Name: combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches
Uploaded: 2017-03-21T13:00:00
Description: Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

在印第安纳大学的印第安纳生物显微镜中心进行了成像, 由 Dr. 的导演邓恩。立体定向装置是由 Soonpaa、水井中心的儿科研究的原型设计和制作的。这项工作得到了 NIH/R01DK097837-09 (nc)、NIH/R01HL068256-05 (nc)、NIH/NIDDK1U54DK106846-01 (nc)、MPN 研究基金会 (nc) 和 CTSI 合作项目 IUSM/圣母院 (nc) 的支持。

所有涉及使用动物的程序都是在印第安纳大学医学院的动物保育和使用委员会的授权下进行的。确保遵守该项工作所在国家的动物实验立法。
1. Mx1CreRBPJ--受体小鼠的制备
<ol><li>交叉 Mx1-Cre+小鼠与 RBPJ 液氧/液氧小鼠10获得 Mx1-Cre 阳性RBPJ液氧/液中小鼠12和 Mx1-Cre 阴性 RBPJ 液氧/液氧窝?...

<ol>
	<li>Carlesso, N., Cardoso, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+regulatory+niches+and+their+role+in+normal+and+malignant+hematopoiesis.">Stem cell regulatory niches and their role in normal and malignant hematopoiesis.</a> Curr Opin Hematol. 17 (4), 281-286 (2010).</li><li>Lo Celso, C., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124 (PT 21), 3529-3535 (2011).</li><li>Calvi, L. 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=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. 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K., Akinduro, O., Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+4-dimensional+tracking+of+hematopoietic+stem+and+progenitor+cells+in+adult+mouse+calvarial+bone+marrow.">In vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow.</a> J Vis Exp. (91), e51683 (2014).</li></ol>

该协议描述了一个实验设计的优化, 以研究活体荧光灯显微镜的造血细胞植入动力学。在这项研究中, 在骨髓细胞或在一个缺口信号有缺陷 bm 的扩张在骨颅骨被跟踪的赖氨酸-GFP 阳性髓细胞后, BMT 到 RBPJWT 或 RBPJKO 受体。这种方法被提出作为一个模型, 可用于解决类似的问题, 如: i), 以确定在 BM 的细胞的其他血统, 如淋巴, 红或巨细胞的扩展和本地化, 通过作为供体细胞造血细胞携带血统特异促进剂驱...

活体多荧光显微镜 (IVFM) 是一种强大的成像技术, 允许高分辨率, real-time 成像的组织与深度高达 1mm, 根据组织。当应用于小鼠颅骨, 它允许观察 BM 内造血细胞的行为, 其非侵入性的方式可达60-100 μ m11。本方法用于测定 RBPJ 敲除小鼠缺乏标准缺口信号的植入的正常髓内祖细胞的动力学。
最近我们小组的工作表明, 在 BM 微环境中有缺陷的标准缺口信号会导致类似骨髓的疾病12。利用 Mx1-Cre 诱导重组10, 通过条件删除 RBPJ 的 DNA 结合域 (标准缺口信号下游的临界转录因子), 得到了缺口信令的损失。在本研究中, 使用了 Mx1-Cre/RBPJ 的液氧液/液氧小鼠模型。有条件地删除 RBPJ 的 DNA 结合母题会导致所有凹槽受体的信号丢失。在 Mx1-Cre 模型中, Mx1 启动后的表达是由激活后的 polyI 管理: C 导致诱导靶基因缺失的血细胞以及基质成分的多个器官, 包括 BM, 脾脏和肝脏。
Mx1-Cre+/RBPJ氧液/液氧和 Mx1-Cre-/RBPJ 液氧/液氧小鼠 polyI: C (在此表明 RBPJKO 和 RBPJWT 分别) 照射和移植正常, 野生型造血细胞。从4周开始移植后, RBPJKO 受体发育显著增多继发脾肿大。虽然 RBPJKO 小鼠在移植后8周和以后的时间点, 骨髓中髓祖细胞的比例增加, 但在4和6周, bm 的分析结果并没有显示出它们与对照组相比的髓内含量显著的差异 RBPJWT收件人.这一观察, 连同 Mx1-Cre 在不同的造血器官中表达的事实, 提出了 BM 微环境是否对骨髓表型的启动有直接影响的问题。
为了确定 bm 是否是疾病发展的一个重要的初始部位, IVFM 的小鼠颅骨与 bm 移植 (BMT)、RBPJ 敲除模型和血统跟踪系统结合使用。在特定溶菌酶启动子 (赖氨酸-GFP)9的控制下表达 EGFP 的转基因小鼠被用来获得在 BM 成像后 BMT 的供体细胞。溶菌酶表达是特定于髓细胞和赖氨酸-GFP 标记细胞从普通的髓系祖 (CMP) 到成熟粒细胞膜13。
IVFM 在不同时间点的 bm 表明, 赖氨酸-GFP 细胞与 RBPJWT 和 RBPJKO 受体的 bm 相似, 但在 RBPJKO 受体的 bm 中扩张和嫁接更快。这个区别是戏剧性的在早先时间点 (星期 2) 和减少随着时间 (星期4和 6)。然而, 在这些后来的时间点, 对同一受体的造血室的评价显示, 在 RBPJKO 小鼠体内的铅和脾内循环的髓细胞数量稳步增加, 表明细胞的产量增加从 BM 到循环。赖氨酸-GFP 细胞在移植小鼠骨髓中的定位分析6周后发现髓细胞在 RBPJKO 微环境中的血管中比对照组更远。
总的来说, IVFM 与这些特定动物模型的结合提供了 RBPJKO BM 微环境中髓细胞植入动力学的洞察。本文所描述的实验设计和定量方法是一种可以应用于解决类似问题的范例。例如, 使用其他特定于单元的沿袭跟踪模型 (如 RAG1-GFP14或 Gata1-GFP15小鼠) 可能允许在 BM 中分别遵循淋巴或红祖细胞的行为。

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			<td height="21" style="height:21px;width:216px;">Ketamine cocktail</td>
			<td style="width:167px;">IU School of Medicine</td>
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			<td style="width:511px;">Ketamine 90-100mg/kg, Xylazine 2.5-5.0 mg/kg, Acepromazine 1.0-2.5 mg/kg</td>
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			<td height="21" style="height:21px;width:216px;">TRITC dextran</td>
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			<td style="width:139px;">TD150-100mg</td>
			<td>Other color dextran may be used.</td>
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			<td height="21" style="height:21px;width:216px;">Andis hair trimmer</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:139px;">CLP-323 75</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Gauze sponge</td>
			<td style="width:167px;">Med Vet International</td>
			<td style="width:139px;">PK224</td>
			<td>4-ply, 2X2</td>
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			<td height="21" style="height:21px;width:216px;">Nair depilatory cream</td>
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			<td>0.9% Sodium Chloride poly bottle</td>
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			<td height="21" style="height:21px;width:216px;">Insulin syringe</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:139px;">14-826-79</td>
			<td>28g, 1/2cc</td>
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			<td height="26" style="height:26px;width:216px;">Fine Forceps</td>
			<td style="width:167px;">Fine Science Tools</td>
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			<td>straight forcep, angled forcep</td>
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			<td height="21" style="height:21px;width:216px;">Scissor</td>
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			<td height="21" style="height:21px;width:216px;">5-0 silk suture</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:139px;">MV-682</td>
			<td>Other non-absorbable suture may be used</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">WillCo- glass bottom dish</td>
			<td style="width:167px;">WillCo</td>
			<td style="width:139px;">GWSt-5040</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optical microscope oil</td>
			<td style="width:167px;">Leica</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic stage insert&#160;</td>
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			<td style="width:139px;">Custom design</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus FV1000 confocal microscope system&#160;</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus XLUMPLFL 20xW, NA 0.95 objective&#160;</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small heating pad</td>
			<td style="width:167px;">Commercial store</td>
			<td style="width:139px;"></td>
			<td>Zoo Med reptile heating pad</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Imaris 8.1 imaging software</td>
			<td style="width:167px;">Bitplane</td>
			<td style="width:139px;"></td>
			<td>3/4 D Image Visualization and Analysis software</td>
		</tr>
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combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches

越来越多的证据表明, 正常的造血是由不同的微提示在 BM, 其中包括专门的细胞龛调节关键造血干细胞 (HSC) 功能1,2。事实上, 更详细的造血微环境的图片现在正在出现, 其中内和内皮龛形成正常 HSC 及其子代的调节功能单元3,4,5.新的研究揭示了血管细胞、脂肪细胞和神经细胞在维持和调节 HSC 功能方面的重要性6,7,8。此外, 有证据表明, 来自不同血统的细胞,即髓细胞和淋巴细胞, 位于 BM 微环境中的特定龛位。但是, BM 微环境及其占用者的完整映射仍在进行中。
表达血统特异荧光标记或转基因小鼠的转基因小鼠菌株, 在 BM 龛的特定细胞中缺乏选定的分子, 现已问世。淘汰赛和血统跟踪模型, 结合移植的方法, 提供了机会, 以完善有关特定的 "利基" 细胞的作用, 明确的造血种群, 如 HSC, B 细胞, T 细胞, 髓细胞和红细胞。这一战略可以进一步强化通过合并使用双光子显微镜的颅骨。通过提供体内高分辨率成像和 bm 颅骨的3维渲染, 我们现在可以精确地确定 bm 中特定的造血亚群的位置, 并评估它们随时间扩展的动力学。在这里, 赖氨酸-GFP 的转基因小鼠 (标记髓细胞)9和 RBPJ 敲除小鼠 (缺乏标准缺口信号)10与 IVFM 结合使用, 以确定髓细胞植入的缺口缺陷 BM 微环境。

2 RBPJKO 和 2 RBPJWT 接受者在不同时间点的单个成像会话中被成像:24 小时和2、4和6周, 移植后的 BM 赖氨酸-GFP 细胞 (工作流在图 1A中说明)。
在每只老鼠中, 图像是从 BM 颅骨的6标准区域获得的, 它们的位置与中心静脉的分岔有关 (图 2A, a) 和冠状动脉缝合 (图 2A, b)。注射葡聚糖得克?...

Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

活体荧光显微镜 (IVFM) 的颅骨是应用与遗传动物模型, 研究造血细胞归巢和植入的骨髓 (BM) 龛。

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

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Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Automated Quantification of Hematopoietic Cell ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“ Stromal Cell Interactions in Histological Images of Undecalcified Bone

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

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酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

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 ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

杏中自体和（内）相容关系的确定结合手授、显微镜和遗传分析

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...