JP6814210B2 - 腎臓の製造方法 - Google Patents
腎臓の製造方法 Download PDFInfo
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- JP6814210B2 JP6814210B2 JP2018525008A JP2018525008A JP6814210B2 JP 6814210 B2 JP6814210 B2 JP 6814210B2 JP 2018525008 A JP2018525008 A JP 2018525008A JP 2018525008 A JP2018525008 A JP 2018525008A JP 6814210 B2 JP6814210 B2 JP 6814210B2
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Description
(1)非ヒト動物の後腎の後腎間葉を組織特異的に除去する工程と、前記後腎にヒト腎臓前駆細胞を移植する工程と、前記後腎の発生を進行させる工程であって、移植した前記ヒト腎臓前駆細胞が分化成熟し、腎臓の一部を形成する工程と、を備える、腎臓の製造方法。
(2)前記後腎の尿管芽を組織特異的に除去する工程と、前記後腎にヒト腎臓前駆細胞を移植する工程と、を更に備える、(1)に記載の腎臓の製造方法。
(3)前記非ヒト動物がブタである、(1)又は(2)に記載の腎臓の製造方法。
(4)前記非ヒト動物がマウスである、(1)又は(2)に記載の腎臓の製造方法。
(5)(1)〜(4)のいずれかに記載の製造方法により製造された、腎臓。
(6)非ヒト動物由来の細胞と、ヒト細胞とを含み、前記ヒト細胞の割合が70質量%以上である、腎臓。
(7)(5)又は(6)に記載の腎臓を有する非ヒト動物。
(8)(5)又は(6)に記載の腎臓と、尿管と、膀胱とを備える移植用臓器。
(9)前記尿管及び前記膀胱が前記非ヒト動物由来である、(8)に記載の移植用臓器。
(10)後腎の後腎間葉を組織特異的に除去することができる、遺伝子改変非ヒト動物。
(11)更に、前記後腎の尿管芽を組織特異的に除去することができる、(10)に記載の遺伝子改変非ヒト動物。
(12)遺伝子改変ブタである、(10)又は(11)に記載の遺伝子改変非ヒト動物。
(13)非ヒト動物の後腎と、前記後腎の後腎間葉を組織特異的に除去する薬剤と、ヒト腎臓前駆細胞とを備える、腎臓製造用キット。
(14)前記後腎の尿管芽を組織特異的に除去する薬剤を更に備える、(13)に記載の腎臓製造用キット。
(15)非ヒト動物の後腎と、前記後腎の後腎間葉を組織特異的に除去する薬剤と、ヒト腎臓前駆細胞とを備える、腎臓再生用医薬。
(16)前記後腎の尿管芽を組織特異的に除去する薬剤を更に備える、(15)に記載の腎臓再生用医薬。
図1は、胎生11日前後のマウスの胎仔を示す模式図である。腎臓は前腎、中腎、後腎の3段階を経て形成される。このうち、前腎及び中腎は後に退行変性する。哺乳類成体において機能する腎臓は後腎である。
本工程では、非ヒト動物の胚の後腎間葉を組織特異的に除去する。非ヒト動物としては、特に制限はなく、例えば、ブタであってもよい。ブタは臓器の大きさがヒト等への移植に適しており、遺伝子改変技術も確立されている。あるいは、非ヒト動物はマウスであってもよい。マウスは、遺伝子改変技術や様々な実験系が確立されているため利用しやすい。
続いて、本工程において、後腎にヒト腎臓前駆細胞を移植する。工程(i)及び工程(ii)は並行して実施してもよい。すなわち、ニッチを空ける時期と、ヒト腎臓前駆細胞を移植する時期は重なっていてもよい。例えば、除去薬剤の添加とヒト腎臓前駆細胞の移植を同時に実施してもよい。
続いて、本工程において、ヒト腎臓前駆細胞を移植した後腎の発生を進行させる。その結果、移植したヒト腎臓前駆細胞が分化成熟し、腎臓の一部を形成する。後腎の発生の進行は、胚から後腎を摘出せずにヒト腎臓前駆細胞を移植した場合には、胚を再び親動物の子宮に戻して成長させることや、全胚培養すること等により実施することができる。あるいは、後腎を摘出してヒト腎臓前駆細胞を移植した場合には、後腎の器官培養を継続することにより実施することができる。あるいは、後腎を患者の傍大動脈領域や体網に移植し、患者の体内でヒト腎臓前駆細胞を移植した場合には、そのまま後腎を成長させることにより実施することができる。
本工程において、非ヒト動物の後腎の尿管芽を組織特異的に除去する。尿管芽の組織特異的な除去は、尿管芽を組織特異的に除去することができる限り特に制限されず、例えば、遺伝子組換え技術を使用して行うことができる。
続いて、本工程において、上記の後腎にヒト腎臓前駆細胞を移植して、尿管芽の発生を進行させる。その結果、移植したヒト腎臓前駆細胞により尿管芽が再生される。工程(iv)及び工程(v)は並行して実施してもよい。すなわち、ニッチを空ける時期と、ヒト腎臓前駆細胞を移植する時期は重なっていてもよい。例えば、除去薬剤の添加とヒト腎臓前駆細胞の移植を同時に実施してもよい。
1実施形態において、本発明は、上述した製造方法により製造された、腎臓を提供する。本実施形態の腎臓は、ホストである非ヒト動物の腎発生ニッチ及び発生プログラムを利用して、所望のヒト腎臓前駆細胞から製造されている。したがって、患者に移植して機能させることができる。
1実施形態において、本発明は、上述したいずれかの腎臓と、尿管と、膀胱とを備える移植用臓器を提供する。
1実施形態において、本発明は、後腎の後腎間葉を組織特異的に除去することができる、遺伝子改変非ヒト動物を提供する。遺伝子改変非ヒト動物は、例えば、遺伝子改変マウスであってもよいし、遺伝子改変ブタであってもよい。また、遺伝子改変の手法は特に限定されず、ES細胞を用いた方法であってもよく、ゲノム編集を用いた方法であってもよい。
1実施形態において、本発明は、非ヒト動物の後腎と、前記胚の後腎間葉を組織特異的に除去する薬剤と、ヒト腎臓前駆細胞とを備える、腎臓製造用キットを提供する。
1実施形態において、本発明は、非ヒト動物の後腎と、前記後腎の後腎間葉を組織特異的に除去する薬剤と、ヒト腎臓前駆細胞とを備える、腎臓再生用医薬を提供する。
1実施形態において、本発明は、上述した腎臓、尿管及び膀胱を備える移植用臓器を患者の体内に移植する工程(a)と、移植から所定期間後に、上記移植用臓器の膀胱と、患者の尿管とを接続する工程(b)と、を備える、腎臓の移植方法を提供する。
(マウス胚における後腎間葉の組織特異的な除去)
Creリコンビナーゼ活性依存的にジフテリア毒素受容体(DTR)を発現するiDTRマウスと、Six2のプロモーターの下流にCreリコンビナーゼ遺伝子を導入したSix2−Creマウスとを交配した。なお、Six2は、後腎間葉で特異的に発現する転写因子である。
(腎臓前駆細胞の移植)
野生型マウス由来の腎臓前駆細胞を、定法により後腎から調製し、細胞解離試薬を用いて単一細胞に解離した。続いて、腎臓前駆細胞1×105個を、実験例1で調製した、後腎間葉が組織特異的に除去された後腎組織に移植した。その後、後腎組織の器官培養を継続した。
(腎臓組織の免疫染色1)
実験例2の後腎組織を、腎臓前駆細胞の移植から5日間培養後に4%パラホルムアルデヒド固定して組織切片標本を作製した。続いて、後腎間葉のマーカーであるSix2及び尿管芽のマーカーであるサイトケラチン8をそれぞれ免疫染色した。図3は免疫染色の結果を示す蛍光顕微鏡写真である。倍率は100倍である。図3において、Six2陽性細胞は移植した腎臓前駆細胞由来である。また、サイトケラチン8陽性細胞はホスト動物(実験例1のF1マウス)由来である。
(腎臓組織の免疫染色2)
緑色蛍光タンパク質(GFP)遺伝子を組み込んだトランスジェニックマウス(GFP tgマウス)由来の腎臓前駆細胞を、定法により後腎から調製し、細胞解離試薬を用いて単一細胞に解離した。続いて、腎臓前駆細胞1×105個を、実験例1と同様にして調製した、後腎間葉が組織特異的に除去された後腎組織に移植した。その後、後腎組織の器官培養を継続した。
(マウスの生体内での腎臓の再生)
野生型マウスの傍大動脈領域に、iDTRマウスとSix2−Creマウスとを交配して得られた、後腎組織で組織特異的にジフテリア毒素受容体を発現する胎仔の後腎及び総排出腔を移植した。
(マウス胚の後腎間葉及び尿管芽の双方の置換)
《尿管芽の組織特異的な除去》
本実験例では、Six2−Cre−ERtg/wtHoxB7−Cretg/wtの遺伝子型を有するiDTRマウスを用いた。まず、実験例1と同様にして、マウス胚から後腎を摘出して器官培養した。続いて、培地にジフテリア毒素を添加して尿管芽を組織特異的に除去した。
続いて、尿管芽を除去したマウス後腎に、実験例2と同様にして、野生型マウス由来の腎臓前駆細胞1×105個を移植した。その後、後腎の器官培養を5日間継続した。
続いて、器官培養の培地にタモキシフェン及びジフテリア毒素を添加した。その結果、後腎間葉特異的なマーカーであるSix2発現細胞において、Cre−ERが核内に移行して組換えを起こし、ジフテリア毒素受容体を発現した。その結果、後腎間葉の細胞がジフテリア毒素により死滅した。これにより、後腎間葉を組織特異的に除去した。
続いて、後腎間葉を除去したマウス後腎に、実験例2と同様にして、野生型マウス由来の腎臓前駆細胞1×105個を再度移植した。その後、後腎の器官培養を3〜5日間継続した。
続いて、器官培養した後腎を4%パラホルムアルデヒド固定して組織切片標本を作製した。続いて、尿管芽を染色する抗カルビンディン抗体で免疫染色して蛍光顕微鏡で観察した。
Claims (2)
- マウスの発生途中の後腎の後腎間葉を組織特異的に除去する工程(i)と、
前記後腎にヒト腎臓前駆細胞を移植する工程(ii)と、
前記後腎の発生を進行させる工程であって、移植した前記ヒト腎臓前駆細胞が分化成熟し、腎臓の一部を形成する工程(iii)と、
を備える、腎臓の製造方法。 - 前記工程(i)〜(iii)の前又は後に、
前記後腎の尿管芽を組織特異的に除去する工程(iv)と、
前記後腎にヒト腎臓前駆細胞を移植する工程(v)と、
を更に備える、請求項1に記載の腎臓の製造方法。
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