Summary

从人类胚胎干细胞中引导肝细胞样细胞诱导的有效方法

Published: May 06, 2021
doi:

Summary

本手稿描述了人类胚胎干细胞(hESC)分化成功能性肝细胞样细胞(HLCs)的详细协议,在hESC分化过程中连续补充Activin A和CHIR99021,以最终内分泌体(DE)。

Abstract

从人类胚胎干细胞中提取的肝细胞样细胞(HMC)的潜在功能对疾病建模和药物筛选应用有很大的希望。如果这里是将 hESC 区分为功能性 HMC 的有效且可重复的方法。建立内皮体血统是区分HLC的关键步骤。通过我们的方法,我们通过在 hESC 分化过程中持续补充 Activin A 和 CHIR99021 来调节关键信号通路,然后生成肝祖细胞,最后在具有完全定义试剂的阶段性方法中使用典型的肝细胞形态学的 HLC。这种方法产生的 hESC 衍生 HLC 表示特定阶段标记(包括白蛋白, HNF4®核受体和陶罗乔拉酸钠共运多肽(NTCP),并表现出与成熟和功能性肝细胞(包括花青绿色染色、糖原储存、血氧林-异丙辛染色和CYP3活性)相关的特殊特性,为肝病研究中基于HLC的应用的发展提供了平台。

Introduction

肝脏是一种高度代谢的器官,起着多种作用,包括脱氧、储存糖原、蛋白质的分泌和合成。各种病原体、药物和羊群可引起肝脏病理变化,影响其功能肝细胞作为肝脏的主要功能单位,在人工肝脏支持系统和药物毒性消除方面发挥着重要作用。然而,在细胞治疗和肝病研究中,原发性人类肝细胞的资源有限。因此,开发新的功能性人类肝细胞来源是再生医学领域的一个重要研究方向。自1998建立HESC以来,由于具有优越的分化潜力(可以在合适的环境中分化成各种组织)和高度的自我更新性,为生物关节肝、肝细胞移植甚至肝组织工程提供了理想的来源细胞,因此,hESC得到了广泛的应用。

目前,通过丰富内分泌物6,可以大大提高肝分化效率。在干细胞分泌成内分泌物的系数中,转化生长因子β(TGF-β)信号和WNT信号通路的水平是内分泌形成阶段节点的关键因素。激活高水平的TGF-β和WNT信号可以促进内分泌体7、8的发展。阿克蒂文A是属于TGF-β超级家庭的细胞因子。因此,Activin A广泛应用于人类诱导多能干细胞(hiPSC)和hESC9、10的内皮体诱导。葛兰素史克3是一种血清素-三氨酸蛋白激酶。研究人员发现,CHIR99021是葛兰素史克3®的特异性抑制剂,可以刺激典型的WNT信号,并在一定条件下促进干细胞分化,表明CHIR99021有可能诱导干细胞分化成内分泌物11、12、13。

在这里,我们报告一种高效和可重复的方法,用于有效诱导 hESC 分化为功能 HHLC。Activin A 和 CHIR99021 的连续添加产生了约 89.7 ±0.8% SOX17 (DE 标记) 阳性细胞。 在体外进一步成熟后,这些细胞表示肝特异性标记,并施加肝细胞状形态(基于血氧林-肌辛染色(H &E))和功能,如吸收花青绿色(ICG)、糖原储存和CYP3活性。结果表明,HESC可以通过这种方法成功分化为成熟的功能HLC,为肝病相关研究和 体外 药物筛选提供依据。

Protocol

1. 干细胞维护 注:下文所述的细胞维护协议适用于在附着单层中维护的 hES03 细胞系。对于本手稿中的所有以下协议,细胞应在生物安全柜下处理。 通过稀释5倍补充介质到mTesR基础介质,准备1xmTesR干细胞培养介质。 通过在冰面上稀释 5 毫升符合 hESC 资格的矩阵凝胶,用 5 毫升的 DMEM/F12 来制备 30 倍 hESC 合格的矩阵介质。存储在-20°C。 通过稀释 33.3 μL 的…

Representative Results

HLC 感应的示意图图和每个分化阶段的代表性亮场图像显示在图 1 中。在第一阶段,Activin A和CHIR99021被添加3天,以诱导干细胞形成内皮细胞。在第二阶段,内皮细胞在用分化介质治疗5天后分化成肝祖细胞。在第三阶段,早期肝细胞在HGF和OSM(图1A)中10天后成熟并分化为HLC。在分化的最后阶段,细胞表现出典型的肝细胞表型(细胞是多边形的,分布均匀…

Discussion

在这里,我们提出了一个分步方法,将 HSC 分为三个阶段。在第一阶段,阿克蒂文A和CHIR99021用于区分氢氟碳化合物到DE。在第二阶段,KO-DMEM和DMSO用于区分DE到肝祖细胞。在第三阶段,HZM加HGF、OSM和氢皮质松21-血氧钠盐用于继续将肝祖细胞分化为HLC。

在使用协议时,需要考虑以下关键步骤。细胞的初始分化密度和中等变化的准确时间是成功分化的重要因素。当我们开始分化时?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了中国国家自然科学基金委员会(第81870432号,81570567 X.L.Z.)(第81571994号)的支持:中国广东省自然科学基金(第2020A1515010054号,中华人民共和国),李嘉诚汕头大学基金会(第2020A15015010054号)。L1111 2008 至 P.N.S.)。我们要感谢汕头大学医学院的林士丹教授的有益建议。

Materials

2-Mercaptoethanol Sigma M7522 For hepatic progenitor differentiation
488 labeled goat against mouse IgG ZSGB-BIO ZF-0512 For IF,second antibody
488 labeled goat against Rabbit IgG ZSGB-BIO ZF-0516 For IF,second antibody
Accutase Stem Cell Technologies 7920 For cell passage
Activin A peproTech 120-14E For definitive endoderm formation
Anti – Albumin (ALB) Sigma-Aldrich A6684 For IF and WB, primary antibody
Anti – Human Oct4 Abcam Ab19587 For IF, primary antibody
Anti – α-Fetoprotein (AFP) Sigma-Aldrich A8452 For IF and WB, primary antibody
Anti -SOX17 Abcam ab224637 For IF, primary antibody
Anti-Hepatocyte Nuclear Factor 4 alpha (HNF4α) Sigma-Aldrich SAB1412164 For IF, primary antibody
B-27 Supplement Gibco 17504-044 For definitive endoderm formation
BSA Beyotime ST023-200g For cell blocking
CHIR99021 Sigma-Aldrich SML1046 For definitive endoderm formation
DAPI Beyotime C1006 For nuclear staining
DEPC-water Beyotime R0021 For RNA dissolution
DM3189 MCE HY-12071 For definitive endoderm formation
DMEM/F12 Gibco 11320-033 For cell culture
DMSO Sigma-Aldrich D5879 For hepatic progenitor differentiation
DPBS Gibco 14190-144 For cell culture
GlutaMAX Gibco 35050-061 For hepatic progenitor differentiation
H&E staining kit Beyotime C0105S For H&E staining
Hepatocyte growth factor (HGF) peproTech 100-39 For hepatocyte differentiation
HepatoZYME-SFM (HZM) Gibco 17705-021 For hepatocyte differentiation
Hydrocortisone-21-hemisuccinate Sigma-Aldrich H4881 For hepatocyte differentiation
Indocyanine Sangon Biotech A606326 For Indocyanine staining
Knock Out DMEM Gibco 10829-018 For hepatic progenitor differentiation
Knock Out SR Multi-Species Gibco A31815-02 For hepatic progenitor differentiation
Matrigel hESC-qualified Corning 354277 For cell culture
MEM NeAA Gibco 11140-050 For hepatic progenitor differentiation
mTesR 5X Supplement Stem Cell Technologies 85852 For cell culture
mTesR Basal Medium Stem Cell Technologies 85851 For cell culture
Oncostatin (OSM) peproTech 300-10 For hepatocyte differentiation
P450 – CYP3A4 (Luciferin – PFBE) Promega V8901 For CYP450 activity
PAS staining kit Solarbio G1281 For PAS staining
Pen/Strep Gibco 15140-122 For cell differentiation
Peroxidase-Conjugated Goat anti-Mouse IgG ZSGB-BIO ZB-2305 For WB,second antibody
Primary Antibody Dilution Buffer for Western Blot Beyotime P0256 For primary antibody dilution
ReverTraAce qPCR RT Kit TOYOBO FSQ-101 For cDNA Synthesis
RNAiso Plus TaKaRa 9109 For RNA Isolation
RPMI 1640 Gibco 11875093 For definitive endoderm formation
Skim milk Sangon Biotech A600669 For second antibody preparation
SYBR Green Master Mix Thermo Fisher Scientific A25742 For RT-PCR Analysis
Torin2 MCE HY-13002 For definitive endoderm formation
Tween Sigma-Aldrich WXBB7485V For washing buffer preparation

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Cite This Article
Zhou, Q., Xie, X., Zhong, Z., Sun, P., Zhou, X. An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells. J. Vis. Exp. (171), e62654, doi:10.3791/62654 (2021).

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