产生来自多能干细胞的自组装人心脏类器官
Özet
在这里，我们描述了一种方案，通过自组织有效地使用人类多能干细胞来创建与发育相关的人类心脏类器官（hHO）。该协议依赖于发育线索的顺序激活，并产生高度复杂，功能相关的人体心脏组织。
Abstract
研究人类心脏在健康和疾病中发育的能力受到 在体外模拟人类心脏复杂性的能力的高度限制。开发更有效的器官样平台，可以模拟复杂的 体内 表型，如类器官和器官芯片，将增强研究人类心脏发育和疾病的能力。本文描述了一种方案，通过使用人类多能干细胞的自组织和使用小分子抑制剂逐步激活发育途径来产生高度复杂的人心脏类器官（hHO）。胚胎体（EB）在具有圆底，超低附着孔的96孔板中产生，促进个体化结构的悬浮培养。
EB通过三步Wnt信号调节策略分化为hHO，该策略涉及初始Wnt通路激活以诱导心脏中胚层命运，第二步Wnt抑制以创建确定的心脏谱系，以及第三个Wnt激活步骤以诱导心前器官组织。这些步骤以96孔的形式进行，效率高，可重复，每次运行产生大量类器官。通过免疫荧光成像从第3天到第11天的分化分析揭示了第15天hHO内的第一和第二心场规格和高度复杂的组织，包括具有心房和心室心肌细胞区域的心肌组织，以及衬有心内膜组织的内腔。类器官还在整个结构和心外膜组织的外衬里中表现出复杂的血管网络。从功能的角度来看，hHO具有强大的水平，并表现出由Flu-4活体成像确定的正常钙活性。总体而言，该协议构成了人体器官样心脏组织 体外 研究的坚实平台。
Introduction
先天性心脏缺陷（CHDs）是人类最常见的先天性缺陷类型，影响约1%的活产1，2，3。在大多数情况下，冠心病的原因仍然未知。在实验室中创建与发育中的人类心脏非常相似的人类心脏模型的能力是直接研究人类而不是代孕动物模型中CHDs的根本原因的重要一步。
实验室培养的组织模型的缩影是类器官，类似于细胞组成和生理功能中感兴趣的器官的3D细胞结构。类器官通常来自干细胞或祖细胞，并已成功用于模拟许多器官，如脑4，5，肾6，7，肠8，9，肺10，11，肝脏12，13和胰腺14，15，仅举几例。最近的研究表明，创造自组装心脏类器官在体外研究心脏发育是可行的。这些模型包括使用小鼠胚胎干细胞（mESCs）对早期心脏发育进行建模16，17直至房室规格18，并使用人类多能干细胞（hPSCs）来生成具有高度复杂细胞组成的多生殖层心内胚类器官19和腔室心形体20。
本文提出了一种新颖的3步WNT调制方案，以高效且具有成本效益的方式生成高度复杂的hHO。类器官在96孔板中生成，从而形成一个可扩展的高通量系统，可以轻松自动化。该方法依赖于创建hPSC聚集体并触发心脏发生的发育步骤，包括中胚层和心脏中胚层形成，第一和第二心场规范，心前器官形成和房室规范。分化15天后，hHO包含心脏中发现的所有主要细胞谱系，明确定义的内腔，心房和心室腔以及整个类器官的血管网络。这种高度复杂且可重复的心脏类器官系统适合在心脏发育，疾病和药理学筛查研究中研究结构，功能，分子和转录组学分析。
Protocol
1. hPSC培养与维护 注意：人诱导的PSCs（hiPSCs）或人胚胎干细胞（hESCs）在解冻后需要培养至少连续传代2次，然后才能用于产生EB进行分化或进一步冷冻保存。hPSCs在PSC培养基（见 材料表）中基底膜 – 细胞外基质（BM-ECM）包被的6孔培养板上培养。当在6孔板中的hPSCs上进行培养基更换时，将培养基直接添加到孔的内侧，而不是直接在细胞顶部，以防止不必要的细胞脱离…
Representative Results
为了在体外实现自组织hHO，我们使用Wnt通路调节剂修改和组合了先前描述的心肌细胞21和心外膜细胞22的2D单层分化方案，以及使用生长因子BMP4和Activin A的3D心前类器官16。，优化了Wnt途径激活剂CHIR99021的浓度和暴露持续时间，以产生来自人类PSC的高度可重复性和复杂的hHO.hPSCs或hESCs在PSC培养基中培养至60-80%的融合在…
Discussion
人类干细胞衍生的心肌细胞和其他心脏来源的细胞的最新进展已被用于模拟人类心脏发育22，24，25 和疾病26，27，28 ，并作为筛选治疗药物的工具29，30 和有毒物质31，32</sup…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院国家心脏，肺和血液研究所的支持，奖项编号为K01HL135464和R01HL151505，美国心脏协会的奖项编号为19IPLOI34660342。我们要感谢MSU高级显微镜核心和密歇根州立大学药理学和毒理学系的William Jackson博士获得共聚焦显微镜，IQ显微镜核心和密歇根州立大学基因组学核心的测序服务。我们还要感谢阿吉雷实验室的所有成员的宝贵意见和建议。
Materials
| Antibodies | |||
| Alexa Fluor 488 Donkey anti- mouse | Invitrogen | A-21202 | 1:200 |
| Alexa Fluor 488 Donkey anti- rabbit | Invitrogen | A-21206 | 1:200 |
| Alexa Fluor 594 Donkey anti- mouse | Invitrogen | A-21203 | 1:200 |
| Alexa Fluor 594 Donkey anti- rabbit | Invitrogen | A-21207 | 1:200 |
| Alexa Fluor 647 Donkey anti- goat | Invitrogen | A32849 | 1:200 |
| HAND1 | Abcam | ab196622 | Rabbit; 1:200 |
| HAND2 | Abcam | ab200040 | Rabbit; 1:200 |
| NFAT2 | Abcam | ab25916 | Rabbit; 1:100 |
| PECAM1 | DSHB | P2B1 | Rabbit; 1:50 |
| TNNT2 | Abcam | ab8295 | Mouse; 1:200 |
| THY1 | Abcam | ab133350 | Rabbit; 1:200 |
| TJP1 | Invitrogen | PA5-19090 | Goat; 1:250 |
| VIM | Abcam | ab11256 | Goat; 1:250 |
| WT1 | Abcam | ab89901 | Rabbit; 1:200 |
| Media and Reagents | |||
| Accutase | Innovative Cell Technologies | NC9464543 | cell dissociation reagent |
| Activin A | R&D Systems | 338AC010 | |
| B-27 Supplement (Minus Insulin) | Gibco | A1895601 | insulin-free cell culture supplement |
| B-27 Supplement | Gibco | 17504-044 | cell culture supplement |
| BMP-4 | Gibco | PHC9534 | |
| Bovine Serum Albumin | Bioworld | 50253966 | |
| CHIR-99021 | Selleck | 442310 | |
| D-(-)-Fructose | Millipore Sigma | F0127 | |
| DAPI | Thermo Scientific | 62248 | 1:1000 |
| Dimethyl Sulfoxide | Millipore Sigma | D2650 | |
| DMEM/F12 | Gibco | 10566016 | |
| Essential 8 Flex Medium Kit | Gibco | A2858501 | pluripotent stem cell (PSC) medium containing 1% penicillin-streptomycin |
| Fluo4-AM | Invitrogen | F14201 | |
| Glycerol | Millipore Sigma | G5516 | |
| Glycine | Millipore Sigma | 410225 | |
| Matrigel GFR | Corning | CB40230 | Basement membrane extracellular matrix (BM-ECM) |
| Normal Donkey Serum | Millipore Sigma | S30-100mL | |
| Paraformaldehyde | MP Biomedicals | IC15014601 | Powder dissolved in PBS Buffer – use at 4% |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Phosphate Buffer Solution | Gibco | 10010049 | |
| Phosphate Buffer Solution (10x) | Gibco | 70011044 | |
| Polybead Microspheres | Polysciences, Inc. | 73155 | 90 µm |
| ReLeSR | Stem Cell Technologies | NC0729236 | dissociation reagent for hPSCs |
| RPMI 1640 | Gibco | 11875093 | |
| Thiazovivin | Millipore Sigma | SML1045 | |
| Triton X-100 | Millipore Sigma | T8787 | |
| Trypan Blue Solution | Gibco | 1525006 | |
| VECTASHIELD Vibrance Antifade Mounting Medium | Vector Laboratories | H170010 | |
| WNT-C59 | Selleck | NC0710557 | |
| Diğer | |||
| 1.5 mL Microcentrifuge Tubes | Fisher Scientific | 02682002 | |
| 15 mL Falcon Tubes | Fisher Scientific | 1495970C | |
| 2 mL Cryogenic Vials | Corning | 13-700-500 | |
| 50 mL Reagent Reservoirs | Fisherbrand | 13681502 | |
| 6-Well Flat Bottom Cell Culture Plates | Corning | 0720083 | |
| 8 Well chambered cover Glass with #1.5 high performance cover glass | Cellvis | C8-1.5H-N | |
| 96-well Clear Ultra Low Attachment Microplates | Costar | 07201680 | |
| ImageJ | NIH | Image processing software | |
| Kimwipes | Kimberly-Clark Professional | 06-666 | laboratory wipes |
| Micro Cover Glass | VWR | 48393-241 | 24 x 50 mm No. 1.5 |
| Microscope Slides | Fisherbrand | 1255015 | |
| Moxi Cell Counter | Orflo Technologies | MXZ001 | |
| Moxi Z Cell Count Cassette – Type M | Orflo Technologies | MXC001 | |
| Multichannel Pipettes | Fisherbrand | FBE1200300 | 30-300 µL |
| Olympus cellVivo | Olympus | For Caclium Imaging, analysis with Imagej | |
| Sorvall Legend X1 Centrifuge | ThermoFisher Scientific | 75004261 | |
| Thermoshaker | ThermoFisher Scientific | 13-687-711PM | |
| Top Coat Nail Varish | Seche Vite | Can purchase from any supermarket |
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Etiketler
Self-assemblingHuman HeartOrganoidsPluripotent Stem Cells3D StructureDeveloping HeartCardiovascular DiseasesPharmacological TreatmentsHigh ThroughputCongenital Heart DefectsEmbryoid BodiesCell CultureDifferentiationHPSCDPBSACUTAPSC MediumThiasCell SuspensionCell CounterHemo CytometerRound Bottom Ultra Low Attachment 96 Well PlateRPMI 1640Insulin Free B27 Supplement
Bu Makaleden Alıntı Yapın
Lewis-Israeli, Y. R., Volmert, B. D., Gabalski, M. A., Huang, A. R., Aguirre, A. Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells. J. Vis. Exp. (175), e63097, doi:10.3791/63097 (2021).