Name: use of trowell-type organ culture to study regulation of dental stem cells
Uploaded: 2021-07-08T13:30:00
Description: Watch this Scientific Journal Video about Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells at JoVE.com

这项研究得到了Jane和Aatos Erkko基金会的支持。

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
<ol><li>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。</li>
	<li>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...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+epithelial+signaling+center+governs+tooth+budding+morphogenesis.">Early epithelial signaling center governs tooth budding morphogenesis.</a> The Journal of Cell Biology. 214 (6), 753-767 (2016).</li><li>Narhi, K., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture+of+embryonic+craniofacial+tissues:+analyzing+effects+of+signaling+molecules+on+gene+expression.">Explant culture of embryonic craniofacial tissues: analyzing effects of signaling molecules on gene expression.</a> Methods in Molecular Biology. 666, 253-267 (2010).</li><li>Yang, Z., Balic, A., Michon, F., Juuri, E., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Wnt/beta-Catenin+signaling+controls+epithelial+stem+cell+homeostasis+in+teeth+by+inhibiting+the+antiapoptotic+effect+of+Fgf10.">Mesenchymal Wnt/beta-Catenin signaling controls epithelial stem cell homeostasis in teeth by inhibiting the antiapoptotic effect of Fgf10.</a> Stem Cells. 33 (5), 1670-1681 (2015).</li><li>Binder, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+distinctive+Ptch+receptors+establish+multimodal+hedgehog+signaling+in+the+tooth+epithelial+stem+cell+niche.">Functionally distinctive Ptch receptors establish multimodal hedgehog signaling in the tooth epithelial stem cell niche.</a> Stem Cells. 37 (9), 1238-1248 (2019).</li><li>Harada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+putative+stem+cells+in+dental+epithelium+and+their+association+with+Notch+and+FGF+signaling.">Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.</a> The Journal of Cell Biology. 147 (1), 105-120 (1999).</li><li>Seidel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+regulates+the+generation+of+ameloblast+progenitors+in+the+continuously+growing+mouse+incisor.">Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor.</a> Development. 137 (22), 3753-3761 (2010).</li><li>Hadjantonakis, A. 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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-mL plastic syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 20/26 gauge hypodermic needles</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>61965-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14287</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>F.S.T.</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12</td>
			<td>Gibco</td>
			<td>31765-027</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS South American (CE)</td>
			<td>LifeTechn.</td>
			<td>10270106</td>
			<td>divide in aliquotes, store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer, Steri 250 Seconds-Sterilizer</td>
			<td>Simon Keller Ltd</td>
			<td>4AJ-6285884</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX-1 (200 mM L-alanyl-L-glutamine dipeptide)</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopore Polycarb.Filters, 0,1 um 25-mm diameter</td>
			<td>MerckMillipore</td>
			<td>VCTP02500</td>
			<td>Store in 70% ethanol at room temperature.</td>
		</tr>
		<tr>
			<td>L-Ascobic Acid</td>
			<td>Sigma</td>
			<td>A4544-25g</td>
			<td>diluted 100 mg/ml in MilliQ, filter strerilized and divided in 20&#956;l aliquotes, store at dark, -20&#176;C</td>
		</tr>
		<tr>
			<td>Low melting agarose</td>
			<td>TopVision</td>
			<td>R0801</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal grids</td>
			<td></td>
			<td></td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>Medicon</td>
			<td>07.60.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000U/ml) sol.</td>
			<td>Gibco</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, Soda-Lime glass</td>
			<td>DWK Life Sciences</td>
			<td>9170442</td>
			<td></td>
		</tr>
		<tr>
			<td>Petridish 35 mm, with vent</td>
			<td>Duran</td>
			<td>237554008</td>
			<td></td>
		</tr>
		<tr>
			<td>Petridish 90 mm, no vent classic</td>
			<td>Thermo Fisher</td>
			<td>101RT/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of trowell-type organ culture to study regulation of dental stem cells

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。

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?...

Watch this Scientific Journal Video about Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells at JoVE.com

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

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。

使用Trowell型器官培养研究牙科干细胞的调节

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Trowell型器官培养用于培养组织外植体。在这里，我们展示了如何使用这种方法来研究连续生长的牙齿中的干细胞。通过这种技术，我们可以研究干细胞及其在生态位中的行为，并筛选各种分子对干细胞存活和维持的影响。这种方法也可以应用于研究其他器官的干细胞，例如皮肤或乳腺。首先在 35 毫米培养皿中放置带有 1 到 2 毫米直径孔的 35 毫米金属网格。用介质填充该位置，直到介质到达网格表面。然后，将板在37摄氏度下孵育，直到组织被分离并准备培养。斩首小鼠后，去除皮肤以露出下颌骨，并切开咬肌以将下颌骨与上颌骨和头部其余部分分开。分离下颌骨后，切除舌头和最大软组织。通过切开联合在中线分开下颌骨。将下颌骨放在冰上含有PBS的培养皿中。使用一次性 20 x 26 号皮下注射针解剖门牙。然后，清洁骨骼表面的软组织和肌肉组织，以获得更好的可视化效果。对于从小于 10 天的动物身上获得的下颌骨，使用一次性 20 x 26 号皮下注射针纵向打开下颌骨的每一半以露出门牙。轻轻地将门牙与周围的骨头分离。切开包含颈环的近端，去除矿化牙釉质和牙本质基质。将收集的近端保存在Dulbecco的PBS中，直到准备好培养。对于超过10天的小鼠，纵向打开下颌骨的每一半以露出门牙。用镊子夹住下颌骨，折断骨头露出牙齿。切开包含颈环的近端。将收集的近端保存在Dulbecco的PBS中，直到准备好培养。小心地将矩形过滤器放在预热培养皿中的网格顶部，并正确定向组织碎片。将板在 37 摄氏度、5% 二氧化碳和 90% 至 95% 湿度下孵育。隔天小心更换培养基，避免形成气泡。每天监测组织生长并捕获图像。从培养皿中取出培养基，小心地将冰冷的甲醇添加到组织中。然后，将其孵育五分钟。将携带组织外植体的过滤器转移到装有固定溶液的采样管中。将外植体固定在PBS中制备的4%多聚甲醛中，最多在4摄氏度下过夜。上皮干细胞位于称为颈环的壁龛中，位于门牙的近端。颈袢包括一个内釉质上皮和外釉质上皮，其包裹着星状网，星状网是松散排列的上皮细胞的核心。每个门牙都有一个唇袢和一个舌颈袢，但只有唇颈袢含有干细胞。捕获从出生后两天的Sox2-GFP小鼠中分离的门牙的近端，从而能够识别和可视化表达Sox2的切牙上皮干细胞。在从Sox2-GFP和Fucci红色转基因小鼠分离的宫颈环中，鉴定出GFP阳性干细胞和Fucci红阳性非增殖细胞。将Sox2-GFP表达作为报告基因分析Wnt/β-连环蛋白信号激活剂BIO对表达Sox2的干细胞以及GFP表达产生负面影响。该技术可以针对干细胞行为的实时成像进行优化。压制或培养的外植体也可以酶处理成单细胞悬浮液以进行进一步分析。

Research

JoVE Journal

Developmental Biology

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

科研

发育生物学

Method of Studying Palatal Fusion using Static Organ Culture

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

人牙髓干细胞神经分化过程中的分离、繁殖及普利翁蛋白表达

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...