HD054347와 VLS에 NS063997 - 01 :이 작품은 국립 보건원에 의해 부분적으로 지원되었다. 이 작품은 또한 건강 계약 # C024324 VLS로의 뉴욕 국무부를 통해 엠파이어 스테이트 줄기 세포 기금에 의해 부분적으로 지원되었다. 의견 여기 표현은 전적으로 저자의 견해이며, 반드시 엠파이어 스테이트 줄기 세포위원회, 건강의 뉴욕 국무부, 또는 뉴욕주 분들을 반영하지 않습니다. VLS는 도리스 듀크 임상 과학자 발달 수상자입니다. 또한 안티 - O1, 안티 - O4 항체 자신의 선물을 교수 티모시 Vartanian 감사합니다.

1. 해부 및 신경 줄기 세포 문화의 유지 관리를위한 솔루션 및 자료 준비 <ol><li> 미리 100ml 해부 매체 (넉아웃 DMEM/F12, Invitogen)을 준비하고 냉동. </li><li> Prepare100 ML 문화 매체 (줄기 프로 NSC SFM, Invitrogen)와 37 ° 보관 물을 욕조에 C. </li><li> 세포의 장기 cryopreservation에 대한 셀 - 냉동 매체 (녹아웃 DMEM/F12 +10 % FBS + 5% DMSO)를 준비합니다. </li><li> 원하는 경우, 조직 고정을위한 4 % paraformaldehyde (PFA)를 준비합니다. </li><li> 핸들과 멸균, autoclaved 포셉와 메스 블레이드는 절개에 사용됩니다. </li><li> 피펫 총 10 ML 전송 pipettes, 그리고 해리 40 μm의 셀 스트레이너 (BD 팰컨 352,340)를 별도로 설정합니다. </li><li> 해부 여러 10cm 문화 요리 (BD), 해리 50 ML의 원심 분리기 튜브 (BD), 냉동 세포 조직 보관 및 냉동 튜브 (BD)에 대한 1.5 ML 원심 분리기 튜브를 따로 설정합니다. </li></ol> 2. 인간 태아 뇌에서 인간 신경 줄기 세포를 분리 <ol><li> 즉시 태아의 종료 후 가능한 조직의 적출하는 것은 절차의 성공에 필수적인 요소입니다. 선택 절차를 보려면 샘플을 얻기 위해 타이밍은 태아의 죽음 이후 시간을 최소화하기 위해 않도록 사전에 준비하실 수 있습니다. 개념의 제품은 2 시간 게시 절차 내에 수확하지만, 이상 분 이내에 선택 절차에 형성될 수 있습니다. 태아 조직은 종종 조각입니다. 그러나, 일반적으로 두뇌의 상당 부분은 시각적인 신원 확인을 위해 그대로 유지됩니다. 임신 성 연령 (GA)의 제한은 법정 법률에 의해 결정되지만 18-22주 GA 사이에이 프로토콜을 사용하여 수행되었습니다. </li><li> 태아 두뇌는 얼음처럼 차가운 마네 DMEM/F12 솔루션을 포함하는 10 ML 페트리 접시에 배치됩니다. 해부 학적 경계표에 의해 피질의 다른 부분을 식별합니다. 전두엽과 parieto - 뒤통수 cortices에 대한 경계는 중앙 고랑과 실비안 균열 (fissure)의 추정 교차로에서 지향하고 있습니다. 뇌실은 그대로 유지하고 손상을 확인하고, 중앙 고랑에 앞쪽에 전두엽 피질로부터 수술 블레이드와 실비안 균열 (fissure)의 경계를 따라 조직을 해부하다. </li><li> 전두엽 피질의 분리 블록에서 잔여 혈액과 meninges를 제​​거합니다. 샘플이 충분 품질 경우, 다양한 목적을 위해 여러 개의 작은 샘플로 블록을 해부하다에 이상적입니다 : 섹션 (PFA, 4 % paraformaldehyde에 고정) 및 단백질 / mRNA assays (빠른 -80 ° C에 냉동). </li><li> 50 ML의 원심 관에 선택한 뇌 블록을 전송하고, 조직의 볼륨의 약 3 시간에 얼음처럼 차가운 마네 DMEM/F12 솔루션을 추가합니다. 부드럽게 10 ML 전송 피펫과 기계 pipetting하여 조직을 떼어 놓다 모든 조직 때까지 (일반적으로 20-30 회) 조각되고, 그리고 단일 또는 가까운 단일 세포를 얻는 40 μm의 셀 스트레이너 (BD 팰컨 352340)를 통해 세포를 필터링 정지. </li><li> 5 분 2000 RPM과 실온에서 세포 현탁액을 원심 분리기, 10 ML 신선한 따뜻한 문화 매체 (줄기 프로 NSC SFM)의 세포 펠렛을 resuspend하고, hemocytometer로 휴대폰 번호를 계산합니다. </li><li> 각 25cm 두 문화 flasks에 5 ML 따뜻한 문화 매체를 추가하고 각 플라스크에 2X10 6 세포를 전송합니다. 문화는 37 유지 ° 분석하기 전에 1 주일 C / 5% CO 2 배양기. 자세한 문화 또는 실험 동안 일주일에 한 번씩은 절반 매체를 변경합니다. </li></ol> 3. 자세한 특성화 또는 experimentations에 대한 신경 줄기 세포의 Manipulaton <ol><li> Neurospheres은 보통 200 400 μm의 사이에 이르기까지 NSC 직경과 추천 문화 조건 1-2주 양식. 이 단계에서 Neurospheres 단일 세포를 얻는 15 분 37 칼슘, 마그네슘 무료 행크스 매체 (행크스)에서 0.2 g / L EDTA (에틸렌 다이아 민 테트라 초산) ° C로 dissociated 수 있습니다. 세포 현탁액은 신선한 행크스에 씻어서 2,000 RPM에서 냈지, 그리고 subculture에 대한 따뜻한 문화 매체 replated 있습니다. </li><li> 차별을 시작하려면, dissociated 전지 coverslip 당 1X10 5 셀 (24mmX24mm)의 밀도에 poly-D-lysine/laminin 한 코팅 coverslips에 도금입니다. Oligodendrocyte 차별은을 위해 마네 DMEM/F12 (Invitrogen, 메인, MD) 2% B27 (50X, Invitrogen, 메인, MD) 10 NG / ML bFGF 100 NG / ML 쉬이잇 + 10ng/ml PDGF - AA의 세포를 유지하여 이루어집니다 이일은 다음 다른 오일에 대한 성장 요인없이 동일한 매체로 전환. 의 연결을 분화는 7 일 동안 마네의 유지 세포 DMEM/F12 2퍼센트 B27 (50X)를 얻을 수 있습니다. Astrocyte 차별화는 일주일 동안 마네 DMEM/F12 1% FBS 안에 culturing 세포에 의해 이루어집니다. </li><li> 유전자와 신경 줄기 세포의 Transfection은 preformed neurospheres에서 dissociated 세포로 할 수 있습니다. 여기, 우리는 electroporation에 의해 EGFP - C1과 transfected 신경 줄기 세포를 보여주었다. EGFP - C1 구조의 electroporation은 (마우스 신경 줄기 세포에 대한 AMAXA Nucleofector 키트를 사용하여 수행되었습니다 VPG -회사의 지시 아래 AMAXA Nucleofector 장치 (Lonza AAD - 1001)과 1004). 즉, 1 X 10 6 세포를 5 μg의 DNA는 100 μl transfection 매체와 혼합되었고, 펄스 electroporation 후, 세포는 더 이상 문화의 중간을 유지하는 신경 줄기 세포에서 resuspended되었습니다. transfected 세포의 분화는 단계 3.2에서 설명한 동일한 절차에 따라 electroporation 후 neurospheres 3~4일의 분리와 처리되었습니다. </li></ol> 4. 냉동 신경 줄기 세포와 비주류 <ol><li> Dissociated 세포 suspensions는 1X10 7 셀 / 유리병 / ML의 농도와 centrifuged 및 냉동 매체 resuspended 있습니다. 천천히 -20 ° C, -80에서 세포를 동결 ° C 후 오랫동안 저장을위한 액체 질소로 전송할 수 있습니다. </li><li> 세포는 빠른 37와 해동 아르 ° C 물 목욕 및 resuspended은 세척을위한 혈청 DMEM/F12 +10 % 따뜻하게 냉동 매체를 제거하는 centrifuged 및 예열 문화 매체에 resuspended. </li></ol> 5. 대표 결과 : 18주 임신 성 나이에 정상적인 태아의 신경 줄기 세포는 설명 방법과 neurospheres가 둥근, 부드러운 테두리와 비교적 균일한 크기 (Fig.2A)와 일주일 뒤에 볼 수있는 다음과 같은 교양되었습니다. 이러한 neurospheres은 EGFP - C1이나 기타 구성과 transfected 및 형광 현미경 (Fig.2B)에 따라 다음 수 있습니다. 설립 neurospheres 다음 EDTA (에틸렌 다이아 민 테트라 초산)과 dissociated 및 코팅 coverslips에 분산된 세포로 도금했다. 각각의 프로토콜에 따라 차별화된 세포는 4 % parafamaldehyde로 고정하고, 다른 세포 유형의 특정 마커와 스테인드되었습니다. Multipotentiality는 (Fig2E, F, rhodamine) astrocytes (Fig2C, D, rhodamine) 뉴런을 나타내는 마커 및 oligodendrocytes (Fig2G, H, rhodamine)의 표현과 관찰이다. EGFP의 electroporation 공사를하지 세포는 다른 세포 유형으로 차별하고 다른 셀에 특정 마커와 스테인드되었습니다. Multipotentiality는 (Fig3C, rhodamine 및 Fig3D, fluoroscein) astrocytes (Fig3A, B, fluoroscein) 뉴런을 나타내는 마커 및 oligodendrocytes (Fig3E, F, rhodamine)의 표현과 관찰이다. <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="그림 1"/> 그림 1. 폐기 인간 태아의 두뇌에서 신경 줄기 세포를 분리하는 실험 절차의 도식 <img src="/files/ftp_upload/2681/2681fig2.jpg" alt="그림 2"/> 그림 2. 체외에서 propogated Undifferentiated와 차별화된 인간의 신경 세포. (A) Neurospheres은 부드럽고 둥근 테두리 1 이상의 주일 동안 문화 이후 급속한 성장을 보여 위상 콘트라스트 현미경 아래에 표시됩니다. (B) 다양한 plasmids과 구성의 소개 transfection을 통해 형성될 수 있습니다. EGFP - C1의 transfection 다음과 같은 세 가지 일, 여러 세포는 fluoroscein의 immunostaining 및 형광 현미경 하에서 본 녹색 형광 단백질의 표현을 보여줍니다. EGFP - C1 transfected neurospheres은 뉴런 (C, D), astrocytes (E, F)와 oligodendrocytes (G, H)로 서로 다른 조건에서 dissociated하고 차별하고 rhodamine의 형광 아래에서 보았다. 동시에, transfected EGFP 긍정적인 세포 fluoroscein 형광 아래 표시됩니다. Transfected 세포 (흰색 화살표 머리) untransfected 셀 (흰색 화살표)에서 구별할 수 있습니다. 세포 핵은 Hoechst33342 물들일 수 있습니다. 스케일 바는 200 μm의, B 100 μm의 및 CH 25 μm의 수 있습니다. <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="그림 3"/> 그림 3. transfection없이 Neurospheres은 뉴런 (A, B, fluoroscein), astrocytes (C, rhodamine, D, fluoroscein)와 oligodendrocytes (E, F, rhodamine)으로 서로 다른 조건에서 dissociated와 차별됩니다. 세포 핵은 Hoechst33342 물들일 수 있습니다. 스케일 바 AF 25 μm의 수 있습니다.

<ol>
	<li>Gage, F. H., Ray, J., Fisher, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+use+of+stem+cells+from+the+CNS.">Isolation, characterization, and use of stem cells from the CNS.</a> Annu. Rev. Neurosci. 18, 159-192 (1995).</li><li>Vescovi, A. L., Snyder, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+properties+of+neural+stem+cell+clones:+plasticity+in+vitro+and+in+vivo.">Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo.</a> Brain Pathol. 9, 569-598 (1999).</li><li>Schwartz, P., Bryant, P., Fuja, T., Su, H., O'Dowd, D., Klassen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+progenitor+cells+from+post-mortem+human+cortex.">Isolation and characterization of neural progenitor cells from post-mortem human cortex.</a> J Neurosci Res. 74, 838-851 (2003).</li><li>Martinez-Serrano, A., Rubio, F. J., Navarro, B., Bueno, C., Villa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+and+progenitor+cells:+in+vitro+and+in+vivo+properties,+and+potential+for+gene+therapy+and+cell+replacement+in+the+CNS.">Human neural stem and progenitor cells: in vitro and in vivo properties, and potential for gene therapy and cell replacement in the CNS.</a> Curr Gene Ther. 1, 279-299 (2001).</li><li>Rajan, P., Snyder, E. <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+cells+and+their+manipulation.">Neural stem cells and their manipulation.</a> Methods Enzymol. 419, 23-52 (2006).</li><li>Ruiz-Lozano, P., Rajan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+in+vitro+models+of+disease.">Stem cells as in vitro models of disease.</a> Curr Stem Cell Res Ther. 2, 280-292 (2007).</li><li>Sheen, V., Ferland, R., Harney, M., Hill, R., Neal, J., Banham, A., Brown, P., Chenn, A., Corbo, J., Hecht, J., Folkerth, R., Walsh, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+proliferation+and+migration+in+human+Miller-Dieker+neural+precursors.">Impaired proliferation and migration in human Miller-Dieker neural precursors.</a> Ann Neurol. 60, 137-144 (2006).</li><li>Bahn, S., Mimmack, M., Ryan, M., Caldwell, M., Jauniaux, E., Starkey, M., Svendsen, C., Emson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+target+genes+of+the+neuron-restrictive+silencer+factor+in+neurospheres+derived+from+fetuses+with+Down's+syndrome:+a+gene+expression+study.">Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study.</a> Lancet. 359, 310-315 (2002).</li><li>Ferland, R. J., Batiz, L. F., Neal, J., Lian, G., Bundock, E., Lu, J., Hsiao, Y. C., Diamond, R., Mei, D., Banham, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+neural+progenitors+along+the+ventricular+and+subventricular+zones+in+periventricular+heterotopia.">Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia.</a> Hum Mol Genet. 18, 497-516 (2009).</li><li>Esposito, G., Imitola, J., Lu, J., De Filippis, D., Scuderi, C., Ganesh, V. S., Folkerth, R., Hecht, J., Shin, S., Iuvone, T., Chesnut, J., Steardo, L., Sheen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+functional+profiling+of+human+Down+syndrome+neural+progenitors+implicates+S100B+and+aquaporin+4+in+cell+injury.">Genomic and functional profiling of human Down syndrome neural progenitors implicates S100B and aquaporin 4 in cell injury.</a> Hum Mol Genet. 17, 440-457 (2008).</li><li>Esposito, G., Scuderi, C., Lu, J., Savani, C., De Filippis, D., Iuvone, T., Steardo, L. J. r., Sheen, V., Steardo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100B+induces+tau+protein+hyperphosphorylation+via+Dickopff-1+up-regulation+and+disrupts+the+Wnt+pathway+in+human+neural+stem+cells.">S100B induces tau protein hyperphosphorylation via Dickopff-1 up-regulation and disrupts the Wnt pathway in human neural stem cells.</a> J Cell Mol Med. 12, 914-927 (2008).</li><li>Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L. L., Jendoubi, M., Sidman, R. L., Wolfe, J. H., Kim, S. U., Snyder, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftable+human+neural+stem+cells+respond+to+developmental+cues,+replace+neurons,+and+express+foreign+genes.">Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.</a> Nat Biotechnol. 16, 1033-1039 (1998).</li><li>Fults, D., Pedone, C. A., Morse, H. G., Rose, J. W., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+primitive+neuroectodermal+tumor+cell+line+from+the+cerebral+hemisphere.">Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere.</a> J Neuropathol Exp Neurol. 51, 272-280 (1992).</li><li>Conti, L., Cattaneo, E. <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+systems:+physiological+players+or+in+vitro+entities?.">Neural stem cell systems: physiological players or in vitro entities?.</a> Nat Rev Neurosci. 11, 176-187 (2010).</li><li>Svendsen, C. N., ter Borg, M. G., Armstrong, R. J., Rosser, A. E., Chandran, S., Ostenfeld, T., Caldwell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> J Neurosci Methods. 85, 141-152 (1998).</li></ol>

관심 없음 충돌 선언하지 않습니다.

문화 신선한 조직 및 생산 인간의 세포 라인에 대한 다양한 접근 방법이 있습니다. 역사적으로, 신선한 조직은 수확되었고 중추 신경계의 여러 세포 유형을 생성하는 즉시 양식. 이 접근법은 그러나 명확하게 얻은 인간 샘플의 경우에는 일반적으로 매우 작은 수있는 샘플의 수에 따라 제한됩니다. 조작의 최소한의 정도로봤을 때, 갓 교양 신경 세포가 확장 culturing에서 잠재적인 유물을 제한함으?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 녹아웃 DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td> 해리 매체 </td></tr><tr><td> 스템 프로 NSC SFM </td><td> Invitrogen </td><td> A10509 - 01 </td><td> 문화 매체 </td></tr><tr><td> 태아 소 혈청 </td><td> Invitrogen </td><td> 10091-148 </td><td> 냉동 매체 </td></tr><tr><td> 행크스 솔루션 (- CA 2 +, - MG 2 +) </td><td> Invitrogen </td><td> 14175-095 </td><td> 해리 매체 </td></tr><tr><td> DMSO </td><td> 시그마 - 알드리치 </td><td> D2650 </td><td> 냉동 매체 </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 시그마 - 알드리치 </td><td> 431788 </td><td> 해리 매체 </td></tr><tr><td> Paraformaldehyde </td><td> 시그마 - 알드리치 </td><td> 158127 </td><td> 고정 솔루션 </td></tr><tr><td> bFGF </td><td> R &amp; D </td><td> 234 - FSE </td><td> 차별화 매체 </td></tr><tr><td> 쉬이잇 </td><td> R &amp; D </td><td> 1845 - SH </td><td> 차별화 매체 </td></tr><tr><td> PDGF - AA </td><td> R &amp; D </td><td> 221 - AA </td><td> 차별화 매체 </td></tr><tr><td> B27 </td><td> Invitrogen </td><td> 17504-044 </td><td> 차별화 매체 </td></tr><tr><td> 마우스 방지 MAP2 </td><td> 시그마 - 알드리치 </td><td> M2320 </td><td> 1:200 </td></tr><tr><td> 토끼 방지 DCX </td><td> 세포 신호 </td><td> 4604s </td><td> 1:200 </td></tr><tr><td> 토끼 방지 GFAP </td><td> DAKO </td><td> Z0334 </td><td> 1:200 </td></tr><tr><td> 토끼 방지 S100B </td><td> DAKO </td><td> Z0311 </td><td> 1:200 </td></tr><tr><td> 토끼 방지 O1 </td><td> 교수 티모시 Vartanian의 선물 * </td><td></td><td> 1시 50분 </td></tr><tr><td> 토끼 방지 O4 </td><td> 교수 티모시 Vartanian의 선물 * </td><td></td><td> 1시 50분 </td></tr><tr><td> 40μm 셀 스트레이너 </td><td> BD 팰컨 </td><td> 352340 </td><td></td></tr></tbody></table> * 티모시 Vartanian, MD, 박사, 신경과 및 신경 과학, 웨일 코넬 메디컬 칼리지, 뉴욕, 미국학과

generation of neural stem cells from discarded human fetal cortical tissue

신경 줄기 세포 (NSCs)은 대뇌 피질 판의 개발 중에 심실 영역 neuroepithelium 함께 거주. 이러한 초기 progenitors 궁극적으로 중급 progenitors을 일으키다 이상, 대뇌 피질을 형성 다양한의 연결 및 glial 세포 subtypes. 폐기 정상 태아 조직에서 인간 NSCs을 (그래서 neurospheres라고도 함) 생성 및 확장하는 능력은 바로 정상적인 인간 NSC 개발 1-5의 기능 측면을 연구하기 위해있는 수단을 제공합니다. 이 접근법은 또한이를 통해 조상의 증식, 이주 및 차별 6-9를 변경 질병 프로세스를 식별할 수있는 기회를 affording, 알려진 신경 질환에서 NSCs의 생성 방향으로 이동하실 수 있습니다. 우리는 빠른 속도로 알츠하이머 질병 표현형 10,11에 기여할 수 있습니다 인간의 다운 증후군의 NSCs의 병리 학적 메커니즘을 식별에 초점을했습니다. 나도 생체내에이나 체외 마우스 모델은 인간의 염색체 21에 위치한 유전자의 동일한 레퍼토리를 복제할 수 없습니다. 여기 중단 인간의 태아 cortices의 증후군의 NSCs 다운 격리와 문화에서 성장하는 간단하고 신뢰할 수있는 메서드를 사용합니다. 방법론이 제한 해부의 명소, 셀 정렬은 도금 인간 NSCs의 passaging과 조직, 절개를 수확의 특정 측면을 제공합니다. 우리는 또한 더 선택적 세포 subtypes에 인간 NSCs의 차별을 유도하는 몇 가지 기본적인 프로토콜을 제공합니다.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

폐기 인간의 태아 대뇌 피질의 조직에서 신경 줄기 세포의 격리와 문화에 대한 간단하고 신뢰할 수있는 방법을 설명합니다. 알려진 인간의 신경 장애에서 파생된 문화가 같은 약리 효능을 평가하기위한 플랫폼을 제공뿐만 아니라, 병적인 세포 및 분자 공정의 특성에 사용될 수 있습니다.

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

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

generation-neural-stem-cells-from-discarded-human-fetal-cortical

Research

JoVE Journal

Neuroscience

15.4K 조회수.  Beth Israel Deaconess Medical Center. 폐기 인간의 태아 대뇌 피질의 조직에서 신경 줄기 세포의 격리와 문화에 대한 간단하고 신뢰할 수있는 방법을 설명합니다. 알려진 인간의 신경 장애에서 파생된 문화가 같은 약리 효능을 평가하기위한 플랫폼을 제공뿐만 아니라, 병적인 세포 및 분자 공정의 특성에 사용될 수 있습니다.

비디오: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

세포 어세를 형성 신경 콜로니 : 신경 전구 세포로부터 타고난 신경 줄기 세포를 차별 어세이

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

연구

신경과학

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 ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

인간 태아 뇌에서 전구 - 파생 Oligodendrocyte 문화 시스템

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

인간의 신경의 집계의 cGMP-해당 확장 방법은 줄기 및 전구 세포는 다 능성 줄기 세포 또는 태아 뇌 조직에서 파생 된

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

인간의 다 능성 줄기 세포에서 도파민 신경 세포의 분화를 감독

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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

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

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

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

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

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

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

신경 줄기 세포의 열거 클론 세이를 이용하여

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...