人胚胎干细胞的单层内胚层分化

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https://www.liankebio.com/article-information_Tech-1533.html

作者:联科生物 发布日期:2017-03-10 09:00

无血清条件下,activin A存在的胚胎干细胞分化,会生成一种共表达Brachyury, Foxa2 和 c-Kit或者c-Kit 和Cxcr4的内胚层祖细胞群。这些含骨形态生成蛋白-4的祖细胞结合碱性成纤维生长因子(FGF-basic)和activin A会使肝细胞群生长,高度富集(45–70%)表达alpha-甲胎蛋白和白蛋白的细胞。

Protocol
1. 准备ES细胞到matrigel plate,去除MEFs

? 准备1:3 matrigel plate:冰上预冷TC 培养板和移液管,冰箱或者冰上解冻1:3 matrigel。小心把matrigel包被在板子上且保证覆盖底部,然后吸出多余的matrigel(在包被 matrigel过程中保持所有东西预冷)。把matrigel plates放在冰上30 min。再次吸出多余的matrigel并把包被matrigel的板子37?C孵育30 min。

? 37?C TrypLE处理融合的ES细胞2-3 min(10 cm dish用4ml TrypLE或者6孔板的每孔1ml),然后吸出TrypLE。用wash media (IMDM + Glu + P/S + 0.1%BSA) 洗2-3次。10 cm dish加4ml hES media或者6孔板的每孔1ml,用细胞刮棒刮去板子底部的ES细胞。上下吹打3-5次,显微镜下观察直到可以得到5-10个细胞团。

? 接种细胞到matrigel-coated板子上

2. 设置T0:5% O2, 5% CO2条件下,当有80%融合时,细胞会发生分化。用wash media洗细胞2次并添加适量体积T0 培养基(如10cm dish加10ml,6孔板的每孔2ml)

3. 用wash media洗细胞并且每天更换培养基直到T5。前几天看到大细胞死亡是正常现象。

4. 进入肝分化,用Accutase 37?C 3min分离T5细胞,wash media洗细胞两次。6孔板的每孔加入1ml wash media并刮下细胞。收集T5细胞,1200 rpm离心3min。用T5 media重悬细胞并且以1:6接种细胞到matrigel-coated 6孔板,250K cells/well。

5. 每2天用不同分化培养基接种细胞(如下),并在18-25天收集用于分析。

注:matrigel的每批内毒素水平和蛋白浓度都有差异,建议内毒素水平不高于2 units/mL,蛋白水平应在7-10mg/mL之间。如果蛋白水平较高,可能要多于1:1稀释,这取决于hESC克隆形态和hESC分化为需要系别的能力。操作matrigel过程中,所有管子、培养板和移液器都必须是预冷的,matrigel室温下回凝固。

附:1) hES Media (500 ml)
DMEM/F12: 400 ml

KOSR: 100 ml

NEAA: 5 ml

L-glutamine: 5 ml

Pen/strep: 5 ml

β-Mercaptoethanol: 3.5 ul

bFGF: 10 ng/ml (50 ul of 100 ug/ml 贮存)

L-Ascorbic Acid: 50 ug/ml –每次培养基更换加新鲜的5 mg/ml贮存液

2) 分化培养基:

T0 Media
Basic Media RPMI
Ingredient Stock Volume Final
Glutamine 100x 10 ul/ml 1x
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5 ×10-4 M
Chir 99021 10 mM 0.2 ul/ml 2 uM
Activin A 100 ug/ml 1 ul/ml 100 ng/ml
T1 and T2 MEDIA
Basic Media RPMI
Ingredient Stock Volume Final
Glutamine 100x 10 ul/ml 1x
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5×10-4 M
Ascorbic Acid 5 mg/ml 10 ul/ml 50 ug/ml
BMP4 10 ug/ml 0.025 ul/ml 0.25 ng/ml
bFGF 10 ug/ml 0.5 ul/ml 5 ng/ml
Activin A 100 ug/ml 1 ul/ml 100 ng/ml
VEGF 25 ug/ml 0.4 ul/ml 10 ng/ml
T3 and T4 MEDIA
Basic Media SFD
Ingredient Stock Volume Final
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5×10-4 M
Ascorbic Acid 5 mg/ml 10 ul/ml 50 ug/ml
BMP4 10 ug/ml 0.025 ul/ml 0.25 ng/ml
bFGF 10 ug/ml 0.5 ul/ml 5 ng/ml
Activin A 100 ug/ml 1 ul/ml 100 ng/ml
VEGF 25 ug/ml 0.4 ul/ml 10 ng/ml

*SFD: 1L750 ml: homemade IMDM (Invitrogen 12200-069) (1×10 L) (+ Glutamine, + 25 mM HEPES+P/S)250 ml: Hams F125 ml: N2-supplement (Invitrogen 17502-048)10 ml: B27 supplement w/o retinoic acid (Invitrogen 12587-010)5 ml: 10% BSA in PBS (3) 肝分化培养基:

DE T5 and T6 MEDIA
Basic Media SFD
Ingredient Stock Volume Final
Glutamine 100x 10 ul/ml 1x
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5×10-4 M
Ascorbic Acid 5 mg/ml 10 ul/ml 50 ug/ml
BMP4 10 ug/ml 1 ul/ml 10 ng/ml
bFGF 10 ug/ml 0.5 ul/ml 5 ng/ml
Activin A 100 ug/ml 0.5 ul/ml 50 ng/ml
VEGF 10 ug/ml 1 ul/ml 10 ng/ml
DE T7-T12 MEDIA
Basic Media SFD
Ingredient Stock Volume Final
Glutamine 100x 10 ul/ml 1x
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5×10-4 M
Ascorbic Acid 5 mg/ml 10 ul/ml 50 ug/ml
BMP4 100 ug/ml 0.5 ul/ml 50 ng/ml
bFGF 100 ug/ml 0.1 ul/ml 10 ng/ml
VEGF 25 ug/ml 0.4 ul/ml 10 ng/ml
EGF 50 ug/ml 0.2 ul/ml 10 ng/ml
TGFα 50 ug/ml 0.4 ul/ml 20 ng/ml
HGF 100 ug/ml 1 ul/ml 100 ng/ml
DEX 50 uM 2 ul/ml 100 nM
T13-T18
Basic Media SFD
Ingredient Stock Volume Final
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5×10-4 M
Ascorbic Acid 5 mg/ml 10 ul/ml 50 ug/ml
bFGF 100 ug/ml 0.1 ul/ml 10 ng/ml
VEGF 25 ug/ml 0.4 ul/ml 10 ng/ml
EGF 50 ug/ml 0.2 ul/ml 10 ng/ml
HGF 100 ug/ml 1 ul/ml 100 ng/ml
OSM 100 ug/ml 0.2 ul/ml 20 ng/ml
DEX 50 uM 4 ul/ml 200 nM
VK(Vitamin K) 10 mg/ml 0.6 ul/ml 6 ug/ml
DMSO dry 10 ul/ml 1%
gSI (γ-Secretase Inhibitor) 1mM 1.5 ul/ml 1.5 uM
Liver T19-T25
Basic Media SFD
Ingredient Stock Volume Final
MTG (MONOTHIOGLYCEROL) 13 ul/ml 3 ul/ml 4.5×10-4 M
Ascorbic Acid 5 mg/ml 10 ul/ml 50 ug/ml
HGF 100 ug/ml 1 ul/ml 100 ng/ml
OSM 100 ug/ml 0.2 ul/ml 20 ng/ml
DEX 50 uM 2 ul/ml 100 nM
VK(Vitamin K) 10 mg/ml 0.6 ul/ml 6ug/ml

以上内容摘自StemBook-protocols

主要产品介绍:

cytokines Supplier/Catalog # Stock Conc.
hBMP-4 PeproTech # 120-05/120-05ET/AF-120-05ET 10 ug/mL
hbFGF PeproTech # 100-18B/AF-100-18B/ 100-18C/AF-100-18C 10 ug/mL
hVEGF PeproTech # 100-20/AF-100-20 5 ug/mL
Activin A PeproTech # 120-14/AF-120-14/120-14E/AF-120-14E/120-14P 10 ug/mL
EGF PeproTech # AF-100-15 100 ug/mL
HGF PeproTech # 100-39/100-39H 10 ug/mL
TGFα PeproTech # 100-16A/AF-100-16A 20 ug/mL
Oncostatin M (OSM) PeproTech # 300-10/300-10T/AF-300-10T 100 ug/ml
细胞培养耗材 Supplier/Catalog #
细胞培养皿, 100 mm, TC 处理, 无菌, 无细胞毒性 Eppendorf # 0030702115
细胞培养板, 6孔, TC处理, 带盖, 平底, 独立包装, 无菌, 无细胞毒性 Eppendorf # 0030720113

其他需要辅助试剂,可点击联科官网或致电400-6721-600查询

Matrigel N2 supplement
DMEM/F12 B27 Supplement
KnockoutTM Serum Replacement (KOSR) Non-Essentail Amino Acids
β-Mercaptoethanol BSA
L-Ascorbic Acid γ -Secretase Inhibitor
Monothioglycerol Vitamin K (VK)
TrypLE Dexamethasone
DNAse I Bovine Pancreas DMSO
Hams F12 media Chir 99021
L-Glutamine L-ASCORBIC ACID (AA)
Penicillin/Streptomycin MONOTHIOGLYCEROL (MTG)