이 작품은 오버 재단 기금에 의해 지원되었다.

1. 전지 도금을하기 전에 준비를해야 항목 : <ol><li> 전체 NSC 매체의 적절한 볼륨이 각각 9시 1분 비율 NeuroCult NSC 기초 중간 및 NeuroCult NSC 확산 보조 식품을 혼합하여 준비가되어 있습니다. </li><li> 크린 시토킨없이 NeuroCult NCFC 세럼 - 무료 매체의 나누어지는가 해동됩니다. </li><li> 매체는 37 ° C의 물을 욕조에 워밍업이다. </li><li> 표피 성장 인자 (EGF), 10 μg / 0.2 %에서 ML과 헤파린의 농도에서 기본 fibroblastic 성장 인자 (B - FGF)의 주식 솔루션은 미리 준비가되어 있습니다. </li><li> 실험의 크기에 따라 여러 35mm 조직 문화 요리 판 세포 하나 150-200cm 플라스틱 페트리 접시도 중복 35mm 요리를 개최하는 데 필요한 물을 위해 제 3 35mm 접시 필요합니다. </li></ol> 2. 셀 준비 : 실험에 따라 세포는 성인이나 배아 소스 (주 dissociated 조직 또는 dissociated neurospheres)에서 준비하실 수 있습니다. 성인 / 마우스 배아 중추 신경계 (CNS)에서 조직을 해부하다 또는 다음 1,2 앞에 설명한 배아 - 유래 / 성인 neurospheres을 떼어 놓다 : <ol><li> 단일 세포 현탁액은 40 μm의 크기의 메쉬 필터를 통해 전달되도록 같은 비 dissociated 대단히 짧은 시간을 제거합니다. </li><li> 세포 현탁액의 10μl는 셀 카운트를 수행하는 Trypan 파랑의 90μl와 혼합됩니다 참고 :. 기타 적절한 세포 dilutions도 사용할 수 있습니다. </li><li> 기본 배아 또는 성인 파생된 신경 세포를 사용하는 경우, 전체 NSC 매체 6.5 X 10 5 세포 / ML의 농도로 세포 현탁액을 희석. 성인이나 배아 신경 세포에서 파생된 dissociated neurospheres의 세포를 사용하는 경우, 전체 NSC 매체에서 2.2 × 10 5 세포 / ML의 농도로 세포 현탁액을 희석. </li></ol> 3. 세미 솔리드 N - CFCA 매체에 도금 셀 : <ol><li> 다음과 같은 구성 요소의 적절한 볼륨 복제의 숫자에 따라 순서에 혼합됩니다. 여기 복제 두 필요한 금액을 혼합하거나 요리를 중복. 복제의 추가 숫자, 표 1을 참조하십시오. <ul><li> 크린 시토킨없이 NeuroCult NCFC 세럼 - 무료 매체의 1.7 ML </li><li> NeuroCult NSC 확산 보조 식품의 330 μL </li><li> EGF의 6.6 μL (10μg/mL) </li><li> 페니실린 / 스트렙토 마이신의 32 μL </li><li> B - FGF (10μg/mL)의 3.3 μL는 culturing 세포가 신경 세포를 성인에서 파생된 경우에만 필요합니다. </li><li> 헤파린 (0.2 %)의 3.3 μL는 culturing 세포가 신경 세포를 성인에서 파생된 경우에만 필요합니다. </li><li> 세포 현탁액의 25μL (dissociated neurospheres 또는 6.5 X 10 5 셀 / dissociated CNS 조직에서 ML 기본 세포에서 2.2 X 10 5 셀 / ML 세포) 참고 :. 도금 최종 셀 번호는 35mm 문화 요리 당 2500에 대한 셀해야합니다 neurosphere 파생된 세포 및 기본 세포 35mm 문화 요리 당 7,500 전지. 어떤 경우에는, 최종 세포 도금 밀도는 셀 적정 실험을 수행하여 조정되어야한다. 150 식민지 - 통계 분석을 위해, 문화, 21 일 후에 감지된 식민지의 총 개수는 적어도 50의 범위 내에서하여야한다. </li></ul></li><li> 세포를 포함하는 매체는 추운 콜라겐 솔루션의 1.3 ML이 세포 현탁액에 전송하고 거품을 도입 피하기 위해 부드러운 pipetting으로 잘 혼합됩니다 부드럽게 그리고 혼합이다. </li><li> 혼합 솔루션은 각각 35mm 문화 요리 (~ 1.5 ML / 요리)의 중심에 적절하고 요리는 부드럽게 혼합이 접시의 표면 위에 균일하게 분산하도록 원형 운동을 사용하여 밀고있다. . </li><li> 중복 35mm 문화 요리는 100mm 페트리 접시에 게재됩니다. 새로운 35mm 요리의 뚜껑을 제거하고 열려있는 요리도 동일한 100mm 페트리 접시에 배치됩니다. 멸균 물이 부화 기간 동안 최적의 습도를 유지하기 위해이 열려 35mm 요리에 추가됩니다. 스퀘어 bioassay 판 (245mm)가 더 많은 복제 35mm 요리가 설정되었을 때 사용됩니다. 다시, 2 또는 멸균 물이 들어있는 3 열 35mm 요리를 포함합니다. </li><li> 번호판이 37 보육 세트에 전송 ° C 5 % CO2와 95 %의 습도. 증가 온도 및 겔 형성하여 콜라겐 확대해는 약 한 시간 이내 발생합니다. 문화는이 시간 동안 방해해서는 안됩니다. </li><li> 교양 세포 21일 (식민지의 크기의 차이가 명확하게 21일 후 구분할 수)에 incubated 수 있습니다. </li></ol> 4. 보충 매체를 준비하고 문화를 먹이 : N - CFCA 문화는 시간 (21 일)의 기간 동안 incubated이므로, 문화는 적절한 전체 NeuroCult와 공급한다 보충 매체는 다음과 같이 신선한 준비 : <ul><li> NeuroCult NSC 기초 중형 4.5 ML은 0.5 m로 혼합NeuroCult NSC 확산 보조 식품 후 성장 요인 L이 추가됩니다 </li><li> 10μg/mL EGF 250 μL (0.5 μg / ML EGF의 최종 농도를 제공) </li><li> 10μg/mL B - FGF (0.25μg/mL B - FGF의 최종 농도를 주다) 125 μL는 culturing 세포가 신경 세포를 성인에서 파생된 경우에만 필요합니다. </li><li> 0.2 % 헤파린 125 μL은 culturing 세포가 신경 세포를 성인에서 파생된 경우에만 필요합니다. </li><li> 적절한 전체 NeuroCult의 보충 매체 (배아 또는 성인 전지)의 60 μL가 전체 NCFC 분석 문화 보육 (21 일) 동안 매 7 일마다 한 각 NCFC 분석 접시의 중앙에 추가됩니다. </li></ul> 5. N - CFC 분석 유래 식민지를 채점하고 타고난 NSCs 및 신경 전구 세포의 빈도를 계산 : <ul><li> 각 35mm 문화 요리는 2.0 mm X 2.0 mm의 격자 크기 gridded 점수 요리에 위치하고 양쪽 요리는 현미경 스테이지에 표시됩니다. </li><li> 객관 렌즈, 각각의 요리가 스캔되고 식민지가 자신의 크기에 따라 점수 아르 - 낮은 전력을 (배 2.5X)를 사용. </li><li> 식민지는 두 가지 주요 범주로 분류됩니다 : <ol><li> 이하 2mm 직경 </li><li> ≥ 2mm 직경 </li></ol></li></ul> 6. 대표 결과 : 칠일 도금 후 (그림 1) - neurosphere 분석에서와 마찬가지로, N - CFC 분석에 도금 세포 3 이내에 세포의 작은 식민지를 분아 따위에 의해 번식하고되기 시작한다. 2 주 시간에서 다양한 크기의 식민지가 구별하실 수 있습니다. 식민지의 대부분의 14 일 후에 성장을 중지하는 경향이 있지만, 일부 식민지는 크기가 증가되고 있습니다. 1mm 직경, 3) 1 - - 2mm 직경 4) ≥ 2mm 직경 미만 0.5 mm 직경, 2) 0.5 1) : 일 21에 의해 식민지는 4 가지 범주 중 하나에 분류 될 수 있습니다. 실제로, 시조가 파생된로 2mm 직경보다 작은 식민지가라고합니다 (그림 2)와 NSC는 (그림 3)과 같은 파생 식민지는 직경 ≥ 2mm가라고합니다. 도금 총 세포마다 직경 식민지 ≥ 2mm의 숫자는, 장기적인 자기 갱신과 다중 잠재적인 기능을 갖춘 실제 타고난 신경 줄기 세포의 빈도를 나타냅니다. 병렬 NSA 실험에서 7~8일 후 특정 세포 인구의 주파수를 형성 총 neurosphere은 N - CFCA 실험 21 일 후 같은 세포 인구의 주파수를 형성 전체 식민지와 유사한 것으로 추산되었습니다. N - CFCA 그러나, 각 세포는 전체 proliferative 가능성을 보여 줄 수 있도록 같은 더 허용 조건을 제공합니다. <table border="1"><tbody><tr><td> 구성 요소 </td><td> 2 복제 </td><td> 3 복제 </td><td> 4 복제 </td></tr><tr><td> 크린 시토킨없이 NeuroCult NCFC 세럼 - 무료 중간 </td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult NSC 확산 보조 식품 </td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF (10 μg / ML) </td><td> 6.6 </td><td> 9.9 </td><td> 13.2 </td></tr><tr><td> 성인용 전지 bFGF (10 μg / ML) </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td> 성인용 세포에 대한 헤파린 솔루션 (0.2 %), </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td> 페니실린 / 스트렙토 마이신 (1:100) </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td> :에서 셀 2.2 X 10 5 교양 세포 / ML 또는 6.5 X 10 5 일차 전지 / ML </td><td> 25 </td><td> 37.5 </td><td> 50 </td></tr><tr><td> 콜라겐 솔루션 </td><td> 1300 </td><td> 1950 </td><td> 2600 </td></tr></tbody></table> 표 1. 완벽한 N - CFC 분석 문화의 구성 요소. <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="그림 1" /> 1. 통로 하나 E14 마우스 NSCs의 N - CFCA 문화의 대표 식민지 칠일 도금 후 그림. 식민지 다른 형태와 크기를 표시 수도 있습니다. 원본 확대, 4X <img src="/files/ftp_upload/2639/2639fig2.jpg" alt="그림 2" /> 그림 2. 통과 N - CFCA 문화에서 다른 크기 21일 도금 후에도 E14 마우스 NSCs 대표 시조 파생 식민지. 그림과 같이, 식민지는 다른 형태가 있지만 모든 크기가 적은 2mm입니다. 원본 확대, 4X <img src="/files/ftp_upload/2639/2639fig3.jpg" alt="그림 3" /> 그림 3. 대표 타고난 줄기 세포는 21일 도금 후 통과 한 E14 마우스 NSCs을의 N - CFCA 문화 식민지를 파생. 줄기 세포 유래 식민지는 (보고 서로 다른 형태를 표시할 수 있습니다비디오)하지만, 모두가 직경 ≥ 2mm입니다. 원본 확대, 4X

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <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+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Azari, H., Sharififar, S., Rahaman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</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>Reynolds, B. A., Rietze, R. L. <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+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+Neural+Stem+and+Progenitor+Cells+in+the+Neural+Colony+Forming+Cell+Assay.">Enumeration of Neural Stem and Progenitor Cells in the Neural Colony Forming Cell Assay.</a> Stem Cells. , (2008).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Louis, S. A., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

저자 중 하나, 샤론 A. 루이스는 StemCell Technologies의 병원, 본 연구에서 사용되는 몇 가지 시약의 제작자와 제휴합니다.

neurosphere 검정 3,1,2은 성인과 배아 CNS 조직과 같은 다양한 소스로부터 신경 줄기 세포를 분리하고 확장하는 가장 일반적인 방법이지만, 정확하게 신경 전구체 세포의 혼합 인구 NSC 주파수를 측정할 수 없다 (줄기와 progenitors) neurospheres의 수와 타고난 줄기 세포 4의 사이의 숫자 1-1 관계가 아니므로. 이 제한을 해결하려면, 원본 NSA는 신경 줄기와 progenitors은 5-7 삼 주 동안 그들의 전체 확산 용량 성장할 수 있도록처럼 맞게되었습니다. 반 고체 콜라겐 매트릭스가 식민지의 한 도금 세포 및 집계의 마이 그 레이션을 방지 등 액체 NSA 달리, N - CFCA의 식민지 실제로 clonally, 파생됩니다. 이 분석과 일치의 결과를 얻으려면 우리는 좋습니다 <ol><li> 원래 세포 현탁액에서 단일 세포 현탁액을 보장합니다. 비 dissociated 대단히 짧은 시간을 제거하는만큼 40 μm의 크기의 메쉬 필터를 통해 하나의 세포 현탁액 패스. </li><li> 얼음 또는 4 ° C 냉장고에있는 콜라겐 솔루션을 유지. 콜라겐은 온도 증가에 의해 확대해으로 세포 현탁액의 혼합에 추가할 수있는 마지막 항목입니다. </li><li> 매주 문화를 공급하는 것을 잊지 마십시오. 매 7 일마다 한 성장 인자 (들)을 포함하는 보충 미디어를 추가하면 세포가 확장 문화 기간 동안 세포 분열 따위에 의해 번식을 계속하실 수 있습니다. </li><li> 최종 셀 도금 밀도는 중 기본 또는 교양 신경 세포에서 유래 세포에 최적화된되었습니다 있도록 문화 21 일 후에 감지된 식민지의 총 개수 NCFC - A는 적어도 50의 범위 이내 - 200 식민지. 통계 분석에 대한 식민지의 충분한 숫자를 제공함과 동시에이 범위는 샘플의 직경에있는 희귀 NSC 파생 식민지&gt; 2mm의 검색이 가능합니다. 시작 셀 소스에 따라 크게 적은 (&lt;50)과 식민지의 높은 숫자가 (&gt; 250) 때로는 얻을 수 있습니다. 너무 많은 식민지가 정확 콜로니 계수의 결과로 성장 매체 구성 요소의 인구 과잉 anddepletion집니다 동안 부족 식민지는 직경이 검출 수준 아래되고있는 식민지&gt; 2mm가 발생합니다. 따라서 세포 도금 밀도 (즉, 세포 도금의 총 수를 늘리거나 감소) 따라서 조정해야합니다 </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
<tr>
<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
</tr>
<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

neurosphere 분석 (NSA)은 분리 확장하고 또한 신경 줄기 세포 (NSCs)의 주파수를 계산하기 위해 가장 자주 사용되는 방법 중 하나입니다. 또한,이 혈청이없는 문화 시스템은 또한 줄기 세포를 확장하고 종양과 정상 조직의 다양한 그들의 주파수를 결정하기 위해 채용되었습니다. 이것은 일대일 관계가 neurosphere 형성과 NSCs 사이에 존재하지 않는다는 것을 최근에 표시되었습니다. 이것은 NSA와 같은 현재 적용가 배아와 성인 포유류의 뇌 모두에서 분리된 신경 전구체 세포의 혼합 인구 NSCs의 빈도를 과대 평가하는 것이 좋습니다. 이 비디오는 거의 세미 솔리드 분석을 기반으로 소설 콜라겐을 보여, 신경 식민지 따라서 자신의 장기 proliferative 가능성에 따라 전구 세포에서 줄기를 차별하는 능력을 가지고 있으며, 세포 분석 (N - CFCA)를 형성하는 방법을 제공합니다 NSC 주파수를 열거할 수 있습니다. 식민지가 &lt;2mm가 progenitors에서 파생하는 동안 N - CFCA에서 직경 식민지 ≥ 2mm는 NSC의 모든 기능 기준을 만족하는 세포에서 파생됩니다. N - CFCA 절차는 기본 및 교양 성인이나 배아 마우스 CNS 세포를 포함하여 서로 다른 소스에서 준비 전지에 사용될 수 있습니다. 여기 통로에서 N - CFCA을 수행하기 위해 배아 일 14 쥐 두뇌에서 발생 한 neurospheres을 준비 세포를 사용합니다. 문화는 도금 세포들이 전체 proliferative 잠재력을 전시 있도록하고 신경 시조와 타고난 신경 줄기 세포의 빈도가 &lt;2mm 아르 콜로니의 수를 계산하여 각각 계산 확산 매체로 삼 주 동안 매일 7 일 보충함 아르 그리고 ≥ 2mm 처음 도금되었습니다 세포의 개수에 대한 언급 손님.

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

이 비디오 프로토콜은 신경 식민지 - 형성 세포 분석을 사용하여 신경 전구체 세포의 혼합 인구의 타고난 신경 줄기 세포를 차별하고 열거하는 방법을 보여줍니다.

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). ...

neural-colony-forming-cell-assay-an-assay-to-discriminate-bona-fide

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Neuroscience

22.0K 조회수.  University of Florida. 이 비디오 프로토콜은 신경 식민지 - 형성 세포 분석을 사용하여 신경 전구체 세포의 혼합 인구의 타고난 신경 줄기 세포를 차별하고 열거하는 방법을 보여줍니다.

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

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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

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 ...

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. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

쥐의 눈으로 인간 망막 안료 상피 세포의 트랜스 scleral Subretinal 이식에 대 한 세련 된 프로토콜의 개발

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

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.

절연 및 마우스 배아 신경 줄기 세포의 문화

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Sialoglycan의 신진 대사 표지에 의하여 1 차적인 신경 줄기 및 전구 세포의 세포 표면 마커를 확인

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...