JP2022188204A - 網膜組織を含む細胞凝集体及びその製造方法 - Google Patents
網膜組織を含む細胞凝集体及びその製造方法 Download PDFInfo
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Abstract
Description
[1]神経網膜を含むコア部と、上記コア部の表面の少なくとも一部を連続的に又は断続的に覆う被覆部とを備える、スフェア状細胞凝集体であって、
(1)上記神経網膜において、少なくとも視細胞層を含む神経網膜層が形成されており、上記視細胞層は少なくとも視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞を含み、上記視細胞層に含まれる細胞が上記コア部の表面の接線方向に連続して存在しており、
(2)上記被覆部は、互いに接触する網膜色素上皮細胞を含み、
(3)上記細胞凝集体は、水晶体、硝子体、角膜及び血管を含まず、かつ
(4)上記網膜色素上皮細胞は、上記神経網膜層とともに連続する上皮構造を構成していない
ことを特徴とするスフェア状細胞凝集体。
[2](1)における視細胞層と、該視細胞層の少なくとも一部を覆う網膜色素上皮細胞との間に、さらに細胞外マトリクスが存在する、[1]のスフェア状細胞凝集体。
[3]細胞外マトリクスが、ヒアルロン酸、ラミニン、IV型コラーゲン、ヘパラン硫酸プロテオグリカン及びエンタクチンからなる群から選択される1又は複数の細胞外マトリクスである、[2]のスフェア状細胞凝集体。
[4]神経網膜を含むスフェア状の細胞凝集体(神経網膜の細胞凝集体)であって、
(I)上記神経網膜の細胞凝集体において、上記神経網膜は上記細胞凝集体の表面に存在しており、かつ
(II)上記神経網膜において少なくとも視細胞層を含む神経網膜層が形成されており、上記視細胞層には網膜前駆細胞、視細胞前駆細胞及び視細胞からなる群から選択される1以上の細胞が存在している、神経網膜の細胞凝集体を調製する工程と、
網膜色素上皮細胞を調製する工程と、
上記神経網膜の細胞凝集体及び上記網膜色素上皮細胞を接触させる工程とを含む、
[1]~[3]のいずれかのスフェア状細胞凝集体の製造方法。
[5]上記神経網膜の細胞凝集体における上記神経網膜におけるChx10陽性細胞の存在割合が20%以上である、[4]の製造方法。
[6]接触工程において、接着因子の存在下で接触させる、[4]又は[5]の製造方法。
[7]接着因子が細胞外マトリクスである、[6]の製造方法。
[8]細胞外マトリクスが、ヒアルロン酸、ラミニン、IV型コラーゲン、ヘパラン硫酸プロテオグリカン及びエンタクチンからなる群から選択される1又は複数の細胞外マトリクスである、[7]の製造方法。
[9]神経網膜の細胞凝集体及び網膜色素上皮細胞の少なくとも一方が、多能性幹細胞由来である、[4]~[7]のいずれかの製造方法。
[10]網膜色素上皮細胞を調製する工程において、網膜色素上皮細胞が、細胞シート又は細胞懸濁液として調製される、[4]~[8]のいずれかの製造方法。
[11]接触工程の後に、網膜色素上皮細胞が多角形又は敷石状の細胞形態を有するまでさらに培養する、[4]~[10]のいずれかの製造方法。
[12][1]~[3]のいずれかのスフェア状細胞凝集体を含有してなる、被験物質の毒性又は薬効評価用試薬。
[13][1]~[3]のいずれかのスフェア状細胞凝集体又は当該細胞凝集体の一部に被験物質を接触させ、被験物質が該スフェア状細胞凝集体又は該スフェア状細胞凝集体に含まれる細胞に及ぼす影響を検定することを含む、被験物質の毒性又は薬効評価方法。
[14][1]~[3]のいずれかのスフェア状細胞凝集体又は当該細胞凝集体の一部を含有してなる、網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の治療薬。
[15][1]~[3]のいずれかのスフェア状細胞凝集体又は当該細胞凝集体の一部の有効量を、移植を必要とする対象に移植することを含む、網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の治療方法。
[16]網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の治療における使用のための[1]~[3]のいずれかのスフェア状細胞凝集体又は当該細胞凝集体の一部。
[17][1]~[3]のいずれかのスフェア状細胞凝集体又は当該細胞凝集体の一部を有効成分として含有する、医薬組成物。
[18][1]~[3]のいずれかのスフェア状細胞凝集体から物理的に切り出されてなる、スフェア状細胞凝集体の一部。
[19]網膜色素上皮細胞及び神経網膜を含む細胞シートである、[18]のスフェア状細胞凝集体の一部。
[20][1]~[3]のいずれかのスフェア状細胞凝集体の一部を物理的に切り出す工程を含む、スフェア状細胞凝集体の一部の製造方法。
[21]上記スフェア状細胞凝集体の一部が、網膜色素上皮細胞及び神経網膜を含む細胞シートである、[20]の製造方法。
本発明の一実施形態のスフェア状細胞凝集体は、神経網膜を含むコア部と、上記コア部の表面の少なくとも一部を連続的に又は断続的に覆う被覆部とを備えるものであり、以下の(1)~(4)の特徴を有する:
(1)上記神経網膜において、少なくとも視細胞層を含む神経網膜層が形成されており、上記視細胞層は少なくとも視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞を含み、上記視細胞層に含まれる細胞が上記コア部の表面の接線方向に連続して存在しており、
(2)上記被覆部は、互いに接触する網膜色素上皮細胞を含み、
(3)上記細胞凝集体は、水晶体、硝子体、角膜及び血管を含まず、かつ
(4)上記網膜色素上皮細胞は、上記神経網膜層とともに連続する上皮構造を構成していない。
本発明の一実施形態のスフェア状細胞凝集体の製造方法は、神経網膜を含むスフェア状の細胞凝集体(神経網膜の細胞凝集体)を調製する工程と、網膜色素上皮細胞を調製する工程と、神経網膜の細胞凝集体及び網膜色素上皮細胞を接触させる工程とを含む。
以下、神経網膜の細胞凝集体およびその製造方法について説明する。なお、「神経網膜の細胞凝集体」とは、神経網膜を含むスフェア状の細胞凝集体を意味する。
(I)神経網膜を含むスフェア状の細胞凝集体(神経網膜の細胞凝集体)において、神経網膜は細胞凝集体の表面に存在している。例えば、神経網膜は神経網膜の細胞凝集体の表面に厚みを持って存在しており、外環境に対して連続的な面で境界を形成している。外環境に面している境界面は視細胞が多数を占めており、その内側に内層の細胞や網膜前駆細胞が存在している。これら網膜層を構成する細胞は互いに接着し連続的に存在している。その内部には空洞、若しくは整然と並んだ層構造が形成されていない空間があるため、その境目に明瞭な影が観察され、上皮構造を形成していることが確認される。神経網膜を含むスフェア状の細胞凝集体は、上述したコア部と被覆部とを備えるスフェア状細胞凝集体のコア部に相当する。
(A)多能性幹細胞を、フィーダー細胞非存在下で、1)TGFβファミリーシグナル伝達経路阻害物質及び/又はソニック・ヘッジホッグシグナル伝達経路作用物質、並びに2)未分化維持因子を含む培地で培養する工程、
(B)無血清培地中で浮遊培養することによって細胞凝集体を形成させる工程、
(C)工程(B)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質を含む培地中でさらに浮遊培養する工程。
網膜色素上皮細胞は、多能性幹細胞由来であることが好ましい。多能性幹細胞から網膜色素上皮細胞を調製する方法はWO2012/173207号、WO2015/053375号、WO2015/053376号、WO2015/068505号、WO2017/043605、Stem Cell Reports, 2(2), 205-218 (2014)及びCell Stem Cell, 10(6), 771-785 (2012)に開示されている方法が挙げられるが、特に限定されない。また、上述したWO2016/063985に記載の方法を改良することで網膜色素上皮細胞の凝集体を調製することも可能である。網膜色素上皮細胞は、細胞シート又は細胞懸濁液として調製されてもよい。
神経網膜の細胞凝集体と網膜色素上皮細胞とを接触させる方法は、神経網膜の細胞凝集体と網膜色素上皮細胞が接着可能な限り特に限定されないが、例えば、神経網膜の細胞凝集体を網膜色素上皮細胞の懸濁液と共に培養し、プレートの底に沈んだ神経網膜の細胞凝集体に網膜色素上皮細胞を接着させる方法、又は、網膜色素上皮細胞シートに神経網膜の細胞凝集体を接触させる方法、さらに、浸透圧差を利用する方法等が挙げられる。
本発明の一実施形態の被験物質の毒性又は薬効評価用試薬は、本発明の一実施形態のスフェア状細胞凝集体又は当該細胞凝集体の一部を含有してなり、本発明の一実施形態の被験物質の毒性又は薬効評価方法は、本発明の一実施形態のスフェア状細胞凝集体又は当該細胞凝集体の一部に被験物質を接触させ、被験物質が該スフェア状細胞凝集体又は該スフェア状細胞凝集体に含まれる細胞に及ぼす影響を検定することを含む。
本発明の一実施形態の治療薬は、スフェア状細胞凝集体又は当該細胞凝集体の一部を含有してなる、網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患(特に視細胞及び網膜色素上皮細胞が同時に障害又は損傷されている重症例)の治療薬である。本発明の一実施形態の治療方法は、スフェア状細胞凝集体又は当該細胞凝集体の一部の有効量を、移植を必要とする対象に移植することを含む、網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患(特に視細胞及び網膜色素上皮細胞が同時に障害又は損傷されている重症例)の治療方法である。本発明の一実施形態の医薬組成物は、本発明に係るスフェア状細胞凝集体又は当該細胞凝集体の一部を有効成分として含有する。本発明の一実施形態の医薬組成物は、網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患(特に視細胞及び網膜色素上皮細胞が同時に障害又は損傷されている重症例)の治療薬として有用である。
本発明の一実施形態のスフェア状細胞凝集体の一部は、本発明のスフェア状細胞凝集体の一部であり、例えば本発明のスフェア状細胞凝集体から物理的に切り出すことによって得ることができる。スフェア状細胞凝集体の一部の形状は問わず、スフェア状でなくてもよい。スフェア状細胞凝集体の一部は、神経網膜とその表面の少なくとも一部を連続的に又は断続的に覆う、互いに接触する網膜色素上皮細胞を含む被覆とを備える細胞凝集体であり、例えば網膜色素上皮細胞及び神経網膜を含む細胞シートであってよい。
Crx::Venusレポーター遺伝子を持つように遺伝子改変したヒトES細胞(Kh-ES1株、Cell Stem Cell,10(6),771-785,(2012)を、「Scientific Reports,4,3594(2014)」に記載の方法に準じてフィーダーフリー条件下で培養した。フィーダーフリー培地としてはStemFit培地(商品名:AK03N、味の素社製)、フィーダー細胞に代わる足場としてLaminin511-E8(商品名、ニッピ社製)を用いた。
実施例1のようにして作成されたRPE細胞とNRを、それぞれ浮遊培養開始後40日目以降Wntシグナル伝達経路作用物質及びFGFシグナル伝達阻害物質を含まない血清培地(NucT0培地とNucT2培地を1:3で混合した培地:以下、NucT1培地と記載する場合がある。なお、NucT2培地とは、Neurobasal Medium(Thermo fisher社製)に10%FBS、B27 supplement w/o V.A.(Thermo fisher社製)、L-Glutamine、Taurin、T3が添加された培地を意味する。)で59日目まで長期培養した。当該浮遊培養開始後60日目以降Wntシグナル伝達経路作用物質及びFGFシグナル伝達阻害物質を含まない血清培地(NucT2培地)で長期培養した。
RPE細胞:PBS(Thermo fisher社製)で洗浄後、Tryple select(Thermo fisher社製)で酵素処理を15~30分間行い、ピペッティングすることでシングルセル化し、40μmのセルストレーナー(Cell strainer)に通した後、Nuc培地に懸濁させた。
NR:NRをチューブに回収し、iMatrix511(ニッピ社製)又はMatrigel(BD社製)でコーティングを15~60分実施し、PBSで洗浄した。
実施例1のようにして作製されたRPE細胞とNRを、それぞれ浮遊培養開始後40日目以降Wntシグナル伝達経路作用物質及びFGFシグナル伝達阻害物質を含まない血清培地(NucT0培地とNucT2培地を1:3で混合したNucT1培地で培養。NucT2培地:Neurobasal Mediumに10%FBS、B27 supplement w/o V.A.、L-Glutamine、Taurin、T3が添加された培地)で59日目まで長期培養した。当該浮遊培養開始後60日目以降Wntシグナル伝達経路作用物質及びFGFシグナル伝達阻害物質を含まない血清培地(NucT2培地)で長期培養した。
実施例2で作製したNR-RPE細胞シートをNuc T2培地にてコンジュゲートさせてから5日間及び50日間培養した(図7A、A’、D、D’)。上記培養したNR-RPE細胞シートからNRとRPE細胞を同時に切り出した(図7B、B’、C、C’、E、E’、F、F’)。顕微鏡及び蛍光顕微鏡で観察したところ、外側から観察するとRPE細胞が接着していることが観察され、内側から観察すると、RPE細胞はほとんど観察されずCRX::Venusの蛍光がよく確認できた。よって、NR-RPE細胞シートにおいて極性をもって切り出すことができた。
Claims (17)
- 神経網膜を含むコア部と、前記コア部の表面の少なくとも一部を連続的に又は断続的に覆う被覆部とを備える、スフェア状細胞凝集体であって、
(1)前記神経網膜において、少なくとも視細胞層を含む神経網膜層が形成されており、前記視細胞層は少なくとも視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞を含み、前記視細胞層に含まれる細胞が前記コア部の表面の接線方向に連続して存在しており、
(2)前記被覆部は、互いに接触する網膜色素上皮細胞を含み、
(3)前記細胞凝集体は、水晶体、硝子体、角膜及び血管を含まず、
(4)前記網膜色素上皮細胞は、前記神経網膜層とともに連続する上皮構造を構成しておらずいない、かつ
(5)(1)における視細胞層と、該視細胞層の少なくとも一部を覆う網膜色素上皮細胞との間に、さらにハイドロゲルが存在する
ことを特徴とするスフェア状細胞凝集体。 - 網膜色素上皮細胞が多角形又は敷石状の細胞形態を示す、請求項1に記載のスフェア状細胞凝集体。
- 神経網膜における網膜前駆細胞及び視細胞前駆細胞の存在割合が20%以上である、請求項1又は2に記載のスフェア状細胞凝集体。
- 神経網膜における網膜前駆細胞及び視細胞前駆細胞の存在割合が30%以上である、請求項1~3のいずれか1項に記載のスフェア状細胞凝集体。
- 網膜前駆細胞及び視細胞前駆細胞がChx10陽性細胞又はCrx陽性細胞である、請求項1~4のいずれか1項に記載のスフェア状細胞凝集体。
- 被覆部がコア部の表面積の50%以上を覆っている、請求項1~5のいずれか1項に記載のスフェア状細胞凝集体。
- 被覆部がコア部の表面積の20%以上を連続的に覆っている、請求項1~6のいずれか1項に記載のスフェア状細胞凝集体。
- 請求項1~7のいずれか1項に記載のスフェア状細胞凝集体を含有してなる、被験物質の毒性又は薬効評価用試薬。
- 請求項1~7のいずれか1項に記載のスフェア状細胞凝集体に被験物質を接触させ、被験物質が該スフェア状細胞凝集体又は該スフェア状細胞凝集体に含まれる細胞に及ぼす影響を検定することを含む、被験物質の毒性又は薬効評価方法。
- 請求項1~7のいずれか1項に記載のスフェア状細胞凝集体を含有してなる、網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の治療薬。
- 網膜色素上皮細胞、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の治療における使用のための請求項1~7のいずれか1項に記載のスフェア状細胞凝集体。
- 請求項1~7のいずれか1項に記載のスフェア状細胞凝集体を有効成分として含有する、医薬組成物。
- 請求項1~7のいずれか1項に記載のスフェア状細胞凝集体から物理的に切り出されてなる、スフェア状細胞凝集体の一部。
- 網膜色素上皮細胞及び神経網膜を含む細胞シートである、請求項13に記載のスフェア状細胞凝集体の一部。
- 請求項1~7のいずれか1項に記載のスフェア状細胞凝集体の一部を物理的に切り出す工程を含む、スフェア状細胞凝集体の一部の製造方法。
- 前記スフェア状細胞凝集体の一部が、網膜色素上皮細胞及び神経網膜を含む細胞シートである、請求項15に記載の製造方法。
- 神経網膜を含むスフェア状の細胞凝集体(神経網膜の細胞凝集体)であって、
(I)前記神経網膜の細胞凝集体において、前記神経網膜は前記細胞凝集体の表面に存在しており、かつ
(II)前記神経網膜において少なくとも視細胞層を含む神経網膜層が形成されており、前記視細胞層には視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞が存在している、神経網膜の細胞凝集体を調製する工程と、
網膜色素上皮細胞を調製する工程と、
前記神経網膜の細胞凝集体及び前記網膜色素上皮細胞をハイドロゲルの存在下で接触させる工程とを含む、請求項1~7のいずれか1項に記載のスフェア状細胞凝集体の製造方法。
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