Name: using primary neurosphere cultures to study primary cilia
Uploaded: 2017-04-14T12:30:00
Description: Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Work in S.M.'s laboratory is funded by recruitment grants from CPRIT (R1220) and NIH (1R01GM113023-01).

1. Isolering af Neurosfærer fra voksne mus Brain <ol><li> Afliv en voksen mus (omkring 2 måneder gammel) med en overdosis af isofluran. Dobbelttjek, at musen er stoppet vejrtrækning og dissekere umiddelbart efter døden. </li><li> Ved hjælp af en saks, lave en midtlinie incision for at åbne kraniet. Fjern hjernen. </li><li> Placer hjernen i koldt PBS i en 10 cm skål på is. Følge hele-mount dissektion metode til at opnå SVZ&#39;en fra den laterale ventrikel 48. </li><li> Placer laterale...

Her beskriver vi en metode til at generere og vedligeholde neurosfære kulturer fra voksne mus SVZ. Et par relevante punkter vedrørende kulturerne er som følger. Første størrelserne af kuglerne er typisk mellem 50 - 200 um. Det er vores erfaring, når en neurosfære bliver større end 300 um i diameter, har det optimale tidspunkt for passage været savnet. Disse større kugler indeholder døde celler i kernen. Sekund, som neurosfærer er almindeligt anvendt til at studere neurale stamceller / progenitorceller, er de...

Den primære cilium er en mikrotubulus-baserede dynamisk subcellulært afsnit, der fungerer som en sensorisk antenne i koordineringen cellulære signalveje, herunder Sonic Hedgehog (Shh) pathway under embryonisk neuronal udvikling 1, 2, og opdelte subcellulære signalering hos voksne neuronal funktion 3, 4 . Signalering komponenter af disse veje såsom Shh receptoren Patched 5; reaktionsvejen aktivator Smoothened (Smo) 6; og Gpr161 7, sjældne G-protein-koblet receptor (GPCR), som negativt regulerer Shh pathway, lokalisere til cilier på en dynamisk måde. Flere GPCR&#39;er er blevet rapporteret at lokalisere til cilia i neuroner i hjernen 7, 8, 9, 10 </sup&gt;, 11, 12, 13, 14, 15, 16. Defekter i cilier og cilia-genererede signalveje påvirker multiple væv og er kollektivt kendt som ciliopathies 17, 18, 19. Den ciliopathy sygdomsspektrum omfatter ofte neuro defekter, såsom kraniofaciale abnormiteter 20, 21, 22. Desuden primære cilier i hypothalamus neuroner regulerer centrale mæthed veje, og defekter resulterer i det centrale fedme 23, spejling fedme i syndromisk ciliopathies såsom Bardet Biedel syndrom 24. Desuden Neuropeptidreceptoren signalering i cilia regulerer central mæthed veje i 11, 14. Ciliære lokalisering af adenylylcyclase III (ACIII) og GPCR&#39;er såsom somatostatinreceptor 3 i hippocampale neuroner resulterer i hidtil ukendte defekter objekt anerkendelse og hukommelsessvigt 25, 26 og parallel manglende ciliær integritet 27. De udviklingsmæssige aspekter af cilier-genererede signalering er nært knyttet til vævshomeostase; navnlig cilier er vigtige for udviklingen af Shh-undertype medulloblastomer forbindelse granula progenitorer i cerebellum 28, 29. Således primære cilier spille vigtige roller i løbet af embryonale, postnatal og voksne neuronal udvikling og funktion. Neurale stamceller (NSC) bosiddende i subventrikulære zone (SVZ) af den laterale ventrikel, den subgranulære zone af gyrus dentatus i hippocampus, ogventrikulær zone af den tredje ventrikel i hypothalamus hos pattedyr 30, 31, 32. NSC er multipotente, besidder evnen til selv-fornyelse, og er vigtige for hjernens udvikling og regenerativ medicin 30. De fleste NSC&#39;er i SVZ&#39;en er hvilende og besidde et ensomt primær cilium, at i mange tilfælde strækker ud til den laterale ventrikel 33. De primære cilium signaler via lokaliseringen af forskellige receptorer, inducere nedstrøms cellulære responser, især i forbindelse med Shh, TGFp, og receptortyrosinkinase pathways 2, 34, 35, 36. Idet primære cilier strækker sig ind i laterale ventrikel, antages det primære cilier detektere cytokiner i cerebrospinalvæsken (CSF) for at aktivere NSC&#39;er 37 </sop&gt;. Nylige undersøgelser tyder på, at Shh signalvejen og primære cilier er kritiske for aktiveringen af stamceller i reparation og regenerering af multiple væv, herunder lugteepitelet, lunge og nyre 38, 39, 40, 41. Imidlertid er de mekanismer, ved hvilke CSF kommunikerer med NSC&#39;er og enten primære cilier er involveret er ikke kendt. Adhærente NSC&#39;er i kultur er cilierede; lokalisere Shh pathway komponenter, såsom Smo og Gpr161 i cilia; og er Shh lydhøre 42. Således kan NSC&#39;er tjene som et vigtigt modelsystem til at studere Shh pathway, ciliær handel, og neuronale differentieringsveje. Desuden kan neuroner differentierede fra NSC&#39;er også anvendes til ciliær trafficking assays. Neurosfærer udgøres af klynger af fritflydende celler i forbindelse med spredning af neural stamceller / progenitorceller, der vokser i nærvær af specifikke vækstfaktorer og ikke-adhæsive overflader 43, 44. Neurosfærer tjener som vigtig in vitro kultur modeller til at studere neurale stamceller / progenitorceller i normal udvikling og sygdom 31, 45, 46, 47. Her beskriver vi en neurosfære-baseret assay til dyrkning neurale stam- / progenitorceller og for differentiering til neuroner / glia. Vi understreger især handel med signalering komponenter til cilia af neurale stam- / progenitorceller og differentierede neuroner (figur 1). I modsætning til dyrkning primære neuroner, primære neurosfærer er forholdsvis let at dyrke, er modtagelige for multiple passager og fryse-tø-cykler, og kan undergå differentiering til neuroner / glia. Vigtigst vi fastslå, at neurosfære-afledte neuralestamceller / progenitorceller og differentierede neuroner cilierede i kultur og lokalisere signalmolekyler relevante for ciliefunktionen i disse rum. Neurosphære-baserede dyrkningsmetoder kan tjene som en ideel model for at studere ciliogenesis og ciliaere handel med NSC&#39;er og differentierede neuroner.

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using primary neurosphere cultures to study primary cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

Efter udpladning af cellerne fra SVZ i NSC medium i en uge, blev de flydende neurosfærer observeret (figur 2A). Størrelserne af kuglerne varierede mellem 50 og 200 um. At undersøge, om kuglerne blev afledt fra neurale stamceller / progenitorceller, blev neurosfærerne udpladet på PLL- og laminin-overtrukne dækglas i NSC medium i 2 dage. De blev derefter immunfarvet mod neurale stam- / progenitorcelle markering, nestin. To dage var nødvendige for at tillade i områd...

Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Using Primary Neurosphere Cultures to Study Primary Cilia

Den primære cilium er fundamentalt vigtigt i neural progenitorcelle proliferation, neuronal differentiering, og voksen neuronal funktion. Her beskriver vi en metode til at studere ciliogenesis og handel med signalproteiner til cilia i neurale stamceller / progenitorceller og differentierede neuroner anvender primære neurosfære-kulturer.

Brug Primære Neurosfære kulturer til at studere Primær Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

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