HD054347 ve NS063997-01 VLS için: Bu çalışma Ulusal Sağlık Enstitüleri tarafından desteklenmektedir. Bu çalışma aynı zamanda New York Dışişleri Bakanlığı aracılığıyla Sağlık Sözleşme # C024324 VLS Empire State Kök Hücre Fonu tarafından desteklenmiştir. Burada ifade edilen görüşler yalnızca yazarın aittir ve ille de Empire State Kök Hücre Kurulu, New York Eyaleti Sağlık Departmanı, ya da New York Devlet yansıtmamaktadır. VLS Doris Duke Klinik Scientist Gelişim Ödülü Alıcı. Ayrıca Profesör Timothy Vartanian Anti-O1, Anti-O4 antikorlar onun hediye için teşekkür ederim.

1. Diseksiyon ve sinir kök hücre kültürü bakımı için çözümler ve malzemelerin hazırlanması <ol><li> 100ml diseksiyonu orta (nakavt DMEM/F12, Invitogen) vaktinden önce hazırlayın ve buzdolabına kaldırın. </li><li> Prepare100 ml kültür ortamı (Stem Pro MGK SFM, Invitrogen) ve 37 tutmak ° C su banyosu. </li><li> Uzun süreli hücre dondurulması için hücre dondurma orta (nakavt DMEM/F12 10% FBS +% 5 DMSO) hazırlayın. </li><li> İsterseniz, doku fiksasyonu için% 4 paraformaldehid (PFA) hazırlamak. </li><li> Kulplu Steril, otoklavlanmış forseps ve bisturi ağızları diseksiyon için kullanılır. </li><li> Bir pipet tabancası, 10 ml transferi pipetler, ve 40 mcM hücre disosiasyon süzgeç (BD Falcon 352.340) bir kenara koyun. </li><li> 10 cm diseksiyon için çeşitli kültür kaplarına (BD), disosiasyon için 50 ml santrifüj tüplerine (BD), donma hücrelerin doku saklama ve donmuş şişeleri (BD) için 1,5 ml santrifüj tüplerine kenara koyun. </li></ol> 2. Insan fetal beyin insan sinir kök hücreleri Yalıtımlı <ol><li> Fetus sona ermesinden hemen sonra canlı doku Hasat prosedürün başarısı için hayati önem taşımaktadır. Seçmeli prosedürleri için, numune almaya zamanlama fetal ölümünden sonra zamanı en aza indirmek amacıyla önceden düzenlenmiş olabilir. 2 saat prosedürü içinde anlayışının ürünleri hasat edilir, ancak ideal bir dakika içinde seçmeli prosedürleri elde edilebilir. Fetal dokularda genellikle parçalanmış. Ancak, genel olarak beynin önemli bir bölümünü görsel kimlik için sağlam kalır. Gebelik yaşı (GA) Sınırlamalar yasal hukuk tarafından belirlenir ancak GA 18-22 hafta arasında bu protokol kullanılarak yapılmıştır. </li><li> Fetal beyin buz nakavt DMEM/F12 çözüm içeren 10 ml Petri kabı yerleştirilir. Anatomik işaretleri korteksin farklı parçalarını tanımak. Frontal ve parieto-oksipital korteks Sınırları merkezi sulkus ve sylvian fissür ekstrapole kesiştiği odaklı. Ventrikül sağlam ve hasarsız tutmak için emin olun, merkezi sulkus ön frontal korteks ve cerrahi bıçaklar ile sylvian fissür sınırındaki doku parçalara ayır. </li><li> Frontal korteks ayrılan blok herhangi bir kalıntı kan ve meninks çıkarın. Örnek yeterli kalitede ise, çeşitli amaçlar için birkaç küçük örnekleri içine blok incelemek için idealdir: bölümler (PFA,% 4 paraformaldehid sabit) ve protein / mRNA testleri (hızlı -80 ° C donmuş). </li><li> Seçilen beyin blok 50 ml santrifüj tüpüne aktarın ve hakkında 3 kez doku hacmi az buz nakavt DMEM/F12 çözüm eklemek. 10 ml transfer pipetle yavaşça mekanik pipetleme doku ayrıştırmaları tüm dokulara kadar (genellikle 20-30 kez) parçalanmış olur ve daha sonra tek ya da yakın tek bir hücre elde etmek için 40 mcM hücre süzgeç (BD Falcon 352.340) ile hücrelerin filtre süspansiyon. </li><li> Hücre süspansiyonu 2000 rpm ve oda sıcaklığında 5 dakika süreyle santrifüj, 10 ml taze sıcak bir kültür ortamı (Stem Pro MGK SFM) hücre pelletini tekrar süspansiyon ve hemasitometre ile hücre sayısını. </li><li> 5 ml sıcak bir kültür ortamı, her biri 25 cm 2 kültür şişeler içine ekleyin ve her balona 2x10 6 hücre transferi. Kültürler 37 korunur ° C /% 5 CO 2 inkübatör analizden önce 1 hafta boyunca. Daha fazla kültür ve deneyler için haftada bir kez yarım orta değiştirin. </li></ol> 3. Nöral kök hücreler daha fazla karakterizasyonu veya deneylerine için Manipulaton <ol><li> Neurospheres genellikle MGK çapı 200 ve 400 mikron arasında değişen, önerilen kültür koşulları altında 1 ila 2 hafta form. Neurospheres bu aşamada tek hücre elde etmek için 15 dakika süreyle 37 kalsiyum ve magnezyum ücretsiz Hanks orta (Tom Hanks) 0.2 g / L EDTA ° C ile ayrıştırıldı olabilir. Hücreler süspansiyonu, taze Hanks durulanır 2000 rpm döndü ve altkültürün sıcak kültür ortamı replated. </li><li> Farklılaşma başlatmak için, ayrışmış hücreleri lamel başına 1x10 5 hücreleri (24mmX24mm) yoğunluğu poly-D-lysine/laminin 1 kaplı lamelleri kaplanmıştır. Oligodendrosit farklılaşması için nakavt DMEM/F12 (Invitrogen, Ana, MD) +2% B27 (50X, Invitrogen, Ana, MD) 10 ng / ml bFGF 100 ng / ml SSH + 10 ng PDGF-AA hücreleri koruyarak elde edilir 2 gün sonra 5 gün süreyle büyüme faktörleri olmadan aynı ortama geçiş. 7 gün boyunca nöronal farklılaşma nakavt muhafaza hücreleri tarafından DMEM/F12 +2% B27 (50X) elde edilir. Astrosit farklılaşma, bir hafta boyunca nakavt DMEM/F12% 1 FBS kültür hücreleri tarafından yapılır. </li><li> Genleri ile nöral kök hücreler Transfeksiyon preformed neurospheres ayrı hücreleri ile yapılabilir. Burada, nöral kök hücreler elektroporasyon EGFP-C1 ile transfekte gösterdi. EGFP-C1 inşa elektroporasyon (Fare Nöral Kök Hücreler AMAXA Nucleofector Kitleri kullanılarak yapıldı VPGŞirketin talimat aşağıdaki AMAXA Nucleofector Device (Lonza AAD-1001), 1004). Kısacası, 1 X 10 6 hücreleri ile 5 mg DNA 100 ul transfeksiyon orta ile karışık, ve darbe elektroporasyon sonra, hücreleri, sinir kök hücreleri daha fazla kültür ortamı sürdürmek yeniden süspanse edildi. Transfekte hücrelerin farklılaşması adım 3.2 &#39;de açıklanan aynı prosedürler izlenerek, neurospheres elektroporasyon sonra 3-4 gün ayrışma ile işlendi. </li></ol> 4. Donma sinir kök hücreleri ve altkültürlerin <ol><li> Grubuyla aynı hücre süspansiyonları 1x10 7 hücre / flakon / ml &#39;lik bir konsantrasyon ile santrifüj ve donma ortamda yeniden süspanse. -20 ° C, -80 hücrelerin yavaş yavaş dondurma ° C için sıvı nitrojen aktarmak sonra da uzun süre depolama. </li><li> Hücreler hızlı 37 ile çözülmüş ° C su banyosu ve yeniden süspanse +10% serum DMEM/F12 bir yıkama için ısındı, donma orta kaldırmak için santrifüj ve ısıtılmış bir kültür ortamı yeniden bekletildi. </li></ol> 5. Temsilcisi Sonuçlar: Sinir kök hücreleri normal bir fetus gebelik yaşı 18 hafta açıklanan yöntem ve neurospheres, yuvarlak, düzgün sınırları ve oldukça homojen bir boyutu (Fig.2A) ile bir hafta sonra görülebilir kültüre edildi. Bu neurospheres EGFP-C1 veya diğer yapıları ile transfekte ve floresan mikroskobu (Fig.2B) altında takip edilebilir. Daha sonra kurulan neurospheres EDTA ile kaplı lamelleri üzerinde dağınık hücreler olarak ayrışmış ve kaplama. % 4 parafamaldehyde ile ilgili protokol çerçevesinde farklılaşmış hücreler sabit ve farklı hücre tipi özel işaretleri ile boyandı. Multipotentiality (Fig2E, F, rodamin) astrositler (Fig2C, D, rodamin) nöronların gösterge işaretleri ve oligodendrosit (Fig2G, H, rodamin) ifadesi ile görülmektedir. EGFP, elektroporasyon geçirmiş hücrelerin farklı hücre türlerine de farklılaşmış ve farklı hücre özel işaretleri ile boyandı. Multipotentiality (Fig3C, rodamin ve Fig3D, fluoroscein) astrositler (Fig3A, B, fluoroscein) nöronların gösterge işaretleri ve oligodendrosit (Fig3E, F, rodamin) ifadesi ile görülmektedir. <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="Şekil 1"/> Şekil 1 atılır insan fetal beyin sinir kök hücreleri izole etmek için deneysel işlemin şematik <img src="/files/ftp_upload/2681/2681fig2.jpg" alt="Şekil 2"/> Şekil 2, in vitro propogated ayrım yapılmamış ve farklı insan sinir hücrelerinin . (A) Neurospheres pürüzsüz, yuvarlak sınırları ve kültür üzerinden 1 hafta sonra hızlı bir büyüme gösteren faz kontrast mikroskobu altında gösterilmiştir. (B) çeşitli plazmid ve yapıları Giriş transfeksiyon ile elde edilebilir. EGFP-C1 transfeksiyon izleyen üç gün, birden fazla hücre fluoroscein immün floresan mikroskobu altında görülebilen yeşil flüoresan protein ekspresyonu göstermektedir. EGFP-C1 transfekte neurospheres nöronları (C, D), astrositler (E, F) ve oligodendrosit (G, H) içine farklı koşullar altında, ayrışmış ve farklılaştırılmış ve rodamin floresan altında görülmektedir. Aynı zamanda, transfekte EGFP pozitif hücrelerin fluoroscein floresan altında gösterilmiştir. Transfekte hücreleri (beyaz ok uçları) untransfected hücreleri (beyaz oklar) ayırt edilemez. Hücre çekirdekleri Hoechst33342 ile boyandı. Ölçeği çubuklar 200 mikron A, B için 100 mikron ve 25 mikron CH için. <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="Şekil 3"/> Şekil 3 transfeksiyon olmadan Neurospheres ayrışmış ve farklı nöronların (A, B, fluoroscein), astrositler (C rodamin, D, fluoroscein) ve oligodendrosit (E, F, rodamin) içine farklı koşullar altında. Hücre çekirdekleri Hoechst33342 ile boyandı. Ölçeği bar AF için 25 mikron.

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Kültür taze doku ve üreten insan hücre hatları yönelik çeşitli yaklaşımlar vardır. Tarihsel olarak, taze doku hasat ve merkezi sinir sisteminde çeşitli hücre tipleri oluşturmak için hemen kültür. Bu yaklaşım, ancak net bir şekilde elde durumda insan örnekleri için, genellikle oldukça küçük olabilir örneklerinin sayısı ile sınırlıdır. Minimal manipülasyon derecesi göz önüne alındığında, taze kültür sinir hücreleri uzun kültür potansiyel eserler sınırlayarak en güvenilir d...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Nakavt DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td> Disosiyasyon orta </td></tr><tr><td> Kök Pro MGK SFM </td><td> Invitrogen </td><td> A10509-01 </td><td> Kültür ortamı </td></tr><tr><td> Fetal sığır serumu </td><td> Invitrogen </td><td> 10091-148 </td><td> Dondurulmuş orta </td></tr><tr><td> Hanks çözümü (-Ca 2 +,-Mg 2 +) </td><td> Invitrogen </td><td> 14175-095 </td><td> Disosiyasyon orta </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D2650 </td><td> Dondurulmuş orta </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> 431788 </td><td> Disosiyasyon orta </td></tr><tr><td> Paraformaldehit </td><td> Sigma-Aldrich </td><td> 158127 </td><td> Fiksasyon çözüm </td></tr><tr><td> bFGF </td><td> Ar-Ge </td><td> 234-FSE </td><td> Farklılaşma orta </td></tr><tr><td> SSH </td><td> Ar-Ge </td><td> 1845-SH </td><td> Farklılaşma orta </td></tr><tr><td> PDGF-AA </td><td> Ar-Ge </td><td> 221-AA </td><td> Farklılaşma orta </td></tr><tr><td> B27 </td><td> Invitrogen </td><td> 17504-044 </td><td> Farklılaşma orta </td></tr><tr><td> Fare anti-map2 </td><td> Sigma-Aldrich </td><td> M2320 </td><td> 1:200 </td></tr><tr><td> Tavşan anti-DCX </td><td> Hücre sinyal </td><td> 4604s </td><td> 1:200 </td></tr><tr><td> Tavşan anti-GFAP </td><td> DAKO </td><td> Z0334 </td><td> 1:200 </td></tr><tr><td> Tavşan anti-S100B </td><td> DAKO </td><td> Z0311 </td><td> 1:200 </td></tr><tr><td> Tavşan anti-O1 </td><td> Profesör Timothy Vartanian hediyeler * </td><td></td><td> 01:50 </td></tr><tr><td> Tavşan anti-O4 </td><td> Profesör Timothy Vartanian hediyeler * </td><td></td><td> 01:50 </td></tr><tr><td> 40μm hücre süzgecinden </td><td> BD Falcon </td><td> 352340 </td><td></td></tr></tbody></table> * Timothy Vartanian, MD, PhD, Nöroloji ve Nörobilim, Weill Cornell Medical College, New York, ABD Bölümü

generation of neural stem cells from discarded human fetal cortical tissue

Nöral kök hücreler (NSC&#39;lerde) kortikal plak gelişimi sırasında ventriküler zon neuroepithelium boyunca bulunur. Bu ilk atalarıdır sonuçta ara atalarıdır yol açan ve daha sonra, serebral korteks oluşturan çeşitli nöronal ve glial hücre alt tipleri. Atılır, normal fetal doku, insan NSC&#39;lerde (böylece neurospheres denir) oluşturmak ve genişletmek için kapasite doğrudan normal insan MGK geliştirme 1-5 fonksiyonel yönlerini incelemek için bir araç sağlar. Bu yaklaşım aynı zamanda, böylece progenitör proliferasyon, göç ve farklılaşma 6-9 değiştiren hastalık süreçleri tanımlamak için fırsat karşılayabilme bilinen nörolojik bozukluklar NSC&#39;lerde nesil doğru olabilir . Biz hızlandırılmış Alzheimer hastalığı fenotip 10,11 katkıda bulunabilecek insan Down sendromu NSC&#39;lerde patolojik mekanizmaların belirlenmesi üzerine odaklanmıştır. Ne, in vivo, ne in vitro fare modellerinde 21 insan kromozomu üzerinde bulunan genlerin aynı repertuar çoğaltabilirsiniz . Burada iptal insan fetal korteks Down sendromu NSC&#39;lerde kültür izole etmek ve onları büyümeye basit ve güvenilir bir yöntemi kullanabilirsiniz. Metodolojisi sınırlı anatomik işaretleri, hücre sıralama, kaplama ve insan NSC&#39;lerde Pasajlanması doku diseksiyonu hasat belirli yönlerine sağlar. Biz de daha seçici hücre alt tiplere insan NSC&#39;lerde farklılaşma inducing için bazı temel protokolleri sağlar.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Atılır insan fetal kortikal doku sinir kök hücrelerin izolasyonu ve kültürü üzerinde basit ve güvenilir bir yöntem açıklanmıştır. Bilinen insan nörolojik bozukluklar türetilen Kültürler farmakolojik etkinliğini değerlendirmek için bir platform sağlamak gibi, hücresel ve moleküler patolojik süreçleri karakterizasyonu için kullanılabilir.

Atılan İnsan Fetal Kortikal Doku Sinir Kök Hücre Üretimi

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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Research

JoVE Journal

Neuroscience

15.4K Görüntüleme.  Beth Israel Deaconess Medical Center. Atılır insan fetal kortikal doku sinir kök hücrelerin izolasyonu ve kültürü üzerinde basit ve güvenilir bir yöntem açıklanmıştır. Bilinen insan nörolojik bozukluklar türetilen Kültürler farmakolojik etkinliğini değerlendirmek için bir platform sağlamak gibi, hücresel ve moleküler patolojik süreçleri karakterizasyonu için kullanılabilir.

Video: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Hücre Testi Şekillendirme Sinir-Koloni: Sinir Progenitör Hücreler Bona Fide Sinir Kök Hücreler Ayırım Bir Testi

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Nörobilim

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 ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

İnsan Fetal Beyin gelen Progenitör-türetilmiş Oligodendrosit Kültür Sistemi

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

İnsan Nöral Agregalarının bir cGMP uygulanabilir Açılım Yöntemi kök hücrelerin pluripotent kök hücreleri veya Fetal Beyin dokusundan türetilen

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit 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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

İnsan Pluripotent Kök Hücreleri dopaminerjik Nöron Farklılaşma Yönetmen

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelleme Astrositom Patogenez<em&gt; İn Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Kortikal Astrositler veya Şartlı, Genetiği fareler ile Nöral Kök Hücreler kullanma

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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. ...