우리는 비디오 지원 마크 워즈 니 악을 인정하고 싶습니다. 우리는 또한 culturing 쥐 NC 세포의 원래 프로토콜 UT 남서에서 숀 모리슨을 인정하고 싶습니다. 이 작품은 밴더빌트 대학 메디컬 센터 아카데미 프로그램 지원을 지원하고 NIH (HD36720과 HD036720-11S109)와 PAL에 AHA 11GRNT7690040 남구로 AHA (0615209B)와 NIH (NS065604)에서 predoctoral 장학금 및 ERP에서 교부금에 의해 있었다 NIH 연수 보조금 T32HD007502에 의해 지원.

1. 준비 플레이트 <ol><li> 항상 무균 기술을 사용합니다. </li><li> Dulbecco의 PBS (dPBS)에서 3.3 ML의 최종 볼륨으로 인간의 플라즈마 FN 주식의 100 μL를 diluting하여 fibronectin (FN)를 준비합니다. 최종 농도가 30 μg / ML이며이는 1 주일 동안 4 ° C에 저장할 수 있습니다. </li><li> FN 솔루션 무균 조직 배양 네 잘 접시의 각 우물의 바닥을 커버하고 15 분 동안 앉아 보자. 전체 표면이 덮여 있는지 확인하십시오. 이 시간 (2 단계와 3 단계) 동안 미디어를 확인합니다. </li><li> 솔루션을 FN과 접시가 건조하도록 제거합니다. 부드럽게 500 μL DMEM, 제거 DMEM으로 우물 린스, 그리고 자기 갱신 500 μL (SR) 매체 (아래 참조)에 추가합니다. CO 2 5 %를 포함한 humidified 배양기에서 37 ° C에서 품어. 접시는 사용하기 전에 건조되도록 해부하기 전에이 약 한 시간을 완료합니다. </li></ol> 2. SR 매체를 준비 <ol><li> vagal 및 트렁크 노스캐롤라이나의 문화에 대한열 배아 (약 쓰레기, 마우스 라인의 유전적 배경에 따라)에서, SR 매체의 25 ML을 준비합니다. 12.5 ML 낮은 포도당 DMEM, 7.5 ML Neurobasal 중간, 25 μL retinoic 산성 (117 μm의 최종 농도), 25 μL 2 - 메르 캅 토 에탄올 (50 MM 최종 농도)를 결합. 잘 섞는다. </li><li> 3.75 ML 여자는 배아 엑기스, 250 μL N이 소금 보충, 500 μL B27 보충제 250 μL 페니실린 - 스트렙토 마이신 (1 % 최종 농도)를 추가합니다. 0.22 μm의 필터를 통해 매체를 필터링합니다. </li><li> 10 μL 멸균 IGF1 (20 μg / ML 최종 농도)와 20 μL 멸균 bFGF (20 μg / ML 최종 농도)를 추가합니다. 반전으로 섞는다. 4 ° C.에 저장 </li></ol> 3. 워시 매체를 준비 <ol><li> 의 경우 약 10 배아는 세척 매체의 50 ML을 준비합니다. 35 ML 낮은 포도당 DMEM, 15 ML Neurobasal 매체, 500 μL 페니실린 - 스트렙토 마이신 (1 % 최종 농도)와 50 밀리그램 BSA이 결합되어 있습니다. 0.22과 무균 필터μm의 필터. </li></ol> 4. Collagenase / dispase 준비 <ol><li> 100 MG / ML collagenase / dispase 5 ML의 dPBS 50 μL를 추가합니다. 잘 섞는다. </li><li> 0.2 μm의과 주사기 필터는 12 잘 접시 세 우물 각각에 1.5 ML을 필터링하고 추가합니다. 나머지 우물에 피펫 약 1 ML 세척 매체. 해부 조직을 소화 준비 때까지 얼음 전체 접시를 보관합니다. </li><li> 멸균 면도날과 p20 및 P1000 피펫 팁 필터의 조언을 자르고. 끝에만의 beveled 가장자리 아래 잘라. P1000 오프 절단은 p20이 고립된 조직의 조각을 전송하는 데 사용됩니다 동안 전체 배아를 전송하는 데 사용됩니다. </li></ol> 5. 9.5 dpc의 태아에서 Vagal 및 간선 신경 튜브를 분리 <ol><li> 시간이 초과 임신의 경우 질 플러그 댐은 아침 플러그가 관찰되고 정오에 0.5 dpc로 간주됩니다. 9.5 dpc에서 자궁을 희생하고 제거합니다. </li><li> 자궁에서 decidua를 제거하고 부드럽게 Rdecidua에서 배아를 emove. 일단이 프로토콜과 경험, 멸균 dPBS에서 한 번에 3-4 9.5 dpc의 배아를 분리. 멸균 기술과 소독을 해부 장비를 사용하십시오. 악기 autoclaving, 열 살균 또는 에탄올에 부화하여 소독 수 있습니다. </li><li> 앞부분은 vagal 노스캐롤라이나의 경우 : 인슐린 바늘을 사용 중반 귀의 placode에 신경 튜브를 잘라. 4 번째 체절의 후부 가장자리에 다시 잘라. pharyngeal 아치와 가슴을 제거하는 신경 튜브로 복부 조직을 손질. </li><li> 트렁크 NC의 경우 : 인슐린 바늘을 사용하여 somites 16-22 (또는 배아 이전 22 체절 무대보다 발달면 마지막 체절) 사이 신경 튜브의 부분을 제거합니다. </li><li> 난황 및 genotyping에 대한 남은 배아 조직을 유지. </li></ol> 6. 비 신경 Ectoderm 및 Mesoderm 제거 <ol><li> 십분 동안 상온에서 collagenase / dispase로 세그먼트를 포함하는 신경 튜브를 배치의. 즉시 세탁 매체에 씻는다. </li><li> 멸균 dPBS로 조직을 반환합니다. 멸균 인슐린 바늘을 사용하여 부드럽게 조직에서 비 신경 ectoderm를 제거하고 멀리 신경 튜브에서 somites을 분리. 체절 조직에서 mesoderm의 마지막 남은 부분 잘라내기 다운 p20 팁을 사용하여 부드럽게 triturating하여 제거할 수 있습니다. 신경 튜브를 손상하지 않도록주의하십시오. 가루약 중에 밀접하게 이것을 관찰. </li><li> 세차 매체의 두 번째 및 세 번째 삼십초 세차를 통해 고립된 신경 튜브를 놓습니다. </li><li> SR 매체에 한 번 씻어, 그리고 (단계 1.3) 이전에 준비되었다 FN-코팅도의 중심에 격리된 신경 튜브를 넣습니다. 멸균 물을 접시와 hypoxia 실 ...을 축이다. hypoxia 챔버로 접시를 놓고 3 % O 2 (1 % 혼합 O 2, 6퍼센트 CO 2, 93% N 2를 포함하는 탱크를 사용)로 혼합 가스와 함께 챔버를 플러시. </li><li> 항상 THA을 보장하기 위해 매우 신중 챔버 처리하지마 explants은 그대로이며, 우물의 중심에 남아 있습니다. </li><li> 37 품어 ° C. 단계 5-6 개요는 그림 1에 표시됩니다. </li></ol> 7. 신경 튜브의 제거 <ol><li> 부화 후 24 시간 후, 부드럽게 떨어져 불임이 인슐린 바늘을 사용하여 마이 그 세포로부터 신경 튜브의 가장자리를 괴롭히고하여 신경 튜브를 제거합니다. 제거하고 단계 6.2에서와 같이 멸균 p20 인하를 사용하여 매체에서 신경 튜브를 버리고 신선한 SR 매체 (그림 2)와 매체를 교체하십시오. 이것은 가장 쉽게 거꾸로 현미경을 사용하여 수행됩니다. </li></ol> 8. 대표 결과 hypoxic 조건에서 ° C, NC 세포는 거의 순수한 인구 (그림 3A)에서 신경 튜브에서 떨어져 마이그레이션한 37에서 부화 후 24 시간에 따라. 때로는 이상적인 문화보다 강력한 outgrowths를 얻을 수 없습니다. 예를 들어, 그 24 시간 이내에 t 가능그는 신경 튜브 자체적으로 웅크리고있다되며 엔씨는 신경 튜브 (그림 3B)에서 떨어져 마이 그 레이션되지 않습니다. 간혹 신경 튜브 fibronectin 코팅 접시에 부착하지 않습니다. 우리의 경험에서는, 하위 최적의 NC 마이 그 레이션 또는 신경 튜브의 부착에 문제가 악영향 각각 normoxia 조건이나 fibronectin의 농도에 의해 영향을받을 수 있습니다. collagenase / dispase의 효소 활동이 배치 및 소화 시간에 따라 약간의 차이가 적절하게 조정해야하지만 15 분 이상 조직을 소화하지 않습니다. collagenase / dispase의 조직을 포함하는 신경 튜브 Overdigestion 또한 결함 outgrowths집니다. 체절 조직이 쉽게 collagenase / dispase에서 부화 후 신경 튜브에서 제거되지 않은 경우 신경 튜브는 열명 분 이상 동안 incubated 수 있습니다. 때로는 신경 튜브 기판에 연결되지 않습니다. 이 경우 fibronec을 두 번 확인주석 농도와 hypoxia 조건. normoxic 조건은 문화, 야생 형 노스캐롤라이나에 사용할 수 있지만, hypoxic 조건보다 밀접하게 생체내 환경 29,30 년을 모방. 우리의 경험에서, hypoxic 조건이 때 culturing 돌연변 노스캐롤라이나 중요했다. 예를 들어, Foxd3 돌연변 노스캐롤라이 normoxic 조건에서 배양해 때, 트렁크 노스캐롤라이 컨트롤에 비해 크게 감소 셀 파생물했다. explants은 hypoxic 조건 (그림 4)에서 배양해 때 가지 크기의이 불균형이 제거되었습니다. 또한, 야생 형 신경 튜브 explants가 normoxia에서 배양해 때, caspase-긍정 세포의 숫자 (데이터가 표시되지 않음) hypoxia에서 배양해 유사한 explants의 사실을 늘어납니다. hypoxia에있는 모든 NC 문화를 유지함으로써, 비교가보다 쉽게​​ 컨트롤의 역학과 돌연변이의 문화 사이에 만들 수 있습니다. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="그림 1" /> <strong&gt; 그림 1. 노스캐롤라이나 절연의 전체 개략도. ) 배아에서 관심 지역을 해부. B) collagenase / 십분 (15 분 초과하지 않는)에 대한 dispase에 신경 튜브를 소화. C) 세탁 매체에 씻는다. D) 비 신경 ectoderm 및 mesoderm 멀리 해부. EF)은 세탁 매체에 두 번 씻는다. 자동 갱신 매체에서 G) 플레이트. 3퍼센트 O 2 hypoxic 조건에서 37 ° C에서 품어. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="그림 2" /> 그림 2. explant에서 신경 튜브의 Stepwise 제거. 신경 튜브가 아닌 노스캐롤라이나 세포와 오염을 방지하기 위해 후 24 ~ 48 시간 제거해야합니다. ) 신경 튜브와 NC의 파생물 (실선) 사이의 경계를 확인할 수 있습니다. B) 인슐린 바늘로 신경 튜브의 가장자리를 따라 잘라. C) 신경 튜브를 취소하고 새로운 자기 갱신 매체와 매체를 교체하십시오. 점선은 자연적인 진전의 정도를 나타냅니다. 약어 : NT, 신경 튜브. 그림 3. 대표적인 결과의 예. hypoxic 조건에서 자기 갱신 배지에서 24 시간 배양 후) 일반 explant의 파생물은 (점선)은 자연적인 진전의 정도를 나타냅니다. 나) 문화의 48 시간 후 노스캐롤라이나의 파생물의보기를 확대했습니다. 낮은 가지 수율과 C) 덜 이상적인 문화. A와 C는 동일한 조건에서 배양해되었다. 이미지는 문화 견고에서 자연 범위를 보여줍니다. 이것은 collagenase / dispase digestions에 신경 튜브 절연, FN의 농도, hypoxic 조건과 시간의 효율성에 의해 영향을받을 수 있습니다. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="그림 4" /> 그림 4. normoxia 대 hypoxia에서 NC의 explant 문화의 체외 분석합니다. normoxic 문화 조건에서 48 시간 후 신경 튜브 explants에서 마이 그 레이션 컨트롤 (야생 타입) 노스캐롤라이나 세포. 대조적으로, Foxd3 돌연변이노스캐롤라이나 크게 normoxia의 세포 outgrowths을 (NC의 outgrowths의 붉은 외곽선 표시 에지) 감소했다. 비교 explants은 hypoxic 조건 하에서 재배 때, Foxd3 돌연변이 노스캐롤라이나의 explants는 후속 분석을 수 있도록 컨트롤 comparably되었다. 참고이 문제는 생체내의 Foxd3 돌연변이 노스캐롤라이나의 동작과 잘 상호.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+property+of+postmigratory+cranial+neural+crest+cells+and+their+utility+in+alveolar+bone+regeneration+and+tooth+development.">Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development.</a> Stem Cells. 27, 866-877 (2009).</li><li>Biernaskie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKPs+derive+from+hair+follicle+precursors+and+exhibit+properties+of+adult+dermal+stem+cells.">SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.</a> Cell Stem Cell. 5, 610-623 (2009).</li><li>Hagedorn, L., Suter, U., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P0+and+PMP22+mark+a+multipotent+neural+crest-derived+cell+type+that+displays+community+effects+in+response+to+TGF-beta+family+factors.">P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors.</a> Development. , 126-3781 (1999).</li><li>Heanue, T. A., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+and+isolation+of+enteric+nervous+system+progenitors+using+Sox2.">Prospective identification and isolation of enteric nervous system progenitors using Sox2.</a> Stem Cells. 29, 128-140 (2011).</li><li>Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+in+reduced+levels+of+oxygen+promotes+clonogenic+sympathoadrenal+differentiation+by+isolated+neural+crest+stem+cells.">Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.</a> J. Neurosci. 20, 7370-7376 (2000).</li><li>Ito, K., Morita, T., Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+clonal+analysis+of+mouse+neural+crest+development.">In vitro clonal analysis of mouse neural crest development.</a> Developmental biology. 157, 517-525 (1993).</li><li>Etchevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+chick,+mouse+or+human+neural+crest+cells.">Primary culture of chick, mouse or human neural crest cells.</a> Nat. Protoc. 6, 1568-1577 (2011).</li><li>Mundell, N. A., Labosky, P. 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+crest+stem+cell+multipotency+requires+Foxd3+to+maintain+neural+potential+and+repress+mesenchymal+fates.">Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates.</a> Development. 138, 641-652 (2011).</li><li>Gammill, L. S., Gonzalez, C., Gu, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+trunk+neural+crest+migration+requires+neuropilin+2/semaphorin+3F+signaling.">Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling.</a> Development. 133, 99-106 (2006).</li><li>Gammill, L. S., Gonzalez, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+2/semaphorin+3F+signaling+is+essential+for+cranial+neural+crest+migration+and+trigeminal+ganglion+condensation.">Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation.</a> Dev. Neurobiol. 67, 47-56 (2007).</li><li>Kasemeier-Kulesa, J. C., Bradley, R., Pasquale, E. B., Lefcort, F., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph/ephrins+and+N-cadherin+coordinate+to+control+the+pattern+of+sympathetic+ganglia.">Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia.</a> Development. 133, 4839-4847 (2006).</li><li>Osborne, N. J., Begbie, J., Chilton, J. K., Schmidt, H., Eickholt, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin/neuropilin+signaling+influences+the+positioning+of+migratory+neural+crest+cells+within+the+hindbrain+region+of+the+the+chick.">Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the the chick.</a> Dev. Dyn. 232, 939-949 (2005).</li><li>Schwarz, Q., Maden, C. H., Vieira, J. M., Ruhrberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+1+signaling+guides+neural+crest+cells+to+coordinate+pathway+choice+with+cell+specification.">Neuropilin 1 signaling guides neural crest cells to coordinate pathway choice with cell specification.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 6164-6169 (2009).</li><li>Simon, M. C., Keith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxygen+availability+in+embryonic+development+and+stem+cell+function.">The role of oxygen availability in embryonic development and stem cell function.</a> Nat. Rev. Mol. Cell Biol. 9, 285-296 (2008).</li><li>Ivanovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+or+in+situ+normoxia:+The+stem+cell+paradigm.">Hypoxia or in situ normoxia: The stem cell paradigm.</a> J. Cell Physiol. 219, 271-275 (2009).</li></ol>

우리는 공개 할게 없다.

주의이 방법의 성공을 보장하기 위해 태아의 발달 단계에 납부하여야한다. 초기 마우스 배아의 somites을 계산하는 것은 쓰레기 내에서 배아와 일치하고 격리를 위해 신경 튜브의 정확한 영역을 결정하는 단계에 대해 모두 중요합니다. 배아 사이에 하나 또는 두 개의 somites의 변형 실시한 실험의 해상도에 따라 개발시기의 합리적인 범위 내에 있습니다. 9 9.5 dpc 사이의 태아 17 및 25 somites 사이가됩?...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> DMEM (낮은 혈당) </td><td> Gibco / Invitrogen </td><td> 11,885 </td><td></td></tr><tr><td> Neurobasal 중간 </td><td> Gibco </td><td> 21,103 </td><td></td></tr><tr><td> BSA </td><td> 시그마 </td><td> A3912-10G </td><td></td></tr><tr><td> dPBS </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BD Biosciences </td><td> 354,037 </td><td> -20 ° C. 50 μg / ML aliquots에 저장 </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354,060 </td><td> -20 ° C. 25 μg / ML aliquots에 저장 </td></tr><tr><td> Fibronectin </td><td> Gibco </td><td> 33016-015 </td><td> 1mg/mL 알에 저장-20 ° C.에 iquots </td></tr><tr><td> Retinoic 산 </td><td> 시그마 </td><td> R2625 </td><td> -20 ° C.에 에탄올에 reconstituting 후 35 μg / ML aliquots에 저장 </td></tr><tr><td> 2 - 메르 캅 토 에탄올 </td><td> 시그마 </td><td> D-5637 </td><td></td></tr><tr><td> N이 보충 </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 보충제 </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip 0.22 μm의 필터 </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0.20 μm의 필터 </td><td> 코닝 </td><td> 431,219 </td><td></td></tr><tr><td> 주사기 (여과 용) </td><td> BD Biosciences </td><td> 301,604 </td><td></td></TR&gt; <tr><td> 네 잘 접시 </td><td> 써모 피셔 과학 </td><td> 176,740 </td><td></td></tr><tr><td> Collagenase / Dispase </td><td> 로슈 </td><td> 269​​ 638 </td><td> 활동은 배치에 따라 다릅니다. -20 ° C. 100 밀리그램 / ML aliquots에 저장 </td></tr><tr><td> 인슐린 바늘 (29 ½ 게이지) </td><td> Becton 딕슨 </td><td> 309,306 </td><td></td></tr><tr><td> Hypoxia 상공 회의소 </td><td> Billups-로텐버그 </td><td></td><td></td></tr><tr><td> 산소 분석기 </td><td> Billups-로텐버그 </td><td></td><td></td></tr><tr><td> 집게 # 5 </td><td> 파인 과학 도구 </td><td></td><td> 자궁과 decidua을 제거하십시오. </td></tr><tr><td> 트립신-EDTA (에틸렌 다이아 민 테트라 초산) (0.25 %) </td><td> Gibco </td><td> 25,200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

배아 신경 능선 (NC)는 신경 튜브 등의 측면에서 유래 multipotent 전구 인구 mesenchymal 전이 (EMT)로 상피를 겪습 다양한 세포 유형에 1-3로 상승을주는, 태아에 걸쳐 마이 그 레이션합니다. NC는 또한 표적 장기 4-6의 분화와 성숙에 영향을주는 독특한 능력이있다. 체외에서 explanted 때, 노스캐롤라이나의 progenitors 자기 갱신을 받다, 마이 그 레이션과 뉴런, glia, 평활근 세포, 연골 및 뼈 포함하여 조직 유형의 다양한으로 차별화. NC의 multipotency 처음. 조류 신경 튜브 7-9의 explants에서 설명한 NC 세포의 체외 절연에서 증식, 이주 및 multipotency 포함하여 노스캐롤라이나 역학 연구를 용이하게되었다. 조류 및 쥐의 시스템의 추가 작업은 배아 10에 다시 이식하면 explanted 노스캐롤라이나 세포들이 노스캐롤라이나 잠재력을 유지 것을 증명-13. 이러한 고유 셀룰러 속성 explanted NC의 progenitors에 보존되기 때문에 신경 튜브 explant 분석은 체외에서 노스캐롤라이나를 공부를위한 매력적인 옵션을 제공합니다. 포유류의 노스캐롤라이나의 더 나은 이해를 달성하기 위해 여러 방법은 NC의 인구를 분리하기 위해 고용되었다. NC-파생 progenitors이 포스트 철새 NC의 progenitors 11,14-20의 역학을 연구하는 배아와 성인 모두에서 사후 철새 위치에서 배양해 수있다 그러나 그들은 신경 튜브에서 이민으로 노스캐롤라이나의 progenitors의 고립은 최적의 보존을 제공 NC 잠재력 전지 및 철새 속성 13,21,22. 일부 프로토콜은 특정 progenitors 11,13,14,17위한 풍부한 NC 인구를 분리하는 형광 활성화된 셀 정렬 (FACS) 채용. 그러나, 초기 단계의 배아로 시작하는 경우, 분석을위한 적절한 셀 번호는 초기 노스캐롤라 populatio의 절연 복잡 FACS로 취득하기가 어렵습니다각각의 배아에서 NS. 여기서는 Wnt1-Cre 활성화 혈통 기자 23 일 기준으로 약 96% 순수 노스캐롤라이나 인구에 FACS 및 결과에 의존하지 않는 방식을 설명합니다. 여기에 제시된 방법은 쥐의 노스캐롤라 11,13의 문화에 최적화된 프로토콜에서 적응하고 있습니다. 이 프로토콜 이전 방법에 비해의 장점은 1) 세포는 FACS)가) 비교적 순수한 노스캐롤라이나 인구, 3을 얻기 위해 필요하지 않습니다 premigratory 노스캐롤라이나 세포가 격리되고 4) 피더 레이어 2에서 재배되지 않는 결과를 쉽게임을 있습니다 계량. 또한이 프로토콜은 서로 다른 유전자 조작과 노스캐롤라이나 특성의 학습을 촉진, 모든 돌연변이 마우스 모델에서 NC의 분리에 사용될 수 있습니다. 이 방법의 한계는 노스캐롤라이 노스캐롤라이나 2,24-28의 생존, 이주 및 분화에 영향을 미치는 것으로 알려져 배아의 맥락에서 제거되는 것입니다.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

신경 튜브의 배아 신경 능선의 분리가의 사용을 용이하게<em&gt; 체외에서</em&gt; 마이 그 레이션, 자동 갱신, 그리고 신경 능선의 multipotency를 공부를위한 방법.

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 ...

isolation-culture-neural-crest-cells-from-embryonic-murine-neural

Research

JoVE Journal

Neuroscience

17.1K 조회수.  Vanderbilt University Medical Center. 신경 튜브의 배아 신경 능선의 분리가의 사용을 용이하게 체외에서 마이 그 레이션, 자동 갱신, 그리고 신경 능선의 multipotency를 공부를위한 방법.

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

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 ...

연구

신경과학

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Murine 신경 크레스트 줄기 세포의 배양 칙의 배아 추출 생산

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

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 ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

운동에 의해 변조된 뉴런의 생리, 형태학의 및 Neurochemical 특성화

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

마우스 배아 전체 문화 신경 튜브 폐쇄

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

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 ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

전체 마운트 근육 분석에 의한 재현성 마우스 좌골 신경 크러시와 중생의 후속 평가

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

크로스 종 이식에 의한 신경 크레스트 마이 그 레이션과 차별화 분석

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

쥐 배아 신경 세포의 분리 및 문화 : 빠른 프로토콜

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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 ...