JPH08116966A - Bovine embryonic stem cell and its preparation - Google Patents
Bovine embryonic stem cell and its preparationInfo
- Publication number
- JPH08116966A JPH08116966A JP6284153A JP28415394A JPH08116966A JP H08116966 A JPH08116966 A JP H08116966A JP 6284153 A JP6284153 A JP 6284153A JP 28415394 A JP28415394 A JP 28415394A JP H08116966 A JPH08116966 A JP H08116966A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- bovine
- blastocyst
- cell
- chromosome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明はウシ胚性幹細胞（Embryo
nic Stem Cells以下ＥＳＣと記す）およびその作製方法
に関する。本発明で得られる細胞株は、継代培養が可能
なため大量に増殖させることが可能であり、キメラウシ
の作製や、核移植による個体発生などに使用可能とな
る。The present invention relates to bovine embryonic stem cells (Embryo
nic Stem Cells (hereinafter referred to as ESC) and a method for producing the same. Since the cell line obtained in the present invention can be subcultured, it can be proliferated in a large amount and can be used for production of chimeric cows, ontogeny by nuclear transfer, and the like.
【０００２】[0002]
【従来の技術】従来のＥＳＣはマウスやラットなどの実
験動物由来のもののみが知られている。このＥＳＣは、
初期発生過程で見られる内部細胞塊から原始外胚葉まで
の細胞に似た性質を保持したまま培養下で継代維持でき
る細胞株である。近年、哺乳動物の遺伝子操作や、キメ
ラ動物の作出に使用する目的で、マウス以外の哺乳動物
のＥＳＣを作製することが検討されている。特表平４─
５００９０９号公報には家畜からＥＳＣを誘導する方法
が開示されているが、この公報に記載されたウシ由来の
細胞は継代培養期間が短く、真のＥＳＣとは言いがた
く、その細胞学的特徴も明らかでない。この先行文献で
は真のＥＳＣはブタ由来の細胞について記載されている
のみである。またこの公報に記載された方法を追試して
もウシ由来のＥＳＣを得ることができなかった。特表平
３─５０３２４１号公報、特開平５─３０４９５１号お
よび特開昭６─３８７４２号公報にはＥＳＣの作製方法
や培養方法が開示されているがいずれもマウスやラット
由来のＥＳＣであって、ウシのＥＳＣではない。このよ
うに従来の技術では、ウシＥＳＣを得ることは不可能で
あり、ウシＥＳＣがどのような細胞学的性質を有するも
のなのか推測されているだけである。ウシＥＳＣが容易
に入手可能となることにより、クローンウシの作出や、
特定遺伝子を導入したウシの作製が容易となるなど家畜
改良の面からもその要求が大きい。2. Description of the Related Art Only conventional ESCs derived from experimental animals such as mice and rats are known. This ESC is
It is a cell line that can be passage-maintained in culture while maintaining the properties similar to those of cells from the inner cell mass to the primitive ectoderm that are found in the early developmental process. In recent years, production of ESCs of mammals other than mice has been studied for the purpose of use in genetic engineering of mammals and production of chimeric animals. Special table flat 4-
500909 discloses a method for inducing ESCs from livestock, but the bovine-derived cells described in this publication have a short subculture period, and it is hard to say that they are true ESCs. The characteristics are also unclear. In this prior document, true ESC is only described for cells of porcine origin. Further, it was not possible to obtain bovine-derived ESC even when the method described in this publication was additionally tested. Japanese Patent Application Laid-Open No. 3-503241, Japanese Patent Application Laid-Open No. 5-304951, and Japanese Patent Application Laid-Open No. 6-38742 disclose a method for producing ESC and a method for culturing ESC, which are all derived from mouse or rat. , Not bovine ESC. As described above, it is impossible to obtain bovine ESC by the conventional technique, and it is only speculated what kind of cytological property bovine ESC has. The availability of bovine ESC facilitates the production of cloned bovine,
There is a great demand for improving livestock, such as facilitating production of cattle into which a specific gene has been introduced.
【０００３】[0003]
【発明が解決しようとする課題】本発明は従来得ること
のできなかったウシＥＳＣを提供することを課題とす
る。またさらに、ウシＥＳＣの細胞学的な特徴を明らか
にし、この細胞学的特徴を有するＥＳＣを作製する方法
を提供することを課題とする。SUMMARY OF THE INVENTION An object of the present invention is to provide a bovine ESC which has hitherto been unavailable. Furthermore, it is an object of the present invention to clarify the cytological characteristics of bovine ESC and to provide a method for producing ESCs having this cytological characteristic.
【０００４】[0004]
【課題を解決するための手段】本発明は、従来の技術で
は得ることのできなかったウシＥＳＣ細胞を初めて提供
するものである。小動物のＥＳＣは現在の技術では比較
的容易に得ることができる。これは例えばマウスの授精
卵が胚盤胞にまで成長した段階で回収し、この胚盤胞を
体外で培養し、内部細胞塊（inner cell mass : 以下Ｉ
ＣＭ）から継代培養可能な未分化の細胞を選択すること
で得られる。しかしウシについてはマウスの場合と同じ
操作をおこなってもＥＳＣが得られない。何らかの工夫
が必要である。この問題を解決するために本発明におい
ては、胚盤胞から栄養外胚葉を含む状態でＩＣＭを取り
出し、これを培養に付し、栄養外胚葉とＩＣＭが共存す
る条件下で培養を行った。胚盤胞の採取は従来の技術と
同様にして授精したウシの子宮洗浄を行うことによって
得ることができる。あるいは、ウシの卵巣から分離した
未成熟卵子を成熟させた後、体外授精を行い、体外で培
養して形成させた胚盤胞等が比較的容易に入手可能であ
る。あるいは、比較的回収数は少ないが、正常の性周期
のウシの授精を確認後、子宮洗浄を行って得ても良い。
培養にあたっては、マイトマイシン処理をしたＳＴＯ細
胞株をフィーダー細胞として用いて、その上で培養す
る。培養液は通常、胚盤胞の培養に用いる培養液を使用
する。例えば特開表３−５０３８４１号公報に記載され
ているアミノ酸を補足した培養液、あるいは市販の細胞
培養液が使用可能であり、たとえばダルベッコＭＥＭな
どを例示できる。この培養液の成分として特にウシ胎児
血清（ＦＣＳ）、２−メルカプトエタノール（２Ｍ
Ｅ）、白血病阻害因子（ＬＩＦ）、塩基性線維細胞増殖
因子（ｂＦＧＦ）が必須成分である。各成分は、それぞ
れＦＣＳ１０％、２ＭＥ0.1 mM 1 ＬＩＦ１０3 unit／m
l、ｂＦＧＦ 10 ng／mlの濃度とすることが好ましい。The present invention provides, for the first time, bovine ESC cells which could not be obtained by conventional techniques. Small animal ESCs are relatively easy to obtain with current technology. For example, this is collected at the stage when a fertilized egg of a mouse has grown to a blastocyst, and this blastocyst is cultured in vitro to give an inner cell mass (hereinafter referred to as I
It is obtained by selecting undifferentiated cells that can be subcultured from CM). However, ESC cannot be obtained for cows even if the same operation as for mice is performed. Some kind of ingenuity is required. In order to solve this problem, in the present invention, ICM was taken out from a blastocyst in a state containing trophectoderm, subjected to culture, and cultured under the condition that trophectoderm and ICM coexist. The blastocyst can be obtained by washing the uterus of a bovine inseminated in the same manner as in the conventional technique. Alternatively, blastocysts and the like formed by in vitro insemination after culturing the immature ova isolated from bovine ovaries and culturing them in vitro are relatively easily available. Alternatively, it may be obtained by performing uterine lavage after confirming the insemination of a cow having a normal sexual cycle, although the number of recovered animals is relatively small.
In culturing, the STO cell line treated with mitomycin is used as a feeder cell, and culturing is performed thereon. As the culture medium, a culture medium used for culturing blastocysts is usually used. For example, a culture medium supplemented with an amino acid described in JP-A-3-503841 or a commercially available cell culture medium can be used, and examples thereof include Dulbecco's MEM. As components of this culture solution, especially fetal calf serum (FCS), 2-mercaptoethanol (2M
E), leukemia inhibitory factor (LIF), and basic fibrocyte growth factor (bFGF) are essential components. Each component is 10% FCS, 2ME 0.1 mM 1 LIF10 3 unit / m
It is preferable that the concentration is l, bFGF 10 ng / ml.
【０００５】この培養液を用いて培養すると細胞がフィ
ーダー層に接着し、次いで伸展してくる。この伸展した
細胞がコロニー形成するまで培養液を３〜７日ごとに交
換しする。形成したコロニーは栄養外胚葉とＩＣＭの混
合コロニーである。このコロニーを顕微鏡下で観察しな
がら細胞の形態から栄養外胚葉とＩＣＭの存在を確認す
る。コロニーはトリプシン処理で細胞を分散させ、新し
いフィーダー細胞を形成させた２４穴プレートに植え継
ぎを行う。植え継ぎを繰り返すことにより増殖を停止す
るものが増加する。これは培養にともなって、分化した
細胞が出現し、これらの分化細胞の増殖がコロニーを形
成しなくなるからである。When cells are cultured using this culture medium, the cells adhere to the feeder layer and then spread. The culture medium is exchanged every 3 to 7 days until the spread cells colonize. The formed colony is a mixed colony of trophectoderm and ICM. The presence of trophectoderm and ICM is confirmed from the cell morphology by observing this colony under a microscope. The colonies are treated with trypsin to disperse the cells, and subcultured on a 24-well plate in which new feeder cells are formed. Repeated subculture increases the number of those that stop growing. This is because differentiated cells appear with culture and the proliferation of these differentiated cells does not form colonies.
【０００６】継代を４代以上繰り返し、安定な未分化細
胞の形態をとる細胞が形成したコロニーに顕微鏡観察、
染色体分析、ＳＳＥＡ−１（Stage-specific embryonic
antigen-1) またはＬｅX 抗原の存在の３項目の試験を
おこなってＥＳＣであることを確認する。ＥＳＣは細胞
を継代培養することができると同時に、その形態を観察
するとＩＣＭと判断できる形態学的特徴を有している。
即ち正常な未分化幹細胞としての形態を有していること
である。このため、細胞核型と形態の観察はＩＣＭとし
ての特徴を確認する上で必須である。本発明で得られる
ウシＥＳＣは、単独でフィーダー細胞上で培養を行った
時、コロニーを形成し、分化した細胞を含まない。また
形態学的にはＩＣＭと同一の形態を有している。この形
態学的な特徴は、仁を含む大きな核を有し、細胞質中に
は液胞状の構造が存在し、細胞間の境界が明瞭である。Microscopic observation of colonies formed by cells having stable undifferentiated cell morphology after repeated passage for 4 generations or more,
Chromosome analysis, SSEA-1 (Stage-specific embryonic
Confirm the presence of ESC by conducting a three-item test for the presence of antigen-1) or Le X antigen. ESC is capable of subculturing cells, and at the same time has morphological characteristics that can be judged as ICM when its morphology is observed.
That is, it has a morphology as a normal undifferentiated stem cell. Therefore, observation of cell karyotype and morphology is essential for confirming the characteristics of ICM. The bovine ESC obtained in the present invention forms a colony when cultured alone on feeder cells and does not contain differentiated cells. Morphologically, it has the same form as ICM. This morphological feature has a large nucleus containing nucleoli, a vacuolar structure exists in the cytoplasm, and the boundaries between cells are clear.
【０００７】また細胞核型は以下のような特徴を有して
いる。本細胞の細胞核型は、２倍体であり 2n=60の正常
な染色体数を示している。またブロモデオキシリウリジ
ン (ＢｒｄＵ) 取り込み後染色体の観察を行うとＸ染色
体が不活性化した細胞の比率が極めて低い値を示す。こ
の染色体の活性状態の判定は、複製時期を指標に、アク
リジンオレンジ染色をおこなって決定するものである。
哺乳類の雌では２本のＸ染色体（ＸＸ）のうち１本の活
性を全体に抑制して、雌雄間の遺伝子発現を調節する。
これは、通常胚発生の初期に起こるが、ＩＣＭ細胞では
不活性化されておらず、マウスなどのＥＳＣも同様であ
る。従ってＸ染色体の染色結果、常染色体と同様に染色
されるものを活性染色体、著しく違った状態に染色され
るものを不活性化染色体とすると、本発明のウシＥＳＣ
のＸ染色体は不活性化されていないと判断できる。この
ような特徴からも、本発明細胞は分化していないＩＣＭ
の特徴を有しているといえる。The cell karyotype has the following characteristics. The cell karyotype of this cell is diploid and shows a normal chromosome number of 2n = 60. In addition, when the chromosome is observed after incorporation of bromodeoxyliuridine (BrdU), the ratio of cells in which the X chromosome is inactivated shows a very low value. The determination of the active state of this chromosome is performed by acridine orange staining using the replication time as an index.
In mammalian females, the activity of one of the two X chromosomes (XX) is totally suppressed to regulate gene expression between males and females.
This usually occurs in the early stage of embryonic development, but it is not inactivated in ICM cells, and ESCs of mice and the like are the same. Therefore, as a result of staining the X chromosome, if the one that stains similarly to the autosomal chromosome is the active chromosome and the one that stains in a significantly different state is the inactivated chromosome, the bovine ESC of the present invention.
It can be judged that the X chromosome of is not inactivated. From such characteristics as well, the cells of the present invention are not differentiated ICM
It can be said that it has the characteristics of.
【０００８】本発明の細胞が未分化のＥＳＣであること
の基本的証明として、未分化特異的マーカーの発現を挙
げることができる。これは未分化細胞に特異的に発現さ
れるマーカーの一つであるＳＳＥＡ−１（Stage-specif
ic embryonic antigen-1) あるいはＬｅx を発現してい
ることを確認することによって行うことができる。ＳＳ
ＥＡ−１は Solter & Knowles , Proc Natl Acad Sci U
SA, Vol.75, 5565, 1978に記載されている細胞分化の重
要マーカーであり、Ｌｅx は Nomoto et al.,Exp. Cell
Res..Vol.164,pp 48-62,1986に記載されている細胞分
化のマーカーであり、それぞれ分化にともなって消失す
る。例えば、本発明実施例でフィーダー細胞として使用
しているＳＴＯ細胞（ＡＴＣＣ ＣＲＬ−１５０３）や
線維芽細胞は通常、形態変化を起こさずに長期間培養が
可能であるが、このＳＳＥＡ−１あるいはＬｅx が存在
しない。これらの抗原は、細胞の分化の指標として重要
な抗原であるが、ＥＳＣの特定や、単離に用いられたこ
とはない。両者はかっては同一の抗原と考えられていた
が、近年になり、両者の抗体の認識に違いがあることが
明らかとなった。しかし、本発明においては、目的とす
るＥＳＣを得るためにはいずれの抗体を使用しても良
い。ＳＳＥＡ−１あるいはＬｅx の発現を確認するもっ
とも簡便な方法は、ＳＳＥＡ−１あるいはＬｅx に対す
る抗体を用いて細胞を抗体染色することである。このＳ
ＳＥＡ−１あるいはＬｅx に対する抗体は市販されてお
り容易に入手可能である。本発明によるウシＥＳＣはこ
のＳＳＥＡ−１あるいはＬｅx 染色陽性となる。Expression of an undifferentiation-specific marker can be mentioned as a basic proof that the cells of the present invention are undifferentiated ESCs. This is one of the markers specifically expressed in undifferentiated cells, SSEA-1 (Stage-specif
ic embryonic antigen-1) or Le x can be confirmed. SS
EA-1 is Solter & Knowles, Proc Natl Acad Sci U
Lex is an important marker of cell differentiation described in SA, Vol.75, 5565, 1978, and Le x is Nomoto et al., Exp. Cell.
Res .. Vol. 164, pp 48-62, 1986, which is a marker for cell differentiation and disappears with differentiation. For example, STO cells (ATCC CRL-1503) and fibroblasts used as feeder cells in the examples of the present invention can usually be cultured for a long period of time without causing morphological changes. x does not exist These antigens are important antigens as indicators of cell differentiation, but have never been used for ESC identification or isolation. Both were once thought to be the same antigen, but in recent years it has become clear that there is a difference in their recognition of antibodies. However, in the present invention, any antibody may be used to obtain the desired ESC. The most convenient way to confirm the expression of SSEA-1 or Le x is to the cells for antibody staining using an antibody against SSEA-1 or Le x. This S
Antibodies to SEA-1 or Le x are commercially available and can be easily obtained. The bovine ESC according to the present invention becomes positive for SSEA-1 or Le x staining.
【０００９】ウシＥＳＣは細胞分化の機能を有してお
り、この細胞を用いたキメラの作製や細胞核移植、遺伝
子導入による形質転換動物の作製などの材料とすること
ができる。[0009] Bovine ESC has a cell differentiation function, and can be used as a material for producing chimeras using these cells, cell nuclear transfer, and producing transgenic animals by gene transfer.
【００１０】以下に実施例を示し、本発明をさらに詳細
に説明する。The present invention will be described in more detail with reference to the following examples.
（１）ウシ胚盤胞の回収 ホルスタイン種の雌牛にあらかじめホルモン刺激を行
い、過剰***を促した後、黒毛和種の***で人工授精を
行った。人工授精後７〜８日目に常法によって子宮をフ
ラッシングして胚盤胞を回収した。この胚盤胞は顕微鏡
下に鋭利な金属ブレードで切断し、胚盤胞を内部細胞塊
（ＩＣＭ）とその周囲の栄養外胚葉のみを取り出した。(1) Collection of bovine blastocysts Holstein cows were stimulated with hormones in advance to promote superovulation, and then artificial insemination was performed using Japanese black sperm. Seven to eight days after the artificial insemination, the uterus was flushed by a conventional method to collect blastocysts. The blastocyst was cut under a microscope with a sharp metal blade, and only the inner cell mass (ICM) and the vegetative ectoderm around the blastocyst were taken out.
【００１１】（２）ＥＳＣの樹立 あらかじめコラーゲンコーティング処理を行った２４ウ
エルの細胞培養プレートに、マイトマイシンＣ（10μg/
ml）で処理したＳＴＯ細胞を２×１０5 ／ウエルの細胞
密度に単層培養して、フィーダー細胞とした。この細胞
培養プレートに前記で分離したＩＣＭとその周囲の栄養
外胚葉を総数９３個を移植し培養を行った。培養は１０
％ＦＣＳ（ＦＢＳ製）、１０-4Ｍ ２ＭＥ（ギブコ製)1
03units/ml、ＬＩＦ（アムラド製）10ng/ml のヒトｂＦ
ＧＦ、１％の非必須アミノ酸（ギブコ製）を含むダルベ
ッコのＭＥＭを用いて培養を行った。培養は３７℃、５
％ＣＯ2 含有空気の条件とし、培地は３〜４日に１度全
量を交換した。培養３〜４日目で観察したところ７１個
がフィーダー層に付着しコロニー形成を行っていた。培
養５日目には円状に広がり（図１ａ）、さらに培養を継
続したところ中心部がやや盛り上がったコロニーを形成
した。ＩＣＭの細胞が中心部のみならず周辺部にも広が
っていた（図１ｂ）。公知のマウスＥＳＣと比較した場
合、仁を含む大きな核は類似しているが、顕著な液胞様
の構造が存在し、細胞間の境界はかなり明瞭で上皮様形
態を示す。またこれとよく似てはいるがコロニー周辺部
にはより上皮様の性質が強い内胚葉細胞が認められた
（図１ｃ）。ＩＣＭ様のコロニーをタングステン針を用
いてはがし、0.25％トリプシン/0.4％ ＥＤＴＡ溶液で
５〜１０分間室温で処理し細胞を分離し、ピペッティン
グで細胞を新しいフィーダー層を形成させた２４ウエル
プレートにで移植した。(2) Establishment of ESC Mitomycin C (10 μg /
ml) treated STO cells were monolayer-cultured at a cell density of 2 × 10 5 / well to obtain feeder cells. A total of 93 ICMs separated above and the vegetative ectoderm around them were transplanted to this cell culture plate and cultured. Culture is 10
% FCS (made by FBS), 10 -4 M 2ME (made by Gibco) 1
0 3 units / ml, LIF (Amrad) 10 ng / ml human bF
The culture was performed using Dulbecco's MEM containing GF and 1% of non-essential amino acids (manufactured by Gibco). Culture at 37 ℃, 5
Under the condition of air containing% CO 2 , the total amount of the medium was exchanged once every 3 to 4 days. When observed on the 3rd to 4th days of culture, 71 colonies adhered to the feeder layer and formed. On the 5th day of culture, it spreads in a circle (Fig. 1a), and when the culture was continued, colonies with a slightly raised central part were formed. The cells of ICM spread not only in the central part but also in the peripheral part (Fig. 1b). When compared to the known mouse ESCs, the large nuclei containing nucleoli are similar, but there are prominent vacuolar-like structures and the cell-cell boundaries are fairly clear and exhibit an epithelial-like morphology. Although similar to this, endoderm cells with strong epithelial-like properties were observed in the periphery of the colony (Fig. 1c). Peel off ICM-like colonies using a tungsten needle, treat with 0.25% trypsin / 0.4% EDTA solution for 5-10 minutes at room temperature to separate cells, and pipet them into a 24-well plate with a new feeder layer formed. Transplanted in.
【００１２】継代２代目以降も同様にして、コロニー形
成を顕微鏡観察下で、分化細胞とＩＣＭ細胞を区別しな
がら培養を継続した。この結果を、図２に示す。図２に
示されるように継代するごとに徐々に未分化コロニーを
含むものが減少する。２０個の胚盤胞を培養して１８個
が未分化コロニーを形成した。これらのコロニー由来の
細胞を継代すると同様のコロニーを生じるものは代を重
ねるごとに減少し、２代目１７株、３代目１１株、４代
目６株、５代目１株となった。培養を継続することがで
きたのはこの株のみで、ＴＳＨＫ−２と命名した。この
株は４代目までは徐々に内胚葉細胞が消失し、５代目移
行はＥＳ様細胞のみとなった。ＴＳＨＫ−２は９代目ま
で培養し、細胞形態的にもＥＳＣであることが確認でき
た。この細胞株ＴＳＨＫ−２は植え継ぎ後コロニーが明
瞭に認められるまでに７〜１０日を必要とするが、その
後は、細胞の増殖が比較的速くなる性質を有している。
この細胞株のコロニー形成の顕微鏡観察像を図３ａ、
ｂ、ｃに示す。ａで観察されたコロニーが２日後にはｂ
のように約２倍以上に増殖し、この状態の細胞は図３ｃ
に示すように非常に密な状態であった。対照としてマウ
ス由来のＥＳ細胞コロニーの顕微鏡観察像を図３ｄに示
した。両者を比較するとその差は歴然としており、本発
明により得たウシＥＳＣは、細胞間の境界が明瞭で小さ
な液胞状の構造が多い点で異なっていた。In the same way, from the second passage onward, the culture was continued while observing the formation of colonies under a microscope while distinguishing differentiated cells from ICM cells. The result is shown in FIG. As shown in FIG. 2, the number of undifferentiated colonies gradually decreased with each passage. 20 blastocysts were cultured and 18 formed undifferentiated colonies. Those that produced similar colonies when cells derived from these colonies were subcultured decreased with the passage of time, and the number of strains became 17 strains of the 2nd generation, 11 strains of the 3rd generation, 6 strains of the 4th generation, 1 strain of the 5th generation. Only this strain was able to continue culturing and was designated TSHK-2. In this strain, endoderm cells gradually disappeared until the 4th generation, and only ES-like cells migrated to the 5th generation. TSHK-2 was cultured up to the 9th generation, and it was confirmed that the cell morphology was ESC. This cell line TSHK-2 requires 7 to 10 days until a colony is clearly recognized after subculturing, but after that, it has a property that cell growth is relatively fast.
A microscopic image of colony formation of this cell line is shown in FIG.
Shown in b and c. The colony observed in a is b after 2 days
As shown in Fig. 3c, cells in this state proliferate about 2 times or more.
It was in a very dense state as shown in. As a control, a microscope image of a mouse-derived ES cell colony is shown in FIG. 3d. When the two were compared, the difference was obvious, and the bovine ESC obtained by the present invention was different in that the boundaries between cells were clear and many small vacuolar structures were present.
【００１３】（３）ウシＥＳＣの細胞核型と染色体分析 継代した細胞の細胞核型の分析と染色体分析を行った。
染色体分析は複製バンド法により行った。培養細胞にＢ
ｒｄＵ 150μg/mlを加え、１１〜１４時間インキュベー
ションを行って細胞内に取り込ませた。細胞をハーベス
トする３時間前に、コルセミド0.3 μg/mlを加えて培養
を行った。細胞を 0.075Ｍ ＫＣｌを用いて室温で１０
分間、低張処理した後、メタノールと酢酸３：１の混合
カルノア固定液で固定した。染色体標本は通常の空気乾
燥法で作製し、直前に調製したアクリジンオレンジ溶液
で染色し、蛍光顕微鏡で観察を行った。培養２代目コロ
ニー６サンプルを任意に選択し、その細胞の雌雄判定、
核型、およびＸ染色体の不活性化を観察したその結果を
表１に示した。(3) Cell karyotype and chromosome analysis of bovine ESC The cell karyotype and chromosome analysis of the passaged cells were performed.
Chromosome analysis was performed by the replica band method. B for cultured cells
rdU (150 μg / ml) was added, and the cells were incubated for 11 to 14 hours for incorporation into cells. Three hours before harvesting the cells, 0.3 μg / ml of colcemid was added and the cells were cultured. Cells were incubated with 0.075M KCl at room temperature for 10
After hypotonic treatment for 3 minutes, it was fixed with a mixed Carnoy's fixative solution of methanol and acetic acid 3: 1. Chromosome specimens were prepared by an ordinary air drying method, stained with the acridine orange solution prepared immediately before, and observed with a fluorescence microscope. 6 samples of the second colony of culture are arbitrarily selected, and sex determination of the cells is performed,
The results obtained by observing the karyotype and inactivation of the X chromosome are shown in Table 1.
【００１４】[0014]
【表１】 [Table 1]
【００１５】培養２代目の細胞は７株中３株が雄（Ｘ
Ｙ）、４株が雌（ＸＸ）であった。観察した細胞の８５
％以上が２倍体（２２＝６０）細胞であり雌雄でこの出
現頻度には差はなかった。哺乳類の雌では２本のＸ染色
体のうち、１本の活性を全体に抑え、雌雄間の遺伝子量
の補正を行っており、この現象をＸ染色体の不活性化と
呼ぶ。雌マウスの例ではＸ染色体の不活性化は胚発生の
初期におこるが、胚盤胞のＩＣＭ細胞では起こっていな
い。またマウス由来のＥＳＣでも２本のＸ染色体は活性
化されている。このマウス由来のＥＳＣは分化に伴って
Ｘ染色体が１本、不活性化することが確認されており、
このことからＸ染色体の活性化をＥＳＣの指標とするこ
とができる。培養２代目の細胞のＸ染色体の不活性化は
染色体の染色状態によって判定される。不活性化Ｘ染色
体はＳ期後期に複製され、一様に染色されるが、活性Ｘ
染色体は常染色体と同様に染色される。表１に示した７
試料のうち雌細胞であって、このようなＸ染色体が活性
化されているものは試料４であった。試料４はＴＳＨＫ
−２の親株であり、ＩＣＭの状態を有した細胞であるこ
とが確認できた。しかし長期継代できなかった細胞の親
株である標本６、７は不活性化Ｘ染色体を持つ細胞の頻
度がそれぞれ３９％、８２％で標本４の５％に比較して
有意に高かったことから、これらの標本の細胞はＩＣＭ
段階からさらに分化したものと考えられた。また雄の細
胞株については性染色体がＸＹとなっており、Ｘ染色体
は活性化されている。なおＴＳＨＫ−２の９代継代後の
細胞は、２代目と同様の細胞核型と活性化Ｘ染色体を有
していることが確認できた。In the second generation of cells, 3 out of 7 cells were male (X
Y), 4 strains were female (XX). 85 cells observed
% Or more were diploid (22 = 60) cells, and there was no difference in the appearance frequency between males and females. In female mammals, the activity of one of the two X chromosomes is suppressed as a whole to correct the gene amount between male and female, and this phenomenon is called inactivation of X chromosome. In the case of female mice, X chromosome inactivation occurs early in embryogenesis, but not in blastocyst ICM cells. In addition, in mouse-derived ESC, two X chromosomes are activated. It has been confirmed that this mouse-derived ESC inactivates one X chromosome with differentiation,
From this, activation of the X chromosome can be used as an index of ESC. Inactivation of the X chromosome of the second generation cultured cells is determined by the staining state of the chromosome. The inactivated X chromosome replicates late in S phase and is uniformly stained, but
Chromosomes stain similarly to autosomes. 7 shown in Table 1
Among the samples, Sample 4 was a female cell in which such an X chromosome was activated. Sample 4 is TSHK
It was confirmed that the cell strain was the parent strain of -2 and had an ICM state. However, since the frequencies of cells with inactivated X chromosome were 39% and 82%, respectively, in Samples 6 and 7, which were the parent strains of cells that could not be passaged for a long period of time, were significantly higher than those in Sample 4 of 5%. , The cells of these specimens are ICM
It was considered that the stage was further differentiated. Further, in the male cell line, the sex chromosome is XY, and the X chromosome is activated. It was confirmed that the cells after the 9th passage of TSHK-2 had the same cell karyotype and activated X chromosome as those of the 2nd passage.
【００１６】（４）ウシＥＳＣの未分化特異的マーカー
の検出 未分化特異的マーカーの検出を９代継代したＴＳＨＫ−
２株、マウスＥＳＣ、フィーダーのＳＴＯ細胞および胚
盤胞の培養８日目の細胞について行った。検出は未分化
表面抗原Ｌｅx または ＳＳＥＡ−１に対するモノクロ
ーナル抗体を用いた免疫細胞学的検出法により行った。
各培養細胞を４％パラホルムアルデヒド溶液を用いて室
温で１０分間固定した。ついで１００倍希釈した抗マウ
スＬｅx モノクローナル抗体ラットＩｇＭ（ＬＥＸ−
２：三井製薬工業製）を加え、３７℃で１時間インキュ
ベートした。次いでビオチン化抗ラットＩｇＭで処理
し、アビジン／ビオチン─パーオキシダーゼ複合体と反
応させた（ＡＢＣキット：ベクター社製）。ついで０．
３％過酸化水素水で３０分間処理して内在性のパーオキ
シダーゼを失活させた後、0.2 mg／mlジアミノベンチジ
ンと0.03％過酸化水素水を含む50mMトリス／塩酸緩衝液
(pH7.6）を用いて室温で処理して発色させた。陽性細胞
は赤く染色される。９代継代したＴＳＨＫ−２株は染色
され未分化抗原ＬＥＸ−２を発現していることが確認さ
れた。またマウスＥＳＣも同様に染色された。しかし分
化した細胞であるＳＴＯ細胞は染色されなかった。この
結果から本発明の細胞は未分化な細胞であることが確認
された。また胚盤胞の初代培養８日目のコロニーは、コ
ロニーの中心部と辺縁部が顕著に染色されていた。この
部位はこれまでの知見から、胚盤胞のＩＣＭに相当する
細胞に由来した細胞集団であることが確認されている。
また栄養外胚葉に相当する細胞は染色されていなかっ
た。以上の結果からＬｅx またはＳＳＥＡ−１を発現し
ている細胞はＩＣＭ由来であることが確認された。(4) Detection of undifferentiated specific marker of bovine ESC TSHK-in which the undifferentiated specific marker was detected for 9 passages
2 strains, mouse ESC, feeder STO cells and blastocysts on day 8 of culture were used. Undifferentiated surface antigen Le x or It was performed by an immunocytological detection method using a monoclonal antibody against SSEA-1.
Each cultured cell was fixed with a 4% paraformaldehyde solution at room temperature for 10 minutes. Then, a 100-fold diluted anti-mouse Le x monoclonal antibody rat IgM (LEX-
2: Mitsui Pharmaceutical Co., Ltd.) was added and incubated at 37 ° C. for 1 hour. Then, it was treated with biotinylated anti-rat IgM and reacted with an avidin / biotin-peroxidase complex (ABC kit: vector). Then 0.
After deactivating the endogenous peroxidase by treating with 3% hydrogen peroxide solution for 30 minutes, 50 mM Tris / HCl buffer containing 0.2 mg / ml diaminobenzidine and 0.03% hydrogen peroxide solution.
(pH 7.6) was used at room temperature for color development. Positive cells stain red. The 9th passage TSHK-2 strain was stained and confirmed to express the undifferentiated antigen LEX-2. Mouse ESC was also stained in the same manner. However, the differentiated cells, STO cells, were not stained. From this result, it was confirmed that the cells of the present invention are undifferentiated cells. In the colonies of the blastocysts on the 8th day of primary culture, the central portion and the peripheral portion of the colonies were markedly stained. From this knowledge, it has been confirmed that this site is a cell population derived from cells corresponding to the ICM of blastocyst.
The cells corresponding to trophectoderm were not stained. From the above results, it was confirmed that the cells expressing Le x or SSEA-1 were derived from ICM.
【００１７】以上の操作によりウシ胚盤胞から継代可能
な細胞を選別し、顕微鏡観察、細胞核学および染色体分
析、未分化抗原の検出を行った結果得られた細胞がウシ
ＥＳＣであることを確認できた。By the above operation, cells that can be passaged from bovine blastocysts were selected, and the cells obtained as a result of microscopic observation, cell nucleology and chromosome analysis, and detection of undifferentiated antigen were confirmed to be bovine ESCs. It could be confirmed.
【００１８】[0018]
【発明の効果】本発明の実施により、これまで提供され
ていなかったウシＥＳＣを得ることができる。ＥＳＣは
大量にしかも継代培養することが可能であり、外来遺伝
子の導入や、ウシの個体発生の材料として使用すること
が可能となる。INDUSTRIAL APPLICABILITY By carrying out the present invention, bovine ESC which has not been provided so far can be obtained. ESC can be subcultured in a large amount and can be used as a material for introducing a foreign gene or for bovine ontogeny.
【図１】生物（ウシ胚盤胞から取り出したＩＣＭと栄養
外胚葉をフィーダー細胞上培養した際に形成するコロニ
ー）の形態の細胞形態の観察像 (100 倍) を示す。ａは
培養５日目のコロニー形態 (200 倍）、ｂは培養１０日
目のＩＣＭ細胞のコロニー形態、ｃは内胚葉細胞の形態
(100 倍）、ｄは分化した細胞の混入状態 (100倍）を
それぞれ示す。FIG. 1 shows an observation image (100 ×) of a cell morphology of an organism (a colony formed when an ICM taken out from a bovine blastocyst and a trophectoderm are cultured on a feeder cell). a is colony morphology (200 times) on day 5 of culture, b is colony morphology of ICM cells on day 10 of culture, and c is morphology of endoderm cells.
(100 times), d shows the mixed state of differentiated cells (100 times).
【図２】20個の胚盤胞を継代培養した時のコロニー形成
能を有する株数の変化を示す。FIG. 2 shows changes in the number of strains capable of forming colonies when 20 blastocysts were subcultured.
【図３】生物（ＴＳＨＫ−２細胞株の形成するコロニ
ー）の顕微鏡観察像 (200 倍）を示す。ａは継代後１９
日目のコロニー、ｂは同じコロニーの２日後の像、ｃは
同じ条件の他のコロニー、ｄは対照としたマウスＥＳＣ
を示す。FIG. 3 shows a microscope image (200 ×) of an organism (colonies formed by the TSHK-2 cell line). a is 19 after passage
Day 1 colony, b 2 day image of the same colony, c another colony under the same conditions, d mouse ESC as control
Indicates.
Claims (4)
核を有し、細胞質中には液胞状の構造が存在し、細胞間
の境界が明瞭である。 （２）細胞核型および染色体の不活性化：***像はＸＹ
またはＸＸ型の２倍体細胞核型を呈し、Ｘ染色体は活性
化されている。 （３）ＳＳＥＡ−１（Stage-specific embryonic antig
en-1) あるいはＬｅx 抗原を発現している。 （４）継代培養が可能である。1. A bovine embryonic stem cell having the following characteristics. (1) Cell morphology: Under microscopic observation, it has a large nucleus containing nucleoli, a vacuolar structure is present in the cytoplasm, and the boundaries between cells are clear. (2) Cell karyotype and chromosome inactivation: division image is XY
Alternatively, it exhibits a diploid cell karyotype of XX type, and the X chromosome is activated. (3) SSEA-1 (Stage-specific embryonic antig
en-1) or the Le x antigen is expressed. (4) Subculture is possible.
部細胞塊と栄養外胚葉を取り出す工程 （２）不活性化したフィーダー細胞上で、内部細胞塊と
栄養外胚葉を少なくともウシ胎児血清、２─メルカプト
エタノール、白血病抑制因子及び塩基性線維細胞増殖因
子を含む培地で培養する工程 （３）顕微鏡観察、染色体染色、抗ＳＳＥＡ−１抗体、
あるいは抗Ｌｅx 抗体との反応性により胚性幹細胞を選
別する工程 からなる請求項１の（１）〜（４）の特性を有するウシ
胚性幹細胞の作製方法。2. (1) A step of extracting an inner cell mass and trophectoderm from a blastocyst derived from a bovine embryo (2) At least bovine inner cell mass and trophectoderm on the inactivated feeder cell Step of culturing in a medium containing fetal serum, 2-mercaptoethanol, leukemia inhibitory factor and basic fibrocyte growth factor (3) Microscopic observation, chromosome staining, anti-SSEA-1 antibody,
Or a method for manufacturing a bovine embryonic stem cells having the characteristics of claim 1 (1) to (4) comprising a step of selecting embryonic stem cells by reactivity with anti-Le x antibody.
塊と栄養外胚葉を取り出す工程が以下のステップからな
る請求項２記載の方法 （１）ホルモン刺激により過剰***を促した雌ウシに人
工授精を行い、その後、胚盤胞を回収する工程 （２）胚盤胞を切断あるいは免疫手術することで、胚盤
胞より内部細胞塊と栄養外胚葉を取り出す工程3. The method according to claim 2, wherein the step of removing the inner cell mass and trophectoderm from the blastocyst derived from a bovine embryo comprises the following steps: (1) A cow accelerating superovulation by hormone stimulation. Artificial insemination and then recovering the blastocyst (2) A step of removing the inner cell mass and trophectoderm from the blastocyst by cutting or immunosurgery the blastocyst
受精により形成されたものである請求項２記載の方法。4. The method according to claim 2, wherein the blastocyst derived from a bovine fertilized egg is formed by in vitro fertilization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6284153A JPH08116966A (en) | 1994-10-25 | 1994-10-25 | Bovine embryonic stem cell and its preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6284153A JPH08116966A (en) | 1994-10-25 | 1994-10-25 | Bovine embryonic stem cell and its preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08116966A true JPH08116966A (en) | 1996-05-14 |
Family
ID=17674869
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6284153A Pending JPH08116966A (en) | 1994-10-25 | 1994-10-25 | Bovine embryonic stem cell and its preparation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08116966A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113125227A (en) * | 2020-12-25 | 2021-07-16 | 上海乐辰生物科技有限公司 | Autumn aqueous solution for preparing high-resolution chromosome for karyotype analysis and application |
-
1994
- 1994-10-25 JP JP6284153A patent/JPH08116966A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113125227A (en) * | 2020-12-25 | 2021-07-16 | 上海乐辰生物科技有限公司 | Autumn aqueous solution for preparing high-resolution chromosome for karyotype analysis and application |
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