نشكر أندرياس Radbruch لإجراء مناقشات قيمة. ونحن ممتنون لسابين Gruczek، باتريك تيمان ومانويلا Ohde للمساعدة في رعاية الحيوان وروبرت غونتر للحصول على مساعدة فنية ممتازة. نشكر المقيمون لدينا مدربين لورا Oehme، Jannike بيات-سرمدي، كارولين بولوك، كاترين روث، فلورنسا Pache وكاتارينا القرن لتقييم عينات الأنسجة وراندي ليندكويست لتصحيح التجارب المطبعية للمخطوطة. نشكر J. وN. لي، مايو كلينيك، سكوتسديل بولاية أريزونا، الولايات المتحدة الأمريكية عن الأجسام المضادة MBP محددة. وأيد هذا العمل من قبل DFG HA5354 / 4-1، قبل JIMI-منحة الشبكة منشأة DFG الأساسية لintravital المجهري وTRR130 / TP17، وDFG FOR 2165 (HA5354 / 6-1) لAEHSZ كان مدعوما من ماكس بلانك الدولية مدرسة للأمراض المعدية والمناعة (IMPRS-IDI)، برلين.

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Juan Escribano Navarro is affiliated with Wimasis GmbH, Munich, Germany. The other authors have no conflicts of interest to declare.

على الرغم من التقدم في أساليب التصوير البصرية الحديثة، لا يزال كثير من الأحيان أعاق تحليل البيانات النسيجي بسبب عدم وجود أدوات القياس الكمي المناسبة والأساليب، أو عن طريق التحليلات المغرضة التي تركز على منطقة صغيرة من الفائدة. نهج التآزر المقدمة هنا يجمع بين تحليل ?...

بسبب التطورات التكنولوجية السريعة التي حدثت مؤخرا في المجهر، بما في ذلك التصوير الضوئي، أصبح تحليل الخلايا داخل سياق الأنسجة كلها يمكن الوصول إليها على نحو متزايد لالمناعة. توصيف الخلايا واحد في تعليق يمثل طريقة قيمة والتي لا غنى عنها لفهم وظيفة الخلوية والجزيئية. ومع ذلك، فإن تحليل الخلايا داخل (الصغرى) بيئتهم -anatomical أمر ضروري لفهم التفاعلات بين مختلف أنواع الخلايا التي تتعاون في العمليات المعقدة مثل تطوير الاستجابات المناعية. في حين أنه من السهل نسبيا على المجاهر للحصول على المعلومات النوعية من الصور، فإنه لا يزال يشكل تحديا لقياس هذه البيانات، ويرجع ذلك جزئيا إلى حقيقة أن طرق التحليل في هذا المجال لا تزال متخلفة بالمقارنة مع ما هو ممكن في الحصول على الصور. لا يزال كثير من الباحثين يعتمدون على العد اليدوي خلية في الصور الأنسجة التي تستغرق وقتا طويلا،وبالتالي إدخال التحيز بين المقيمون المختلفة والتي تعيق التكرار من قبل جماعات أخرى. في كثير من الأحيان، يتم اختيار صورة تمثيلية واحدة للتأكيد بيان على موقف الخلوي أو زملاء التعريب في منشور، مما يجعل من الصعب على القارئ للحكم على أهمية الإحصائية لمثل هذا الحدث. جنبا إلى جنب مع حقيقة أن محتوى المعلومات كاملة من بيانات الصورة ونادرا ما تستغل، وهذا يؤكد على الحاجة إلى نهج أكثر متحيز وأسرع وشامل لتحليل الصور النسيجية. نخاع العظم هو نسيج المعقدة، والتي تأخذ على الوظائف الحيوية الهامة بوصفها جهاز تكون الدم في الفقاريات الكبار. وبالإضافة إلى كونها مهد للخلايا المكونة للدم 1،2 ولعب دورا هاما في التنمية اللمفاويات B 3، فإنه يعمل أيضا كموقع حيث بدأت التفاعلات المناعية 4 ويدعم، وخلايا إعادة تدوير B الناضجة 5. وبالإضافة إلى ذلك، ودورها في الحفاظ على طأصبح ذاكرة mmunological تقدير متزايد في العقد الماضي، حيث تم العثور على عدة أنواع من الخلايا التي تشكل ذاكرة مناعية في الإقامة هناك 6-9. العلاقة بين العمارة الأنسجة المعقدة للنخاع العظام وظائفها لا تزال بعيدة المنال. على عكس الأجهزة اللمفاوية الثانوية، التي يتم تنظيمها في مقصورات الكلية مثل مناطق خلية T و B، ونخاع العظام يفتقر إلى اضحة التقسيم الكلي. وحتى الآن تم تعريفها مقصورات متميزة في نخاع العظام بواسطة قربها من قشرة العظام أو الأوعية الدموية. أهمية مختلف السكان الخلية انسجة المقيمين في نخاع العظام لعدد من العمليات مثل دعم الخلايا الجذعية، وتطوير الخلايا البائية أو صيانة السكان خلية ذاكرة المناعة (مثل خلايا البلازما المعمرة (أجهزة الكمبيوتر)، CD4 + و CD8 + خلايا الذاكرة T) يشير بوضوح إلى أن هناك درجة معينة من التقسيم الجزئي في نخاع العظام. <pالطبقة = &quot;jove_content&quot;&gt; وقد أدت هذه الملاحظات إلى مفهوم محاريب microanatomical متميزة، وهي متخصصة في بعض وظائف (الجذعية صيانة الخلية، وتطوير الخلايا B في مراحل مختلفة، والحفاظ على الذاكرة المناعية) في نخاع العظام. على الرغم من أن يبدو أن هناك درجة معينة من عدم التجانس بين المنافذ التي تخدم وظائف مختلفة، وبعض من العوامل التي تنتجها الخلايا اللحمية، مثل CXC-chemokine يجند 12 (CXCL12) أو انترلوكين 7 (IL-7)، هي العناصر الحاسمة ل العديد من هذه محاريب 10. التصور وتوصيف الخلايا اللحمية في نخاع العظام أمر صعب نظرا لخصائصها المورفولوجية مع طويلة، ملحقات شجيري رقيقة تشكيل شبكة في جميع أنحاء نخاع العظام، وعدم وجود علامات المناسبة لتميز القطعان اللحمية. وليس من الواضح لبعد ما مدى تتقاسم هذه المنافذ السمات المشتركة فيما يتعلق تكوين. من الخلوية والجزيئيةن، والتي تجعل عناصر مكانة معينة فريدة من نوعها. بالإضافة إلى خلايا انسجة، وقد ثبت أن أنواع الخلايا المكونة للدم للعب دورا حاسما من خلال توفير إشارات معينة على الأقل لبعض المنافذ. ومن الواضح أن تعقيد التركيبة المتخصصة يتطلب تحليلهم في الموقع، وأنها أصبحت ذات أهمية متزايدة للالمناعة وأمراض الدم لزوم في نخاع العظام المعمارية المصغرة، على سبيل المثال، من خلال تحليل العلاقات المكانية بين المكونات الخلوية لها. هنا، ووضع استراتيجية لقياس شارك في توطين وحي علاقات الخلوية في نخاع العظام في طريقة تلقائية وغير متحيزة وتقدم. سير عمل مفصلة بما في ذلك جيل من الفئران خيالية، بإيواء الخلايا الفلورسنت انسجة والخلايا المكونة للدم غير الفلورسنت، وإعداد أقسام النسيجية من عظام undecalcified، والحصول على الصور مبائر تغطي العظم كله، فضلا عن تحليل الصور الآلي للج الخلويةوتقدم س التعريب ولها التحقق من صحة / التمييز من المواقع عشوائي من قبل أداة المحاكاة (الشكل 8).

<table border="0" cellpadding="0" cellspacing="0" width="1864">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Name of Reagent/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:1200px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Neomycin</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:155px;">N6386 SIGMA</td>
			<td>Neomycin trisulfate salt hydrate, EU hazard code: GHS08; www.sigmaaldrich.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:365px;">Ursovit AD3EC</td>
			<td style="width:145px;">Serumwerke Bernburg</td>
			<td style="width:155px;"></td>
			<td>1 ml contains: 50.000 I.E retinyl palmitate,&#160; 5.000 I.E. cholecalciferol, 30 mg tocopheryl acetate, 100 mg ascorbic acid, 1 mg sorbic acid, 200 mg polyoxyl 35 castor oil, 0,5 mg propyl gallate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:365px;">Transfer buffer (100ml PBS, 1ml 1M HEPES, 50U/ml Penicillin/Streptomycin)</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:155px;">P4333&#160;&#160; Sigma&#160;&#160;&#160;&#160; H3375&#160;&#160; Sigma&#160;&#160;</td>
			<td>www.sigmaaldrich.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:365px;">4-Hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin)</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Chicken gamma globulin (CGG) 100 mg</td>
			<td style="width:145px;">Rockland</td>
			<td style="width:155px;">D602-0100</td>
			<td>www.rockland-inc.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">20 % Paraformaldehyde solution (EM-grade)</td>
			<td style="width:145px;">Science Services</td>
			<td style="width:155px;">15713</td>
			<td>EU hazard codes: GHS02, GHS05, GHS07, GHS08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">D(+)-Sucrose</td>
			<td style="width:145px;">Carl Roth</td>
			<td style="width:155px;">4621.1</td>
			<td>www.carlroth.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Dry ice</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:365px;">Acetone</td>
			<td style="width:145px;">Sigma.Aldrich</td>
			<td style="width:155px;">179124 SIGMA-ALDRICH</td>
			<td>EU hazard codes: GHS02, GHS07; www.sigmaaldrich.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:365px;">Hexane</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:155px;">208752 SIGMA-ALDRICH</td>
			<td>EU hazard codes: GHS02, GHS07, GHS08, GHS09; www.sigmaaldrich.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Tissue-Tek cryomolds (standard)</td>
			<td style="width:145px;">Sakura&#160;</td>
			<td style="width:155px;">4557</td>
			<td>25 x 20 x 5 mm; www.sakuraus.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Tissue-Tek O.C.T.</td>
			<td style="width:145px;">Sakura&#160;</td>
			<td style="width:155px;">4583</td>
			<td>&#160;www.sakuraus.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Kawamoto&#39;s SCEM embedding medium</td>
			<td style="width:145px;">Section-Lab, JP</td>
			<td style="width:155px;"></td>
			<td>http://section-lab.jp/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Kawamoto&#39;s cryosection preparation kit&#160;</td>
			<td style="width:145px;">Section-Lab, JP</td>
			<td style="width:155px;"></td>
			<td>http://section-lab.jp/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Kawamoto&#39;s cryofilm type 2C(9)</td>
			<td style="width:145px;">Section-Lab, JP</td>
			<td style="width:155px;"></td>
			<td>http://section-lab.jp/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Microtome blade MX35 Premier Plus, Low Profile</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:155px;">3052835</td>
			<td>L X W: 80 x 8 mm (31.5 x 3.13&#34;), thickness: 0.25 mm (0.01&#34;);www.thermoscientific.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">polyclonal rabbit anti-RFP antibody, biotinylated</td>
			<td style="width:145px;">Rockland</td>
			<td style="width:155px;">600-406-379</td>
			<td>www.rockland-inc.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Alexa Fluor 555 streptavidin</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:155px;">S-32355</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">rat anti-MBP</td>
			<td style="width:145px;">J. and N. Lee</td>
			<td style="width:155px;"></td>
			<td>available from: J. and N. Lee, Mayo Clinic, Scottsdale, AZ , U.S.A., clone MT-14.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">goat anti-rat-Alexa Fluor 647</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:155px;">A-21247</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">rat anti-B220 - Alexa Fluor 594</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>produced and coupled in-house (DRFZ), clone RA3.6B2, Alexa Fluor 594 from Life Technologies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">mouse anti-l1 light chain -FITC</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>produced and coupled in-house (DRFZ), clone LS136</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">rat anti-k light chain - FITC</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>produced and coupled in-house (DRFZ), clone 187.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">DAPI (4&#8242;,6-diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:155px;">D9542 SIGMA</td>
			<td>www.sigmaaldrich.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Fluorescent mounting medium</td>
			<td style="width:145px;">DAKO</td>
			<td style="width:155px;">S3023</td>
			<td>www.dako.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Cover slips (24mm x 24mm x 0.13-0.16 mm)</td>
			<td style="width:145px;">Carl Roth</td>
			<td style="width:155px;">H875.2</td>
			<td>www.carlroth.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Superfrost slides glasses (75 mm x 25 mm)</td>
			<td style="width:145px;">VWR</td>
			<td style="width:155px;">48311-703</td>
			<td>www.vwr.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Laser scanning confocal microscope&#160;</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>equipped with laser lines of 405, 488, 561, 594, 633 nm and a 20x/0.8 NA air objective lens; We used a Zeiss LSM710 and Zen 2010 Version 6.0 software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Automated image analysis tools for bone marrow</td>
			<td style="width:145px;">Wimasis, Munich</td>
			<td style="width:155px;"></td>
			<td>The cell contact tool and cell vicinity tool will be made available by Wimasis upon request: www.wimasis.de</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VC2012 Runtime</td>
			<td style="width:145px;">Microsoft</td>
			<td style="width:155px;"></td>
			<td>free download: http://www.microsoft.com/en-us/download/details.aspx?id=30679</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Simulation tool for random cell positioning</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>available from us, upon request</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:365px;">Image analysis software with image segmentation functions</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>We used Volocity (Perkin Elmer) for measuring cell size distributions of hematopoietic cell types in bone marrow (Figure 5). Alternatives are Definiens Image Analysis (Definiens) or Cell Profiler (free download: http://www.cellprofiler.org)&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:365px;">Fiji image analysis software&#160;</td>
			<td style="width:145px;"></td>
			<td style="width:155px;"></td>
			<td>free download: http://fiji.sc/Fiji. Fiji was used by trained raters for manual cell count (Figure 3).</td>
		</tr>
	</tbody>
</table>

automated quantification of hematopoietic cell &#8211; stromal cell interactions in histological images of undecalcified bone

المجهر متحد البؤر هو الأسلوب المفضل لتحليل توطين أنواع الخلايا متعددة داخل أنسجة معقدة مثل نخاع العظام. ومع ذلك، فإن التحليل النوعي والكمي لتوطين الخلوية أمر صعب، لأنها تعتمد في كثير من الحالات على العد والفرز اليدوي، وبالتالي تحمل مخاطر إدخال تحيز تعتمد على التصنيفات والحد من الموثوقية interrater. وعلاوة على ذلك، غالبا ما يكون من الصعب الحكم على ما إذا كان في توطين التعاون بين خليتين النتائج من المواقع عشوائي، وخصوصا عندما تختلف أنواع الخلايا بقوة في وتيرة حدوثها. هنا، هو عرض طريقة لتقدير غير متحيز للخلوي شارك في التعريب في نخاع العظام. يصف بروتوكول إعداد العينة المستخدمة للحصول على أقسام النسيجية من بأكملها العظام الطويلة الفئران بما في نخاع العظام، وكذلك بروتوكول تلطيخ واكتساب صور عالية الدقة. سير عمل تحليل تمتد من الاعتراف المكونة للدم وغير همت-يتم تقديم أنواع الخلايا opoietic في 2 الأبعاد صور نخاع (2D) العظم لالكمي لاتصالات مباشرة بين تلك الخلايا. وهذا يشمل أيضا تحليلا الحي، للحصول على معلومات حول المكروية الخلوية المحيطة نوع من الخلايا معين. من أجل تقييم ما إذا كان المشارك توطين أنواع الخلايا اثنين هو مجرد نتيجة لتحديد المواقع خلية عشوائي أو يعكس الجمعيات التفضيلية بين الخلايا، وهي أداة المحاكاة الذي هو مناسبة لاختبار هذه الفرضية في حالة المكونة للدم وكذلك خلايا انسجة، هو المستخدمة. وهذا النهج لا يقتصر على نخاع العظام، ويمكن أن تمتد إلى الأنسجة الأخرى للسماح للتكرار، والتحليل الكمي للبيانات النسيجية.

قطع cryosections من العظام undecalcified مع أسلوب الشريط كاواموتو يسمح العظم كله لا بد من تخفيض باعتباره القسم سليمة، مع نخاع العظم من منطقة الطعوم لا تزال تعلق حتى العظم المعدنية، سواء في diaphysis وكذلك في مناطق المشاشية مع هم كثافة عالية من تربيقية العظام (الشكل 1). تلطيخ ا...

Watch this Scientific Journal Video about Automated Quantification of Hematopoietic Cell â€“ Stromal Cell Interactions in Histological Images of Undecalcified Bone at JoVE.com

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

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

الكمي الآلي للخلية المكونة للدم - التفاعلات خلية انسجة في صور النسيجية من Undecalcified العظام

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

automated-quantification-hematopoietic-cell-stromal-cell-interactions

Research

JoVE Journal

Developmental Biology

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

11.4K Views.  German Rheumatism Research Center, a Leibniz Institute. A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

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

Quantification of  Colonic Stem Cell Mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 &#181;m thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 &#181;m apart were prepared to assess a total of &#62;104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts &#247; by the number of wild type (blue) crypts in a treatment group.

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

تقدير حجم الجذعية القولون الطفرات خلية

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

زرع تنافسية لتقييم المكونة للدم للياقة الخلايا الجذعية

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

الترتيب الزمني للبيانات التعبير الحيوي من التعبير المكاني مفصل خرائط

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

عزل وتوصيف الخلايا الجذعية الوسيطة اللحمية من الحبل السري البشري والجنين المشيمة

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

تقنية زرع الثدي الثديية الرواية جديدة لتصور الجيل سفينة من الخلايا الجذعية البطانية الأوعية الدموية

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

الهندسة الحيوية من أنسنة نخاع العظام ميكرونفيرونمنتس في الماوس والتصور من خلال التصوير لايف

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

هندسة أنسجة القرنية: في المختبر نموذجا للتفاعلات Stromal العصب القرنية البشرية

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

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

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

على أساس رانكل "اوستيوكلاست الثقافة المقايسة للماوس نخاع العظام" التحقيق في دور mTORC1 في "تشكيل اوستيوكلاست"

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

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

خلية واحدة القياس الكمي لمعدلات تحلل البروتين بالأسفار الوقت الفاصل بين الفحص المجهري في ثقافة الخلية ملتصقة

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

تحليل الاستنساخ من سلائف الخلايا الجذعية الجنينية المكونة للدم باستخدام مؤشر وحيد الخلية الفرز جنبا إلى جنب مع الخلية البطانية مكانة ثقافة المشارك

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...