JPWO2017069222A1 - 細胞の調製方法 - Google Patents
細胞の調製方法 Download PDFInfo
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Abstract
Description
本出願は、2015年10月21日に出願された、日本国特許出願第2015-207529号明細書(その開示全体が参照により本明細書中に援用される)に基づく優先権を主張する。
マウス線維芽細胞→軟骨細胞(SOX9 + Klf4 + c-Myc遺伝子を導入)
マウス線維芽細胞→心筋細胞(GATA4 + Mef2c + Tbx5遺伝子を導入)
マウス線維芽細胞→肝細胞(Hnf4α+(Foxa1またはFoxa2またはFoxa3)遺伝子を導入)
マウス線維芽細胞→神経幹細胞(Sox2 + FoxG1遺伝子を導入など)、
マウス、ヒト細胞→造血幹細胞など。
本発明の方法において原材料として用いる哺乳動物の分化した体細胞としては、哺乳動物由来であれば、特に限定されない。体細胞とは、細胞のうち生殖細胞以外の細胞を意味する。
本発明の方法により調製される他の体細胞は、原材料として用いる体細胞以外の体細胞である。また、原材料として用いる体細胞から、生理的な条件下(特に、生体内)で分化する体細胞は、「他の体細胞」には含まれない。例えば、生体内で間葉系幹細胞から骨芽細胞へと分化するため、間葉系幹細胞を原材料として用いる場合は、骨芽細胞は「他の体細胞」には該当しない。
本発明の方法において、分化した体細胞を、分化した体細胞以外の他の体細胞を誘導するための培地(分化誘導培地)中で培養する。分化誘導培地としては、目的の調製される体細胞に応じて、公知の分化誘導培地を使用することができる。
(1-1) M.D. Hoffman and D.S.W. Benoit, J Tissue Eng Regen Med. 2013 Apr 1. doi: 10.1002/term.1736
(1-2) C-Y Li, et al., Stem Cell Res Ther. 2015 Apr 13;6:55. doi: 10.1186/s13287-015-0066-5.
(1-3) B. Gharibi and F.J. Hughes, Stem Cells Transl Med. 2012 Nov;1(11):771-82. doi: 10.5966/sctm.2010-0031. Epub 2012 Oct 23
(1-4) S.K. Both et al. Tissue Eng. 2007 Jan;13(1):3-9.
(1-5) F. Ng et al., Blood. 2008 Jul 15;112(2):295-307. doi: 10.1182/blood-2007-07-103697. Epub 2008 Mar 10.
(1-6) Hynes K. et al., J Dent Res. 2013 Sep;92(9):833-9。
(2-2)G.-I. Im, et al. Tissue Engineering. March 2006, 12(3): 527-536. doi:10.1089/ten.2006.12.527.
(2-3) H-J. Kim and G.-I. Im, Journal of Orthopaedic Research,Volume 27, Issue 5, pages 612-619, May 2009
(2-4) A.M. Ibrahim et al., Microscopy Research and Technique, Volume 78, Issue 8, pages 667-675, August 2015。
(3-1)Kingham PJ, DF Kalbermatten, D Mahay,et al: Adipose-derived stem cells differentiate into a Schwann cell phenotype andpromote neurite outgrowth in vitro. ExpNeurol, 2007; 207:267-274.
(3-2)Liu Y, Zhang Z, Qin Y, Wu H, Lv Q, Chen X, Deng W: A new method for Schwann-like cell differentiation of adipose derived stem cells. Neurosci Lett. 2013 Sep 13;551:79-83.。
本発明の方法において、他の体細胞を誘導するための培地中で、TGF-βパスウェイ阻害剤の存在下に培養する。
TGF-β/SMADパスウェイ阻害剤は、TGF-β/SMADパスウェイに属するタンパク質の活性を阻害できる化合物を意味する。TGF-β/SMADパスウェイは図16(Chen G et al, Int J Biol Sci, 2012より引用)に模式的に示す、当業者に公知のシグナル経路である。
R1はH、メチル基又はハロゲン(例えば、フッ素、塩素、臭素又はヨウ素)である。
R2はH又はメチル基である。]
TGF-β/ERKパスウェイ阻害剤は、TGF-β/ERKパスウェイに属するタンパク質の活性を阻害できる化合物を意味する。TGF-β/ERKパスウェイは図17(Y. E. Zhang,“Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009より引用)に模式的に示す、当業者に公知のシグナル経路である。
TGF-β/JNKパスウェイ阻害剤は、TGF-β/JNKパスウェイに属するタンパク質の活性を阻害できる化合物を意味する。TGF-β/JNKパスウェイは図18(Y. E. Zhang,“Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009より引用)に模式的に示す、当業者に公知のシグナル経路である。
本発明の方法において、他の体細胞を誘導するための培地中で、TGF-β/p38パスウェイ阻害剤の存在下に培養する。
本発明の方法において、他の体細胞を誘導するための培地中で、TGF-β/RhoAパスウェイ阻害剤の存在下に培養する。
本発明の方法において、哺乳動物の分化した体細胞を誘導培地中、TGF-βパスウェイ阻害剤の存在下に培養する。
本発明の方法により調製される体細胞は、生体に移植することで、種々の疾患又は状態の予防又は治療に使用することができる。本明細書において、特に明示のない限り、「治療」という用語は、患者が特定の疾患又は障害を患っている間に行う処置を意図し、これによって疾患若しくは障害の重症度、又は1つ若しくは複数のその症状が軽減されるか、又は疾患若しくは障害の進行が遅延又は減速することを意味する。本明細書において、「治療」には「予防」を含むものとする。
本発明は、TGF-βパスウェイ阻害剤を含む、分化した体細胞を他の体細胞にコンヴァートさせるための誘導剤をも提供する。また、分化した体細胞を他の体細胞にコンヴァートさせるための、TGF-βパスウェイ阻害剤の使用をも提供する。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、または各化合物を加えた石灰化誘導培地を、500 μL/well加えた。
D4476: 2 μM
ALK5 inhibitor II: 2 μM
SB431542: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、石灰化誘導培地、または各化合物を加えた石灰化誘導培地を、500 μL/well加えた。
D4476: 2μM
SB431542: 2μM
ALK5 inhibitor II: 2μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、石灰化誘導培地、または各化合物を加えた石灰化誘導培地を、500 μL/well加えた。
D4476: 2μM
SB431542: 2μM
ALK5 inhibitor II: 2μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、またはALK5 inhibitor IIを加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSC)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、またはALK5 inhibitor IIを加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを10×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。4日日に培養上清を吸引除去し、50 mM ALK5 Inhibitor IIを添加した誘導培地(a)(下記)を加えて培養を行った。6日目に誘導培地(a)を除去し、50 mM ALK5 Inhibitor IIを添加した誘導培地(b)(下記)を加えて培養を続けた。その後、9,12,14,16日目に、50 mM ALK5 Inhibitor IIを添加した誘導培地(b)を用いて同様に培地交換を繰り返した。
[誘導培地(a)]
DMEM (High Glucose):500 mL
FETAL BOVINE SERUM (FBS):50 mL
MEM Non‐Essential Amino Acids Solution:5 mL100 mM‐Sodium Pyruvate Solution:5 mL
Penicillin‐Streptomycin Mixed Solution:5 mLInsulin:170 nM
3,3',5‐Triiodo‐L‐thyronine:1 nM
Rosiglitazone:1 uM
IBMX(3-isobutyl-1-methylxanthine):0.5 mMIndomethacin:62.5 nM
Dexamethasone:1 uM
[誘導培地(b)]
DMEM (High Glucose):500 mL
FOETAL BOVINE SERUM (FBS):50 mL
MEM Non‐Essential Amino Acids Solution:5 mL100 mM‐Sodium Pyruvate Solution:5 mL
Penicillin‐Streptomycin Mixed Solution:5 mLInsulin:170 nM
3,3',5‐Triiodo‐L‐thyronine:1 nM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104 cells/wellの濃度で12-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、Cyclic Pifithrin-αまたはALK5 inhibitor IIを添加した軟骨誘導培地(StemPro Chondrogenesis Differentiation Kit : ThermoFisher Scientific社製)を1000 μL/well加えた。
Cyclic Pifithrin-α:5μM
ALK5 inhibitor II: 2μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104 cells/wellの濃度で12-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、軟骨誘導培地、または2μM ALK5 inhibitor IIを添加した軟骨誘導培地を、1000 μL/well加えた。
この細胞をPBS(−)で2回洗浄したのち3%酢酸溶液で1回洗浄したのちナカライテスク社のPH2.5 アルシアンブルー染色液を加えて1時間、室温で染色した。PBS(-)で3回洗浄したのち顕微鏡で観察した。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104 cells/wellの濃度で12-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、2μM ALK5 inhibitor II と2μM D4476を添加した骨格筋細胞増殖培地(5% FBS, 50 microG/ml Bovine Fetuin, 10 nG/ml hEGF, 1 nG/ml bFGF, 10 microG/ml Insulin, 0.4 microG Dexamethasoneを添加したHam's/F10培地)を1000 μL/well加えた。2日に1度、培養液をフレッシュなものに置換し、day 28まで培養した。
ヒト正常皮膚線維芽細胞株HDFsを、5×103 細胞/ウェルの濃度で24 well plateに播種した(day 0)。翌日、各wellの培養液を棄て、500 μl/wellの新しい培地に交換した。骨芽細胞誘導培地はDulbecco’s modified Eagle’s medium (DMEM)、50 μg/mlアスコルビン酸、10 mM β-glycerophosphate、100 nM Dexamethasoneに10% ウシ胎仔血清(FBS)を添加したものである。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM
Pifithrin alpha[p53阻害剤]: 5 μM
SB431542: 2 μM
ALK5 Inhibitor II: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2 /95% humid air 37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または各化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM
Pifithrin alpha[p53阻害剤]: 5 μM
SB431542: 2 μM
ALK5 Inhibitor II: 2 μM。
Day 14に、各wellから培養液を吸引除去し、PBS(-)で洗浄後、細胞からISOGEN IIにてtotal RNAを抽出した。このRNAから、Rever Tra Ace qPCR RT Master Mixを用いてcDNAを合成した。このcDNAにReal-time PCR Master Mixと、CIDEA遺伝子またはβアクチン遺伝子に特異的なprimersとTaqman probeを混和した。AB7300 Real-time PCR systemを用いてqRT-PCRを行った。CIDEA遺伝子のmRNAレベルをβアクチン遺伝子mRNAに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco's modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2 /95% humid air 37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または各小分子化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM。
Pifithrin alpha[p53阻害剤]: 5 μM
PD0325901: 1 μM
SB431542: 2 μM
ALK5 Inhibitor II: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco's modified minimum essential medium; DMEM)に縣濁した。これを1×104 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または各小分子化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM
SB431541: 2 μM
ALK5 inhibitor II: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト間葉系幹細胞(human mesenchymal stem cells; MSC)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地(Normal medium)、石灰化誘導培地(OB medium)、またはALK5 inhibitor II(ALK5 i II)を4 μMの濃度で加えた石灰化誘導培地(OB medium+ ALK5 i II)を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIおよび/またはTGF-βを加えた石灰化誘導培地を、500 μL/well加えた。
ALK5 inhibitor II: 4 μM
TGF-β: 50 ng/ml。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地(Normal medium)、MDI medium(線維芽細胞用の脂肪細胞誘導培地)、またはALK5 inhibitor IIを4 μMの濃度で加えたMDI培地(MDI medium + ALK5 i II)を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)(Normal medium)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、MDI培地、またはALK5 inhibitor IIを4 μMの濃度で加えたMDI培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度でfibronectin coated 24-well plateの中心部に播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、軟骨誘導培地(Chondrocyte medium)、またはALK5 inhibitor IIを4 μMの濃度で加えた軟骨誘導培地(Chondrocyte medium + ALK5 i II)を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度でfibronectin coated 24-well plateの中心部に播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、軟骨誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた軟骨誘導培地を、500 μL/well加えた。
ヒト正常歯肉由来線維芽細胞(human gingival fibroblasts; GFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常歯肉由来線維芽細胞(human gingival fibroblasts; GFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた石灰化誘導培地を、500 μL/well加えた。
動物実験は所属機関の認可を得て行った。8週齢オスのNOD/SCIDマウス(Charles River)を麻酔した。注水下に歯科ドリルを用いて左大腿骨骨幹に直径約7mmの部分骨欠損を作成した。後述の実施例16と同様の方法で、HDFsをALK5 inhibitor II存在下で13日間培養した細胞(CdOBs; Chemical-mediated directly converted osteoblasts)を、マトリゲル (BD Bioscience, San Jose, CA)とともに懸濁し、骨欠損部とその周辺の骨表面に5×105cells/マウスの濃度で移植した。また、同様に骨欠損を作ったのち、線維芽細胞をマトリゲルに懸濁し移植したマウスも準備した。21日後にマウスを安楽死させ、大腿を切除し、中性ホルマリンで固定後、X-ray CT device (Scan Xmate-L090, Com Scan Techno, Yokohama, Japan)を用いてマイクロ・コンピューター断層撮影(μCT)を行った。
実施例25のμCT像を3次元構築したイメージを図30に示す。ALK5 inhibitor IIを添加して培養した細胞は、生体内で骨形成能を有することが示された。CdOBがマウスの体内で骨欠損部において骨形成を行ったことがわかる。
動物実験は所属機関の認可を得て行った。上記実施例25と同様に移植実験を行った。また、線維芽細胞を移植したマウスも準備した。21日後にマウスを安楽死させ、実施例25と同様に大腿を切除し、中性ホルマリンで固定後、骨組織をSCEM (Leica Microsystem) compoundで包埋し、急速凍結した。6 μmの切片にスライス後、連続切片をヘマトキシリン・エオジン (H&E)染色およびAlizarin Red Sで染色した。
ヒト正常皮膚線維芽細胞株HDFsを、60 mm培養ディシュに培養し、通常培地で培養した(-)。また、HDFsを4 μMのALK5 inhibitor IIを加えた通常培地で3または7日間培養した。これらの細胞からISOGEN IIにてtotal RNAを回収した。各細胞のmRNA発現パターンをAffymetrix 社のDNAチップを用いてゲノムワイドに解析した。MSCマーカーの発現量を表したヒートマップを図32に示す。ALK5 inhibitor IIを添加した培地で培養することにより線維芽細胞が間葉系幹細胞(MSCs)にコンヴァートしたことが分かる。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、または各小分子化合物を加えた石灰化誘導培地を、500 μL/well加えた。
I-BET151: 2μM
Pifthrin-α: 5μM
PD0325901:2μM
2-Me-5HT:2μM
CX4945:2μM
CHIR99021:2μM
Forskolin:2μM
DZnep:50nM
D4476:2μM
SB431542:2μM
ALK5 i II:2μM。
実施例29の実験において、Alizarin Red S染色液で染色した後の液を、96 well plateに回収し、吸光度計にて吸光度(OD550nm)を測定した。
結果を図34に示す。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地(OB培地)、または各小分子化合物あるいはサイトカインを添加した石灰化誘導培地を、500 μL/well加えた。
D4476: 1または4 μM
LY2157299: 1または4 μM
LY364947: 1または4 μM
SB431542: 1または4 μM
SB525334: 1または4 μM
SD208: 1または4 μM
ALK5 i II: 1または4 μM
TGF-β: 10 ng/ml。
実施例31の実験において、Alizarin Red S染色液で染色した後の液を、96 well plateに回収し、吸光度計にて吸光度(OD550nm)を測定した。
結果を図36に示す。
さまざまな個人に由来するヒト正常皮膚由来線維芽細胞(HDF45(KURABOより購入)、HDF69(PromoCellより購入)、HDF22(PromoCellより購入))を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 i IIを4μM添加した石灰化誘導培地を、500 μL/well加えた。
さまざまな個人に由来するヒト正常皮膚由来線維芽細胞(HDF45、HDF69、HDF22)を用いて、実施例33と同様の培養を行った。
結果を図38に示す。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、石灰化誘導培地(OB培地)、または各小分子化合物および/またはサイトカインを添加した石灰化誘導培地を、500 μL/well加えた。
ALK5 i II: 4 μM
1α,25-dihydroxy Vitamin D3 (VD3): 5 nM
Human insulin-like growth factor-1 (IGF-1): 100 ng/ml。
Values are means ± S.D. (n=4).)。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、またはALK5 i II、VD3、IGF-1を、それぞれ4 μM 、5 nM、100 ng/mlの濃度で添加した石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBS を添加したDulbecco's modified minimum essential medium; DMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図に示すように、群1には通常培地を1 mL/well 加えた。群2にはRosiglitazoneを含まない脂肪細胞誘導培地(脂肪細胞誘導培地(R-))を1 mL/well 加えた。群3には脂肪細胞誘導培地を1 mL/well 加えた。群4にはALK5 inhibitor II を4 μMの濃度で添加した脂肪細胞誘導培地(R-)を1 mL/well 加えた。群5〜9にはALK5 inhibitor II を4 μMの濃度で添加した脂肪細胞誘導培地を1 mL/well加えた。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、群1には通常培地を1 mL/well 加えた(Control)。群2には脂肪細胞誘導培地を1 mL/well 加えた。群3〜8にはALK5 inhibitor II を4 μMの濃度で添加した脂肪細胞誘導培地を1 mL/well加えた。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、脂肪細胞誘導培地(BA medium)、または化合物 ALK5 inhibitor II、LY2157299、SB431542、D4476を添加した脂肪細胞誘導培地を、1mL/well 加えた。化合物の添加濃度は4 μM、8 μM、12 μM、または16 μMである。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 − Day 8まで培養した。その後、Day 9 − Day 14までは、ALK5 inhibitor II、LY2157299、SB431542、D4476をいずれも含まない培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(−)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、UCP-1遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を、混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。UCP1 遺伝子のmRNA レベルをβ アクチン遺伝子mRNAレベルに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図のように群1には通常培地を、群2には脂肪細胞誘導培地を、群3〜6には化合物ALK5 inhibitor II (4 μM)、LY2157299 (8 μM)、SB431542 (4 μM)、D4476 (4 μM)をそれぞれ加えた脂肪細胞誘導培地を、1mL/well 加えた。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 - Day 8まで培養した。その後、Day 9からDay 14は、ALK5 inhibitor II、LY2157299、SB431542、D4476 をいずれも含まない脂肪細胞誘導培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(−)で洗浄した。4%パラホルムアルデヒドで固定後、PBS(−)にて洗浄し、Perm Buffer(0.2% Triton-X 添加PBS)を加えて15 分間インキュベートした。PBS(−)にて3 回洗浄した後、Blocking One を加えて、室温で60 分間インキュベートした。抗UCP-1 抗体(RD MAB6158)を加えて室温で2 時間反応させた後、Wash buffer にて3 回wash した。CF488-conjugated anti-mouse Ig 抗体(Biotum 20014)を加えて室温で2 時間反応させた後、PBS (-) にて3 回wash した。Lifetechnology社製SlowFade Gold antifade reagent with DAPIで核染色したのち蛍光顕微鏡を用いて倍率100 倍で写真撮影を行った。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、脂肪細胞誘導培地、あるいは化合物 ALK5 inhibitor II(4μM)またはLY2157299(8μM)を加えた脂肪細胞誘導培地を、1 mL/well 加えた。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および 10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 - Day 8まで培養した。その後、Day 9 - Day 14までは、ALK5 inhibitor IIもLY2157299を含まない培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(−)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、UCP-1遺伝子、CIDEA遺伝子、KCNK3遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。UCP1 遺伝子のmRNA レベルをβ アクチン遺伝子mRNAに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、脂肪細胞誘導培地、またはALK5 inhibitor IIを、4 μMの濃度で加えた脂肪細胞誘導培地を、1 mL/well 加えた。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 − Day 8まで培養した。その後、Day 9 − Day 14までは、ALK5 inhibitor IIを含まない培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(−)で洗浄した。4%パラホルムアルデヒドで固定後、PBS(−)にて洗浄し、Perm Buffer(0.2% Triton-X 添加PBS)を加えて15 分間インキュベートした。PBS(−)にて3 回洗浄した後、Blocking One を加えて、室温で60 分間インキュベートした。抗UCP-1 抗体(RD MAB6158)を加えて室温で2 時間反応させた後、Wash buffer にて3 回wash した。CF488-conjugated anti-mouse Ig 抗体(Biotum 20014)を加えて室温で2 時間反応させた後、PBS(-) にて3 回wash した。Lifetechnology社製SlowFade Gold antifade reagent with DAPIで核染色したのち蛍光顕微鏡を用いて倍率100 倍で写真撮影を行った。撮影した画像をKeyence BZ-H3A softwareを用いてDAPI陽性細胞中のUCP1陽性細胞のパーセンテージを算出した。
正常ヒト表皮角化細胞(Human Epidermal Keratinocyte; NHEK(AD))を正常ヒト表皮角化細胞増殖用無血清液体培地(HuMedia-KG2 ; Kurabo社製)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、HuMedia-KG2培地(Ctrl medium)、K-脂肪細胞誘導培地(ケラチノサイト用脂肪細胞誘導培地)、または、ALK5 inhibitor II(4 μM)あるいはLY2157299(8 μM)を添加したK-脂肪細胞誘導培地を、1mL/well 加えた。
正常ヒト表皮角化細胞(Human Epidermal Keratinocyte; NHEK(AD))を正常ヒト表皮角化細胞増殖用無血清液体培地(HuMedia-KG2 ; Kurabo社製)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、HuMedia-KG2培地(Ctrl medium)、K-骨芽細胞誘導培地(ケラチノサイト用骨芽細胞誘導培地)、または化合物 ALK5 inhibitor II(4 μM)を添加したK-骨芽細胞誘導培地を、1mL/well 加えた。K-骨芽細胞誘導培地は、50 μg/mL ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM dexamethasone を添加したHuMedia-KG2である。2 日に1 度、培養液をフレッシュなものに置換し、 Day 14まで培養した。その後、Day 14 − Day 28までは、いずれの細胞もALK5 inhibitor IIを含まない培地で培養した。Day 28 に、各well から培養液を吸引除去し、PBS(−)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、Runx2遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を、混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。Runx2遺伝子のmRNA レベルをβ アクチン遺伝子mRNAレベルに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
実施例45 (図49)
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で48-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを5群に分け、図中に記載の培地を加えた。すなわち、
群1:Control培地
群2:WA培地
群3:1 μM Rosiglitazoneを添加したWA培地
群4:16 μMのALK5 inhibitor IIを添加したWA培地
群5:16 μMのALK5 inhibitor II と1 μM Rosiglitazoneを添加したWA培地。
Control培地は、10% FBS、100 mM non-essential amino acids (NEAA), 100 U/ml penicillin, および 100 μg/mL streptomycinを添加したDulbecco’s modified minimum essential medium; DMEMである。
WA培地は、10% FBS、100 mM non-essential amino acids (NEAA), 100 U/ml penicillin, 100 μg/mL streptomycin、0.5 mM IBMX(3-isobutyl-1-methylxanthine)、62.5 nM Indomethacin、1 μM Dexamethasone、および170 nM Insulinを添加したDulbecco’s modified minimum essential medium; DMEMである。
(1)ウェル内の培養液を除去後、PBSで各ウェルを1回洗浄。
(2)各ウェルに4%ホルムアルデヒド液を50μL加え、室温で一晩固定。
(3)PBSで2 回洗浄。
(4)各ウェルにBODIPY染色液を50μLずつ加え、室温で30 分間静。
(5)ウェル内から染色液を除去後、室温で30 分間静置。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で48-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを7群に分け、図中に記載の培地を加えた。すなわち、
群1:Control培地
群2:WA培地
群3:1 μM Rosiglitazoneを添加したWA培地
群4:8 μMのALK5 inhibitor IIを添加したWA培地
群5:16 μMのALK5 inhibitor IIを添加したWA培地
群6:8 μMのALK5 inhibitor II と1 μM Rosiglitazoneを添加したWA培地。
群7:16 μMのALK5 inhibitor II と1 μM Rosiglitazoneを添加したWA培地。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で6-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを5群に分け、実施例45と同様に図中に記載の培地を加えた。培地の組成は実施例45と同じである。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で48-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを10群に分け、図中に記載の培地を加えた。すなわち、
群1:WA培地
群2:16 μMのALK5 inhibitor II(ALK5 i II)を添加したWA培地
群3:16 μMのLY2157299を添加したWA培地
群4:16 μMのSB431542を添加したWA培地
群5:16 μMのD4476を添加したWA培地
群6:1 μM Rosiglitazoneを添加したWA培地。
群7:16 μMのALK5 inhibitor II (ALK5 i II)と1 μM Rosiglitazoneを添加したWA培地。
群8:16 μMのLY2157299と1 μM Rosiglitazoneを添加したWA培地。
群9:16 μMのSB431542と1 μM Rosiglitazoneを添加したWA培地。
群10:16 μMのD4476と1 μM Rosiglitazoneを添加したWA培地。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で6-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを10群に分け、実施例48と同様に、図中に記載の培地を加えた。培地の組成は実施例45と同じである。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で6-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを5群に分け、図中に記載の培地を加えた。すなわち、
群1:1 μM Rosiglitazone を添加したWA培地
群2:1 μM Rosiglitazone と16 μMのALK5 inhibitor II(ALK5 i II)を添加したWA培地
群3:1 μM Rosiglitazone と16 μMのLY2157299(LY)を添加したWA培地
群4:1 μM Rosiglitazone と16 μMのSB431542(SB)を添加したWA培地
群5:1 μM Rosiglitazone と16 μMのD4476(D4)を添加したWA培地。
ヒト線維芽細胞aHDF45を24ウェルプレートに1.0×104 cells/500 μL/wellずつ播種した。翌日、ウェルを2群に分け、1群は培地を下記のControl培地に交換し、別の1群は4 μMのAlk5 i IIを添加した下記のSC培地に交換した。37℃、5% CO2/95% 大気下で14日間培養した。培地交換は週2回行った。
Control培地:10% FBS、100 mM non-essential amino acids (NEAA), 1 mM Sodium Pyruvate、100 U/ml penicillin, および 100 μg/mL streptomycinを添加したDulbecco’s modified minimum essential medium; DMEM。
SC(Schwann cell)培地:10% FBS、10 ng/mL rhbFGF(basic fibroblast growth factor)、5.7 μg/mL Forskolin 、200 ng/mL rhHeregulin beta-1、5 ng/mL rhPDGF-AA 、100 mM non-essential amino acids (NEAA), 1 mM Sodium Pyruvate、100 U/ml penicillin, および 100 μg/mL streptomycinを添加したDulbecco’s modified minimum essential medium; DMEM。
正常ヒト表皮角化細胞(Human Epidermal Keratinocyte ; NHEK(AD))を正常ヒト表皮角化細胞増殖用無血清液体培地(HuMedia-KG2 ; Kurabo社製)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、HuMedia-KG2培地、ヒト表皮基底細胞増殖用培地(CnT-PR;CELLnTEC社製)、またはALK5 inhibitor IIを1 μMまたは10 μMの濃度で添加したヒト表皮基底細胞増殖様培地を、それぞれ1 mL/well 加えた。2 日に1 度、培養液をフレッシュなものに置換し、day 21まで培養した。Day 21 に各well から培養液を吸引除去し、PBS(−)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、Uroplakin 1b遺伝子、Uroplakin 3b遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を、混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。Uroplakin 1b 遺伝子とUroplakin 3b遺伝子のmRNA レベルをβ アクチン遺伝子mRNAレベルに対する比として定量し、HuMedia-KG2培地で培養したヒト表皮角化細胞の値を1として算出した。その結果を図56に示す。ALK5 inhibitor IIを添加したCnT-PR培地で培養することにより、ヒト表皮角化細胞が、Uroplakin 1b遺伝子とUroplakin 3b遺伝子を発現する尿路上皮細胞にコンヴァートしたことがわかる。
ヒト正常末梢血単核細胞(human peripheral blood mononuclear cells; PBMCs)をcontrol medium(10% FBS を添加したRPMI1640培地)に縣濁した。これを 2.5×105cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、群1にはControl mediumを1 mL/well 加えた。群2にはP-脂肪細胞誘導培地(末梢血単核細胞用脂肪細胞誘導培地)を1 mL/well加えた。群3にはALK5 inhibitor II を4 μMの濃度で添加したP-脂肪細胞誘導培地を1 mL/well加えた。P-脂肪細胞誘導培地は、10% FBS、30 U/ml recombinant hIL-2、1 nM T3、1 μM Rosiglitazone、 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasoneおよび1 μg/mL Insulinを添加したRPMI1640培地である。2 日に1 度、培養液の50%をフレッシュなものに置換した。
Claims (15)
- 哺乳動物の分化した体細胞を、前記分化した体細胞以外の他の体細胞を分化誘導するための培地中で、TGF-βパスウェイ阻害剤の存在下に培養して、前記分化した体細胞を他の体細胞にコンヴァートさせることを特徴とする、体細胞を調製する方法。
- TGF-βパスウェイ阻害剤が、D4476、SB431542、LY2157299、SD208、又はALK5 inhibitor IIである、請求項1に記載の方法。
- TGF-βパスウェイ阻害剤が、ALK5 inhibitor IIである、請求項1又は2に記載の方法。
- 線維芽細胞を間葉系の細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- 線維芽細胞又はケラチノサイトを骨芽細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- 線維芽細胞又は末梢血単核細胞を白色脂肪細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- 線維芽細胞又はケラチノサイトを褐色脂肪細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- 体細胞を分化誘導するための培地が、Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ)アゴニストを含む、請求項6又は7に記載の方法。
- 線維芽細胞を軟骨細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- 線維芽細胞を筋芽細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項記載の方法。
- 線維芽細胞をシュワン細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- ケラチノサイトを尿路上皮細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- 線維芽細胞を間葉系幹細胞にコンヴァートさせることを特徴とする、請求項1〜3のいずれか1項に記載の方法。
- TGF-βパスウェイ阻害剤を含む、分化した体細胞を他の体細胞にコンヴァートさせるための誘導剤。
- 分化した体細胞を他の体細胞にコンヴァートさせるためのキットであって、TGF-βパスウェイ阻害剤及び前記他の体細胞を分化誘導するための培地を含むキット。
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WO2019228705A1 (en) * | 2018-05-28 | 2019-12-05 | Societe Des Produits Nestle S.A. | Production of brown adipocytes |
WO2020086667A1 (en) | 2018-10-25 | 2020-04-30 | American University | Method for promoting adipocyte differentiation and obesity-related disease treatment |
KR102304483B1 (ko) * | 2019-05-08 | 2021-09-24 | 성균관대학교산학협력단 | 저분자 화합물을 이용한 갈색 지방 세포 유사 세포로의 직접분화 방법 및 조성물 |
CN110218699B (zh) * | 2019-06-30 | 2020-07-28 | 北京佑仁生物科技集团有限公司 | 脂肪干细胞快速培养及分化方法 |
WO2021039972A1 (ja) * | 2019-08-30 | 2021-03-04 | セルアクシア株式会社 | 尿路上皮細胞への誘導剤及び尿路上皮細胞の誘導方法 |
WO2021106698A1 (ja) * | 2019-11-25 | 2021-06-03 | 株式会社片岡製作所 | 培地用組成物 |
CN111849884B (zh) * | 2020-07-30 | 2021-02-19 | 山东天川精准医疗科技有限公司 | 一种人胎盘羊膜干细胞向肝细胞定向分化的诱导方法 |
CN116867892A (zh) * | 2021-02-15 | 2023-10-10 | 凸版印刷株式会社 | 三维组织体的制造方法和脂肪来源干细胞的分化促进方法 |
CN114317401B (zh) * | 2021-12-07 | 2022-10-04 | 浙江大学 | 一种促进胰岛前体细胞长期稳定传代的方法 |
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