Die Autoren möchten Chantal Cros für hilfreiche technische Unterstützung zu danken. Diese Forschung wurde finanziert durch Zuschüsse aus den "Commissariat À Hang Atomique et Aux alternativen Energien" (URANOs - Programm transversale de Toxicologie du CEA und CPRR CEA-AREVA), und von ANR (Toxizität von Uran: Multi-level-Ansatz der Biomineralisation Prozess in Knochen, ANR-16-CE34-0003). Diese Arbeit wurde auch von der Universität Nizza Sophia-Antipolis und CNRS unterstützt.

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	<li>Keith, S., Faroon, O., Roney, N., Scinicariello, F., Wilbur, S., Ingerman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+profile+for+uranium.">Toxicological profile for uranium.</a> Agency for Toxic Substances and Disease Registry. , (2013).</li><li>Ballou, J. E., Gies, R. A., Case, A. C., Haggard, D. L., Buschbom, R. L., Ryan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+and+early+disposition+of+inhaled+233UO2(NO3)2+and+232UO2(NO3)2+in+the+rat.">Deposition and early disposition of inhaled 233UO2(NO3)2 and 232UO2(NO3)2 in the rat.</a> Health Phys. 51 (6), 755-771 (1986).</li><li>Kathren, R. L., McInroy, J. F., Moore, R. H., Dietert, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteoclast+and+its+unique+cytoskeleton.">The osteoclast and its unique cytoskeleton.</a> Ann N Y Acad Sci. 1240, 14-17 (2011).</li><li>Nesbitt, S. A., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+matrix+collagens+through+bone-resorbing+osteoclasts.">Trafficking of matrix collagens through bone-resorbing osteoclasts.</a> Science. 276 (5310), 266-269 (1997).</li><li>Salo, J., Lehenkari, P., Mulari, M., Metsikko, K., Vaananen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+osteoclast+bone+resorption+products+by+transcytosis.">Removal of osteoclast bone resorption products by transcytosis.</a> Science. 276 (5310), 270-273 (1997).</li><li>Pierrefite-Carle, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+natural+uranium+on+the+UMR-106+osteoblastic+cell+line:+impairment+of+the+autophagic+process+as+an+underlying+mechanism+of+uranium+toxicity.">Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.</a> Arch. Toxicol. 91 (4), 1903-1914 (2017).</li><li>Ubios, A. M., Guglielmotti, M. B., Steimetz, T., Cabrini, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+inhibits+bone+formation+in+physiologic+alveolar+bone+modeling+and+remodeling.">Uranium inhibits bone formation in physiologic alveolar bone modeling and remodeling.</a> Environ. Res. 54 (1), 17-23 (1991).</li><li>Bozal, C. B., Martinez, A. B., Cabrini, R. L., Ubios, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ethane-1-+hydroxy-1,1-bisphosphonate+(EHBP)+on+endochondral+ossification+lesions+induced+by+a+lethal+oral+dose+of+uranyl+nitrate.">Effect of ethane-1- hydroxy-1,1-bisphosphonate (EHBP) on endochondral ossification lesions induced by a lethal oral dose of uranyl nitrate.</a> Arch. Toxicol. 79 (8), 475-481 (2005).</li><li>Kurttio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+as+a+possible+target+of+chemical+toxicity+of+natural+uranium+in+drinking+water.">Bone as a possible target of chemical toxicity of natural uranium in drinking water.</a> Environ. Health Perspect. 113 (1), 68-72 (2005).</li><li>Collin-Osdoby, P., Osdoby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-mediated+osteoclast+formation+from+murine+RAW+264.7+cells.">RANKL-mediated osteoclast formation from murine RAW 264.7 cells.</a> Methods Mol. Biol. 816, 187-202 (2012).</li><li>Beranger, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+poly(ADP-Ribose)+polymerase-1+and+JunD/Fra2+accounts+for+RANKL-induced+Tcirg1+gene+expression+during+osteoclastogenesis.">Differential binding of poly(ADP-Ribose) polymerase-1 and JunD/Fra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesis.</a> J. Bone Miner. Res. 22 (7), 975-983 (2007).</li><li>Mirto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+uranium(VI)+speciation+for+the+evaluation+of+in+vitro+uranium+cytotoxicity+on+LLC-PK1+cells.">Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells.</a> Hum. Exp. Toxicol. 18 (3), 180-187 (1999).</li><li>Carri&#232;re, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+uranium+speciation+on+normal+rat+kidney+(NRK-52E)+proximal+cell+cytotoxicity.">Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.</a> Chem. Res. Toxicol. 17 (3), 446-452 (2004).</li><li>Milgram, S., Carri&#232;re, M., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+accumulation+and+distribution+of+uranium+and+lead+in+osteoblastic+cells+as+a+function+of+their+speciation.">Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.</a> Toxicology. 252 (1-3), 26-32 (2008).</li><li>Milgram, S., Carri&#232;re, M., Thiebault, C., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+and+phenotypic+effects+of+uranium+and+lead+on+osteoblastic+cells+are+highly+dependent+on+metal+speciation.">Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.</a> Toxicology. 250 (1), 62-69 (2008).</li><li>Studzinski, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Growth, Differentiation and Senescence A Practical Approach. , (1999).</li><li>Gritsaenko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+uranium+impairs+the+differentiation+and+the+resorbing+function+of+osteoclasts.">Natural uranium impairs the differentiation and the resorbing function of osteoclasts.</a> Biochim Biophys Acta. 1861 (4), 715-726 (2017).</li><li>Motiur Rahman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation-coupled+osteoclast+differentiation+by+RANKL:+Cell+density+as+a+determinant+of+osteoclast+formation.">Proliferation-coupled osteoclast differentiation by RANKL: Cell density as a determinant of osteoclast formation.</a> Bone. 81, 392-399 (2015).</li></ol>

Die Autoren haben nichts preisgeben

Soweit wir wissen, ist dies das erste Mal, das ein detailliertes Verfahren mit dem Ziel, die Wirkung des natürlichen Urans auf Knochen resorbing Zellen untersuchen beschrieben wird. Dieser Ansatz wird nützlich sein, um ein besseres Verständnis der Uran-Auswirkungen auf die Physiologie des Knochens zu erreichen und bieten ein interessantes neues Tool für das Screening von Uran Chelatoren. Darüber hinaus glauben wir, dass das hier beschriebene Protokoll angewendet werden könnte, um die Auswirkungen von anderen Schwer...

Uran ist ein natürlich vorkommendes radioaktives Element in Böden, Luft und Wasser vorhanden; Tiere und Menschen sind als solche Natururan in ihrer Ernährung ausgesetzt. Zusätzlich zu den natürlichen Quellen stammt Uran aus anthropogenen Aktivitäten erhöht seine Häufigkeit in der Umgebung. Uran stellt chemische und radiologische Gefahren. Jedoch weil natürliches Uran (das ist ein Isotopen Mischung mit 99.27 % 238U 0,72 % 235U und 0,006 % 234U) hat eine niedrige spezifische Aktivität (25.10.3 Bq.g-1), deren Auswirkungen auf die Gesundheit sind zurückzuführen auf seine chemische Toxizität.
Was auch immer seine Eintrag Route (Einatmen, verschlucken oder dermale Exposition), die meisten von Uran in den Körper eindringen mit Kot ausgeschieden ist und nur ein kleiner Teil erreicht den Körperkreislauf. Ca. 67 % des Urans im Blut wird wiederum durch die Nieren gefiltert und verlässt den Körper im Urin innerhalb 24 h1. Der Rest lagert sich vor allem in Nieren und Knochen, die zwei wichtigsten Zielorgane von Uran Toxizität2,3,4. Da das Skelett als der primäre Standort von Uran langfristige Aufbewahrung2,3,4,5,6identifiziert wurde, wurden mehrere Studien durchgeführt, um zu erkunden die Wirkung von Uran auf Knochen Physiologie7.
Knochen ist ein mineralisierten Gewebe, die während seiner Lebensdauer kontinuierlich umgebaut ist. Knochenaufbau ist ein komplexer Prozess, der spezialisierte Zelltypen abhängig und besteht meistens aus zwei Phasen: Resorption von bereits vorhandenen alten Matrix durch Osteoklasten gefolgt von de-Novo-Knochen-Bau von Osteoblasten. Osteoklasten sind große Multikern Zellen, die aus der Verschmelzung von Vorläuferzellen hämatopoetischen Ursprungs, die zur Resorption Seiten wandern wo sie8Knochen zu befestigen. Ihre Befestigung erfolgt gleichzeitig mit eine umfassende Reorganisation der ihre Zytoskelett-9. Diese Reorganisation ist erforderlich für die Errichtung einer isolierten Fach zwischen der Zelle und der Knochenoberfläche, in denen die Osteoklasten Protonen sondert, führt zur Auflösung der Hydroxylapatit und Proteasen, die in den Abbau von, der organische Matrix. Die entstehenden Abbauprodukte sind Endocytosed, transportiert durch die Zelle, die Membranfläche gegenüber der Knochenoberfläche und abgesondert, einen Prozess namens Transcytosis10,11.
Ergebnisse von in Vivo und in Vitro Studien zeigen, dass Uran hemmt die Knochenbildung und die Anzahl und die Aktivität der Osteoblasten7,12 verändert. Im Gegensatz dazu haben die Auswirkungen von Uran auf Knochenabbau und Osteoklasten schlecht erforscht. Mehrere in-Vivo -Studien haben eine Verbesserung der Knochenabbau nach Verabreichung Uranyl Nitrat zu Mäusen oder Ratten13,14berichtet. Darüber hinaus schlug eine epidemiologische Untersuchung, dass die Zunahme der Uran-Aufnahme über das Trinkwasser tendenziell einen Anstieg der Serumspiegel eines Knochen-Resorption-Markers in Männer15zugeordnet werden. Zusammen genommen, diese Erkenntnisse führte zu dem Schluss, dass Uran, die reichert sich in Knochen Knochenabbau fördern könnte. Allerdings bleiben die zellulären Mechanismen beteiligt diese potenzielle Wirkung von Uran eine offene Frage. Aus diesem Grund haben wir beschlossen, die Auswirkungen von Uran auf Verhalten von resorbierbarem Knochenzellen zu untersuchen.
Hier beschreiben wir das Protokoll wir haben zur Charakterisierung und Quantifizierung der Auswirkungen des natürlichen Urans auf Pre Osteoklasten Lebensfähigkeit und Osteoklasten Differenzierung und resorptiven Aktivität. Die hier beschriebenen Experimente wurden durchgeführt mit RAW 264,7 murinen transformierten Makrophagen-Zelllinie, die können leicht unterscheiden in Osteoklasten wenn in Anwesenheit der Zytokin RANKL 4 oder 5 Tage lang kultiviert, und wird klassisch zu studieren Osteoklasten Differenzierung und Funktion16. Die entwickelten Verfahren sind zuverlässig, hoch reproduzierbare Ergebnisse und gelten uneingeschränkt für primäre Osteoklasten zu geben. Aus all diesen Gründen glauben wir, dass diese Methode für ein besseres Verständnis der molekularen Mechanismen der Toxizität von Uran in den Knochen immer nützlich ist. Darüber hinaus denken wir, dass dieser Ansatz als Screening-Instrument zur Identifizierung von neuen Uranium Komplexbildner angepasst werden könnte.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Lonza</td>
			<td>BE12-169F</td>
			<td></td>
		</tr>
		<tr>
			<td>EMEM without phenol red</td>
			<td>Lonza</td>
			<td>12-668E</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for cell culture</td>
			<td>Lonza</td>
			<td>BE17-724F</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum</td>
			<td>GE Life Sciences</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% sodium bicarbonate aqueous solution</td>
			<td>Sigma-Aldrich</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide (MTT) powder</td>
			<td>Sigma-Aldrich</td>
			<td>M5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S sodium salt, 1% w/v aq. sol.</td>
			<td>Alfa Aeros</td>
			<td>42746</td>
			<td></td>
		</tr>
		<tr>
			<td>Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell 24 well plates</td>
			<td>Falcon</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask 75 cm2</td>
			<td>Falcon</td>
			<td>353133</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Conical Tubes 50 ml</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers 30 cm</td>
			<td>TPP</td>
			<td>90003</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for analyzing the impacts of natural uranium on in vitro osteoclastogenesis

Uran hat gezeigt, dass Knochen Physiologie stören und es ist allgemein bekannt, dass dieses Metall in den Knochen ansammelt. Jedoch ist wenig bekannt über die Wirkung des natürlichen Urans auf das Verhalten von Knochenzellen. Insbesondere ist die Wirkung von Uran auf Osteoklasten, die Zellen verantwortlich für die Resorption der Knochenmatrix nicht dokumentiert. Um dieses Problem zu untersuchen, haben wir ein neues Protokoll mit Uranyl Acetat als Quelle des natürlichen Urans und murinen Zelllinie RAW 264,7 als Modell der Osteoklasten-Vorläufer etabliert. Hierin, detaillierte wir alle Tests erforderlich, um Uran Zytotoxizität auf Osteoklasten-Vorläufer zu testen und bewerten die Auswirkungen auf die Osteoclastogenesis und die resorbing Funktion der Reifen Osteoklasten. Die Bedingungen, die wir entwickelt haben, insbesondere für die Herstellung von Nährmedien Uranyl-haltigen und für die Aussaat des rohen 264,7 Zellen ermöglichen zuverlässige und hoch reproduktiver Ergebnisse zu erzielen. Darüber hinaus haben wir den Einsatz von Software-Tools zur Erleichterung der Analysis verschiedener Parameter wie die Größe der Osteoklasten oder den Prozentsatz resorbiert Matrix optimiert.

Tartrat-resistente saure Phosphatase Färbung wurde verwendet, um die Osteoklasten als große lila Zellen mit 3 oder mehr Kerne zu visualisieren. Repräsentative Bilder von Osteoklasten aus rohen 264,7 Zellen kultiviert in Anwesenheit von RANKL und Uranyl Ionen sind in Abbildung 1dargestellt. Änderungen in der Anzahl und Größe der Osteoklasten in Reaktion auf Uran sind gut sichtbar in zusammengesetzte Bilder von ganzen Brunnen und vergrößerte Bilder....

Watch this Scientific Journal Video about Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis at JoVE.com

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uran ist bekannt, Knochenstoffwechsel beeinflussen. Hier präsentieren wir Ihnen ein Protokoll zur Untersuchung der Wirkung von natürlichem Uranexposition auf die Lebensfähigkeit, die Differenzierung und die Funktion der Osteoklasten, die Zellen verantwortlich für Knochenabbau.

Methoden zur Analyse der Auswirkungen des natürlichen Urans auf In-vitro- Osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

methods-for-analyzing-impacts-natural-uranium-on-vitro

Research

JoVE Journal

Developmental Biology

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

8.1K Aufrufe.  Université Nice Sophia-Antipolis. Uran ist bekannt, Knochenstoffwechsel beeinflussen. Hier präsentieren wir Ihnen ein Protokoll zur Untersuchung der Wirkung von natürlichem Uranexposition auf die Lebensfähigkeit, die Differenzierung und die Funktion der Osteoklasten, die Zellen verantwortlich für Knochenabbau.

Serie: Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Herstellung und Verwendung von Lentivirus, um selektiv zu transduzieren Primäre Oligodendrozyten Vorläuferzellen für<em&gt; In-vitro-</em&gt; Myelinisierung Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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Entwicklungsbiologie

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Alginat Encapsulation pluripotenter Stammzellen Mit einer Co-Axial Düsen

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Protokolle für die Analyse der Rolle der Paneth-Zellen bei der Regeneration des Murine Darm mit Conditional<em&gt; Cre-lox</em&gt; Mausmodelle

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Methoden, um die Lymphdrüsen und Blutzellen zu prüfen, die in<em&gt; Drosophila</em&gt; Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Methoden für Imaging intrazellulären pH der Follikel Stem Cell Linie in Live Drosophila Eierstockgewebe

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Anwendung von RNAi und Hitze-Schock-induzierte Transkription Faktor Ausdruck zu programmieren Keimzellen Neuronen in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Methoden für die Untersuchung der Regeneration im Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC Tube-Formation Assay zur Bewertung der Auswirkungen natürlicher Produkte auf die Angiogenese

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Methoden zur Prüfung der endokkrinen Disruption bei Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Isolierung von mesenchymalen Stammzellen des Fettgewebes von Ratten zur Differenzierung in insulinproduzierende Zellen

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...