이 연구는 밴더빌트 - 잉그램 암 센터 지원 그랜트 (P30 CA068485), 아동 뇌종양 재단과 국립 보건원 (NS042205)에서 보조금에 의해 지원되었다.

1. 종양 - 베어링 소뇌의 마이크로 절개, 종양 조직과 도금의 해리 <ol><li> 종양 조직 검색 <ol><li> 아픈 쥐 베어링 medulloblastoma가 자주 runted 있었다 hydrocephaly 및 사후 마비 및 전복시 자세를 회복하기 위해 실패를 포함하여 전형적인 신경 증상이 표시됩니다. 종양 조직을 검색하려면, 이산화탄소 흡입하여 쥐를 안락사. 자궁 경부 전위, 뒷부분 두개골에 압력을 생성하고 종양 조직 무결성을 손상 수있는 절차를 수행하지 않는 것이 중요합니다. </li><li> 잘린은 두개골의 좋은 시각화를위한 머리 근육 조직 최대한 제거, 가위를 사용하여 죽은 후 즉시 수행됩니다. 95 % 에탄올로 적셔 Kimwipe와 두개골의 표면을 청소하십시오. </li><li> 두개골의 정중선을 따라 개방 잘라하고, 어느 시점에 종양 베어링 소뇌를 포함하여 전체 두뇌가 노출, 괜찮아요 핀셋을 사용하여 두개골 조직을 제거하는 미세 가위를 사용합니다. </li><li> 건강한 성인의 cerebella 잘 정의 반구와 vermis를 표시하지만, 종양이 베어링 생쥐의 cerebella은 종종 매끄러운 표면과 눈에 혈관과 비정질, 확대하고 있습니다. 무균 기법을 사용하여, Mg2 않고 얼음처럼 차가운 PBS에 핀셋과 장소를 사용하여 소뇌 종양을 검색 + 및 CA 2 +. </li></ol> 참고 : 모든 악기가 사용하기 전에 95 %의 에탄올로 소독하고 있습니다. </li><li> 종양 조직의 분리 <ol><li> 4 분간 37 인큐베이션 다음 실온에서 3 분 고급 가위, ° C로 조직을 말하다, PBS에서 종양 조직의 4 배 볼륨 약 50 % Accutase (PBS에 희석)에 종양 조직을 전송 , 그 이후 조직 추가 3 분 1 - ML의 Pipetman와 반복 pipeting를 겪습. 이 방법은 단일 세포와 작은 세포 집합체의 혼합물을 얻을 것입니다. </li><li> 펠렛 세포에 1천그램에서 5 분 동안 휴대폰 정지 PBS로 3 배와 원심 분리기를 희석. gelatinized 60mm의 Primaria 조직 문화 요리에 신선한 신경 줄기 세포 배양 매체와 플레이트의 세포 펠렛을 Resuspend. 우리가 먼저 도금에서 향상된 첨부 파일 Primaria 요리를 사용하여, 후속 단락은 일반적인 조직 문화 요리에 도금 수 있습니다. </li></ol></li></ol> 참고 : 적어도 30 분 젤라틴 0.1 %로 코트 문화 요리. 글루타민, 펜 - Strep, N2, B27, 인간 EGF (25 NG / ML)과 기본 FGF (25ng / ML)로 Neurobasal 매체의 구성 신선한 신경 줄기 세포 배양 매체를 준비합니다. 2. 강화, 유지 보수 및 시리얼 항로로 Medulloblastoma 종양 세포 확장 우리는 일반적으로 초기 도금의 일주합니다 (그림 1) 이후 많은 식민지를 얻을. 이들 식민지는 dissociated 및 종양 세포 인구의 농축을위한 새로운 gelatinized 요리에 replated 수 있습니다. 새로운 문화 매체 먼저 변경 사항은 다음 단순히 기계적으로 세포 현탁액 (아무 Accutase가 필요하지)를 항복 4 분 동안 반복 pipeting 다음 식민지를 분리하기 위해 1 - ML Pipetman를 사용합니다. 대형 세포 대단히 짧은 시간이 dissociated되었는지 확인하기 위해 현미경으로 확인합니다. 그런 다음 추가 culturing에 대한 새로운 gelatinized 요리에 세포 현탁액을 조제하고 추가 구절에 대해 동일한 절차를 통해 이동합니다. 무소속의 종양 세포, 혈액 세포와 다른 세포 유형이 부착된 종양 세포가 급속하게 확장 떠나, 다시 platings 직렬에 의해 제거됩니다. 문화 초기 시딩 다음 두 번째 날이 매체 및 이후 매일을 변경합니다. 3. Immunofluorescent 스테 이닝 및 교양 세포의 시험 1. 유리 coverslips에 종양 세포를 성장 1 일, 6 잘 접시의 각 놓으 유리 coverslip에 다음 37 30 분 젤라틴 0.1 % ° C.를 추가 ML 당 종자는 약 2 - 4X 10 5 종양 세포. 세포가 하룻밤 사이에 첨부해야하고 신경 줄기 세포 매체는 하루에 3 바뀌었습니다. 얼룩은 4 일 수행됩니다. 이 Immunofluorescent 얼룩 얼음처럼 차가운 PBS로 두 번 배지 및 세척 세포를 제거합니다. 15 분 동안 갓 만든 4퍼센트 PFA로 세포를 수정. 간단히 PBS로 두 번 세포를 씻어 5 분 0.3 % 트리톤 (PBS)와 permeablize. 간단히 40 분 10 % 염소 혈청과 PBS와 블록과 두 세포를 씻으십시오. 90 분간 일차 항체와 세포를 부화 후, 오분 각, PBS로 세포를 세 번 씻는다. 60 분 차 항체와 세포를 부화 후, 오분 각각, PBS로 세 번 씻어. 수성 설치 매체의 5 ML과 슬라이드에 coverslips를 탑재하고 공촛점 현미경을 수행합니다. 4. 종양 세포의 분화 n을 제거eural의 줄기 세포 매체와 짧게 두 번 PBS로 세포를 씻어. 일 1 차별 매체를 추가, 일 4 매체를 변경하고 일 7 immunofluorescence 염색법에 의해 분화의 수준을 결정합니다. 차별화 매체 DMEM, 펜 - Strep와 10 % 태아 소 혈청으로 구성되어 있습니다. 5. 대표 결과 형태 결과 Accutase 처리 및 부드러운 반복 pipeting 후, 종양 조직은 작은 세포 집계 및 단일 세포로 대부분 dissociated해야합니다. 많은 상당한 식민지는 그림 1과 같이 초기 도금 후 5 일 빠르면 볼 수 있습니다. 높은 proliferative 종양 세포가 높은 핵 / 세포질 비율로 종종 이중 극성입니다. 이러한 세포는 일반적으로 gelatinized 표면에 부착된 작은 세포 집합체에서 발산 볼 수 있습니다. 작고 둥근 혈액 세포는, 일반적으로 후속 매체 변경 및 시리얼 platings에 의해 제거됩니다. 몇 구절 후, 하나는 볼 수 균일하게 분산, proliferative Ki67 + 종양 세포와 다른 세포 유형 (그림 2)와 작은 오염. 신경 줄기 세포의 마커, clonal 분석 및 여러 lineages로 분화 표현 proliferative 세포 종양 줄기 세포의 dissociated 소뇌 종양 조직 쇼 특성에서 파생된 여부를 확인하기 위해, 우리는 줄기 세포 마커 표현, clonal 분석 및 차별화의 힘에 의해 더욱 특성화를 수행했습니다. 우리는이 분리된 종양 세포가 높은 등 Sox2과 Nestin (그림 3)과 같은 신경 줄기 세포의 마커를 표현하는 것으로 나타났습니다. 우리가 SmoM2 - YFP 종양에서 조직을 검색할 때, 모든 종양 세포 표현 YFP. 그 기본 속눈썹을보고 최근의 연구와 일치는 쉬 경로에 의존 medulloblastoma 12 SmoM2 - YFP 표현이 명확하게 Sox2의 속눈썹 + 종양 세포 (그림 3) 지역화된했습니다의 발전을 위해 중요합니다. 문화 매체의 ML 300 세포의 clonal 밀도에 도금 때, 우리는 하나의 종양 세포에서 상당한 식민지 (그림 4) clonal 확장을 관찰했다. 또한, 이들 세포는 프로 차별화 조건 (그림 5)에서 양식 때 여러 가지의 연결 및 glial 세포 유형으로 구별 수있었습니다. <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="그림 1" /> 그림 1. dissociated medulloblastoma 조직의 첫 번째 도금 후에 높은 proliferative 식민지의 형태. 일반적으로, 5 dissociated 종양 조직의 초기 시딩 며칠 후에, 그리고 대표 하나에 상당한 크기의 식민지 양식은 밝은 분야에서 여기에 그림입니다. 고밀도 코어 셀 집합에서 BI - 극지, 길쭉한 세포의 방출. 점진적으로 일련 platings에 의해 고갈 작은 둥근 적혈구가있는 종양 세포에 첨부. <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="그림 2" /> 그림 2. 세 구절 이상 설립 medulloblastoma 세포의 형태. 이 명시야 이미지는 몇 구절 후 설립 medulloblastoma 세포의 전형적인 모습을 보여줍니다. 세포가 높은 세포질 / 핵의 비율로 대부분 이중 극성 남아 있습니다. 우리는 종양 세포의 acetylated tubulin의 얼룩을 표시,이 중 대부분은 강력한 Ki67 표현으로 표시된 사이클링됩니다. <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="그림 3" /> 그림 3. 설립 medulloblastoma 세포는 신경 줄기 세포 마커를 표현한다. SmoM2 - YFP는 constitutively 활성화 SMO, 따라서 쉬 통로의 활동을 표현하는 세포를 표시합니다. 설립 medulloblastoma 세포 라인의 모든 세포가 YFP을 표현하고, 그들의 대부분은 Nestin 및 Sox2 등 다양한 신경 줄기 세포 마커, 높은 수준의 공동 표현. 흥미롭게도, 우리가 공촛점 현미경 동안 최소한의 레이저 전원과 YFP 신호 감지를 수행할 때, 우리는 Sox2 + 세포의 속눈썹에 집중 YFP 신호를 발견했습니다. 이 관찰 쉬 경로에 의존 medulloblastoma 개발에 솜털의 필수적인 역할을 설명한 최근 보고서 12 일치합니다. <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="그림 4" /> 그림 4. 설립 종양 세포가 clonal 확장을 받고있다. 분리 후 단일 세포 현탁액에, 종양 세포는 배지의 ML 300 세포의 clonal 밀도에서 24 잘 판에 도금했다. 각 물론 우리는 clonally 확대 식민지를 관찰했다. 명시야 이미지의이 시리즈는 문화의 10 일간에 걸쳐 전형적인 변화를 보여줍니다. <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="그림 5" /> 그림 5. 설립 medulloblastoma 세포의 차별화 분석. 종양 세포는 EGF / bFGF가 DMEM/10 % FBS, YFP + 종양 세포는 크게 자신의 형태를 변경하고 다양한 셀 유형, 병원에 차별에 혈청이없는 매체를 포함에서 전환되었을 때Tuj1를 luding + 또는 NeuN + 뉴런, GFAP + astroglial 셀 또는 CNPase + oligodendrocytes.

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관심 없음 충돌 선언하지 않습니다.

우리는 constitutively 적극적인 헤지 호그 신호와 돌연변이 생쥐에서 가져온 기본 종양 조직에서 medulloblastoma의 줄기 세포를 분리 풍부하고 유지하는 효율적인 방법을 설명합니다. 우리는 성공적으로 건강한 종양의 줄기 세포 라인을 확립 한 중요한 단계는 기본 종양 조직의 분리시 사용되는 Accutase 치료되고있는 것으로 나타났습니다. 우리의 경험에서 때 소뇌에서 첫번째 dissociating 종양 조직, 37에?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
<tr>
<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
</tr>
<tr>
<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
</tr>
<tr>
<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50%</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1393</td>
<td> 0.1%</td>
</tr>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
</tr>
<tr>
<td>Primaria dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>087724C</td>
<td>&nbsp;</td>
<tr>
<td>Sox2 antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB4343</td>
<td>Mouse, 1:1000</td>
</tr>
<tr>
<td>Nestin antibody</td>
<td>&nbsp;</td>
<td>DSHB</td>
<td>rat401</td>
<td>Mouse, 1:200</td>
</tr>
<tr>
<td>YFP antibody</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>A11122</td>
<td>Rabbit, 1:2000</td>
</tr>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Neuromics</td>
<td>CH22102</td>
<td>Chicken, 1:1000</td>
</tr>
<tr>
<td>Tuj1 antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5076</td>
<td>Mouse, 1:2000</td>
</tr>
<tr>
<td>NeuN antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB377</td>
<td>Mouse, 1:2000</td>
</tr>
<tr>
<td>CNPase antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB326</td>
<td>Mouse, 1:1000</td>
</tr>		</tbody>
		</table>
		</div>

isolation, enrichment, and maintenance of medulloblastoma stem cells

뇌 종양은 tumorigenesis의 근본 원인 아르 줄기 세포의 작은 인구를 보유하기 위해 제안되었습니다. Neurosphere의 assays는 일반적으로 정상과 tumorous 조직에서 추출한 포함 신경 줄기 세포의 특성을 연구하기 위해 채택되었습니다. 그러나, 분화 및 세포 사망의 감지할 수있을 정도의 금액은 문화 매체 영역을 합산 내의 모든 세포의 접근성 등 하위 최적의 상태로 가능성 때문에 교양 neurospheres에서 흔히 있습니다. Medulloblastoma, 가장 일반적인 소아 CNS 종양은 그 빠른 진행과 디즈멀 임상 결과와 전체 뇌 - 척추 축을 따라 확산 경향이 특징입니다. Medulloblastoma은 각각 1 20 %와 아동기에 intracranial 및 사후 포사 종양의 40 %에 대한 회계, 소뇌의 neuroepithelial 종양이다. 그것은 지금은 잘 쉬 신호는 소뇌 발달 2-4 중 소뇌 과립 신경 세포의 엽 성의 전구 물질 (CGNPs)의 확산을 자극한다는 걸 입증합니다. 쉿 경로가 constitutively 활성화되는 마우스 모델을 사용하여 많은 연구가, medulloblastoma 5-9로 쉬 신호를 연결했습니다. 최근 보고서는 Patched1 LacZ / + 마우스에서 파생된 medulloblastoma 세포의 일부가 종양 10 시작하고 propogating 능력이 암 줄기 세포,있는 것으로 나타났습니다. 여기서 우리는 효율적인 방법 Smoothened에 돌연변이 (11, hereon가 SmoM2로 함), 쉬를 위해 중요합니다 GPCR로 인해 constitutively 활성화 쉬 경로와 medulloblastoma의 여러 마우스 모델에서 파생된 종양 줄기 세포를 분리 풍부 및 유지 보수를 설명 경로 활성화. 모든 격리된 medulloblastoma 조직에서 우리는 많은 높은 proliferative 식민지를 설정할 수 있었다. 견고 같은 Nestin 및 Sox2 등 여러 신경 줄기 세포의 마커를 표현이 세포는, 일련 구절을 받다 (20보다 큰)과 clonogenic되었습니다 수 있습니다. 이러한 교양 종양의 줄기 세포가 줄기 세포의 성장을 좋아하는 조건에서 양식 때 세포질 비율이 높은 핵과 자주 bipoar 비교적 작은 동안, 그들은 극적으로, 그들의 형태를 변경 여러 세포 프로세스를 확장 평평하고 세포로 전환시 세포주기에서 전학을 가겠다고 문화 매체 10 % 태아 소 혈청과 보완. 더 중요한 것은, 이러한 종양 줄기 세포가 Tuj1로 차별 + 또는 NeuN + 뉴런, GFAP + astrocytes 및 CNPase + oligodendrocytes, 따라서 그들의 멀티 힘을 강조. 또한, 이들 세포는 orthotopically 호스트 마우스에 이식하면 보조 medulloblastomas을 전파 수 있었다.

Watch this Scientific Journal Video about Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells at JoVE.com

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

이 프로토콜은 절연, 강화하고, 이소성 소닉 헤지 호그 경로 활동과 돌연변이 생쥐에서 파생된 medulloblastoma 종양의 줄기 세포의 유지 보수를 설명합니다.

절연, 강화하고, Medulloblastoma의 줄기 세포의 유지 보수

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

isolation-enrichment-and-maintenance-of-medulloblastoma-stem-cells

Research

JoVE Journal

Neuroscience

16.2K 조회수.  Vanderbilt University. 이 프로토콜은 절연, 강화하고, 이소성 소닉 헤지 호그 경로 활동과 돌연변이 생쥐에서 파생된 medulloblastoma 종양의 줄기 세포의 유지 보수를 설명합니다.

비디오: Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

Immunocompromised 마우스의 Medulloblastoma 세포 Intracranial Orthotopic Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

연구

신경과학

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 ...

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 ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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

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

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

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

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

Isolation 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 ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

성인 얼룩말 피시의 무딘 힘 부상의 효과를 연구하는 확장 가능한 모델

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

성인 얼룩말 물고기를 사용하여 학습 및 메모리 연구에서 인지 평가를위한 연관 학습 도구로 셔틀 박스 분석

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...