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><tbody><tr><td>Accutase (1x)</td><td>Sigma</td><td>A6964</td><td>Enzymatic cell dissociation reagent</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>A6283</td><td></td></tr><tr><td>All-trans retinoic acid (RA)</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>Anti-CD31 (PECAM-1)</td><td>Thermo Scientific (Labvision)</td><td>RB-10333</td><td></td></tr><tr><td>Anti-Claudin-5</td><td>Invitrogen</td><td>4C3C2</td><td></td></tr><tr><td>Anti-Glut-1</td><td>Thermo Scientific (Labvision)</td><td>SPM498 (MA5-11315)</td><td></td></tr><tr><td>Anti-Occludin</td><td>Invitrogen</td><td>33-1500</td><td></td></tr><tr><td>Anti-VE-cadherin</td><td>Santa Cruz</td><td>sc-52751</td><td></td></tr><tr><td>Anti-ZO-1</td><td>Invitrogen</td><td>33-9100</td><td></td></tr><tr><td>Bacto Proteose Peptone</td><td>BD</td><td>211684</td><td></td></tr><tr><td>b-Mercaptoethanol</td><td>Merck (Sigma-Aldrich)</td><td>805740</td><td></td></tr><tr><td>Cell culture plates and flasks</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>Centrifuge (Heraeus Megafuge 1.0R)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Class II biosafety cabinet</td><td>Nuaire</td><td>NU-437-400E</td><td></td></tr><tr><td>CO2 Incubator (DHD Autoflow CO2 Air-Jacketed Incubator)</td><td>Nuaire</td><td></td><td></td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C5533</td><td></td></tr><tr><td>Columbia ager + 5 % sheep blood</td><td>Biomerieux</td><td>43049</td><td></td></tr><tr><td>Costar Transwell polyester filters (12- or 24-well)</td><td>Corning</td><td>3460, 3470</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Merck (Sigma-Aldrich)</td><td>G8270</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>31330-038</td><td></td></tr><tr><td>DMSO</td><td>ROTH</td><td>A994.1</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>21600-069</td><td></td></tr><tr><td>Epithelial Volt-Ohm Meter (Millicell ERS-2) with STX electrode</td><td>Merck (Millipore)</td><td>MERS00002</td><td></td></tr><tr><td>Fe(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;</td><td>ROTH</td><td>5632.1</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Fluoresence microscope (Eclipse Ti)</td><td>Nikon</td><td></td><td></td></tr><tr><td>Hemacytometer (Neubauer)</td><td>A. Hartenstein</td><td>ZK06</td><td></td></tr><tr><td>Human basic fibroblast growth factor (bFGF)</td><td>PeproTech</td><td>100-18B</td><td></td></tr><tr><td>Human Endothelial Serum Free Medium (hESFM)</td><td>Gibco</td><td>11111-044</td><td></td></tr><tr><td>Inverted microscope (Wilovert)</td><td>Hund (Will Wetzlar)</td><td></td><td></td></tr><tr><td>iPS(IMR90)-4 cells</td><td>WiCell</td><td></td><td></td></tr><tr><td>Kellogg&#039;s supplement</td><td></td><td></td><td>To prepare 110 ml of Kellogg&#039;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(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt; and combine the solutions. Filter sterilize and store aliquoted at -20 &amp;deg;C.</td></tr><tr><td>Knockout serum replacement (KOSR)</td><td>Gibco</td><td>10828-028</td><td></td></tr><tr><td>L-glutamine (GlutaMAX)</td><td>Invitrogen</td><td>35050-038</td><td></td></tr><tr><td>LunaScript RT SuperMix Kit</td><td>NEB</td><td>E3010L</td><td>cDNA synthesis kit</td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Methanol</td><td>ROTH</td><td>4627.5</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ROTH</td><td>KK36.1</td><td></td></tr><tr><td>Micropipettes (Research Plus)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>ROTH</td><td>6329</td><td></td></tr><tr><td>Nonessential amino acids (NEAA)</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>NucleoSpin RNA isolation kit</td><td>Machery-Nagel</td><td>740955</td><td>RNA isolation kit</td></tr><tr><td>Pipette boy (Accu-Jet Pro)</td><td>Brand</td><td></td><td></td></tr><tr><td>Platelet poor plasma-derived serum, bovine (PDS)</td><td>Fisher</td><td>50-443-029</td><td></td></tr><tr><td>PowerUp SYBR Green Master Mix</td><td>Applied Biosystems</td><td>A25742</td><td>qPCR master mix</td></tr><tr><td>qPCR film (MicroAmp Optical Adhesive Film)</td><td>Applied Biosystems</td><td>4211971</td><td></td></tr><tr><td>qPCR plates (MicroAmp Fast 96-well)</td><td>Applied Biosystems</td><td>4346907</td><td></td></tr><tr><td>ROCK inhibitor, Y27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>RT-PCR thermo cycler (StepOnePlus)</td><td>Applied Biosystems</td><td>4376600</td><td></td></tr><tr><td>Serological pipettes</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>StemFlex basal medium + 50x StemFlex supplement</td><td>Gibco</td><td>A3349401</td><td>Stem-cell maintenance medium</td></tr><tr><td>Swinging Bucket Rotor (Heraeus #2704)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Thiamine pyrophosphate</td><td>Sigma</td><td>C8754-5G</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Versene</td><td>Gibco</td><td>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

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

인간 답게 된 마우스 모델은 미세 혈관을 대상으로 세균 감염을 연구하는

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

연구

면역학 및 감염병학

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

만성 EAE와 마우스의 신경 줄기 / 전구 세포의 전신 주입

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

<meta charset="utf8"></head><body>iPSC 유래 HPC를 사용하여 상주 미세아교세포를 이용한 인간 뇌 오가노이드 생성

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

<meta charset="utf8"></head><body>Microglia 발달을 통한 인간 뇌 오가노이드의 유도

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Microglia 발달을 통한 인간 뇌 오가노이드의 유도

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

발생학

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

인트스타스타스테어링 전달을 통해 마우스에서 수막구균 수막염 혈청계S C유도

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

인간 유도 만능 줄기 세포를 성숙한 면역 표현형을 가진 뇌 미세혈관 내피 세포 유사 세포로 분화

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

생체공학

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

내분비주주입을 통한 렙토메닝 세포 변형 유도

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

신경과학

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

인간 유도 만능 줄기 세포에서 뇌 미세 혈관 내피 세포의 파생, 확장, 냉동 보존 및 특성화

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Neisseria meningitidis 유도만능줄기세포 유래 뇌내피세포의 감염

요약

더 많은 비디오 탐색

이 비디오의 챕터

인간 답게 된 마우스 모델은 미세 혈관을 대상으로 세균 감염을 연구하는

만성 EAE와 마우스의 신경 줄기 / 전구 세포의 전신 주입

Microglia 발달을 통한 인간 뇌 오가노이드의 유도

Microglia 발달을 통한 인간 뇌 오가노이드의 유도

Microglia 발달을 통한 인간 뇌 오가노이드의 유도

인트스타스타스테어링 전달을 통해 마우스에서 수막구균 수막염 혈청계S C유도

인간 유도 만능 줄기 세포를 성숙한 면역 표현형을 가진 뇌 미세혈관 내피 세포 유사 세포로 분화

내분비주주입을 통한 렙토메닝 세포 변형 유도

인간 유도 만능 줄기 세포에서 뇌 미세 혈관 내피 세포의 파생, 확장, 냉동 보존 및 특성화

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포