JP2022033871A - 網膜色素上皮細胞の調製法 - Google Patents
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Abstract
Description
本発明は、そのいくつかの態様において、多能性幹細胞から網膜色素上皮細胞を調製する方法に関する。
網膜色素上皮(RPE)とは、神経網膜と脈絡毛細管板との間に位置する色素細胞の単層である。RPE細胞は、網膜およびその光受容体の維持および機能において決定的な役割を演じる。そのような役割は、血液網膜関門の形成、迷光の吸収、神経網膜への栄養素の供給、視色素の再生ならびに落屑した光受容体外節の取り込みおよび再循環を含む。
(a)分化誘導剤を含む培地中、接着性表面上で未分化ヒト多能性幹細胞の細胞集団を培養して、分化細胞を得る工程であって、該細胞集団の細胞の少なくとも50%がOct4+TRA-1-60+である、工程;および
(b)TGFβスーパーファミリーの1つまたは複数のメンバーを含む培地中、接着性表面上で分化細胞を培養して、RPE細胞を得る工程
を含む、方法が提供される。
(a)本明細書に記載される方法にしたがってRPE細胞を産生する工程;および
(b)収集後、治療有効量のRPE細胞を対象の眼の中に移植し、それによって疾患を処置する工程
を含む、方法が提供される。
[本発明1001]
網膜色素上皮(RPE)細胞を産生する方法であって、
(a)分化誘導剤を含む培地中、接着性表面上で未分化ヒト多能性幹細胞の細胞集団を培養して、分化細胞を得る工程であって、該細胞集団の細胞の少なくとも50%がOct4+TRA-1-60+である、工程;および
(b)TGFβスーパーファミリーの1つまたは複数のメンバーを含む培地中、該接着性表面上で該分化細胞を培養して、RPE細胞を得る工程
を含む、方法。
[本発明1002]
工程(a)の前に未分化ヒト多能性幹細胞の集団を分化誘導剤の非存在下、接着性表面上で増殖させる工程をさらに含む、本発明1001の方法。
[本発明1003]
増殖させる工程が分化誘導剤の非存在下で行われる、本発明1002の方法。
[本発明1004]
工程(b)の後に接着性表面からRPE細胞を単離する工程をさらに含む、本発明1001の方法。
[本発明1005]
単離後のRPE細胞をさらなる接着性表面上で培養して、増殖したRPE細胞集団を産生する工程をさらに含む、本発明1004の方法。
[本発明1006]
RPE細胞を収集する工程をさらに含む、本発明1005の方法。
[本発明1007]
単離する工程が酵素的に行われる、本発明1004の方法。
[本発明1008]
工程(a)の前にヒト多能性幹細胞をヒトフィーダー細胞上で増殖させる工程をさらに含む、本発明1001の方法。
[本発明1009]
接着性表面上で増殖させる工程の前にヒト多能性幹細胞をヒトフィーダー細胞上で増殖させる工程をさらに含む、本発明1002の方法。
[本発明1010]
前記増殖したRPE細胞集団の細胞の90%超がCRALBP+PMEL17+である、本発明1005の方法。
[本発明1011]
接着性表面が、ラミニン、フィブロネクチン、ビトロネクチン、コラーゲンIおよびコラーゲンIVからなる群から選択される、本発明1001~1010のいずれかの方法。
[本発明1012]
接着性表面がラミニンまたはビトロネクチンである、本発明1011の方法。
[本発明1013]
ラミニンがラミニン521である、本発明1012の方法。
[本発明1014]
さらなる接着性表面が、ゼラチン、ポリ-d-リジン、ラミニン、コラーゲンIおよびコラーゲンIVからなる群から選択される、本発明1005の方法。
[本発明1015]
さらなる接着性表面がゼラチンまたはポリ-d-リジンである、本発明1014の方法。
[本発明1016]
少なくとも3週間行われる、本発明1001の方法。
[本発明1017]
収集後のRPE細胞を凍結保存する工程をさらに含む、本発明1006の方法。
[本発明1018]
凍結保存する工程が、90%HS/10%DMSO、CryoStor 2%、CryoStor 5%およびCryoStor 10%、ならびにStem Cell Bankerからなる群から選択される培地中で行われる、本発明1017の方法。
[本発明1019]
ヒトフィーダー細胞がヒト帯線維芽細胞を含む、本発明1009の方法。
[本発明1020]
ヒト多能性幹細胞がヒト胚性幹細胞を含む、本発明1001~1019のいずれかの方法。
[本発明1021]
分化誘導剤がニコチンアミドを含む、本発明1001~1019のいずれかの方法。
[本発明1022]
工程(a)の培地がアクチビンAを欠いている、本発明1021の方法。
[本発明1023]
TGFβスーパーファミリーのメンバーが、TGFβ1、TGFβ3およびアクチビンAからなる群から選択される、本発明1001~1019のいずれかの方法。
[本発明1024]
工程(b)の培地がニコチンアミドおよびアクチビンAを含む、本発明1001~1019のいずれかの方法。
[本発明1025]
工程(b)の後に、ニコチンアミドを含みかつアクチビンAを欠く培地中でRPE細胞を培養する工程をさらに含む、本発明1024の方法。
[本発明1026]
工程(a)が少なくとも5日間行われる、本発明1001~1025のいずれかの方法。
[本発明1027]
工程(b)が少なくとも1週間行われる、本発明1001~1026のいずれかの方法。
[本発明1028]
網膜疾患の処置を、それを必要とする対象において行う方法であって、
(a)本発明1001~1027のいずれかの方法にしたがってRPE細胞を産生する工程;および
(b)収集後、治療有効量の該RPE細胞を該対象の眼の中に移植し、それによって該疾患を処置する工程
を含む、方法。
[本発明1029]
分化したRPE細胞の移植が眼の網膜下腔で行われる、本発明1028の方法。
[本発明1030]
RPE細胞が、懸濁液の状態で、またはマトリックス上もしくは基材上に固定化された細胞の単層として、移植される、本発明1028の方法。
[本発明1031]
網膜疾患または網膜障害が、網膜色素変性症、レーバー先天性黒内障、遺伝性または後天性の黄斑変性症、加齢黄斑変性症(AMD)、ベスト病、網膜剥離、脳回転状萎縮症、脈絡膜欠如、パターンジストロフィー、RPEジストロフィー、シュタルガルト病、光傷害、レーザー傷害、炎症性傷害、感染性傷害、放射線傷害、新生血管傷害または外傷性傷害のいずれか1つによって生じた損傷に起因するRPE損傷および網膜損傷の少なくとも1つから選択される、本発明1028の方法。
[本発明1032]
本発明1001~1027のいずれかの方法にしたがって産生されたRPE細胞の集団。
[本発明1033]
網膜疾患または網膜障害の処置を、それを必要とする対象において行う方法であって、治療有効量の本発明1032のRPE細胞を該対象に投与し、それによって該網膜疾患または網膜障害を処置する工程を含む、方法。
本発明は、そのいくつかの態様において、多能性幹細胞から網膜色素上皮細胞を調製する方法に関する。
(a)分化誘導剤を含む培地中、接着性表面上で未分化ヒト多能性幹細胞の細胞集団を培養して、分化細胞を得る工程であって、該細胞集団の細胞の少なくとも50%がOct4+TRA-1-60+である、工程;および
(b)TGFβスーパーファミリーの1つまたは複数のメンバーを含む培地中、接着性表面上で分化細胞を培養して、RPE細胞を得る工程
を含む、方法が提供される。
(a)第一の分化誘導剤(たとえばニコチンアミド)を含む培地中での、ESCの培養;および
(b)TGFβスーパーファミリーのメンバー(たとえばアクチビンA)および第一の分化誘導剤(たとえばニコチンアミド)を含む培地中での、工程(a)から得られた細胞の培養。
-血清または血清代替品を含有する培地、たとえば非限定的に、ノックアウト血清代替品(knock out serum replacement)(KOSR)、Nutridoma-CS、TCH(商標)、N2、N2誘導体もしくはB27、または組み合わせ;
-細胞外マトリックス(ECM)成分、たとえば非限定的に、フィブロネクチン、ラミニン、コラーゲンおよびゼラチン。この場合、ECMは、TGFβスーパーファミリーの増殖因子の1つまたは複数のメンバーを担持するために使用され得る;
-抗菌剤、たとえば非限定的に、ペニシリンおよびストレプトマイシン;ならびに
-非必須アミノ酸(NEAA)、培養中のSCの生存を促進する際に役割を果たすことが知られているニューロトロフィン、たとえば非限定的にBDNF、NT3、NT4。
糖質コルチコイド、細胞増殖抑制剤(たとえばアルキル化剤または代謝拮抗物質)、抗体(ポリクローナルまたはモノクローナル)、イムノフィリンに作用する薬剤(たとえばシクロスポリン、タクロリムスまたはシロリムス)。さらなる薬物は、インターフェロン、オピオイド、TNF結合タンパク質、ミコフェノレートおよび小さな生物学的薬剤を含む。
スフェロイド体の産生なしでのRPE細胞の誘導(ラミニン)
材料および方法
HAD-C 102 hESCを、センターウェルプレート中、ヒト臍帯フィーダー(hUCF;コード008)上に手作業で増殖させたのち、コラゲナーゼを使用して、フィーダーフリーのラミニン521(5μg/mlを37℃で2時間;Biolamina、Lam-521)でコートされたフラスコ上で継代し、bFGFおよびTGFβを含有するHSA培地を含有するNutristem(商標)(Biological Industries、05-110-1A)で増殖させて、RPE細胞を製造した。ラミニン521上でのhESC増殖の6日目、分化を開始し、培地を、10mMニコチンアミド(Sigma、N-5535)を含有するNutristem Minus(bFGFおよびTGFβなし;Biological Industries、06-5102-01-1A)に換えて1週間(9日目に培地を交換した)、その後、10mMニコチンアミドおよび140ng/mlアクチビンA(Peprotech、G-120-14E)を含有するNutristem Minusに換えて2週間、10mMニコチンアミドを含有するNutristem Minusに換えて約1週間で、色素細胞のパッチが認められた。次いで、TrypLE select(Invitrogen、12563-011)を使用して細胞を収集し、40μmストレーナに通してろ過し、遠心分離した。生細胞をカウントし、3つの組み換えヒトゼラチン(rhゼラチン、Fibrogen、RhG100-001)でコートされたウェル(6ウェルプレートの)中、20%ヒト血清(Akron、AK9905)を含有するDMEM(HyClone、SH30081)の存在下で3日間、Nutristem Minusの存在下で11日間、播種した。次いで、継代0(P0、10日目)終了時に生細胞を収集し、20%ヒト血清培地を含むrhゼラチンなしのT75フラスコ中、20%ヒト血清を含有するDMEMの存在下で最大3日間、Nutristem Minusの存在下で最大11日間、播種した。この工程をもう1回繰り返し、継代2終了時の細胞を収集し、凍結保存した。プロセスの概要は図1に見ることができる。
分化プロセスの終了時、色素細胞のパッチが産生された(図2A、黄色の矢印)。TrypLE Selectによる全細胞の収集、40μmストレーナに通すろ過および1つの増殖工程ののち、多角形形態を有する細胞が細胞培養プレート全体を覆っていた(図2B、P0終了時)。
スフェロイド体の産生なしでのRPE細胞の誘導(ビトロネクチン)
材料および方法
HAD-C 102 hESCを、センターウェルプレート中、ヒト臍帯フィーダー(hUCF;コード008)上に手作業で増殖させたのち、コラゲナーゼを使用して、フィーダーフリーのビトロネクチン(6ウェルプレートの1ウェルあたり5~10μg/200ml PBS)でコートされたフラスコ上で継代し、bFGFおよびTGFβを含有するHSA培地を含有するNutristem(商標)(Biological Industries、05-110-1A)で増殖させて、RPE細胞を製造した。いくつかの実験においては、未分化hESCをコラゲナーゼで継代し、さらに増殖させるために、同じ培地中、ビトロネクチン上で再びプレーティングした。ビトロネクチン上でのhESC増殖の6日目または7日目、分化を開始し、培地を、10mMニコチンアミド(Sigma、N-5535)を含有するNutristem Minus(bFGFおよびTGFβなし;Biological Industries、06-5102-01-1A)に換えて2週間(9日目に培地を交換した)、その後、10mMニコチンアミドおよび140ng/mlアクチビンA(Peprotech、G-120-14E)を含有するNutristem Minusに換えて2週間、10mMニコチンアミドを含有するNutristem Minusに換えて約1週間で、色素細胞のパッチが認められた。Triple Selectを使用して培養物全体を収集した。生細胞をカウントし、3つの組み換えヒトゼラチン(rhゼラチン、Fibrogen、RhG100-001)でコートされたウェル(6ウェルプレートの)中、20%ヒト血清(Akron、AK9905)を含有するDMEM(HyClone、SH30081)の存在下で最大3日間、Nutristem Minusの存在下で最大11日間、播種した。次いで、継代0(P0、10日目)終了時に生細胞を収集し、20%ヒト血清培地を含むrhゼラチンなしのT75フラスコ中、20%ヒト血清を含有するDMEMの存在下で3日間、Nutristem Minusの存在下で11日間、播種した。この工程をもう1回繰り返し、継代2終了時の細胞を収集し、凍結保存した。実験を4回(低酸素濃度下で2回、通常酸素濃度下で2回)繰り返した。
分化プロセスの終了時、色素細胞のパッチが産生された。培養物全体を酵素消化によって収集し、さらなる培養および増殖のために、RPE細胞増殖を促進する条件下で、再びプレーティングした。多角形形態を有する細胞が細胞培養プレート全体を覆っていた。細胞の大部分が濃い色素を宿している(図5A~C)。免疫染色は、細胞の大部分がRPE細胞のマーカー、たとえばベストロフィン、CRALBP、MITF、PEDFおよびZO-1を発現することを示した(図6A~E)。
Claims (27)
- 網膜色素上皮(RPE)細胞を産生する方法であって、
(a)分化誘導剤としてニコチンアミドを含みかつアクチビンAを欠いている培地中、ラミニン、フィブロネクチン、ビトロネクチン、コラーゲンIおよびコラーゲンIVからなる群から選択される接着性表面上で、かつ、フィーダー細胞フリー条件下で、未分化ヒト多能性幹細胞の細胞集団を培養して、分化細胞を得る工程であって、該細胞集団の細胞の少なくとも50%がOct4+TRA-1-60+である、工程;および
(b)ニコチンアミド並びに、TGFβ1、TGFβ3およびアクチビンAからなる群から選択されるTGFβスーパーファミリーの1つまたは複数のメンバーを含む培地中、該接着性表面上で該分化細胞を培養して、RPE細胞を得る工程
を含む、方法。 - 工程(a)の前に未分化ヒト多能性幹細胞の集団を分化誘導剤の非存在下、接着性表面上で増殖させる工程をさらに含む、請求項1記載の方法。
- 増殖させる工程が分化誘導剤の非存在下で行われる、請求項2記載の方法。
- 工程(b)の後に接着性表面からRPE細胞を単離する工程をさらに含む、請求項1記載の方法。
- 単離後のRPE細胞をさらなる接着性表面上で培養して、増殖したRPE細胞集団を産生する工程をさらに含む、請求項4記載の方法。
- RPE細胞を収集する工程をさらに含む、請求項5記載の方法。
- 単離する工程が酵素的に行われる、請求項4記載の方法。
- 工程(a)の前にヒト多能性幹細胞をヒトフィーダー細胞上で増殖させる工程をさらに含む、請求項1記載の方法。
- 接着性表面上で増殖させる工程の前にヒト多能性幹細胞をヒトフィーダー細胞上で増殖させる工程をさらに含む、請求項2記載の方法。
- 前記増殖したRPE細胞集団の細胞の90%超がCRALBP+PMEL17+である、請求項5記載の方法。
- 接着性表面がラミニンまたはビトロネクチンである、請求項1記載の方法。
- ラミニンがラミニン521である、請求項11記載の方法。
- さらなる接着性表面が、ゼラチン、ポリ-d-リジン、ラミニン、コラーゲンIおよびコラーゲンIVからなる群から選択される、請求項5記載の方法。
- さらなる接着性表面がゼラチンまたはポリ-d-リジンである、請求項13記載の方法。
- 前記増殖させる工程が少なくとも3週間行われる、請求項2記載の方法。
- 収集後のRPE細胞を凍結保存する工程をさらに含む、請求項6記載の方法。
- 凍結保存する工程が、90%HS/10%DMSO、CryoStor 2%、CryoStor 5%およびCryoStor 10%、ならびにStem Cell Bankerからなる群から選択される培地中で行われる、請求項16記載の方法。
- ヒトフィーダー細胞がヒト臍帯線維芽細胞を含む、請求項9記載の方法。
- ヒト多能性幹細胞がヒト胚性幹細胞を含む、請求項1~18のいずれか一項記載の方法。
- 工程(b)の後に、ニコチンアミドを含みかつアクチビンAを欠く培地中でRPE細胞を培養する工程をさらに含む、請求項1記載の方法。
- 工程(a)が少なくとも5日間行われる、請求項1~20のいずれか一項記載の方法。
- 工程(b)が少なくとも1週間行われる、請求項1~21のいずれか一項記載の方法。
- 請求項1~22のいずれか一項記載の方法にしたがって調製されたRPE細胞の、網膜疾患の処置のための薬剤の製造における使用。
- RPE細胞が、懸濁液の状態、またはマトリックス上もしくは基材上に固定化された細胞の単層としての細胞である、請求項23記載の使用。
- 網膜疾患または網膜障害が、網膜色素変性症、レーバー先天性黒内障、遺伝性または後天性の黄斑変性症、加齢黄斑変性症(AMD)、ベスト病、網膜剥離、脳回転状萎縮症、脈絡膜欠如、パターンジストロフィー、RPEジストロフィー、シュタルガルト病、光傷害、レーザー傷害、炎症性傷害、感染性傷害、放射線傷害、新生血管傷害または外傷性傷害のいずれか1つによって生じた損傷に起因するRPE損傷および網膜損傷の少なくとも1つから選択される、請求項23記載の使用。
- 請求項1~22のいずれか一項記載の方法にしたがって産生されたRPE細胞の集団。
- 請求項26記載のRPE細胞の、網膜疾患または網膜障害の処置のための薬剤の製造における使用。
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