Name: neisseria meningitidis infection of induced pluripotent stem-cell derived brain endothelial cells
Uploaded: 2020-07-14T15:00:00
Description: Watch this Scientific Journal Video about Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells at JoVE.com

L.M.E. è supportato dal programma di formazione alla ricerca DFG GRK2157 dal titolo "3D Tissue Models for Studying Microbial Infections by Human Pathogens" assegnato ad A.S-U. B.J.K. è sostenuto da una borsa di studio post-dottorato della Fondazione Alexander von Humboldt. Inoltre, ringraziamo Lena Wolter per la sua assistenza tecnica nella generazione delle iPSC-BEC in cultura.

NOTA: Tutte le fasi di preparazione del terreno/reagente, del mantenimento delle cellule staminali e della differenziazione sono adattate da Stebbins et al.22.
1. Preparazione dei materiali necessari per la coltura delle iPSC e il differenziamento delle BEC.
<ol><li>Rivestimento della matrice della plastica per coltura tissutale (TC) per coltura iPSC IMR90-4<ol><li>Aliquote di gel di matrice di membrana basale (ad es. Matrigel) in aliquote da 2,5 mg e conservare a -20...

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	<li>Rua, R., McGavern, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Meningeal+Immunity.">Advances in Meningeal Immunity.</a> Trends in Molecular Medicine. 24 (6), 542-559 (2018).</li><li>Abbott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+blood-brain+barrier.">Structure and function of the blood-brain barrier.</a> Neurobiology of Disease. 37 (1), 13-25 (2010).</li><li>Rouphael, N. G., Stephens, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis:+Biology,+microbiology,+and+epidemiology.">Neisseria meningitidis: Biology, microbiology, and epidemiology.</a> Methods in Molecular Biology. , (2012).</li><li>Stephens, D. S., Greenwood, B., Brandtzaeg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemic+meningitis,+meningococcaemia,+and+Neisseria+meningitidis.">Epidemic meningitis, meningococcaemia, and Neisseria meningitidis.</a> Lancet. 369 (9580), 2196-2210 (2007).</li><li>Le Guennec, L., Coureuil, M., Nassif, X., Bourdoulous, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+used+by+bacterial+pathogens+to+cross+the+blood-brain+barrier.">Strategies used by bacterial pathogens to cross the blood-brain barrier.</a> Cellular Microbiology. , (2020).</li><li>Doran, K. 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=Host-pathogen+interactions+in+bacterial+meningitis.">Host-pathogen interactions in bacterial meningitis.</a> Acta Neuropathologica. 131 (2), 185-209 (2016).</li><li>Cunha, C. S. E., Griffiths, N. J., Virji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+opc+invasin+binds+to+the+sulphated+tyrosines+of+activated+vitronectin+to+attach+to+and+invade+human+brain+endothelial+cells.">Neisseria meningitidis opc invasin binds to the sulphated tyrosines of activated vitronectin to attach to and invade human brain endothelial cells.</a> PLoS Pathogens. , (2010).</li><li>Coureuil, 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=Meningococcus+hijacks+a+&#946;2-adrenoceptor/&#946;-arrestin+pathway+to+cross+brain+microvasculature+endothelium.">Meningococcus hijacks a &#946;2-adrenoceptor/&#946;-arrestin pathway to cross brain microvasculature endothelium.</a> Cell. 143 (7), 1149-1160 (2010).</li><li>Bernard, S. 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>

La modellazione dei BEC e della BBB ha avuto delle sfide, poiché i BEC umani primari e immortalizzati, in vitro, tendono a mancare di fenotipi di barriera robusti. L'avvento delle tecnologie delle cellule staminali umane ha permesso la generazione di cellule BEC derivate da iPSC che mantengono i fenotipi tipici della BBB come i marcatori endoteliali, l'espressione della giunzione stretta, le proprietà di barriera, la risposta ad altri tipi di cellule del SNC e i trasportatori funzionali dell'efflusso 12,13,14,...

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

La barriera emato-encefalica (BBB) e la barriera meningea sangue-liquido cerebrospinale (mBCSFB) sono barriere cellulari estremamente strette che separano la circolazione dal sistema nervoso centrale (SNC) e sono costituite principalmente da cellule endoteliali cerebrali (BEC) altamente specializzate1,2. Insieme, i BEC mantengono una corretta omeostasi cerebrale regolando i nutrienti e i prodotti di scarto dentro e fuori il cervello, escludendo molte tossine, farmaci e agenti patogeni 1,2. La meningite batterica si verifica quando i batteri trasmissibili per via ematica sono in grado di interagire e penetrare la barriera formata dai BEC e causare infiammazione. Neisseria meningitidis (Nm, meningococco) è un batterio Gram-negativo che colonizza il rinofaringe del 10-40% degli individui sani, ma in alcuni casi può causare gravi malattie sistemiche3. Negli individui affetti, Nm può ottenere l'accesso al flusso sanguigno dove può causare porpora fulminante e penetrare i BEC ottenendo l'accesso al SNC causando la meningite3. La NM è una delle principali cause di meningite batterica in tutto il mondo e, nonostante gli sforzi di vaccinazione, è ancora una delle cause primarie di meningite4. L'intervento medico moderno, come il trattamento antibiotico, ha reso queste condizioni sopravvissute, tuttavia le persone colpite da meningite spesso rimangono con danni neurologici permanenti 5,6.
Studi precedenti hanno identificato i fattori batterici e la segnalazione dell'ospite che contribuiscono alle interazioni Nm-BEC 7,8,9,10,11. Le adesine e le invasine identificate, come la proteina di opacità Opc e i pili di tipo IV, così come i recettori come CD147, sono stati condotti su vari modelli BEC in vitro, tuttavia questi modelli mancano di molte proprietà di definizione della BBB 7,9,11,12. La comprensione completa delle interazioni Nm-BEC rimane elusiva a causa in parte dell'incapacità di utilizzare modelli in vivo, della protezione vaccinale incompleta e della mancanza di robusti modelli BEC umani in vitro.
La modellazione degli hBEC in vitro è stata impegnativa a causa delle proprietà uniche dei BEC. Rispetto alle cellule endoteliali periferiche, le BEC hanno una serie di fenotipi che migliorano le loro proprietà di barriera, come l'elevata resistenza elettrica trans-endoteliale (TEER) dovuta a complesse giunzioni strette12. Una volta rimossi dal microambiente cerebrale, i BEC perdono rapidamente le loro proprietà di barriera, limitando l'utilità di modelli primari o immortalizzati in vitro che formano solo una debole barriera12,13. La combinazione della specificità umana delle infezioni da NM, la mancanza di robusti modelli in vivo e le sfide che modellano i BEC umani in vitro crea la necessità di modelli migliori per comprendere la complessa interazione ospite-patogeno tra Nm e BAC. Recentemente, utilizzando tecnologie di cellule staminali pluripotenti indotte umane (iPSC) modello, le cellule simili a BEC sono state derivate da iPSC che imitano meglio le BEC in vivo12,13,14,15. Le iPSC-BEC sono di origine umana, facilmente scalabili e possiedono i fenotipi BEC attesi rispetto alle loro controparti primarie o immortalizzate12,13,14,15. Inoltre, noi e altri abbiamo dimostrato che le iPSC-BEC sono utili per modellare varie malattie del SNC come l'interazione ospite-patogeno, la malattia di Huntington e il deficit di MCT8 che causa la sindromedi Allan-Hurndon-Dudley 16,17,18,19,20,21 . Qui, dimostriamo come derivare le iPSC-BEC da fonti rinnovabili di iPSC e l'infezione delle iPSC-BEC con Nm che porta all'attivazione della risposta immunitaria innata. Riteniamo che questo modello sia utile per interrogare l'interazione ospite-patogeno che non può essere ricapitolata in altri modelli in vitro ed è particolarmente utile quando si esaminano le interazioni con patogeni specifici per l'uomo come il Nm.

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		<col />
		<col />
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	</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

La meningite meningococcica è un'infezione pericolosa per la vita che si verifica quando Neisseria meningitidis (meningococco , Nm) può accedere al sistema nervoso centrale (SNC) penetrando nelle cellule endoteliali cerebrali (BEC) altamente specializzate. Poiché Nm è un patogeno specifico per l'uomo, la mancanza di solidi sistemi modello in vivo rende difficile lo studio delle interazioni ospite-patogeno tra Nm e BEC e stabilisce la necessità di un modello basato sull'uomo che imiti i BEC nativi. I BEC possiedono proprietà di barriera più strette rispetto alle cellule endoteliali periferiche caratterizzate da complesse giunzioni strette e da un'elevata resistenza elettrica trans-endoteliale (TEER). Tuttavia, molti modelli in vitro , come i BEC primari e i BEC immortalizzati, mancano o perdono rapidamente le loro proprietà di barriera dopo la rimozione dal microambiente neurale nativo. I recenti progressi nelle tecnologie delle cellule staminali umane hanno sviluppato metodi per derivare cellule endoteliali simili al cervello da cellule staminali pluripotenti indotte (iPSC) che fenopiano meglio le BEC rispetto ad altri modelli umani in vitro . L'uso di BEC derivati da iPSC (IPSC-BEC) per modellare l'interazione Nm-BEC ha il vantaggio di utilizzare cellule umane che possiedono proprietà di barriera BEC e può essere utilizzato per esaminare la distruzione della barriera, l'attivazione immunitaria innata e l'interazione batterica. Qui dimostriamo come derivare le iPSC-BEC dalle iPSC oltre alla preparazione batterica, all'infezione e alla raccolta del campione per l'analisi.

Il protocollo qui descritto è adattato da Stebbins et al. ed evidenzia il processo per differenziare le iPSC in cellule endoteliali simili al cervello che possiedono proprietà BBB e come utilizzare questo modello per gli studi di infezione utilizzando iPSC-BEC con Nm19,22. Le iPSC-BEC, se differenziate correttamente, mostrano proprietà di barriera strette misurate da TEER che sono spesso superiori a 2000 Ω·cm2 ed esprimono marcatori endoteliali co...

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

Il protocollo qui descritto evidenzia i principali passaggi nella differenziazione delle cellule endoteliali cerebrali derivate da cellule staminali pluripotenti indotte, nella preparazione di Neisseria meningitidis per l'infezione e nella raccolta di campioni per altre analisi molecolari.

Meningitidis di Neisseria Infezione di cellule endoteliali cerebrali derivate da cellule staminali pluripotenti indotte

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

This protocol demonstrates the usage of iPSC-derived brain-like endothelial cells to examine host pathogen interactions at the blood-brain barriers, such as the meningeal blood-CSF barriers, in a model that can better mimic brain endothelial cells in vivo. In vitro examination of brain endothelial cell barrier properties and interactions with human-specific pathogens can be challenging due to limitations in modeling. iPSC-derived brain endothelial cells are particularly useful for these applications as they possess superior barrier phenotypes and are of human origin.Maintain the induced pluripotent stem cells, or iPSCs, at 37 degrees Celsius and 5%carbon dioxide in six-well plates with two milliliters of stem cell maintenance medium. To passage the cells, aspirate the medium from a well that is not confluent and add one milliliter of non-enzymatic cell dissociation reagent. Then incubate the plate at 37 degrees Celsius for seven minutes.Meanwhile, replace the matrix gel solution on a new six-well plate with two milliliters of fresh stem cell maintenance medium per well. Aspirate the cell dissociation reagent, taking care not to aspirate cells that are still attached to the plate. Rinse the bottom of the well with six milliliters of stem cell maintenance medium until all cells are completely detached.Seed the new six-well plate with varying densities, typically one to six, or one to 12 for normal maintenance. Shake the plate back and forth and left to right, pausing in between alternating shaking motions until the medium has settled. Then put it in the incubator.Add one milliliter of enzymatic cell dissociation reagent into each well and incubate the plate at 37 degrees Celsius for seven minutes. To deactivate the cell dissociation reagent, transfer one milliliter of the cell suspension into a 15-milliliter tube with at least two milliliters of fresh stem cell maintenance medium. Spin down the cell suspension at 1500 G for five minutes.Then resuspend the cell pellet in one milliliter of stem cell maintenance medium and count the cells with a hemocytometer. Resuspend 7.5 times 10 to the fifth cells in 12 milliliters of stem cell maintenance medium and 10 micromolar rock inhibitor. Aspirate matrix from a T75 flask and transfer the cell suspension to the flask.Shake the flask to distribute the cells and place it in the incubator. Refresh the media every day for the next two days to remove rock inhibitor and support the growth of stem cell colonies. On the third day, begin differentiation by changing the media to an unconditioned medium.Change the medium daily for the next five days. Six days after starting differentiation, selectively expand the endothelial cell population by switching to endothelial cell or EC medium with 20 nanograms per milliliter bFGF and 10 micromolar retinoic acid. Incubate the cells for two days.Prepare collagen IV and fibronectin solution according to manuscript directions, and use it to coat cell culture plates and membrane inserts. Aspirate the EC medium from the cells and add 12 milliliters of cell dissociation reagent. Incubate the flask at 37 degrees Celsius until 90%of the cells have detached.During the incubation time, remove the coating solution from the previously prepared plates and inserts, and let them dry in a sterile hood. Once the cells have detached, use a 10-milliliter pipette to create a single cell suspension. Transfer the cells to a 50-milliliter tube and dilute them with an equal volume of human endothelial serum-free medium.Then count the cells with a hemocytometer. Pellet the cells at 1500 G for 10 minutes. Then resuspend them in freshly prepared EC medium with bFGF and retinoic acid for a concentration of 2 million cells per milliliter.Add 500 microliters of the cell suspension to the top of a 12-well insert and 1.5 milliliters of medium to the bottom. Distribute the cells evenly across the insert and incubate the plate at 37 degrees Celsius and 5%carbon dioxide. On the next day, change the media to EC without bFGF or retinoic acid.Place the epithelial volt/ohm meter in the biosafety hood and connect the electrodes. Disinfect the electrodes by submerging them in 70%ethanol for at least five minutes and let them dry completely. Remove the cells from the incubator and immediately measure the transendothelial electrical resistance, or TEER, by placing the shorter electrode on top of the insert and the longer electrode into the medium surrounding the insert.On the day before the infection experiment, start an overnight culture of the bacteria from frozen stock. Streak Neisseria meningitidis onto Columbia Agar with 5%sheep blood and incubate at 37 degrees Celsius and 5%carbon dioxide overnight. Pathogenic bacteria, such as Neisseria meningitidis, have the potential to cause disease in humans.It's important to properly train individuals working with such microorganisms and always use best practices and wear proper PPE. On the next day, prepare fresh PPM plus media according to manuscript directions and inoculate 10 milliliters of the media with the bacteria and incubate the culture. While the bacteria are incubating, replace the medium in the iPSC-derived brain endothelial cells, then centrifuge the bacterial culture at 4, 000 G for 10 minutes.Aspirate the media and resuspend the pellet in 250 microliters of PBS. Adjust to an OD600 of approximately 0.4, which is roughly one times 10 to the eighth colony-forming units per milliliter. Dilute the bacteria to the desired multiplicity of infection with EC medium and infect the cells with 100 microliters of the prepared bacterial suspension per well.Then incubate the cells for the desired time of infection. When differentiated properly, the iPSC-derived brain endothelial cells exhibit tight barrier properties that are often greater than 2000 ohm centimeter squared and endothelial markers, such as VE-Cadherin and CD-31. Additionally, they express and localize the tight junction markers Claudin-5, Occludin, and ZO-1, as well as transporters, such as Glut-1.Upon infection with Neisseria meningitidis, the cells upregulate neutrophilic, pro-inflammatory cytokines, such as CXCL8, CXCL1, CXCL2, CCL20, and IL6. When performing cell differentiation, daily maintenance and achieving the initial optimal seeding density when splitting the cells is critical. Following the steps outlined here, researchers can collect a variety of samples to investigate many aspects of post-pathogen interaction from inflammatory responses to brain endothelial cell dysfunction.The advent of brain endothelial cells derived from iPSCs have opened the doors for the brain barriers field to investigate a wide range of diseases where endothelial cells are compromised. As certain CNS pathogens are human-specific, we demonstrate the use of a human-based system with the human-specific pathogen Neisseria meningitidis

Research

JoVE Journal

Immunology and Infection

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

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

Visualizzazione e quantificazione delle interazioni intracellulari di Neisseria meningitidis E umano α-actinina da Imaging confocale

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

Ricerca

Immunologia e infezioni

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

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

Selezione di Plasmodium falciparum Parassiti per Cytoadhesion alle cellule cerebrali umane endoteliali

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

Differenziazione Regia di cellule staminali pluripotenti indotte verso Linfociti T

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

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

PRP come un nuovo approccio per prevenire l&#39;infezione: Preparazione e<em&gt; In vitro</em&gt; Proprietà antimicrobiche di PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

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

Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità

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

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

Utilizzando organoidi intestinali derivati da cellule staminali pluripotenti indotta dall'uomo per studiare e modificare la protezione delle cellule epiteliali contro la Salmonella e altri patogeni

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

Differenziazione indotta di cellule M in monostrati Enteroidi Ileal Imbecilli derivati da cellule staminali umane

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

Le cellule staminali derivate dall'ag virale specifico t linfociti sopprimeno la replica HBV nei topi

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

Infezione da pneumococco delle cellule primarie endoteliali nel flusso costante

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

Analisi della permeabilità della barriera sangue-encefalica attraverso l'attraversamento microbico attraverso le cellule endoteliali microvascolari

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 ...