Name: the specification of telencephalic glutamatergic neurons from human pluripotent stem cells
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Description: Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

Författarna vill tacka Dr Y. Sasai för generöst ge FOXG1 antikroppen. Detta arbete stöddes av Connecticut Stem Grants Cell Research (08-SCB-UCHC- 022 och 11-SCB24) och spastisk paraplegi Foundation.

1. Generering av Human Cell pluripotenta stamceller aggregat (D1-D4) <ol><li> Humana pluripotenta stamceller odlas på mus embryonala fibroblast (MEF) matare i närvaro av hESC medium kompletterat med basisk fibroblasttillväxtfaktor (bFGF, 4 ng / ml). Efter 5-7 dagar i odling, när kolonierna är stora men fortfarande differentierade, är de redo för nästa steg. </li><li> Enzymlösningen bör först beredas. I en 50 ml rör, upplösa dispas (eller kollagenas) vid en 1 mg / ml koncentration i DMEM/F12-medium. Efters...

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Det finns flera viktiga steg under den neurala differentiering processen. Det är viktigt att säkerställa att de humana PSC är pluripotenta eftersom annars cellerna kan redan vara förspänd mot att bli en icke-neuronal härstamning. Detta kan bekräftas genom färgning av humana PSC med antikroppar mot pluripotensbestämmande markörer såsom Oct4, Sox2, nanog, och Tra-1-60 1-3. Om de mänskliga PSC inte fäster mycket väl efter passage dem, kan ROCK-hämmare (Y27632) läggas att hjälpa till. För dem s...

Mänskliga pluripotenta stamceller (PSC), både mänskliga embryonala stamceller (hESCs) och inducerade pluripotenta stamceller (iPSCs), har kapacitet att generera alla celltyp i kroppen, inklusive nervceller 1-3. Riktad differentiering av olika neuronala subtyper från mänskliga PSC är nyckeln för tillämpningen av dessa celler i regenerativ medicin. Den generation av funktionella neuronala subtyper under utveckling är en komplex process som involverar induktion av neurala härstamning, specifikation av regionala stamfäder längs Rostro-caudal axeln och differentieringen av post-mitotiska neuron typer från de regionala stamceller 4,5. Från och med 2001 har flera system etablerats för att generera neurala härkomst från hESCs som har gett en plattform för nästa generation av neuronala subtyper 6,7. Baserat på utvecklingsmässiga principer, flera neuron typer såsom spinal motoriska neuroner 8-12, mitthjärnan DOPaminergic neuroner 13-15 och neurala näthinnans celler 16,17 har effektivt anges från humana PSC. Detta gjordes genom att kritiska morfogener som är viktiga för att specificera dessa neuron typer under in vivo-utvecklingen. Andra protokoll har också utvecklats för att främja differentiering av hESCs till nervceller med hjälp av antingen ytterligare faktorer 18-20 såsom små molekyler eller genom samodling med andra celltyper för att främja differentiering 21. Den mänskliga neocortex är högt utvecklad och innehåller många celltyper, inklusive glutamaterga nervceller som spelar en viktig roll i inlärning, minne och kognitiv funktion 22,23. Det första steget vid generering av glutamaterga neuroner i odling är att ange telencephalic progenitorceller. Yoshiki Sasai grupp rapporterade först riktade differentiering av telencephalic prekursorer från mus ekonomiska och sociala råd (mESCs) med en serumfritt suspensioN-kulturen i närvaro av DKK1 (som inhiberar Wnt signalering) liksom LeftyA (som inhiberar nodal signalering) 24. Därefter har flera grupper inklusive vår rapporterade också specifikationen av telencephalic prekursorer från mänskliga PSC i serumfritt medium 25-27. Den generation telencephalic prekursorer från mänskliga PSC kräver inte användning av exogena morfogener och effektivitet i att generera dessa prekursorer är mycket högre än den från mESCs 26,27. Här har ett kemiskt definierat system för neural induktion som väl grundades av Zhang grupp 7 har beskrivits. Utan tillsats av exogena caudalizing faktorer genererar detta protokoll effektivt telencephalic prekursorer från mänskliga PSC 27. Dessa stamfäder kan sedan delas in i dorsala eller ventrala föregångare genom att reglera signalering av Wnt-och Sonic hedgehog (SHH). Den dorsala stamfäder vidare kan differentiera till glutamaterga neuron efficiently 27. Dessutom fungerar detta protokoll också väl för generering av glutamaterga neuroner från humant iPSCs 28, som möjliggör generering av patientspecifika neuroner som kan utnyttjas för att undersöka verkningsmekanismen samt potentiella terapier för en stor mängd sjukdomar . Dessutom ger vårt system också en plattform för att undersöka möjligheten att utarbeta och specifikation av olika neuronala typer i telencephalon.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>1140-050</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (14.3 M)</td>
			<td>Sigma</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine)</td>
			<td>Sigma</td>
			<td>116K5103</td>
		</tr>
		<tr>
			<td>Laminin (human)</td>
			<td>Sigma</td>
			<td>L-6274</td>
		</tr>
		<tr>
			<td>Laminin (mouse)</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Stemgent</td>
			<td>04-0012</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
		</tr>
		<tr>
			<td>Foxg1 antibody</td>
			<td>Dr. Y. Sasai</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoxb4 antibody (1:50)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>I12</td>
		</tr>
		<tr>
			<td>Pax6 antibody (1:5000)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>PAX6</td>
		</tr>
		<tr>
			<td>Nkx2.1 antibody (1:200)</td>
			<td>Chemicon</td>
			<td>MAB5460</td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
		</tr>
		<tr>
			<td>vGLUT1 antibody (1:100)</td>
			<td>Synaptic Systems</td>
			<td>135302</td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-02</td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (GDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-10</td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>PrepoTech Inc.</td>
			<td>100-11</td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma</td>
			<td>D-0260</td>
		</tr>
		<tr>
			<td>Sonic hedgehog (SHH)</td>
			<td>R&amp;D</td>
			<td>1845-SH</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Becton Dickinson (BD)</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352097</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>BD</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>BD</td>
			<td>353047</td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>BD</td>
			<td>353009</td>
		</tr>
		<tr>
			<td>T75 flasks (untreated)</td>
			<td>BD</td>
			<td>353133</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>BD</td>
			<td>353004</td>
		</tr>
		<tr>
			<td>9'' glass pipetes</td>
			<td>Fisher</td>
			<td>13-678-20D</td>
		</tr>
		<tr>
			<td>Steriflip filters (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>Stericup filters 1,000 ml (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
		</tr>
		<tr>
			<td>Phase contrast microscope (Observer A1)</td>
			<td>Zeiss</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (Hera Cell 150)</td>
			<td>Thermo Electron Corporation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety hood (Sterilgard III Advance)</td>
			<td>The Baker Company</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge (5702 R)</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

Här har en stegvis procedur för att effektivt generera telencephalic glutamaterga nervceller från mänskliga pluripotenta stamceller (PSC) har beskrivits. Differentieringen initieras genom att bryta de mänskliga PSC till klumpar som avrunda att bilda aggregat när cellerna placeras i en suspensionskultur. Aggregaten odlas sedan i hESC medium från dag 1-4 för att medge spontan differentiering. Under denna tid, cellerna har förmåga att bli någon av de tre groddblad. Från dag 5-8, är cellerna placerade i en neural induktion medium för knuffa dem i neurala linjen. Omkring dag 8, cellerna fick fästa på 6 brunnars plattor och differentiera under vilken tid neuroepitelceller formuläret. Dessa neuroepitelceller kan isoleras på dag 17. Cellerna kan därefter hållas så neurosfärer tills de är redo att plattades på täckglas. Med hjälp av ett basiskt medium utan caudalizing faktorer neuroepitelceller är specified till telencephalic prekursorer, som sedan kan ytterligare differentieras i rygg telencephalic föregångare och glutamaterga neuroner effektivt. Sammantaget ger vårt system ett verktyg för att generera humana glutamaterga nervceller för forskare att studera utvecklingen av dessa neuroner och de sjukdomar som påverkar dem.

Här, till ett protokoll differentiera mänskliga PSC till telencephalic glutamaterga nervceller genom flera viktiga steg: bildandet av PSC aggregat har induktion av neuroepitelceller, generering av telencephalic föregångare, och terminal differentiering av dessa stamfäder till telencephalic neuroner (figur 1) beskrivits. Detta system är robust och effektiv vid generering av telencephalic progenitorer och glutamaterga neuroner. Som ett exempel (figur 2), utan tillsats av caudalizing...

Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Detta förfarande ger telencephalic nervceller genom att gå igenom checkpoints som liknar de som observerats under människans utveckling. Cellerna får spontant differentiera, utsätts för faktorer som driver dem mot det neurala härstamning, isoleras och stryks ut på täckglas för att möjliggöra terminal differentiering och mognad.

Specifikationen av Telencephalic glutamaterg nervceller från mänskliga pluripotenta stamceller

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Research

JoVE Journal

Neuroscience

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

Effektiv Derivering av mänskliga neurala stamceller och nervceller från Pluripotenta mänskliga embryonala stamcellslinjer med liten molekyl Induktion

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

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

En cGMP-tillämplig Expansion Metod för aggregat av mänskliga neurala Stem och stamceller härledas från pluripotenta stamceller eller fosterhjärnvävnad

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

Feeder fria Härledning av neurallisten progenitorceller från Human pluripotenta stamceller

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

Riktad dopaminerga nerv Differentiering från Human pluripotenta stamceller

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

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

Enkla Generation av en hög avkastning kultur av inducerade nervceller från mänsklig vuxen hud fibroblaster

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

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Metod för hög hastighet Stretch skada av mänskliga inducerade pluripotenta stamceller-derived nervceller i en 96-väl Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Efter differentiering Replating av mänskliga pluripotenta stamceller-härledda nervceller för hög innehåll screening av Neuritogenes och synapsen mognad

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Omvandling av mänskliga inducerade pluripotenta stamceller (iPSCs) till funktionella spinal och kranial motor neuron använda PiggyBac vektorer

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Analysera mitokondriell transport och morfologi i mänskliga inducerad pluripotent stamcell-härledda nervceller i ärftliga spastisk paraplegi

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Härledning, expansion, kryopreservation och karakterisering av hjärnans mikrovaskulära endotelceller från mänskliga inducerade pluripotenta stamceller

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...