人诱导多潜能干细胞衍生肝细胞的药物发现方法
Summary
这里提出的协议描述了一个平台, 用于鉴别肝脏疾病的小分子。本文详细介绍了如何将 iPSCs 分化为96井板中具有肝细胞特征的细胞, 并利用细胞筛选出具有潜在治疗活性的小分子。
Abstract
将人类诱导的多能干细胞 (iPSCs) 与肝细胞样细胞 (HLCs) 进行鉴别的能力为研究肝代谢中先天错误提供了新的机会。然而, 为了提供一个平台, 支持识别可能用于治疗肝病的小分子, 该程序需要一种与筛选数以千计的化合物相兼容的区域性格式。在这里, 我们描述了一个协议使用完全定义的文化条件, 使人类 iPSCs 的可再生分化为肝样细胞在96井组织培养板。我们还提供了一个例子, 使用该平台, 以筛选化合物的能力, 以降低载脂蛋白 B (APOB) 产生的 iPSC 肝细胞产生的家庭高胆固醇血症患者。与药物发现相兼容的平台的可用性应该允许研究人员确定新的治疗方法, 用于影响肝脏的疾病。
Introduction
成功地确定可用于治疗罕见疾病的药物依赖于可用于筛查的化验方法的发展。假设或基于目标的屏幕 (反向药理学) 是有用的, 但需要详细了解该疾病的分子基础。表型筛 (经典药理学) 避免了对生物化学途径的详细理解, 而是依赖于精确地反映疾病病理生理学的模型的发展。尽管对以目标为基础的方法的热情, FDA 批准的一流药物的分析显示, 表型屏幕已远远超过成功的1。该方法的总体目标是建立一个高通量筛选平台, 可用于鉴别代谢性肝病的小分子。一些体外模型被描述包括原发肝细胞、肝癌干细胞和肝祖细胞2。然而, 这些模型大多有局限性, 需要新的模型, 可以准确地重述代谢性肝脏缺陷的病理生理学的文化。最近, 人类多潜能干细胞结合基因编辑提供了一个机会, 以模型甚至罕见的稀有疾病的文化, 而不需要直接访问病人的3。虽然使用病人特定的 iPSCs 作为一种工具来发现小分子治疗罕见肝病的概念是合理的, 但只有少数报告表明这种方法的可行性4。然而, 我们最近建立了一个平台, 使用 iPSC 的肝细胞成功地识别药物, 可用于治疗肝代谢缺陷5。
该协议解释了将人类 iPSCs 与96井板中的肝细胞样细胞进行鉴别的过程, 并利用它们来筛选小分子库。并以高胆固醇血症为例, 描述了代谢性肝病的终点分析。这种方法应有助于研究小分子在感染性肝病、代谢性肝病、药物毒性和其他肝病中的作用和应用。
Protocol
1. 人类诱导多潜能干细胞的培养 涂层重组人 e-钙黏蛋白 fc 融合蛋白质 (e cad fc) 或其他适合 hPSC 文化的矩阵6 将 Dulbecco 的磷酸盐缓冲盐水 (DPBS (+)) 稀释为15微克/毫升。 涂层100毫米悬浮组织培养皿与5毫升稀释的 E cad Fc 和孵育在37˚C 至少 1 h. 移除基板, 并用5毫升的介质 (例如, mTeSR1 称为 M 介质从今以后)7。注意:</st…
Representative Results
生成肝细胞-类似于单元格:图 1描述了在将人类 iPSCs 分化为类似肝细胞时发生的变化的时间范围。iPSCs 的文化在 E Cad Fc 提供大约2毫米直径殖民地表达多能标记 OCT4 (图 1A-B)。在 e-钙黏蛋白基体上生长的细胞的形态学与其他表面培养的不同。他们倾向于有一个扁平的形态学与更大…
Discussion
基于目标的药物发现, 小分子被发现影响特定蛋白质的活性, 已成为许多现有筛查工作的重点。尽管这种方法提供了大量的药物, 但基于反转表型的屏幕, 经典药理学, 在识别临床上有效的1的第一类化合物方面更成功。表型药物发现的一个缺点是它依赖于适当的疾病模型的可用性。这可能是一个挑战, 当细胞模型的疾病是不可用的, 或当共同的研究动物物种未能概括的临床症状。然?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到国家卫生研究院 (DK55743、DK087377、DK102716 和 HG006398) 的支持。我们要感谢 Behshad Pournasr 博士, 詹姆斯 Heslop 博士, 并为他们的贡献而奔跑。
Materials
| 100 mm x 20 mm sterile tissue culture dishes | Corning | 430167 | |
| 100 mm x 20 mm sterile suspension culture dishes | Corning | 430591 | |
| 96-wells tissue culture plate | Corning | 3595 | |
| Anti-human Albumin | Dako | A 0001 | |
| Anti-human FOXA2(6C12) | Novus Biological | H00003170-M12 | |
| Anti-human HNF4 alpha | Santa Cruz | SC-6556 | |
| Anti-human Oct-3/4 antibody | Santa Cruz | SC-9081 | |
| Anti-human SOX17 | R&D | AF1924 | |
| Anti-human TRA-1-60 FITC conjugated | Millipore | FCMAB115F | |
| Activin A Recombinant Human Protein | Invitrogen | PHC9563 | |
| B-27 Supplement, minus insulin | Invitrogen | 0050129SA | |
| B-27 Supplement, serum free | Invitrogen | 17504044 | |
| BMP4 Recombinant Human Protein | Invitrogen | PHC9533 | |
| Cell Dissociation Reagent StemPro Accutase | Invitrogen | A1110501 | |
| CellTiter-Glo Luminescent Cell Viability Assay | Promega | 7572 | |
| DPBS+(calcium, magnesium) | Invitrogen | 14040-133 | |
| DPBS-(no calcium, no magnesium) | Invitrogen | 14190-144 | |
| DMEM/F-12, HEPES | Invitrogen | 11330057 | |
| ELISA human APOB ELISA development kit | Mabtech | 3715-1H-20 | |
| Fibroblast Growth Factor 2 (FGF2) | Invitrogen | PHG0023 | |
| Hepatocyte Culture Medium (HCM Bullet Kit) | Lonza | CC-3198 | |
| Hepatocyte Growth Factor (HGF) | Invitrogen | PHC0321 | |
| L-Glutamine | Invitrogen | 25030081 | |
| MEM Non-Essential Amino Acids Solution | Invitrogen | 11140076 | |
| Oncostatin M (OSM) Recombinant Human Protein | Invitrogen | PHC5015 | |
| Penicillin-Streptomycin | Invitrogen | 15140163 | |
| Feeder free pluripotent stem cell medium: mTesR1 | STEMCELL technologies | 5850 | |
| Reduced Growth Factor Basement Membrane Matrix | Invitrogen | A1413301 | |
| RPMI 1640 Medium, HEPES | Invitrogen | 22400105 | |
| StemAdhere Defined Matrix for hPSC (E-cad-Fc) | Primorigen Biosciences | S2071 | |
| TMB-ELISA Substrate Solution | Thermo Scientific | 34022 | |
| Anti-TRA-1-60 FITC conjugated | Millipore | FCMAB115F | |
| Versene (EDTA) 0.02% | Lonza | 17-711E | |
| Y-27632 ROCK inhibitor | STEMCELL Technologies | 72302 |
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Cite This Article
Liu, J., Lamprecht, M. P., Duncan, S. A. Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery. J. Vis. Exp. (135), e57194, doi:10.3791/57194 (2018).