JP6777323B2 - 毛細血管由来幹細胞、その用途、及び、その製造方法 - Google Patents
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Description
本明細書は、本願の優先権の基礎である特願2015−151601号(2015年7月31日出願)の明細書に記載された内容を包含する。
[技術分野]
本発明は、ヒトに由来する特定の表面マーカーを発現する間葉系幹細胞様細胞集団と前記細胞集団を含む組織又は器官再生用の医療材料(医薬組成物、医療機器、及び医療製品)に関する。
(1)EphA7陽性であることを特徴とする、単離された間葉系幹細胞様のヒト多能性(multipotent)幹細胞集団。
(2)1以上の周細胞マーカー、好ましくはNG2、PDGFRβ、CD13、CD146、NGFR、α-smooth muscle actin、Desmin、及びRGS5から選ばれる1以上の周細胞マーカー、より好ましくはNG2が陽性である、上記(1)記載の細胞集団。
(3)1以上の神経幹細胞マーカー、好ましくはNestin、β3Tubulin、S100A、RC2、Sox2、CD133、及びFABP7から選ばれる1以上の神経幹細胞マーカー、より好ましくはNestin、β3Tubulin、及びS100Aから選ばれる1以上が陽性である、上記(1)又は(2)記載の細胞集団。
(4)CD34及びCD45陰性である、上記(1)〜(3)のいずれかに記載の細胞集団。
(5)脳由来ではない、上記(1)〜(4)のいずれかに記載の細胞集団。
(6)毛細血管を含む組織に由来する、上記(1)〜(5)のいずれかに記載の細胞集団。
(7)スフェア形成能を有する、上記(1)〜(6)のいずれかに記載の細胞集団。
(8)間葉系細胞に分化可能である、上記(1)〜(7)のいずれかに記載の細胞集団。
(9)神経系細胞に分化可能である、上記(1)〜(7)のいずれかに記載の細胞集団。
(10)血管形成能を有する、上記(1)〜(9)のいずれかに記載の細胞集団。
(11)上記(1)〜(10)のいずれかに記載の細胞集団を含む、組織又は器官再生用の医療材料(前記医療材料には、医薬組成物、医療機器、及び医療製品を含む)。
(12)組織又は器官が間葉系の組織又は器官(たとえば、骨、軟骨、腱、脂肪、血管、骨格筋、及び心筋)及び神経系の組織又は器官から選ばれる、上記(11)記載の医療材料。
(13)毛細血管を含む組織に由来する付着性細胞からEphA7陽性の細胞群を分離することを特徴とする、多能性(multipotent)幹細胞集団の製造方法。
(14)毛細血管を含む組織の細胞から付着性の細胞群を分離する工程、前記付着細胞群から周細胞マーカー、好ましくはNG2、PDGFRB、及びCD146から選ばれる1以上、より好ましくはNG2の周細胞マーカー陽性細胞群を分離する工程、及び、前記周細胞マーカー陽性細胞群からEphA7陽性の細胞群を分離する工程を含む、上記(13)記載の方法。
(15)毛細血管を含む組織が皮下脂肪組織、骨格筋組織、心臓組織、腎臓組織、及び内臓脂肪組織から選ばれるいずれかである、上記(13)又は(14)記載の方法。
本発明の細胞集団は、高い脈管形成・再生能を有し、栄養・酸素の供給路である脈管系を構築し、幹細胞機能維持のための環境(Vascular Niche)を提供することができる。すなわち、組織再生・維持に必要不可欠な要素をそれ自体で供給できる細胞集団である。
本発明の細胞集団は多分化能(multipotency)を有し、脈管形成とともに、さまざまな組織実質細胞を供給する。すなわち、脈管構築して組織再生の基礎を作るとともに、組織幹細胞としてはたらき、間葉系細胞(脂肪、骨、軟骨、骨格筋等)、神経系細胞(グリア細胞、シュワン細胞等)に分化する。
本発明は、EphA7陽性で特徴づけられる、単離された間葉系幹細胞様の多能性(multipotent)幹細胞集団に関する。後述するように、本発明の細胞集団は毛細血管を含む組織から単離されたため、本明細書ではこの細胞集団をCapSCs (capillary stem cells)と記載することもある。
本発明の細胞集団はEphA7陽性で特徴づけられる。「EphA7(Ephrin type-A receptor 7)」は、プロテインキナーゼファミリーのephrin受容体サブファミリーに属するタンパクである。ephrin及びephrin関連受容体は、発生、特に神経系の発生に関連すると考えられている。ephrin受容体は、細胞外ドメイン配列とそのリガンドへの結合親和性から2つのグループ:ephrin-Aとephrin-Bに大別される。「EphA7」は、脳・神経系の発生に重要であることが知られており、幹細胞、特に間葉系幹細胞や周細胞での発現は報告されていない。発明者らは、樹立した周幹細胞株において、この「EphA7」が多分化能に関連した遺伝子であることを特定した。
本発明の細胞集団は、ヒトの全身に広く存在する組織幹細胞であり、その由来は特に限定されないが、脳由来でないことが好ましい。本発明の細胞集団は、組織毛細血管あるいは毛細血管を含む組織から効率的に単離することができ、「毛細血管を含む組織」としては、例えば、皮下脂肪組織、骨格筋組織、心臓組織、腎臓組織、内臓脂肪組織等をあげることができる。
本発明の細胞集団は、後述する間葉系幹細胞に適した条件の培養下において優れた増殖能を有し、また培養容器として細胞非接着性のものを用いる等、接着しない条件下で培養することによりスフェアを形成する(以下、「スフェア形成能」と言う)。
本発明の細胞集団は、「間葉系幹細胞様」の多能性幹細胞であり、間葉系細胞(脂肪、骨、軟骨、骨格筋等)に分化可能であるが、神経系細胞(グリア細胞、シュワン細胞など)にも分化可能であり、さらに、毛細血管幹細胞としての血管形成能も有する分化多能性(multipotent)の幹細胞である。なお、本明細書において「多能性(multipotent)」とは、複数の限定的な数の系統の細胞へ分化できる能力を意味し、三胚葉の全てに分化可能(pluripotent)であることを必ずしも意味しない。分化能は、たとえば、前掲Kabara M, Kawabe J, et al. Lab Invest. 2014;94:1340-1354に基づいて確認することができる。
細胞の形態は、間葉系幹細胞に特徴的な紡錘形ないし多角形を示し、接着性であるが、前述のとおり接着しない条件下で培養すると、増殖性が高い幹細胞の特徴である「スフェア形成能」を示す。
本発明の細胞集団は、毛細血管を含む組織に由来する付着性の細胞からEphA7陽性の細胞群を分離することにより調製することができる。
まず、毛細血管を含む組織から細胞を単離する。「毛細血管を含む組織」としては、前述のとおり、皮下脂肪組織、骨格筋組織、心臓組織、腎臓組織、内臓脂肪組織等を挙げることができる。細胞は、常法にしたがい、酵素(コラゲナーゼ、プロテアーゼ等)あるいは市販の細胞分散バッファー等でこれらの組織を処理することにより単離する。
単離した細胞は、間葉系細胞の培養に通常使用される培地に播種し、3〜4日間、好ましくは少なくとも2代以上継代培養する。得られた付着性の細胞は、トリプシン又はトリプシン・EDTA等で処理して容器から分離・回収する。所望により、得られた付着性の細胞から、さらに周細胞マーカー、たとえばNG2、PDGFRB、及びCD13から選ばれる1以上、好ましくはNG2陽性の細胞を分離してもよい。細胞の分離方法については後述する。
次いで、分離した付着性細胞、又は周細胞マーカー、たとえばNG2、PDGFRB、及びCD13から選ばれる1以上、好ましくはNG2陽性の付着性細胞から、EphA7陽性の細胞を分離する。NG2、PDGFRB、CD13、EphA7はいずれも細胞表面抗原(マーカー)であるため、該抗原に特異的な抗体を利用することで、これらの表面マーカーを発現している細胞を簡便に分離することができる。換言すれば、本発明の細胞集団は、上記周細胞マーカー及びEphA7の発現により純化され、富化された細胞集団である。抗体を利用した分離方法としては、例えば、細胞分離ビーズ、磁気細胞分離、蛍光細胞分離等による方法等を挙げることができる。
本発明の細胞集団は、臓器再生の基本となる毛細血管を構築する一方で、幹細胞として組織実質細胞(間葉系細胞及び神経系細胞)を再生することができる、臓器再生にとって合理的な幹細胞である。それゆえ、本発明の細胞集団は組織又は器官再生用の医療材料として利用することができる。なお、本発明の「医療材料」には、国や地域の法令・規格上、医薬品(医薬組成物)、医療機器、医療製品に分類されるものの全てを含む。
虚血部位の再生については、例えば、床ずれ・皮膚潰瘍、手術瘢痕、難治性消化性潰瘍を含む創傷、潰瘍性大腸炎、クローン病などの慢性炎症性腸疾患を含む炎症性疾患、重症四肢虚血、心筋梗塞・狭心症・心不全を含む虚血性心疾患、脳梗塞、糖尿病性ニューロパチー、重症虚血を伴うがん等、血管や虚血部位の再生を必要とするあらゆる疾患が含まれる。とくに、通常の医薬では治療が困難な、重症慢性下肢虚血(閉塞性動脈硬化症、バージャー病)、治療不応性虚血性心疾患、重症虚血を伴うがん、網膜症を含めた糖尿病性血管障害等が対象疾患として好ましい。
温度感受性SV40T抗原発現マウスから樹立した、末梢組織毛細血管由来の10系統のクローン周細胞株(WO2013/118786参照)から、分化能の程度が異なる3種の細胞株(CapSCs #7(高分化), CapSCs #9(低分化)、CapSCs #3(中間),)を選び、RNA発現アレイ(東レ 3D-Gene array)を用いて発現遺伝子の網羅的アレイ比較解析を行った。
1.特異的細胞マーカーによるマウスCapSCsの分離(図1)
マウス皮下脂肪組織(0.5g wet)からコラゲナーゼI/IIとAccumax処理により細胞を単離し、10%FBSを含むDMEM培地を用いて2〜3日培養した。さらに培地を交換して3〜4日培養し、付着細胞(脂肪間質細胞:ASC)をトリプシン処理により回収した。
(1)脂肪細胞分化能及び神経細胞分化能
前項で調製したNG2陽性EphA7陽性細胞(マウスCapSCs)の脂肪細胞及び神経細胞への分化能を評価した。
脂肪細胞への分化能は、Stem Cell Kits, #SC010(R&D systems)を使用し、adipogenic supplement(hydrocortisone, isobutylmethilxanthine, indomethacin)を添加したαMEM培地(10%FBS及び抗生剤含有)で、細胞を7-14日間培養し、抗FABP-4抗体による免疫染色、Oil Red O染色、BODIPY(boron-dipyrromethene)による蛍光染色を行って確認した。
神経細胞への分化は、Human/Mouse/Rat Neural Lineage Functional Identification Kit, #SC028(R&D systems)を使用し、maintenance supplement(Recombinant human FGF-basic, recombinant human EGF)を添加したDMEM培地(N2-MAX Media Supplementと抗生剤含有)で細胞を2日間培養した後、differentiation supplement(IGF-1, fetal bovine serum )を含む前記DMEM培地で7-10日間培養し、Nestin、β3Tubulin、marker O4(OligoM4)、S100抗体による免疫染色を行って確認した。対照として、crude PCs(NG2陽性EphA7陰性細胞)及びcrude ASCsについても同様に培養し染色を行った。
NG2陽性EphA7陽性細胞(マウスCapSCs)の骨芽細胞への分化能を評価した。Stem Cell Kits, #SC010(R&D systems)を使用し、osteogenic supplement(ascorbate-phosphate, β-glycerolphosphate, recombinant human BMP-2)を添加したαMEM培地(10%FBS及び抗生剤含有)で、細胞を14-21日間培養し、抗オステオポンチン抗体による免疫染色、alizarin-red染色を行った。対照として、周細胞を同様に培養して染色を行った。その結果、マウスCapSCs細胞は、対照に比較して、より効率的にオステオポンチン陽性骨芽細胞へ分化することが確認された(図2B)。
NG2陽性EphA7陽性細胞(マウスCapSCs)を、上記(1)と同様に、Human/Mouse/Rat Neural Lineage Functional Identification Kit, #SC028(R&D systems)を使用して培養し、Nestin(図2C)、GFAP及びヒツジIgG AF594(図2D)、β3Tubulin及びマウスIgG AF594(図2D)に対する抗体を用いた免疫染色を行った。対照として、周細胞を同様に培養して免疫染色した。その結果、マウスCapSCs細胞は、対照に比較して、より効率的に神経細胞へ分化することが確認された。
NG2陽性EphA7陽性細胞(マウスCapSCs)をFGF(10ng/ml)を加えたDMEM培地で7代継代培養した。マウスCapSCs細胞は、対照に比べて、より効率的に増殖してスフェアを形成することが確認された(図3)。
マウスCapSCsの遺伝子発現プロファイルを定量的RT−PCRで解析した。対照として、crude PCs(NG2陽性EphA7陰性細胞)及びcrude ASCsについても同様に遺伝子発現プロファイルを解析した。その結果、既知のPCマーカー(NG2、PDGFRβ、CD146)の発現は、マウスCapSCs と対照との間で差異はなく、これらのマーカーでは分別できないことが確認された(図4A上)。
1.特異的細胞マーカーによるヒトCapSCsの分離(図5)
ヒト神経芽腫(1 month-old, female)由来の皮下脂肪組織(0.3g wet)をコラゲナーゼI/IIとAccumaxで処理して細胞を単離し、10%FBSを含むDMEM培地を用いて2代継代培養し、付着細胞(脂肪間質細胞:hAPCs)をトリプシン処理により回収した。
(1)脂肪細胞分化能
前項で調製したNG2陽性EphA7陽性細胞(ヒトCapSCs)の脂肪細胞への分化能を評価した。Human/Mouse/Rat Neural Lineage Functional Identification Kit, #SC028(R&D systems)を使用し、adipogenic supplement(hydrocortisone, isobutylmethylxanthine, indomethacin)を添加したαMEM培地(10%FBS及び抗生剤含有)で細胞を7-21日間培養し、抗FABP4抗体による免疫染色を行った。対照として、crude hPCsについても同様に培養し、免疫染色した。NG2陽性EphA7陽性細胞は、対照に比較して、脂肪細胞により効率的に分化することが確認された(図6A)。
NG2陽性EphA7陽性細胞(ヒトCapSCs)の神経細胞分化能を評価した。Human/Mouse/Rat Neural Lineage Functional Identification Kit, #SC028(R&D systems)を使用し、maintenance supplement(Recombinant human FGF-basic, recombinant human EGF)を添加したDMEM培地(N2-MAX Media Supplementと抗生剤含有)で2日間培養した後、differentiation supplement(IGF-1, fetal bovine serum )を含む前記DMEM培地で7-10日間培養し、Nestin、β3Tubulin、marker O4(OligoM4)、S100抗体による免疫染色を行った。対照として、NG2陽性EphA7陰性細胞を同様に培養して免疫染色した。その結果、ヒトCapSCs細胞は神経細胞に分化するが、対照であるNG2陽性EphA7陰性は神経細胞に分化しないことが確認された(図6B)。
それぞれ5ng/ml, 10ng/ml, 50ng/mlのVEGFを含む3次元ゲル培養下で、NG2陽性EphA7陽性細胞(ヒトCapSCs)を培養したときの顕微鏡観察像を示す(図7上)。対照として、crude hPCsを5ng/mlのVEGFを含む3次元ゲル培養したときの顕微鏡観察像と同様に培養したヒトCapSCsの顕微鏡観察像を比較して示す(図7下)。この結果から、ヒトCapSCsは優れた血管形成能を有することが確認できた。
NG2陽性EphA7陽性細胞(ヒトCapSCs)をFGF(10ng/ml)を加えたDMEM培地で30代まで継代培養した。ヒトCapSCs細胞は効率よく増殖し、単一細胞からスフェアを形成した(図8)。
ヒトCapSCsの表面マーカー発現プロファイルを2 color フローサイトメトリーで解析した。解析した。その結果、既知のPCマーカー(NG2、CD146)、MSCマーカー(CD44、CD90、CD105)の発現は確認されたが、HSCマーカー(CD45、CD34)の発現は認められなかった(図9左)。
12週齢、オスのBalb/c nude mouseを用いて左大腿動静脈を結紮・摘出して下肢虚血モデルを作成し、3日後に各群(n=8)1 x104個のマウスCapSCsを5カ所に分けて虚血肢に局注した。 手術後、経時的(1〜28日)にレーザードップラーによる下腿の血流評価(治療直後の虚血肢/健側肢比の改善割合)を実施した。また、NG2陽性EphA7陰性細胞、及び細胞浮遊用の生理食塩水(対照)を投与した群についても同様の評価を行った。
8-10週齢、オスのSCIDマウスの腓腹筋にcardiotoxin(CTX、Sigma-Aldrich; 100 μl of 0.25 mg/ml in saline per mouse)を局注し、骨格筋を障害させた(各群 n=7)。4時間後に1x105個のDsRed発現マウスCapSCsを200μlの生理食塩水に懸濁して障害腓腹筋に局注した。対照として、NG2陽性EphA7陰性周細胞(PCs)を同様に障害腓腹筋に局注した。障害から2-3週間後に尾静脈からRhodamine-Lectinを注入し、パラホルムアルデヒトで還流固定後に腓腹筋を摘出した。
網膜症モデルとして酸素誘発網膜血管新生(Oxygen-induced retinopathy: OIR)モデルを用いた。日齢7日目のC57BL/6J マウスに75%酸素を5日間与えることにより、日齢12日目に広範な網膜無還流領域が形成される。その後75%酸素から通常の酸素濃度下の環境に戻して5日間飼育することにより日齢17日目までに病的網膜新生血管が発生する。本研究では日齢12日目に同一個体のOIRマウスの片眼のみに1x104個のマウスCapSCsを投与し(投与眼)、反対側は、細胞非投与=対照眼とした。日齢17日目に尾静脈からRhodamine-Lectinを注入し循環血管を可視化した後、眼球を摘出し、フォールマウント標本を作成して病的網膜血管形成に対するCapSCsの効果を検討した。
Claims (13)
- 単離された間葉系幹細胞様のヒト多能性(multipotent)幹細胞集団であって、毛細血管を含む組織に由来し、NG2陽性及びEphA7陽性について富化されている、単離された細胞集団。
- 1以上の神経幹細胞マーカーが陽性である、請求項1記載の単離された細胞集団。
- CD34及びCD45陰性である、請求項1又は2に記載の単離された細胞集団。
- 脳由来ではない、請求項1〜3のいずれか1項に記載の単離された細胞集団。
- スフェア形成能を有する、請求項1〜4のいずれか1項に記載の単離された細胞集団。
- 間葉系細胞に分化可能である、請求項1〜5のいずれか1項に記載の単離された細胞集団。
- 神経系細胞に分化可能である、請求項1〜5のいずれか1項に記載の単離された細胞集団。
- 血管形成能を有する、請求項1〜7のいずれか1項に記載の単離された細胞集団。
- 請求項1〜8のいずれか1項に記載の単離された細胞集団と、足場材料及び/又は医薬的に許容しうる担体とを含む、組織又は器官再生用の医療材料。
- 組織又は器官が間葉系及び神経系の組織又は器官から選ばれる、請求項9記載の医療材料。
- 毛細血管を含む組織に由来する付着性細胞からEphA7陽性であることを指標として細胞群を分離することを特徴とする、多能性(multipotent)幹細胞集団の製造方法。
- 毛細血管を含む組織の細胞から付着性の細胞群を分離する工程、前記付着細胞群から周細胞マーカー陽性細胞群を分離する工程、及び、前記周細胞マーカー陽性細胞群からEphA7陽性であることを指標として細胞群を分離する工程を含む、請求項11記載の方法。
- 毛細血管を含む組織が皮下脂肪組織、骨格筋組織、心臓組織、腎臓組織、及び内臓脂肪組織から選ばれるいずれかである、請求項11又は12記載の方法。
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