JPWO2020080270A1 - 栄養組成物 - Google Patents
栄養組成物 Download PDFInfo
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- JPWO2020080270A1 JPWO2020080270A1 JP2020553137A JP2020553137A JPWO2020080270A1 JP WO2020080270 A1 JPWO2020080270 A1 JP WO2020080270A1 JP 2020553137 A JP2020553137 A JP 2020553137A JP 2020553137 A JP2020553137 A JP 2020553137A JP WO2020080270 A1 JPWO2020080270 A1 JP WO2020080270A1
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Abstract
Description
細胞治療や再生医療では、in vitroでiPS細胞などの幹細胞を、目的とする細胞またはそれを含む細胞集団(組織)に分化誘導させた後、それを移植または投与して疾患の治療や病変組織を再生することが図られる。しかしながら、移植する細胞または細胞集団に未分化の幹細胞(例、iPS細胞)や目的細胞に分化できなかった細胞(例、内胚葉、中胚葉、外胚葉)が残存している場合、移植後に生体内でテラトーマ(奇形腫)が形成されるおそれや、目的細胞に分化できなかった細胞が増殖するおそれがある。移植等に用いる細胞集団からこのような事象が起きないようにするためには、(1)移植前の細胞集団の培養の段階において、未分化の幹細胞が残存したり、幹細胞から目的細胞に分化できなかった細胞が形成および/または残存したりすることを極力防ぐか、(2)移植後の細胞集団において、未分化の幹細胞のテラトーマ化や、目的細胞に分化できなかった細胞の増殖を抑制する必要がある。幹細胞から分化した細胞を含む細胞集団においては、幹細胞由来の目的外細胞の形成および/または増殖を抑制することは、細胞治療や再生医療の安全性や有効性を向上させる上で重要である。
すなわち本発明は、上記課題解決のため、以下の[1]〜[11]を提供する。
[1] バリンを除く、イソロイシン、ロイシン、メチオニン、リジン、フェニルアラニン、トリプトファン、スレオニンおよびヒスチジンからなる群より選択される1以上の必須アミノ酸を含有し、非必須アミノ酸を任意で含有する、幹細胞から分化した細胞を含む細胞集団において幹細胞由来の目的外細胞の形成および/または増殖を抑制するための、栄養組成物。
[2] 必須アミノ酸として、少なくともメチオニンを含有する、項[1]に記載の栄養組成物。
[3] 非必須アミノ酸を含有しない、項[1]に記載の栄養組成物。
[4] アルギニン、グリシン、セリン、アスパラギンおよびグルタミンからなる群より選択される1以上の非必須アミノ酸を含有する、項[1]に記載の栄養組成物。
[5] アミノ酸以外の栄養素をさらに含む、項[1]に記載の栄養組成物。
[6] 11日間以上摂取される、項[1]に記載の栄養組成物。
[7]栄養組成物が、1)固形食、固形剤、半固形食、半固形剤、飲料、液剤から選ばれるものであるか、または2)培地である、項[1]に記載の栄養組成物。
[8] 項[1]記載の栄養組成物、および幹細胞から分化した細胞を含む細胞集団を含むキット。
[9] 幹細胞から分化した細胞を含む細胞集団に、項[1]に記載の栄養組成物を摂取させることを含む、幹細胞由来の目的外細胞の形成および/または増殖の抑制方法。
[10] 幹細胞から分化した細胞を含む細胞集団において幹細胞由来の目的外細胞の形成および/または増殖を抑制するための、項[1]に記載の栄養組成物の使用。
[11] 幹細胞から分化した細胞を含む細胞集団が移植または投与された哺乳動物に、項[1]に記載の栄養組成物を摂取させることを含む、生体内での、幹細胞由来の目的外細胞の形成および/または増殖の抑制方法。
本発明の栄養組成物は、バリンを除く、イソロイシン、ロイシン、メチオニン、リジン、フェニルアラニン、トリプトファン、スレオニンおよびヒスチジンからなる群(特定必須アミノ酸)より選択される1以上の必須アミノ酸を含有し、非必須アミノ酸を任意で含有する、幹細胞から分化した細胞を含む細胞集団において幹細胞由来の目的外細胞の形成および/または増殖を抑制するためのものである。
「バリンを除く、イソロイシン、ロイシン、メチオニン、リジン、フェニルアラニン、トリプトファン、スレオニンおよびヒスチジンからなる群より選択される1以上の必須アミノ酸を含有」するとは、必須アミノ酸のうち、バリンは含有していないこと、かつ「イソロイシン、ロイシン、メチオニン、リジン、フェニルアラニン、トリプトファン、スレオニンおよびヒスチジンからなる群」(特定必須アミノ酸群)のうち、選択される1以上(1種のみでも、複数種でも、全てであってもよい。)を含有し、選択されなかったものは含有しないことを意味する。
「幹細胞から分化した細胞を含む細胞集団」は、典型的には、幹細胞を目的細胞に分化誘導させるための培養工程の途中段階または最終段階において生成する細胞集団であって、幹細胞から分化誘導した目的細胞と、目的細胞に分化できなかった目的外細胞(例、未分化の幹細胞(例、iPS細胞)、培養の途中段階で目的細胞への分化が止まった細胞(例、内胚葉、中胚葉、外胚葉))とが混合した状態の細胞集団である。
ヒト等の哺乳動物に摂取させるための第1栄養組成物の形状は、経口投与または非経口投与が可能な形状であれば特に限定されるものではないが、例えば、固形食、固形剤(例、経口摂取用固形剤、粉末剤)、半固形食(例、ゼリー、流動食)、半固形剤(例、経口摂取用ゼリー剤)、飲料、液剤(例、経口摂取用液剤、非経口摂取用液剤(例、輸液製剤))が挙げられる。
バリン:0mg/kg体重;
イソロイシン:0mg/kg体重〜30mg/kg体重、好ましくは0mg/kg体重〜20mg/kg体重;
ロイシン:0mg/kg体重〜50mg/kg体重、好ましくは0mg/kg体重〜40mg/kg体重;
メチオニン:0mg/kg体重〜30mg/kg体重、好ましくは0mg/kg体重〜15mg/kg体重;
リジン:0mg/kg体重〜50mg/kg体重、好ましくは0mg/kg体重〜30mg/kg体重;
フェニルアラニン:0mg/kg体重〜50mg/kg体重、好ましくは0mg/kg体重〜25mg/kg体重;
トリプトファン:0mg/kg体重〜30mg/kg体重、好ましくは0mg/kg体重〜5mg/kg体重;
スレオニン:0mg/kg体重〜30mg/kg体重、好ましくは0mg/kg体重〜15mg/kg体重;
ヒスチジン:0mg/kg体重〜30mg/kg体重、好ましくは0mg/kg体重〜10mg/kg体重。
第1栄養組成物は、アミノ酸以外の栄養素(その他成分)をさらに含んでいてもよい。幹細胞から分化した細胞を含む細胞集団を移植または投与された哺乳動物が、その後一定期間にわたって第1栄養組成物を摂取し続けることを考慮すると、第1栄養組成物は所定のアミノ酸に加えて、アミノ酸以外の栄養素をさらに含み、第1栄養組成物のみでその哺乳動物にとって必要な栄養素をすべて摂取できるような形態とすることが好ましい。
ブタン酸(酪酸、4:0)、ヘキサン酸(カプロン酸、6:0)、ヘプタン酸(7:0)、オクタン酸(カプリル酸、8:0)、デカン酸(カプリン酸、10:0)、ドデカン酸(ラウリン酸、12:0)、トリデカン酸(13:0)、テトラデカン酸(ミリスチン酸、14:0)、ペンタデカン酸(15:0)、ヘキサデカン酸(パルミチン酸、16:0)、ヘプタデカン酸(17:0)、オクタデカン酸(ステアリン酸18:0)、イコサン酸(アラキジン酸、20:0)、ドコサン酸(ベヘン酸、22:0)、テトライコサン酸(リグノセリン酸、24:0)、デセン酸(10:1)、テトラデセン酸(ミリストレイン酸、14:1)、ペンタデセン酸(15:1)、ヘキサデセン酸(パルミトレイン酸、16:1)、ヘプタデセン酸(17:1)、オクタデセン酸(オレイン酸、18:1、n−9)、オクタデセン酸(シス−バクセン酸、18:1、n−7)、イコセン酸(エイコセン酸、20:1)、ドコセン酸(22:1)、テトラコセン酸(24:1)、ヘキサデカジエン酸(16:2)、ヘキサデカトリエン酸(16:3)、ヘキサデカテトラエン酸(16:4)、ヘプタデカジエン酸(17:2)、オクタデカジエン酸(18:2)、オクタデカジエン酸(リノール酸、18:2、n−6)、オクタデカトリエン酸(18:3)、オクタデカトリエン酸(α−リノレン酸、18:3、n−3)、オクタデカトリエン酸(γ−リノレン酸、18:3、n−6)、オクタデカテトラエン酸(18:4、n−3)、イコサジエン酸(エイコサジエン酸、20:2、n−6)、イコサトリエン酸(エイコサトリエン酸、20:3、n−6)、イコサテトラエン酸(エイコサテトラエン酸、20:4、n−3)、イコサテトラエン酸(アラキドン酸、20:4、n−6)、イコサペンタエン酸(エイコサペンタエン酸、20:5、n−3)、ヘンイコサペンタエン酸(21:5、n−3)、ドコサジエン酸(22:2)、ドコサテトラエン酸(22:4、n−6)、ドコサペンタエン酸(22:5、n−3)、ドコサペンタエン酸(22:5、n−6)、ドコサヘキサエン酸(22:6、n−3)。
幹細胞から分化した細胞を含む細胞集団の培養に使用するための第2栄養組成物は、代表的には、細胞の培地の形状をとる。すなわち、第2栄養組成物の形状は、細胞の培養(特に幹細胞から目的細胞を分化誘導するための培養)に一般的に用いられている培地またはその他の公知の培地の形状に準じたものとすることができる。
本発明によるキットは、本発明の栄養組成物、および幹細胞から分化した細胞を含む細胞集団を含む。本明細書において、本発明の栄養組成物との関係で記載した技術的事項は、当該栄養組成物を使用する本発明のキットとの関係においても同様に適用することができる。例えば、本発明のキットは、細胞治療または再生医療の手術において移植のために用いられる細胞集団と、その手術を受けたヒト(患者)またはヒト以外の哺乳動物(実験動物)が移植手術後に摂取するための第1栄養組成物とを含むものとすることができる。
本試験例において用いるマウスは、ヒトiPS細胞を移植するに際して、異種細胞移植となることから、免疫不全マウス雄性NOD/Shi-scid-IL2Rγnullマウス(以下、「NOGマウス」)を利用する。これらはヒトiPS細胞の移植実験で広く用いられている動物種である(K.Miura, et al. Nat Biotechnol (2009), 27:743-5)。該マウスは実験動物中央研究所から入手した。
以下の試験例において、固形飼料のコントロールとしてリサーチダイエット社A10021Bを用いた。この飼料をベースに各種アミノ酸を抜いた固形飼料、A05080209(バリン欠乏飼料)(リサーチダイエット社)、A05080220(非必須アミノ酸欠乏飼料)(リサーチダイエット社)を作製した。これらA10021B、A05080209、A05080220は3.87 kCal/gでエネルギー量は統一されている。セリンおよびグリシン欠乏飼料としてはTest diet社(Mod TestDiet (登録商標) 5CC7 w/ No Added Serine or Glycine, 5BJX 5CC7, 3.97 kCal/g)を用いた。各飼料の組成を下記表に示す。
*2 Vitamin Mix V10001の組成(当該Mix 10gあたりの各成分の重量):ビタミンAパルミテート(20,000 IU)、ビタミンD3(1,000 IU)、ビタミンE酢酸塩(50 IU)、メナジオンナトリウムビサルファイト(0.5 mg)、ビオチン(0.3 mg)、シアノコバラミン(10 μg)、葉酸(6 mg)、ニコチン酸(30 mg)、パントテン酸カルシウム(30 mg)、ピリドキシン塩酸塩(6 mg)、リボフラビン(6 mg)、チアミン塩酸塩(6 mg)、アスコルビン酸(500 mg)、スクロース(9.7842 g)
*4 Baker Amino Acid Mineral Premixの成分:コーンスターチ、リン酸カルシウム、リン酸カリウム、塩化ナトリウム、炭酸カルシウム、硫酸マグネシウム、クエン酸鉄、硫酸マグネシウム、炭酸亜鉛、モリブデン酸ナトリウム、ホウ酸、ヨウ化カリウム、硫酸銅、セレン酸ナトリウム、硫酸コバルト
*5 Baker AA Premix / No Ser or Gly:L-リシン一塩酸塩、L-ロイシン、L-アルギニン-HCl、L-アラニン、L-アスパラギン、グルタミン酸、L-グルタミン、L-プロリン、L-フェニルアラニン、L-バリン、L-スレオニン、L-イソロイシン、L-メチオニン、L-ヒスチジン-HCl-H2O、L-チロシン、L-システイン、L-トリプトファン
実験に使用するNOGマウスは6週齢で入荷し、入荷後1週間の馴化飼育期間を経て使用した。体重を測定し、1.5-2.0%のイソフルラン吸入麻酔下において背部中央右側または背部中央左側より切開し、腎臓を露出させた。腎被膜下に注射針を用いてヒトiPS細胞1383D2株(京都大学iPS細胞研究所から入手)(100万細胞)を腎被膜下に移植した。合計18匹のマウスにiPS細胞を移植し、4匹のマウスは手術処置は同様にするが、移植のみおこなわないSham群として用いた。その後、腎臓を腹部に戻して縫合した。麻酔から覚醒したことを確認してケージに戻した(3匹/ケージ, Sham群のみ2匹/ケージ)。その後移植群18匹をそれぞれコントロール飼料(6匹)、バリン欠乏飼料2群(6匹×2)に分割し、Sham群4匹にはバリン欠乏飼料を給餌した。1週間毎に飼料を新しいものに取替え、3週間目に体重を測定し、体重低下の回復のためバリン欠乏飼料2群(6匹×2)の飼料をコントロール飼料に変更した。翌週移植4週後にバリン欠乏飼料1群(6匹)の飼料を再びバリン欠乏飼料に戻した。その後この群は実験終了まで1週間毎にバリン欠乏飼料とコントロール飼料を交互に給餌した。3週前でコントロール飼料に戻した別のバリン欠乏飼料1群(6匹)についてはこの後実験終了までコントロール飼料を給餌した。移植後68日目に該マウスを麻酔下で解剖し、移植腎と移植していない反対側の腎臓の双方の重量を測定し、その差をテラトーマ重量として算出した。
実験に使用するNOGマウスは6週齢で入荷し、入荷後1週間の馴化飼育期間を経て使用した。体重を測定し、1.5-2.0%のイソフルラン吸入麻酔下において背部中央右側または背部中央左側より切開し、腎臓を露出させた。腎被膜下に注射針を用いてヒトiPS細胞1383D2株(京都大学iPS細胞研究所から入手)(500万細胞)を腎被膜下に移植した。合計18匹のマウスにiPS細胞を移植し、4匹のマウスは手術処置は同様にするが、移植のみおこなわないSham群として用いた。その後、腎臓を腹部に戻して縫合した。麻酔から覚醒したことを確認してケージに戻した(3-4匹/ケージ, Sham群のみ2-4匹/ケージ)。その後移植群20匹をそれぞれコントロール飼料(10匹)、セリン・グリシン欠乏飼料群(10匹)に分割し、Sham群5匹にはセリンおよびグリシン欠乏飼料を給餌した。1週間毎に飼料を新しいものに取替え、移植後7, 14, 28, 48日目に体重を測定し、移植後48日目に該マウスを麻酔下で解剖し、移植腎と移植していない反対側の腎臓の双方の重量を測定し、その差をテラトーマ重量として算出した。
実験に使用するNOGマウスは8週齢で入荷し、入荷後1週間の馴化飼育期間を経て使用した。体重を測定し、1.5-2.0%のイソフルラン吸入麻酔下において背部中央右側または背部中央左側より切開し、腎臓を露出させた。腎被膜下に注射針を用いてヒトiPS細胞1383D2株(京都大学iPS細胞研究所から入手)(500万細胞)を腎被膜下に移植した。合計15匹のマウスにiPS細胞を移植した。その後、腎臓を腹部に戻して縫合した。麻酔から覚醒したことを確認してケージに戻した(2-4匹/ケージ)。その後移植群15匹をそれぞれコントロール飼料(5匹)、非必須アミノ酸(アスパラギン、アスパラギン酸、アラニン、アルギニン、グリシン、グルタミン、グルタミン酸、システイン、セリン、チロシンおよびプロリン)欠乏飼料群(10匹)に分割した。1週間毎に飼料を新しいものに取替え、移植後3, 10, 24, 43, 50日目に体重を測定し、移植後50日目に該マウスを麻酔下で解剖し、移植腎と移植していない反対側の腎臓の双方の重量を測定し、その差をテラトーマ重量として算出した。
(1)ヒトiPS細胞の培養
ヒトiPS細胞(1383D2;京都大学iPS研究所)をLaminin-511コーティング上にStemFit AK02Nで播種1日後に、以下の培地に変更し、培地交換2日後に細胞生存率をCell-Titer Glo(Promega)で測定した。
バリン含有培地:DMEM/F-12(Gibco),E8 supplement(Thermo)。
バリン不含培地:DMEM/F-12(-Val)(機能ペプチド研究所、特注),E8 supplement(Thermo)。
結果を図4に示す。バリン不含培地で培養したヒトiPS細胞は培地交換2日後にはほとんど生存が認められなかった。
(1)ヒト血管内皮細胞(human endothelial cell;EC)の作製
ヒトiPS細胞(1383D2;京都大学iPS研究所)を、DMEM/F-12(Gibco)(10ml)に1% B-27 Supplements(GIBCO)、BMP4(25ng/ml)およびCHIR99021(8μM)を添加した培地中、5%CO2、37℃で3日間培養することで中胚葉系細胞を誘導した。得られた中胚葉系細胞をさらに、Stempro-34 SFM(Gibco)(10ml)にVEGF(200ng/ml)およびFolskolin(2μM)を添加した培地中、5%CO2、37℃で7日間培養することで、CD31陽性、CD73陽性およびCD144陽性のヒト非造血性血管内皮細胞集団を得た。
ヒトiPS細胞(1383D2)を、RPMI 1640(富士フィルム)(2ml)にWnt3a(50ng/mL)およびアクチビンA(100ng/ml)を添加した培地中、5%CO2、37℃で5日間培養することで、内胚葉系細胞を誘導した。得られた内胚葉系細胞を、同培地に1%B27 Supplements(GIBCO)およびFGF2(10ng/ml)を添加した上で、5%CO2、37℃で、さらに5日間培養することで、AFP、ALBおよびHNF4αが陽性のヒト肝臓内胚葉細胞集団を得た。
ヒトiPS細胞(1383D2)を、DMEM/F-12(Gibco)(10ml)に1% B-27 Supplement(GIBCO)、BMP4(25ng/ml)およびCHIR99021(8μM)を添加した培地中、5%CO2、37℃で3日間培養することで中胚葉系細胞を誘導した。得られた中胚葉系細胞を、同培地にPDGFBB(10ng/ml)およびアクチビンA(2 ng/ml)を添加した上で、5%CO2、37℃で、さらに3日間培養した。その後、DMEM/F-12(Gibco)(10ml)に1% B-27 Supplements(GIBCO)、FGF2(10 ng/ml)およびBMP4(12ng/ml)を添加した培地中、5%CO2、37℃で、さらに3日間培養することで、ヒト間葉系幹細胞を得た。
作製したヒト肝臓内胚葉細胞(HE)、ヒト血管内皮細胞(EC)、ヒト間葉系幹細胞(MC)およびヒトiPS細胞(1383D2)を10:7:1:5の割合の細胞数(総数2.3×106個)で混合し、三次元培養容器Elplasia(クラレ)上で一日間、5%CO2、37℃で共培養することで凝集体を作製した。この共培養において、培養培地は、HCM(Lonza)にFBS(5%)、HGF(10ng/ml)、OSM(20ng/ml)およびDex(100nM)を加えた肝臓細胞用培地(A)と、Stempro-34 SFM(Gibco)にVEGF(50ng/ml)およびFGF2(10ng/ml)を加えた血管内皮細胞用培地(A)を、1:1の体積割合で混合したもの(本明細書中「オルガノイド用培地(A)」という)を用いた。共培養1日後にオルガノイド用培地(A)を以下の培地に変更し、培地交換1日後に細胞生存率をFACS fortessaで測定した。
バリン含有培地:DMEM/F-12(Gibco)にKSR(5%)、HGF(10ng/ml)、OSM(20ng/ml)およびDex(100nM)を加えた肝臓細胞用培地(B)に、上記血管内皮細胞用培地(A)を、1:1の体積割合で混合したもの。
バリン不含培地:DMEM/F-12(-Val)(機能ペプチド研究所、特注)にKSR(5%)、HGF(10ng/ml)、OSM(20ng/ml)およびDex(100nM)を加えた肝臓細胞用培地(B’)に、上記血管内皮細胞用培地(A)を、1:1の体積割合で混合したもの。
結果を図5に示す。オルガノイドとともに培養したヒトiPS細胞は、バリン不含培地では培地交換1日後でバリン含有培地の1/3に減少していた。
Claims (11)
- バリンを除く、イソロイシン、ロイシン、メチオニン、リジン、フェニルアラニン、トリプトファン、スレオニンおよびヒスチジンからなる群より選択される1以上の必須アミノ酸を含有し、非必須アミノ酸を任意で含有する、幹細胞から分化した細胞を含む細胞集団において幹細胞由来の目的外細胞の形成および/または増殖を抑制するための、栄養組成物。
- 必須アミノ酸として、少なくともメチオニンを含有する、請求項1に記載の栄養組成物。
- 非必須アミノ酸を含有しない、請求項1に記載の栄養組成物。
- アルギニン、グリシン、セリン、アスパラギンおよびグルタミンからなる群より選択される1以上の非必須アミノ酸を含有する、請求項1に記載の栄養組成物。
- アミノ酸以外の栄養素をさらに含む、請求項1に記載の栄養組成物。
- 11日間以上摂取される、請求項1に記載の栄養組成物。
- 栄養組成物が、1)固形食、固形剤、半固形食、半固形剤、飲料、液剤から選ばれるものであるか、または2)培地である、請求項1に記載の栄養組成物。
- 請求項1記載の栄養組成物、および幹細胞から分化した細胞を含む細胞集団を含む、キット。
- 幹細胞から分化した細胞を含む細胞集団に、請求項1に記載の栄養組成物を摂取させることを含む、幹細胞由来の目的外細胞の形成および/または増殖の抑制方法。
- 幹細胞から分化した細胞を含む細胞集団において幹細胞由来の目的外細胞の形成および/または増殖を抑制するための、請求項1に記載の栄養組成物の使用。
- 幹細胞から分化した細胞を含む細胞集団が移植または投与された哺乳動物に、請求項1に記載の栄養組成物を摂取させることを含む、生体内での、幹細胞由来の目的外細胞の形成および/または増殖の抑制方法。
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