JP5856949B2 - 人工多能性幹細胞の製造方法 - Google Patents
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- JP5856949B2 JP5856949B2 JP2012510720A JP2012510720A JP5856949B2 JP 5856949 B2 JP5856949 B2 JP 5856949B2 JP 2012510720 A JP2012510720 A JP 2012510720A JP 2012510720 A JP2012510720 A JP 2012510720A JP 5856949 B2 JP5856949 B2 JP 5856949B2
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Description
本出願は、2010年4月16日付で出願した日本国特許出願2010−95404に基づく優先権を主張するものであり、当該基礎出願を引用することにより、本明細書に含めるものとする。
まず、末梢血から調製した単核球集団を、抗CD3抗体およびインターロイキンの存在下で3〜14日間、好ましくは3〜7日間培養する。この培養により、単核球のうち、CD3陽性T細胞の増殖が特異的に促進されると考えられる。
次に、単核球集団に対して脱分化処理を行う。
以上のようにして脱分化処理した単核球集団を10〜30日、好ましくは14〜25日、より好ましくは20日間、iPS細胞を培養する一般的な条件下で培養する。例えば、増殖因子含有DMEM/F12を用い、5%CO2存在下、35℃〜40℃、好ましくは37℃で培養すればよい。あるいは、DMEM等の培地を用いてもよく、MEFやSNL等で作製したフィーダー細胞を用いてもよい。また、増殖因子は特に制限されず、周知の増殖因子から当業者が適宜選択できるが、例えば、繊維芽細胞増殖因子(FGF)や上皮成長因子(EGF)であってもよい。
慶應大学病院倫理委員会で承認されたプロトコールに従って、インフォームドコンセントを行った各健常人ボランティア(11歳〜66歳、男女、計5名)から、1〜20mlの末梢血を採血した。フィコール・ハイパック(GE Healthcare 社)を比重液とし、遠心分離(30分間、400×g)を行い、末梢血から単核球分画を単離した。
以下(1)〜(4)の脱分化因子遺伝子を公知の方法(WO2010/008054)に従って、SeV18+/TSΔFベクター(WO2010/008054)に導入したセンダイウイルスの調製を行った。
(1)Oct3/4遺伝子
(2)Klf4遺伝子
(3)c−Myc遺伝子
(4)Sox2遺伝子
具体的には、(1)〜(4)の遺伝子を含む、マイナス鎖RNAウイルスゲノムRNA(マイナス鎖)またはその相補鎖(プラス鎖)をコードするcDNAの組み換えウイルスゲノムを発現するプラスミドベクター、及びウイルスの自己複製に必要な蛋白質(F、N、P、L、T7RNAポリメラーゼ)を発現するプラスミドベクターを293T/17細胞に導入し、さらにFタンパク質を発現するLLC−MK2/F/A細胞を重層して培養を行い、生成したウイルスを含む培養上清を回収することにより製造した。
(株化マウス胚性繊維芽細胞、SNL)
ディッシュに0.1%ゼラチンを加え、37℃で約1時間静置し、ディッシュをコーティングした。SNL(EGACC社)を1.5×105細胞/mlの密度に調製し、1ディッシュ(直径10cm)あたり10mlを加え、一晩培養し、フィーダー細胞を調製した。
抗CD3抗体(BD Biosciences 社)をPBSで10μg/mlに調製した。この抗体希釈液で6ウェルディッシュの底面を覆い、37℃で30分間〜3時間インキュベートした。使用直前に抗体希釈液を取り除き、PBSで洗浄して抗CD3抗体結合ディッシュとした。
(脱分化処理1日目)
抗CD3抗体を用いて培養した単核球集団を7.5×105細胞/mlの密度に調製し、ここにMOI 1、3、5、10、あるいは20でセンダイウイルスを添加することによって、組み換えベクターを導入した後、KBM502培地中で24時間培養した。
セルスクレーパーで細胞を剥がし取り、細胞を含む培地を、ウェル毎にチューブに回収した。20℃、800〜1000rpmで5分間遠心した後、ペレットにKBM502培地2mlを加え、数回ピペッティングし、ペレットをシングルセルにならない程度に破壊し、懸濁した。この懸濁液を、元のディッシュのウェルに戻し、KBM502培地中で24時間培養した。
セルスクレーパーで細胞を剥がし取り、細胞を含む培地をウェル毎にチューブに回収した。ピペッティングによって、細胞をシングルセルにし、細胞数を計数した。20℃、800〜1000rpmで5分間遠心した後、ペレットに適量のKBM502培地を加えた。ピペッティングによって、ペレットをシングルセルにし、10cmディッシュに調製したフィーダー細胞(SNL)上に、5×104、5×105、5×106/ディッシュの密度で単核球を播種し、KBM502培地中で24時間培養した。
上記のように脱分化処理した細胞を、フィーダー細胞(SNL)上に播種し、培地を10ng/mlヒト塩基性繊維芽細胞増殖因子(bFGF、和光純薬工業)添加iPS細胞培地(10ml/10cmディッシュ)に交換した。その後、48時間毎に培地を交換し、20日間培養を続けた。なお、iPS細胞培地は、DMEM/F12(Invitrogen 社)、20%knockout serum replacement (Invitrogen 社)、2mM L−グルタミン、(Invitrogen 社)、1×10-4M 非必須アミノ酸(Invitrogen 社)、1×10-4M2−メルカプトエタノール(Invitrogen 社)、0.5%ペニシリン−ストレプトマイシン(和光純薬工業)から成る。
脱分化処理により得られたコロニーに対し、アルカリフォスファターゼ染色、および、クリスタルバイオレット染色を行った。まず、コロニーを10%中性緩衝ホルマリン液(和光純薬工業)で固定した後、1-Step NBT/BCIP(Pierce 社)で染色した。さらに、クリスタルバイオレットをメタノールに溶解して4%クリスタルバイオレット溶液を調製し、細胞に添加して30分間染色した。なお、アルカリフォスファターゼは幹細胞で発現することが知られており、幹細胞のマーカーとして用いられている(例えば、Riekstina U. et al., Stem Cell Rev. 2009 Dec 5(4): 378-386 参照)。また、クリスタルバイオレット染色により生細胞のみが染色される。さらに、DAPI(Molecular Probes 社)を用いて適宜核の対比染色を行った。
上記アルカリフォスファターゼ陽性細胞のコロニーのうち、3コロニーをランダムにクローニングし、これらの細胞がiPS細胞であることを確認するため、DAPI染色およびアルカリフォスファターゼ染色を行い形態学的観察を行ったところ、3つのクローンとも、胚性幹細胞あるいはiPS細胞に典型的な形態を有し、アルカリフォスファターゼ陽性であった。
さらに、各クローンの細胞について各種幹細胞マーカーのタンパク質および遺伝子発現を、免疫組織学的染色および逆転写ポリメラーゼ連鎖反応(RT−PCR)法により解析した。RT−PCR法では、脱分化処理前の単核球、および、脱分化処理後の単核球について同様に解析を行い、陽性コントロール細胞としてヒト胚性幹細胞を用いた(KhES−2、京都大学より入手)。
プライマー:
Nanog-F: CAGCCCCGATTCTTCCACCAGTCCC(配列番号1)
Nanog-R: CGGAAGATTCCCAGTCGGGTTCACC(配列番号2)
Oct 3/4-F: GACAGGGGGAGGGGAGGAGCTAGG(配列番号3)
Oct 3/4-R: CTTCCCTCCAACCAGTTGCCCCAAAC(配列番号4)
Sox 2-F: GGGAAATGGGAGGGGTGCAAAAGAGG(配列番号5)
Sox 2-R: TTGCGTGAGTGTGGATGGGATTGGTG(配列番号6)
Klf 4-F: ACGATCGTGGCCCCGGAAAAGGACC(配列番号7)
Klf 4-R: TGATTGTAGTGCTTTCTGGCTGGGCTCC(配列番号8)
cMyc-F: GCGTCCTGGGAAGGGAGATCCGGAGC(配列番号9)
cMyc-R: TTGAGGGGCATCGTCGCGGGAGGCTG(配列番号10)
GDF 3-F: CTTATGCTACGTAAAGGAGCTGGG(配列番号11)
GDF 3-R: GTGCCAACCCAGGTCCCGGAAGTT(配列番号12)
Rex 1-F: CAGATCCTAAACAGCTCGCAGAAT(配列番号13)
Rex 1-R: GCGTACGCAAATTAAAGTCCAGA(配列番号14)
DPPA 4-F: GGAGCCGCCTGCCCTGGAAAATTC(配列番号15)
DPPA 4-R: TTTTTCCTGATATTCTATTCCCAT(配列番号16)
DPPA 2-F: CCGTCCCCGCAATCTCCTTCCATC(配列番号17)
DPPA 2-R: ATGATGCCAACATGGCTCCCGGTG(配列番号18)
GAPDH-F: CAGAACATCATCCCTGCCTCTAG(配列番号19)
GAPDH-R: TTGAAGTCAGAGGAGACCACCTG(配列番号20)
比較例では、FACSにより末梢血単核球分画からT細胞を選別し、iPS細胞を製造する。
本比較例では、実施例に記載の単核球集団からiPS細胞を作製する際、抗CD3抗体およびインターロイキン2の存在下で単核球集団を培養する工程が必要であることを示す。
Claims (6)
- ヒト末梢血由来の単核球集団を材料とする、人工多能性幹細胞の製造方法であって、
(イ)ヒト末梢血由来の単核球集団を抗CD3抗体およびインターロイキン2の存在下で3〜14日間培養する工程と、
(ロ)培養後の前記単核球集団に対して脱分化処理を行う工程と
を含むことを特徴とする方法。 - 請求項1に記載の人工多能性幹細胞の製造方法であって、
前記工程(ロ)において、前記単核球集団に脱分化因子の導入操作を行うことを特徴とする方法。 - 請求項2に記載の人工多能性幹細胞の製造方法であって、
前記脱分化因子の導入操作において、前記脱分化因子を発現する組み換え発現ベクターの導入操作を行うことを特徴とする方法。 - 請求項3に記載の人工多能性幹細胞の製造方法であって、
前記組み換え発現ベクターがセンダイウイルスベクターであることを特徴とする方法。 - 請求項2〜4のいずれかに記載の人工多能性幹細胞の製造方法であって、
前記工程(ロ)において、前記脱分化因子がSox2、Oct3/4、Klf4およびc−Mycであることを特徴とする方法。 - 請求項1〜5のいずれかに記載の人工多能性幹細胞の製造方法であって、
(ハ)脱分化処理を行った前記単核球集団を、増殖因子の存在下で14〜25日間培養する工程をさらに含むことを特徴とする方法。
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CA2796599A1 (en) | 2011-10-20 |
WO2011129446A1 (ja) | 2011-10-20 |
US9447432B2 (en) | 2016-09-20 |
EP2559757B1 (en) | 2017-12-06 |
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