L.M.E.는 A.S-U.에 수여된 "인간 병원체에 의한 미생물 감염 연구를 위한 3D 조직 모델"이라는 제목의 DFG 연구 교육 프로그램 GRK2157 지원을 받습니다. B.J.K.는 알렉산더 폰 훔볼트 재단(Alexander von Humboldt Foundation)의 박사후 연구원의 지원을 받고 있습니다. 또한, 문화 분야에서 iPSC-BEC를 생성하는 데 기술적 도움을 준 Lena Wolter에게 감사의 뜻을 전합니다.

<ol>
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저자는 공개할 것이 없습니다.

시험관 내 1차 및 불멸화된 인간 BEC는 강력한 장벽 표현형이 부족한 경향이 있기 때문에 BEC와 BBB를 모델링하는 데 어려움이 있었습니다. 인간 줄기 세포 기술의 출현으로 내피 마커, 단단한 접합 발현, 장벽 특성, 다른 CNS 세포 유형에 대한 반응 및 기능적 유출 수송체와 같은 예상되는 특징적인 BBB 표현형을 유지하는 iPSC 유래 BEC 유사 세포의 생성이 가능해졌습니다 12,13,14,15,22<...

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

혈액-뇌 장벽(BBB)과 수막 혈액-CSF 장벽(mBCSFB)은 중추신경계(CNS)에서 순환을 분리하는 매우 단단한 세포 장벽이며 주로 고도로 전문화된 뇌 내피 세포(BEC)로 구성되어 있습니다1,2. BEC는 뇌 안팎의 영양소와 노폐물을 조절하고 많은 독소, 약물 및 병원균을 배제하여 적절한 뇌 항상성을 유지합니다 1,2. 세균성 뇌수막염은 혈액 매개 박테리아가 BEC에 의해 형성된 장벽과 상호 작용하고 이를 뚫고 들어가 염증을 일으킬 때 발생합니다. Neisseria meningitidis(Nm, 수막구균)는 건강한 사람의 10-10%의 비염을 집락화하는 그람 음성 박테리아이지만 경우에 따라 심각한 전신 질환을 유발할 수 있습니다3. 영향을 받은 개인에서 Nm은 자반증을 유발할 수 있는 혈류에 접근할 수 있을 뿐만 아니라 BEC를 관통하여 뇌수막염을 유발하는 중추신경계에 접근할 수 있다3. Nm은 전 세계적으로 세균성 뇌수막염의 주요 원인이며, 백신 접종 노력에도 불구하고 여전히 뇌수막염의 주요 원인이다4. 항생제 치료와 같은 현대 의학적 개입으로 이러한 질환은 생존할 수 있게 되었지만, 뇌수막염에 걸린 사람들은 종종 영구적인 신경학적 손상을 입게 된다 5,6.
이전 연구에서는 Nm-BEC 상호 작용에 기여하는 박테리아 요인 및 숙주 신호 전달을 확인했습니다 7,8,9,10,11. CD147과 같은 수용체뿐만 아니라 불투명도 단백질 Opc 및 type-IV pili와 같은 확인된 부착 및 침습은 다양한 BEC 모델에서 시험관 내에서 수행되었지만 이러한 모델에는 BBB 특성을 정의하는 많은 것이 부족합니다 7,9,11,12. Nm-BEC 상호 작용에 대한 완전한 이해는 부분적으로 in vivo 모델을 활용할 수 없고, 불완전한 백신 보호 및 in vitro에서 강력한 인간 BEC 모델의 부족으로 인해 여전히 어렵습니다.
시험관 내 hBEC를 모델링하는 것은 BEC의 고유한 특성으로 인해 어려운 일이었습니다. 말초 내피 세포와 비교하여, BEC는 복잡한 밀착 접합부로 인한 높은 경내피 전기 저항(trans-endothelial electrical resistance, TEER)과 같은 장벽 특성을 강화하는 다수의 표현형을 가지고 있다12. 일단 뇌 미세환경으로부터 제거되면, BEC는 그들의 장벽 특성을 빠르게 상실하여 약한 장벽만을 형성하는 1차 또는 불멸화된 시험관 내 모델의 유용성을 제한한다12,13. Nm 감염의 인체 특이성, 강력한 in vivo 모델의 부족, in vitro에서 인간 BEC를 모델링하는 데 어려움이 결합되어 Nm과 BEC 간의 복잡한 숙주-병원체 상호 작용을 이해하기 위한 더 나은 모델이 필요합니다. 최근에는 모델 인간 유도만능줄기세포(iPSC) 기술을 사용하여 생체 내 BEC를 더 잘 모방하는 iPSC로부터 BEC 유사 세포를 유도했습니다 12,13,14,15. iPSC-BEC는 인간에서 유래하고, 쉽게 확장할 수 있으며, 1차 또는 불멸 표현형에 비해 예상되는 BEC 표현형을 가지고 있습니다12,13,14,15. 또한, iPSC-BEC가 숙주-병원체 상호작용, 헌팅턴병, 앨런-허른던-더들리 증후군을 유발하는 MCT8 결핍과 같은 중추신경계의 다양한 질병을 모델링하는 데 유용하다는 것을 입증했습니다 16,17,18,19,20,21. 여기서는 재생 가능한 iPSC 공급원에서 iPSC-BEC를 유도하는 방법과 Nm에 의한 iPSC-BEC의 감염이 선천성 면역 반응의 활성화를 유도하는 방법을 보여줍니다. 우리는 이 모델이 다른 in vitro 모델에서 재현할 수 없는 숙주-병원체 상호작용을 조사하는 데 유용하며 Nm과 같은 인간 특이적 병원체와의 상호작용을 검사할 때 특히 유용하다고 믿습니다.

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

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

수막구균성 뇌수막염은 나이세리아 수막염(Neisseria meningitidis , 수막구균, Nm)이 고도로 전문화된 뇌 내피 세포(BEC)에 침투하여 중추신경계(CNS)에 접근할 수 있을 때 발생하는 생명을 위협하는 감염입니다. Nm은 인간 특이적 병원체이기 때문에 강력한 in vivo 모델 시스템이 없기 때문에 Nm과 BEC 간의 숙주-병원체 상호 작용에 대한 연구가 어려워지고 네이티브 BEC를 모방하는 인간 기반 모델이 필요합니다. BEC는 복잡한 밀착 접합부와 높은 경내피 전기 저항(TEER)을 특징으로 하는 말초 내피 세포와 비교할 때 더 단단한 장벽 특성을 가지고 있습니다. 그러나 1차 BEC 및 불멸화된 BEC와 같은 많은 in vitro 모델은 기본 신경 미세환경에서 제거된 후 장벽 특성이 부족하거나 빠르게 손실됩니다. 최근 인간 줄기세포 기술의 발전으로 유도만능줄기세포(iPSC)에서 뇌와 유사한 내피세포를 유도하는 방법이 개발되어 다른 체외 인간 모델과 비교할 때 BEC를 더 잘 표현할 수 있습니다. Nm-BEC 상호작용을 모델링하기 위해 iPSC 유래 BEC(iPSC-BEC)를 사용하면 BEC 장벽 특성을 가진 인간 세포를 사용할 수 있다는 이점이 있으며, 장벽 파괴, 선천성 면역 활성화 및 박테리아 상호작용을 검사하는 데 사용할 수 있습니다. 여기서는 분석을 위한 박테리아 준비, 감염 및 샘플 수집 외에도 iPSC에서 iPSC-BEC를 도출하는 방법을 보여줍니다.

여기에 설명된 프로토콜은 Stebbins et al.에서 채택한 것으로, iPSC를 BBB 특성을 가진 뇌 유사 내피 세포로 분화하는 과정과 Nm19,22의 iPSC-BEC를 사용하는 감염 연구에 이 모델을 활용하는 방법을 강조합니다. iPSC-BEC는 적절하게 분화될 경우 TEER로 측정한 2000 Ω·cm2보다 큰 단단한 장벽 특성을 나타내며 VE-cadherin 및 CD31(PECAM)과 같은 내피 마커를 발현합니...

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

여기에 설명된 프로토콜은 유도만능줄기세포 유래 뇌유사 내피세포의 분화, 감염을 위한 Neisseria meningitidis 준비 및 기타 분자 분석을 위한 샘플 수집의 주요 단계를 강조합니다.

Neisseria meningitidis 유도만능줄기세포 유래 뇌내피세포의 감염

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

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

Research

JoVE Journal

Immunology and Infection

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

2.3K 조회수.  University of Würzburg. 여기에 설명된 프로토콜은 유도만능줄기세포 유래 뇌유사 내피세포의 분화, 감염을 위한 Neisseria meningitidis 준비 및 기타 분자 분석을 위한 샘플 수집의 주요 단계를 강조합니다.

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

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

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

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

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

의 세포 상호 작용 Visualisation 및 부량 Neisseria meningitidis</em공촛점 이미징에 의해 α - actinin>과 인간

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

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면역학 및 감염병학

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

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

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

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

선정 Plasmodium falciparum</em인간 두뇌 내피 세포 Cytoadhesion위한> 기생충

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

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

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

T Lymphocytes쪽으로 유도 Pluripotent 줄기 세포의 분화 감독

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

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

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

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

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

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

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

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

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

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

인간 유도 만능 줄기 세포 유래 장 소기관을 사용 하 여 살 모 넬 라 및 기타 병원 균에 대 한 상피 세포 보호 연구 및 수정

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

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

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

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

인간 줄기 세포 유래 Ileal 장용 단층에서 M 세포 유사 세포의 분화 유도

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

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

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

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

줄기 세포 유래 바이러스 Ag 특이적 T 림프구는 마우스에서 HBV 복제를 억제

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

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

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

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

일정한 흐름에 있는 1 차적인 인간 내피 세포의 폐렴구균 감염

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

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

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

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

미세 혈관 내피 세포를 통해 미생물 통과에 의해 혈액 - 뇌 장벽의 투과성 분석

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

Isolation of Primary Mouse Lung Endothelial Cells

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

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

일차 마우스 폐 내피 세포의 분리

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