Name: differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells
Uploaded: 2020-05-15T15:00:00
Description: Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

Dit werk werd ondersteund door een subsidie van de NIH (1R35GM133496-01) aan Z. Gao. We willen Dr. Ryan Hobbs bedanken voor de hulp bij het sectieren. We bedanken de kernfaciliteiten van Penn State College of Medicine, waaronder de Genome Sciences en Bioinformatics, de Advanced Light Microscopy Imaging en de Flow Cytometry. We bedanken ook Dr. Yuka Imamura voor de hulp bij RNA-seq analyse.

1. mESC cultuur
<ol><li>Bestrijk een met weefselkweek behandelde plaat van 10 cm met 0,1% gelatine en laat de gelatine minstens 15-30 minuten uitharden voordat u deze opzuigt.</li>
	<li>Zaad γ doorstraalde embryonale fibroblasten van muizen (MEF's) één dag voordat de mESCs worden gekweekt in het voorverwarmde mESC-medium (Dulbecco's gemodificeerd Eagle-medium (DMEM) met 15% foetaal runderserum (FBS), niet-essentiële aminozuren, β-mercaptoethanol, L-glutamine, penicilline / streptomycine, natriumpyruvaat, LIF, PD03...

<ol>
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De methode voor neurale differentiatie van embryonale stamcellen van muizen is al tientallen jaren vastgesteld en onderzoekers zijn doorgegaan met het wijzigen van de vorige protocollen of het creëren van nieuwe protocollen voor verschillende doeleinden7,10,11. We gebruikten een reeks assays om de efficiëntie en voortgang van de differentiatiestadia van mESCs naar neuronen uitgebreid te analyseren, die kunnen worden gebruikt b...

Sinds hun eerste afleiding van de binnenste celmassa van de zich ontwikkelende muizenblastocysten1,2,zijn embryonale stamcellen van muizen (mESC) gebruikt als krachtige hulpmiddelen om zelfvernieuwing en differentiatie van stamcellen te bestuderen3. Bovendien leidt het bestuderen van mESC-differentiatie tot een enorm begrip van moleculaire mechanismen die de efficiëntie en veiligheid in op stamcellen gebaseerde therapie bij de behandeling van ziekten zoals neurodegeneratieve aandoeningen kunnen verbeteren4. In vergelijking met diermodellen biedt dit in vitro systeem veel voordelen, waaronder eenvoud in praktijk en beoordeling, lage kosten bij het onderhouden van cellijnen in tegenstelling tot dieren en relatief gemak bij genetische manipulaties. De efficiëntie en kwaliteit van gedifferentieerde celtypen worden echter vaak beïnvloed door verschillende lijnen van mESCs en de differentiatiemethoden5,6. Ook zijn de traditionele assays om de efficiëntie van differentiatie te evalueren afhankelijk van kwalitatief onderzoek van geselecteerde markergenen die geen robuustheid hebben en daarom geen wereldwijde veranderingen in genexpressie begrijpen.
Hier willen we een batterij testen gebruiken voor systematische beoordeling van de neuronale differentiatie. Met behulp van zowel traditionele in vitro analyses op geselecteerde markers als RNA-seq, zetten we een platform op voor het meten van de differentiatie-efficiëntie en de transcriptomische veranderingen tijdens dit proces. Op basis van een eerder vastgesteld protocol7genereerden we embryoïde lichamen (EB's) via de hanging drop-techniek, gevolgd door inductie met behulp van suprafysiologische hoeveelheid retinoïnezuur (RA) om neurale voorlopercellen (NPC's) te genereren, die vervolgens werden gedifferentieerd naar neuronen met neuraal inductiemedium. Om de efficiëntie van de differentiatie te onderzoeken, voerden we, naast traditionele RT-qPCR- en immunofluorescentie (IF) -assays, RNA-seq- en flowcytometrie uit. Deze analyses bieden uitgebreide meting van de progressie van de stadiumspecifieke differentiatie.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.05% Trypsin + 0.53mM EDTA 1X</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-052-CV</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.1% Gelatin</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">G1890-100G</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">16% Paraformaldehyde</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">28908</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">40-&#956;m cell strainer</td>
			<td style="width:148px;">Falcon</td>
			<td style="width:127px;">352340</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Albumax</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11020021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">AlexaFluor 488 goat anti-mouse IgG (H+L)</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">A11001</td>
			<td>Antibody was diluted at 1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Alkaline Phosphatase Staining Kit II</td>
			<td style="width:148px;">Stemgent</td>
			<td style="width:127px;">00-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">AzuraQuant Green Fast qPCR Mix LoRox</td>
			<td style="width:148px;">Azura Genomics</td>
			<td style="width:127px;">AZ-2105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">B27 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BD FACSCanto</td>
			<td style="width:148px;">BD</td>
			<td style="width:127px;">657338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">bFGF</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">11123149001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BioAnalyzer High Sensitivity DNA Kit</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:127px;">5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chir99021</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chloroform</td>
			<td style="width:148px;">C298-500</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DAPI</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">R37606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DMEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">DMEM/F12 medium</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">EB buffer</td>
			<td style="width:148px;">Qiagen</td>
			<td style="width:127px;">19086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethanol</td>
			<td style="width:148px;">111000200</td>
			<td style="width:127px;">Pharmco</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Fetal bovine serum</td>
			<td style="width:148px;">Atlanta Biologicals</td>
			<td style="width:127px;">S10250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Fisherbrand Superfrost Plus Microscope Slides</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">HiSeq 2500 Sequencing System</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">SY-401-2501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Isopropanol</td>
			<td style="width:148px;">BDH1133-4LG</td>
			<td style="width:127px;">BDH VWR Analytical</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">L-glutamine</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">25030024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">LIF</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Collected from MEF supernatant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">m18srRNA primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-GCAATTATTCCCCATGAACG-3&#39; 
			5&#39;-GGCCTCACTAAACCATCCAA-3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MEM Non-essential amino acids</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mNanog primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGGCTTTGGAGACAGTGAGGTG-3&#39; 
			5&#39;-TGGGTAAGGGTGTTCAAGCACT-3&#39;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">mNes primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGTGCCCAGTTCTAGTGGTGTCC-3&#39; 
			5&#39;-CCTCTAAAATAGAGTGGTGAGGGTTG-3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">mNeuroD1 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CGAGTCATGAGTGCCCAGCTTA-3&#39; 
			5&#39;-CCGGGAATAGTGAAACTGACGTG-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mOct4 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGATCACTCACATCGCCAATCA-3&#39; 
			5&#39;-CGCCGGTTACAGAACCATACTC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mPax6 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CTTGGGAAATCCGAGACAGA-3&#39; 
			5&#39;-CTAGCCAGGTTGCGAAGAAC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">N2 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Nestin primary antibody</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:127px;">MAB5326</td>
			<td>Antibody was diluted at 1:200 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Neural basal</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">21103049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Neurofilament primary antibody</td>
			<td style="width:148px;">DSHB</td>
			<td style="width:127px;">2H3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit</td>
			<td style="width:148px;">BioO Scientific</td>
			<td style="width:127px;">NOVA-5138-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">PD0325901</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Penicillin/streptomycin</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">- Potassium chloride</td>
			<td style="width:148px;">P217-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Potassium phosphate monobasic anhydrous</td>
			<td style="width:148px;">0781-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium chloride</td>
			<td style="width:148px;">BP358-10</td>
			<td style="width:127px;">Fisher Bioreagents</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium phosphate, dibasic, heptahydrate</td>
			<td style="width:148px;">SX0715-1</td>
			<td style="width:127px;">Milipore</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Random hexamer primer</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">SO142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Retinoic acid</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">R2625</td>
			<td>Prepared in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium pyruvate</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-000-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sucrose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">84097</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">SuperScript III Reverse Transcriptase</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">18064022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:148px;">Sakura</td>
			<td style="width:127px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TriPure Isolation Reagent</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:127px;">11667165001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TruSeq Rapid</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">20020616</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">&#946;-mercaptoethanol</td>
			<td style="width:148px;">Fisher BioReagents</td>
			<td style="width:127px;">BP176-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells

We beschrijven de stapsgewijze procedure voor het kweken en differentiëren van embryonale stamcellen van muizen in neuronale afstammingslijnen, gevolgd door een reeks testen om de gedifferentieerde cellen te karakteriseren. De embryonale stamcellen van de E14-muis werden gebruikt om embryoïde lichamen te vormen via de hanging drop-methode en vervolgens geïnduceerd om te differentiëren in neurale voorlopercellen door retinoïnezuur en uiteindelijk gedifferentieerd in neuronen. Kwantitatieve reverse transcriptie polymerase kettingreactie (RT-qPCR) en immunofluorescentie-experimenten onthulden dat de neurale voorlopers en neuronen overeenkomstige markers vertonen (nestine voor neurale voorlopers en neurofilament voor neuronen) op respectievelijk dag 8 en 12 postdifferentiatie. Flowcytometrie-experimenten op een E14-lijn die een Sox1-promotorgestuurde GFP-verslaggever tot expressie bracht, toonden aan dat ongeveer 60% van de cellen op dag 8 GFP-positief is, wat wijst op de succesvolle differentiatie van neurale voorlopercellen in dit stadium. Ten slotte werd RNA-seq-analyse gebruikt om de wereldwijde transcriptomische veranderingen te profileren. Deze methoden zijn nuttig voor het analyseren van de betrokkenheid van specifieke genen en paden bij het reguleren van de celidentiteitstransitie tijdens neuronale differentiatie.

Als weergave van onze methode voerden we een EB-, NPC- en neurondifferentiatie-experiment uit op E14-cellen. E14-cellen werden gekweekt op γ bestraalde MEF's (figuur 1A) totdat de γ bestraalde MEF-populatie verdunde. We bevestigden de pluripotentie van de E14-cellen door alkalische fosfatase (AP) kleuring(Figuur 1B)en later RT-qPCR (zie hieronder) uit te voeren voor Nanog en Oct4 markers. De γ bestraalde MEF-vrije E14-cellen werden vervolgen...

Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We beschrijven de procedure voor de in vitro differentiatie van embryonale stamcellen van muizen in neuronale cellen met behulp van de hanging drop-methode. Verder voeren we een uitgebreide fenotypische analyse uit via RT-qPCR, immunofluorescentie, RNA-seq en flowcytometrie.

Differentiatie en karakterisering van neurale voorlopercellen en neuronen uit embryonale stamcellen van muizen

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Research

JoVE Journal

Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Neurale differentiatie van muis embryonale stamcellen in serum-vrije monolaagkweek

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Epigenetische regulatie van Cardiac Differentiatie van embryonale stamcellen en weefsels

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Regie van differentiatie van primitieve en definitieve hematopoietische progenitorcellen van menselijke pluripotente stamcellen

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficiënte Differentiatie van pluripotente stamcellen tot NKX6-1<sup&gt; +</sup&gt; Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Analyse van retinoïnezuur geïnduceerde neurale differentiatie van muis embryonale stamcellen in twee en drie-dimensionale embryoiden organen

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Snelle, gerichte differentiatie van de epitheliale cellen van de retinale Pigment van menselijke embryonale of geïnduceerde pluripotente stamcellen

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Isolatie, cultuur en differentiatie van beenmerg stromale cellen en Osteoclast voorlopercellen van muizen

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Generatie van geïnduceerde neurale stamcellen uit perifere mononucleaire cellen en differentiatie naar precursoren voor Dopaminerge neuron voor transplantatie studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Efficiënte Neurale differentiatie met behulp van eencellige cultuur van menselijke embryonale stamcellen

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Voorbereiding van kleine RNA-bibliotheken voor sequencing van vroege muisembryo's

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...