Name: efficient differentiation of mouse embryonic stem cells into motor neurons
Uploaded: 2012-06-09T14:00:00
Description: Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Dette håndskrift er dedikeret til mindet om Dr. Wenlan Wang, der gik bort den 26. maj 2011. Vi takker Dr. Douglas A. Kerr for generøst tilvejebringe de HBG3 MES celler anvendt i denne undersøgelse. Dette arbejde blev finansieret af Nemours, et tilskud (2 RR016472-10) under INBRE program National Center for Research Resources (NCRR), og en Cobre Grant Award fra NCRR (5 P20 RR020173-05) til støtte for Center for Pediatric Forskning på Alfred I. DuPont Hospital for Children, Wilmington, Delaware, USA.

1. Trin 1: Mouse embryonale stamceller (MES) Cell Culture (Timing: 3 dage) 1. Udpladning primære embryoniske musefibroblaster (PMEF) <ol><li> Coat fire 100 mm vævskulturskåle med gelatine til PMEF adhæsion. Tilsættes 8 ml 0,1% gelatine-opløsning (StemCell Technologies) til hver skål, og inkuberes i 30 minutter ved stuetemperatur. </li><li> Fjern overskydende gelatine fra retter. Må ikke skylle opvasken. </li><li> Fortyndes et hætteglas af mitomycin C-inaktiveret PMEF (Millipore), der...

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	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+motoneurons+derived+from+mouse+embryonic+stem+cells.">Functional properties of motoneurons derived from mouse embryonic stem cells.</a> J. Neurosci. 24, 7848-7858 (2004).</li><li>Wichterle, H., Peljto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+to+spinal+motor+neurons.">Differentiation of mouse embryonic stem cells to spinal motor neurons.</a> Curr. Protoc. Stem Cell. Biol. Chapter 1, Unit 1H.1.1-Unit 1H.1.9 (2008).</li><li>Kiris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem Cell Res. 6, 195-205 (2011).</li><li>Wu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+assessment+of+a+cell+model+of+spinal+muscular+atrophy.">Proteomic assessment of a cell model of spinal muscular atrophy.</a> BMC Neurosci. 12, 25 (2011).</li><li>McMahon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin-mediated+antagonism+of+BMP+signaling+is+required+for+growth+and+patterning+of+the+neural+tube+and+somite.">Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite.</a> Genes Dev. 12, 1438-1452 (1998).</li><li>Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+posterior+neural+tissue+is+induced+by+FGF+in+the+chick+embryo.">Early posterior neural tissue is induced by FGF in the chick embryo.</a> Development. 125, 473-484 (1998).</li><li>Launay, C., Fromentoux, V., Shi, D. L., Boucaut, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+FGF+receptor+blocks+neural+induction+by+endogenous+Xenopus+inducers.">A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.</a> Development. 122, 869-880 (1996).</li><li>Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purmorphamine+activates+the+Hedgehog+pathway+by+targeting+Smoothened.">Purmorphamine activates the Hedgehog pathway by targeting Smoothened.</a> Nat. Chem. Biol. 2, 29-30 (2006).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14071-14076 (2002).</li><li>Lee, S. M., Danielian, P. S., Fritzsch, B., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+FGF8+signaling+from+the+midbrain-hindbrain+junction+regulates+growth+and+polarity+in+the+developing+midbrain.">Evidence that FGF8 signaling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.</a> Development. 124, 959-969 (1997).</li></ol>

Kvaliteten af ​​MES-celler er den mest kritiske parameter for effektiv produktion af motorneuroner. MES-celler skal dyrkes på PMEF at forhindre spontan differentiering. Tilsætningen af ​​LIF hjælper med at bevare MES cellerne i en udifferentieret tilstand. Som MES cellerne deler hver 12-15 timer, dyrkningsmediet bliver nedbrydes hurtigt og skal udskiftes dagligt. Effektiv aktivering af Shh pathway er en kritisk parameter for at inducere motorneuroner specifikation. Shh-protein (R ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Slutkoncentration </td></tr><tr><td> PMEF </td><td> Millipore </td><td> PMEF-H </td><td> NA </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF medium </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965-118 </td><td> NA </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16140-071 </td><td> 10% </td></tr><tr><td> L-glutamin </td><td> Invitrogen </td><td> 25030-081 </td><td> 1% </td></tr><tr><td> Penicillin / streptomycin </td><td> Invitrogen </td><td> 15240-063 </td><td> 1% </td></tr><tr><td></td><td></td&gt; <td></td><td></td></tr><tr><td> MES medium </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cell-Kvalificeret FBS </td><td> Invitrogen </td><td> 16141-079 </td><td> 15% </td></tr><tr><td> Glutamax-I </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> Ikke-essentielle aminosyrer </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Nukleosider </td><td> Millipore </td><td> ES-008-D </td><td> 1% </td></tr><tr><td> β-mercaptoethanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Penicillin / streptomycin </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> LIF </td><td> Millipore </td><td&gt; LIF2010 </td><td> 10 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Neural differentiering medium </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cult FBS </td><td> StemCell Technologies </td><td> 06905 </td><td> 15% </td></tr><tr><td> Ikke-essentielle aminosyrer </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Mono-thio glycerol </td><td> Sigma-Aldrich </td><td> M-6145 </td><td> 1mM </td></tr><tr><td> Noggin </td><td> Invitrogen </td><td> PHC1506 </td><td> 50 ng / ml </td></tr><tr><td> FGF-8 </td><td> Invitrogen </td><td> PHG0274 </td><td> 20 ng / ml </td></tr><tr><td> bFGF </td><td> Invitrogen </td><td> PHG0024 </td><td> 20 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> ALS medium (differentiering) </td><td></td><td></td><td></td></tr><tr><td> ES-Cult Basal Medium-A </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Knockout serum udskiftning </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 supplement </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> ITS Supplement-B </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Ascorbinsyre </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> Penicillin / streptomycin </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> Glutamax-I </td><td>Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-glucose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% i D 2 H2O </td></tr><tr><td> Fibronektin </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 ug / ml </td></tr><tr><td> Heparin </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml i d 2 H2O </td></tr><tr><td> β-mercaptoethanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Retinsyre </td><td> Sigma-Aldrich </td><td> R-2625 </td><td> 1 uM </td></tr><tr><td> SAG </td><td> EMD Kemikalier </td><td> 566660 </td><td> 1 uM </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND medium (Motor Neuron kultur) </td><td></td><td></td><td></td></tr><tr><td> ES-Cult Basal Medium-A </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Knockout serum udskiftning </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 supplement </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> ITS Supplement-B </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Ascorbinsyre </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> Penicillin / streptomycin </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> Glutamax-I </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-glucose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% i D 2 H2O </td></tr><tr><td> Fibronektin </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 ug / ml </td></tr><tr><td> Heparin </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml i d 2 H2O </td></tr><tr><td> β-mercaptoethanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> BDNF </td><td> R &amp; D Systems </td><td> 248-BD-005/CF </td><td> 10 ng / ml </td></tr><tr><td> CNTF </td><td> R &amp; D Systems </td><td> 257-NY-010/CF </td><td> 10 ng / ml </td></tr><tr><td> GDNF </td><td> R &amp; D Systems </td><td> 212-GD-010/CF </td><td> 10 ng / ml </td></tr><tr><td> NT-3 </td><td> R &amp; D Systems </td><td> 267-N3-005/CF </td><td> 10 ng / ml </td></tr></tbody></table> NA = ikke-gældende. Tabel 1. PMEF og Medier for MES cellekultur eller differentiering. <table border="1"><tbody><tr><td> &lt;strong&gt; Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Slutkoncentration </td></tr><tr><td> 0,1% gelatine </td><td> StemCell Technologies </td><td> 07903 </td><td> NA </td></tr><tr><td> Poly-DL-ornithin </td><td> Sigma-Aldrich </td><td> P0421 </td><td> 0,1 mg / ml i d 2 H2O </td></tr><tr><td> Muselaminin </td><td> Millipore </td><td> CC095 </td><td> 2 ug / ml i PBS </td></tr><tr><td> 0,25% Trypsin / EDTA </td><td> StemCell Technologies </td><td> 07901 </td><td> NA </td></tr><tr><td> Accumax </td><td> Millipore </td><td> SCR006 </td><td> NA </td></tr></tbody></table> NA = ikke-gældende. Tabel 2 nedenfor. Reagenser til coating og dissociation.

efficient differentiation of mouse embryonic stem cells into motor neurons

Direkte differentiering af embryoniske stamceller (ES)-celler i funktionelle motorneuroner repræsenterer en lovende ressource til at studere sygdomsmekanismer, at screene nye lægemiddelforbindelser, og at udvikle nye terapier for motoriske neuronsygdomme, såsom spinal muskulær atrofi (SMA) og amyotrofisk lateral sklerose ( ALS). Mange nuværende protokoller anvender en kombination af retinsyre (RA) og sonisk hedgehog (Shh) til at differentiere muse embryonale stamceller (MES)-celler i motorneuroner 1-4. Imidlertid har differentiering effektiviteten af ​​MES celler i motorneuroner kun mødt med begrænset succes. Vi har udviklet et to-trins differentiering protokol 5, der væsentligt forbedrer differentiering effektivitet sammenlignet med i dag etablerede protokoller. Det første trin er at forbedre neuralization processen ved tilsætning af noggin og fibroblastvækstfaktorer (FGF&#39;er). Noggin er et knoglemorfogenetisk protein (BMP)-antagonist og er impliceret i neural induktion enccording til standard model neurogenese og resulterer i dannelsen af anterior neural mønsterdannelse 6. FGF signalering virker synergistisk med Noggin i at fremkalde nervevæv dannelsen ved at fremme en posterior neural identitet 7-9. I dette trin blev MES celler primet med noggin, bFGF og FGF-8 i to dage for at fremme differentiering til neurale cellelinier. Det andet skridt er at fremkalde motorisk neuron specifikation. Noggin / FGF&#39;er eksponerede MES-celler blev inkuberet med RA og Shh-agonist, Smoothened agonist (SAG) i yderligere 5 dage for at lette motorneuroner generation. At overvåge differentiering af masse ved motoriske neuroner, anvendte vi en ES-cellelinie afledt fra en transgen mus, der udtrykker EGFP under kontrol af motorneuroner promotor Hb9 1. Anvendelse af denne robuste protokol, opnåede vi 51 ± 0,8% af differentiering virkningsgrad (n = 3, p &lt;0,01, Student t-test) 5. Resultaterne fra immunofluorescensfarvning viste, at GFP +-celler udtrykker motorneuroner specifikke markører, ø-1 og cholinacetyltransferase (ChAT). Vores to-trins differentiering protokol giver en effektiv måde at differentiere MES celler i spinale motoriske neuroner.

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Vi har udviklet en ny protokol for at forbedre effektiviteten af ​​in vitro differentiering af muse embryonale stamceller i motoriske neuroner. De differentierede ES-celler opnået motoriske neuroner har som vist ved ekspressionen af ​​neuronale og motorisk neuron markører under anvendelse immunohistokemiske teknikker.

Effektiv differentiering af stamceller fra mus i Motoriske neuroner

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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