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

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

1. hiPSC Differentiering till neurala stamceller <ol><li> Växa och expandera hiPSCs i mänskliga embryonala stamceller (HES) media (tabell 1) co-odlade på en mus embryonala fibroblast (MEF) feeder lager tills stora (men subkonfluenta) kolonier är redo för neural differentiering via en embryoid kropp (EB) mellan (Figur 2). Rutin hiPSC odlingsbetingelser är väl beskrivna på annat håll 16,17; kort, odla hiPSCs i HES media på ett MEF matarlager tills sammanflytande, d?...

<ol>
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Vi har beskrivit metoder genom vilka att skilja hiPSCs till NPC, en neural celltyp där en betydande andel av genen undertecknande av hiPSC-härledda neuroner bevaras och som kan fungera som en proxy för utvecklingsvägar potentiellt bidrar till sjukdoms patogenes 8, 11. Dessutom, som vi har detaljerade, NPCs är en robust replika och enkelt omvandlade neurala befolkningen, vilket vi tror kan vara lämpliga för molekylära och biokemiska studier av sjukdoms anlag. Även om vi ha...

Genuttryck studier av nervceller differentierade in vitro från humana inducerade pluripotenta stamceller (hiPSCs) av oss 1 och övriga 2,3 indikerar att hiPSC nervceller liknar foster snarare än vuxna hjärnvävnad. För närvarande kan hiPSC-baserade modeller vara lämpligare för studier av anlag för, snarare än sena funktioner i, neurologisk sjukdom. Vi har tidigare rapporterat att en betydande fraktion av genen tecknandet av schizofreni hiPSC härledda neuron är konserverad i schizofreni hiPSC härledda neurala stamceller (NPC), vilket indikerar att NPC kan vara en användbar celltyp för att studera de molekylära vägar som bidrar till schizofreni 1 . Vi och andra har rapporterat avvikande migration, ökad oxidativ stress och reaktiva syreradikaler, känslighet för under gränsvärdena miljöpåfrestningar och nedsatt mitokondriefunktion vid schizofreni hiPSC NPCs 1,4-6, samt minskad neuronal canslutning och synapserna i schizofreni hiPSC nervceller 5,7-10. Om de molekylära faktorer som bidrar till avvikande migration och / eller oxidativ stress i schizofreni hiPSC NPCs bygger också reducerade nervkopplingar i schizofreni hiPSC-härledda neuron, skulle NPCs vara ett robust och mycket replika neurala befolkningen med att studera de mekanismer som är ansvariga för sjukdomen. Vidare, eftersom ett snabbt kan alstra stora mängder celler och behöver inte vänta veckor eller månader för neuronal mognad, NPC-baserade analyser är lämpliga för studier av större patient kohorter och är mer mottagliga för hög genommatningsscreening. Vi tror att hiPSC NPCs kan fungera som en proxy för utvecklingsvägar potentiellt bidrar till sjukdoms patogenes, som redan visats i sjukdomar så olika som schizofreni 1 och Huntingtons sjukdom 11. För att skilja NPCs från hiPSCs, initial neurala iproduktionen sker genom dubbla SMAD inhibition (0,1 mM LDN193189 och 10 mM SB431542) 12. Genom antagonisera BMP och TGF signalering med dessa små molekyler, är endoderm och mesoderm specifikation blockerad, accelererande neuronal differentiering och leder till bildandet av synliga neurala rosetter inom en vecka plätering. Neural mönstring inträffar tidigt i denna process, förmodligen under perioden av neural rosettbildning och omedelbart därefter. I avsaknad av andra signaler, dessa primitiva neurala celler antar en främre framhjärnan liknande öde 13. Omedelbart efter neural rosettbildning, och pågående hela NPC expansionen, framhjärnan NPCs odlas med FGF2 8,14. De har dubbla härstamning potential och kan differentieras till neurala populationer av 70-80% III-tubulin-positiva neuroner och 20-30% gliafibrillärt surt protein (GFAP) -positiva astrocyter (Figur 1). Majoriteten av framhjärnan hiPSC nervceller är VGLUT1 positiva, Och så är förmodligen glutamaterga, men ca 30% av nervceller är GAD67-positiva (GABAergic) 8. NPCs rutinmässigt passe mer än tio gånger in vitro, samtidigt som konsekvent differentiering profiler, och utan att ackumulera karyotyp avvikelser. Grupper har rapporterat aging NPCs mer än 40 gånger 15, dock finner vi att bortom tio passager, visar NPCs ökad benägenhet för astrocyternas differentiering. NPCs väl tolerera flera frys-tinar och kan gått att växa som neurospheres genom att helt enkelt odling i icke-vidhäftande plattor. NPCs effektivt transduceras av virala vektorer, möjliggör snabb utvärdering av de molekylära och cellulära konsekvenser av genetisk störning, och lätt att bygga för att ge tillräckligt med material för biokemiska studier. Dessutom eftersom virala vektorer medger robust överuttryck och / eller knockdown av sjukdomsrelevanta gener, antingen kontroll eller patienten härlett neural-celler, kan man använda denna plattform för att testa effekten av genetiska bakgrunden på dessa manipulationer. Även om det inte lämpar sig för synaptic eller aktivitetsbaserade analyser som kräver mogna nervceller, kan NPCs vara ett praktiskt alternativ för många enkla molekylära eller biokemiska analyser av patientgenererade neurala celler.

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			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:216px;">Catalog Number</td>
			<td style="width:500px;">Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#11330</td>
			<td>for HES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Glutamax</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#35050</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">NEAA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#11140</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#21985-023</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">FGF2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0074</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">#450-02</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td style="width:216px;">&#160;#05832</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Accutase</td>
			<td style="width:171px;">Innovative Cell Technologies</td>
			<td style="width:216px;">AT-104</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#17104019</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">CF1 mEFs</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">#PMEF-CF</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

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

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

Neurala rosetter kan identifieras morfologiskt, med hjälp av en ljusfältsmikroskop, med deras karakteristiska utseende som runda kluster av neuroepiteliala celler med apico-basal polaritet (Figur 1). Även NPCs är vanligtvis odlas vid mycket hög celltäthet, omedelbart efter passage, är något pyramidformad soma och bipolär neurite struktur synlig (Figur 1D). Validerade NPC uttrycka nestin och Sox2 i flertalet celler, men III-tubulin färgningen är också synliga i alla NPC popul...

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

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

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

En guide till Generera och använda hiPSC Härledda NPCs för studien av Neurologiska sjukdomar

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

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