Name: isolating nasal olfactory stem cells from rodents or humans
Uploaded: 2011-08-22T12:00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Bu çalışma, mali ANR (Agence Nationale de la Recherche), AFM (Association Française contre les Miyopatiler), PACA ve Irme Feder (Institut de Recherche sur la Moelle épinière et l&#39;Encéphale) tarafından desteklenmiştir. Marie Pierre Blanchard (Jean Roche Enstitüsü) minnetle zaman atlamalı kayıt sırasında onu etkin yardım için teşekkür ederim.

1. Sıçanlar Koku Mukoza Koleksiyonu <ol><li> Temiz bir kültür kaput DMEM / HAM F12 kültür ortamı ile dolu üç adet 35 mm Petri kapları hazırlayarak başlayın. </li><li> Herhangi bir ötenazi yöntemi kurumun hayvan bakımı ve kullanımı komitesi tarafından önceden onaylanmış ve kalifiye personel tarafından yürütülen olmalıdır. Sıçan gibi ketamin / xylazine anestezi sodyum pentobarbital veya diğer enjektabl formları ile derin anestezi girdikten sonra, başını kesmek ve cilt çıkarın. Uçucu anestezikler kaçınılmalıdır. Anestezi yeterliliği dekapitasyon önce ayak tutam tarafından değerlendirilecektir. Makas ile alt çene çıkarın ve bir rongeur yardımı ile, her iki tarafta da yüz kasları yok. </li><li> Dişlerin arkasından başlayarak, bir rongeur nazal kavite, bir defada bir tarafı kaplayan kemik çıkarın. Turuncu / kahverengi organları burun arkasında bulunan koku alma konkalar görüş haline gelir. </li><li> İncelikli bir forseps ile konkalar atın. Dik plaka, kribriform plaka ve burun boşluğunun tavan ark: 26 iğne kullanarak, üç satır boyunca doku keserek septum yatarken koku mukozası izole. </li><li> Her iki tarafta biyopsileri toplayın ve bir DMEM / HAM F12 dolu Petri kabı aktarabilirsiniz. Bu prosedür, başlangıcından ötenazi itibaren 10 dakikadan daha uzun sürmemelidir. </li><li> Şimdi, mukus ortadan kaldırmak amacıyla, orta dolu Petri kutularına biyopsileri iki kez transfer. </li></ol> 2. İnsanlar Koku Mukoza Koleksiyonu <ol><li> Bu prosedür, ilgili yerel etik kurul (lar) uygun bir Kulak Burun Boğaz (KBB) cerrah tarafından gerçekleştirilen olmalı, ve her ayaktan bilgilendirilmiş onam formu imzalamanız gerekir. </li><li> 0 ° veya 30 ° rijit endoskop (4 mm çapında) kullanarak, her iki burun boşlukları varsayılan polip ya da herhangi bir inflamatuar lezyon varlığı incelemek ve değerlendirmek. Septum sapma dikkate alarak, en iyi nazal kavite seçin. </li><li> Bir pamuk aplikatör kullanarak, 10 dakika boyunca, adrenalin, lidokain gibi lokal anestezi uygulanır. </li><li> Throughcut etmoid forseps ile orta konka medial kök veya dorsomedial alanda septum ya bir iki milimetre kare biyopsi toplamak. </li><li> Olfaktör biyopsi sonra 1 ml DMEM / HAM F12 ile dolu 2 ml steril bir tüp içine, steril bir iğne kullanılarak aktarılır. Ters tüp İpucu biyopsi kültür ortamında dalmış olduğundan emin olun. </li><li> Buzdolabında bir kap içinde tüp takın ve araştırma laboratuvarı taşıma. Bu aşamada, biyopsi karşılaştırmalı moleküler çalışmalar için özel beyin hastalıkları odaklanmış veya kök hücre üretmek için işlenen tek başına kullanılabilir. </li></ol> 3. İnsan ve Rat Mukoza Koku Kök Hücre İzolasyonu <ol><li> DMEM / HAM F12 biyopsileri yıkayın. 37 1 saat için 1 ml dispase II çözeltisi (2.4 IU / ml) ile dolu bir Petri kabındaki biyopsileri inkübe ° C </li><li> Sonra, bir Kırınan ters ışık ile bir diseksiyon mikroskobu altında, mikro spatula kullanarak temel lamina propria olfaktör epitel kaldırılır. </li><li> Olfaktör epitel çizgili olan lamina propria ile karşılaştırıldığında daha ince ve siyah arka plan üzerine yarı saydam görünen turuncu / kahverengi. Beyaz bir arka plan üzerinde, epitel ve lamina propria, kahverengi, gri görünüyor. </li><li> Saflaştırılmış sonra, lamina propria DMEM / HAM F12 ile dolu bir Petri kabı içine aktarın. </li><li> Doku kemirgen ise, lamina propria, iki adet 25 gauge iğne ile küçük parçalar halinde kesin. Daha sonra, 1 ml kollajenaz IA ile dolu bir 15 ml tüp parçaları aktarın. </li><li> Tüp içinde, steril bir plastik pipet kullanarak, doku ayrıştırmaları. Sonra az 10 dakika, 37 tüp inkübe ° C </li><li> Ayrışma sonlandırmak için tüp hafifçe rock ve 9, Ca-özgür ve Mg-free PBS ml ve 5 dakika boyunca 200 g santrifüj ekleyin. </li><li> Plastik kültür kaplarına% 10 fetal sığır serumu, antibiyotik ve plaka ile desteklenmiş DMEM / HAM F12 kültür ortamı içinde hücre pelletini tekrar. </li><li> Şimdi, doku, insan ise, sonra 200 ila 500 mikron arasında değişen, kalınlığı 3 ila 4 adet lamina propria dilim. </li><li> Kendi 2 cm çapında kültür çanak içine her şerit yerleştirin ve steril 1.3 cm çapında cam kapak fişleri ile doku kapağı. </li><li> Sonra, her kültürün çanak kültür ortamı ul 500 (% 10 fetal sığır serumu ve antibiyotik ile desteklenmiş DMEM / HAM F12) ekleyin. </li><li> Ya da doku tipi için, her 2 ila 3 gün kültür ortamı yenilemek. </li><li> Beş ila yedi gün sonra, kök hücre kültürü çanak işgal etmeye başlayacak ve iki hafta sonra konfluent olmalıdır. , Geçiş ulaştı ve kültür şişeler hücrelere transfer olduğunda confluency. </li></ol> 4. Küre Oluşumu ve Nöronal differeKoku Kök Hücreler, ntiation <ol><li> Kök hücre küre oluşturmak için, 37 şişeler az iki saat inkübe ° C poli-L-lisin. (METİN: 5 g/cm2). </li><li> Tedavi şişeler santimetre kare başına 16.000 hücre yoğunluğu hücreleri Plaka. </li><li> Her iki günde tamamlanan orta santimetrekare başına 0.2ml (DMEM / HAM F12, insülin, transferrin, selenyum (ITS-X,% 1), EGF (50 ng / ml) ve FGF2 (takviye ile hücreleri beslemek, 50 ng / ml)). </li><li> Iki ila beş gün sonra, kayan hücre küre ve ya yeniden plakalı toplamak veya hücre tedavisi hayvan modelleri aşılama önce onları ayrıştırmaları. </li><li> Nöron-benzeri hücreler içine koku alma kök hücrelerin ayırt etmek için, B-27, penisilin, streptomisin, glutamin ve glutamat içeren Neurobasal ortamda 21 gün için onları yetiştirmek. </li><li> Daha sonra her 3 günde bir orta değişiklikler yapmak. Nöron benzeri hücreler 2-3 hafta sonra görünmelidir. </li></ol> 5. Temsilcisi Sonuçlar: Burun insan eksplant outgrowing kök hücreler (Şekil 2A) hızlı bölünen ve confluency 1-2 hafta içinde ulaşılabilir. Stemness, bir anahtar özelliği, nestin ifade değerlendirildi (Şekil 2B). EGF (50 ng / ml) ve FGF2 (50 ng / ml) ile desteklenmiş bir serum serbest kültür ortamı ile poli-L-lizin büyüdüğü zaman, koku kök hücreler küre (Şekil 2C) neden olmaktadır. Serum içeren kültür ortamında yetişen yeni kaplama küre GFAP-ifade hücreleri (~% 50), tubulin ifade hücreleri (~% 10-15) ve O4 ifade hücreleri (~% 2-5) 9 (Rakamlar ortaya çıkmasına neden 2D-F). Ancak, küre-kökenli hücreler kaderi değiştirilebilir. Örneğin, B27 ve glutamat, β-III tubulin (Şekil 2G) ve map2 (Şekil 2H) ifade nöron-benzeri hücreler içine burun koku kök hücrelerin çoğu ile desteklenmiş bir Neurobasal kültür ortamında büyüdü. <img src="/files/ftp_upload/2762/2762fig1.jpg" alt="Şekil 1"/> Şekil 1, deney Genel şeması . Olfaktör mukoza biyopsileri, sıçan ve insan nazal kavite eksize. Eksplantlarındaki beyin hastalıkları biyobelirteçleri belirlenmesine yönelik karşılaştırmalı moleküler çalışmalar için başlı başına kullanılabilir. Lamina propria ve nöro-epitel arasındaki etkileşimler, koku alma kök hücreleri izole etmek için, 45 dakika sonra, bir mikro-spatula ile epitel kaldırılır dispase II enzim bozulur ve. Kemirgen koku alma kök hücreler daha kollajenaz IA ile lamina propria dissociating tarafından seçilir. Outgrowing kök hücrelerin bütün istila kadar insan doku, koku alma lamina propria adet cam lamel altında kültüre. Çoğalması sonra, uygun bir kültür ortamı kullanılarak, koku kök hücreler küre üretebilir veya nöron hücrelerine ayırt. Olfaktör kök hücreler için kullanılabilir) onarım beyin hastalıkları ya da travma veya ii) merkezi sinir sistemi hastalıkları moleküler belirteçleri tespit. Çizimler Servier Tıp Sanat yardımı ile yapılır. <img src="/files/ftp_upload/2762/2762fig2.jpg" alt="Şekil 2"/> Şekil 2, burun, insan koku alma kök hücreleri Kültür ve farklılaşma . Serum içeren ortamda yetiştirilen lamina propria eksplant (A) büyüyen insan kök hücreleri, hızla bölünerek. Kök hücreler, stemness işaretleyici nestin (B) ifade eder. Poli-L-lisin kaplı plastik kaplama ve EGF ve FGF2 ile desteklenen bir serum serbest kültür ortamında yetiştirilen, koku kök hücreler küre (C) oluşturur. Küre kökenli hücrelerin, serum içeren kültür ortamında kaplama, doğuran GFAP-ifade hücreleri (~% 50), tubulin ifade hücreleri (~% 10-15) ve O4 ifade hücreleri (~% 2-5) 9 (DF). B27 ve glutamat ile desteklenmiş bir Neurobasal kültür ortamında yetişen, β-III tubulin (G) ve map2 (H) ifade nöron-benzeri hücrelere farklılaşırlar.

<ol>
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Çıkar çatışması ilan etti.

Burada sunulan teknikleri nörogelişimsel ve nörodejeneratif hastalıkların yanı sıra, patolojik ya da travmatize beyin tamiri için bir araç nedenleri içine kemirgen ve insan olfaktör mukoza klinik araştırmalar için yararlı bir model. Protokol nispeten basittir ve deneyimli bir hücre biyoloğu tarafından kolayca yapılabilir. Biyopsi ve kültür teknikleri için başarı oranı yüksektir. Kritik adımlar <ul><li> Kemirgen olfaktör mukoza toplanması için, ötanazi ve son koku doku eksizyonu arasında 10 dakikalık bir zaman sınırı geçmemek üzere tavsiye edilir. </li><li> Kemirgen koku alma lamina propria ayrılma genellikle 10 dakika sonra elde edilir. kollajenaz IA kuluçka. Değilse, lamina propria float undissociated bit, 200 g santrifüj ve mekanik bir zamanda yeni hücreler replating önce Pasteur pipeti kullanarak kayan lamina ayrıştırmaları süpernatantı gün sonra toplamak için önerilir. Zaten bağlı hücreleri içeren ilk sıra taze kültür ortamı ile doludur. </li><li> Kemirgen doku daha küçük olan insan lamina propria, için, biz bir enzimatik ayrılma önermiyoruz. Doku dilimlenmiş ve her eksplant, plastik tabak alt ve cam lamel arasına yerleştirilir. Başarılı kültürler, kalınlık aralığı 200 ila 500 mikron eksplantlar içerir. </li></ul> Olası değişiklikler <ul><li> Geçerli protokol in vitro olfaktör nöronların üretmek için biraz değiştirilmiş olabilir. Bu amaçla, nöro-epitel kaldırılmaz ve tüm koku mukozası bir McIlwain kıyıcı (200 mikron kalınlığında) ile dilimlenmiş. Her eksplant, bir tabak içinde kaplama, kısmen bir saat süreyle kurutulur ve sonra FCS içeren kültür ortamı ile sulandırılır. Ilk gün kaplama sırasında, epitelyal ve mezenkimal hücreler eksplant büyür. Daha sonra, bu hücre tabakasının üstüne nöron atalarıdır göç ve nöronlar farklılaşırlar. </li><li> Koku alma kök hücrelerin Arıtma flow sitometri kullanılarak elde edilebilir. Özgül yüzey belirteçleri kağıdı 11 karakterizasyonu yayınlanan listeye alınabilir. </li><li> Nakli deneyleri için, koku alma kök hücre GFP pozitif sıçanlarda bir yük kullanmak mümkündür. Bu sıçan suşu (Sprague Dawley, eGFP PGK organizatörü tarafından yönlendirilen) ITERT (Nantes, Fransa) mevcuttur. Koku alma, insan kök hücreleri, GFP geninin eklemek için, biz lentivirüs enfeksiyon dayalı bir yöntem kullanabilirsiniz. </li><li> Bu yazıda, koku alma ekto mezenkimal kök hücreleri üzerinde odaklanmıştır. Ancak, faiz, koku alma ensheathing hücreleri, başka bir hücre tipi, aynı doku arındırılmalıdır. Biz onların toplama ve arıtma 1,17 için bir yöntem tanımlanmış ve paraplejik hastaların 18 faz I / IIa klinik çalışma için insan burun ensheathing hücreleri kullandı. </li><li> Mezenkimal kök hücre süper ailesinin bir üyesi olarak, OE-MKH adipositler, osteosit ve miyositler 11 ayırt etmek için, uygun kültür koşulları altında edebiliyoruz. </li></ul> Gelecekteki uygulamalar <ul><li> Biz zaten, otizm, bipolar bozukluk, ailesel disotonomi, Parkinson hastalığı, Alzheimer ve şizofreni 2-7 hastalarda hücresel ve moleküler anomaliler çalışma insan koku alma biyopsi kullanmışlardır . Teorik olarak, tüm beyin hastalıkları, burun biyopsileri ya da periferik koku alma kök hücreler kullanılarak incelenebilir. </li><li> Kemirgen ve insan burun koku alma kök hücreler zaten amnezi, Parkinson hastalığı, koklear hasar ve omurilik travması 12-16 hayvan modellerinde aşılı olmuştur. Alzheimer hastalığı, beyin iskemi, Multipl skleroz hayvan modellerinde bu hücrelerin nakli için akla. </li></ul>

<table border="1"><tbody><tr><td> Reaktifin Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td colspan="3"> Koku mukozasının sıçanlarda Tahsil </td></tr><tr><td> DMEM / HAM F12 </td><td> Invitrogen </td><td> 31331-028 </td></tr><tr><td> Pentobarbital Sodyum </td><td></td><td></td></tr><tr><td> Rongeur </td><td> FST </td><td></td></tr><tr><td> 26 iğne </td><td> Terumo </td><td> NN-2613R </td></tr><tr><td> Forseps </td><td></td><td></td></tr><tr><td colspan="3"> İnsanlarda olfaktör mukoza Koleksiyonu </td></tr><tr><td> Rijit endoskop </td><td> Karl Storz veya Richard Wolf Tıp </td><td></td></tr><tr><td> Lidokain </td><td></td><td></td></tr><tr><td> Epinefrin </td><td></td><td></td></tr><tr><td> Throughcut etmoid forseps </td><td> Karl Storz veya Richard Wolf Tıp </td><td></td></tr><tr><td colspan="3"> Koku alma kök hücrelerinin izolasyonu </td></tr><tr><td> Dispase II. </td><td> Roche </td><td> 10 295 825 001 </td></tr><tr><td> Diseksiyon mikroskobu </td><td></td><td></td></tr><tr><td> Mikro spatula </td><td> FST </td><td></td></tr><tr><td> Kollajenaz IA </td><td> Sigma-Aldrich </td><td> C9891 </td></tr><tr><td> Ca-free/Mg-free PBS </td><td> Invitrogen </td><td> 14190-250 </td></tr><tr><td> Fetal buzağı serumu </td><td> Invitrogen </td><td> 10270098 </td></tr><tr><td> Cam lamel </td><td> Knittel&#39;in Glaser </td><td> 001/35 </td></tr><tr><td colspan="3"> Küre oluşumu ve nöronal farklılaşma </td></tr><tr><td> Poli-L-lisin </td><td> Sigma-Aldrich </td><td> P-1274 </td></tr><tr><td> İnsülin transferrin selenyum (ITS) </td><td> Invitrogen </td><td> 51500056 </td></tr><tr><td> AGF </td><td> Ar-Ge Sistemleri </td><td> 236-EG </td></tr><tr><td> FGF2 </td><td> Ar-Ge Sistemleri </td><td> 233-FB </td></tr><tr><td> Neurobasal orta </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B-27 Serum-Alerjik Supplement </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> Penisilin / streptomisin </td><td> Invitrogen </td><td> 15140122 </td></tr><tr><td> Glutamin </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Glutamat </td><td> Sigma-Aldrich </td><td></td></tr></tbody></table>

isolating nasal olfactory stem cells from rodents or humans

Olfaktör mukoza, burun boşluğunda bulunan tespit kokuları sorumlu. Aynı zamanda dış ortama maruz kalan ve yaşayan her bireyin kolayca erişilebilir sadece sinir doku. Sonuç olarak, bu doku patolojik beyin moleküler anomalileri tespit veya hücre tedavisi için yetişkin kök hücrelerin izole amaçlayan herkes için eşsizdir. Beyin hastalıklarının moleküler anormallikler genellikle post-mortem toplanan sinir doku örnekleri kullanılarak çalışıldı. Ancak, bu malzeme, çeşitli sınırlamalar vardır. Buna karşılık, koku mukoza, kolay erişilebilir ve güvenli bir şekilde, koku alma 1 duyusunun kaybı olmadan biyopsi olabilir. Buna göre, koku mukozasının bir gelişimsel çalışmada hangi yetişkin insan (örneğin, otizm, şizofreni) 2-4 veya nörodejeneratif (örneğin Parkinson, Alzheimer) 4,5 hastalıkların bir &quot;açık pencere&quot; sağlar. Karşılaştırmalı moleküler çalışmalar nöron 3,7 4,6 ya da in vitro deneylerde ya Olfaktör mukoza için kullanılabilir . Olfaktör epitel de bir sinir doku viral enfeksiyonlar bakteri, kirlilikten zarar görmüş olduğunu, yerini her geçen gün yeni nöron üretir. Bu kalıcı nöron atalarıdır sürekli değil, aynı zamanda, yani mukoza, neuroepithelium ve altta yatan lamina propria 8-10 iki bölmeleri içinde ikamet eden kök hücre . Yakın zamanda lamina propria bulunan yetişkin kök hücreleri arındırmak için bir yöntem geliştirdi ve kemik iliği mezenkimal kök hücreleri (BM-MSC) yakından ilişkili olduğunu göstermiştir ettikten sonra, biz (onlara koku alma ekto mezenkimal kök hücreleri adında OE- MSC) 11. İlginçtir ki, BM-MKH karşılaştırıldığında, OE-MKH yüksek proliferasyon oranı yükseltilmiş clonogenicity ve sinir hücrelerine ayırt etmek için bir eğim gösterilecek. Biz, şizofreni ve Parkinson hastalığı 4 yeni aday genler açıklayacak adanmış çalışmalar yapmak, bu özellikleri avantaj sağladı. Biz ve diğerleri de omurilik travma 12,13, koklear hasar 14 OE-MKH sonra ya da Parkinson hastalığı 15 veya 16 amnezi bir hayvan modellerinde, hücre tedavisi için aday umut verici olduğunu göstermiştir . Bu çalışmada, sıçanlarda ve insanlarda koku alma mukoza biyopsi yöntemleri mevcut. Toplandıktan sonra, lamina propria enzimatik bir enzimatik veya enzimatik olmayan bir yöntem kullanarak saflaştırılmış epitel ve kök hücreleri ayrılır. Koku alma kök hücreler Saf sonra ya çok sayıda büyümüş ve sıvı azot banked veya küreler oluşturacak şekilde uyarılmış veya sinir hücrelerine ayırt edilebilir. Bu kök hücreler karşılaştırmalı omics (proteomik, genomik transcriptomic epigenomic) çalışmaları için de kullanılabilir.

Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Biz burada, sıçan ve insan burun boşlukları olfaktör mukoza biyopsi için bir yöntem açıklanmaktadır. Bu biyopsiler, beyin hastalıkları moleküler anomaliler tespit edilmesi ya da beyin travması / hastalık hayvan modellerinde hücre nakli için kullanılabilir multipotent yetişkin kök hücreleri izole ya için de kullanılabilir.

Kemirgenler ve İnsanlar Burun Koku Kök Hücreler Yalıtımlı

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

isolating-nasal-olfactory-stem-cells-from-rodents-or-humans

Research

JoVE Journal

Neuroscience

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Güve Koku Sistemi Elektrofizyolojik Ölçümler

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Nörobilim

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Yalıtımlı LacZ ifade Sitometrisi kullanarak Fare İç Kulak Dokular Hücreler

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Tarafından Embriyonik ve Erişkin Nöral Kök Hücre Genişletme<em&gt; In Utero</em&gt; Elektroporasyon veya Viral Stereotaksik Enjeksiyon

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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

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

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

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

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

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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

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

İnsan Pluripotent Kök Hücreleri dopaminerjik Nöron Farklılaşma Yönetmen

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

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

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

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

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

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

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

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 ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Fare Embriyonik Kök Hücre in vitro nöronal farklılaşma

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...