘａ Ｆｌｕｏｒ（登録商標） ５５５を結合した抗ヤギＩｇＧウサギ抗体を二次抗体として用いて、蛍光免疫染色を行った。その結果、図７のＡ～Ｃに示すように、ほとんどの細胞が効率良く（９０％以上）、外胚葉、中胚葉または内胚葉に分化していることを確認した。 To the culture flask, 0.8 mL of Trip-LE Select solution (Gibco) was added, the cells were peeled off, the cells were placed in 10 mL of PBS solution, and then the cells were collected by centrifugation (800 rpm, 3 minutes). After removing the supernatant, the same operation was performed using 10 mL of PBS solution. The obtained cells were cultured for 2 days using DEF-CS 500 Culture System (Takara Bio Inc.) to repair the cells and at the same time, stable PIS cells were obtained. Then, the PIS cells were differentiated into ectoderm, mesoderm or endoderm using Stem XVivo Ectoderm Kit, Stem XVivo Mesoderm Kit, and Stem XVivo Endoderm Kit (R & D Systems). Differentiated cells used the anti-human Oct2 goat antibody, anti-human Brachyury goat antibody and anti-human SOX17 goat antibody contained in these kits as primary antibodies, and used Alexa Fluor® 555-bound anti-goat IgG rabbit. Fluorescent immunostaining was performed using the antibody as a secondary antibody. As a result, as shown in FIGS. 7C, it was confirmed that most of the cells were efficiently differentiated into ectoderm, mesoderm or endoderm (90% or more).
さらに、ＰＩＳ細胞に対し、ＰＳ－ｄｉｆ ＢＡ Ｂｒｏｗｎ Ａｄｉｐｏｃｙｔｅ Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ Ｋｉｔ（ベリタス社）を用いて、中胚葉への誘導および褐色脂肪細胞への分化を行った。得られた細胞は、抗ＵＣＰ１ウサギ抗体（Ｂｉｏｓｉｓ社：ｂｓ１９２Ｒ）を用いて免疫染色した。また、０．１ｍＭのオレイン酸を添加して培養した。その結果、分化した細胞は、褐色を呈するだけでなく、図８のＡに示すようにＵＣＰ１を発現しており、図８のＢに示すように脂肪を蓄積する性質があった。このことから、ＰＩＳ細胞が褐色脂肪細胞に分化していることを確認した。また、生存した細胞の褐色脂肪細胞へ分化効率は９０％以上であり、非常に高効率で分化できることを確認した。 Furthermore, PIS cells were induced into mesoderm and differentiated into brown adipocytes using PS-dif BA Brown Adipose Tifferentiation Kit (Veritas). The obtained cells were immunostained with an anti-UCP1 rabbit antibody (Biosis: bs192R). In addition, 0.1 mM oleic acid was added and cultured. As a result, the differentiated cells not only exhibited brown color, but also expressed UCP1 as shown in A of FIG. 8, and had the property of accumulating fat as shown in B of FIG. From this, it was confirmed that the PIS cells were differentiated into brown adipocytes. Moreover, it was confirmed that the differentiation efficiency of the surviving cells into brown adipocytes was 90% or more, and that the cells could be differentiated with extremely high efficiency.
実施例７：脂肪細胞からＰＩＳ細胞を作製でき、高効率で分化できることの確認 Example 7: Confirmation that PIS cells can be produced from adipocytes and can be differentiated with high efficiency.
この実験は、体内に多量に存在し、且つ脂肪吸引等で多量に得られる脂肪細胞から、効率よくＰＩＳ細胞を作製および分化させることが可能であることを証明するために実施した。 This experiment was carried out to prove that PIS cells can be efficiently produced and differentiated from adipocytes which are present in a large amount in the body and can be obtained in a large amount by liposuction or the like.
マウス脂肪細胞３Ｔ３－Ｌ２４（N.Shiomi et al.(2011) JBiSE 4, 684）を、６ｍＬのＤＭＥＭを用いて、２５ｃｍ２の培養フラスコで１日間培養し、フラスコの底面に付着させた。培養フラスコに、Ｒｏｃｋ阻害剤である２０μＭのＹ－２７６３２および５μＭのチアゾビビンと、アポトーシス阻害剤である６０μＭのｐ５３阻害剤（Ｃｙｃｌｉｃ ｐｉｆｔｈｒｉｎ－α－ｈｙｄｒｏｂｔｏｍｉｄｅ）および３０μＭのＢａｘ阻害剤（Ｂａｘ Ｉｎｈｉｂｉｔｏｒ Ｐｅｐｔｉｄｅ Ｖ５）と、を添加して８時間培養し、この４種類の阻害剤を十分に細胞内に取り込ませた。その後、１００ｍＭのスペルミン溶液を添加して、２日間培養した。Ｔｒｙｐ－ＬＥ Ｓｅｌｅｃｔ溶液（Ｇｉｂｃｏ）１ｍＬを添加して細胞をはがし、ＰＢＳ溶液１０ｍＬに入れた後、遠心分離（８００ｒｐｍ、３分間）により細胞を回収した。上澄みを取り除いた後、ＰＢＳ溶液１０ｍＬを用いて同様の操作を行った。さらに、得られた細胞は、ＳｔｅｍＭｅｄｉｕｍ（登録商標）マウスＥＳ細胞用無血清培地（ＤＳファーマバイオメディカル株式会社）を用いて２日間培養し、細胞の修復を行うと同時に、形質の安定したＰＩＳ細胞を得た。Mouse adipocytes 3T3-L24 (N. Shiomi et al. (2011)
ＳｔｅｍＳｕｒｅ（登録商標） ０．１ｗ・ｖ％ゼラチン溶液（和光純薬工業株式会社）を用いてコーティングされた２４ウェルの培養プレートの各ウェルに、神経分化培地ＮＤｉｆｆ（登録商標） ２２７（タカラバイオ株式会社：Ｙ４０００２）を２ｍＬ入れ、ＰＩＳ細胞を３日間培養して神経幹細胞に誘導した。さらに、これらの細胞を、Ｎｅｕｒｏｃｕｌｔ（登録商標） Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ Ｍｅｄｉｕｍ （ベリタス社）を用いて神経細胞やグリア細胞に分化させた。得られた神経系の細胞に対し、抗β―チューブリンIIIウサギ抗体とＭＡＰ２ウサギ抗体を一次抗体、Ｃｙ３結合抗ウサギヤギ抗体を二次抗体として、免疫染色を行った。その結果、図９のＡに示すように、ほぼ１００％の細胞が神経幹細胞に分化した。図９のＢとＣに示すように、さらに神経幹細胞から分化した細胞も、ほぼ１００％が成熟神経細胞であることを確認した。 Nerve differentiation medium NDiff® 227 (Takara Biostock) in each well of a 24-well culture plate coated with StemSure® 0.1w v% gelatin solution (Wako Pure Chemical Industries, Ltd.). Company: Y40002) was added, and PIS cells were cultured for 3 days to induce neural stem cells. Furthermore, these cells were differentiated into nerve cells and glial cells using Neurocult® Differation Medium (Veritas). The obtained nervous system cells were immunostained using anti-β-tubulin III rabbit antibody and MAP2 rabbit antibody as the primary antibody and Cy3-binding anti-rabbit goat antibody as the secondary antibody. As a result, as shown in A of FIG. 9, almost 100% of the cells differentiated into neural stem cells. As shown in B and C of FIG. 9, it was confirmed that almost 100% of the cells further differentiated from the neural stem cells were mature neurons.
今回開示された実施形態および実施例はすべての点で例示であって制限的なものではないと考えられるべきである。本発明の範囲は上記した説明ではなくて請求の範囲によって示され、請求の範囲と均等の意味および範囲内でのすべての変更が含まれることが意図される。 The embodiments and examples disclosed this time should be considered to be exemplary and not restrictive in all respects. The scope of the present invention is shown by the scope of claims rather than the above description, and is intended to include all modifications within the meaning and scope of the claims.
本発明の多能性幹細胞の製造方法は、ｉＰＳの作製よりも、はるかに簡便、迅速かつ高効率である。したがって、再生医療の分野の様々な治療に利用可能であり、産業上の利用価値は極めて高い。 The method for producing pluripotent stem cells of the present invention is much simpler, faster and more efficient than the production of iPS. Therefore, it can be used for various treatments in the field of regenerative medicine, and its industrial utility value is extremely high.
Claims (1)
ポリアミンを含む培地中でヒトまたはマウスの体細胞を培養することにより、多能性幹細胞を取得する、多能性幹細胞の製造方法であって、
前記ポリアミンは、スペルミン、スペルミジン、プトレシン、および、それらのアセチル化体、並びに、それらポリアミンの２つ以上の重合体、からなる群から選択される少なくとも１種である、製造方法。 Does not include the step of performing genetic recombination
A method for producing pluripotent stem cells, which obtains pluripotent stem cells by culturing human or mouse somatic cells in a medium containing polyamine.
The production method, wherein the polyamine is at least one selected from the group consisting of spermine, spermidine, putrescine, an acetylated product thereof, and two or more polymers of the polyamines.
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| WO2010004989A1 (en) | 2008-07-07 | 2010-01-14 | タカラバイオ株式会社 | Method for production of pluripotent stem cell |
| JP2011050379A (en) | 2009-08-07 | 2011-03-17 | Kyoto Univ | CANINE iPS CELL AND METHOD OF PRODUCING THE SAME |
| WO2013100140A1 (en) | 2011-12-29 | 2013-07-04 | 国立大学法人 京都大学 | Induction of pluripotent stem cell cells from germ cells |
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