Name: a guide to generating and using hipsc derived npcs for the study of neurological diseases
Uploaded: 2015-02-21T15:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC Farklılaşma Sinir Progenitör Hücreler için <ol><li> Büyümek ve insan embriyonik kök hücre hiPSCs genişletin (HES) medya (Tablo 1) eş-kültüre bir fare embriyonik fibroblast (MEF), büyük (ama SUBCONFLUENT) koloniler bir embriyoid vücudun (EB) ara aracılığıyla nöral farklılaşma için hazır olana kadar besleyici tabaka üzerinde (Şekil 2). Rutin hiPSC kültür koşulları iyi yerde 16,17 tarif edilmiştir; 3 Her 7 gün: kısaca, birbirine karışana kadar bir MEF besleyici tabaka üzerinde HES medyada hiPSCs büyümek, daha sonra enzimatik kollajenaz (DMEM 1mg / ml) ile geçit ve yaklaşık 1 genişletin. </li><li> Poli-L-Ornitin (cam yüzeyler için 50 ug / ml), plastik yüzeyler için steril su içinde ml / g, 10 ile Poli-L-Ornitin / laminin kaplanmış plakalar, kaplama plakaları hazırlamak ve 3-24 saat boyunca oda sıcaklığında inkübe etmek. 3-24 saat boyunca oda sıcaklığında steril PBS içerisinde 5 g / mi Laminin steril su ve daha sonra kaplama ile en az bir kez yıkayın. NOT: plastik sarılı ise, plakalar olabiliraltı aya kadar -20 ° C &#39;de saklanır. </li><li> 1. Günde enzimatik DMEM / F12 içinde 1 mg / ml kollajenaz IV kullanılarak plaka gibi büyük koloniler (örneğin, Matrigel TeSR 18 olarak tanımlanmış ortamlarda yetiştirilen, genellikle MEF&#39;ler yetiştiler ya da ancak hiPSCs) kaynaşma hiPSCs kaldırın. NOT: 37 ºC inkübasyon 1-2 saat sonra koloniler çanak yüzen olacak. </li><li> Yavaşça DMEM / F12, 2 ml / yuva N2 / B27 ortamı (Tablo 1) ve yapışkan olmayan 6 oyuklu tabaklara transfer tekrar süspansiyon ile 1-2X (değil santrifüj, çökeltilerek) hiPSC koloniler yıkama (1 içine 3-kuyu kombine eh) 19. NOT: Gecelik, koloniler yüzen küresel kümeler oluşturacak &quot;embriyoid organları&quot; (EBS) olarak adlandırılan. Önemli hücresel ölüm bekliyoruz. </li><li> Ve Gün 2, eğim plakaları üzerinde EBS yerleşmek için izin verir. Dikkatle ortamı çıkarın ve enkaz kaldırmak için DMEM / F12 ile bir kez yıkayın. N2 ile besleyin / çift SMAD inhibitörleri (0.1M LDN193189 ve 10M SB431542) 12 ile desteklenmiş B27 medya </&gt; sup. </li><li> Gün 3-7, neuralization çift SMAD inhibe bağlamında oluşacak; EBS yuvarlak ama kistik olmadığını kontrol edin. EBS 0.1M LDN193189 ve 10M SB431542 ile desteklenmiş N2 / B27 medya ile her ikinci gün besleyin. </li><li> Nöral rozet (apico-bazal kutupları nöroepitelyal hücrelerin yuvarlak kümeleri olarak karakterize bir kaç gün içinde görünmeye başlar ki Günleri 7-14, kaplanmış plakalar Poli-L-Ornithine / Laminin için EBS kaplama izleyen, bir aydınlık mikroskop kullanılarak kontrol edin; Şekil 1). Yapışkan EBS 0.1 M LDN193189, 10M SB431542 ve 1 ug / ml, laminin ile takviye N2 / B27 ortamı ile her iki günde bir beslemeye devam edin. </li></ol> Sinir rozet 2. Hasat NOT: Biz nöral rozet enzimatik Sinir Rozet Seçim Reaktif 20 veya benzeri seçim reaktif kullanılarak hasat öneririz. Nöral rozet elle 6-kuyu Poli-L-Ornithine / laminin kaplı levha, thi içine alınabilir rağmenmetodolojisi ayrıca NPC zenginleştirmek ve nöral olmayan hücre tiplerini tüketmek için günde 20 toplama bir ikinci tur gerektirebilir, kullanıcı beceri bağımlı, usta kapsamlı eğitim alır, ve. <ol><li> 14. günde nöral rozet enzimatik seçimi için, uyulması EBS aspire maddeleri ve 6-delikli plaka için oyuk başına sinir rozet seçimi reaktif 1 ml. 1 saat süre ile 37 ° C&#39;de inkübe edin. </li><li> P1000 Pipetman ile, yavaşça her kuyudan enzimi çıkarın. Yıkamak için oyuk başına DMEM / F12 içinde 1 ml ilave edilir. </li><li> Bir P1000 ile DMEM / F12 1 ml toplamak ve hızlı bir şekilde, böylece plaka rozet ayrılmakta, iyi geri DMEM / F12 sınırdışı. Bir şahin tüp içine rozetler toplayın. </li><li> Pipet başka 1 DMEM / F12 ml ve hızlı kalan rozet ayırmak için aynı kuyunun içine sınırdışı. (Çiğnemek etmeyin. Rozet agrega kırmak için çalışın). Aynı şahin tüp içine sinir rozetler toplayın. </li><li> Adımı tekrarlayın gerekli 2.4. Rozetler kolayca ayırmak istemiyorsanız, mo ekleyinenzimi yeniden ve işlemi tekrarlayın (2,1-2,4 adımları) </li><li> 3 dakika boyunca 300 xg&#39;de hücreleri Spin. Aspire yıkama ve NPC medya (Tablo 1) 2 ml rozet yeniden askıya. Aktarım hücreleri (1: 1) ile Poli-L-Ornitin / laminin içine 6 plaka kaplama. </li><li> Gün 15-21 itibaren, nöral rozet genişletmek; yem rozet NPC medya ile her ikinci gün. </li><li> Nöral rozet kalitesini değerlendirmek ve düz fibroblast benzeri hücreler kültür içinde genişleyen değil emin olun. NOT: sigara rozet devam ederse, NPC kültürü bazen Accutase içine aldı rozet sadece en iyi seçerek, manuel toplama ile kurtarılabilir. 37 ºC 15 dakika ayırmak. Pelet, yıkama ve NPC ortam levha 24 oyuklu poli-L-ornitin / laminin kaplanmış plaka üzerine. </li></ol> Sinir Progenitör Hücreler 3. Genişleme Not: hiPSC NPC Matrigel- veya Poli-L-Ornitin / laminin kaplanmış plakalar ya da üzerinde yetiştirilir. Biz genellikle Matrige kullanınl-plakalar daha çabuk hazırlanan ve düşük maliyetle edilebilir. <ol><li> Hızlı Matrigel kaplı plakalar hazırlamak çözülmesi ve 24 ml Matrigel, 1 mg donmuş bir kısım tekrar süspansiyon soğuk DMEM / F12 ve hemen iki altı oyuklu plakaların her bir oyuğuna 2 ml dağıtmak için. 37 ° C&#39;de en az 1 saat süreyle inkübe edin. NOT: Matrigel sadece kullanımdan hemen önce, yıkanmış değil, aspire edilmesi gerekiyor. </li><li> Ikinci günde NPC Yem ve çok yüksek yoğunlukta korumak veya kendiliğinden nöronlara ayırt edecektir. NOT: rağmen daha kurulmuş NPC genellikle 1 split: 2 seyrek olarak bir kez her iki haftada: Her hafta 4, çok düşük geçiş NPC 1 bölünebilir. </li><li> , Bölme birinci aspirasyon ortam ve 6-çukurlu plaka içinde çukur başına 1 ml sıcak Accutase (1X) ekleyin. 10-15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Yavaşça mümkün olduğu kadar az mekanik stres ile DMEM / F12 ihtiva eden bir 15 ml tüp içine müstakil hücreleri aktarın. Enzim ise hücreleri titre etmeyin. Spinni Pelet hücreleri5 dakika boyunca 1000 x g&#39;de ng. </li><li> Yıkama aspire ve özgün kuyu başına ~ 1 ml NPC medyada NPC yeniden askıya 6-iyi. NOT: 6-iyi bir çanak bir birleşik kuyu 5-10.000.000 hücreleri vardır ki bekleyin; Bu hematositometre önce yeniden kaplama kullanılarak bir hücre 10l kesir hesabıyla doğrulanabilir. </li><li> NPC, nöronlar veya neurospheres olarak yeniden süspansiyon, yeniden plakalı NPC&#39;ler ardından. 6 oyuklu plakanın her bir kuyucuğu başına yaklaşık 1-2000000 hücreleri plaka, NPC korumak için. neurosphere oluşumu verimliliği deneyleri ve hücre hatları arasında değişir, ancak 200.000 ila 1.000.000 hücre yapışkan olmayan bir 6-yuvalı plaka içinde çukur başına tohumlanır halinde genellikle en iyi ortaya olabilir; neurospheres topaklanma meydana gelirse, tohumlanmış hücrelerinin sayısını azaltabilir. Nöron ortamı ile NPC ortamı yerine 6-çukurlu plaka içinde çukur başına nöronlara ayırt etmek için, plaka yaklaşık olarak 200,000 hücre. </li><li> İmmünohistokimyasal her kurulan NPC hattını doğrulamak. Yeterli miktarda hücre malzemesi olmuştur expanNestin ve Sox2 antikorların Değerlendirme aracı, etiket NPC. Onaylı NPC hatları da 4-6 hafta boyunca nöronların içine farklılaştırılmış ve III-tübülinin ve MAP2AB için antikorlar ile etiketlenmiş olmalıdır. <ol><li> 10 dakika boyunca 4 ° C&#39;de PBS içinde% 4 paraformaldehid içinde hücreleri saptamak. PBS içinde% 1.0 Triton, 15 dakika boyunca oda sıcaklığında NPC geçirgenliği ve daha sonra 30 dakika boyunca oda sıcaklığında% 0.1 Triton ile% 5 eşek serumu içinde engeller. </li><li> Gece boyunca 4 ° C&#39;de,% 0.1 Triton ile% 5 eşek serumu, birincil antikor ile inkübe edin. Not: Aşağıdaki antikorlar tavsiye: keçi anti-Sox2, 1: 200; Fare anti-insan Nestin, 1: 200; tavşan anti-βIII-tübülin, 1: 500; fare anti-βIII-tübülin, 1: 500; fare anti-MAP2AB, 1: 200. (Şekil 2). </li><li> Oda sıcaklığında 1-2 saat süre ile,% 0.1 Triton ile% 5 eşek serumu, 300: PBS ile yıkandıktan sonra, 1 ile keçi, fare ya da tavşan uygun konjuge sekonder antikor ile ikincil antikorlar hücreleri inkübe edin. 0.5 ug / ml DAPI (4 &#39;ile çekirdek, leke hücreleri görselleştirmek için6-diamidino-2-fenilindol sonra) ve Vectashield ile monte edin. </li></ol></li></ol> 4. NPC İletimi <ol><li> Transfekte edilmiş hücrelerin 21 yüzdesini arttırmak için (1 saat süre ile 1000 x g de virüsün mevcudiyetinde hücre kültür plakalarının santrifüj) spinfection kullanın. NPC medyada seyreltilmiş enfeksiyon istenen çokluğu (genellikle 1-10) (İB), titre aspire medya ve ilgili aşırı ifadesinin (veya kontrol) lentivirüsler (veya retrovirüsler) ile değiştirin. 1.5 mi, 6-çukurlu plaka içinde çukur başına hacmi (ya da doku kültürü plakaları diğer türleri için, uygun bir şekilde ölçekli hacmi) kullanın. 1000 xg&#39;de Spin ve bir plaka santrifüj içinde 1 saat süre ile 37 ° C. NOT: İdeal, bölme 1-2 gün içinde lentiviral veya retroviral vektörlerle NPC transduce. Ticari veya laboratuvar hazırlanan virüsleri kullanarak olsun, uygun seri seyreltme ile önceden virüsün toplu titre etmek yararlı olabilir. (Şekil 3). </li><li> Cel azaltmak içinlular ölüm, spinfection 8 saat içinde ortam değiştirin. Not: Viral birleştirme ve transgen sentezleme raportör gen floresan ile 24 saat içerisinde tespit edilebilir. (Virüs bir floresan muhabiri eksiği varsa, bu değerlendirmek daha zordur; başarılı transfeksiyon iyi ekspresyonu ürünleri için immünoblot, immünohistokimyada veya qPCR tarafından onaylanmış olabilir.) Tam anlatım 3-7 gün sürebilir; Ek virüs tekrar spinfections transfekte hücrelerin yüzdesini arttırmak için gerekli olabilir. Tekrarlayan spinfections günlük oluşabilir ama çok sık olması gerekmez. İdeal olarak, bir flüoresan raportör kullanılıyorsa,&gt; hücrelerin% 80 etiketlenmelidir. </li></ol> 5. Neurosphere değiştirme tahlili Not: NPC enzimatik ayrışma ardından kendiliğinden Neurospheres bir şekilde, - hücre NPC ortam içinde süspansiyon halinde kültürlenen ise, (NPC genişleme &#39;de kullanılana benzer bir şekilde tarafından 3,3-3,6 adım). <ol><li> Kısaca, ayırmak büyümekneurospheres oluşturmak için yapışkan olmayan plakalar içinde 48 saat için NPC ortam d NPC. (Şekil 4). </li><li> Neurosphere geçişi için, bir 96-yuvalı plaka içinde, oldukça DMEM daha çekme Neurosphere önce 1-2 saat soğuk NPC ortamı kullanılarak taze Matrigel plakaları hazırlamak. Kuyulardan Matrigel aspire etmeyin. </li><li> Ilk olarak, embriyonik kök hücre-türevli neurospheres 22 için yayınlanan gibi, el, selüler artığın ayrılması için NPC medya içinde bir defa yıkandıktan sonra, mikroskop altında NPC türetilmiş neurospheres al. Bir Matrigel kaplı 96 oyuklu plakanın her bir oyuğuna, bir neurosphere aktarın. </li><li> Her bir 96-çukurlu plaka içindeki neurospheres soğuk NPC bir ortamda seyreltilir, ek olarak 0.5 mg Matrigel ekleyin. </li><li> Elle iyi bir pipet ucu kullanarak ortasında her neurosphere merkezi. NOT: Bu sonuçlar değişkenliği azaltmak amacıyla, benzer büyüklükte neurospheres almak önemlidir. </li><li> Göç 48 saat için ortaya ve daha sonra w hücreleri düzeltmek için izinistenen immünohistokimyasal işaretleri ile i% 4 paraformaldehid ve leke. </li><li> Radyal geçiş bunların tamamen 4x objektif mikroskop kullanarak, fotoğraf neurospheres tespit edilmesi gerekebilir. Kuyunun kenarına veya analiz ikinci bir neurosphere temas herhangi neurospheres hariç. NOT: göç daha uzun 48 saat oluşursa, ortaya çıkan radyal göç olasılıkla bir 4x objektif kullanarak görüntü çok büyük olacaktır. </li><li> NIH ImageJ yazılımı (Şekil 5) kullanılarak her bir neurosphere ortalama geçiş ölçün. Bu aşağıda tarif edilen analiz yöntemleri kullanılarak yapılabilir: <ol><li> Radyal göç: <ol><li> El ile daha sonra oluşan şeklin alanını hesaplamak için ölçün (Ctrl + M) işlevini kullanabilirsiniz serbest seçim aracını kullanarak uzak göç hücrelerin kenarına iz. Ölçme önce alan Alanı Analiz sekmesi altında bulunan Set Ölçümleri penceresinde işaretli olduğundan emin olun. KullanAlan ölçümü ve bir çevreye (A = πr 2) alanı için denklem değeri çapındaki (dış) belirlemek gerekmektedir. </li><li> Orijinal neurosphere kenarına Trace alanını ölçmek ve aynı şekilde yarıçapı (iç) hesaplayın. Dış ve iç yarıçapı arasındaki fark olarak toplam radyal göç hesaplayın. </li></ol></li><li> Büyük hücresel göç: <ol><li> Tedbir (Ctrl + M) işlevi, ardından düz çizgi seçim aracını kullanarak iç neurosphere kenarından beş uzak hücrelerinin hareket mesafeyi ölçün. </li></ol></li></ol></li></ol>

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	<li>Brennand, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+differences+in+hiPSC+NPCs+derived+from+patients+with+schizophrenia.">Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia.</a> Mol Psychiatry. , (2014).</li><li>Nicholas, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+hPSC-derived+forebrain+interneurons+requires+an+extended+timeline+and+mimics+human+neural+development.">Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development.</a> Cell Stem Cell. 12, 573-586 (2013).</li><li>Mariani, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+cortical+development+in+vitro+using+induced+pluripotent+stem+cells.">Modeling human cortical development in vitro using induced pluripotent stem cells.</a> Proc Natl Acad Sci U S A. 109, 12770-12775 (2012).</li><li>Hashimoto-Torii, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+heat+shock+factor+1+in+neuronal+response+to+fetal+environmental+risks+and+its+relevance+to+brain+disorders.">Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders.</a> Neuron. 82, 560-572 (2014).</li><li>Robicsek, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+neuronal+differentiation+and+mitochondrial+dysfunction+in+hair+follicle-derived+induced+pluripotent+stem+cells+of+schizophrenia+patients.">Abnormal neuronal differentiation and mitochondrial dysfunction in hair follicle-derived induced pluripotent stem cells of schizophrenia patients.</a> Mol Psychiatry. 18 (10), 1067-1076 (2013).</li><li>Paulsen, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+oxygen+metabolism+associated+to+neurogenesis+of+induced+pluripotent+stem+cells+derived+from+a+schizophrenic+patient.">Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient.</a> Cell Transplant. 21 (7), 1547-1559 (2012).</li><li>Yu, D. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+hippocampal+neurogenesis+using+human+pluripotent+stem+cells.">Modeling hippocampal neurogenesis using human pluripotent stem cells.</a> Stem Cell Reports. 2, 295-310 (2014).</li><li>Brennand, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+schizophrenia+using+human+induced+pluripotent+stem+cells.">Modelling schizophrenia using human induced pluripotent stem cells.</a> Nature. 473 (7346), 221-225 (2011).</li><li>Wen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+dysregulation+in+a+human+iPS+cell+model+of+mental+disorders.">Synaptic dysregulation in a human iPS cell model of mental disorders.</a> Nature. , (2014).</li><li>Zhang, L., Song, X., Mohri, Y., Qiao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+pf+Inflammation+and+Tumor+Microenvironment+in+the+Development+of+Gastrointestinal+Cancers:+What+Induced+Pluripotent+Stem+Cells+Can+Do.">Role pf Inflammation and Tumor Microenvironment in the Development of Gastrointestinal Cancers: What Induced Pluripotent Stem Cells Can Do.</a> Current stem cell research &amp; therapy. , (2014).</li><li>An, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Correction+of+Huntington's+Disease+Phenotypes+in+Induced+Pluripotent+Stem+Cells.">Genetic Correction of Huntington's Disease Phenotypes in Induced Pluripotent Stem Cells.</a> Cell Stem Cell. 11 (2), 253-263 (2012).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nat Biotechnol. 27, 275-280 (2009).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Marchetto, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+neural+development+and+treatment+of+rett+syndrome+using+human+induced+pluripotent+stem+cells.">A model for neural development and treatment of rett syndrome using human induced pluripotent stem cells.</a> Cell. 143, 527-539 (2010).</li><li>Sareen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+7+and+19+trisomy+in+cultured+human+neural+progenitor+cells.">Chromosome 7 and 19 trisomy in cultured human neural progenitor cells.</a> PLoS One. 4, e7630 (2009).</li><li>Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Cowan, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+embryonic+stem-cell+lines+from+human+blastocysts.">Derivation of embryonic stem-cell lines from human blastocysts.</a> N Engl J Med. 350, 1353-1356 (2004).</li><li>Ludwig, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+in+defined+conditions.">Derivation of human embryonic stem cells in defined conditions.</a> Nat Biotechnol. 24, 185-187 (2006).</li><li>Coufal, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+in+human+neural+progenitor+cells.">L1 retrotransposition in human neural progenitor cells.</a> Nature. 460, 1127-1131 (2009).</li><li>Xia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells+to+model+spinocerebellar+ataxia+type+2+in+vitro.">Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro.</a> Journal of molecular neuroscience : MN. 51, 237-248 (2013).</li><li>Aasen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26, 1276-1284 (2008).</li><li>Delaloy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-9+coordinates+proliferation+and+migration+of+human+embryonic+stem+cell-derived+neural+progenitors.">MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors.</a> Cell Stem Cell. 6, 323-335 (2010).</li><li>Kriks, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Maroof, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+and+functional+maturation+of+cortical+interneurons+from+human+embryonic+stem+cells.">Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells.</a> Cell Stem Cell. 12, 559-572 (2013).</li><li>Shi, Y., Kirwan, P., Smith, J., Robinson, H. P., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+cortex+development+from+pluripotent+stem+cells+to+functional+excitatory+synapses.">Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses.</a> Nat Neurosci. 15, 477-486 (2012).</li><li>Espuny-Camacho, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+neurons+derived+from+human+pluripotent+stem+cells+integrate+efficiently+into+mouse+brain+circuits+in+vivo.">Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo.</a> Neuron. 77, 440-456 (2013).</li></ol>

The authors have nothing to disclose.

Biz, NPC içine hiPSCs, bir sinir hücre tipi olan hiPSC türetilmiş nöronların gen imzası önemli bir kısmı korunur ve bu potansiyel hastalık patogenezinde 8 katkıda gelişimsel yollar için bir vekil olarak hizmet edebilir ayırt etmek ile yöntemler tarif etmişlerdir 11. Biz detaylı gibi Ayrıca, NPC biz hastalık yatkınlık moleküler ve biyokimyasal çalışmalar için uygun olabilir inanıyoruz sağlam Replikatif ve kolayca transdük nöral nüfus vardır. <p class="...

Bizim 1 insan uyarılmış pluripotent kök hücreler (hiPSCs) in vitro farklılaşmış nöronların ve diğerleri 2,3 Gen ifadesi çalışmaları hiPSC nöronlar fetal ziyade yetişkin beyin dokusu benzer olduğunu göstermektedir. Şu anda, hiPSC-tabanlı modeller yerine geç özellikleri, nörolojik hastalığı dışında, yatkınlık çalışmaları için daha uygun olabilir. Daha önce NPC şizofreni 1 katkıda moleküler yolları araştırılmasında faydalı bir hücre tipi olabileceğini göstermektedir, şizofreni hiPSC türetilen nöron gen imza önemli bir kısmının şizofreni hiPSC türetilmiş sinir progenitör hücrelerinin (NPC) muhafaza edilir bildirmişlerdir . Biz ve diğerleri anormal göç, artmış oksidatif stres ve reaktif oksijen türleri, alt eşik çevresel streslere ve şizofrenide bozulmuş mitokondriyal fonksiyonu hiPSC NPC 1,4-6 hassasiyeti yanı sıra azalmış nöronal c bildirmiştironnectivity ve şizofreni hiPSC nöronlar 5,7-10 sinaptik fonksiyon. Şizofreni hiPSC NPC anormal göç ve / veya oksidatif strese katkıda moleküler faktörler de şizofreni hiPSC kaynaklı nöronlar azalmış nöronal bağlantı temelini ise, NPC hastalıktan sorumlu mekanizmaları incelemek için sağlam ve yüksek replikatif nöral nüfus olabilir. Bir hızla hücrelerin çok sayıda oluşturabilir ve nöronal olgunlaşması için hafta veya ay beklemek gerekmez, çünkü Ayrıca, NPC-tabanlı deneyler daha büyük hasta gruplarını çalışma için uygundur ve yüksek verimli tarama daha yatkındır. Biz zaten şizofreni 1 ve Huntington hastalığı 11 gibi farklı hastalıkların ortaya konmuştur olarak hiPSC NPC, potansiyel hastalık patogenezinde katkıda gelişimsel yollar için bir vekil olarak hizmet verebilir inanıyoruz. HiPSCs, ilk nöral gelen NPC ayırt etmektim çift SMAD inhibisyon (0.1 mM LDN193189 ve 10 mM SB431542) 12 tarafından gerçekleştirilir. Bu küçük moleküller ile sinyalizasyon BMP ve TGF antagonize ederek, endoderm ve mezoderm özellikleri nöronal farklılaşma hızlandırılması ve kaplama bir hafta içinde görünür nöral rozet oluşumuna yol açan, bloke edilir. Sinir desenlendirme muhtemelen hemen sonra nöral rozet oluşumu ve döneminde, erken bu süreçte oluşur. Diğer ipuçları yokluğunda, bu ilkel nöral hücreler anterior ön beyin-benzeri kaderi 13 varsayıyorum. Hemen nöral rozet oluşumu takip ve NPC genişleme boyunca devam eden, ön beyin NPC FGF2&#39;nin 8,14 ile kültür. Onlar çift soy potansiyeline sahip ve% 70-80 III-tubulin-pozitif nöronlar ve% 20-30 glial fibriler asidik protein (GFAP) pozitif astrositler (Şekil 1) nöral nüfusa ayırt edilebilir. ön-beyin hiPSC nöronlarının çoğunluğu VGLUT1-pozitifNöronların yaklaşık% 30 GAD67 pozitif (GABA) rağmen ve böylece, muhtemelen glutamaterjik 8. NPC rutin tutarlı farklılaşma profilleri muhafaza ve karyotip anomalileri biriken olmadan iken, in vitro fazla on kez geçişli. Gruplar Pasajlanması NPC&#39;lerin 15, ancak, biz on pasajlar ötesinde, NPC astrosit farklılaşma eğilimini göstermektedir artmış olduğunu bulmak 40&#39;tan fazla kez bildirilmiştir. NPC birden donma-çözülür iyi tolere ve sadece yapışmayan plakalarda kültürleme yoluyla neurospheres olarak büyümeye geçiş yapılabilir. NPC etkili bir biyokimyasal çalışmalar için yeterli malzeme elde etmek için hızlı bir genetik müdahalelerin moleküler ve hücresel sonucu değerlendirmeyi ve kolayca genişletilebilir sağlayan, viral vektörler tarafından iletilir. Ayrıca, çünkü viral vektörler izin sağlam aşırı ekspresyonu kontrolü veya hasta türetilmiş neur birinde ve / veya hastalık-ilgili genlerin demonte,El hücreleri, kimse bu manipülasyonlar genetik kökenli etkisini test etmek için bu platformu kullanabilirsiniz. Sinaptik veya olgun nöronlar gerektiren faaliyet tabanlı deneyleri için uygun olmasa da, NPC hasta kaynaklı sinir hücrelerinin çoğu basit moleküler veya biyokimyasal analizler için pratik bir alternatif olabilir.

<table><tbody><tr><td>DMEM/F12</td><td>Life Technologies</td><td>#11330</td><td>for HES media</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>#10565</td><td>for neural media</td></tr><tr><td>KO-Serum Replacement</td><td>Life Technologies</td><td>#10828</td><td>Needs to be lot tested</td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>#35050</td><td></td></tr><tr><td>NEAA</td><td>Life Technologies</td><td>&amp;nbsp;#11140</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>&amp;nbsp;#17502-048</td><td>Needs to be lot tested</td></tr><tr><td>B27-RA</td><td>Life Technologies</td><td>&amp;nbsp;#12587-010</td><td>Needs to be lot tested</td></tr><tr><td>FGF2</td><td>Life Technologies</td><td>#13256-029</td><td>Resuspend in PBS + 1% BSA</td></tr><tr><td>LDN193189</td><td>Stemgent</td><td>#04-0074</td><td></td></tr><tr><td>SB431542</td><td>Stemgent</td><td>#04-0010</td><td></td></tr><tr><td>BDNF</td><td>Peprotech</td><td>#450-02</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>GDNF&amp;nbsp;</td><td>Peprotech</td><td>&amp;nbsp;#450-10</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>Dibutyryl cyclic-AMP</td><td>Sigma</td><td>&amp;nbsp;#D0627</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>#A0278</td><td>Resuspend in H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>STEMdiff Neural Rosette Selection Reagent</td><td>Stemcell Technologies</td><td>&amp;nbsp;#05832</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td></td></tr><tr><td>Collagenase IV</td><td>Life Technologies</td><td>#17104019</td><td></td></tr><tr><td>CF1 mEFs</td><td>Millipore</td><td>#PMEF-CF</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma</td><td>P3655</td><td></td></tr><tr><td>Laminin, Natural Mouse 1 mg</td><td>Life Technologies</td><td>#23017-015</td><td></td></tr><tr><td>BD Matrigel</td><td>BD</td><td>#354230</td><td>Resuspend on ice in cold DMEM at 10 mg/ml, use 1 mg per two 6-well plate equivalent</td></tr><tr><td>Tissue culture treated 6-well plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Ultra low attachment 6-well plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>goat anti-Sox2&amp;nbsp;</td><td>Santa Cruz</td><td>sc&amp;shy;17320</td><td>use at 1:200</td></tr><tr><td>mouse anti-human Nestin</td><td>Millipore</td><td>MAB5326</td><td>use at 1:200</td></tr><tr><td>rabbit anti-&amp;beta;III-tubulin</td><td>Covance</td><td>PRB&amp;shy;435P</td><td>use at 1:500</td></tr><tr><td>mouse anti-&amp;beta;III-tubulin</td><td>Covance</td><td>MMS&amp;shy;435P</td><td>use at 1:500</td></tr><tr><td>mouse anti-MAP2AB</td><td>Sigma</td><td>M1406</td><td>use at 1:200</td></tr><tr><td>Plate centrifuge</td><td>Beckman Coulter</td><td>Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td><td></td></tr></tbody></table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Sinir rozet apico-bazal kutupları (Şekil 1) ile nöroepitelyal hücrelerin yuvarlak kümeleri olarak karakteristik görünüşü, bir aydınlık mikroskop kullanarak, morfolojik tespit edilebilir. NPC çok yüksek hücre yoğunluğu tipik olarak kültürlenir rağmen, hemen gerçekleşen pasajlanmasını da sonra biraz piramit şeklinde soma ve bipolar nörit yapısı görülebilir (Şekil 1D) &#39;dir. III-tubulin boyama NPC bazı oranı sürekli kültür (Şekil 1F)...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Nörolojik Hastalıklar Çalışmaları Yaratma ve Kullanma hiPSC Türetilmiş NPC A Guide

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

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18.2K Görüntüleme.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Video: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Mikro-elektrot dizileri Ağ Aktivite Ölçüm uyarılmış pluripotent kök hücrelerin Rapid Nöronal Farklılaşma

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Gelişim Biyolojisi

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Hızlı algılama nörogelişimsel fenotipleri insan sinir habercisi hücrelerdeki (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Kronik EAE ile Fare nöral kök / progenitör hücrelerinin sistemik enjeksiyonundan

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

İmmünoloji ve Enfeksiyon

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Kültür farklılaştırılmış insan Neuroprogenitor Hücreler Nöronlar Lazer Yakalama Mikrodisseksiyon

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Nörobilim

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

İnsan Nöral Progenitor Hücre Kaynaklı Nöronlar ile Bir Neurite Outgrowth Assa ve Nörotoksisite Değerlendirmesi

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Differentiation of Porcine Induced Pluripotent Stem Cells (piPSCs) into Neural Progenitor Cells (NPCs)

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models used in pre-clinical research, such as rodents, are not able to meet the criteria to translate the findings to the clinic. Non-human primates, canines, and porcine are considered more adequate models for biomedical research and drug development purposes, mainly due to their physiological, genetic, and anatomical similarities to humans. The swine model has gained particular interest in translational neuroscience, enabling safety and allotransplantation testing. Herein the generation of porcine iPSCs is described along with its further differentiation into neural progenitor cells (NPCs). The generated cells expressed NPC markers Nestin and GFAP, confirmed by RT-qPCR, and were positive for Nestin, b-Tubulin III, and Vimentin by immunofluorescence. These results show the evidence for the generation of NPC-like cells after in vitro induction with chemical inhibitors from a large animal model, an interesting and adequate model for regenerative and translational medicine research.

Differentiation of Porcine Induced Pluripotent Stem Cells piPSCs into Neural Progenitor Cells NPCs

This protocol describes a method for chemical differentiation and culture of neural progenitor cells derived from porcine induced pluripotent stem cells (piPSCs).

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Yetişkin İnsan Fibroblastları türetilmesi ve Genişletilebilir Sinir Progenitör Hücreler içine Doğrudan Dönüşüm

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Kan Türevi Nöronal Hücrelerin GM'den Arındırılmış Üretimi

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Fibroblastlardan Nöronal Progenitör Hücrelere Doğrudan Dönüşüm ve Astrositlere Farklılaşma Kullanarak Nörolojik Hastalıklar için İn vitro Modelleme

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

Üç Boyutlu Süspansiyon Biyoreaktöründe İnsan Nörojenin 2-İndüklenebilir Nöronların Üretimi

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...