JP7473571B2 - 多能性幹細胞の懸濁培養のための培養培地 - Google Patents
多能性幹細胞の懸濁培養のための培養培地 Download PDFInfo
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Description
多能性幹細胞(PSC)の接着(2次元、2D)培養: 本発明者らによって開発された新規培養培地上での培養の前に、HESC系I-6細胞およびI-3細胞[Amit and Itskovitz-Eldor, 2002]を、(i)不活化ヒト包皮線維芽細胞(HFF)との培養、(ii)公知の方法(Amit et al, 2000、Amit et al 2003)によるマウス胚性幹細胞(MEF)との培養、または(iii)公知の方法(Amit et al, 2001、Amit et al 2003)によるマトリゲル(商標)マトリックスとの培養を実施した。
(1)Wnt3A培地(10ng/mlのWnt3a): 15%のノックアウト-SR(血清代替物、Gibco、カタログ番号10828-028)、2mMのL-グルタミン、0.1mMのβ-メルカプトエタノール、1%の非必須アミノ酸ストック、10ng/mlのWnt3a(R&D systems社製、カタログ番号5036-WN)を添加した85%DMEM/F12。
(13)CHIR99021培地のみ(LIFなし): 15%のノックアウト-SR、2mMのL-グルタミン、0.1mMのβ-メルカプトエタノール、1%の非必須アミノ酸ストック、および3μMのCHIR99021を添加した85%のDMEM/F12。
(15)PMSF培地(単独): 15%のノックアウト-SR、2mMのL-グルタミン、0.1mMのβ-メルカプトエタノール、1%の非必須アミノ酸ストック、および0.1mMのPMSFを添加した85%のDMEM/F12。
(28)TLCK培地-15%のノックアウト-SR、2mMのL-グルタミン、0.1mMのβ-メルカプトエタノール、1%の非必須アミノ酸ストック、50μMのTLCKを添加した85%のDMEM/F12。
(1)PSCをフィーダー細胞上での培養から懸濁培養へと適応させるに際して、1回目の継代は、以下の酵素:TrypLEx(米国、ニューヨーク州、グランドアイランド、Gibco-Invitrogen Corporation社製)、トリプシンEDTA(BI)、アキュターゼ、Gentle dissociator(SCT)、Releaser(SCT)またはIV型コラゲナーゼ(Worthington社製)のいずれかを用いる酵素的分割を実施した。その後、細胞をペトリ皿に移した。
実験結果
Wnt3aに基づくいくつかの培地の処方について、bFGFを添加せずに、それらが懸濁液中のhPSCのフィーダー層不含培養を支援する能力について試験した。試験した培地の処方は、未分化hESCのフィーダー層不含増殖を支援するのに好適であることがわかった。
新規培養培地は2次元培養システム上でのPSCの連続培養を支援することもできる
試験培地と共に、マトリゲルマトリックス、フィブロネクチン、ラミニンまたはヒト包皮線維芽細胞無細胞マトリックスで被覆した培養皿を使用して、PSCを接着細胞として培養した。IV型コラゲナーゼを使用して、4~7日毎に細胞を継代した。培地を毎日交換した。連続的増殖を20回継代した後、細胞を、Oct4、Tra160およびTA181などの多能性マーカーについて特徴付けた。結果を図21A~Cに示す。ここに示されるように、新規培養培地は、2次元培養システム上での未分化且つ多能性の幹細胞の増殖を支援することもできる。
様々なプロテアーゼ阻害剤を含む無血清限定培養培地は、ヒト多能性幹細胞の未分化増殖を支援する
いくつかの新規培養培地の処方(「一般的材料および実験方法」の項に記載した培養培地)について、PSCの未分化の懸濁培養を支援する能力を試験した。PMSF、TLCK、またはWnt3aを添加し、bFGFを添加しない培地を使用しながら、hESCを18回を超える継代数にわたって、PSCの特徴を発現しながらも、未分化のものとして培養することができた。細胞形態は、懸濁培養したPSCについて報告された形態と類似し(図23A~B)、細胞の凝集体は明確な境界を有し、形状は円盤状であった(Amit et al., 2010)。FACS分析は、懸濁液中での6回の継代後には、培養したPSC(I3)の92パーセントがOct4とSSEA4とを同時発現することを示した。試験細胞の95%超がSSEA4について陽性であった(図23B)。同様に、追加の生物学的反復の結果は、図24Bに示したように、83パーセントの細胞がこれらのマーカーを同時発現したことを示す。動的システム(スピナー)を用いる細胞培養により、細胞濃度が1mlあたり600万個を超える細胞が得られた(データは示さない)。スピナーフラスコを用いて連続的に1ヶ月(4~5回の継代)を超えて培養した細胞は、多能性マーカーであるOct4およびSSEA4の発現を維持し、2回の生物学的反復において、95%または86%の同時発現を維持した(図25および図26)。さらに、試験したバージョンの培地を使用して懸濁培養した細胞は、EBを形成し、図27A~Cに例示したような異なる細胞系列に分化することができた。
(さらなる参考文献は本文中に引用している。)
1. Amit M, Carpenter MK, Inokuma MS, Chiu C-P, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000; 227:271-278.
2. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, and Itskovitz-Eldor J. Human feeder layers for human embryonic stem cells. Biology of Reproduction 68: 2150-2156, 2003.
3. Michal Amit, Ilana Laevsky, Yael Miropolsky, Kohava Shariki, Meital Peri, Joseph Itskovitz-Eldor. Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nature protocols, 6(5): 572-579, 2011.
4. Amit M, Chebath J, Marguletz V, Laevsky I, Miropolsky Y, Shariki K, Peri M, Revel M, Itskovitz-Eldor J. Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells. Stem Cells Reviews and Reports. 6(2):248-259, 2010.
5. Daheron L, Opitz SL, Zaehres H, Lensch WM, Andrews PW, Itskovitz-Eldor J, Daley GQ. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells. 2004;22(5):770-8.
6. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med. 2004 Jan;10(1):55-63.
7. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001; 19:971-974.
8. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002; 20:933-936.
9. Amit M, Margulets V, Segev H, Shariki C, Laevsky I, Coleman R, and Itskovitz-Eldor J. Human feeder layers for human embryonic stem cells. Biol Reprod 2003; 68:2150-2156.
10. Amit M, Shariki K, Margulets V, and Itskovitz-Eldor J. "Feeder and serum-free culture system for human embryonic stem cells". Biol Reprod 70:837-845, 2004.
11. Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod. 2003 Jul;18(7):1404-9.
12. Thomson JA, Itskovitz-Eldor, J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282:1145-1147 [erratum in Science 1998;282:1827].
13. Reubinoff BE, Pera MF, Fong C, Trounson A., Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000; 18:399-404.
14. Amit M. & Itskovitz-Eldor J. Derivation and spontaneous differentiation of human embryonic stem cells. J Anat. 2002; 200:225-232.
15. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med 2000; 6:88-95.
16. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 2005; 2(3):185-190.
17. Xu C, Rosler E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells. 2005b 23:315-23.
Claims (17)
- GSK3β阻害剤および血清代替物から実質的になる限定培養培地であって、基質接着のない懸濁培養法による培養において、少なくとも5回の継代にわたり、ヒト多能性幹細胞を未分化且つ多能性状態に維持することができる限定培養培地。
- JNK阻害剤を含まない、請求項1に記載の限定培養培地。
- p38阻害剤を含まない、請求項1に記載の限定培養培地。
- 1ng/mlを超える量の塩基性線維芽細胞増殖因子(bFGF)を含まない、請求項1~3のいずれか一項に記載の限定培養培地。
- 塩基性線維芽細胞増殖因子(bFGF)を含まない、請求項1~3のいずれか一項に記載の限定培養培地。
- 無血清である、請求項1~5のいずれか一項に記載の限定培養培地。
- 前記GSK3β阻害剤が、CHIR99021、BIO(AXONMEDCHEM-Axon1693)、またはケンパウロン(TOCRIS-カタログ番号1398)である、請求項1~5のいずれか一項に記載の限定培養培地。
- 前記GSK3β阻害剤が、CHIR99021である、請求項1~7のいずれか一項に記載の限定培養培地。
- 前記CHIR99021の使用濃度が、少なくとも0.1μMである、請求項8に記載の限定培養培地。
- 前記CHIR99021の使用濃度が、0.1~50μM/mlの濃度範囲内である、請求項8に記載の限定培養培地。
- 基質接着のない懸濁培養法による培養において、少なくとも5回の継代にわたって多能性幹細胞を多能性状態に維持することができる、請求項1~10のいずれか一項に記載の限定培養培地。
- 前記血清代替物の使用濃度が、前記限定培養培地に対して、5%(v/v)~30%(v/v)である、請求項1~11のいずれか一項に記載の限定培養培地。
- 多能性幹細胞と、請求項1~12のいずれか一項に記載の限定培養培地とを含む細胞培養物。
- 前記多能性幹細胞が誘導多能性幹細胞である、請求項13に記載の細胞培養物。
- 前記多能性幹細胞が胚性幹細胞である、請求項13に記載の細胞培養物。
- 前記多能性幹細胞がヒト多能性幹細胞である、請求項13~15のいずれか一項に記載の細胞培養物。
- 基質接着のない懸濁培養法によって多能性幹細胞を培養する方法であって、
請求項1~12のいずれか一項に記載の限定培養培地中で前記多能性幹細胞を培養し、それによって、前記多能性幹細胞の懸濁培養を実施することを含む、方法。
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KR20230031998A (ko) | 2023-03-07 |
AU2017301040A1 (en) | 2019-02-28 |
IL303869A (en) | 2023-08-01 |
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JP2022062164A (ja) | 2022-04-19 |
IL264283B2 (en) | 2023-12-01 |
EP3487987A4 (en) | 2020-02-19 |
KR20190027922A (ko) | 2019-03-15 |
WO2018015954A1 (en) | 2018-01-25 |
CA3030252A1 (en) | 2018-01-25 |
AU2017301040B2 (en) | 2023-01-05 |
KR102536879B1 (ko) | 2023-05-26 |
IL264283B1 (en) | 2023-08-01 |
EP3487987A1 (en) | 2019-05-29 |
JP2019520848A (ja) | 2019-07-25 |
US20190284526A1 (en) | 2019-09-19 |
SG11201900231RA (en) | 2019-02-27 |
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