JP4905719B2 - Neural stem cell preparation method - Google Patents
Neural stem cell preparation method Download PDFInfo
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- JP4905719B2 JP4905719B2 JP2007502657A JP2007502657A JP4905719B2 JP 4905719 B2 JP4905719 B2 JP 4905719B2 JP 2007502657 A JP2007502657 A JP 2007502657A JP 2007502657 A JP2007502657 A JP 2007502657A JP 4905719 B2 JP4905719 B2 JP 4905719B2
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Description
本発明は、神経幹細胞の調製法に関し、より詳細には、骨髄から神経幹細胞を誘導・調製する方法と、神経再生治療への応用に関するものである。 The present invention relates to a method for preparing neural stem cells, and more particularly to a method for inducing and preparing neural stem cells from bone marrow and application to nerve regeneration therapy.
脳梗塞などの脳血管障害や神経変性疾患は、超高齢社会を迎えつつあるわが国にとって解決すべき最重要課題のひとつである。しかし、これまでの神経細胞死抑制などに焦点をあてた治療法開発の努力にもかかわらず、有効な治療法の開発に結びつく成果は未だ十分に得られていない。 Cerebrovascular disorders such as cerebral infarction and neurodegenerative diseases are one of the most important issues to be solved for Japan, which is facing a super-aged society. However, in spite of the efforts to develop therapeutic methods focusing on the suppression of neuronal cell death and the like so far, results that have led to the development of effective therapeutic methods have not yet been obtained sufficiently.
神経再生療法は、既に障害をうけている脳機能を再生神経により改善できる可能性があり、新たな神経疾患治療法として期待されている。既に米国では、パーキンソン病などの神経変性疾患において胎児脳より採取された神経幹細胞移植による神経再生治療が行われている。しかし、この治療法には倫理上の問題など解決すべき課題が少なくない。 Nerve regeneration therapy has the potential to improve brain functions that have already been damaged by regenerating nerves, and is expected as a new treatment method for neurological diseases. Already in the United States, nerve regeneration treatment is performed by transplanting neural stem cells collected from fetal brain in neurodegenerative diseases such as Parkinson's disease. However, there are many problems to be solved such as ethical problems in this treatment method.
脳梗塞治療を目的としたＥＳ細胞由来の神経幹細胞移植も行われているが、ＥＳ細胞の機能解析が不十分で、かつその入手が倫理的にも制度的にも現状困難という問題がある。また、ＥＳ細胞の神経への分化誘導法は未だ確立しておらず、生体内で生着・機能するかどうかも不確かである。 Although ES cell-derived neural stem cell transplantation for the treatment of cerebral infarction has also been carried out, there is a problem that functional analysis of ES cells is inadequate and acquisition is difficult ethically and institutionally. In addition, a method for inducing differentiation of ES cells into nerves has not yet been established, and it is uncertain whether or not it will engraft and function in vivo.
神経再生医療への利用が期待される神経幹細胞は、上述のように胎児脳やＥＳ細胞に由来するもののほか、成熟脳由来の脳室下帯組織（subventricular zone：ＳＶＺ）からも採取可能である。胎児脳由来の神経幹細胞は、神経細胞に分化する。一方、成熟脳ＳＶＺ由来の神経幹細胞は、グリア細胞に高率に分化し、通常の培養条件では神経細胞に分化しない。成熟脳ＳＶＺ由来の神経幹細胞を神経細胞に分化させるためには栄養因子の添加や、分化誘導因子（Ｎｏｔｃｈ１など）の遺伝子導入が必要である。 Neural stem cells expected to be used in nerve regeneration medicine can be collected from fetal brain and ES cells as described above, as well as from subventricular zone (SVZ) derived from mature brain. . Neural stem cells derived from fetal brain differentiate into neurons. On the other hand, neural stem cells derived from mature brain SVZ differentiate into glial cells at a high rate, and do not differentiate into neurons under normal culture conditions. In order to differentiate neural stem cells derived from mature brain SVZ into neurons, it is necessary to add trophic factors and introduce genes for differentiation-inducing factors (such as Notch1).
神経再生医療において、このような培養神経幹細胞を移植する方法としては、直接細胞を脳内に注入・移植する方法が主に施行されている。しかし、この方法は手技が煩雑であり、移植法は未だ確立されたものとはいえない。一方、このように直接脳内に移植する方法以外に、脳梗塞急性期に骨髄単核球細胞を静脈内に投与する治療法が試みられている。もっとも、この治療法は神経幹細胞を投与するものではなく、未分化の骨髄細胞を静脈内投与するものであるので、神経再生の効果がどの細胞によって生じているのかなどメカニズムが不明で効果の予測・評価が困難である。 In nerve regenerative medicine, as a method of transplanting such cultured neural stem cells, a method of directly injecting and transplanting cells into the brain is mainly performed. However, this method is complicated and the transplantation method has not been established yet. On the other hand, in addition to the method of transplanting directly into the brain in this way, a therapeutic method in which bone marrow mononuclear cells are administered intravenously during the acute phase of cerebral infarction has been attempted. However, this treatment method does not administer neural stem cells but administers undifferentiated bone marrow cells intravenously, so the mechanism such as which cell is causing the nerve regeneration effect is unknown and the effect is predicted・ Evaluation is difficult.
骨髄間質細胞に遺伝子導入することによって、あるいは数種類の成長因子を添加しながら骨髄間質細胞を培養することによって、骨髄細胞から神経細胞を作り出すことができたとの報告がある。また最近になって、ヒトやマウスの骨髄細胞から神経幹細胞であるニューロスフェア（neurosphere）が得られたとの報告がある（下記の非特許文献１・２参照）。しかし、これらの方法は、（１）ニューロスフェアおよび分化可能な神経幹細胞を作成するまでの培養期間が２ヶ月から６ヶ月程度かかり、長期間を要する、（２）最終的に神経細胞あるいはグリア細胞に分化させるのにＢＤＮＦやＰＤＧＦなどの栄養因子による刺激が必要である、などの難点がある。 There are reports that nerve cells could be created from bone marrow cells by introducing genes into bone marrow stromal cells or by culturing bone marrow stromal cells while adding several types of growth factors. Recently, it has been reported that neurospheres, which are neural stem cells, were obtained from human or mouse bone marrow cells (see Non-Patent Documents 1 and 2 below). However, in these methods, (1) the culture period required to produce neurospheres and differentiable neural stem cells takes about 2 to 6 months, and it takes a long time. (2) Ultimately, neurons or glial cells However, there is a drawback that stimulation with a nutritional factor such as BDNF or PDGF is required to differentiate the cells.
また、本発明者らは、ヒト臍帯血由来の幹細胞（ＣＤ３４陽性細胞）を脳梗塞モデルマウスに静脈内投与することで、脳内での血管再生、さらに神経再生が起こることを見出し、これを報告している（下記の非特許文献３参照）。 In addition, the present inventors have found that human umbilical cord blood-derived stem cells (CD34 positive cells) are intravenously administered to cerebral infarction model mice to cause vascular regeneration and further nerve regeneration in the brain. (See Non-Patent Document 3 below).
脳梗塞などの脳血管障害やその後遺症、パーキンソン病やアルツハイマー病などの神経変性疾患に対しては、神経機能を回復させる有効な治療法がない。神経機能の再生をめざした治療法確立のためには再生する神経細胞が提供されなければならない。神経再生治療への応用が期待される神経幹細胞は、上述したように現在まで、胎児脳由来の神経幹細胞、成体脳由来の神経幹細胞、ＥＳ細胞由来の神経幹細胞、骨髄成体多能性幹細胞（multipotent adult progenitor cell：ＭＡＰＣ）由来の神経幹細胞などが報告されている。しかし、その供給源や神経細胞への分化効率、さらに移植脳への生着性、機能発揮性など問題点が山積している。また、骨髄細胞に遺伝子を導入するなど、神経細胞を人為的に作成する手法なども考案されているが、発がん性や生着、機能性などに問題がある。 For cerebrovascular disorders such as cerebral infarction and its sequelae, and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, there is no effective treatment for restoring neurological function. In order to establish a therapy aimed at regenerating nerve function, regenerating nerve cells must be provided. As described above, neural stem cells that are expected to be applied to nerve regeneration treatment have been fetal brain-derived neural stem cells, adult brain-derived neural stem cells, ES cell-derived neural stem cells, adult bone marrow pluripotent stem cells (multipotent Adult stem cells derived from adult progenitor cells (MAPC) have been reported. However, there are many problems such as the supply source, efficiency of differentiation into nerve cells, engraftment to the transplanted brain, and function performance. In addition, methods for artificially creating nerve cells, such as introducing genes into bone marrow cells, have been devised, but there are problems with carcinogenicity, engraftment, functionality and the like.
理想的には、まず倫理的にも、また移植片対宿主病（graft-versus-host disease：ＧＶＨＤ）回避のためにも、神経再生治療に使用される神経幹細胞は、患者本人の細胞であること、つまり自家移植（Autograft）であることが望ましい。また、神経細胞に高率に分化し（Neurogenesis）、神経機能を保ち（Functional）、かつ必要な時期に（Timely）、簡単に（eaSY）、大量に（Amplify）、作成される幹細胞であること、即ち、これらの特徴をあわせもったいわば「FANTASY」であることが望ましい。 Ideally, neural stem cells used for nerve regeneration therapy are the patient's own cells, first ethically and for avoiding graft-versus-host disease (GVHD). That is, autograft is desirable. It must also be a stem cell that differentiates into neurons at a high rate (Neurogenesis), maintains nerve function (Functional), and is easily (eaSY), abundant (Amplify), in large quantities That is, it is desirable to combine these characteristics with “FANTASY”.
ところで、生体には組織あるいは臓器障害時に際して様々な修復機能が備わっている。これらは主に消化管上皮や肝臓、血管内皮細胞など組織固有の細胞の分裂や増殖を介して行われているものであるが、最近種々の骨髄由来の幹細胞がこれに関与することが知られてきた。しかしながら、ほとんどの病態において骨髄が組織修復にどの程度関与するかの検討はなされていないのが実情である。神経幹細胞移植による神経再生治療法の開発に際して、このような生体内で通常起こっているホメオスターシス維持機構の一環としての修復機構のメカニズムを研究した上で、その機構に添った治療法を考案することが有効・適切と考えられる。 By the way, the living body has various repair functions in the case of tissue or organ failure. These are mainly performed through the division and proliferation of tissue-specific cells such as gastrointestinal epithelium, liver, and vascular endothelial cells. Recently, various bone marrow-derived stem cells are known to be involved in this process. I came. However, in reality, the degree to which bone marrow is involved in tissue repair in most pathological conditions has not been studied. When developing a nerve regeneration therapy by neural stem cell transplantation, we will study the mechanism of the repair mechanism as part of the homeostasis maintenance mechanism that normally occurs in vivo, and then devise a therapy according to that mechanism. Is considered effective and appropriate.
本発明は、上記の問題点に鑑みなされたものであって、その目的は、脳梗塞などの脳血管障害や神経変性疾患に対する新たな神経再生治療法を開発・確立すること、そのため、神経再生治療又はその治療法開発において好適に用いられ、簡便かつ短期間に調製可能であり、しかもその移植により良好に神経細胞を脳患部に生着、機能させることが期待できる神経幹細胞の調製法等を提供することにある。 The present invention has been made in view of the above-mentioned problems, and its purpose is to develop and establish a new nerve regeneration treatment method for cerebrovascular disorders such as cerebral infarction and neurodegenerative diseases. A method for preparing neural stem cells that are suitably used in treatment or development of therapies, can be prepared easily and in a short period of time, and can be expected to engraft and function neuronal cells well in the affected area by transplantation. It is to provide.
本発明者らは、上記の課題に鑑み鋭意研究を進めた結果、まず免疫不全のＳＣＩＤマウスを処置して脳梗塞モデルマウスを作出し、脳梗塞１週間後の当該マウスより採取した骨髄細胞を培養することで、骨髄細胞から神経幹細胞（ニューロスフェア）に分化誘導できることを明らかにした。さらに、後述するように再生と免疫に関する重要な知見が得られ、骨髄から短期間（２週間程度）に神経幹細胞を誘導・調製する技術を確立した。この方法によって、実際にヒトの正常骨髄から神経幹細胞を誘導・調製することにも成功した。この方法により、患者骨髄から神経幹細胞を調製し、これを自家移植として静脈内投与することによって、再生神経を良好に脳患部に生着、機能させる神経再生治療法が実現可能であること等を見出し、本発明を完成させるに至った。 As a result of diligent research in view of the above problems, the present inventors first treated an immunodeficient SCID mouse to produce a cerebral infarction model mouse, and obtained bone marrow cells collected from the mouse one week after the cerebral infarction. It was clarified that differentiation can be induced from bone marrow cells to neural stem cells (neurospheres) by culturing. Furthermore, as described later, important knowledge about regeneration and immunity was obtained, and a technique for inducing and preparing neural stem cells from bone marrow in a short period (about 2 weeks) was established. By this method, neural stem cells were actually derived and prepared from human normal bone marrow. By preparing neural stem cells from patient bone marrow by this method and administering them intravenously as autologous transplants, it is possible to realize a nerve regeneration treatment method for engrafting and functioning regenerative nerves well in affected brain areas, etc. The headline and the present invention have been completed.
即ち、本発明は、産業上および医学・医療上有用な発明として、下記（１）〜（４８）の発明を包含するものである。
（１）免疫抑制剤、および、脳梗塞その他脳障害後のヒト又は動物から採取した血清を添加して骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。
ここで、脳障害とは、脳の血管の狭窄、閉塞、結紮又はその他の原因で脳の一部が虚血状態になっていること、すなわち脳虚血障害を意味する。
（２）脳梗塞その他脳障害後のヒト又は動物から採取した骨髄細胞を、免疫抑制剤を添加して培養することにより分化誘導し、神経幹細胞を調製する方法。
（３）免疫抑制剤、および、ケモカイン（もしくはその他のサイトカイン）を添加して骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。
（４）ケモカインとして、インターロイキン−８（ＩＬ−８）ファミリーのケモカインを添加する、上記（３）記載の方法。
（５）サイトカインとして、ＴＮＦα（tumor necrosis factor-α：腫瘍壊死因子）を添加する、上記（３）記載の方法。
（６）ケモカイン（もしくはその他のサイトカイン）であり、骨髄細胞から神経幹細胞への分化を直接的又は間接的に誘導する分化誘導因子。
（７）上記（６）記載の分化誘導因子を用いて、培養細胞から神経幹細胞を調製する方法。
（８）上記（６）記載の分化誘導因子と免疫抑制剤とからなる神経幹細胞分化誘導剤。
（９）神経幹細胞として、ニューロスフェア（neurosphere）またはニューロスフェア様細胞塊を調製する、上記（１）〜（５）又は（７）のいずれかに記載の方法。
（１０）上記（１）〜（５）又は（７）のいずれかに記載の方法により神経幹細胞を生産する方法。
（１１）上記（１０）記載の神経幹細胞をさらに培養することにより分化誘導し、神経細胞を生産する方法。
（１２）上記（１０）記載の神経幹細胞もしくは上記（１１）記載の神経細胞を、脳梗塞治療または他の神経再生治療に使用する方法。
（１３）上記（１０）記載の神経幹細胞もしくは上記（１１）記載の神経細胞を、静脈内投与などの方法により投与する、脳梗塞治療法または他の神経再生治療法。
（１４）上記（１）記載の方法において、神経幹細胞の調製に用いる骨髄細胞および血清には、治療対象の患者から採取したものを使用する方法。
（１５）上記（２）〜（５）又は（７）のいずれかに記載の方法において、神経幹細胞の調製に用いる骨髄細胞には、治療対象の患者から採取したものを使用する方法。
（１６）上記（１）〜（５）又は（７）のいずれかに記載の方法において、神経幹細胞の調製に用いる免疫抑制剤に、ＦＫ５０６（タクロリムス）、シクロスポリン、抗ＣＤ２８抗体および抗ＩＣＯＳ抗体などのＴ細胞機能を抑制する免疫抑制剤を使用する方法。
ここで、抗ＣＤ２８抗体および抗ＩＣＯＳ抗体とは、それぞれＣＤ２８、ＩＣＯＳの作用を阻害し、Ｔ細胞機能を抑制するブロッキング抗体を意味し、Ｔ細胞を刺激し活性化するものではない。
（１７）上記（１３）記載の神経再生治療法において、神経（幹）細胞の投与と同時または異時に、免疫抑制剤を投与する方法。
（１８）上記（１３）記載の神経再生治療法において、神経（幹）細胞の投与と同時または異時に、ヒト臍帯血由来ＣＤ３４陽性細胞、血管内皮前駆細胞（ＥＰＣ）などの血管形成能を有する細胞を投与する方法。
（１９）上記（１０）記載の神経幹細胞もしくは上記（１１）記載の神経細胞を含む脳梗塞治療剤。
（２０）上記（１０）記載の神経幹細胞もしくは上記（１１）記載の神経細胞を含む神経再生治療剤。
（２１）免疫不全マウス（ＳＣＩＤマウスおよびヌードマウスを含む）、その母系マウス、又はこれらのマウスから作成した脳梗塞モデルマウスの骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。
（２２）免疫不全マウスの骨髄細胞を、脳梗塞モデルマウスから採取した血清、又はケモカイン（もしくはその他のサイトカイン）を添加して培養する、上記（２１）記載の方法。
（２３）免疫不全マウスの母系マウスの骨髄細胞を、脳梗塞モデルマウスから採取した血清（又は、ケモカインもしくはその他のサイトカイン）と免疫抑制剤とを添加して培養する、上記（２１）記載の方法。
（２４）免疫不全マウスの母系マウスから作成した脳梗塞モデルマウスの骨髄細胞を、免疫抑制剤を添加して培養する、上記（２１）記載の方法。
（２５）免疫不全マウスから作成した脳梗塞モデルマウスの骨髄細胞を培養することにより神経幹細胞を調製する、上記（２１）記載の方法。
（２６）免疫不全マウスの母系マウスに免疫抑制剤を投与して免疫不全状態にした後作成した脳梗塞モデルマウスの骨髄細胞を培養することにより神経幹細胞を調製する、上記（２１）記載の方法。
（２７）免疫不全マウスが、C.B-17/IcrCrlBRマウスを母系とするＳＣＩＤマウスである、上記（２１）〜（２６）のいずれかに記載の方法。
（２８）免疫不全マウスが、BALB/cAJclマウスを母系とするヌードマウスである、上記（２１）〜（２６）のいずれかに記載の方法。
（２９）上記（２１）〜（２８）のいずれかに記載の方法により調製された神経幹細胞。
（３０）ＳＣＩＤマウスの母系マウスの脳の血管を結紮してなる脳梗塞モデル動物。
（３１）ＳＣＩＤマウスの母系マウスの脳の血管が、中大脳動脈である上記（３０）記載の脳梗塞モデル動物。
（３２）ＳＣＩＤマウスの母系マウスが、C.B-17/IcrCrlBRマウスである上記（３０）又は（３１）記載の脳梗塞モデル動物。
（３３）ヌードマウス又はその母系マウスの脳の血管を結紮してなる脳梗塞モデル動物。
（３４）ヌードマウス又はその母系マウスの脳の血管が、中大脳動脈である上記（３３）記載の脳梗塞モデル動物。
（３５）ヌードマウスの母系マウスが、BALB/cAJclマウスである上記（３３）又は（３４）記載の脳梗塞モデル動物。
（３６）上記（３０）〜（３５）のいずれかに記載の脳梗塞モデル動物に、被検薬物を投与して、当該被検薬物の脳梗塞に対する有効性をスクリーニングする方法。
（３７）上記（３０）〜（３５）のいずれかに記載の脳梗塞モデル動物に、神経（幹）細胞又は他の細胞を移植して、当該移植治療の脳梗塞に対する有効性をスクリーニングする方法。
（３８）免疫抑制作用を有する物質を有効成分とする神経再生治療剤。
（３９）免疫抑制作用を有する物質が、Ｔ細胞機能を抑制する物質である、上記（３８）記載の神経再生治療剤。
（４０）免疫抑制作用を有する物質が、ＦＫ５０６（タクロリムス）、シクロスポリン、抗ＣＤ２８抗体および抗ＩＣＯＳ抗体のいずれかである、上記（３９）記載の神経再生治療剤。
（４１）ケモカインその他のサイトカイン、又はこれらの骨髄細胞から神経幹細胞への分化を誘導する作用を促進する物質を有効成分とする神経再生治療剤。
（４２）ケモカインが、インターロイキン−８（ＩＬ−８）ファミリーのケモカインである、上記（４１）記載の神経再生治療剤。
（４３）サイトカインが、ＴＮＦα（tumor necrosis factor-α：腫瘍壊死因子）である、上記（４１）記載の神経再生治療剤。
（４４）脳梗塞又は他の神経疾患の治療に用いられる、上記（３８）〜（４３）のいずれかに記載の神経再生治療剤。
（４５）脳梗塞又は他の神経疾患の治療に用いられる神経再生治療剤のスクリーニング方法であって、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進する作用を有するかどうかを指標に、候補物質を探索することを特徴とする神経再生治療剤のスクリーニング方法。
（４６）ヒト又は動物から採取した骨髄細胞の培地に被検物質を投与し、当該被検物質が、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進する作用を有するかどうかを調べることを特徴とする上記（４５）記載の神経再生治療剤のスクリーニング方法。
（４７）免疫抑制作用を有する物質群の中から、候補物質を探索することを特徴とする上記（４５）又は（４６）記載の神経再生治療剤のスクリーニング方法。
（４８）ケモカインその他のサイトカイン、又はこれらの活性もしくは産生を調節する作用を有する物質群の中から、候補物質を探索することを特徴とする上記（４５）又は（４６）記載の神経再生治療剤のスクリーニング方法。That is, the present invention includes the following inventions (1) to (48) as industrially useful medical and medical inventions.
(1) A method for preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressant and serum collected from humans or animals after cerebral infarction or other brain injury.
Here, cerebral disorder means that a part of the brain is in an ischemic state due to stenosis, occlusion, ligation or other causes of cerebral blood vessels, that is, cerebral ischemic disorder.
(2) A method of preparing neural stem cells by inducing differentiation of bone marrow cells collected from humans or animals after cerebral infarction or other cerebral disorders by adding an immunosuppressant and culturing them.
(3) A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressant and a chemokine (or other cytokine).
(4) The method according to (3) above, wherein a chemokine of interleukin-8 (IL-8) family is added as a chemokine.
(5) The method according to (3) above, wherein TNFα (tumor necrosis factor-α: tumor necrosis factor) is added as a cytokine.
(6) A differentiation-inducing factor that is a chemokine (or other cytokine) and directly or indirectly induces differentiation from bone marrow cells to neural stem cells.
(7) A method for preparing neural stem cells from cultured cells using the differentiation-inducing factor according to (6) above.
(8) A neural stem cell differentiation inducer comprising the differentiation inducer according to (6) above and an immunosuppressant.
(9) The method according to any one of (1) to (5) or (7) above, wherein a neurosphere or a neurosphere-like cell mass is prepared as a neural stem cell.
(10) A method for producing neural stem cells by the method according to any one of (1) to (5) or (7) above.
(11) A method for inducing differentiation by further culturing the neural stem cell according to (10) to produce a neural cell.
(12) A method of using the neural stem cell according to (10) or the neural cell according to (11) for cerebral infarction treatment or other nerve regeneration treatment.
(13) A cerebral infarction treatment method or other nerve regeneration treatment method, wherein the neural stem cell according to (10) or the nerve cell according to (11) is administered by a method such as intravenous administration.
(14) In the method described in (1) above, the bone marrow cells and serum used for the preparation of neural stem cells use those collected from the patient to be treated.
(15) The method according to any one of (2) to (5) or (7) above, wherein bone marrow cells used for the preparation of neural stem cells are those collected from a patient to be treated.
(16) In the method according to any one of (1) to (5) or (7) above, FK506 (tacrolimus), cyclosporine, anti-CD28 antibody, anti-ICOS antibody and the like are used as immunosuppressive agents for preparing neural stem cells. A method of using an immunosuppressive agent that suppresses T cell function.
Here, the anti-CD28 antibody and the anti-ICOS antibody mean blocking antibodies that inhibit the action of CD28 and ICOS, respectively, and suppress the T cell function, and do not stimulate and activate T cells.
(17) A method of administering an immunosuppressive agent in the nerve regeneration treatment method according to the above (13), simultaneously or at the same time as administration of nerve (stem) cells.
(18) In the nerve regeneration treatment method according to the above (13), the blood vessel forming ability of human umbilical cord blood-derived CD34-positive cells, vascular endothelial progenitor cells (EPCs) or the like is simultaneously or different from the administration of nerve (stem) cells. A method of administering cells.
(19) A therapeutic agent for cerebral infarction comprising the neural stem cell according to (10) above or the neural cell according to (11) above.
(20) A nerve regeneration therapeutic agent comprising the neural stem cell according to (10) or the neural cell according to (11).
(21) A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells of immunodeficient mice (including SCID mice and nude mice), their maternal mice, or cerebral infarction model mice prepared from these mice.
(22) The method according to (21) above, wherein bone marrow cells of an immunodeficient mouse are cultured by adding serum or chemokine (or other cytokine) collected from a cerebral infarction model mouse.
(23) The method according to (21) above, wherein bone marrow cells of a maternal mouse of an immunodeficient mouse are cultured by adding serum (or chemokine or other cytokine) collected from a cerebral infarction model mouse and an immunosuppressive agent. .
(24) The method according to (21) above, wherein bone marrow cells of a cerebral infarction model mouse prepared from a maternal mouse of an immunodeficient mouse are cultured by adding an immunosuppressive agent.
(25) The method according to (21) above, wherein neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared from an immunodeficient mouse.
(26) The method according to (21) above, wherein neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared after administering an immunosuppressant to a maternal mouse of an immunodeficient mouse to make it immunocompromised. .
(27) The method according to any one of (21) to (26) above, wherein the immunodeficient mouse is a SCID mouse having a CB-17 / IcrCrlBR mouse as a maternal line.
(28) The method according to any one of (21) to (26) above, wherein the immunodeficient mouse is a nude mouse having a BALB / cAJcl mouse as a maternal line.
(29) A neural stem cell prepared by the method according to any one of (21) to (28) above.
(30) A cerebral infarction model animal obtained by ligating the blood vessels of the brain of a SCID mouse maternal mouse.
(31) The cerebral infarction model animal according to the above (30), wherein the blood vessel of the brain of the maternal mouse of the SCID mouse is a middle cerebral artery.
(32) The cerebral infarction model animal according to (30) or (31) above, wherein the maternal mouse of the SCID mouse is a CB-17 / IcrCrlBR mouse.
(33) A cerebral infarction model animal obtained by ligating the blood vessels of the brain of a nude mouse or its mother mouse.
(34) The cerebral infarction model animal according to the above (33), wherein the blood vessel of the brain of the nude mouse or its mother mouse is the middle cerebral artery.
(35) The cerebral infarction model animal according to (33) or (34) above, wherein the nude mouse is a BALB / cAJcl mouse.
(36) A method for screening the effectiveness of the test drug against cerebral infarction by administering the test drug to the cerebral infarction model animal according to any one of (30) to (35).
(37) A method for screening the effectiveness of the transplantation treatment for cerebral infarction by transplanting nerve (stem) cells or other cells into the cerebral infarction model animal according to any one of (30) to (35) above .
(38) A nerve regeneration therapeutic agent comprising a substance having an immunosuppressive action as an active ingredient.
(39) The nerve regeneration therapeutic agent according to (38), wherein the substance having an immunosuppressive action is a substance that suppresses T cell function.
(40) The therapeutic agent for nerve regeneration according to the above (39), wherein the substance having an immunosuppressive action is any one of FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody.
(41) A nerve regeneration therapeutic agent comprising as an active ingredient a chemokine or other cytokine, or a substance that promotes the action of inducing differentiation from bone marrow cells into neural stem cells.
(42) The nerve regeneration therapeutic agent according to the above (41), wherein the chemokine is an interleukin-8 (IL-8) family chemokine.
(43) The nerve regeneration therapeutic agent according to the above (41), wherein the cytokine is TNFα (tumor necrosis factor-α: tumor necrosis factor).
(44) The nerve regeneration therapeutic agent according to any one of (38) to (43), which is used for treatment of cerebral infarction or other neurological diseases.
(45) A screening method for a nerve regeneration therapeutic agent used for the treatment of cerebral infarction or other neurological diseases, which has an action of inducing differentiation from bone marrow cells to neural stem cells or promoting the differentiation. A screening method for a therapeutic agent for nerve regeneration characterized by searching for a candidate substance as an index.
(46) Whether a test substance is administered to a medium of bone marrow cells collected from a human or an animal, and the test substance induces differentiation from bone marrow cells to neural stem cells or has an action of promoting the differentiation (45) The screening method of the nerve regeneration therapeutic agent according to the above (45).
(47) The screening method for a therapeutic agent for nerve regeneration according to (45) or (46), wherein a candidate substance is searched from a group of substances having an immunosuppressive action.
(48) The nerve regeneration therapeutic agent according to the above (45) or (46), wherein a candidate substance is searched from a chemokine or other cytokine, or a substance group having an action of regulating activity or production thereof. Screening method.
本発明の神経幹細胞調製法は、簡易かつ短期間に、骨髄細胞から神経幹細胞を調製することができる。得られた神経幹細胞は、神経細胞に高効率に分化可能であり、生体への移植により脳患部において良好に神経細胞を生着、機能発揮させることが期待できるので、神経再生治療、あるいは脳梗塞モデルマウス等を用いた治療法開発の目的に使用できる。また、材料となる骨髄細胞は生体からの採取が比較的容易であるので、例えば脳梗塞後の患者から骨髄を採取し、免疫抑制剤の存在下に培養を行って神経幹細胞を調製し、これを静脈内投与などで患者に移植する自家移植治療に本発明を利用することができる。 The neural stem cell preparation method of the present invention can prepare neural stem cells from bone marrow cells in a simple and short period of time. The obtained neural stem cells can be differentiated into nerve cells with high efficiency, and can be expected to engraft and function well in the affected area of the brain by transplanting to the living body, so that nerve regeneration treatment or cerebral infarction It can be used for the purpose of developing treatments using model mice. In addition, since bone marrow cells used as a material are relatively easy to collect from a living body, for example, bone marrow is collected from a patient after cerebral infarction and cultured in the presence of an immunosuppressant to prepare neural stem cells. The present invention can be used for autologous transplantation treatment in which a patient is transplanted into a patient by intravenous administration or the like.
以下、本発明の具体的態様、技術的範囲等について説明する。
［１］骨髄からの新たな神経幹細胞調製法の開発
本発明者らは最近、良好な再現性を示し、しかも長期間生存可能な脳梗塞モデルマウスを開発した（特願２００４−１０８５００号）。この脳梗塞モデルマウスは、後述するように、免疫不全マウスであるＳＣＩＤマウスの中大脳動脈を結紮することにより、中大脳動脈の皮質枝の血流を選択的に途絶させ、再現性のよい均一な脳梗塞を作成したものである。興味深いことに、この脳梗塞ＳＣＩＤマウスでは、虚血後の脳梗塞の進展（delayed infarct expansion）は脳梗塞後３日で終了し、その後は脳萎縮の進展ではなく、脳形態上はむしろ回復する。免疫組織化学による検討の結果、このマウスの脳梗塞下組織（脳梗塞周囲の瘢痕部位）において脳梗塞後多数の神経幹細胞の出現が観察され、これが正常神経組織に生着して脳再生、脳機能改善に貢献していること、さらに、この神経幹細胞は骨髄に由来するものであることを明らかにした。Hereinafter, specific embodiments and technical scope of the present invention will be described.
[1] Development of a New Neural Stem Cell Preparation Method from Bone Marrow The present inventors recently developed a cerebral infarction model mouse that exhibits good reproducibility and can survive for a long period of time (Japanese Patent Application No. 2004-108500). As will be described later, this cerebral infarction model mouse selectively disrupts the blood flow of the cortical branch of the middle cerebral artery by ligating the middle cerebral artery of the SCID mouse, which is an immunodeficient mouse, and is uniform with good reproducibility. Created a cerebral infarction. Interestingly, in this cerebral infarction SCID mouse, the delayed infarct expansion after ischemia ended 3 days after the cerebral infarction, and after that, rather than the development of cerebral atrophy, the brain morphology is rather recovered . As a result of examination by immunohistochemistry, the appearance of numerous neural stem cells after cerebral infarction was observed in the subcerebral infarcted tissue (the scar site around the cerebral infarction) of this mouse. It has been clarified that it contributes to functional improvement and that the neural stem cells are derived from bone marrow.
そこで、上記脳梗塞ＳＣＩＤマウスから脳梗塞１週間後に採取した骨髄細胞を培養したところ、上述のように、神経幹細胞（ニューロスフェア）に分化誘導することができた（図１Ｄ）。またその後も培養を続けると、神経幹細胞は分裂・増殖し、神経細胞に分化誘導された（同図Ｅ）。さらに、神経再生と免疫に関する下記（１）〜（６）の重要な知見が得られた（なお、各実験の詳細については後述する）。 Thus, when bone marrow cells collected from the cerebral infarction SCID mouse one week after cerebral infarction were cultured, differentiation into neural stem cells (neurospheres) could be induced as described above (FIG. 1D). Further, when the culture was continued thereafter, the neural stem cells were divided and proliferated, and differentiation was induced into the nerve cells (Fig. E). Furthermore, the following important findings (1) to (6) regarding nerve regeneration and immunity were obtained (details of each experiment will be described later).
（１） 脳梗塞を起こしていない正常ＳＣＩＤマウスの骨髄を培養しても神経幹細胞は誘導されないが、この骨髄に脳梗塞モデルマウスの血清を添加すると神経幹細胞が誘導され（図２、図５Ａ・Ｂ）、神経幹細胞は神経細胞にも分化した（図５Ｃ・Ｄ）。このことより、脳梗塞後の血清中に、骨髄細胞を神経幹細胞に分化誘導させる刺激因子（differentiation factor）が存在し、生体ではこの刺激因子が血流を介して骨髄に伝えられ、神経幹細胞が産生されると考えられる。
さらに解析の結果、この刺激因子、換言すれば神経幹細胞への分化誘導因子のひとつは、インターロイキン−８（ＩＬ−８）ファミリーのケモカインである、ＣＩＮＣ−１／ＧＲＯ（cytokine-induced neutrophil chemoattractant-1／growth-related oncogene）であることを明らかにした（図２２、２３）。(1) Although neural stem cells are not induced by culturing the bone marrow of normal SCID mice that have not developed cerebral infarction, addition of serum from cerebral infarction model mice to this bone marrow induces neural stem cells (FIGS. 2, 5A, B) Neural stem cells also differentiated into neurons (FIGS. 5C and D). As a result, there is a differentiation factor in the serum after cerebral infarction that induces bone marrow cells to differentiate into neural stem cells. In vivo, this stimulating factor is transmitted to the bone marrow via the bloodstream, It is thought to be produced.
Furthermore, as a result of analysis, one of the stimulating factors, in other words, one of the differentiation inducing factors into neural stem cells, is a chemokine of the interleukin-8 (IL-8) family, CINC-1 / GRO (cytokine-induced neutrophil chemoattractant- 1 / growth-related oncogene) (FIGS. 22 and 23).
（２） ＳＣＩＤマウスの母系である免疫機能が正常なC.B-17/IcrCrlBRマウスの中大脳動脈を結紮して脳梗塞モデルを作成した後、その骨髄を採取して培養すると、神経幹細胞が一旦形成されるが（図１０Ｃ・Ｄ）、これらは２週間以内にすべて消失してしまう（図１５）。 (2) After creating a cerebral infarction model by ligating the middle cerebral artery of a CB-17 / IcrCrlBR mouse with normal immune function, which is the mother system of SCID mice, neural stem cells are formed once the bone marrow is collected and cultured. However, these all disappear within 2 weeks (FIG. 15).
（３） 一方、上記C.B-17/IcrCrlBRマウスに対して、免疫抑制剤ＦＫ５０６（タクロリムス）を投与して免疫不全状態にし、脳梗塞を起こさせた後も免疫抑制剤の投与を続け、その後当該マウスから採取した骨髄細胞を培養したところ、神経幹細胞に分化誘導させることができた（図１３）。他の免疫抑制剤であるシクロスポリンＡ（ＣｓＡ）を投与した場合も同様の結果が得られた（図２４）。また、C.B-17/IcrCrlBRマウスを脳梗塞後、その骨髄を採取し、免疫抑制剤ＦＫ５０６を添加して培養すると、神経幹細胞が形成された（図１４、図１５）。 (3) On the other hand, the immunosuppressive agent FK506 (tacrolimus) was administered to the CB-17 / IcrCrlBR mouse to make it immunodeficient, and administration of the immunosuppressive agent was continued after cerebral infarction was caused. When bone marrow cells collected from mice were cultured, differentiation into neural stem cells could be induced (FIG. 13). Similar results were obtained when cyclosporin A (CsA), another immunosuppressant, was administered (FIG. 24). When C.B-17 / IcrCrlBR mice were cerebral infarcted, their bone marrow was collected and cultured with the addition of the immunosuppressant FK506 to form neural stem cells (FIGS. 14 and 15).
（４） 脳梗塞を起こしていない正常C.B-17/IcrCrlBRマウスの骨髄細胞を異なる条件下で培養したところ、免疫抑制剤ＦＫ５０６および脳梗塞モデルマウスの血清を添加した場合にのみ、神経幹細胞が形成された（図１６）。ＦＫ５０６の代わりに、Ｔ細胞機能を抑制する抗体（抗Ｔ細胞抗体）を添加しても同様に神経幹細胞が形成された（図２１、図２７）。 (4) When bone marrow cells of normal CB-17 / IcrCrlBR mice without cerebral infarction were cultured under different conditions, neural stem cells formed only when the immunosuppressant FK506 and sera of cerebral infarction model mice were added. (FIG. 16). In place of FK506, even when an antibody that suppresses T cell function (anti-T cell antibody) was added, neural stem cells were similarly formed (FIGS. 21 and 27).
（５） さらに、ヒトの正常骨髄細胞を、免疫抑制剤ＦＫ５０６および脳梗塞患者の血清を添加して培養したところ、神経幹細胞に分化誘導させることができた（図１９Ａ）。これをさらに培養すると、神経細胞に分化した（同図Ｂ）。 (5) Furthermore, when human normal bone marrow cells were cultured with the addition of immunosuppressant FK506 and serum from a cerebral infarction patient, differentiation into neural stem cells could be induced (FIG. 19A). When this was further cultured, it differentiated into neurons (Fig. B).
（６） 脳梗塞ＳＣＩＤマウスの骨髄から調製された神経幹細胞を、別の脳梗塞ＳＣＩＤマウスに静脈内投与してその脳梗塞治療効果を調べたところ、脳形態上の回復が観察され、神経再生の促進効果が認められた（図２０）。 (6) When neural stem cells prepared from the bone marrow of a cerebral infarction SCID mouse were intravenously administered to another cerebral infarction SCID mouse and examined for the therapeutic effect of the cerebral infarction, recovery in brain morphology was observed, and nerve regeneration Was observed (FIG. 20).
本発明は、これらの知見に基づき、（１）正常骨髄細胞を神経幹細胞へ分化させるためには、脳梗塞後の血清中に存在する刺激因子と、骨髄培養液中の免疫反応を抑制させる免疫抑制剤の添加が重要であり、（２）脳梗塞後の患者又は免疫正常なモデル動物から採取した骨髄細胞を神経幹細胞へ分化させるためには、免疫抑制剤の添加が重要であること、および、（３）上記の刺激因子（分化誘導因子）のひとつは、ＩＬ−８ファミリーのケモカインＣＩＮＣ−１／ＧＲＯであることを見出し、以下の第１〜第３の神経幹細胞調製法を提供するものである。 Based on these findings, the present invention is based on these findings. (1) In order to differentiate normal bone marrow cells into neural stem cells, stimulating factors present in serum after cerebral infarction and immunity that suppresses immune reactions in bone marrow cultures The addition of an inhibitor is important. (2) In order to differentiate bone marrow cells collected from a patient after cerebral infarction or an immunonormal model animal into neural stem cells, the addition of an immunosuppressant is important, and (3) One of the above stimulating factors (differentiation inducing factors) is found to be IL-8 family chemokine CINC-1 / GRO, and provides the following first to third neural stem cell preparation methods It is.
第１の神経幹細胞調製法：免疫抑制剤、および、脳梗塞その他脳障害後のヒト又は動物から採取した血清を添加して骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。
第２の神経幹細胞調製法：脳梗塞その他脳障害後のヒト又は動物から採取した骨髄細胞を、免疫抑制剤を添加して培養することにより分化誘導し、神経幹細胞を調製する方法。
第３の神経幹細胞調製法：免疫抑制剤、および、ケモカイン（好ましくは、ＩＬ−８ファミリーのケモカイン）を添加して骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。First neural stem cell preparation method: A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressant and serum collected from humans or animals after cerebral infarction or other brain injury.
Second neural stem cell preparation method: A method of preparing neural stem cells by inducing differentiation by adding bone marrow cells collected from humans or animals after cerebral infarction or other cerebral disorders to which an immunosuppressant is added and culturing.
Third neural stem cell preparation method: A method for preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressant and a chemokine (preferably, IL-8 family chemokine).
第１（および第３）の調製法において、常法にしたがってヒト又は動物から採取した骨髄は、免疫抑制剤および脳障害後の血清（又は、当該血清の代わりにケモカイン）を添加する以外、従来の神経幹細胞の培養方法と同様の方法により培養可能である。また、第２の調製法においても、免疫抑制剤を添加する以外、従来の神経幹細胞の培養方法と同様の方法により培養可能である。従来の培養方法としては、ニューロスフェア（neurosphere）法、低密度単層培養法、高密度単層培養法などが例示され、このうち好ましい培養法としては、ニューロスフェア法が挙げられる。後述の実施例においては、このニューロスフェア法を用いて、塩基性線維芽細胞成長因子（ｂＦＧＦ，５０μｇ／ｍｌ）および上皮成長因子（ＥＧＦ，２０μｇ／ｍｌ）存在下骨髄細胞を培養し、神経幹細胞を調製した。 In the first (and third) preparation method, bone marrow collected from humans or animals according to a conventional method is conventionally used except that an immunosuppressant and serum after brain injury (or chemokine instead of the serum) are added. The neural stem cells can be cultured by the same method as described above. Further, in the second preparation method, it can be cultured by the same method as the conventional neural stem cell culture method except that an immunosuppressant is added. Examples of conventional culture methods include a neurosphere method, a low-density monolayer culture method, and a high-density monolayer culture method, and among these, a preferred culture method includes the neurosphere method. In Examples described later, using this neurosphere method, bone marrow cells are cultured in the presence of basic fibroblast growth factor (bFGF, 50 μg / ml) and epidermal growth factor (EGF, 20 μg / ml), and neural stem cells Was prepared.
以下、本発明の調製法において使用する免疫抑制剤、血清および分化誘導因子であるケモカインについて説明する。
［1-A］免疫抑制剤
培養液に添加する免疫抑制剤は、実施例で用いたＦＫ５０６（タクロリムス）およびシクロスポリンを使用することができるが、そのほかに、バシリキシマブ、アザチオプリン、ムロモナブＣＤ３、ミゾリビン、ミコフェノール酸モフェチル、など公知の免疫抑制剤の使用が考えられる。免疫抑制剤は、Ｔ細胞機能など細胞性免疫を抑制する作用・性質を有するものが好ましく、免疫抑制機能を有する限りにおいて、抗体（例えば抗ＣＤ２８抗体、抗ＩＣＯＳ抗体）などの物質も広く本発明の「免疫抑制剤」に含まれる。
培養液への免疫抑制剤の添加量は、特に限定されないが、０．０１μｇ／ｍｌ〜１．０μｇ／ｍｌ程度の濃度で添加することが好ましい。後述の実施例では、０．１μｇ／ｍｌの濃度でＦＫ５０６を培養液に添加した。Hereinafter, the immunosuppressant used in the preparation method of the present invention, serum, and chemokine that is a differentiation-inducing factor will be described.
[1-A] Immunosuppressant FK506 (tacrolimus) and cyclosporin used in the Examples can be used as immunosuppressants to be added to the culture solution. In addition, basiliximab, azathioprine, muromonab CD3, mizoribine, mycobin Use of known immunosuppressive agents such as phenolate mofetil is conceivable. The immunosuppressive agent preferably has an action / property that suppresses cellular immunity such as T cell function. As long as it has an immunosuppressive function, substances such as antibodies (for example, anti-CD28 antibody and anti-ICOS antibody) are widely used in the present invention. In "immunosuppressive agents".
The amount of the immunosuppressive agent added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 0.01 μg / ml to 1.0 μg / ml. In the examples described later, FK506 was added to the culture solution at a concentration of 0.1 μg / ml.
［1-B］血清
第１の調製法において、培養液へ添加する血清の量も特に限定されるものではないが、５μｌ／ｍｌ〜２５μｌ／ｍｌ程度の濃度で添加することが好ましい。上述のように、脳梗塞後の血清中には、骨髄細胞を神経幹細胞に分化誘導させる分化誘導因子が存在し、この分化誘導因子は、虚血侵襲や他の原因により脳神経細胞が一部障害を受けることによって、生体の有する修復機構の一環として産生されるものと考えられる。したがって、脳梗塞後の血清に限らず、事故等により脳組織が一部障害を受けた後の血清中にもこの分化誘導因子は出現し、当該血清を添加して骨髄細胞を培養することで同様の効果が得られると考えられる。
また、例えば脳梗塞後の血清を使用して調製した本発明の神経幹細胞を、脳梗塞の治療に限らず、他の神経疾患の再生治療に利用してもよい。
「動物」から採取した血清を使用して、治療法の開発用などに神経幹細胞を調製する場合、当該動物としてはマウスやラットのほか、ウシ、ブタ、ヒツジ、ヤギ、ウサギ、イヌ、ネコ、モルモット、ハムスターなどの哺乳動物が例示される。マウスは、ＳＣＩＤマウス以外のものを使用してもよい。
なお、後述の実施例に示すように、ヒトおよびマウスの場合、脳梗塞後１週間目に採取した血清を使用することで良好に神経幹細胞を調製できたので、本発明の第１の調製法においては、脳梗塞後（または他の脳障害後）５日〜１０日の血清を使用することが好ましい。[1-B] Serum In the first preparation method, the amount of serum added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 5 μl / ml to 25 μl / ml. As described above, there is a differentiation-inducing factor in the serum after cerebral infarction that induces differentiation of bone marrow cells into neural stem cells. This differentiation-inducing factor is partially damaged by cerebral neurons due to ischemic invasion or other causes. Is considered to be produced as part of the repair mechanism of the living body. Therefore, this differentiation-inducing factor appears not only in sera after cerebral infarction but also in sera after brain tissue is partially damaged due to an accident, etc. It is considered that the same effect can be obtained.
For example, the neural stem cells of the present invention prepared using serum after cerebral infarction may be used not only for treatment of cerebral infarction but also for regenerative treatment of other neurological diseases.
When preparing neural stem cells for the development of therapeutic methods using serum collected from "animals", in addition to mice and rats, the animals include cattle, pigs, sheep, goats, rabbits, dogs, cats, Mammals such as guinea pigs and hamsters are exemplified. A mouse other than the SCID mouse may be used.
In the case of humans and mice, neural stem cells were successfully prepared by using serum collected one week after cerebral infarction, as shown in the examples described later, so the first preparation method of the present invention In this case, it is preferable to use serum for 5 to 10 days after cerebral infarction (or after other cerebral injury).
［1-C］分化誘導因子
第３の調製法は、免疫抑制剤とともに、分化誘導因子としてケモカインを添加して骨髄細胞を培養する方法である。
上述のように、脳梗塞後の血清中には、骨髄細胞を神経幹細胞へ分化させる何らかの分化誘導因子が存在し、この分化誘導因子が血流を介して骨髄に伝えられ、神経幹細胞が産生されると考えられる。本発明者は、この分化誘導因子の探索に際して、ＳＣＩＤマウスの免疫反応の特徴に注目した。すなわち、ＳＣＩＤマウスはＴ細胞が欠落しているがＮＫ細胞は十分存在しているため、ＳＣＩＤマウスがＣ．Ｂ１７マウスより骨髄での神経幹細胞産生に適した条件を備えているとすると、ＮＫ細胞にその原因を求めるのが妥当と考えた。さらに、骨髄細胞の分化に関与し、かつ虚血などの生体への侵襲刺激に際して血中に放出される因子を探索した。[1-C] Differentiation Inducing Factor The third preparation method is a method of culturing bone marrow cells by adding a chemokine as a differentiation inducing factor together with an immunosuppressant.
As described above, there is some differentiation inducing factor that differentiates bone marrow cells into neural stem cells in the serum after cerebral infarction, and this differentiation inducing factor is transmitted to the bone marrow via the bloodstream to produce neural stem cells. It is thought. The present inventor paid attention to the characteristics of the immune response of SCID mice when searching for the differentiation-inducing factor. That is, SCID mice lack T cells but sufficient NK cells. If conditions suitable for neural stem cell production in the bone marrow were prepared from B17 mice, it was considered appropriate to determine the cause for NK cells. Furthermore, the present inventors searched for factors that are involved in bone marrow cell differentiation and that are released into the blood upon invasive stimulation of a living body such as ischemia.
ＣＩＮＣ−１／ＧＲＯ（cytokine-induced neutrophil chemoattractant-1／growth-related oncogene）は、インターロイキン−８（ＩＬ−８）ファミリーのケモカインであり、コルヒチン刺激などのnoxious stimulationにより脳の視床下部神経で産生亢進され、下垂体後葉を経て血中に放出される（Neurosci. Res. (1997) 27:181-184.）。ＣＩＮＣ−１は、ＮＫ細胞からも産生されて子宮の脱落膜を介した受精卵の免疫反応に関与し（Biochem. Biophys. Res. Commun. (1994) 200:378-383.）、また骨髄間質（stroma）細胞からも産生される（Exp. Cell Res. (2004) 299:383-392.）。さらに、本発明者は独自に、脳虚血負荷に際しても視床下部や下垂体後葉においてＣＩＮＣ−１産生が亢進するという調査結果を得ていた。
そこで、ＣＩＮＣ−１が神経幹細胞への分化誘導因子である可能性を検討した。その結果、脳梗塞を起こしていない正常C.B-17/IcrCrlBRマウスから採取した骨髄に、免疫抑制剤ＦＫ５０６およびＣＩＮＣ−１を添加して培養すると、骨髄から神経幹細胞へ分化誘導させることができた（図２２、２３）。この結果から、ＣＩＮＣ−１が分化誘導因子のひとつであることが示された。また、ＣＩＮＣ−１は、培養中の骨髄細胞を凝集させる作用を有しており（図２３Ｂ）、この凝集した細胞塊が核となってニューロスフェアが形成されると考えられる。したがって、このような凝集作用を有する他のケモカインもＣＩＮＣ−１と同様に分化誘導因子として作用すると考えられる。このようなケモカインの作用が骨髄中の幹細胞に直接作用しているのか、あるいは間質細胞などを介して間接的に作用しているのかは今後更なる解析を必要とするが、いずれにせよ、ケモカインを免疫抑制剤とともに培養骨髄中に添加することで、神経幹細胞への分化誘導が可能である。CINC-1 / GRO (cytokine-induced neutrophil chemoattractant-1 / growth-related oncogene) is a chemokine of the interleukin-8 (IL-8) family, and is produced in the hypothalamic nerve of the brain by noxious stimulation such as colchicine stimulation. Increased and released into the blood via the posterior lobe of the pituitary gland (Neurosci. Res. (1997) 27: 181-184.). CINC-1 is also produced from NK cells and is involved in the immune response of fertilized eggs via the uterine decidua (Biochem. Biophys. Res. Commun. (1994) 200: 378-383.) It is also produced from stroma cells (Exp. Cell Res. (2004) 299: 383-392.). Furthermore, the inventor has independently obtained a survey result that CINC-1 production is enhanced in the hypothalamus and the posterior pituitary gland during cerebral ischemia.
Therefore, the possibility of CINC-1 being a differentiation inducing factor into neural stem cells was examined. As a result, when bone marrow collected from normal CB-17 / IcrCrlBR mice without cerebral infarction was added and cultured with immunosuppressants FK506 and CINC-1, differentiation from bone marrow to neural stem cells could be induced ( 22 and 23). From this result, it was shown that CINC-1 is one of the differentiation-inducing factors. CINC-1 has an action of aggregating bone marrow cells in culture (FIG. 23B), and it is considered that the aggregated cell mass serves as a nucleus to form a neurosphere. Therefore, it is considered that other chemokines having such an aggregating action also act as a differentiation-inducing factor like CINC-1. Whether such chemokine actions directly act on stem cells in the bone marrow or indirectly through stromal cells will require further analysis in the future. By adding a chemokine together with an immunosuppressant into cultured bone marrow, differentiation into neural stem cells can be induced.
ケモカインはＩＬ−８ファミリーのケモカインの使用が好ましく、ヒト、ラット又はマウス由来のＩＬ−８ファミリーのケモカイン、あるいは他の哺乳動物のカウンターパートから選ばれるケモカインを添加する方法が挙げられる。ヒト由来ＩＬ−８ファミリーのケモカインとしては、ＩＬ−８／ＣＸＣＬ８、ＧＲＯ−α、ＧＲＯ−β及びＧＲＯ−γなどが例示され、ラット由来は、ＣＩＮＣ−１、ＣＩＮＣ−２α、ＣＩＮＣ−２β、ＣＩＮＣ−３、マウス由来は、ＫＣ、ＭＩＰ−２などが例示される。その他のＣＸＣケモカインの使用も考えられる。 The chemokine is preferably an IL-8 family chemokine, and includes a method of adding a chemokine selected from human, rat or mouse-derived IL-8 family chemokines or other mammalian counterparts. Examples of human-derived IL-8 family chemokines include IL-8 / CXCL8, GRO-α, GRO-β, and GRO-γ, and rat-derived chemokines include CINC-1, CINC-2α, CINC-2β, and CINC. -3, examples of mouse origin include KC and MIP-2. The use of other CXC chemokines is also conceivable.
培養液へのケモカインの添加量は、特に限定されないが、０．１μｇ／ｍｌ〜１．０μｇ／ｍｌ程度の濃度で添加することが好ましい。後述の実施例では、１０-5Ｍの濃度でＣＩＮＣ−１を培養液に添加した。
上述のように、ケモカインは、免疫抑制剤とともに培養骨髄中に添加することによって、神経幹細胞への分化誘導が可能であるので、ケモカインと免疫抑制剤とを組み合わせ、神経幹細胞分化誘導剤として提供できる。また、免疫抑制剤の添加は培養下のＴ細胞機能を抑制するためであるから、あらかじめＴ細胞を除去するなどして他の方法でＴ細胞機能を抑制することができれば、免疫抑制剤を添加せずにケモカインのみで培養細胞から神経幹細胞を調製することも可能と考えられる。The amount of chemokine added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 0.1 μg / ml to 1.0 μg / ml. In the examples described later, CINC-1 was added to the culture solution at a concentration of 10 −5 M.
As described above, chemokine can be induced to differentiate into neural stem cells by adding it to cultured bone marrow together with an immunosuppressive agent. Therefore, a chemokine and an immunosuppressive agent can be combined and provided as a neural stem cell differentiation inducing agent. . Moreover, since the addition of the immunosuppressive agent is to suppress the T cell function in the culture, the immunosuppressive agent is added if the T cell function can be suppressed by other methods, such as by removing the T cell in advance. It is also possible to prepare neural stem cells from cultured cells without using chemokines alone.
さらに後述するように、本発明者らによる探索の結果、上記ケモカイン以外に、サイトカインであるＴＮＦα（tumor necrosis factor-α）が、骨髄細胞から神経幹細胞への分化誘導作用を有することを明らかにした（図２６）。従って、本発明の上記第３の調製法において、ケモカインの代わりに、ＴＮＦα又はＩＬ１８などその他のサイトカインを分化誘導因子として使用してもよい。ＴＮＦαなどのサイトカインを分化誘導因子として使用する場合も、上記ケモカインと同様の方法・条件で使用可能であり、骨髄細胞から神経幹細胞への調製に利用することができる。 As will be described later, as a result of the search by the present inventors, it has been clarified that in addition to the chemokine, TNFα (tumor necrosis factor-α), which is a cytokine, has an action of inducing differentiation from bone marrow cells to neural stem cells. (FIG. 26). Therefore, in the third preparation method of the present invention, other cytokines such as TNFα or IL18 may be used as a differentiation inducing factor instead of the chemokine. When a cytokine such as TNFα is used as a differentiation-inducing factor, it can be used in the same method and conditions as the chemokine and can be used for preparation from bone marrow cells to neural stem cells.
本発明の神経幹細胞調製法は、次のような利点・特徴を有する。
（１） 簡便かつ短期間に、神経幹細胞の誘導・調製が可能である。後述の実施例に示すように、実際に骨髄から短期間（２週間程度）で神経幹細胞を誘導・調製することができ、得られた神経幹細胞はその後分裂・増殖して、神経細胞に高効率に分化した。
神経再生治療に応用する場合、治療開始は２週間以内（即ち、幹細胞の産生および生着に最も適した時期である急性期）が好ましく、従来この期間内に大量の幹細胞を準備・調製することは困難であった。短期間に幹細胞の調製可能な本発明の方法は、神経再生治療の臨床応用に最も適した方法といえる。The neural stem cell preparation method of the present invention has the following advantages and features.
(1) Neural stem cells can be induced and prepared easily and in a short period of time. As shown in the examples described later, neural stem cells can be induced and prepared from bone marrow in a short period of time (about 2 weeks), and the obtained neural stem cells are then divided and proliferated for high efficiency in nerve cells. Differentiated.
When applied to nerve regeneration treatment, the start of treatment is preferably within 2 weeks (that is, the acute phase, which is the most suitable time for stem cell production and engraftment), and a large number of stem cells are conventionally prepared and prepared within this period. Was difficult. The method of the present invention that can prepare stem cells in a short time can be said to be the most suitable method for clinical application of nerve regeneration therapy.
（２） 脳患部において良好に神経細胞を生着、機能発揮させることが期待できる。実際に、本方法により調製された神経幹細胞を移植（静脈内投与）したところ、脳形態上の回復が観察され、幹細胞移植により神経細胞が脳組織に生着したものと考えられる。
上述のように、本発明者らは、脳梗塞ＳＣＩＤマウスにおいて、脳梗塞後多数の神経幹細胞の出現が観察され、これが正常神経組織に生着して脳再生、脳機能改善に貢献していること、および、この神経幹細胞は骨髄に由来するものであることを明らかにした。本発明は、このように脳梗塞修復時に特異的に産生され、神経を再生する骨髄由来の神経幹細胞を調製するものであり、生体が要求する細胞を大量に提供することができ、生体機構に沿った移植治療が可能になると考えられる。(2) It can be expected that the nerve cells are engrafted and functioned well in the affected area of the brain. Actually, when neural stem cells prepared by this method were transplanted (intravenous administration), recovery in brain morphology was observed, and it is considered that the nerve cells were engrafted in the brain tissue by the stem cell transplantation.
As described above, in the cerebral infarction SCID mouse, the present inventors observed the appearance of a large number of neural stem cells after cerebral infarction, which engrafted in normal nerve tissue and contributed to brain regeneration and improvement of brain function. And that these neural stem cells are derived from bone marrow. The present invention prepares bone marrow-derived neural stem cells that are specifically produced during cerebral infarction repair and regenerate nerves, and can provide a large amount of cells required by the living body. It is thought that transplantation treatment along the line will be possible.
（３） 骨髄細胞は比較的採取が容易であるので、例えば脳梗塞後の患者から骨髄を採取し、免疫抑制剤の存在下に培養を行って神経幹細胞を調製し、これを静脈内投与などで患者に移植する自家移植も十分実現可能である。
細胞移植は、基本的に自家移植が倫理的にも医学的にも最良の方法と考えられる。本発明は、生体への生着性と機能発揮性の点で最良ともいえる患者自身の骨髄由来神経幹細胞を提供することができ、再生医療に適している。ただし、本発明は、自家移植への利用のみに限定されるものではなく、他家移植用に他者由来の骨髄から神経幹細胞を調製する方法にも利用可能である。(3) Since bone marrow cells are relatively easy to collect, for example, bone marrow is collected from a patient after cerebral infarction and cultured in the presence of an immunosuppressant to prepare neural stem cells, which are administered intravenously, etc. Thus, autotransplantation for transplantation to patients is also feasible.
Cell transplantation is basically considered to be the best ethical and medical method for autotransplantation. The present invention can provide the patient's own bone marrow-derived neural stem cells, which can be said to be the best in terms of engraftment in a living body and function, and is suitable for regenerative medicine. However, the present invention is not limited to use for autologous transplantation, and can also be used for a method for preparing neural stem cells from bone marrow derived from another person for other transplantation.
（４） 分化誘導因子であるケモカイン（もしくはその他のサイトカイン）の使用によって、より簡便かつ安全性を向上できる。また、慢性期などの患者で骨髄又は血清中に分化誘導因子としてのケモカインやサイトカインがまったく或いは殆ど存在しない場合に、ケモカインなどのサイトカインを添加して神経幹細胞を調製する方法は極めて効果的である。そのほか、他家移植用に他者由来の骨髄から神経幹細胞を調製する方法においても、血清の代わりにケモカインやサイトカインを使用することは、安全性の面から非常に望ましい方法である。 (4) Use of a chemokine (or other cytokine), which is a differentiation-inducing factor, can improve safety more easily. In addition, when there is no or almost no chemokine or cytokine as a differentiation-inducing factor in bone marrow or serum in patients such as chronic stage, a method for preparing neural stem cells by adding a cytokine such as chemokine is extremely effective. . In addition, in the method of preparing neural stem cells from bone marrow derived from another person for transplantation, it is highly desirable from the viewpoint of safety to use chemokines or cytokines instead of serum.
（５） 本発明は、脳梗塞の治療に限らず、その他の脳血管障害、脳虚血性疾患、神経変性疾患に対する神経移植治療にも利用可能であり、神経再生治療に広く利用可能性を有する。 (5) The present invention can be used not only for the treatment of cerebral infarction but also for nerve transplantation treatment for other cerebrovascular disorders, cerebral ischemic diseases, and neurodegenerative diseases, and has wide applicability for nerve regeneration treatment. .
（６） 本発明は、神経再生治療へ直接利用可能であるほか、治療法開発においても利用可能であり、例えば本発明により得られた神経幹細胞を様々な条件下、脳梗塞モデルマウスに移植してその治療効果を確認することで、更なる治療法開発・研究進展が期待できる。 (6) The present invention can be used not only for nerve regeneration treatment but also in the development of therapeutic methods. For example, neural stem cells obtained by the present invention are transplanted into cerebral infarction model mice under various conditions. By confirming the therapeutic effect, further development of research and research progress can be expected.
また、本発明により調製した神経（幹）細胞を神経再生治療剤として患者に移植・投与する方法としては、例えば、静注、点滴静注などによって投与する。このような注射剤は常法に従って製造され、希釈剤として一般に生理食塩水、細胞培養液などを用いることができる。さらに必要に応じて、殺菌剤、防腐剤、安定剤、等張化剤、無痛化剤などを加えてもよい。これら製剤中の神経（幹）細胞の配合量は特に限定されるものではなく、疾患の種類、症状の程度、患者の年齢、体重などに応じて決定すればよい。また、本発明の神経（幹）細胞を、数回にわたり患者に投与してもよいし、１回当たりの投与量、投与間隔などを診断結果に応じて決定してもよい。 Moreover, as a method of transplanting / administering nerve (stem) cells prepared according to the present invention to a patient as a nerve regeneration therapeutic agent, for example, intravenous administration, intravenous infusion, or the like is used. Such an injection is produced according to a conventional method, and generally a physiological saline, a cell culture solution or the like can be used as a diluent. Further, if necessary, bactericides, preservatives, stabilizers, tonicity agents, soothing agents and the like may be added. The compounding amount of nerve (stem) cells in these preparations is not particularly limited, and may be determined according to the type of disease, the degree of symptoms, the age of the patient, the weight, and the like. In addition, the nerve (stem) cells of the present invention may be administered to a patient several times, and the dose per administration, the administration interval, etc. may be determined according to the diagnosis result.
神経（幹）細胞に対する患者の免疫応答を抑制するため、神経（幹）細胞の投与と同時またはその投与前（もしくは投与後）に、免疫抑制剤を投与することが望ましい。投与方法は、神経（幹）細胞の投与と異なる方法であってもよい。 In order to suppress a patient's immune response to nerve (stem) cells, it is desirable to administer an immunosuppressant simultaneously with or before (or after) administration of nerve (stem) cells. The administration method may be different from the administration of nerve (stem) cells.
また、脳内での血管再生、神経再生を向上させるため、神経（幹）細胞の投与と同時またはその投与前（もしくは投与後）に、ヒト臍帯血由来ＣＤ３４陽性細胞（J.Clin.Invest. 114:330-338（2004）参照）、血管内皮前駆細胞（ＥＰＣ）などの血管形成能を有する細胞を投与する方法は、好ましい方法である。 In order to improve blood vessel regeneration and nerve regeneration in the brain, human umbilical cord blood-derived CD34-positive cells (J. Clin. Invest. 114: 330-338 (2004)), and a method of administering cells having angiogenic ability such as vascular endothelial progenitor cells (EPC) is a preferred method.
以上のように、神経（幹）細胞を神経再生治療剤として用いることができるが、これ以外に、後述の実施例の結果から、in vitroにおいて、またin vivoにおいても、骨髄由来神経幹細胞の形成を維持・促進し、神経再生治療を実現するためには、免疫機能を抑制すること、特にＴ細胞機能を抑制することが重要であることが示されたので、免疫抑制物質（特にＴ細胞機能を抑制する物質）を神経再生治療剤として用いることが可能である。 As described above, nerve (stem) cells can be used as therapeutic agents for nerve regeneration, but in addition to this, formation of bone marrow-derived neural stem cells in vitro and in vivo based on the results of Examples described later. It has been shown that it is important to suppress immune function, particularly to suppress T cell function, in order to maintain / promote and realize nerve regeneration therapy. Can be used as a therapeutic agent for nerve regeneration.
例えば、ＦＫ５０６（タクロリムス）、シクロスポリン、抗ＣＤ２８抗体および抗ＩＣＯＳ抗体など、Ｔ細胞の活性を抑制する物質を神経再生治療剤として用いることができる。 For example, substances that suppress the activity of T cells, such as FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody, can be used as a therapeutic agent for nerve regeneration.
そのほか、前述のように、骨髄細胞から神経幹細胞への分化誘導作用を有するＣＩＮＣ−１、ＴＮＦαといったケモカイン、サイトカインについても、これらの分化誘導因子を投与することによって、in vitro と同様に、in vivoでの骨髄由来神経幹細胞の形成が促進されると考えられるので、神経再生治療剤としての応用が可能である。 In addition, as described above, chemokines such as CINC-1 and TNFα, which have an action of inducing differentiation from bone marrow cells to neural stem cells, and cytokines can be administered in vivo in the same manner as in vitro by administering these differentiation inducers. It is thought that the formation of bone marrow-derived neural stem cells is promoted in this case, and therefore, it can be applied as a nerve regeneration therapeutic agent.
さらに、これらケモカイン、サイトカインの分化誘導作用を促進する物質（これらケモカイン、サイトカインの産生を促進する物質を含む）も神経再生治療剤としての応用が可能である。このような物質は、例えば後述する本発明のスクリーニング系を利用して探索することができる。 Furthermore, these chemokines and substances that promote the differentiation-inducing action of cytokines (including these chemokines and substances that promote the production of cytokines) can also be applied as therapeutic agents for nerve regeneration. Such a substance can be searched using the screening system of the present invention described later, for example.
本発明のこれら免疫抑制物質、ケモカイン、サイトカイン等を有効成分とする神経再生治療剤は、経口剤でもよいし、注射剤、坐剤、塗布剤等の非経口剤でもよい。錠剤、カプセル剤、顆粒剤、細粒剤、散剤等の経口剤は、例えば、デンプン、乳糖、白糖、トレハロース、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩類等を用いて常法に従って製造される。これらの製剤中の有効成分の配合量は特に制限されるものではなく適宜設定できる。この種の製剤には、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料等を適宜に使用することができる。 The nerve regeneration therapeutic agent containing these immunosuppressive substances, chemokines, cytokines and the like of the present invention as active ingredients may be oral agents, or parenteral agents such as injections, suppositories, and coating agents. Oral preparations such as tablets, capsules, granules, fine granules, powders and the like are produced according to a conventional method using, for example, starch, lactose, sucrose, trehalose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like. The compounding amount of the active ingredient in these preparations is not particularly limited and can be appropriately set. In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrances and the like can be appropriately used.
非経口剤の場合は、例えば、静注、点滴静注、皮下注射、筋肉注射などによって投与する。この非経口剤は常法に従って製造され、希釈剤として一般に注射用蒸留水、生理食塩水等を用いることができる。また、この非経口剤は安定性の点から、バイアル等に充填後冷凍し、通常の凍結乾燥処理により水分を除き、使用直前に凍結乾燥物から液剤を再調製することもできる。さらに必要に応じて、殺菌剤、防腐剤、安定剤、等張化剤、無痛化剤などを加えてもよい。これら製剤中の有効成分の配合量は特に制限されるものではなく、前述の神経（幹）細胞を投与する場合と同様に、疾患の種類、症状の程度、患者の年齢、体重などに応じて決定すればよい。また、本発明の神経再生治療剤を、数回にわたり患者に投与してもよいし、１回当たりの投与量、投与間隔などを診断結果に応じて決定してもよい。 In the case of a parenteral agent, for example, it is administered by intravenous injection, intravenous infusion, subcutaneous injection, intramuscular injection or the like. This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use. Further, if necessary, bactericides, preservatives, stabilizers, tonicity agents, soothing agents and the like may be added. The compounding amount of the active ingredient in these preparations is not particularly limited, and depends on the type of disease, the degree of symptoms, the age of the patient, the body weight, etc., as in the case of administering the nerve (stem) cells described above. Just decide. Further, the therapeutic agent for nerve regeneration of the present invention may be administered to the patient several times, and the dose per administration, the administration interval, etc. may be determined according to the diagnosis result.
［２］脳梗塞モデル動物の開発
上述した本発明の神経幹細胞調製法の開発に伴い、免疫不全マウス（ＳＣＩＤマウス）の母系マウスの脳の血管を結紮してなる新たな脳梗塞モデル動物を開発した。この脳梗塞モデル動物は、ＳＣＩＤマウスの代わりに、その母系マウスを使用すること以外、特願２００４−１０８５００号記載の方法と同様に作製可能である。[2] Development of a cerebral infarction model animal Along with the development of the neural stem cell preparation method of the present invention described above, a new cerebral infarction model animal is developed by ligating the blood vessels of the maternal mouse of an immunodeficient mouse (SCID mouse). did. This cerebral infarction model animal can be prepared in the same manner as in the method described in Japanese Patent Application No. 2004-108500, except that the maternal mouse is used instead of the SCID mouse.
ＳＣＩＤマウスの母系マウスとしては、現在市販されている（例えば、FOX Chase Cancer Centerなど参照のこと）ＳＣＩＤマウス、または、この改良型のマウスの母系マウスであればよい。結紮する脳の血管としては、脳内の脳梗塞を発症させることができる血管であれば、いずれの血管であっても特に制限はないが、発症例の多い血管や、また実験の行いやすい表皮側の血管が好ましい。好ましい血管としては、中大脳動脈、内頚動脈、椎骨脳底動脈などが例示される。 The maternal mouse of the SCID mouse may be a SCID mouse that is currently commercially available (see, for example, FOX Chase Cancer Center) or a maternal mouse of this improved mouse. The blood vessels in the ligation are not particularly limited as long as they are capable of developing cerebral infarction in the brain. Lateral blood vessels are preferred. Examples of preferable blood vessels include middle cerebral artery, internal carotid artery, vertebral basilar artery and the like.
また、結紮する部位としても特に制限はないが、結紮部位によっては、虚血領域の選択性が悪くなることもあることから、選択性を確保できる部位を設定することも必要である。例えば、結紮部位として、中大脳動脈が嗅索を通過した直後、すなわち遠位側Ｍ１部位（distal M1 portion）を選択することにより、中大脳動脈の皮質枝の血流を選択的に途絶させることが可能である。 Moreover, although there is no restriction | limiting in particular as a site | part to ligate, Since the selectivity of an ischemia area | region may worsen depending on a ligation site | part, it is also necessary to set the site | part which can ensure selectivity. For example, the blood flow in the cortical branch of the middle cerebral artery is selectively disrupted by selecting the ligation site immediately after the middle cerebral artery passes through the olfactory cord, that is, the distal M1 portion. Is possible.
脳の血管を結紮する手法としては、脳梗塞が発症できる結紮方法であれば特に制限はなく、例えば、クリップ法、血管の凝固・切断法、血管内栓子法などの各種の手法を使用することができる。虚血を一過性にするか、永久的にするかにより、結紮方法を選定することも必要である。例えば、凝固用ピンセットにて電気凝固後に切断する永久結紮法、動脈瑠結紮用クリップを用いた一過性結紮法などが挙げられる。 The method of ligating the blood vessels of the brain is not particularly limited as long as it is a ligation method capable of developing cerebral infarction, and various methods such as a clip method, a blood vessel coagulation / cutting method, and an intravascular embolization method are used. be able to. It is also necessary to select a ligation method depending on whether ischemia is transient or permanent. For example, a permanent ligation method of cutting after electrocoagulation with coagulation tweezers, a transient ligation method using a clip for arterial ligation, and the like can be mentioned.
具体的な結紮方法としては、例えば、ハロセンなどでマウスを麻酔し、マウスの左頬骨を切除し、頭蓋底を露出させ、中大脳動脈走行部位に直径１〜５ｍｍ程度の骨窓を歯科用ドリルで作成し、硬膜、クモ膜を剥離し、中大脳動脈を分離して結紮を行うことができる。 As a specific ligation method, for example, the mouse is anesthetized with halothane or the like, the left cheekbone of the mouse is excised, the skull base is exposed, and a bone window having a diameter of about 1 to 5 mm is formed on the running site of the middle cerebral artery. In this method, the dura mater and the arachnoid membrane can be detached, and the middle cerebral artery can be separated and ligated.
上記方法にしたがって、実際にＳＣＩＤマウスの母系マウスであるC.B-17/IcrCrlBRマウスの脳の血管を結紮することにより、良好な再現性を示し、しかも長期間生存可能な脳梗塞モデルマウスを作製することができた（後述の実施例２参照）。 By ligating the blood vessels of the brain of CB-17 / IcrCrlBR mouse, which is actually a maternal mouse of SCID mouse, according to the above method, a cerebral infarction model mouse that exhibits good reproducibility and can survive for a long period of time is prepared. (See Example 2 below).
さらに、上記脳梗塞モデルマウスの作製方法と同様に、無胸腺マウスであるヌードマウス（BALB/cAJcl-nu）およびその母系マウスの左中大脳動脈をそれぞれ結紮することにより、良好な再現性で脳梗塞モデルマウスを作製することができた（後述の実施例７参照）。 Furthermore, in the same way as the method for preparing the cerebral infarction model mouse, the cerebral infarction mouse was ligated to the nude mouse (BALB / cAJcl-nu), which is an athymic mouse, and the left middle cerebral artery of the mother mouse. An infarct model mouse could be prepared (see Example 7 described later).
ＳＣＩＤマウスと同様に、ヌードマウスは、そのＴ細胞機能が抑制された免疫不全マウスであり、上記方法でヌードマウスから脳梗塞モデルを作製後、その骨髄および脳梗塞下組織（脳梗塞瘢痕部位）から採取した細胞をそれぞれ培養したところ、nestin陽性のニューロスフェアが多数形成された（図２５）。対照的に、その母系マウス（コントロール）から脳梗塞モデルを作製後、その骨髄および脳梗塞下組織から採取した細胞をそれぞれ培養したところ、nestin陽性のニューロスフェアは殆ど形成されなかった。この結果からも、Ｔ細胞が骨髄あるいは脳梗塞下組織におけるニューロスフェア形成に抑制的機能を果たしていることがわかる。 Similar to the SCID mouse, the nude mouse is an immunodeficient mouse in which the T cell function is suppressed. After preparing a cerebral infarction model from the nude mouse by the above method, the bone marrow and the tissue under cerebral infarction (cerebral infarction scar site) When the cells collected from each were cultured, many nestin-positive neurospheres were formed (FIG. 25). In contrast, when a cerebral infarction model was prepared from the maternal mouse (control) and then cells collected from the bone marrow and the tissue under the cerebral infarction were cultured, almost no nestin-positive neurospheres were formed. This result also shows that T cells have an inhibitory function on neurosphere formation in bone marrow or subcerebral infarcted tissue.
従って、（１）Ｔ細胞機能を抑制する免疫抑制剤を利用することによって、ＳＣＩＤマウスの母系マウスの骨髄細胞から神経幹細胞を調製したのと同様の方法で、ヌードマウスの母系マウスの骨髄細胞から神経幹細胞を調製することができると考えられる。さらに、（２）ＳＣＩＤマウスおよびヌードマウス以外のＴ細胞機能不全マウスの骨髄からも、ＳＣＩＤマウス等からと同様の方法で、神経幹細胞を調製することができると考えられる。 Therefore, (1) by using an immunosuppressive agent that suppresses T cell function, a neural stem cell was prepared from a bone marrow cell of a maternal mouse of a SCID mouse in the same manner as that of a bone marrow cell of a nude mouse. It is believed that neural stem cells can be prepared. Furthermore, it is considered that (2) neural stem cells can be prepared from the bone marrow of T cell dysfunctional mice other than SCID mice and nude mice in the same manner as from SCID mice.
また、本発明は、（１）本発明の脳梗塞モデル動物に、被検薬物を投与して、当該被検薬物の脳梗塞に対する有効性をスクリーニングする方法、および、（２）本発明の脳梗塞モデル動物に、神経（幹）細胞又は他の細胞を移植して、当該移植治療の脳梗塞に対する有効性をスクリーニングする方法、を提供するものである。 The present invention also includes (1) a method of screening a test drug by administering the test drug to the cerebral infarction model animal of the present invention, and (2) the brain of the present invention. The present invention provides a method for screening the effectiveness of cerebral infarction of the transplantation treatment by transplanting nerve (stem) cells or other cells into an infarct model animal.
本発明のスクリーニング方法としては、例えば、本発明の脳梗塞モデル動物に、被検薬物を投与、あるいは神経幹細胞等を移植して、カーボンブラック灌流法、脳の大きさの測定、ＭＲＩなどによる機器分析などにより脳梗塞の病巣部の大きさや容積の変化、形態学的検討（左右大脳皮質幅の比、ＴＵＮＥＬ染色によるアポトーシスの程度、ＢｒｄＵ標識による再生神経や再生血管内皮細胞数）、行動テスト（オープンフィールドテストや驚愕反射、迷路学習、回避学習）などを測定し、これを対照群と比較して脳梗塞病巣部の拡大の阻止、脳機能の回復などの程度を評価する方法が挙げられる。 As the screening method of the present invention, for example, a test drug is administered to the cerebral infarction model animal of the present invention, or a neural stem cell or the like is transplanted, and a carbon black perfusion method, brain size measurement, MRI, or the like is used. Analysis of changes in the size and volume of cerebral infarction lesions, morphological examination (ratio of left and right cerebral cortex width, degree of apoptosis by TUNEL staining, number of regenerative nerves and regenerative vascular endothelial cells by BrdU labeling), behavior test ( Measurement of open field test, startle reflex, labyrinth learning, avoidance learning, etc.) and comparing this with the control group can be used to evaluate the extent of inhibition of cerebral infarction lesion, recovery of brain function, etc.
さらに、本発明は、脳梗塞などの脳虚血性疾患、又はその他の神経疾患の治療に用いられる神経再生治療剤のスクリーニング方法として、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進する作用を有するかどうかを指標に、候補物質を探索するスクリーニング方法を提供するものである。 Furthermore, the present invention is a method for screening a neuroregenerative therapeutic agent used for the treatment of cerebral ischemic diseases such as cerebral infarction, or other neurological diseases, or induces differentiation from bone marrow cells to neural stem cells. The present invention provides a screening method for searching for a candidate substance using as an index whether or not it has a promoting action.
例えば、後述の実施例記載の培養系において、ヒト又は動物から採取した骨髄細胞の培地に被検物質を投与し、当該被検物質が、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進する作用を有するかどうかを調べることによって、候補物質を探索する。培養条件は、使用する骨髄細胞の起源などに応じて、（１）免疫抑制剤を添加したもの、（２）分化誘導因子（又は脳障害後に採取した血清）を添加したもの、（３）免疫抑制剤と分化誘導因子（又は脳障害後血清）の両者を添加したもの、（４）両者とも添加しないもの、など種々の条件の中から適切な条件を設定すればよい。 For example, in the culture system described in the Examples below, a test substance is administered to a medium of bone marrow cells collected from humans or animals, and the test substance induces differentiation from bone marrow cells to neural stem cells, or A candidate substance is searched by investigating whether it has the effect | action which accelerates | stimulates differentiation. Depending on the origin of the bone marrow cells used, the culture conditions are (1) those with an immunosuppressant added, (2) those with a differentiation-inducing factor (or serum collected after brain injury), (3) immunity Appropriate conditions may be set from various conditions such as those in which both an inhibitor and a differentiation-inducing factor (or serum after brain injury) are added, and (4) those in which neither is added.
実際に、後述の実施例記載の培養系を用いて、骨髄細胞から神経幹細胞への分化を誘導する物質を探索したところ、サイトカインであるＴＮＦαがこのような分化誘導作用をもつことを見出すことができ、本発明のスクリーニング方法の有効性が認められた。 In fact, when a substance that induces differentiation from bone marrow cells to neural stem cells was searched using the culture system described in the Examples below, it was found that TNFα, a cytokine, had such a differentiation-inducing action. The effectiveness of the screening method of the present invention was confirmed.
本発明のスクリーニング方法において、免疫抑制作用を有する物質群の中から、神経再生に有用な候補物質の探索を行ってもよいし、ケモカインなどのサイトカイン、あるいはこれらの活性や産生を調節する物質群の中から、神経再生に有用な候補物質の探索を行ってもよい。 In the screening method of the present invention, a candidate substance useful for nerve regeneration may be searched from a group of substances having an immunosuppressive action, or a cytokine such as a chemokine, or a group of substances that regulate their activity or production A candidate substance useful for nerve regeneration may be searched from among them.
また、本発明のスクリーニング方法は、後述の実施例記載の培養系を用いたスクリーニング系に制限されるものではなく、その他のスクリーニング系を用いても勿論よい。例えば、分化誘導因子として同定されたＣＩＮＣ−１やＴＮＦαなどの物質を標的分子として、これらの分子の活性や産生を調節する物質を既存の様々な系で調べることができ、これにより、骨髄細胞から神経幹細胞への分化を促進し、神経再生治療に有効な候補物質を探索することができる。 Further, the screening method of the present invention is not limited to the screening system using the culture system described in the examples described later, and other screening systems may of course be used. For example, using substances such as CINC-1 and TNFα identified as differentiation-inducing factors as target molecules, substances that regulate the activity and production of these molecules can be examined in various existing systems. It is possible to search for candidate substances that promote differentiation into neural stem cells and are effective in nerve regeneration treatment.
以下、図面を参照しながら本発明の実施例について説明するが、本発明はこれら実施例によって何ら限定されるものではない。
〔実施例１：脳梗塞ＳＣＩＤマウス骨髄由来の神経幹細胞の調製〕
［1-1］脳梗塞ＳＣＩＤマウスの作成
特願２００４−１０８５００号の明細書記載の方法にしたがって、免疫不全マウスであるＳＣＩＤマウス（５週齢）の中大脳動脈を結紮し、脳梗塞モデルマウスを作成した。
具体的には、３％ハロセン麻酔下にマウス左頬骨を切除し、頭蓋底を露出した。中大脳動脈走行部位に直径１．５ｍｍの骨窓を歯科用ドリルで作成した。硬膜、クモ膜を剥離し、中大脳動脈を分離して結紮準備とした。中大脳動脈結紮法としては、凝固用ピンセットにて電気凝固後に切断する永久結紮法、動脈瑠結紮用クリップを用いた一過性結紮法などが可能である。結紮部位は、動脈が嗅索を通過した直後、すなわちdistal M1 portionである。この部位を結紮することにより、中大脳動脈の皮質枝の血流を選択的に途絶させることが可能である。Examples of the present invention will be described below with reference to the drawings, but the present invention is not limited to these examples.
[Example 1: Preparation of neural stem cells derived from cerebral infarction SCID mouse bone marrow]
[1-1] Preparation of cerebral infarction SCID mouse According to the method described in the specification of Japanese Patent Application No. 2004-108500, the middle cerebral artery of an SCID mouse (5 weeks old) which is an immunodeficient mouse is ligated, and a cerebral infarction model mouse It was created.
Specifically, the left cheekbone of the mouse was excised under 3% halothane anesthesia to expose the skull base. A bone window having a diameter of 1.5 mm was created with a dental drill at the running site of the middle cerebral artery. The dura mater and arachnoid membrane were removed, and the middle cerebral artery was separated to prepare for ligation. As the middle cerebral artery ligation method, a permanent ligation method of cutting after electrocoagulation with coagulation tweezers, a transient ligation method using an arterial ligation clip, and the like are possible. The ligation site is immediately after the artery passes through the olfactory tract, that is, the distal M1 portion. By ligating this site, it is possible to selectively disrupt the blood flow in the cortical branch of the middle cerebral artery.
このような中大脳動脈結紮法により、ＳＣＩＤマウス左中大脳動脈のdistal M1 portionを結紮して作成した脳梗塞モデルマウス（脳梗塞ＳＣＩＤマウス）の脳梗塞領域を、実際に２，３，５−トリフェニルテトラゾリウム（2,3,5-triphenyltetrazolium（ＴＴＣ））染色法にて検討した。ＴＴＣ染色法は、中大脳動脈結紮後（ＭＣＯ）１，３，７日目にそれぞれマウス脳を摘出し、brain slicerにて作成した冠状脳スライスを用いて施行した。図７左には、この染色法によって、結紮後（ＭＣＯ）１日目に摘出した脳の染色結果が示される。この方法により、左中大脳動脈皮質枝領域に選択的に梗塞（白い部分）が観察され、再現性のよい均一な脳梗塞を作成することができる（特願２００４−１０８５００号参照）。なお脳梗塞は、上記方法による結紮後（ＭＣＯ）３日でほぼ完成する。 The cerebral infarction region of a cerebral infarction model mouse (cerebral infarction SCID mouse) prepared by ligating the distal M1 portion of the left middle cerebral artery of the SCID mouse by such a middle cerebral artery ligation method is actually 2, 3, 5- It examined by the triphenyltetrazolium (2,3,5-triphenyltetrazolium (TTC)) dyeing | staining method. The TTC staining method was performed using coronal brain slices prepared by brain slicers after excision of mouse brains on days 1, 3, and 7 after middle cerebral artery ligation (MCO). The left side of FIG. 7 shows the result of staining the brain extracted on the first day after ligation (MCO) by this staining method. By this method, an infarction (white portion) is selectively observed in the left middle cerebral artery cortical branch region, and a uniform cerebral infarction with good reproducibility can be created (see Japanese Patent Application No. 2004-108500). Cerebral infarction is almost completed 3 days after ligation (MCO) by the above method.
［1-2］ニューロスフェア（neurosphere）様細胞塊の形成
上記方法により作成した脳梗塞ＳＣＩＤマウスを結紮後７日目にクリーンベンチ内で断頭し、大腿骨から骨髄を採取した。その後、ＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞を３ｍｌの培養液にて再浮遊させ、ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）存在下にlow cell bindingプレート上で１０−２８日間培養を行った。その結果、図１Ｄに示すように、７日目以降にはニューロスフェア（neurosphere）様細胞塊が細胞顕微鏡下で観察された。[1-2] Formation of Neurosphere-Like Cell Mass Cerebral infarction SCID mice prepared by the above method were decapitated in a clean bench 7 days after ligation, and bone marrow was collected from the femur. Thereafter, pipetting was performed in the basic culture solution (250 μl) of DMEM and N-2 until single cells were added, 10 ml of the culture solution was added, and the mixture was centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on a low cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. As a result, as shown in FIG. 1D, a neurosphere-like cell cluster was observed under the cell microscope after the seventh day.
比較のため、胎生２週目のＣ５７Ｂ／６マウスから採取した線条体細胞をlow cell binding プレート上で１０日間培養を行ったところ、図１Ａに示すように、ニューロスフェアが形成された。これをhigh bindingプレート上でさらに培養すると３日後には分化し（同図Ｂ）、発現蛋白を免疫組織化学法（二重染色間接蛍光抗体法）にて検討すると、ＭＡＰ２陽性の神経細胞と、ＧＦＡＰ陽性のグリア細胞に分化していた（同図Ｃ参照）。 For comparison, striatal cells collected from C57B / 6 mice at 2 weeks of gestation were cultured on a low cell binding plate for 10 days. As a result, neurospheres were formed as shown in FIG. 1A. When this was further cultured on a high binding plate, it differentiated after 3 days (Fig. B). When the expressed protein was examined by immunohistochemistry (double staining indirect fluorescent antibody method), MAP2-positive neurons and Differentiated into GFAP-positive glial cells (see FIG. C).
一方、脳梗塞ＳＣＩＤマウスから採取した骨髄細胞をlow cell binding プレート上で１０日間培養すると、上述のように、胎生期マウス脳（線条体）由来のニューロスフェアとよく似たニューロスフェア様細胞塊が形成された（同図Ｄ）。これをhigh bindingプレート上でさらに培養すると３日後には突起を持った細胞に分化し（同図Ｅ）、一部はＭＡＰ２陽性の神経細胞と、ＧＦＡＰ陽性のグリア細胞に分化していた（同図Ｆ参照）。 On the other hand, when bone marrow cells collected from cerebral infarction SCID mice were cultured on a low cell binding plate for 10 days, as described above, neurosphere-like cell clusters resembling neurospheres derived from embryonic mouse brain (striatum) Was formed (Fig. D). When this was further cultured on a high binding plate, it differentiated into cells with protrusions after 3 days (Fig. E), and partly differentiated into MAP2-positive neurons and GFAP-positive glial cells (same as above). (See FIG. F).
さらに、ＳＣＩＤマウスに対して偽手術（骨窓まで開き、結紮はしない処置）を施行した脳梗塞を生じさせていない偽手術ＳＣＩＤマウス（sham SCIDマウス）から骨髄を採取し、同様の条件で培養を行ったが、ニューロスフェア様細胞塊はほとんど形成されなかった。 In addition, bone marrow was collected from sham-operated SCID mice (sham SCID mice) that had not undergone cerebral infarction in which SCID mice were subjected to sham surgery (a procedure that opened to the bone window and not ligated), and were cultured under the same conditions. The neurosphere-like cell cluster was hardly formed.
［1-3］ニューロスフェア様細胞塊の形成効率
脳梗塞ＳＣＩＤマウス骨髄由来のニューロスフェア様細胞塊形成に関して、脳梗塞後（結紮後）骨髄を採取するまでの期間や骨髄細胞の培養期間が、細胞塊形成にどのように影響するかを検討した。[1-3] Neurosphere-like cell mass formation efficiency Regarding the formation of neurosphere-like cell mass derived from cerebral infarction SCID mouse bone marrow, the period until cerebral infarction (after ligation) bone marrow collection and bone marrow cell culture period are We examined how it affects cell mass formation.
脳梗塞作成後（結紮後）１，２，３週目のＳＣＩＤマウスから採取した骨髄をそれぞれ２８日間培養し、新たに形成されるニューロスフェア様細胞塊数を数えた。その結果、図２に示すように、脳梗塞後１週目（ＭＣＯ１Ｗ）に採取した骨髄が最も多くのニューロスフェア様細胞塊を形成し、培養後１０−１３日目に形成能がピークとなることが明らかになった。 Bone marrow collected from SCID mice at 1, 2, and 3 weeks after the creation of cerebral infarction (after ligation) was cultured for 28 days, and the number of newly formed neurosphere-like cell clusters was counted. As a result, as shown in FIG. 2, the bone marrow collected in the first week after cerebral infarction (MCO1W) forms the most neurosphere-like cell mass, and the forming ability reaches a peak on day 10-13 after the culture. It became clear.
一方、偽手術ＳＣＩＤマウスの培養骨髄からは、ニューロスフェア様細胞塊は培養１ヶ月を経過してもほとんど形成されなかった（Sham control）。しかし、偽手術ＳＣＩＤマウスの骨髄に脳梗塞ＳＣＩＤマウス（ＭＣＯ１Ｗ）から採取した血清を添加して培養すると、ニューロスフェア様細胞塊が形成された（Sham control + Serum）。 On the other hand, neurosphere-like cell clusters were hardly formed from cultured bone marrow of sham-operated SCID mice even after one month of culture (Sham control). However, when serum collected from cerebral infarction SCID mice (MCO1W) was added to the bone marrow of sham-operated SCID mice and cultured, neurosphere-like cell clusters were formed (Sham control + Serum).
［1-4］神経幹細胞・神経細胞への分化能
脳梗塞ＳＣＩＤマウス（ＭＣＯ１Ｗ）の骨髄を培養し、１０日目に培養液に浮遊しているニューロスフェア様細胞塊を採取してhigh bindingプレート上でさらに培養した。３−７日後に分化した細胞をパラフォルムアルデヒドを含む固定液にて固定して免疫染色を行い、発現蛋白の検討を行った。その結果、細胞塊の中心にはＮｅｓｔｉｎ陽性の神経幹細胞が高率に観察された（図３左）。また、周囲には分化したＭＡＰ２陽性の神経細胞（同図中央）、ＧＦＡＰ陽性のグリア細胞（同図右）が観察された。[1-4] Differentiation ability into neural stem cells / neuronal cells Bone marrow of cerebral infarction SCID mice (MCO1W) is cultured, and on the 10th day, the neurosphere-like cell mass floating in the culture medium is collected and a high binding plate is collected. Further culture on above. After 3-7 days, the differentiated cells were fixed with a fixative containing paraformaldehyde and immunostained to examine the expressed protein. As a result, Nestin-positive neural stem cells were observed at a high rate in the center of the cell mass (FIG. 3 left). In addition, differentiated MAP2-positive neurons (middle of the figure) and GFAP-positive glial cells (right of the figure) were observed in the vicinity.
次に、骨髄由来のニューロスフェア様細胞塊の神経細胞への分化能、分化効率を検討した。その結果、図４に示すように、脳梗塞ＳＣＩＤマウス（ＭＣＯ１Ｗ）の骨髄を培養して得られたニューロスフェア様細胞塊の約６０％がＮｅｓｔｉｎ陽性の神経幹細胞であり、そのうち６７％（全体のニューロスフェア様細胞塊の約４０％）がＭＡＰ２陽性、ＮｅｕＮ陽性の神経細胞に分化した。また、ＭＡＰ２陽性の神経細胞を産生した細胞塊には、ほぼ１００％の確率で同時にＧＦＡＰ陽性のグリア細胞が観察された。 Next, the differentiation ability and differentiation efficiency of neurosphere-like cell clusters derived from bone marrow into neurons were examined. As a result, as shown in FIG. 4, about 60% of neurosphere-like cell clusters obtained by culturing bone marrow of cerebral infarction SCID mice (MCO1W) are Nestin-positive neural stem cells, of which 67% (total About 40% of the neurosphere-like cell mass) differentiated into MAP2-positive and NeuN-positive neurons. In addition, GFAP-positive glial cells were simultaneously observed with a probability of almost 100% in the cell mass that produced MAP2-positive neurons.
［1-5］神経幹細胞へ分化誘導させる刺激因子の存在
上述のように、脳梗塞ＳＣＩＤマウスの骨髄を培養することで、神経幹細胞が産生された。このように脳梗塞ＳＣＩＤマウスの骨髄から神経幹細胞が分化誘導されるメカニズムを検討する目的で、脳梗塞を起こしていない偽手術ＳＣＩＤマウスの培養骨髄液に別のマウスの血清（３０μｌ）を添加した。[1-5] Presence of stimulating factor that induces differentiation into neural stem cells As described above, neural stem cells were produced by culturing the bone marrow of cerebral infarction SCID mice. Thus, for the purpose of examining the mechanism by which neural stem cells are induced to differentiate from the bone marrow of cerebral infarction SCID mice, the serum (30 μl) of another mouse was added to the cultured bone marrow fluid of sham-operated SCID mice without cerebral infarction. .
その結果、偽手術ＳＣＩＤマウスの骨髄に別の偽手術ＳＣＩＤマウスの血清を添加してもニューロスフェア様細胞塊はまったく形成されなかったが、偽手術ＳＣＩＤマウスの骨髄に脳梗塞ＳＣＩＤマウス（ＭＣＯ１Ｗ）の血清を添加するとニューロスフェア様細胞塊ができ（図５Ａ・Ｂ、図２）、これは神経細胞にも分化した（図５Ｃ・Ｄ）。以上の結果から、脳梗塞を起こした後の血清には、骨髄細胞を神経幹細胞へ分化させる何らかの刺激因子（differentiation factor）が存在し、この刺激因子が血流を介して骨髄に伝えられ、神経幹細胞が産生されると考えられる。 As a result, neurosphere-like cell clusters were not formed at all even when serum from another sham-operated SCID mouse was added to the bone marrow of sham-operated SCID mice, but cerebral infarction SCID mice (MCO1W) When the serum was added, neurosphere-like cell clusters were formed (FIGS. 5A and B, FIG. 2), which also differentiated into neurons (FIGS. 5C and D). From the above results, the serum after cerebral infarction has some differentiation factor that differentiates bone marrow cells into neural stem cells, and this stimulation factor is transmitted to the bone marrow via the bloodstream, Stem cells are thought to be produced.
［1-6］本実施例の結果と考察
以上のように、免疫不全マウスであるＳＣＩＤマウスの中大脳動脈を結紮して脳梗塞モデルマウスを作成し、脳梗塞発症後（結紮後）に骨髄細胞を採取、培養することで、神経幹細胞塊（ニューロスフェア）を誘導・調製することができた。また、このニューロスフェアが神経細胞に分化することを明らかにした。[1-6] Results and Discussion of this Example As described above, a cerebral infarction model mouse was prepared by ligating the middle cerebral artery of an SCID mouse, which is an immunodeficient mouse, and bone marrow after the onset of cerebral infarction (after ligation). By collecting and culturing cells, neural stem cell clusters (neurospheres) could be induced and prepared. In addition, it was clarified that this neurosphere differentiates into nerve cells.
一方、脳梗塞を起こしていない偽手術ＳＣＩＤマウスの骨髄からはニューロスフェアは形成されなかったが、これに脳梗塞モデルマウスの血清を添加して培養することでニューロスフェア形成がみられたことから、ニューロスフェア形成には脳梗塞が誘因となって産生される刺激因子が必要と考えられる。 On the other hand, neurospheres were not formed from the bone marrow of sham-operated SCID mice that did not have cerebral infarction, but neurosphere formation was observed by adding serum from cerebral infarction model mice to culture. In addition, it is thought that a stimulating factor produced by cerebral infarction is necessary for neurosphere formation.
〔実施例２：脳梗塞後骨髄からの神経幹細胞産生と免疫不全との関連性検討〕
［2-1］免疫正常マウスからの脳梗塞モデルの作成
上述のように、免疫不全マウスであるＳＣＩＤマウスの脳梗塞モデルから採取した骨髄を培養することで、多くの神経幹細胞（ニューロスフェア）が形成された。そこで、この現象がマウスの免疫不全と関係があるかどうかを検討する目的で、ＳＣＩＤマウスの母系マウスである免疫正常なC.B-17/IcrCrlBRマウス（以下、「Ｃ．Ｂ１７マウス」という。）に対して、同様の方法で脳梗塞モデルを作成して脳梗塞ＳＣＩＤマウスと比較し、その骨髄から神経幹細胞が分化誘導されるか等について検討した。[Example 2: Examination of relationship between neural stem cell production from bone marrow after cerebral infarction and immunodeficiency]
[2-1] Creation of a cerebral infarction model from immune-normalized mice As described above, by culturing bone marrow collected from a cerebral infarction model of SCID mice that are immunodeficient mice, many neural stem cells (neurospheres) can be obtained. Been formed. Therefore, for the purpose of examining whether this phenomenon is related to the immunodeficiency of mice, CB-17 / IcrCrlBR mice (hereinafter referred to as “C.B17 mice”), which are maternal mice of SCID mice, are normal mice. On the other hand, a cerebral infarction model was prepared by the same method, compared with cerebral infarction SCID mice, and examined whether neural stem cells were induced to differentiate from the bone marrow.
Ｃ．Ｂ１７マウスからの脳梗塞モデルの作成は、上述の脳梗塞ＳＣＩＤマウスの作成方法と同様である。すなわち、３％ハロセン麻酔下にマウス左頬骨を切除し、頭蓋底を露出した。中大脳動脈走行部位に直径１．５ｍｍの骨窓を歯科用ドリルで作成した。硬膜、クモ膜を剥離し、中大脳動脈を分離して結紮準備とした。中大脳動脈結紮法としては、凝固用ピンセットにて電気凝固後に切断する永久結紮法、動脈瑠結紮用クリップを用いた一過性結紮法などが可能である。結紮部位は、動脈が嗅索を通過した直後、すなわちdistal M1 portionである。この部位を結紮することにより、中大脳動脈の皮質枝の血流を選択的に途絶させることが可能である。 C. The creation of the cerebral infarction model from the B17 mouse is the same as the method for creating the cerebral infarction SCID mouse described above. That is, the left cheekbone of the mouse was excised under 3% halothane anesthesia to expose the skull base. A bone window having a diameter of 1.5 mm was created with a dental drill at the running site of the middle cerebral artery. The dura mater and arachnoid membrane were removed, and the middle cerebral artery was separated to prepare for ligation. As the middle cerebral artery ligation method, a permanent ligation method of cutting after electrocoagulation with coagulation tweezers, a transient ligation method using an arterial ligation clip, and the like are possible. The ligation site is immediately after the artery passes through the olfactory tract, that is, the distal M1 portion. By ligating this site, it is possible to selectively disrupt the blood flow in the cortical branch of the middle cerebral artery.
このような中大脳動脈結紮法により、Ｃ．Ｂ１７マウス左中大脳動脈のdistal M1 portionを結紮して作成した脳梗塞モデルマウス（脳梗塞Ｃ．Ｂ１７マウス）の脳梗塞領域を、実際にＴＴＣ染色法にて検討した。ＴＴＣ染色法は、結紮後（ＭＣＯ）１，３，７日目にそれぞれマウス脳を摘出し、brain slicerにて作成した冠状脳スライスを用いて施行した。その結果、各群４匹、計１２匹はすべて左中大脳動脈皮質枝領域に選択的に梗塞が作成され、脳梗塞部位は極めて均一であった。図７には、結紮後（ＭＣＯ）１日目に摘出した脳の染色結果が、脳梗塞ＳＣＩＤマウスのものと対比して示される（白い部分が梗塞部位）。 By such a middle cerebral artery ligation method, C.I. The cerebral infarction region of the cerebral infarction model mouse (cerebral infarction C.B17 mouse) prepared by ligating the distal M1 portion of the B17 mouse left middle cerebral artery was actually examined by the TTC staining method. The TTC staining method was performed using coronal brain slices prepared by brain slicer after excision of mouse brains on days 1, 3 and 7 after ligation (MCO). As a result, in each group of 4 animals, a total of 12 animals, infarcts were selectively created in the left middle cerebral artery cortical branch region, and the cerebral infarction sites were extremely uniform. FIG. 7 shows the result of staining of the brain extracted on the first day after ligation (MCO), as compared with that of the cerebral infarction SCID mouse (the white part is the infarct site).
Ｃ．Ｂ１７マウス、およびＳＣＩＤマウスにおける中大脳動脈閉塞後の脳梗塞の様子を摘出脳で観察した。マウス左中大脳動脈のdistal M1 portionを結紮し、３，７，１６，２８日目にマウスを経心的にＰＬＰ固定液で灌流固定後、脳を摘出した。その結果、摘出脳は全例中大脳動脈領域の欠損が観察され（図６には、結紮後１６日目のものが示される）、ＳＣＩＤマウスの母系であるＣ．Ｂ１７マウスを用いた場合も、ＳＣＩＤマウスと同様に再現性のよい均一な脳梗塞モデルを作成できることが明らかになった。 C. The state of cerebral infarction after middle cerebral artery occlusion in B17 mice and SCID mice was observed in the isolated brain. The distal M1 portion of the left middle cerebral artery of the mouse was ligated, and the mouse was transcardially fixed with a PLP fixative solution on days 3, 7, 16, and 28, and then the brain was removed. As a result, in the isolated brain, a defect in the middle cerebral artery region was observed in all cases (FIG. 6 shows the 16th day after ligation). It was revealed that even when B17 mice were used, a uniform cerebral infarction model with good reproducibility could be created as with SCID mice.
ところで、ＳＣＩＤマウスから作成した脳梗塞モデルマウスでは、虚血後の脳梗塞の進展（delayed infarct expansion）は脳梗塞後３日で終了し、その後は脳萎縮の進展ではなく、脳形態上はむしろ回復することが認められている（特願２００４−１０８５００号参照）。このことは、脳梗塞ＳＣＩＤマウスが脳梗塞モデルマウスとしてのみならず、脳再生モデルとしても適していることを示している。 By the way, in the cerebral infarction model mouse prepared from the SCID mouse, the delayed infarct expansion after ischemia is completed in 3 days after the cerebral infarction, and after that, it is not the development of cerebral atrophy but rather in the brain form. Recovery is permitted (see Japanese Patent Application No. 2004-108500). This indicates that the cerebral infarction SCID mouse is suitable not only as a cerebral infarction model mouse but also as a brain regeneration model.
そこで次に、脳梗塞Ｃ．Ｂ１７マウスにおいても、このような脳形態上の回復が認められるか検討した。上述のように、Ｃ．Ｂ１７マウスおよびＳＣＩＤマウスの左中大脳動脈のdistal M1 portionを結紮し、３，７，１４，２８日目にマウスを経心的にＰＬＰ固定液で灌流固定後、脳を摘出し、残存大脳皮質の大きさを定量化することで、脳欠損の程度、回復・再生の有無を観察した。具体的には、梗塞側大脳皮質の大脳裂から梗塞部位までの幅（ａ）を正常側（ｂ）と比較し、その比（ａ／ｂ）をcortical width index（ＣＩ値）として算出した（図２０参照）。その結果、図８に示すように、脳梗塞ＳＣＩＤマウス、脳梗塞Ｃ．Ｂ１７マウスともＣＩ値は３日目から７日目までは０．３４とほぼ一定であった。しかし、ＳＣＩＤマウスでは２８日目に０．３７と増加し、回復・再生が認められたのに対し、Ｃ．Ｂ１７マウスでは２８日目でも０．３４と一定であった。この結果から、脳梗塞Ｃ．Ｂ１７マウスでは、脳梗塞ＳＣＩＤマウスとは異なり、脳形態上の回復は認められず、脳再生モデルとはいえなかった。 Therefore, next, cerebral infarction C.I. Whether or not such a recovery in brain morphology was also observed in B17 mice was examined. As described above, C.I. The distal M1 portion of the left middle cerebral artery of B17 mice and SCID mice was ligated, and the mice were transcardially fixed with PLP fixative solution on the 3rd, 7th, 14th, and 28th days, and then the brain was removed and the remaining cerebral cortex was removed. By quantifying the size of the brain, the degree of brain defect and the presence or absence of recovery / regeneration were observed. Specifically, the width (a) from the cerebral cleft of the infarcted cerebral cortex to the infarcted site was compared with the normal side (b), and the ratio (a / b) was calculated as the cortical width index (CI value) ( FIG. 20). As a result, as shown in FIG. The CI value of B17 mice was almost constant at 0.34 from the third day to the seventh day. However, SCID mice increased to 0.37 on the 28th day, and recovery / regeneration was observed. In the B17 mouse, it was constant at 0.34 even on the 28th day. From this result, cerebral infarction C.I. In B17 mice, unlike cerebral infarction SCID mice, recovery in brain morphology was not observed, and it was not a brain regeneration model.
［2-2］脳梗塞Ｃ．Ｂ１７マウスにおける神経幹細胞等の発現検討
脳梗塞Ｃ．Ｂ１７マウスは、ＣＩ値では脳再生が認められなかったが、梗塞後のマウス脳に神経幹細胞等が形成されているかどうかを、神経幹細胞や神経細胞、グリア細胞の各種マーカーを用いた免疫組織化学で検討した。[2-2] Cerebral infarction C.I. Examination of expression of neural stem cells and the like in B17 mice In B17 mice, brain regeneration was not observed with CI values, but whether or not neural stem cells or the like were formed in the mouse brain after infarction was determined by immunohistochemistry using various markers of neural stem cells, neural cells, and glial cells. I examined it.
具体的には、神経幹細胞に対しては抗ムサシ１（Musashi1）抗体を用い、未熟神経細胞の同定にはDoublecortin（ＤＣＸ）を用いた。オリゴデンドロサイト前駆細胞はPlatelet-derived Growth Factor Receptorα（ＰＤＧＦＲα）とＮＧ２をマーカーとし、前および未熟オリゴデンドロサイトに対してはＯ４とMyelin-associated Glycoprotein（ＭＡＧ）を用いた。アストロサイトは抗ＧＦＡＰ抗体で同定した。さらに、未熟神経細胞や軸索伸長過程にある神経のマーカーとしてＰＳＡ−ＮＣＡＭを用いた。ＰＳＡ−ＮＣＡＭは，培養神経幹細胞の細胞膜に発現することが確認されている。また、Ｎカドヘリン（N-cadherin）もニューロスフェアに発現することが確認されている。これらは、ニューロスフェアの分化誘導シグナル調節に深く関与している。 Specifically, anti-musashi 1 antibody was used for neural stem cells, and Doublecortin (DCX) was used for identification of immature neural cells. Oligodendrocyte progenitor cells used Platelet-derived Growth Factor Receptor α (PDGFRα) and NG2 as markers, and O4 and Myelin-associated Glycoprotein (MAG) were used for pre- and immature oligodendrocytes. Astrocytes were identified with anti-GFAP antibody. Furthermore, PSA-NCAM was used as a marker for immature neurons and nerves in the process of axonal elongation. It has been confirmed that PSA-NCAM is expressed in the cell membrane of cultured neural stem cells. It has also been confirmed that N-cadherin is expressed in the neurosphere. These are deeply involved in the regulation of neurosphere differentiation-inducing signals.
上述の各種マーカーを用いて、脳梗塞Ｃ．Ｂ１７マウスの脳室下帯組織（subventricular zone：ＳＶＺ）、および脳梗塞下の組織における神経幹細胞等の産生について検討した。なお、脳梗塞下の組織とは、梗塞組織（脳梗塞瘢痕部位）および梗塞組織と白質（脳梁）とが接する脳梗塞周囲の部位のことである。この脳梗塞下の組織は、脳室下帯組織（ＳＶＺ）とともに、脳梗塞ＳＣＩＤマウスにおいて多数の神経幹細胞の出現（即ち、脳再生）が観察された部位である（特願２００４−１０８５００号参照）。また、この脳梗塞下の組織における神経幹細胞は、骨髄に由来するものであった。 Using the various markers described above, cerebral infarction C.I. The production of neural stem cells and the like in subventricular zone (SVZ) of B17 mice and tissues under cerebral infarction was examined. The tissue under cerebral infarction is an infarcted tissue (cerebral infarction scar site) and a site around the cerebral infarction where the infarcted tissue and white matter (corpus callosum) contact. The tissue under cerebral infarction is a site where appearance of a large number of neural stem cells (ie, brain regeneration) was observed in cerebral infarction SCID mice together with subventricular zone tissue (SVZ) (see Japanese Patent Application No. 2004-108500). ). The neural stem cells in the tissue under cerebral infarction were derived from bone marrow.
［2-3］脳室下帯組織（ＳＶＺ）における神経幹細胞等の発現
脳梗塞Ｃ．Ｂ１７マウス作成後１，７，１４，３５日目にマウスを経心的にＰＬＰ固定液で灌流固定後、脳を摘出し、ビブラトームを用いて脳切片を作成し、その後、免疫組織化学を行った。[2-3] Expression of neural stem cells and the like in subventricular zone tissue (SVZ) On days 1, 7, 14, and 35 after the creation of B17 mice, the mice were transperfusionally fixed with PLP fixative solution, the brain was removed, brain sections were prepared using a vibratome, and then immunohistochemistry was performed. It was.
梗塞後１日目には、ＳＶＺの各種マーカー発現はすべて正常側と差はなかった。７日目になると、Musashi1陽性、ＰＳＡ−ＮＣＡＭ陽性の細胞が、ＳＶＺに増殖するようになった。１４日目になると、これらの神経幹細胞は脳室膨大部から白質内に侵入するようにみられた。３５日目には、ＳＶＺでの発現細胞は減少しコントロールレベルとなったが、脳室下の白質には多数のMusashi1陽性、ＰＳＡ−ＮＣＡＭ陽性の細胞が認められた。同部位には、ＤＣＸ陽性の未熟神経細胞や、小型のＮｅｕＮ陽性細胞もみられた。また、梗塞後７日目よりＳＶＺにはＮＧ２陽性、ＰＤＧＦＲα陽性のオリゴデンドロサイト前駆細胞も発現するようになった。これらは成熟オリゴデンドロサイトマーカーを有しておらず、１４日目までみられたが、３５日目には減少した。 On the first day after infarction, the expression of various markers of SVZ was not different from the normal side. On day 7, Musashi1-positive and PSA-NCAM-positive cells began to proliferate into SVZ. On the 14th day, these neural stem cells appeared to invade the white matter from the enormous part of the ventricle. On day 35, SVZ-expressing cells decreased to a control level, but many Musashi1-positive and PSA-NCAM-positive cells were observed in the subventricular white matter. At the same site, DCX-positive immature neurons and small NeuN-positive cells were also observed. In addition, NG2 positive and PDGFRα positive oligodendrocyte progenitor cells were also expressed in SVZ from day 7 after infarction. These had no mature oligodendrocyte markers and were seen until day 14 but decreased on day 35.
このように、ＳＶＺでは梗塞後７日目以降、１４日目をピークに神経幹細胞の増殖がみられ、以後減少する。神経幹細胞としては脳室下の白質に３５日目までは存在するが、以後は成熟して神経となるか、あるいは死滅してしまうと考えられる。ＳＶＺには梗塞後７日目にはＮｏｔｃｈ１陽性細胞がみられたが、それ以降はみられなかったことから、ＳＶＺ細胞の分化増殖シグナルは、梗塞後１週間程度で終了すると考えられた。 Thus, in SVZ, proliferation of neural stem cells is observed peaking on the 14th day after the 7th day after the infarction, and decreases thereafter. Neural stem cells are present in the white matter under the ventricle until the 35th day, but after that they are considered to mature and become nerves or die. In SVZ, Notch1-positive cells were observed on the 7th day after infarction, but were not observed thereafter. Therefore, the differentiation / proliferation signal of SVZ cells was considered to be terminated in about 1 week after infarction.
［2-4］Ｃ．Ｂ１７マウス梗塞下組織における神経幹細胞の発現抑制
Ｃ．Ｂ１７マウスおよびＳＣＩＤマウスに対して脳梗塞作成後２１日目のマウス脳のビブラトーム切片を用いて、ＮｅｕＮに対する免疫組織化学を施行した。その結果を図９に示す。脳梗塞ＳＣＩＤマウスの梗塞下組織（白質領域）には多数の小型ＮｅｕＮ陽性細胞（神経前駆細胞）が観察されたが（同図Ａ）、脳梗塞Ｃ．Ｂ１７マウスの梗塞下組織は、ほとんどマクロファージで占拠されており、ＰＳＡ−ＮＣＡＭ陽性の神経幹細胞や小型のＮｅｕＮ陽性細胞は梗塞後３５日目までほとんど認められなかった（同図Ｂ）。このことは、Ｃ．Ｂ１７マウスの正常な免疫反応が、梗塞下組織（脳梗塞瘢痕部位を含む）における神経幹細胞発現・神経再生に抑制的に作用していることを示唆するものである。[2-4] C.I. Inhibition of neural stem cell expression in B17 mouse infarcted tissue Immunohistochemistry for NeuN was performed on B17 mice and SCID mice using vibratome sections of the mouse brain 21 days after the creation of cerebral infarction. The result is shown in FIG. A number of small NeuN positive cells (neural progenitor cells) were observed in the infarcted tissue (white matter region) of cerebral infarction SCID mice (FIG. A). The infarcted tissue of B17 mice was mostly occupied by macrophages, and PSA-NCAM positive neural stem cells and small NeuN positive cells were hardly observed until 35 days after infarction (Fig. B). This is because C.I. This suggests that the normal immune response of B17 mice acts to suppress neural stem cell expression and nerve regeneration in infarcted tissues (including cerebral infarction scar sites).
次に、脳梗塞下組織から採取した細胞を培養して神経幹細胞が形成されるかどうか検討した。結紮後７日目の脳梗塞Ｃ．Ｂ１７マウスおよび脳梗塞ＳＣＩＤマウスの梗塞下組織を採取し、その後、ＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞を３ｍｌの培養液にて再浮遊させ、ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）存在下にlow cell bindingプレート上で２９日間培養を行い、新たに形成されるニューロスフェア様細胞塊数を数えた。その結果、図１２に示すように、脳梗塞ＳＣＩＤマウスの梗塞下組織からは培養５日目以降にニューロスフェア様細胞塊の形成が観察されたが、脳梗塞Ｃ．Ｂ１７マウスの梗塞下組織からはニューロスフェア様細胞塊は全く形成されなかった。 Next, it was examined whether neural stem cells were formed by culturing cells collected from cerebral infarcted tissue. Cerebral infarction 7 days after ligation B17 mice and cerebral infarction SCID mouse subinfarcted tissues are collected, then pipetted into single cells in DMEM and N-2 basic culture solution (250 μl), 10 ml culture solution is added and 600 rpm for 5 minutes. Centrifuged. Cells are resuspended in 3 ml of culture medium, cultured for 29 days on a low cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml), and newly formed neurosphere-like cell clusters I counted the number. As a result, as shown in FIG. 12, formation of neurosphere-like cell clusters was observed from the infarcted tissue of cerebral infarction SCID mice after the fifth day of culture. No neurosphere-like cell cluster was formed from the infarcted tissue of B17 mice.
［2-5］Ｃ．Ｂ１７マウスにおける骨髄由来ニューロスフェア様細胞塊の形成
脳梗塞作成後７日目のＳＣＩＤマウスとＣ．Ｂ１７マウスの骨髄を２８日間培養し、ニューロスフェア様細胞塊が形成されるかどうかを観察した。その結果、ＳＣＩＤマウスの骨髄からは培養２週間目までにニューロスフェア様細胞塊が形成された（図１０Ａ・Ｂ）。一方、Ｃ．Ｂ１７マウスの骨髄からは培養開始７日目までにわずかにニューロスフェア様細胞塊が形成されたが（同図Ｃ・Ｄ）、これらは２週間以内にすべて消失した。[2-5] C.I. Formation of Bone Marrow-Derived Neurosphere-Like Cell Mass in B17 Mice SCID mice and C.I. The bone marrow of B17 mice was cultured for 28 days, and it was observed whether a neurosphere-like cell mass was formed. As a result, neurosphere-like cell clusters were formed from the bone marrow of SCID mice by the second week of culture (FIGS. 10A and 10B). On the other hand, C.I. From the bone marrow of the B17 mouse, a neurosphere-like cell cluster was slightly formed by the 7th day from the start of culture (FIG. CD), but all of these disappeared within 2 weeks.
そこで、ニューロスフェア様細胞塊における細胞死のメカニズムを探る目的で、培養後同時期のＳＣＩＤマウス由来とＣ．Ｂ１７マウス由来のニューロスフェア様細胞塊をＡｎｅｘｉｎＶで標識してアポトーシスを検討した。その結果、ＳＣＩＤマウス由来のニューロスフェア様細胞塊では、細胞中心部が少しネクローシスに陥っているのみであったが（図１１Ａ参照）、Ｃ．Ｂ１７マウス由来のニューロスフェア様細胞塊では、内側の細胞はほとんどがネクローシスで、外側の細胞はすべてアポトーシスに陥っていた（同図Ｂ参照）。この結果から、脳梗塞Ｃ．Ｂ１７マウス由来のニューロスフェア様細胞塊は、アポトーシスで消滅することが示された。 Therefore, for the purpose of investigating the mechanism of cell death in the neurosphere-like cell cluster, it is derived from SCID mice at the same time after culture and C.I. Neurosphere-like cell clusters derived from B17 mice were labeled with Annexin V to examine apoptosis. As a result, in the neurosphere-like cell cluster derived from SCID mice, the cell center was only slightly necrotic (see FIG. 11A). In the neurosphere-like cell cluster derived from B17 mice, the inner cells were mostly necrotic and the outer cells were all apoptotic (see Fig. B). From this result, cerebral infarction C.I. Neurosphere-like cell clusters derived from B17 mice were shown to disappear upon apoptosis.
また、脳梗塞を起こしていない偽手術Ｃ．Ｂ１７マウスの骨髄からは、ニューロスフェア様細胞塊は全く形成されなかった。さらに、偽手術Ｃ．Ｂ１７マウスから採取した骨髄を、脳梗塞Ｃ．Ｂ１７マウスの血清を添加して培養すると、ニューロスフェア様細胞塊が一旦形成されるがすぐに排除され、ニューロスフェア様細胞塊を得ることはできなかった。 In addition, sham operation without cerebral infarction C.I. No neurosphere-like cell clusters were formed from the bone marrow of B17 mice. In addition, sham surgery C.I. Bone marrow collected from B17 mice was used for cerebral infarction. When serum of B17 mice was added and cultured, a neurosphere-like cell mass was once formed, but was immediately eliminated, and a neurosphere-like cell mass could not be obtained.
［2-6］本実施例の結果と考察
以上のように、免疫不全マウスであるＳＣＩＤマウスの脳梗塞モデルから採取した脳梗塞下組織（脳梗塞瘢痕部位）や骨髄からは、大量のニューロスフェアが形成された。これに対して、その母系マウスである免疫正常なＣ．Ｂ１７マウスの脳梗塞モデルから採取した脳梗塞瘢痕部位や骨髄からは、ニューロスフェアはほとんど形成されなかった。これらの結果から、正常な免疫反応が骨髄および脳梗塞瘢痕部位における神経幹細胞（ニューロスフェア）の発現に抑制的に作用していると考えられる。[2-6] Results and discussion of this example As described above, a large amount of neurospheres were obtained from subcerebral infarction tissues (cerebral infarction scar sites) and bone marrow collected from cerebral infarction models of SCID mice, which are immunodeficient mice. Formed. On the other hand, the immunogenic normal C.I. From the cerebral infarction scar site | part and bone marrow extract | collected from the cerebral infarction model of B17 mouse, the neurosphere was hardly formed. From these results, it is considered that a normal immune response suppresses the expression of neural stem cells (neurospheres) in bone marrow and cerebral infarction scar sites.
〔実施例３：免疫抑制剤を用いた骨髄・脳梗塞下組織（脳梗塞瘢痕部位）からの神経幹細胞調製法〕
上述のように、脳梗塞後の神経再生（神経幹細胞の発現・形成）に免疫の関与が考えられたので、この推論を確かめるため、脳梗塞後の骨髄・脳梗塞瘢痕部位からの神経幹細胞の形成に対する免疫抑制の効果をin vivoとin vitro双方で検討した。[Example 3: Preparation of neural stem cells from bone marrow / cerebral infarction tissue (cerebral infarction scar site) using an immunosuppressant]
As mentioned above, immunity is considered to be involved in nerve regeneration (expression and formation of neural stem cells) after cerebral infarction. To confirm this reasoning, neural stem cells from bone marrow and cerebral infarction scar sites after cerebral infarction were confirmed. The effect of immunosuppression on formation was investigated both in vivo and in vitro.
［3-1］免疫抑制剤を投与したＣ．Ｂ１７マウス骨髄・脳梗塞下組織（脳梗塞瘢痕部位）からのニューロスフェア形成
Ｃ．Ｂ１７マウスに免疫抑制剤ＦＫ５０６（１．０ｍｇ／ｋｇ）を前投与すなわち３日間連日腹腔内投与し、その後、左中大脳動脈閉塞による脳梗塞を作成した。脳梗塞後（結紮後）もＦＫ５０６（１．０ｍｇ／ｋｇ）投与を継続し、脳梗塞後７日目に骨髄と脳梗塞瘢痕部位とを採取してＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞を３ｍｌの培養液にて再浮遊させ、ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）存在下にlow cell bindingプレート上で１０−２８日間培養を行った。図１３に示すように、培養７日目以降には骨髄（Ａ・Ｂ）と脳梗塞瘢痕部位（Ｃ）からニューロスフェア様細胞塊が細胞顕微鏡下で観察された。
一方、偽手術Ｃ．Ｂ１７マウスにＦＫ５０６（１．０ｍｇ／ｋｇ）を１０日間連日腹腔内投与しただけの培養骨髄からはニューロスフェア様細胞塊はほとんど形成されなかった。[3-1] C. administration of immunosuppressant C. Formation of neurospheres from B17 mouse bone marrow / subcerebral infarcted tissue (cerebral infarction scar site) B17 mice were pre-administered with the immunosuppressant FK506 (1.0 mg / kg), that is, intraperitoneally administered daily for 3 days, and then a cerebral infarction due to left middle cerebral artery occlusion was created. After cerebral infarction (after ligation), administration of FK506 (1.0 mg / kg) was continued. On the seventh day after cerebral infarction, bone marrow and cerebral infarction scar sites were collected and contained in a basic culture solution of DMEM and N-2 ( 250 μl) was pipetted until single cells were added, 10 ml of culture medium was added, and the mixture was centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on a low cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. As shown in FIG. 13, neurosphere-like cell clusters were observed under the cell microscope from the bone marrow (A / B) and the cerebral infarction scar site (C) after the seventh day of culture.
On the other hand, sham operation C.I. A neurosphere-like cell cluster was hardly formed from cultured bone marrow in which FK506 (1.0 mg / kg) was intraperitoneally administered daily for 10 days to B17 mice.
［3-2］脳梗塞Ｃ．Ｂ１７マウス骨髄に免疫抑制剤を添加した場合のニューロスフェア形成
次に、脳梗塞作成後７日目のＣ．Ｂ１７マウス骨髄を培養する際に免疫抑制剤ＦＫ５０６（０．１μｇ／ｍｌ）を培養液に添加してニューロスフェア様細胞塊が形成されるかどうかを観察した。すなわち、骨髄細胞をＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞をＦＫ５０６（０．１μｇ／ｍｌ）を含む３ｍｌの培養液にて再浮遊させ、low cell bindingプレート上で１０−２８日間培養を行った。すると、培養４日目以降にニューロスフェア様細胞塊が細胞顕微鏡下で観察された（図１４Ａ・Ｂ）。[3-2] Cerebral infarction C.I. Neurosphere formation when an immunosuppressant is added to B17 mouse bone marrow Next, C.I. When culturing B17 mouse bone marrow, immunosuppressant FK506 (0.1 μg / ml) was added to the culture medium to observe whether a neurosphere-like cell mass was formed. That is, bone marrow cells were pipetted into single cells in DMEM and N-2 basic culture solution (250 μl), 10 ml of culture solution was added, and the mixture was centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of a culture solution containing FK506 (0.1 μg / ml) and cultured on a low cell binding plate for 10-28 days. Then, after the fourth day of culture, neurosphere-like cell clusters were observed under a cell microscope (FIGS. 14A and 14B).
即ち図１５に示すように、脳梗塞作成後７日目のＣ．Ｂ１７マウス骨髄を培養しても、ほとんどニューロスフェア様細胞塊は形成されなかったが（C.B (MCO1W)）、上述のように、ＦＫ５０６（０．１μｇ／ｍｌ）を培養液に添加するとニューロスフェア様細胞塊が形成された（C.B (MCO1W) + FK506）。これは培養４日目以降に観察され、ＳＣＩＤマウスの脳梗塞後における形成（SCID (MCO1W)）よりも早期に見られた。 That is, as shown in FIG. Even though B17 mouse bone marrow was cultured, almost no neurosphere-like cell mass was formed (CB (MCO1W)). As described above, when FK506 (0.1 μg / ml) was added to the culture solution, A cell mass was formed (CB (MCO1W) + FK506). This was observed after the 4th day of culture, and was observed earlier than the formation of SCID mice after cerebral infarction (SCID (MCO1W)).
一方、脳梗塞を起こしていない偽手術Ｃ．Ｂ１７マウス骨髄に対しては、
（１）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）のみ、
（２）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目のＳＣＩＤマウスの血清（５０μｌ）、
（３）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目のＳＣＩＤマウスの血清（５０μｌ）＋ＦＫ５０６（０．１μｇ／ｍｌ）、
の３種類の培養液を用意した。On the other hand, sham operation without cerebral infarction For B17 mouse bone marrow
(1) Only bFGF (50 μg / ml) and EGF (20 μg / ml),
(2) bFGF (50 μg / ml) and EGF (20 μg / ml) + sera of SCID mice one week after cerebral infarction (50 μl),
(3) bFGF (50 μg / ml) and EGF (20 μg / ml) + sera of SCID mice one week after cerebral infarction (50 μl) + FK506 (0.1 μg / ml),
Three types of culture solutions were prepared.
すると、培養４日目以降に上記（３）の培養液中にのみニューロスフェア様細胞塊が細胞顕微鏡下で観察された（図１６Ｂ・Ｃ）。一方、上記（２）の培養液からはニューロスフェア様細胞塊が一旦形成されるが、すぐに排除されニューロスフェアは得られなかった。これらのことから、骨髄におけるニューロスフェア形成に抑制的に作用している免疫反応を排除することにより神経幹細胞が形成されること、骨髄から異所性に神経幹細胞が形成されるためにはＴ細胞機能など細胞性免疫が抑制されなければならないことが明らかになった。 Then, after the fourth day of culture, a neurosphere-like cell cluster was observed only in the culture solution of (3) above under a cell microscope (FIGS. 16B and 16C). On the other hand, a neurosphere-like cell cluster was once formed from the culture solution of (2) above, but it was eliminated immediately and no neurosphere was obtained. From these facts, it is possible to form neural stem cells by eliminating an immune reaction that suppresses neurosphere formation in the bone marrow, and to form ectopically from bone marrow, T cells It became clear that cellular immunity such as function must be suppressed.
［3-3］Ｃ．Ｂ１７マウス骨髄に抗Ｔ細胞抗体を添加した場合のニューロスフェア形成
ＣＤ２８およびＩＣＯＳ（inducible costimulatory molecule）は、多くのＴ細胞依存性免疫反応における受容体であり、そのブロッキング抗体である抗ＣＤ２８抗体および抗ＩＣＯＳ抗体は、Ｔ細胞機能を抑制することが知られている（Am. J. Respir. Cell Mol. Biol. (1997) 16:335-342.；J. Immunol. (2000) 165:5035-5040.等）。そこで、ＦＫ５０６以外に、このようなＴ細胞機能を抑制する抗体（抗Ｔ細胞抗体）を添加した場合も、ＦＫ５０６を添加した場合と同様に神経幹細胞が形成されるかどうかを検討した。[3-3] C.I. Neurosphere formation when anti-T cell antibody is added to B17 mouse bone marrow CD28 and ICOS (inducible costimulatory molecule) are receptors in many T cell-dependent immune responses and their blocking antibodies, anti-CD28 antibody and anti-CD28 antibody ICOS antibodies are known to suppress T cell function (Am. J. Respir. Cell Mol. Biol. (1997) 16: 335-342 .; J. Immunol. (2000) 165: 5035-5040 .etc). Therefore, in addition to FK506, it was examined whether or not neural stem cells were formed in the same manner as when FK506 was added when such an antibody that suppresses T cell function (anti-T cell antibody) was added.
脳梗塞を起こしていない偽手術Ｃ．Ｂ１７マウス骨髄に対して、
（１）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目のＳＣＩＤマウスの血清（５０μｌ）＋ＦＫ５０６（０．１μｇ／ｍｌ）、
（２）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目のＳＣＩＤマウスの血清（５０μｌ）＋抗ＣＤ２８抗体（２μｇ／ｍｌ，BD Biosciences）、
（３）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目のＳＣＩＤマウスの血清（５０μｌ）＋抗ＩＣＯＳ抗体（４μｇ／ｍｌ，BD Biosciences）、
および、コントロールとして、
（４）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目のＳＣＩＤマウスの血清（５０μｌ）＋生理食塩水（saline）、
の４種類の培養液を用意した。Sham surgery without cerebral infarction For B17 mouse bone marrow
(1) bFGF (50 μg / ml) and EGF (20 μg / ml) + sera of SCID mice one week after cerebral infarction (50 μl) + FK506 (0.1 μg / ml),
(2) bFGF (50 μg / ml) and EGF (20 μg / ml) + sera of SCID mice one week after cerebral infarction (50 μl) + anti-CD28 antibody (2 μg / ml, BD Biosciences),
(3) bFGF (50 μg / ml) and EGF (20 μg / ml) + sera of SCID mice one week after cerebral infarction (50 μl) + anti-ICOS antibody (4 μg / ml, BD Biosciences),
And as a control,
(4) bFGF (50 μg / ml) and EGF (20 μg / ml) + sera of SCID mice one week after cerebral infarction (50 μl) + saline (saline),
Four types of culture solutions were prepared.
すると、培養４日目以降に上記（４）以外の培養液中にニューロスフェア様細胞塊が細胞顕微鏡下で観察された（図２１）。さらに、浮遊しているニューロスフェア様細胞塊を採取してhigh bindingプレート上で更に培養した。７日後に分化した細胞をパラフォルムアルデヒドを含む固定液にて固定し、nestin陽性のニューロスフェア数を計測したところ、１dishあたりのnestin陽性ニューロスフェア数は、上記（４）のsaline群に比しＦＫ５０６添加群（１）や抗ＣＤ２８抗体添加群（２）、抗ＩＣＯＳ抗体添加群（３）で有意に多かった（図２７）。
以上の結果から、Ｔ細胞機能を抑制する抗Ｔ細胞抗体を添加することにより、ＦＫ５０６を添加した場合と同様に神経幹細胞が形成されること、および、骨髄から神経幹細胞が形成されるためにはＴ細胞機能を抑制することが重要であることが明らかになった。Then, after the 4th day of culture, neurosphere-like cell clusters were observed under a cell microscope in the culture solutions other than the above (4) (FIG. 21). Furthermore, floating neurosphere-like cell clusters were collected and further cultured on a high binding plate. Cells differentiated after 7 days were fixed with a fixative containing paraformaldehyde, and the number of nestin-positive neurospheres was measured. The number of nestin-positive neurospheres per dish was higher than that of the (saline group) in (4) above. There were significantly more in the FK506 addition group (1), the anti-CD28 antibody addition group (2), and the anti-ICOS antibody addition group (3) (FIG. 27).
From the above results, by adding an anti-T cell antibody that suppresses T cell function, neural stem cells are formed as in the case of adding FK506, and neural stem cells are formed from bone marrow. It became clear that it was important to suppress T cell function.
［3-4］Ｃ．Ｂ１７マウスから調製されたニューロスフェア様細胞塊の神経細胞への分化能
脳梗塞を起こしていない偽手術Ｃ．Ｂ１７マウス骨髄にＦＫ５０６と血清を添加して培養し、１０日目に浮遊しているニューロスフェア様細胞塊を採取してhigh bindingプレート上でさらに培養した。３−７日後に分化した細胞をパラフォルムアルデヒドを含む固定液にて固定して、ＮｅｓｔｉｎとＭＡＰ２に対する免疫組織化学（二重染色間接蛍光抗体法）を行った。その結果、これらの中にはＮｅｓｔｉｎ陽性の神経幹細胞が高率に観察された（図１７）。同図に示すように、細胞塊の中心はＮｅｓｔｉｎ陽性（明灰色）で神経幹細胞であり、周囲にはより分化したＭＡＰ２陽性の神経細胞（暗灰色）が観察された。また、上記ニューロスフェア様細胞塊について神経への分化能を検討した結果、骨髄培養から得られたニューロスフェア様細胞塊の約６０％がＮｅｓｔｉｎ陽性の神経幹細胞で、そのうち６７％がＭＡＰ２陽性の神経細胞に分化した。[3-4] C.I. Differentiation ability of neurosphere-like cell clusters prepared from B17 mice into nerve cells Sham surgery without cerebral infarction BK mouse bone marrow was cultured with FK506 and serum added, and the neurosphere-like cell mass floating on day 10 was collected and further cultured on a high binding plate. Cells differentiated after 3-7 days were fixed with a fixative containing paraformaldehyde, and immunohistochemistry (double-staining indirect fluorescent antibody method) against Nestin and MAP2 was performed. As a result, Nestin-positive neural stem cells were observed at a high rate among these (FIG. 17). As shown in the figure, the center of the cell cluster was Nestin-positive (light gray) and neural stem cells, and more differentiated MAP2-positive nerve cells (dark gray) were observed around it. Further, as a result of examining the differentiation ability of the neurosphere-like cell cluster into nerves, about 60% of neurosphere-like cell clusters obtained from bone marrow culture are Nestin-positive neural stem cells, of which 67% are MAP2-positive nerves. Differentiated into cells.
［3-5］Ｃ．Ｂ１７マウスの骨髄と脳梗塞下組織（脳梗塞瘢痕部位）から形成されるニューロスフェアの相違
上述のように、Ｃ．Ｂ１７マウスの脳梗塞後の骨髄からはニューロスフェア様の細胞塊が一旦は形成されるが、脳梗塞瘢痕部位からはニューロスフェア様細胞塊の痕跡すら認められなかった。このことは、骨髄では脳梗塞後に血清中の刺激因子を介して神経幹細胞産生への誘導が生じるものの、正常なＴ細胞機能があるとその影響により幹細胞が排除され、脳に到達できないものと考えられる（図１２）。[3-5] C.I. Differences in neurospheres formed from bone marrow and subcerebral infarcted tissue (cerebral infarct scar site) in B17 mice. Neurosphere-like cell clusters were once formed from bone marrow after cerebral infarction in B17 mice, but no traces of neurosphere-like cell clusters were observed from cerebral infarction scar sites. This is because bone marrow induces neural stem cell production via a stimulating factor in serum after cerebral infarction, but if normal T cell function is present, stem cells are excluded due to the effect and cannot reach the brain. (FIG. 12).
［3-6］本実施例の結果と考察
以上のように、Ｃ．Ｂ１７マウスに免疫抑制剤であるＦＫ５０６を前投与し、Ｔ細胞機能を十分抑制してから脳梗塞を作成すると、骨髄および脳梗塞瘢痕部位からニューロスフェアが形成された。また、脳梗塞を起こしていない偽手術Ｃ．Ｂ１７マウスの骨髄に脳梗塞後１週間目のＳＣＩＤマウスの血清とＦＫ５０６を添加して培養するとニューロスフェアが形成された。さらに、Ｔ細胞機能抑制の目的でＦＫ５０６の代わりに抗ＣＤ２８抗体あるいは抗ＩＣＯＳ抗体を添加して培養すると、同様に骨髄からニューロスフェアが形成された。[3-6] Results and Discussion of this Example As described above, C.I. When FK506, which is an immunosuppressant, was pre-administered to B17 mice and cerebral infarction was created after sufficiently suppressing T cell function, neurospheres were formed from bone marrow and cerebral infarction scar sites. In addition, sham operation without cerebral infarction C.I. Neurospheres were formed when the serum of SCID mice 1 week after cerebral infarction and FK506 were added to the bone marrow of B17 mice and cultured. Furthermore, when anti-CD28 antibody or anti-ICOS antibody was added instead of FK506 for cultivation for the purpose of suppressing T cell function, neurospheres were similarly formed from bone marrow.
このことより、骨髄から異所性に神経幹細胞が形成されるためにはＴ細胞機能など細胞性免疫が抑制されなければならないこと、正常マウスでも免疫を調節（抑制）すれば骨髄で神経再生が生じ、アポトーシスによる細胞死を回避して、脳梗塞瘢痕部位に移行生着し、そこで分化して神経になることが示唆された。 Therefore, in order to form ectopic neural stem cells from the bone marrow, cell immunity such as T cell function must be suppressed, and even in normal mice, nerve regeneration can be achieved in the bone marrow by regulating (suppressing) immunity. It was suggested that cell death due to apoptosis was avoided, and the cells migrated to the cerebral infarction scar site, where they differentiated into nerves.
〔実施例４：脳梗塞ＳＣＩＤマウス骨髄由来の神経幹細胞の移植と脳組織への生着・分化〕
脳梗塞ＳＣＩＤマウスの骨髄由来の神経幹細胞が生理的に脳梗塞巣に侵入して脳組織へ生着、分化するかどうかを確認するため、以下の実験を行った。[Example 4: Transplantation of neural stem cells derived from cerebral infarction SCID mouse bone marrow and engraftment / differentiation into brain tissue]
In order to confirm whether neural stem cells derived from the bone marrow of cerebral infarction SCID mice physiologically enter the cerebral infarction lesion and engraft and differentiate into brain tissue, the following experiment was performed.
即ち、骨髄由来の神経幹細胞がＳＣＩＤマウスの脳梗塞後に生じる神経再生に関与するかどうかを、緑色蛍光タンパク質（green fluorescence protein：ＧＦＰ）を発現するＧＦＰトランスジェニックマウスの骨髄を移植したＳＣＩＤマウス脳梗塞モデルで検討した。 That is, whether or not bone marrow-derived neural stem cells are involved in nerve regeneration that occurs after cerebral infarction of SCID mice, SCID mouse cerebral infarction transplanted with bone marrow of GFP transgenic mice expressing green fluorescence protein (GFP) The model was examined.
まず、ＳＣＩＤマウスに２００ｒａｄの放射線照射による骨髄抑制を施した後にＧＦＰトランスジェニックマウスの骨髄細胞（１０万個）を移植した。移植後２週間目に脳梗塞を作成し、さらに脳梗塞後１６日目にマウスを経心的にＰＬＰ固定液で灌流固定した。脳を摘出し、ビブラトームを用いて脳切片を作成した。その後脳切片をＧＦＰとＰＳＡ−ＮＣＡＭに対する二重染色免疫組織化学に供した。 First, SCID mice were subjected to bone marrow suppression by irradiation with 200 rads, and then transplanted with bone marrow cells (100,000) of GFP transgenic mice. A cerebral infarction was created 2 weeks after the transplantation, and the mouse was transcardially fixed with a PLP fixative solution 16 days after the cerebral infarction. The brain was removed and brain sections were prepared using a vibratome. The brain sections were then subjected to double staining immunohistochemistry for GFP and PSA-NCAM.
具体的には、脳切片をラットモノクローナル抗ＧＦＰ抗体とマウスモノクローナル抗ＰＳＡ−ＮＣＡＭ抗体を含むリン酸緩衝液（２０００倍希釈）にて１２時間反応させ（第一反応）、洗浄後ＦＩＴＣ標識ヤギ抗ラットＩｇＧとビオチン標識ヤギ抗マウスＩｇＧ（第二反応）、次いでアビジン標識Ｃｙ３と反応させた。ＧＦＰとＰＳＡ−ＮＣＡＭの可視化は共焦点レーザー蛍光顕微鏡にて観察した。ＧＦＰ陽性細胞は脳梗塞下組織に広く認められ、一部はＰＳＡ−ＮＣＡＭ陰性でミクログリアの形態を示していたが（図１８Ｂ参照）、梗塞下の白質外側および大脳皮質断面（本発明者らがステムロード（Stem Road）と称する部位）に沿って広く認められたＧＦＰ陽性細胞群は、同時にＰＳＡ−ＮＣＡＭ陽性で神経前駆細胞と考えられる（同図Ｃ参照）。ＰＳＡ−ＮＣＡＭ陽性部位は他に脳室周囲組織にもみられたが、ここはＧＦＰ陰性であった。 Specifically, brain sections were reacted for 12 hours in a phosphate buffer solution (diluted 2000 times) containing rat monoclonal anti-GFP antibody and mouse monoclonal anti-PSA-NCAM antibody (first reaction). After washing, FITC-labeled goat anti-goat Rat IgG and biotin-labeled goat anti-mouse IgG (second reaction) were reacted with avidin-labeled Cy3. Visualization of GFP and PSA-NCAM was observed with a confocal laser fluorescence microscope. GFP-positive cells were widely observed in subcerebral infarcted tissues, and some of them were PSA-NCAM negative and showed microglial morphology (see FIG. 18B). A group of GFP-positive cells widely recognized along the stem road) is considered to be PSA-NCAM-positive and neural progenitor cells (see FIG. C). Other PSA-NCAM positive sites were also found in periventricular tissues, but they were GFP negative.
以上の結果から、上記ステムロードには骨髄細胞由来の神経幹細胞が梗塞後に発現して脳実質内に侵入すること、すなわち、骨髄由来の神経幹細胞が脳梗塞下の組織へ生着し、神経に分化することが示された。 From the above results, it is shown that bone marrow cell-derived neural stem cells are expressed after infarction and invade into the brain parenchyma, that is, bone marrow-derived neural stem cells have engrafted in the tissue under cerebral infarction and entered the nerve. It was shown to differentiate.
〔実施例５：ヒトへの応用と神経幹細胞移植による脳梗塞治療効果〕
［5-1］ヒトの骨髄由来神経幹細胞調製法
以上の知見をヒトに応用し、ヒト骨髄由来の神経幹細胞の調製が可能かどうかを検討した。
脳梗塞を起こしていない正常ヒト骨髄を採取し、その後、ＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞を３ｍｌの培養液にて再浮遊させ、low cell bindingプレート上で１０−２８日間培養を行った。培養液は、
（１）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）のみ、
（２）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目の患者の血清（５０μｌ）、
（３）ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）＋脳梗塞後１週間目の患者の血清（５０μｌ）＋ＦＫ５０６（０．１μｇ／ｍｌ）
の３種類を用意した。[Example 5: Application to human and treatment effect of cerebral infarction by neural stem cell transplantation]
[5-1] Human bone marrow-derived neural stem cell preparation method The above knowledge was applied to humans to examine whether human bone marrow-derived neural stem cells could be prepared.
Normal human bone marrow without cerebral infarction was collected, then pipetted into single cells in DMEM and N-2 basic culture solution (250 μl), added with 10 ml culture solution, and centrifuged at 600 rpm for 5 minutes. . Cells were resuspended in 3 ml of culture and cultured on a low cell binding plate for 10-28 days. The culture solution is
(1) Only bFGF (50 μg / ml) and EGF (20 μg / ml),
(2) bFGF (50 μg / ml) and EGF (20 μg / ml) + serum of patient 1 week after cerebral infarction (50 μl),
(3) bFGF (50 μg / ml) and EGF (20 μg / ml) + serum of patient 1 week after cerebral infarction (50 μl) + FK506 (0.1 μg / ml)
Three types were prepared.
すると、培養７日目以降に上記（３）の培養液中にのみニューロスフェア様細胞塊が細胞顕微鏡下で観察された。培養１０日目に形成されたニューロスフェア様細胞塊（図１９Ａ）をhigh bindingプレート上でさらに５日間培養すると、神経細胞に分化した（同図Ｂ）。 Then, neurosphere-like cell clusters were observed under the cell microscope only in the culture solution of (3) above after the seventh day of culture. When the neurosphere-like cell cluster (FIG. 19A) formed on the 10th day of culture was further cultured on a high binding plate for 5 days, it differentiated into neurons (FIG. 19B).
［5-2］脳梗塞ＳＣＩＤマウス骨髄由来の神経幹細胞の移植
脳梗塞ＳＣＩＤマウスから得られた骨髄由来の神経幹細胞を、別の脳梗塞ＳＣＩＤマウスに移植した場合の脳再生に与える影響、脳梗塞治療効果について検討した。[5-2] Transplantation of neural stem cells derived from bone marrow derived from cerebral infarction SCID mice Effect of bone marrow-derived neural stem cells obtained from cerebral infarction SCID mice on brain regeneration when transplanted to another cerebral infarction SCID mouse, cerebral infarction The therapeutic effect was examined.
即ち、脳梗塞ＳＣＩＤマウスの骨髄から調製されたニューロスフェア様細胞塊を単細胞に分離して、その１０万個を別の脳梗塞ＳＣＩＤマウス（脳梗塞後２日目）に静脈内投与した。そして、その脳梗塞後１６日目に当該マウスを経心的にＰＬＰ固定液で灌流固定後、脳を摘出した。摘出脳は、全例中大脳動脈領域の欠損が観察された。 That is, neurosphere-like cell clusters prepared from the bone marrow of cerebral infarction SCID mice were separated into single cells, and 100,000 of them were intravenously administered to another cerebral infarction SCID mouse (2 days after cerebral infarction). On the 16th day after the cerebral infarction, the mouse was transcardially fixed with a PLP fixative solution, and the brain was removed. In the isolated brain, defects in the middle cerebral artery region were observed in all cases.
梗塞側大脳皮質の大脳裂から梗塞部位までの幅（ａ）を正常側（ｂ）と比較し、その比（ａ／ｂ）をcortical width index（ＣＩ値）として算出すると、ＣＩ値は、骨髄由来神経幹細胞（ＢＭ−ＮＳＣ）投与マウスでは０．４８で、ＰＢＳを投与したコントロールマウスでは０．３６であった（図２０）。このように、神経幹細胞を移植されたマウスの脳は、コントロールマウスの脳より残存大脳皮質が拡大していた。 When the width (a) from the cerebral cleft of the infarcted cerebral cortex to the infarcted site is compared with the normal side (b) and the ratio (a / b) is calculated as the cortical width index (CI value), the CI value is It was 0.48 in the mouse treated with the derived neural stem cell (BM-NSC) and 0.36 in the control mouse administered with PBS (FIG. 20). Thus, the brain of the mouse transplanted with neural stem cells had a larger residual cerebral cortex than the brain of the control mouse.
上述のように、ＳＣＩＤマウスから作成した脳梗塞モデルマウスでは、虚血後の脳梗塞の進展（delayed infarct expansion）は脳梗塞後３日で終了し、その後は脳萎縮の進展ではなく、脳形態上はむしろ回復するが、神経幹細胞を移植するとさらに回復が進むことを示している。このことは、本神経幹細胞の移植が脳梗塞後の神経再生を促進することを示している。 As described above, in the cerebral infarction model mouse prepared from the SCID mouse, the delayed infarct expansion after ischemia is completed in 3 days after the cerebral infarction, and the brain morphology is not progressed thereafter. Although the above is rather a recovery, it shows that the recovery proceeds further when neural stem cells are transplanted. This indicates that transplantation of this neural stem cell promotes nerve regeneration after cerebral infarction.
〔実施例６：分化誘導因子の発見とこれを用いた骨髄由来神経幹細胞の調製〕
ＣＩＮＣ−１が神経幹細胞への分化誘導因子である可能性が考えられたので、以下の実験によりＣＩＮＣ−１が骨髄細胞分化に及ぼす影響を検討した。[Example 6: Discovery of differentiation-inducing factor and preparation of bone marrow-derived neural stem cells using the same]
Since there was a possibility that CINC-1 was a differentiation inducing factor into neural stem cells, the effect of CINC-1 on bone marrow cell differentiation was examined by the following experiment.
脳梗塞を起こしていない偽手術Ｃ．Ｂ１７マウス骨髄にＣＩＮＣ−１（１０-5Ｍ：ペプチド研究所）とＦＫ５０６（０．１μｇ／ｍｌ）を添加して培養した。すると培養５日目以内にすでに多くの細胞塊が形成された（図２２Ａ・Ｂ）。これらはまだ細胞密度が低い細胞凝集塊であり、ニューロスフェアとは異なっていたが、培養７日目以降になると、ニューロスフェア様細胞塊が形成された（図２２Ｃ・Ｄ）。Sham surgery without cerebral infarction CINC-1 (10 −5 M: Peptide Institute) and FK506 (0.1 μg / ml) were added to B17 mouse bone marrow and cultured. Then, many cell clusters were already formed within 5 days of culture (FIGS. 22A and 22B). These were cell aggregates having a low cell density and were different from neurospheres, but after 7 days of culture, neurosphere-like cell clusters were formed (FIGS. 22C and 22D).
また、ＦＫ５０６の影響を検討した結果、ＣＩＮＣ−１（１０-5Ｍ）とＦＫ５０６（０．１μｇ／ｍｌ）を添加して培養した場合には、上述のように、培養７日目以降にニューロスフェア様細胞塊が形成された（図２３Ａ）のに対して、ＣＩＮＣ−１（１０-5Ｍ）のみを添加し、ＦＫ５０６を添加しない場合は、細胞塊は形成されるが、殆どが排除されてしまうか、あるいは細胞塊の状態で止まっており（図２３Ｂ）、ニューロスフェア形成は認められなかった。Moreover, as a result of examining the influence of FK506, when CINC-1 (10 −5 M) and FK506 (0.1 μg / ml) were added and cultured, as described above, the neuron was observed after the seventh day of culture. When sphere-like cell clusters are formed (FIG. 23A), when only CINC-1 (10 −5 M) is added and FK506 is not added, cell clusters are formed, but most are eliminated. Or stopped in the state of a cell mass (FIG. 23B), and no neurosphere formation was observed.
以上の結果は、脳虚血刺激で産生亢進し、ＮＫ細胞でも作られるＣＩＮＣ−１が骨髄細胞の神経幹細胞への分化誘導因子のひとつであることを示すと同時に、Ｔ細胞機能の抑制もその形成維持には重要であることを示している。 The above results show that CINC-1, which is increased in production by cerebral ischemic stimulation and is also produced by NK cells, is one of the differentiation-inducing factors of bone marrow cells into neural stem cells, and also suppresses T cell function. It is important to maintain the formation.
〔実施例７：神経幹細胞の形成における免疫抑制の重要性と免疫抑制物質の神経再生治療への応用〕
これまでの実験結果から、in vitroにおいて、またin vivoにおいても、骨髄由来神経幹細胞の形成を維持・促進し、神経再生治療を実現するためには、免疫機能を抑制すること、特にＴ細胞機能を抑制することが重要であると考えられた。このことをさらに確かめるため、以下の実験を行った。[Example 7: Importance of immunosuppression in the formation of neural stem cells and application of immunosuppressive substances to nerve regeneration therapy]
From the results of experiments so far, in order to maintain and promote the formation of bone marrow-derived neural stem cells in vitro and in vivo, and to realize nerve regeneration treatment, it is necessary to suppress immune function, particularly T cell function. It was thought that it was important to suppress this. In order to confirm this further, the following experiment was conducted.
［7-1］ＦＫ５０６以外の免疫抑制剤を用いたニューロスフェアの形成
Ｃ．Ｂ１７マウスにＦＫ５０６（tacrolimus hydrate: 1.0 mg/kg/day）あるいはＦＫ５０６と同じくＴ細胞機能を抑制する免疫抑制剤であるシクロスポリンＡ（ＣｓＡ: 10mg/kg/day）を３日間連日腹腔内投与し、左中大脳動脈閉塞による脳梗塞を作成した（３匹ずつ）。その後もＦＫ５０６（1.0 mg/kg）あるいはＣｓＡ（10mg/kg/day）投与を継続し、脳梗塞後７日目に骨髄と脳梗塞下組織（脳梗塞瘢痕部位）を採取してＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞を３ｍｌの培養液にて再浮遊させ、ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）存在下にlow cell bindingプレート上で１０−２８日間培養を行った。７日目以降にはＦＫ５０６とＣｓＡ投与群の骨髄（ＢＭ）と脳梗塞下組織からニューロスフェア様細胞塊が細胞顕微鏡下で観察された。一方、生理食塩水（saline）を１０日間連日腹腔内投与しただけの培養骨髄からはニューロスフェア様細胞塊はほとんど形成されなかった。浮遊しているニューロスフェア様細胞塊を採取してhigh bindingプレート上でさらに培養した。７日後に分化した細胞をパラフォルムアルデヒドを含む固定液にて固定し、nestin陽性のニューロスフェア数を計測したところ、ＦＫ５０６およびＣｓＡ投与群ではコントロールに比し、有意にnestin陽性ニューロスフェアが多かった（図２４）。
以上のことより、Ｔ細胞機能を抑制する免疫抑制剤を投与することで脳梗塞後に骨髄で神経再生が生じ、アポトーシスによる細胞死を回避して、脳梗塞部位に移行生着し、そこで分化して神経になることが示唆された。[7-1] Formation of neurospheres using immunosuppressive agents other than FK506 B17 mice were intraperitoneally administered with FK506 (tacrolimus hydrate: 1.0 mg / kg / day) or cyclosporin A (CsA: 10 mg / kg / day), which is an immunosuppressant that suppresses T cell function as with FK506, for 3 days. Cerebral infarction due to left middle cerebral artery occlusion was created (3 animals each). Thereafter, administration of FK506 (1.0 mg / kg) or CsA (10 mg / kg / day) was continued, and on the 7th day after cerebral infarction, bone marrow and cerebral infarction tissue (cerebral infarction scar site) were collected and DMEM and N- Pipetting up to single cells in 2 basic cultures (250 μl), adding 10 ml cultures and centrifuging at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on a low cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. From day 7 onward, neurosphere-like cell masses were observed under the cell microscope from bone marrow (BM) and cerebral infarcted tissue in the FK506 and CsA administration groups. On the other hand, almost no neurosphere-like cell clusters were formed from cultured bone marrow that had been intraperitoneally administered with saline for 10 days every day. The suspended neurosphere-like cell mass was collected and further cultured on a high binding plate. Cells differentiated after 7 days were fixed with a fixative containing paraformaldehyde, and the number of nestin-positive neurospheres was measured. In the FK506 and CsA-administered groups, there were significantly more nestin-positive neurospheres than the control. (FIG. 24).
From the above, administration of an immunosuppressive agent that suppresses T cell function causes nerve regeneration in the bone marrow after cerebral infarction, avoids cell death due to apoptosis, migrates to the cerebral infarction site, and differentiates there. It was suggested to become nervous.
［7-2］Ｔ細胞機能欠如マウス（ヌードマウス）のニューロスフェア形成
無胸腺マウスであるヌードマウス（BALB/cAJcl-nu）とその母系マウス（コントロール）の左中大脳動脈閉塞による脳梗塞作成後、７日目に骨髄細胞と脳梗塞下組織（脳梗塞瘢痕部位）を採取、培養して神経幹細胞塊（ニューロスフェア）を作成した。すなわち、マウスをクリーンベンチ内で断頭し、大腿骨から骨髄細胞を、脳梗塞瘢痕部より細胞を採取した。その後、ＤＭＥＭとＮ−２の基本培養液内（２５０μｌ）で単細胞になるまでピペッティングし、１０ｍｌの培養液を加えて６００ｒｐｍで５分間遠心した。細胞を３ｍｌの培養液にて再浮遊させ、ｂＦＧＦ（５０μｇ／ｍｌ）とＥＧＦ（２０μｇ／ｍｌ）存在下にlow cell bindingプレート上で１０−２８日間培養を行った。７日目以降にはニューロスフェア様細胞塊が細胞顕微鏡下で観察されたが、Ｔ細胞が正常なコントロールマウスの培養骨髄と脳梗塞瘢痕部からはニューロスフェア様細胞塊はほとんど形成されなかった。浮遊しているニューロスフェア様細胞塊を採取してhigh bindingプレート上でさらに培養した。７日後に分化した細胞をパラフォルムアルデヒドを含む固定液にて固定し、nestin陽性のニューロスフェア数を計測したところ、ヌードマウスではコントロールに比し、有意にnestin陽性ニューロスフェアが多かった（図２５）。
以上のことより、Ｔ細胞の存在がなければ、脳梗塞後に骨髄で神経再生が生じ、アポトーシスによる細胞死を回避して、脳梗塞部位に移行生着し、そこで分化して神経になることが示唆された。[7-2] Neurosphere formation in T cell function-deficient mice (nude mice) After creation of cerebral infarction due to occlusion of the left middle cerebral artery in nude mice (BALB / cAJcl-nu) that are athymic mice and their maternal mice (control) On day 7, bone marrow cells and tissue under cerebral infarction (cerebral infarction scar site) were collected and cultured to prepare a neural stem cell mass (neurosphere). That is, the mouse was decapitated in a clean bench, and bone marrow cells were collected from the femur and cells were collected from the cerebral infarction scar. Thereafter, pipetting was performed in the basic culture solution (250 μl) of DMEM and N-2 until single cells were added, 10 ml of the culture solution was added, and the mixture was centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on a low cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. From day 7 onward, neurosphere-like cell clusters were observed under a cell microscope, but almost no neurosphere-like cell clusters were formed from cultured bone marrow and cerebral infarct scars of control mice with normal T cells. The suspended neurosphere-like cell mass was collected and further cultured on a high binding plate. Cells differentiated after 7 days were fixed with a fixative containing paraformaldehyde, and the number of nestin-positive neurospheres was measured. Nude mice had significantly more nestin-positive neurospheres than controls (FIG. 25). ).
From the above, in the absence of T cells, nerve regeneration occurs in the bone marrow after cerebral infarction, avoids cell death due to apoptosis, migrates and engrafts at the cerebral infarction site, and then differentiates into nerves. It was suggested.
本実施例の結果から、Ｔ細胞が骨髄あるいは脳梗塞瘢痕部におけるニューロスフェア形成に抑制的機能を果たしていること、換言すれば、ニューロスフェアの形成を維持・促進するためにはＴ細胞機能を抑制することが重要であることが示された。すなわち、in vivoでのＦＫ５０６やＣｓＡなどの免疫抑制剤の効果、ＳＣＩＤマウス、ヌードマウスの効果などは、免疫抑制が神経幹細胞作成に重要であることを示すものであると同時に、in vivoで免疫抑制が脳梗塞治療効果に結びつくことを明らかとしている点で、免疫抑制物質が脳梗塞治療など神経再生治療に有効であることを示すものといえる。 From the results of this Example, it can be seen that T cells have a suppressive function on the formation of neurospheres in the bone marrow or cerebral infarction scar, in other words, the T cell function is suppressed in order to maintain and promote the formation of neurospheres. It was shown that it is important to do. That is, the effects of immunosuppressants such as FK506 and CsA in vivo, the effects of SCID mice and nude mice indicate that immunosuppression is important for the generation of neural stem cells. It can be said that immunosuppressive substances are effective for nerve regeneration treatment such as cerebral infarction treatment because it is clarified that suppression leads to cerebral infarction treatment effect.
〔実施例８：新たな分化誘導因子の発見と本スクリーニングの有効性〕
実施例６で分化誘導因子のひとつとして見出されたＣＩＮＣ−１は、ケモカインに分類されるサイトカインであるが、分化誘導因子として作用する分子が他のサイトカインにも存在する可能性が考えられたので、前記と同様の培養系を用いたスクリーニングによって他の分化誘導因子を探索した結果、以下のように、サイトカインのＴＮＦαが、骨髄細胞から神経幹細胞への分化誘導作用を有することを見出した。[Example 8: Discovery of new differentiation-inducing factor and effectiveness of this screening]
CINC-1 found as one of differentiation inducers in Example 6 is a cytokine classified as a chemokine, but it was considered that molecules acting as differentiation inducers may also be present in other cytokines. Therefore, as a result of searching for other differentiation-inducing factors by screening using the same culture system as described above, it was found that the cytokine TNFα has a differentiation-inducing action from bone marrow cells to neural stem cells as follows.
脳梗塞を起こしていない偽手術ＳＣＩＤマウス骨髄にＣＩＮＣ−１（10-5Ｍ）を添加、あるいはＴＮＦα(0.1 ng/ml)を添加して培養した。ＣＩＮＣ−１、ＴＮＦαを添加すると培養5日目以内にすでに細胞塊が形成され７日目以降にはニューロスフェア様細胞塊が形成されたが、これらを添加していないもの（saline添加）はほとんど細胞塊形成がみられなかった。浮遊しているニューロスフェア様細胞塊を採取してhigh bindingプレート上でさらに培養した。７日後に分化した細胞をパラフォルムアルデヒドを含む固定液にて固定し、nestin陽性のニューロスフェア数を計測したところ、ＣＩＮＣ−１のみならずＴＮＦα添加群でもコントロールに比し、有意にnestin陽性ニューロスフェアが多かった（図２６）。CINC-1 (10 −5 M) was added to sham-operated SCID mouse bone marrow without cerebral infarction, or TNFα (0.1 ng / ml) was added and cultured. When CINC-1 and TNFα were added, cell clusters were already formed within 5 days of culture, and neurosphere-like cell clusters were formed after 7 days, but most of them did not contain these (saline added) No cell clump formation was observed. The suspended neurosphere-like cell mass was collected and further cultured on a high binding plate. Cells differentiated after 7 days were fixed with a fixative containing paraformaldehyde, and the number of nestin-positive neurospheres was measured. As a result, not only CINC-1 but also the TNFα-added group was significantly more nestin-positive neuron than the control. There were many spheres (FIG. 26).
以上のことから、ＴＮＦαで骨髄が刺激されると、骨髄で神経再生が生じることが示され、ＴＮＦαも骨髄細胞から神経幹細胞への分化誘導因子のひとつであること、および、神経幹細胞培養系を用いた本スクリーニング法が、神経幹細胞の誘導物質のスクリーニング、さらには当該物質を用いた神経再生治療剤の開発に有効であることが示された。 From the above, it has been shown that when bone marrow is stimulated with TNFα, nerve regeneration occurs in the bone marrow. TNFα is also one of the differentiation inducing factors from bone marrow cells to neural stem cells, and the neural stem cell culture system It was shown that this screening method used is effective for screening a neural stem cell inducer, and further for developing a nerve regeneration therapeutic agent using the substance.
以上のように、本発明は、骨髄から簡易かつ短期間に神経幹細胞を調製する方法等を提供するものであり、細胞移植による脳梗塞発症後の速やかな脳機能の回復、脳梗塞以外の脳血管障害、神経変性疾患に対する神経再生治療、またはそのための治療法開発など神経再生医療の分野において広く利用可能性を有し、再生医療などに取り組む各種の医療関連産業において有用である。
As described above, the present invention provides a method for preparing neural stem cells from bone marrow easily and in a short period of time, and the rapid recovery of brain function after the onset of cerebral infarction by cell transplantation, brain other than cerebral infarction It has wide applicability in the field of nerve regenerative medicine such as nerve regeneration treatment for vascular disorders and neurodegenerative diseases, or the development of treatments therefor, and is useful in various medical related industries tackling regenerative medicine.
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