JP5856029B2 - 間葉系幹細胞を未分化増殖させる方法、および間葉系幹細胞を濃縮する方法 - Google Patents
間葉系幹細胞を未分化増殖させる方法、および間葉系幹細胞を濃縮する方法 Download PDFInfo
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Description
上述の通り、本発明に係る方法は、臍帯血における間葉系幹細胞を濃縮する工程を包含している。当該工程において、間葉系幹細胞は、血球細胞を特異的に認識する抗体を用いて臍帯血から血球細胞の大部分を除去することによって濃縮される。当該抗体は、磁気ビーズなどの担体に結合している。抗体と結合している担体を、臍帯血(由来のサンプル)と混合した後に、抗体を介して血球細胞と結合している担体を回収することによって、間葉系幹細胞を濃縮したサンプルが得られる。代替的な方法として、上記抗体はビオチンによって標識され得る。当該方法では、上記抗体を標識しているビオチンに対するストレプトアビジンの相互作用を利用して、血球細胞を選択的に除去し得る。
本発明に係る方法は、間葉系幹細胞を濃縮したサンプルを培養する工程を包含している。当該工程において、上記サンプルは、増殖因子としてSHH、FGF1またはFGF2を含んでいる培地を用いて培養される。当該培地を用いることによって、Flt−3L、IL−3およびIL−6を添加した培地では増殖しなかった(実施例を参照)間葉系幹細胞を、未分化増殖させ得る。
フィコール溶液(GEヘルスケア社)を用いた密度勾配遠心分離(30分間、900g、25℃)によって、25mLの臍帯血から単核球画分を分離した。RoboSep装置(Stem Cell Technologies社)を用いて、得られた単核球画分から血球細胞をさらに取り除いて、残りの細胞集団を濃縮した。この単核球画分からの血球細胞の除去において、Negative Selection Progenitor Cell Enrichment kit(Stem Cell Technologies社)を試薬として用いた。当該試薬には、磁気ビーズと結合している抗Glycophorin A抗体、抗CD2抗体、抗CD3抗体、抗CD11c抗体、抗CD11b抗体、抗CD14抗体、抗CD16抗体、抗CD19抗体、抗CD24抗体、抗CD56抗体、抗CD61抗体および抗CD66b抗体が含まれている。したがって、当該試薬は、これらの抗体によって認識される抗原を表面発現している細胞を除去可能である。当該分離における手順および必要な他の試薬のすべてについて、上記試薬に対応しているRoboSep装置の動作プログラム、および付属のユーザマニュアルにしたがった。
(2−1.臍帯血由来の細胞の増殖に対するSHHの作用)
1.において入手した細胞を次のように増殖させた。培養の0〜14日にわたって、Flt−3L(Cellgenix社)、SCF(Cellgenix社)、IL−3(Miltenyi Biotec社)およびIL−6(Miltenyi Biotec社)およびSHH(Miltenyi Biotec社)を補ったMesenCult-XF Medium(Stem Cell Technologies社)を用いて、5%CO2の存在下の37℃において細胞を増殖させた。上記培地における各サイトカインの最終濃度はそれぞれ、2000IU/mL(Flt−3L)、100ng/mL(SCF)、1000IU/mL(IL−3)、1000IU/mL(IL−6)および100ng/mL(SHH)であった。上記サイトカインカクテルは造血幹細胞の増殖用のサイトカインカクテルとして提供されている。培地に添加した因子の有効性を確認するために、上記培養と平行して、Flt−3L、IL−3およびIL−6のみを補ったMesenCult-XF Medium、およびMesenCult-XF Mediumのみを用いて、1.において入手した細胞をそれぞれ培養した。培養の0、3、6、8、11および14日目のそれぞれにおける総細胞数を、トリパンブルー染色によって決定した。決定した細胞数の経時的変化を図2に示した。
2−1.の培養によって増殖した細胞が間葉系幹細胞であることを確認するために、培養の14日目において増殖している細胞の種類をFACS解析によって確認した。14日目の培養物の一部は、蛍光標識された抗CD105抗体(Biolegend社)および蛍光標識された抗CD73抗体(Biolegend社)を用いて調べた。すべてのFACS解析は、BD FACSCalibur(登録商標)フローサイトメーター(日本BD社)を用いて実施した。これらのFACS解析の結果を図3(抗CD105抗体)および図4(抗CD73抗体)に示す。図3は、培養14日目におけるCD105陽性細胞についてFACS解析した、シングルリニアリージョンのヒストグラムを示す図である。図4は、培養前および培養14日目におけるCD73陽性細胞についてFACS解析した、シングルリニアリージョンのヒストグラムを示す図である。
1.において入手した細胞を96穴プレートにおいて次のように培養した。後述の表1の組合せのサイトカイン(5ng/mLのFGF1(Miltenyi Biotech社)、5ng/mLのFGF2(Miltenyi Biotech社)、100ng/mLのSCF(Cellgenix社))を補ったMesenCult-XF Mediumを用いて、5%CO2の存在下の37℃において細胞を増殖させた。7日間の培養の後に、顕微鏡によって細胞数を確認して、サイトカインの各組合せによる間葉系幹細胞の増殖に対する作用を評価した。
Claims (5)
- 間葉系幹細胞を未分化増殖させる方法であって、
血球細胞を特異的に認識する抗体を用いて、臍帯血における間葉系幹細胞を濃縮したサンプルを生成する工程と、
SHH、FGF1またはFGF2を含んでいる培地において、上記サンプルを培養する工程とを包含しており、
上記培地はSCFをさらに含んでいる、方法。 - 上記培地は、Flt−3L、IL−3およびIL−6をさらに含んでいる、請求項1に記載の方法。
- 上記臍帯血は、ヒト臍帯血である、請求項1または2に記載の方法。
- 間葉系幹細胞を濃縮する方法であって、
血球細胞と特異的に結合する抗体ビーズを用いて、臍帯血における間葉系幹細胞を濃縮する工程を包含しており、
上記抗体ビーズは、抗Glycophorin A抗体、抗CD2抗体、抗CD3抗体、抗CD11c抗体、抗CD11b抗体、抗CD14抗体、抗CD16抗体、抗CD19抗体、抗CD24抗体、抗CD56抗体、抗CD61抗体、および抗CD66b抗体と結合している、方法。 - 上記臍帯血は、ヒト臍帯血である、請求項4に記載の方法。
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JP2012192560A JP5856029B2 (ja) | 2012-08-31 | 2012-08-31 | 間葉系幹細胞を未分化増殖させる方法、および間葉系幹細胞を濃縮する方法 |
CN201380033421.8A CN104395460B (zh) | 2012-08-31 | 2013-08-08 | 使间质干细胞不分化地增殖的方法、及浓缩间质干细胞的方法 |
PCT/JP2013/071559 WO2014034407A1 (ja) | 2012-08-31 | 2013-08-08 | 間葉系幹細胞を未分化増殖させる方法、および間葉系幹細胞を濃縮する方法 |
US14/342,975 US9670461B2 (en) | 2012-08-31 | 2013-08-08 | Method for undifferentiated growth of mesenchymal stem cell and method for concentration of mesenchymal stem cell |
KR1020147036218A KR101813269B1 (ko) | 2012-08-31 | 2013-08-08 | 간엽계 줄기세포를 미분화 증식시키는 방법 및 간엽계 줄기세포를 농축하는 방법 |
SG11201407568YA SG11201407568YA (en) | 2012-08-31 | 2013-08-08 | Method for undifferentiated growth of mesenchymal stem cell and method for concentration of mesenchymal stem cell |
EP13833725.8A EP2891713B1 (en) | 2012-08-31 | 2013-08-08 | Method for undifferentiated proliferation of mesenchymal stem cells, and method for concentrating mesenchymal stem cells |
CA2872852A CA2872852C (en) | 2012-08-31 | 2013-08-08 | Method for undifferentiated proliferation of mesenchymal stem cell and method for concentration of mesenchymal stem cell |
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WO2006128029A2 (en) * | 2005-05-25 | 2006-11-30 | University Of Virginia Patent Foundation | Production of osteoclasts from adipose tissues |
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CA2872852C (en) | 2017-05-16 |
US20140220686A1 (en) | 2014-08-07 |
JP2014045733A (ja) | 2014-03-17 |
KR20150022895A (ko) | 2015-03-04 |
WO2014034407A1 (ja) | 2014-03-06 |
KR101813269B1 (ko) | 2017-12-29 |
EP2891713A4 (en) | 2016-01-27 |
EP2891713B1 (en) | 2018-11-07 |
US9670461B2 (en) | 2017-06-06 |
EP2891713A1 (en) | 2015-07-08 |
CA2872852A1 (en) | 2014-03-06 |
SG11201407568YA (en) | 2015-01-29 |
CN104395460B (zh) | 2019-06-18 |
HK1205187A1 (en) | 2015-12-11 |
CN104395460A (zh) | 2015-03-04 |
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