이 작품은 (SP에) 재향 군인회에서 공로 검토 교부금 (NEUD-004-07F)에 의해 지원되었다.

1. 인간의 NPC의 확장 (그림 1) <ol><li> 냉동 태아의 뇌 (론자, 워커, MD, USA) 및 확산 보충제, EGF (10 NG / ML)를 포함 neurobasal 매체 neurosphere를 (그림 1A)로 T-75 플라스크 현탁 배양을에서 인간의 NPC의 주식을 회복 FGF (10 NG / ㎖). </li><li> 문화의 3 일 후에, 5 분 동안 500 rpm에서 15 ML 튜브와 원심 분리기에 neurosphere를 전송할 수 있습니다. </li><li> 세포 펠렛 위 ~ 100 ㎕의 매체를 남겨두고 뜨는을 취소하고 에펜 도르프 튜브에 전송할 수 있습니다. 100 μL의 세포 펠렛은 2 T-75 플라스크로 분할 충분하다. neuroprogenitor 세포 얇은 분할면 증식 실패로 이하, 플라스크의 수를 줄이는 경우. </li><li> 튜브의 바닥에 대하여 200 μL 팁을 유지하면서, 200 μL 팁 50X로 펠렛을 씹다. 똑같이 두 부분으로 세포 현탁액을 나누고 2 T-75 플라스크 (1-2 분할), 계속 하나에 추가차별화를위한 확장과 다른. </li><li> neurosphere를이 300 ~ 500 μM의 원래 크기에 도달 할 때까지 모든 4~5일 매체를 변경하여 확장 (첫 번째 플라스크)에 대한 neuroprogenitor 세포의 문화를 계속합니다. (500 rpm으로, 5 분) 원심 분리하여 매체를 변경, 기존의 매체를 폐기하고 새로운 매체를 추가 할 수 있습니다. </li></ol> 2. 신경 풍부한 문화에 인간의 NPC의 분화 (그림 2) <ol><li> 신경 세포의 풍부한 문화에 neuroprogenitor 세포의 분화를 들어, 각 웰에 500 μl를 추가하여 폴리-L-라이신 100 μg / ㎖로 24 - 웰 플레이트와 코트의 각 웰에 하나의 커버 슬립을 배치합니다. RT에서 30 분 동안 접시를 품어. </li><li> 폴리-L-라이신 솔루션을 기음과 멸균 물 접시를 씻어. </li><li> 다음, 코트는 5 ㎍ / ㎖의 마우스 라미닌 500 μL와 접시의 우물과는 30 분 동안 품어. 배양 한 다음, PBS로 우물을 씻어. </li><li> 사일 splitti 후세포 (단계 1.4) 겨, 24 웰 플레이트의 웰 당 ~ 500을 neurospheres의 밀도로 코팅 된 요리로 &#39;차별화&#39;라는 플라스크에있는 작은 neurosphere를 전송합니다. </li><li> neurosphere를이 접시에 부착하면 6 시간 후, 증식 배지를 제거하고 neurobasal 매체로 구성된 차별화 매체를 추가, B27 보충제, NGF (20 NG / ML), BDNF (10 NG / ML), DBC (100 ㎜), 및 레티노 산 (2 μM). </li></ol> 3. 차별화의 NPC의 면역 형광 염색법 (그림 3) <ol><li> 두 주 동안 신경 세포 분화 배지에서 neuroprogenitor 세포의 문화에 따라, 신경 세포의 풍부한 문화를 얻을 수있다. 한 번 PBS에 신경을 씻어 다음 30 분 동안 4 % 파라 포름 알데히드로 수정합니다. 고정되면, PBS로 세포를 세 번 씻어. </li><li> 60 분 동안 RT에서 permeabilization 완충액 (5 %의 BSA와 PBS에서 0.2 % 트리톤 X-100)으로 세포를 배양한다. </li><li> 4 ° C의 난에서 요리를 O / N을 품어NA의 다음 PBS에서 3 % BSA의 클론 및 단클론 항체의 조합으로 통 : 노이 앤 (1:250) 및 synapsin (1:250), 콜린 에스 테라 아세틸 (1:500) 및 시납 (1:250), BDNF (1 : 500) 및 GAP43 (1:250), STAT3 (1:500) 및 GFAP (1:1,000). </li><li> PBS와 커버 슬립을 세 번 씻어 준 후 90 분 동안 실온의 어두운 곳에서 안티 - 토끼를 Cy3 및 안티 마우스 FITC 차 항체를 품어. 배양 한 다음, PBS로 커버 슬립을 세 번 씻는다. </li><li> 집게로 배양 접시에서 커버 슬립을 꺼내 설치 매체에 거꾸로 배치, 유리 슬라이드에 장착 매체의 10 μl를 놓습니다. 부드럽게, 초과 설치 매체를 닦아 매니큐어와 가장자리를 밀봉. </li><li> 형광 현미경의 면역 염색 신경을 검사합니다. </li></ol> 4. 뉴런의 레이저 캡처 미세 절제 (그림 4) <ol><li> PEN 막 슬라이드에 신경 세포의 풍부한 문화에 NPC의 차별화도 2에 대해 기술 된 절차에 따라 폴리-L-라이신 및 마우스 라미닌으로 코팅들. </li><li> RNase가없는 상태에서 이후의 모든 단계를 수행합니다. </li><li> 세포를 시각화하는 HistoGene 스테인 (악튜러스)를 사용하여 문화를 얼룩. </li><li> 베리타스 LCM 시스템 전체 슬라이드의 도로지도 이미지를 사용하여 성상 세포의없는 순수한 신경 세포 인구의 영역을 찾을 수 있습니다. 그리기 도구를 사용하여 신경을 표시합니다. </li><li> 이 단계 과정에서 컴퓨터 제어 정밀도 및 자동화를 사용하여 이러한 영역의 레이저 캡처를 수행한다. 첫째, IR (적외선)은 멤브레인 캡에 부착 얻기 위해 70 ㎿가 2,500 마이크로 초 펄스의 레이저 전원 설정으로 여러 장소에서 발생합니다. 다음으로, 표시 영역 (10)은 측정 값의 낮은 레벨 설정에서 UV 절삭 공구를 사용하여 절개된다. </li><li> 이러한 프로세스의 결합을 선택적으로 CapSure LCM 매크로 모자에 PEN 막에서 표시된 영역을 캡처합니다. 상기 캡을 들어 올릴 때, n 인 멤브레인eurons 캡을 준수합니다. </li><li> PicoPure RNA 분리 키트 (악튜러스)를 사용하여 LCM 샘플에서 총 RNA를 분리하고 DNA 분해 효소로 처리. </li><li> 키트의 지시에 따라 RiboAMP RNA 증폭 키트를 사용하여 격리 된 RNA를 증폭. </li><li> 인간의 신경 미세 섬유 중쇄 (hNFHc)을 감지하는의 TaqMan 프로브를 사용하여 실시간 RT-PCR 분석을 수행합니다. </li></ol> 약어 : AD, 알츠하이머 병, BDNF, 뇌 유래 신경 영양 인자, CREB, 사이 클릭 AMP 반응 요소 결합 단백질, DBC, Dibutyryl 사이 클릭 AMP, EGF, 표피 성장 인자, FGF, 섬유 아세포 성장 인자, GFAP, 아교 섬유 성 산성 단백질, LCM, 레이저 미세 절제를 캡처; NGF 신경 성장 인자; NPC, neuroprogenitor 셀; PEN, 폴리에틸렌 나 프탈레이트.

<ol>
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선언에 관심 없음 충돌.

우리는이 연구에서 자기 갱신 인간 neuroprogenitor 세포의 분화에 의해 신경 세포가 풍부한 세포 배양 모델과 레이저 캡처 미세 절제에 의해 신경 세포의 순수한 인구를 분리하는 방법을 설명합니다. 우리는 NGF, BDNF, DBC와의 NPC의 신경 세포 분화 레티노 산의 조합을 사용했습니다. DBC는 CREB, 신경 (12)를 강화하는 전사 인자를 활성화하는 데 사용됩니다. 레티노 익산은 세포주기의 종료를 유도?...

평생 신경은 측면 심실의 subventricular 영역과 성인 포유류의 뇌 (1)의 치아 이랑의 subgranular 층에서 발생하는 것으로 알려져 있습니다. Neuroprogenitor 세포 (NPC들)이이 지역에서 발생이 신경 세포, 성상 세포 및 희소 돌기 아교 세포 (2)로 분화 할 수있는 다 능성 세포입니다. NPC들 때문에 파킨슨 병, 근 위축성 측삭 경화증, 뇌졸중, 알츠하이머 병 (AD) 3 등 다양한 퇴행성 신경 질환을 가진 환자에 이식 할 잠재력의 관심을 생성했다. 의 NPC와 연구는 일반적으로 이식 각도에 집중했지만 신경 퇴행의 메커니즘을 결정하는 세포 배양 모델로서 NPC 유래 신경의 전위는 충분히 이용되지 않았다. 그렇지 않은으로 이전 연구는 일반적으로 각 실험에 대한 격리 할 필요가 쥐의 뇌 조직에서 분리 된 후 유사 분열 신경을 사용했습니다자기 갱신. SH-SY5Y 및 SK-N-MC 세포를 포함한 인간 신경 아세포종 세포주는 확장 될 수 있지만, 그들은 차 신경의 특성이 없다. 그들은 여러 계대 확장 될 수 있고, 차 뉴런 4,5의 특성과 함께 세포 집단을 생성하기 위해 구별 될 수 있기 때문에 인간의 NPC는 반면에, 양쪽의 이점을 제공한다. 현재의 연구에서, 우리는 인간 태아의 뇌에서 분리 된 시판의 NPC에서 일관성있는 신경 세포의 풍부한 인구를 얻기 위해 새로운 분화 프로토콜을 설명합니다. 이러한 문화가 glial 세포의 작은 퍼센트를 포함 않기 때문에, 우리는 분자 특성에 대한 신경 세포의 순수한 인구를 분리하기 위해 추가적인 방법이 필요합니다. 레이저 캡처의 microdissection (LCM)는 조직 절편에서 세포의 동종 집단을 선택적으로 유전자 발현 분석 (6)에 대해 포착 될 수있는 새로운 기술이다. 뇌는 신경 세포, 아교 및 다른 세포 타이로 구성된 이종 조직이다PES. LCM은 신경 세포 특이 유전자의 발현을 결정하는 데 사용되었다 70-10을 분석한다. 우리는 이전에 구체적으로 해마의 신경 세포 11 감소 사이 클릭 AMP 반응 요소 결합 단백질의 발현 (CREB)과 BDNF를 보여 AD (Tg2576) 마우스 뇌 섹션에서 해마의 뉴런의 LCM을 수행했습니다. 본 연구에서는 PEN 막 슬라이드에서 배양 된 신경 세포의 분리에 대한 인간의 NPC의 확장, 신경 세포의 분화, 신경 세포 마커에 대한 면역 형광 염색 및 LCM에 대한 절차를 설명합니다.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Neuroprogenitor cells (NPCs) </td>
			<td>Lonza</td>
			<td>PT-2599</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A human basal medium</td>
			<td>Stem cell technology</td>
			<td>5750</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Proliferation supplement</td>
			<td>Stem cell technology</td>
			<td>5753</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Differentiation supplement</td>
			<td>Stem cell technology</td>
			<td>5754</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B-27 Supplement (50x) </td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human epidermal growth factor</td>
			<td>Stem cell technology</td>
			<td>2633</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fibroblast growth factor </td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brain Derived Neurotrophic Factor </td>
			<td>Cell signaling </td>
			<td>3897S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nerve Growth Factor </td>
			<td>Invitrogen</td>
			<td>13257-019</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibutyryl cyclic AMP</td>
			<td>Sigma</td>
			<td>D-0627</td>
			<td></td>
		 </tr>
		 <tr>
			<td>poly-L-lysine </td>
			<td>Sigma</td>
			<td>P-5899</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse laminin </td>
			<td>Sigma</td>
			<td>L-2020</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>STAT3 antibody</td>
			<td>Cell signaling</td>
			<td>9132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFAP antibody</td>
			<td>Cell signaling</td>
			<td>3670</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GAP43 antibody</td>
			<td>Transduction laboratories</td>
			<td>612262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BDNF antibody</td>
			<td>Millipore</td>
			<td>AB1534SP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetyl cholinesterase antibody </td>
			<td>Santz cruz</td>
			<td>Sc-11409</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synaptophysin antibody</td>
			<td>Abcam</td>
			<td>ab18008-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NeuN antibody</td>
			<td>Chemicon</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synapsin antibody</td>
			<td>Novus</td>
			<td>NB300-104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse FITC</td>
			<td>Jackson Immuno </td>
			<td>115-095-146</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti Rabbit Cy3</td>
			<td>Jackson Immuno </td>
			<td>711-165-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X 100</td>
			<td>Acros</td>
			<td>21568-2500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehye </td>
			<td>Fisher</td>
			<td>4042</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip (Big circle cover slip)</td>
			<td>Fisherbrand</td>
			<td>12-545-102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting medium (Prolong Gold)</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen membrane</td>
			<td>Applied biosystems</td>
			<td>LCM0521</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histogene LCM frozen section staining kit</td>
			<td>Applied biosystems</td>
			<td>KIT0401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboAmp RNA Amplification kit</td>
			<td>Applied biosystems</td>
			<td>KIT0201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picopure RNA Isolation kit</td>
			<td>Applied biosystems</td>
			<td>KIT0202</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CapsureMacro LCM caps</td>
			<td>Applied biosystems</td>
			<td>LCM0211</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

인간 태아의 뇌에서 분리 Neuroprogenitor 세포 (NPC들)는 표피 성장 인자 (EGF) 및 세포의 풍부한 공급을 제공하는 섬유 아세포 성장 인자 (FGF)의 존재하에 증식 조건 하에서 확장 하였다. 의 NPC는 신경 성장 인자 (NGF)의 새로운 조합의 존재하에 분화 된, 뇌 유래 신경 영양 인자 (BDNF), dibutyryl 캠프 (DBC) 및 폴리-L-라이신 및 마우스 라미닌 코팅 접시에 레티노 산은 신경 세포를 얻는 풍부한 문화. NPC들도 neurotrophins과의 부재, DBC 및 레티노 산 및 성상 세포가 풍부한 배양을 수득 모양체 신경 영양 인자 (CNTF)의 존재하에 분화되었다. 차별화의 NPC는 노이 앤, synapsin, 아세틸 콜린, 시납과 GAP43 등의 신경 세포 마커의 패널에 대한 면역 형광 염색을 특징으로했다. 아교 섬유 성 산성 단백질 (GFAP)와 STAT3, 성상 세포 마커는 차별화 된 NPC의 10 ~ 15 %가 검출되었다. 용이하게하기 위해셀 타입의 특정 분자 특성은 레이저 캡처 미세 절제는 폴리에틸렌 나 프탈레이트 (PEN) 막 슬라이드에서 배양 신경 세포를 분리 하였다. 이 연구에서 설명하는 방법은 신경 퇴행의 분자 메커니즘에 대한 우리의 이해를 사전에 가치있는 도구를 제공합니다.

NPC가 확대 (그림 1) 이 neurosphere를 저작하여 단일 세포 현탁액으로 세분화 할 때, 피펫 팁은 현탁액 상하 피펫 일부 저항이 존재하도록 튜브의 바닥을 터치 할 필요가있다. 분쇄 횟수는 개인과 현미경으로 얻어진 세포 현탁액을 검사하여 시행 착오에 의해 결정되어야 사이에서 변화 할 것이다. 의 NPC가 밀착되면 증식이 빠른 속도로있을 것이기 때문에, 그것은 세포 ?...

Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

인간 neuroprogenitor 세포 (NPC들)은 증식 조건 하에서 확장 하였다. 의 NPC는 neurotrophins과의 조합의 존재에 신경이 풍부한 문화로 분화되었다. 신경 세포 마커는 면역 형광 염색으로 검출되었다. 뉴런의 순수한 인구를 분리하기 위해,의 NPC는 PEN 막 슬라이드에 차별화 된 레이저 캡처 미세 절제를 행 하였다.

문화의 차별화 된 인간 Neuroprogenitor 세포에서 신경 세포의 레이저 캡처 미세 절제

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

laser-capture-microdissection-neurons-from-differentiated-human

Research

JoVE Journal

Neuroscience

12.1K 조회수.  Denver VA Medical Center.  인간 neuroprogenitor 세포 (NPC들)은 증식 조건 하에서 확장 하였다. 의 NPC는 neurotrophins과의 조합의 존재에 신경이 풍부한 문화로 분화되었다. 신경 세포 마커는 면역 형광 염색으로 검출되었다. 뉴런의 순수한 인구를 분리하기 위해,의 NPC는 PEN 막 슬라이드에 차별화 된 레이저 캡처 미세 절제를 행 하였다.

비디오: Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

의 레이저 캡처 Microdissection Drosophila 주변 뉴런

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

외상성 뇌 손상 후 뉴런 또는 유전자 발현 분석을위한 단일 뉴런의 강화 인구의 레이저 캡처 Microdissection

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

인간 만능의 줄기 세포에서 Telencephalic Glutamatergic 뉴런의 사양

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

레이저 포획 미세 절에 의해 마우스 척수 모터 신경 세포 체에서 전사 분석을위한 RNA의 생산

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

레이저 캡처 이외에 Microdissection과 코 생검 결합을 통해 획득 후각 뉴런 : 정신 장애의 치료 반응을 연구하기 위해 잠재적 인 접근

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

레이저 캡처 이외에 Microdissection - 개별 도파민 신경 세포의 분리 및 전체 복부 피개 영역의 데모

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

루트 신경절 신경 세포와 차별화 된 지방 유래 줄기 세포를 이룬다 :<em&gt; 체외</em&gt; 공동 문화 모델은 주변 신경 재생을 연구하는

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

문화의 차별화 된 인간 Neuroprogenitor 세포에서 신경 세포의 레이저 캡처 미세 절제

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