从人类胚胎干细胞中引导肝细胞样细胞诱导的有效方法
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等。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年建立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 |
References
- Hou, Y., Hu, S., Li, X., He, W., Wu, G. Amino Acid Metabolism in the Liver: Nutritional and Physiological Significance. Advances in Experimental Medicine and Biology. 1265, 21-37 (2020).
- Huang, C., Li, Q., Xu, W., Chen, L. Molecular and cellular mechanisms of liver dysfunction in COVID-19. Discovery Medicine. 30, 107-112 (2020).
- Todorovic Vukotic, N., Dordevic, J., Pejic, S., Dordevic, N., Pajovic, S. B. Antidepressants- and antipsychotics-induced hepatotoxicity. Archives of Toxicology. , (2021).
- Thomson, J. A., et al. Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-1147 (1998).
- Li, Z., et al. Generation of qualified clinical-grade functional hepatocytes from human embryonic stem cells in chemically defined conditions. Cell Death & Disease. 10, 763 (2019).
- Rassouli, H., et al. Gene Expression Patterns of Royan Human Embryonic Stem Cells Correlate with Their Propensity and Culture Systems. Cell Journal. 21, 290-299 (2019).
- Loh, K. M., et al. Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell Stem Cell. 14, 237-252 (2014).
- Mukherjee, S., et al. Sox17 and beta-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network. Elife. 9, (2020).
- Ang, L. T., et al. A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells. Cell Reports. 22, 2190-2205 (2018).
- Carpentier, A., et al. Engrafted human stem cell-derived hepatocytes establish an infectious HCV murine model. Journal of Clinical Investigation. 124, 4953-4964 (2014).
- Gomez, G. A., et al. Human neural crest induction by temporal modulation of WNT activation. 发育生物学. 449, 99-106 (2019).
- Lee, J., Choi, S. H., Lee, D. R., Kim, D. S., Kim, D. W. Generation of Isthmic Organizer-Like Cells from Human Embryonic Stem Cells. Molecules and Cells. 41, 110-118 (2018).
- Matsuno, K., et al. Redefining definitive endoderm subtypes by robust induction of human induced pluripotent stem cells. Differentiation. 92, 281-290 (2016).
- Mathapati, S., et al. Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells. Current Protocols in Stem Cell Biology. 38, 1-18 (2016).
- Hay, D. C., et al. Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proceedings of the National Academy of Sciences of the United States of America. 105, 12301-12306 (2008).
- Siller, R., Greenhough, S., Naumovska, E., Sullivan, G. J. Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. 4, 939-952 (2015).
- Yu, J. S., et al. PI3K/mTORC2 regulates TGF-beta/Activin signalling by modulating Smad2/3 activity via linker phosphorylation. Nature Communications. 6, 7212 (2015).
- Borowiak, M., et al. Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell. 4, 348-358 (2009).
- Tahamtani, Y., et al. Treatment of human embryonic stem cells with different combinations of priming and inducing factors toward definitive endoderm. Stem Cells and Development. 22, 1419-1432 (2013).
- Song, Z., et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Research. 19, 1233-1242 (2009).
- Sullivan, G. J., et al. Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology. 51, 329-335 (2010).
- Xia, Y., et al. Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions. Journal of Hepatology. 66, 494-503 (2017).
- Li, S., et al. Derivation and applications of human hepatocyte-like cells. World Journal of Stem Cells. 11, 535-547 (2019).
- Ogawa, S., et al. Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development. 140, 3285-3296 (2013).
- Kim, D. E., et al. Prediction of drug-induced immune-mediated hepatotoxicity using hepatocyte-like cells derived from human embryonic stem cells. Toxicology. 387, 1-9 (2017).
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).