CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.

The authors have nothing to disclose.

가장 중요한 단계, 2), 3) 철저히 세포를 떼어 놓다, 수막을 제거하는 뇌량 아래 조직을 고려하지 않고 피질을 절제하는 적절한 수확 및 대뇌 피질의 성상 세포의 배양 아르 일)을 보장하기 위해, 4) GFAP에 대한 풍부 + 성상 교세포. 우리는 GFAP + 성상 세포에 대한 풍부 방법 (GFAP 프로모터의 제어하에 (지배적 인 변형 유전자의 Ad5GFAPCre 벡터 또는 활용과 TgGZT 121)를 ...

성상 세포종은 가장 흔한 일차 뇌종양 및 아교 모세포종 (GBM), 등급 IV의 성상 세포종 아르, 12-15개월 1,2의 평균 생존과 가장 일반적이고 공격적인 서브 타입이다. 미만성 성상 세포종, 특히 GBM의 침공, 완전한 수술 적 절제를 배제 보조 요법의 효과를 제한하고, 필연적으로 치료 후 재발 세 리드. 드 노보 (주) GBM 또는 필연적으로 (보조) GBM 사에 진행 낮은 뇌 성상 세포종 중 처음 본 환자. GBM은 유 전적으로 이기종 세 가지 핵심 신호 전달 경로 제어하는 유전자에 상호 배타적와 공동으로 발생하는 돌연변이에 의해 특징입니다 : G 1 / S (굽은) 세포주기 체크 포인트, 수용체 티로신 키나제 (RTK)를, 그리고 TP53 5-7 경로. GBM은 GBM의 하위 유형 influ 것을 제안, 서로 다른 뇌 세포 유형과 유사한 별개의 발현 양상과 네 게놈 하위 유형으로 구성원점 6,8,9의 그것의 세포에 의해 추측되는. 성상 세포종 나은 모델은 성상 세포종 병인 중에 특정 세포 유형의 돌연변이의 특정 조합의 역할을 정의해야한다. 보다 효율적인 임상 약물 개발을위한 이러한 모델을 활용하여 궁극적으로 환자 결과를 개선하는 데 도움이됩니다. 성상 세포종 전류 모델은 인간 세포주, 환자 유래의 이종 이식 (PDX), 유전자 변형 정상적인 인간 성상 세포와 신경줄 기세포 (NSC), 유전자 조작 및 마우스 (GEM) 10-14 확립 포함한다. 대뇌 피질의 성상 및 NSC - - floxed 종양 대립 유전자의 다양한 조합을 품고 GEM에서 수확 우리는 차 뇌 세포를 활용 대안, 비 생식 GEM (nGEM) 모델 15을 개발했다. 목적 표현형 I에서 전임상 약물 개발을위한 시험 관내 및 생체 내 모두에서 특징 및 잠재적으로 이용 될 수있는 유 전적으로 정의 성상 세포종 세포 모델을 생성 할 수 있었다mmune 능력있는 마우스. 설립 인간의 세포 라인 그들은 곧장 앞으로, 기술적으로 널리 사용할 수 있습니다. 성상 세포종의 발병 기전 및 생체 외 및 생체 내에서 약물 반응의 가장 일반적으로 사용되는 모델이며, 면역 결핍 쥐 10,11,16-18에서 소성을 xenografting에 따라 반응 속도와 발암을 정의한 . 이들 단점은 높은 성상 세포종에 연구를 제한 뇌 성상 세포종에서 확립 세포주를 생성하는 무능력을 포함한다; 기원의 정의 셀의 부족; 종종 원래의 환자 샘플에서 현저하게 차이가 게놈 프로파일 복잡한 게놈 이상의 존재; 그리고 감수성 혈청 11,17,19-22 직렬 문화 중 표현형 및 유전자형 드리프트. 설립 인간의 GBM 세포 라인의 개별 종양 돌연​​변이의 표현형의 결과는 종종 eluc을 배제 실제로 존재하는 이상,의 무리에 의해 마스크 될 수있다직접 유전자형 - 표현형 결과의 idation. PDX는 면역 결핍 마우스 또는 면역 결핍 생쥐 12,23의 뇌에 소성을 주입 이전에 정의 된 무 혈청 배지에서 비 부착 회전 타원체로 그들의 문화를 통해 환자 격리 성상 세포종 세포의 피하 통로를 통해 생성됩니다. PDX보다 정확하게 인간 성상 세포종의 게놈 프리를 유지하지만, 인간의 확립 세포주 유사한 개별 발암 돌연변이 표현형 효과는 그들의 게놈 19,24 복잡성으로 인해 마스크 할 수있다. 특히 신규 한 치료법에 대한 응답으로, 특정 종양 돌연​​변이 표현형 결과를 정의하는 확립 인간 세포주 또는 PDX의 패널은 자주, 유전자형 표현형의 상관 관계를 확립 일반화를 보여주고, 세포주 특정 효과의 가능성을 최소화하기 위해 이용된다. PDX 정확하게 인간의 별아교 세포종, INC의 조직 병리학 적 특징을 요점을 되풀이하는 동안내습 luding 확립 인간 세포주의 동 소성은 일반적으로 이종 이식편을 수행하지 21,23,25. 또한, 정상적인 인간의 성상 및 NSC는 시험관 및 생체 내 13,14,26에서 성상 세포종의 종양을 모델로 정의 된 발암 돌연변이 유전자 설계되었다. 이러한 세포는 인간의 확립 세포주 및 PDX 게놈의 복잡성이 부족하고 정확하게 인간 성상 세포종의 조직 병리학 요점을 되풀이하지만, 생체 내에서 면역 결핍 설치류 xenografting을 필요로한다. 인간의 모든 세포 모델은 면역 매개 이종 이식 거부 반응을 방지하기 위해 면역 결핍 쥐 호스트를 요구하기 때문에,이 모델은 기질 타겟팅 및 면역 조절 성 전임상 연구를 제한 동계 체제의 기본 종양 기질 상호 작용을 요점을 되풀이하는 데 실패하고 그대로 면역 체계 부족 10, 11를 치료. 종양 무타 소정 조합의 표현형 결과의 GEM 허가 심사현장 종양에있는 동안 생체 내에서 tions. 비 조건부 GEM 개발을 통해 모든 조직 내에서 돌연변이를 가지고있는 반면, 조건부 GEM은 세포 유형 - 특이 적 프로모터 10,11,15,18의 사용을 통해 특정 세포 유형에 Cre 호텔 매개 재결합을 제한하여 돌연변이의 대상으로 사용 발암 대립 유전자를 floxed있다. 조건부 성상 세포종의 GEM은 그대로 뇌 (11) 내에서 서로 다른 종류의 세포에 발암 성 변이의 기능적 역할을 규명하기 위해 이용되고있다. 조건부 GEM을 사용 시츄 gliomagenesis에서의 전임상 유틸리티 1) 생체 외 상관 관계의 부족, 복합 유전형과 시츄 종양 발생에서의, 3) 긴 레이턴시를 마우스의 큰 코호트를 생성하는 2)의 어려움을 포함하여 많은 요인에 의해 제한된다 4) 확률 적 종양 진행. 반응계에서 종양이 대응 관내 모델이 부족하기 때문에, 시험 관내 시험은 약물 w 수행 할 수없는기존의 i 번째 조건 GEM 모델. 다른 암과는 대조적으로, 성상 세포종의 조건부 GEM 모델은 거의 하나의 종양 유전자 돌연변이 (11)에 의해 유도되지 않는다. 따라서, 복잡한 사육 방식은 여러 종양 돌연​​변이 조건부 GEM을 생성해야합니다. 높은 성상 세포종으로의 진행은 일반적으로 불균일, 확률 적으로 발생하고 궁극적 복잡한 게놈 경관과 빠른 성장 동역학 (27)과 종양 상승을 제공하면서 또한, 성상 세포종 개시는이 모델에서 오랜 지연 기간 후에 가변 투과도 발생, 28. 조건부 GEM 모델에서 변수 투과도 및 악성 진행의 확률 적 특성은 개별 쥐가 임상 약물 시험에서 자신의 등록 전에 높은 성상 세포종의 존재 및 위치를 검출하기 위해 방사선 촬영으로 검사를해야합니다. 함께 찍은, 이러한 제한은 홍보에 필요한 생성 및 조건 GEM의 대규모 코호트 시험을 방해e 임상 약물 검사. 특정 신경 세포 유형의 바이러스 성 수용체 (TVA)를 표현하기 위해 설계 GEM 감염 조류 레트로 바이러스 (RCA 단자) 벡터를 이용하여 RCA 단자-TVA의 GEM 시스템은 광범위하게 성상 세포종의 종양 (11)를 모델로 활용되고 있습니다. 조건부 GEM과 대조적으로,이 모델 시스템은 복잡한 육종 계획에 대한 요구없이 특정 세포 유형에서의 다중 발암 돌연변이의 도입을 가능하게한다. 그러나, 가변 투과도 적극적 바이러스 통합을 달성하기 위해 세포를 분할하기위한 요구, 및 숙주 게놈 (29)에 도입 유전자의 임의의 삽입에 의해 제한된다. 그들은 다른 모델 시스템 (15)의 많은 한계를 극복하기 때문에 GEM에서 수확 세포를 활용하는 비 생식 GEM (nGEM) 모델은 점점 더 중요 해지고있다. 성상 세포종의 병인 세포 유형과 공동 발생 돌연변이를 개시 역할 억제하기 어렵다이들은 악성 진행 중에 미정 세포 유형에서의 광범위한 유전 적 돌연변이를 축적 말기 종양으로부터 유도되기 때문에 내 인간 GBM 세포주 또는 PDX 설립 사용. 대조적으로, 성상 세포종 모든 등급은 특정 정제 뇌 세포 유형 11,30 내에 유전 변이를 유도함으로써 nGEM 정의를 사용하여 모델링 될 수있다. 따라서, 특정 유전자 돌연변이 세포 및 분자 표현형에 세포 유형의 영향을 시험 관내 및 생체 내에서 결정될 수있다. 설립 된 인간 GBM 세포주와 유사 nGEM를 사용 초기 시험 관내 약물 테스트는 동일한 셀을 이용한 생체 내 테스트에 대한 약물의 우선 순위를 사용할 수있다. 생체 내에서 종양 형성 후 orthotopically 면역 능력 한배 새끼 동계 (30)의 뇌에 nGEM 세포 동종 이식에 의해 결정될 수있다. 이러한 동 소성 이식 모델은 따라서뿐만 아니라 종래의 세포 상해의 생체 내 시험에서 허용차 치료를 대상으로하지만, 면역 조절 성 간질 - 표적으로 한 치료뿐만 아니라. 마지막으로, 종양의 개시 및 진행에 미세 환경의 역할은 동일 세포 유형에서 동일한 변이를 사용 nGEM 종래 GEM 모델 사이의 결과를 비교함으로써 결정될 수있다. 우리와 다른 일차 전지를 사용하여 성상 세포종 nGEM을 개발했다 - 성상, NSC, 또는 희소 돌기 아교 세포 전구 세포 (OPC) - GEM 30-34에서 수확을. 성상 세포종 nGEM 개발 배후의 이론적 근거는 잠재적 면역 유능한 동물에서 시험 관내 약물 전임상 시험 및 생체 내에서 사용될 수있는 특정 유형의 세포에서 발암 돌연변이 표현형 결과를 결정하는 모델을 생성 하였다. 사포닌에 loxP /에 loxP 또는 TgGZT - 우리는 표현 표현형 WT 대뇌 피질의 성상 세포 및 비 Cre 호텔에서 NSC, floxed RB 경로와&gt; 94 % C57 / BL6 배경에 유지 조건 GEM 수확121 - 다양한 조합 35-39에서 유전자 - NF1에 loxP /에 loxP, Kras은 G12D, PTEN을에 loxP /에 loxP - 및 RTK / RAS / PI3K 통로를 floxed. 우리는 Cre 호텔 재조합 효소를 암호화하는 아데노 바이러스 벡터를 사용하여 시험 관내에서 유전자 재조합을 유도. 대뇌 피질의 성상 세포 수확은 세포 유형의 혼합물을 포함하기 때문에, 우리는이 문화 GFAP + 대뇌 피질의 성상 세포에 풍부하게, 같은 TgGZT 인간 GFAP 프로모터에서 쫓겨 121 Ad5GFAPCre 벡터 지배적 발암 유전자를 사용했다. 우리는 생체 외 및 생체 내에서 대뇌 피질의 성상 및 NSC의 표현형 G 1 / S의 결과 (굽은), MAPK와 PI3K 통로 돌연변이를 정의했다. MAPK 및 PI3K 경로 활성 G1 / S 불량 아스트로 분자 모방 proneural 인간의 GBM과, 소성을 주입하면, 균일 한 성장 동역학, 짧은 대기 시간을 가진 사전 정의 된 위치에 형성된 종양 및 조직 병리학인간의 GBM (30)의 알 특징. 생체 내에서 종양 성장의 종 모니터링 치료 40 님의 질문에 답변 처리 동료와 종양 성장의 정량 분석의 정상화를 통해 전임상 약물 검사를 도움이됩니다. 우리는 루시퍼 라제는 대뇌 피질의 성상을 표현 주입 한 쥐의 종 생물 발광 영상으로 종양의 성장 속도론을 결정했다. 따라서 조건부 GEM에서 파생 된 대뇌 피질의 성상 및 NSC는 성상 세포종 관련 돌연변이의 기능적인 결과와 임상 약물 개발을위한 잠재적 인 모델 시스템의 정의에 대한 다루기 쉬운 모델 시스템을 제공한다.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:233px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">11995065</td>
			<td style="width:233px;">DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Fetal Bovine Serum, Regular</td>
			<td style="width:191px;">Cellgro</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">15140122</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">08-395</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Cartilage Thumb Forceps, Curved</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">1631</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4</td>
			<td style="width:191px;">Miltex</td>
			<td style="width:119px;">17-301</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Ethanol (200 proof)</td>
			<td style="width:191px;">Decon Labs</td>
			<td style="width:119px;">2710</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Razor Blades</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14175-095</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">TrypLE Express (1X), Phenol Red</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">12605-010</td>
			<td style="width:233px;">Other Trypsin solutions are also suitable for cortical astrocyte havest and culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">CULTURE DISH, 60 x 15 mm</td>
			<td style="width:191px;">Thomas Scientific</td>
			<td style="width:119px;">9380H77</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">15 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">50 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352070</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Adenovirus stock</td>
			<td style="width:191px;">Gene Transfer Vector Core, U. Iowa</td>
			<td style="width:119px;">Ad5CMVCre</td>
			<td style="width:233px;">Store in 5 &#181;l aliquots at -80 C. Hazardous.&#160; Use Bsl2 safetyy&#160; precautions</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium sulfate (Na2SO4)&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">238597</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Potassium sulfate (K2SO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">221325</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium chloride (MgCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">HEPES potassium salt</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0527</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">D-(+)- Glucose</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Phenol Red</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3532</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Sodium hydroxide (NaOH)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Papain</td>
			<td style="width:191px;">Worthington</td>
			<td style="width:119px;">LS003127</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">L-Cysteine-HCl</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1276</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Syringe filter (0.22 &#181;m pore size)</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLGP033NS</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:241px;">Neurocult proliferation kit, mouse</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5702</td>
			<td style="width:233px;">This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">0.2% Heparin solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">7980</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PMG8041</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">bFGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (10X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">E-64</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3132</td>
			<td style="width:233px;">Make 10 mM stock in DMSO, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">6-well plates</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-83</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Cell strainer (40 &#181;m pore size)</td>
			<td style="width:191px;">Corning</td>
			<td style="width:119px;">352340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Stem cell dissociation solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5707</td>
			<td style="width:233px;">Alternatively, use gentle enzyme solutions such as Accutase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Methyl cellulose 15 cP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7027</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dulbecco&#39;s Modified Eagle&#39;s Media 2X</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLM-202-B</td>
			<td style="width:233px;">For making 5% methyl cellulose solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">1.7mL Snap Cap Microcentrifuge Tube</td>
			<td style="width:191px;">Corning</td>
			<td align="right" style="width:119px;">3620</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hamilton syringe, 250 &#181;l LT no needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-815-92</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">PB600-1 Antigen Dispenser</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:119px;">&#160;83700</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Disposable 18 ga needles&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9015638&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">27 ga 1/2&#34; luer tip needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-48</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">T48402</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Betadine</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9386574</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Puralube Opthalmic Ointment</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9689910</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">&#160;Model 900 Stereotaxic frame</td>
			<td style="width:191px;">Kopf Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">VETBOND</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9259532&#160;</td>
			<td style="width:233px;">Tissue adhesive&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Lidocaine&#160;</td>
			<td style="width:191px;">ShopMedVet</td>
			<td style="width:119px;">RXLIDO-EPI</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;">G3580</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Anti-Sox2</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">AB5603</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)</td>
			<td style="width:191px;">Dako</td>
			<td style="width:119px;">Z0334&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">IVIS Kinetic</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td style="width:233px;">For in vivo imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">122796</td>
			<td style="width:233px;">For in vivo bioluminescence imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EdU Imaging Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">C10340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">MSCV Luciferase PGK-hygro</td>
			<td style="width:191px;">Addgene</td>
			<td style="width:119px;">18782</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

우리는 표현형 생체 외 및 생체 특징으로 할 수있다 조건부 종양 대립 유전자 (그림 1) floxed 신생아 GEM의 숨겨에서 수확 대뇌 피질의 성상 및 NSC와 nGEM 모델 시스템을 개발했다. 구체적으로는 시험 관내에서 피질 별아교 세포의 발암 돌연변이의 영향을 조사하기 위하여, 성상 세포에 대한 제 풍부하는 것이 중요하다. 두피 성상 세포는 미세 아교 세포 수확, 아스트로 사이?...

Watch this Scientific Journal Video about Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice at JoVE.co

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

모델링 성상 세포종 병인<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 대뇌 피질의 성상이나 조건, 유전자 조작 쥐에서 신경 줄기 세포를 사용하여

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

modeling-astrocytoma-pathogenesis-vitro-vivo-using-cortical

Research

JoVE Journal

Neuroscience

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

13.3K 조회수.  University of North Carolina School of Medicine. Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

비디오: Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

연구

신경과학

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

스프 레딩 우울증을 사용하여 에피소드와 만성 두통의 신경 면역 신호를 모델링 체외에서</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

의 배아와 성인 신경 줄기 세포의 확장<em&gt; 자궁에</em&gt; Electroporation 또는 바이러스 성 Stereotaxic 주입

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

이미징 세포 내 칼슘<sup&gt; 2 +</sup&gt; 유전자 인코딩 된 칼슘 표시기를 사용하여 성인 마우스의 선조체 성상의 신호

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

신경 줄기 세포와 쥐의 뇌에서 5-하이드록시메틸시토신의 검출

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

시험관 내 마우스 배아 줄기 세포로부터의 신경 분화

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

섬유아세포에서 신경 전구세포로의 직접 변환을 이용한 신경질환 시험관 모델링 및 성상세포로의 분화

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...