Markus Pajtler ve Robert Bohrer destek ve danışmanlık, bu protokol görselleştirme için paha biçilemez.

Hayvanlar üzerinde yapılan deneylerde, hayvanların bakımı ve kullanımı ile ilgili deneysel prosedürleri ve Kurumsal Hayvan tarafından belirlenen düzenlemelere NIH Rehberi ardından, Avrupa Toplulukları Konsey Direktifi (86/609/EEC) uygun olarak yapıldığını ve bu yazarlar Bakım ve Duisburg-Essen Üniversitesi (Almanya) Komitesi (IACUC) kullanın. Bölüm 1: Kurma (Video gösterilir) GEREÇ VE ÇÖZÜMLER HAZIRLAMA (kuluçka yumurta alarak önce) <ul><li> Yumurta nemlendirilmiş bir inkübatör 37 az 11 gün inkübe olması ° C (100 ° F). </li><li> % 70 etanol sprey, temiz plakaları, hassas, temiz forseps, 4 ° C (40 ° F) soğuk steril buz DMEM ve 60 ml steril şırıngalar </li><li> 50 ml Steril Corning tüpler 4 ° C soğuk DMEM 1:1 oranında (DMEM yumuşatılır kitle) elma sirkesine embriyolarının karışımı ile yarım dolu olması. , Yumurta, civciv embriyo inkübatör diseksiyon dışarı alınır gibi kısa sürede başlatılmalıdır. </li><li> Diseksiyon araçları sterilize otoklav veya diseksiyon gün, 20 dakika boyunca% 70 etanol araçları bırakın. </li></ul> Bölüm 2: Civciv Embriyolar Diseksiyon (Video gösterdiği) <ol><li> En az 30 saniye boyunca% 70 etanol sprey ile inkübe yumurta Islak ve aşırı yumurta sallayarak olmasa da temiz yumuşak kağıt havlu ile iyice kurulayın. Not: Aynı anda 60&#39;dan fazla civciv embriyolarının diseksiyon değil tavsiye ederiz . </li><li> Keskin bir kenarına şık dikkatlice açın ve yumurta sarısı çuval ya da civciv embriyo kendisi yok dikkatli embriyo. </li><li> Göbek kordonu hızla başında arayın ve sarısı çuval ya da herhangi bir küçük damarları tahrip etmeden net bir sarma açık. Sonra kesin temiz forseps ile göbek kordonu teşrih. </li><li> Yumurta sarısı çuval embriyo dışarı atın. Not: Embriyolar (video gösterilmez) hızlı dekapite içinde olmalıdır . </li><li> 4 asgari temel orta embriyoların toplayın ° C </li></ol> Bölüm 3: Civciv Embriyolar Maserasyon <ol><li> 60 ml steril enjektör pistonu atın. </li><li> Biri 60 ml şırınga içine yaklaşık 10 embriyo transferine. </li><li> Pistonu elma sirkesine malzeme kaybetme riskine girmemek için şırıngaya dikkatle geri koyun. </li><li> Pistonu aşağı iterek ıslatarak yumuşatmak. </li><li> Hazırlanan Corning tüpler 1:1 (: DMEM yumuşatılır kitle) bir oranında DMEM ile yarı dolu elma sirkesine kitle toplayın. 10 embriyolar yaklaşık 25 ml hacmi kullanılarak üretilir. </li><li> 45 dakika 4 ° C için çalkalayıcı karışımı yerleştirin. </li></ol> Bölüm 4: Civciv Embriyo Santrifüj - DMEM Süspansiyon <ol><li> Civciv embriyo transferi - DMEM süspansiyon santrifüj tüplerine. </li><li> Embriyo başına 25 mg 1 mg konsantrasyonda steril hiyalüronidaz ekleyin. </li><li> Çok hassas Dara santrifüj tüpleri. Not: Bu adım çok yüksek g-kuvveti ve malzeme ya da dengesiz tüpler kullanılarak, santrifüj oluşabilir zarar santrifüj çalıştırmak gibi dikkatli yapılmalıdır. Yüksek doğruluk ile ilgili makine uygun DMEM ile kayıp miktarını ayarlayarak tarafından alınan tüm tüpler için ondalık noktadan sonra üç pozisyonda ağırlığındaki. </li><li> - 4 ° C santrifüj sıcaklığı azaltın Not: Ayrıca santrifüj rotor gece boyunca soğutma oda ya da buzdolabında saklanan olmalıdır . </li><li> En az 180.000 xgx saat 6 saat Santrifüj Not: tek faz ayrılması konusunda üstün sonuçlar için 266.000 xgx h. Mümkünse, vakum ayarı kullanın. </li></ol> Bölüm 5: Civciv Embriyo Özü Filtrasyon Santrifüj sonra, üç aşamadan fark edilebilir: yağ parçaları, ortasında berrak sıvı faz ve santrifüj tüpünün dibinde pelet ile üst pis sıvı faz Not: Titreme veya tempolu santrifüje karışım hareket Bu merkezi berrak sıvı faz kaybederek, santrifüj adım sonucu yok olduğu gibi kesinlikle kaçınılmalıdır. Not: Tüm aşağıdaki adımları laminer hava akışı tezgah kullanarak steril koşullar altında yapılması gereken: <ol><li> 0.45 mikron gözenekli bir filtre sistemi içine berrak sıvı faz Pipet. Sonra bağlı vakum pompası geçin. Not: altta pelet dokunmayın. Bu, özü ve filtre sistemi bir tıkanma kontaminasyona yol açacak. </li><li> 0.22 mikron ve bağlı vakum pompası anahtarı tekrar gözenekli bir filtre sistemi içine ön-özü aktarın. </li><li> Kısım elde civciv embriyo ekstresisteril 15 ml Corning tüpler. </li><li> Hızla -80 alikotları saklamak ° C (-62 ° F). Not: -80 saklandığı takdirde iki yıla kadar son CEE ° 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>

Bu protokol, geçmişte 10,11 tarif edilmiştir prosedürlerin değişiklikler dayanmaktadır . Eski yayınların yanı sıra burada bireysel farklı sürecinin adımları daha genişletilmiş göstermektedir. Bu, doğru davası ve güvenilir sonuçlar sağlayan önemli detayları ile deneyi destekler. Ayrıca, diseksiyon sürecinin vazgeçilmez bir parçası CEE çıkarma gibi biz her detayında sarısı çuval embriyoların yaptırmayı prosedürü açıklar. Aşağıda anlatıldığı gibi Unutulmaması ge...

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

Nöro-ektoderm nöral krest embriyogenez sırasında ortaya çıkan ve sadece geçici bir süre devam. İlk deneyler zaten nöral krest 1 ayrılmaz bir parçası olmak pluripotent progenitör hücrelerin geçirmez. Fenotipik, nöral krest kök hücreler (NCSC) nöral krest 2,3,4,5 göç sırasında aynı anda P75 (düşük afin sinir büyüme faktörü reseptörü LNGFR) ve SOX10 ifade ile tanımlanır . Bu progenitör hücreler, düz kas hücreleri, kromafin hücreler nöronlar ve glial hücrelerin yanı sıra, melanosit, kıkırdak ve kemik 6,7,8,9 ayırt edebilirsiniz . 10 in vitro, özel nöral krest kök hücre orta (NCSCM) NCSC yetiştirmek için gereklidir. NCSCM en karmaşık parçası NCSC yanı sıra, sinir eksplant diğer türleri için büyüme faktörlerinin önemli bir kaynağı temsil civciv embriyo ekstraktı (CEE) hazırlanması. CEE yanında diğer NCSCM maddeler ticari olarak kullanılabilir. Izolasyon, maserasyon, santrifüj ve filtrasyon işlemleri gibi zorlu detayları hakkında bilmek büyük önem CCE kullanarak laboratuvarda standart ekipman üretimi. Bu protokolde, saf ve yüksek kaliteli CEE maksimize miktarda üretmek için doğru teknikleri anlatıyoruz.

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

In vitro nöral krest kök hücreler (NCSC) yetiştirmek için özel bir orta (NCSCM) gereklidir. NCSCM önemli bir parçası civciv embriyo ekstraktı (CEE). Biz burada, izolasyon, maserasyon, santrifüj ve filtrasyon işlemleri gibi ayrıntıları da dahil olmak üzere saf ve kaliteli CEE maksimize miktarda üretmek için doğru teknikleri anlatıyoruz.

Civciv Embriyo Özü murin Sinir Crest Kök Hücre Yetiştiriciliği Üretimi

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 Görüntüleme.  University Children's Hospital Essen. In vitro nöral krest kök hücreler (NCSC) yetiştirmek için özel bir orta (NCSCM) gereklidir. NCSCM önemli bir parçası civciv embriyo ekstraktı (CEE). Biz burada, izolasyon, maserasyon, santrifüj ve filtrasyon işlemleri gibi ayrıntıları da dahil olmak üzere saf ve kaliteli CEE maksimize miktarda üretmek için doğru teknikleri anlatıyoruz.

Video: 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.

İnsan Saç köklerinin arasından Sinir Crest Kök Hücre İzolasyonu ve Kültür

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Nörobilim

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 Test: Akım Sitometri Kullanımı Farklılaşan Nöral Kök Hücre Döl nöronal Progenitör Hücrelerinin üretilmesi ve zenginleştirilmesi için bir Testi

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.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

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.

Civciv Embriyo aksonal Yörüngeler ve Synaptic Hedefler Trace için Beyin elektroporasyon

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.

Yetişkin Kemirgen Beyin konvansiyonel ve konvansiyonel olmayan Bölgelerinden Nöral Kök Hücreler Büyüyen

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.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

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.

Nöral Kök Hücre Sayım klonal Tahliller Kullanma

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.

Sıçan gözlerinin içine insan retina Pigment epitel hücrelerinin Trans-scleral Subretinal nakli için rafine bir protokol geliştirilmesi

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.

Yalıtım ve kültür embriyonik fare nöral kök hücrelerin

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.

Primer Nöral Kök ve Progenitor Hücrelerinin Hücre Yüzey Belirteçlerinin Sialoglycan'ın Metabolik Etiketlemesi ile Belirlenmesi

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