JP5687059B2 - 筋肉誘導前駆体組成物を利用した骨格筋の強化およびその処置 - Google Patents
筋肉誘導前駆体組成物を利用した骨格筋の強化およびその処置 Download PDFInfo
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Description
本発明の目的は、移植後長期間生存性を示す新規な筋肉誘導前駆細胞(MDC)およびMDC組成物を提供することである。本発明のMDCおよびMDCを含有する組成物は、早期前駆筋肉細胞、即ちデスミン、M−カドヘリン、MyoD、ミオゲニン、CD34、およびBcl−2を包含するがこれらに限定されない前駆細胞マーカーを発現する筋肉誘導幹細胞を含む。さらにまた、これらの早期前駆筋肉細胞はFlk−1、Sca−1、MNF、およびc−met細胞マーカーを発現するが、CD45またはc−Kit細胞マーカーは発現しない。
(項目1)
骨格筋の強化を必要とする哺乳類において骨格筋の強化を行う方法であって、
a)MDCを単離する工程、
b)単離されたMDCを10〜20日間培養物中で増殖させる工程、および、
c)増殖されたMDCの治療有効部分を、骨格筋の強化を必要とする哺乳類の骨格筋に投与する工程、
を含むことにより、骨格筋の強化を必要とする哺乳類において骨格筋の強化を行う、方法。
(項目2)
骨格筋の強化を必要としている哺乳類が筋肉の病理学的状態に罹患している、項目1記載の方法。
(項目3)
哺乳類がヒトである、項目1記載の方法。
(項目4)
(a)30〜120分間、第1の細胞培養容器中にヒト骨格筋細胞を懸濁する工程、
(b)第2の細胞培養容器に第1の細胞培養容器から培地を移す工程、
(c)第2の細胞培養容器の壁部に培地中の残存細胞を付着させる工程、および、
(d)第2の細胞培養容器の壁部から細胞を単離する工程、ここで単離された細胞はMDCであること、
を含むことにより、MDCを単離する方法によってMDCが単離される、項目1記載の方法。
(項目5)
(a)骨格筋細胞懸濁液の線維芽細胞が接着する第1の容器中で骨格筋組織由来の骨格筋細胞の懸濁液をプレーティングする工程、
(b)第2の容器中に工程(a)由来の非接着細胞を再プレーティングする工程、ここで再プレーティングの工程は細胞の約15〜約20%が第1の容器に接着した後であること、および、
(c)工程(b)を少なくとも1回反復する工程、
を含むことにより、MDCを単離する方法によってMDCが単離される、項目1記載の方法。
(項目6)
哺乳類の骨格筋内にMDCを注射することによりMDCが投与される、項目1記載の方法。
(項目7)
哺乳類の筋肉が欠陥を有する、項目1記載の方法。
(項目8)
MDCを上記欠陥に適用することによりMDCが投与される、項目1記載の方法。
(項目9)
MDCが注射を介して適用される、項目8記載の方法。
(項目10)
骨格筋の強化を必要とする哺乳類において骨格筋の強化を行う方法であって、
a)MDCを単離する工程、
b)単離されたMDCを約−25℃〜−90℃の温度に凍結する工程、
c)凍結した単離されたMDCを解凍する工程、および、
d)解凍されたMDCを、骨格筋の強化を必要とする哺乳類の骨格筋に投与する工程、
を含むことにより、骨格筋の強化を必要とする哺乳類において骨格筋の強化を行う、方法。
(項目11)
骨格筋の強化を必要とする哺乳類が筋肉の病理学的状態に罹患している、項目10記載
の方法。
(項目12)
哺乳類がヒトである、項目10記載の方法。
(項目13)
(a)30〜120分間、第1の細胞培養容器中にヒト骨格筋細胞を懸濁する工程、
(b)第2の細胞培養容器に第1の細胞培養容器から培地を移す工程、
(c)第2の細胞培養容器の壁部に培地中の残存細胞を付着させる工程、および、
(d)第2の細胞培養容器の壁部から細胞を単離する工程、ここで単離された細胞はMDCであること、
を含むことにより、MDCを単離する方法によってMDCが単離される、項目10記載の方法。
(項目14)
(a)骨格筋細胞懸濁液の線維芽細胞が接着する第1の容器中で骨格筋組織由来の骨格筋細胞の懸濁液をプレーティングする工程、
(b)第2の容器中に工程(a)由来の非接着細胞を再プレーティングする工程、ここで再プレーティングの工程は細胞の約15〜約20%が第1の容器に接着した後であること、および、
(c)工程(b)を少なくとも1回反復する工程、
を含むことにより、MDCを単離する方法によってMDCが単離される、項目10記載の方法。
(項目15)
哺乳類の骨格筋内にMDCを注射することによりMDCが投与される、項目10記載の方法。
(項目16)
哺乳類の筋肉が欠陥を有する、項目10記載の方法。
(項目17)
MDCを上記欠陥に適用することによりMDCが投与される、項目10記載の方法。
(項目18)
MDCが注射を介して適用される、項目17記載の方法。
追加的な目的および本発明により得られる利点は、以下に記載する詳細な説明および実施例から明らかになる。
本発明は、ヒトMDCおよびそのような細胞を使用することにより、骨格筋組織を形成して損傷した骨格筋を修復または骨格筋の体積および/もしくは強度を野生型レベルより高値に上昇させる方法を提供する。本発明はさらに、骨格筋の障害、例えば限定しないが骨格筋の脆弱化、筋ジストロフィー、筋萎縮症、痙性、ミオクローヌスおよび筋肉痛を治療する方法を提供する。成人組織からのヒト筋肉誘導細胞(MDC)の単離によって、これらの細胞を投与されたヒト被験体内で増大した骨格筋密度および骨格筋体積を達成することができる。
本発明は身体組織、好ましくは骨格筋内部への移植の後に長期の生存率を示す初期前駆細胞(本明細書においては筋肉誘導前駆細胞または筋肉誘導幹細胞とも称する)よりなるMDCを提供する。本発明のMDCを得るためには、筋肉の体外移植組織、好ましくは骨格筋を、動物ドナー、好ましくはヒトを包含する哺乳類より得る。この体外移植組織は筋肉前駆細胞の「残余」を包含する構造的および機能的なシンシチウムとして機能する(T.A.Partridge等、1978,Nature 73:306〜8;B.H.Lipton等、1979,Science205:12924)。
本発明の1つの実施形態において、MDCは骨格筋源から単離され、目的の筋肉もしくは非筋肉の軟組織内に、または骨格筋内に導入または移植される。好都合には、本発明のMDCは単離され、移植後長期の生存を示す前駆細胞を多数含有するように富化される。さらにまた、本発明の筋肉誘導前駆細胞はデスミン、CD34、およびBcl−2のような特徴的な細胞マーカーを多数発現する。さらにまた、本発明の筋肉誘導前駆細胞はSca−1、およびFlk−1細胞マーカーを発現するが、CD45またはc−Kit細胞マーカーを発現しない(実施例1参照)。
本発明の別の態様において、本発明のMDCは活性な生物学的分子1つ以上をコードする核酸配列を含有し、かつその生物学的分子、例えばタンパク質、ポリペプチド、ペプチド、ホルモン、代謝産物、薬剤、酵素等を発現するために、遺伝子操作してよい。そのようなMDCはヒトを包含するレシピエントに対して組織適合性(自系)であるか非組織適合性(同種異系)であってよい。この細胞は、種々の治療のため、例えば限定しないが骨格筋の脆弱化、筋ジストロフィー、筋萎縮症、痙性、ミオクローヌスおよび筋肉痛を包含する骨格筋の疾患ならびに病理状態の治療のための長期間の局所送達系として機能できる。
MDCは記載されている通り調製した(Chancellor等の米国特許6,866,842号)。筋肉の体外移植組織は多くの源、即ち、3週齢のmdx(ジストロフィー)マウス(C57BL/10ScSn mdx/mdx,Jackson Laboratories)、4〜6週齢の正常雌性SD(スプラーグドーリー)ラット、またはSCID(重度複合免疫不全)マウスの後肢から得た。動物源の各々に由来する筋肉組織を切開して骨があれば除き、粉砕してスラリーとした。次にスラリーを37℃において0.2%XI型コラゲナーゼ、ジスパーゼ(II等級、240単位)および0.1%トリプシンと共に1時間連続インキュベートすることにより消化した。得られた細胞懸濁液を18,20および22ゲージの針を通過させ、5分間3000rpmで遠心分離した。その後、細胞を生育培地(DMEMに10%ウシ胎児血清、10%ウマ血清、0.5%ニワトリ胚抽出液、および2%ペニシリン/ストレプトマイシンを補給したもの)中に懸濁した。次に細胞をコラーゲンコーティングフラスコ(Chancellor等の米国特許6,866,842号)中に予備プレーティングした。約1時間後、上澄みをフラスコから取り出し、新しいコラーゲンコーティングフラスコ内に再プレーティングした。この1時間のインキュベーション内で急速に接着した細胞は、大部分が線維芽細胞であった(上出のZ.Qu等;Chancellor等の米国特許6,866,842号)。細胞の30〜40%が各フラスコに接着した後に上澄みを取り出し、再プレーティングした。約5〜6回の連続プレーティングの後、培養物は小型丸型の細胞、即ちPP6細胞と命名されるものが富化されており、これを出発細胞集団から単離し、その後の試験に使用した。早期のプレーティングにおいて単離された接着細胞をプールし、PP1−4細胞と命名した。
急速接着MDCおよび緩徐接着MDCの集団を哺乳類被験体の骨格筋から単離した。被験体はヒト、ラット、イヌまたは他の哺乳類であってよい。生検試料サイズは42〜247mgの範囲であった。
予備プレーティング技術を用いてヒト筋肉生検試料から単離したヒト筋肉誘導細胞(hMDC)の集団を試験することにより、hMDCがそのネズミ相応物と同様の筋原性特徴および再生特徴を有することを示した。
予備プレーティング技術:この技術は出願全体を通して、特に上記実施例1において開示している。
本発明者等は3つの予備プレーティング画分、即ち予備プレーティング1および2(pp1−2)および予備プレーティング2〜3(pp2〜3)および予備プレーティング5〜6)を検査した。全ての集団はCD34およびCD144に対して陰性であり、CD56およびCD146に対して陽性であった。経時的にCD56およびCD146の低下が観察された。免疫染色によれば、細胞は多核筋管内に融合する能力を呈し、デスミン、ミオシン、およびジストロフィンの発現を示したことから、その筋原性能力が明らかになった。
Claims (14)
- 骨格筋の強化を必要とする哺乳類において骨格筋の強化を行うための医薬の製造方法であって、該医薬は、増殖された筋肉誘導前駆細胞(MDC)の治療有効部分を含み、該MDCは、
a)哺乳類から単離された骨格筋細胞を、4℃で、4〜7日間保存する工程、
b)該骨格筋細胞を、細胞培養培地を含む第1の細胞培養容器に5%CO 2 下37℃で30分〜120分懸濁し、該第1の細胞培養容器の壁部に急速接着細胞を接着させ、それによって緩徐接着細胞を該培養培地中に残す工程、
c)該第1の細胞培養容器から第2の細胞培養容器に、緩徐接着細胞を含む該培地を移す工程、
d)移された該第2の細胞培養容器中の細胞をインキュベートして、該細胞を該第2の細胞培養容器の壁部に接着させる工程であって、このインキュベーションは5%CO 2 下37℃で1日〜3日である、工程、
e)1日〜3日のインキュベーション後に、該第2の細胞培養容器から該培地を取り除いて新しい培地と取り換える工程、
f)該第2の細胞培養容器の壁部から該細胞を単離する工程であって、該単離された細胞がMDCである、工程、および
g)該単離されたMDCを10〜20日間培養物中で増殖させる工程
を含む方法によって単離されるものであることを特徴とする、方法。 - 前記骨格筋の強化を必要とする哺乳類が骨格筋の病理学的状態に罹患している、請求項1記載の方法。
- 前記哺乳類がヒトである、請求項1記載の方法。
- 前記MDCは、前記哺乳類の骨格筋内への注射による投与に適している、請求項1記載の方法。
- 前記哺乳類の骨格筋が欠陥を有する、請求項1記載の方法。
- 前記MDCが、前記欠陥への適用により投与されるのに適している、請求項1記載の方法。
- 前記MDCが注射を介する適用に適している、請求項6記載の方法。
- 骨格筋の強化を必要とする哺乳類において骨格筋の強化を行うための医薬の製造方法であって、該医薬は、解凍された筋肉誘導前駆細胞(MDC)を含み、該MDCは、
a)哺乳類から単離された骨格筋細胞を、4℃で、4〜7日間保存する工程、
b)該骨格筋細胞を、細胞培養培地を含む第1の細胞培養容器に5%CO 2 下37℃で30分〜120分懸濁し、該第1の細胞培養容器の壁部に急速接着細胞を接着させ、それによって緩徐接着細胞を該培養培地中に残す工程、
c)該第1の細胞培養容器から第2の細胞培養容器に、緩徐接着細胞を含む該培地を移す工程、
d)移された該第2の細胞培養容器中の細胞をインキュベートして、該細胞を該第2の細胞培養容器の壁部に接着させる工程であって、このインキュベーションは5%CO 2 下37℃で1日〜3日である、工程、
e)1日〜3日のインキュベーション後に、該第2の細胞培養容器から該培地を取り除いて新しい培地と取り換える工程、
f)該第2の細胞培養容器の壁部から該細胞を単離する工程であって、該単離された細胞がMDCである、工程、
g)単離されたMDCを10〜20日間培養物中で増殖させる工程、
h)該単離されたMDCを−25℃〜−90℃の温度に凍結する工程、および
i)該凍結した単離されたMDCを解凍する工程
を含む方法によって単離されるものであることを特徴とする、方法。 - 前記骨格筋の強化を必要とする哺乳類が骨格筋の病理学的状態に罹患している、請求項8記載の方法。
- 前記哺乳類がヒトである、請求項8記載の方法。
- 前記MDCが、前記哺乳類の骨格筋内への注射による投与に適している、請求項8記載の方法。
- 前記哺乳類の骨格筋が欠陥を有する、請求項8記載の方法。
- 前記MDCが、前記欠陥への適用により投与されるのに適している、請求項12記載の方法。
- 前記MDCが注射を介する適用に適している、請求項13記載の方法。
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |