Name: directed dopaminergic neuron differentiation from human pluripotent stem cells
Uploaded: 2014-09-15T13:00:00
Description: Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

1. Herstellung von Nährmedien <ol><li> Bereiten embryonalen Maus-Fibroblasten (MEF) Medium durch die Kombination der folgenden: 445 ml DMEM, 50 ml fetales Rinderserum (FBS) und 5 ml 100x Penicillin / Ampicillin-Stammlösung. Halten sterilfiltriert Medium bei 4 ° C für nicht mehr als 14 Tage. </li><li> Bereiten serumhaltigem HPSC Kulturmedium durch die Kombination der folgenden: 385 ml DMEM / F12, 100 ml Knockout Serumersatz (KSR), 5 ml 100x nicht-essentielle Aminosäure-Stammlösung, 5 ml 100x Penicillin / Ampicill...

<ol>
	<li>Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Will+embryonic+stem+cells+be+a+useful+source+of+dopamine+neurons+for+transplant+into+patients+with+Parkinson's+disease.">Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 1755-1757 (2002).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 12543-12548 (2004).</li><li>Park, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+analyses+of+human+embryonic+stem+cell-derived+dopamine+neurons.">In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.</a> Journal of Neurochemistry. 92, 1265-1276 (2005).</li><li>Buytaert-Hoefen, K. A., Alvarez, E., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tyrosine+hydroxylase+positive+neurons+from+human+embryonic+stem+cells+after+coculture+with+cellular+substrates+and+exposure+to+GDNF.">Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF.</a> Stem Cells. 22, 669-674 (2004).</li><li>Zeng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+differentiation+of+human+embryonic+stem+cells.">Dopaminergic differentiation of human embryonic stem cells.</a> Stem Cells. 22, 925-940 (2004).</li><li>Yan, Y., et al. <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+dopaminergic+neuronal+subtypes+from+human+embryonic+stem+cells.">Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.</a> Stem Cells. 23, 781-790 (2005).</li><li>Schulz, T. C., et al. <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+human+embryonic+stem+cells+to+dopaminergic+neurons+in+serum-free+suspension+culture.">Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.</a> Stem Cells. 22, 1218-1238 (2004).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+in+vitro+from+human+embryonic+stem+cells+treated+with+neurotrophic+factors.">Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.</a> Neuroscience Letters. 359, 99-103 (2004).</li><li>Cho, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 3392-3397 (2008).</li><li>Cooper, O., et al. <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+human+ES+and+Parkinson's+disease+iPS+cells+into+ventral+midbrain+dopaminergic+neurons+requires+a+high+activity+form+of+SHH,+FGF8a+and+specific+regionalization+by+retinoic+acid.">Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.</a> Molecular and Cellular Neurosciences. 45, 258-266 (2010).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27, 275-280 (2009).</li><li>Sanchez-Danes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+A9+midbrain+dopaminergic+neurons+by+lentiviral+delivery+of+LMX1A+in+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells.</a> Human Gene Therapy. 23, 56-69 (2012).</li><li>Fasano, C. A., Chambers, S. M., Lee, G., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+derivation+of+functional+floor+plate+tissue+from+human+embryonic+stem+cells.">Efficient derivation of functional floor plate tissue from human embryonic stem cells.</a> Cell Stem Cell. 6, 336-347 (2010).</li><li>Kriks, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Xi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+midbrain+dopamine+neurons+from+primate+pluripotent+stem+cells.">Specification of midbrain dopamine neurons from primate pluripotent stem cells.</a> Stem Cells. 30, 1655-1663 (2012).</li><li>Kirkeby, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+regionally+specified+neural+progenitors+and+functional+neurons+from+human+embryonic+stem+cells+under+defined+conditions.">Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions.</a> Cell Reports. 1, 703-714 (2012).</li><li>Mendez, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+analysis+of+functional+fetal+dopamine+cell+suspension+transplants+in+the+striatum+and+substantia+nigra+of+patients+with+Parkinson's+disease.">Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.</a> Brain: a Journal of Neurology. 128, 1498-1510 (2005).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Nishimura, K., Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+application+of+stem+cell+technology+toward+the+treatment+of+Parkinson's+disease.">Therapeutic application of stem cell technology toward the treatment of Parkinson's disease.</a> Biological &amp; Pharmaceutical Bulletin. 36, 171-175 (2013).</li><li>Smidt, M. P., Burbach, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+mesodiencephalic+dopaminergic+neuron.">How to make a mesodiencephalic dopaminergic neuron.</a> Nature Reviews. Neuroscience. 8, 21-32 (2007).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin+and+bFGF+cooperate+to+maintain+the+pluripotency+of+human+embryonic+stem+cells+in+the+absence+of+feeder+layers.">Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.</a> Biochemical and Biophysical Research Communications. 330, 934-942 (2005).</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> Proceedings of the National Academy of Sciences of the United States of America. 99, 14071-14076 (2002).</li></ol>

Menschlichen pluripotenten Stammzellen (hES und einschließlich der beiden hiPSCs) in vitro differenziert werden, um die meisten zu erzeugen, wenn nicht alle, Zellarten des Körpers einschließlich der DA-Neuronen, die in früheren Studien 19 nachgewiesen wurde. Hier, auf der Kenntnis von embryonalen dopaminergen Neuronen-Entwicklung 20 und veröffentlichte Protokolle von anderen Laboratorien 14,15 optimierten wir die Kulturbedingungen für die Erzeugung von DA-Neuronen aus HES ...

Dopaminergen (DA) Neuronen in verschiedenen Hirnregionen, einschließlich des Mittelhirns, dem Hypothalamus, der Netzhaut und Riechkolben finden. Die A9 DA Neurone in der Substantia nigra pars compacta (SNpc) Regelverhalten und Bewegung durch die Projektion zum Striatum im Vorderhirn und Formen des extrapyramidal-motorischen Systems. Degeneration der A9 DA Neuronen führt zu Parkinson-Krankheit (PD), die die zweithäufigste neurodegenerative Erkrankung menschlichen und derzeit unheilbar ist. Zellersatztherapie ist eine der vielversprechendsten Strategien für die Behandlung von PD 1, daher besteht ein großes Interesse bei der Ableitung A9 DA-Neuronen von menschlichen pluripotenten Stammzellen (hPSCs), die sowohl menschliche embryonale Stammzellen (hES) und jüngsten Menschen induzierten pluripotenten Stammzellen (hiPSCs). Viel Forschung hat versucht, A9 DA Neuronen aus hPSCs mit verschiedenen Methoden abzuleiten. Die frühesten Berichte alle generierten DA Neuronen durch ein neuronalesRosette Vorläuferstadium. Durch Co-Kultivierung mit MS5 2 oder PA6 3-5 Stromazellen mit oder ohne externe Wachstumsfaktoren für 2-4 Wochen, L. Studer und Kollegen und drei anderen Gruppen erfolgreich induzierte neuronale hES Rosetten zu produzieren. Sie bereicherten dann diese Rosetten durch mechanische Präparation oder enzymatische Verdauung zur weiteren Differenzierung. In anderen Berichten, erzeugt Forscher neuralen Rosetten durch Embryoidkörper (EB) schwimmenden Kultur Differenzierung 6-9. Später etablierte Forscher eine Monoschicht auf Basis Differenzierung Methode 10,11, wo sie vergoldet und hES hiPSCs auf extrazellulären Matrix, fügte verschiedene Wachstumsfaktoren in der Kultur, die Differenzierung von hPSCs DA Neuronen, die die In-vivo-embryonalen Entwicklung DA Neuronen imitiert induzieren . Obwohl alle diese Studien erhalten Tyrosinhydroxylase (TH) exprimierenden Zellen mit einigen Merkmalen der DA-Neuronen, die gesamte Prozesszeit Differenzierung und arbeitsaufwendig,im allgemeinen ineffizient, und noch wichtiger, sind die A9 Identität dieser Neuronen in den meisten Studien, außer dem einen mit LMX1A ektopische Expression 12 gezeigt. Kürzlich wurde eine neue Bodenplatte (FP) basierenden Protokoll wurde entwickelt, 13-16, bei der die FP-Vorstufen mit DA Neuron Potential wurden zuerst durch Aktivierung des Sonic-Hedgehog und Wnt-Signalwege während der frühen Phase der Differenzierung erzeugt wird, und dann Die FP-Zellen wurden weiter auf DA-Neuronen angegeben. Obwohl dieses Protokoll ist effizienter, gibt es noch einige Probleme; zum Beispiel, dauert der gesamte Differenzierung lange Zeit (mindestens 35 Tage) und feeder-Zelle abhängig 15 oder 16 abhängig ist, EB oder A9 Identität nicht nachgewiesen wurde 14. Hier, auf der Basis der Kenntnis von in vivo embryonalen DA Neuronenentwicklung und andere Forscher veröffentlichten Ergebnisse haben wir die Kulturbedingungen für die eff optimiertiziente Generation von DA Neuronen von beiden HES-Zellen und hiPSCs. Zunächst erzeugt FP-Vorläuferzellen durch Aktivierung des kanonischen Wnt-Signalmolekül mit kleinen CHIR99021 und Sonic Hedgehog-Signal mit kleinen Molekülen und SAG purmorphamine. Diese FP-Zellen exprimieren FOXA2, LMX1A, CORIN, OTX2 und NESTIN. Wir angegebene dann diese FP-Zellen zu DA Neuronen mit Wachstumsfaktoren, einschließlich BDNF, GDNF, etc. Die erzeugten DA Neuronen der A9 Zelltyp, wie sie sind für GIRK2 positive und negative für Calbindin 17. Dieses Protokoll ist Feederzell-oder EB-unabhängigen, hocheffiziente und reproduzierbar. Mit diesem Protokoll kann man DA Neuronen in weniger als 4 Wochen aus hES-Zellen oder hiPSCs von Normalpersonen für Zelltransplantation Studie oder von hiPSCs von PD-Patienten für die in vitro Modellierung der PD oder Test potentielle therapeutische Mittel für PD abzuleiten.

<table border="0" cellpadding="0" cellspacing="0" width="778">
	<tbody>
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			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">26140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Collagenase IV</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Growth factor reduced matrigel</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">Accutase</td>
			<td style="width:184px;">MP Biomedicals</td>
			<td style="width:119px;">1000449</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SB431542</td>
			<td style="width:184px;">Tocris Bioscience</td>
			<td style="width:119px;">1614</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
			<td style="width:184px;">Stemgent</td>
			<td style="width:119px;">04-0074</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">423-F8-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">CHIR99021</td>
			<td style="width:184px;">Cellagen Technology</td>
			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

directed dopaminergic neuron differentiation from human pluripotent stem cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

Ein Überblick über die Differenzierung Protokoll ist in 1 dargestellt. Die Effizienz der hier vorgestellten Differenzierungsprotokoll beruht auf den Status der Ausgangszellen. Daher ist es entscheidend, um sicherzustellen, dass alle ersten differenzierten Kolonien entfernt, bevor Dissoziieren hPSCs in einzelne Zellen zur Differenzierung und zweitens verbraucht eine meisten, wenn nicht alle, der MEF-Feeder-Zellen durch Inkubation der Zellen auf Gelatine-beschichteten Platten 30 Minuten, und die dritte,...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Regie dopaminergen Neuron Differenzierung von humanen pluripotenten Stammzellen

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Research

JoVE Journal

Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Generierung von neuralen Stammzellen aus menschlichen Föten Ausrangierte Rindengewebe

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Forschung

Neurowissenschaften

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Effiziente Ableitung von humanen neuronalen Vorläuferzellen und Neuronen aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Die Spezifikation der telencephalen glutamatergen Neuronen von menschlichen pluripotenten Stammzellen

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Ein cGMP-Verfahren zur Erweiterung anwendbar Aggregate von humanen neuralen Stamm-und Vorläuferzellen, abgeleitet von pluripotenten Stammzellen oder fetalen Hirngewebe

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Feeder freie Ableitung von Neural Crest Vorläuferzellen aus menschlichen pluripotenten Stammzellen

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

Differenzierung der embryonalen Stammzellen der Maus in kortikalen Interneuron Vorstufen

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Post-Differenzierung Replating von Human Pluripotent Stem Cell abgeleiteten Neuronen für High-Content Screening von Neuritogenese und Synapse Reifung

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Neuronale Differenzierung von Maus embryonalen Stammzellen In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Ableitung, Expansion, Kryokonservierung und Charakterisierung von mikrovaskulären Endothelzellen des Gehirns aus humaninduzierten Pluripotenten Stammzellen

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

Generierung humaner Neuronen und Oligodendrozyten aus pluripotenten Stammzellen zur Modellierung von Neuron-Oligodendrozyten-Interaktionen

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...