Differentiering og karakterisering af neurale forfædre og neuroner fra museembryst stamceller
Summary
Vi beskriver proceduren for in vitro differentiering af mus embryonale stamceller i neuronale celler ved hjælp af hængende dråbe metode. Derudover udfører vi en omfattende fænotypisk analyse gennem RT-qPCR, immunofluorescence, RNA-seq og flowcytometri.
Abstract
Vi beskriver den trinvise procedure for dyrkning og differentiering af museembrystiske stamceller i neuronale slægter, efterfulgt af en række analyser for at karakterisere de differentierede celler. E14 mus embryonale stamceller blev brugt til at danne embryonale organer gennem hængende drop metode, og derefter induceret til at differentiere sig til neurale stamfader celler ved retinsyre, og endelig differentieret i neuroner. Kvantitative reverse transskription polymerase kædereaktion (RT-qPCR) og immunofluorescence eksperimenter viste, at de neurale forfædre og neuroner udviser tilsvarende markører (nestin for neurale forfædre og neurofilament for neuroner) på dag 8 og 12 post-differentiering, henholdsvis. Flow cytometri eksperimenter på en E14 linje udtrykker en Sox1 promotor-drevet GFP reporter viste, at omkring 60% af cellerne på dag 8 er GFP positive, hvilket indikerer en vellykket differentiering af neurale stamfader celler på dette stadium. Endelig blev RNA-seq-analysen brugt til at profilere de globale transskriptomiske ændringer. Disse metoder er nyttige til at analysere inddragelsen af specifikke gener og veje i reguleringen af celleidentitetsovergangen under neuronal differentiering.
Introduction
Siden deres første afledning fra den indre cellemasse af de udviklende muse blastocyster1,2, mus embryonale stamceller (mESC) er blevet brugt som kraftfulde værktøjer til at studere stamcelle selvfornyelse og differentiering3. Desuden fører undersøgelse af mESC-differentiering til en enorm forståelse af molekylære mekanismer, der kan forbedre effektiviteten og sikkerheden i stamcellebaseret terapi til behandling af sygdomme som neurodegenerative lidelser4. Sammenlignet med dyremodeller giver dette in vitro-system mange fordele, herunder enkelhed i praksis og vurdering, lave omkostninger ved at opretholde cellelinjer i modsætning til dyr og relativ lethed i genetiske manipulationer. Effektiviteten og kvaliteten af differentierede celletyper påvirkes dog ofte af forskellige linjer af mESC’er samt differentieringsmetoderne5,6. Desuden er de traditionelle analyser til evaluering af differentieringseffektivitet afhængige af kvalitativ undersøgelse af udvalgte markørgener, som mangler robusthed, og de forstår derfor ikke globale ændringer i genekspression.
Her sigter vi mod at bruge et batteri af analyser til systematisk vurdering af den neuronale differentiering. Ved hjælp af både traditionelle in vitro-analyser på udvalgte markører og RNA-seq etablerer vi en platform til måling af differentieringseffektiviteten samt de transskriptomiske ændringer under denne proces. Baseret på en tidligere etableret protokol7genererede vi embryonale organer (EBs) gennem den hængende dråbeteknik, efterfulgt af induktion ved hjælp af suprafysiologisk mængde retinsyre (RA) til at generere neurale stamfaderceller (NPC’er), som efterfølgende blev differentieret til neuroner med neural induktion medium. For at undersøge effektiviteten af differentiering, ud over traditionelle RT-qPCR og immunofluorescence (IF) assays, vi udførte RNA-seq og flow cytometri. Disse analyser giver en omfattende måling af udviklingen af den fasespecifikke differentiering.
Protocol
Representative Results
Discussion
Metoden til neural differentiering af museememinnale stamceller er blevet etableret i årtier , og forskere har fortsat med at ændre de tidligere protokoller eller skabe nye til forskellige formål7,10,11. Vi brugte en række analyser til omfattende at analysere effektiviteten og fremskridtene af differentieringsstadierne af mESCs til neuroner, som kan bruges til analyse af andre slægtsdifferentiering af mus eller menneskelige…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af et tilskud fra NIH (1R35GM133496-01) til Z. Gao. Vi vil gerne takke Dr. Ryan Hobbs for hjælpen i afsnittet. Vi takker Penn State College of Medicine’s kernefaciliteter, herunder Genom Sciences og Bioinformatik, Advanced Light Microscopy Imaging, og Flow Cytometri. Vi takker også Dr. Yuka Imamura for hjælpen i RNA-seq analyse.
Materials
| 0.05% Trypsin + 0.53mM EDTA 1X | Corning | 25-052-CV | |
| 0.1% Gelatin | Sigma | G1890-100G | Prepared in de-ionized water |
| 16% Paraformaldehyde | Thermo Scientific | 28908 | Diluted in 1X PBS |
| 40-μm cell strainer | Falcon | 352340 | |
| Albumax | Thermo Fisher Scientific | 11020021 | |
| AlexaFluor 488 goat anti-mouse IgG (H+L) | Invitrogen | A11001 | Antibody was diluted at 1:500 for IF |
| Alkaline Phosphatase Staining Kit II | Stemgent | 00-0055 | |
| AzuraQuant Green Fast qPCR Mix LoRox | Azura Genomics | AZ-2105 | |
| B27 supplement | Thermo Fisher Scientific | 17504044 | |
| BD FACSCanto | BD | 657338 | |
| bFGF | Sigma | 11123149001 | |
| BioAnalyzer High Sensitivity DNA Kit | Agilent | 5067-4626 | |
| Chir99021 | Cayman Chemicals | 13122 | |
| Chloroform | C298-500 | Fisher Chemical | |
| DAPI | Invitrogen | R37606 | |
| DMEM | Corning | 10-017-CM | |
| DMEM/F12 medium | Thermo Fisher Scientific | 11320033 | |
| EB buffer | Qiagen | 19086 | |
| Ethanol | 111000200 | Pharmco | Diluted in de-ionized water |
| Fetal bovine serum | Atlanta Biologicals | S10250 | |
| Fisherbrand Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
| HiSeq 2500 Sequencing System | Illumina | SY-401-2501 | |
| Isopropanol | BDH1133-4LG | BDH VWR Analytical | Diluted in de-ionized water |
| L-glutamine | Thermo Fisher Scientific | 25030024 | |
| LIF | N/A | N/A | Collected from MEF supernatant |
| m18srRNA primers | IDTDNA | N/A | 5'-GCAATTATTCCCCATGAACG-3' 5'-GGCCTCACTAAACCATCCAA-3' |
| MEM Non-essential amino acids | Corning | 25-025-Cl | |
| mNanog primers | IDTDNA | N/A | 5'-AGGCTTTGGAGACAGTGAGGTG-3' 5'-TGGGTAAGGGTGTTCAAGCACT-3' |
| mNes primers | IDTDNA | N/A | 5'-AGTGCCCAGTTCTAGTGGTGTCC-3' 5'-CCTCTAAAATAGAGTGGTGAGGGTTG-3' |
| mNeuroD1 primers | IDTDNA | N/A | 5'-CGAGTCATGAGTGCCCAGCTTA-3' 5'-CCGGGAATAGTGAAACTGACGTG-3' |
| mOct4 primers | IDTDNA | N/A | 5'-AGATCACTCACATCGCCAATCA-3' 5'-CGCCGGTTACAGAACCATACTC-3' |
| mPax6 primers | IDTDNA | N/A | 5'-CTTGGGAAATCCGAGACAGA-3' 5'-CTAGCCAGGTTGCGAAGAAC-3' |
| N2 supplement | Thermo Fisher Scientific | 17502048 | |
| Nestin primary antibody | Millipore | MAB5326 | Antibody was diluted at 1:200 for IF |
| Neural basal | Thermo Fisher Scientific | 21103049 | |
| Neurofilament primary antibody | DSHB | 2H3 | |
| NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit | BioO Scientific | NOVA-5138-07 | |
| PD0325901 | Cayman Chemicals | 13034 | |
| Penicillin/streptomycin | Corning | 30-002-Cl | |
| Phosphate-buffered saline (PBS) | N/A | N/A | Prepared in de-ionized water |
| – Potassium chloride | P217-500G | VWR | |
| – Potassium phosphate monobasic anhydrous | 0781-500G | VWR | |
| – Sodium chloride | BP358-10 | Fisher Bioreagents | |
| – Sodium phosphate, dibasic, heptahydrate | SX0715-1 | Milipore | |
| Random hexamer primer | Thermo Scientific | SO142 | |
| Retinoic acid | Sigma | R2625 | Prepared in DMSO |
| Sodium pyruvate | Corning | 25-000-Cl | |
| Sucrose | Sigma | 84097 | Diluted in 1X PBS |
| SuperScript III Reverse Transcriptase | Invitrogen | 18064022 | |
| Tissue-Tek O.C.T. compound | Sakura | 4583 | |
| TriPure Isolation Reagent | Sigma-Aldrich | 11667165001 | |
| TruSeq Rapid | Illumina | 20020616 | |
| β-mercaptoethanol | Fisher BioReagents | BP176-100 |
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