将人类牙齿中的类器官确立为机械研究和再生疗法的有力工具
Summary
我们提出了一种从人类牙齿开始开发上皮类器官培养物的方案。类器官具有强大的可膨胀性，可重现牙齿的上皮干细胞，包括其阿美巴分化能力。独特的类器官模型为研究人类牙齿（干细胞）生物学提供了一种有前途的工具，并为牙齿再生方法提供了前景。
Abstract
牙齿在生活中不仅对食物咀嚼和言语至关重要，而且对心理健康也至关重要。关于人类牙齿发育和生物学的知识很少。特别是，人们对牙齿的上皮干细胞及其功能知之甚少。我们成功地开发了一种从人类牙齿组织（即从拔出的智齿中分离出来的牙齿毛囊）开始的新型类器官模型。类器官具有稳健和长期可扩展性，并在标记表达和功能活性方面概括所提出的人类牙齿上皮干细胞区室。特别是，类器官能够展开在变色过程中 在体内 发生的阿美母细胞分化过程。这种独特的类器官模型将提供一种强大的工具，不仅可以研究人类牙齿发育，还可以研究牙齿病理学，并可能为牙齿再生疗法铺平道路。用基于这种新的类器官模型的生物牙齿替换丢失的牙齿可能是当前合成材料标准植入的一种有吸引力的替代方案。
Introduction
牙齿在食物咀嚼，言语和心理健康（自我形象）中起着至关重要的作用。人牙由不同密度和硬度的高度矿化组织组成1.牙釉质是牙冠的主要成分，是人体内矿化程度最高的组织。在牙釉质形成（amelogenesis）期间，当牙齿发育时，牙齿上皮干细胞（DESCs）分化成牙釉质形成细胞（ameloblasts）。一旦形成，由于在牙齿萌出开始时，阿米伯父细胞凋亡性丧失，珐琅质很少修复或更新1。由创伤或细菌性疾病引起的受损牙釉质组织的修复目前使用合成材料完成;然而，这些都存在重要的缺点，例如微裂，下骨整合和锚固，有限的寿命以及缺乏功能齐全的修复2。因此，具有产生阿米母细胞能力和产生矿化组织潜力的人类DEC的稳健可靠的培养将是牙科再生领域向前迈出的重要一步。
关于人类DESC表型和生物学功能的知识稀缺3，4，5。有趣的是，人类牙齿的DESC已被提出存在于Malassez的上皮细胞休息（ERM）中，该细胞簇存在于牙齿卵泡（DF）内，其周围是未脱出的牙齿，并且一旦牙齿萌出，它们仍然存在于根部周围的牙周韧带中1。已经发现与牙髓共培养的ERM细胞分化成阿美母细胞样细胞并产生牙釉质样组织6。然而，由于缺乏可靠的研究模型7，对ERM细胞在牙釉质（再）生成中的特异性作用的深入研究受到限制。目前的ERM体外培养系统受到有限的寿命和标准使用的2D条件下表型快速丧失的阻碍8，9，10，11，12。因此，迫切需要一个易于处理的体外系统来忠实地扩展，研究和区分人类DESC。
在过去的十年中，一种 在体外 生长上皮干细胞的强大技术已成功应用于几种类型的（人类）上皮组织，以研究其生物学以及疾病13，14，15，16。该技术使组织上皮干细胞能够自我发育成3D细胞结构（即类器官），当接种到细胞外基质（ECM）模拟支架（通常为Matrigel）中并在定义的培养基中培养，复制组织的干细胞位信号传导和/或胚胎发生。类器官发育所需的典型生长因子包括表皮生长因子（EGF）和无翼型MMTV整合位点（WNT）激活剂14，15，16。所得类器官的特征在于模仿组织原始上皮干细胞的持久保真度，以及高可膨胀性，同时保持其表型和功能特性，从而克服了从诊所获得的通常有限的原代人体组织可用性。为了建立类器官，不需要在培养之前从异质组织（即包含其他细胞类型，如间充质细胞）中分离上皮干细胞，因为间充质细胞不附着或在ECM中茁壮成长，最终导致纯上皮类器官13，16，17，18，19.这种有前途的多功能技术导致了来自各种人体上皮组织的歧管类器官模型的发展。然而，对于深入研究牙齿发育，再生和疾病有价值的人类牙齿衍生类器官尚未建立20，21。我们最近成功地开发了这样一种新的类器官模型，从从青少年患者中提取的第三磨牙（智齿）的DF组织开始19。
在这里，我们描述了从成人人类牙齿（即从第三磨牙的DF）开发上皮类器官培养物的方案（图1A）。所得类器官表达ERM相关的干性标志物，同时具有长期可扩展性。有趣的是，与大多数其他类器官模型相反，通常需要的EGF对于健壮的类器官发育和生长是多余的。有趣的是，茎性类器官显示出阿美母细胞的分化特性，从而模仿 了体内发生的ERM / DESC特征和过程。这里描述的新的和独特的类器官模型允许探索DESC生物学，可塑性和分化能力，并为迈出牙齿再生方法的第一步打开了大门。
Protocol
此处描述的所有方法均已获得鲁汶伦理委员会研究（13/0104U）的批准。提取的第三磨牙（智齿）是在患者知情同意后获得的。 1. 准备工作 在37°C的1.9%CO 2培养箱中预热48孔培养板15-20 小时。 液化一种基质胶等分试样（生长因子还原;无酚红;进一步称为基底膜基质;BMM）在步骤2.1之前在冰上（4°C）上至少2小时。注意：避免 BMM 的冻融循环。?…
Representative Results
牙齿类器官发育我们提供了一个详细的方案，用于从智齿拔除后获得的人类DF组织中建立类器官培养物（图1A）。分离的DF被酶促和机械解离。获得的细胞在BMM中培养，这些培养基根据经验被定义为最佳的类器官发育和生长（牙齿类器官培养基;汤姆）19. 类器官通常在DF细胞接种后2周内发育（P0; 图 2A…
Discussion
该协议描述了从人类牙齿开始的类器官的高效和可重复的产生。据我们所知，这是从人类牙齿组织开始建立当前概念（上皮）类器官的第一种方法。类器官可长期扩张，并显示牙齿上皮干性表型，与先前在DF7的ERM室中报道的DSC重复。此外，类器官复制了功能性DESC / ERM特征，包括展开阿美巴分化过程7，25，26。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢UZ Leuven口腔颌面外科（MKA）的所有工作人员以及患者在收集新鲜提取的第三磨牙方面提供的宝贵帮助。我们还要感谢Reinhilde Jacobs博士和Elisabeth Tijskens博士在样本收集方面的帮助。这项工作得到了鲁汶大学（BOF）和FWO-Flanders（G061819N）的资助。L.H.是FWO博士研究员（1S84718N）。
Materials
| 1.5 mL Microcentrifuge tube | Eppendorf | 30120.086 | |
| 15 mL Centrifuge tube | Corning | 430052 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M-6250 | |
| 48-well flat bottom plates | Corning | 3548 | |
| 50 mL Centrifuge tube | Corning | 430290 | |
| A83-01 | Sigma-Aldrich | SML0788 | |
| Agarose | Lonza | 50004 | |
| Albumin Bovine (cell culture grade) | Serva | 47330.03 | |
| AMELX antibody | Santa Cruz | sc-365284 | |
| Amphotericin B | Gibco | 15200018 | |
| B27 (without vitamin A) | Gibco | 12587-010 | |
| Cassette | VWR | 7202191 | |
| Catalase from bovine liver | Sigma-Aldrich | C100 | |
| CD44 antibody | Abcam | ab34485 | |
| Cell strainer, 40 µm | Falcon | 352340 | |
| Cholera Toxin | Sigma-Aldrich | C8052 | |
| Citric acid | Sigma-Aldrich | C0759 | |
| CK14 antibody | Thermo Fisher Scientific | MA5-11599 | |
| Collagenase IV | Gibco | 17104-019 | |
| Cover glass | VWR | 6310146 | |
| Cryobox | Thermo Scientific | 5100-0001 | |
| Cryovial | Thermo Fisher Scientific | 375353 | |
| Dimethylsulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
| Dispase II | Sigma-Aldrich | D4693 | |
| DMEM 1:1 F12 without Fe | Invitrogen | 074-90715A | |
| DMEM powder high glucose | Gibco | 52100039 | |
| Dnase | Sigma-Aldrich | D5025-15KU | |
| Donkey serum | Sigma-Aldrich | D9663 – 10ML | |
| Embedding workstation, 220 to 240 Vac | Thermo Fisher Scientific | 12587976 | |
| Ethanol absolute, ≥99.8% (EtOH) | Fisher Chemical | E/0650DF/15 | |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
| FGF10 | Peprotech | 100-26 | |
| FGF2 (= basic FGF) | R&D Systems | 234-FSE-025 | |
| FGF8 | Peprotech | AF-100-25 | |
| GenElute Mammaliam Total RNA Miniprep Kit | Sigma-Aldrich | RTN350-1KT | Includes 1% β-mercaptoethanol dissolved in lysis buffer |
| Glass Pasteur pipette | Niko Mechanisms | 170-40050 | |
| Glycine | VWR | 101194M | |
| HEPES | Sigma-Aldrich | H4034 | |
| IGF-1 | PeproTech | 100-11 | |
| InSolution Y-27632 (ROCK inhibitor, RI) | Sigma-Aldrich | 688001 | |
| Insulin from bovine pancreas | Sigma-Aldrich | I6634 | |
| ITGA6 antibody | Sigma-Aldrich | HPA012696 | |
| L-Glutamine | Gibco | 25030024 | |
| Matrigel (growth factor-reduced; phenol red-free) | Corning | 15505739 | |
| Microscope slide | Thermo Fisher Scientific | J1800AMNZ | |
| Millex-GV Syringe Filter Unit, 0.22 μm | Millipore | SLGV033R | |
| Minimum essential medium eagle (αMEM) | Sigma-Aldrich | M4526 | |
| mouse IgG (Alexa 555) secondary antibody | Thermo Fisher Scientific | A-31570 | |
| N2 | Gibco | 17502-048 | |
| N-acetyl L-cysteine | Sigma-Aldrich | A7250 | |
| Nicotinamide | Sigma-Aldrich | N0636 | |
| Noggin | PeproTech | 120-10C | |
| P63 antibody | Abcam | ab124762 | |
| Pap Pen | Sigma-Aldrich | Z377821-1EA | Marking pen |
| Paraformaldehyde (PFA), 16% | Merck | 8.18715 | |
| Penicillin G sodium salt | Sigma-Aldrich | P3032 | |
| Penicillin-streptomycin (Pen/Strep) | Gibco | 15140-122 | |
| Petri dish | Corning | 353002 | |
| Phosphate buffered saline (PBS) | Gibco | 10010-015 | |
| Pipette (P20, P200, P1000) | Eppendorf or others | 2231300006 | |
| Plastic transfer pipette (3.5 mL) | Sarstedt | 86.1171.001 | |
| Rabbit IgG (Alexa 488) secondary antibody | Thermo Fisher Scientific | A21206 | |
| RSPO1 | PeproTech | 120-38 | |
| SB202190 (p38i) | Biotechne (Tocris) | 1264 | |
| Scalpel (surgical blade) | Swann-Morton | 207 | |
| SHH | R&D Systems | 464-SH-200 | |
| Silicone molds (Heating block) | VWR | 720-1918 | |
| Sodium Chloride (NaCl) | BDH | 102415K | |
| Sodium Hydrogen Carbonate (NaHCO3) | Merck | 106329 | |
| Sodium-pyruvate (C3H3NaO3) | Sigma-Aldrich | P-5280 | |
| SOX2 antibody | Abcam | ab92494 | |
| StepOnePlus | Thermo Fisher Scientific | Real-Time PCR System | |
| Stericup-GP, 0.22 µm | Millipore | SCGPU02RE | |
| Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 μm | Millipore | SCGP00525 | |
| Sterile 1000 μL pipette tips with filter | Greiner | 740288 | |
| Sterile 20 μL pipette tips with filter | Greiner | 774288 | |
| Sterile 200 μL pipette tips with and without filter | Greiner | 739288 | |
| Sterile H2O | Fresenius | B230531 | |
| Streptomycin sulfate salt | Sigma-Aldrich | S6501 | |
| Superscript III first-strand synthesis supermix | Invitrogen | 11752-050 | Reverse transcription kit |
| Tissue processor | Thermo Scientific | 12505356 | |
| Transferrin | Serva | 36760.01 | |
| Triton X-100 | Sigma | T8787-50ML | |
| TrypLE express | Gibco | 12605-010 | |
| Vectashield mounting medium+DAPI | Labconsult NV | H-1200 | Antifade mounting medium with DAPI |
| WNT3a | Biotechne (Tocris) | 5036-WN-500 | |
| Xylenes, 99%, for biochemistry and histology | VWR | 2,89,75,325 |
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Citer Cet Article
Hemeryck, L., Lambrichts, I., Bronckaers, A., Vankelecom, H. Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy. J. Vis. Exp. (182), e63671, doi:10.3791/63671 (2022).