JP2017147993A - Cultivation method of primordial germ cells and media additive for primordial germ cell culture - Google Patents
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本発明は、始原生殖細胞の培養方法及び始原生殖細胞の培養用培地添加物に関する。 The present invention relates to a method for culturing primordial germ cells and a culture medium additive for culturing primordial germ cells.
ニワトリ等の鳥類では、一羽から１個しか受精卵（一細胞期受精卵）が得られない。しかも受精卵が卵管にあるため、遺伝子組換え等の遺伝子操作で改変された鳥類を作成することが困難であった。そこで、鳥類の改変研究では、生殖細胞へ分化することのできる始原生殖細胞（Ｐｒｉｍｏｒｄｉａｌ Ｇｅｒｍ Ｃｅｌｌ、以下単に「ＰＧＣ」ともいう）に対して遺伝子操作が行われてきた。 In birds such as chickens, only one fertilized egg (one cell stage fertilized egg) can be obtained. Moreover, since fertilized eggs are located in the oviduct, it has been difficult to produce birds that have been modified by genetic manipulation such as genetic recombination. Thus, in avian modification studies, genetic manipulation has been performed on primordial germ cells (hereinafter also simply referred to as “PGC”) that can differentiate into germ cells.
遺伝子操作の標的とするＰＧＣは、例えば、非特許文献１に開示されたように、ｂｕｆｆａｌｏ ｒａｔ ｌｉｖｅｒ（ＢＲＬ）細胞等の支持細胞を用いて培養することができる。また、非特許文献２には、塩基性線維芽細胞増殖因子を含む培地を用いた、支持細胞を使用しない培養方法が報告されている。 As disclosed in Non-Patent Document 1, for example, PGC targeted for genetic manipulation can be cultured using supporting cells such as buffalo rat river (BRL) cells. Non-Patent Document 2 reports a culture method using a medium containing basic fibroblast growth factor and not using feeder cells.
しかし、非特許文献１に開示された支持細胞を用いた培養方法の場合、支持細胞の動物種がＰＧＣの動物種と異なる異種細胞となるため、コンタミネーションが避けられなかった。また、非特許文献２に開示された支持細胞を使用しない培養方法では、ＰＧＣを安定に増殖させられず、効率が良くないという不都合があった。 However, in the case of the culturing method using feeder cells disclosed in Non-Patent Document 1, contamination is inevitable because the animal species of the feeder cells are different types of cells from the animal species of PGC. Further, the culture method disclosed in Non-Patent Document 2 that does not use feeder cells has the disadvantage that PGC cannot be stably grown and is not efficient.
本発明は、上記実情に鑑みてなされたものであり、支持細胞を使用せずに極めて簡便かつ安定に始原生殖細胞を増殖させることができる始原生殖細胞の培養方法及び始原生殖細胞の培養用培地添加物を提供することを目的とする。 The present invention has been made in view of the above circumstances, and a primordial germ cell culture method and a primordial germ cell culture medium capable of proliferating primordial germ cells extremely simply and stably without using supporting cells The object is to provide additives.
ニワトリ及びマウス等のＰＧＣは、発生早期から血流に乗って生殖隆起へ移動する。移動の間に本来の経路から外れてしまったＰＧＣ、及び生殖隆起に到達してもストローマ細胞と細胞間シグナルをやりとりできなかったＰＧＣは速やかにアポトーシスする。当該知見に着目した本発明者は鋭意研究を重ね、本発明を完成させた。すなわち、本発明の第１の観点に係る始原生殖細胞の培養方法は、
アポトーシス阻害剤を含む培地で始原生殖細胞を培養する培養ステップを含む。
PGCs such as chickens and mice move to the genital ridge in the bloodstream from the early stages of development. PGCs that have deviated from their original pathway during migration, and PGCs that have failed to exchange cell signals with stromal cells even when reaching the genital ridge, are rapidly apoptotic. The inventor who paid attention to the knowledge has made extensive studies and completed the present invention. That is, the method for culturing primordial germ cells according to the first aspect of the present invention comprises:
Culturing a primordial germ cell in a medium containing an apoptosis inhibitor.
この場合、前記アポトーシス阻害剤は、
ミオシンII阻害剤である、
こととしてもよい。
In this case, the apoptosis inhibitor is
A myosin II inhibitor,
It is good as well.
また、前記ミオシンII阻害剤は、
ブレビスタチンである、
こととしてもよい。
The myosin II inhibitor is
Is blebbistatin,
It is good as well.
また、前記ブレビスタチンの前記培地における濃度は、
２．５μＭ以上１０．０μＭ以下である、
こととしてもよい。
The concentration of blebbistatin in the medium is
2.5 μM or more and 10.0 μM or less,
It is good as well.
また、前記アポトーシス阻害剤は、
Ｒｈｏキナーゼ阻害剤である、
こととしてもよい。
The apoptosis inhibitor is
A Rho kinase inhibitor,
It is good as well.
また、前記Ｒｈｏキナーゼ阻害剤は、
Ｙ−２７６３２又はＨ１１５２である、
こととしてもよい。
The Rho kinase inhibitor is
Y-27632 or H1152.
It is good as well.
また、前記アポトーシス阻害剤は、
カスパーゼ阻害剤である、
こととしてもよい。
The apoptosis inhibitor is
A caspase inhibitor,
It is good as well.
また、前記カスパーゼ阻害剤は、
ｚＶＡＤ−ＦＭＫである、
こととしてもよい。
The caspase inhibitor is
zVAD-FMK.
It is good as well.
また、前記培養ステップで培養する始原生殖細胞は、
鳥類の初期胚の胚血液から分離される始原生殖細胞である、
こととしてもよい。
In addition, primordial germ cells cultured in the culture step,
Primordial germ cells isolated from the embryonic blood of early avian embryos,
It is good as well.
本発明の第２の観点に係る始原生殖細胞の培養用培地添加物は、
アポトーシス阻害剤を含む。
The culture medium additive for culturing primordial germ cells according to the second aspect of the present invention comprises:
Contains an apoptosis inhibitor.
本発明によれば、支持細胞を使用せずに極めて簡便かつ安定に始原生殖細胞を増殖させることができる。 According to the present invention, it is possible to proliferate primordial germ cells very simply and stably without using feeder cells.
本発明に係る実施の形態について説明する。なお、本発明は下記の実施の形態及び図面によって限定されるものではない。 Embodiments according to the present invention will be described. In addition, this invention is not limited by the following embodiment and drawing.
（実施の形態）
本実施の形態に係るＰＧＣの培養方法は、アポトーシス阻害剤を含む培地でＰＧＣを培養する培養ステップを含む。本実施の形態では、ニワトリのＰＧＣを例に培養方法を説明する。培養するニワトリのＰＧＣは、特に限定されないが、例えば、Ｅｙａｌ−Ｇｉｌａｄｉ ａｎｄ Ｋｏｃｈａｖの発生ステージ（Ｉ〜ＸＩＶ）のステージＸの胚盤葉上層に含まれる。ニワトリの胚盤葉は、ニワトリの受精卵から公知の方法で分離できる。
(Embodiment)
The method for culturing PGC according to the present embodiment includes a culturing step of culturing PGC in a medium containing an apoptosis inhibitor. In the present embodiment, a culture method will be described using a chicken PGC as an example. The chicken PGC to be cultured is not particularly limited, and is included in, for example, the upper stage blastoderm of the developmental stage (I to XIV) of Eyal-Giladi and Kochav. The blastoderm of the chicken can be separated from the fertilized egg of the chicken by a known method.
好適には、ＰＧＣは、ニワトリの初期胚の胚血液から分離される。初期胚の胚血液は、孵卵開始から４８〜６０時間、好ましくは５０〜５８時間、特に好ましくは５５時間経過後の受精卵から採取できる。特に、ニワトリの初期胚の胚血液は、Ｈａｍｂｕｒｇｅｒ−Ｈａｍｉｌｔｏｎ（ＨＨ）ステージ１３〜１５のニワトリ胚から採取するのが好ましい。 Preferably, the PGC is isolated from embryonic blood of a chicken early embryo. The embryonic blood of the early embryo can be collected from a fertilized egg after 48 to 60 hours, preferably 50 to 58 hours, particularly preferably 55 hours after the start of incubation. In particular, the embryonic blood of an early chicken embryo is preferably collected from a hamburger-Hamilton (HH) stage 13-15 chicken embryo.
ＰＧＣは、例えば、密度勾配遠心法により胚血液から単離精製できる。密度勾配遠心法では、高濃度の密度勾配遠心分離媒体を含む培地、該培地よりも低濃度の密度勾配遠心分離媒体を含む培地に続いて、胚血液を含む培地の順に遠沈管内に重層し、該遠沈管を遠心する。遠心によって、高濃度の密度勾配遠心分離媒体を含む培地と低濃度の密度勾配遠心分離媒体を含む培地との界面にＰＧＣが分離される。したがって、界面の培地を回収することでＰＧＣを精製できる。 PGC can be isolated and purified from embryonic blood, for example, by density gradient centrifugation. In density gradient centrifugation, a medium containing a high concentration density gradient centrifugation medium, a medium containing a density gradient centrifugation medium having a lower concentration than the medium, and then a medium containing embryo blood are layered in the centrifuge tube in this order. The centrifuge tube is centrifuged. Centrifugation separates PGCs at the interface between a medium containing a high concentration density gradient centrifugation medium and a medium containing a low concentration density gradient centrifugation medium. Therefore, PGC can be purified by recovering the interface medium.
上記ニワトリの種類は特に限定されず、例えば、Ｗｈｉｔｅ Ｌｅｇｈｏｒｎ、Ｂｒｏｗｎ Ｌｅｇｈｏｒｎ、Ｂａｒｒｅｄ Ｒｏｃｋ、Ｓｕｓｓｅｘ、Ｎｅｗ Ｈａｍｐｓｈｉｒｅ、Ｒｈｏｄｅ Ｉｓｌａｎｄ、Ａｕｓｓｔｒａｌｏｒｐ、Ｍｉｎｏｒｃａ、Ａｍｒｏｘ、Ｃａｌｉｆｏｒｎｉａ Ｇｒａｙ、Ｉｔａｌｉａｎ Ｐａｒｔｉｄｇｅ ｃｏｌｏｒｅｄ及びＫｏｒｅａｎ Ｏｇｅ等が挙げられる。 The type of the chicken is not particularly limited, and examples thereof include White Leghorn, Brown Leghorn, Barred Rock, Sussex, New Hampshire, Rhode Island, Australia, Minorca, Amrox, California, and others.
アポトーシス阻害剤は、アポトーシスを阻害できれば特に限定されない。アポトーシスの阻害では、例えば、アポトーシスシグナル経路に関与する酵素等を阻害すればよい。特に細胞間接着を失った単一細胞におけるアポトーシスシグナル経路の阻害が好ましい。アポトーシス阻害剤は、例えば、ミオシンII阻害剤、Ｒｈｏキナーゼ（Ｒｈｏ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ、以下単に「ＲＯＣＫ」ともいう）阻害剤及びカスパーゼ阻害剤等である。 The apoptosis inhibitor is not particularly limited as long as it can inhibit apoptosis. In the inhibition of apoptosis, for example, an enzyme involved in the apoptosis signal pathway may be inhibited. In particular, inhibition of the apoptotic signal pathway in single cells that have lost cell-cell adhesion is preferred. Examples of the apoptosis inhibitor include a myosin II inhibitor, a Rho kinase (Rho-associated protein kinase, hereinafter simply referred to as “ROCK”) inhibitor, a caspase inhibitor, and the like.
ミオシンII阻害剤としては、ブレビスタチン等が好ましい。ＲＯＣＫ阻害剤としては、Ｙ−２７６３２、Ｈ１１５２及びリパスジル等が好ましい。カスパーゼ阻害剤としては、ｚＶＡＤ−ＦＭＫ等が好ましい。 As the myosin II inhibitor, blebbistatin and the like are preferable. As the ROCK inhibitor, Y-27632, H1152, Ripasudil and the like are preferable. As the caspase inhibitor, zVAD-FMK and the like are preferable.
培養ステップでは、例えば、アポトーシス阻害剤を添加した培地でＰＧＣを培養すればよい。アポトーシス阻害剤の培地における濃度は、特に限定されないが、０．０１μＭ以上１００μＭ以下、０．１μＭ以上２０μＭ以下、又は０．５μＭ以上１０μＭ以下が好ましい。 In the culture step, for example, PGC may be cultured in a medium to which an apoptosis inhibitor is added. The concentration of the apoptosis inhibitor in the medium is not particularly limited, but is preferably 0.01 μM to 100 μM, 0.1 μM to 20 μM, or 0.5 μM to 10 μM.
アポトーシス阻害剤として、好ましくはブレビスタチンが用いられる。ブレビスタチンの培地における濃度は、０．１５μＭ以上１２μＭ以下、好ましくは２μＭ以上１１μＭ以下、特に好ましくは、２．５μＭ以上１０．０μＭ以下である。好適には、ブレビスタチンの培地における濃度は、５．０μＭである。 As an apoptosis inhibitor, blebbistatin is preferably used. The concentration of blebbistatin in the medium is 0.15 μM or more and 12 μM or less, preferably 2 μM or more and 11 μM or less, and particularly preferably 2.5 μM or more and 10.0 μM or less. Preferably, the concentration of blebbistatin in the medium is 5.0 μM.
上記培地の組成は、ＰＧＣを培養できれば特に限定されない。例えば培地は、ラット、ニワトリ等の肝細胞の馴化培地及びダルベッコ改変イーグル培地（Ｄｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ ｅａｇｌｅ ｍｅｄｉｕｍ、以下単に「ＤＭＥＭ」ともいう）に、ニワトリ血清、各種アミノ酸、幹細胞用試薬及び線維芽細胞増殖因子（Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ、以下単に「ＦＧＦ」ともいう）等を含む。 The composition of the medium is not particularly limited as long as PGC can be cultured. For example, the medium is a conditioned medium of liver cells such as rats and chickens and Dulbecco's modified Eagle medium (hereinafter also simply referred to as “DMEM”), chicken serum, various amino acids, reagents for stem cells and fibroblasts. Growth factors (Fibroblast growth factor, hereinafter also simply referred to as “FGF”) and the like are included.
好ましくは、ＦＧＦは培地中で徐放されるように培地に加えられる。ＦＧＦを培地中で徐放させるには、例えば、乳酸とグリコール酸との共重合体であるＰＬＧＡ等のポリマーにＦＧＦを封入すればよい。ＰＬＧＡポリマーに封入されたＦＧＦは、所定期間、持続的に培地中に徐放されるため、ＦＧＦが培地に安定的に供給される。 Preferably, FGF is added to the medium so that it is slowly released in the medium. In order to release FGF slowly in the medium, for example, FGF may be encapsulated in a polymer such as PLGA, which is a copolymer of lactic acid and glycolic acid. Since the FGF encapsulated in the PLGA polymer is continuously released into the medium continuously for a predetermined period, the FGF is stably supplied to the medium.
本実施の形態に係る培養方法では、支持細胞ではなく、細胞基底膜様の基材上でＰＧＣを培養できる。当該基材としては、例えば、細胞外マトリックスタンパク質を含む基材及びゼラチンが挙げられる。好ましくは、当該基材には、ラミニン、コラーゲンＩＶ、エンタクチン及びヘパラン硫酸プロテオグリカン、ＴＧＦ−β、ＦＧＦ及び組織プラスミノーゲン活性化因子等が含まれる。基材を用いる場合、培養器の表面を基材で被覆すればよい。 In the culturing method according to the present embodiment, PGC can be cultured on a cell basement membrane-like substrate instead of supporting cells. Examples of the substrate include a substrate containing extracellular matrix protein and gelatin. Preferably, the substrate includes laminin, collagen IV, entactin and heparan sulfate proteoglycan, TGF-β, FGF, tissue plasminogen activator, and the like. When using a substrate, the surface of the incubator may be coated with the substrate.
以上詳細に説明したように、本実施の形態に係る培養方法によれば、下記実施例３に示すように、支持細胞を使用せずに安定にＰＧＣを増殖させることができる。本培養方法は、支持細胞を調製する必要がないため、極めて簡便にＰＧＣを増殖させることができる。 As described above in detail, according to the culture method according to the present embodiment, as shown in Example 3 below, PGC can be stably grown without using supporting cells. Since this culture method does not require the preparation of feeder cells, PGC can be proliferated very easily.
また、本実施の形態に係る培養方法の培養ステップでは、ニワトリの初期胚の胚血液から分離されるＰＧＣを培養してもよいこととした。胚血液に含まれるＰＧＣは浮遊しているため、例えば、胚盤葉上層から分離する場合のプロテアーゼ処理をすることなく簡便に分離することができる。また、プロテアーゼ処理が不要であるため、プロテアーゼがＰＧＣに及ぼす影響を懸念しなくてよい。 In addition, in the culturing step of the culturing method according to the present embodiment, PGC separated from the embryonic blood of the chicken early embryo may be cultured. Since PGC contained in embryonic blood is floating, for example, it can be easily separated without protease treatment when it is separated from the upper layer of the blastoderm. Further, since protease treatment is unnecessary, there is no need to worry about the effect of protease on PGC.
本実施の形態に係る培養方法で培養したＰＧＣは、長期培養しても生体内でのＰＧＣの特性を維持している。また、当該ＰＧＣは、生殖腺への移動能を保持している。このため、当該ＰＧＣは、ゲノム編集等の対象として有用であり、遺伝子改変動物の作出が容易となる。 The PGC cultured by the culture method according to the present embodiment maintains the characteristics of PGC in vivo even after long-term culture. Further, the PGC retains the ability to move to the gonads. For this reason, the PGC is useful as a target for genome editing and the like, and the creation of genetically modified animals is facilitated.
なお、本実施の形態に係るＰＧＣの培養方法は、ニワトリ以外の鳥類のＰＧＣの培養にも適用できる。鳥類は、特に限定されず、例えば、アヒル、シチメンチョウ、カモ、ガン、ウズラ、キジ、オウム、フィンチ、タカ、ダチョウ、エミュー及びヒクイドリ等である。また、本実施の形態に係るＰＧＣの培養方法は、げっ歯類等の鳥類以外の動物種のＰＧＣの培養にも適用できる。 In addition, the culture | cultivation method of PGC which concerns on this Embodiment is applicable also to culture | cultivation of PGCs of birds other than a chicken. The birds are not particularly limited, and examples thereof include duck, turkey, duck, cancer, quail, pheasant, parrot, finch, hawk, ostrich, emu and cassowary. Moreover, the culture | cultivation method of PGC which concerns on this Embodiment is applicable also to culture | cultivation of PGC of animal species other than birds, such as rodents.
なお、上述のアポトーシス阻害剤は、塩酸塩等の塩であってもよい。例えば、アポトーシス阻害剤として、Ｙ−２７６３２、Ｈ１１５２及びリパスジルの塩を使用してもよい。また、アポトーシス阻害剤は、アポトーシスに関与するタンパク質の遺伝子発現を抑制するアンチセンスＲＮＡ等であってもよいし、アポトーシスに関与するタンパク質の機能を阻害するアプタマー等であってもよい。 In addition, the above-mentioned apoptosis inhibitor may be a salt such as hydrochloride. For example, a salt of Y-27632, H1152, and ripaspil may be used as an apoptosis inhibitor. The apoptosis inhibitor may be an antisense RNA that suppresses gene expression of a protein involved in apoptosis, or an aptamer that inhibits the function of a protein involved in apoptosis.
また、本実施の形態に係るＰＧＣの培養方法は、培地の成分として、ＢＲＬの馴化培地のみならずニワトリ肝細胞の馴化培地を用いることができる。下記実施例５に示すように、ＢＲＬの馴化培地よりも少量の１０％程度のニワトリ肝細胞の馴化培地を培地に加えることで、ＰＧＣの十分な増殖活性が得られる。 In addition, the PGC culturing method according to the present embodiment can use not only the conditioned medium of BRL but also the conditioned medium of chicken liver cells as a medium component. As shown in Example 5 below, sufficient growth activity of PGC can be obtained by adding a small amount of conditioned medium of about 10% chicken hepatocytes to the medium than the conditioned medium of BRL.
別の実施の形態では、ＰＧＣの培養用培地添加物が提供される。該培養用培地添加物は、上記アポトーシス阻害剤を含む。当該培養用培地添加物は、アポトーシス阻害剤に加え、上述の各種培地成分を含んでもよい。ＰＧＣの培養用培地添加物が、例えば、２．５μＭ以上１０．０μＭ以下、好ましくは５．０μＭとなるように、ＰＧＣの培地に添加されることで、支持細胞を使用せずに安定にＰＧＣを増殖させることができる。 In another embodiment, a culture medium supplement for PGC is provided. The culture medium additive contains the apoptosis inhibitor. The culture medium additive may contain the above-mentioned various medium components in addition to the apoptosis inhibitor. By adding the culture medium additive for culture of PGC to PGC medium so that it is, for example, 2.5 μM or more and 10.0 μM or less, preferably 5.0 μM, PGC can be stably used without using supporting cells. Can be grown.
また、別の実施の形態では、ＰＧＣの培養キットが提供される。該培養キットは、少なくともアポトーシス阻害剤を含む。必要に応じて、当該培養キットは、細胞基底膜様の基材と、各種培地成分と、を含んでもよい。 In another embodiment, a PGC culture kit is provided. The culture kit includes at least an apoptosis inhibitor. If necessary, the culture kit may include a cell basement membrane-like base material and various medium components.
以下の実施例により、本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The following examples further illustrate the present invention, but the present invention is not limited to the examples.
（実施例１：ＰＧＣの分離方法）
ＨＨステージ１３〜１５の横斑プリマスロック胚（祓川エッグファーム社製）から採取したＰＧＣを含む胚血液を、５００μｌの１０％ウシ胎児血清（ＦＢＳ、Ｈｙｃｌｏｎｅ社製）含有ＫｎｏｃｋＯｕｔ ＤＭＥＭ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）へ懸濁した。ＰＧＣの精製は、Ｎｙｃｏｄｅｎｚ（商標、Ａｘｉｓ−Ｓｈｉｅｌｄ社製）を用いた密度勾配遠心法により行った。詳細には、まず、１５ｍｌ遠沈管の中に、１１％Ｎｙｃｏｄｅｎｚ及び１０％ＦＢＳを含有する５ｍｌのＤＭＥＭ、５．５％Ｎｙｃｏｄｅｎｚ及び１０％ＦＢＳを含有する５ｍｌのＤＭＥＭ、続いて胚血液及び１０％ＦＢＳを含有するＤＭＥＭの順に重層し、８００×ｇで１５分間、遠心操作を行った。遠心操作後、Ｎｙｃｏｄｅｎｚの濃度差で生じた界面を中心に５ｍｌの密度勾配遠心溶液を回収し、精製ＰＧＣを回収した。
(Example 1: PGC separation method)
Embryonic blood containing PGCs collected from HH stage 13-15 side patch Plymouth Rock embryo (manufactured by Yodogawa Egg Farm) was added to 500 μl of 10% fetal calf serum (FBS, Hyclone) KnockOut DMEM (Thermo Fisher Scientific) To the product). PGC was purified by density gradient centrifugation using Nycodenz (trademark, manufactured by Axis-Shield). Specifically, first, in a 15 ml centrifuge tube, 5 ml DMEM containing 11% Nycodenz and 10% FBS, 5 ml DMEM containing 5.5% Nycodenz and 10% FBS, followed by embryonic blood and 10% The layers were layered in the order of DMEM containing FBS, and centrifuged at 800 × g for 15 minutes. After centrifugation, 5 ml of a density gradient centrifugal solution was collected around the interface produced by the Nycodenz concentration difference, and purified PGC was collected.
（実施例２：ＰＧＣの培養）
まず、ＰＧＣの培養に用いるＢＲＬ−馴化培地を次のように調製した。１５０ｍｍのディッシュに約８０〜９０％コンフルエントの状態に培養したＢＲＬを準備し、３０ｍｌの５％ＫｎｏｃｋＯｕｔ（商標）Ｓｅｒｕｍ Ｒｅｐｌａｃｅｍｅｎｔ（ＫＳＲ、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）、２ｍＭ ＧｌｕｔａＭＡＸ（商標、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）含有ＤＭＥＭにて５％ＣＯ２、３７℃の環境下で３日間培養した。この培養上清を回収し、一次バッチ馴化培地とした。新たに５％ＫＳＲ、２ｍＭ ＧｌｕｔａＭＡＸ含有ＤＭＥＭを添加しさらに培養を行い、この培養上清を回収し、二次バッチ馴化培地とした。同様に三次バッチ馴化培地まで回収を行い、一〜三次バッチ馴化培地を混合し、０．２μｍのポアサイズのＮａｌｇｅｎｅ（商標）Ｒａｐｉｄ−Ｆｌｏｗ（商標）ＰＥＳメンブレンフィルターユニット（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製）でろ過した後、ＰＧＣの培養に使用した。
(Example 2: PGC culture)
First, a BRL-conditioned medium used for PGC culture was prepared as follows. A BRL cultured in a state of about 80-90% confluence in a 150 mm dish is prepared, and 30 ml of 5% KnockOut (trademark) Serum Replacement (KSR, manufactured by Thermo Fisher Scientific), 2 mM GlutaMAX (trademark, manufactured by Thermo Fischer). ) It was cultured for 3 days in an environment of 5% CO 2 and 37 ° C. in DMEM containing. The culture supernatant was collected and used as a primary batch conditioned medium. 5% KSR, 2 mM GlutaMAX-containing DMEM was newly added and further cultured, and the culture supernatant was collected and used as a secondary batch conditioned medium. Similarly, the third batch conditioned medium was collected, mixed with the first to third batch conditioned medium, and filtered with a 0.2 μm pore size Nalgene ™ Rapid-Flow ™ PES membrane filter unit (manufactured by Thermo Scientific). Later, it was used for PGC culture.
ＰＧＣの培養には、５０倍希釈マトリゲル（商標）基底膜マトリックス（Ｃｏｒｎｉｎｇ社製）又は０．１％ゼラチン（Ｓｉｇｍａ Ａｌｄｒｉｃｈ社製）水溶液でコートした培養器を用いた。密度勾配遠心法により精製したＰＧＣを、９６ウェルプレートの１ウェル当たりに１００個加え、培養を開始した。培養は、表１に示す基本培地を用いて３８℃、５％ＣＯ２、３％Ｏ２環境下で行った。細胞の増殖に合わせて２〜４日おきに継代培養を行った。アポトーシス阻害剤として、ブレビスタチン、Ｙ−２７６３２、Ｈ１１５２及びＺ−ＶＡＤ−ＦＭＫ（すべて和光純薬工業社製）を添加した培地を用いて培養した。 For incubating PGC, an incubator coated with 50-fold diluted Matrigel ™ basement membrane matrix (Corning) or 0.1% gelatin (Sigma Aldrich) aqueous solution was used. 100 PGCs purified by density gradient centrifugation were added per well of a 96-well plate, and culture was started. The culture was performed using a basic medium shown in Table 1 in an environment of 38 ° C., 5% CO 2 , 3% O 2 . Subculture was performed every 2 to 4 days according to cell growth. As an apoptosis inhibitor, culture was performed using a medium supplemented with blebbistatin, Y-27632, H1152, and Z-VAD-FMK (all manufactured by Wako Pure Chemical Industries, Ltd.).
（結果）
密度勾配遠心法で精製したＰＧＣの培養開始直後の観察像を図１（Ａ）に示す。矢印がＰＧＣを指している。観察されたＰＧＣは、球形で、細胞質の一部にグリコーゲン顆粒を有するという典型的なＰＧＣの特徴を有していた。また、継代１９回、４５日間培養後のＰＧＣの観察像を図１（Ｂ）に示す。一部の細胞は接着細胞へ変化しているものの、長期培養後でも多くの細胞が上述したＰＧＣの特徴を有したまま増殖していることが確認された。なお、図１（Ａ）、（Ｂ）中のスケールバーは、それぞれ１００μｍに相当する。
(result)
An observation image immediately after the start of culture of PGC purified by density gradient centrifugation is shown in FIG. The arrow points to PGC. The observed PGCs were spherical and had typical PGC characteristics with glycogen granules in part of the cytoplasm. In addition, an observation image of PGC after 19 passages and 45 days of culture is shown in FIG. Although some cells were changed to adherent cells, it was confirmed that many cells proliferated with the above-mentioned PGC characteristics even after long-term culture. Note that the scale bars in FIGS. 1A and 1B each correspond to 100 μm.
（実施例３：ＰＧＣの増殖活性の評価）
培養したＰＧＣの増殖活性を次のように測定することで各アポトーシス阻害剤の効果を比較した。ファルコン９６ウェルホワイトプレート（Ｃｏｒｎｉｎｇ社製）で、１ウェル当たり５０００個のＰＧＣ（継代１８回、４６日間培養）の培養を、３８℃、５％ＣＯ２、３％Ｏ２環境下で開始した。培地には、ブレビスタチン、Ｙ−２７６３２、Ｈ１１５２及びＺ−ＶＡＤ−ＦＭＫをそれぞれ０．１６〜１０μＭで添加した。各アポトーシス阻害剤存在下で培養したＰＧＣについて、培養３日後にＣｅｌｌＴｉｔｅｒ−Ｇｌｏ（商標）Ｌｕｍｉｎｅｓｃｅｎｔ Ｃｅｌｌ Ｖｉａｂｉｌｉｔｙ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製）を用いて増殖活性を測定した。本キットで得られる化学発光の検出には、２０３０Ｍｕｌｔｉｌａｂｅｌ Ｒｅａｄｅｒ ＡＲＶＯ Ｘ４（ＰｅｒｋｉｎＥｌｍｅｒ社製）を用いた。
(Example 3: Evaluation of proliferative activity of PGC)
The effect of each apoptosis inhibitor was compared by measuring the proliferation activity of the cultured PGC as follows. Cultivation of 5000 PGCs (18 passages, 46 days culture) per well in a Falcon 96 well white plate (Corning) was started in a 38 ° C., 5% CO 2 , 3% O 2 environment. . Brevistatin, Y-27632, H1152, and Z-VAD-FMK were added to the medium at 0.16 to 10 μM, respectively. The proliferation activity of PGC cultured in the presence of each apoptosis inhibitor was measured using CellTiter-Glo (trademark) Luminescent Cell Viability Assay (manufactured by Promega) after 3 days of culture. For detection of chemiluminescence obtained with this kit, 2030 Multilabel Reader ARVO X4 (manufactured by PerkinElmer) was used.
（結果）
各アポトーシス阻害剤存在下での増殖活性を比較した結果を図２に示す。全てのアポトーシス阻害剤がＰＧＣの増殖活性に影響した。各アポトーシス阻害剤の至適濃度（ブレビスタチンが５又は１０μＭ、Ｙ−２７６３２が５μＭ、Ｈ１１５２が１．２５μＭ及びｚＶＡＤ−ＦＭＫが１０μＭ）の条件では、アポトーシス阻害剤を添加していない培地での培養に比べ、高い増殖活性を示した。最も増殖活性が高かったブレビスタチンにおいてはアポトーシス阻害剤を添加していない培地での培養に比べ、３．６倍高い増殖活性を示した。
(result)
The results of comparing the proliferation activity in the presence of each apoptosis inhibitor are shown in FIG. All apoptosis inhibitors affected the proliferative activity of PGC. Cultivation in a medium not added with an apoptosis inhibitor under conditions of optimal concentrations of each apoptosis inhibitor (5 or 10 μM for blebbistatin, 5 μM for Y-27632, 1.25 μM for H1152 and 10 μM for zVAD-FMK) Compared to the above, it showed high proliferative activity. Blebbistatin, which had the highest growth activity, showed a 3.6-fold higher growth activity compared to culture in a medium not added with an apoptosis inhibitor.
５μＭのブレビスタチン存在下での上記実施例２に係る培養方法と、Ｌａｖｏｉｒらにより確立された上記非特許文献１に記載の従来法とで、ダブリングタイムを比較した。ダブリングタイムは、Ｃｅｌｌ Ｃａｌｃｕｌａｔｏｒ Ｄｏｕｂｌｉｎｇ Ｔｉｍｅ（ｈｔｔｐ：／／ｗｗｗ．ｄｏｕｂｌｉｎｇ−ｔｉｍｅ．ｃｏｍ／ｃｏｍｐｕｔｅ．ｐｈｐ）で算出した。 The doubling time was compared between the culture method according to Example 2 in the presence of 5 μM blebbistatin and the conventional method described in Non-Patent Document 1 established by Lavoir et al. The doubling time was calculated by Cell Calculator Doubling Time (http://www.doubling-time.com/compute.php).
ＢＲＬを支持細胞として用いて、かつアポトーシス阻害剤を培地に加えない従来法のダブリングタイムは６．２５日で、約１００個の細胞が４０日間で８．４×１０３個まで増殖した。一方、実施例２に係る培養方法のダブリングタイムは４．０１日で、約１００個の細胞が４８日間で４．０×１０５個まで増殖した。したがって、上記実施例２に係る培養方法は、従来法より約１．６倍速くダブリングすることが示された。 The doubling time of the conventional method using BRL as a supporting cell and not adding an apoptosis inhibitor to the medium was 6.25 days, and about 100 cells grew to 8.4 × 10 3 cells in 40 days. On the other hand, the doubling time of the culture method according to Example 2 was 4.01 days, and about 100 cells grew to 4.0 × 10 5 cells in 48 days. Therefore, it was shown that the culture method according to Example 2 doubling about 1.6 times faster than the conventional method.
（実施例４：培養ＰＧＣの特性評価）
上記実施例３で選定した培養条件で培養したＰＧＣの特性を、免疫染色及び生殖腺への移動能アッセイにより評価した。５×１０５個のＰＧＣ（継代１９回、４８日間培養）を１５ｍｌチューブに回収し、２００×ｇ、５分間遠心した後、１％ウシ血清アルブミン（ＢＳＡ、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）含有リン酸緩衝生理食塩水（ＰＢＳ、日水製薬社製）で３回洗浄した（以降、各操作の間に同様に遠心操作を行った）。洗浄したＰＧＣを４％パラホルムアルデヒド（和光純薬工業社製）含有ＰＢＳで３０分間、室温で固定し、１ｍＭグリシン（和光純薬工業社製）及び１％ＢＳＡ含有ＰＢＳで３回洗浄後、０．１％Ｔｒｉｔｏｎ−Ｘ １００（ナカライテスク社製）含有ＰＢＳを加え５分間、室温で透過処理を行った。
(Example 4: Characteristic evaluation of cultured PGC)
The characteristics of PGCs cultured under the culture conditions selected in Example 3 above were evaluated by immunostaining and assay for migration to the gonad. 5 × 10 5 PGCs (19 passages, 48 days culture) were collected in a 15 ml tube, centrifuged at 200 × g for 5 minutes, and then phosphorous containing 1% bovine serum albumin (BSA, manufactured by Thermo Fisher Scientific). The plate was washed 3 times with acid buffered saline (PBS, manufactured by Nissui Pharmaceutical Co., Ltd.). The washed PGC was fixed with PBS containing 4% paraformaldehyde (Wako Pure Chemical Industries) for 30 minutes at room temperature, washed 3 times with PBS containing 1 mM glycine (Wako Pure Chemical Industries) and 1% BSA, and then 0 PBS containing 1% Triton-X 100 (manufactured by Nacalai Tesque) was added and permeabilized for 5 minutes at room temperature.
透過処理したＰＧＣを１％ＢＳＡ含有ＰＢＳにて３回洗浄後、５０倍希釈抗ニワトリＮａｎｏｇウサギポリクローナル抗体を含む抗ニワトリｖａｓａ ｈｏｍｏｌｏｇ（Ｃｖｈ）マウスモノクローナル抗体産生ハイブリドーマ培養上清を加え１時間、３７℃にて一次抗体反応を行った。 The permeabilized PGC was washed 3 times with PBS containing 1% BSA, and then an anti-chicken vasa homolog (Cvh) mouse monoclonal antibody-producing hybridoma culture supernatant containing a 50-fold diluted anti-chicken Nanog rabbit polyclonal antibody was added for 1 hour at 37 ° C. The primary antibody reaction was carried out.
なお、上記抗ニワトリＮａｎｏｇウサギポリクローナル抗体は、ニワトリＮａｎｏｇ組換え体（ｒｃｈＮａｎｏｇ）をウサギに免疫することで取得した。より詳細には、まず、ｒｃｈＮａｎｏｇを、メルトース結合タンパク質（ＭＢＰ）遺伝子を含むｐＭＡＬ−ｃ２Ｘプラスミド（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ社製）及びグルタチオンＳトランスフェラーゼ（ＧＳＴ）遺伝子を含むｐＧＥＸ−６Ｐ−１プラスミド（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）で、製造者の説明書に従って、融合タンパク質であるＭＢＰ−ｒｃｈＮａｎｏｇ又はＧＳＴ−ｒｃｈＮａｎｏｇとして発現させた。３００μｇのＭＢＰ−ｒｃｈＮａｎｏｇを、同量の完全フロイントアジュバントとともに雌ＮＺＷウサギの皮下に２週おきに計４回注射した。ウサギから採取した抗血清を、ＧＳＴ−ｒｃｈＮａｎｏｇ結合アガロースビーズを用いたアフィニティークロマトグラフィーで精製することで抗ニワトリＮａｎｏｇウサギポリクローナル抗体を得た。 The anti-chicken Nanog rabbit polyclonal antibody was obtained by immunizing a rabbit with a chicken Nanog recombinant (rchNanog). More specifically, first, rchNanog is converted into a pMAL-c2X plasmid (manufactured by New England Biolabs) containing a meltose-binding protein (MBP) gene and a pGEX-6P-1 plasmid (GE Healthcare) containing a glutathione S transferase (GST) gene. Produced) as a fusion protein MBP-rch Nanog or GST-rch Nanog according to the manufacturer's instructions. 300 μg of MBP-rch Nanog was injected subcutaneously into female NZW rabbits with the same amount of complete Freund's adjuvant every 2 weeks for a total of 4 times. Anti-serum collected from rabbits was purified by affinity chromatography using GST-rch Nanog-conjugated agarose beads to obtain anti-chicken Nanog rabbit polyclonal antibodies.
また、抗Ｃｖｈマウスモノクローナル抗体産生ハイブリドーマに関しては、Ｃｖｈ組換え体（ｒＣｖｈ）をｒｃｈＮａｎｏｇと同様にＭＢＰ−ｒＣｖｈ又はＧＳＴ−ｒＣｖｈとして発現させた。次に５０μｇのＧＳＴ−ｒＣｖｈを、同量の完全フロイントアジュバントとともに８週齢の雌ＢＡＬＢ／ｃマウスの腹腔内に注射した。２週おきに計３回、同じ抗原を含む０．１ｍｌのＰＢＳでブーストした。当該マウスに５０μｇのＧＳＴ−ｒＣｖｈを含むＰＢＳを静脈内投与することでブーストし、３日後に、当該マウスの脾細胞をＳＰ２／９ Ａｇ１４骨髄腫細胞と融合させ、抗Ｃｖｈマウスモノクローナル抗体産生ハイブリドーマを得た。当該ハイブリドーマを無血清培地又は１０％ＦＢＳ−ＩＭＤＭで培養し、コンフルエントになった段階で培養上清を回収した。回収した培養上清を、孔径が０．４５μｍのフィルターで濾過し、上述の一次抗体反応に用いた。 As for the anti-Cvh mouse monoclonal antibody-producing hybridoma, the Cvh recombinant (rCvh) was expressed as MBP-rCvh or GST-rCvh in the same manner as rchNanog. 50 μg of GST-rCvh was then injected intraperitoneally into 8 week old female BALB / c mice with the same amount of complete Freund's adjuvant. Boosted with 0.1 ml PBS containing the same antigen 3 times every 2 weeks. The mice were boosted by intravenous administration of PBS containing 50 μg of GST-rCvh. Three days later, the spleen cells of the mice were fused with SP2 / 9 Ag14 myeloma cells, and an anti-Cvh mouse monoclonal antibody-producing hybridoma was obtained. Obtained. The hybridoma was cultured in a serum-free medium or 10% FBS-IMDM, and the culture supernatant was collected when it became confluent. The collected culture supernatant was filtered through a filter having a pore size of 0.45 μm and used for the primary antibody reaction described above.
一次抗体反応後のＰＧＣを１％ＢＳＡ含有ＰＢＳで３回洗浄し、２００倍希釈Ａｌｅｘａ５９４標識抗ウサギＩｇＧ（Ｈ＋Ｌ）ヤギ抗体、Ａｌｅｘａ４８８標識抗マウスＩｇＧ（Ｈ＋Ｌ）ヤギ抗体及び１％ＢＳＡ含有ＰＢＳ（共にＴｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を添加し、１時間、３７℃にて二次抗体反応を行った。二次抗体反応後のＰＧＣを１％ＢＳＡ含有ＰＢＳで３回洗浄し、１０μｌ ＤＡＰＩ含有ＶＥＣＴＡＳＨＩＥＬＤ（商標）Ｍｏｕｎｔｉｎｇ Ｍｅｄｉｕｍ（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ社製）へ懸濁してスライド標本化した。得られたスライド標本を蛍光顕微鏡ＢＸ５１（Ｏｌｙｍｐｕｓ社製）で観察した。 The PGC after the primary antibody reaction was washed 3 times with PBS containing 1% BSA, diluted 200-fold with Alexa594-labeled anti-rabbit IgG (H + L) goat antibody, Alexa488-labeled anti-mouse IgG (H + L) goat antibody and PBS containing 1% BSA (both Thermo Fisher Scientific) was added, and a secondary antibody reaction was performed at 37 ° C. for 1 hour. The PGC after the secondary antibody reaction was washed three times with PBS containing 1% BSA, suspended in 10 μl DAPI-containing VECTASHIELD (trademark) Mounting Medium (manufactured by Vector Laboratories), and prepared as a slide specimen. The obtained slide specimen was observed with a fluorescence microscope BX51 (manufactured by Olympus).
生殖腺への移動能アッセイを次の方法で行った。ＺｓＧｒｅｅｎ１レンチウイルスベクターを、Ｌｅｎｔｉ−Ｘ（商標）Ｅｘｐｒｅｓｓｉｏｎ Ｓｙｓｔｅｍ（タカラバイオ社製）を利用して作製し、ＰＧＣへの感染に用いた。ＰＧＣへのＺｓＧｒｅｅｎ１レンチウイルスベクターの感染は、本ベクターを含む４μｇ／ｍｌポリブレン溶液（タカラバイオ社製）を用いて４時間、３７℃でインキュベートすることで行った。ＺｓＧｒｅｅｎ１にて蛍光標識したＰＧＣを、１０００個ずつステージＸ白色レグホン胚（アキタ社製）の胚盤葉下腔へインジェクションし、Ｐｅｒｒｙ及びＮａｉｔｏらにより確立された全胚培養系を用いて７日胚まで培養した。培養した７日胚より生殖腺を回収し、蛍光実体顕微鏡ＳＺＸ１２（Ｏｌｙｍｐｕｓ社製）にてＺｓＧｒｅｅｎ１により蛍光標識されたＰＧＣの局在を観察した。 The gonad mobility assay was performed as follows. A ZsGreen1 lentiviral vector was prepared using Lenti-X ™ Expression System (manufactured by Takara Bio Inc.) and used for infection of PGC. Infection of PGC with the ZsGreen1 lentiviral vector was carried out by incubating at 37 ° C. for 4 hours using a 4 μg / ml polybrene solution (manufactured by Takara Bio Inc.) containing this vector. 1000 PGCs fluorescently labeled with ZsGreen1 were injected into the subblastoidal space of stage X white leghorn embryos (manufactured by Akita) one by one, and 7-day embryos using the whole embryo culture system established by Perry and Naito et al. Until cultured. The gonads were collected from the cultured 7-day embryos, and the localization of PGC fluorescently labeled with ZsGreen1 was observed with a fluorescent stereomicroscope SZX12 (manufactured by Olympus).
（結果）
多能性マーカーであるＮａｎｏｇ及び生殖細胞マーカーであるＣｖｈの発現を、長期培養したＰＧＣにおいて免疫染色によって確認した結果を図３に示す。図３において核の位置を示すＤＡＰＩの位置とＮａｎｏｇの位置とが重なっているため、ＰＧＣは、核でＮａｎｏｇを発現し、細胞質でＣｖｈを発現していることが観察された。この結果、長期培養したＰＧＣは生体内でのＰＧＣの特性を維持している可能性が示された。なお、図中のスケールバーは、それぞれ５０．０μｍを示す。
(result)
The results of confirming the expression of Nanog, which is a pluripotency marker, and Cvh, which is a germ cell marker, by immunostaining in PGCs cultured for a long time are shown in FIG. In FIG. 3, since the position of DAPI indicating the position of the nucleus and the position of Nanog overlap, it was observed that PGC expressed Nanog in the nucleus and Cvh in the cytoplasm. As a result, it was shown that PGCs cultured for a long time may maintain the characteristics of PGCs in vivo. In addition, the scale bar in a figure shows 50.0 micrometers, respectively.
長期培養したＰＧＣの生殖腺への移動能アッセイの結果を図４に示す。回収した生殖腺の中で緑色蛍光タンパク質であるＺｓＧｒｅｅｎ１により標識されたＰＧＣが生殖腺の中に散在していることが観察された（図４右）。この結果、培養したＰＧＣも本来ＰＧＣが持つ生殖腺への移動能を保持している可能性が示唆された。 FIG. 4 shows the results of an assay for the ability of PGCs cultured for a long time to migrate to the gonads. It was observed that PGCs labeled with green fluorescent protein ZsGreen1 were scattered in the gonads in the collected gonads (right side of FIG. 4). As a result, it was suggested that the cultured PGC may also retain the ability of the PGC to move to the gonad.
（実施例５：ニワトリ肝細胞−馴化培地を用いたＰＧＣの培養）
まず、上記実施例２のＢＲＬ−馴化培地のＢＲＬの代わりにレグホン雄肝細胞（ｌｅｇｈｏｒｎ−ｍａｌｅ ｈｅｐａｔｏｍａ、ＬＭＨ）を用いて、ＬＭＨ−馴化培地を調製した。ＬＭＨとして医薬基盤・健康・栄養研究所の細胞番号ＪＣＲＢ０２３７を用いた。
(Example 5: Chicken hepatocytes-PGC culture using conditioned medium)
First, instead of BRL in the BRL-conditioned medium of Example 2 above, LGH-conditioned medium was prepared using leghorn male hepatoma (LMH). Cell number JCRB0237 from the Institute for Pharmaceutical Bases, Health and Nutrition was used as the LMH.
ＰＧＣの培養には、５０倍希釈マトリゲル（商標）基底膜マトリックス（Ｃｏｒｎｉｎｇ社製）又は０．１％ゼラチン（Ｓｉｇｍａ Ａｌｄｒｉｃｈ社製）水溶液でコートした培養器を用いた。ＨＨステージ１３〜１５の横斑プリマスロック胚（祓川エッグファーム社製）から採取したＰＧＣを含む胚血液を、リン酸緩衝液にて洗浄した。表１のＢＲＬ−馴化培地の代わりに、ＬＭＨ−馴化培地を９．２〜５４．６％とした基本培地を用いて、２４ウェルプレートの１ウェル当たりに、洗浄した胚血液から１胚を加え、３８℃、５％ＣＯ２、３％Ｏ２環境下で培養した。細胞の増殖に合わせて２〜４日おきに継代培養を行った。 For incubating PGC, an incubator coated with 50-fold diluted Matrigel ™ basement membrane matrix (Corning) or 0.1% gelatin (Sigma Aldrich) aqueous solution was used. Embryonic blood containing PGCs collected from the HH stage 13-15 side patch Plymouth Rock embryo (manufactured by Yodogawa Egg Farm) was washed with a phosphate buffer. In place of the BRL-conditioned medium in Table 1, one embryo was added from the washed embryo blood per well of a 24-well plate using a basic medium containing 9.2-54.6% LMH-conditioned medium. The cells were cultured in an environment of 38 ° C., 5% CO 2 and 3% O 2 . Subculture was performed every 2 to 4 days according to cell growth.
Ｆａｌｃｏｎ ９６ウェルホワイトプレート（Ｃｏｒｎｉｎｇ社製）の１ウェル当たり１０００個のＰＧＣ（継代１５回、４５日間培養）を、ＬＭＨ−馴化培地を９．２〜５４．６％とした表２に示す基本培地において、３８℃、５％ＣＯ２、３％Ｏ２環境下で培養開始した。培養したＰＧＣは、培養３日後にＣｅｌｌＴｉｔｅｒ−Ｇｌｏ Ｌｕｍｉｎｅｓｃｅｎｔ Ｃｅｌｌ Ｖｉａｂｉｌｉｔｙ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製）を用いて増殖活性を測定した。本キットで得られる化学発光の検出には、２０３０Ｍｕｌｔｉｌａｂｅｌ Ｒｅａｄｅｒ ＡＲＶＯ Ｘ４（ＰｅｒｋｉｎＥｌｍｅｒ社製）を用いた。 1000 PGCs (15 passages, 45 days culture) per well of a Falcon 96-well white plate (Corning) were used, and the basics shown in Table 2 with LMH-conditioned medium at 9.2-54.6% In the medium, the culture was started in an environment of 38 ° C., 5% CO 2 , 3% O 2 . The cultured PGCs were measured for proliferation activity using CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega) after 3 days of culture. For detection of chemiluminescence obtained with this kit, 2030 Multilabel Reader ARVO X4 (manufactured by PerkinElmer) was used.
（結果）
図５は、様々な濃度のＬＭＨ−馴化培地を含む基本培地におけるＰＧＣの増殖活性を示す図である。ＬＭＨ−馴化培地を、９．２％から２２．４％の濃度で用いた場合に、ＰＧＣの増殖活性が亢進した。ＬＭＨ−馴化培地を、１１．４％の濃度で用いた時に最も高い増殖活性を示した。増殖活性を比較すると、ＬＭＨ−馴化培地は約１０％と比較的低濃度で、４０％ＢＲＬ−馴化培地と同等の増殖活性を示した。
(result)
FIG. 5 is a diagram showing the proliferation activity of PGCs in a basic medium containing various concentrations of LMH-conditioned medium. When LMH-conditioned medium was used at a concentration of 9.2% to 22.4%, the proliferation activity of PGC was enhanced. LMH-conditioned medium showed the highest growth activity when used at a concentration of 11.4%. When the growth activity was compared, LMH-conditioned medium had a relatively low concentration of about 10%, and showed growth activity equivalent to 40% BRL-conditioned medium.
上述した実施の形態は、本発明を説明するためのものであり、本発明の範囲を限定するものではない。すなわち、本発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等の発明の意義の範囲内で施される様々な変形が、本発明の範囲内とみなされる。 The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.
本発明は、始原生殖細胞、特にはニワトリの始原生殖細胞の生産に好適である。 The present invention is suitable for the production of primordial germ cells, particularly chicken primordial germ cells.
Claims (10)
始原生殖細胞の培養方法。 Culturing primordial germ cells in a medium containing an apoptosis inhibitor,
Primordial germ cell culture method.
ミオシンII阻害剤である、
請求項１に記載の始原生殖細胞の培養方法。 The apoptosis inhibitor is
A myosin II inhibitor,
The method for culturing primordial germ cells according to claim 1.
ブレビスタチンである、
請求項２に記載の始原生殖細胞の培養方法。 The myosin II inhibitor is
Is blebbistatin,
The method for culturing primordial germ cells according to claim 2.
２．５μＭ以上１０．０μＭ以下である、
請求項３に記載の始原生殖細胞の培養方法。 The concentration of blebbistatin in the medium is
2.5 μM or more and 10.0 μM or less,
The method for culturing primordial germ cells according to claim 3.
Ｒｈｏキナーゼ阻害剤である、
請求項１に記載の始原生殖細胞の培養方法。 The apoptosis inhibitor is
A Rho kinase inhibitor,
The method for culturing primordial germ cells according to claim 1.
Ｙ−２７６３２又はＨ１１５２である、
請求項５に記載の始原生殖細胞の培養方法。 The Rho kinase inhibitor is
Y-27632 or H1152.
The method for culturing primordial germ cells according to claim 5.
カスパーゼ阻害剤である、
請求項１に記載の始原生殖細胞の培養方法。 The apoptosis inhibitor is
A caspase inhibitor,
The method for culturing primordial germ cells according to claim 1.
ｚＶＡＤ−ＦＭＫである、
請求項７に記載の始原生殖細胞の培養方法。 The caspase inhibitor is
zVAD-FMK.
The method for culturing primordial germ cells according to claim 7.
鳥類の初期胚の胚血液から分離される始原生殖細胞である、
請求項１から８のいずれか一項に記載の始原生殖細胞の培養方法。 Primordial germ cells cultured in the culturing step,
Primordial germ cells isolated from the embryonic blood of early avian embryos,
The method for culturing primordial germ cells according to any one of claims 1 to 8.
始原生殖細胞の培養用培地添加物。 Including an apoptosis inhibitor,
Medium supplement for culturing primordial germ cells.
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