Yazarlar, mükemmel teknik destek için pHPL ve Margaretha Frühwirth ve Daniela Thaler sağlamak için Dr. Katharina Schallmoser teşekkür ederim.

A.) ECFC ISOLATON 1. GÜN: <ol><li> Deney başlamadan önce, hücre kültür ortamı Endotel Büyüme Orta-2 (EGM-2) hazırlar. (Sigma, St Louis, MO 500ml Endotel Bazal heparin (1000 U / ml 1 ml) ile Orta-2, 5ml 100x çözüm penisilin / streptomisin Ek <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamin (2 mM nihai konsantrasyonu ) ve 50 ml toplanmış insan trombosit lizatı (pHPL). Ardından 0.2 ml hidrokortizon, 2 ml hFGF-B, 0.5 ml VEGF, 0.5 ml EGF, 0.5 ml IGF-1 ve 0.5 ml askorbik asit (SingleQuots olarak verilmiştir, tüm Lonza, Walkersville, MD eklemek <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience. com /</a> ). Steril süzüntü 0.2 m gözenek boyutu 500 ml Millipore vakum filtresi ile orta. PHPL küçük pıhtıları formları için bir orta ve iki filtreler gerekebilir. Pre-sıcak süzülmüş EGM-2 37 ° C su banyosunda </li><li> Yetişkin insan periferik kan kübital venden 5 ml 6ml koruyucu sodyum heparin kan vacutainer tüp içine çekerek kan örnekleri toplayın. En fazla iki saat içinde kan örnekleri işlemek gerekir. </li><li> Bacalı kap, bir adet 25 ml pipet ve bir laminer akış tezgahı iki adet 5 ml pipetler şişesi, bir adet 75 cm 2 (Costar T75) hazırlayarak başlayın. </li><li> Ön dolgu 15 ml, 25 ml pipet ile önceden ısıtılmış EGM-2 balonun içine desteklenmiş. Şimdi kan flakon ve 5 ml ölçülü balona pipetle 5 ml örnek açın. Ikinci 5 ml pipetle 5 ml ilave EGM-2, boş bir kan şişe ile durulayın. T75 içinde toplam hacim 25 ml. 37 ° ° C,% 5 CO 2 ve% 95 nem, 24 saat için bir kuluçka şişesi ve yer Bacalı-kapağını kapatın . </li></ol> 2. GÜN: <ol><li> Yaklaşık 24 saat sonra (gece boyunca) yapışık olmayan hücrelerin kurtulmak için orta-kan karışımı kaldırmanız gerekir. Laminer akış hazırlamak bu iki 25 ml pipetler ve üç adet 5 ml pipetler için, ön-sıcak 20 ml, bir su banyosunda 37 ° C EGM-2 (önceki gün hazırlanmıştır) ve 30 ml PBS desteklenmiş. </li><li> Inkübatör örnek şişesi çıkarın ve 25 ml pipet ile tüm süpernatant kaldırmak. Herhangi bir potansiyel kolonisi zarar görmesini önlemek için balon yüzeyinde çiziklerin oluşmasına özen gösterin. Diğer bir tarafı hafifçe önceden ısıtılmış PBS ve devirme şişesi 10 ml ekleyerek iç şişesi hücre temas yüzeyi üç kez yıkayın. PBS, hemen her zaman ve atın çıkarın. </li><li> Balona 20 ml taze önceden ısıtılmış EGM-2 ve inkübatör içine yerleştirin. Bu süreç, ayırmak için değil, bağlı hücreler için mümkün olduğunca hızlı ve yumuşak olarak yapılmalıdır. PBS içinde ya da oda sıcaklığında uzun süre için tutulur ECFCs kolayca ayırmak. </li><li> Ekran sistemik her ikinci gün balondaki yoluyla düşük bir büyütme ECFC akıbet için arayan bir ışık mikroskobu kullanılarak iki gün başlayan. Bir koloni koloni konumunu gösteren balonun dış tarafında üst üste işareti bulunduğunda. Flaks kuluçka dışında 5 dakikadan fazla olmaması gerektiğini unutmayın. </li></ol> 5. Gün: <ol><li> Beşinci gününde tohumlama sonra yapışmaz hücreleri çıkarmak için tam bir orta değişim gerçekleştirmek. Bu amaçla ön-sıcak 20 ml EGM-2 37 ° C su banyosu ve iki adet 25 ml pipetler hazırlamak. </li><li> 25 ml pipet kullanarak tam orta çıkarın ve mümkün olan en kısa sürede taze, önceden ısıtılmış takviye EGM-2 ekleyin. Hücreler kurutma önlemek için bir dakika daha fazla orta olmadan bırakılmamalıdır. Inkübatör içine yerleştirin ve şişeyi günlük tarama tekrarlayın. </li><li> Taze takviye EGM-2 haftada iki kez,% 30 orta yerine hücreleri tarafından besleyin. </li></ol> GÜN 14-28: <ol><li> Arnavut kaldırımı taş morfolojisi ile tipik kolonilerin etrafında 12 günde görünmelidir. Her koloninin konumunu belirtmek için balonun üst dış tarafında işaretleyin. Tek hücreleri ayırmak için sürece koloniler 28 gün kadar büyümeye izin verin. Sonra, birincil koloniler, büyüklüğüne göre 14 ile 28 gün arasında hasat edilebilir tespit edilmiştir. </li></ol> B.) ECFC HASAT <ol><li> Pre-sıcak 5 ml tripsin 0.05% / EDTA, 20 ml PBS ve 5ml taze takviye EGM-2. </li><li> Tamamen (herhangi bir potansiyel kolonisi zarar görmesini önlemek için balonun yüzeyine dokunmamaya özen) bir Falcon tüp içine şişeden orta kaldırmak ve 10 ml önceden ısıtılmış PBS ile hücreleri dikkatli bir şekilde yıkayın. </li><li> Inkübatör 5 dakika için 5 ml tripsin / EDTA ve inkübe ekleyin. Toksik hale yana hücreleri fazla 7 dakika tripsin maruz olmamalıdır. Balonun tarafı iyice dokunarak hücreleri ayırmak için yardımcı olur. Korunmuş kullanılan kültür ortamı yaklaşık 10 ml ekleyerek tripsin proteaz aktivitesi Quench. </li><li> 50 ml Falco içine hücre süspansiyonu çıkarınn tüp. Hasat edilen hücre süspansiyonu daha sonra eklenir şişeyi kez 10 ml PBS ile yıkayın. 4 7 dakika için 300 g hücre süspansiyonu Santrifüj ° C hücreleri toplamak ve tripsin kaldırmak için. Süpernatantı atın ve hemen 1 mL PBS içinde hücre pelletini tekrar süspansiyon haline getirin. Buz üzerinde tutun. </li><li> Vallahi bir protokolde R. Ricardo ve K. Phelani tarafından açıklandığı gibi hücre sayısını belirler. 5 </li></ol> C.) ECFC GENLEŞME <ol><li> Ek 500 ml EGM-2) A 1.1 &#39;de tanımlanmıştır. Ön-sıcak - 37 ° C su banyosu. </li><li> Nunc, Naperville, IL; yaklaşık 2500 cm 2 kültür alanı için 11 T220 (225 cm 2) veya 15 T175 (175 cm 2) ya da birinin dört katmanlı hücre fabrikası (CF-4 <a href="http://www.nuncbrand.com/">hazırlamak</a> http://www.nuncbrand <a href="http://www.nuncbrand.com/">com</a> ). Ayrıca iki tatlı havalandırma kapakları ve laminer akış tezgah özel steril huni gerekecektir. </li><li> 100 bir başlangıç ​​hücre yoğunluğu tohum ECFCs için hücre sayısı c / cm 2 hesaplanması gerekmektedir. Biri için CF-4 500 ml taze EGM-2 252.800 hücrelerinin yeniden süspanse ve steril huni kullanarak CF-4 dökülür. Bir havalandırma havalandırma kapağı ile kapatın ve 4 unları arasında orta eşit dağılımını sağlayan, CF-4 bir boylam yan yatırmak. Sonra odasına geri eğme ve havalandırma kapağının kapağı açın. CF-4 inkübatöre yerleştirin. </li><li> CF-4 kadar en az haftada bir kez orta% 20 değiştirin konfluent. </li><li> Hasat hücreler tripsin / EDTA 70 ml ve yaklaşık 200 ml PBS kullanarak) B. açıklanan. </li></ol> TEMSİLCİSİ SONUÇLAR Bu izolasyon yöntemi kullanarak 12 gün az ilkokul koloni oluşumu gözlemledi. Önceki bir çalışmada, ortalama 4.0 ± periferik kan her ml edilen 0,8 ECFC-sömürgelerde kurtarmak için başardık 4 ECFCs tipik bir Arnavut kaldırımı taş görünümü gösterdi . Büyük ölçekli genişleme sonra, ekim yoğunluğu 100 hücre / cm 2, 3 erkek ve 1 kadın gönüllüler (26 ve 50 yılları arasında yaş) yaklaşık 30 gün olmak üzere toplam&gt; 1 x 10 8 hücre elde. Kültür ECFC bu koşullar altında, proliferatif, fonksiyonel ve genomik istikrarlı olması gösterildi 4 Ayrıca hücrelerin daha sonra kullanmak üzere sıvı azot (% 10 DMSO) saklanabilir. 3,4

<ol>
	<li>Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+endothelial+cells+in+sickle+cell+anemia.">Circulating activated endothelial cells in sickle cell anemia.</a> N Engl J Med. 337, 1584-1590 (1997).</li><li>Yoder, M. C., Mead, L. E., Prater, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.</a> Tissue Eng Part C Methods. 14, 185-196 (2008).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. , (2009).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. , (2008).</li></ol>

Bu roman izolasyon ve genişleme yöntemi, daha verimli ve daha az zaman alıcı ve herhangi bir hücre ayırma herhangi bir yoğunluk gradiyenti (gerekli) kaçınarak, geleneksel yöntemlere göre daha daha az pahalı bir basit ve verimli bir süreç. Büyükbaş antijen ve varsayılan sonraki xenoimmunization pHPL temas kullanarak dışındadır. Kültür dönemde küçük pıhtıları pHPL neden oluşturabilir. Tecrübelerimize dayanarak bu pıhtıları koloni oluşumu ve hücre çoğalması veya fonksiyon etkisi yok...

<table><tbody><tr><td>Endothelial basal Medium-2</td><td>Lonza Inc.</td><td></td><td>500ml</td></tr><tr><td>EGM-2 SingleQuots</td><td>Lonza Inc.</td><td></td><td>EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid</td></tr><tr><td>preservative-free Heparin</td><td>Biochrom AG</td><td></td><td>10U/ml per EGM-2</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td></td><td>2mM per EGM-2</td></tr><tr><td>Penicillin and Streptomycin</td><td>Sigma-Aldrich</td><td></td><td>100U/mL Penicillin and&lt;br/&gt;100&amp;#x03BC;g/mL Streptomycin per&lt;br/&gt;EGM-2</td></tr><tr><td>Trypsin/1mM EDTA </td><td>Invitrogen</td><td></td><td>7ml for T75 flask&lt;br/&gt;70ml for CF-4</td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Four-layered cell factory (CF-4) </td><td>Corning</td><td></td><td></td></tr><tr><td>Sterile Funnel</td><td>Corning</td><td></td><td></td></tr><tr><td>T75cm&lt;sup&gt;2&lt;/sup&gt; culture flask (with vented cap)</td><td>Corning</td><td></td><td></td></tr><tr><td>6 mL vacutainer tubes (preservative-free sodium heparin) </td><td>BD Biosciences</td><td></td><td>preloaded with 108 IU/6ml of&lt;br/&gt;preservative-free sodium heparin</td></tr><tr><td>50ml Falcon tubes</td><td>Falcon BD</td><td></td><td></td></tr><tr><td>5ml Stripetts</td><td>Costar</td><td></td><td></td></tr><tr><td>25ml Stripetts</td><td>Costar</td><td></td><td></td></tr><tr><td>Squeezer</td><td>Costar</td><td></td><td></td></tr><tr><td>0,2 pore size sterile filter</td><td>EMD Millipore</td><td></td><td>2x 500ml per EGM-2</td></tr></tbody></table>

isolation and large scale expansion of adult human endothelial colony forming progenitor cells

Bu kağıt heparinize gelen endotel progenitör hücrelerin koloni oluşturan (ECFCs) için yeni bir kurtarma stratejisi tanıttı ama aksi takdirde ortalama 12 gün içinde yetişkin insan periferik kan unmanipulated. &gt; 1x10 8 ECFCs büyük ölçekli bir genişleme sonra, daha ileri testler için elde edilebilir. Avantajlı pHPL ile sığır serum antijenleri ile insan hücrelerinin kişi hariç tutulabilir. Akım sitometri ve immünohistokimyasal olarak izole hücreler ECFC ve in vitro işlevselliği gibi vasküler yapılar oluşturmak için bir matrigel tayininde test edilebilir olarak karakterize edilebilir . Ayrıca bu hücrelerin subkutan bir fare modelinde in vivo olarak fonksiyonel, perfüze damarları oluşturmak için enjekte edilebilir. Uzun vadeli büyüme ve kriyoprezervasyon çoğalması sonra, fonksiyon ve genomik istikrar korunmuş gibi görünüyor. 3,4 Bu hayvansal protein izolasyonu ve genişleme yöntemi ECFCs bir büyük miktarda üretmek için kolay uygulanabilir.

Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Endotel progenitör hücrelerin koloni oluşturan (ECFCs) vasküler homeostazı ve onarım çalışması için bir gelecek vaat eden bir araçtır.<sup&gt; 1,2</sup&gt; Bu kağıt, izolasyon ve anti-sığır aşılama riski azalan toplanmış insan trombosit lizatı (pHPL) heparinli yetişkin insan periferik kan ECFC genişlemesi için bir roman hayvan-serum ücretsiz bir yöntem.

İzolasyon ve Büyük Ölçekli Genişleme Yetişkin İnsan Endotel Koloni Oluşturan Progenitör Hücreler

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

isolation-large-scale-expansion-adult-human-endothelial-colony

Research

JoVE Journal

Biology

10.7K Görüntüleme.  Medical University of Graz, Austria. Endotel progenitör hücrelerin koloni oluşturan (ECFCs) vasküler homeostazı ve onarım çalışması için bir gelecek vaat eden bir araçtır. 1,2 Bu kağıt, izolasyon ve anti-sığır aşılama riski azalan toplanmış insan trombosit lizatı (pHPL) heparinli yetişkin insan periferik kan ECFC genişlemesi için bir roman hayvan-serum ücretsiz bir yöntem.

Video: Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Yalıtım Normal kolon ve kolorektal Karsinom - gelişmiş bir iletişim kuralı üzerinden insan endotel hücrelerinin

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Kanser Araştırmaları

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Yalıtım endotelyal Progenitor Hücre insan göbek kordon kanı

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Gelişim Biyolojisi

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

İnsan Göbek kordonu İzolasyon ve Hayvan Serum Genişleme Mezenkimal stromal hücreler (MKH) ve endotel Koloni Oluşturan Progenitör Hücreler (ECFCs) Türetilmiş

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Biyoloji

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Endotel Kolonisi Oluşturan Hücreler ve Mezenkimal Kök Hücreler tarafından Oluşturulan Eş Kültür Sfaoidler Kullanılarak Üç Boyutlu Anjiyogenez Tsay Sistemi

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Hemojenik Endotel Hücrelerinin İnsan Pluripotent Kök Hücrelerinden Yönlendirilmiş Farklılaşması

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Tıp

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Kalp Damar Cerrahisi sonra serum Örnekleri etkilenerek Sağlıklı Gönüllüler ve Bunların Göçmen Potansiyelden Endotel Ata Hücreleri İzolasyon

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

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.

Endotel Colony Forming Hücrelerinin Fenotipik ve Fonksiyonel Karakterizasyonu İnsan Göbek Kordonu Kanı türetilen

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

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Embriyonik Ventriküler Endotel Hücre İzolasyonu

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

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.

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.