JPWO2019189640A1 - 幹細胞由来涙腺組織の作製方法 - Google Patents
幹細胞由来涙腺組織の作製方法 Download PDFInfo
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- JPWO2019189640A1 JPWO2019189640A1 JP2020511023A JP2020511023A JPWO2019189640A1 JP WO2019189640 A1 JPWO2019189640 A1 JP WO2019189640A1 JP 2020511023 A JP2020511023 A JP 2020511023A JP 2020511023 A JP2020511023 A JP 2020511023A JP WO2019189640 A1 JPWO2019189640 A1 JP WO2019189640A1
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Abstract
Description
〔1〕 多能性幹細胞から得られたSEAM細胞集団(self-formed ectodermal autonomous multi-zone細胞集団)から、SSEA4及びCD104共陽性の細胞を単離し、得られた細胞をEGF(Epidermal Growth Factor)及びROCK阻害剤を含む培地中で三次元培養することにより、涙腺の関連タンパクを発現する細胞集団を取得することを特徴とする、幹細胞由来涙腺組織の作製方法。
〔2〕 単離した細胞が、さらにCD200陰性である、前記〔1〕記載の作製方法。
〔3〕 輪部幹細胞をEGF及びROCK阻害剤を含む培地中で三次元培養することにより、涙腺の関連タンパクを発現する細胞集団を取得することを特徴とする、幹細胞由来涙腺組織の作製方法。
〔4〕 培地がさらにTGF-βを含む、前記〔1〕〜〔3〕いずれか記載の作製方法。
〔5〕 関連タンパクがAQP5、LYZ、CNN1、BARX2、SOX9、SOX10、RUNX1、TFCP2L1、LTF及びHTN1から選ばれる1種以上である、前記〔1〕〜〔4〕いずれか記載の作製方法。
〔6〕 前記〔1〕〜〔5〕いずれか記載の作製方法により得られた涙腺上皮細胞を培養する工程を含む、移植用涙腺オルガノイドの製造方法。
〔7〕 前記〔1〕〜〔5〕いずれか記載の作製方法により得られた涙腺上皮細胞を培養する工程を含む、涙腺に関連する疾患の薬剤スクリーニング方法。
〔8〕 前記〔6〕記載の方法により得られた涙腺上皮細胞オルガノイドから分泌される涙液。
〔9〕 前記〔8〕記載の涙液を含有する、医薬組成物。
〔10〕 前記〔1〕〜〔5〕いずれか記載の作製方法において形成された細胞集団の形状を指標として選択することを特徴とする、幹細胞由来涙腺組織へ分化誘導しやすい細胞の選択方法。
〔11〕 前記〔1〕〜〔6〕いずれか記載の方法により得られた涙腺オルガノイド。
〔12〕 前記〔10〕記載の方法により選択された細胞を培養して得られる、涙腺オルガノイド。
iPS細胞から眼周囲細胞の分化誘導には、self-formed ectodermal autonomous multi-zone(SEAM)法を用いた(Hayashi et al. Nature. 2016 Mar 17;531(7594):376-80., Hayashi et al., Nature Protocols, 2017, 12(4), 683-696, doi: 10.1038/nprot.2017.007)。
実施例1で得られたSEAMから、CD200-、SSEA4+、CD104+の眼表面幹細胞を回収した。
実施例1と同様にして、ヒトiPS細胞をStemFitで10日間培養したサンプル(iPSC)、SEAM誘導後8〜10週間で得られたSSEA4/CD104共陽性の画分をソートしたサンプル(Sort)、実施例2と同様にして、spheroidを形成させたサンプル(Spheroid)、オルガノイドを形成させたサンプル(Organoid)、それぞれをQIAzol Lysis Reagent(QIAGEN)を用いて回収し、qRT-PCRに供し、それぞれの段階での各種マーカー発現を評価した。結果を図3に示す。
培養した涙腺オルガノイドをそのまま、あるいは4% PFAで固定処理したものの切片を、5%NST (5% Normal Donkey Serum, 3% Tritonを含むTBS)でブロッキング後に、抗PAX6抗体(sc-53108)、抗HTN1抗体(sc-28110)、抗CAL抗体(ab46794)で1次抗体反応を行い、洗浄後、Alexa Fluor 488、あるいは Alexa Fluor 568で標識した2次抗体を1時間処理して染色した。さらに二次抗体反応の最後の10分間はHoechst 33342で核を染色した。結果を図4に示す。
実施例2と同様にして三次元培養する際に、EGF 10ng/mLを添加した効果、さらには、Y-27632 10μMや、涙腺誘導に関与すると報告のあるKGF 20ng/mL、FGF10 100ng/mL、BMP7 100ng/mLを加えて培養を行った。結果を図5Aに示す。
実施例2と同様にして三次元培養する際に、EGF 10ng/mL、Y-27632 10μMを加えた培地に、TGF-β3をそれぞれ4ng/mL、20ng/mL、100ng/mLで加えて培養を行った。結果を図6Aに示す。
実施例2で回収したCD200-、SSEA4+、CD104+の眼表面幹細胞を、100〜200cells/well(12well plate)で播種し、DMEM/F-12に2% B27 Supplementと20ng/mL KGFと10μM Y-27632を添加した培地で29日間培養した。コロニー形成の様子をタイムラプスで撮影したものを図7Aに示す。
ヒト輪部幹細胞を、iMatrix-511でコートしたディッシュに播種し、DMEM/F-12にB27 Supplementを加えたものに、KGF 20ng/mL、Y-27632 10μMを加えたもので16日間二次元培養した(Sheet)。また、ヒト輪部幹細胞20,000cellsを用いて、実施例2と同様にして形成したspheroidをマトリゲルGFRの中に包埋し、DMEM/F-12にB27 Supplementを加えたものに、EGF 10ng/mL、Y-27632 10μMを加えたもの(YE)、さらにTGF-β3 100ng/mLを加えたもの(YET3)でそれぞれ三次元培養を行った。結果を図8Aに示す。
F344/NJcl-rnu/rnu(ヌードラット)5週齢、メスの左眼窩外涙腺を摘出し、その場所へ、iPS細胞由来眼表面幹細胞50,000cellsからDMEM/F-12にB27 Supplement、EGF 10ng/mL、Y-27632 10μMを加えた培地で25日間培養を行って誘導した涙腺オルガノイドを移植した。34日後に安楽殺後、移植片(図9A、B、C)を摘出して解析を行った。
実施例1、2と同様にして、オルガノイドを形成させた。具体的には、ヒトES細胞(KhES-1)を用いて実施例1と同様にしてSEAM細胞集団を調製し、実施例2と同様にして眼表面幹細胞を取得後、得られた眼表面幹細胞を、100,000cells/spheroidで、DMEM/F-12にB27 Supplementを加えたものに、Y-27632 10μM、KGF 20ng/mLで添加した培地を用いて、PrimeSurface(登録商標)96Uプレートに播種して1日間培養してspheroidを形成した。次に、これをマトリゲルGFRの中に包埋し、さらにDMEM/F-12にB27 Supplementを加えたものに、Y-27632 10μM、EGF 10ng/mLを添加した培地を用いて2週間培養を行い、Day1、day3、Day14にて明視野の写真を撮影した。結果を図11に示す。
Claims (12)
- 多能性幹細胞から得られたSEAM細胞集団(self-formed ectodermal autonomous multi-zone細胞集団)から、SSEA4及びCD104共陽性の細胞を単離し、得られた細胞をEGF(Epidermal Growth Factor)及びROCK阻害剤を含む培地中で三次元培養することにより、涙腺の関連タンパクを発現する細胞集団を取得することを特徴とする、幹細胞由来涙腺組織の作製方法。
- 単離した細胞が、さらにCD200陰性である、請求項1記載の作製方法。
- 輪部幹細胞をEGF及びROCK阻害剤を含む培地中で三次元培養することにより、涙腺の関連タンパクを発現する細胞集団を取得することを特徴とする、幹細胞由来涙腺組織の作製方法。
- 培地がさらにTGF-βを含む、請求項1〜3いずれか記載の作製方法。
- 関連タンパクがAQP5、LYZ、CNN1、BARX2、SOX9、SOX10、RUNX1、TFCP2L1、LTF及びHTN1から選ばれる1種以上である、請求項1〜4いずれか記載の作製方法。
- 請求項1〜5いずれか記載の作製方法により得られた涙腺上皮細胞を培養する工程を含む、移植用涙腺オルガノイドの製造方法。
- 請求項1〜5いずれか記載の作製方法により得られた涙腺上皮細胞を培養する工程を含む、涙腺に関連する疾患の薬剤スクリーニング方法。
- 請求項6記載の方法により得られた涙腺上皮細胞オルガノイドから分泌される涙液。
- 請求項8記載の涙液を含有する、医薬組成物。
- 請求項1〜5いずれか記載の作製方法において形成された細胞集団の形状を指標として選択することを特徴とする、幹細胞由来涙腺組織へ分化誘導しやすい細胞の選択方法。
- 請求項1〜6いずれか記載の方法により得られた涙腺オルガノイド。
- 請求項10記載の方法により選択された細胞を培養して得られる、涙腺オルガノイド。
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