마커스 Pajtler과 로버트 Bohrer의 지원과 조언이 프로토콜의 시각화를위한 귀중한되었습니다.

동물 실험 기관에 의해 규정된 동물 실험 절차와 규정에 대한 동물의 보호 및 사용에 관한 NIH의 지침에 따라, 유럽 커뮤니티 이사회 지침 (86/609/EEC)에 따라 수행다고 저자 상태 관리 및 뒤스부르크 - 에센 대학 (독일)에서위원회 (IACUC)를 사용합니다. 1 부 : (비디오에 표시되지 않음) 설정 (인큐베이터 밖으로 알을 복용하기 전에) 자료 및 솔루션 준비 <ul><li> 계란은 37 11일위한 humidified 인큐베이터에 incubated해야 ° C (100 ° F). </li><li> 70 % 에탄올 스프레이, 깨끗한 접시, 정밀 청소 집게, 4 ° C (40 ° F) 추운 살균 얼음 DMEM 60 ML 무균 주사기 </li><li> 50 ML 무균 코닝 튜브 ° C 감기 DMEM가 1시 1분의 비율 (DMEM 분해 질량)에서 분해 배아를 섞어 4 반 가득해야합니다. 즉시 계란이 여자는 태아의 인큐베이터의 해부 밖으로 가져옵니다 같은 것은 시작되어야한다. </li><li> 압력솥이나 해부 당일 해부 도구를 소독, 20 분 70 %의 에탄올에있는 도구를 잠그다. </li></ul> 2 부 : 칙 태아의 해부 (비디오에서 보여준) <ol><li> 최소한 30 초 동안 70 % 에탄올 스프레이로 incubated 알을 습식 및 과도하게 계란을 흔들림없는 동안 깨끗하고 부드러운 종이 타올로 신중하게 건조. 참고 : 우리는 동시에 60 개 이상의 여자는 배아를 해부하지 권장합니다. </li><li> 날카로운 모서리로부터적으로하여 신중하게 계란을 열고 노른자의 자루 또는 여자는 배아 자체를 파괴하지주의와 배아를 꺼내. </li><li> 빨리 탯줄의 시작에 대한 검색 및 노른자의 자루 또는 작은 혈관을 파괴하지 않고 맑은 포장을 엽니다. 그런 다음 정확한 깨끗한 포셉과 탯줄을 해부하다. </li><li> 난황 자루의 배아를 꺼내주세요. 참고 : 태아가 (비디오에 표시되지 않음) 신속하게 참수해야합니다. </li><li> 4 최소한의 필수적인 매체 배아를 수집 ° C. </li></ol> 파트 3 : 칙 태아의 물에 담가서 부드럽게 함 <ol><li> 60 ML 멸균 주사기 밖으로 플런저를 가져가라. </li><li> 한 60 ML 주사기로 약 10 배아를 전송합니다. </li><li> 분해 물질을 잃을 위험하지 위해 신중하게 다시 주사기로 플런저를 넣어. </li><li> 플런저를 밀어 물에 담가서 부드럽게. </li><li> 준비 코닝 튜브 1시 1분 (DMEM 분해 질량)의 비율로 DMEM과 절반 채워진에서 분해 덩어리를 수집합니다. 10 배아에게 약 25 ML의 볼륨을 사용하는 것은 생산이다. </li><li> 4 45 분 ° C. 위해 쉐이크에 혼합 플레이스 </li></ol> 파트 4 : 칙 배의 원심 분리 - DMEM 서스펜션 <ol><li> 여자의 배아를 전송 - DMEM 서스펜션을 원심 분리 튜브에. </li><li> 배아의 25 MG 당 1 밀리그램의 농도에서 살균 hyaluronidase를 추가합니다. </li><li> 매우 정확하게 테어 원심 분리 튜브. 참고 : 원심 분리가 매우 높은 G - 포스 및 재료 또는 불균형 튜브를 사용하는 경우 원심 분리기가 발생할 수의 손상에서 실행되는이 단계는주의해서 수행해야합니다. 모든 튜브에 대한 소수점은 DMEM으로 누락된 금액을 조정하여 접수해야 다음 세 위치에 대한 기계에 따라 무게 높은 정확도. </li><li> 4 원심 분리기의 온도를 줄이기 ° C. 참고 : 또한 원심 분리의 회전자는 밤 동안 냉각 챔버 또는 냉장고에 보관해야합니다. </li><li> 적어도 180.000 xgx H. 6 시간 원심 분리기 참고 : 단일 단계의 분리에 관한 뛰어난 결과를 얻으려면이 266.000 xgx H.를 사용하여 가능하면 진공 조정을 사용합니다. </li></ol> 5 부 : 칙의 배아 추출의 여과 원심 분리 후, 세 단계가 발견하실 수 있습니다 지방산 조각, 중간에 투명 액상 및 원심 분리 관의 하단에있는 펠렛과 함께 상위 거무죽죽한 액상 참고 사항 :. 떨려 또는 활발한 centrifuged 혼합물의 이동하면되어야합니다 이것은 명확한 중앙 액체 단계를 잃는하여 원심 분리 단계의 결과를 파괴처럼 엄격히 피해야. 참고 : 모든 다음 단계는 층류 공기 흐름 벤치를 사용하여 무균 조건 하에서 수행되어야 : <ol><li> 0.45 μm의의 모공과 필터 시스템에 투명한 액체 단계를 피펫. 다음 연결된 진공 펌프 스위치. 참고 : 하단에있는 펠렛를 만지지 마십시오. 이것은 추출하고 필터 시스템의 차단의 오염으로 이어질 것입니다. </li><li> 0.22 μm의와 연결된 진공 펌프의 스위치를 다시의 모공과 필터 시스템에 사전 추출물을 전송합니다. </li><li> 나누어지는 획득 여자는 배아 추출물에게무균 15 ML 코닝 튜브 인치 </li><li> 신속 -80에서 aliquots를 저장할 ° C (-62 ° F). 참고 : -80에 저장된 경우 최대 2 년 동안 마지막시 ° C. </li></ol>

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. 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+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <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+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜은 과거 10,11에서 설명했습니다 절차의 변경에 따라 달라집니다. 전 출판물 이외에도 우리는 여기 개별 서로 다른 프로세스의 단계 더 확장을 보여줍니다. 이것은 올바른 절차와 신뢰할 수있는 결과를 확보 중요한 세부 실험자을 지원합니다. 해부 과정시 추출의 필수적인 부분이므로 나아가, 우리는 모든 세부 사항에 노른자의 자루의 배아를 복용의 절차를 설명합니다. 아래?...

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		<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>11 days incubated chicken eggs</td>
<td>&nbsp;</td>
<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Alcoholic disinfection spray</td>
<td>&nbsp;</td>
<td>Sch&uuml;lke &amp; Mayr GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEMGlutamax</td>
<td>&nbsp;</td>
<td>GIBCO Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe sterile (60 ml)</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tubes sterile (50; 15 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipet tips sterile</td>
<td>&nbsp;</td>
<td>Starlab GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase typeIV-S</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
<td>&nbsp;</td>
<td>Fisher Scientific AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipettes sterile (25; 10; 5 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge L5-50</td>
<td>&nbsp;</td>
<td>Rotor type Ti-45</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

production of chick embryo extract for the cultivation of murine neural crest stem cells

신경 능선은 embryogenesis 동안 신경 ectoderm에서 발생에만 일시적으로 지속. 초기 실험은 이미 신경 문장 하나의 불가분의 일부로 pluripotent 전구 세포를 침대. Phenotypically, 신경 크레스트 줄기 세포 (NCSC)는 동시에 신경 문장 2,3,4,5으로부터 마이 그 레이션하는 동안 p75 (낮은 affine 신경 성장 인자 수용체, LNGFR)와 SOX10 표현에 의해 정의됩니다. 이러한 전구 세포는 평활근 세포, chromaffin 세포, 뉴런과 glial 세포뿐만 아니라, melanocytes, 연골과 뼈 6,7,8,9로 구별하실 수 있습니다. 시험 관내, 특별한 신경 크레스트 줄기 세포 매체 (NCSCM)에 NCSC를 육성하기 위해서는 10 필요합니다. NCSCM의 가장 복잡한 부분은 NCSC뿐만 아니라 신경 explants 다른 유형의 성장 요인의 필수 소스를 대표하는 여자는 배아 추출물 (시)의 준비입니다. 시 옆에 다른 NCSCM 재료는 상업적으로 사용할 수 있습니다. 그것은 고립, 물에 담가서 부드럽게 함, 원심 분리 및 여과 공정으로 도전하는 세부 사항에 대해 알고 높은 중요 CCE 사용하여 실험실 표준 장비를 생산. 이 프로토콜에서는 순수하고 고품질시의 최대 금액을 생산하는 정확한 방법을 설명합니다.

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

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

체외에서 신경 크레스트 줄기 세포 (NCSC)을 육성하기 위해 특수 매체 (NCSCM)가 필요합니다. NCSCM의 필수적인 부분은 여자의 배아 추출물 (시)입니다. 우리는 여기서 고립, 물에 담가서 부드럽게 함, 원심 분리 및 여과 공정과 같은 세부 사항을 포함하여 순수하고 고품질시의 최대 금액을 생산하는 정확한 방법을 설명합니다.

Murine 신경 크레스트 줄기 세포의 배양 칙의 배아 추출 생산

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

production-chick-embryo-extract-for-cultivation-murine-neural-crest

Research

JoVE Journal

Neuroscience

12.7K 조회수.  University Children's Hospital Essen. 체외에서 신경 크레스트 줄기 세포 (NCSC)을 육성하기 위해 특수 매체 (NCSCM)가 필요합니다. NCSCM의 필수적인 부분은 여자의 배아 추출물 (시)입니다. 우리는 여기서 고립, 물에 담가서 부드럽게 함, 원심 분리 및 여과 공정과 같은 세부 사항을 포함하여 순수하고 고품질시의 최대 금액을 생산하는 정확한 방법을 설명합니다.

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

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 ...

연구

신경과학

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

병아리 배아에서 축삭 궤적과 시냅스 대상을 추적하는 후뇌의 일렉트로

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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

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

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

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

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

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 ...