L.M.E. wird durch das DFG-Graduiertenkolleg GRK2157 "3D-Gewebemodelle zur Untersuchung mikrobieller Infektionen durch menschliche Pathogene" gefördert, das an A. S-U. B.J.K. wird durch ein Postdoktorandenstipendium der Alexander von Humboldt-Stiftung gefördert. Darüber hinaus danken wir Lena Wolter für ihre technische Unterstützung bei der Generierung der iPSC-BECs in Kultur.

<ol>
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C., 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=Pathogenic+Neisseria+meningitidis+utilizes+CD147+for+vascular+colonization.">Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization.</a> Nature Medicine. 20 (7), 725-731 (2014).</li><li>Slanina, H., Hebling, S., Hauck, C. R., Schubert-Unkmeir, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+invasion+by+neisseria+meningitidis+requires+a+functional+interplay+between+the+focal+adhesion+kinase,+Src+and+cortactin.">Cell invasion by neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin.</a> PLoS ONE. 7 (6), (2012).</li><li>Unkmeir, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+Opc-dependent+internalization+of+Neisseria+meningitidis+in+human+brain+microvascular+endothelial+cells.">Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells.</a> Molecular Microbiology. 46 (4), 933-946 (2002).</li><li>Helms, H. C., 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=In+vitro+models+of+the+blood-brain+barrier:+An+overview+of+commonly+used+brain+endothelial+cell+culture+models+and+guidelines+for+their+use.">In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use.</a> Journal of Cerebral Blood Flow and Metabolism. , (2015).</li><li>Lippmann, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+Blood-Brain+Barrier+Endothelial+Cells+from+Human+Pluripotent+Stem+Cells.">Derivation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells.</a> Nature Biotechnology. 30 (8), 783-791 (2012).</li><li>Lippmann, E. S., Al-Ahmad, A., Azarin, S. M., Palecek, S. P., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retinoic+acid-enhanced,+multicellular+human+blood-brain+barrier+model+derived+from+stem+cell+sources.">A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources.</a> Scientific Reports. 4, 4160 (2014).</li><li>Hollmann, E. K., Bailey, A. K., Potharazu, A. V., Neely, M. D., Bowman, A. B., Lippmann, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+differentiation+of+human+induced+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells.">Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.</a> Fluids and Barriers of the CNS. , (2017).</li><li>Kim, B. J., 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=Modeling+Group+B+Streptococcus+and+Blood-Brain+Barrier+Interaction+by+Using+Induced+Pluripotent+Stem+Cell-Derived+Brain+Endothelial+Cells.">Modeling Group B Streptococcus and Blood-Brain Barrier Interaction by Using Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells.</a> mSphere. , (2017).</li><li>Kim, B. J., 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=Streptococcus+agalactiae+disrupts+P-glycoprotein+function+in+brain+endothelial+cells.">Streptococcus agalactiae disrupts P-glycoprotein function in brain endothelial cells.</a> Fluids and Barriers of the CNS. 16 (1), 26 (2019).</li><li>Kim, B. J., Shusta, E. V., Doran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past+and+Current+Perspectives+in+Modeling+Bacteria+and+Blood&#8211;Brain+Barrier+Interactions.">Past and Current Perspectives in Modeling Bacteria and Blood&#8211;Brain Barrier Interactions.</a> Frontiers in Microbiology. , (2019).</li><li>Martins Gomes, S. F., 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=Induced+Pluripotent+Stem+Cell-Derived+Brain+Endothelial+Cells+as+a+Cellular+Model+to+Study+Neisseria+meningitidis+Infection.">Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection.</a> Frontiers in Microbiology. , (2019).</li><li>Vatine, G. D., 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=Modeling+Psychomotor+Retardation+using+iPSCs+from+MCT8-Deficient+Patients+Indicates+a+Prominent+Role+for+the+Blood-Brain+Barrier.">Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier.</a> Cell Stem Cell. , (2016).</li><li>Lim, R. G., 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=Huntington's+Disease+iPSC-Derived+Brain+Microvascular+Endothelial+Cells+Reveal+WNT-Mediated+Angiogenic+and+Blood-Brain+Barrier+Deficits.">Huntington's Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits.</a> Cell Reports. 19 (7), 1365-1377 (2017).</li><li>Stebbins, M. J., Wilson, H. K., Canfield, S. G., Qian, T., Palecek, S. P., Shusta, E. V. <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+characterization+of+human+pluripotent+stem+cell-derived+brain+microvascular+endothelial+cells.">Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.</a> Methods. 101, 93-102 (2016).</li><li>Neal, E. 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=A+Simplified,+Fully+Defined+Differentiation+Scheme+for+Producing+Blood-Brain+Barrier+Endothelial+Cells+from+Human+iPSCs.">A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs.</a> Stem Cell Reports. 12 (6), 1380-1388 (2019).</li><li>Wilson, H. K., Canfield, S. G., Hjortness, M. K., Palecek, S. P., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+effects+of+cell+seeding+density+on+the+differentiation+of+human+pluripotent+stem+cells+to+brain+microvascular+endothelial+cells.">Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells.</a> Fluids and Barriers of the CNS. , (2015).</li><li>Wilson, H. K., Faubion, M. G., Hjortness, M. K., Palecek, S. P., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+brain+endothelial+cells+derived+from+human+induced+pluripotent+stem+cells+is+enhanced+by+rho-associated+coiled+coil-containing+kinase+inhibition.">Cryopreservation of brain endothelial cells derived from human induced pluripotent stem cells is enhanced by rho-associated coiled coil-containing kinase inhibition.</a> Tissue Engineering - Part C: Methods. , (2016).</li><li>Qian, 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=Directed+differentiation+of+human+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells.">Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells.</a> Science Advances. , (2017).</li><li>Sances, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-Derived+Endothelial+Cells+and+Microengineered+Organ-Chip+Enhance+Neuronal+Development.">Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development.</a> Stem Cell Reports. , (2018).</li><li>Canfield, S. G., 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=An+isogenic+blood-brain+barrier+model+comprising+brain+endothelial+cells,+astrocytes,+and+neurons+derived+from+human+induced+pluripotent+stem+cells.">An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.</a> Journal of Neurochemistry. , (2017).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Rho-associated+protein+kinase+(ROCK)+inhibitor+to+human+pluripotent+stem+cells.">Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells.</a> Journal of Bioscience and Bioengineering. , (2012).</li><li>Schubert-Unkmeir, A., Sokolova, O., Panzner, U., Eigenthaler, M., Frosch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+pattern+in+human+brain+endothelial+cells+in+response+to+Neisseria+meningitidis.">Gene expression pattern in human brain endothelial cells in response to Neisseria meningitidis.</a> Infection and Immunity. 75 (2), 899-914 (2007).</li><li>Sokolova, O., 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=Interaction+of+Neisseria+meningitidis+with+human+brain+microvascular+endothelial+cells:+Role+of+MAP-+and+tyrosine+kinases+in+invasion+and+inflammatory+cytokine+release.">Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: Role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release.</a> Cellular Microbiology. 6 (12), 1153-1166 (2004).</li><li>Alimonti, J. B., 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=Zika+virus+crosses+an+in+vitro+human+blood+brain+barrier+model.">Zika virus crosses an in vitro human blood brain barrier model.</a> Fluids and Barriers of the CNS. , (2018).</li><li>Kim, B. J., Schubert-unkmeir, A. <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+Models+for+Studying+the+Interaction+of+Neisseria+meningitidis+with+Human+Brain+Endothelial+Cells.">In vitro Models for Studying the Interaction of Neisseria meningitidis with Human Brain Endothelial Cells.</a> Neisseria meningitidis: Methods and Protocols, Methods in Molecular Biology. 1969, 135-148 (2019).</li><li>Kim, B. J., 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=Bacterial+induction+of+Snail1+contributes+to+blood-brain+barrier+disruption.">Bacterial induction of Snail1 contributes to blood-brain barrier disruption.</a> Journal of Clinical Investigation. 125 (6), 2473-2483 (2015).</li></ol>

Die Autoren haben nichts zu verraten.

Die Modellierung von BECs und der BHS war mit Herausforderungen verbunden, da primäre und immortalisierte humane BECs in vitro dazu neigen, robuste Barrierephänotypen zu vermissen. Das Aufkommen menschlicher Stammzelltechnologien hat die Erzeugung von iPSC-abgeleiteten BEC-ähnlichen Zellen ermöglicht, die die erwarteten charakteristischen BHS-Phänotypen wie Endothelmarker, Tight-Junction-Expression, Barriereeigenschaften, Reaktion auf andere ZNS-Zelltypen und funktionelle Efflux-Transporter beibehalten 12,1...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Larvae</a>. The Authors section was updated from:
Leo M. Endres1 
Alexandra Schubert-Unkmeir1 
Brandon J. Kim1,2 
1Institute for Hygiene and Microbiology, University of W&#252;rzburg 
2Department of Biological Sciences, University of Alabama
to:
Leo M. Endres1 
Sarah F. Hathcock2 
Alexandra Schubert-Unkmeir1 
Brandon J. Kim1,2 
1Institute for Hygiene and Microbiology, University of W&#252;rzburg 
2Department of Biological Sciences, University of Alabama

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Larvae</a>. The Authors section was updated.

Erratum: Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Die Blut-Hirn-Schranke (BHS) und die meningeale Blut-Liquor-Schranke (mBCSFB) sind extrem dichte zelluläre Barrieren, die den Kreislauf vom zentralen Nervensystem (ZNS) trennen und hauptsächlich aus hochspezialisierten Gehirnendothelzellen (BECs) bestehen1,2. Zusammen sorgen BECs für eine ordnungsgemäße Homöostase des Gehirns, indem sie Nährstoffe und Abfallprodukte in und aus dem Gehirn regulieren und gleichzeitig viele Toxine, Medikamente und Krankheitserreger ausschließen 1,2. Eine bakterielle Meningitis tritt auf, wenn durch Blut übertragene Bakterien in der Lage sind, mit der von BECs gebildeten Barriere zu interagieren und diese zu durchdringen und Entzündungen zu verursachen. Neisseria meningitidis (Nm, Meningokokken) ist ein gramnegatives Bakterium, das den Nasenraum von 10 bis 40 % der gesunden Menschen besiedelt, aber in einigen Fällen schwere systemische Erkrankungen verursachen kann3. Bei betroffenen Personen kann Nm Zugang zum Blutkreislauf erhalten, wo es Purpura fulminans verursachen und in BECs eindringen kann, um Zugang zum ZNS zu erhalten, was zu Meningitisführt 3. Nm ist weltweit eine der Hauptursachen für bakterielle Meningitis und trotz Impfbemühungen immer noch eine der Hauptursachen für Meningitis4. Moderne medizinische Eingriffe, wie z. B. die Behandlung mit Antibiotika, haben diese Erkrankungen überlebensfähig gemacht, aber die Betroffenen mit Meningitis bleiben oft mit bleibenden neurologischen Schäden zurück 5,6.
Frühere Studien haben bakterielle Faktoren und Wirtssignale identifiziert, die zu Nm-BEC-Interaktionen beitragen 7,8,9,10,11. Die identifizierten Adhäsine und Invasine wie das Opazitätsprotein Opc und Typ-IV-Pili sowie Rezeptoren wie CD147 wurden an verschiedenen BEC-Modellen in vitro durchgeführt, diesen Modellen fehlen jedoch viele definierende BHS-Eigenschaften7,9,11,12. Ein vollständiges Verständnis der Nm-BEC-Wechselwirkungen ist nach wie vor schwer fassbar, was zum Teil auf die Unfähigkeit, In-vivo-Modelle zu verwenden, den unvollständigen Impfschutz und das Fehlen robuster menschlicher BEC-Modelle in vitro zurückzuführen ist.
Die Modellierung von hBECs in vitro war aufgrund der einzigartigen Eigenschaften von BECs eine Herausforderung. Im Vergleich zu peripheren Endothelzellen weisen BECs eine Reihe von Phänotypen auf, die ihre Barriereeigenschaften verbessern, wie z. B. einen hohen transendothelialen elektrischen Widerstand (TEER) aufgrund komplexer Tight Junctions12. Einmal aus der Mikroumgebung des Gehirns entfernt, verlieren BECs schnell ihre Barriereeigenschaften, was die Nützlichkeit von primären oder immortalisierten In-vitro-Modellen, die nur eine schwache Barriere bilden, einschränkt12,13. Die Kombination aus der menschlichen Spezifität von Nm-Infektionen, dem Mangel an robusten In-vivo-Modellen und den Herausforderungen bei der Modellierung menschlicher BECs in vitro schafft einen Bedarf an besseren Modellen, um die komplexe Wirt-Pathogen-Interaktion zwischen Nm und BECs zu verstehen. In jüngster Zeit wurden BEC-ähnliche Zellen aus iPS-Zellen abgeleitet, die BECs in vivo besser nachahmen 12,13,14,15. iPSC-BECs sind menschlichen Ursprungs, leicht skalierbar und besitzen im Vergleich zu ihren primären oder immortalisierten Gegenstücken die erwarteten BEC-Phänotypen12,13,14,15. Darüber hinaus haben wir und andere gezeigt, dass iPSC-BECs nützlich sind, um verschiedene Erkrankungen des ZNS zu modellieren, wie z.B. Wirt-Pathogen-Interaktion, Chorea Huntington und MCT8-Mangel, der das Allan-Hurndon-Dudley-Syndrom verursacht 16,17,18,19,20,21. In dieser Arbeit zeigen wir, wie iPSC-BECs aus erneuerbaren iPSC-Quellen abgeleitet werden können und wie die Infektion von iPSC-BECs mit Nm zur Aktivierung der angeborenen Immunantwort führt. Wir glauben, dass dieses Modell nützlich ist, um Wirt-Pathogen-Interaktionen zu untersuchen, die in anderen In-vitro-Modellen nicht rekapituliert werden können, und besonders nützlich ist, wenn Interaktionen mit humanspezifischen Krankheitserregern wie Nm untersucht werden.

<table border="0" cellpadding="0" cellspacing="0" style="width:1301px;" width="1300">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Accutase (1x)</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">A6964</td>
			<td style="width:465px;">Enzymatic cell dissociation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Acetic acid</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">A6283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">All-trans retinoic acid (RA)</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-CD31 (PECAM-1)</td>
			<td style="width:216px;">Thermo Scientific (Labvision)</td>
			<td style="width:161px;">RB-10333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-Claudin-5</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">4C3C2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-Glut-1</td>
			<td style="width:216px;">Thermo Scientific (Labvision)</td>
			<td style="width:161px;">SPM498 (MA5-11315)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-Occludin</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">33-1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-VE-cadherin</td>
			<td style="width:216px;">Santa Cruz</td>
			<td style="width:161px;">sc-52751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-ZO-1</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">33-9100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Proteose Peptone</td>
			<td style="width:216px;">BD</td>
			<td style="width:161px;">211684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Mercaptoethanol</td>
			<td style="width:216px;">Merck (Sigma-Aldrich)</td>
			<td style="width:161px;">805740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Cell culture plates and flasks</td>
			<td style="width:216px;">Sarstedt</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Centrifuge (Heraeus Megafuge 1.0R)</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Class II biosafety cabinet</td>
			<td style="width:216px;">Nuaire</td>
			<td style="width:161px;">NU-437-400E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">CO2 Incubator (DHD Autoflow CO2 Air-Jacketed Incubator)</td>
			<td style="width:216px;">Nuaire</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Collagen IV</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">C5533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Columbia ager + 5 % sheep blood</td>
			<td style="width:216px;">Biomerieux</td>
			<td style="width:161px;">43049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Costar Transwell polyester filters (12- or 24-well)</td>
			<td style="width:216px;">Corning</td>
			<td style="width:161px;">3460, 3470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">D(+)-Glucose</td>
			<td style="width:216px;">Merck (Sigma-Aldrich)</td>
			<td style="width:161px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">DAPI</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">DMEM/F12</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">31330-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">DMSO</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">A994.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">21600-069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Epithelial Volt-Ohm Meter (Millicell ERS-2) with STX electrode</td>
			<td style="width:216px;">Merck (Millipore)</td>
			<td style="width:161px;">MERS00002</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">Fe(NO3)3</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">5632.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Fibronectin</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Fluoresence microscope (Eclipse Ti)</td>
			<td style="width:216px;">Nikon</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Hemacytometer (Neubauer)</td>
			<td style="width:216px;">A. Hartenstein</td>
			<td style="width:161px;">ZK06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Human basic fibroblast growth factor (bFGF)</td>
			<td style="width:216px;">PeproTech</td>
			<td style="width:161px;">100-18B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Human Endothelial Serum Free Medium (hESFM)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">11111-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Inverted microscope (Wilovert)</td>
			<td style="width:216px;">Hund (Will Wetzlar)</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">iPS(IMR90)-4 cells</td>
			<td style="width:216px;">WiCell</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">Kellogg&#39;s supplement</td>
			<td style="width:216px;"></td>
			<td style="width:161px;"></td>
			<td>To prepare 110 ml of Kellogg&#39;s supplement, prepare 100 ml of 4 g/ml glucose, 0.1 g/ml glutamine, and 0.2 mg/ml thiamine pyrophosphate and 10 ml of 5 mg/ml Fe(NO3)3 and combine the solutions. Filter sterilize and store aliquoted at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Knockout serum replacement (KOSR)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">L-glutamine (GlutaMAX)</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LunaScript RT SuperMix Kit</td>
			<td style="width:216px;">NEB</td>
			<td style="width:161px;">E3010L</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Matrigel Matrix</td>
			<td style="width:216px;">Corning</td>
			<td style="width:161px;">354230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Methanol</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">4627.5</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">KK36.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Micropipettes (Research Plus)</td>
			<td style="width:216px;">Eppendorf</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">6329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Nonessential amino acids (NEAA)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucleoSpin RNA isolation kit</td>
			<td>Machery-Nagel</td>
			<td style="width:161px;">740955</td>
			<td>RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Pipette boy (Accu-Jet Pro)</td>
			<td style="width:216px;">Brand</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Platelet poor plasma-derived serum, bovine (PDS)</td>
			<td style="width:216px;">Fisher</td>
			<td style="width:161px;">50-443-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PowerUp SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td style="width:161px;">A25742</td>
			<td>qPCR master mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">qPCR film (MicroAmp Optical Adhesive Film)</td>
			<td style="width:216px;">Applied Biosystems</td>
			<td style="width:161px;">4211971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">qPCR plates (MicroAmp Fast 96-well)</td>
			<td style="width:216px;">Applied Biosystems</td>
			<td style="width:161px;">4346907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">ROCK inhibitor, Y27632 dihydrochloride</td>
			<td style="width:216px;">Tocris</td>
			<td style="width:161px;">1254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">RT-PCR thermo cycler (StepOnePlus)</td>
			<td style="width:216px;">Applied Biosystems</td>
			<td style="width:161px;">4376600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Serological pipettes</td>
			<td style="width:216px;">Sarstedt</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">StemFlex basal medium + 50x StemFlex supplement</td>
			<td style="width:216px;">Gibco</td>
			<td>A3349401</td>
			<td>Stem-cell maintenance medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Swinging Bucket Rotor (Heraeus #2704)</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Thiamine pyrophosphate</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">C8754-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Versene</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">15040-033</td>
			<td>Non-enzymatic cell dissociation reagent (EDTA)</td>
		</tr>
	</tbody>
</table>

neisseria meningitidis infection of induced pluripotent stem-cell derived brain endothelial cells

Meningokokken-Meningitis ist eine lebensbedrohliche Infektion, die auftritt, wenn Neisseria meningitidis (Meningokokken, Nm) Zugang zum zentralen Nervensystem (ZNS) erlangen kann, indem sie in hochspezialisierte Gehirnendothelzellen (BECs) eindringt. Da es sich bei Nm um einen humanspezifischen Krankheitserreger handelt, macht der Mangel an robusten In-vivo-Modellsystemen die Untersuchung der Wirt-Pathogen-Interaktionen zwischen Nm und BECs schwierig und begründet die Notwendigkeit eines humanbasierten Modells, das native BECs nachahmt. BECs besitzen im Vergleich zu peripheren Endothelzellen, die durch komplexe Tight Junctions und einen erhöhten transendothelialen elektrischen Widerstand (TEER) gekennzeichnet sind, engere Barriereeigenschaften. Viele In-vitro-Modelle , wie z. B. primäre BECs und immortalisierte BECs, fehlen jedoch entweder oder verlieren schnell ihre Barriereeigenschaften, nachdem sie aus der nativen neuronalen Mikroumgebung entfernt wurden. Jüngste Fortschritte in der humanen Stammzelltechnologie haben Methoden zur Ableitung gehirnähnlicher Endothelzellen aus induzierten pluripotenten Stammzellen (iPS-Zellen) entwickelt, die BECs im Vergleich zu anderen In-vitro-Humanmodellen besser phänokopieren. Die Verwendung von iPSC-abgeleiteten BECs (iPSC-BECs) zur Modellierung der Nm-BEC-Interaktion hat den Vorteil, dass menschliche Zellen verwendet werden, die BEC-Barriereeigenschaften besitzen, und kann zur Untersuchung der Barrierezerstörung, der Aktivierung des angeborenen Immunsystems und der bakteriellen Interaktion verwendet werden. Hier zeigen wir, wie man iPSC-BECs aus iPS-Zellen ableiten kann, zusätzlich zur bakteriellen Vorbereitung, Infektion und Probenentnahme für die Analyse.

Das hier beschriebene Protokoll wurde von Stebbins et al. adaptiert und beleuchtet den Prozess zur Differenzierung von iPS-Zellen in gehirnähnliche Endothelzellen, die BHS-Eigenschaften besitzen, und wie dieses Modell für Infektionsstudien mit iPSC-BECs mit Nm19,22 verwendet werden kann. Die iPSC-BECs weisen bei richtiger Differenzierung enge Barriereeigenschaften auf, die durch TEER gemessen werden und oft größer als 2000 Ω·cm2 sind, und exprimi...

Watch this Scientific Journal Video about Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells at JoVE.com

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Das hier beschriebene Protokoll zeigt die wichtigsten Schritte bei der Differenzierung von induzierten pluripotenten Stammzellen aus gehirnähnlichen Endothelzellen, der Vorbereitung von Neisseria meningitidis auf eine Infektion und der Probenentnahme für andere molekulare Analysen.

Neisseria meningitidis Infektion von induzierten pluripotenten Stammzell-abgeleiteten Gehirnendothelzellen

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

neisseria-meningitidis-infection-induced-pluripotent-stem-cell

Research

JoVE Journal

Immunology and Infection

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

2.3K Aufrufe.  University of Würzburg. Das hier beschriebene Protokoll zeigt die wichtigsten Schritte bei der Differenzierung von induzierten pluripotenten Stammzellen aus gehirnähnlichen Endothelzellen, der Vorbereitung von Neisseria meningitidis auf eine Infektion und der Probenentnahme für andere molekulare Analysen.

Serie: Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Visualisierung und Quantifizierung der intrazellulären Wechselwirkungen von Neisseria meningitidis And Human α-Actinin durch konfokale Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Forschung

Immunologie und Infektion

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Auswahl der Plasmodium falciparum Parasiten für Cytoadhesion zu Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Gerichtete Differenzierung von induzierten pluripotenten Stammzellen in Richtung T-Lymphozyten

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Entwicklung von Stammzellen abgeleiteten Antigen-spezifische regulatorische T-Zellen gegen Autoimmunität

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

Verwendung von humaninduzierten, zellabgeleiteten Intestarz-Organoiden zur Untersuchung und Modifizierung des epithelialen Zellschutzes gegen Salmonellen und andere Krankheitserreger

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

Induzierte Differenzierung von M-Zell-ähnlichen Zellen in menschlichen Stammzell-abgeleiteten Ileal Enteroid Monolayern

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Stammzell-abgeleitete virale Ag-spezifische T-Lymphozyten unterdrücken HBV-Replikation bei Mäusen

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Pneumokokken-Infektion von primären humanen Endothelzellen im konstanten Fluss

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

Analyse der Durchlässigkeit der Blut-Hirn-Barriere durch mikrobielle Traversale durch mikrovaskuläre Endothelzellen

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolierung von primären Lungenendothelzellen der Maus

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...