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

Forfatterne ønsker å takke dr. Y. Sasai for sjenerøst gi FOXG1 antistoff. Dette arbeidet ble støttet av Connecticut stamcelleforskning Grants (08-SCB-UCHC- 022 og 11-SCB24) og spastisk paraplegi Foundation.

1. Generasjon av Human pluripotent Stem Cell Aggregater (D1-D4) <ol><li> Humane pluripotent stamceller dyrkes i mus embryonale fibroblast (MEF) forer i nærvær av hESC medium supplert med grunnleggende fibroblast vekstfaktor (bFGF, 4 ng / ml). Etter 5-7 dager i kultur når koloniene er stor, men likevel udifferensiert, er de klare for neste trinn. </li><li> Enzym-oppløsningen bør først forberedt. I en 50 ml rør, oppløse dispase (eller kollagenase) ved 1 mg / ml konsentrasjon i DMEM/F12 medium. Siden disse løsni...

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Det er flere kritiske trinnene under nevrale differensiering prosessen. Det er viktig å sikre at de menneskelige PSCs er pluripotent fordi ellers cellene kan allerede være forutinntatt mot å bli en ikke-nevronale avstamning. Dette kan bekreftes ved farging menneskelige PSCs med antistoffer mot pluripotency markører som Oct4, Sox2, Nanog, og Tra-1-60 1-3. Hvis de menneskelige PSCs ikke feste veldig godt etter passaging dem, kan ROCK inhibitor (Y27632) legges til hjelp. For de som har vanskeligheter med å ...

Menneskelige pluripotente stamceller (PSCs), inkludert både menneskelige embryonale stamceller (hESCs) og indusert pluripotent stamceller (iPSCs), har kapasitet til å generere hver celletype i kroppen, inkludert nerveceller 1-3. Rettet differensiering av ulike nevrale subtyper fra menneskelige PSCs har nøkkelen for anvendelse av disse cellene i regenerativ medisin. Den generasjonen av funksjonelle nevrale subtyper under utvikling er en kompleks prosess som involverer induksjon av nevrale avstamning, spesifikasjonen av regionale stamfedre langs Rostro-caudal aksen, og differensiering av post-mitotiske Nevron typer fra de regionale forfedre 4,5. Starten i 2001, har flere systemer er etablert for å generere nevrale avstamning fra hESCs, som har gitt en plattform for den påfølgende generasjonen av nevrale subtyper 6,7. Basert på utviklingsmessige prinsipper, flere neuron som for eksempel spinal motor 8-12 nevroner, midbrain dopaminergic nevroner 13-15, og nevrale retinal celler 16,17 er effektivt spesifisert fra menneskelige PSCs. Dette ble gjort ved å bruke kritiske morphogens som er viktige for spesifisering av disse neuron typer under in vivo utvikling. Andre protokoller er også utviklet for å fremme differensieringen av hESCs i nevroner ved hjelp av enten ytterligere faktorer 18-20 for eksempel små molekyler, eller ved ko-dyrking med andre celletyper å bidra til å fremme differensiering 21. Den menneskelige neocortex er høyt utviklet og inneholder mange celletyper, inkludert glutamatergic nevroner som spiller en viktig rolle i læring, hukommelse og kognitiv funksjon 22,23. Det første trinnet i å generere glutamatergic nevroner i kultur er å spesifisere telencephalic stamceller. Yoshiki Sasai gruppe først rapporterte rettet differensiering av telencephalic forløpere fra mus ESCs (mESCs) med en serum-free suspension kulturen i nærvær av NOK1 (som hemmer Wnt signalering) samt LeftyA (som hemmer nodal signalering) 24. Senere har flere grupper inkludert vår også rapportert spesifisering av telencephalic forløpere fra menneskelige PSCs i serum fritt medium 25-27. Generering av telencephalic forløpere fra menneskelige PSCs krever ikke bruk av eksogene morphogens og effektiviteten i å generere disse forløperne er mye høyere enn det fra mESCs 26,27. Her har et kjemisk definert system for neural induksjon som var godt etablert av Zhang gruppe 7 blitt beskrevet. Uten tilsetning av eksogene caudalizing faktorer, genererer denne protokollen effektivt telencephalic forløpere fra menneskelige PSCs 27. Disse stamfedre kan deretter differensiert i dorsal eller ventral stamfedre ved å regulere signalering av Wnt og sonic hedgehog (ssh). De dorsale stamfedre kan videre skille ut glutamatergic nevroner efficiently 27. I tillegg fungerer denne protokollen også godt for generering av glutamatergic nevroner fra menneske 28 iPSCs, som gjør det mulig for generering av pasient-spesifikke nevroner som kan benyttes til å utforske virkningsmekanismen så vel som potensielle terapi for et stort utvalg av sykdommer . Dessuten gir systemet vårt også en plattform for å utforske utvikling og spesifisering av ulike nevrale 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

Her har en trinnvis prosedyre for effektivt å generere telencephalic glutamatergic nevroner fra menneskelige pluripotente stamceller (PSCs) blitt beskrevet. Differensieringen prosessen initiert ved å bryte menneskelige PSCs inn klumper som runder opp til å danne aggregater når cellene er plassert i en suspensjon kultur. Aggregatene dyrkes så i hESC medium fra dag 1-4 for å tillate spontan differensiering. I løpet av denne tid, vil cellene har evne til å bli en av de tre bakterie lag. Fra dager 5-8, blir cellene plassert i et nevralt induksjon middels å presse dem inn i den nevrale avstamning. Rundt dag 8, blir cellene tillatt å feste på 6 brønners plater og skille hvorav tid neuroepithelial celler form. Disse neuroepithelial celler kan isoleres ved dag 17. Cellene kan deretter holdes så neurospheres før de er klare til å bli belagt på dekkglass. Ved hjelp av en enkel medium uten caudalizing faktorer, neuroepithelial celler er specified til telencephalic forløpere, som deretter kan ytterligere differensiert i dorsal telencephalic forfedre og glutamatergic nevroner effektivt. Samlet gir systemet vårt et verktøy for å generere menneskelige glutamatergic nevroner for forskere å studere utviklingen av disse nevronene og sykdommer som påvirker dem.

Her, med en protokoll differensiere menneskelige PSCs inn telencephalic glutamatergic nevroner gjennom flere kritiske trinn: dannelsen av PSC tilslag, har induksjon av neuroepithelial celler, produksjon av telencephalic progenitorer og terminal differensiering av disse stamfedre inn telencephalic nevroner (figur 1) blitt beskrevet. Dette systemet er robust og effektivt i produksjon av telencephalic forfedre og glutamatergic nevroner. Som et eksempel (figur 2), uten tilsetning av caudali...

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

Denne prosedyren gir telencephalic nevroner ved å gå gjennom sjekkpunkter som er lik de som ble observert i løpet av menneskelig utvikling. Cellene tillates å spontant skille, er utsatt for forhold som presse dem mot den nevrale avstamning, er isolert, og er belagt på dekkglass for å tillate terminal differensiering og modning.

Spesifikasjonen av Telencephalic glutamatergic Neurons fra menneskelige pluripotente 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 Utledning av Human nevrale stamceller og nevroner fra pluripotent humane embryonale stamceller med lite molekyl Induksjon

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-relevant Expansion Metode for Aggregater of Human Neural Stem og stamceller avledet fra pluripotent stamceller eller Fetal hjernevev

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-free Utledning av Neural Crest stamceller fra menneskelige pluripotent 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

Regissert dopaminerge Neuron Differensiering fra Menneskelig Pluripotent 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

Enkel generasjon av en høy avkastning kultur indusert neurons fra menneskelige voksne huden 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

Metode for høyhastighets strekk skade av menneskelige indusert Pluripotent Stamcelle-avledet Neurons i 96-brønnen 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

Post-differensiering Replating av Human pluripotent Stem Cell-avledet neurons for High-Content screening av Neuritogenesis og synapse modning

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

Konvertering av Human indusert pluripotent Stem Cells (iPSCs) i funksjonell spinal og skallen motor neurons bruke 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

Analysere mitokondrietransport og morfologi i humant indusert pluripotente stamcelleavledede nevroner i arvelig 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

Avledning, utvidelse, kryopreservasjon og karakterisering av mikrovaskulære endotelceller fra humane induserte pluripotente stamceller

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