This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

재료 및 문화 매체의 1. 준비 <ol><li> SCZ의 해부와 해리를 들어, 알루미늄 호일로 뇌 매트릭스, 양날 면도날, 그리고 집게를 포장 한 다음 고압 증기 멸균하여 그들을 소독. </li><li> 전체 마우스 뇌를 씻어 차가운 PBS 버퍼 50 ㎖를 준비합니다. </li><li> 해부 현미경을 설정하고 뇌 (멸균 가위와 집게)의 해부에 필요한 수술 도구와 SCZ (1 ML의 주사기, 30 G 바늘, 뇌 매트릭스, 및 미세 집게)의 분리를 준비합니다. </li><li> N2 매체의 제조 : <ol><li> F-12 / DMEM (+ L - 글루타민, + 탄산 수소 나트륨) 2 % B27, 1 % N2 보충, 1 % 페니실린 - 스트렙토 마이신과 매체를 준비합니다. 주 : 성장 배지는 N2 배지 및 성장 인자로 구성 (20 NG / ㎖의 정제 된 표피 성장 인자 (EGF), 20 ng의 / ㎖ 염기성 섬유 아세포 성장 인자 (bFGF2)). </li></ol></li><li> 소화 버퍼를 준비합니다 (40 단위 / ml의 파파인, 2.4 단위 / ㎖ dispas전자 II, 2 % 페니실린 - 스트렙토 마이신 PBS에서) 조직 소화. </li><li> 코팅 플레이트 및 커버 슬립의 제조 : <ol><li> (0.01 % PLO) 및 라미닌 (DH 2 O에 용해 10 ㎍ / ㎖) 폴리 -L- 오르니 틴을 준비합니다. 단층 또는 면역 염색 18 밀리 된 커버로 SCZ-aNSCs의 유지 보수를 위해 코트 6 웰 플레이트, 4 ℃에서 하룻밤 PLO으로 그들을 품어. 그런 O.는 판과 커버 슬립 마지막 세척 후 건조하도록 허용 DH 2로 3 회 씻는다. </li><li> 다음으로, 하룻밤 4 ℃에서 라미닌과 번호판을 품어. 그런 다음, DH 2 O.로 3 회 세척 주의 : 세포 부착에 영향을 미칠 것이다, 라미닌을 건조하지 마십시오. 주 : 코팅 용액을 3 번 재사용 될 수있다. </li></ol></li></ol> 2. 분리 및 성인 SCZ의 해리 <ol><li> 문화 준비하기 전에, 얼음에 뇌 매트릭스와 양날 면도기를 배치합니다. 참고 : 뇌 행렬을 동결 BR 때문에하지 마십시오아인은 부착 할 수있다. </li><li> CO 2 질식 또는 자궁 전위에 의해 (8 주 이전) 마우스를 희생. <ol><li> 70 % 에탄올을 분사 한 후 날카로운 가위로 머리를 잘라. 머리를 고정하기 위해 단단히 머리의 양쪽을 잡고. 꼬리 - 주동이의 방향으로 중간 선에서 가위로 피부를 잘라. 이것은 두개골에서 피부의 완전한 제거를 촉진한다. </li><li> 지느러미 중간 선 위치에서 두개골과 뇌 사이의 집게를 삽입 먼저 두개골 (람다에 후부)의 꼬리 부분을 제거하고. 왼쪽 (또는 오른쪽) 정수리 뼈를 잡고 조심스럽게 벗겨. 다른 정수리 뼈의 제거에 대해이 절차를 반복합니다. 참고 : 시신경과 뇌의 수막 제거의 단선이 쉽게 두개골에서 뇌를 분리 할 수 ​​있습니다. </li><li> 차가운 PBS 버퍼 (25 ㎖)에 뇌를 전송하고 여분의 혈액을 제거하기 위해 두 번 씻어. </li></ol></li><li> 얼음과 엄마의 뇌 매트릭스로 뇌를 배치KE 관상 삭감 1mm 두께의 슬라이스를 얻었다. 35mm의 플라스틱 페트리 접시에 차가운 PBS로 뇌의 후방 부에 위치하는 SCZ 영역을 포함하는 뇌 조각 (1mm)으로 옮긴다. 주의 : 뇌 균열이 뇌 매트릭스의 중심선에 배치되도록, 관상 부의 평행 평면을 얻기 위해; 이 부분에서 불필요한 변형을 방지하는 것이 중요합니다. 참고 : 브레 그마에 2-3mm 후방에서 뇌 조각을 얻습니다; 이것은 SVZ에서 SCZ의 분리를 허용한다. </li><li> 저배율로 해부 현미경, 절곡 30G 바늘 6,9와 피질 및 해마의 백질 영역으로부터 SCZ를 마이크로 - 해부. 그런 다음, SCZ 위의 피질 영역을 제거합니다. 차가운 PBS없이 얼음에 35mm 플라스틱 페트리 접시에 슬라이스의 해부 SCZ 영역을 배치합니다. 주 : 성숙 뉴런을 포함하는 여분의 영역의 오염 및 aNSCs neurosphere 형성 B의 생존에 영향을 미칠 수있는ecause 그들은 aNSC 배양 조건에서 세포 사멸을 겪는다. </li><li> 구부러진 30 G 바늘을 사용하여 즉시 작은 조각으로 해부 조직을 잘라. 주 : 긴 시간이 SCZ 조직을 절개 필요한 경우 초핑 전에 차가운 PBS의 해부 조직 몰입. </li><li> 다시 일시 분해 완충액 1 ㎖와 다진 조직을 분해 완충액 2 ml를 함유하는 15 ㎖의 튜브에 조직을 전송하고, 37 ℃ 수조에 30 분 정도 배양한다. 참고 : 잘 혼합 튜브마다 10 분 흔들어. </li></ol> 3. 뇌량 밑 영역에서 파생 된 성인 신경 줄기 세포 배양 <ol><li> 소화 조직을 떼어 놓다 약간 튜브를 누른 다음 5 분 동안 145 XG에 튜브를 원심 분리기. 소화 버퍼 씻어 예열 N2 배지 1 ㎖로 상등액 재현 탁 분해 된 조직을 폐기하고, 부드럽게 검액에게 P1000 피펫을 사용하여 5 회 최대 피펫. 참고 : 오버 분쇄하여 좁은로피펫 팁은 세포 생존 및 후속 성장을 감소시킬 수있다. </li><li> 5 분 동안 145 XG에서 튜브를 원심 분리기. 상청액을 폐기 한 후, N2 배지 1 ㎖의 세포 펠렛을 다시 일시. </li><li> 비 코팅 된 6- 웰 플레이트에 N2 배지 1 ㎖를 제조하고이 용액의 최종 부피를 위해 현탁 한 세포를 가하여. </li><li> 각 웰에 EGF (20 NG / ㎖)과의 bFGF (20 NG / ㎖)를 추가합니다. 부드럽게 도금 셀에 추가 성장 인자를 혼합하여 손으로 6 웰 배양 접시를 흔들. 37 ° C, 5 % CO 2 배양기에서 6 웰 플레이트를 유지합니다. </li><li> 팔일 동안 매일 각 웰에 EGF (20 NG / ㎖)과의 bFGF (20 NG / ㎖)를 추가합니다. 모든 세 번째 날, 배지의 대략 2 mL의 부피를 유지하기 위해 N2 배지 200 μl를 추가한다. </li></ol> neurosphere를 같이하고 단일 플라이 문화에 NSCs의 4.과 Passaging <ol><li> neurosphere를 수집하고 새로운 15 ML 원뿔 튜브로 전송. 참고 : neurosphere를 수 (&gt; 50 μm의 직경) 체육를물론 한 번의 마우스 뇌의 SCZ에서 r은 SVZ의보다 작은 64.3 ± 7.31이었다 (190.5 ± 6.33) 9. </li><li> 하나의 세포로 neurosphere를을 해리 37 ° C의 물을 욕조에 5 분 해리 버퍼의 0.5 ml의와 neurosphere를 품어. 기본 neurosphere를 단일 세포로 해리와 neurosphere 또는 단일 층 문화 등 여러 통로를 통해 유지 될 수있다. 참고 SCZ-aNSCs가 neurosphere 배양 형식 단층 배양 포맷&gt; 10 배이지만 &lt;5 회 계대 수 있기 때문에 단층으로 SCZ-aNSCs neurosphere를 확대하여 우수하다. 주의 : SCZ-aNSCs 강한 응집을 나타내고, 하나의 세포로 해리를하기 어렵다. 따라서, neurosphere 문화 형식은 일상적인 세포 확장하지 않는 것이 좋습니다. </li><li> 부드럽게 5 분 동안 145 XG에 위 아래로 P1000 피펫 미만 5 회 원심 분리 튜브를 시료 용액을 피펫. 상층 액과 다시 스와 폐기N2 배지 1 ㎖로 neurosphere를 쓴다. 참고 : 세포 생존과 이후의 성장을 감소 할 수있는 좁은 피펫 팁으로 분쇄하여 이상. </li><li> 세포 현탁액 (10 μL) 및 0.4 % 트리 판 블루 용액을 1 혼합물, 다음에 hematocytometer를 생균 수를 계산 : 세포를 계산하는 일을 만든다. PLO / 라미닌 6- 웰 플레이트를 코팅 한 후, 각 웰에 대한 N2 배지 2 ml를 2.5 × 105 세포 / ml의 세포를 접시. 참고 : 세포 밀도의 변화가 자신의 상태 및 분화 가능성에 영향을 줄 수 있습니다. </li><li> 오일에 대한 성장 인자의 매일 치료 SCZ-aNSCs (2 ㎖, 20 NG / ㎖)을 유지하고, 이들 통로들을. </li></ol> 뇌량 밑 영역에서 파생 된 성인 신경 줄기 세포의 분화 (5) <ol><li> SCZ-aNSCs의 분화 성장 인자와 함께 1 ml의 N2 (20 NG / ㎖) 1 × 105 세포 / ml와 PLO / 라미닌 코팅 18mm 커버 슬립 위에 평판 aNSCs. </li><li> 다음날,세포가 단단히 커버 슬립에 부착 될 때, 성장 인자를 제거하기 위해 N2와 함께 성장 배지를 교환한다. </li><li> 6일 후, 세포 파편을 제거하고 면역 그들을 수정 PBS 1 ㎖에 분화 세포를 세척 하였다. 주 : BrdU의 증식은 세포의 신규 합성 DNA에 혼입 될 수있다. 필요에 따라서, BrdU의 (10 μg의 / ㎖) 세포를 고정하기 전에 세포 살에 첨가 될 수있다. </li></ol> 6. 면역 염색 성인 신경 줄기 세포와 차별화 된 전구 세포 <ol><li> 면역 염색을 위해, PBS로 세포를 세척하고, 실온에서 20 분 동안 4 % PFA로 고정합니다. 주의 : PFA는 매우 독성; 피부와 눈에 접촉을 피하십시오. </li><li> 의 4 % PFA를 제거 PBS로 3 회 고정 된 세포를 씻어 후 4 ° Cuntil가 면역 염색에 필요한에 저장합니다. </li><li> (3 % 소 혈청 알부민, 0.1 % 트리톤 X-100, 1X P 용액을 차단하여 커버 슬립에 세포를 부화실온에서 적어도 30 분 동안 BS). </li><li> 신선한 차단 솔루션의 기본 항체를 준비하고 4 ℃에서 하룻밤 샘플을 품어. <ol><li> aNSCs를 면역 염색 <ol><li> 안티 네 스틴 및 BrdU의 항 - 항체를 사용 aNSCs 얼룩. 고정하기 전에 aNSCs에 BrdU의 20 μg의를 추가하고 2 시간 동안 그들을 품어. 차단 단계를 수행하는 37 Cbefore에서 20 분 동안 2 N 염산을 사용하여 변성 단계를 수행합니다. </li></ol></li><li> 차별화 된 전구 세포를 면역 염색 : <ol><li> 차별화 된 전구 세포에 라벨을 방지 O4, 안티 bIII - 튜 불린, 및 안티 glial fibrillary 산성 단백질 (GFAP) 항체를 사용합니다. </li></ol></li></ol></li><li> PBS로 3 번 샘플을 세척하고, 형광 염료 (1 : 500)에 접합 된 이차 항체로 배양 한 30 분 동안 실온에서 용액을 차단한다. 그런 다음, PBS로 샘플을 3 회 반복한다. 참고 : 차의 호스트와 일치를 사용하여 이차 항체를항체. Hoechest33343 (1 : 2000)은 핵 염색에 사용됩니다. </li><li> 슬라이드 글라스에 장착 솔루션을 추가하고 설치를 진행합니다. 여러 파장에서 공 초점 현미경에 샘플을 관찰하고 이미지 : FITC (488 나노 미터)를 Cy3 (543 ㎚), Cy5에 (647 ㎚), 및 Hoechest33343 (405 nm의). </li></ol>

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B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Molecular neurobiology. 34, 153-161 (2006).</li><li>Suslov, O. N., Kukekov, V. G., Ignatova, T. N., Steindler, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+heterogeneity+demonstrated+by+molecular+phenotyping+of+clonal+neurospheres.">Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14506-14511 (2002).</li><li>Richards, L. J., Kilpatrick, T. J., Bartlett, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+generation+of+neuronal+cells+from+the+adult+mouse+brain.">De novo generation of neuronal cells from the adult mouse brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 89, 8591-8595 (1992).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Gritti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+and+fibroblast+growth+factors+behave+as+mitogenic+regulators+for+a+single+multipotent+stem+cell-like+population+from+the+subventricular+region+of+the+adult+mouse+forebrain.">Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 19, 3287-3297 (1999).</li><li>Wachs, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficacy+of+clonal+growth+and+expansion+of+adult+neural+stem+cells.">High efficacy of clonal growth and expansion of adult neural stem cells.</a> Laboratory investigation; a journal of technical methods and pathology. 83, 949-962 (2003).</li><li>Xiong, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+time+for+passaging+neurospheres+based+on+primary+neural+stem+cell+cultures.">Optimal time for passaging neurospheres based on primary neural stem cell cultures.</a> Cytotechnology. 63, 621-631 (2011).</li><li>Lee, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Matrigel-based+culture+for+expansion+of+neural+stem+cells.">Optimization of Matrigel-based culture for expansion of neural stem cells.</a> Animal Cells and Systems. 19, 175-180 (2015).</li><li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse.">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nature protocols. 7, 2005-2012 (2012).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mouse,+two+cultures:+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice.">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> JoVE (Journal of Visualized Experiments). , e51225 (2014).</li></ol>

The authors declare no conflicts of interest.

본 논문은 성인 마우스 SCZ에서 NSCs의를 생성하는 다양한 응용 프로그램을 유지하기 위해 상세한 프로토콜을 설명합니다. SCZ-NSCs을 정화하고 확장하는 데 필요한 체외 배양 시스템을 구축하기위한 세 가지 중요한 단계가 있습니다. 첫째, SCZ 영역 정확하게 다른 잠재적 신경성 영역 (도 1b)로부터 절개하는 것이 중요하다. SCZ 영역을 포함하는 두꺼운 정확한 섹션은 1mm 간격 뇌 매트릭스 수득 한 후, 미세 바늘이 다른 피질 영역 (도 1a)에서 SCZ의 미세 절개에 사용되었다. 비 NSCs의는 대뇌 피질과 같은 인접 조직에서 SCZ-aNSCs와 함께 배양하면, 치명적인 죽음에 부정적인 가능성 및 SCZ-NSCs의 16-18,22 분야 형성에 영향을 발생합니다. 꼬리 SCZ (정수리 2-3 밀리미터 후방)는 별개 SCZ-aNSCs를 생성하기위한 최선의 영역으로 확인되었다. 둘째, approp채취 및 계대 단계 riate 효소 처리는 세포의 높은 수율을 달성하는데 중요하다. 디스 파제 II와 파파인은 트립신보다 aNSCs을 분리하는 더 효과적이었다. 해리 버퍼 대신 트립신 계대에 대한 세포의 분리는 생존 (19)을 강화. 피펫과 기계 분리 및 분쇄 최소화해야한다. 셋째, 셀 스트레이너는 일반적으로 조직을 분해 한 후 세포 파편을 제거하기 위해 일차 배양 시스템에서 사용된다. 그러나 SCZ-aNSCs의 국산화로 인해,이 세포는 효소 소화와 기계적 분쇄에 의해 흰색 물질의 파손 후 얻을 수있다. 세포 여과기 여과 중에 세포의 상당한 양이 손실 될 것이다. 따라서, 셀 스트레이너를 사용하지 않고 SCZ-aNSCs를 배양하면 세포의 높은 수율을 얻을 수있는 좋은 방법입니다. neurosphere 문화와 단층 문화 모두 NSCs을의 유지, 신경의 한 제한을 적용 할 수 있지만,구 문화는 분할 무작위로 집계 할 수 있습니다 후 단일 NSCs의이 해리 것입니다. neurosphere를 다른 크기의 집계 결과. neurosphere의 크기가 특정 임계 값에 도달하면, neurosphere 인해 코어 (20)에서 영양 부족, 성장 인자, 및 산소로 이종 구조로 성장한다. 또한, 큰 크기 neurosphere를 쉽게 분리 완충액으로 해리되지 않고 낮은 세포 생존에 이르는 광범위한 기계적 분쇄와 이상 효소 처리 시간을 필요로한다. 따라서, 단층 배양 시스템은 복수의 통로를 통해 SCZ-aNSCs를 유지하도록 권장한다. 단층 배양 시스템에서 aNSCs 안정적 같은 조상 (타입 C와 A) (도 3a)와 같은 특정 세포로 분화없이 자발적 NSCs의 (유형 B)으로 유지된다. SCZ-aNSCs은 오랜 기간, 단층으로 &lt;A neurosphere 형식의 5 구절과&gt; 10 구절에 대한 계대 하였다. 이 단점입니다단층 배양 시스템은 장기 배양 21 시험관 NSCs의 제안을 유지 이전 결과와 istent. 그러나, 증식 속도는 감소하고 죽는 세포의 일부는 통로 (5) 이상 증가 하였다. 확장 계대가 능성 증가 염색체 이상 (22)와 신경 세포의 분화에 영향을 미칩니다. 따라서, 연장 된 계대 효과를 방지하기 위해서는 증식 및 분화를 분석 초기 통로 (&lt;통로 5) 세포를 사용하는 것이 권장된다. SCZ-aNSCs의 자기 갱신의 가능성이 SVZ-aNSCs과 유사하지만, 덜 신경 세포의 분화는 SCZ-aNSCs 9에 나타내었다. SVZ 및 치아 이랑 (DG)을 포함하여 성인 뇌의 신경 인성 영역에서 aNSCs를 분리하는 방법은, (22)를 설립했다. 이러한 프로토콜은 분리 및 체외에서 aNSCs의 재배를 추진했지만, 높은 수를 얻기 몇 가지 제한 사항이 있습니다세포. 대부분의 프로토콜은 마 과정에서 뇌 조직의 손실을 야기 할 수있는 뇌 조직 초퍼를 이용한다. 신경 인성 영역에서 aNSCs를 분리하는 또 다른 방법은 메스를 사용하여 두뇌를 통해 코로나 컷을 사용합니다. 이것은 SVZ의 미세 절개에 의해 또는 종 방향 균열 (23)을 따라 DG로 이어진다. 다른 뇌 영역의 존재는 다른 종류의 세포가 시험관 내에서 세포의 생존에 영향을 줄 수있는 aNSC 배양을 오염시킬 수있다. 현재의 프로토콜을 통해 여러 샘플을 실험군의 다양한 컨트롤을 포함하여 대 단일 실험 내에서 관리 할 수있다. 그것은 높은 셀 수율 다양한 뇌 영역에서 NSCs의 달성을 허용 로서도, 뇌 매트릭스를 이용 슬라이싱 것은 뇌 자르고 도구 우수하다. 또한 같은 뇌의 여러 지역 aNSCs의 비교를 가능하게한다. 내생 NSCs의 이식 및 엔지니어링은 considere있는되었습니다줄기 세포 치료를위한 가능한 전략으로서 D. 이를 위해 aNSCs의 특성에 대한 시험 관내 연구는 포괄적으로 탐구해야한다. 따라서, 시험 관내 배양 시스템에서 잘 특성화의 설립 NSCs의 적용을 촉진하는데 도움이 될 것이다. 적절한 조건에서 자기 갱신 및 여러 혈통 분화에 의해 입증이 문화 시스템에서, SCZ-aNSCs의 줄기 세포 특성을 잘 유지되었다. 따라서,이 배양 시스템은 생체 연구 및 치료 애플리케이션 SCZ-aNSCs의 확장에 사용될 수있다.

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>+L-glutamin, +Sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td>1X dilution</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociattion buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>04-942-078-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>WPI</td>
			<td>555229F</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Storz</td>
			<td>E3321-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain matrix (1mm)</td>
			<td>RWD</td>
			<td>68707</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-edged razor</td>
			<td>DORCO</td>
			<td>ST-300</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</td>
			<td>Deckglaser</td>
			<td>111580</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well dish</td>
			<td>SPL</td>
			<td>32012</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>6 well dish</td>
			<td>SPL</td>
			<td>32006</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>Coating materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Primary antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>Abcam</td>
			<td>ab2430</td>
			<td>rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>DCX</td>
			<td>Santa Cruz</td>
			<td>SC8066</td>
			<td>goat (1:500)</td>
		</tr>
		<tr>
			<td>Tuj1</td>
			<td>Sigma</td>
			<td>T2200</td>
			<td>rabbit (1:2000)</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>rat (1:1000)</td>
		</tr>
		<tr>
			<td>O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>mouse (1:500)</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>Rat (1:500)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td rowspan="12">1:500</td>
		</tr>
		<tr>
			<td>anti-mouse cy3</td>
			<td>Jackson</td>
			<td>715-165-151</td>
		</tr>
		<tr>
			<td>anti-mouse 647</td>
			<td>Jackson</td>
			<td>715-606-150</td>
		</tr>
		<tr>
			<td>anti-rabbit 488</td>
			<td>Alexa</td>
			<td>A21206</td>
		</tr>
		<tr>
			<td>anti-rabbit cy3</td>
			<td>Jackson</td>
			<td>711-165-152</td>
		</tr>
		<tr>
			<td>anti-rabbit 647</td>
			<td>Jackson</td>
			<td>711-605-152</td>
		</tr>
		<tr>
			<td>anti-goat 488</td>
			<td>Alexa</td>
			<td>A11055</td>
		</tr>
		<tr>
			<td>anti-goat cy3</td>
			<td>Jackson</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>anti-goat 647</td>
			<td>Invitrogen</td>
			<td>A21447</td>
		</tr>
		<tr>
			<td>anti-rat 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
		</tr>
		<tr>
			<td>anti-rat cy3</td>
			<td>Jackson</td>
			<td>712-166-150</td>
		</tr>
		<tr>
			<td>anti-rat 647</td>
			<td>Jackson</td>
			<td>712-605-153</td>
		</tr>
		<tr>
			<td>Immunostaining materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Millipore</td>
			<td>82-100-6</td>
			<td>3% BSA with 0.1% Triton X-100 in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>usb</td>
			<td>22686</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Biosesang</td>
			<td>P2031</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechest33342</td>
			<td>Life Technology</td>
			<td>H3570</td>
			<td>Dye for staining nuclei</td>
		</tr>
	</tbody>
</table>

isolation and culture of adult neural stem cells from the mouse subcallosal zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

알 수없는 신경성 지역에서 aNSCs을위한 문화 시스템을 정의하는 것은이 세포를 이해하기위한 뇌 수리 (12)에 자신의 잠재적 인 사용을 개발하기위한 필수적이다. 다른 발달 단계에서 또는 다른 지역에서 NSCs을 다르게 3,4 행동 것으로 알려져있다. 최근에는 SCZ 유래 세포는 SVZ 유래 세포 7-9에 비해 생체 내 및 시험관 내에서 신경 세포의 분화, 차동 전위를 나타내는 것으로보고되었다. 따라서 정확하게 각 신경성 영역을 분리하기 위해 상기 SCZ 포함 뇌 조각은 1mm 뇌 매트릭스 (도 1A)를 사용하여 절개 하였다. 성장 인자와 함께 배양 8 일 후, SCZ로부터 유도 aNSCs는 세포이어서 (도 1b)를 유지할 수있는 neurosphere를을 형성 할 수있다. neurosphere를에 NSCs의 서브 세트가 될 수 있기 때문에 SPOntaneously 단층 배양 시스템은 또한 SCZ-aNSCs의 비교적 균일 한 인구의 유지 보수를 위해 도움이된다, (13) 차별화. 성장 인자 및 트립신으로 분해 효소 쉽게 neurosphere 14, 15의 깊은 내부에 침투하지 않습니다. 단층 문화 NSCs의 확장에 더도 조건을 제공합니다. 기본 neurosphere를에서 SCZ-aNSCs는 소화 버퍼 처리하여 단일 세포로 분해되었다. 어느 날 세포 파종 후, aNSCs는 코팅 된 플레이트와 전시 세포 증식 (그림 2A)에 부착 하였다. 확산의 자신의 힘을 확인하려면 BrdU의 미디어에 추가되었습니다. 2 시간 동안 BrdU를 배양 한 후, 세포를 용이 SCZ-aNSCs 활발히 증식 및 주요 특성을 유지하고 있음을 나타내는, 항 BrdU의 (증식 마커), 항 네 스틴 항체 (신경 줄기 세포 마커) 염색 하였다 줄기 세포 (도 2B). 따라서, 그들은 EXH하지 않았다아이 비트 차별화 된 (일시적으로 증폭 세포에서 발현) 같은 EGFR과 같은 세포 및 (neuroblasts로 표현) DCX (그림 2C)에 대한 마커. SCZ-aNSCs의 다수의 분화 잠재력을 확인하기 위해, 성장 인자를 배양 배지에서 제거 하였다. 육일 후, 세포는 분화 된 세포에 대한 다양한 업체와 면역 염색 하였다. 사용 된 다른 aNSCs의 자손, 마커 신경 세포에 대한 (Tuj1), 성상 교세포 (GFAP), 및 희소 돌기 아교 세포 (O4)를 전시하는 단계; 이들 세포 유형은 모두 SCZ-aNSCs에서 (도 3)을 생성 하였다. <img alt="그림 1" src="/files/ftp_upload/54929/54929fig1.jpg" /> 그림 1 : neurosphere의 SCZ 영역 및 형성의 분리. 성체 마우스의 뇌에서 SCZ 영역의 해부 (A) 절차. 문화 SCZ-내지 An희주는 8 주령의 마우스의 뇌는 뇌 매트릭스 (1mm 간격)에 배치됩니다. (빨간 점선) 해부 된 SCZ 영역 (브레 그마 2-3mm)를 포함, 1mm의 뇌 조각을 절편 후. (B) Neurosphere 형성. 체외 문화, 차 neurosphere를 (통로 0) 이후 여덟 일을 형성하고 계대 하였다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54929/54929fig2.jpg" /> 그림 2 : 단일 층 문화와 SCZ-aNSCs의 유지. (A) 해부 세포를 파종 후 어느 날, SCZ-aNSCs 부착 및 단층 방식 (왼쪽)에 코팅 된 요리로 확장되었다. 사흘 유지 후 SCZ-aNSCs의 수 (오른쪽) 증가 하였다. (B)의 BrdU (빨강, 세포 증식 마커), 네 스틴 (녹색, 신경 줄기 세포 마커) 및 Hoechest33343 (블루, 핵에 대한 마커)에 면역 염색. 신경 줄기와 면역 염색 (C) / 전구 세포 마커 네 스틴 (빨간색, B 형 신경 줄기 세포 마커), EGFR (녹색, C 형 과도 증폭 세포 마커), 및 DCX (파란색, A 형 마커 neuroblasts). 핵은 Hoechest33343 (흰색)으로 대조 하였다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54929/54929fig3.jpg" /> 그림 3 : SCZ-aNSCs에서 분화 된 세포의 면역 염색. 분화 마커 Tuj1 (녹색, 미성숙 신경 세포 마커), O4 (빨강, 희소 돌기 아교 세포 마커), 및 GFAP와 면역 염색하면 (소리흐름, 성상 세포 마커). 확대 이미지는 세트로 표시됩니다. Hoechest33343 (파란색) 카운터 오염성 핵 사용 하였다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54929/54929fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.0K 조회수.  Korea University College of Medicine. Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described. 

비디오: Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

절연 및 Neurosphere 어세를 사용하여 성인 마우스 신경 줄기 세포의 확장

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

연구

신경과학

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

인간의 모공에서 신경 크레스트 줄기 세포의 분리와 문화

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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 ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

성인 쥐의 뇌의 기존 및 비 인습적 인 지역에서 신경 줄기 세포 성장

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

배아 전뇌에서 내피 세포의 분리 및 문화

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

하나의 마우스, 두 문화 : 분리 및 성인 신경의 문화가 개인 마우스의 두 신경 인성 영역에서 줄기 세포

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

셀 성인 마우스 뇌실 영역에서 신경 줄기 및 전구 세포의 정렬과 세포주기 역학의 라이브 이미징

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.