פרויקט זה מומן בחלקו תחת מענקים מהמוסד הלאומי לבריאות (R01AI121180, R21AI109239 ו K18CA151798), ה- American Diabetes Association (1-16-IBS-281), ואת מחלקת פנסילבניה הבריאות (קופות להתיישבות טבק) כדי JS

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+concepts+of+rheumatoid+arthritis.">Evolving concepts of rheumatoid arthritis.</a> Nature. 423, 356-361 (2003).</li><li>Ferraro, 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=Interindividual+variation+in+human+T+regulatory+cells.">Interindividual variation in human T regulatory cells.</a> Proc Natl Acad Sci U S A. 111, E1111-E1120 (2014).</li><li>Tang, Q., 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-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J Exp Med. 199, 1455-1465 (2004).</li><li>van Herwijnen, M. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry:+paraffin+sections+using+the+Vectastain+ABC+kit+from+vector+labs.">Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs.</a> J Vis Exp. , (2007).</li><li>Lu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+all-trans+retinoic+acid+in+stabilizing+human+natural+regulatory+T+cells+under+inflammatory+conditions.">Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions.</a> Proc Natl Acad Sci U S A. 111, E3432-E3440 (2014).</li><li>Wu, 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=Galectin-9-CD44+interaction+enhances+stability+and+function+of+adaptive+regulatory+T+cells.">Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells.</a> Immunity. 41, 270-282 (2014).</li><li>Di Stasi, 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=Inducible+apoptosis+as+a+safety+switch+for+adoptive+cell+therapy.">Inducible apoptosis as a safety switch for adoptive cell therapy.</a> N Engl J Med. 365, 1673-1683 (2011).</li><li>Ramos, C. 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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=Mixed+T+cell+receptor+dimers+harbor+potentially+harmful+neoreactivity.">Mixed T cell receptor dimers harbor potentially harmful neoreactivity.</a> Proc Natl Acad Sci U S A. 107, 10972-10977 (2010).</li><li>Kim, Y. 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=Engineered+antigen-specific+human+regulatory+T+cells:+immunosuppression+of+FVIII-specific+T-+and+B-cell+responses.">Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses.</a> Blood. 125, 1107-1115 (2015).</li><li>Himburg, H. 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The authors have nothing to disclose.

בפרוטוקול זה, צעד קריטי הוא בידול במבחנה של iPSCs TCR / FoxP3-transduced הגן. איתות Notch חוץ גופית גורם להתפתחות כלפי השושלת תא T. כדי לבדל iPSCs לתוך CD4 + FoxP3 + Tregs, השתמשנו תאים ב OP9-DL1 / DL4 / IA, אשר מאוד מפורשת MHC II מולקולות (IA ב). רוב iPSCs להתמיין CD4 + תאים. עם זאת, לאחר ביטוי TCR שטח, תאים טרום-T בדיל רבים מאבדים את היכולת להבדיל ובסופו של דבר למות. כתוצאה מכך, את המספר הסלולרי של Tregs הפונקציונלי הנגזרות iPSC מפחית באופן דראמטי לאחר ארבעה שבועות של בידול במבחנה. כדי למנוע זאת, תוספת של IL-2 יכולה לשפר הישרדות תא באותם מועדים לפי שעון. שיטה חלופית לנהוג התבגרות והישרדות של Ag הספציפי מראש Tregs היא להעביר את טרום Tregs כי יש בדיל במבחנה במשך שבוע לעכברים. טרום Tregs אלה יכולים לחיות בדוntinue הבחנה והתבגרות in vivo לעוד שלושה שבועות. באמצעות שיטה זו, אוכלוסיית Ag הספציפית Treg פונקציונלית ניתן להפיק מן iPSCs, שהן nTregs הדמוית ויש להם את היכולת לדכא דלקת אוטואימונית במודל בעכברים. כדי לשפר את היעילות של in vivo פיתוח של Ag ספציפי iPSC-Tregs, תרכובות שונות (למשל, חומצה רטינואית, gelectin) 13,14 או ציטוקינים מדכאים (למשל, TGF-β, IL-10) ניתן להשתמש לאחר העברת המאמצת של-Tregs מראש. בנוסף להגדלת הישרדות התא, גישה משולבת זו יכולה לשמור ביטוי FoxP3 14,15 ולשפר את איכות Ag ספציפי iPSC-Tregs. בעיה פוטנציאלית שעלולה להתעורר לפיתוח in vivo Treg הוא דיכוי חיסוני הכוללת, וכתוצאה מכך סיבוכים במהלך זיהומים או הרזיה שלאחר מכן. מספר רב של Tregs Ag ספציפי המתפתחות viVO עלולה להחמיר זיהומים. במקרה זה, גן ההתאבדות, caspase המושרה 9 (ICasp9) 15,16, ניתן לשלב לתוך וקטור TCR / FoxP3. גישה זו מאפשרת הסרת תאי גזע שמקורם Tregs ידי הזרקה של מולקולות קטנות bioinert dimerizing סוכן (AP1903) כדי &quot;לכבות&quot; את הדור של תאי גזע שמקורם Tregs, אשר להתגבר על בעיה זו פוטנציאל. עצמית Ag הוא חלבון אופייני המוכר על ידי המערכת החיסונית של מארחים סובלים מהפרעת אוטואימונית פרט. עצמית Ag זהו היעד של מערכת החיסון, ואת תאי T בקורלציה אינם נמחקים. כדי ליצור עצמית Ag הספציפי Tregs, שימוש תמרת TCR הוא בחירה טובה. עצמית Ag (למשל, Heat shock protein) הספציפי TCR ניתן transduced לתוך Tregs בוגרת CD4 + CD25 + מתאי הדם היקפיים mononuclear (PBMCs), וגישה זו נוצלה בניסויים קליניים. לחלופין, כפי described בפרוטוקול זה, באמצעות תמרת הגן של-Ag העצמי ספציפי TCR עם FoxP3 וגירוי עם Notch signaling, גזע Tregs תאים שמקורם יכול להיות Tregs העצמית Ag הספציפי. טיפולים מבוססי תאים למחלות אוטואימוניות ניצול Tregs מהונדסים עשוי להיות שימושי 17. למרות תמרת TCR בתאי T הוכיחה את עצמו בטוח, ריאליים, החלים בניסויים קליניים, יש עדיין חששות בטיחות גדולים בשל אוטואימוניות הנגרמת על ידי תגובתיות צולבת עם רקמות בריאות 18. יתר על כן, באמצעות שיטות קיימות, מהונדסים גנטיים Tregs בדרך כלל יש התמדה לטווח קצר in vivo 19. לחלופין, מקור מבטיח לפיתוח מספר רב של חד השבטי Ag הספציפי Tregs הוא תאי גזע. תאי גזע עובריים (ESCs) יש את pluripotency עצמי התחדשות הטובה ביותר, אבל זה לא ריאלי להשיג אותם מחולים. למרות בקלות לבודד מן הדם ההיקפי, בתאי גזע hematopoietic (HSCs) הם רבתאי גזע חזק ניתן להרחיבו תרבית תאים בדומה ESCs 20. טכנולוגית הווה iPSC התקדמה דור יעיל יותר של PSC מן התאים הסומטיים של המטופלים על ידי תמרה של גורמי תעתוק שונים. מספר שיפורים מתודולוגיים פותחו בשנים האחרונות לייצר iPSCs ידי צמצום מפגעים פוטנציאליים מקסימאלי כגון immunogenicity ו tumorigenicity. לעומת ESCs, יש iPSCs pluripotency זהה עצמית והתחדשות. כתוצאה מכך, טכנולוגית iPSC עשויה להקנות יתרון בפיתוח PSCs למטופל ו / או ספציפית למחלה. השימוש iPSCs לפתח Ag ספציפי Tregs עשוי לקדם את תחום טיפולים מבוססי תאים למחלות אוטואימוניות 10,11.

Autoimmune arthritis is a systemic disease characterized by hyperplasia of synovial tissue and progressive destruction of articular cartilage, bone, and ligaments1. The defective generation or function of Tregs in autoimmune arthritis contributes to chronic inflammation and tissue injury because Tregs play a crucial role in preventing the development of auto-reactive immune cells. 
Manipulation of Tregs is an ideal strategy for the development of therapies to suppress inflammation in an Ag-dependent manner. For Treg-based immunotherapy, the specificity of the transferred Tregs is important for the treatment of ongoing autoimmunity2. To exhibit the suppressive activity, Tregs need to migrate and be retained at the afflicted region, which can be directed by the specificity of the TCR for the Ag at that location3. Although polyclonal Tregs may contain a small population containing this Ag specificity from their TCRs, the numbers of these Ag-specific Tregs are usually low. Consequently, cell-based therapies using polyclonal Tregs against autoimmune disorders require adoptive transfers of a large number of Tregs4,5. Because pluripotent stem cells (PSCs) have the ability to develop into any type of cell, Ag-specific PSC-Tregs may prove to be good candidates for Treg-based immunotherapy. Previous studies have shown the successful development of PSC-derived T cells, including Tregs6-8. 
Here, we describe a protocol to develop Ag-specific iPSC-Tregs. We further describe a cell-based therapy of autoimmune arthritis in a murine model using such Tregs. This method is based upon genetically modifying murine iPSCs with Ag-specific TCRs and the transcriptional factor FoxP3. The engineered iPSCs then differentiate into Ag-specific Tregs on the OP9 stromal cells expressing Notch ligands DL1, DL4, and MHC-II (I-Ab) molecules in the presence of cytokines mFlt3L and mIL-7. These Ag-specific iPSC-Tregs can produce suppressive cytokines, such as TGF-&#946; and IL-10, when stimulated with the Ag, and adoptive transfer of such Tregs has the ability to suppress AIA development in a murine model. The described protocol can be used to develop stem cell-derived Ag-specific Tregs for potential therapeutic interventions.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">C57BL/6j mice</td>
			<td style="width:71px;">Jackson Laboratory</td>
			<td style="width:119px;">664</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">B6.129S7 Rag1tm1Mom/J</td>
			<td style="width:71px;">Jackson Laboratory</td>
			<td style="width:119px;">2216</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-CD3 (2C11) antibody</td>
			<td style="width:71px;">BD Pharmingen</td>
			<td style="width:119px;">553058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-CD28 (37.51) antibody</td>
			<td style="width:71px;">BD Pharmingen</td>
			<td style="width:119px;">553295</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD4 (GK1.5) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">100417</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD8 (53&#8211;6.7) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">100714</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD25 (3C7) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">101912</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-TCR-&#946; (H57597) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">109220</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-IL10</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">505010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-TGF&#946;</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">141402</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">DMEM</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">ABCD1234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">&#945;-MEM</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A10490-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FBS</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH3007.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Brefeldin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B7651</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polybrene</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">107689</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Genejammer</td>
			<td style="width:71px;">Integrated science</td>
			<td style="width:119px;">204130</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">ACK Lysis buffer</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">10-548E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">mFlt-3L</td>
			<td style="width:71px;">peprotech</td>
			<td style="width:119px;">250-31L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">mIL-7</td>
			<td style="width:71px;">peprotech</td>
			<td style="width:119px;">217-17</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Gelatin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P6148-500G</td>
			<td style="width:167px;">Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Permeabilization buffer</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">421002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">mBSA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A7906</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ova albumin</td>
			<td style="width:71px;">Avantor</td>
			<td style="width:119px;">0440-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">CFA</td>
			<td style="width:71px;">Difco</td>
			<td style="width:119px;">2017014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tailveiner restrainer</td>
			<td style="width:71px;">Braintree scientific</td>
			<td style="width:119px;">RTV 150-STD</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

development of stem cell-derived antigen-specific regulatory t cells against autoimmunity

מחלות אוטואימוניות להתעורר עקב אובדן סובלנות עצמית אימונולוגית. בתאי T רגולטוריים (Tregs) הם מתווכים חשובים של סובלנות עצמית אימונולוגית. Tregs מייצג כ 5 - 10% של תת-אוכלוסיית תא CD4 + T הבוגר בעכברים ובבני אדם, עם כ 1 - 2% מאלה Tregs במחזור הדם ההיקפי. תאי גזע מושרים (iPSCs) יכול להיות מובחן לתוך Tregs תפקודית, אשר יש פוטנציאל לשמש עבור טיפולים מבוססי תאים של מחלות אוטואימוניות. כאן, אנו מציגים שיטה לפתח אנטיגן (Ag) Tregs -specific מ iPSCs (כלומר, iPSC-Tregs). השיטה מבוססת על שילוב גורם שעתוק FoxP3 ו קולטן תא Ag-T ספציפי (TCR) לתוך iPSCs ולאחר מכן הבחנה על תאי סטרומה OP9 להביע Notch הליגנדים דלתא דמוית (DL) 1 ו DL4. בעקבות הבידול חוץ גופית, iPSC-Tregs להביע CD4, CD8, CD3, CD25, FoxP3, ו Ag ספציפי TCR והם מסוגלים להגיב לגירוי Ag.שיטה זו יושמה בהצלחה לטיפול מבוסס תאים של דלקת אוטואימונית מודל בעכברים. העברת המאמצת של Ag ספציפי iPSC-Tregs אלה לתוך מפרקים הנגרמת Ag (AIA) עכברים -bearing יש את היכולת להפחית דלקת מפרקים ונפיחות וכדי למנוע איבוד מסת העצם.

כפי שניתן לראות כאן, ביום 28, Ag הספציפי Tregs משמעותי הביע CD3 ו Ag הספציפי TCR, שני סמנים של תאי T. אוכלוסיית CD3 + TCRVβ5 + הביע CD4. רוב תאי CD3 + TCRVβ5 + CD4 + הביע גם CD25, CD127, ו CTLA-4, אשר באים לידי ביטוי בדרך כלל ברמות גבוהות טבעי ובתקנות T (nTregs) ו בתאי T להביע FoxP3 ectopically. ביטוי FoxP3 בתאי שמקורם iPSC נמשך גם לאחר לטווח ארוך בגירוי חוץ גופייה עם ליגנד Notch, כפי שזוהה על ידי מכתים תאי ונותח על ידי cytometry זרימה (איור 1). בנוסף, Ag הספציפי iPSC-Tregs מיוצר ציטוקינים מדכאים, כגון TGF-β ו- IL-10, כאשר מומרץ במבחנה עם splenocytes Ag-פעם (איור 2), המציין את iPSC-Tregs הייתה פעילות מדכאת פוטנציאלית. מיקרוסקופ פלואורסצנטי גילה<html> כי יותר FoxP3 + תאים נכחו שטופלו OVA מאשר הברכיים שטופלו PBS; לא היו FoxP3 + תאים קיימים בברכיים בעכברים קבלת iPSCs DsRed +-transduced וקטור. רבים יותר CD4 + FoxP3 + TCRVβ5 + תאים מוצגים בברכיים של עכברים שקיבלו iPSCs transduced עם MiDR-TCR-FoxP3 מאשר MiDR-FoxP3 (איור 3). מקרים אלה מצביעים כי Ag ספציפי iPSC-Tregs להגר הברך AIA לאחר העברת המאמצת לעכברים הנמען. תאי iPSC הנגזרות מועברים באופן משמעותי ירד הברך הדלקתית תופחת כאשר OVA היה נוכח, אבל לא הייתה כל השפעה על הברך המלאה שהוזרקה רק עם MBSA במודל בעכברים (איור 4); אובדן עצם מופחתת בברך תא העברה דמיינו ידי מערכת מיקרו CT ברזולוציה גבוהה (איור 5). tp_upload / 54,720 / 54720fig1.jpg &quot;/&gt; איור 1:. דיפרנציאציה של Ag ספציפי iPSC-Tregs Murine iPSCs היו transduced עם מבנה: MiDR-TCRα-2A-TCRβ-2A-FoxP3 שיישאו את הגנים של OVA ספציפי TCR ו FoxP3. התאים-transduced גן (DsRed +) היו שיתוף תרבותי על תאי B OP9-DL1-DL4-IA בנוכחות mFlt3L ו- MIL-7. (א) מורפולוגיה של T ובתקנות התמיינות תאים ביום 0, 7, 14 ו 22. (ב) ניתוח תזרים cytometric עבור ביטוי החלבון של תאים שמקורם iPSC על CD3 יום 28. + TCRVβ5 + תאים היו מגודרים כמצוין (R1) ונותחו על מנת לתת ביטוי CD4 ו- CD8, עם CD25, CD127, CTLA- 4, ו FoxP3 המוצגים עבור תאים המגודרים כמו CD4 + CD8 - תאים (R2). (קווים כהים, אזורים מוצלים מצביעים שולטים isotype) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">אנא לחץ כאן כדי להציג larg</a>גרסה אה של נתון זה. <img alt="איור 2" src="/files/ftp_upload/54720/54720fig2.jpg" /> איור 2: ניתוח פונקציונלי של לי n Vitro הבדיל Ag ספציפי Tregs Murine iPSC-Tregs היו מגורה על ידי splenocytes. (נגמ&quot;שים; ובתקנות T / נגמ&quot;שים = 1: 4) ו פעמו עם פפטיד OVA 323-339. ייצור ציטוקינים תאי (TGF-β ו- IL-10) נותחו על ידי זרימת cytometry לאחר gating על חי CD4 + CD25 + תאים. (קווים כהים, אזורים מוצלים מצביעים שולטים isotype) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/54720/54720fig3.jpg" /> איור 3:g-ספציפי iPSC-Tregs לחדור לתוך המפרקים בברך. Ag ספציפי iPSC-Tregs הועברו adoptively לעכברים C57BL / 6. זמן קצר לאחר אינדוקציה מפרקים (ימים 7 - 14), הברכיים הוסרו מוכתם עבור immunohistology (ברים בקנה מידה: 20 מיקרומטר). עכברים שקיבלו יש OVA ספציפי iPSC-Tregs מספר גדול של תאים TCRVβ5 + בברכיים. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 4" src="/files/ftp_upload/54720/54720fig4.jpg" /> איור 4: העברת המאמצת של Ag ספציפי iPSC-Tregs מקילה AIA עכברים iPSCs Murine היו transduced עם מבנה retroviral MiDR, MiDR-FoxP3, או MiDR-TCR-FoxP3 והיו שיתוף תרבותי על OP9-DL1 / DL4 /. תאי B IA. ביום 7, התאים transduced-גן (3 × 10 6 / עכבר) היוdoptively מועבר לתוך עכברות C57BL / 6 שהיו מושרות עם AIA השבועי לאחר העברת התא. על אינדוקצית המפרקים למחרת, חומרת דלקת הפארקים הייתה פיקוח על ידי מדידת קוטר הברך. (AC) אחוז גידול בקוטר ברך. גידול בקוטר ברך חושב על בסיס בקוטר ברך preinjection עבור כל עכבר לפני הזריקה על ציון מפרק יום 0. הוערך על ידי בחינת שני הברכיים באופן עיוור; כל ברך הוצב ציון (0: אין נפיחות או שינוי צבע גלויות; 1: גלוי נפיחות עם או בלי שינוי צבע; 2: מיתון נפיחות עם שינוי צבע; 3: חמור נפיחות עם שינוי צבע). בכל קבוצה, חמישה עכברים שימשו, ונתונים מייצגים שלושה ניסויים בלתי תלויים. נתונים מיוצגים כממוצע ± SD. (ד) ממוצע הבקיע ביום 7 עבור שתי הברכיים מחמש עכברים. נתונים מיוצגים כממוצע ± SD משלושה ניסויים עצמאיים (** p &lt;0.01, *** p &lt;0.001, דו כיוונית ANOVA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">נא ללחוץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 5" src="/files/ftp_upload/54720/54720fig5.jpg" /> איור 5:. Ag ספציפי iPSC-Tregs להגר עד הברך מוזרק OVA ולצמצם את איבוד מסת העצם iPSC-Tregs הועברו adoptively לעכברים C57BL / 6. בתוך 21 ימים אינדוקציה שלאחר דלקת פרקים, העכברים היו מורדמים ואת הארכיטקטורה העצם סביב הברכיים העכבר היה צילמו על ידי מערכת מיקרו CT. עכברים שקיבלו OT-II TCR / FoxP3 גן-transduced murine iPSC-Tregs להפגין הפחתה משמעותית של איבוד מסת העצם לעומת לשלוט בעכברים שקיבלו iPSCs-transduced וקטור. (א) סריקות בוצעו עם אורך 2.2 מ&quot;מ, ותמונות נתפסו לאחר עיבוד תמונה נוסף (טיוח וטרנספורמציה נפח;ברים בקנה מידה:. 1 מ&quot;מ) (B) נפח העצם סביב מפרק הברך הוערכה באמצעות שחזור תלת ממדי של התמונות מיקרו-CT, ונפח עצם בתוך נפח של עניין חושבה (ברים בקנה מידה:. 1 מ&quot;מ) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">נא ללחוץ כאן לצפות בגרסה גדולה יותר של דמות זו.</a>

Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

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

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.

פיתוח תאי גזע שמקורם אנטיגן ספציפי תקינים בתאי T נגד אוטואימוניות

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

development-stem-cell-derived-antigen-specific-regulatory-t-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.6K Views.  The Pennsylvania State University College of Medicine. 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. 

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

דור של מושרה תאים T רגולטוריים של האדם תאים ראשוניים טי נאיביות וזיכרון

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

כלים חדשים כדי להרחיב תאי T רגולטוריים מאנשים HIV-1 נגוע-

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

תאי מציגי הרוצח המלאכותי Antigen (KaAPC) ליעיל<em&gt; במבחנה</em&gt; דלדול של תאי T אנושי אנטיגן ספציפי

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

יצירה<em&gt; דה נובו</em&gt; לקולטנים המצויים בתאי אנטיגן ספציפי האדם T על ידי retroviral התמרה של Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

במבחנה בידול של האדם CD4 + FOXP3 + מושרה תקינה בתאי T (iTregs) מ נאיבית CD4 + T תאים באמצעות פרוטוקול TGF-β-המכיל

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

קוונפיקציה של מתרבים אנטיגן אנושי-ספציפי CD4+ T תאים באמצעות Carboxyפלואורומיתאת הדיקן אסתר

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

יעילות גבוהה הדור של העכבר העיקרי של אנטיגן בתאי T ציטוטוקסיים לבדיקות פונקציונליות במודל סוכרת אוטואימוניות

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

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.

גזע תא נגזר ויראלי Ag-לימפוציטים T שכפול HBV לדכא בעכברים

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

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

ב Vivo הגדלת האינדוקציה תא T הרגולציה התקינה של הבטן

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

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

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

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

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Neisseria meningitidis זיהום של תאי גזע פלוריפוטנטיים המושרים שמקורם בתאי אנדותל במוח

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