JP2016518831A - ブラストイド、細胞株に基づく人工胚盤胞 - Google Patents
ブラストイド、細胞株に基づく人工胚盤胞 Download PDFInfo
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Abstract
Description
Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff SL, Embryonic stem cell potency fluctuates with endogenous retrovirus activity Nature 2012 Jul 5; 487 (7405): 57-63; doi: 10.1038/nature11244
Morgani SM, Canham MA, Nichols J, Sharov AA, Migueles RP, Ko MS, Brickman JM, Totipotent embryonic stem cells arise in ground-state culture conditions. Cell Rep. 2013 Jun 27;3(6):1945-57. doi: 10.1016/j.celrep.2013.04.034. Epub 2013 Jun 6.)
Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. Obokata H, Sasai Y, Niwa H, Kadota M, Andrabi M, Takata N, Tokoro M, Terashita Y, Yonemura S, Vacanti CA, Wakayama T. Nature. 2014 Jan 30;505(7485):676-80. doi: 10.1038/nature12969
− 少なくとも1つの栄養芽細胞と、少なくとも1つの多能性および/または全能性細胞とを組み合わせることによって初期細胞凝集塊を形成する工程と、
− 前記初期細胞凝集塊を培養培地で培養し、内側細胞層と外側細胞層とを含む少なくとも二重層の細胞凝集塊を得る工程であって、内側細胞層が、前記少なくとも1つの多能性および/または全能性細胞に由来し、胚を形成することができる内部細胞を含み、外側細胞層が、前記少なくとも1つの栄養芽細胞に由来し、少なくとも栄養外胚葉を形成することができる外部細胞を含む工程と、
− 好ましくは、前記少なくとも二重層の細胞凝集塊を培養し、ブラストイドを得る工程とを含む。
− 少なくとも1つの栄養芽細胞と、少なくとも1つの多能性および/または全能性細胞とを組み合わせることによって初期細胞凝集塊を形成することと、
− 前記細胞凝集塊が少なくとも二重層の細胞凝集塊に組織化することを誘導することと、
− 好ましくは、少なくとも二重層の細胞凝集塊を細胞構造体の形成をすることができるように培養し、栄養外胚葉様組織が内部細胞塊と胞胚腔とを囲むこととによって、in vitro において少なくとも二重層の細胞凝集塊を得るための方法にも関する。
Morgani SM, Canham MA, Nichols J, Sharov AA, Migueles RP, Ko MS, Brickman JM, Totipotent embryonic stem cells arise in ground-state culture conditions. Cell Rep. 2013 Jun 27;3(6):1945-57. doi: 10.1016/j.celrep.2013.04.034. Epub 2013 Jun 6.)
Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. Obokata H, Sasai Y, Niwa H, Kadota M, Andrabi M, Takata N, Tokoro M, Terashita Y, Yonemura S, Vacanti CA, Wakayama T. Nature. 2014 Jan 30;505(7485):676-80. doi: 10.1038/nature12969 に記載されており、その内容は参照によって本明細書に組み込まれる。
・20μM Y27632
・3μM CHIR99021
・10ng/ml IGF2
・1mM 8Br-cAMP
・5ng/ml TGFb1
・15ng/ml FGF4
・1nM 007-AM
・1μM インドラクタムV
・300ng/ml Il6
・10%ウシ胎児血清(FBS)
・B27N2培地=当該技術分野において公知の細胞培養培地であり、その組成は上述された。
・E8培地=市販の細胞培養培地であり、その組成はNat. Methods. 2011,“Chemically defined conditions for human iPSC derivation and culture”・Chen G1, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA に記載されている。
・TX培地=細胞培養培地であり、その組成は、Kubaczka C, Senner C, Arauo-Bravo MJ, Sharma N, Kuckenberg P, Becker A, Zimmer A, Br・tle O, Peitz M, Hemberger M, Schorle H. Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions.Stem Cell Reports. 2014 Jan 30;2(2):232-42. doi: 10.1016/j.stemcr.2013.12.013. eCollection 2014 Feb 11 に記載されている。
・ES培地=市販の細胞培養培地であり、その組成は上述された。
・CHIR99021=GSK3aおよびGSK3βを抑制する化合物であり、化学名は、6-{2-[4-(2,4-ジクロロフェニル)-5-(4-メチル-1H-イミダゾール-2-イル)-ピリミジン-2-イルアミノ]‐エチルアミノ}-ニコチンニトリル(CAS 252917-06-9)である。
・LIF:白血病阻害因子は、当該技術分野で公知のタンパク質および増殖因子であり、マウスおよびラット胚性幹細胞の多能性を維持する。
・Y27632=(1R,4r)-4-((R)-1-アミノエチル)-N-(ピリミジン-4-イル)シクロヘキサンカルボキシアミドである。当該技術分野において公知の分子であり、Rho/ROCK経路を抑制する(CAS146986-50-7)。
・アクチビン=当該技術分野で公知のタンパク質および増殖因子であり、TGFシグナル経路を活性化する。
・形質転換増殖因子ベータ1(TGFb1)=当該技術分野で公知のタンパク質および増殖因子であり、TGFシグナル経路を活性化する。
・インドラクタムV=当該技術分野で公知の分子であり、PKC経路を調節する(90365-57-4)。
・Il6=当該技術分野で公知のタンパク質および増殖因子であり、STATシグナル経路を活性化する。
・繊維芽細胞増殖因子4(FGF4)=当該技術分野で公知のタンパク質および増殖因子であり、MAPKシグナル経路を活性化する。
・ヘパリン結合EGF様増殖因子(HB-EGF)=当該技術分野で公知のタンパク質および増殖因子であり、MAPKシグナル経路を活性化する。
・インスリン様増殖因子2(IGF2)=当該技術分野で公知のタンパク質および増殖因子であり、Akt経路を活性化する。
・インスリン=当該技術分野で公知のタンパク質およびホルモンであり、AKTシグナル経路を活性化する。
・17β-エストラジオール=当該技術分野で公知のホルモンであり、エストロゲン受容体とGPR30とを介してHippo経路を調節する(CAS79037-37-9)。
・タモキシフェン=当該技術分野で公知の分子であり、エストロゲン受容体とGPR30とを介してHippo経路を調節する(CAS10540-29-1)。
・G-1=当該技術分野で公知の分子であり、GPR30を介してHippo経路を調節する(CAS881639-98-1)。
・Pen/Strep 50μg/ml(Invitrogen社から入手可能である抗生物質、カタログ番号15070-063)
・L-グルタミン 1mM
・5ml 非必須アミノ酸(Invitrogen社 #11140-035、×100)
・ベータ-メルカプトエタノール 0.1mM
・75ml(=15%) FBS 75ml
・500mlまでのDMEM
・白血病阻害因子、500〜1000ユニット/ml
・1μM PD0325901 CAS番号 39210-10-9
・3μM CHIR99021 CAS番号 252917-06-9
・250mlのRPMI培地1640(Invitrogen 61870または11875)
・65mlのウシ胎児血清
・Pen/Strep,50μg/ml
・ピルビン酸ナトリウム 1mM
・L-グルタミン,2mM
・ベータ-メルカプトエタノール 0.1mM
・FGF-4,25ng/ml
・ヘパリン,1μg/ml
・234ml DMEM/F12(Invitrogen社 #11320-074,[-] L-グルタミン)
・234ml Neurobasal(Invitrogen社 #21103-049,[+] L-グルタミン)
・2.5ml (-1mM) L-グルタミン(Invitrogen社 #25030,×100)
・5ml 非必須アミノ酸(Invitrogen社 #11140-035,×100)
・5ml Pen/Strep(Invitrogen社 #15140)
・5mg/ml BSA(Sigma社)
・5ml ピルビン酸ナトリウム、最終濃度1mM(Invitrogen社 #11360-039,×100)
・2.3μl(=0.1mM) ベータ-メルカプトエタノール(Sigma社)
・5ml N2サプリメント(Invitrogen社)
・10ml B27サプリメント(Invitrogen社)
培養培地に好ましい成分を供給するための保存液は、
・10%FBS(Greiner Bio one社)
・Y27632:20μM
・CHIR99021(Axon Medchem社、axon 1386) 3μM
・8Br-cAMP(8Br-cAMP エコノミーグレード、Biolog社、B007-50E) 1mM
・007-AM(Biolog社 C051) 1nM
・IGF2(R&D system社、292-G2-050) 10ng/ml
・TGFb1(R&D system社 338-AC-010)、10ng/ml
・FGF4(R&D system社 235-F4-025)、15ng/ml
・HB-EGF(R&D system社 259-HE-050)、50ng/ml
・17βエストラジオール(491187 Aldrich社)、10ng/ml
・インドラクタムV(3651 Tocris社)、1μM
-4日目
マウス胚性繊維芽細胞(EF)-細胞は、組織培養ポリスチレンプレートにおけるES培地中に15,000細胞/cm2で播種された。細胞は、37℃に設定された培養器において1日間増殖させられた。
マウスES細胞は、LIF(500ユニット/ml)を含む「2i」培地中に15,000細胞/cm2でマウスEF層上に播種された。マウスES細胞は、37℃に設定された培養器において2日間培養された。
マウスTS細胞は、37℃に設定された培養器において、TS培地中に10000細胞/cm2の密度でマウスEF細胞層上に播種された。
マウスES細胞は、トリプシン処理され、マウスEF細胞は、4mlのES培地を用いて1つの15cmディッシュにおいて2×20分間培養することによって除去された。マウスES細胞は、150μLのES培地において7細胞/マイクロウェルで播種された。ES細胞は、20分間定着させられ、その後1mlのES培地を添加され、培養物は、37℃の培養器に静置され、マウスES細胞を1日間凝集させた。
上述のように得られたマウスTS細胞培養物はトリプシン処理され、マウスEF細胞は、4mlのTS培地を用いて1つの15cmディシュにおいて2×20分間培養することによって除去された。得られたマウスTS細胞は、B27N2培地中に再懸濁された。
B27N2培地は、以下の成分を添加され取り換えられる。
・FGF4(15ng/ml)
・TGFb1(5ng/ml)
アガロースチップを有する12ウェルプレートは、約110時間後に培養器から取り出された。これまでに人工胚盤胞がES-TS細胞クラスタの空洞化によって形成されていた。この時点で、非接着性足場を接着性足場(たとえば、ポリスチレン組織培養プレート)に置き換えることが可能であり、ブラストイドを in vitro で増やし続けるか、またはブラストイドを偽妊娠マウスの子宮に移植することが可能である。
二重層形成:TS細胞の外層とES細胞の内層とを有する(少なくとも)二重層の細胞凝集塊は、遺伝子導入された細胞株を用いて評価される。特に、ES細胞は、サイトメガロウイルスプロモータの制御下でヒストン2Bと赤色蛍光タンパク質とを導入され、クローンに由来する。H2B-RFP ES細胞は、したがって、それらの核内に赤色蛍光を恒常的に発現し、それらの追跡を可能にする。TS細胞は、全ての細胞でサイトメガロウイルスプロモータの制御下で緑色蛍光タンパク質(GFP)を恒常的に発現するマウスに由来するものであった。GFP TS細胞は、したがって、それらの細胞質において緑色蛍光を発現し、それらの追跡を可能にする。二重層凝集塊の形成は、したがって、細胞の緑色外層と、細胞の赤色内層との観察によって評価される。取り囲みの効率性は、ES-TS細胞クラスタ培養の24時間後、赤色投影面積(ES細胞の内部凝集塊を表す)の周囲に描かれる人工的な3ピクセル幅の環と重複する緑色投影面積(TS細胞層を表す)の割合として測定される。図4に記載された、上述の取り囲みの効率性は、約200個のES-TS細胞クラスタから得られた平均値を表す。
空洞化:空洞形成は、細胞核によって占められていない凝集塊内の体積形成によって評価される。空洞形成は、核特異的色素(たとえば、DAPI)によって染色された細胞凝集塊の横断面切片を用いて好ましく評価される。空洞形成は、細胞核を含まず、細胞凝集塊の総表面積の20%以上を覆う、細胞凝集塊の横断面内の表面の存在によってより好ましく評価される。
CDX2、Oct4、Nanog発現:転写因子CDX2の発現は、GFP発現TS細胞を含むES-TS凝集塊において従来の免疫細胞化学によって評価される。cdx2の発現は、10μmの顕微鏡切片あたりのcdx2陽性の核を有するGFP陽性細胞の数として評価される。転写因子Oct4とNanogとの発現は、H2B-RFPを発現するES細胞を含むES-TS凝集塊において従来の免疫細胞染色によって評価される。Oct4とNanogとの発現は、10μmの顕微鏡切片あたりのOct4またはNanog陽性核を有するH2B-REP陽性細胞の数として評価される。
in vitro で増殖されたマウスES細胞と、培養培地における細胞株に由来するマウスTS細胞とを組み合わせた後、当該培養培地は、好ましくは、Rho/ROCKシグナル調節因子、Wntシグナル調節因子、PKAシグナル調節因子、PKCシグナル調節因子、MAPKシグナル調節因子、STATシグナル調節因子、Aktシグナル調節因子、Tgfシグナル調節因子、Hippoシグナル調節因子およびEPAC1シグナル活性化因子の群から1つ以上を含み、ES細胞とTS細胞とはES-TS細胞クラスタに組み合わされ、約110時間後に空洞化、上皮化、多能性の維持、および原始内胚葉の分化を示す。したがって、ブラストイドが形成される。
Claims (17)
- 少なくとも2重層の細胞凝集塊、またはブラストイドを作製するためのin vitro法であって、
少なくとも1つの栄養芽細胞と、少なくとも1つの多能性および/または全能性細胞とを組み合わせることによって初期細胞凝集塊を形成する工程と、
培養培地において前記初期細胞凝集塊を培養し、内側細胞層と外側細胞層とを含む少なくとも2重層の細胞凝集塊を得る工程であって、前記内側細胞層は、前記少なくとも1つの多能性および/または全能性細胞に由来し、胚を形成することができる内部細胞を含み、前記外側細胞層は、前記少なくとも1つの栄養芽細胞に由来し、少なくとも栄養外胚葉を形成することができる外部細胞を含む工程と、
好ましくは、前記少なくとも2重層の細胞凝集塊を培養し、ブラストイドを得る工程とを含むことを特徴とする方法。 - 前記少なくとも2重層の細胞凝集塊は、胚性幹細胞と栄養膜幹細胞とを含むことを特徴とする請求項1に記載の方法。
- 前記少なくとも2重層の細胞凝集塊は、非接着性足場において形成されることを特徴とする請求項1または2に記載の方法。
- 前記非接着性足場は、マイクロウェルを含むことを特徴とする請求項3に記載の方法。
- 前記少なくとも2重層の細胞凝集塊またはブラストイドは、Rho/ROCK抑制因子の添加によって得られることを特徴とする請求項1〜4のいずれか1項に記載の方法。
- 前記培養は、空洞化、上皮化および/または多能性の維持に関するシグナル経路を調節することを含むことを特徴とする請求項1〜5のいずれか1項に記載の方法。
- 前記少なくとも2重層の細胞凝集塊またはブラストイドの、空洞化、上皮化および/または多能性の維持は、Wnt経路、PKA経路、PKC経路、MAPK経路、STAT経路、Akt経路、Tgf経路および/またはHippo経路の少なくとも1つの調節によって行われることを特徴とする請求項6に記載の方法。
- 前記Wnt経路の調節は、Wnt経路の活性化によって行われることを特徴とする請求項7に記載の方法。
- 前記Wnt経路の活性化は、グリコーゲンシンターゼキナーゼ抑制因子(GSK3抑制因子)の添加によって、Wntアゴニストの添加によって、または遺伝子組み換えによって行われることを特徴とする請求項8に記載の方法。
- 前記PKA経路の調節は、PKA活性化因子を用いるPKA経路の活性化によって、または遺伝子組み換えによって行われることを特徴とする請求項7に記載の方法。
- 前記培養培地は、さらに血清を含むことを特徴とする請求項1〜10のいずれか1項に記載の方法。
- 請求項1〜11のいずれか1項に記載の方法によって得られるブラストイド。
- 請求項1〜12のいずれか1項に記載の方法によって得られる少なくとも二重層の細胞凝集塊。
- 胚を成長させる方法であって、少なくとも2重層の細胞凝集塊またはブラストイドをin vitroで成長させることを含むことを特徴とする方法。
- 胚を成長させる方法であって、少なくとも2重層の細胞凝集塊またはブラストイドを子宮内で成長させることを含むことを特徴とする方法。
- Rho/ROCK抑制因子、Wnt経路調節因子、PKA経路調節因子、PKC経路調節因子、MAPK経路調節因子、STAT経路調節因子、Akt経路調節因子、Tgf経路調節因子、Hippo経路調節因子の1つ以上を含み、さらに少なくとも2重層の細胞凝集塊を含むことを特徴とする細胞培養物。
- Rho/ROCK抑制因子、Wnt経路調節因子、PKA経路調節因子、PKC経路調節因子、MAPK経路調節因子、STAT経路調節因子、Akt経路調節因子、Tgf経路調節因子、Hippo経路調節因子の1つ以上を含み、ブラストイドをさらに含むことを特徴とする細胞培養物。
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WO2021079992A1 (ja) * | 2019-10-25 | 2021-04-29 | 国立大学法人京都大学 | 多能性幹細胞からの胚盤胞様構造体の作製法 |
WO2022114188A1 (ja) * | 2020-11-30 | 2022-06-02 | 国立大学法人京都大学 | 二層性胚盤モデルおよびその製造方法 |
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CA2908443A1 (en) | 2013-04-16 | 2014-10-23 | Koninklijke Nederlandse Akademie Van Wetenschappen | Blastoid, cell line based artificial blastocyst |
CN111793608B (zh) * | 2017-07-28 | 2022-05-17 | 杨涛 | 定向诱导hiPSC分化为神经细胞体系的HS5条件培养基 |
CN108913654B (zh) * | 2018-07-06 | 2022-08-05 | 南宁市第二人民医院 | Hippo-YAP通路与Wnt通路相互作用在脂肪干细胞细胞谱命运选择中的用途 |
WO2021067854A1 (en) * | 2019-10-03 | 2021-04-08 | Salk Institute For Biological Studies | Blastocyst-like structures from extended pluripotent stem cells |
JP2023550180A (ja) * | 2020-11-24 | 2023-11-30 | モナシュ ユニバーシティ | 誘導幹細胞 |
EP4029932A1 (en) | 2021-01-13 | 2022-07-20 | IMBA-Institut für Molekulare Biotechnologie GmbH | Blastocyst-like cell aggregate and methods |
WO2022152774A1 (en) | 2021-01-13 | 2022-07-21 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Blastocyst-like cell aggregate and methods |
CN112831461B (zh) * | 2021-02-26 | 2023-08-08 | 澳门大学 | 一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物 |
CN113201500A (zh) * | 2021-05-07 | 2021-08-03 | 华夏源细胞工程集团股份有限公司 | 一种培养iPS细胞的无滋养层培养基 |
TW202328432A (zh) * | 2021-08-26 | 2023-07-16 | 清華大學 | 誘導全能性幹細胞及其製備方法 |
WO2024030443A1 (en) * | 2022-08-01 | 2024-02-08 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Bovine blastocyst like structures and uses thereof |
CN115927168A (zh) * | 2023-01-13 | 2023-04-07 | 广州国家实验室 | 全能样细胞高效产生类囊胚的方法及其应用 |
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