Bu proje, Kanada Multipl Skleroz Derneği RKRWO bir hibe ile finanse edildi Kanada Multipl Skleroz Derneği Bursu bir alıcı. SDR, Kanada ve Kanada Sağlık Araştırma Enstitüleri Multipl Skleroz Derneği Doktora Sonrası Araştırma Bursu bir alıcı.

<ol>
	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> J Neurosci Res. 45, 558-570 (1996).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> Cell. 70, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> J Cell Biol. 185, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> Neuron. 49, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> Glia. 56, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> J Neurosci. 12, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> Dev Biol. 58, 106-113 (1977).</li></ol>

Çıkar çatışması ilan etti.

Bu rapor, kültürleri ya da OL-zenginleştirilmiş DRGN / OL ortak kültürler farklılaşma fare OPC izole etmek için bir yöntem açıklanır. Yalnız kültür, OPC MBP içine ayırt + Ve EKK, miyelin benzeri membranöz levha üretim. Neurite yatak DRGN eklendiği zaman, OLS MBP DRGN neurites sarmak + ve membran. Bu model, OL-aracılı aksonal ensheathment yöneten karmaşık temellerinin araştırılması yarar sağlar. Büyük bir değer olsa da, bu tür kültürlerin kurulması, teknik olarak çok zordur. Özellikle zorlu yönlerini etkili doku sindirim / disosiasyon, dengeli bir kültür ortamı pH bakım ve DRGN medya değişiklikleri içerir. Sindirim uzunluğu, doku miktarı sindirilir ve öğütme miktarı doku ayrılma etkinliği ve sonuçta etkiler olduğunu göz önünde bulundurmak önemlidir. Ayrışmış sinir sistemi dokularında düşük hücresel verimi elde etmek için deneyimli araştırmacılar için alışılmadık bir durum değildir. Buna ek olarak, fare OPC, özellikle alkali koşullar altında, kültür ortamı pH değişikliklerine duyarlı olma eğilimindedirler. % 8.5 CO 2 kültürlerin bakım OPC temel üzerinde hafif asidik koşullarda daha iyi tolere görünen bu yana, bu önlemek için amaçlamaktadır. Ancak, medya değişiklikleri, beslenme DRGNs ile ilgili olarak, nöronlar ile kurutun olarak hızlı bir şekilde gerçekleştirilebilir olmalıdır gelişmekte neurite yatak bozmayacak gibi hafif olmalıdır. Ani medya değişiklikleri yüzey neurite yatak, ve lamel tam ayrılma olası sonuç yerinden olabilir. Bu model sisteminin potansiyel liyakat, teknik açıdan zorlu doğa büyük ölçüde düşer. Bu sistemin bir avantajı, embriyonik doku hasat üreme kadın feda etmeye gerek engellemeyi, hücre kültürü derivasyon için post-natal farelerin kullanılması. Diğer bir avantajı OPC genişlemesi için büyüme faktörleri için gereğinin olmaması (GDS). Karışık glial kültürler astrosit türetilen trofik faktörlerin varlığı nedeniyle muhtemelen OPC yayılmasını destekleyen bir ortam sağlar. Türetme yoluyla neurospheres 7,8 gibi diğer yöntemler, temel fibroblast büyüme faktörü (bFGF), epidermal büyüme faktörü (EGF) ve OPC genişleme için trombosit kökenli büyüme faktörü (PDGF) gibi GF&#39;lerin mitojenik özellikleri güveniyor. Benzer şekilde, postnatal (P5-10) DRGNs için fareler kullanarak, sinir büyüme faktörü (NGF), embriyonik DRGNs 9, 10, in vitro hayatta kalmak için gerekli bir nörotrofik faktör ile kültür ortamı tamamlayan gereği önler . Ilgi DRGNs 4 ile kültüre EKK&#39;nın myelinating kapasitesini olumsuz etkiler olarak NGF kullanmaktan kaçının. Kullanımı kaçınmak GF-desteklenmiş büyük ölçekte kullanılan bu reaktifler pahalı gibi medya da, ekonomik faydaları vardır. Belki de bu kültürün modelin en önemli yararı, bu nedenle, transgenik fare hatları çok çeşitli OPC ve DRGNs elde etmek için fırsatlar sağlayarak, sadece fare dokuları türevidir. Bu her iki DRGN ve / veya miyelinasyonun yöneten OPC-spesifik özellikleri çalışma için izin verir. Bu aksonlar OL-aracılı miyelinasyonun düzenleyen reseptör / ligand etkileşimleri nedenlerine açıklık için özellikle önemli olacaktır. Bu teknikte, miyelinasyonun altında yatan moleküler ipuçları anlamaya yönelik uygulamaları nedeniyle nörolojik araştırma açısından büyük bir değer taşımaktadır.

<html><table border="1"><tbody><tr><td> Ürün Adı </td><td> Şirket </td><td> Ürün Numarası </td></tr><tr><td> Dulbecco&#39;nun Modifiye Kartal Medium (DMEM) </td><td> Çok hücreli </td><td> 319-005-CL </td></tr><tr><td> Hank Dengeli Tuz Çözeltisi (HBSS) </td><td> Invitrogen </td><td> 14170-112 </td></tr><tr><td> Minimum Essential Medya (MEM) </td><td> Gibco </td><td> 12360-038 </td></tr><tr><td> Fetal sığır serumu (FBS) </td><td> Gibco </td><td> 10091-148 </td></tr><tr><td> Penisilin-Streptomisin (Pen / Strep) </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> GlutaMAX </td><td> Invitrogen </td><td> 35050-061 </td></tr><tr><td> Poli-L-lisin </td><td> Sigma-Aldrich </td><td> P2636 </td></tr><tr><td> Sığır Serum Albumin (BSA) </td><td> Sigma-Aldrich </td><td> A4503 </td></tr><tr><td> İnsan merosin saflaştırılmış protein (LN2) </td><td> Millipore </td><td> CC085 </td></tr><tr><td> Rekombinant sıçan siliyer nörotrofik faktör (CNTF) </td><td> PeproTech </td><td> 450-50 </td></tr><tr><td> L-tiroksin </td><td> BioChemika </td><td> 89430 </td></tr><tr><td> Holo-transferrin </td><td> Sigma-Aldrich </td><td> T0665 </td></tr><tr><td> B27 takviyesi </td><td> Gibco </td><td> 0080085-SA </td></tr><tr><td> Sığır insülin </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> 3,3 &#39;,5-Triiodo-L-thyronine </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> Progesteron </td><td> Sigma-Aldrich </td><td> P8783 </td></tr><tr><td> Putrescine </td><td> Sigma-Aldrich </td><td> P7505 </td></tr><tr><td> Sodyum Selenit </td><td> Sigma-Aldrich </td><td> S5261 </td></tr><tr><td> 5-Fluoro-2&#39;-dezoksiuridin (FuDR) </td><td> Sigma-Aldrich </td><td> F0503 </td></tr><tr><td> Papain çözüm </td><td> Worthington </td><td> LS003126 </td></tr><tr><td> DNaseI </td><td> ROCHE </td><td> 1010159001 </td></tr><tr><td> L-sistein </td><td> Sigma-Aldrich </td><td> C7352 </td></tr><tr><td> 24-iyi doku kültürü yemekleri </td><td> Cellstar </td><td> 662-160 </td></tr><tr><td> Havalandırma kapağı ile T25-doku kültürü şişeleri </td><td> Corning </td><td> 430639 </td></tr><tr><td> 10 cm doku kültürü yemekleri </td><td> Corning </td><td> 430167 </td></tr><tr><td> 10 cm&#39;lik petri </td><td> Fisher Scientific </td><td> 0875713 </td></tr><tr><td> Kollajenaz A </td><td> ROCHE </td><td> 103578 </td></tr><tr><td> CellTrics 50 mikron filtre (opsiyonel) </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td> Miyelin Temel Protein (MBP) Antikor </td><td> Abd Serotec </td><td> MCA409S </td></tr><tr><td> NG2 antikor </td><td> Millipore </td><td> AB5320 </td></tr><tr><td> Miyelin İlişkili Glikoprotein (MAG) antikor </td><td> Millipore </td><td> MAB1567 </td></tr><tr><td> 2 &#39;, 3&#39;-Siklik nükleotid 3&#39;-fosfodiesteraz (CNP) antikor </td><td> Covance </td><td> SMI-91R-100 </td></tr><tr><td> Aktin pan Ab-5 antikor </td><td> Fitzgerald </td><td> 10R-A106AX </td></tr><tr><td> Nörofilaman-200 (NF) antikor </td><td> Sigma-Aldrich </td><td> N4142 </td></tr><tr><td> Tubulin beta-3 zinciri (Tuj1) antikoru </td><td> Millipore </td><td> MAB5544 </td></tr><tr><td> Alexa Fluor 488 keçi anti-tavşan IgG (H + L) sekonder antikoru </td><td> Invitrogen </td><td> A11008 </td></tr><tr><td> Alexa Fluor 555 keçi anti-fare IgG (H + L) sekonder antikoru </td><td> Invitrogen </td><td> A21422 </td></tr><tr><td> Alexa Fluor 647 keçi anti-sıçan (IgG) (H + L) sekonder antikoru </td><td> Invitrogen </td><td> A21247 </td></tr><tr><td> Keçi anti-fare IgG (H + L)-HRP konjuge sekonder antikor </td><td> BioRad </td><td> 170-6516 </td></tr><tr><td> Keçi anti-sıçan IgG (H + L)-HRP konjuge sekonder antikor </td><td> Santa Cruz Biyoteknoloji </td><td> SC-2065 </td></tr><tr><td> 4 &#39;,6-diamidino-2-phenylindole (DAPI) </td><td> Sigma-Aldrich </td><td> D9542 </td></tr></tbody></table> Medya Tarifler OL-100X Ek * <table border="1"><tbody><tr><td> Bileşen </td><td> Eklenecek tutar </td></tr><tr><td> DMEM </td><td> 100 mL </td></tr><tr><td> BSA </td><td> 1.02 g </td></tr><tr><td> Progesteron </td><td> 0.6 mg </td></tr><tr><td> Putrescine </td><td> 161 mg </td></tr><tr><td> Sodyum Selenit </td><td> 0.05 mg </td></tr><tr><td> 3,3 &#39;,5-Triiodo-L-thyronine </td><td> 4 mg </td></tr></tbody></table> * Mağaza -80 ° C 250 mcL alikot &quot;t&gt; OL medya <table border="1"><tbody><tr><td> Bileşen </td><td> Eklenecek tutar </td></tr><tr><td> DMEM </td><td> 23.75 ml </td></tr><tr><td> 100X OL-Ek </td><td> 250 mcL </td></tr><tr><td> Sığır insülin (1 mg / ml stok) </td><td> 125 mcL </td></tr><tr><td> GlutaMAX </td><td> 250 mcL </td></tr><tr><td> Holo-transferrin (33 mg / ml stok) </td><td> 37.5 mcL </td></tr><tr><td> B27 Supplement </td><td> 500 mcL </td></tr><tr><td> FBS </td><td> 125 mcL </td></tr><tr><td> CNTF (50 mcL ng / stok) </td><td> 25 mcL </td></tr></tbody></table> Karışık glial kültür ortamı (DMEM içinde oluşur) <table border="1"><tbody><tr><td> Bileşen </td><td> Final konsantrasyonu </td></tr><tr><td> FBS </td><td> % 10 </td></tr><tr><td> Pen / Strep (% 0.33 stok) </td><td> 33 adet / ml penisilin ve 33 mg / ml Streptomisin </td></tr><tr><td> GlutaMAX </td><td> % 1 </td></tr></tbody></table> DRGN medya (DMEM içinde oluşan) <table border="1"><tbody><tr><td> Bileşen </td><td> Final konsantrasyonu </td></tr><tr><td> FBS </td><td> % 10 </td></tr><tr><td> Pen / Strep (stok% 1) </td><td> 100 birim / ml penisilin ve 100 mg / ml Streptomisin </td></tr></tbody></table> Sindirim Çözüm Tarifler: OPC papain solüsyonu (MEM yılında yapılan) <table border="1"><tbody><tr><td> Bileşen </td><td> Final konsantrasyonu </td></tr><tr><td> Papain çözüm </td><td> 1.54 mg / ml </td></tr><tr><td> L-sistein </td><td> 360 mcg / mL </td></tr><tr><td> DNaseI </td><td> 60 mcg / mL </td></tr></tbody></table> DRG papain solüsyonu (HBSS içinde oluşan) <table border="1"><tbody><tr><td> Bileşen </td><td> Final konsantrasyonu </td></tr><tr><td> Papain </td><td> 1.54 mg / ml </td></tr><tr><td> L-sistein </td><td> 360 mcg / mL </td></tr></tbody></table> DRG kollajenaz (HBSS içinde yapılmış bir çözüm) <table border="1"><tbody><tr><td> Bileşen </td><td> Final konsantrasyonu </td></tr><tr><td> Kollajenaz A </td><td> 4 mg / ml </td></tr></tbody></table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

OL geliştirme altında yatan moleküler mekanizmaları belirleme OL biyoloji bilgimizi ilerletmek için yalnızca kritik değil, Multipl Skleroz (MS) gibi demiyelinizan hastalıkların patogenezinde anlamak için de etkileri vardır. Hücresel gelişimi sık primer hücre kültür modelleri ile çalışılmaktadır. Primer hücre kültürü, in vivo olarak bulunan gereksiz değişkenlerin kontrollü bir ortam sağlayarak, belirli bir hücre tipi değerlendirilmesi kolaylaştırır . Sıçanlarda türetilen OL kültürler OL biyoloji içgörü büyük bir miktar olsa da, fareler OL kültürler kurmayı benzer çabalar büyük engellerden ile karşılandı. Transgenik fare hatları yararlanmak amacıyla kültür fare primer OLS için yöntemler geliştirmek zorunludur. Neurosphere türev, diferansiyel yapışma arıtma ve 1-3 immunopurification arasında değişen, kemirgen dokusundan OPC çıkarılması için birden çok yöntem tarif edilmiştir. Pek çok yöntem başarı sunarken, en kapsamlı kültür ve / veya pahalı ekipman / reaktifler gerektirir. Bunu aşmak için, başlangıçta McCarthy &amp; de Vellis 2 tarafından açıklanan yöntemi bir adaptasyon fare doku OPC arındırıcı tercih edilir. Bu yöntem, fiziksel neonatal kemirgen korteks türetilen karışık bir glial kültüründen ayıran OPC içerir. Sonuç, bir saflaştırılmış OPC nüfusu OL-zenginleştirilmiş kültür ayrılabilir. Bu yaklaşım, görece kısa bir kültür zaman ve büyüme faktörleri veya immunopanning antikorlar için gereksiz gereksinimi nedeniyle itiraz ediyor. , Saflaştırılmış bir kültür OL gelişme mekanizmaları keşfetmek bilgilendirici olmasına rağmen, miyelin kılıf oluşumu değerlendiren için en önemli fizyolojik bir ortam sağlamaz. Co-kültür OLS nöron aksonları OL-aracılı miyelinasyonun düzenleyen moleküler temellerinin içgörü ödünç. / OL nöron ko-kültür çalışmaları birçok insan için, dorsal kök ganglion nöronlar (DRGNs) seçim nöron türü olduğu kanıtlanmış. Ekstraksiyon, kirlenmesine neden olan hücreler az miktarda ve yoğun neurite yatak oluşumu kolaylığı nedeniyle OLS ile ko-kültür için idealdir. 4-6 xenocultures myelinating sıçan / fare kullanarak çalışmalar yayınlanmış olsa da, böyle derivasyon için bir yöntem DRGN / OL post-natal fare doku ortak kültürler myelinating tanımlanmıştır değil. Burada etkili beklenen sonuçlar örnekleri ile birlikte, bu tür kültür üretmek için nasıl ayrıntılı yöntemler sunuyoruz. Bu yöntemler OL geliştirme / myelinating fonksiyonu ile ilgili sorulara yanıt için yararlı ve faydalı araçlar sinirbilim alanında.

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Bu makale, olgun oligodendrosit (EKK) üretmek için ayırt primer kültür fare oligodendrosit öncü hücreler (OPC) zenginleştirilmiş nüfus, elde etmek için yöntemler anlatılmaktadır. Buna ek olarak, bu rapor üretmek için teknikler açıklar fare fare dorsal kök ganglion nöronlar (DRGNs) neurite yatağa fare OPC ekim ortak kültürler myelinating.

Post-natal murin Dokular Co-kültürler Myelinating zenginleştirilmiş oligodendrosit Kültürleri ve oligodendrosit / Nöron türetilmesi

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.2K Görüntüleme.  Ottawa Hospital Research Institute. Bu makale, olgun oligodendrosit (EKK) üretmek için ayırt primer kültür fare oligodendrosit öncü hücreler (OPC) zenginleştirilmiş nüfus, elde etmek için yöntemler anlatılmaktadır. Buna ek olarak, bu rapor üretmek için teknikler açıklar fare fare dorsal kök ganglion nöronlar (DRGNs) neurite yatağa fare OPC ekim ortak kültürler myelinating.

Video: Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Yüksek zenginleştirilmiş Nöronal Kültürlerin Üretimi için Kortikal Doku Blokları, Kriyoprezervasyon

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Nörobilim

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

E13 farelerin Glial Yasak Öncüleri türetilmesi

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

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

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

İnsan Fetal Beyin gelen Progenitör-türetilmiş Oligodendrosit Kültür Sistemi

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

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitokondri ilişkili ER Membranlar (MAM) ve Glycosphingolipid zenginleştirilmiştir Microdomains (GEM&#39;lerle): Fare Beyin izolasyon

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Eşzamanlı Öncesi ve Sonrası-sinaptik Elektrofizyolojik Recording<em&gt; Xenopus</em&gt; Sinir-kas Co-kültürlerin

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Tek Hücre Hassas Nevronik Co-kültürler hazırlanması

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotipik Dilim Kültürler Oligodendrosit Dinamikler ve miyelinasyonun incelemek için

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Çok elektrot Dizileri Yetiştirilen Organotipik Omurilik Co-kültürler Fonksiyonel Rejenerasyon incelenmesi

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.