The authors would like to acknowledge funding from the Horserace Betting Levy Board U.K. (grant number 721) and VetCell BioScience Ltd, U.K. and by Consejer&#236;a de Innovaci&#242;n, Ciencia y Empresa, Junta de Andaluc&#236;a, Spain.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equine+embryonic+stem-like+cells+and+mesenchymal+stromal+cells+have+different+survival+rates+and+migration+patterns+following+their+injection+into+damaged+superficial+digital+flexor+tendon.">Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon.</a> Equine Veterinary Journal. 42, 636-642 (2010).</li><li>Sole, A., 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=Scintigraphic+evaluation+of+intra-arterial+and+intravenous+regional+limb+perfusion+of+allogeneic+bone+marrow-derived+mesenchymal+stem+cells+in+the+normal+equine+distal+limb+using+(99m)+Tc-HMPAO.">Scintigraphic evaluation of intra-arterial and intravenous regional limb perfusion of allogeneic bone marrow-derived mesenchymal stem cells in the normal equine distal limb using (99m) Tc-HMPAO.</a> Equine Veterinary Journal. 44, 594-599 (2012).</li><li>Carvalho, A. 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=Evaluation+of+mesenchymal+stem+cell+migration+after+equine+tendonitis+therapy.">Evaluation of mesenchymal stem cell migration after equine tendonitis therapy.</a> Equine Veterinary Journal. 46, 635-638 (2014).</li><li>Welling, M. M., Duijvestein, M., Signore, A., van der Weerd, L. <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+biodistribution+of+stem+cells+using+molecular+nuclear+medicine+imaging.">In vivo biodistribution of stem cells using molecular nuclear medicine imaging.</a> Journal of Cellular Physiology. 226, 1444-1452 (2011).</li><li>Dowling, B. A., Dart, A. J., Hodgson, D. R., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superficial+digital+flexor+tendonitis+in+the+horse.">Superficial digital flexor tendonitis in the horse.</a> Equine Veterinary Journal. 32, 369-378 (2000).</li><li>Kasashima, Y., Ueno, T., Tomita, A., Goodship, A. E., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+bone+marrow+aspiration+from+the+equine+sternum+for+the+safe+recovery+of+mesenchymal+stem+cells.">Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells.</a> Equine Veterinary Journal. 43, 288-294 (2011).</li><li>Avella, C. S., 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=Ultrasonographic+assessment+of+the+superficial+digital+flexor+tendons+of+National+Hunt+racehorses+in+training+over+two+racing+seasons.">Ultrasonographic assessment of the superficial digital flexor tendons of National Hunt racehorses in training over two racing seasons.</a> Equine Veterinary Journal. 41, 449-454 (2009).</li><li>de Vries, E. F., Roca, M., Jamar, F., Israel, O., Signore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+labelling+of+leucocytes+with+(99m)Tc-HMPAO.+Inflammation/Infection+Taskgroup+of+the+European+Association+of+Nuclear+Medicine.">Guidelines for the labelling of leucocytes with (99m)Tc-HMPAO. Inflammation/Infection Taskgroup of the European Association of Nuclear Medicine.</a> European Journal of Nuclear Medicine and Molecular Imaging. 37, 842-848 (2010).</li><li>Trela, J. 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=Scintigraphic+comparison+of+intra-arterial+injection+and+distal+intravenous+regional+limb+perfusion+for+administration+of+mesenchymal+stem+cells+to+the+equine+foot.">Scintigraphic comparison of intra-arterial injection and distal intravenous regional limb perfusion for administration of mesenchymal stem cells to the equine foot.</a> Equine Veterinary Journal. 46, 479-483 (2014).</li><li>Barbash, I. 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JD and RKS are Scientific Advisory Board members to ReCellerate Inc.

بالإضافة إلى نخاع العظام، والخلايا الجذعية المعزولة من مصادر مثل الأنسجة الدهنية هي مناسبة لوصفها مع هذا البروتوكول. وعلاوة على ذلك، الخلايا من حالة تجمد يمكن إحياء وتوسيع في الثقافة إلى الأرقام المطلوبة للدراسات وسم 12. ا?...

وتستند استراتيجيات التجدد لإصلاح الأنسجة المريضة أو التالفة على الخلايا الجذعية متعددة القدرات المستمدة من مجموعة متنوعة من الأنسجة وزرعها في المنطقة المصابة. وقد أظهرت التطورات الأخيرة في تطبيق ذاتي BMMSC لعلاج وتر وإصابات الرباط في الحصان تحسين مقاييس النتائج في كل تجريبي 1-5 والدراسات السريرية 6. الحصان هو نموذج جاذبية خاصة لتقييم فعالية الخلايا الجذعية العلاجات المتعلقة لأنها تعاني من العمر وإرهاق الإصابات المتعلقة الأوتار من forelimb البعيدة، فمن حيوان الرياضي، وأنه هو كبير، وتسهيل الانتعاش نخاع العظام و غرس دقيقة. إصابات الأوتار تلتئم بشكل طبيعي مع التليف ولكن وتر تلتئم هو أدنى وظيفيا 7 ولها مخاطر عالية من إعادة الإصابة 8. يتأثر المثنية الرقمي سطحية وتر (SDFT) الأكثر شيوعا وتطورت لتكون بمثابة الطاقة المرنةويؤكد مخزن والخبرات تحميل عالية لتحقيق كفاءة الطاقة وسرعة عالية الحركة. استعادة وظيفة بعد الاصابة ولذلك حرجة. هذه الإصابات هي مماثلة لتلك التي تؤثر على وتر أخيل في البشر الذي يؤدي وظيفة مماثلة 9. لا توجد خيارات جيدة العلاج لعلاج أو تحقيق حالة جيدة لمثل هذه الإصابات، وتوفر استراتيجيات التجدد بالتالي، يستند خلية فرصة جذابة لتحسين النتائج والحد من إعادة الإصابة. في معظم الدراسات 5 - يتم حقن 20 مليون BMMSC ذاتي مباشرة إلى الآفة التي تحدث عادة في غضون جوهر الجسم وتر التي بالتالي بمثابة وعاء للخلايا. مصير الخلايا مرة واحدة حقن ليست طرق وضع العلامات الخلية واضحة ومختلفة لتعقب وقد وصفت الخلايا مؤخرا. عرضت الخلايا المسمى مع علامة مضان من أجل البقاء فقط في أعداد صغيرة نسبيا (&lt;10٪) 10،11. تسميات مضان تستلزماستخراج الأنسجة وباجتزاء للتحليل النسيجي الذي هو مضيعة للوقت و لا تسهل بسهولة التحليل الزمني في نموذج حيواني كبير أو في الحالات السريرية. في مزيد من العمل مؤخرا استخدمنا 99m النظائر المشعة ح لتسمية الخلايا واتبع مصيرهم جاما مضان 1. يسمح هذا الأسلوب مقارنات سريعة ليتم بين طرق مختلفة من تسليم خلية، بما في ذلك داخل الآفة، عن طريق الوريد عن طريق الوريد الوداجي 1 أو نضح الإقليمي عبر داخل الشرايين 12 أو عن طريق الوريد 1،12 الحقن. استمرار وتوزيع الخلايا ومن ثم يمكن تصويرها من قبل جاما مضان من مختلف الأجهزة. ويبين هذا أن 24٪ فقط من حقن الخلايا ظلت intralesionally في الآفة التي مدار 24 ساعة (1) وهذا معتمد من قبل دراسة أخرى باستخدام الآفات خلق تجريبيا وباستخدام نفس radiolabel 5. وعلاوة على ذلك، تظهر الخلايا قدرة محدودة على منزل في الآفات وتر عندما delivereد كتبها نضح الإقليمي أو عن طريق الوريد ولكنها تنتشر في الرئتين عن طريق طرق الأخيرة (4). BMMSC المسمى مع النانوية الحديد هو طريقة بديلة لتعقب الخلايا المزروعة في الأوتار forelimb 13. على الرغم من جسيمات متناهية الصغر الحديد الخلايا المسمى تسمح تتبع خلية في الجسم الحي بواسطة التصوير بالرنين المغناطيسي، دراسات الزمنية في الحيوانات الكبيرة تقتصر على عدد المرات التي يمكن أن تدار التخدير في كل نقطة زمنية لتنفيذ فحوصات الرنين المغناطيسي. وعلاوة على ذلك، النانوية الحديد هي hypointense على التصوير بالرنين المغناطيسي الذي يحد من المعلومات عن هجرة الخلايا المسمى في الجسم وتر. النظائر المشعة الأخرى التي يمكن استخدامها وتشمل الإنديوم-111 ولكن هذا يعاني من عيب أطول عمر النصف من TC-99m (2.8 أيام مقابل 6.0 ساعة) وارتفاع الطاقة انبعاث أشعة غاما. بالإضافة إلى ذلك، تم الإبلاغ عن بقاء الخلية أن تنخفض عندما وصفت مع الإنديوم-111 14. TC-99m، من ناحية أخرى، ويستخدم بشكل روتيني في كل من الخيول والإنسان النوويةالطب لتسمية الخلايا وحيدة النواة في الدم المحيطي ومتابعة توزيعها في الجسم الحي من قبل مضان. ويمكن بسهولة نسبيا التي اتخذت من قبل الخلايا باستخدام HMPAO كناقل رابط لربط تكنيتيوم، كما TC-99m-HMPAO، إلى الخلايا. وصفت TC-99m-HMPAO BMMSC تظهر قابلية جيدة، ويمكن أن تتكاثر في المختبر 4. هذا البروتوكول تفاصيل وضع العلامات وتتبع BMMSC ذاتي الخيول زرعها في الآفات التي تحدث بشكل طبيعي في SDFT forelimb. ومن المهم أن نلاحظ أن البروتوكول يهدف فقط لاستخدامها كأداة بحثية. لا ينصح استخدامه كطريقة علاجية سريرية تأثير radiolabel على النمط الظاهري الخلوية لم توضح تماما.

<table>
	<tbody>
		<tr>
			<td>Technetium99m&#160;</td>
			<td></td>
			<td></td>
			<td>Please enquire with local ionisation radiation supplier in accordance with legal requirements.&#160; The isotope must be used within 2 h of elution from the molybdenum-99 generator</td>
		</tr>
		<tr>
			<td>Ceretec - Hexamethylpropyleneamine oxime (HMPAO)&#160;</td>
			<td>GE HealthCare</td>
			<td></td>
			<td>Please enquire directly with GE HealthCare</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge, Minispin/Minispin Plus</td>
			<td>Ependorf</td>
			<td>22620100</td>
			<td></td>
		</tr>
		<tr>
			<td>18G and 19G Needles</td>
			<td>Terumo Medical</td>
			<td>NN-1838R (18G);&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NN1938R (19G)</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 1 mL and 2 mL</td>
			<td>Scientific Laboratory Supplies Ltd</td>
			<td>SYR6200 (1 mL); SYR6003 (2 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Greiner Bio-One Ltd</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS - Phosphate-Buffered Saline</td>
			<td>LifeTechnologies</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze Swabs</td>
			<td>Shermond Ltd</td>
			<td>UNG602</td>
			<td></td>
		</tr>
		<tr>
			<td>CoflexVet self adhering bandage</td>
			<td>Andover Healthcare, Inc.</td>
			<td>3540RB-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound imaging software</td>
			<td>Scion Image, Scion Corporation, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicasXplus Scintigram processing software</td>
			<td>Bartec Technologies Ltd</td>
			<td></td>
			<td>http://www.bartectechnologies.com/veterinaryscintigraphy.html</td>
		</tr>
		<tr>
			<td>Field isotope counter for monitoring isotope</td>
			<td>John Caunt U.K.</td>
			<td>GMS1800a</td>
			<td>http://www.johncaunt.com/</td>
		</tr>
		<tr>
			<td>Well counter for isotope measurements, dose calibrator</td>
			<td>Capintec Southern Scientific</td>
			<td>CRC-25R</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo imaging and tracking of technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy

التطورات الحديثة في تطبيق خلايا نخاع العظم الجذعية الوسيطة (BMMSC) لعلاج إصابات الأوتار والأربطة في الحصان أقترح تحسين مقاييس النتائج في كل من الدراسات التجريبية والسريرية. على الرغم من أن BMMSC يتم زرعها في آفة وتر بأعداد كبيرة (عادة 10 حتي 20000000 الخلايا)، سوى عدد قليل نسبيا البقاء على قيد الحياة (&lt;10٪) على الرغم من أن هذه يمكن أن تستمر لمدة تصل إلى 5 أشهر بعد الزرع. هذا يبدو أن المراقبة المشتركة في الأنواع الأخرى حيث تم زرع BMMSC إلى الأنسجة الأخرى وأنه من المهم أن نفهم عند حدوث هذه الخسارة، وكيفية البقاء على قيد الحياة كثير عملية زرع الأولية وما إذا كان يتم مسح الخلايا في الأجهزة الأخرى. تتبع مصير الخلايا يمكن تحقيقه عن طريق radiolabeling وBMMSC قبل الزرع التي تسمح غير الغازية في التصوير المجراة من موقع الخلية النوعي والكمي لأعداد الخلايا. يصف هذا البروتوكول على labeli الخليةالإجراء نانوغرام يستخدم التكنيتيوم-99m (TC-99m)، وتتبع هذه الخلايا التالية زرع في الأوتار المثنية اصابة في الخيول. TC-99m هو (ر 1/2 من 6.01 ساعة) النظائر قصيرة الأجل التي تنبعث أشعة جاما، ويمكن أن يكون داخليا من قبل الخلايا في وجود hexamethylpropyleneamine أوكسيم مجمع محبة للدهون (HMPAO). هذه الخصائص تجعله مثاليا للاستخدام في عيادات الطب النووي لتشخيص كثير من الأمراض المختلفة. يمكن اتباعها مصير الخلايا المسمى في المدى القصير (تصل إلى 36 ساعة) من خلال جاما مضان لتحديد كل من عدد من الخلايا الاحتفاظ بها في الآفة وتوزيع الخلايا في الرئتين والغدة الدرقية وغيرها من الأجهزة. ويتم تكييف هذه التقنية من وضع العلامات الكريات البيض في الدم، ويمكن استخدامها لصورة مزروع BMMSC في الأجهزة الأخرى.

<html> TC-99m-HMPAO إدماجها BMMSs لا يؤثر سلبيا على قدرتهم على التمسك البلاستيك زراعة الأنسجة وعلى الرغم من أنها تظهر القدرة على الانتشار لتشكيل الطبقات الوحيدة (الشكل 1) لدينا لم يحدد تماما ما إذا كانت معدلات انتشار أو الظواهر الخلوية الأخرى تتأثر. مورفولوجيا يشبه خلا?...

Watch this Scientific Journal Video about In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy at JoVE.com

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

في فيفو التصوير وتتبع التكنيتيوم-99m إعتبر نخاع العظم الوسيطة الخلايا الجذعية في فرسي Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

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In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

9.7K Views.  The Royal Veterinary College. This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Video: In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

تقييم ما قبل السريرية من التيروزين كيناز مثبطات لعلاج اللوكيميا الحادة

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

في الجسم الحي 19 F التصوير بالرنين المغناطيسي لتتبع الخليوي

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

تسلسلي<em&gt; في الجسم الحي</em&gt; التصوير من الخلايا الجذعية المكونة للعظم / السلف خلال اصلاح الكسر

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

الفخذ نخاع العظم الطموح في الفئران لايف

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

دراسة سرطان البنكرياس الجذعية خصائص الخلايا لتطوير استراتيجيات علاجية جديدة

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

العزلة والخلوية Phenotyping من الخلايا الجذعية الوسيطة المشتقة من نخاع الزلالية السائل والعظام من Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

نضوب خلايا الفأر من Xenografts ورم الإنسان يحسن بشكل ملحوظ تحليل المصب من الخلايا المستهدفة

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

العصب استرشادا مشجعا حقن الخلايا الجذعية الذاتي قرب عصب حنجري المتكررة الأيسر الخيلي

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

تحليل كمي [18F]-NAF-PET-MRI لتقييم دوران العظام الديناميكية في مريض يعاني من آلام الظهر المنخفضة في Facetogenic

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.