Die Autoren bedanken sich bei Dr. Klaus Kaestner für das Geschenk des murinen Ngn3-EGFP embryonale Stammzell-Linie, Mrs. Lucy Brown und die Analytische Cytometry Core-Unterstützung bei der Zellsortierung danken. Diese Arbeit wurde zum Teil durch NIH gewährt R21DK069997 und R01DK081587 verliehen HTK und vom California Institute for Regenerative Medicine (CIRM) Ausbildung Zuschüsse, MW-und LS-finanziert

<ol>
	<li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Committing+embryonic+stem+cells+to+early+endocrine+pancreas+in+vitro.">Committing embryonic stem cells to early endocrine pancreas in vitro.</a> Stem cells. 22, 1205-1217 (2004).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-expressing+colonies+developed+from+murine+embryonic+stem+cell-derived+progenitors.">Insulin-expressing colonies developed from murine embryonic stem cell-derived progenitors.</a> Diabetes. 56, 921-929 (2007).</li><li>Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+in+vitro+differentiation+assay+for+pancreatic-like+cell+development+from+murine+embryonic+stem+cells:+detailed+gene+expression+analysis.">Characterization of an in vitro differentiation assay for pancreatic-like cell development from murine embryonic stem cells: detailed gene expression analysis.</a> Assay. Drug. Dev. Technol. 9, 403-419 (2011).</li><li>Lee, C. S., Perreault, N., Brestelli, J. E., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin+3+is+essential+for+the+proper+specification+of+gastric+enteroendocrine+cells+and+the+maintenance+of+gastric+epithelial+cell+identity.">Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity.</a> Genes. 16, 1488-1497 (2002).</li><li>Gu, G., Dubauskaite, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+pancreatic+lineage:+NGN3++cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors.">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129, 2447-2457 (2002).</li><li>Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+proliferation+of+hematopoietic+stem+cells.">Differentiation and proliferation of hematopoietic stem cells.</a> Blood. 81, 2844-2853 (1993).</li><li>Gittes, G. K., Galante, P. E., Hanahan, D., Rutter, W. J., Debase, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-specific+morphogenesis+in+the+developing+pancreas:+role+of+mesenchymal+factors.">Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors.</a> Development. 122, 439-447 (1996).</li><li>Miralles, F., Battelino, T., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+plays+a+key+role+in+morphogenesis+of+the+pancreatic+islets+of+Langerhans+by+controlling+the+activity+of+the+matrix+metalloproteinase+MMP-2.">TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.</a> J. Cell. Biol. 143, 827-836 (1998).</li><li>Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cultivation+of+human+islets+from+expanded+ductal+tissue.">In vitro cultivation of human islets from expanded ductal tissue.</a> Proc. Natl. Acad. Sci. U. S. A. 97, 7999-8004 (2000).</li><li>Gao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+endocrine+progenitor+cells+and+critical+factors+for+their+differentiation+in+human+adult+pancreatic+cell+culture.">Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.</a> Diabetes. 52, 2007-2015 (2003).</li><li>Gao, R., Ustinov, J., Korsgren, O., Otonkoski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neogenesis+of+human+islets+reflects+the+plasticity+of+differentiated+human+pancreatic+cells.">In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.</a> Diabetologia. 48, 2296-2304 (2005).</li><li>Suarez-Pinzon, W. L., Lakey, J. R., Brand, S. J., Rabinovitch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+with+epidermal+growth+factor+and+gastrin+induces+neogenesis+of+human+islet+beta-cells+from+pancreatic+duct+cells+and+an+increase+in+functional+beta-cell+mass.">Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet beta-cells from pancreatic duct cells and an increase in functional beta-cell mass.</a> J. Clin. Endocrinol. Metab. 90, 3401-3409 (2005).</li><li>Hao, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+differentiation+from+nonendocrine+epithelial+cells+of+the+adult+human+pancreas.">Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas.</a> Nature Medicine. 12, 310-316 (2006).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+pancreatic+progenitor+cells--recent+studies.">Minireview: pancreatic progenitor cells--recent studies.</a> Endocrinology. 149, 4312-4316 (2008).</li><li>Seaberg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nature Biotechnology. 22, 1115-1124 (2004).</li><li>Rovira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+centroacinar/terminal+ductal+progenitor+cells+in+adult+mouse+pancreas.">Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 75-80 (2010).</li></ol>

Wir haben nichts zu offenbaren.

Colony Assays, die quantitative und funktionale Bewertung der einzelnen Pankreas-Vorläuferzellen ermöglichen wurden bisher begrenzt, die stark die Fortschritte in den Studien von Pankreas-Stamm-und Vorläuferzellen Zellbiologie behindert. Das Wesen einer Kolonie Assay ist, dass sie die intrinsische Fähigkeit von Vorläuferzellen, anstatt ausschließlich deren Phänotyp Maßnahmen, indem sie die Funktionalität und die Linie Potenziale der Vorläuferzellen zu beachten. Es ist auch ein quantitatives Werkzeug, dass die Zahl der Vorfahren in einer bestimmten Population bestimmen können. In der hämatopoetischen Stammzelltransplantation Bereich haben Kolonie-Assays eine wichtige Rolle bei der Identifizierung spielte, sowie die Entschlüsselung der Wachstumsfaktor Anforderungen, Wechselwirkungen und Mechanismen, die die Linie Engagement dieser Zellen. 6 In dem Gebiet der Pankreas-Vorläuferzellen Zellbiologie, Bauchspeicheldrüsen-Orgel 7 , 8 oder Bulk-Kulturen 9-13 entwickelt worden, aber solche Tests nicht auf biologische questions in der einzelnen Zelle Ebene. Ohne einzelne Zelle analysiert, kann die Existenz von mehreren möglichen Bauchspeicheldrüse Stammzellen / Vorläuferzellen nicht zweifelsfrei nachgewiesen werden. 14 Hier zeigen wir, dass Insulin-exprimierenden koloniebildenden Vorläuferzellen können untersucht unter Verwendung einer dreidimensionalen, Matrigel-haltigen, halbfeste Kultur-System. Obwohl eine Suspensionskultur System 15,16 entwickelt worden ist, die es erlaubt einzelne erwachsene Bauchspeicheldrüse Vorläuferzellen zur Proliferation und Differenzierung in &quot;pancreatospheres&quot; bietet unsere Kolonie Assay weitere Vorteile. Erstens ermöglicht es unseren halbfesten Kulturmedien System Beschichtung von bis zu 25.000 sortierten Zellen pro 500 ul Kulturmedium pro 24-well und gleichzeitig die daraus resultierenden Koloniebildung im linearen Bereich. 2 Im Gegensatz, die Aussetzung pancreatosphere Test die Verwendung einzelner erfordert Zellablagerungsvorrichtung Methode aus der Zell-Sorter, damit einzelne Zelle analysiert, und somit ein größeres Volumen von Kulturmedien und Platz im Inkubatorerforderlich sind. Deshalb ist unsere Kolonie Assay relativ einfach und kostengünstig durchzuführen, wenn eine große Anzahl von verschiedenen Zellen gleichzeitig für Vorläuferzellen Aktivitäten verglichen werden müssen. Zweitens ermöglicht es unseren halbfesten Medien gleichmäßige Verteilung der Komponenten der extrazellulären Matrix und Wachstumsfaktoren, die sowohl die Proliferation und Differenzierung in der gleichen auch auftreten können. Im Gegensatz dazu ist es schwieriger, eine solche Umgebung, vor allem die Matrix, die Zellen in Suspension Kultur. Wir sind uns bewusst, dass wir mit unserer Methode ist es nicht endgültig bewiesen werden, dass jede Kolonie aus einer einzigen Zelle abgeleitet ist, als zwei Vorläufern kann zufällig nahe beieinander und bilden eine Kolonie positioniert werden. Allerdings kann dieses Problem durch die sekundäre Analyse mit einzelnen Zelle Manipulation überwunden werden. Direkte Handlese der sortierten Einzelzellen konnte endgültig zeigen die einzelne Zelle Ursprung jeder Kolonie. Zusammenfassend ist der Insulin-exprimierenden koloniebildenden Aktivität progenitors in halbfesten Medien hierin beschriebenen bietet einzigartige Möglichkeiten für mechanistische Untersuchungen von Pankreas-Vorläuferzellen an der einzelnen Zelle Ebene. Zum Beispiel, in einem früheren Bericht haben wir festgestellt, dass neben der Matrigel, eine krude Mischung aus Proteine ​​der extrazellulären Matrix, in die Kulturmedien für die Bildung des Insulin-exprimierenden Kolonien von Ngn3-EGFP +-Zellen, 2 was auf die extrazelluläre Matrix wird benötigt wichtig für die Vorfahren. Mit dieser Kolonie Assay, wird es nun möglich sein, weitere sezieren und definieren spezifische Matrix Anforderungen für eine solche Tätigkeit. Neben dem Studium Vorläuferzellen aus embryonalen Stammzellen der Maus gewonnen, untersuchen wir derzeit, ob diese in-vitro-Assay-System unterstützt auch die Bildung von anderen charakteristischen Kolonien aus der Bauchspeicheldrüse und der Leber von perinatalen und erwachsenen Mäusen stammen, sowie von menschlichen Leichen Pankreasgewebe ohne von Inselchen.

<table border="1"><tbody><tr><td> Material / Reagent </td><td> Firma </td><td> Katalog Anzahl </td><td> Kommentare </td></tr><tr><td> 1X DMEM / F-12 (1:1) </td><td> Mediatech </td><td> 15 bis 090-CV </td><td></td></tr><tr><td> Fetal Calf Serum </td><td> Tissue Culture Biologicals </td><td> 201 </td><td> Siehe Abschnitt 1,1 </td></tr><tr><td> L-Glutamin </td><td> Gibco </td><td> 25030-081 </td><td></td></tr><tr><td> 1X Dulbecco PBS </td><td> Mediatech </td><td> 21 bis 031-CV </td><td></td></tr><tr><td> Bovine Serum Albumin </td><td> Sigma </td><td> A8412 </td><td> 7,5% </td></tr><tr><td> Matrigel </td><td> Becton Dickinson </td><td> 356231 </td><td> Reduziertes Wachstum Faktor </td></tr><tr><td> Methylcellulose </td><td> Sinetsu Chemical, Tokyo, Japan </td><td></td><td> 1.500 centipoise (High-viscosity) </td></tr><tr><td> Nicotinamid </td><td> Sigma </td><td> N0636 </td><td></td></tr><tr><td> Mensch Rekombinante Activin βB </td><td> R &amp; D Systems </td><td> 659-AB-025 </td><td></td></tr><tr><td> Exendin-4 </td><td> Sigma </td><td> E7144 </td><td></td></tr><tr><td> Mensch Rekombinante VEGF-A </td><td> R &amp; D Systems </td><td> 293-VE-010 </td><td></td></tr><tr><td> 24-Well-Platten </td><td> Costar </td><td> 3473 </td><td> Ultra-low Befestigung </td></tr><tr><td> 60mm Petrischale mit Grid </td><td> Nunc </td><td> 169558 </td><td></td></tr></tbody></table>

a quantitative assay for insulin-expressing colony-forming progenitors

Das Gebiet der Pankreas-Stamm-und Vorläuferzellen der Zellbiologie hat durch einen Mangel an in vitro funktionale und quantitative Tests, die für die Analyse der einzelnen Zelle ermöglichen behindert. Analysen der einzelnen Vorfahren sind von entscheidender Bedeutung, weil sie definitive Möglichkeiten, eindeutig beweisen die Abstammung Potenzial der einzelnen Vorfahren geben. Obwohl Methoden entwickelt worden, um &quot;pancreatospheres&quot; in Suspensionskultur aus einzelnen Zellen zu generieren, gibt es einige Einschränkungen. Erstens ist es zeitaufwendig, einzelne Zelle Deposition für eine große Zahl von Zellen, was wiederum Befehle große Mengen von Nährmedien und Raum. Durchführen Zweitens, Nummerierung der resultierenden pancreatospheres ist arbeitsintensiv, vor allem, wenn die Frequenz des pancreatosphere-Einleitung Vorfahren niedrig ist. Drittens ist die pancreatosphere Assay keine effiziente Methode, um damit sowohl die Proliferation und Differenzierung von Vorläuferzellen der Bauchspeicheldrüse in der gleichen Kultur gut, restricting die Nützlichkeit des Tests. Um diese Einschränkungen zu überwinden, hat eine halbfeste Medien basieren Kolonie Assay für Pankreas-Vorläuferzellen entwickelt worden und wird in diesem Bericht vorgestellt. Diese Methode nutzt eine bestehende Konzept von der hämatopoetischen Kolonie-Assay, in dem Methylcellulose verwendet, um die Viskosität zu den Medien geben wird, so dass die Vorläuferzellen in drei-dimensionalen Raum bleiben, wie sie die Proliferation sowie Differenzierung zu unterziehen. Zur Bereicherung Insulin-exprimierenden koloniebildenden Vorläuferzellen aus einer heterogenen Bevölkerung, verwendeten wir Zellen, die Express Neurogenin (NGN) 3, ein Pankreas endokrine Vorläuferzellen Zell-Marker. Murine embryonale Stammzellen (ES)-Zellen abgeleiteten Ngn3 exprimierenden Zellen mit dem Enhanced Green Fluorescent Protein Reporter getaggt wurden sortiert und bis zu 25.000 Zellen pro Vertiefung ausplattiert in low-Anlage 24-Well-Kulturschalen. Jedes Well enthielt 500 ul von halbfesten Medien mit den folgenden Hauptkomponenten: methylcellulose, Matrigel, Nicotinamid, Exendin-4, Activin βB und konditionierten Medien von murinen ES-Zellen abgeleiteten Pankreas-ähnlichen Zellen gesammelt. Nach 8 bis 12 Tagen Kultur, Insulin-exprimierenden Kolonien mit ausgeprägter Morphologie wurden gebildet und könnte weiter für Pankreas-Genexpression mittels quantitativer RT-PCR und immunoflourescent Färbung der Linie Zusammensetzung der einzelnen Kolonie bestimmen analysiert werden. Zusammenfassend können unsere Kolonie Assay leicht Detektion und Quantifizierung von funktionellen Vorfahren innerhalb einer heterogenen Population von Zellen. Darüber hinaus ermöglicht der halbfesten Medien-Format einheitliche Darstellung der Komponenten der extrazellulären Matrix und Wachstumsfaktoren auf Zellen, so dass Vorläuferzellen zu vermehren und in vitro zu differenzieren. Diese Kolonie Assay bietet einzigartige Möglichkeiten für mechanistische Untersuchungen von Pankreas-Vorläuferzellen an der einzelnen Zelle Ebene.

Watch this Scientific Journal Video about A Quantitative Assay for Insulin-expressing Colony-forming Progenitors at JoVE.com

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Eine dreidimensionale klonogene Test, dass Pankreas-like Vorläuferzellen in Insulin-exprimierenden Kolonien unterscheiden können beschrieben. Diese Methode nutzt die Vorteile von halbfesten Medien mit Methylcellulose, Matrigel und Wachstumsfaktoren, in denen einzelne Vorläuferzellen proliferieren und differenzieren<em&gt; In-vitro-</em&gt;, Erlauben die Quantifizierung der Anzahl der funktionellen Vorfahren in einer Population.

A Quantitative Assay für Insulin-exprimierenden Koloniebildende Vorläufern

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

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13.4K Aufrufe.  California State University Channel Islands. Eine dreidimensionale klonogene Test, dass Pankreas-like Vorläuferzellen in Insulin-exprimierenden Kolonien unterscheiden können beschrieben. Diese Methode nutzt die Vorteile von halbfesten Medien mit Methylcellulose, Matrigel und Wachstumsfaktoren, in denen einzelne Vorläuferzellen proliferieren und differenzieren In-vitro-, Erlauben die Quantifizierung der Anzahl der funktionellen Vorfahren in einer Population.

Serie: A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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Biologie

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolation und großräumige Ausdehnung der erwachsenen Menschen Endothelial Colony Forming Progenitorzellen

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

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.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

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

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

Identifikation und Analyse von Maus Erythroid Vorläufern mit dem CD71/TER119 Durchflusszytometrische Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

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.

Phänotypische und funktionelle Charakterisierung von Endothelial Kolonie-bildenden Zellen aus humanem Nabelschnurblut

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

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

A Quantitative Analysis Fitness-Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Ein hoher Durchsatz MHC II-Bindungsassay für die quantitative Analyse von Peptid-Epitope

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Eine Strategie für einfühlsam, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Immunfluoreszenz-Assay, um die Variation der Protein-Ebene an Zentrosomen Messen

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Entwicklung eines Quantitative Rekombinase Polymerase Amplification Assay mit einem internen positiven Kontrolle

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...