JPWO2016204298A1 - Method for producing canine iPS cells - Google Patents
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Abstract
【課題】長期にわたり安定して継代できるイヌiPS細胞株を、極めて簡単な組成の培養液を用いて作製する。【解決手段】 核初期化因子であるOCT3/4、SOX2、KLF4及びC-MYCの４つの転写因子を、例えばドキシサイクリンにより前記転写因子の発現を制御できる薬剤誘導性のベクターにより導入したイヌ体細胞を、塩基性線維芽細胞増殖因子（bFGF）の存在下でLIFのような分化多能性維持因子（塩基性線維芽細胞増殖因子は含まれない）、さらに好ましくはMEK抑制因子、GSK3β抑制因子などの分化抑制因子を含まない培養液で培養して、イヌiPS細胞を得る。【選択図】図１A canine iPS cell line that can be stably passaged over a long period of time is prepared using a culture solution having an extremely simple composition. SOLUTION: Canine somatic cells into which four transcription factors OCT3 / 4, SOX2, KLF4 and C-MYC, which are nuclear reprogramming factors, are introduced by a drug-inducible vector capable of controlling the expression of the transcription factor by doxycycline, for example. Pluripotency maintenance factor (not including basic fibroblast growth factor) such as LIF in the presence of basic fibroblast growth factor (bFGF), more preferably MEK inhibitor, GSK3β inhibitor Canine iPS cells are obtained by culturing in a culture medium that does not contain a differentiation inhibitor such as. [Selection] Figure 1
Description
本発明はイヌｉＰＳ細胞の作製方法に関する。 The present invention relates to a method for producing canine iPS cells.
イヌは、マウスやラットなどのげっ歯類と比較して生理学的にヒトに近いことに加え、大型であり、また寿命も長いため、比較的多量の薬物などの投与が可能で、長期間の実験にも適している。また、ヒトと同様の環境で生活していることからもヒトの疾患モデル動物としてより重宝されている。さらに、イヌで見られる遺伝性疾患の半数以上が同じ遺伝子の変異によるヒト疾患に類似し、イヌゲノム配列が解明されたことからも、疾患モデルとしてのイヌの需要はさらに広がる可能性がある。 Dogs are physiologically close to humans compared to rodents such as mice and rats, and are large and long-lived, so they can be administered relatively large amounts of drugs. Suitable for experiments. Moreover, it is more useful as a human disease model animal because it lives in the same environment as humans. Furthermore, since more than half of the inherited diseases seen in dogs are similar to human diseases caused by mutations in the same gene, and the canine genomic sequence has been elucidated, the demand for dogs as a disease model may further expand.
これまでに、イヌiPS細胞の作製例として種々の報告があるが、これらの報告においては、基本培地であるDMEM/F12（Dulbecco's Modified Eagle 培地とHam's F-12培地とを等量ずつ混合した培地）に、FBSなどの血清やKSRなどの血清代替物、さらにLIFのような分化抑制剤を添加した培養液が用いられていた。例えば、特許文献１では、基本培地にMAPKK（マイトジェン活性化タンパク質キナーゼキナーゼ）阻害剤であるPD0325901とALK（アクチビンレセプター様キナーゼ）阻害剤であるA-83-01とGSK（グリコーゲン合成酵素キナーゼ）阻害剤であるCHIR99021とLIF（白血病抑制因子）とHDAC（ヒストン脱アセチル化酵素）阻害剤であるバルプロ酸を混合した培養液が使用されている。また、特許文献２では、基本培地に血清代替物であるKSRとLIFとbFGF（塩基性線維芽細胞増殖因子）を混合した培養液が使用されている。さらに非特許文献１には、基本培地にKSRとLIFとbFGF、さらにはGSK3β（グリコーゲン・シンターゼ・キナーゼ３β）抑制因子、MEK（ERK-MAP キナーゼ経路遮断剤）抑制因子、TGF-β（ベータ型変異増殖因子）拮抗薬を混合した培養液が用いられている。 So far, there are various reports as preparation examples of canine iPS cells. In these reports, basic medium DMEM / F12 (Dulbecco's Modified Eagle medium and Ham's F-12 medium mixed in equal amounts) ), A culture medium to which serum such as FBS, a serum substitute such as KSR, and a differentiation inhibitor such as LIF were added was used. For example, in Patent Document 1, MAPKK (mitogen activated protein kinase kinase) inhibitor PD0325901, ALK (activin receptor-like kinase) inhibitor A-83-01 and GSK (glycogen synthase kinase) inhibition are included in the basic medium. A culture solution in which CHIR99021, a drug, LIF (leukemia inhibitory factor) and valproic acid, an inhibitor of HDAC (histone deacetylase), are mixed is used. In Patent Document 2, a culture medium is used in which KSR, LIF, and bFGF (basic fibroblast growth factor), which are serum substitutes, are mixed in a basic medium. Furthermore, Non-Patent Document 1 includes KSR, LIF and bFGF in the basic medium, GSK3β (glycogen / synthase / kinase 3β) inhibitor, MEK (ERK-MAP kinase pathway blocker) inhibitor, TGF-β (beta type) A culture solution in which a mutant growth factor) antagonist is mixed is used.
一方、ヒトiPS細胞やマウスiPS細胞においては、bFGFを用いずに、基本培地であるDMEM/F12培地に神経細胞培養に使用されるN2サプリメント（商品名）を混合した培地とneurobasal培地にB27サプリメント（商品名）を混合した培地の混合培地にLIF、MEK阻害剤、GSK3β阻害剤を加えた培地が使用されている（非特許文献２、３）。これらの文献では、得られたiPSコロニーを酵素処理することで単一細胞を得ることができ、当該単一細胞も５０代程度の継代培養ができたとある。 On the other hand, in human iPS cells and mouse iPS cells, without using bFGF, the basic medium DMEM / F12 medium is mixed with N2 supplement (trade name) used for neuronal cell culture, and neurobasal medium is B27 supplement. A medium in which LIF, MEK inhibitor, and GSK3β inhibitor are added to a mixed medium of a medium mixed with (trade name) is used (Non-patent Documents 2 and 3). According to these documents, single cells can be obtained by enzymatic treatment of the obtained iPS colonies, and the single cells can be subcultured for about 50 generations.
さらに、非特許文献４や５では、より未分化なマウスiPS細胞に類似した立体的なコロニー形成能を持つものことが報告されてはいるが、酵素を用いた継代法に耐えうることができず、各阻害剤を培地に添加しなければその形態は維持できない。また、長期間の継代培養ができていないことから、これらのiPS細胞の株化には至っていないと考えられる。 Furthermore, in Non-Patent Documents 4 and 5, it has been reported that it has a three-dimensional colony-forming ability similar to that of undifferentiated mouse iPS cells, but it can withstand passage methods using enzymes. The form cannot be maintained unless each inhibitor is added to the medium. In addition, it is considered that these iPS cells have not yet been established because long-term subculture is not possible.
これまでのところ、LIFやSK3β阻害剤のような分化多能性維持因子やMEK阻害剤などの分化抑制因子を用いることなく、bFGFのような細胞増殖促進因子のみの存在下で、体細胞からイヌiPS細胞が作製され、しかも長期にわたって継代培養できるiPS細胞が作製されたとの報告はない。 So far, without using pluripotency maintenance factors such as LIF and SK3β inhibitors and differentiation suppression factors such as MEK inhibitors, somatic cells can be isolated from somatic cells in the presence of only cell growth promoting factors such as bFGF. There is no report that canine iPS cells have been produced and iPS cells that can be subcultured over a long period of time have been produced.
本発明は、長期にわたり安定して継代できるイヌiPS細胞株を、極めて簡単な組成の培養液を用いて作製することを課題とする。 An object of the present invention is to produce a canine iPS cell line that can be stably passaged over a long period of time using a culture solution having an extremely simple composition.
本発明は、イヌの体細胞からイヌiPS細胞株を作製する方法であって、イヌ体細胞に核初期化因子を導入する工程と、核初期化因子を導入したイヌ体細胞を、塩基性維芽細胞増殖因子の存在下で分化多能性維持因子を含まない培養液を用いて培養をする工程を含む方法である。 The present invention relates to a method for producing a canine iPS cell line from canine somatic cells, comprising a step of introducing a nuclear reprogramming factor into a canine somatic cell, and a canine somatic cell into which a nuclear reprogramming factor has been introduced. It is a method comprising a step of culturing using a culture solution containing no differentiation pluripotency maintenance factor in the presence of a blast growth factor.
本発明によると、極めて簡単な組成の培養液で、長期にわたり安定して継代できるイヌiPS細胞株が製造される。また、この細胞を分化誘導すればイヌの間葉系幹細胞（ＭＳＣ）やその他イヌの幹細胞、体細胞がin vitroで製造できる。 According to the present invention, a canine iPS cell line that can be stably passaged over a long period of time is produced using a culture solution having a very simple composition. In addition, if these cells are induced to differentiate, canine mesenchymal stem cells (MSC) and other canine stem cells and somatic cells can be produced in vitro.
本発明に係るイヌiPS細胞の作製方法は、イヌ体細胞に核初期化因子を導入する工程と、核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子の存在下で分化多能性維持因子を含まない培養液を用いて培養する工程を含む。 The method for producing canine iPS cells according to the present invention comprises a step of introducing a nuclear reprogramming factor into a canine somatic cell, and differentiation of a canine somatic cell into which a nuclear reprogramming factor has been introduced in the presence of a basic fibroblast growth factor. A step of culturing using a culture solution containing no pluripotency maintenance factor.
本発明において使用され得るイヌ体細胞は、イヌから採取される体細胞であれば特に限定されず、例えば、胎子期の体細胞であり、成熟した体細胞でもあり得る。具体的には、神経幹細胞や造血幹細胞、間葉系幹細胞、***幹細胞などの幹細胞、リンパ球、上皮細胞、筋肉細胞、線維芽細胞等の既に分化した細胞等であり得る。 The canine somatic cell that can be used in the present invention is not particularly limited as long as it is a somatic cell collected from a dog. For example, it can be a fetal somatic cell or a mature somatic cell. Specifically, stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells and sperm stem cells, and already differentiated cells such as lymphocytes, epithelial cells, muscle cells and fibroblasts can be used.
核初期化因子は体細胞の核初期化を誘導する因子である。核初期化因子を体細胞に導入することでイヌ体細胞に分化多能性及び自己複製能が備えられる。該核初期化因子はこれらの機能を有する限り特に限定されず、例えば、転写因子に代表される核酸（遺伝子）、ペプチド、タンパク質、有機化合物、無機化合物又はこれらの混合物等であり、好ましくは転写因子である。転写因子としても、イヌ体細胞を核初期化する因子であれば特に限定されず、公知の転写因子、例えば、OCT3/4やSOX2、KLF4、C-MYCなどの遺伝子が示される。また、その他の因子として、NANOG、LIN28、TERT、SV40ラージＴ抗原が例示される。これらの核初期化因子の１種又は２種以上が用いられ、例えば、iPS細胞の作製に汎用されているOCT3/4やSOX2、KLF4、C-MYCの４つの因子の組み合わせや、発癌のリスク低減する観点から、この組み合わせからC-MYCを除いたOCt3/4、SOX2及びKLF4の３因子の組み合わせが好ましく用いられる。また、転写因子は、好ましくはイヌ由来の因子であるが、ヒトやマウスなどイヌ以外の動物種の転写因子でもあり得る。Nuclear reprogramming factors are factors that induce nuclear reprogramming of somatic cells. By introducing a nuclear reprogramming factor into somatic cells, canine somatic cells have pluripotency and self-renewal ability. The nuclear reprogramming factor is not particularly limited as long as it has these functions, and is, for example, a nucleic acid (gene) represented by a transcription factor, a peptide, a protein, an organic compound, an inorganic compound, or a mixture thereof, preferably transcription. Is a factor. The transcription factor is not particularly limited as long as it can reprogram canine somatic cells, and known transcription factors such as genes such as OCT3 / 4 , SOX2 , KLF4 , and C- MYC are shown. Other factors include NANOG , LIN28 , TERT , and SV40 large T antigen. One or more of these nuclear reprogramming factors are used. For example, the combination of the four factors OCT3 / 4 , SOX2 , KLF4 , and C- MYC , which are widely used for the production of iPS cells, and the risk of carcinogenesis From the viewpoint of reduction, a combination of three factors of OCt3 / 4 , SOX2 and KLF4 excluding C-MYC from this combination is preferably used. The transcription factor is preferably a dog-derived factor, but may also be a transcription factor of animal species other than dogs such as humans and mice.
核初期化因子の体細胞への導入方法も体細胞の核初期化が可能な方法であれば特に限定されるものではなく、公知の方法が使用され得る。例えば、転写因子をコードする核酸であれば、該核酸を発現することが可能なベクターを用いてイヌ体細胞に導入する方法が示される。２種以上の核酸（転写因子）を用いる場合には、使用される全ての核酸を１つのベクターに組み込んでイヌ体細胞内で同時に発現させてもよく、複数のベクターを用いてイヌ体細胞内で同時に発現させてもよい。 A method for introducing a nuclear reprogramming factor into a somatic cell is not particularly limited as long as it is a method capable of nuclear reprogramming of a somatic cell, and a known method can be used. For example, if it is a nucleic acid which codes a transcription factor, the method of introduce | transducing into a canine somatic cell using the vector which can express this nucleic acid is shown. When two or more kinds of nucleic acids (transcription factors) are used, all the nucleic acids to be used may be incorporated into one vector and expressed in canine somatic cells at the same time. May be expressed simultaneously.
当該ベクターとしては、例えば、レトロウイルス（レンチウイルスを含む）、アデノウイルス、アデノ随伴ウイルス、センダイウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シルビスウイルス、ラブドウイルス、パラミクソウイルス、オルソミクソウイルス等のウイルスベクター；YAC（Yeast artificial chromosome）ベクター、BAC（Bacterial artificial chromosome）ベクター、PAC(P1-derived artificial chromosome)ベクター等の人工染色体ベクター；プラスミドベクター；宿主細胞内で自律複製可能なエピゾーマルベクター等が挙げられる。導入した転写因子の発現を人為的に制御することで、効率的に体細胞を初期化することができ、また、継続的に転写因子を発現することで、安定してiPS細胞を培養維持することができるとの観点から、薬剤の添加により転写因子を発現させる薬剤誘導性ベクター、例えばドキシサイクリンを用いるドキシサイクリン誘導性Tet-Onレンチウイルスベクター(TetO-FUW-OSKM:Addgene社製)が示される。ベクターの導入方法も特に限定されず、リポフェクション法、マイクロインジェクション法、ＤＥＡＥデキストラン法、遺伝子銃法、エレクトロポレーション法、リン酸カルシウム法等の公知の方法が利用され得る。 Examples of such vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, Sendai virus, herpes virus, vaccinia virus, pox virus, poliovirus, silbis virus, rhabdovirus, paramyxovirus, ortho Virus vectors such as myxoviruses; artificial chromosome vectors such as YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors; plasmid vectors; epis that can replicate autonomously in host cells Examples thereof include somal vectors. By artificially controlling the expression of the introduced transcription factor, somatic cells can be efficiently initialized, and by continuously expressing the transcription factor, iPS cells can be stably maintained in culture. From the viewpoint of being able to do so, a drug-inducible vector that expresses a transcription factor by adding a drug, for example, a doxycycline-inducible Tet-On lentiviral vector (TetO-FUW-OSKM: manufactured by Addgene) using doxycycline is shown. The method for introducing the vector is not particularly limited, and a known method such as a lipofection method, a microinjection method, a DEAE dextran method, a gene gun method, an electroporation method, or a calcium phosphate method can be used.
核初期化因子を導入されたイヌ体細胞は培養液にて培養され、初期化される。本発明においては、塩基性線維芽細胞増殖因子の存在下でLIFなどの分化多能性維持因子を含まない培養液が用いられる。この培養液の基礎培地には、動物細胞の培養に用いられる培地が用いられる。基礎培地は、例えば、BME培地であり、BGJb培地であり、GLASGOW MEM培地であり、IMDM培地であり、Eagle MEM培地であり、DMEM培地であり、F12培地であり、Neuro Basal Mediumであり得る。 Canine somatic cells into which a nuclear reprogramming factor has been introduced are cultured in a culture medium and reprogrammed. In the present invention, a culture solution containing no differentiation pluripotency maintenance factor such as LIF in the presence of a basic fibroblast growth factor is used. As the basal medium of the culture solution, a medium used for culturing animal cells is used. The basal medium is, for example, a BME medium, a BGJb medium, a GLASGOW MEM medium, an IMDM medium, an Eagle MEM medium, a DMEM medium, an F12 medium, or a Neuro Basal Medium.
本発明に用いられる培養液は、分化多能性維持因子を含まない培地であって、さらに好ましくは分化抑制因子も含まない培地である。本明細書において、分化多能性維持因子とは、得られたiPS細胞の分化多能性を維持する、すなわち、幹細胞の分化多能性を維持するために人為的に添加される因子をいう。分化多能性維持因子は、幹細胞が分化多能性を保つのに必要な細胞シグナル経路を活性化させる因子であり、例えば、これまで汎用されているLIFやActivinなどが示される。また、本明細書において、分化抑制因子とは、幹細胞が分化するのに必要な細胞シグナルを阻害するものをいい、得られたiPS細胞が自ら分化、すなわち個々の細胞が種々の機能を発揮し、異種の細胞に変化することを阻害する。例えば、これまで汎用されているMEK阻害薬剤である。分化多能性維持因子や分化抑制因子は、その作用機序において分化多能性維持因子としても分化抑制因子としても利用し得る場合には、分化多能性維持因子として取り扱えば足りる。また、両者を含まない培地を用いる場合には、本発明の性質上両者を区別する必要もない。本発明は、そのような因子を加えることなく、iPS細胞を作製、又はiPS細胞として維持可能することを目的とするものだからである。従って、本発明の培養液は、特許文献１に記載されたようなMAPKK阻害薬剤やALK阻害薬剤、GSK阻害薬剤であるCHIR99021、HDAC阻害薬剤など、その作用機序から見て分化多能性維持因子と同じ作用を発揮する因子を含まず、さらに好ましくは分化抑制因子と同じ作用を発揮する因子も含まない。なお、塩基性線維芽細胞増殖因子は分化多能性維持因子の範疇に入る因子ではあるが、本願発明では塩基性線維芽細胞増殖因子（bFGF）は必須成分として取り扱われるので、本願発明における「分化多能性維持因子」には塩基性線維芽細胞増殖因子（bFGF）を含めないものとする。 The culture medium used in the present invention is a medium that does not contain a differentiation pluripotency maintenance factor, and more preferably a medium that does not contain a differentiation inhibitory factor. In the present specification, the pluripotency maintenance factor refers to a factor that is artificially added to maintain the differentiation pluripotency of the obtained iPS cells, that is, to maintain the pluripotency of stem cells. . The differentiation pluripotency maintenance factor is a factor that activates a cell signal pathway necessary for stem cells to maintain differentiation pluripotency, and examples thereof include LIF and Activin that have been widely used so far. In the present specification, the differentiation inhibitor refers to a substance that inhibits a cell signal necessary for a stem cell to differentiate, and the obtained iPS cell differentiates itself, that is, each cell exhibits various functions. Inhibits changing to a heterologous cell. For example, MEK inhibitors that have been widely used so far. A differentiation pluripotency maintenance factor and a differentiation inhibitory factor need only be handled as a differentiation pluripotency maintenance factor when they can be used as a differentiation pluripotency maintenance factor or a differentiation inhibition factor in their action mechanism. Moreover, when using the culture medium which does not contain both, it is not necessary to distinguish both from the property of this invention. This is because the object of the present invention is to produce iPS cells or maintain them as iPS cells without adding such factors. Therefore, the culture solution of the present invention is capable of maintaining pluripotency in terms of its action mechanism, such as MAPKK inhibitor, ALK inhibitor, GSK inhibitor CHIR99021, and HDAC inhibitor as described in Patent Document 1. It does not include a factor that exhibits the same action as the factor, and more preferably does not include a factor that exhibits the same action as the differentiation inhibiting factor. Although basic fibroblast growth factor is a factor that falls into the category of pluripotency maintenance factor, in the present invention, basic fibroblast growth factor (bFGF) is treated as an essential component. The “pluripotency maintenance factor” does not include basic fibroblast growth factor (bFGF).
また、本発明においては無血清の培養液が使用される。無血清の培養液とは、培養液が無調整又は未精製の血清、例えばFBSや組成の不明な血清代替物、例えばKSRを含まない培地を意味する。 In the present invention, a serum-free culture solution is used. A serum-free culture medium means a medium containing no serum or an unpurified serum such as FBS or a serum substitute of unknown composition, such as KSR.
一方、培養液には塩基性線維芽細胞増殖因子（bFGF）が必須成分として添加される。bFGFは公知の成分である。bFGFの添加量は、適宜当業者によって決定され得る。その添加量は、例えば培養液中１mlに対して、その下限量は例えば0.01ngであり、0.1ngであり、0.5ngであり、1ngであり得る。また、その上限量は例えば10,000ngであり、5,000ngであり、1,000ngであり、500ngであり、200ngであり得る。bFGFの由来は問わず、例えばヒト由来であり、ウシ由来であり、イヌ由来でもあり得る。 On the other hand, basic fibroblast growth factor (bFGF) is added to the culture medium as an essential component. bFGF is a known component. The amount of bFGF added can be appropriately determined by those skilled in the art. The lower limit amount is, for example, 0.01 ng, 0.1 ng, 0.5 ng, and 1 ng with respect to 1 ml of the culture solution, for example. Moreover, the upper limit amount is, for example, 10,000 ng, 5,000 ng, 1,000 ng, 500 ng, or 200 ng. Regardless of the origin of bFGF, for example, it is human, bovine, and canine.
培養液は、さらに、bFGF以外の細胞の増殖や生育の維持に関する因子を含み得る。当該因子は、例えば、脂肪酸又は脂質、アミノ酸（例えば、非必須アミノ酸）、アルブミン、ビタミン、増殖因子、抗酸化剤、２−メルカプトエタノール、ピルビン酸、緩衝剤、無機塩類であり得る。具体的には、いわゆるサプリメントと称されるものであって、細胞増殖に寄与し得る因子である。培養液は、１種又は２種以上の因子を含み、例えば、N2サプリメント(Invitorogen社）、B27サプリメント（Invitorogen社）のような混合物が好ましく用いられる。 The culture solution may further contain factors related to the growth and growth maintenance of cells other than bFGF. The factor can be, for example, fatty acids or lipids, amino acids (eg, non-essential amino acids), albumin, vitamins, growth factors, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts. Specifically, it is a so-called supplement and is a factor that can contribute to cell proliferation. The culture solution contains one or more factors, and for example, a mixture such as N2 supplement (Invitorogen) and B27 supplement (Invitorogen) is preferably used.
本発明において好ましく用いられる培養液は、上記の基礎培地に必須の成分であるbFGFのみを添加した培養液であり、さらには、DMEM/F12培地に、N2サプリメント、B27サプリメントの少なくとも１つのサプリメント、好ましくは２つのサプリメント（N2及びB27）を含むN2B27培地に必須成分であるbFGFのみを加えた培養液、より望ましくはこれにアルブミンを加えた培養液である。 The culture medium preferably used in the present invention is a culture liquid in which only bFGF, which is an essential component of the above-mentioned basal medium, is added. Furthermore, the DMEM / F12 medium contains at least one supplement of N2 supplement and B27 supplement, A culture solution in which only bFGF, which is an essential component, is added to an N2B27 medium containing two supplements (N2 and B27) is preferable, and a culture solution in which albumin is added to this is more preferable.
核導入されたイヌ体細胞は、フィーダー細胞上又はフィーダーレスで培養される。フィーダー細胞も、これまでのイヌiPS細胞やヒトiPS細胞、マウスiPS細胞の作製に使用された各種の細胞が用いられ、例えば、イヌ胎子由来線維芽細胞やマウス胎子由来線維芽細胞が示される。フィーダーレスとは、フィーダー細胞を用いずにウェルやディッシュなどの培養器具上で培養することを意味する。フィーダーレスの場合には、好ましくはマトリゲルのような人工基底膜マトリックスをコートした培養器具が用いられる。 Nuclei introduced canine somatic cells are cultured on feeder cells or without feeders. As the feeder cells, various cells used in the production of conventional canine iPS cells, human iPS cells, and mouse iPS cells are used, and examples include canine fetal derived fibroblasts and mouse fetal derived fibroblasts. Feederless means culturing on a culture instrument such as a well or a dish without using feeder cells. In the case of feederless, a culture device coated with an artificial basement membrane matrix such as Matrigel is preferably used.
本発明により得られるイヌiPS細胞は、イヌの体細胞由来の細胞であって、内因性のOCT3/4、KLF4、SOX2の少なくとも１つ、好ましくは全ての遺伝子とNANOG遺伝子の発現が見られる細胞である。さらに好ましくは内因性のC-MYCやGBX2の遺伝子の発現が見られる細胞である。そして、さらに望ましくはドキシサイクリンの存在下で、外因性の遺伝子である４つの遺伝子OCT3/4（例えばE2A-OCT3/4）やSOX2（例えばE2A-SOX2）、KLF4（例えばE2A-KLF4）のうち少なくとも１つの遺伝子、好ましくはこれらの３つ遺伝子の発現が見られる細胞である。そして、C-MYC（例えばE2A-OCT3/4）が導入された場合には、これら４つの外因性遺伝子のうち少なくとも１つの遺伝子、好ましくはこれら４つの遺伝子の発現が見られる細胞である。The canine iPS cell obtained by the present invention is a cell derived from a somatic cell of a dog, and is a cell in which expression of at least one of endogenous OCT3 / 4 , KLF4 , and SOX2 , preferably all genes and NANOG gene is observed. It is. More preferred are cells in which endogenous C-MYC or GBX2 gene expression is observed. More preferably, in the presence of doxycycline, at least one of the four exogenous genes OCT3 / 4 (eg, E2A-OCT3 / 4 ), SOX2 (eg, E2A-SOX2) , and KLF4 (eg, E2A-KLF4 ) A cell in which expression of one gene, preferably these three genes is observed. When C-MYC (for example, E2A-OCT3 / 4 ) is introduced, it is a cell in which expression of at least one of these four exogenous genes, preferably these four genes is observed.
核導入されたイヌ体細胞の培養条件は、適宜当業者により設定され得る。例えば、３０〜４０℃の培養温度で、１〜１０％程度のＣＯ2存在下で、５日〜１週間、好ましくは１週間〜２週間の培養期間である。その一例を挙げると、約５％のＣＯ2存在下、３７℃、２週間の培養である。Culture conditions for canine somatic cells into which nuclei have been introduced can be appropriately set by those skilled in the art. For example, the culture period is 5 days to 1 week, preferably 1 week to 2 weeks in the presence of about 1 to 10% CO 2 at a culture temperature of 30 to 40 ° C. One example is culture at 37 ° C. for 2 weeks in the presence of about 5% CO 2 .
得られるiPS細胞は、自己複製能と分化多能性を有する。自己複製能は、分化多能性を保持した状態（未分化状態）で増殖できる能力であり、自己複製能を有することは継代が可能であることを意味する。特に、本発明の作製方法により得られたイヌiPS細胞は、従来のイヌiPS細胞に比べて長い継代、つまり少なくとも１０代、３０代、好ましくは５０代以上の継代が可能である。また、当該作製方法により得られたイヌiPS細胞は、酵素処理によりその凝集体（細胞塊）を単一細胞に分割することができ、単一細胞の状態で継代培養できる。酵素処理は、例えばトリプシンによる処理であり、アクターゼによる処理であり得る。酵素処理は、酵素溶液と得られたiPS細胞の細胞塊を接触させることで行われる。処理の条件は当業者により適宜決定できる事項であって、例えば０.０１％以上１.０％のトリプシン溶液、好ましくはさらにＥＤＴＡを含む溶液が用いられる。当該酵素処理による分割は、いわゆるピペッティングやセルナイフによる分割などの物理的方法に変わる分割方法である。特許文献１に記載の方法で得られるイヌiPS細胞の細胞塊は酵素処理により単一細胞に分割できなかったが、本発明の作製方法によれば酵素処理により単一細胞への分割が可能な細胞塊（細胞集団）が得られ、継代培養の際の効率化が図られる。 The resulting iPS cells have self-renewal ability and pluripotency. The self-replicating ability is the ability to proliferate in a state that retains pluripotency (undifferentiated state), and having self-replicating ability means that passage is possible. In particular, canine iPS cells obtained by the production method of the present invention can be passaged longer than conventional canine iPS cells, that is, at least 10 generations, 30 generations, preferably 50 generations or more. In addition, canine iPS cells obtained by the production method can divide the aggregate (cell mass) into single cells by enzyme treatment, and can be subcultured in a single cell state. The enzyme treatment is, for example, treatment with trypsin, and can be treatment with actase. The enzyme treatment is performed by bringing the enzyme solution into contact with the resulting cell mass of iPS cells. Treatment conditions can be appropriately determined by those skilled in the art. For example, 0.01% to 1.0% trypsin solution, preferably a solution further containing EDTA is used. The division by the enzyme treatment is a division method that changes to a physical method such as so-called pipetting or cell knife division. Although the cell mass of canine iPS cells obtained by the method described in Patent Document 1 could not be divided into single cells by enzyme treatment, the production method of the present invention can be divided into single cells by enzyme treatment. A cell mass (cell population) is obtained, and efficiency in subculture is improved.
分化多能性は三胚葉（外胚葉、内胚葉、中胚葉）系の幹細胞、すなわち、あらゆる幹細胞に分化できる能力であり、分化多能性を有することはイヌＥＳ細胞と同様にあらゆる幹細胞に分化することを意味する。従って、得られたイヌiPS細胞は、これを分化誘導することで、種々の細胞、例えば、骨格筋細胞、心筋細胞、血液細胞、神経細胞、生殖細胞などが作製され得る。また、作製されたイヌiPS細胞を分化して得られた細胞、例えば幹細胞や体細胞は、さらにiPS細胞へ戻すことも可能である。これは、例えば前記の薬剤誘導性ベクターが導入された細胞に当該薬剤を作用させることや、新たに核初期化因子を導入することで達成され得る。 Pluripotency is the ability to differentiate into three germ layers (ectodermal, endoderm, mesoderm) stem cells, that is, any stem cell. Differentiating into pluripotency differentiates into any stem cell like canine ES cells It means to do. Therefore, various cells such as skeletal muscle cells, cardiomyocytes, blood cells, nerve cells, germ cells and the like can be produced by inducing differentiation of the obtained canine iPS cells. In addition, cells obtained by differentiating the prepared canine iPS cells, such as stem cells and somatic cells, can be further returned to iPS cells. This can be achieved, for example, by allowing the drug to act on cells into which the drug-inducible vector has been introduced, or by newly introducing a nuclear reprogramming factor.
継代培養条件は、上記核初期化する際に用いた培養液と同様の培養液が用いられ得る。また、継代培養に際しては、初期化する際に用いた培養液組成に対して、分化多能性維持因子や分化抑制因子を用いても差し支えない。フィーダー細胞の有無も問わず、フィーダー細胞上でも、またフィーダーレスの条件でも差し支えない。 As the subculture conditions, a culture solution similar to the culture solution used for the nuclear reprogramming can be used. In subculturing, a differentiation pluripotency maintenance factor or a differentiation inhibitory factor may be used for the culture solution composition used for initialization. Regardless of the presence or absence of feeder cells, it can be on feeder cells or feeder-less conditions.
分化誘導方法も特に制限されず、公知であるiPS細胞からの分化誘導方法が適用され得る。例えば、間葉系幹細胞（MSC)への分化には、DMEM/F12培地にウシ胎子血清（FBS）やNon essential amino acid(NEAA)を加えた培地で浮遊培養して胚様体を作製した後、これをMEM-α Glutamax（商品名：GIBCO社製）にMEM NEAAやFBS、bFGFを加えた培地で接着培養する方法が、骨様細胞には、作製した胚様体をDMEM培地にFBS、β−glycerophosphateを加えた骨分化培地で培養する方法が例示されるが、これらに限られるものではない。 The differentiation induction method is not particularly limited, and a known differentiation induction method from iPS cells can be applied. For example, for differentiation into mesenchymal stem cells (MSC), embryonic bodies were prepared by suspension culture in a medium containing fetal bovine serum (FBS) or non essential amino acid (NEAA) in DMEM / F12 medium. , This is a method of adhering culture in MEM-α Glutamax (trade name: manufactured by GIBCO) in a medium containing MEM NEAA, FBS, or bFGF. For bone-like cells, the prepared embryoid body is prepared in DMEM medium with FBS, Although the method of culture | cultivating in the bone differentiation medium which added (beta) -glycerophosphate is illustrated, it is not restricted to these.
以上のように本発明の方法によれば、従来の方法に比べてより少ない因子、特にLIFのような分化多能性維持因子、さらには分化抑制因子を用いずにイヌ体細胞からイヌiPSを作製できる。得られたイヌiPS細胞は、多世代の継代可能な自己複製能と三胚葉系の幹細胞、さらには各種の体細胞や生殖細胞に分化する分化多能性を有する。 As described above, according to the method of the present invention, canine iPS can be obtained from canine somatic cells without using a factor that is less than that of the conventional method, in particular, a pluripotency maintenance factor such as LIF, or a differentiation inhibitory factor. Can be made. The obtained canine iPS cells have a multi-generation passable self-renewal ability, a tridermal stem cell, and a pluripotency that differentiates into various somatic cells and germ cells.
以下、以下の実施例に基づいて本発明についてさらに詳細に説明するが、本発明は下記実施例に限定されないのは言うまでもない。 Hereinafter, although the present invention will be described in more detail based on the following examples, it is needless to say that the present invention is not limited to the following examples.
〔イヌiPS細胞株の作製〕
イヌ胎子線維芽細胞（CEF）に、ドキシサイクリンの添加により遺伝子を発現させることができる薬剤誘導性レンチウイルスを用いて遺伝子導入を行い、イヌiPS細胞の作製を試みた。N2B27培地を基礎培地として、添加因子としてLIF、bFGF、GSK3β阻害薬剤（CHIR99021）及びMEK阻害薬剤（PD0325901）の要否について検討を行った。また、フィーダー細胞（マウス胎子線維芽細胞（mouse embryonic fibroblast：MEF）の要否についても検討を加えた。なお、以下において、試薬の濃度は全て最終濃度である。[Preparation of canine iPS cell line]
Gene induction was performed on canine fetal fibroblasts (CEF) using a drug-induced lentivirus capable of expressing a gene by addition of doxycycline to try to produce canine iPS cells. The necessity of LIF, bFGF, GSK3β inhibitor (CHIR99021) and MEK inhibitor (PD0325901) as additive factors was examined using N2B27 medium as a basal medium. In addition, the necessity of feeder cells (mouse embryonic fibroblast (MEF)) was also examined. In the following, all reagent concentrations are final concentrations.
１.フィーダー細胞（マウス胎子線維芽細胞（mouse embryonic fibroblast：MEF））の作製
妊娠14.5日齢のICRマウス（Slc:ICR、日本SLC社製）から胎子を摘出し、常法に従って得た胎子組織を、DMEM培地（SIGMA社製）に10％FBS（PAA Laboratories GmbH 社製、Ontario、Canada）、2mM L-glutamine（SIGMA社製）、1％ペニシリン/ストレプトマイシン混合溶液（SIGMA社製）を添加した培養液で37℃、5％CO2下で培養した。この初代培養細胞は増殖後、継代を行い、3代以内の継代細胞を実験に使用した。また、実験には、10μg/mlマイトマイシン注用（協和発酵工業社製）を添加した前記培養液で、37℃、5％CO2下で2.5時間培養してマイトマイシンC処理を行った。D-PBS(-)5mlで細胞を洗浄し、0.25％トリプシン-EDTA溶液（SIGMA社製）で回収し、フィーダー細胞とした。1. Preparation of feeder cells (mouse embryonic fibroblasts (MEF)) Fetal tissues obtained by extracting fetuses from 14.5 day-old ICR mice (Slc: ICR, manufactured by SLC, Japan) and following conventional methods 10% FBS (PAA Laboratories GmbH, Ontario, Canada), 2 mM L-glutamine (SIGMA), 1% penicillin / streptomycin mixed solution (SIGMA) was added to DMEM medium (SIGMA) The culture was incubated at 37 ° C. and 5% CO 2 . The primary cultured cells were subcultured after being proliferated, and subcultured cells within 3rd generation were used for the experiment. Further, in the experiment, mitomycin C treatment was performed by culturing at 37 ° C. under 5% CO 2 for 2.5 hours with the above culture solution to which 10 μg / ml mitomycin injection (manufactured by Kyowa Hakko Kogyo Co., Ltd.) was added. The cells were washed with 5 ml of D-PBS (−) and collected with a 0.25% trypsin-EDTA solution (manufactured by SIGMA) to prepare feeder cells.
２．イヌ胎子線維芽細胞（CEF）へのレンチウイルスによる遺伝子の導入
１）パッケージング細胞の準備
パッケージング細胞として293FT cell line（Invitrogen社製）を用いた。DMEMに10％FBS、2mM L-glutamine、1％ペニシリン/ストレプトマイシン混合溶液、0.1mM MEM NEAA（GIBCO社製）、1mM sodium pyruvate（SIGMA社製）、500μg/ml geneticin（Invitrogen社製、培養初日および遺伝子導入時は除去）を添加した培養液を用いた。100mm組織培養用ディッシュに培養液10mlを入れ、細胞数が2×106個の濃度で、37℃、5％CO2下で培養した。この培養細胞は増殖後、0.25％トリプシン-EDTA溶液を用いて回収した。得られた293FT細胞を100mm組織培養用ディッシュに、上記の293FT細胞用の培養液を入れ、1ディッシュ当たりの細胞数が2×106個となるように播種し、37℃、5％CO2下で一晩培養した。2. Introduction of genes by lentivirus into canine fetal fibroblasts (CEF) 1) Preparation of packaging cells 293FT cell line (Invitrogen) was used as packaging cells. DMEM, 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 0.1 mM MEM NEAA (GIBCO), 1 mM sodium pyruvate (SIGMA), 500 μg / ml geneticin (Invitrogen, culture first day) A culture solution to which (removed at the time of gene introduction) was added was used. 10 ml of the culture solution was placed in a 100 mm tissue culture dish and cultured at a concentration of 2 × 10 6 cells at 37 ° C. and 5% CO 2 . The cultured cells were recovered after growth using a 0.25% trypsin-EDTA solution. The obtained 293FT cells were put into a 100 mm tissue culture dish, and the above-mentioned culture solution for 293FT cells was put in, and seeded so that the number of cells per dish was 2 × 10 6 , 37 ° C., 5% CO 2. Incubate overnight under.
２）核初期化因子導入用ウイルス液の調整
予め準備しておいたテトラサイクリン制御性トランス活性化因子を発現するFUW-M2rtTAレンチウイルスベクター（Addgene社製）とレンチウイルス構成タンパク発現ベクター（psPAX2およびpMD2.G（Addgene社製））を、 Lipofectamin 2000（Invitrogen社製）を用いたリポフェクタミン法により、１）で準備した293FT細胞に導入した。さらに、４つの多能性維持遺伝子（マウスOCT3/4、KLF4、SOX2、C-MYC）が組み込まれてテトラサイクリン応答因子を持ったTetO-FUW-OSKMレンチウイルスベクター（Addgene社製）とレンチウイルス構成タンパク発現ベクターを上記と同様にして新たな293FT細胞に導入した。24時間後にそれぞれの培養液を交換し、さらに24時間後、0.45μm cellulose acetate filter (Whatman社製、Kent、ME)を用いてFUW-M2rtTA及びTetO-FUW-OSKMのウイルス液を回収した。2) Preparation of the virus solution for nuclear reprogramming factor introduction The FUW-M2rtTA lentiviral vector (manufactured by Addgene) and the lentiviral component protein expression vectors (psPAX2 and pMD2) that express tetracycline-regulated transactivator prepared in advance. .G (manufactured by Addgene)) was introduced into the 293FT cells prepared in 1) by the lipofectamine method using Lipofectamin 2000 (manufactured by Invitrogen). In addition, TetO-FUW-OSKM lentiviral vector (manufactured by Addgene) with four pluripotency maintenance genes (mouse OCT3 / 4, KLF4, SOX2, C-MYC) and tetracycline response factor The protein expression vector was introduced into new 293FT cells as described above. After 24 hours, the respective culture solutions were replaced. After 24 hours, FUW-M2rtTA and TetO-FUW-OSKM virus solutions were collected using a 0.45 μm cellulose acetate filter (Whatman, Kent, ME).
３）CEFの準備とレンチウイルスの感染
妊娠30週齢のビーグル犬（日本SLC社製）から胎子（雄）を摘出し、常法に従って得た胎子組織からCEFを、前記MEFと同様の条件で培養した。培養には、DMEM培地に10％FBS、2mM L-glutamine、100IU/mlペニシリン-100μg/mlストレプトマイシン混合溶液を添加した培養液を用いた。CEFを35mm組織培養用ディッシュ（IWAKI社製）に、1ディッシュ当たりの細胞数が9.5×104個となるように播種し、37℃、5％CO2下で一晩培養した。ディッシュ一面に増殖したCEFに、上記２つのウイルス液の混合液に8μg/mlポリブレン（Nacalai Tesque社製）を添加したものを加え、CEFにウイルスを感染させた。3) CEF preparation and lentiviral infection The fetus (male) was extracted from a 30-week-old beagle dog (manufactured by SLC, Japan), and CEF was obtained from the fetal tissue obtained according to a conventional method under the same conditions as the MEF. Cultured. For the culture, a culture solution in which 10% FBS, 2 mM L-glutamine, 100 IU / ml penicillin-100 μg / ml streptomycin mixed solution was added to DMEM medium was used. CEF was seeded in a 35 mm tissue culture dish (IWAKI) so that the number of cells per dish was 9.5 × 10 4 and cultured overnight at 37 ° C. and 5% CO 2 . To the CEF grown on the entire dish, a mixture of the above two virus solutions with 8 μg / ml polybrene (Nacalai Tesque) added was added to infect the virus with CEF.
３．イヌiPS細胞作製
１）イヌiPS細胞の作製条件の検討
イヌiPS細胞の作製条件を調べるために、基材条件及び培地条件（添加因子）について、表１に示す条件で検討した。また、対照として特許文献１に記載された条件を採用した。対照にはドキシサイクリンのみを添加したN2B27培地を用いた。3. Canine iPS cell production 1) Examination of canine iPS cell production conditions In order to examine canine iPS cell production conditions, substrate conditions and medium conditions (addition factors) were examined under the conditions shown in Table 1. Moreover, the conditions described in Patent Document 1 were adopted as a control. As a control, N2B27 medium supplemented only with doxycycline was used.
ウイルス感染の24時間後に培養液を交換し、さらに24時間後0.25%トリプシン-EDTA溶液を用いて継代し、準備しておいたMEF細胞（フィーダー細胞）又はマトリゲル（Becton Dickinson Bioscience bioscience社製）をコーティングしたディッシュに、２．で得られた感染済みCEFを、CEF培養液を用いて、1つの35mm組織培養用ディッシュ当たりの細胞数が2〜3×104個となるように播種し、37℃、5％CO2下で培養した。The culture solution was changed 24 hours after virus infection, and further 24 hours later, the cells were subcultured with 0.25% trypsin-EDTA solution, and prepared MEF cells (feeder cells) or Matrigel (Becton Dickinson Bioscience bioscience) On a dish coated with 2. Inoculate the infected CEF obtained in step 1 with a CEF culture solution so that the number of cells per dish for 35 mm tissue culture is 2 to 3 × 10 4 cells, at 37 ° C. and 5% CO 2. In culture.
播種翌日より、N2B27培地［DMEM/F12培地（Lifetechnologies社製）に1×N2(Invitrogen社製)を添加した培地とNeuro Basal Mediumに1×B27(Invitrogen社製)を添加した培地を１対１（容量比）に混合した培地］に、5mg/mlアルブミン(ウシ血清由来アルブミン：BSA)、1mM L-グルタミン、1％ペニシリン/ストレプトマイシン混合溶液、1％NEAAと、0.1mM 2-メルカプトエタノール（SIGMA社製）と、4μg/ml ドキシサイクリン(Clontech)と、表１に従って、4ng/ml basic fibroblast growth factor（bFGF：Peprotech Rocky社製）、1000U/ml leukemia inhibitory factor（LIF：和光純薬工業社製）、２つ阻害薬剤(3μM GSK3βinhibitor:CHIR99021、1μM MEK inhibitor:PD0325901、各TOCRIS社製)を加えた培地に交換し、その後毎日培地を交換した。bFGFとLIFは培地交換ごとに添加した。また、対照として、市販のイヌiPS培地（DMEM/F12培地、SIGMA社製）に20％KSR（GIBCO社製）と、2mM L-グルタミンと、1％ペニシリン/ストレプトマイシン混合溶液と、0.1mM NEAAと、1.14μM 2-メルカプトエタノールと、4ng/ml bFGFと、1000U/ml LIFを加えた培地（特許文献１参照）を用いて、培養を行った。 From the day after seeding, there is a one-to-one relationship between N2B27 medium [DMEM / F12 medium (Lifetechnologies) supplemented with 1 × N2 (Invitrogen)] and Neuro Basal Medium with 1 × B27 (Invitrogen). Medium mixed with (volume ratio)], 5 mg / ml albumin (bovine serum-derived albumin: BSA), 1 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 1% NEAA, 0.1 mM 2-mercaptoethanol (SIGMA 4μg / ml doxycycline (Clontech) and 4ng / ml basic fibroblast growth factor (bFGF: Peprotech Rocky), 1000U / ml leukemia inhibitory factor (LIF: Wako Pure Chemical Industries) The medium was replaced with a medium supplemented with two inhibitors (3 μM GSK3β inhibitor: CHIR99021, 1 μM MEK inhibitor: PD0325901, each manufactured by TOCRIS), and thereafter the medium was replaced every day. bFGF and LIF were added every time the medium was changed. As a control, commercially available dog iPS medium (DMEM / F12 medium, manufactured by SIGMA), 20% KSR (GIBCO), 2 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 0.1 mM NEAA, 1.14 μM 2-mercaptoethanol, 4 ng / ml bFGF, and a culture medium containing 1000 U / ml LIF (see Patent Document 1) were used for culture.
２）イヌiPS細胞の継代
MEF細胞上で培養したCEF細胞については、出現した初代コロニーをウイルス感染後8〜20日後に、ガラスピペットの細い先端をバーナーで熱し、45度に曲げたセルナイフを用いて物理的にコロニーを分割し、MEFコートした新たな35mm組織培養用ディッシュに、前記１）でiPS細胞の作製に用いたN2B27培地を用いて播種した。以後、３〜６日間隔で同様にして継代した。また、安定して培養できたコロニーについては、0.05%トリプシン-EDTA溶液を用いた単一細胞での酵素継代を行った。一方、マトリゲル上に播種したCEF細胞についてもウイルス感染後8〜20日後に、同様の方法で継代し、フィーダーレス下で培養を行った。2) Passage of canine iPS cells
For CEF cells cultured on MEF cells, 8 to 20 days after virus infection of the emerged primary colonies, heat the thin tip of a glass pipette with a burner and physically divide the colonies using a cell knife bent at 45 degrees Then, a new 35 mm tissue culture dish coated with MEF was seeded using the N2B27 medium used in the preparation of iPS cells in 1) above. Thereafter, the cells were passaged in the same manner at intervals of 3 to 6 days. Moreover, about the colony which was able to culture | cultivate stably, the enzyme passage by the single cell using 0.05% trypsin-EDTA solution was performed. On the other hand, CEF cells seeded on Matrigel were also passaged in the same manner 8 to 20 days after virus infection, and cultured under feederless conditions.
フィーダー細胞上で培養した場合、培養開始５日後で初代コロニーが出現した。フィーダー細胞上で培養した場合の出現コロニーの顕微鏡観察による画像を図１に示す。図１に示すようにコロニーの形態はドーム方から扁平型で多様であった。また、このときのコロニー作製効率（作製数／培養細胞数）は表２に示すとおりであり、LIFのみを添加した場合はもちろんのこと、bFGFのみを添加したN2B27培地でもiPS細胞が作製された。しかしながら、コロニーの作製効率はbFGFのみを用いた場合は、LIFのみを用いた場合に比べておおよそ２倍の細胞数となり作製効率がよかった。なお、表２の分母には３ディッシュの合計細胞数を表している。 When cultured on feeder cells, primary colonies appeared 5 days after the start of the culture. The image by the microscope observation of the appearance colony at the time of culture | cultivating on a feeder cell is shown in FIG. As shown in FIG. 1, the form of the colony was flat and varied from the dome direction. The colony production efficiency (number of cells produced / cultured cells) at this time is as shown in Table 2. Of course, iPS cells were produced not only when LIF alone was added but also with N2B27 medium containing only bFGF. . However, the colony production efficiency was approximately twice as high as the number of cells when bFGF alone was used, compared to when LIF alone was used. The denominator in Table 2 represents the total number of cells in 3 dishes.
継代では、MEF上でbFGFのみを用いた場合の継代培養数は５０回以上にも及んだが、その他の場合にはわずか数回でしかなかった。15回継代培養した細胞の顕微鏡画像を図２に示す。また、フィーダー細胞を使用せずにマトリゲル上で培養した場合にもコロニーが作製されることが確認された。MEF上で得られたコロニーはMEF上およびマトリゲル上の双方で、酵素処理により単一細胞の継代が可能となった（図３）。しかも、これら単一細胞から５０回以上の長期にわたる継代も行えた。 In subculture, the number of subcultures using bFGF alone on MEF reached more than 50 times, but in other cases it was only a few times. A microscopic image of the cells subcultured 15 times is shown in FIG. It was also confirmed that colonies were produced even when cultured on Matrigel without using feeder cells. Colonies obtained on MEF were able to pass single cells by enzyme treatment on both MEF and Matrigel (FIG. 3). Moreover, it was possible to perform passages of these single cells over 50 times.
〔イヌiPS細胞株の特性解析〕
フィーダー細胞上で得られたイヌiPS細胞株の細胞特性として、未分化マーカーの発現、分化能、核型分析、ＡＬＰ活性及び増殖率を調べた。[Characteristic analysis of canine iPS cell line]
As cell characteristics of the canine iPS cell line obtained on feeder cells, the expression of undifferentiated markers, differentiation ability, karyotype analysis, ALP activity and proliferation rate were examined.
１.未分化マーカーの発現
免疫染色及び量的リアルタイムＰＣＲにより未分化マーカー（OCT3/4、NANOG、SSEA4等)の発現を調べた。
１）免疫染色
３０代継代された細胞株を4％パラホルムアルデヒドで室温、5分間固定した。D-PBS(-)（Nacalai Tesque社製）で洗浄後、0.5％ Triton X-100（Nacalai Tesque社製）で5分間透過処理をした。さらに、1％BSA含有D-PBS(-)中で30分間のブロッキングを行なった。その後、一次抗体としてヤギ抗NANOGポリクロナール抗体（Abcam社製）、ウサギ抗OCT3/4モノクロナール抗体（SANTA CRUZ社製）、マウス抗SSEA4モノクロナール抗体（Millipore社製）をNANOGは100倍希釈、OCT3/4とSSEA4は1,000倍希釈したものを加えて、4℃で一晩培養した。1. Expression of undifferentiated marker The expression of undifferentiated markers (OCT3 / 4, NANOG, SSEA4, etc.) was examined by immunostaining and quantitative real-time PCR.
1) Immunostaining Cell lines that had been passaged for 30 generations were fixed with 4% paraformaldehyde at room temperature for 5 minutes. After washing with D-PBS (-) (Nacalai Tesque), it was permeabilized with 0.5% Triton X-100 (Nacalai Tesque) for 5 minutes. Furthermore, blocking was performed in D-PBS (-) containing 1% BSA for 30 minutes. Then, goat anti-NANOG polyclonal antibody (Abcam), rabbit anti-OCT3 / 4 monoclonal antibody (SANTA CRUZ), mouse anti-SSEA4 monoclonal antibody (Millipore) as NANOG 100-fold diluted as primary antibodies, OCT3 / 4 and SSEA4 were diluted 1,000 times and cultured at 4 ° C overnight.
0.1％BSA／PBSで洗浄後、二次抗体を使用した。二次抗体はNANOGに対してはPE標識ロバ抗ヤギIgG抗体（NOVUS社製)を、OCT3/4に対してはAlexa546標識ヤギ抗マウスIgG抗体（Invitrogen社製）を、SSEA4に対してはCy3標識ヤギ抗マウスIgM抗体（Chemicon社製）をそれぞれ1,000倍に希釈して使用した。30〜60分間染色後、ProLong Gold antifade reagent with DAPI（Invitrogen社製）を用いて封入し、共焦点レーザー顕微鏡（Nikon Clsi型、ニコン社製）にて観察した。その結果を図４に示した。 After washing with 0.1% BSA / PBS, the secondary antibody was used. Secondary antibodies are PE-labeled donkey anti-goat IgG antibody (NOVUS) for NANOG, Alexa546-labeled goat anti-mouse IgG antibody (Invitrogen) for OCT3 / 4, and Cy3 for SSEA4. Labeled goat anti-mouse IgM antibody (Chemicon) was diluted 1,000 times and used. After staining for 30 to 60 minutes, it was encapsulated using ProLong Gold antifade reagent with DAPI (Invitrogen) and observed with a confocal laser microscope (Nikon Clsi type, Nikon). The results are shown in FIG.
２）量的リアルタイムPCRによる未分化マーカーの解析
３０代継代されたイヌiPS細胞から、RNeasy（登録商標） Micro Kit（QIAGEN社製）を用いてtotal RNAの抽出を行った。内因性遺伝子であるcanine OCT3/4、C-MYC、KLF4及びNANOGに対しては、すでに明らかになっているイヌの塩基配列（OCT3/4：Accession：XM_538830.1、C-MYC：Accession：NM_001003246.2、KLF4：Accession：XM_005626996.1、NANOG：Accession：XM_005642425）を基にプライマーを設計した。また、Canine SOX2に対してはLeeら（J Biol Chem. 286:32697-704）が報告したプライマーを、GBX2に対してはVaagsら（Stem Cells. 27:329-40）が報告したプライマーを使用した。外因性遺伝子であるE2A-C-MYCに対して、addgene社から報告されているベクター配列を基にプライマー（センス鎖とアンチセンス鎖）を設計した。2) Analysis of undifferentiated marker by quantitative real-time PCR Total RNA was extracted from canine iPS cells passaged for thirty using RNeasy (registered trademark) Micro Kit (manufactured by QIAGEN). For the endogenous genes canine OCT3 / 4 , C-MYC , KLF4, and NANOG , the already known canine base sequences (OCT3 / 4: Accession: XM_538830.1, C-MYC: Accession: NM_001003246) .2, KLF4: Accession: XM — 005626996.1, NANOG: Accession: XM — 005642425). Also, Lee et al (J Biol Chem 286: 32697-704. ) For Canine SOX2 primers reported, Vaags et (Stem Cells 27: 329-40.) For GBX2 using primers reported by did. For the exogenous gene E2A-C-MYC , primers (sense strand and antisense strand) were designed based on the vector sequence reported by addgene.
次に、抽出したtotal RNAから逆転写反応により、cDNAの合成を行った。合成には、My Cycler（商品名：バイオ・ラッドラボラトリーズ社製）を用いた。合成されたcDNA溶液1μlにつき、SsoFastTM EvaGreen（登録商標） Supermix（Bio Rad社製）5μl、外因性及び内因性未分化マーカーの25pmol/μl センス鎖とアンチセンス鎖のプライマー溶液各0.2μl、を加え、滅菌蒸留水で総量10μlとした。その後、MiniOpticon（登録商標） System（Bio Rad社製）を用いて反応させた。Canine OCT3/4、C-MYC、KLF4、NANOG、SOX2およびGBX2にはCEF、iPS細胞株7:26代継代培養（P26）、iPS細胞株9:P21、iPS細胞株9マトリゲル上培養:P35から得られたmRNAよりcDNAを作製し用いた。E2A-C-MYCには、iPS細胞株9:P15を継代後にドキシサイクリンを除いて２日目、４日目、６日目のコロニーから得られたmRNAからcDNAを作製し用いた。内部標準マーカーとしてβACTINを使用した。ネガティブコントロールにはCEFを使用した。Next, cDNA was synthesized from the extracted total RNA by a reverse transcription reaction. For the synthesis, My Cycler (trade name: manufactured by Bio-Rad Laboratories) was used. For each 1 μl of the synthesized cDNA solution, add 5 μl of SsoFast ™ EvaGreen (registered trademark) Supermix (Bio Rad), 25 μmol / μl of extrinsic and endogenous undifferentiated markers, and 0.2 μl each of the primer solution for the sense strand and the antisense strand. The total volume was made up to 10 μl with sterile distilled water. Then, it was made to react using MiniOpticon (registered trademark) System (manufactured by Bio Rad). Canine OCT3 / 4, C-MYC , KLF4, NANOG, SOX2 and the gbx2 CEF, iPS cell lines 7:26 passages culture (P26), iPS cell lines 9: P21, iPS cell lines 9 matrigel the culture: P35 CDNA was prepared from the mRNA obtained from the above and used. For E2A-C-MYC , iPS cell line 9: P15 was passaged and doxycycline was removed, and cDNA was prepared from mRNA obtained from the second, fourth and sixth day colonies. ΒACTIN was used as an internal standard marker. CEF was used as a negative control.
免疫染色ではNanog、OCT3/4およびSSEA4に陽性であり（図４）、リアルタイムPCRでは内因性未分化マーカーであるC-MYC、OCT3/4、KLF4、GBX2、NANOG、SOX2の各遺伝子の発現がみられた（図５）。また、ドキシサイクリン非存在下では、イヌiPS細胞は増殖を止め、外因性未分化マーカーであるE2A-C-MYCの遺伝子はドキシサイクリン非存在下でその発現は急速に抑制された（図６）。Immunostaining is positive for Nanog, OCT3 / 4, and SSEA4 (Figure 4), and real-time PCR shows the expression of endogenous undifferentiated markers C-MYC , OCT3 / 4 , KLF4 , GBX2 , NANOG , and SOX2 It was seen (FIG. 5). In the absence of doxycycline, canine iPS cells stopped growing and the expression of the exogenous undifferentiated marker E2A-C-MYC was rapidly suppressed in the absence of doxycycline (FIG. 6).
２．分化能
分化能として、胚様体の形成及び外胚葉マーカーであるTUJ1、中胚葉マーカーであるDESMIN、 内胚葉マーカーであるGATA4の遺伝子発現を調べた。
１）胚様体の形成
得られたイヌiPS細胞を、4〜6日間の浮遊培養を行った。なお、培養液はDMEM/F12培地に20％FBS、2 mM L-グルタミン、1％ペニシリン/ストレプトマイシン混合溶液、0.1mM NEAA、1.14μM 2-メルカプトエタノールを添加した培地を使用した。形成した胚様体は、0.1％ゼラチンコートした35mm組織培養用ディッシュで、同様の培地を用いて約1〜2週間接着培養を行った。2. As differentiation potential differentiation ability, which is formed and ectoderm markers embryoid bodies TUJ1, examined mesoderm markers in a Desmin, the gene expression of GATA4 is endoderm markers.
1) Formation of embryoid body The obtained canine iPS cells were cultured in suspension for 4 to 6 days. As the culture solution, a medium obtained by adding 20% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin mixed solution, 0.1 mM NEAA, 1.14 μM 2-mercaptoethanol to DMEM / F12 medium was used. The formed embryoid body was a 35 mm tissue culture dish coated with 0.1% gelatin and cultured for about 1 to 2 weeks using the same medium.
２）遺伝子発現
前記未分化マーカーの測定時と同様の方法で、１）で作製された胚様体からtotal RNAを回収した。回収したRNAから逆転写反応でcDNAを作製してPCRを行った後、2.0％アガロースゲルで、100V 20分間電気泳動した。オリゴヌクレオチドプライマーにはGATA4、DESMINおよびTUJ1を使用した。DESMINに対するプライマーは、公知であるイヌの塩基配列（DESMIN：Accession：NM_001012394.1）を基に設計した。GATA4に対するプライマーはVaagsら（Stem Cells. 27:329-40）が報告したプライマーを使用した。TUJ1に対するプライマーはHayesら（Stem Cells. 26:465-73）が報告したプライマーを使用した。2) Gene expression Total RNA was recovered from the embryoid body prepared in 1) in the same manner as in the measurement of the undifferentiated marker. A cDNA was prepared from the recovered RNA by reverse transcription reaction and subjected to PCR, and then electrophoresed on a 2.0% agarose gel at 100 V for 20 minutes. GATA4 , DESMIN and TUJ1 were used as oligonucleotide primers. Primers for Desmin, the base sequence of canine known (DESMIN: Accession: NM_001012394.1) were designed based on. As a primer for GATA4, a primer reported by Vaags et al. (Stem Cells. 27: 329-40) was used. As a primer for TUJ1, a primer reported by Hayes et al. (Stem Cells. 26: 465-73) was used.
得られたイヌiPS細胞を4〜6日間浮遊培養すると図７（Ａ）に示すように胚様体が形成された。また、これらの胚体様を接着培養することで、種々の細胞へと分化し、これらの分化細胞では、外胚葉マーカーであるTUJ1、中胚葉マーカーであるDESMIN、 内胚葉マーカーであるGATA4の遺伝子発現がみられた（同図（Ｂ））。When the obtained canine iPS cells were cultured in suspension for 4 to 6 days, embryoid bodies were formed as shown in FIG. 7 (A). Further, by bonding culturing these embryos-like, differentiate into various cells, these differentiated cells, ectodermal marker TUJ1, a mesoderm marker Desmin, GATA4 gene is endodermal marker Expression was observed (Fig. (B)).
３．核型分析
得られたイヌiPS細胞コロニーを分割せずに、0.1％ゼラチンコートした35mm組織培養ディッシュに継代した。３５代継代した細胞を簡易ヘマカラーキット（Merk chemicals社製、Darmstad、Germany）を用いてギムザ染色して、光学顕微鏡下で観察した。図８に示されたように、７８本のイヌ常染色体２ｎが観察され、性染色体がＸＹ型で観察された。3. Karyotype analysis The resulting canine iPS cell colonies were subdivided into 35 mm tissue culture dishes coated with 0.1% gelatin. Cells passaged for 35 passages were stained with Giemsa using a simple hema color kit (Merk chemicals, Darmstad, Germany) and observed under an optical microscope. As shown in FIG. 8, 78 canine autosomes 2n were observed, and sex chromosomes were observed in the XY type.
４．ＡＬＰ活性
得られたイヌiPS細胞株（フィーダー細胞上で培養された細胞株）について、bFGF濃度を4,10,20,100ng/mlで添加し、未分化状態に対する影響を未分化マーカーであるアルカリフォスファターゼ（ALP）活性を指標にして調べた。ALP活性はStemgent（登録商標） Alkaline Phosphatase Staining Kit II（Stemgent社製）を用いて酵素染色した後に判定した。その結果を図９に示す。この結果から、bFGF濃度を上げることで陽性を示す細胞数が増加し、未分化状態の維持が向上することが示唆された。図９はマトリゲル上で培養したイヌiPS細胞の場合を示すが、ＭＥＦ上での培養では100ng/mlのbFGFを含む培地で培養することが、未分化状態の維持には好ましいと言える（図示せず）。4). ALP activity
For the obtained canine iPS cell line (cell line cultured on feeder cells), bFGF concentration was added at 4, 10, 20, 100 ng / ml, and the effect on the undifferentiated state was determined by alkaline phosphatase (ALP), an undifferentiated marker. ) The activity was used as an index. ALP activity was determined after enzyme staining using Stemgent (registered trademark) Alkaline Phosphatase Staining Kit II (manufactured by Stemgent). The result is shown in FIG. This result suggests that increasing the bFGF concentration increases the number of positive cells and improves the maintenance of the undifferentiated state. FIG. 9 shows the case of canine iPS cells cultured on Matrigel. In culture on MEF, it can be said that culturing in a medium containing 100 ng / ml bFGF is preferable for maintaining an undifferentiated state (not shown). )
５．増殖率測定
得られたイヌiPS細胞株（フィーダー細胞上で培養された細胞株）について、bFGF濃度を4,10,20,100ng/mlで添加してマトリゲル上で培養し、その増殖性に対する影響を調べた。単一細胞継代後96時間における増殖率をワケンカウンター（和研薬株式会社製）にて測定した。その結果を図１０に示す。4ng/ml以上の添加はイヌiPS細胞の増殖率に影響を与えることはなかった。また、ＭＥＦ上での培養でも4ng/ml以上の添加はイヌiPS細胞の増殖率に影響を与えることはなかった（図示せず）。5. Growth rate measurement The obtained canine iPS cell line (cell line cultured on feeder cells) was added at bFGF concentration of 4, 10, 20, 100 ng / ml and cultured on Matrigel. Examined. The proliferation rate at 96 hours after single cell passage was measured with a Waken counter (manufactured by Wakken Pharmaceutical Co., Ltd.). The result is shown in FIG. Addition of 4 ng / ml or more did not affect the proliferation rate of canine iPS cells. In addition, even when cultured on MEF, addition of 4 ng / ml or more did not affect the proliferation rate of canine iPS cells (not shown).
６．まとめ
上記のように、ドキシサイクリン誘導性レンチウイルスベクターを用いて、N2B27培地にbFGFのみを添加する培地により、長期継代可能であり、かつ、酵素分割で単一細胞継代が可能なイヌiPS細胞を作製できた。このiPS細胞は、フィーダー細胞上ではもちろんのことフィーダーレスでも維持することが可能であり、凍結融解によってもその形態及び増殖能に変化はみられなかった（図示せず）。マウスiPS細胞などの未分化な幹細胞は単一細胞継代で継代できることが報告されていることから、得られたイヌiPS細胞はマウス型iPS細胞と同様に未分化度が高いと考えられる。6). Summary As described above, canine iPS cells that can be passaged for a long time using a doxycycline-inducible lentiviral vector and a medium in which only bFGF is added to N2B27 medium, and that can be subcultured by single cells by enzyme splitting. Could be made. This iPS cell can be maintained on a feeder cell as well as without a feeder, and its morphology and proliferation ability were not changed by freezing and thawing (not shown). Since it has been reported that undifferentiated stem cells such as mouse iPS cells can be passaged by single cell passage, the obtained canine iPS cells are considered to have a high degree of undifferentiation in the same manner as mouse-type iPS cells.
〔間葉系幹細胞への分化誘導〕
次に実施例１で作製したイヌiPS細胞から間葉系幹細胞（MSC）へ分化誘導し、その形態的構造や機能について解析を行った。[Induction of differentiation into mesenchymal stem cells]
Next, differentiation was induced from canine iPS cells prepared in Example 1 to mesenchymal stem cells (MSC), and the morphological structure and function thereof were analyzed.
１.イヌiPS細胞からMSCへの分化誘導
イヌiPS細胞を実施例１と同様にして浮遊培養によって胚様体を形成させ、4日後に60mm細胞培養用ディッシュ(IWAKI社製)接着培養した。接着時に培地をMEM-α Glutamax（GIBCO社製）に1％ペニシリン/ストレプトマイシン混合溶液、1％MEM NEAA、10％FBS、および8ng/ml bFGFを添加したMSC培地に変更し、37℃、5％ CO2下で培養した。培地交換は2日に1回行ない、bFGFの添加は毎日行なった。次に70〜80％コンフルエントまで増殖した細胞を0.05％トリプシン-EDTA溶液で処理して細胞を回収し、新しいディッシュに播種することで継代培養を行った。また、対照として、成犬由来の骨髄間質細胞（BMSC）を同様にして継代培養を行った。1. Induction of differentiation from canine iPS cells to MSCs Embryoid bodies were formed from canine iPS cells by suspension culture in the same manner as in Example 1, and after 4 days, a 60 mm cell culture dish (manufactured by IWAKI) was attached and cultured. At the time of adhesion, the medium was changed to MSC medium supplemented with 1% penicillin / streptomycin mixed solution, 1% MEM NEAA, 10% FBS, and 8ng / ml bFGF to MEM-α Glutamax (GIBCO), 37 ° C, 5% CO 2 and cultured under. The medium was changed once every two days, and bFGF was added every day. Next, cells grown to 70-80% confluence were treated with a 0.05% trypsin-EDTA solution to recover the cells, and subcultured by seeding in a new dish. In addition, as a control, bone marrow stromal cells (BMSC) derived from adult dogs were subcultured in the same manner.
接着培養で得られたMSCの顕微鏡観察における画像を図１１に、また、継代培養したMSCの顕微鏡観察における画像を図１２に示す。図１２に示す点線囲みの部分に、胚様体の周囲に分化してできたMSCが観察された。また、図１２に示すように、成犬由来のBMSCの継代培養と同様に、継代を重ねても得られたイヌiPS細胞由来MSCの継代においても変化は見られなかった。 FIG. 11 shows an image of the MSC obtained by adhesion culture in the microscopic observation, and FIG. 12 shows an image of the subcultured MSC in the microscopic observation. MSCs that were differentiated around the embryoid body were observed in the area surrounded by the dotted line shown in FIG. Moreover, as shown in FIG. 12, the change was not seen also in the passage of the canine iPS cell origin MSC obtained by repeated passage similarly to the passage culture of BMSC derived from an adult dog.
２.フローサイトメトリーによる細胞表面マーカーの解析
分化誘導したMSCを0.05％トリプシン-EDTA溶液で処理して細胞を回収し、PE標識マウス抗イヌCD34抗体（Becton Dickinson Bioscience社製）、FITC標識ラット抗イヌCD45抗体（AbD Serotec社製）、PE標識ラット抗イヌCD44抗体（Becton Dickinson Bioscience社製）及びビオチン化マウス抗イヌCD90抗体（AbD Serotec社製）を加え、氷上で30分静置して反応させた。さらに抗CD90抗体に対しては、静置後、浮遊細胞をFCN液（D-PBS(-)に2％FBSと1mg/ml sodium azideを添加した溶液）にて洗浄した後、streptavidin-PE-Cy5 (Becton Dickinson Bioscience社製)を加えて氷上で30分静置して反応させた。染色後、各細胞表面マーカーの発現をフローサイトメーター（FACSCaliburTM、Becton Dickinson社製）を用いて測定した。また、対照としてイヌ骨髄間質細胞由来MSC(BMSC)についても測定を行った。得られたイヌiPS細胞はBMSCと同様に、CD90とCD44に陽性であり（図１３）、CD45とCD34陰性であった（図１４）。2. Analysis of Cell Surface Markers by Flow Cytometry Differentiated MSCs were treated with 0.05% trypsin-EDTA solution to collect cells, PE-labeled mouse anti-canine CD34 antibody (Becton Dickinson Bioscience), FITC-labeled rat anti-antibody Canine CD45 antibody (AbD Serotec), PE-labeled rat anti-canine CD44 antibody (Becton Dickinson Bioscience) and biotinylated mouse anti-canine CD90 antibody (AbD Serotec) were added and allowed to stand for 30 minutes on ice. I let you. Furthermore, for anti-CD90 antibody, after standing, the suspended cells were washed with FCN solution (a solution of 2% FBS and 1 mg / ml sodium azide added to D-PBS (-)), and then streptavidin-PE- Cy5 (Becton Dickinson Bioscience) was added and allowed to stand for 30 minutes on ice for reaction. After staining, the expression of each cell surface marker was measured using a flow cytometer (FACSCalibur ™, manufactured by Becton Dickinson). As a control, measurement was also performed on canine bone marrow stromal cell-derived MSC (BMSC). The obtained canine iPS cells were positive for CD90 and CD44 (FIG. 13) and negative for CD45 and CD34 (FIG. 14), as in BMSC.
３.未分化マーカーおよび神経系マーカーに対する免疫染色
分化誘導したMSCを4％パラホルムアルデヒド中で10分間固定した。D-PBS(-)(Nacalai Tesque社製)で洗浄後、0.5％Triton X-100（Nacalai Tesque社製）で5分間透過処理をした。さらに、1％BSA含有D-PBS(-)中で30分間のブロッキングを行なった。その後、一次抗体として、ウサギ抗OCT3/4ポリクローナルIgG抗体（Santa Cruz社製）、ヤギ抗NANOGポリクロナール抗体（Abcam社製）、ウサギ抗NESTINポリクローナル抗体（Millipore社製）、及びウサギ抗glial fibrillary acidic protein（GFAP）ポリクローナル抗体（Millipore社製）を、抗NANOG抗体は100倍希釈、他は1000倍希釈したものを室温で60分間反応させた。二次抗体は、OCT3/4、NESTIN及びGFAPに対しては、Alexa 546標識抗ウサギIgG抗体（Invitrogen社製）を1000倍希釈、NANOGに対してはPE標識ロバ抗ヤギIgG抗体（NOVUS社製）を100倍希釈して、室温で30分間反応させた。染色後、ProLong（登録商標） Gold antifade reagent with DAPI（Invitrogen社製）を用いて封入し、共焦点レーザー顕微鏡（Nikon clsi型：ニコン社製）にて観察した。イヌiPS細胞由来のMSCは神経幹細胞マーカーであるNESTINに陽性であり、グリア細胞マーカーであるGFAPに陰性であった（図１５）。さらに未分化マーカーであるOCT3/4及びNANOGに陰性を示した（図１６）。3. Immunostaining for undifferentiated markers and nervous system markers Differentiated MSCs were fixed in 4% paraformaldehyde for 10 minutes. After washing with D-PBS (-) (Nacalai Tesque), it was permeabilized with 0.5% Triton X-100 (Nacalai Tesque) for 5 minutes. Furthermore, blocking was performed in D-PBS (-) containing 1% BSA for 30 minutes. Then, as primary antibodies, rabbit anti-OCT3 / 4 polyclonal IgG antibody (Santa Cruz), goat anti-NANOG polyclonal antibody (Abcam), rabbit anti-NESTIN polyclonal antibody (Millipore), and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (manufactured by Millipore) was reacted for 60 minutes at room temperature with anti-NANOG antibody diluted 100-fold and others diluted 1000-fold. Secondary antibodies are 1000-fold diluted Alexa 546-labeled anti-rabbit IgG antibody (Invitrogen) for OCT3 / 4, NESTIN and GFAP, and PE-labeled donkey anti-goat IgG antibody (NOVUS) for NANOG. ) Was diluted 100 times and allowed to react at room temperature for 30 minutes. After staining, it was encapsulated using ProLong (registered trademark) Gold antifade reagent with DAPI (Invitrogen) and observed with a confocal laser microscope (Nikon clsi type: Nikon). MSCs derived from canine iPS cells were positive for the neural stem cell marker NESTIN and negative for the glial cell marker GFAP (FIG. 15). Furthermore, it was negative for undifferentiated markers OCT3 / 4 and NANOG (FIG. 16).
〔骨様細胞への分化誘導〕
分化誘導して得られたMSCから骨様細胞へ分化誘導を行った。得られたMSCを12wellプレート（IWAKI社製）に細胞数が4×103個/cm2となるように播種した。1日後から培地を、DMEM培地（1.0g/L glucose）（Nacalai Tesque社製）に10％FBS、1％ペニシリン/ストレプトマイシン混合溶液、50μMアスコルビン酸（和光純薬工業社製）、10mM β-glycerophosphate（Nacalai Tesque社製）、100nM Dexamethasone（SIGMA社製）を添加したものに変更した。培地交換は2日に1回半量行ない、28日間37℃、5％CO2下で培養した。分化誘導した細胞について、骨芽細胞で陽性となるアルカリフォスファアターゼ（ALP）染色を、Stemgent Alkaline Phosphatase Staining Kit II（Stemgent社製）を用いて行った。また、骨芽細胞による石灰沈着の確認を行なうため、Von Kossa染色を行なった。細胞を95％エタノール（SIGMA社製）で15分間室温にて固定した。超純水で洗浄後、5％硝酸銀（和光純薬工業社製）溶液を加え、室温、蛍光灯下で1〜2時間反応させた。次に、超純水で洗浄後、5％チオ硫酸ナトリウム（和光純薬工業社製）溶液を加えて3分間反応させ、洗浄後、光学顕微鏡によって観察した。分化誘導された細胞（図１７(Ａ））は、骨芽細胞マーカーであるALP染色に陽性を示し（同図(Ｂ)）、VonKossa染色に陽性を示した（同図(Ｃ)）。[Induction of differentiation into bone-like cells]
Differentiation was induced from MSCs obtained by differentiation induction to bone-like cells. The obtained MSC was seeded on a 12-well plate (manufactured by IWAKI) so that the number of cells was 4 × 10 3 cells / cm 2 . After 1 day, the medium was added to DMEM medium (1.0 g / L glucose) (Nacalai Tesque), 10% FBS, 1% penicillin / streptomycin mixed solution, 50 μM ascorbic acid (Wako Pure Chemical Industries), 10 mM β-glycerophosphate. (Nacalai Tesque) and 100nM Dexamethasone (SIGMA) were added. The medium was exchanged half a day once every two days, and cultured for 28 days at 37 ° C. and 5% CO 2 . The differentiation-induced cells were subjected to alkaline phosphatase (ALP) staining that became positive in osteoblasts using Stemgent Alkaline Phosphatase Staining Kit II (manufactured by Stemgent). In addition, Von Kossa staining was performed to confirm calcification by osteoblasts. The cells were fixed with 95% ethanol (SIGMA) for 15 minutes at room temperature. After washing with ultrapure water, a 5% silver nitrate solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted at room temperature under a fluorescent lamp for 1-2 hours. Next, after washing with ultrapure water, a 5% sodium thiosulfate solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added, reacted for 3 minutes, washed, and then observed with an optical microscope. Differentiated cells (FIG. 17 (A)) were positive for ALP staining, which is an osteoblast marker (FIG. (B)), and positive for VonKossa staining (FIG. (C)).
〔神経様細胞への分化誘導〕
分化誘導して得られたMCSから神経様細胞に分化誘導した。得られたMSCをアストロサイト培養上清添加培地（Neuron Culture Medium）（住友ベークライト社製）に20ng/ml bFGFを添加した培養液の入った無処理60mmディッシュ（IWAKI社製）に移し、37℃、5％CO2下で培養した。培地交換は2日に1回、半量行ない、bFGFの添加は毎日行なった。10日間の培養後、形成した浮遊細胞塊を10μg/mlラミニン（AGCテクノグラス社製）をコートしたpoly-L-lysine-coated 35mmディッシュ（IWAKI社製）に移し、Neurobasal Mediumに2mM L-glutamine、1％ペニシリン/ストレプトマイシン混合溶液、B27 Supplement（50x、GIBCO社製）を添加した培地にて37℃、5％CO2下で接着培養した。培地交換は2日に1回半量行ない、毎日、終濃度20ng/mlのbFGFおよびヒト組換え上皮細胞増殖因子（EGF、R&D Systems社製）を添加した。得られたMSCを神経幹細胞分化培地で培養することで、Neuroshpereが得られた（図１８(Ａ)）。このNeurosphereを接着培養することで、Neurosphereの周囲から神経幹細胞様の形態を示した細胞が出現した（図１８(Ｂ））。[Induction of differentiation into nerve-like cells]
Differentiation was induced from the MCS obtained by inducing differentiation into nerve-like cells. The obtained MSC was transferred to an untreated 60 mm dish (IWAKI) containing a culture solution containing 20 ng / ml bFGF in an astrocyte culture supernatant-supplemented medium (Neuron Culture Medium) (manufactured by Sumitomo Bakelite). Incubated under 5% CO 2 . Medium exchange was performed once every two days in half volume, and bFGF was added every day. After culturing for 10 days, the formed floating cell mass was transferred to a poly-L-lysine-coated 35 mm dish (IWAKI) coated with 10 μg / ml laminin (AGC Techno Glass), and 2 mM L-glutamine in Neurobasal Medium. Adhesion culture was performed at 37 ° C. and 5% CO 2 in a medium supplemented with 1% penicillin / streptomycin mixed solution and B27 Supplement (50 ×, manufactured by GIBCO). The medium was exchanged once and twice every two days, and bFGF and human recombinant epidermal growth factor (EGF, manufactured by R & D Systems) at a final concentration of 20 ng / ml were added every day. Neuroshpere was obtained by culturing the obtained MSC in a neural stem cell differentiation medium (FIG. 18 (A)). By adhesion culture of this Neurosphere, cells showing a neural stem cell-like morphology appeared from around the Neurosphere (FIG. 18B).
〔MSCのiPS細胞への再初期化〕
実施例２においてイヌiPS細胞を分化誘導することで得られたMSCを10回以上継代したのち、再び実施例１のbFGF添加N2B27培地を用いたイヌiPS細胞の作製方法と同様にして、ドキシサイクリンを培養液に添加することで、一度分化誘導させたMSCから再びイヌiPS細胞コロニーを作製することができた（図示せず）。しかも、MSCに核初期化因子を再度導入することなくiPS細胞への再初期化が行えた。[Re-initialization of MSC to iPS cells]
After MSC obtained by inducing differentiation of canine iPS cells in Example 2 is passaged 10 times or more, doxycycline is again produced in the same manner as in the method for producing canine iPS cells using the N2B27 medium supplemented with bFGF in Example 1. Was added to the culture medium, and canine iPS cell colonies could be produced again from MSCs once induced to differentiate (not shown). Moreover, iPS cells could be re-initialized without reintroducing nuclear reprogramming factors into the MSC.
本発明によれば、簡単な培地組成で、長期にわたり継代が可能なイヌiPS細胞が作製される。 According to the present invention, canine iPS cells that can be passaged over a long period of time with a simple medium composition are produced.
Claims (21)
イヌ体細胞に核初期化因子を導入する工程と、
核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子の存在下で分化多能性維持因子を含まない培養液を用いて培養をする工程を含む方法。A method of producing a canine iPS cell line from canine somatic cells,
Introducing nuclear reprogramming factors into canine somatic cells;
A method comprising culturing canine somatic cells into which a nuclear reprogramming factor has been introduced, using a culture solution containing no differentiation pluripotency maintenance factor in the presence of a basic fibroblast growth factor.
イヌ体細胞に核初期化因子を導入する工程と、
核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子のみを含むN2B27培地を用いて培養をする工程を含む方法。A method of producing a canine iPS cell line from canine somatic cells,
Introducing nuclear reprogramming factors into canine somatic cells;
A method comprising culturing canine somatic cells into which a nuclear reprogramming factor has been introduced using an N2B27 medium containing only basic fibroblast growth factor.
核初期化因子を導入したイヌ体細胞を、塩基性線維芽細胞増殖因子の存在下で分化多能性維持因子を含まない培養液を用いて培養して、イヌiPS細胞株を得る工程と、
培養で得られた細胞を分化誘導する工程を含む、イヌ体細胞の製造方法。Introducing nuclear reprogramming factors into canine somatic cells;
Culturing canine somatic cells into which a nuclear reprogramming factor has been introduced using a culture solution that does not contain a differentiation pluripotency maintenance factor in the presence of a basic fibroblast growth factor to obtain a canine iPS cell line;
A method for producing canine somatic cells, comprising a step of inducing differentiation of cells obtained by culture.
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| PCT/JP2016/068194 WO2016204298A1 (en) | 2015-06-19 | 2016-06-19 | METHOD FOR PRODUCING CANINE iPS CELLS |
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| CN111534481A (en) * | 2020-05-18 | 2020-08-14 | 扬州大学 | Method for improving efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into iPS cells |
| EP4170018A1 (en) | 2021-10-20 | 2023-04-26 | Roslin Technologies Limited | Ipsc induction and progeny derivation |
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