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 manuskriptet er dedikert til minne om Dr. Wenlan Wang som døde 26. mai 2011. Vi takker Dr Douglas A. Kerr for sjenerøst gi HBG3 MES cellene brukt i denne studien. Dette arbeidet ble finansiert av Nemours, en bevilgning (2 RR016472-10) under INBRE program National Center for Research Resources (NCRR), og en Cobre Grant Award fra NCRR (5 P20 RR020173-05) å støtte Center for Pediatric Forskning ved Alfred I. duPont Hospital for Children, Wilmington, Delaware, USA.

1. Trinn 1: Mouse Embryonic Stem (MES) Cell Culture (Timing: 3 dager) 1. Kontaktflate primære embryonale mus fibroblaster (PMEF) <ol><li> Coat fire 100-mm vevskulturstudier retter med gelatin for PMEF vedheft. Legg 8 ml av 0,1% gelatin løsning (StemCell Technologies) til hver rett og Inkuber i 30 min ved romtemperatur. </li><li> Fjern overflødig gelatin fra retter. Ikke skyll oppvasken. </li><li> Fortynn en ampulle med mitomycin C-inaktivert PMEF (Millipore) som inneholder ca. 5 x 10 ...

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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 av MES celler er den mest kritiske parameteren for effektiv produksjon av motoriske nerveceller. MES cellene må dyrkes på PMEF å hindre spontan differensiering. Tilsetting av LIF bidrar til å opprettholde beskjeden cellene i en udifferensiert tilstand. Som MES celler deler hver 12-15 timer, kulturen medium blir utarmet raskt og må skiftes daglig. Effektiv aktivering av Hysj veien er en kritisk parameter for å indusere motor neuron spesifikasjonen. Hysj protein (R &amp; D Sys...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Endelig konsentrasjon </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-Kvalifisert 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-essensielle 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 Differensiering 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-essensielle aminosyrer </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Mono-Thio glyserol </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> MND medium (differensiering) </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 erstatning </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> Askorbinsyre </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-glukose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% i d 2 H 2 O </td></tr><tr><td> Fibronectin </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 mikrogram / ml </td></tr><tr><td> Heparin </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 mikrogram / ​​ml i d 2 H 2 O </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 mM </td></tr><tr><td> SAG </td><td> EMD Kjemikalier </td><td> 566660 </td><td> 1 mM </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 erstatning </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> Askorbinsyre </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-glukose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% i d 2 H 2 O </td></tr><tr><td> Fibronectin </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 mikrogram / ml </td></tr><tr><td> Heparin </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 mikrogram / ​​ml i d 2 H 2 O </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 = Non-aktuelt. Tabell 1. PMEF og media for MES cellekultur eller differensiering. <table border="1"><tbody><tr><td> &lt;strong&gt; Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Endelig konsentrasjon </td></tr><tr><td> 0,1% Gelatin </td><td> StemCell Technologies </td><td> 07903 </td><td> NA </td></tr><tr><td> Poly-DL-ornithine </td><td> Sigma-Aldrich </td><td> P0421 </td><td> 0,1 mg / ml i d 2 H 2 O </td></tr><tr><td> Mus laminin </td><td> Millipore </td><td> CC095 </td><td> 2 mikrogram / 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 = Non-aktuelt. Tabell 2. Reagenser for belegg og dissosiasjon.

efficient differentiation of mouse embryonic stem cells into motor neurons

Direkte differensiering av embryonale stamceller (ES) celler i funksjonelle motoriske nevroner representerer en lovende ressurs for å studere sykdomsmekanismer, for å skjerme nye stoffet forbindelser, og å utvikle nye behandlingsmetoder for motor neuron sykdommer som spinal muskelatrofi (SMA) og amyotrofisk lateral sklerose ( ALS). Mange nåværende protokoller bruke en kombinasjon av retinsyre (RA) og sonic hedgehog (Hysj) for å skille mus embryonale stamceller (MES) celler inn i motor nevroner 1-4. Imidlertid har differensiering effektiviteten av MES celler i motor nevroner bare møtt med moderat suksess. Vi har utviklet et to-trinns differensiering protokoll 5 som vesentlig forbedrer differensiering effektivitet sammenlignet med for tiden etablerte protokoller. Det første trinnet er å forbedre neuralization prosessen ved å legge Noggin og fibroblast vekstfaktorer (FGFs). Noggin er en Benmorfogenetiske protein (BMP) antagonist og er innblandet i nevrale induksjon enccording til standard modell av neurogenesis og resulterer i dannelse av fremre nevrale mønster seks. FGF-signalering virker synergistisk med Noggin i inducing nevrale vev dannelse ved å fremme en posterior neural identitet 7-9. I dette trinnet, ble MES celler primet med Noggin, bFGF, og FGF-8 for to dager for å fremme differensiering mot nevrale linjene. Det andre trinnet er å indusere motor neuron spesifikasjonen. Noggin / FGFs utsatte MES celler ble inkubert med RA og en Shh agonist, glattes agonist (SAG) for ytterligere 5 dager for å lette motoriske nervecellen generasjon. For å overvåke differensiering av rotet til motoriske nevroner, brukte vi en ES celle linje stammer fra en transgen mus som uttrykker eGFP under kontroll av den motoriske nervecellen spesifikk promoter Hb9 en. Ved hjelp av denne robuste protokollen, oppnådde vi 51 ± 0,8% av differensiering effektivitet (n = 3, p &lt;0,01, Student t-test) 5. Resultater fra immunofluorescentfarging viste at GFP + celler uttrykker de motoriske nervecellen spesifikke markører, Islet-1 og kolin acetyltransferase (chat). Vår to-trinns differensiering protokollen gir en effektiv måte å skille MES celler i spinal motoriske nerveceller.

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 utviklet en ny protokoll for å forbedre effektiviteten av in vitro differensiering av mus embryonale stamceller inn i motoriske nevroner. De differensierte ES celler kjøpt motor nevroner har bevist ved uttrykk av nevrale og motor neuron markører med immunhistokjemiske teknikker.

Effektiv Differensiering av mus embryonale stamceller i motoriske nevroner

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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