JP2019205448A - 多能性幹細胞から血小板を生産するための方法およびその組成物 - Google Patents
多能性幹細胞から血小板を生産するための方法およびその組成物 Download PDFInfo
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Abstract
Description
本出願は、2012年12月21日に出願された米国仮特許出願第61/740,699号および2013年3月15日に出願された米国仮特許出願第61/787,476号(両方とも「多能性幹細胞から血小板を生産するための方法およびその組成物」と題される)に対する利益を主張し、これら両方の内容全体が参照によって本明細書中に組み込まれる。
血小板は、血液凝固という重要な高度に特化した機能を果たす小さな血液細胞である。平均的なヒトの血液ではほぼ1兆個の血小板が循環し、そのターンオーバーは、全血小板集団が10日毎に置き換えられるようなものである。これは、莫大な量の進行中の血小板生成を示す。血小板は、高度に組織化された細胞骨格および300を超えるタンパク質の細胞内貯蔵を有し、血管傷害の部位においてこれらを分泌する。血小板は、炎症、血管増殖および腫瘍転移においても役割を果たす。
ヒト胚性幹細胞(hESC)は、in vitroで無制限に繁殖および拡大増殖され得、ヒト治療のための細胞の、潜在的に無尽蔵のドナーなしの供給源を提供する。in vitroでのhESCの造血細胞への分化は、過去10年間にわたって広範囲に調査されてきた。hESCの方向付けられた造血分化は、2つの異なる型の培養系によってin
vitroで首尾よく達成されている。これらのうち1つは、血清含有培地中での、h
ESCと間質フィーダー細胞との共培養を使用する(3;4)。第2の型の手順は、血清あり/なしのサイトカインの存在下での、超低細胞結合プレートにおける懸濁培養条件を使用する(5〜7);その終点は、細胞凝集体または胚様体(「EB」)の形成である。造血先駆体ならびに赤血球、骨髄、マクロファージ、巨核球およびリンパ系の系列を示す成熟した機能的子孫が、上記分化するhESC培養系の両方において同定されている(3〜6:8〜14)。以前の研究でもまた、血清の存在下で間質細胞と共培養することによって、hESCから巨核球/血小板が生成した(15;16)。しかし、上記研究における巨核球/血小板の収量は低かった(15;16)。
本開示は、多能性幹細胞、例えば、ヒト胚性幹細胞(hESC)および人工多能性幹細胞(iPSCまたはiPS細胞)、例えばヒト人工多能性幹細胞(hiPSCまたはhiPS細胞)からの血小板の生成のための方法を提供する。これらの方法は、胚様体を形成することなく実施され得、間質インデューサー細胞の使用を伴わずに実施され得る。さらに、収量および/または純度は、多能性幹細胞から血小板を生成する以前の方法について報告されているよりも高くなり得る。血小板は、より高い効率およびより大きい規模で生成され得るので、本開示の方法および組成物は、医療上の輸血目的での使用についての大きな可能性を有する。さらに、血小板は、核を有さず、最小の遺伝子材料のみを含むので、本開示の調製物は、任意の汚染する有核細胞、例えば未分化hESCを効果的に排除するために、輸血前に照射され得る。したがって、有核細胞の起こり得る存在は、安全性の問題を示さないはずである。
であって、前記調製物が白血球を実質的に含まず、前記血小板の実質的に全てが機能的である、医薬調製物。
O非ペプチド模倣物のうち1つまたは複数を含む。
E)を形成するステップ;(b)前記造血性内皮細胞を培養して、MLPを形成するステップ;および任意選択で(c)前記MLPを培養して、巨核球を形成するステップを含むステップによって生成され得る。この多能性幹細胞はヒトのものであり得る。
血小板の分化および成熟化を誘発するために使用され得る。TPO受容体活性化ペプチドおよび融合タンパク質(即ち、Fab 59、ロミプロスチム/AMG 531またはペグ化体(Peg−TPOmp))が、TPOの代わりに使用され得る。非ペプチド模倣物(エルトロンボパグ(SB497115、Promacta)およびAKR−501)は、TPOとは異なる機構によってTPO受容体と結合してそれを活性化し、TPOに対して相加効果を有し得る。TPO受容体を活性化するTPOアゴニスト抗体(即ち、MA01G4G344)またはミニボディ(minibody)(即ち、VB22B sc(Fv)2)もまた、TPOの効果を模倣するために使用され得る。例示的なTPOアゴニストは、各々その全体が参照によって本明細書に組み込まれるStasiら、Blood Reviews 24巻(2010年)179〜190頁およびKuter、Blood. 2007年;109巻:4607〜4616頁に開示されている。例示的なTPOアゴニストには以下が含まれる:ADP、エピネフリン、トロンビンおよびコラーゲン、ならびにTPO−RアゴニストもしくはTPO模倣物として文献中で同定されている他の化合物、第2世代血小板新生剤、ロミプロスチム、エルトロンボパグ(SB497115、Promacta)、第1世代血小板新生増殖因子、組換えヒトトロンボポエチン(TPO)、ペグ化組換えヒト巨核球増殖発達因子(PEG−rHuMGDF)、TPOペプチド模倣物、Fabの相補性決定領域中に挿入されたTPO受容体活性化ペプチド(Fab
59)、AMG 531(2つのジスルフィド結合したヒトIgG1−HC定常領域(Fc断片)(その各々がポリグリシンを介して連結された2つの同一のペプチド配列と残基228において共有結合している)から構成される「ペプチボディ(peptibody)」)、Peg−TPOmp(ペグ化TPOペプチドアゴニスト)、経口投与可能なTPOアゴニスト、TPO非ペプチド模倣物、AKR−501、モノクローナルTPOアゴニスト抗体、ポリクローナルTPOアゴニスト抗体、TPOミニボディ、例えばVB22B sc(Fv)2、ドメインサブクラス変換TPOアゴニスト抗体、例えばMA01G4G344、組換えヒトトロンボポエチン、または組換えTPO融合タンパク質、TPO非ペプチド模倣物。TPOが本発明の一実施形態としてどの場面で使用されても、例示的なTPOアゴニストは本発明のさらなる実施形態においてTPOの代用になり得る。
mSpam(商標)CC220サイトカインカクテル(CC220)(StemCell
Technologies Inc.から入手可能)として提供され得る。この造血拡大増殖培地は、ROCKインヒビターおよび/またはヘパリンを任意選択で含み得る。TPO、SCF、IL−6、IL−9およびIL−11は、公知の巨核球発達因子および成熟化因子である(Stasiら、Blood Reviews 24巻(2010年)179〜190頁)。
され得る。
vivo寿命、投与後の生着の欠如、およびホーミング特性に起因して、血小板は、薬物担体として有用であり得ると考えられる。hESC、hiPCおよびMLPは遺伝子改変され得、疾患の処置のための所望の薬物を発現する血小板を生成するために使用され得る。一態様では、hESC、hiPCまたはMLPは、抗腫瘍剤を発現するように遺伝子改変され得る。かかる遺伝子改変されたhESC、hiPSCおよびMLPから生成された血小板は、新生物疾患の処置のために腫瘍にかかる抗腫瘍剤を送達するために使用され得る。
えば小分子薬物、アプタマーもしくは他の核酸薬剤、または組換えタンパク質を含むように操作されている。
本開示において使用する定義および略号のリストは、詳細な説明の最後に提供する。
胞接着分子CD62pおよびαIIbβIIIについて陽性になった(活性化の際のαIIbβIIIのコンフォメーション変化後のPAC−1結合部位の露出によって生じると考えられるが、CD62pは顆粒放出に起因して外側膜上に露出されると考えられる)、機能的または活性化された血小板を生成した。これらのマーカーは両方とも、活性化された血小板の表面上に発現されることが公知であり、PAC−1およびCD62p(p−セレクチン)結合アッセイを使用して、hiPSC−PLT上で検出された。間質インデューサー細胞は巨核球生成に必要とされないので、本明細書に記載される方法は、フィーダーフリーの血小板生成、例えば、異種細胞のいずれの使用も伴わない血小板生成を提供し得る。
LTおよびhESC−PLTが、血液血小板と同様、脈管傷害後にαIIbβIIIインテグリンを介して発達中のマウス血小板血栓中に取り込まれることが実証されている(図12A)。hiPSC−PLTおよびhESC−PLTの機能的能力は、αIIbβIIIに特異的に結合して血小板機能を阻害するヒト−マウスキメラモノクローナル抗体のFab断片ReoProによる事前処理によって、αIIbβIIIによって媒介されることが決定された(図12B)。これらの結果は、hESC−PLTが脈管傷害の部位においてin vivoで機能的であるという証拠を提供する。重要なことに、血清フリー条件およびフィーダーフリー条件下で多能性幹細胞から誘導された血小板は、凝血および血栓形成をin vivoで促進することができることが最初に示された。
ブリドーマ混合物II(PFHMII)、アスコルビン酸2ホスフェート、Glutamax I(L−アラニル−L−グルタミン)、ペニシリン/ストレプトマイシンを含み、幹細胞因子(SCF)(例えば、25ng/ml)、トロンボポエチン(TPO)(例えば、25ng/ml)、Fms関連チロシンキナーゼ3リガンド(FL)(例えば、25ng/ml)、インターロイキン−3(IL−3)(例えば、10ng/ml)、インターロイキン−6(IL−6)(例えば、10ng/ml)および任意選択でヘパリン(例えば、5単位/ml)をさらに含む培養培地中でPVE−HE細胞を培養するステップを含む方法を介して、PVE−HE細胞から生成され得る。この培養期間は、平均して3〜4日間持続し得る。最後の48時間、最後の36時間、最後の24時間、最後の18時間、最後の12時間または最後の6時間を含む培養の後半で、BETのインヒビターが、培養物に、好ましくは細胞毒性未満のレベルで添加され得る。これらの培養物から回収されたMLPは、血小板生成またはその他分析のために凍結保存されてもよいし、または直ぐに使用されてもよい。
ならびに任意選択で、ROCKインヒビター、例えばY27632(例えば、5μM)および/またはヘパリン(例えば、2.5〜25単位/ml)のうち1種または複数をさらに含む培地中でMLPまたはMKを培養するステップを含む方法を介して、MLPまたはMKから生成され得る。
を回避し、剪断培養の利益をなおも得るために、本開示は、CD42bの剪断または喪失を防止するプロテアーゼインヒビターを使用することを企図する。かかるインヒビターの例には、メタロプロテアーゼインヒビターおよびより具体的にはマトリックスメタロプロテアーゼ(MMP)インヒビターが含まれる。剪断培養において使用され得るインヒビターの別の例は、プラスミノーゲン活性化因子インヒビターである。これらのインヒビターは、汎インヒビターであり得、これは、単一のインヒビターが、そのクラス内の1種よりも多くのおよび場合によっては全てのプロテアーゼを阻害し得ることを意図する。あるいは、これらは特異的インヒビターであり得、これは、単一のインヒビターが、そのクラス内の1種のプロテアーゼを完全にまたは大部分阻害することを意図する。
よびアクチノニンが含まれる。
T出願WO/13063331;WO/1316974;および参考文献Fortenberry YM. Plasminogen activator inhibitor−1 inhibitors: a patent review (2006−present). Expert Opin Ther Pat. 2013年7月;23巻(7号):801〜15頁;およびPannekoekら、EMBO J. 1986年;5巻(10号):2539〜44頁に記載されるものが含まれる。
Genomics Consortium)、OTX015(Mitsubishi Tanabe Pharma Corporation)、BzT−7、GSK525762A(iBET、GlaxoSmithKline)、JQ1(Cell 2011年146巻(6号):904〜17頁)および以下の化合物(WO 2011054851、GlaxoSmithKline):
巨核球培養物中における前血小板形成性細胞の最初の出現の3〜5日後から収集される。ある特定の実施形態では、血小板は、密度勾配遠心分離を使用して精製される。さらなる実施形態では、この密度勾配遠心分離はPercoll媒体を使用する。さらなる実施形態では、この密度勾配遠心分離はBSA/HSA媒体を使用する。別の一実施形態では、この血小板精製法は、CD41a陰性である粒子を分離する。別の一実施形態では、この血小板精製法は、CD42b陰性である粒子を分離する。別の一実施形態では、この血小板精製法は、細胞の生存度および形態学的完全性を保持する。他の実施形態では、血小板は、CD41aおよびCD42bを発現する。他の実施形態では、血小板は、トロンビン刺激に対して応答性である。別の一実施形態では、血小板は、フィブリノーゲンおよびフォン・ヴィルブランド因子(vWF)表面上に伸展することができる。さらなる実施形態では、血小板は、PAC−1結合およびインテグリン活性化の能力を有する。別の一実施形態では、血小板は、ミクロ凝集体を形成し、血餅の形成および退縮を促進する。別の一実施形態では、血小板は、アピラーゼおよび/またはEDTAの存在下では活性化されない。
シアリダーゼ活性を低減させ、血小板生成物調製物における1種もしくは複数の細菌の増殖を(任意選択で)阻害する条件下で血小板を誘導することを企図する。この方法は、血小板生成物調製物をある量のシアリダーゼインヒビターと接触させ、それによって、シアリダーゼ処理された血小板生成物調製物を取得するステップを含み得る;ここで、シアリダーゼインヒビターに供されていない血小板生成物調製物と比較して、シアリダーゼ活性は低減され、1種または複数の細菌の増殖は阻害される。
sp、Micrococcus sp、Peptostreptococcus、Proteus vulgaris、Pseudomonas sp、Pseudomys oxalis、Propionibacterium sp、Salmonella sp、Serratia sp、Serratia marcescens Staphylococcus sp(コアグラーゼ陰性Staphylococcus、Staphylococcus epidermidis、Staphylococcus aureus)、Streptococcus sp、(S.gallolyticus、S.bovis、S.pyogenes、S.viridans)およびYersinia enterocolitica。
スである。
フルオキセチン、パロキセチン、バルプロ酸、カルバマゼピン、ブロモクリプチン、モルヒネ、フェンタニル、ナルトレキソン、ナロキソン);炎症応答をモジュレートする薬物(例えば、アスピリン、インドメタシン、イブプロフェン、ナプロキセン、ステロイド、クロモリンナトリウム、テオフィリン);腎機能および/または心血管機能に影響する薬物(例えば、フロセミド、チアジド、アミロライド、スピロノラクトン、カプトプリル、エナラプリル、リシノプリル、ジルチアゼム、ニフェジピン、ベラパミル、ジゴキシン、イソルジル(isordil)、ドブタミン、リドカイン、キニジン、アデノシン、ジギタリス、メバスタチン、ロバスタチン、シンバスタチン、メバロネート);胃腸機能に影響する薬物(例えば、オメプラゾール、スクラルファート);抗生物質(例えば、テトラサイクリン、クリンダマイシン、アンホテリシンB、キニーネ、メチシリン、バンコマイシン、ペニシリンG、アモキシシリン、ゲンタマイシン、エリスロマイシン、シプロフロキサシン、ドキシサイクリン、アシクロビル、ジドブジン(AZT)、ddC、ddI、リバビリン、セファクロル、セファレキシン、ストレプトマイシン、ゲンタマイシン、トブラマイシン、クロラムフェニコール、イソニアジド、フルコナゾール、アマンタジン、インターフェロン);抗がん剤(例えば、シクロホスファミド、メトトレキセート、フルオロウラシル、シタラビン、メルカプトプリン、ビンブラスチン、ビンクリスチン、ドキソルビシン、ブレオマイシン、マイトマイシンC、ヒドロキシウレア、プレドニゾン、タモキシフェン、シスプラチン、ダカルバジン(decarbazine));免疫調節剤(例えば、インターロイキン、インターフェロン、GM−CSF、TNFα、TNFβ、シクロスポリン、FK506、アザチオプリン、ステロイド);血液および/または血液形成性器官に対して作用する薬物(例えば、インターロイキン、G−CSF、GM−CSF、エリスロポエチン、ビタミン、鉄、銅、ビタミンB12、葉酸、ヘパリン、ワルファリン、クマリン);ホルモン(例えば、成長ホルモン(GH)、プロラクチン、黄体形成ホルモン、TSH、ACTH、インスリン、FSH、CG、ソマトスタチン、エストロゲン、アンドロゲン、プロゲステロン、ゴナドトロピン放出ホルモン(GnRH)、チロキシン、トリヨードサイロニン);ホルモンアンタゴニスト;石灰化および骨のターンオーバーに影響する薬剤(例えば、カルシウム、ホスフェート、副甲状腺ホルモン(PTH)、ビタミンD、ビスホスホネート、カルシトニン、フッ化物)、ビタミン(例えば、リボフラビン、ニコチン酸、ピリドキシン、パントテン酸、ビオチン、コリン、イノシトール、カルニチン(camitine)、ビタミンC、ビタミンA、ビタミンE、ビタミンK)、遺伝子治療剤(例えば、ウイルスベクター、核酸保有リポソーム、DNA−タンパク質コンジュゲート、アンチセンス剤);または標的化剤などの他の薬剤。
外科的手順(椎弓切除術、椎間板切除術、関節手術、腹部手術または胸部手術から生じるものなどの外科的病変)を含む閉鎖(内部)創傷の処置において使用され得る。MK細胞における発現および血小板顆粒における貯蔵が本発明に従って達成され得る他の創傷治癒性タンパク質、特に、非線維性増殖因子には、インスリン様増殖因子1(IGF−1);塩基性線維芽細胞増殖因子(bFGF);トランスフォーミング増殖因子β−3(TGFβ−3)、顆粒球コロニー刺激因子(GCSF)、顆粒球マクロファージコロニー刺激因子(GMCSF)、ケラチノサイト増殖因子(KGF)、フィブロネクチン、ビトロネクチン、トロンボスポンジン、ラミニン、テネイシン(tenasin)が含まれる。
ラーゼインヒビター、5−HTアゴニスト、3−ヒドロキシ−3−メチルグルタリル補酵素A(HMG−CoA)インヒビター、H2アンタゴニスト、抗新生物剤およびシクロオキシゲナーゼ−2インヒビターが含まれる。
デキサメタゾン)、非ステロイド性薬剤(サリチル酸誘導体、即ちアスピリン;パラ−アミノフェノール誘導体、即ちアセトアミノフェン;インドールおよびインデン酢酸(インドメタシン、スリンダクおよびエトドラク(etodalac))、ヘテロアリール酢酸(トルメチン、ジクロフェナクおよびケトロラク)、アリールプロピオン酸(イブプロフェンおよび誘導体)、アントラニル酸(メフェナム酸およびメクロフェナム酸)、エノール酸(ピロキシカム、テノキシカム、フェニルブタゾンおよびオキシフェンタトラゾン(oxyphenthatrazone))、ナブメトン、金化合物(オーラノフィン、オーロチオグルコース、金チオリンゴ酸ナトリウム);免疫抑制薬:(シクロスポリン、タクロリムス(FK−506)、シロリムス(ラパマイシン)、アザチオプリン、ミコフェノール酸モフェチル);血管新生剤:血管内皮増殖因子(VEGF)、線維芽細胞増殖因子(FGF);アンジオテンシン受容体ブロッカー;一酸化窒素供与体;アンチセンスオリゴヌクレオチドおよびそれらの組合せ;細胞周期インヒビター、mTORインヒビターおよび増殖因子受容体シグナル伝達キナーゼインヒビター;レチノイド(retenoid);サイクリン/CDKインヒビター;HMG補酵素レダクターゼインヒビター(スタチン);ならびにプロテアーゼインヒビター。
ル抗体、bFGFアンタゴニスト、プロブコール、プロスタグランジン、1,11−ジメトキシカンチン−6−オン、1−ヒドロキシ−11−メトキシカンチン−6−オン、スコポレクチン(scopolectin)、コルヒチン、NO供与体、四硝酸ペンタエリスリトール、シンドノエイミン(syndnoeimine)、S−ニトロソ誘導体、タモキシフェン、スタウロスポリン、β−エストラジオール、α−エストラジオール、エストリオール、エストロン、エチニルエストラジオール、ホスフェストロール、メドロキシプロゲステロン、シピオン酸エストラジオール、安息香酸エストラジオール、トラニラスト、カメバカウリン(kamebakaurin)および他のテルペノイド、これはがんの治療に適用される、ベラパミル、チロシンキナーゼインヒビター、チルホスチン、シクロスポリンA、パクリタキセルおよびその誘導体、バッカチン、タキソテール、ならびに他の合成により取得されたおよびネイティブ供給源から取得された亜酸化炭素(MCS)の大環状オリゴマーならびにそれらの誘導体、モフェブタゾン、アセメタシン、ジクロフェナク、ロナゾラク、ダプソン、o−カルバモイルフェノキシ酢酸、リドカイン、ケトプロフェン、メフェナム酸、ピロキシカム、メロキシカム、リン酸クロロキン、ペニシラミン、ヒドロキシクロロキン、オーラノフィン、金チオリンゴ酸ナトリウム、オキサセプロール、セレコキシブ、β−シトステリン(β−sitosterin)、アデメチオニン、ミルテカイン(myrtecaine)、ポリドカノール、ノニバミド(nonivamide)、レボメントール、ベンゾカイン、エスチン、エリプチシン、Calbiochem D−24851、コルセミド、サイトカラシンA〜E、インダノシン(indanocine)、ノコダゾール(nocadazole)、S 100タンパク質、バシトラシン、ビトロネクチン受容体アンタゴニスト、アゼラスチン、グアニリル(guanidyl)シクラーゼ刺激因子 金属プロテイナーゼ−1および−2の組織インヒビター、遊離核酸、ウイルストランスミッター中に組み込まれた核酸、DNAおよびRNA断片、プラスミノーゲン(plaminogen)活性化因子インヒビター−1、プラスミノーゲン活性化因子インヒビター−2、アンチセンスオリゴヌクレオチド、VEGFインヒビター、IGF−1、抗生物質、抗血栓薬、アルガトロバン、アスピリン、アブシキシマブ、合成アンチトロンビン、ビバリルジン、クマジン、エノキサパリン(enoxoparin)、脱硫酸化およびN−再アセチル化ヘパリン、組織プラスミノーゲン活性化因子、GpIIb/IIIa血小板膜受容体、第Xa因子インヒビター抗体、ヒルジン、r−ヒルジン、PPACK、プロタミン、プロウロキナーゼ、ストレプトキナーゼ、ワルファリン、ウロキナーゼ、血管拡張薬、ジピラミドール(dipyramidole)、トラピジル、ニトロプルシド、PDGFアンタゴニスト、トリアゾロピリミジンおよびセラミン(seramin)、ACEインヒビター、カプトプリル、シラザプリル、リシノプリル、エナラプリル、ロサルタン、チオプロテアーゼインヒビター、プロスタサイクリン、バピプロスト、インターフェロンα、βおよびγ、ヒスタミンアンタゴニスト、セロトニンブロッカー、アポトーシスインヒビター、アポトーシス調節因子、p65 NF−κ.BおよびBcl−xLアンチセンスオリゴヌクレオチド、ハロフジノン、ニフェジピン、トコフェロール、トラニラスト(tranirast)、モルシドミン、茶ポリフェノール、没食子酸エピカテキン、没食子酸エピガロカテキン、ボスウェル酸およびその誘導体、レフルノミド、アナキンラ、エタネルセプト、スルファサラジン、エトポシド、ジクロキサシリン、テトラサイクリン、トリアムシノロン、ムタマイシン(mutamycin)、プロカインイミド(procainimid)、レチノイン酸、キニジン、ジソピリミド(disopyrimide)、フレカイニド、プロパフェノン、ソタロール(sotolol)、アミドロン(amidorone)、天然および合成により取得されたステロイド、ブリオフィリン(bryophyllin)A、イノトジオール(inotodiol)、マキロシド(maquiroside)A、ガラキノシド(ghalakinoside)、マンソニン(mansonine)、ストレブロシド(strebloside)、ヒドロコルチゾン、ベタメタゾン、デキサメタゾン、フェノプロフェン(fenoporfen)、イブプロフェン、インドメタシン、ナプロキセン、フェニルブタゾン、抗ウイルス剤、抗真菌薬、抗原虫剤、天然テルペノイド、ヒポカエスクリン(hip
pocaesculin)、バリントゲノール−C21−アンゲレート(barringtogenol−C21−angelate)、14−デヒドロアグロスチスタチン(14−dehydroagrostistachin)、アグロスケリン(agroskerin)、アグロスチスタチン(agrostistachin)、17−ヒドロキシアグロスチスタチン、オバトジオリド(ovatodiolid)、4,7−オキシシクロアニソメリック酸(4,7−oxycycloanisomelic acid)、バッカリノイド(baccharinoid)B1、B2、B3およびB7、ツベイモシド(tubeimoside)、ブルセアノール(bruceanol)A、BおよびC、ブルセアンチノシド(bruceantinoside)C、ヤダンジオシド(yadanzioside)NおよびP、イソデオキシエレファントピン(isodeoxyelephantopin)、トメンファントピン(tomenphantopin)AおよびB、コロナリン(coronarin)A、B、CおよびD、ウルソール酸、ヒプタチン酸(hyptatic acid)A、ゼオリン(zeorin)、イソイリドゲルマナール(iso−iridogermanal)、メイテンフォリオール(maytenfoliol)、エフサンチン(effusantin)A、エクシサニン(excisanin)AおよびB、ロンギカウリン(longikaurin)B、スカルポネアチン(sculponeatin)C、カメバウニン(kamebaunin)、ロイカメニン(leukamenin)AおよびB、13,18−デヒドロ−6−α−セネシオイルオキシカパリン(13,18−dehydro−6−α−senecioyloxychaparrin)、タキサマイリン(taxamairin)AおよびB、レジェニロール(regenilol)、トリプトライド、シマリン、アポシマリン、アリストロキア酸、アノプテリン(anopterin)、ヒドロキシアノプテリン、アネモニン、プロトアネモニン、ベルベリン、塩化ケリブリン(cheliburin chloride)、シクトキシン(cictoxin)、シノコクリン(sinococuline)、ボンブレスタチン(bombrestatin)AおよびB、クドライソフラボン(cudraisoflavone)A、クルクミン、ジヒドロニチジン(dihydronitidine)、塩化ニチジン(nitidine chloride)、12−β−ヒドロキシプレグナジエン−3,20−ジオン、ビロボール、ギンコール、ギンコール酸、ヘレナリン、インジシン、インジシン−N−オキシド、ラシオカルピン(lasiocarpine)、イノトジオール、グリコシドla、ポドフィロトキシン、ジュスチシジン(justicidin)AおよびB、ラレアチン(larreatin)、マロテリン(malloterin)、マロトクロマノール(mallotochromanol)、イソブチリルマロトクロマノール、マキロシドA、マルカンチンA、メイタンシン、リコリジシン(lycoridicin)、マルゲチン(margetine)、パンクラチスタチン、リリオデニン、ビスパルテノリジン(bisparthenolidine)、オキソウシンスニン(oxoushinsunine)、アリストラクタム(aristolactam)−AII、ビスパルテノリジン、ペリプロコシド(periplocoside)A、ガラキノシド、ウルソール酸、デオキシプソロスペルミン(deoxypsorospermin)、サイコルビン(psycorubin)、リシンA、サンギナリン、マヌーコムギ酸(manwu wheat acid)、メチルソルビフォリン(methylsorbifolin)、スファテリアクロメン(sphatheliachromen)、
スチゾフィリン(stizophyllin)、マンソニン、ストレブロシド、アカゲリン(akagerine)、ジヒドロウサンバレンシン(dihydrousambaraensine)、ヒドロキシウサンバリン(hydroxyusambarine)、ストリクノペンタミン(strychnopentamine)、ストリクノフィリン(strychnophylline)、ウサンバリン(usambarine)、ウサンバレンシン(usambarensine)、ベルベリン、リリオデニン、オキソウシンスニン、ダフノレチン(daphnoretin)、ラリシレシノール、メトキシラリシレシノール、シリンガレシノール、ウンベリフェロン、アフロモソン(afromoso
n)、アセチルビスミオン(acetylvismione)B、デスアセチルビスミオン(desacetylvismione)A、またはビスミオン(vismione)AおよびB。
ゾール(acodazole)、アクロニシン(acronycine)、アドゼレシン、アラノシン、アルデスロイキン、アロプリノールナトリウム、アルトレタミン、アミノグルテチミド、アモナフィド、アンプリジェン、アムサクリン、アンドロゲン、アングイジン、アフィジコリングリシネート、アサレイ(asaley)、アスパラギナーゼ、5−アザシチジン、アザチオプリン、カルメット・ゲラン桿菌(BCG)、ベーカーズアンチフォール(Baker’s Antifol)(可溶性)、ベータ−2’−デオキシチオグアノシン、ビサントレンHCl、硫酸ブレオマイシン、ブスルファン、ブチオニンスルホキシミン、BWA 773U82、BW 502U83.HCl、BW 7U85メシレート、セラセミド(ceracemide)、カルベチマー、カルボプラチン、カルムスチン、クロラムブシル、クロロキノキサリン−スルホンアミド、クロロゾトシン、クロモマイシンA3、シスプラチン、クラドリビン、コルチコステロイド、Corynebacterium parvum、CPT−11、クリスナトール、シクロシチジン、シクロホスファミド、シタラビン、シテンベナ(cytembena)、ダビスマレアート(dabis maleate)、ダカルバジン、ダクチノマイシン、ダウノルビシンHCl、デアザウリジン、デクスラゾキサン、ジアンヒドロガラクチトール、ジアジコン(diaziquone)、ジブロモダルシトール、ジデムニンB、ジエチルジチオカルバメート、ジグリコアルデヒド、ジヒドロ−5−アザシチジン、ドキソルビシン、エキノマイシン、エダトレキセート、エデルフォシン(edelfosine)、エフロルニチン、Elliott溶液、エルサミトルシン(elsamitrucin)、エピルビシン、エソルビシン、リン酸エストラムスチン、エストロゲン、エタニダゾール、エチオフォス(ethiofos)、エトポシド、ファドラゾール(fadrazole)、ファザラビン、フェンレチニド、フィルグラスチム、フィナステリド、フラボン酢酸、フロクスウリジン、リン酸フルダラビン、5−フルオロウラシル、フルオゾール(Fluosol.)、フルタミド、硝酸ガリウム、ゲムシタビン、酢酸ゴセレリン、ヘプスルファム(hepsulfam)、ヘキサメチレンビスアセトアミド、ホモハリントニン、硫酸ヒドラジン、4−ヒドロキシアンドロステンジオン、ヒドロキシウレア(hydrozyurea)、イダルビシンHCl、イホスファミド、インターフェロンアルファ、インターフェロンベータ、インターフェロンガンマ、インターロイキン−1アルファおよびベータ、インターロイキン−3、インターロイキン−4、インターロイキン−6、4−イポメアノール(4−ipomeanol)、イプロプラチン、イソトレチノイン、ロイコボリンカルシウム、酢酸リュープロリド、レバミゾール、リポソームダウノルビシン、リポソーム封入ドキソルビシン、ロムスチン、ロニダミン、メイタンシン、メクロレタミン塩酸塩、メルファラン、メノガリル、メルバロン(merbarone)、6−メルカプトプリン、メスナ、カルメット・ゲラン桿菌のメタノール抽出残渣、メトトレキサート、N−メチルホルムアミド、ミフェプリストン、ミトグアゾン、マイトマイシン−C、ミトタン、ミトキサントロン塩酸塩、単球/マクロファージコロニー刺激因子、ナビロン、ナフォキシジン、ネオカルチノスタチン、酢酸オクトレオチド、オルマプラチン、オキサリプラチン、パクリタキセル、パラ(pala)、ペントスタチン、ピペラジンジオン、ピポブロマン、ピラルビシン、ピリトレキシム、ピロキサントロン塩酸塩、PIXY−321、プリカマイシン、ポルフィマーナトリウム、プレドニムスチン、プロカルバジン、プロゲスチン、ピラゾフリン(pyrazofurin)、ラゾキサン、サルグラモスチム、セムスチン、スピロゲルマニウム、スピロムスチン、ストレプトニグリン、ストレプトゾシン、スロフェヌル、スラミンナトリウム、タモキシフェン、タキソテール、テガフール、テニポシド、テレフタルアミジン、テロキシロン(teroxirone)、チオグアニン、チオテパ、チミジン注射、チアゾフリン、トポテカン、トレミフェン、トレチノイン、トリフロペラジン塩酸塩、トリフルリジン、トリメトレキサート、腫瘍壊死因子、ウラシルマスタード、硫酸ビンブラスチン、硫酸ビンクリスチン、ビンデシン、ビノレルビン、ビンゾリジン(vinzolidine)、Yoshi 864、ゾルビシン、およびそれらの混合物から選択され得る抗がん薬などが充填され得る。
れ、これはMKによって生成される血小板内に含まれ、次いで活性化の際に循環血小板から放出される。
するための、精子および卵細胞の融合、核移植、単為生殖、雄性発生、またはクロマチンの再プログラミングおよび再プログラミングされたクロマチンの原形質膜中への引き続く取り込みによって生成された、接合体、卵割球または胚盤胞段階の哺乳動物胚から誘導された細胞である。胚性幹細胞は、その供給源またはそれらを生成するために使用される特定の方法に関わらず、(i)3種全ての胚葉の細胞へと分化する能力、(ii)少なくともOct4およびアルカリホスファターゼの発現、ならびに(iii)免疫不全動物中に移植した場合に奇形腫を生成する能力、に基づいて同定され得る。本発明の実施形態において使用され得る胚性幹細胞には、ヒトES細胞(「ESC」または「hES細胞」)、例えばMA01、MA09、ACT−4、No.3、H1、H7、H9、H14およびACT30胚性幹細胞が含まれるがこれらに限定されない。さらなる例示的な細胞株には、NED1、NED2、NED3、NED4、NED5およびNED7が含まれる。NIH
Human Embryonic Stem Cell Registryもまた参照のこと。使用され得る例示的なヒト胚性幹細胞株は、MA09細胞である。MA09細胞の単離および調製は、Klimanskayaら(2006年)「Human Embryonic Stem Cell lines Derived from Single Blastomeres」Nature 444巻:481〜485頁中に以前に記載された。本開示に従って使用され得る他のES細胞の単離および調製もまた、Chungら(2008年)「Human Embryonic Stem Cell Line
Generated Without Embryo Destruction」、Cell Stem Cell、2巻:113〜117頁中に以前に記載されている。本発明の例示的な実施形態に従って使用されるヒトES細胞は、GMP標準に従って誘導および維持され得る。
おいて発現される。他の実施形態では、少なくとも3種の再プログラミング因子が、体細胞を首尾よく再プログラミングするために、体細胞において発現される。他の実施形態では、少なくとも4種の再プログラミング因子が、体細胞を首尾よく再プログラミングするために、体細胞において発現される。他の実施形態では、さらなる再プログラミング因子が同定され、体細胞を多能性幹細胞へと再プログラミングするために、単独で、または1種もしくは複数の公知の再プログラミング因子と組み合わせて使用される。人工多能性幹細胞は、機能的に定義され、これには、種々の方法(組込みベクター、非組込みベクター、化学的手段など)のいずれかを使用して再プログラミングされた細胞が含まれる。多能性幹細胞は、寿命、有効性、ホーミングを増加させるため、同種免疫応答を予防もしくは低減させるため、またはかかる多能性細胞から分化した細胞(例えば、血小板)中に所望の因子を送達するために、遺伝子改変または他の方法で改変され得る。
イにおいて試験され得る。例えば、この細胞は、ES細胞マーカーの発現について試験され得る;この細胞は、SCIDマウス中に移植した場合に奇形腫を生成する能力について評価され得る;この細胞は、3種全ての胚葉の細胞型を生成するように分化する能力について評価され得る。多能性iPSCが取得されると、これは、巨核球細胞および血小板を生成するために使用され得る。
場合、造血性細胞を生じる。
の血液細胞を生成することが可能である。MK生成のための主要なシグナルはTPOである。TPOは、骨髄中で最終的なMK表現型に向かう前駆細胞の分化を誘発する。MKは、以下の系列を介して発生する:CFU−ME(多能性造血幹細胞または血球芽細胞)、巨核芽球、前巨核球、巨核球。この細胞は、最終的に巨核芽球段階に達し、分裂する能力を喪失する。しかし、この細胞は、そのDNAを依然として複製でき、発生を続けて、倍数性になる。細胞質は、拡大し続け、DNA相補体は、64Nよりも大きくなるまで増加し得る。
る。その細胞質ドメイン内に6つのITIMを有するPECAM1は、このサブファミリーのメンバーである。
異的前駆細胞は、フィーダーフリー条件下で維持され得る。
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多能性由来造血性内皮細胞(PVE−HE)の生成。
ヒトiPS由来PVE−HE細胞から、剥離した巨核球系列特異的前駆体(MLP)を生成する。
リセロール(a−MTG)、rhインスリン−トランスフェリン−セレン−エタノールアミン溶液、タンパク質フリーハイブリドーマ混合物II(PFHMII)、アスコルビン酸2ホスフェート、Glutamax I(L−アラニル−L−グルタミン)、ペニシリン/ストレプトマイシン、幹細胞因子(SCF)25ng/ml、トロンボポエチン(TPO)25ng/ml、Fms関連チロシンキナーゼ3リガンド(FL)25ng/ml、インターロイキン−3(IL−3)10ng/ml、インターロイキン−6(IL−6)10ng/mlおよびヘパリン5単位/mlから構成されるMLP誘導および拡大増殖培地(MLP−DEM、APELまたはStemline IIとも呼ばれる、図1に示すとおり)で置き換えた。次いで、細胞を、5%CO2中37℃で最大8日間インキュベートした。MLP分化を8日間維持した。
MSOを含む細胞凍結培地CS10(Sigma)を使用して達成した。
ヒトiPS−PVE−HE−MLP由来巨核球(MK)からの成熟血小板の生成。
iPS−PVE−HE−MLP由来巨核球(MK)由来の成熟血小板の分析。
in vivo環境においてhiPSC−PLTが血餅形成に寄与することを示す。hiPSC−PLTの機能的能力もまた、αIIbβIIIによって媒介されることが決定された。具体的には、hiPSC−PLTを、αIIbβIIIに特異的に結合し血小板機能を阻害するヒト−マウスキメラモノクローナル抗体のFab断片ReoProで事前処理した(図12B)。アスタリスクは、ReoProで処理していない対照と比較した統計的に有意な差異を示す(対照と比較してp<.01、スチューデントt検定)。
多能性細胞からの血小板の生成。
ば、1〜6)で標識する。播種したプレートを、37℃、5%CO2インキュベーター中に配置する。培養物を、立体顕微鏡および倒立光学顕微鏡を使用して毎日点検する。コロニーサイズ、分化の程度を含む細胞形態および培地の色に関する観察を、ワークシート上に記録する。mTeSR1培地を、典型的には、培養物が60〜90%コンフルエントになるまで、1〜2日毎に交換する。形態およびコンフルエンス評価がその培養物の継代の必要性を示す場合、コロニーを細胞解離緩衝液を使用して回収する。細胞を凝集体として維持するように再度注意を払う。培養物を、mTeSR1培地中のMatrigel被覆容器上に再播種する。回収されたcm2および播種されるcm2に基づいて、細胞を、典型的には、必要とされる幹細胞の収量に依存して、3〜7日毎に1:4〜1:8の比率で分割する。播種した培養物を、37℃、5%CO2インキュベーター中に戻す。幹細胞は、必要とされるロットサイズに依存して、造血性分化の誘発の前に1〜6回継代され得る。
によって、達成する。
Jose、CA)を含むDPBS+3%FBS(FACS緩衝液)中に再懸濁する。蛍光コンジュゲート化抗体および適切なアイソタイプ対照(マウスIgG1k−APCおよびマウスIgG1k−PE)を、室温で15分間存在下でインキュベートする。次いで、標識された細胞を、FACS緩衝液中に希釈し、遠心分離し、FACS緩衝液中に再懸濁する。FACS分析を、10,000の事象をモニタリングすることによって実施する。二重陽性(CD41a+CD42b+)MLPを発現する細胞の許容可能な百分率を有する培養物を、回収し、凍結保存する。暫定の最低仕様は、10%二重陽性細胞である。hiPSCから誘導されたMLPについての代表的な2次元ドットプロットが、図15に示される。この図では、細胞集団は、二重陽性染色集団29.9%で、86.4%CD41a+;31.2%CD42b+である。
0)。
理する(好ましくは少なくとも10%の二重陽性細胞)。
1)60μL/チューブの最終反応容量中に、既知の数の蛍光ビーズおよびフィコエリ
トリンコンジュゲート化抗CD61 IgGと共に凍結乾燥ペレットを含む1つのTruCount(BDカタログ番号340334)チューブ、ならびに
2)60μL/チューブの最終反応容量中に、非特異的一次抗体結合を説明するためにPEコンジュゲート化マウスIgGを含む1つのアイソタイプ対照チューブ。
FACSによって)確認する。感度を増強するために、検出方法は、低速遠心分離と併せたフィルター、マトリックスまたは勾配で捕捉することによって潜在的な細胞汚染物を濃縮して細胞をペレット化する一方、上清中に血小板を残す手順と組み合わされ得る。
血小板潜在力およびPAC−1結合についてのアッセイ。
ンに曝露させた活性化PLTおよび対照試料を、室温で15〜20分間インキュベートする。試料は、対照としてヒト血液血小板を使用して決定される前方散乱対側方散乱ゲーティングを用いたFACS分析を受ける。活性化したものにおけるCD41aおよびPAC−1陽性事象の数を、非活性化対照におけるCD41aおよびPAC−1陽性事象の数と比較することによって、PAC−1結合(活性化)を定量する。
剪断力下で生成された血小板の収量および純度を改善するためのMMPインヒビターなどのプロテアーゼの使用。
が剪断力培養下でMKから生成されたことを実証している。
静置培養での血小板形成において有用な保護的インヒビターとしての、MMP8特異的インヒビター。
昇温でのiPS血小板生成。
iBETは、c−myc遺伝子の下方調節を介して、巨核球傾倒を促進し、全体的血小
板収量を増加させる。
Claims (73)
- 少なくとも108個の血小板を含む、ヒト患者における使用に適切な医薬調製物であって、該調製物が白血球を実質的に含まず、該血小板の実質的に全てが機能的である、医薬調製物。
- 109〜1014個の血小板、任意選択で、109個、1010個、1011個、1012個、1013個または1014個の血小板を含む、請求項1に記載の医薬調製物。
- 前記血小板が、以下の属性:9.7〜12.8fLの平均血小板容量範囲;前記調製物中のサイズの単峰型分布;および/または1標準偏差が2μm3未満(好ましくは、1.5μm3未満、1μm3未満またはさらには0.5μm3未満)である対数正規型血小板容量分布のうち1つまたは複数を有する、請求項1〜2のいずれか一項に記載の医薬調製物。
- 前記血小板が、以下のマーカー:CD41aおよびCD42bのうち少なくとも1つについて陽性である、請求項1〜3のいずれか一項に記載の調製物。
- 前記血小板がヒト血小板である、請求項1〜4のいずれか一項に記載の調製物。
- 前記血小板の少なくとも50%、60%、70%、80%または90%が、室温での貯蔵後少なくとも2、3または4日間にわたって機能的である、請求項1〜5のいずれか一項に記載の調製物。
- フィーダー細胞なしに機能的血小板を生成する弱い接着性のまたは非接着性の巨核球を有するバイオリアクター。
- 少なくとも109個のMLPを含む組成物。
- MLPを含む凍結保存組成物。
- 凍結保存されたMLPを含むバンク。
- 前記MLPが、規定されたHLA型のものである、請求項9または10に記載の凍結保存組成物またはバンク。
- 患者に対してHLAがマッチしている、請求項9に記載の凍結保存組成物。
- 109〜1014個のMLP、任意選択で109個、1010個、1011個、1012個、1013個または1014個のMLPを含む、請求項9〜12のいずれか一項に記載の凍結保存組成物またはバンク。
- 巨核球から血小板を生成するための方法であって、
a)巨核球の非接着性培養物を提供するステップ;
b)該巨核球をTPOまたはTPOアゴニストと接触させて、培養において前血小板の形成を引き起こすステップであって、該前血小板が血小板を放出するステップ;および
c)該血小板を単離するステップ
を含む方法。 - 巨核球から血小板を生成するための方法であって、
a)巨核球の非接着性培養物を提供するステップ;
b)該巨核球を、造血拡大増殖培地ならびに任意選択で(1)TPOもしくはTPOアゴニスト、SCF、IL−6およびIL−9または(2)TPOもしくはTPOアゴニスト、SCFおよびIL−11と接触させて、培養中において前血小板の形成を引き起こすステップであって、該前血小板が血小板を放出するステップ;および
c)該血小板を単離するステップ
を含む方法。 - 前記TPOアゴニストが、ADP、エピネフリン、トロンビン、コラーゲン、TPO−Rアゴニスト、TPO模倣物、第2世代血小板新生剤、ロミプロスチム、エルトロンボパグ(SB497115、Promacta)、組換えヒトトロンボポエチン(TPO)、ペグ化組換えヒト巨核球増殖発達因子(PEG−rHuMGDF)、Fab 59、AMG 531、Peg−TPOmp、TPO非ペプチド模倣物、AKR−501、モノクローナルTPOアゴニスト抗体、ポリクローナルTPOアゴニスト抗体、TPOミニボディ、VB22B sc(Fv)2、ドメインサブクラス変換TPOアゴニスト抗体、MA01G4G344、組換えヒトトロンボポエチン、組換えTPO融合タンパク質またはTPO非ペプチド模倣物のうち1つまたは複数を含む、請求項14または15に記載の方法。
- 実質的に全ての前記単離された血小板が機能的である、請求項14〜16のいずれか一項に記載の方法。
- 巨核球の前記非接着性培養物が、フィーダーフリーの培養物である、請求項14〜17のいずれか一項に記載の方法。
- ステップ(b)における前記培養が、0.5〜100ng/mlの幹細胞因子(SCF)、10〜100ng/mlのトロンボポエチン(TPO)および10〜100ng/mlのインターロイキン−11(IL−11)、少なくとも1種のROCKインヒビター、ならびに/または2.5〜25単位/mlのヘパリンのうち1つまたは複数を含む培地中である、請求項14〜18のいずれか一項に記載の方法。
- ステップ(b)における前記培養が、10〜100ng/mlのTPO、0.5〜100ng/mlのSCF、5〜25ng/mlのIL−6、5〜25ng/mlのIL−9、少なくとも1種のROCKインヒビター、および/または2.5〜25単位/mlのヘパリンのうち1つまたは複数を含む培地中である、請求項14〜18のいずれか一項に記載の方法。
- 前記少なくとも1種のROCKインヒビターがY27632を含む、請求項19または20に記載の方法。
- 前記Y27632が、2〜20μM、約3〜10μM、約4〜6μMまたは約5μMの濃度内である、請求項21に記載の方法。
- 前記巨核球を剪断力に供するステップをさらに含む、請求項14〜22のいずれか一項に記載の方法。
- 巨核球1個当たり少なくとも2個、3個、4個または5個の血小板が生成される、請求項14〜23のいずれか一項に記載の方法。
- 巨核球1個当たり少なくとも50個の血小板が生成される、請求項24に記載の方法。
- 巨核球1個当たり少なくとも100個、500個、1000個、2000個、5000個または10000個の血小板が生成される、請求項25に記載の方法。
- 前記血小板の少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、少なくとも90%または少なくとも95%が、CD41a+およびCD42b+である、請求項14〜26のいずれか一項に記載の方法。
- 前記血小板が、フィーダー細胞または間質フィーダー細胞の非存在下で生成される、請求項14〜27のいずれか一項に記載の方法。
- 前記血小板が、全ての異種細胞の非存在下で生成される、請求項14〜28のいずれか一項に記載の方法。
- 前記血小板がヒトのものである、請求項14〜29のいずれか一項に記載の方法。
- 前記巨核球が、外因的に添加されたプロテアーゼインヒビターの存在下で培養される、請求項14〜30のいずれか一項に記載の方法。
- 前記巨核球が、外因的に添加されたMMPインヒビターの存在下で培養される、請求項14〜30のいずれか一項に記載の方法。
- 前記巨核球が、外因的に添加されたMMP8インヒビターの存在下で培養される、請求項14〜30のいずれか一項に記載の方法。
- 前記巨核球が、外因的に添加されたMMP8特異的インヒビターおよび汎MMPインヒビターの存在下で培養される、請求項14〜30のいずれか一項に記載の方法。
- 前記巨核球が、約39℃の温度で培養される、請求項14〜34のいずれか一項に記載の方法。
- 前記巨核球が、
(a)多能性幹細胞を培養して、造血性内皮細胞(PVE−HE)を形成するステップ;
(b)該造血性内皮細胞を培養して、MLPを形成するステップ;および
(c)該MLPを培養して、巨核球を形成するステップ
を含むステップによって生成される、請求項14〜35のいずれか一項に記載の方法。 - BETインヒビターが、ステップ(b)における前記培養物に添加される、請求項36に記載の方法。
- 前記BETインヒビターがIBET151である、請求項37に記載の方法。
- 前記多能性幹細胞がヒトのものである、請求項36〜38のいずれか一項に記載の方法。
- 前記造血性内皮細胞が、胚様体形成なしに誘導される、請求項36〜39のいずれか一項に記載の方法。
- 前記多能性幹細胞が、人工多能性幹細胞(iPSC)である、請求項36〜40のいずれか一項に記載の方法。
- 前記iPSCがヒトのものである、請求項41に記載の方法。
- 前記造血性内皮細胞が、胚様体形成なしに誘導される、請求項41または42に記載の方法。
- 前記造血性内皮細胞が、1%〜10%の酸素、2%〜8%の酸素、3%〜7%の酸素、4%〜6%の酸素または約5%の酸素を含む低酸素条件下で、前記多能性幹細胞から分化させられる、請求項36〜43のいずれか一項に記載の方法。
- 前記MLPが、38〜40セルシウス度の間の温度、または約39セルシウス度の温度で培養されて、巨核球を形成する、請求項36〜44のいずれか一項に記載の方法。
- 請求項14〜45のいずれか一項に記載の方法によって生成された血小板を含む医薬調製物。
- ヒト患者における使用に適切であり、少なくとも108個の血小板を含む、請求項46に記載の調製物。
- ヒト患者における使用に適切であり、白血球を実質的に含まない、請求項46に記載の調製物。
- 前記請求項のいずれかに記載の組成物または前記請求項のいずれかに記載の方法によって生成された組成物の使用であって、それを必要とする患者あるいは凝固に影響する疾患もしくは障害に罹患している患者またはそれによって処置可能な疾患もしくは障害に罹患している患者の処置のための医薬の製造における使用。
- 前記疾患または障害が、血小板減少症、外傷、血液媒介寄生生物またはマラリアを含む、請求項49に記載の使用。
- 血小板輸血を必要とする患者を処置する方法であって、前記請求項のいずれかに記載の組成物または前記請求項のいずれかに記載の方法によって生成された組成物を、該患者に投与するステップを含む方法。
- 血小板減少症、外傷、血液媒介寄生生物またはマラリアを含む疾患または障害を処置するために有効である、請求項51に記載の方法。
- 巨核球またはMLPから血小板を生成するための方法であって、
プロテアーゼインヒビターの存在下において、剪断力条件下で、MLPの巨核球の非接着性集団を培養するステップ、および
該培養物から血小板を回収し、任意選択で単離するステップ
を含む方法。 - 前記プロテアーゼインヒビターがMMPインヒビターである、請求項53に記載の方法。
- 前記剪断力条件が一定の剪断力条件である、請求項53または54に記載の方法。
- 前記剪断力条件が、1〜4.1ダイン/cm2の剪断力を含む、請求項53〜55のいずれか一項に記載の方法。
- 前記巨核球またはMLPが、マイクロ流体デバイス中で培養される、請求項53〜56のいずれか一項に記載の方法。
- 前記巨核球またはMLPが、iPS細胞、ES細胞、または天然に存在するCD34+細胞、任意選択で、骨髄もしくは臍帯血CD34+細胞から誘導される、請求項53〜57のいずれか一項に記載の方法。
- 前記プロテアーゼインヒビターがGM6001である、請求項53〜58のいずれか一項に記載の方法。
- 前記プロテアーゼインヒビターがMMP8特異的インヒビターである、請求項53〜58のいずれか一項に記載の方法。
- 前記MMP8特異的インヒビターが、MMP8−I((3R)−(+)−[2−(4−メトキシベンゼンスルホニル)−1,2,3,4−テトラヒドロイソキノリン−3−ヒドロキサメート])である、請求項60に記載の方法。
- 2種またはそれより多くのプロテアーゼインヒビターが使用される、請求項53〜61のいずれか一項に記載の方法。
- 前記2種のプロテアーゼインヒビターが、MMP一般的インヒビターおよびMMP8特異的インヒビターである、請求項62に記載の方法。
- 前記プロテアーゼインヒビターが、前記培養物内の血小板のピーク生成の時点で添加される、請求項53〜63のいずれか一項に記載の方法。
- 前記巨核球またはMPLが、TPOまたはTPOアゴニストの存在下で培養されて、前血小板の形成を引き起こし、該前血小板が血小板を放出する、請求項53〜64のいずれか一項に記載の方法。
- 前記巨核球またはMPLが、造血拡大増殖培地中、および任意選択で
(1)TPOもしくはTPOアゴニスト、SCF、IL−6およびIL−9または
(2)TPOもしくはTPOアゴニスト、SCFおよびIL−11
中で培養されて、培養において前血小板の形成を引き起こし、該前血小板が血小板を放出する、請求項53〜65のいずれか一項に記載の方法。 - 前記巨核球またはMPLが、37℃よりも高く40℃と等しいかまたはそれより低い温度で培養される、請求項53〜66のいずれか一項に記載の方法。
- 前記巨核球またはMPLが約39℃の温度で培養される、請求項67に記載の方法。
- 巨核球またはMPLから血小板を生成するための方法であって、
37℃よりも高く40℃と等しいかまたはそれより低い温度で、iPS細胞またはES細胞から誘導された巨核球またはMPLの非接着性集団を培養するステップ、および
該培養物から血小板を回収し、任意選択で単離するステップ
を含む方法。 - 前記巨核球またはMPLが約39℃の温度で培養される、請求項69に記載の方法。
- PVE−HE細胞からMPLを生成するための方法であって、
BETのインヒビターの存在下で、iPS細胞またはES細胞から誘導されたPVE−HE細胞の集団を培養するステップ、および
該培養物からMPLを回収し、任意選択で単離するステップ
を含む方法。 - BETの前記インヒビターがI−BET151である、請求項71に記載の方法。
- PVE−HE細胞からMPLを生成するための方法であって、
c−mycサプレッサーの存在下で、iPS細胞またはES細胞から誘導されたPVE−HE細胞の集団を培養するステップ、および
該培養物からMPLを回収し、任意選択で単離するステップ
を含む方法。
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