Bu çalışmada, Vanderbilt-Ingram Kanser Merkezi Destek Hibe (P30 CA068485), Çocukluk Çağı Beyin Tümörü Vakfı ve Ulusal Sağlık Enstitüsü (NS042205) hibe ile desteklendi.

1. Tümör taşıyan Serebellum Mikro-disseksiyon, Tümör Doku ve Kaplama, Ayrılma <ol><li> Tümör dokusu alımı <ol><li> Hasta fareler taşıyan medulloblastom genellikle runted, hidrosefali ve posterior felç ve devrik zaman duruş yeniden yetmezliği de dahil olmak üzere tipik bir nörolojik semptomlar görüntülenir. Tümör dokusu almak için, karbon dioksit Solunduğunda fareler euthanize. Servikal dislokasyon, posterior kafatasına basınç oluşturur ve tümör dokusu bütünlüğünü tehlikeye düşürebilecek bir işlem gerçekleştirmek için önemlidir. </li><li> Decapitation kafatası iyi bir görünüm için saç ve kas dokusu mümkün olduğunca ortadan kaldırarak, bir makas kullanarak ölümünden sonra hemen yapılır. Kafatası yüzeyi,% 95 etanol ile ıslatılmış Kimwipe ile temizleyin. </li><li> Ince makas, kafatasının orta hat boyunca bir açılış kesmek ve kafatası doku tümör taşıyan serebellum da dahil olmak üzere tüm beyin maruz kalmaktadır, bu noktada ince cımbız kullanarak kaldırmak için kullanın. </li><li> Sağlıklı yetişkinlerin cerebella iyi tanımlanmış hemisferlerin ve vermis görüntüler, Tümör taşıyan farelere cerebella genellikle düz bir yüzey ve göze çarpan kan damarları ile amorf, büyümüş. Steril teknikleri kullanarak, serebellar tümör Mg2 olmadan buz gibi soğuk PBS cımbız kullanarak ve yer almak + ve Ca 2 +. </li></ol> Not: tüm araçları kullanmadan önce% 95 etanol sterilize edilir. </li><li> Tümör dokusu Fototerapisi <ol><li> 4 dakika boyunca oda sıcaklığında 3 dakika 37 inkübasyon tarafından ince makas, ° C ile doku kıyma, PBS den 4 kez tümör dokusu hacmi yaklaşık% 50 Accutase (PBS içinde seyreltilmesi) tümör dokusu transferi sonra doku ek 3 dakika için 1 ml Pipetman tekrarlayan pipeting uğrar. Bu yöntem, tek hücre ve küçük hücresel agrega karışımı boyun eğmek zorundadır. </li><li> 1000 gr az 5 dakika pelet hücreleri hücresel süspansiyon PBS ile 3 kat ve santrifüj sulandırınız. Jelatinize 60 mm primaria doku kültürü çanak üzerine taze sinir kök hücre kültür ortamı ve plaka hücre pelletini tekrar. Biz ilk kaplama gelişmiş eklenti primaria yemekleri kullanır; sonraki geçişleri normal doku kültürü yemekleri üzerine kaplama olabilir. </li></ol></li></ol> Not: en az 30 dakika süreyle% 0,1 jelatin ile Kat kültür yemekleri. Glutamin, Pen-Strep, N2, B27, insan EGF (25 ng / ml) ve temel FGF (25ng / ml) ile Neurobasal orta oluşan taze sinir kök hücre kültür ortamı hazırlayın. 2. Zenginleştirme, Bakım ve Seri Yolculukları Medulloblastom Tümör Hücreleri genişletilmesi Biz genellikle ilk kaplama (Şekil 1) 1 hafta sonra birçok koloniler. Bu koloniler, ayrışmış ve tümör hücre popülasyonunu zenginleşmesi için yeni jelatinize yemekleri üzerine replated olabilir. Yeni bir kültür ortamına ilk değişiklik, o zaman sadece mekanik bir hücresel süspansiyon (Accutase gerekli) verim, 4 dakika boyunca tekrarlayan pipeting takip koloniler ayırmak için 1 ml Pipetman kullanabilirsiniz. Büyük hücre kümeleri ayrışmış sahip olmasını sağlamak için bir mikroskop altında kontrol edin. Sonra daha fazla kültür için yeni jelatinize yemekler üzerine hücre süspansiyonu dağıtır ve ek geçişleri için aynı prosedürü geçmesi. Ekli olmayan tümör hücreleri, kan hücreleri ve diğer hücre tipleri, bağlı tümör hücreleri hızla genişletmek için bırakarak, yeniden kaplamalar seri kaldırılır. Kültürün ilk tohumlama izleyen ikinci gününde, orta ve bundan sonra her gün değiştirin. 3. Doku ve Hücre Immunofluorescent Boyama ve Sınav 1. Cam lamelleri tümör hücrelerinin büyümesini 1. gün, 6-plaka her yer bir cam lamel, daha sonra 30 dakika boyunca 37 ° jelatin% 0.1 ° C ML başına Tohum yaklaşık 2-4X 10 5 tümör hücreleri. Hücreler gecede eklemek gerekir ve sinir kök hücre orta 3. günde değişti. Boyama, 4. günde yapılır. 2 Immunofluorescent boyama Buz gibi soğuk PBS ile iki kez kültür ortamı ve yıkama hücreleri çıkarın. 15 dakika boyunca taze hazırlanmış% 4 PFA hücreleri saptamak. Kısaca PBS ile iki kez hücreleri yıkayın ve 5 dakika boyunca% 0.3 Triton (PBS) ile permeablize. Kısaca hücreler PBS ve blok ile iki kez 40 dakika boyunca% 10 keçi serumu ile yıkayın. Hücreleri için primer antikor ile 90 dakika inkübe edin, sonra her biri için 5 dakika PBS ile hücreler üç kez yıkayın. Hücreleri için ikincil antikor ile 60 dakika inkübe edin, her biri 5 dakika sonra, PBS ile üç defa yıkamak. 5 ml sulu bir montaj orta Slaytlarınıza lamelleri Dağı ve konfokal mikroskobi gerçekleştirmek. 4. Tümör Hücreleri farklılaşma N çıkarıneural kök hücre orta ve kısaca hücreleri PBS ile iki kez yıkayın. Farklılaşması orta 1 gün, günde 4 orta değiştirmek ve 7 gün immünofloresan boyama farklılaşma düzeyini belirlemek. Farklılaşması orta DMEM, Kalem-Strep ve% 10 fetal sığır serumu oluşur. 5. Temsilcisi Sonuçlar Morfoloji sonuçları Accutase tedavi ve nazik tekrarlayan pipeting sonra, tümör dokusu küçük hücresel agrega ve tek hücre içine çoğunlukla ayrışmış olmalıdır. Birçok büyük kolonileri Şekil 1&#39;de gösterildiği gibi, 5 gün sonra ilk kaplama gibi erken bir zamanda görülebilir. Yüksek proliferatif tümör hücreleri genellikle yüksek çekirdek / sitoplazma oranı ile bi-polar. Bu hücreler genellikle jelatinize yüzeye bağlı küçük hücresel agrega yayılan görülür. Kan hücreleri, küçük ve yuvarlak, genellikle sonraki ortam değişiklikleri ve seri kaplamalar kaldırılabilir. Çok ayetle sonra, bir gözlemleyebilirsiniz düzgün yayılı, proliferatif Ki67 + tümör hücreleri ve diğer hücre tipleri (Şekil 2) çok az kontaminasyon. Sinir kök hücre belirteçleri, klonal analiz ve birden fazla soy içine farklılaşma Açıklanması Proliferatif hücreler, tümör kök hücrelerinin ayrışmış serebellar tümör dokusu özelliklerini gösterdikleri türetilmiş olup olmadığını belirlemek için, kök hücre işaretleyici ifade, klonal analiz ve farklılaşma potens ileri karakterizasyonu yapıldı. Biz bu izole tümör hücreleri son derece sinir kök hücre belirteçleri Sox2 ve Nestin (Şekil 3) olarak ifade ettikleri bulundu. SmoM2 YFP tümör dokuları alınan tüm tümör hücrelerinin ifade YFP. Birincil kirpikler raporlama son çalışma ile tutarlı Shh yolağı bağımlı medulloblastom 12, SmoM2-YFP ifadesi açıkça Sox2 silya + tümör hücreleri (Şekil 3) lokalize gelişimi için önemlidir. Başına 300 ml kültür ortamı hücrelerinin klonal yoğunlukta kaplama, biz tek bir tümör hücresi oldukça büyük bir koloni (Şekil 4) klonal genişleme görülmektedir. Ayrıca, bu hücreler yanlısı farklılaşma koşullar altında (Şekil 5) kültüre çeşitli nöronal ve glial hücre tipleri ayırt etmek mümkün. <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="Şekil 1" /> Şekil 1. Ayrışmış medulloblastom doku ilk kaplama sonra yüksek proliferatif koloni morfolojisi. Genel olarak, parlak bir alan içinde oldukça büyük bir koloni formları, 5 ayrışmış tümör dokusunun ilk tohumlama gün sonra ve bir temsilcisi olarak burada gösterilmiştir. Bi-polar, uzamış yoğun bir çekirdek hücrenin toplam hücreleri yayar. Seri kaplamalar yavaş yavaş tükenmiş küçük, yuvarlak, kırmızı kan hücreleri, tümör hücrelerinin tutunmuştur. <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="Şekil 2" /> Şekil 2. Üç geçişleri dışında kurulan medulloblastom hücrelerinin morfolojisi. Bu parlak alan görüntü çok ayetle sonra kurulan medulloblastom hücrelerinin tipik görünümü gösterir. Hücreleri çok yüksek sitoplazmik / nükleer oranı ile bi-polar kalır. Biz tümör hücrelerinin asetillenmiş tubulin boyama, en güçlü Ki67 ifade tarafından gösterildiği gibi bisiklet vardır. <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="Şekil 3" /> Şekil 3. Kurulan medulloblastom hücreleri sinir kök hücre işaretleri ifade eder. SmoM2 YFP ifade hücreleri yapısal olarak aktif Smo, dolayısıyla Shh yolağı faaliyet işaretler. Kurulan medulloblastom hücre hatları tüm hücreler YFP ifade ve çoğu Nestin ve Sox2 gibi çeşitli sinir kök hücre belirteçleri, yüksek düzeyde ortak dile getirdi. İlginçtir ki, biz konfokal mikroskopi sırasında en az lazer gücü ile YFP sinyal algılama yapıldığında, Sox2 + hücrelerin silya konsantre YFP sinyal tespit edildi. Bu gözlem, Shh yolağı bağımlı medulloblastom gelişimi kirpikler temel rol açıklanan son raporunda 12 ile tutarlıdır . <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="Şekil 4" /> Şekil 4. Kurulan tümör hücrelerinin klonal genişleme uğrarlar. Disosiasyon sonra tek bir hücre süspansiyonu içine, tümör hücrelerinin klonal bir kültür ortamı ml başına 300 hücre yoğunluğu az 24 iyi plaka üzerine kaplanmıştır. Her bir kuyunun klonal genişletilmiş koloniler gözlemledi. Bu dizi parlak bir alan görüntüler 10 günlük süre içinde bir kültür tipik değişiklikleri göstermektedir. <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="Şekil 5" /> Şekil 5. Farklılaşma kurulan medulloblastom hücrelerinin analiz eder. Tümör hücrelerinin EGF / bFGF DMEM/10% FBS, YFP + tümör hücrelerinin kendi morfolojisi önemli ölçüde değişmiş ve çeşitli hücre tipleri, inc ayrılabilir serum orta içeren geçişTuj1 luding + veya Neun + nöronlar, GFAP + Astroglial hücreleri veya CNPase + oligodendrosit.

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Medulloblastom kök hücreleri yapısal olarak aktif Kirpi sinyalizasyon ile mutant farelerde alınan birincil tümör dokusundan izole zenginleştirmek ve korumak için etkili bir şekilde açıklar. Biz, başarılı, sağlıklı bir tümör kök hücre hattı kurulması bir kritik adım birincil tümör dokusu disosiasyon sırasında kullanılan Accutase bir tedavi olduğunu bulundu. Bizim tecrübelerimize göre, beyincik ilk dissociating tümör dokusu, 4 dakika 37% 50 Accutase tedavi ° C, 1 ml Pipetman kullanarak g...

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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>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>
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isolation, enrichment, and maintenance of medulloblastoma stem cells

Beyin tümörleri Tümörgenez kök neden olan kök hücreler küçük bir nüfusa sahip öne sürülmüştür. Neurosphere testleri genellikle normal ve tümöral dokuların elde edilenler dahil olmak üzere nöral kök hücreler, doğayı incelemek için kabul edilmiştir. Ancak, kayda değer miktarda farklılaşması ve hücre ölümüne kürenin agrega içinde tüm hücrelerin kültür ortamı erişilebilirlik gibi sub-optimal durum muhtemelen nedeniyle kültür neurospheres yaygındır. Medulloblastom, en sık görülen pediatrik merkezi sinir sistemi tümörü, hızlı ilerlemesi ve kasvetli klinik sonuç ile tüm beyin-omurilik ekseni boyunca yayılma eğilimi ile karakterize. Medulloblastom 1 sırasıyla% 20 ve% 40, çocukluk çağında intrakranial ve posterior fossa tümörü için muhasebe, beyinciğin bir nöroepitelyal tümör . Şimdi iyi Shh sinyalizasyon serebellar gelişim 2-4 sırasında serebellar granül nöron öncüleri (CGNPs) yayılması uyarır kurulmuştur. Çeşitli çalışmalar, Shh yolu yapısal olarak aktif olan fare modelleri kullanarak medulloblastom 5-9 Shh sinyalizasyon bağlantılı. Yeni bir rapor Patched1 LacZ / + fareler türetilen medulloblastom hücrelerinin tümörleri 10 başlatılması ve propogating yeteneğine kanser kök hücreleri, bir alt kümesi olduğunu göstermiştir. Burada Smoothened içinde bir mutasyon (11 hereon SmoM2 olarak anılacaktır), Shh için kritik bir GPCR nedeniyle yapısal olarak aktive Shh yolağı, medulloblastom birkaç fare modelleri türetilmiş tümör kök hücreleri izole zenginleştirmek ve korumak için etkili bir yöntem tarif yolağı aktivasyonu. Her izole medulloblastom dokusu, çok sayıda yüksek proliferatif koloniler kurmayı başardık. Bu hücreler, sağlam Nestin ve Sox2 gibi çeşitli sinir kök hücre belirteçleri ifade seri pasajlar tabi (20&#39;den fazla) ve clonogenic edildi. Bu kültür tümör kök hücreler, kök hücre büyüme lehine koşullar altında kültüre sitoplazmik oranı yüksek nükleer ile sık sık bipoar nispeten küçük iken, bu ölçüde, kendi morfolojisi değiştirdi birden fazla hücre sürecinin genişletilmiş, basık ve bir hücreye geçiş üzerine hücre döngüsü çekildi kültür ortamı,% 10 fetal sığır serumu ile desteklenmiştir. Daha da önemlisi, bu tümör kök hücreler Tuj1 ayrılabilir + veya Neun + nöronlar, GFAP + astrositler ve CNPase + oligodendrosit, böylece onların çok potens vurgulayarak. Ayrıca, bu hücreler orthotopically ana farelere nakledilen zaman ikincil medülloblastomlar yayma yeteneğine sahip.

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

Bu protokol, izolasyon, zenginleştirme, ektopik Sonic hedgehog yolu aktivite ile mutant farelerde elde edilen medulloblastom tümör kök hücreler bakım ve açıklar.

Medulloblastom Kök Hücreleri İzolasyon, Zenginleştirme ve Bakım

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 Görüntüleme.  Vanderbilt University. Bu protokol, izolasyon, zenginleştirme, ektopik Sonic hedgehog yolu aktivite ile mutant farelerde elde edilen medulloblastom tümör kök hücreler bakım ve açıklar.

Video: 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.

Medulloblastom Hücre bağışıklık Fare İntrakranial Ortotopik Allografting

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

Nörobilim

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.

Neurosphere Testi Yetişkin Fare Nöral Kök Hücre İzolasyonu ve Genişleme

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.

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 ...

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 ...

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.

Embriyonik Kemirgen Brain itibaren İlköğretim Mesensefalik dopaminerjik Nöronlar İzolasyonu, Kültür ve Uzun Vadeli Bakım

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.

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

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.

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 ...

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.

Yetişkin Zebra Balıklarında Künt Kuvvet Yaralanmasının Etkilerini Incelemek İçin Ölçeklenebilir Bir Model

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.

Yetişkin Zebra Balığı Kullanarak Öğrenme ve Bellek Çalışmalarında Bilişsel Değerlendirme için İlişkisel Bir Öğrenme Aracı Olarak Mekik Kutusu Tahlil

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