使用Trowell型器官培养研究牙科干细胞的调节
Summary
Trowell型器官培养方法已被用于解开控制牙齿发育的复杂信号网络，最近还用于研究与持续生长的小鼠门牙干细胞有关的调节。荧光报告动物模型和实时成像方法有助于对牙科干细胞及其特定的生态位微环境进行深入分析。
Abstract
器官发育、功能和再生取决于干细胞，干细胞存在于称为干细胞生态位的离散解剖空间内。不断生长的小鼠门牙为研究组织特异性干细胞提供了一个极好的模型。门牙的上皮组织特异性干细胞位于牙齿的近端，位于称为颈环的壁龛中。它们提供持续的细胞流入，以抵消牙齿自锐尖端的持续磨损。这里介绍的是小鼠门牙近端分离和培养的详细方案，该门牙容纳干细胞及其生态位。这是一种改进的Trowell型器官培养方案，可以对组织碎片（外植体）以及由金属网格支撑的过滤器上的液体/空气界面处的厚组织切片进行 体外 培养。这里描述的器官培养方案使组织操作在 体内 不可行，并且当与荧光报告基因结合使用时，它为随着时间的推移鉴定和跟踪活组织中的离散细胞群（包括干细胞）提供了一个平台。可以在该系统中测试各种调节分子和药理化合物对干细胞及其生态位的影响。这最终为研究干细胞调控和维持提供了有价值的工具。
Introduction
由于干细胞（SC）的终身保存，小鼠门牙持续生长，这些干细胞支持牙齿成分的不断生产。这些包括产生产生牙釉质的釉质母细胞的上皮SCs和产生产生牙本质的牙母细胞的间充质干细胞（MSCs）以及其他细胞1。连续生长的门牙中的上皮SCs最初被鉴定为标记保留细胞2，3，此后已被证明表达许多众所周知的干性基因，包括Sox24。这些细胞与其他器官中的上皮SC具有共同特征，并且位于门牙唇侧称为颈环的SC壁龛内。生态位是由细胞和细胞外基质组成的动态实体，控制SC活性5。谱系追踪研究表明，Sox2+上皮SCs可以再生牙齿的整个上皮隔室，并且它们对于连续牙齿的形成至关重要6，7。具有牙本质修复或再生潜力的间充质干细胞主要通过血管和神经从器官外部招募8，9，10，11，因此，为研究MSC人群的招募，迁移和住房提供了合适的模型。
在体内研究SCs并不总是可行的，因为许多遗传和/或药物操作会影响器官稳态和/或具有致命的后果。因此，器官培养为体外研究SCs及其生态位的调控提供了极好的工具。利用金属网格的器官培养系统最初由Trowell12开发，用于研究器官发育，并由Saxen13进一步修改以研究肾脏发育中的感应信号。从那时起，这种培养整个或部分器官的体外方法已成功应用于不同领域。在牙齿发育领域，该方法已被广泛用于研究控制牙齿发育14和连续牙齿形成的上皮 – 间充质相互作用15。Thesleff实验室的工作已经证明了该系统在牙齿生长和形态发生的时间分析，分析各种分子和生长因子对牙齿生长的影响以及牙齿发育的延时实时成像方面的实用性16，17。最近，该方法已被用于研究门牙SC及其生态位18，19的调节，本文对此进行了详细描述。
Protocol
该协议涉及动物的使用，所有程序均已获得赫尔辛基大学动物使用和护理伦理委员会以及动物设施的批准。 1. 器官培养皿的制备 在层流罩中执行所有程序。用70%乙醇清洁工作表面，并使用高压灭菌的玻璃仪器和溶液。在玻璃珠消毒器中对剪刀和其他金属设备进行消毒。 通过在1x PBS中洗涤三次来制备通常储存在70%乙醇中的过滤器以除去乙醇（图1…
Representative Results
上皮SCs位于称为颈环的壁龛中，该壁龛位于门牙的近端（图3A）。颈袢是形态上不同的结构，由内部和外部牙釉质上皮组成，包裹着星状网，星状网是松散排列的上皮细胞的核心（图3B，C）。每个门牙有两个颈袢（图3A），但只有唇颈袢包含SC。上皮SCs定位于星状网和颈袢尖端的相邻珐琅质上皮20?…
Discussion
体外 器官培养已被广泛用于研究控制器官生长和形态发生的诱导电位和上皮-间充质相互作用。Thesleff实验室已经展示了如何适应Trowell型器官培养的Saxén修饰，并将其用于研究牙齿发育14。荧光报告基因的可重复条件和进步使其成为监测牙齿形态发生和其中不同细胞群的有用方法。本文描述了如何应用该协议来研究上皮干细胞及其所在的微环境。
本文?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了Jane和Aatos Erkko基金会的支持。
Materials
| 1-mL plastic syringes | |||
| Disposable 20/26 gauge hypodermic needles | Terumo | ||
| DMEM | Gibco | 61965-026 | |
| Dulbecco's Phosphate-Buffered Saline | Gibco | 14287 | |
| Extra Fine Bonn Scissors | F.S.T. | 14084-08 | |
| F-12 | Gibco | 31765-027 | |
| FBS South American (CE) | LifeTechn. | 10270106 | divide in aliquotes, store at -20°C |
| Glass bead sterilizer, Steri 250 Seconds-Sterilizer | Simon Keller Ltd | 4AJ-6285884 | |
| GlutaMAX-1 (200 mM L-alanyl-L-glutamine dipeptide) | Gibco | 35050-038 | |
| Isopore Polycarb.Filters, 0,1 um 25-mm diameter | MerckMillipore | VCTP02500 | Store in 70% ethanol at room temperature. |
| L-Ascobic Acid | Sigma | A4544-25g | diluted 100 mg/ml in MilliQ, filter strerilized and divided in 20μl aliquotes, store at dark, -20°C |
| Low melting agarose | TopVision | R0801 | |
| Metal grids | Commercially available, or self-made from stainless-steel mesh (corrosion resistant, size of mesh 0.7 mm). Cut approximately 30 mm diameter disk and bend the edges to give 3 mm height. Use nails to make holes. | ||
| Micro forceps | Medicon | 07.60.03 | |
| Paraformaldehyde | Sigma-Aldrich | ||
| Penicillin-Streptomycin (10,000U/ml) sol. | Gibco | 15140-148 | |
| Petri dishes, Soda-Lime glass | DWK Life Sciences | 9170442 | |
| Petridish 35 mm, with vent | Duran | 237554008 | |
| Petridish 90 mm, no vent classic | Thermo Fisher | 101RT/C | |
| Small scissors |
Referencias
- Balic, A. Biology explaining tooth repair and regeneration: A mini-review. Gerontology. 64 (4), 382-388 (2018).
- Harada, H., et al. Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. The Journal of Cell Biology. 147 (1), 105-120 (1999).
- Smith, C. E., Warshawsky, H. Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine. The Anatomical Record. 183 (4), 523-561 (1975).
- Balic, A., Thesleff, I. Tissue interactions regulating tooth development and renewal. Current Topics in Developmental Biology. 115, 157-186 (2015).
- Scadden, D. T. The stem-cell niche as an entity of action. Nature. 441 (7097), 1075-1079 (2006).
- Juuri, E., et al. Sox2 marks epithelial competence to generate teeth in mammals and reptiles. Development. 140 (7), 1424-1432 (2013).
- Juuri, E., et al. Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ progenitors. Developmental Cell. 23 (2), 317-328 (2012).
- Feng, J., Mantesso, A., De Bari, C., Nishiyama, A., Sharpe, P. T. Dual origin of mesenchymal stem cells contributing to organ growth and repair. Proceedings of the National Academy of Sciences of the United States of America. 108 (16), 6503-6508 (2011).
- Kaukua, N., et al. Glial origin of mesenchymal stem cells in a tooth model system. Nature. 513 (7519), 551-554 (2014).
- Zhao, H., et al. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor. Cell Stem Cell. 14 (2), 160-173 (2014).
- Vidovic, I., et al. alphaSMA-expressing perivascular cells represent dental pulp progenitors in vivo. Journal of Dental Research. 96 (3), 323-330 (2017).
- Trowell, O. A. A modified technique for organ culture in vitro. Experimental Cell Research. 6 (1), 246-248 (1954).
- Saxen, L., Vainio, T., Toivonen, S. Effect of polyoma virus on mouse kidney rudiment in vitro. Journal of the National Cancer Institute. 29, 597-631 (1962).
- Thesleff, I., Sahlberg, C. Organ culture in the analysis of tissue interactions. Methods in Molecular Biology. 461, 23-30 (2008).
- Jarvinen, E., Shimomura-Kuroki, J., Balic, A., Jussila, M., Thesleff, I. Mesenchymal Wnt/beta-catenin signaling limits tooth number. Development. 145 (4), (2018).
- Ahtiainen, L., Uski, I., Thesleff, I., Mikkola, M. L. Early epithelial signaling center governs tooth budding morphogenesis. The Journal of Cell Biology. 214 (6), 753-767 (2016).
- Narhi, K., Thesleff, I. Explant culture of embryonic craniofacial tissues: analyzing effects of signaling molecules on gene expression. Methods in Molecular Biology. 666, 253-267 (2010).
- Yang, Z., Balic, A., Michon, F., Juuri, E., Thesleff, I. Mesenchymal Wnt/beta-Catenin signaling controls epithelial stem cell homeostasis in teeth by inhibiting the antiapoptotic effect of Fgf10. Stem Cells. 33 (5), 1670-1681 (2015).
- Binder, M., et al. Functionally distinctive Ptch receptors establish multimodal hedgehog signaling in the tooth epithelial stem cell niche. Stem Cells. 37 (9), 1238-1248 (2019).
- Harada, H., et al. Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. The Journal of Cell Biology. 147 (1), 105-120 (1999).
- Seidel, K., et al. Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development. 137 (22), 3753-3761 (2010).
- Hadjantonakis, A. K., Nagy, A. FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis. 27 (3), 95-98 (2000).
- Yu, Y. A., Szalay, A. A., Wang, G., Oberg, K. Visualization of molecular and cellular events with green fluorescent proteins in developing embryos: a review. Luminescence. 18 (1), 1-18 (2003).
- D’Amour, K. A., Gage, F. H. Genetic and functional differences between multipotent neural and pluripotent embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 100, 11866-11872 (2003).
- Chavez, M. G., et al. Isolation and culture of dental epithelial stem cells from the adult mouse incisor. Journal of Visualized Experiments: JoVE. (87), (2014).
- Binder, M., et al. Novel strategies for expansion of tooth epithelial stem cells and ameloblast generation. Science Reports. 10 (1), 4963 (2020).
Citar este artículo
Juuri, E., Balic, A. Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells. J. Vis. Exp. (173), e62462, doi:10.3791/62462 (2021).