肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Summary

本文将着重从人类胚胎干细胞群的人肝胚层代。

Abstract

尽管在模拟人类药物的毒性取得了进展，许多化合物在由于预见的副作用临床试验失败。临床研究的成本是巨大的，因此它是至关重要的，更预测毒理学屏幕在药物开发（Greenhough等2010）早期开发和部署。人肝细胞的代表，目前黄金标准模型评价药物毒性，而且是一种有限的资源，表现出变量的函数。因此，使用永生细胞株和动物组织模型是经常雇由于其丰富度。虽然这两个来源的信息，他们是有限的功能较差，物种变异和/或文化的不稳定（Dalgetty等2009）。多能干细胞（PSCS）是一个有吸引力的替代来源的人类肝细胞样细胞（HLCs）（Medine等2010）。物业服务公司的自我更新和分化，在成人中发现所有的体细胞类型的能力，从而代表分化细胞的一个潜在的不竭源泉。我们已经开发出一种程序，是简单，高效，适合自动化和产量人类功能HLCs（Hay等人2008;弗莱彻等人2008; Hannoun等2010;佩恩等人2011年和干草等2011）。我们相信我们的技术会导致可扩展性生产的药物研发，疾病模型，额外的有形的设备建设和可能基于细胞移植治疗HLCs。

Protocol

1。所有化工股的前期准备工作和文化的塑料制品的涂层在一个组织文化罩在无菌条件下进行的所有步骤。 制备碱性成纤维细胞生长因子（hbFGF） 准备10％BSA在PBS和过滤解决方案，通过一个0.22微米的过滤器。 准备从10％BSA溶液0.2％的BSA溶液。 加入10毫升0.2％BSA solution/100微克hbFGF。 5 mL 10％的BSA溶液通过过滤器过滤，预湿用0.22微米的过滤器。丢?…

Discussion

我们已经开发出一种简单，同质化和高度在体外重现的模型生成可扩展人类HLCs水平。我们的模式已经被验证由外部的合作实验室。我们经常干细胞HLCs使用我们在内部发展的标志和具体肝功能试验（其中大部分是市售）工具盒的特点。在我们的进程的关键阶段是：维持干细胞多能性;直接明确的内胚层干细胞分化能力;肝胚层的同质文化的规范，并能够推导出成熟的肝胚层参展在体外的…

Materials

Matrigel coating plates and flasks

1. Matrigel (10 mL, BD Biosciences, UK); store at -20°C.
2. KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4°C.
3. Tissue culture plates (6 well, 12 well, Corning, UK)
4. Tissue culture flask (25 cm2 vented, Corning, UK)

hESC Maintenance

1. Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R & D Systems, USA); store at -20°C.
2. BSA solution (50 mL, Sigma Aldrich, UK); store at 4°C.
3. Human basic fibroblast growth factor (100 μg, Peprotech, USA); store at -20°C.

Passaging hESCs with collagenase

1. Confluent well or flask of hESCs.
2. Matrigel coated wells or flasks as appropriate.
3. Phospate buffered saline (-MgCl₂, -CaCl₂) (500 mL, Gibco, Invitrogen, UK); store at room temperature.
4. Collagenase IV (1 g, Gibco, Invitrogen, UK); store at 4°C.
5. Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R & D Systems, USA).
6. Human basic fibroblast growth factor (100 μg, Peprotech, USA).

Differentiation of hESCs to hepatic endoderm

1. RPMI 1640 (500 mL, Gibco, Invitrogen, UK); store at 4°C.
2. B27 Supplement (10 mL, Gibco, Invitrogen, UK); store at -20°C.
3. Activin A (2 μg, Peprotech, USA); store at -20°C.
4. Recombinant mouse Wnt3a (2 μg, R & D Systems, USA); store at -20°C.
5. KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4°C.
6. KO-SR (500 mL, Gibco, Invitrogen, UK); store at -20°C.
7. Non-essential amino acids (100 mL, Gibco, Invitrogen, UK); store at 4°C.
8. β-Mercaptoethanol (10 mL, Gibco, Invitrogen, UK); store at 4°C.
9. DMSO (Sigma Aldrich, UK); store at room temperature
10. Leibovitz L-15 culture medium (500 mL, Sigma Aldrich, UK); store at 4°C.
11. Tryptose phosphate broth (100 mL, Sigma Aldrich, UK); store at 4°C.
12. Foetal bovine serum, heat inactivated (500 mL, Gibco, Invitrogen, UK); store at -20°C.
13. Hydrocortisone 21-hemisuccinate (100 mg, Sigma Aldrich, UK); store at -20°C.
14. Insulin (bovine pancreas) (100 mg, Sigma Aldrich, UK); store at -20°C.
15. L-Glutamine (100 mL, Gibco, Invitrogen, UK); store at -20°C.
16. Ascorbic acid (25 g, Sigma Aldrich, UK); store at -20°C.
17. Human HGF (10 μg, Peprotech, USA); store at -20°C.
18. Recombinant Human Oncostatin M (OSM) (50 μg, R & D Systems, USA); store at -20°C.
19. Syringe driven filter unit 0.22 μm (Millipore, UK)

Characterisation of hESC derived Hepatic Endoderm

Immunostaining

1. Phosphate buffer saline (-MgCl₂, -CaCl₂) (500 mL, Gibco, Invitrogen, UK); store at room temperature.
2. PBST, PBS made up with 0.1% TWEEN 20 (Sigma-Aldrich, UK).
3. Paraformaldehyde (PFA) (Sigma-Aldrich, UK) is made up in PBS, store -20°C.
4. Glycerol (Sigma-Aldrich, UK), store at room temperature.
5. Tris Base (Sigma-Aldrich, UK), store at room temperature.
6. Ethanol
7. Serum (AbD Serotech, UK), store at -20°C.
8. Secondary Antibody, Alexa Fluorophores (Molecular Probes, Invitrogen, UK).
9. MOWIOL 4-88 (Polysciences Inc, USA) is made up in Tris HCL and glycerol as per manufacturers instructions. DAPI (Pierce, Thermo Fisher Scientific, UK) is added to the MOWIOL solution at a 1:1000 dilution.

Primary antibodies
Antigen*	Tipo	Supplier	Dilution
ALB	Mouse Monoclonal	Sigma Aldrich	1/500
E-Cadherin	Mouse Monoclonal	Millipore	1/100
α-fetoprotein	Mouse Monoclonal	Sigma	1/500
SSEA-4 FITC	Mouse Monoclonal	Biolegend	1/100
IgG	Mouse Monoclonal	DAKO	1/500
Secondary antibodies
Anti-mouse FITC conjugate	Goat Monoclonal	Invitrogen	1/400

Table 2. The antibodies used for hESC derived hepatic endoderm immunostaining, the concentrations used, the species developed in and the companies they are purchased from.

Functional Analysis of Hepatic Endoderm and Normalisation (per mg protein)

Cytochrome P450 Assays

1. p4-GLO CYP3A4, CYP1A2, Kits and luminometer (Promega, USA).
1. White flat bottom 96 well assay plate (BD Biosciences, UK).
2. BCA Assay Kit (Pierce, Thermo Fisher Scientific, UK).
3. Transparent 96 well assay plate (IWAKI, UK)