| 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) |
|
