Bu çalışma (SP) Gaziler İdaresi Liyakat İnceleme hibe (NEUD-004-07F) tarafından desteklenmiştir.

1.. İnsan NPC genişleme (Şekil 1) <ol><li> Dondurulmuş fetal beyin (Lonza, Walkersville, MD, USA) ve proliferasyon takviyeleri, EGF (10 ng / ml) ihtiva eden bir ortam içinde neurobasal neurospheres (Şekil 1A) gibi T-75 şişelerinde süspansiyon kültür bunları insan NPC&#39;nin stok ve canlandırmak FGF (10 ng / ml). </li><li> Kültür içinde 3 gün sonra, 5 dakika boyunca 500 rpm&#39;de bir 15 ml tüp ve santrifüj neurospheres aktarın. </li><li> Hücre peleti ~ 100 | il yukarıda orta geride bırakarak Süpernatant atılır ve bir Eppendorf tüpüne aktarılır. A 100 ul hücre peleti 2 T-75 şişelerinde bölmek yeterlidir. Neuroprogenitor hücreler ince bölerseniz çoğalırlar başarısız olarak daha az, şişelerin sayısını azaltın. </li><li> Tüpün alt karşı 200 ul ucu tutarken bir 200 ul 50x ucu ile pelet Karışım. Iki eşit parçaya bölün ve hücre süspansiyonu 2 T-75 şişelerinde (1-2 split), sürekli için birine eklemenizfarklılaşması için genişleme ve başka. </li><li> Neurospheres 300-500 mikron orijinal boyutuna ulaşana kadar her 4-5 gün orta değiştirerek genişleme (ilk şişe) için neuroprogenitor hücrelerin kültürü devam etmektedir. (500 rpm, 5 dk) santrifüj ile orta değiştirin, eski orta atmak ve yeni orta ekleyin. </li></ol> 2. Bir Nöron-zengin Kültür içine İnsan NPC Farklılaşma (Şekil 2) <ol><li> Bir nöron zengin kültürüne neuroprogenitor hücrelerin farklılaşması, her bir kuyuya 500 ul ilave edilerek poli-L-lisin, 100 ug / ml, 24-çukurlu plaka ve ceket her oyuğuna tek bir lamel yerleştirin. Oda sıcaklığında 30 dakika için yemekler inkübe edin. </li><li> Poli-L-lisin solüsyonu aspire ve steril su ile plaka yıkayın. </li><li> Daha sonra, kat 5 ug / ml fare laminin 500 ul plakanın kuyuları ve 30 dakika boyunca inkübe edin. İnkübasyondan sonra, PBS ile kuyu yıkayın. </li><li> 4 gün sonra, splittiHücreleri (adım 1.4) ng, 24 çukurlu levha başına ~ 500 neurospheres yoğunluğunda kaplı kaplar içine &#39;ayrımı için,&#39; etiketlenmiş şişenin küçük neurospheres aktarın. </li><li> Neurospheres çanak iliştirdiğinizde 6 saat sonra, yayılması orta kaldırmak ve neurobasal orta oluşan farklılaşma orta ekleyin, B27 takviyesi, NGF (20 ng / ml), BDNF (10 ng / ml), DBC (100 mM), ve retinoik asit (2 uM). </li></ol> 3. Farklılaştırılmış NPC İmmünofloresan boyanması (Şekil 3) <ol><li> Iki hafta içinde nöronal farklılaşma ortam içinde neuroprogenitor hücrelerinin kültürü takiben, nöron zengin bir kültür elde edilir. Bir kere PBS içinde nöronları yıkayın ve daha sonra 30 dakika boyunca% 4 paraformaldehid içinde tespit edin. Sabit sonra, hücreler PBS ile üç kez yıkayın. </li><li> 60 dakika boyunca oda sıcaklığında permeabilizasyon tamponu (% 5 BSA ve PBS içinde% 0.2 Triton X-100) ile hücrelerin inkübe edin. </li><li> 4 ° C i de yemekler O / N inkübena aşağıdaki PBS içinde% 3 BSA içinde poliklonal ve monoklonal antikorların bir kombinasyonu ile çalkalama: neun (1:250) ve synapsin (1:250) asetil kolinesteraz (1:500) ve sinaptofizin (1:250), BDNF (1 : 500) ve GAP43 (1:250); STAT3 (1:500) ve GFAP (1:1000). </li><li> PBS ile lamelleri üç kez yıkayın ve daha sonra 90 dakika süre ile karanlıkta, oda sıcaklığında anti-tavşan-Cy3 ve anti-fare-FITC sekonder antikor ile inkübe edin. İnkübasyondan sonra, PBS ile lamelleri üç kez yıkayın. </li><li> Forseps ile kültürü çanak lamel dışarı almak ve montaj ortamına ters yerleştirin, bir cam slayt üzerine montaj orta 10 ul yerleştirin. Yavaşça, herhangi bir aşırı montaj orta siliyorum ve oje ile kenarları mühür. </li><li> Bir floresan mikroskobunda immunosteyn nöronlar inceleyin. </li></ol> 4. Nöronlar Lazer Yakalama Mikrodisseksiyon (Şekil 4) <ol><li> PEN membran slayt üzerinde nöron-zengin kültürüne NPCleri ayırtŞekil 2 için tarif edilen yöntemler kullanılarak poli-L-lisin ve laminin ile kaplanmış fare var. </li><li> RNAse içermeyen koşullar altında tüm sonraki basamakları gerçekleştirir. </li><li> Hücreleri görselleştirmek için HistoGene Leke (Arcturus) kullanarak kültürleri Leke. </li><li> Veritas LCM sistemi ile tüm slayt yol haritası görüntü kullanılarak astrositlerde yoksun saf nöronal nüfus alanları bulabilirsiniz. Çizim araçlarını kullanarak nöronlar işaretleyin. </li><li> Iki aşamalı bir işlemde, bilgisayar kontrollü hassas ve otomasyon kullanılarak bu alanların lazer yakalama yapın. İlk olarak, IR (Kızıl Ötesi) membran kapağa bağlamak için 70 mW ve 2500 mikro-saniye bir darbenin bir lazer güç ayarına çok yerlerde ateşlenir. Daha sonra, işaretli alanı 10 mV düşük seviyede UV ayarda bir kesme aleti ile eksize edilir. </li><li> Bu süreçlerin kombinasyonu seçici CapSure LCM makro kapaklar üzerine PEN zardan İşaretli alanların yakalar. Kapak kaldırıldığında olduğunda, N ile zareurons kapağa bağlıdır. </li><li> Bir PicoPure RNA izolasyon kiti (Arcturus) kullanarak LCM örneklerinden total RNA izole ve DNase ile tedavi. </li><li> Kiti talimatları izleyerek RiboAMP RNA amplifikasyon kiti kullanılarak izole RNA yükseltin. </li><li> İnsan nörofilaman ağır zinciri (hNFHc) algılamak için TaqMan problar kullanılarak gerçek zamanlı RT-PCR analizi yapın. </li></ol> Kısaltmalar: AD, Alzheimer hastalığı, BDNF, beyin türevli nörotrofik faktör, CREB, siklik AMP yanıt elemanı bağlayıcı protein; DBC, dibutiril siklik AMP, EGF, epidermal büyüme faktörü; FGF, fibroblast büyüme faktörü; GFAP, gliyal fibriler asidik protein, LCM, lazer mikrodiseksiyon yakalama, NGF, sinir büyüme faktörü, NPC, neuroprogenitor hücre, PEN, polietilen naftalat.

<ol>
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Ilan etmek hiçbir çıkar çatışması.

Bu çalışmada, kendi kendini yenileyen insan neuroprogenitor hücrelerin farklılaşması ile nöron-zengin bir hücre kültürü modeli ve lazer yakalama mikro-kesim ile nöronların saf popülasyonu izole etmek için bir yöntem tarif eder. Biz NGF, BDNF, DBC ve NPC nöronal farklılaşma için retinoik asit bir arada kullandık. DBC CREB, nöron 12 arttıran bir transkripsiyon faktörü aktif hale getirmek için kullanılır. Retinoik asit, hücre döngüsü çıkışına neden ve glial nüfusu 13</...

Yaşam boyu nöron yan karıncıkların subventricular bölgesinde ve yetişkin memeli beyin 1 kıvrımlarının subgranular tabakasında meydana geldiği bilinmektedir. Neuroprogenitor hücreler (NPC) bu bölgelerde kaynaklanan nöronları, astrositlerde ve oligodendrositlerin 2 farklılaşabildiği multipotent hücrelerdir. NPC nedeniyle, Parkinson hastalığı, amiyotrofik lateral skleroz, felç ve Alzheimer hastalığı (AD) 3 de dahil olmak üzere, çeşitli nörodejeneratif bozukluğu olan hastalarda nakledilen için potansiyel ilgi yarattı. NPC&#39;ler ile çalışmalar genellikle bu nakli açısına odaklanmış ama Nörodejenerasyonun mekanizmasını belirlemek için hücre kültürü modeli olarak NPC-türetilmiş nöronların potansiyeli tam olmamıştır. Değillerse olarak daha önceki çalışmalarda, genellikle, her bir deney için izole edilmesi gerekli kemirgen beyin dokularından izole post-mitotik nöronlar kullandıkkendini yenileyen. SH-SY5Y ve SK-N-MC hücreleri de dahil olmak üzere insan nöroblastoma hücre çizgileri genişletilebilir de, primer nöron özelliklerine sahip değildir. Birden çok geçişleri için genişletilebilir ve primer nöron 4,5 olan özelliklere sahip bir hücre popülasyonu elde etmek için farklı olabilir, çünkü insan NPC, diğer taraftan, iki avantajlar sunmaktadır. Bu çalışmada, insan fetal beyninden izole edilmiş ticari olarak temin edilebilen NPC&#39;lerden tutarlı bir nöron zengin nüfus elde etmek için yeni bir farklılaşma protokol açıklar. Bu kültürler, glial hücrelerin küçük bir yüzde içeren olduğundan, biz moleküler karakterizasyonu için nöron saf popülasyonu izole etmek için ek yöntemler gerekir. Lazer yakalama mikrodiseksiyon (LCM) bir doku bölümünden hücrelerinin homojen bir popülasyon seçici gen ekspresyon analizi 6 yakalanabilir hangi bir yeni bir tekniktir. Beyin nöronları, glia ve diğer hücre ty oluşan heterojen bir dokudurpes. LCM nörona özgü gen ekspresyonunu tespit etmek için kullanılmıştır 7-10 analiz eder. Daha önce, özellikle hipokampal nöronlar 11 azalma siklik AMP yanıt elemanı bağlayıcı proteinin ekspresyonunu (CREB) ve BDNF göstermek için AD (Tg2576) fare beyin kesitlerden hipokampal nöronlar LCM gerçekleştirdik. Bu çalışmada, biz PEN membran slaytlar kültüre nöronların izolasyonu için insan NPC genişlemesi, nöronal farklılaşma, nöronal belirteçler için immunofluorescent boyama ve LCM için prosedürler açıklanmaktadır.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Neuroprogenitor cells (NPCs) </td>
			<td>Lonza</td>
			<td>PT-2599</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A human basal medium</td>
			<td>Stem cell technology</td>
			<td>5750</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Proliferation supplement</td>
			<td>Stem cell technology</td>
			<td>5753</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Differentiation supplement</td>
			<td>Stem cell technology</td>
			<td>5754</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B-27 Supplement (50x) </td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human epidermal growth factor</td>
			<td>Stem cell technology</td>
			<td>2633</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fibroblast growth factor </td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brain Derived Neurotrophic Factor </td>
			<td>Cell signaling </td>
			<td>3897S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nerve Growth Factor </td>
			<td>Invitrogen</td>
			<td>13257-019</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibutyryl cyclic AMP</td>
			<td>Sigma</td>
			<td>D-0627</td>
			<td></td>
		 </tr>
		 <tr>
			<td>poly-L-lysine </td>
			<td>Sigma</td>
			<td>P-5899</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse laminin </td>
			<td>Sigma</td>
			<td>L-2020</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>STAT3 antibody</td>
			<td>Cell signaling</td>
			<td>9132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFAP antibody</td>
			<td>Cell signaling</td>
			<td>3670</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GAP43 antibody</td>
			<td>Transduction laboratories</td>
			<td>612262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BDNF antibody</td>
			<td>Millipore</td>
			<td>AB1534SP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetyl cholinesterase antibody </td>
			<td>Santz cruz</td>
			<td>Sc-11409</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synaptophysin antibody</td>
			<td>Abcam</td>
			<td>ab18008-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NeuN antibody</td>
			<td>Chemicon</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synapsin antibody</td>
			<td>Novus</td>
			<td>NB300-104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse FITC</td>
			<td>Jackson Immuno </td>
			<td>115-095-146</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti Rabbit Cy3</td>
			<td>Jackson Immuno </td>
			<td>711-165-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X 100</td>
			<td>Acros</td>
			<td>21568-2500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehye </td>
			<td>Fisher</td>
			<td>4042</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip (Big circle cover slip)</td>
			<td>Fisherbrand</td>
			<td>12-545-102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting medium (Prolong Gold)</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen membrane</td>
			<td>Applied biosystems</td>
			<td>LCM0521</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histogene LCM frozen section staining kit</td>
			<td>Applied biosystems</td>
			<td>KIT0401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboAmp RNA Amplification kit</td>
			<td>Applied biosystems</td>
			<td>KIT0201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picopure RNA Isolation kit</td>
			<td>Applied biosystems</td>
			<td>KIT0202</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CapsureMacro LCM caps</td>
			<td>Applied biosystems</td>
			<td>LCM0211</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

Insan fetal beyninden izole Neuroprogenitor hücreleri (NPC) epidermal büyüme faktörü (EGF) ve hücrelerin bol kaynağı sağlamak için fibroblast büyüme faktörü (FGF) mevcudiyetinde çoğalma koşulları altında genişletilmiştir. NPC sinir büyüme faktörü (NGF), yeni bir kombinasyonunun mevcudiyetinde ayırt edildi, beyin-türevi nörotrofik faktör (BDNF), dibutiril cAMP (DBC) ve poli-L-lisin ve laminin ile kaplanmış fare yemekler retinoik asit nöron elde edilmesi için zengin kültürleri. NPC da nörotrofinlerin olmaması, DBC ve retinoik asit ve astrosit zengin kültürler elde etmek için, siliar nörotropik faktör (CNTF) mevcudiyetinde ayırt edildi. Farklılaştırılmış NPC Neun, synapsin, asetilkolinesteraz, sinaptofizin ve GAP43 dahil nöronal belirteçlerin bir panel için immunofloresan boyama ile karakterize edildi. Glial fibriller asidik protein (GFAP) ve STAT3, astrosit belirteçleri, farklı NPC&#39;nin 10-15% olarak tespit edilmiştir. Kolaylaştırmakhücre tipi özel moleküler karakterizasyonu, lazer yakalama mikrodiseksiyon polietilen naftalat (PEN) membran slaytlar üzerinde kültürlenmiş nöronlar izole etmek için yapıldı. Bu çalışmada açıklanan yöntemler nörodejenerasyonun moleküler mekanizması anlayışımızı ilerletmek için değerli bir araç sağlar.

NPC genişleme (Şekil 1) Neurospheres tritürasyon ile tek bir hücre süspansiyonu için ayrılmış zaman, pipet ucu süspansiyon yukarı ve aşağı pipetle bir direnç, böylece tüpün alt dokunun gerekiyor. Öğütülmesinden sayısıdır bireyler ve bir mikroskop altında elde edilen hücre süspansiyonu incelenerek deneme yanılma ile karar verilmesi gereken arasında değişir. NPC yakın temas sırasında proliferasyon, bir daha hızlı olacaktır, çünkü bu hücre...

Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

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

İnsan neuroprogenitor hücreleri (NPC) çoğalan koşullar altında genişletilmiştir. NPC nörotrofinlerin bir kombinasyonunun mevcudiyetinde nöron zengin kültürler içine ayırt edildi. Nöronal belirteçler immunofloresan boyama ile tespit edilmiştir. Nöron saf bir popülasyonu izole etmek için, NPC PEN membran slaytlar üzerinde farklılaştırılmış ve lazer yakalama mikrodiseksiyon uygulandı.

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 ...

laser-capture-microdissection-neurons-from-differentiated-human

Research

JoVE Journal

Neuroscience

12.1K Görüntüleme.  Denver VA Medical Center.  İnsan neuroprogenitor hücreleri (NPC) çoğalan koşullar altında genişletilmiştir. NPC nörotrofinlerin bir kombinasyonunun mevcudiyetinde nöron zengin kültürler içine ayırt edildi. Nöronal belirteçler immunofloresan boyama ile tespit edilmiştir. Nöron saf bir popülasyonu izole etmek için, NPC PEN membran slaytlar üzerinde farklılaştırılmış ve lazer yakalama mikrodiseksiyon uygulandı.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Lazer Yakalama Mikrodisseksiyon Drosophila Periferik Nöronlar

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Nöronal atalarıdır ve Nöronlar Verimli türetilmesi

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Travmatik Beyin Hasarı Sonrası Nöronlar veya gen ekspresyon analizi için Tek Nöron zenginleştirilmiştir Populasyonlarının Lazer Yakalama Mikrodisseksiyon

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Lazer Yakalama Mikroseksiyonu ile Fare Omurilik Motor Nöron Hücre Gövdelerinden Transkriptomik Analiz için RNA Üretimi

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Lazer Yakalama Mikrodisseksiyon ile Burun Biyopsi Kombine yoluyla elde Koku Nöronlar: Mental Bozuklukların Tedavi Tepki Eğitim Bir Potansiyel Yaklaşım

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Lazer Yakalama Mikrodisseksiyon - Bireysel Dopamin Nöronlar İzolasyonu ve Tüm ventral tegmental Area bir Gösteri

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Kök Ganglia Nöronlar ve Farklılaştırılmış Yağ-türetilmiş Kök Hücreler Dorsal: bir<em&gt; İn Vitro</em&gt; Co-kültür Modeli Periferik Sinir Rejenerasyon Eğitim

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Stereotaksik Atlas güdümlü lazer yakalama mikrodiseksiyon travma yaralanma tarafından etkilenen beyin bölgeleri

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

İnsan yetişkin cilt fibroblastlar indüklenen Neurons yüksek verimli kültürünün basit üretimi

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...