This work was supported by grants funded by the British Heart Foundation (BHF), Dinosaur Trust, McAlpine Foundation, Fondation Leducq, Fight for Sight, the Cambridge Biomedical Research Centre, National Institute of Health Research including (i) the BHF Oxbridge Centre of Regenerative Medicine [RM/13/3/30159], (ii) the BHF Cambridge Centre of Research Excellence, (iii) Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust and (iv) Papworth Hospital NHS Foundation Trust, and supported the Cambridge NIHR BRC Cell Phenotyping Hub. MLO is funded by a BHF Intermediate Fellowship. FNK is funded by a BHF PhD Studentship.

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The authors have nothing to disclose.

We present a detailed protocol that allows for the robust and efficient derivation of BOECs from adult peripheral blood mononuclear cells (PBMNCs). Our protocol includes two important refinements that represent advances on previous methods of BOEC isolation.14-16 These include the absence of heparin in the initial PBMNC culture medium and the use of defined, embryonic stem cell-qualified serum. This latter refinement is of particular importance. Embryonic stem cell (ESC)-qualified serum is a more consistent grade of serum and, although it is not known yet what component(s) are enriched in the serum that benefit BOEC isolation, the impact of this defined serum on the efficiency of BOEC generation is clear in our hands. In addition, we have also had success in isolating BOECs using human serum, thereby allowing for the generation of BOECs for clinical translation. In our hands, this refined protocol results in the successful isolation of stable BOEC cultures from greater than 90% of donors, making it one of the most reliable BOEC generation methods reported thus far. Although the use of particular sera is critical to BOEC generation, it also represents a primary limitation of the current protocol. Future improvements to the technique could include the generation of these cells in serum-free, defined culture conditions.
Critical Steps in the protocol include processing blood samples as soon as possible after collection, complete harvesting of the buffy coat cells after density gradient centrifugation and the timely passaging of initial colonies from P0 to P1. This passaging step is critical to establishment of a stable isolation. Like other endothelial cells, BOECs appear to be very sensitive to plating density. If the plating density after passaging is too low, the BOECs will not proliferate. Conversely, if the colonies are allowed to become overconfluent before passaging, the cells will also cease to proliferate and have the tendency to convert into an elongated, mesenchymal cell phenotype. If few colonies appear from days 7 to 14, or if the colonies are small in size, troubleshooting can include increasing cell density by passaging P0 colonies into a T-25 flask instead of a T-75.
Once the technique is mastered, the resultant BOECs can be used in several applications, including in vitro studies of endothelial cell biology, disease modeling and drug screening, as well as in vivo cell transplantation therapies. An important consideration for the development of any cell therapy process is to use cells that are free from pathogenic mutations. We have previously shown that BOECs isolated using our protocol possess genomes that are free from copy number variations and are thus representative of the individual from which they were collected. In addition, we have also demonstrated that the majority of BOEC-derived iPSC lines are free from copy number variations.13 This contrasts with previous reports of copy number variation in fibroblast-derived iPSCs. To date, these cells remain the only iPSCs for which this degree of genomic fidelity has been reported. This feature is important for the field of iPSC biology and the use of iPSCs in disease modeling, drug screening and future cell transplantation therapies.

حتى وقت قريب، كان يعتقد أن جيل ما بعد الولادة الأوعية الدموية الجديدة أن تحدث بشكل حصري من خلال عملية تعرف باسم الأوعية الدموية، الذي يعرف بأنه تنتشر سفن جديدة من الخلايا البطانية للأوعية موجودة من قبل. 1 هذه العملية تتناقض من تكون الأوعية، أو تشكيل دي نوفو الأوعية الدموية من أسلاف البطانية، الذي كان يعتقد أن يحدث حصرا أثناء التطور الجنيني. 2 ومع ذلك، وقد حددت الدراسات الأكثر حداثة وعزل تعميم الخلايا الاصلية البطانية (EPCS) في الدم المحيطي الكبار. هذه الخلايا تمتلك القدرة على التمايز إلى خلايا البطانية ناضجة في الثقافة ويعتقد على المشاركة في تكون الأوعية بعد الولادة. 3،4 بروتوكولات للعزلة وتوسيع هذه EPCS وعادة ما تشمل ثقافة الخلايا الطرفية وحيدة النواة في الدم (PBMNCs) في وسائل الإعلام التي تحتوي على عوامل النمو البطانية، بما في ذلك vasculعامل AR البطانية النمو (VEGF)، وعامل نمو الخلايا الليفية-2. 5-8 الثقافات EPC تنتج مجموعة متنوعة من أنواع مختلفة من الخلايا بشكل كبير. الثقافات الأولية (&lt;7 أيام) ويغلب عليها نوع من الخلايا الوحيدات، والمعروف في الأدب بأنه &quot;في وقت مبكر&quot; EPCS. على الرغم من اسمها، وهذه الخلايا تعبر عن CD14 الوحيدات علامة، هي سلبية للCD34 السلف علامة والتعبير عن مستويات الحد الأدنى فقط من الكلاسيكية البطانية علامات CD31 وVEGF مستقبلات 2 (VEGFR2). 5 الثقافة استمرار يثير سكان الثانوي من الخلايا، المعروفة باسم EPCS أواخر ثمرة أو الخلايا البطانية ثمرة الدم (BOECs)، والتي تظهر المستعمرات سرية اعتبارا من خلايا تشبه الخلايا البطانية. وخلافا للEPCS مبكرة الوحيدات، BOECs، والتي أيضا تم استدعاء مستعمرة تشكيل الخلايا البطانية (ECFCs)، الخلايا البطانية ثمرة أو الخلايا البطانية في وقت متأخر من ثمرة، يحمل التشكل المرصوفة بالحصى التي هي الحال في الطبقات الوحيدة الخلية البطانية وتشبه إلى حد كبير في علامة السطح5 والتعبير الجيني 9 إلى أن تنضج الخلايا البطانية. توليد خلايا تشبه الخلايا البطانية من الدم المحيطي يوفر العديد من المزايا، وخاصة لدراسة الخلل الخلايا البطانية المرتبطة اضطرابات الأوعية الدموية مثل ارتفاع ضغط الدم الرئوي الشرياني (PAH) 10 أو فون ويلبراند المرض. 11 قبل توافر BOECs، البطانية لا يمكن إلا أن تكون مشتقة من خلايا أجهزة explanted في وقت الوفاة أو زرع الأعضاء، أو معزولة من الوريد السري عند الولادة. هذا انخفاض توافر يمثل قيدا خطيرا على فهم بيولوجيا الخلايا البطانية من المرضى الذين يعانون من اضطرابات القلب والأوعية الدموية، وكذلك التفاعلات بين الخلايا البطانية وإما خلايا الدم أو الخلايا الجدارية. وعلاوة على ذلك، عزل وزراعة السكان نقية من الخلايا البطانية من هذه المصادر يشكل تحديا فنيا والخلايا التي يحصل عليها هذه الأساليب يحمل سوى يميتالقدرة التكاثري د. لذا BOECs تقديم بديل قيمة للعزلة وثقافة من الخلايا البطانية الأولية المستمدة من المريض. بالإضافة إلى المختبر في طلباتهم، BOECs أيضا يمكن أن تكون مفيدة في علاج ذاتي زرع الخلايا. وتشمل هذه التطبيقات على حد سواء زرع البطانية خلية لتعزيز اتساع الأوعية الدموية (انظر 12 والمراجع فيه)، فضلا عن توليد الخلايا الجذعية المحفزة (iPSCs). 13 iPSCs BOEC المشتقة يمكن أن تستخدم لنمذجة المرض وتوفر إمكانات هائلة مثل الانطلاق المواد اللازمة لعلاج الخلايا ذاتي. BOECs برمجة أسرع وبكفاءة أعلى من الخلايا الليفية الجلد. وعلاوة على ذلك، BOECs أيضا السماح لتوليد iPSCs أن تكون خالية من العيوب النمط النووي، الذي هو سمة أساسية من سمات أي تكنولوجيا من شأنها أن تكون مناسبة لتطبيقات متعدية. القدرة على توليد iPSCs من عينة دم المريضيسوتو يلغي الحاجة لخزعة الجلد وتوليد الخلايا الليفية الجلد، مما يسهل توليد الخلايا من المرضى الذين يعانون من اضطرابات التئام الجروح، أو الصغار جدا. بروتوكول المفصلة أدناه، التي وافقت عليها والتي تتم وفقا للمبادئ التوجيهية للجنة الخدمة أخلاقيات القومي للبحوث (شرق انجلترا)، يوفر طريقة بسيطة وموثوق بها لتوليد BOECs مع كفاءة أكبر من 90٪ من حجم صغير نسبيا (60 مل) من الدم المحيطي. هذه الخلايا هي التكاثري للغاية ويمكن passaged مرارا وتكرارا، مما يسمح لتوليد مئات الملايين من الخلايا من عينة دم واحدة.

<table>
	<tbody>
		<tr>
			<td colspan="4">For blood collection</td>
		</tr>
		<tr>
			<td>60 mL syringe with luer-lok tip</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>19G Surflo Winged Infusion Set</td>
			<td>Terumo</td>
			<td>SV-19BL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>StarLab</td>
			<td>E1450</td>
			<td>2 per donor</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Martindale Pharmaceuticals</td>
			<td>270541</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For buffy coat isolation</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile wrapped plastic transfer pipettes</td>
			<td>Appleton Woods</td>
			<td>KC231</td>
			<td></td>
		</tr>
		<tr>
			<td>Turk&#8217;s Solution</td>
			<td>Millipore</td>
			<td>1.093E+09</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For cell culture, passaging and freezing cells</td>
		</tr>
		<tr>
			<td>Type 1 Collagen (derived from rat tail)</td>
			<td>BD Biosciences</td>
			<td>35-4236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.02M Acetic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>prepared in reagent grade water</td>
		</tr>
		<tr>
			<td>Endothelial Growth Medium-2MV 
			(containing Bullet Kit, but not serum)</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Note: It is essential that the medium does not contain heparin.&#160;&#160; 
			Do not use EGM-2.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (U.S.), Defined</td>
			<td>Hyclone</td>
			<td>SH30070</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Trypsin EDTA</td>
			<td>Gibco</td>
			<td>T4174</td>
			<td>Dilute to 1x in PBS prior to use</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>C1562</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For flow cytometric characterization</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD14</td>
			<td>BD Biosciences</td>
			<td>555397</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD31</td>
			<td>BD Biosciences</td>
			<td>555445</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-human CD34</td>
			<td>BD Biosciences</td>
			<td>555824</td>
			<td>Mouse IgG1k, Clone: 581/CD34 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>560976</td>
			<td>Mouse IgG1k, Clone: HI30 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>&#160;APC-conjugated mouse anti-human VEGFR2</td>
			<td>R&#38;D Systems</td>
			<td>FAB357A</td>
			<td>Mouse IgG1, Clone: 89106 
			Dilution: 1:10</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555751</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>R&#38;D Systems</td>
			<td>IC002A 
			Dilution: 1:10</td>
			<td>Clone: 11711</td>
		</tr>
		<tr>
			<td>EDTA, 0.5M solution</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For immunofluorescent microscopy&#160;</td>
		</tr>
		<tr>
			<td>Corning Costar 24-well tissue culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD34 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB72271</td>
			<td>Clone 756510, IgG1, use at 10 &#956;g/ml</td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-human VE-cadherin (CD144)</td>
			<td>R&#38;D Systems</td>
			<td>AF938</td>
			<td>Antigen affinity- purified IgG, use at 1:300</td>
		</tr>
		<tr>
			<td>Monoclonal rabbit anti-human Von Willebrand Factor (vWF)</td>
			<td>Abcam</td>
			<td>ab154193</td>
			<td>Clone EPSISR15, use at 1:250</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
			<td>Polyclonal, 2 mg/ml, use at 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11055</td>
			<td>Polyclonal, 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 568 conjugate</td>
			<td>Life Technologies</td>
			<td>A-10042</td>
			<td>Polyclonal,&#160; 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use at 1 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

generation and culture of blood outgrowth endothelial cells from human peripheral blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

عزل الشهباء الخلايا وحيدة النواة معطف من 60 مل من الدم وعادة ما ينتج 100-150x10 6 مجموع خلايا الدم البيضاء. عندما مطلي في واحدة T-75 قارورة، وعدد كبير من الخلايا في تعليق خلية unlysed يجعل من الصعب حل الخلايا الملتصقة الفردية باستخدام brightfield المجهري. تغييرات المتوسطة المتكررة تؤدي إلى إزالة الخلايا غير ملتصقة وتسمح لتصور من السكان ملتصقة من وحيدي &quot;في وقت مبكر&quot; EPCS. في يوم 7، سوف تحتوي على T-75 حوالي 7x10 6 من هذه الخلايا الملتصقة ممدود. من يوم 7-14، يجب أن تظهر مستعمرات BOECs داخل قارورة (الشكل 2A). لأول مرة تظهر المستعمرات كما 3-5 الخلايا المجاورة. عدد مستعمرة ثمرة يمكن أن تختلف 1-10 المستعمرات في 60 مل من الدم. عموما، والجهات المانحة الأصغر سنا (أي 20-25 سنة) تميل إلى إنتاج عدد أكبر من المستعمرات ثمرة، والتي تظهر أيضا في الثقافة في وقت سابق.BOECs تتكاثر من هذه المجموعة المركزية من الخلايا لتشكل مستعمرات دائرية تتكون من عدة مئات من الخلايا. الخلايا داخل هذه المستعمرات يحمل مورفولوجيا الحصوه البطانية الكلاسيكية. فمن الأفضل أن مرور المستعمرات الأولية عندما كانت تحتوي على حوالي 1000 خلية لكل مستعمرة. وعادة ما تصل المستعمرات ثمرة هذا الحجم بعد 7 أيام المظهر الأصلي في الثقافة. مرة واحدة و passaged المستعمرات الأصلية في جديدة T-75 قارورة، ينبغي لها أن تتكاثر لتشكيل أحادي الطبقة متموجة (3-5x10 6 خلايا في قارورة) خلال 5 أيام أو أقل (الشكل 2B). في نسبة صغيرة من العزلة (&lt;10٪)، تفشل المستعمرات ثمرة لتظهر أو لا تتكاثر بما فيه الكفاية بعد هذه الخطوة الركض الأولية. BOECs التي تظهر انخفاض انتشار بعد أول الركض بها نادرا ما تستمر لتصبح العزلة BOEC مستقرة، وكثيرا ما وقف المتكاثرة في غضون شهرين إلى ثلاثة مقاطع. وحيديEPCS الأولى الواردة في الثقافات BOEC غير قابلة للالتكاثري وعادة ما يتم تطهيرها من الثقافات في غضون 1-2 الممرات. هو إزالة هذه الخلايا في وقت مبكر يرجع إلى موت الخلايا، فشل الخلايا الأولى لإعادة الالتزام بعد الركض والتخفيف من EPCS غير التكاثري في وقت مبكر مع الركض المتكرر. إما أن مرور 1 الخلايا يتم passaged كذلك في الثقافة أو تجميد أسفل لاستخدامها لاحقا. مرة واحدة يتم إنشاء العزلة مستقرة، والخلايا يمكن passaged بمعدل 1 متكدسة قارورة إلى 3-5 جديدة T-75 قوارير حتى مرور 8 أو 9 قبل أن يصبح هادئة. مرة أخرى، كثافة الخلية هي الحاسمة في جميع أنحاء الثقافة. في تجربتنا، ينبغي مطلي ما لا يقل عن 750،000 الخلايا في T-75 قارورة، وأقل كثافة الخلية يمكن أن يسبب توقف نمو. وعلى الرغم من إمكانية التكاثري عالية من BOECs، وينبغي أيضا عدم السماح الخلايا لتصبح overconfluent (أي.،&gt; 5X10 6 خلايا في قارورة) وهذا يمكن أن يسبب CONVERS<html> أيون من BOECs إلى النمط الظاهري غير التكاثري. ونقترح أن أي خلية وصفها بأنها ينبغي أن BOEC عرض سطح الخلية المناسب وتلطيخ الخلايا البطانية للعلامات. لا يمكن أن يتحقق توصيف BOECs التدفق الخلوي (الشكل 3) أو المجهري مضان (الشكل 4)، وبعد تلطيخ لعلامات الخلايا البطانية نموذجية. BOECs إيجابية لCD31 علامات سطح البطانية وVEGFR2 وهي سلبية للCD14 الوحيدات علامة وعموم الكريات البيض علامة CD45. كما posess BOECs الهيئات ويبل-بالادي وبالتالي التعبير عن عامل فون ويلبراند (VWF) كما منفصلة، ​​وتلطيخ حشوية منقط. خلافا لغيرها من أنواع الخلايا البطانية ناضجة، مثل الشريان الرئوي أو الخلايا البطانية الأورطي، BOECs أيضا أن أعرب عن السلف وتفعيل علامة CD34 على سطحها. ftp_upload / 53384 / 53384fig1.jpg &quot;/&gt; الشكل 1. رسم تخطيطي من BOEC بروتوكول جيل. يتم عزل الخلايا وحيدة النواة في الدم المحيطي من الدم الوريدي قبل التدرج الكثافة الطرد المركزي ومثقف على لوحات المغلفة الكولاجين في البطانية المتوسطة النمو التي تحتوي على تعريف FBS. تظهر المستعمرات BOEC في غضون 7-14 أيام للثقافة. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الرقم 2" src="/files/ftp_upload/53384/53384fig2.jpg" /> الشكل 2. صور Brightfield الثقافات BOEC التمثيلية. (A) تظهر المستعمرات ثمرة في الثقافات بين أيام 7 و 14. المستعمرات الحالية إلى مجموعات من الخلايا مثل الخلايا البطانية، التي يتم ترتيبها في أحادي الطبقة المرصوفة بالحصى وتتكاثر شعاعيا الخروج من نقطة مركزية. المحيطة المستعمرات ثمرة هي أحادية تمسكاإختر التصميم الخلايا التي تشكل الغالبية العظمى من الخلايا في الحضارات القديمة. هذه الخلايا، التي سبق وصفها بأنها &quot;في وقت مبكر&quot; الخلايا الاصلية البطانية، لديها التشكل مثل مغزل والتعبير عن علامة CD14 الوحيدات. (B) وبعد الركض، وBOECs التكاثري للغاية تولي الثقافات، والخلايا الوحيدات غير التكاثري إما تموت أو تفشل في إعادة نعلق بعد الركض. مقياس شريط 250μm. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/53384/53384fig3.jpg" /> الشكل 3. توصيف BOECs التدفق الخلوي. تم trypsinized BOECs وملطخة الأجسام المضادة isotype السيطرة fluorescently مترافق (الرمادي شغل الذروة) أو الأجسام المضادة الموجهة ضد علامات سطح معينة (خط أحمر). علامات سطح لتوصيف cytometricوتشمل العلامات المكونة للدم CD45 CD14 و، علامات البطانية CD31 وVEGFR2 وprogentior والبطانية تنشيط علامة CD34. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الرقم 4" src="/files/ftp_upload/53384/53384fig4.jpg" /> الرقم 4. مناعي تلطيخ BOECs للعلامات الخلايا البطانية. الصور المناعي التمثيلية للBOECs immunostained مع الأجسام المضادة الموجهة ضد البطانية علامة سطح الخلية CD144 (VE كادهيرين، أعلى اللوحة اليمنى) وبروتين سكري الدم عامل فون ويلبراند (VWF، لوحة اليمنى السفلى) . وأظهرت المقابلة لوحات تبين تلطيخ دابي النووي إلى اليسار. مقياس شريط 50μm. لCD144. <a href="https://www.jove.com/files/ftp_upload/53384/53384fig4large.jpg" target="_blank">الرجاء انقر هنا لعرض أكبر</a>نسخة من هذا الرقم.

Watch this Scientific Journal Video about Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood at JoVE.com

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

جيل وثقافة الدم ثمرة غشائي خلايا من الدم المحيطي الإنسان

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

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18.8K Views.  University of Cambridge. This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Video: Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

تخصيب خلايا NK من دم الانسان مع مجموعة من تقنيات RosetteSep StemCell

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

العزلة والثقافة في الخلايا البطانية الرئوي من الفئران حديثي الولادة

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

توصيف المظهري والوظيفي للخلايا غشائي مستعمرة تشكيل المستمدة من الإنسان دم الحبل السري

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

جيل من صنع الإنسان من الخلايا الجذعية المحفزة الدموية المحيطية باستخدام Lentiviral STEMCCA المتجهات

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

تنقية الرنا الميكروي والتنميط من Exosomes المشتقة من الدم والثقافة وسائل الإعلام

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

كفاءة الجذع الجيل خلية من الدم عن طريق Episomes وHDAC المانعون

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

التصوير ثنائي الفوتون من الكالسيوم داخل الخلايا<sup&gt; 2+</sup&gt; المناولة واكسيد النيتريك الإنتاج في البطانية وخلايا العضلات الملساء لعزل الجرذ الأبهر

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

البروتينات التحليل المناعي من الداخلية والخارجية السنترومير-الحيز الحركي

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

عزل وتوصيف Microvesicles من الدم المحيطي

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

فحص القص الموحد للصفيحات البشرية ومستقبلات سطح الخلية عبر اللوحة المخروطية

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...