Specifikationen af Telencephalic glutamaterge neuroner fra humane pluripotente stamceller
Summary
Denne fremgangsmåde giver telencephalic neuroner ved at gå gennem checkpoints, der ligner dem observeret under den menneskelige udvikling. Cellerne får lov til spontant differentierer, udsættes for faktorer, der skubber dem mod det neurale afstamning, isoleres, og udplades på dækglas for at muliggøre terminal differentiering og modning.
Abstract
Her har en trinvis procedure for effektiv frembringelse telencephalic glutamaterge neuroner fra humane pluripotente stamceller (PSC) blevet beskrevet. Differentieringsprocessen initieres ved at bryde de humane PSC'er i klumper, som runde op til dannelse af aggregater, når cellerne er placeret i en suspensionskultur. Aggregaterne dyrkes derefter i hESC medium fra dag 1-4 for at tillade spontan differentiering. I denne periode har cellerne kapacitet til at blive en af de tre kimlag. Fra dag 5-8, cellerne anbringes i et neuralt induktionsmedium at skubbe dem ind i neurale afstamning. Omkring dag 8 cellerne lov til at fæstne på 6-brøndsplader og differentiere i hvilket tidsrum neuroepitelceller form. Disse neuroepitelceller kan isoleres på dag 17. Cellerne kan derefter holdes som neurosfærer, indtil de er klar til at blive udpladet på dækglas. Ved hjælp af et basisk medium uden caudalizing faktorer, neuroepitelceller er specified i telencephalic forstadier, som derefter kan yderligere differentieret i dorsale telencephalic progenitorer og glutamaterge neuroner effektivt. Samlet set vores system giver et værktøj til at generere menneskelige glutamaterge neuroner for forskere at studere udviklingen af disse neuroner og de sygdomme, der påvirker dem.
Introduction
Humane pluripotente stamceller (PSC), herunder både humane embryonale stamceller (hESCs) og induceret pluripotente stamceller (iPSCs), har kapacitet til at generere alle celletyper i kroppen, herunder neuroner 1-3. Instrueret differentiering af forskellige neuronale undertyper fra humane PSC har nøglen for anvendelse af disse celler i regenerativ medicin. Den generation af funktionelle neuronale undertyper under udvikling er en kompleks proces, der involverer induktion af neurale afstamning, specifikationen af regionale progenitorer langs Rostro-caudale akse, og differentieringen af post-mitotiske neuron typer fra de regionale progenitorer 4,5. Begyndende i 2001, har flere systemer blevet oprettet for at generere neurale afstamning fra hESCs, som har ydet en platform for den efterfølgende generation af neuronale undertyper 6,7. Baseret på udviklingsmæssige principper, flere neuron typer såsom spinal motor neuroner 8-12, midthjernen DOPaminerge neuroner 13-15, og neurale nethinde celler 16,17 er effektivt angivet fra humane PSC. Dette blev gjort ved at anvende kritiske morfogener, som er vigtige for specifikationen af disse neuron typer under de vivo udvikling. Andre protokoller er også blevet udviklet til at fremme differentiering af hESCs til neuroner under anvendelse af enten yderligere faktorer 18-20, såsom små molekyler eller ved co-dyrkning med andre celletyper til at fremme differentiering 21.
Den menneskelige neocortex er højt udviklet og indeholder mange celletyper, herunder glutamaterge neuroner, som spiller en vigtig rolle i læring, hukommelse og kognitiv funktion 22,23. Det første trin i frembringelse glutamaterge neuroner i kultur er at specificere telencephalic progenitorceller. Yoshiki Sasai gruppe først rapporteret den styrede differentiering af telencephalic precursorer fra mus ESC (mESCs) under anvendelse af et serumfrit suspension kultur i nærvær af DKK1 (som inhiberer Wnt signalering) samt LeftyA (som inhiberer nodal signalering) 24. Efterfølgende har flere grupper, herunder vores rapporterede også specifikationen af telencephalic precursorer fra humane PSC'er i serumfrit medium 25-27. Frembringelsen af telencephalic precursorer fra humane PSC kræver ikke anvendelse af exogene morfogener og effektiviteten i at generere disse prækursorer er meget højere end den fra mESCs 26,27. Her har en kemisk defineret system for neural induktion der var veletableret af Zhang gruppe 7 blevet beskrevet. Uden tilsætning af exogene caudalizing faktorer, effektivt denne protokol genererer telencephalic precursorer fra humane PSC 27. Disse progenitorer kan derefter opdeles i dorsale eller ventrale progenitorer ved at regulere signalering af Wnt og sonic pindsvin (SHH). Den dorsale progenitorer kan yderligere differentiere sig til glutamaterge neuroner efficiently 27. Desuden denne protokol fungerer godt til dannelse af glutamaterge neuroner fra human iPSCs 28, som muliggør dannelsen af patientspecifikke neuroner, som kan anvendes til at undersøge virkningsmekanismen samt potentielle terapier for en lang række sygdomme . Desuden vores system giver også en platform til at undersøge mulighederne for udvikling og specifikation af diverse neuronale typer i telencephalon.
Protocol
Representative Results
Discussion
Der er flere kritiske trin i den neurale differentieringsprocessen. Det er vigtigt at sikre, at de humane PSC er pluripotente for ellers cellerne allerede kan være forspændt mod at blive en ikke-neuronal afstamning. Dette kan bekræftes ved farvning af humane PSC'er med antistoffer mod pluripotency markører, såsom Oct4, Sox2, Nanog, og Tra-1-60 1-3. Hvis de humane PSC'er ikke lægger meget godt efter passage dem, kan ROCK inhibitor (Y27632) tilsættes for at hjælpe. For dem har problemer med at h…
Declarações
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Dr. Y. Sasai for gavmildt at give den FOXG1 antistof. Dette arbejde blev støttet af Connecticut Stamcelleforskning Tilskud (08-SCB-UCHC- 022 og 11-SCB24) og Spastisk Paraplegi Foundation.
Materials
| Reagent | Supplier | Catalog # |
| Dulbecco’s modified eagle medium with F12 nutrient mixture (DMEM/F12) | Gibco | 11330-032 |
| Knockout Serum Replacer | Gibco | 10828-028 |
| L-glutamine (200 mM) | Gibco | 25030 |
| Non Essential Amino Acids | Gibco | 1140-050 |
| 2-Mercaptoethanol (14.3 M) | Sigma | M-7522 |
| Neurobasal medium | Gibco | 21103-049 |
| N2 | Gibco | 17502-048 |
| B27 | Gibco | 12587-010 |
| Heparin | Sigma | H3149 |
| Poly-L-ornithine hydrobromide (polyornithine) | Sigma | 116K5103 |
| Laminin (human) | Sigma | L-6274 |
| Laminin (mouse) | Invitrogen | 23017-015 |
| FBS | Gemini | 100-106 |
| Bovine serum albumin (BSA) | Sigma | A-7906 |
| Dispase | Gibco | 17105-041 |
| Collagenase | Invitrogen | 17104-019 |
| Accutase | Innovative Cell Technologies | AT104 |
| ROCK Inhibitor | Stemgent | 04-0012 |
| SB431542 | Stemgent | 04-0010 |
| Dorsomorphin | Stemgent | 04-0024 |
| Fibroblast growth factor 2 (FGF2, bFGF) | Invitrogen | 13256-029 |
| Trypsin inhibitor | Gibco | 17075 |
| 0.1% gelatin | Millipore | ES-006-B |
| Foxg1 antibody | Dr. Y. Sasai | |
| Hoxb4 antibody (1:50) | Developmental Studies Hybridoma Bank | I12 |
| Pax6 antibody (1:5000) | Developmental Studies Hybridoma Bank | PAX6 |
| Nkx2.1 antibody (1:200) | Chemicon | MAB5460 |
| Tbr1 antibody (1:2000) | Chemicon | AB9616 |
| vGLUT1 antibody (1:100) | Synaptic Systems | 135302 |
| Brain derived neurotrophic factor (BDNF) | PrepoTech Inc. | 450-02 |
| Glial derived neurotrophic factor (GDNF) | PrepoTech Inc. | 450-10 |
| Insulin growth factor 1 (IGF1) | PrepoTech Inc. | 100-11 |
| Cyclic AMP (cAMP) | Sigma | D-0260 |
| Sonic hedgehog (SHH) | R&D | 1845-SH |
| 50 ml tubes | Becton Dickinson (BD) | 352098 |
| 15 ml tubes | BD | 352097 |
| 6 well plates | BD | 353046 |
| 24 well plates | BD | 353047 |
| T25 flasks (untreated) | BD | 353009 |
| T75 flasks (untreated) | BD | 353133 |
| Coverslips | Chemiglass Life Sciences | 1760-012 |
| 6 cm Petri dishes | BD | 353004 |
| 9” glass pipetes | Fisher | 13-678-20D |
| Steriflip filters (0.22 μM) | Millipore | SCGP00525 |
| Stericup filters 1,000 ml (0.22 μM) | Millipore | SCGPU10RE |
| Phase contrast microscope (Observer A1) | Zeiss | R2625 |
| Carbon dioxide incubator (Hera Cell 150) | Thermo Electron Corporation | |
| Biosafety hood (Sterilgard III Advance) | The Baker Company | |
| Centrifuge (5702 R) | Eppendorf |
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